Facultade de Bioloxía
Departamento de Bioloxía Vexetal e Ciencia do Solo
Phytotoxic effect of allelochemicals, herbicides and
Acacia melanoxylon R. Br. on physiological processes
in four plant species
Memoria presentada por el Licenciado
Muhammad Iftikhar Hussain
Para aspirar al grado de Doctor por la Universidad de Vigo
con la Mención de Doctor Europeo
Vigo, Octubre 2010
Manuel Joaquín Reigosa, Catedrático de Fisiología Vegetal en el Departamento de
Bioloxía Vexetal e Ciencia do Solo de la Universidade de Vigo,
INFORMA:
La memoria presentada por el Licenciado Muhammad Iftikhar Hussain para aspirar
al grado de Doctor por la Universidad de Vigo con la Mención de Doctor Europeo
que lleva por titulo ―Phytotoxic effect of allelochemicals, herbicides and
Acacia melanoxylon R. Br. on physiological processes in four plant
species‖ fue realizada bajo mi dirección. Considerando que tiene suficiente
entidad para constituir un trabajo de Tesis Doctoral, autorizo su presentación para
ser sometida a los trámites oportunos.
Y para que así conste, firmo el presente informe en Vigo a 18 de Octubre de 2010.
Fdo. Manuel Joaquín Reigosa
To Uzma, you’re indefinite LOVE....
And to my father, for his affectionate guidance
“Idea of the self is not logically dependent on any physical thing, and that the soul
should not be seen in relative terms, but as a primary given, a substance”
Avicenna
(980-1037)
LIST OF THE RESEARCH ARTICLES
This thesis is based on the following original research articles. Additionally, some
unpublished results are presented.
Hussain, M.I., González, L., and Reigosa, M.J. 2008. Germination and growth response
of four plant species towards different allelochemicals and herbicides. Allelopathy
Journal. 22 (1): 101-110.
Hussain, M.I., González, L., and Reigosa, M.J. 2010. Phytotoxic effect of
allelochemicals and herbicides on photosynthesis, growth and carbon isotope
discrimination in Lactuca sativa. Allelopathy Journal. 26 (2): 157-174.
Hussain, M.I., González, L., and Reigosa, M.J. 2010. Allelopathic potential of Acacia
melanoxylon R. Br. on the germination and root growth of native grasses. Weed
Biology and Management, in press
Hussain, M.I., Chiapusio, G., González, L., and Reigosa, M.J. 2010. Benzoxazolin2(3H)-one (BOA) induced changes in leaf water relations, photosynthesis and carbon
isotope discrimination in Lactuca sativa. Plant Physiology and Biochemistry (accepted)
Hussain, M.I., González, L., Souto, C., and Reigosa, M.J. 2010. Ecophysiological
responses of three native grass species to phytotoxic effect of Acacia melanoxylon R.
Br. Agroforestry System (under review).
Hussain, M.I., and Reigosa, M.J. 2010. Cinnamic acid inhibited photosynthetic
efficiency, yield and non-photochemical fluorescence quenching in leaves of Lactuca
sativa L. Environmental and Experimental Botany (under review).
i
POSTER COMMUNICATIONS
Hussain, M.I., González, L., Reigosa, M.J. 2009. Inhibition of photosynthetic
efficiency, leaf water relations, growth and stable isotopes composition (δ13C, δ15N) in
lettuce under trans-cinnamic acid stress. First International Conference of Asian
Allelopathy Society. South China Agricultural University, Guangzhou, China.
December 18 – 22, 2009.
Hussain, M.I., González, L., Reigosa, M.J. 2009. Invasive Acacia melanoxylon R. Br.
inhibited photosynthetic efficiency, yield and non-photochemical quenching in Lactuca
sativa. First International Conference of Asian Allelopathy Society. South China
Agricultural University, Guangzhou, China. December 18 – 22, 2009.
Lorenzo, P., González, L., Hussain, M.I., Reigosa, M.J. 2009. The invasive Acacia
dealbata Link: an overview of its invasive process. First International Conference of
Asian Allelopathy Society. South China Agricultural University, Guangzhou, China.
December 18 – 22, 2009.
Hussain, M.I. González, L., Sanchez-Moreiras, A.M., Pedrol, N., Reigosa, M.J. 2009.
Carbon and nitrogen isotopes discrimination in Lactuca sativa under allelopathic stress
of Acacia melanoxylon R. Br. XI Congreso Hispano-Luso de Fisiologia Vegetal
(SEFV), University of Zaragoza, Spain. September 8-11, 2009.
Hussain, M.I., González, L., Reigosa, M.J. 2008. Describing phytotoxic effects of
Blackwood (Acacia melanoxylon) on cumulative germination. 5th World Congress on
Allelopathy, “Growing Awareness of the Role of Allelopathy in Ecological,
Agricultural, and Environmental Processes, Saratoga Springs, New York, USA.
September, 21-25, 2008.
Hussain, M.I., González, L., Reigosa, M.J. 2008. Potencial alelopático de la especie
invasora Acacia melanoxylon en Galicia: efecto sobre los índices de germinación. II
congreso de agroecoloxía e agricultura ecolóxica en Galiza. Spanish Society of
Ecological Agriculture, Monforte de Lemos (Lugo), May, 2-4, 2008.
ii
Acknowledgement
I would like to express my deep and sincere gratitude to Prof. Dr. Manuel J.
Reigosa for his kindness, admirable supervision, direct guidance and fruitful and
interesting discussions throughout this work. He assisted in all matters, provided
solutions to different problems and always gave input even at times of personal
anxieties.
My special thanks to my father, Muhammad Hussain, who all the time respect my
ideas and gave me opportunity to go abroad for this study and my wife (Uzma) for
lovely smiles that can relieve any kind of tiredness. Thanks to my brother
(Zulfiqar), my sisters (Sughran, Parveen, Sofia, Samina) for their indispensible
love and kindness who ever gave me encouragement to get higher ideas of life.
Deepest gratitude is due to Dr. Luis González who supported and helped me from
beginning till the end of the study. He always helped me in the Lab., in the field
for collecting Acacia branches in the forests, and administration work in the third
cycle office of the University. I am highly thankful to Dr. Nuria Pedrol for help in
water potential measurements, SPSS analysis and Dr. Adela for valuable
discussions. I am extremely thankful to Dr. Carlos Souto for conducting HPLC
analysis of Acacia melanoxylon flowers and phyllodes.
My deepest thanks to Paula, Tamara, Joanna, Marga, Aldo and Carlos for giving me
company and tiredness help throughout this study and helping me in the Lab and in
the glasshouse. I am grateful to Jesús Estévez Sío and Jorge Millos for their
technical assistance in isotope ratio mass spectroscopy analysis. I am grateful and
deeply indebted to the staff and members of the Faculty of Biology for their
generous encouragement and kind help during my stay in the faculty.
I would like to express my deep thanks to the Vice Rector of Research, University
of Vigo for providing a fellowship to conduct research on Sphagnum moss in
University of Franche-Comte, Montbeliard, France. I am also thankful to them to
provide me a travel grant to attend Asian Allelopathy conference in Guangzhou,
China.
I am highly thankful to Dr. Genevieve Chiapusio to welcome me in her lab for
specific project of Sphagnum and friends from Montbeliard, Raheel, Mohannad,
Noeful, Muhammad, Dehlia, Khado, Noe, for giving me very enjoyable and
unforgettable company.
iii
Last but not least, I express my gratitude to all those friends from Taj Mahal,
Mumtaz, Yaser, Liaquat, Raja Tahir, Aziz Sahib and Asjad that provide me
unforgettable company and supported me when I needed. And among others,
especially Maite, Sonia and Chus with you because I do not need to explain
anything, Ravi, Chema, Lazhar, Sar, Jurjen, Hamdi and Markus for their ability to
make me unwind and enjoy the little things, and Rodolfo, Manuel, Alberto, Jose
and Josera, who always welcome me in their homes with love.
To all of you, thank you very much.
Muhammad Iftikhar Hussain
Vigo, España
iv
CONTENTS
Chapter I. Introduction
Background ......................................................................................................... 2
Allelopathy and resource competition ................................................................ 4
Allelopathy phenomena ...........................................................................................5
Production and release of allelochemicals .......................................................... 6
Classification of allelochemicals ........................................................................ 6
Phenolic compounds ........................................................................................... 6
Simple phenolics................................................................................................. 6
Flavonoides ......................................................................................................... 8
Usnic acid and lichen metabolites .................................................................... 10
Terpenoids ........................................................................................................ 10
Monoterpenoids ................................................................................................ 10
Sesquiterpene lactones ...................................................................................... 11
Quassinoids ....................................................................................................... 15
Benzoxazinoids ................................................................................................. 16
Glucosinolates .................................................................................................. 18
Chapter II. Germination and growth response of four plant species towards
different allelochemicals and herbicides
Abstract ............................................................................................................. 29
Introduction ...................................................................................................... 30
Materials and Methods ..................................................................................... 31
Results and discussion ...................................................................................... 32
Literature cited .................................................................................................. 34
v
Chapter III. Phytotoxic effect of allelochemicals and herbicides on photosynthesis,
growth and carbon isotope discrimination in Lactuca sativa
Abstract ............................................................................................................. 42
Introduction ....................................................................................................... 43
Materials and Methods ...................................................................................... 44
Results ............................................................................................................... 47
Discussion ......................................................................................................... 54
Literature cited .................................................................................................. 58
Chapter IV. Allelopathic potential of Acacia melanoxylon R. Br. on the
germination and root growth of native grasses
Abstract ............................................................................................................. 64
Introduction ....................................................................................................... 65
Materials and Methods ...................................................................................... 66
Results ............................................................................................................... 67
Discussion ......................................................................................................... 69
Literature cited .................................................................................................. 71
Chapter V. Ecophysiological responses of native grass species to phytotoxic effect
of Acacia melanoxylon R. Br.
Abstract ............................................................................................................. 86
Introduction ....................................................................................................... 88
Materials and Methods ...................................................................................... 89
Results ............................................................................................................... 94
Discussion ......................................................................................................... 96
Literature cited .................................................................................................. 99
Chapter VI. Benzoxazolin-2(3H)-one (BOA) induced changes in leaf water
relations, photosynthesis and carbon isotope discrimination in Lactuca sativa
Abstract ........................................................................................................... 121
Introduction ..................................................................................................... 123
vi
Materials and Methods ................................................................................... 130
Results ............................................................................................................ 124
Discussion ....................................................................................................... 126
Literature cited ................................................................................................ 134
Chapter VII. Cinnamic acid inhibited photosynthetic efficiency, yield and nonphotochemical fluorescence quenching in leaves of Lactuca sativa L.
Abstract ........................................................................................................... 151
Introduction .................................................................................................... 152
Materials and Methods ................................................................................... 153
Results ............................................................................................................ 156
Discussion ....................................................................................................... 158
Literature cited ................................................................................................ 161
Chapter VIII. General Discussion
General Discussion ......................................................................................... 174
Conclusion ...................................................................................................... 179
Literature cited ................................................................................................ 180
Chapter IX. Summary
Summary ......................................................................................................... 184
Suggestion for future research lines ............................................................... 186
Résumé ........................................................................................................... 187
Literature cited ................................................................................................ 191
vii
viii
Introduction
Chapter I
Chapter I. Introduction
Background
Herbicides are used to limit reduction in crop yield and quality due to weed competition, yield
contamination and interference with harvesting. Herbicide use has undoubtedly contributed to
crop yield increases and the efficiency of production. However, their widespread use may have
detrimental and unexpected effects on wildlife both within crops and in associated semi-natural
habitats in farmland. Moreover, Herbicide-resistant weeds have been identified in 21 European
countries, with the highest number of resistant biotypes found in France (30), Spain (26), United
Kingdom (24), Belgium (18) and Germany (18). Consequently, herbicide-resistance appears to
be mainly a problem in western-Europe. These 21 European countries represent 36 % of the 59
countries world-wide in which herbicide resistant weeds have so far been detected (Heap, 2004;
Moss, 2004). Resistance has evolved in 55 species in Europe and while there is no clear
relationship between plant families or genera and their tendency to evolve resistance, grass
weeds tend to be over-represented in the list of resistant biotypes both within Europe and worldwide. The number of species that have evolved resistance and the number of countries in which
they occur is a useful indication of the frequency of occurrence of resistance.
Nowadays, we know that these compounds (dichlobenil, glyphosate, 2,4-D, sulfonylurea,
bromoxynil, etc.) produce not only dangerous consequences for the human health altering the
hormonal homeostasis and provoking gene damages (Cox, 1991; Allsopp et al., 1995), but also
pest and weed resistance (Gill, 1995; Gressel and Baltazar, 1996; Stroud, 1998), sea and
continental water contamination, and soil erosion (Garrity, 1993; Crosby, 1996). And even so,
around 40% of crops are lost each year because of weeds, insects or pathogens. For example,
more than 8 billion dollars are lost each year in USA due to weeds invading crops (Putnam and
Weston, 1986). Moreover, the critical situation of productivity motivated that some industries
related with the production of pesticides started to produce resistant crops to these pesticides. It
avoids that crops are affected by the use of these synthetic compounds, but it kills the rest of the
plants present in the field (harmful as well as beneficial) and decreases, even more, the species
biodiversity. That is the case of transgenic alfalfa, canola, maize, cotton, potato, rice or beet,
between others, which are resistant to one or more herbicides (Steinbrecher, 1996; Steyer, 1996;
Altieri, 1998).
At this point, it seems necessary to make drastic changes in the agricultural concept, but
reducing productivity for reducing pesticide use is not the solution. United Nation Organization
estimates that the population living in our planet will increase to 9,000 million people in 50
years. Therefore, the challenge for this century must be to increase agricultural production
without environmental degradation. In the recent years a new concept of agriculture "sustainable
agriculture" is starting to result familiar in our ears. "Agroecology" pretends to establish the
ecological bases for the conservation of biodiversity in agriculture, of the ecological equilibrium
in agroecosystems and ensures a decrease in the contamination by culture practices (Altieri and
Nicholls, 2000). Some of the proposed friendly practices are the use of crop rotation, crop
associations, organic fertilizers or sustainable weed management (Altieri and Doll, 1978; Anaya
et al., 1987; Blacklow, 1997). Since agriculture began, the farmers used crop association and
crop rotation in an intuitive way for improving their crops, taking advantage of chemical
2
Introduction
interference among plants to control the cultivations. The base of these associations takes root
of plant interference. In some demonstrated cases this interference was allelopathic. Biological
control methods are a useful option for longer-term control of some specific weed species. In
this way, a natural compound harmful to a weed‘s growth or reproduction can provide ongoing
weed management.
The recent tendencies in weed management defend the damage and/or displacement of
weeds in the agro-ecosystem but not the total eradication of them (as usually happens with
commercial herbicides). At this point, the role of allelopathic compounds can be determinant
because of their high potential as possible natural herbicides (Dayan et al., 1999; Duke et al.,
2000). Weed management must protect environmental quality and human health, and
allelochemicals released from live crops and crop residues can be used this way to damage
weeds and improve crop performance (Liebman, 2001). In the last years, different
allelochemicals have been investigated and assayed to act as new environmentally friendly
herbicides in the field. In an ecological point of view, natural herbicides will have some
important advantages over the commercial herbicides because they could be biodegradable
(Einhellig, 1993; Macías, 1995) and probably more toxicologically safe than synthetic
herbicides. They can be used directly in the field via living crops (e.g. Bialaphos from
Streptomyces spp., tentoxin from Alternaria alternata, artemisinin from Artemisia annua,
sorgoleone from Sorghum bicolor, and other compounds (Duke and Lydon, 1987; Lydon and
Duke, 1999; Dayan et al., 1999; Streibig et al., 1999) or as crop residues (Barnes and Putnam,
1983; Shilling et al., 1985). Natural herbicides can be used also to generate new synthetic
herbicides (Knudsen et al., 2000) or for using transgenes to generate higher concentrations of
allelochemical in the plant (Lydon, 1996).
Secondary metabolites produced and accumulated by plants can induce both inhibitory and
stimulatory effects on organisms and may play roles in shaping plant and microbial
communities (Pennacchio et al., 2005). Germination inhibitory compounds and allelochemicals
are phytotoxic compounds produced by plants that aid them in both interspecific and
intraspecific competitions (Meyer et al., 2007). The search for allelochemicals/phytotoxins is a
growing research field, because these compounds have a great potential for controlling noxious
weeds and could be used as herbicides in agriculture (Singh et al., 2003). Concerns about
ecological, environmental, and health problems possibly associated with synthetic chemicals
have increased interest in the development of new classes of environmentally safe herbicides
(Dayan et al., 1999). The ability of a plant species to inhibit the germination of other plants is an
untapped resource for weed control in crops that could revolutionize organic crop production.
The study of the phytotoxic potential offers useful clues in the investigation of new models of
natural herbicides that could be more specific and less harmful than the synthetic substances
used in agriculture (Singh et al., 2003; Bogatek et al., 2006; Hachinohe and Matsumoto, 2007).
The natural plant products have a number of advantages over synthetic herbicides as they
usually possess complex structures, exhibit structural diversity and hence are invaluable sources
of lead compounds; have more chiral centers; have high molecular weight with no or low
amount of halogens or heavy atoms; are environmentally benign as they degrade rapidly in the
environment; and have novel target sites of action different from the synthetic herbicides (Duke
3
Chapter I. Introduction
et al., 2000, 2002; Singh et al., 2003). Like so, the occurrence in nature of the phenomenon of
‗Allelopathy‘ must depend on any of the following conditions (Reigosa et al., 1999):
a) Phytotoxins are continuously released to the environment
b) The existence of special soil conditions that prevent allelochemical degradation
c) The release of the allelochemical simultaneous to the most sensitive life cycle event of
the target species
d) A non-native species releasing the allelochemicals that are new for the receptor species
and for the soil microorganisms (Reigosa et al., 1996).
Duke et al. (2002) clarifies the potential role of allelochemicals as herbicides for their
direct or bio-activated use against the weeds, as well as the alternatives in the transgenic
management of allelopathic plants to obtain a more available, active and stable concentration of
allelochemicals in agro-ecosystems. Duke displays in this review the advantages
(environmentally-friendly, reduced toxicity, high chemical structure diversity, new molecular
target sites, less resistances, etc.) and the inconveniences (sometimes very costly, environmental
half-life too short, difficult uptake and translocation by plants or reduced concentrations in
nature) of adapting these natural phytotoxins for their use in the field. They conclude that more
efforts must be done in the future for the production by transgenic techniques of highly
allelopathic crops in the search of an efficient natural herbicide. This must be carefully accepted
because of the serious consequences that the extensive use of transgenic crops could represent in
the agro-ecosystem (Altieri, 1998).
ALLELOPATHY AND RESOURCE COMPETITION
Changes in the ecosystem may be driven by autogenic (change conducted by plants or it comes
from within the environment), allogenic (change from the outside as in continued disturbance)
or a combination of both factors (a more ecosystem approach rather than a plant species
approach) (Tansley, 1935). They will determine the succession as a sequential response to
disturbance, where plants develop chemical and physical methods, as well as physiological and
biochemical systems to cope with the environment. This succession will have multiple causes
with contributing processes and modifying factors, which determine the diversity and the
evolution in the plant community. Any minimum and imperceptible change can be determinant
in the ecological succession (Shelley and Rinella, 2001).
Focusing on interference (Muller, 1966), plant interactions such as resource competition or
allelopathy play an important role in the species performance and their establishment in the
field. Dynamics for resource competition in plants (specially light, nutrients and water) is
essential in the understanding of ecological succession (Tilman, 1990; Chapin, 1993), but
chemical interaction among plants plays also a very important role in this primary succession
(Rice, 1984). Their effects on plant metabolism are well known as isolated mechanisms (Mahall
and Callaway, 1992), but their effects on plant community have not been deeply investigated. In
4
Introduction
this way, different works suggested also that allelopathy and resource competition could act
synergically (Rasmussen and Einhellig, 1977; Einhellig, 1986; Mallik, 1998).
ALLELOPATHY PHENOMENA
The idea that plants affect neighboring plants by releasing chemicals in the environment has
been known since c. 370 BC (Willis 1997). Greeks and Romans used this knowledge in
practicing agriculture as early as 64 AD. The antagonistic effects of certain tree species such as
walnut tree (Juglan spp.) on understory plants and nearby crops were also known to humans
centuries ago (Rizvi and Rizvi 1992; Willis 2000, 2002, 2004). Poor yield of repeated
cultivation of certain crops and fruits due to so-called ‗soil sickness‘ has been known and
investigated since the beginning of horticulture. Although this form of plant–plant interference
has been known for quite some time, it is only recently (1937) that the Austrian plant
physiologist, Hans Molisch, gave it a formal name, allelopathy (Molisch 1937, 2001). During its
long history, allelopathy was perceived as a donor–receiver phenomenon where one plant
releases chemicals that affect the growth of the neighboring plants mostly in agricultural and
horticultural settings. The idea of allelopathy as an ecological phenomenon structuring plant
communities is rather recent. In the 1960s and 1970s through a series of field and
complimentary laboratory studies; several authors provided data showing that certain plants can
influence their neighboring community of plants directly by releasing allelochemicals and
indirectly by affecting the activity of rhizosphere microbes (Muller 1966; Whittaker and Feeny,
1970).
Rice (1984) defined allelopathy as the effect of one plant (including microorganisms) on growth
of another plant through the release of chemical compounds into the environment. Both positive
and negative effects are included in this definition. The definition given by Rice is so broad that
it covers almost all aspects of chemical ecology of plants. However, their assertion was met
with skepticism following a demonstration that bare ground created around allopathic shrubs
can be invaded by plants after removal of herbivores (Bartholomew 1970) generating the
famous criticism from J.L. Harper who described allelopathy as a complex ‗undeniably natural
phenomenon‘ but ‗nearly impossible to prove‘ (Harper 1975).
The following four decades experienced a great deal of skepticism by the general plant
ecologists in accepting almost any results suggesting the presence of allelopathy as a viable
explanation of plant–plant interaction principally on the ground that effects of other factors have
not been removed in demonstrating allelopathy. Unreasonable ‗burden of proof‘ was imposed
on the researchers proposing allelopathy as a plant–plant interference mechanism (Williamson
1992). By the end of the last century several authors suggested that allelopathy can not only
affect neighboring plants and influence plant community structuring, but it can also induce a
broader ecosystem level change when it coincides with disturbance (Zackrisson et al. 1997;
Wardle et al. 1997; Mallik, 1995).
Blum et al. (1999) proposed three criteria to establish evidence for allelopathy; These are: (1)
The putative aggressor plant or its debris must produce/contain and release chemicals that
5
Chapter I. Introduction
ultimately will be capable of inhibiting growth or function of another plant when these
chemicals are released into the soil environment, (2) Distribution and accumulation of organic
complexes (i.e., promoter-inhibitor) dominated by inhibitor (i.e., phenolic acids) in soil must be
of sufficient concentration to inhibit nutrient and/or water uptake by roots and/or energy fixation
by the sensitive plants, and (3) The observed field patterns of plant inhibition cannot be
explained solely by physical factors or other biotic factors. These authors, however, recognized
the difficulties in implementing these criteria in field situations.
PRODUCTION AND RELEASE OF ALLELOCHEMICALS
Allelopathic active compounds from terrestrial plants generally act as natural herbicides and
often have multiple effects on the metabolism of target organisms (Einhelling, 2001). Some
have been explored as natural substitutes for commercial herbicides (Duke et al., 2000).
Allelochemicals are present in specialized organs of plants and their amount varied in different
plant organs (Tongma et al., 2001), plant variety (Czarnota et al., 2001) and have the potential
as either herbicides or templates for new herbicide classes (Inderjit and Duke, 2003).
Allelopathic substances may be released from plant tissue in a variety of ways, including
exudation of volatile chemicals from living plant parts (Abrahim et al., 2000); leaching of water
soluble chemicals from aboveground parts in response to action of rain, fog, or dew (Carballeira
and Reigosa, 1999) and release of toxic chemicals from nonliving plant parts through leaching
of toxins from litter, plant cells/tissue decomposed by microorganisms (Souto et al., 2001).
CLASSIFICATION OF ALLELOCHEMICALS
Phenolic compounds
These constitute a wide group of allelochemicals comprising structures with different degrees of
chemical complexity: simple benzoic and cinnamic derivatives, flavonoids, polyphenols and,
recently, depsides, depsidones and other aromatic compounds of lichen origin. The most recent
advances in each category will be commented upon briefly.
Simple phenolics
Phenol derivatives ranging to furano- and piranocoumarins have often been reported as
allelopathic agents (Einhellig, 2004). Benzoic and cinnamic acids are among the most
commonly referred to allelopathic agents (Fig. 1). They have been considered as primary factors
of allelopathy in forest ecosystems, being responsible for successional effects (Sidari and
Muscolo, 2004) and difficulties in reforestation, Singh et al., 1999) as well as autotoxicity in
economic plants such as asparagus (Miller et al., 1991) and coffee (Chou and Waller, 1980). In
the case of tea and coffee, autotoxicity has been related to the caffeine released from litter and
seed senescence and decomposition (Anaya et al., 2002).
6
Introduction
Figure 1. Common benzoic and cinnamic acid derivatives described as allelopathic agents
More recently, polyphenols such as ellagic, gallic and pyrogallic acids along with the flavonoid
(+)-catechin (Fig. 1) isolated from the macrophyte Myriophyllum spicatum L. have been
reported as growth inhibitors of the blue-green alga Microcystis aeruginosa Kütz., (Nakai et al.,
2000) thus depicting the growing interest in the study of allelopathic phenomena in aquatic
ecosystems. Application of these compounds in lakes, ponds and fisheries could be of interest in
solving problems as diverse as algal blooms in eutrophised systems or musty odour in fishes
owing to accumulation of certain microalgae in their skins (Schrader et al., 1999).
However, few authors in allelopathy acknowledge that benzoic and/or cinnamic acid derivatives
are widespread in the plant kingdom and possess important structural functions, as they are key
steps in the biosynthetic pathway of lignins (e.g. in their reduced forms of p-coumaryl, coniferyl
and sinapyl alcohols) and have important roles in plant physiology (Siegler, 2003). Salicylic
acid itself is a mediator for the development of systemic acquired (induced) resistance in a
plant's defense against disease, among other functions (Klessig et al., 1994). In addition,
phenolic acids in soils are subjected to many chemical and biochemical transformations that
deplete their real concentrations: irreversible binding to humic acids, sorption onto soil
particles, ionization, chemical oxidation to quinones and use by bacteria as a carbon source are
among the most important (Blum, 2004). These processes might deplete the concentrations in
the rhizosphere and soil, so many authors suggest that allelopathic effects by phenolic acids are
highly unlikely (Smith and Ley, 1999) and the levels reached are not high enough to reproduce
the levels of phytotoxicity obtained in vitro. Consequently, many cases of suspected allelopathy
attributed to benzoic and cinnamic acids should be reviewed.
It is the present authors' opinion that compounds appearing early in plant evolution - like the
phenolic acids - provided the plant with an evolutionary advantage at that time. It is
7
Chapter I. Introduction
symptomatic that evolutionarily old plants such as certain gymnosperms (e.g. conifers) exude
phenolic acids in large enough amounts to reach phytotoxic concentrations in soil (Djurdjevic et
al., 2003; Gallet and Pellissier, 1997). Co-evolution probably overcame this first defense barrier
with the development of more sophisticated chemical weapons. Although phenolics are still
present as part of the chemical composition of many plants, most have other important functions
(structural, hormonal, etc.). A good example of functional allelopathic behaviour (Wu et al.,
2001, 2002) due to phenolics can be seen in wheat and other Gramineae. They contain in their
chemical composition hydroxamic and phenolic acids, but benzoxazinoids have been finally
acknowledged as being mainly responsible for the allelopathic effects (Wu et al., 2001, 2002;
Huang et al., 2003).
Quinones (Fig. 2) constitute a different case, in spite of their structural similarity to simple
phenolics. Juglone (Black Walnut tree, Juglans nigra L.) is one of the first examples of an
allelochemical to be described. Bacterial degradation does not seem to remove soil juglone
completely, and consequently it has been shown to accumulate in the soil at high enough
concentrations to be toxic to associated plant species (Shibu, 2002). Sorgoleone and related
compounds (Kagan et al., 2003) are the allelochemicals responsible for the allelopathic effect of
sorghum. They are exuded in large amounts from root hairs as oily droplets (Weidenhamer.
2005; Yang et al., 2004) and accumulate in the soil. Half-life times of 10 days have been
recorded, depending on the soil composition (Demuner et al., 2005), but sorgoleone can be
detected up to 7 weeks later (Weston and Czarnota, 2001). The anthraquinones emodin and
physcion have been described as allelochemicals in Polygonum sachalinense Schmidt (Inoue et
al.,
1992).
Figure 2. Benzo-, naphtho- and anthraquinones reported as allelopathic agents
Flavonoids
Flavonoids have roles associated with colour and pollination (flavones, flavonols, chalcones and
catechins) and disease resistance (phytoalexins). They also have weak oestrogenic activity
(isoflavones and coumestanes, e.g. coumestrol). However, few examples can be found of
allelopathic flavonoids. Examples include kaempferol which has been isolated from the soils
beneath Quercus mongolica Fisch. ex Ledeb. along with other simple phenolic acids (Li et al.,
1993) and ceratiolin, a non-phytotoxic dihydrochalcone present in the leaves of the dominant
shrub of the Florida sandhill community Ceratiola ericoides Michx. that easily degrades under
acid or soil conditions to the phytotoxic dihydrocinnamic acid (Fig. 3) (Williamsom et al.,
8
Introduction
1992). However, the most prominent examples are those regarding invasive species where
flavonoids have been reported as responsible: (-)-catechin in Centaurea maculosa Lam. (Fig.
1), (Bais et al., 2003), robinetin and other flavonoids in Robinia pseudoacacia L. (Nasir et al.,
2005) and the flavonoids isolated from mosses that show inhibition of spore germination against
other moss species (Sorbo et al., 2004). The most often cited allelopathic flavonoids (as free
aglucones or glycosilated) are kaempferol, quercetin and naringenin (Fig. 3) (Berhow and
Vaughn, 1999). It seems, however, that, except in certain special cases, flavonoids are not
usually allelopathic agents and exhibit other roles in the plant.
Figure 3. Some allelopathic flavonoids and thermic degradation of ceratioline to the
allelochemical dihydrocinnamic acid
One of the latest and most enlightening examples is the case of Centaurea maculosa (spotted
knapweed), a European native weed that is causing serious trouble in North America owing to
its high invasive potential (Hook et al., 2004). Recent publications support the belief that its
invasive potential is due to the exudation of the flavan-3-ol (-)-catechin. This compound is
exuded as a racemic mixture where only one of the two enantiomers is phytotoxic while the
other has antimicrobial properties. The concentrations of (-)-catechin detected in North America
soils where C. maculosa was found and were twice in corresponding European soils and
dependent on the distance from knapweed roots (Bais et al., 2003). Moreover, North American
grasses were more susceptible to (-)-catechin than the corresponding European species.
Biochemical and mode-of-action studies showed that (-)-catechin is incorporated into the target
plant through the roots, and it triggers a cascade of events that leads to changes in gene
expression and root system death. All of these data strongly support the theory that the success
of C. maculosa outside its native ecosystem is mainly due to the exudation of a chemical
(catechin) to which native plants are not adapted (Fitter, 2003).
9
Chapter I. Introduction
Usnic acid and lichen metabolites
Usnic acid (Fig. 4) has attracted much attention for several years past, as it is a unique and
relatively abundant chemical in lichens - never reported in higher plants - with a wide array of
biological activities (Ingolfsdottir, 2002). The chemical composition of lichens is usually simple
in comparison with higher plants, having a relatively low number of secondary metabolites,
most of them aromatic. They can be divided into four main classes: depsides, depsidones,
depsones and dibenzofurans (Fig. 4). Quinones, anthraquinones and simple phenols are also
common constituents in lichens. The primary ecological roles of secondary lichen substances
have been classified into four groups: protection against damaging light conditions; chemical
weathering compounds; allelopathic compounds (including antibiotic compounds); and antiherbivore defense compounds (Rundel, 1978). Lichen allelopathy is just starting to be explored,
and little has been done in this field. Other lichen metabolites with phytotoxic activity are the
anthraquinones rhodocladonic acid and emodin. Rhodocladonic acid is a typical anthraquinone
occurring in many lichen species of the family Roccellaceae (Huneck and Yoshimura, 1996),
while emodin (Fig. 3) is a widely distributed anthraquinone in higher plants, fungi and lichens.
The phytotoxic activity of these compounds and several derivatives has been recently reported
(Romagni et al., 2004). Also, the phytotoxic activities of the mixture of major orsellinates,
orsellinic acid dimers (antranorin and evernic acid derivatives) and tetramers (prunastrin)
isolated from the lichen Evernia prusnatri L. have been described (Fig. 4) (Gonzalez et al.,
2002).
Terpenoids
Terpenoids are secondary metabolites present in many organisms, and they have a wide range of
structural diversity and biological activity. Their allelopathic activity has recently resulted in
them being considered as possible leads in the development of new natural product based
agrochemicals (Singh et al., 2003). The most recent and significant examples are discussed
below.
Monoterpenoids
Monoterpenoids are common volatiles that are major constituents of essential oils. They have
been described as responsible for the allelopathic interactions in some plant communities,
especially those comprising aromatic plants in arid or semi-arid zones (Fig. 5) (Angelini et al.,
2003; Williamson et al., 1989). Inhibition of germination has been reported for several
monoterpenes (camphor, pinenes, cineoles) in pure form (Vokou et al., 2003), or as essential oil
mixtures. In spite of the fact that they are lipophilic compounds, their solubility levels (alone or
with natural surfactants such as ursolic acid) are enough to exert phytotoxicity (Weidenhamer,
1993), and they do not need to be dissolved or suspended in water, as they are effective using
air as a carrier (Weidenhammer et al., 1994; Won-Yun et al., 1993). When considering their
possibilities of being herbicidal lead compounds or giving rise to these, it is important to note
the high structural similarity between the monoterpenes 1,4- and 1,8-cineole and the herbicide
cinmethylin. Moreover, it has been demonstrated that cinmethylin acts as a proherbicide that
10
Introduction
suffers metabolic breakdown to give rise to 2-hydroxy-1,4-cineole, the true herbicide (Romagni
et al., 2000).
Figure 4. Lichen metabolites reported to have phytotoxic activity.
Sesquiterpene lactones
Sesquiterpene lactones (SLs) constitute an interesting and well-documented group of
compounds. They present a wide range of biological activities, including insecticidal (Datta and
Saxena, 2001), antibacterial (Saroglou et al., 2005) and antifungal (Ahmed and Abdelgaleil,
2005). SLs are responsible for the bitter taste in the plants containing them and seem to act as
antifeedants, being promising compounds in the development of new insect antifeedants
(Paruch, 2001).
SLs have been reported as allelopathic agents in many cases (Macias et al., 1999; Pandey et al.,
1993) with high levels of activity (Batish et al., 2001; Macias et al., 2000), being particularly
abundant in plants of the family Compositae (Fraga, 2005). Many of the most noxious weeds
contain SLs as allelochemicals, and their invasive potential could be directly related to their
chemical content. Members of the families Centaurea, such as Russian knapweed C. repens L.
[now Acroptilon repens (L.) DC] and yellow starthistle (C. solstitialis L.), contain phytotoxic
11
Chapter I. Introduction
guaianolides such as centaurepensin, solstitiolide, acroptillin and repin, or germacranolides such
as cnicin, cnicin acetate, salonitenolide and salonitenolide angelate (C. derventana Vis & Pan i
and C. kosaninii Hayek) (Fig. 6) (Stevens and Merrill, 1985). Many other examples of invasive
noxious weeds containing SLs could be given that are beyond the scope of this review.
Figure 5. Typical monoterpenes present in essential oils and reported to have allelopathic
and/or phytotoxic activity. Note that 1,4-cineole and 1,8-cineole present structures closely
related to that of the herbicide cinmethylin.
Plants other than invasive weeds may also be considered as allelopathic. In this sense, the case
of sunflower is worth noting because of the economical importance of this crop. The importance
of the ecological roles of SLs and other secondary metabolites as plant defense compounds in
sunflower has been reviewed (Macías et al., 1999).
12
Introduction
Figure 6. Some examples of SLs identified as allelopathic agents in members of Centaurea
(now Acroptilon) species.
Many of the above-mentioned compounds present other biological activities related to a plant
defense role. For example, a strong synergistic insecticidal effect can be observed in the larvae
of Manduca sexta (Joh.) when typical Asteraceae SLs are co-administered with the
photooxidant polyacetylene α-terthienyl, resulting in enhanced lipid peroxidation and larval
mortality (Guillet et al., 2000). However, as mentioned above, most defense activities reported
for SLs are fungicidal and phytotoxic. In any case, these few examples illustrate two basic
concepts:
1. They support the importance of the allelopathic factors in the invasive potential of plant
species.
2. The importance of the economy principle: in order to save energy and carbon resources,
plants tend to synthesise multifunctional compounds that may serve as defence against
several enemies; this leads to synthesis of compounds having a wide array of biological
activities that eventually may end up in the discovery of useful compounds for mankind,
as in the case of artemisinin.
The importance of lipophilicity and other physical properties in agrochemical formulation has
been acknowledged (Barbosa et al., 2004), as they determine an easy membrane crossing or an
adequate water solubility. This has been previously demonstrated for the citotoxicity of SL, as
the latter has been shown to be directly related to the size and lipophilicity of the ester side
chain in 1,1,α-13-dihydrohelenanolides (Beekman et al., 1997), but it has also been shown to
occur with phytotoxic activity (Macias et al., 2005). The model proposed fits Lipinski's rule of
5, a common and well-known rule in pharmacological studies (Lajiness, 2004) and Tice's
modification (Tice, 2001) for herbicides.
13
Chapter I. Introduction
In summary, the bioactivity and results obtained with SLs so far make it possible to propose
them as leads for future natural product based herbicide development. Efforts need to be made
to obtain compounds in high amounts (through synthesis or plant extraction) at reasonable costs,
perform soil stability studies and improve physical/chemical properties and formulation.
However, the activities currently reported support them as possible candidates providing new
modes of action. In this way, SL has been shown to be effective in controlling herbicideresistant biotypes of several weeds (Galindo, et al., 2000).
Diterpenes: the case of momilactones
Momilactones (Fig. 7) are pimarane diterpenes initially isolated from rice husks as growth
inhibitors (Kato et al., 1977). Diterpenes are not usually reported as allelopathic agents. Rather,
their ecological role has been associated more with insecticidal, antifeedant and deterrent
activity. Other diterpenes such as giberrellins act as important plant hormones involved in
growth regulation. At first, momilactones were considered as phytoalexins since their presence
in plant tissues was detected and enhanced under Pyricularia oryzae Cavara attack (Cartwright
et al., 1977), UV irradiation (Kodama et al., 1988) and exogenous N-acetylchitooligosaccharides
(Yamada et al., 1993). Not until recently has their possible role as allelopathic agents been
studied in depth.
Figure 7. Rice allelochemicals: momilactones and oryzalexins. Oryzalexins and momilactones
are reported as phytoalexins, whereas momilactone B is the only member of this family being
identified as an allelopathic agent.
At first, momilactones were considered as phytoalexins since their presence in plant tissues was
detected and enhanced under Pyricularia oryzae Cavara attack, UV irradiation, and exogenous
N-acetylchitooligosaccharides. Not until recently has their possible role as allelopathic agents
been studied in depth. In a recent study, exudation of momilactone B from the roots of young
rice seedlings into the environment was described in culture solution (Kato-Noguchi and Ino,
2003) and soil (Kong et al., 2004) in quantities sufficient to inhibit the growth of neighboring
plants (Kato-Noguchi, 2004). Most of the recent work on momilactones as allelopathic agents
refers to momilactone B, which has been found in shoots and roots of rice plants over the entire
14
Introduction
plant life cycle, yielding the highest levels just before flowering initiation (Kato-Noguchi,
2005). The levels of momilactone B vary among different rice cultivars and correlate well with
the amounts of momilactone B found in the corresponding culture solutions.
Allelopathic compounds are usually considered as constitutive chemicals that are continuously
produced and exuded into the environment. However, the definition of allelopathy does not state
that allelopathic compounds need to be constitutive. Consequently, a phytoalexin can be
considered, at the same time, to be an allelochemical. Recently, a brilliant work by Wilderman
et al., 2004, showed that the mRNA levels encoding a specific syn-copalyl diphosphate 9 pimara-7,15-diene synthase were upregulated in leaves subjected to conditions where
phytoalexin production was stimulated, but it is constitutively expressed in roots, where
momilactones are constantly synthesised and released into the soil. Differential expression of
the genome is not uncommon, but this case constitutes a good example of how genomics can
help to explain apparently contradictory facts: how an allelopathic agent can also be, at the same
time and in the same plant, a phytoalexin, thus making a point for the economy of resources
principle that could be operating in diterpenoid metabolism in rice. Many approaches to
momilactone synthesis have been published as previous steps to the finally accomplished first
total synthesis of momilactone A, (Germain and Deslongchamps, 2002), thus showing the
importance of momilactones as promising leads for herbicide and fungicide development.
Quassinoids
Quassinoids are another good example of the increasing interest and work on allelochemicals.
Quassinoids are decanortriterpenes known to occur in the family Simaroubaceae (Polonsky,
1983). As in many other cases, they became of interest because of their wide spectrum of
medicinal activities: antileukemic (Itokawa et al., 1992) and antimalarial (O'Neill et al., 1985)
among others. Consequently, interest in their potential applications in agriculture as insecticidal
(Lidert et al., 1987), fungicidal (Hoffmann et al., 1992) and herbicidal (Lin et al., 1995; Dayan
et al., 1999) agents has arisen. Their high structural complexity, owing to the presence of many
chiral centers, and the low amounts usually obtained from natural sources made their total
synthesis attractive, and many attempts have been made, (Guo et al., 2005) thus illustrating their
importance. The first quassinoid identified as an allelopathic agent was ailanthone, a chemical
obtained from the tree-of-heaven (Ailanthus altissima Swingle), (Heisey, 1996) a Chinese tree
used in traditional medicine that has become an invasive species in Europe. The high invasive
potential of this tree fits well with Callaway's novel weapons hypothesis,(Callaway and
Aschehoug, 2000) as it presents a whole array of bioactive indole alkaloids and quassinoids.
Although ailanthone was originally determined to be the compound responsible for allelopathic
activity, bioassay-directed isolation procedures have recently identified the quassinoids
ailanthinone, chaparrine and ailanthinol B as phytotoxic quassinoids also present in the roots
(Fig. 8) (De Feo et al., 2003). To the authors' knowledge, neither the synthesis of ailanthone nor
any of the other bitter principles of A. altissima has been published so far.
15
Chapter I. Introduction
Figure 8. Structures of allelopathic/phytotoxic quassinoids.
No structure-activity relationship (SAR) studies have been accomplished with the quassinoids
obtained from the tree-of-heaven, although the in vitro phytotoxicity of other chaparrinone- and
picrasane-type quassinoids has been tested, showing that only chaparrinones (chaparrinone,
gaucarubolone and holocanthone) (Fig. 8) were phytotoxic (Dayan et al., 1999). This difference
was correlated with the presence of a hemiketal bridge in the active compounds. According to
this study, quassinoids appear to have a different but still unknown mode of action in plants
when compared with effects in mammal cells. No further studies have been done with
quassinoids regarding their phytotoxic activity.
Benzoxazinoids
Hydroxamic acids (Hx) are among the latest and more promising examples of allelochemicals
isolated from plants with potential uses in agriculture as weed control agents. Hx (Fig. 9) are
benzoxazines, produced by many species within Gramineae in significant amounts, which have
a protective role (Niemeyer and Perez, 1995) showing antifungal, (Hofman and Hofmanová,
1971), antimicrobial (Bravo and Lazo, 1993), insecticidal (Argandoña, 1980) and phytotoxic
(Macias et al. 2005) activities. They can also be found in members of the families Acanthaceae
(Kanchanapoom et al. 2001), Ranunculaceae (Ozden et al. 1992) and Scrophulariaceae (Chen
and Chen 1976). Hx are stored in the plant as inactive glucosides to avoid autotoxicity (Sicker et
al. 2004). Root exudation or tissue injuring by insect wounding causes their release into the
environment where enzymatic and/or microbial degradation breaks down the glucosidic bond
and liberates the aglucones (Cambier et al. 1999). These compounds are unstable and undergo
hydrolysis and ring contraction into the corresponding benzoxazolinones with short half-lifes (1
day for DIMBOA) (Fig 9) (Macias et al. 2004; Fomsgaard et al. 2004). Benzoxazolinones might
be further transformed, either chemically or by soil microbes, into more toxic degradation
products. Consequently, their chemistry has been extensively studied owing to their high degree
of bioactivity and potential for agricultural applications as weed and pest controls (Macias et al.
2006).
16
Introduction
Figure 9. Allelopathic benzoxazinones and their direct degradation products, the
benzoxazolinones.
These compounds have attracted much attention in the past few years, and much work has been
done on their phytotoxic activity, looking for:
(a) development of new models for herbicide design
(b) allelopathic varieties of crops with high Hx content
(c) breeding of crops to improve their chemical arsenal
(d) genetic engineering for Hx-allelopathic trait transfer
However, the use of benzoxazinoids as agrochemical leads is challenged by several facts
concerning their safety with regard to human health and their soil stability. The arylhydroxamic
group is known to be a mutagenic group, and therefore 1,4-benzoxazinoids isolated from cereals
present mutagenicity (Hashimoto et al. 1979).
Regarding their soil stability, several soil degradation studies have been performed with these
compounds, with interesting results. The degradation routes observed for many benzoxazinones
are known to have a determining role in the allelopathic interactions modifying the overall
defense capacity of the plant in a highly significant manner. Moreover, knowledge of the
stability and degradation routes is necessary if the compound is going to be used as an
agrochemical lead. The degradation pathways of several Hx have been extensively studied,
showing the importance of the chemical structure, soil microbes and conditions (Fig. 10)
(Macias et al. 2005). Regarding their mechanism of action, it has been shown that the
combination of cyclic hemiacetal and cyclic hydroxamic acid is necessary for high bioactivity,
17
Chapter I. Introduction
as reactions with the electrophilic ring-opened aldehyde form of the hemiacetal and with a
multicentred cation generated from N-O fission are likely to occur with bionucleophiles (Macias
et al. 2006). All of this research shows the interest that these compounds have generated and
their possibilities as herbicides (Macias et al. 2005), insecticides or pest control agents.
Figure 10. Degradation pathways proposed for benzoxazinoids: (a) DIMBOA derivatives; (b)
DIBOA derivatives.
Glucosinolates
Glucosolinates are a small group of defense compounds comprising around 120 structures and
found only in plants of the families Brassicaceae, Resedaceae and Capparidaceae (Fahey et al.
2001). They are non-toxic compounds that, upon tissue disruption, are enzymatically degraded
by endogenous β-thioglucosidases (myrosinases) into active compounds such as nitriles,
isothiocyanates, oxazolidinethiones and thiocyanate salts (Rask et al. 2000; Oleszek 1987).
Glucosinolates have been described as allelochemicals involved in defensive roles against
insects (acting as repellents) and microorganisms, and also as volatile attractants of specialized
insects such as specific butterfly species (Wittstock and Halkier 2002).
Glucosinolates have been reported also as allelopathic agents in several cases. Glucohirsutin,
hirsutin, arabin, camelinin and 4-methoxyindole-3-acetonitrile have been isolated from the roots
of the Cruciferae Rorippa sylvestris (L.) Bess. (yellow fieldcress) (Yamane et al. 1992), the
invasive weed Alliaria petiolata Bieb. (garlic mustard) (Vaughn and Berhow 1999), and other
Brassicaceae (Lazzeri and Manici 2001) (Fig. 11). Seasonal variations in Glucosinolate content
were recorded and positively correlated with phytotoxic and allelopathic effects of R. sylvestris
(Yamane et al. 1992). Other studies documenting the role of Glucosinolates in Brassica napus
L. contradict this, (Choesin and Boerner 1991) possibly owing to volatilization or degradation.
The possible use of glucosinolates and glucosinolate-containing tissues as bioherbicides for
weed control has been proposed (Brown and Morra 1995). However, their high volatility
18
Introduction
induces a fast disappearance from soils amended with allyl Glucosinolates (Borek et al. 1995).
Although this is good for the environment, it also constitutes a problem, as the compounds do
not last enough time to exert their herbicidal action.
Figure 11. Allelopathic glucosinolates and isotyiocyanates.
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26
Germination and growth response of four
plant species towards different allelochemical
and herbicides
Chapter II
Chapter section published as an original article to SCI journal:
Hussain, M.I., González, L., and Reigosa, M.J. 2008. Germination and growth
response of four plant species towards different allelochemicals and
herbicides. Allelopathy Journal. 22 (1): 101 – 110.
Allelochemicals and herbicides effects
Allelopathy Journal 22 (1): 101-108 (2008)
Table: -, Figs : 2
0971-4693/94 US $ 5.00
© International Allelopathy Foundation 2008
Germination and growth response of four plant species to different
allelochemicals and herbicides
M. IFTIKHAR HUSSAIN*, L. GONZALEZ-RODRIGUEZ and M. J. REIGOSA
Laboratory of Plant Ecophysiology, Faculty of Biology,
University of Vigo, Campus Lagoas-Marcosende s/n, 36310 Vigo, Spain
E. Mail: [email protected]
(Received in revised form: June 4, 2008)
ABSTRACT
Laboratory bioassays were conducted to test the phytotoxicity of
6 allelochemicals (ferulic acid, p-hydroxybenzoic acid, 2-benzoxazolinone, Lmimosine, juglone, trans-cinnamic acid), 6 allelochemicals mixture and 2 herbicides
(pendimethalin and S-metolachlor) on germination and seedling growth of 4 plants
species (Rumex acetosa L., Lolium perenne L., Lactuca sativa L. and Dactylis
glomerata L.). Data obtained were used to compare four germination indices viz., (total
germination index (GT), speed of germination index (S), accumulated speed of
germination index (AS), and coefficient of rate of germination index (CRG). The
allelochemicals 2-benzoxazolinone and trans-cinnamic acid inhibited the total
germination index of L. perenne L. at 1 M concentration. The L- mimosine and six
allelochemicals mixture (1 M concentration) inhibited GT, S, AS, in L. perenne and
also inhibited GT, S, AS in D. glomerata at 1 M and 0.1 M concentration.
L- mimosine also inhibited the CRG in R. acetosa at 0.01 M and in D. glomerata at
0.001 M concentration. The six allelochemicals mixture (1 M concentration)
inhibited the speed of germination (S) of D. glomerata L. while trans-cinnamic acid
inhibited the S in R. acetosa at 0.001 M concentration. All other allelochemicals at
0.1, 0.01, 0.001 M concentrations showed non-significant behavior to S all four plant
species. Juglone inhibited GT of L. perenne at 1 M concentration and in
D. glomerata at 1 M, 0.01 M and CRG in R. acetosa at 0.01 M concentration. The
application of both herbicides strongly inhibited GT, S, AS, CRG in L. perenne L. and
D. glomerata at concentration of 104 M. These results indicate that each index led to a
different interpretation of allelochemicals effect on germination.
Key Words- Allelochemicals, commercial herbicides, germination inhibition, Rumex
acetosa L., Lolium perenne L., Lactuca sativa L., Dactylis glomerata L,
phytotoxicity, germination indices.
INTRODUCTION
The overuse of synthetic agrochemicals for pest control has increased
environmental pollution, unsafe agricultural products and human health concerns (53).
Furthermore, herbicide resistant weeds have become major problem in last 20 years
(16,18,38,42). FAO (15) Expert Consultation Group on Weed Ecology and Management
has expressed great concern about the problem associated with the use of herbicides for
*Correspondence author
28
Chapter II. Germination bioassays
ABSTRACT
Laboratory bioassays were conducted to test the phytotoxicity of 6 allelochemicals
(ferulic acid, p-hydroxybenzoic acid, 2-benzoxazolinone, L-mimosine, juglone, transcinnamic acid), 6 allelochemicals mixture and 2 herbicides (pendimethalin and Smetolachlor) on germination and seedling growth of 4 plants species (Rumex acetosa L.,
Lolium perenne L., Lactuca sativa L. and Dactylis glomerata L.). Data obtained were
used to compare four germination indices viz., (total germination index (GT), speed of
germination index (S), accumulated speed of germination index (AS), and coefficient of
rate of germination index (CRG). The allelochemicals 2-benzoxazolinone and transcinnamic acid inhibited the total germination index of L. perenne L. at 1
M
concentration. The L- mimosine and six allelochemicals mixture (1 M concentration)
inhibited GT, S, AS, in L. perenne and also inhibited GT, S, AS in D. glomerata at 1 M
and 0.1 M concentration. L- mimosine also inhibited the CRG in R. acetosa at 0.01 M
and in D. glomerata at 0.001 M concentration. The six allelochemicals mixture (1 M
concentration) inhibited the speed of germination (S) of D. glomerata L. while transcinnamic acid inhibited the S in R. acetosa at 0.001
M concentration. All other
allelochemicals at 0.1, 0.01, 0.001 M concentrations showed non-significant behavior to
S all four plant species. Juglone inhibited GT of L. perenne at 1 M concentration and in
D. glomerata at 1 M, 0.01 M and CRG in R. acetosa at 0.01 M concentration. The
application of both herbicides strongly inhibited GT, S, AS, CRG in L. perenne L. and D.
glomerata at concentration of 104 M. These results indicate that each index led to a
different interpretation of allelochemicals effect on germination.
Key Words- Allelochemicals, commercial herbicides, germination inhibition, Rumex
acetosa L., Lolium perenne L., Lactuca sativa L., Dactylis glomerata L, phytotoxicity,
germination indices.
29
Allelochemicals and herbicides effects
INTRODUCTION
The overuse of synthetic agrochemicals for pest control has increased environmental
pollution, unsafe agricultural products and human health concerns (53). Furthermore,
Herbicides resistant weeds have become major problem in last 20 years (16,18,38,42).
FAO (15) Expert Consultation Group on Weed Ecology and Management has expressed
great concern about the problem associated with the use of herbicides for weed control
and has recommended minimizing or eliminating the use of herbicides with alternative
strategies like allelopathy.
Allelopathy is an emerging branch of applied science, which studies any process
primarily involving secondary metabolites produced by plants, algae, bacteria, and fungi
that influence the growth and development of biological and agricultural systems,
including positive and negative effects (20). Allelochemicals are synthesized in plants as
secondary metabolites. They are present in specialized organs of plants and their amount
vary in different plant organs (43), plant variety (11) and have potential as either
herbicides or templates for new herbicide classes (21). Allelopathic chemicals are
released from plant tissue in many: volatilization from living plant parts (1, 36), root
exudates (12); leachates from above-ground parts during rain, fog, or dew (8) and
decomposition crop residues/ little by microorganisms (40).
The possible use of secondary metabolites from plants as herbicides has long been
discussed (30,44). Although, the physiological and ecological actions of allelochemicals
are weaker than commercial herbicides (34). However, these secondary metabolites have
ecological advantages like, greater biodegradability (30). Allelochemicals affects many
physiological processes in target organisms, including inhibition of; seed germination
(50), growth of plant species (33), ion uptake (26), inhibition of electron transport in
photosynthesis and respiratory chain (1, 24) and involved in root growth inhibition (32).
Some allelochemicals rapidly depolarize the cell membrane, increasing membrane
permeability, inducing lipid per oxidation and causing a generalized cellular disruption
that ultimately leads to cell death (17,29,56,55).
Bioassays for such studies typically include seed germination, radicle growth and a
photosynthetic activity test (19, 3). Attempts to describe germination responses in
phytotoxic studies include the use of indices such as speed of germination (48),
accumulated speed of germination (14), coefficient of rate of germination (13). More
recently, Chiapusio et al., (9) compared four indices and comparisons between the control
and treatments at each time of counts for their ability to test physiological hypothesis in
allelopathic studies.
This study aimed to investigate the phytotoxic effect of 6 allelochemicals and 6
allelochemicals mixtures compared to 2 herbicides on seed germination of four plants
species (Rumex acetosa L., Lolium perenne L., Lactuca sativa L. and Dactylis glomerata
L). The same data was used to compare four germination indices (GT, S, AS, and CRG)
examined by Chiapusio et al., (9) to discuss their physiological meaning.
30
Chapter II. Germination bioassays
MATERIALS AND METHODS
This study investigated potential phytotoxic effects of 6 plant secondary metabolites
(ferulic acid, p-hydroxybenzoic acid, 2-benzoxazolinone, L-mimosine, juglone, transcinnamic acid), 6 allelochemicals mixture and two herbicides (pendimethalin, Smetolachlor) on germination and seedling growth of Rumex acetosa L., Lolium perenne
L., Lactuca sativa L. and Dactylis glomerata L. The test species were selected based on
utilization capacity, handling and availability for experiment (45) and included mono and
dicotyledonous plant species with different physiological responses. The experiment was
arranged in Randomized Complete Block Design and replicated thrice under controlled
conditions. The seeds of test plant species (Rumex acetosa L., Lolium perenne L.,
Lactuca sativa L.) were purchased from Semillas Fito (Barcelona, Spain), and that of
Dactylis glomerata L. were obtained from Herbiseed (Twyford, England).
Phenolic Compounds and Herbicide Solutions
The phenolic compounds (ferulic acid, p-hydroxybenzoic acid, 2-benzoxazo-linone, Lmimosine, juglone, and trans-cinnamic acid) were obtained from Sigma Chemical
Company, St. Louis, MO, USA. It should be noted that all phenolic tested in this study
are phytotoxins but all phytotoxins are not allelochemicals. The herbicide Pendimethalin
(33% p/v) was obtained from Probelt S.A. (Murcia, Spain), while, S-metolachlor (96%
p/v) was purchased from Syngenta Agro S.A. (Madrid, Spain). The selection of
allelochemicals was based on their allelopathic activity described previously (10, 27, 37,
33, 52). Stock solutions of allelochemicals were made in methanol: water (20:80). The
control was prepared with distilled water and methanol in same proportion. Methanol was
evaporated in a rotary vapourizer and remaining aqueous solution was adjusted to 10 M
concentration. These stock solutions were diluted to 1, 0.1, 0.01, 0.001
M. The
equimolar mixtures of six phenolic compounds were prepared in 1, 0.1, 0.01, 0.001 M
concentration. The pH of each phenolic solution was adjusted to 6.0 with NaOH (23). The
herbicide solutions were prepared in distilled water in 104, 102, 1, 10-2 M concentrations.
Germination Bioassays
Twenty-five seeds of each species were placed on Whatman 3 MM paper in a 9 cm dia
petri dish to which 3 ml of solution was added at the start. An additional one ml of each
solution was added every 48 h. Three replicates of each treatment were incubated in a
germination chamber with the following germination conditions: R. acetosa (day/night
temperature: 28/20 0C and -9/15 h of light and darkness); L. perenne (day/night
temperature: 25/15 0C and -12/12 h of light and darkness), L. sativa (day/night
temperature: 23/17 0C and -18/6 h of light and darkness) and D. glomerata (day/night
temperature: 25/20 0C and -14/10 h of light and darkness) and a relative humidity of 80
%. The light was provided by cool white fluorescent tubes with an irradiance of 35 mol
m-2sec-1. The germination was recorded after every 24 h by counting the number of
germinated seeds: rupture of seed coats and emergence of radicle
1mm, (31), until no
31
Allelochemicals and herbicides effects
further seeds germinated. The total germinated seeds (%) were calculated with the
cumulative germination data after one week (49). The same data were used to calculate
the germination indices [GT: total germination index, S: Speed of germination; AS: Speed
of cumulative germination; CRG: coefficient of germination rate as described by (9)].
Statistical analysis
Data from germination bioassay were analysed using one-way analysis of variance
(ANOVA) (39) to compare means of treatments in each species. The Levene test was
used, when variance was homogeneous. The LSD and post hoc test were used to
determine main differences between the treatment means. The Kruscal-Wallis test was
applied when variance was not homogeneous. All statistical analysis was done using
SPSS (version 14.0) for Windows. Significant differences between treatment means were
compared at 5% probability level.
RESULTS AND DISCUSSION
The phenolic compounds tested in this study are well known phytotoxins. The use of
germination indices helps in determining the allelochemical effects on the physiological
germination process (9). Results showed that each index leads to different interpretations
of allelochemical effects on seed germination of four plant species. Thus, two aspects
could be discussed: germination capacity (GT) and germination progress or germination
indices (S, AS and CRG).
Total germination index (GT)
It is most common index to interpret of germination and useful to obtain ecological
information in plant succession (22). Allelochemicals L- mimosine, juglone, six
allelochemicals mixture (1 M concentration) inhibited the GT of L. perenne (Fig 1 a),
while, 2-benzoxazolinone, L-mimosine, juglone, trans-cinnamic acid, six allelochemical
mixture have shown inhibition on GT at 1 M concentration in D. glomerata (Fig. 1 b).
This inhibition is stronger than that reported by Li et al., (28) on seedling germination for
L. sativa at 10-3 M. Similarly, Reigosa and Malvido (33) reported that four compounds
(vanillic acid, L-mimosine, juglone, and trans-cinnamic acid) delayed the A. thaliana
germination at the highest concentration tested. However, pendimethalin and
S-metolachlor strongly inhibited the GT of L. perenne and D. glomerata at 104 M
concentration, respectively (Fig 1a and b). Allelochemicals and herbicides did not
influence GT in case of R. acetosa and L. sativa at the same concentration (data not
shown). These two plants were not sensitive to low concentrations of test allelochemicals
and herbicides in this study.
The allelochemicals L-mimosine and six allelochemicals mixture at 0.1 M concentration
significantly inhibited the GT in D. glomerata, but pendimethalin and
S-metolachlor herbicides proved most deleterious and strongly inhibited the GT of
D. glomerata (100 µM) (Fig 1c). However, juglone is the only molecule that had
phytotoxic effect on total germination index in L. perenne even at very low concentration
(0.01 µM) (Fig 1d). This compound is potent allelochemical (41) and also influences the
32
Chapter II. Germination bioassays
four germination indices. However, Angela et al., (4) reported that juglone between 10
and 40 µM inhibited the Lemna minor growth, chlorophyll content and net
photosynthesis.
The phytotoxic effects of molecules were concentration dependent. At 0.001µM
concentration, none of the allelochemicals or herbicides (0.01 µM) inhibited the GT of
any plant species. These results agree with Reigosa et al., (34) who concluded that
allelochemicals have a stimulatory effect or no action on various weeds at lower
concentration. In this study, ferulic acid and p-hydroxybenzoic acid did not inhibit total
germination at any concentration. The p-hydroxybenzoic and ferulic acids did not
influence the germination of Poa annua L. (51).
Speed of germination index (S)
S measures retardation or acceleration in the germination process (7,25,46,47) and is
advantageous over GT, because it is more sensitive indicator of allelopathic effects (2),
which occurred during the germination process. The application of allelochemicals Lmimosine and six allelochemical mixtures (1 M concentration) inhibited the S of
L. perenne (Fig 1e). Similarly, vanillic acid, L-mimosine, juglone, and trans-cinnamic
acid—delayed the A. thaliana germination at the highest concentration tested (33).
However, the pendimethalin and S-metolachlor at 104 µM concentration proved most
inhibitory to S of L. perenne respectively (Fig 1e). All the allelochemicals at 0.1 M
concentration had non-significant effects on S in all four test plant species. However, both
herbicides at 100 µM concentration proved most inhibitory to S of L. perenne (Fig 1f).
At 0.01 µM concentration, none of allelochemicals or herbicides (1µM) controlled the S
of any plant species (data not shown). However, in R. acetosa there was significant
difference in inhibition of S between the different allelochemicals at 0.001 M
concentration. At this concentration, only trans-cinnamic acid was a good inhibitor of S
in R. acetosa (Fig. 1g). These results are in agreement with the view that different plant
species can vary enormously in their response to potentially allelopathic substances (5).
All allelochemicals and herbicides have shown non-significant effect on S in L. sativa at
any concentration (data not shown).
Speed of cumulative germination index (AS)
It involves the cumulative number of germinated seeds at each exposure time. It was
adapted from (7,25,47). The application of allelochemicals (L- mimosine, juglone, six
allelochemical mixtures) at 1 M concentration inhibited the AS of L. perenne (Fig. 2a).
However, pendimethalin and S-metolachlor drastically inhibited the AS in L. perenne at
104 µM concentration (Fig 2a). The six allelochemicals mixture had good inhibitory effect
on AS in D. glomerata at 0.1 M (Fig.2b). The pendimethalin and S-metolachlor
inhibited AS in D. glomerata at 100 µM (Fig 2b). The allelochemicals (ferulic acid, phydroxybenzoic acid, BOA, juglone and trans-cinnamic acid) inhibited the AS at 0.001
M concentration in R. acetosa (Fig 2c). These results are in agreement with literature
(33,35,54).
33
Allelochemicals and herbicides effects
Coefficient of germination rate (CRG)
CRG was established by Bewley and Black (6) and its significance is similar to AS index.
The allelochemical BOA (1 M concentration) inhibited the CRG of R. acetosa (Fig. 2d).
However, Reigosa et al., (35) reported that growth of Rumex crispus was inhibited by the
allelochemicals (gallic, ferulic, vanillic, p-coumaric, p-hydroxybenzoic acid, p-vanillin
and phenolic mixture) at concentration of 0.01 M. The pendimethalin and S-metolachlor
proved most inhibitory CRG in R. acetosa at 104 µM concentration (Fig 2d). The
allelochemical L-mimosine inhibited the CRG in D. glomerata at 1 M concentration
(Fig 2 e). Similarly, pendimethalin and S-metolachlor herbicide proved most deleterious
to CRG in D. glomerata at 104 M (Fig 2e) and in L. perenne at 102 M (fig 2f)
concentration.The allelochemicals, L-mimosine, juglone, trans-cinnamic acid and six
allelochemicals mixture (0.01 M) significantly inhibited the CRG in R. acetosa (Fig 2g),
while, only S-metolachlor inhibited the CRG at 1 M concentration (Fig 2g). Only Lmimosine at 0.001 M concentration inhibited the CRG of L. perenne (Fig 2h).
ACKNOWLEDGEMENTS
We are grateful to Dr. A. Barreiro, C. Bolano, A. Justo, M. Ricart and P. Lorenzo for help
in field and laboratory work. We are also thankful to two anonymous reviewers for their
constructive and critical comments on the manuscript.
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37
Allelochemicals and herbicides effects
Total Germination Index (GT)
(a)
*
a
*
(b)
*
(c)
*
*
b
*
*
(d)
*
d
c
*
*
*
*
*
*
* *
*
Speed of Germination Index
(S) Index (GT)
e
g
f
(e)
(f)
(g)
* *
*
*
* *
*
* *
* *
Figure 1.Effect of allelochemicals and herbicides on total germination indices (GT) and speed of germination (S) of Lolium perenne, Dactylis glomerata and Rumex acetosa. Whereas treatments
(1= Control, 2 = Ferulic acid, 3 = p-Hydroxybenzoic acid, 4 = 2-Benzoxazolinone (BOA), 5 = L-Mimosine, 6 = Juglone, 7 = trans-Cinnamic acid, 8 = Pendimethalin, 9 = S-Metolachlor, 10 =
Six allelochemicals mixture). Concentration (allelochemicals (ALC) 1 µM, herbicides (HBC) 104 µM: a, b, M, HBC 102 µM: c, f) (ALC 0.01 µM, HBC 1 µM: d); (ALC 0.001 µM, HBC 10-2
µM: g).
38
Chapter II. Germination bioassays
Speed of Cumulative Germination Index (AS)
a
c
b
*
* *
*
*
*
*
*
*
*
* * *
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
Coefficient of Rate of Germination Index (CRG)
d
e
f
* * *
*
*
*
g
h
*
*
*
*
* *
*
*
*
*
*
*
*
* *
*
*
*
*
*
*
*
Figure 2.Effect of allelochemicals and herbicides on speed of cumulative germination (AS) and coefficient of rate of germination (CRG) of Lolium perenne, Dactylis glomerata and Rumex
acetosa. Where as treatments (1= Control, 2 = Ferulic acid, 3 = p-Hydroxybenzoic acid, 4 = 2-Benzoxazolinone (BOA), 5 = L-Mimosine, 6 = Juglone, 7 = trans-Cinnamic acid, 8 =
Pendimethalin, 9 = S-Metolachlor, 10 = Six allelochemicals mixture). Concentration (allelochemicals (ALC) 1 µM, herbicides (HBC) 104 µM: a, d, e); (ALC 0.1 µM, HBC 102 µM: b, f) (ALC 0.01 µM, HBC 1 µM:
g); (ALC 0.001 µM, HBC 10-2 µM: c, h).
39
Allelochemicals and herbicides effects
40
Phytotoxic effect of allelochemicals and
herbicides on photosynthesis, growth and
carbon isotope discrimination in Lactuca
sativa
Chapter III
Chapter section published as an original article to SCI journal:
Hussain, M.I., González, L., and Reigosa, M.J. 2010. Phytotoxic effect of
allelochemicals and herbicides on photosynthesis, growth and carbon isotope
discrimination in Lactuca sativa. Allelopathy Journal. 26 (2): 157-174.
Phytotoxic effects of Allelochemicals and herbicides
Allelopathy Journal 26 (2): xx-xx (2010)
Tables: 3, Figs : 7
0971-4693/94 US $ 5.00
International Allelopathy Foundation 2010
Phytotoxic effects of allelochemicals and herbicides on
photosynthesis, growth and carbon isotope discrimination in
Lactuca sativa
M. IFTIKHAR HUSSAIN*, L. GONZÁLEZ, and M. J. REIGOSA
Department of Plant Biology and Soil Science, University of Vigo,
Campus Lagoas-Marcosende, E-36310, Vigo, España
E. Mail: [email protected], [email protected]
(Received in revised form: September 16, 2010)
ABSTRACT
We compared the effects of two allelochemicals [ferulic (FA) and
p-hydroxybenzoic
acids
(HBA)]
and
two
herbicides
[pendimethalin
(PML)
and
S-metolachlor (MTR)] on physiological processes (growth, photosynthetic efficiency, yield, nonphotochemical fluorescence quenching, leaf water relations, carbon isotope discrimination and leaf
protein contents) in Lactuca sativa cv. Great Lakes. HBA and FA significantly reduced the shoot length,
leaf, root fresh biomass and root length. There was no significant effect of both allelochemicals and
herbicides on relative water content (RWC) and leaf osmotic potential (LOP) in lettuce. The
allelochemical FA significantly reduced the quantum efficiency (Fv/Fm) of open photosystem II (PSII)
reaction centers in dark-adapted leaves and this reduction was stronger than herbicides. Both
allelochemicals significantly decreased the effective quantum yield (Ф PSII) of PSII, while herbicides did
not effect the Ф PSII. Both allelochemicals also drastically decreased (3-folds less than control) the
photochemical fluorescence quenching (qP) and non-photochemical fluorescence quenching (NPQ). In
HBA treated plants, carbon isotope ratios (δ13C) were significantly less negative (-26.93) than control (27.61). Lettuce plants treated with FA and MTR, δ13C was significantly less negative (-26.90) and (26.88), respectively as compared to control (-27.61). Carbon isotope discrimination (Δ 13C) values were
significantly less (19.40) after treatment with FA and HBA (19.25) at 1.5 mM concentration over the
control (20.17). The ratios of intercellular CO2 concentration from leaf to air (ci/ca) were significantly
less after treatment with FA, HBA and MTR than control. The FA, HBA (1.5 mM), PML and MTR (10 -1
mM) significantly reduced the leaf protein contents of lettuce.
Key words: Allelochemicals, carbon isotope discrimination, ferulic acid, growth, Lactuca sativa, leaf
water relations, pendimethalin, p-hydroxybenzoic acid, physiological responses, Smetolachlor,
INTRODUCTION
Environmental stress factors limit the agricultural productivity and many of these
factors, are related to the metabolic processes. The plants response to stress depends on the
duration, severity and rate of stress imposed (44). Under field conditions, multiple stresses
develop progressively and gradually, elicits morphological, physiological and biochemical
responses. Allelopathic interactions are mediated by secondary metabolites released from
*
Correspondence Author
42
Chapter III. Physiological response of lettuce
INTRODUCTION
Environmental stress factors limit the agricultural productivity and many of these factors, are
related to the metabolic processes. The plants response to stress depends on the duration,
severity and rate of stress imposed (44). Under field conditions, multiple stresses develop
progressively and gradually, elicits morphological, physiological and biochemical responses.
Allelopathic interactions are mediated by secondary metabolites released from the donor plants
in to the environment and influence the growth and development in both natural and agroecosystems (30). The harmful or phytotoxic effects of allelochemicals are considered as biotic
stress called 'allelochemical stress' (46).
Allelochemicals directly affects many physiological and biochemical reactions and thereby,
influence the growth and development of plants (35,59). The allelochemicals reduces the cell
division (54) and have several phytotoxic effects (15). Ferulic acid (a cinnamic acid derivative)
is abundant in soil (14,30,59). The ferulic acid stress on plant roots, reduces the water use,
inhibits foliar expansion and root elongation, reduces rates of photosynthesis and inhibits
nutrient uptake. At cellular level, it induces lipid peroxidation, affects certain enzymatic
activities and rapidly depolarizes the root cell membrane thereby increasing the membrane
permeability, thus blocking plant nutrients uptake (59). Ferulic acid also inhibits the seed
germination and seedling growth of several species including weeds (22,28). Ferulic acid
esterifies with cell wall polysaccharides, get incorporated into the lignin structure, or form
bridges to connect lignin with wall polysaccharides, thereby making the cell walls rigid and
restricting the cell growth (34,52).
The p-hydroxybenzoic acid (hereafter HBA), is a widespread phenolic acid released into soil by
root exudates, leaf leachate and decomposed tissues of different plants e.g. wheat (Triticum
aestivum L.) (11,61). It is one of several allelopathic Sorghum compounds found in aqueous
extracts of plant material and in the decomposing crop residues, germinating seeds and root
exudates (2,16,37). Other plants implicated in allelopathic release of HBA include Camelina
alyssum, several members of genus Althaea (24), and grass Imperata cylindrica (29). HBA in
root exudates of wild oat (Avena fatua) reduces the root and coleoptiles growth of wheat
seedlings (47). HBA application delays the germination and growth of several plant species
(39). It inhibits the growth of Rumex crispus at 0.01 M concentration (49). Vaughan and Ord
(58) observed that p-coumaric and p-hydroxybenzoic acids (1 mM) inhibited the root growth in
pea (Pisum sativum L.). Doblinski et al. (13) observed that p-coumaric and p-hydroxybenzoic
acids (0.5 -1.0 mM) decreased the root length and fresh weight of soybean. In target plants, it
mainly interferes with nucleic acid and protein metabolism (6). The seedling growth of lettuce
and mung bean (Vigna radiata Wilcz.) was significantly inhibited, when barnyard grass
(Echinochloa crusgalli L. Beauv.) was cultured in donor pots. The p-hydroxybenzoic acid and
p-hydroxybenzaldehyde were isolated as the main allelochemicals from the culture solution of
barnyard grass (38).
43
Phytotoxic effects of Allelochemicals and herbicides
The herbicides are toxic to various crops (31). The magnitude of herbicides toxicity, however,
depends on type and dose of compounds, duration of exposure, species and age of plants, and
other environmental factors. Aamil et al. (1), reported that recommended dose of 2,4-D and
isoproturon adversely affected the plant growth, photosynthetic pigments, nodulation, N content
and yield of chickpea. Pendimethalin controls some annual broadleaved weeds (56) and in leafy
vegetables, it causes germination inhibition, growth retardation, malformation (36), and severe
stunting (23). Metolachlor and napropamide herbicides are currently recommended for either
early post- or pre-emergence small-seeded weeds control in tomato (57).
This study aimed to compare the biochemical and physiological effects of two allelochemicals
(ferulic and p-hydroxybenzoic acids) with two commercial herbicides (pendimethalin and Smetolachlor) to better understand their mechanisms of growth inhibition in lettuce plant. This
study may provide us new clues regarding the mode of action of allelochemicals to assess their
suitability as natural herbicides, because these allelochemicals being biodegradable may be used
for weed control with less problems for ecosystem.
MATERIALS AND METHODS
I. Phenolic Compounds and Herbicides: The phenolic compounds [Ferulic acid (FA) and phydroxybenzoic acid (HBA)] were obtained from Sigma Chemical Company (St. Louis, MO,
USA). Stock solutions of allelochemicals were made in Methanol: Water (20:80). The control
was prepared with distilled water and methanol. The methanol was evaporated in a rotary
vaporizer and solution was adjusted to a concentration of 3 mM. These stock solutions were
diluted to 1.5, 1.0, 0.5, 0.1 mM concentrations. The pH of these solutions was adjusted to 6.0
with NaOH (40). The herbicide Pendimethalin (33% p/v) was obtained from Probelt S.A.
(Murcia, Spain), and S-metolachlor (96% p/v) was purchased from Syngenta Agro S.A.
(Madrid, Spain). The herbicides were prepared in distilled water and their concentrations were
10-1, 10-3, 10-5 and 10-7 mM.
II. Plant Material and Growth Conditions: Lettuce (Lactuca sativa L. cv. Great Lakes
California) was used as test crop due to its fast germination and homogeneity (28,53,54). The
seeds of test species were purchased from Semillas Fito (Barcelona, Spain). The seeds were
placed in plastic trays (32 x 20 x 6 cm) with a 5 cm deep layer of perlite (500 g/tray). The trays
were irrigated on alternate days with tap water until germination of seeds and thereafter, with
500 mL 1:1 Hoagland solution/tray, twice in a week. For seedling growth, the environmental
conditions were temperature: 18/8 oC (day/night) and 12/12 h (light/darkness) photoperiod, 80
% relative humidity and 200 mol m–2 sec-1 irradiance. One-month-old seedlings (when plants
have 3-fully expanded leaves), were transferred to pots (10 cm) containing perlite (70 g) to
stimulate the development of root system and shifted to the glass house with same growing
condition and nutrient solution (100 ml/pot). One week later, roots were exposed to 100 mL
treatment solution thrice (days 1, 3 and 5) containing 0.1, 0.5, 1.0 or 1.5 mM allelochemicals or
44
Chapter III. Physiological response of lettuce
four different concentrations (10-1, 10-3, 10-5 or 10-7 mM) of the two commercial herbicides. The
temperature in the glass house was maintained at 21±2 oC with a relative humidity of 75%.
III. Chlorophyll Fluorescence Measurement: Chlorophyll fluorescence was measured in
glass house with a portable, pulse-modulated instrument fluorescence monitoring system (FMS)
(Hansatech, Norfolk, England) using method of Weiss and Reigosa (60), on randomly selected
leaves of lettuce. Following 20 min dark adaptation, the minimum chlorophyll fluorescence (Fo)
was determined using a measuring beam of 0.2 μmol m−2 s−1 intensity. A saturating pulse (1 s
white light with a PPFD of 7500 μmol. m−2 s−1) was used to obtain the maximum fluorescence
(Fm) of the dark-adapted state. The quantum efficiency (Fv/Fm) of open PSII reaction centers in
dark-adapted plants was calculated from (Fm− Fo)/ Fm. Light-induced changes in chlorophyll
fluorescence following actinic illumination (300 μmol m−2 s−1) were recorded prior to
measurement of F′o (minimum chlorophyll fluorescence in light saturated state) and F′m
(maximum fluorescence in light saturated stage). The quantum yield (ФPSII) of open PSII
reaction centers in the light-adapted state, referred to as (F′m−Fs/ F′m), was determined from F′m
and Fs (steady state fluorescence) values according to Genty et al. (21). The fraction of open
PSII reaction centers as qP and non-photochemical quenching NPQ were also calculated (7, 33).
IV. Carbon Isotope Composition Analysis : Collected plant leaf samples were immediately
dried at 70 °C for 72 h in a forced-air oven (Gallenkamp oven, Loughborough, Leicestershire,
UK) to constant weight and ground in Ball Mills (Retsch MM 2000, Haan, Germany). Dry
ground plant material was weighed (1700-2100 µg) with analytical balance (Metler Toledo
GmbH: Greifensee Switzerland), filled in tin capsules (5 x 3.5 mm, Elemental Microanalysis
Limited, U.K.). Each tin capsule was entered automatically in combustion oven at 1600-1800 0C
in the presence of oxygen and converted to CO2. Subsequently isotope ratios were determined in
an Isotopic Ratio Mass Spectrometer (Finnegan: Thermo Fisher Scientific, model MAT-253,
Swerte Germany) coupled with an Elemental Analyzer (Flash EA-1112, Swerte Germany). The
isotopic ratio mass spectrometer has an analytical precision better than 0.3‰ for 13C.
Carbon isotope compositions were calculated as:
δ (‰) = [(Rsample/Rstandard) —1)] x 1000
(i)
Where, Rsample is the ratio of 13C/12C and Rstandard is Vienna PeeDee Belemnite (VPDB), standard
for carbon. The standard carbon R is equal to 1.1237 x10-2. Pure CO2 (δ13C = -28.2 ± 0.1‰) gas
calibrated against standard CO2 (-10.38‰) served as reference gas for δ13C. The accuracy and
reproducibility of the measurements of δ13C checked with an internal reference material (NBS
18 and IAEA-C6 for C), and Acetanilide for C %age ratios, respectively.
Carbon isotope discrimination is a measure of C isotopic composition in plant material
relative to the value of the same ratio in the air on which plants feed:
45
Phytotoxic effects of Allelochemicals and herbicides
Δ (‰) = [(δa – δp)/(1 + δp)] x 1000
(ii)
Where, Δ represents carbon isotope discrimination, δa represents C isotope composition in the
source air, and δp represents C isotope composition in the plant tissue. Theory of Farquhar et al.
(17) and Farquhar and Richards (19) indicates that carbon discrimination in plants leaves can be
expressed in relationship to CO2 concentrations inside and outside the leaves in its simplest
form as:
Δ = a + (b - a) ci/ca
(iii)
Δ = 4.4 + (27 - 4.4) ci/ca
Where, a is discrimination that occurs during diffusion of CO2 through the stomata (4.4‰), b is
discrimination by Rubisco (27‰), and ci/ca is the ratio of the leaf intercellular CO2
concentration to that in the atmosphere. Equation [iii] shows a direct and linear relationship
between Δ and ci/ca. Therefore, measurement of Δ gives an estimation of the assimilation-rate–
weighted value of ci/ca. A lower ci/ca ratio may result either from higher rates of photosynthetic
activity or from stomatal closure induced by environmental stress.
Intrinsic water use efficiency (iWUE) was calculated according to the following
equations (42):
iWUE = A/g = ca [1-(ci/ca)] x (0.625)
(iv)
Where, A is the rate of CO2 assimilation and g is the stomatal conductance. Data of δ 13Cair, ci
and ca were obtained from McCarroll and Loader (42) who used the high precision records of
atmospheric Δ 13C from Antarctic ice cores (20), and the atmospheric CO2 concentration (ppm)
from Robertson et al. (51). Carbon isotope measurements were performed in CACATI (Centro
de Apoio Cientifico Tecnologico a la Investigacion), University of Vigo, Spain.
V. Plant Growth Measurements: Information about plant height/root length was obtained with
a ruler and values were expressed in cm. Fresh and oven dry plant leaves and roots weight were
obtained by first weighting independently fresh leaves and roots then after drying these samples
in a circulatory air oven at 70 oC for 72 h. The samples were weighed again to get dry weight.
VI. Leaf Water Relations : The leaf relative water content (RWC) was calculated by
measuring fresh (Wf), saturated (Wt) and dry (Wd) weights of plant (48). Leaf plant pieces from
three replicates per treatment were weighted in fresh saturates at 4 °C for 3 h, weighted again,
oven dried at 70 °C for 72 h, and finally weighted once more for obtaining three necessary
weights required for the following equation:
46
Chapter III. Physiological response of lettuce
RWC = (Wf – Wd)/(Wt – Wd) x 100
(v)
Leaf osmotic potential (LOP) was determined according to González (23) in milliosmol
kg . One leaf per replicate was transferred into 10 mL syringes and kept frozen at -80 °C until
further analysis. During analysis, syringes were thawed until sample reached room temperature
and osmotic potential was measured on the second drop from collected sap by using a calibrated
vapor pressure Osmometer (Automatic Cryoscopic Osmometer, Osmomat – 030, GmbH,
Gonatec, Berlin, Germany) according to manufacturer‘s instructions.
-1
VII. Protein Content Determination: Total protein was quantified by using the
Spectrophotometric Bradford (9) assays. Lettuce leaves (200 mg fresh weight) were crushed in
liquid nitrogen in cooled mortar with 0.05 g PVPP, and powder was resuspended in 1 mL Tris
buffer containing 0.05 M Tris base, 0.1% w/v ascorbic acid, 0.1% w/v cysteine hydrochloride,
1% w/v PEG 4000, 0.15% w/v citric acid, and 0.008% v/v 2-mercaptoethanol (3). Samples were
centrifuged at 19000 gn and 4 °C for 20 min after resuspension. The supernatant (0.1 mL) was
mixed for protein dye-binding reaction with 3 mL Bradford reagent containing 0.01%
Coomassie® Brilliant Blue G-250, 4.7% v/v ethanol 95%, and 8.5% v/v phosphoric acid 85%.
Absorbance was recorded at 595 nm after 5 min for quantification of the protein content.
Commercial bovine serum albumin (BSA) was used as standard and values were expressed per
gram of dry weight.
VIII. Statistical Analysis: Statistical analysis was performed based on the randomized
complete block design with three replicates using SPSS® 15.00 (SPSS Inc., Chicago, IL, USA).
Multiple comparisons of means were performed by LSD test at 0.05 significance level (when
variance was homogeneous) or Kruskal-Wallis test (when heterogeneous).
RESULTS
Plant growth and water status
Effects of HBA on plant appearance were visible one week after exposure (Tables 1, 2, 3).
Application of 1.5 mM HBA decreased the plant height and fresh biomass than control. HBA
significantly decreased the shoot and root length at all concentrations (Tables 1 and 2).
Similarly, FA also significantly reduced shoots length, root length, leaf and root fresh biomass
as compared to control. Both herbicides (PML, MTR) also decreased the shoot and root growth
and biomass of lettuce but PML was more toxic at 10-1 mM that drastically decreased the leaf,
root fresh biomass and shoot length 2-folds than control (Tables 1, 2, 3).
47
Phytotoxic effects of Allelochemicals and herbicides
Table 1. Effects of allelochemicals [ferulic, p-hydroxybenzoic acid (HBA)] and herbicides
(pendimethalin, S-metolachlor) on leaf growth and development of lettuce
Growth characteristics
Treatment concentration
Treatments
Leaf fresh weight (g)
I
II
III
IV
Control
10.58±0.60 10.58±0.60 10.58±0.60 10.58±0.60
Ferulic acid
7.00±0.51* 7.15±1.10
8.6±1.14
9.81±1.52
HBA
5.25±1.23* 7.28±0.21
7.97±0.46
8.56±1.52
Pendimethalin 4.68±1.02* 4.93±1.73* 5.16±0.54* 6.22±0.73*
S-metolachlor 6.55±0.57* 6.99±0.83* 7.47±1.36* 8.62±1.29
Leaf dry weight (g)
Control
1.63±0.15
1.63±0.15
1.63±0.15
1.63±0.15
Ferulic acid
1.56±0.13
1.37±0.35
1.97±0.311 1.88±0.44
HBA
1.15±0.42
1.71±0.45
1.87±0.15
Pendimethalin 1.47±0.18
1.05±0.38
1.75±0.090 1.09±0.46
S-metolachlor 1.64±0.16
1.92±0.18
1.63±0.33
1.98±0.17
1.97±0.27
Doses of Allelochemicals: I: 1.5, II: 1.0, III: 0.5, IV: 0.1 mM, Doses of Herbicides: I: 10-1, II:
10-3, III: 10-5, IV: 10-7 mM. Each value represents the mean (± S.E.) of three replicates.
*Asterisks indicate significant differences as compared to control for p < 0.05 according to LSD
test.
However, both allelochemicals (FA, HBA) and herbicides (PMR, MTR) did not affect the RWC
and Osmotic potential in leaves of treated plants at any tested concentrations (data not shown).
Chlorophyll fluorescence
Allelochemical FA reduced the quantum efficiency (Fv/Fm) of open PSII reaction centers in dark
adapted states (Fig. 1). Reduction in ratio of Fv/Fm was stronger than both herbicides. Similarly,
HBA also reduced the quantum efficiency (Fv/Fm) in lettuce seedlings on all days but the effect
was stronger on day 5 and 6. Both herbicides (PML and MTR) slightly reduced the F v/Fm
activity on day 2 and afterwards there was Stimulation (Fig. 1).
48
Chapter III. Physiological response of lettuce
Table 2. Effects of allelochemicals [ferulic, p-hydroxybenzoic acid (HBA)] and herbicides
(pendimethalin, S-metolachlor) on shoot and root length of lettuce
Growth characteristics
Treatment concentration
Treatments
Shoot length (cm)
Root length (cm)
I
II
III
15.30±0.23
IV
Control
15.30±0.23
15.30±0.23
15.30±0.23
Ferulic acid
8.10±0.86*
8.73±0.933* 9.60±1.22*
10.93±2.69*
HBA
8.10±0.56*
7.93±0.34*
9.17±0.01*
7.17±0.16*
Pendimethalin 6.93±0.23*
7.03±1.72*
10.02±2.36* 10.47±0.549*
S-metolachlor 8.50±1.75*
9.50±1.25*
10.83±1.87* 10.50±0.764*
Control
27.77±0.22
27.77±0.22
27.77±0.22
Ferulic acid
19.00±1.75* 20.33±0.88* 20.50±1.04* 21.00±0.57*
HBA
18.10±0.78* 18.83±1.01* 20.17±0.72* 21.33±0.88*
27.77±0.22
Pendimethalin 17.83±2.92* 18.42±2.64* 18.88±3.39* 19.08±2.76*
S-metolachlor 15.27±3.24* 17.78±3.72* 19.64±4.93* 23.00±1.52*
Doses of Allelochemicals: I: 1.5, II: 1.0, III: 0.5, IV: 0.1 mM, Doses of Herbicides: I: 10-1, II:
10-3, III: 10-5, IV: 10-7 mM. Each value represents the mean (± S.E.) of three replicates.
*Asterisks indicate significant differences as compared to control for p < 0.05 according to LSD
test.
Both allelochemicals significantly decreased the effective quantum yield (Ф PSII) of
photosystem II from 3-6 days (Fig. 2). Contrarily there was no effect of herbicides on ФPSII
levels. The application of FA drastically decreased (3-folds) the photochemical fluorescence
quenching (qP) compared to control (Fig. 3).
The allelochemicals HBA also drastically reduced (3-folds) qP values over control from 3-6
days than control. On the contrary, there was non-significant effect of both herbicides on qP in
lettuce. The application of FA also drastically decreased (3-folds) the non-photochemical
fluorescence quenching (NPQ) as compared to control on third day (Fig. 4). The allelochemical
HBA also significantly reduced the NPQ values than control on 1-4 days but there was nonsignificant effect of both herbicides on NPQ in lettuce on all days (Fig. 5). However, on third
day, herbicide PML significantly stimulated the NPQ in lettuce.
49
Phytotoxic effects of Allelochemicals and herbicides
Table 3. Effects of allelochemicals [ferulic, p-hydroxybenzoic acid (HBA)] and herbicides
(pendimethalin, S-metolachlor) on root growth and development of lettuce
Growth characteristics
Treatment concentration
Treatments
Root fresh weight (g)
Root dry weight (g)
I
II
6.31±0.34
III
Control
6.31±0.34
Ferulic acid
3.18±1.002* 4.04±0.378* 4.16±0.124* 4.63±0.65*
HBA
2.53±0.44*
6.31±0.34
4.26±0.30*
4.33±0.18*
Pendimethalin 2.95±0.72*
3.29±0.785* 3.48±0.75*
3.62±0.40*
S-metolachlor 2.70±0.31*
3.04±0.39*
4.10±0.27*
4.15±0.072*
Control
0.45±0.042
0.45±0.042
0.45±0.042
0.45±0.042
Ferulic acid
0.44±0.013
0.37±0.07
0.40±0.023
0.377±0.052
HBA
0.28±0.01
0.29±0.06
0.77±0.36
0.35±0.04
0.30±0.04
0.32±0.063
0.37±0.04
S-metolachlor 0.317±0.049 0.35±0.06
0.39±0.038
0.43±0.036
Pendimethalin 0.28±0.030
2.76±0.63*
6.31±0.34
IV
Doses of Allelochemicals: I: 1.5, II: 1.0, III: 0.5, IV: 0.1 mM, Doses of Hebicides: I: 10-1, II: 103
, III:10-5, IV: 10-7 mM. Each value represents the mean (± S.E.) of three replicates. *Asterisks
indicate significant differences as compared to control for p < 0.05 according to LSD test.
The allelochemicals HBA also drastically reduced (3-folds) qP values over control from 3-6
days than control. On the contrary, there was non-significant effect of both herbicides on qP in
lettuce. The application of FA also drastically decreased (3-folds) the non-photochemical
fluorescence quenching (NPQ) as compared to control on third day (Fig. 4). The allelochemical
HBA also significantly reduced the NPQ values than control on 1-4 days but there was nonsignificant effect of both herbicides on NPQ in lettuce on all days (Fig. 5). However, on third
day, herbicide PML significantly stimulated the NPQ in lettuce.
50
Chapter III. Physiological response of lettuce
control
1
*
Fv/Fm
0.8
*
HBA
*
*
0.8
*
0.7
0.7
*
0.6
0.6
0.5
0.5
Fv/Fm
control
0.9
*
0.9
1
FA
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0
0
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
*
control
2
3
Days
4
PML
5
6
*
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
control
*
1
2
3
4
MTR
5
6
Days
Figure 1. Quantum efficiency of open PSII reaction centers in the dark-adapted leaves (Fv/Fm) of
lettuce from days 1 to 6 following exposure to allelochemicals [ferulic (FA), phydroxybenzoic acid (HBA) (1.5 mM)] and herbicides [pendimethalin (PML), Smetolachlor (MTR) (10-1 mM)]. Every column in each graph represents the mean (±
S.E.) of three replicates. Asterisks indicate significant differences at level 0.05 with
respect to control.
Carbon isotope ratio analysis
The FA, HBA, MTR and PML treatments did not significantly affect the carbon %age. The
lettuce seedlings treated with HBA, carbon isotope ratio (δ13C) was significantly less negative (26.93) than control (-27.61) (Fig. 5 B). Similarly, lettuce plants treated with FA and MTR, the
δ13C values were significantly less negative (-26.90) and
(-26.88), respectively, as
compared to control (-27.61) (Fig. 5 B).
Carbon isotope discrimination (Δ13C) values in lettuce leaves were significantly less after
treatment with FA (19.40) and HBA (19.25) at 1.5 mM concentration than control (20.17) (Fig.
5C). Carbon isotope discrimination (Δ13C) values were significantly less (19.40) after treatment
with MTR as compared to control (20.17) (Fig. 5 C). The ratio of intercellular CO2
51
Phytotoxic effects of Allelochemicals and herbicides
concentration from leaf to air (ci/ca) were significantly reduced by FA (1.5, 1.0, 0.5, 0.1 mM),
HBA (1.5, 1.0, 0.5 mM), MTR (10-1, 10-3, 10-5 and 10-7 mM) in treated plants at compared to
control (Fig. 6 A). There was non-significant effect of FA, HBA, MTR and PML on intrinsic
water use efficiency (iWUE) (Fig. 6 B), however, the lower concentration of FA, HBA, and
MTR significantly stimulated the iWUE.
Ф PS II
0.8
control
FA
0.7
0.7
0.6
0.6
0.5
0.5
0.4
*
*
0.3
*
0
0
control
PML
0.7
*
*
0.2
0.1
HBA
*
*
0.3
0.1
0.8
control
0.4
*
0.2
ФPS II
0.8
0.8
control
MTR
0.7
0.6
0.6
0.5
0.5
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0
0
1
2
3
Days
4
5
6
1
2
3
4
5
6
Days
Figure 2. Quantum yield of photosystem II (ФPSII) in lettuce leaves from days 1 to 6 following
exposure to allelochemicals [ferulic (FA), p-hydroxybenzoic acid (HBA) 1.5 mM)] and
herbicides [pendimethalin (PML), S-metolachlor (MTR) (10-1 mM)]. Every column in
each graph represents the mean (± S.E.) of three replicates. Asterisks indicate
significant differences at level 0.05 with respect to control.
52
Chapter III. Physiological response of lettuce
qP
0.9
control
FA
0.8
0.8
0.7
0.7
0.6
0.6
0.5
0.5
*
0.4
*
*
control
*
0.1
0
0
0.9
control
0.8
PML
*
0.2
0.1
0.9
control
0.8
0.7
0.6
0.7
0.5
0.5
0.4
0.4
0.3
0.2
0.3
0.1
0.1
*
*
0.3
0.2
HBA
*
0.4
0.3
qP
0.9
MTR
0.6
0.2
0
0
1
2
3
4
Days
5
6
1
2
3
Days
4
5
6
Figure 3. Changes in chlorophyll fluorescence quenching (qP) in lettuce leaves from days 1 to 6
following exposure to allelochemicals [ferulic (FA), p-hydroxybenzoic acid (HBA) (1.5
mM)] and herbicides [(pendimethalin (PML), S-metolachlor (MTR) (10-1 mM)]. Every
column in each graph represents the mean (± S.E.) of three replicates. Asterisks indicate
significant differences at level 0.05 with respect to control.
Protein contents
Both FA and HBA at 1.5 mM concentration significantly reduced the leaf protein contents,
(0.72 mg g -1) and (0.67 mg g -1), respectively than control leaves (1.03 mg g -1). Herbicide MTR
reduced the leaf protein contents (0.70 mg g -1) at 10-1 mM concentration, while PML
significantly decreased protein contents (0.83 mg g -1) than control (1.03 mg g -1) (Figure 7).
The reduction of total protein contents in lettuce leaves after HBA application, proved that HBA
was most toxic than all other treatments.
53
Phytotoxic effects of Allelochemicals and herbicides
DISCUSSION
Allelochemicals alters the plant growth and development through multiple effects on
physiological processes because there are hundreds of different structures and many compounds
have several phytotoxic effects (15). In this study, p-hydroxybenzoic acids (HBA) significantly
reduced the root and shoot length, leaf and root fresh weight of
9
control
FA
8
7
7
6
6
NPQ
8
control
*
5
5
4
*
3
*
3
*
2
1
0
0
control
*
2
1
14
*
4
*
HBA
PML
12
12
control
MTR
10
10
8
8
6
6
4
4
2
2
0
0
1
2
3
Days
4
5
6
1
2
3
4
Days
5
6
Figure 4. Changes in non-photochemical fluorescence quenching (NPQ) in lettuce leave from
days 1 to 6 following exposure to allelochemicals [ferulic (FA), p-hydroxybenzoic acid
(HBA) (1.5 mM)] and herbicides [pendimethalin (PML), S-metolachlor (MTR) (10-1
mM)]. Every column in each graph represents the mean (± S.E.) of three replicates.
Asterisks indicate significant differences at level 0.05 with respect to control.
lettuce. The reduction in root growth is considered as one of the first effects of allelochemicals
associated with reduction in plant growth and development. Similarly, Reigosa et al. (49),
reported that HBA inhibits the root growth of Rumex crispus at 0.01 M concentration. Doblinski
et al. (13), observed that p-coumaric and p-hydroxybenzoic acids (0.5-1.0 mM) caused a
significant decrease in root length and root fresh weight of soybean. Treatment with FA
54
Chapter III. Physiological response of lettuce
decreased both root and shoot length of lettuce and the effect was concentration dependent. The
ferulic acid affects the root growth of cucumber, tomato and bean (8) and maize (12).
Herbicides (PML and MTR), also significantly inhibited the lettuce leaf and root fresh weight at
all test concentrations. At any concentration, both allelochemicals and herbicides did not
significantly affect RWC and LOP. Contrary, it was reported that phenolic acids (p-coumaric,
caffeic,
(A)
47
46
control
HBA
MTR
(B)
FA
PML
-25.5
-26.0
43
**
*
-26.5
44
δ 13C
C%
45
-27.0
42
-27.5
41
40
-28.0
39
-28.5
38
21.0
20.5
20.0
Δ13C
19.5
19.0
* *
*
*
* *
II
III
IV
18.5
18.0
17.5
17.0
I
concentrations
(C )
Figure 5. Changes in leaf carbon (C %) (A), carbon isotope ratios (δ 13C) (B) and carbon isotope
discrimination (Δ13C) (C) in lettuce following exposure to allelochemicals [ferulic (FA),
p-hydroxybenzoic acid (HBA) at (I= 1.5, II= 1.0, III= 0.5, IV= 0.1 mM)] and herbicides
[pendimethalin (PML), S-metolachlor (MTR) at I= 10-1, II= 10-3, III= 10-5, IV= 10-7
mM)]. Every column in each graph represents the mean (± S.E.) of three replicates.
Asterisks indicate significant differences at level 0.05 with respect to control.
ferulic, and salicylic acid), cause water stress in plants (4,5). Herbicides are effective to control
target weeds but they are continuously contaminating non-target aquatic environments through
55
Phytotoxic effects of Allelochemicals and herbicides
surface runoff (32). Metolachlor and propachlor applied before transplanting were damaging the
lettuce, and pendimethalin severely reduced the lettuce growth when applied after transplanting
(27).
The best-characterized phytotoxic mechanisms induced by allelochemicals are the inhibition of
photosynthesis and oxygen evolution through interactions with components of photosystem II
(50). The quantum efficiency (Fv/Fm) of open PSII reaction centers in dark- adapted states in
lettuce was significantly reduced by both allelochemicals at 1.5 mM concentration. A sustained
decrease of Fv/Fm may indicate the damage to photosystem II reaction centers (10, 41).
0.74
control
FA
HBA
PML
MTR
9.00
*
*
c i/c a
0.70
0.68
*
control
*
*
*
HBA
PML
MTR
*
*
7.00
* *
*
FA
* *
8.00
*
*
0.66
iWUE
0.72
6.00
5.00
4.00
3.00
0.64
2.00
0.62
1.00
0.00
0.60
I
II
concentrations
III
IV
I
II
III
IV
concentrations
Figure 6. Changes in ratio of intercellular to air CO2 concentration (ci/ca) and intrinsic water use
efficiency (iWUE) in lettuce following exposure to allelochemicals [ferulic (FA), phydroxybenzoic acid (HBA) at (I= 1.5, II= 1.0, III= 0.5, IV= 0.1 mM)] and herbicides
[pendimethalin (PML), S-metolachlor (MTR) at I= 10-1, II= 10-3, III= 10-5, IV= 10-7
mM)]. Every bar in each graph represents the mean (± S.E.) of three replicates.
Asterisks indicate significant differences at level 0.05 with respect to control.
Recently, Zhou and Yu (62), reported that allelochemicals can significantly affect the
performance of three main processes of photosynthesis: stomatal control of CO2 supply,
thylakoid electron transport (light reaction) and carbon reduction cycle (dark reaction). Both
allelochemicals significantly reduced effective quantum yield of PSII (ФPSII). The inhibition of
ФPSII values could be due to poor efficiency of PSII reaction centers, and it may indicate the
decreasing rate of linear electron transport (45), suggesting that the proportion of photons
absorbed by PSII and used for photosynthesis was less than control.
Changes in the chlorophyll fluorescence quenching parameters are good indicators of
photosynthetic electron flow (55). After the onset of illumination, maximal chlorophyll
fluorescence declines as the photosynthetic reaction proceeds due to the photosynthetic electron
flow, photochemical quenching (qP), and non-photochemical quenching (NPQ) (21).
56
Chapter III. Physiological response of lettuce
1.20
1.00
Protein contents
*
*
0.80
*
*
0.60
0.40
0.20
0.00
Control
FA
HBA
PML
MTR
Treatments
Figure 7. Effects of allelochemicals [ferulic (FA), p-hydroxybenzoic acid (HBA) (1.5 mM)] and
herbicides [pendimethalin (PML), S-metolachlor (MTR) (10-1 mM)] on leaf protein
contents (mg g-1) on lettuce. Every bar in each graph represents the mean (± S.E.) of
three replicates. Asterisks indicate significant differences at level 0.05 with respect to
control.
The photochemical fluorescence quenching (qP) was significantly decreased after FA and HBA
treatment (values were 3-folds less than control), indicating that the balance between excitation
rate and electron transfer rate has changed leading to a more reduced state of PSII reaction
centers. The decrease in qP induced by the allelochemicals indicates a higher proportion of
closed PSII reaction centers (21), which probably decreases the proportion of available
excitation energy used for photochemistry. Non-photochemical quenching (NPQ) was also
significantly reduced in allelochemicals treated lettuce plants. The decreased qP can be seen as
the regulatory response to down-regulate the quantum yield of PSII electron transport (ΦPSII)
(21). There was no inhibitory effect of herbicides (PML, MTR) on qP and NPQ, rather PML
significantly stimulated NPQ on third day.
Plant photosynthesis discriminates against the stable 13C isotope (17,18,19) when atmospheric
CO2 passes through stomata and during CO2 carboxylation in Rubisco. In this study, carbon
isotope ratios (δ13C) were significantly less negative (-26.93) by HBA (1.0 mM), FA (-26.90)
(0.1 mM) and MTR (-26.88) (10-5 mM) treated seedlings as compared to control (-27.61).
Barkosky and Einhellig (4), reported that carbon isotope ratio (δ 13C) in leaf tissue of soybean at
termination of the 28-day experiment showed significantly less discrimination (less negative δ
13
C) against 13C in plants grown with 0.75 mM. p-hydroxybenzoic acid. Handley et al. (26),
reported a decrease in foliar δ15N because of salt stress in barley (Hordeum spp.). Similarly,
Barkosky et al. (5), concluded that dried leaf tissue from leafy spurge (Euphorbia sula) treated
with 0.25 mM Caffeic acid had a less negative δ 13C compared to controls, indicating less
discrimination against 13C in these plants. Carbon isotope discrimination (Δ 13C), were
significantly lower (19.40) after treatment with both FA and HBA as compared to control
57
Phytotoxic effects of Allelochemicals and herbicides
(20.17). The ratios of intercellular CO2 concentration from leaf to air (ci/ca) were significantly
less after treatment with allelochemicals. Carbon isotope discrimination decreases with a
decrease in intercellular CO2 concentration due to stomatal closure and consequently with water
use efficiency. In C3 species, leaf level 13C Δ during photosynthetic gas exchange primarily
reflects the balance between CO2 supply by diffusion through stomata and CO2 demand by
biochemical reactions in chloroplasts, most importantly catalysis by Rubisco (18). Both
processes discriminate against the heavier isotope, but the fractionation occurs during
carboxylation by Rubisco (25). The allelochemicals (FA, HBA) significantly reduced the leaf
protein contents of lettuce following exposure to 1.5 mM concentration. Similarly, Mersie and
Singh (43), reported reduction in total protein contents after p-hydroxybenzoic acid application.
Both herbicides (PML, MTR) also significantly decreased the protein contents of lettuce at 10 -1
mM concentration.
CONCLUSIONS
These results show that FA and HBA, both are growth inhibitors of lettuce and mechanism of
inhibition is due to damage in PSII reaction centers. Furthermore, both allelochemicals
significantly reduced the shoot length, root length, leaf and root fresh weight of lettuce. This
leads to imbalance in photosynthesis, which stunted the leaf, reduced the root growth and plant
biomass. Allelochemicals also decreased protein contents of lettuce and ratio of stable carbon
isotope was significantly less negative in allelochemical treated plants.
ACKNOWLEDGEMENTS
Many thanks are due to Dr. Aldo Barreiro, Carlos Bolano, Alfredo Justo, Maite Ricart, P.
Lorenzo and Jesús Estévez Sío, for their assistance in the field and laboratory work. Authors
also thanks with regards to Dr. Nuria Pedrol for help in statistical analysis and useful
discussions. We are extremely thankful to anonymous referees for their help in the improvement
of the manuscript.
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61
Phytotoxic effects of Allelochemicals and herbicides
62
Allelopathic potential of Acacia melanoxylon
R. Br. on the germination and root growth of
native grasses
Chapter IV
Chapter section published as an original article to SCI journal:
Hussain, M.I., González, L., and Reigosa, M.J. 2010. Allelopathic potential of Acacia
melanoxylon R. Br. on the germination and root growth of native grasses. Weed
Biology and Management, in press
Germination response of native herbs
Water extracts obtained from the flowers and phyllodes of Acacia melanoxylon were used to
determine their allelopathic potential on germination and seedling growth of native grasses,
cocksfoot (Dactylis glomerata), perennial ryegrass (Lolium perenne), common sorrel (Rumex
acetosa) and general biotest specie, lettuce (Lactuca sativa) in the laboratory. Flowers and
phyllodes of A. melanoxylon were soaked separately in the water in a ratio of 1:1 (w/v) for 24 h
to prepare aqueous extracts and distilled water was used as control. The seeds of target species
were germinated in petri dishes and counted daily up to 7 days. A. melanoxylon flowers (100%,
75%, and 50%) aqueous extract significantly inhibited seed germination in D. glomerata, R.
acetosa, L. perenne and in L. sativa while phyllodes (100%, 75%) extract completely inhibited
germination of R. acetosa, L. perenne, and L. sativa during different days. There was a tendency
of stimulation of germination in R. acetosa and L. perenne by acacia phyllodes extract. The
flower extract caused most reduction in germination index and germination speed of D.
glomerata, L. perenne and L. sativa. The mean LC50-value of A. melanoxylon flowers and
phyllodes extract in the germination inhibition of L. perenne was 43%, 41%, in R. acetosa
(40%, 38%), and in L. sativa was 53%, 41%. The LC50-value of A. melanoxylon phyllodes
extract in the germination inhibition of D. glomerata was 46%. The seeds of four plant species
were sown in glass petri dishes to record the root length. All the four concentration of A.
melanoxylon flowers extract proved to be more toxic that significantly reduced the root length
of D. glomerata, L. perenne, R. acetosa and L. sativa while phyllodes extract decreased the root
length of L. perenne (83%) and R. acetosa (74%) at 100% concentration. Inhibition of seed
germination of target species showed a species-specific and dose-dependent response with
highest inhibition occurred at concentration of 100% and 75%. The L. perenne and D.
glomerata grass seeds were more sensitive regarding germination as compared to L. sativa and
R. acetosa. Water extract of A. melanoxylon flowers were more phytotoxic as compared to
phyllodes even at lower concentration (25%).
Keywords: allelopathy, Dactylis glomerata, Lolium perenne, germination, Acacia melanoxylon,
Rumex acetosa, seedling growth.
64
Chapter IV. Allelopathic potential of A. melanoxylon
In 19th century, some Acacia species were introduced as an ornamental plants in Southern
Europe; have naturalized and become invasive in Mediterranean and Atlantic regions from
Portugal to Italy (Sheppard et al. 2006). Acacia species affects crop growth by competing
different environmental resources, as their litter interferes with the establishment and growth of
adjoining crop plants (Kohli et al. 2006) as well as release of numerous chemical substances
including phenolic compounds in litter (Seigler 2003). Carballeira and Reigosa (1999) have
demonstrated that leachate from Acacia dealbata showed strong inhibitory effects on
germination and growth of Lactuca sativa during acacia‘s blossom. Allelopathy was implicated
by Duhan and Lakshminarayana (1995), Hoque et al. (2003), El-Khawas and Shehata (2005),
Al-Wakeel et al. (2007), and Lorenzo et al. (2008) when they observed that water extracts of
different acacia species inhibited the germination, root/shoot length and dry weight of different
crops/weeds. However, allelopathic plants might have inhibitory, stimulatory, or no effect on
the germination and growth of other plants (Reigosa et al. 1999).
Blackwood (Acacia melanoxylon R. Br.) is an introduced species that has its origin in the
temperate forests of south-east Australia and Tasmania. It is versatile and highly adaptive tree
specie which has spread all over the world (Knapic et al. 2006). It covers a considerable area in
coastal zone of the north-western Iberian Peninsula, both in monocultures and in mixed stands
with Eucalyptus globulus, characterized by vigorous tree or root sprouts and seed germination
stimulated by fire. Upon invasion, it establishes quickly in the alien environment, thereby
resulting in changes in the structure and dynamics of native ecosystems. Although the species is
found mostly in wastelands, it also grows well in cultivated fields, pastures and along roadsides.
This dominance might be due to chemical interference or allelopathy that gives it an additional
advantage over native plants. The forest plantations of A. melanoxylon started in north-western
Iberian Peninsula at the beginning of twentieth century (Areses 1953) and presently considered
as invasive (Xunta de Galicia 2007). Its leaves have allelopathic effects on the germination and
seedling growth of L. sativa (Souto et al. 2001). The allelochemicals are found in different parts
of a plant body; roots, stems, flowers, leaves and likely enter the soil from foliar leaching in
rainwater, exudation from roots into the soil water, or after leaf fall and subsequent
incorporation in the soil (Inderjit & Duke 2003). Different parts of the same plant also vary in
their allelopathic effect on the germination and growth of crops. However, there is no
information about the allelopathic effect of water extract of flowers and phyllodes of A.
melanoxylon on grass species present near the acacia stands.
Seed germination and seedling growth bioassays are widely used for the study of depletion of
germination and stunting of growth of a susceptible plant due to the release of allelochemicals
from a donor plant (Lottina-Hennsen et al. 2006). The aim of present investigation was to
assess the allelopathic potential of flowers and phyllodes extract of A. melanoxylon on
germination, emergence, root elongation and seedling growth of three native grass species.
This can provide basic information about the management of A. melanoxylon.
65
Germination response of native herbs
MATERIALS AND METHODS
Target species
Three grass species (1) Dactylis glomerata L., cv Amba (Poaceae); (2) Lolium perenne L. cv.
Belida (Poaceae); (3) Rumex acetosa L. cv Belleville (Polygonaceae), were used as a
representative grass species because they naturally grow in Galician forest ecosystem, (NW
Spain) and under the canopy stands of A. melanoxylon. The Lactuca sativa L. cv. Great Lakes
California (Asteraceae) was selected as a general biotest species because it is frequently used as
model specie in allelopathic bioassays (Macías et al. 2000). The use of L. sativa could
demonstrate the possible mechanisms by which A. melanoxylon competes with other species
while the results concerning three grass species have more ecological significance. The seeds of
test species were purchased commercially from Semillas Fito (Barcelona, Spain).
Water extraction of Acacia melanoxylon
Fresh shoots of Acacia melanoxylon were collected from natural population in the surrounding
area of Lagoas Marcosende campus of University of Vigo, during flowering period. The flowers
and phyllodes (expanded petioles that form simple lamina; Atkin et al. 1998) were separated
from branches and soaked in distilled water in the ratio of 1:1 (w/v). Similar extraction
techniques were used by Molina et al. (1991) in their studies of Eucalyptus spp. and Lorenzo et
al. (2008) in their studies of allelopathic effects of Acacia dealbata L. The extract was prepared
at room temperature and left in laboratory for 24 hours. The extract was collected, filtered
through filter paper and described as 100%. Distilled water was added in this solution to make
different dilution (75%, 50% and 25%).
Germination bioassays
The glass petri dishes were used (9 cm diameter) having Whatman 3 MM papers. Twenty-five
seeds of each species were placed in petri dishes to which 3 ml of solution was added at the start
and control received 3 ml distilled water. An additional one ml of each solution was added
every 48 hours thereafter. Three replicates of each treatment were incubated in a germination
chamber with following germination conditions: L. perenne (day/night temperature 25/15 0C,
12/12 h of photoperiod), L. sativa 18/8 °C (day/night temperature) and 12/12 h (light/darkness)
photoperiod, R. acetosa (day/night temperature: 28/20 0C and 9/15 h of light/darkness and D.
glomerata (day/night temperature: 25/20 0C and 14/10 h of light and darkness) and a relative
humidity of 80% (Hussain et al. 2008). The light was provided by cool white fluorescent tubes
with an irradiance of 35 mol m-2sec-1. The germination assessed after every 24 hours by
counting the number of germinated seeds (rupture of seed coats and emergence of radicle
1mm) up till 7-day.
66
Chapter IV. Allelopathic potential of A. melanoxylon
Seedling growth bioassays
The petri dishes were placed in cold chamber at 4 0C after one week to stop seedling growth and
radicle length was measured with a measuring tape (Reigosa & Malvido-Eva 2007).
Statistical analysis
The germination rate index (GT) was determined as described by Jäderlund et al. (1996) and
Speed of germination was calculated as proposed by Ahmad and Wardle (1994).
The collected data were statistically analysed by using one-way analysis of variance (ANOVA)
(Sokal & Rohlf 1995) and Dunnett test was used to determine differences between treatment
means at 5% probability level. The mean LC 50 was calculated using probit analysis as described
by Finney (1971). A logistic equation was fitted to the germination data as a function of
logarithm of A. melanoxylon flower and phyllodes concentration using SPSS (version 15.0) for
Windows:
Y = a + bX
Y = Probit value
a = Intercept
b = Slope of the line
X = Log 10 concentration
The value of X was obtained to calculate the LC50 value of A. melanoxylon flower and phyllodes
concentration, the dose for 50% inhibition in seedling growth.
RESULTS
Effect of A. melanoxylon extract on germination at each exposure time
The laboratory test showed that A. melanoxylon flowers (100%, 75%, and 50%) aqueous extract
significantly inhibited germination in D. glomerata, R. acetosa, L. perenne and in L. sativa
(Tables 1, 2, 3, and 4). A. melanoxylon phyllodes (100%, 75%) extract completely inhibited
germination process in R. acetosa during second, third, sixth day, in L. perenne during fourth,
fifth, sixth day and in L. sativa during first and second day. However, there was a tendency of
stimulation in germination in R. acetosa and L. perenne by different concentration of phyllodes
extract.
67
Germination response of native herbs
Effect of A. melanoxylon extract on germination indices
As shown in Table 5 the inhibitory effect of the water extract on total germination index (GT)
and germination speed (S) depended on the extract concentration and the plant species. In D.
glomerata the GT and S was completely inhibited by A. melanoxylon flowers extract at all four
concentrations. Both indices were less sensitive to the phyllodes extract at lower concentration
that was not significantly affected in D. glomerata. In R. acetosa, aqueous extract of A.
melanoxylon flowers and phyllodes did not significantly inhibited GT and S indices at any
concentration tested (Table 5). The flowers extract (100%, 75%, 50%, and 25%) significantly
suppressed GT and S indices in L. perenne and L. sativa (Table 5) while phyllodes extract of A.
melanoxylon significantly decreased GT index in L. perenne at 100% and 75% concentration. In
L. sativa, both GT and S indices were significantly decreased by flower and phyllodes extract at
concentration of 100%, 75%, and 50% (Table 5).
Effect of A. melanoxylon extract on germination of native grasses
The effect of A. melanoxylon flower and phyllodes extract on germination of native grasses was
presented in Table 6 after probit analysis. The total number of seeds, non-germinated seeds,
expected response and probability were determined against the four different concentrations
(100%, 75%, 50%, 25%) of Acacia melanoxylon flowers and phyllodes extract. Non-germinated
seeds data were fitted to the probit model. Non-germinated seeds data were best fitted to the
probit model after log transformation of the data. The respective results of the Chi-Square tests
for goodness of fit were d = 10 (at 95% confidence limit) for non-germinated seeds. The
respective regression equation were Y = -0.091 + 0.675 X in D. glomerata germination data
after exposure of phyllodes extract of A. melanoxylon. No regression equation was computed in
D. glomerata after exposure to flowers aqueous extract of A. melanoxylon because there was no
seed germination. The concentration of 46% of phyllodes of A. melanoxylon was a diagnosed
concentration that inhibited 50% of the seed germination of D. glomerata (Table 6). In L.
perenne, the respective regression equation was Y = 3.276+ 4.688 X following exposure to
flowers aqueous extract of A. melanoxylon. The concentration of 43% of flowers of A.
melanoxylon inhibited 50% of the seed germination of L. perenne. The regression equation was
Y = -0.016 + 1.177 X following exposure to A. melanoxylon phyllodes extract. The
concentration of 41% of phyllodes of A. melanoxylon inhibited 50% of the seed germination of
L. perenne (Table 7). The respective results of the Chi-Square tests for goodness of fit were d =
10 (at 95% confidence limit) for non-germinated seeds in R. acetosa, and respective regression
equation was Y = 0.447+ 0.204 X following exposure to flowers aqueous extract of A.
melanoxylon. The concentration of 40% of A. melanoxylon flowers inhibited 50% of the seed
germination in R. acetosa. The regression equation was Y = 0.180 + 0.079 X following
exposure to A. melanoxylon phyllodes extract. The concentration of 38% of phyllodes of A.
melanoxylon inhibited 50% of the seed germination in R. acetosa (Table 8). The regression
equation was Y = 1.673+1.855X following exposure to flowers aqueous extract of A.
melanoxylon. The concentration of 53% of flower of A. melanoxylon inhibited 50% of the seed
germination in L. sativa. The regression equation was Y = 0.211+ 0.764 X following exposure
68
Chapter IV. Allelopathic potential of A. melanoxylon
to A. melanoxylon phyllodes extract. The concentration of 41% of phyllodes of A. melanoxylon
inhibited 50% of the seed germination of L. sativa (Table 9).
Effect of Acacia melanoxylon extract on root growth
The effect of A. melanoxylon flower and phyllodes extract on root length of plant species was
concentration and species dependent. The root length of L. perenne and R. acetosa were
significantly reduced due to A. melanoxylon flowers extract at all concentration (Fig. 1).
Aqueous extract of A. melanoxylon phyllodes significantly reduced the root length in R. acetosa
at all concentration tested while the same extract significantly decreased the root growth in L.
perenne only at 100% and 75% concentration. A. melanoxylon flower extract significantly
reduced root length in D. glomerata and L. sativa at all concentrations (100%, 75%, 50% and
25%). No significant variation was detected due to A. melanoxylon phyllodes extract on root
length of D. glomerata and L. sativa at any concentration tested (Fig. 2).
DISCUSSION
Allelopathic effects of exotic plants on germination and seedling growth of native species have
received more and more attention in the past decades (Tawaha & Turk 2003; Wakjira et al.
2005; Dorning & Cipollini 2006). The present study reveals that A. melanoxylon has
significantly reduced germination and seedling growth of native grass species. Compared to
distilled water, continuous application of A. melanoxylon flower/phyllodes extract up to 7 days
significantly reduced seed germination of native grass species. This finding is supported by the
results of Souto et al. (2001) who identified several phenolic compounds in the A. melanoxylon
phyllodes extracts. These compounds present in phyllodes extract might be responsible for
retardation of germination and other growth parameter of L. perenne, D. glomerata, and R.
acetosa in the present study. Phenolics are widely recognized for their allelopathic potential in
plants, and can be found in a variety of plant tissues (Djurdjevic et al. 2004). Many other
species of Genus Acacia (Mimosaceae), like Acacia dealbata Link (Carballeira & Reigosa
1999; Lorenzo et al. 2008); Acacia confusa Merr (Chou et al. 1998); Acacia auriculiformis
A.Cunn. ex Benth (Hoque et al. 2003; Oyun 2006); Acacia nilotica (El-Khawas & Shehata
2005; Al-Wakeel et al. 2007) are also known to exhibit allelopathic activity. The effects of
allelochemicals have been studied mostly on seed germination and the suggested mechanisms
for its inhibition are the disruption of mitochondrial respiration (Abrahim et al. 2003) through
the influence of allelochemicals on glycolysis, Krebs cycle, electron transport and oxidative
phosphorylation (Muscolo et al. 1999) and mitochondrial membrane.
In L. perenne the LC50 values of A. melanoxylon flowers and phyllodes extract were 43% and
41% respectively. The values for R. acetosa were 40% and 38% while in L. sativa were 53%
and 41% respectively. The mean LC50-value of A. melanoxylon phyllodes extract in the
germination inhibition of D. glomerata was 46%. Generally, seed germination decreased with
increasing concentration of extract, indicating that seed germination was quantitatively related
to extract concentration. Increasing inhibitory rates with increasing concentration was in
69
Germination response of native herbs
accordance with previous reports (El-darier & Youssef 2000; Singh et al. 2003; Batish et al.
2006) for other allelopathic species. Among the flower and phyllodes extracts, flowers extract
was observed to be inhibitorier. The delayed seed germination with the flowers extract of A.
melanoxylon, compared with the phyllodes extract, could be related to the more inhibitory effect
of the allelochemicals present in the flower parts of the plant. These results are supported by the
findings of Kill and Yun (1992), who reported that the effect of the aerial parts of Artemisia
princeps on the germination and growth of different plant species was greater than the effect of
the sub aerial parts. The maximum decrease in the germination percentage of the grass seeds,
when treated with flower extract of A. melanoxylon, indicates the presence of water-soluble
allelochemicals in maximum concentration. Dana and Domingo (2006), also reported that
Acacia retinodes flowers aqueous extract (100%) decreased the germination of Carrichtera
annua (33%) and Lactuca sativa (86%), however, there was no effect on Conyza albida. The
lower concentration of phyllodes extracts actually promoted seed germination in some bioassay
species, but higher extract concentrations significantly reduced the germination which suggests
that stimulatory or inhibitory effect is a function of concentration (Saxena & Sharma 1996).
Similarly, Reigosa et al. (1999) concluded that certain allelochemicals have a stimulatory effect
or no action on various plant species at lower concentration.
Inhibition of seed germination was recorded in all test species, although the extent of inhibition
varied across species and treatments. The aqueous extracts of flowers and phyllodes of A.
melanoxylon suppressed the germination and seedling growth of L. perenne, D. glomerata, R.
acetosa and inhibitory effect increased with increasing concentrations of the extract. Marginal
improvement in the germination at low concentrations of phyllodes extract could be the result of
detoxification of allelochemical(s) through conjugation, sequestration or secretion of
carbohydrates, and oxidation of phytotoxic compounds (Inderjit & Duke 2003). Al-Wakeel et
al. (2007) reported that lower doses of Acacia nilotica leaf residue (0.25 and 0.5%, w/w)
stimulated the growth of pea shoot and root, but the higher doses (0.75, 1.0, 1.5 and 2%, w/w)
were inhibitory to seedling growth and the effect was concentration dependent. Although, the
flower extract of A. melanoxylon appeared to have a more negative effect on seed germination
than phyllodes extracts. This could be due to a greater concentration of diffusible active
compound in the flowers than in phyllodes, or variation in chemical composition between the
tissues.
Because of the dramatic effects of allelochemicals on seed germination than on the growth and
viability of adult plants (Weir et al. 2004), seed germination bioassay for establishing
allelopathic activity of plant extracts has been commonly used. However, in such bioassays,
most of the workers have used only one germination index or total germination when Bewley
and Black (1985), proposed the use of more than one index to reflect the germination process
more accurately. Thus, in the present study, two indices were used to assess the allelopathic
effects on germination of these species. It is evident from the data that index of total
germination (GT), commonly used in allelopathic bioassays, was not sensitive enough at lower
concentration of phyllodes extract to conclusively confirm allelopathic activity. It is because
this index gives a global interpretation of germination (Jäderlund et al. 1996) and does not take
70
Chapter IV. Allelopathic potential of A. melanoxylon
into account the speed of germination. In view of the failure of GT index to convincingly
demonstrate allelopathic activity of the flowers and phyllodes extract of A. melanoxylon, speed
of germination (S), was used to monitor the germination behaviour of seeds of the target species
because of the sensitivity of S index (Chiapusio et al. 1997). The limitations of any index to
adequately reflect the effect of allelochemicals on germination behaviour is evident from
present studies, and use of multiple indices seems necessary to validate allelochemical activity
of the plant extracts.
Root lengths in all target species were significantly suppressed by different concentration of A.
melanoxylon flowers as compared to phyllodes. Jadhav and Gaynar (1992), concluded that the
percentage of germination, plumule and radicle length of rice and cowpea were decreased with
increasing concentration of Acacia auriculiformis A. Cunn. ex Benth leaf leachates. Similarly,
Kamal et al. (1997) found that A. auriculiformis A. Cunn. ex Benth significantly inhibited
germination and growth of rice and radicle growth of cowpeas. Carballeira and Reigosa (1999),
have demonstrated that leachate from A. dealbata showed strong inhibitory effects on
germination and radical growth of Lactuca sativa during acacia‘s blossom.
CONCLUSION
The results obtained in this study show that aqueous extracts of flowers and phyllodes of A.
melanoxylon possesses allelochemicals thus suppressed the germination and root growth of
native herbs, D. glomerata, R. acetosa and L. perenne while inhibition was concentration
dependent. These results were obtained under laboratory conditions. The target species were
usually present in Galician forest environment and in the adjacent fields. The evaluation of
allelochemicals and their isolation, identification, release, and movement under field conditions
are important future research guidelines. The effective natural products could be used as
environmentally friendly herbicides to control weeds.
ACKNOWLEDGEMENTS
Many thanks are due to Dr. Aldo Barreiro, Dr. Nuria Pedrol, Carlos Bolano, Alfredo Justo,
Maite Ricart, and Paula Lorenzo for their assistance in the field and laboratory work. We
gratefully acknowledge the help of two anonymous referees in the improvement of manuscript.
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73
Germination response of native herbs
Table 1. Effects of Acacia melanoxylon flowers and phyllodes water extract on germination of D. glomerata at each exposure time during one week.
Exposure Time (Days after sowing)
A. melanoxylon
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Control
0.00±0.00
0.42±0.33
4.92±0.90
4.75±0.65
3.50±0.89
3.09±0.80
2.33±0.63
100%
0.00±0.00
0.00±0.00
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
75%
0.00±0.00
0.00±0.00
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
50%
0.00±0.00
0.00±0.00
0.00±0.00*
0.oo±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
25%
0.00±0.00
0.00±0.00
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
Control
0.00±0.00
0.42±0.33
4.92±0.90
4.75±0.65
3.50±0.89
3.09±0.80
2.33±0.63
100%
0.00±0.00
0.00±0.00
2.00±1.15
4.00±2.00
3.00±0.57
0.00±0.00
1.67±0.88
75%
0.00±0.00
0.00±0.00
4.00±1.15
4.00±1.15
2.67±0.66
2.67±1.76
3.33±2.02
50%
0.00±0.00
0.00±0.00
3.33±0.41
2.00±2.00
2.22±0.83
3.33±2.02
2.00±0.57
25%
0.00±0.00
0.00±0.00
3.67±0.88
4.33±1.20
2.13±0.76
1.33±0.66
1.67±0.88
Flowers aqueous extract
Phyllodes aqueous extract
Results represents the mean (± S.E.) of three replicates.
*Significant differences with respect to control for p < 0.05 according to Dunnett test.
74
Chapter IV. Allelopathic potential of A. melanoxylon
Table 2. Effects of Acacia melanoxylon flowers and phyllodes water extract on germination of R. acetosa at each exposure time during one week.
Exposure Time (Days after sowing)
A. melanoxylon
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Control
0.00±0.00
2.08±0.417
2.25±0.55
2.17±0.405
1.25±0.37
0.27±0.19
0.00±0.00
100%
0.00±0.00
0.00±0.00*
1.67±0.33
1.67±0.33
2.00±0.57
1.33±0.33
0.33±0.33
75%
0.00±0.00
0.00±0.00*
1.33±0.33
4.33±0.88*
2.00±1.52
0.67±0.33
0.00±0.00
50%
0.00±0.00
0.00±0.00*
1.33±0.34
2.33±0.33
2.67±0.33
0.67±0.33
0.67±0.45
25%
0.00±0.00
0.00±0.00*
2.67±1.66
3.33±0.88
2.33±0.88
0.33±0.33
0.00±0.00
Control
0.00±0.00
2.08±0.417
2.25±0.55
2.17±0.405
1.25±0.37
0.27±0.19
0.00±0.00
100%
0.00±0.00
0.00±0.00*
3.33±0.88
2.00±1.00
1.67±0.66
1.67±0.88*
0.33±0.33
75%
0.00±0.00
0.00±0.00*
5.00±0.57*
3.67±0.33
2.33±0.33
2.33±1.20*
0.67±0.66
50%
0.00±0.00
0.00±0.00*
5.00±0.15*
1.33±0.33
2.00±1.15
1.00±1.00*
0.00±0.00
25%
0.00±0.00
0.00±0.00*
5.33±0.88*
2.67±0.33
1.67±0.88
1.67±0.33*
0.33±0.33
Flowers aqueous extract
Phyllodes aqueous extract
Results represents the mean (± S.E.) of three replicates.
*Significant differences with respect to control for p < 0.05 according to Dunnett test.
75
Germination response of native herbs
Table 3. Effects of Acacia melanoxylon flowers and phyllodes water extract on germination of L. perenne at each exposure time during one week.
Exposure Time (Days after sowing)
A. melanoxylon
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Control
0.00±0.00
5.42±1.60
5.50±1.04
3.33±0.87
4.58±1.01
1.55±0.63
0.50±0.337
100%
0.00±0.00
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00
75%
0.00±0.00
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00
50%
0.00±0.00
0.33±0.333*
0.00±0.00*
0.00±0.00*
0.67±0.666*
0.00±0.00*
0.00±0.00
25%
0.00±0.00
1.00±0.57
0.67±0.33*
2.67±1.45
3.33±3.333
0.33±0.333
0.00±0.00
Control
0.00±0.00
5.42±1.60
5.50±1.04
3.33±0.87
4.58±1.01
1.55±0.63
0.50±0.337
100%
0.00±0.00
2.00±1.52
3.33±3.33
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00
75%
0.00±0.00
2.33±1.45
4.00±1.73
2.00±0.57
0.33±0.333*
0.00±0.00*
0.00±0.00
50%
0.00±0.00
5.00±2.64
5.33±3.38
3.33±2.02
0.60±0.66*
0.00±0.00*
0.00±0.00
25%
0.00±0.00
5.67±4.70
14.00±3.51*
4.33±2.96
3.67±0.33
1.67±1.66
0.00±0.00
Flowers aqueous extract
Phyllodes aqueous extract
Results represent the mean (± S.E.) of three replicates.
*Significant differences with respect to control for p < 0.05 according to Dunnett test.
76
Chapter IV. Allelopathic potential of A. melanoxylon
Table 4. Effects of A. melanoxylon flowers and phyllodes water extract on germination of L. sativa at each exposure time during one week.
Exposure Time (Days after sowing)
A. melanoxylon
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Control
7.25±1.58
6.58±1.60
2.67±0.73
1.00±0.57
0.75±0.37
0.82±0.423
0.67±0.337
100%
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00
0.00±0.00
0.00±0.00
0.00±0.00
75%
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00
0.00±0.00
0.00±0.00
0.00±0.00
50%
0.00±0.00*
0.00±0.00*
0.33±0.33*
0.67±0.66
0.00±0.00
0.00±0.00
0.00±0.00
25%
0.00±0.00*
0.67±0.667*
1.33±1.333
2.33±2.333
0.00±0.00
0.00±0.00
0.00±0.00
Control
7.25±1.58
6.58±1.60
2.67±0.73
1.00±0.57
0.75±0.37
0.82±0.423
0.67±0.337
100%
0.00±0.00*
2.33±1.85*
1.00±1.04
1.67±1.66
1.67±1.66
0.00±0.00
0.00±0.00
75%
1.67±0.88*
2.00±1.15*
2.00±1.15
0.33±0.33
0.26±0.06
0.00±0.00
0.00±0.00
50%
3.67±1.85*
3.67±0.33*
1.00±1.00
1.33±0.88
0.33±0.33
0.00±0.00
0.67±0.66
25%
5.00±0.57
5.33±2.90
1.67±1.20
0.33±0.33
2.00±2.00
0.33±0.33
0.67±0.33
Flowers aqueous extract
Phyllodes aqueous extract
Results represent the mean (± S.E.) of three replicates.
*Significant differences with respect to control for p < 0.05 according to Dunnett test.
77
Germination response of native herbs
Table 5. Effect of different concentration of Acacia melanoxylonflower and phyllodes aqueous extract on germination indices (G T, S)
of D. glomerata, R. acetosa, L. perenne and L. sativa.
D. glomerata
R. acetosa
L. perenne
L. sativa
A. melanoxylon
GT
S
GT
S
GT
S
GT
S
Control
82.67±7.05
1.24±0.207
40.00±8.00
0.94±0.20
84.67±4.05
1.79±0.348
78.67±8.74
5.13±2.08
100%
0.00±0.00*
0.00±0.00*
28.00±4.00
0.66±0.52
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
75%
0.00±0.00*
0.00±0.00*
37.33±5.33
0.55±0.68
0.00±0.00*
0.00±0.00*
0.00±0.00*
0.00±0.00*
50%
0.00±0.00*
0.00±0.00*
38.67±6.67
0.71±0.19
4.00±4.000*
0.08±0.078*
4.00±4.000*
0.06±0.061*
25%
0.00±0.00*
0.00±0.00*
34.67±3.52
0.67±0.08
32.00±21.16*
0.47±0.251*
17.33±17.333*
0.33±0.338*
Control
82.67±7.05
1.24±0.20
40.00±8.00
0.94±0.20
84.67±4.05
1.79±0.348
78.67±8.74
5.13±2.08
100%
42.67±8.11*
0.70±0.11*
36.00±10.06
0.52±0.13
21.33±19.36*
0.61±0.53
22.67±9.61*
0.02±0.09*
75%
66.67±2.66*
1.16±0.10
37.33±13.92
0.83±0.11
70.67±9.33*
1.61±0.66
29.33±7.33*
0.60±0.15*
50%
70.67±11.85
1.22±0.21
35.33±2.66
0.57±0.18
80.00±10.06
2.17±0.54
28.00±6.11*
1.20±0.53*
25%
70.67±11.85
0.93±0.17
46.00±6.11
0.72±0.46
76.00±4.61
1.67±0.10
77.33±22.66
3.91±0.82
Flowers aqueous extract
Phyllodes aqueous extract
Results represent the mean (± S.E.) of three replicates.
*Significant differences with respect to control for p < 0.05 according to Dunnett test.
78
Chapter IV. Allelopathic potential of A. melanoxylon
Table 6. Probit analysis for seed germination of Dactylis glomerata exposed to four
concentration (100%, 75%, 50%, 25%) of Acacia melanoxylon R. Br. phyllodes aqueous extract.
Total no.
Non-germinated
Expected
of seeds
seeds
Response
100
25
15
11.59
0.464
75
25
9
10.75
0.430
50
25
8
9.60
0.384
25
25
7
7.76
0.309
Acacia phyllodes
Concentration (%)
Probability
Regression Line parameters
Y = A + BX
Y = -0.091 + 0.675 X
LC50 values = 3.48
Diagnostic concentration = 46%
79
Germination response of native herbs
Table 7. Probit analysis for seed germination of Lolium perenne exposed to four concentrations
(100%, 75%, 50%, 25%) of Acacia melanoxylon R. Br. aqueous extract.
Total no.
Non-germinated
Expected
of seeds
seeds
Response
100
25
25
24.98
0.999
75
25
25
24.91
0.996
50
25
24
24.22
0.969
25
25
17
16.87
0.675
100
25
20
12.34
0.494
75
25
7
10.88
0.435
50
25
6
8.88
0.356
25
25
5
5.85
0.234
A. flowers Conc.(%)
Probability
A. phyllodes Conc.(%)
Regression Line parameters (Acacia flowers)
Regression Line parameters (Acacia phyllodes)
Y = A + BX
Y = A + BX
Y = 3.276 + 4.688X
Y = -0.016 + 1.177X
LC50 values = 2.33
LC50 values = 4.26
Diagnostic concentration = 43%
Diagnostic concentration = 41%
80
Chapter IV. Allelopathic potential of A. melanoxylon
Table 8. Probit analysis for seed germination of Rumex acetosa exposed to four
concentration (100%, 75%, 50%, 25%) of Acacia melanoxylon R. Br. aqueous extract.
Total no.
Non-germinated
Expected
of seeds
seeds
Response
100
25
18
16.81
0.673
75
25
16
16.58
0.663
50
25
16
16.25
0.650
25
25
15
15.67
0.627
100
25
16
14.28
0.571
75
25
16
14.18
0.567
50
25
14
14.04
0.562
25
25
11
13.81
0.553
A. flowers Conc.(%)
Probability
A. phyllodes Conc.(%)
Regression Line parameters (Acacia flowers)
Regression Line parameters (Acacia phyllodes)
Y = A + BX
Y = A + BX
Y = 0.447 + 0.204X
Y = 0.180 + 0.079X
LC50 values = 2.23
LC50 values = 6.10
Diagnostic concentration = 40%
Diagnostic concentration = 38%
81
Germination response of native herbs
Table 9. Probit analysis for seed germination of Lactuca sativa exposed to four
concentration (100%, 75%, 50%, 25%) of Acacia melanoxylon R. Br. aqueous extract.
Total no.
Non-germinated
Expected
of seeds
seeds
Response
100
25
25
23.82
0.953
75
25
25
23.12
0.925
50
25
21
21.68
0.867
25
25
16
17.77
0.711
100
25
19
14.58
0.584
75
25
18
13.65
0.546
50
25
8
12.31
0.492
25
25
6
10.04
0.402
A. flowers Conc.(%)
Probability
A. phyllodes Conc.(%)
Regression Line parameters (Acacia flowers)
Regression Line parameters (Acacia phyllodes)
Y = A + BX
Y = A + BX
Y = 1.673 + 1.855X
Y = 0.211 + 0.764X
LC50 values = 1.8
LC50 values = 6.26
Diagnostic concentration = 53%
Diagnostic concentration = 41%
82
Chapter IV. Allelopathic potential of A. melanoxylon
(a)
(b)
Fig. 1. Effect of A. melanoxylon (a) flowers and (b) phyllodes aqueous extract on root length of R. acetosa and
L. perenne. Every column in each graph represents the mean value ± S.E. from three replicates. Asterisks indicate
significant differences at 0.05 probability level with respect to control according to Dunnett test.
83
Germination response of native herbs
(a)
(b)
Fig. 2. Effect of A. melanoxylon (a) flowers and (b) phyllodes aqueous extract on root length of D. glomerata and
L. sativa. Every column in each graph represents the mean value ± S.E. from three replicates. Asterisks indicate
significant difference at 0.05 probability level with respect to control according to Dunnett test.
84
Ecophysiological response of native grass
species to phytotoxic effect of Acacia
melanoxylon R. Br.
Distilled
water
Chapter V
Chapter section submitted as an original article to SCI journal:
Hussain, M.I., González, L., Souto, C., and Reigosa, M.J. 2010. Ecophysiological
responses of three native grass species to phytotoxic effect of Acacia melanoxylon
R. Br. Agroforestry System (under review)
Ecophysiological response of native herbs
Abstract
Acacia melanoxylon R. Br. (Blackwood) is a native Australian species that has invaded
woodlands and degraded natural habitats in the north western Iberian Peninsula (Galicia) in
Spain. Several phenolic (p-hydroxybenzoic, vanillic, p-coumaric, syringic, protocatequic, ferulic
acids) and flavonoids (catechin, luteolin, rutin, apigenin, and quercetin) were identified from
methanol extracts of flowers and phyllodes of A. melanoxylon by HPLC. Flowers and phyllodes
of A. melanoxylon were soaked separately in the water in a ratio of 1:1 (w/v) for 24 h to prepare
aqueous extracts (100%, 75%, 50%, 25%) and distilled water was used as control. The seeds of
three native grass weeds, cocksfoot (Dactylis glomerata), perennial ryegrass (Lolium perenne),
common sorrel (Rumex acetosa) and a crop lettuce (Lactuca sativa) were grown in perlite
culture and aqueous extracts of A. melanoxylon (flowers and phyllodes) were applied
exogenously at various concentrations. A. melanoxylon flowers extract (100%) inhibited the
shoot length of D. glomerata and L. perenne by 31% and 20% of the control, respectively. Leaf
fresh weights of L. perenne, D. glomerata, and L. sativa were reduced after treatment with
extracts of acacia flowers and phyllodes at all concentrations. A. melanoxylon (flowers extract)
decreased root fresh weight of D. glomerata, L. perenne and L. sativa. Leaf relative water
content of D. glomerata and L. perenne was reduced after treatment with acacia flowers and
phyllodes extract (100%, 75%, 50%, 25%). A. melanoxylon flowers and phyllodes extract
significantly reduced leaf osmotic potential in D. glomerata, L. perenne and L. sativa. Quantum
efficiency of open PSII reaction centers (Fv/Fm) and quantum yield (ΦPSII) of photosystem II
were decreased in L. perenne, D. glomerata, R. acetosa and L. sativa after treatment with acacia
flowers extract (100%). Acacia flowers and phyllodes extract (100%) inhibited the qP level
during all six days in D. glomerata, L. perenne, R. acetosa and L. sativa. A significant reduction
in NPQ was observed during different days in all four plant species due to Acacia flowers and
phyllodes extract (100%). The δ 13C/12C ratio was significantly less negative in L. perenne, D.
glomerata and L. sativa as compared to the control. A. melanoxylon flowers and phyllodes
extract (100%) significantly reduced protein contents in D. glomerata, L. perenne and in L.
sativa.
86
Chapter V. Phytotoxic effects of A. melanoxylon
Key Words Allelopathy . Phenolics . Acacia melanoxylon . Dactylis glomerata . Lolium perenne .
Carbon isotopes discrimination . Rumex acetosa Flavonoids
Abbreviations:
C% - Carbon concentration; ci/ca - ratio of intercellular CO2 concentration from leaf to air; F′m maximal fluorescence level from light adapted leaves; F′o - minimal fluorescence level from
light adapted leaves; F′v - variable fluorescence level from light adapted state; F m - maximal
fluorescence level from dark adapted leaves; Fo - initial fluorescence level from dark adapted
leaves; Fv - variable fluorescence level from dark adapted leaves; Fv/Fm - efficiency of
photosystem II photochemistry in the dark-adapted state; LOP - leaf osmotic potential; NPQ non-photochemical quenching; qP - photochemical quenching; RWC - relative water content; δ
13
C - composition of carbon isotope ratios; Δ13C - carbon isotope discrimination; ΦPSII maximum quantum yield of PSII electron transport.
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Ecophysiological response of native herbs
Introduction
Allelopathic interactions are mediated by secondary metabolites (allelochemicals) released from
donor plants into the environment in several ways, including volatilization from leaf tissues,
leaching of nonvolatile molecules from foliage by rainfall, exudation from living roots, or
decomposition of residues by soil microorganisms (Shiraishi et al. 2002; Rai et al. 2003;
Kobayashi 2004). Persistence and activity of allelochemicals released under natural conditions
or field settings are influenced by the environment, specifically meteorological and rhizospheric
conditions, as well as physical distance from the source of production (Jose and Gillespie 1998).
Some invasive plants threaten the persistence of native assemblages, change the composition of
plant communities and alter ecosystem functioning (D‘Antonio and Meyerson 2002; Kourtev et
al. 2003).
Acacia species are distributed primarily in the dry tropics and several Australian Acacia
species have become highly invasive weeds around the world (Blakesley et al. 2002). Acacia
species were introduced in Southern Europe as an ornamental plant in the 19th century;
naturalized in the local habitat and slowly become invasive species in Atlantic climates and
Mediterranean regions from Portugal to Italy (Sheppard et al. 2006). Black wood (Acacia
melanoxylon R. Br.) is an introduced species that has its origin in the temperate forests of southeast Australia and Tasmania. It is a versatile and highly adaptive tree species which has spread
all over the world (Knapic et al. 2006) and currently covers a considerable area in the coastal
region of the north-western Iberian Peninsula, both in monocultures and in mixed stands with
Eucalyptus globulus. Forest plantations of this species started in the northwestern Iberian
Peninsula at the beginning of the twentieth century (Areses 1953) and are currently considered
as invasive (Xunta de Galicia 2007), characterized by vigorous tree or root sprouts and with
seed germination stimulated by fire. Upon invasion, it establishes quickly in the alien
environment, thereby resulting in changes to the structure and dynamics of native ecosystems.
Although the species is found mostly in wastelands, it also grows well in cultivated fields,
pastures and along roadsides. Based on the field observation and previous phytotoxicity study of
A. melanoxylon phyllodes against Lactuca sativa, Dactylis glomerata, (Souto et al. 1994; Souto
et al. 2001), it was suggested that Acacia melanoxylon releases allelopathic compounds through
decomposition of litter that inhibit the growth of other plants.
Allelochemicals have several molecular target sites and affect several physiological
processes (Weir et al. 2004; Gniazdowska and Bogatek 2005). They include alteration of
enzyme activities (Politycka 1998), inhibition of cell division (Anaya and Pelayo-Benavides
1997), ion uptake (Lehman and Blum 1999), and inhibition of electron transport in both
photosynthetic and the respiratory chain (Czarnota et al. 2001). It has also been postulated that
allelopathy stress might lead to an imbalance between antioxidant defenses and the amount of
reactive oxygen species (ROS), resulting in oxidative stress (Cruz-Ortega et al. 2002; RomeroRomero et al. 2005). However, under field conditions, multiple stress factors develops
progressively and gradually, eliciting morphological, physiological and biochemical responses.
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Chapter V. Phytotoxic effects of A. melanoxylon
These responses can be synergistically or antagonistically modified by the superimposition of
other stress (Chaves et al. 2002).
A. melanoxylon is an invader in the coastal region of the north-western Iberian
Peninsula. The use of L. sativa could demonstrate the possible mechanisms by which A.
melanoxylon competes with other species while the results concerning L.perenne, D.
glomerata and R. acetosa, which grows in the study area, have more ecological significance.
The phytotoxicity of acacia aqueous extracts makes them good candidates as a source of
natural herbicide against weeds and plant species (Dana and Domingo 2006; Al-Wakeel et al.
2007; Lorenzo et al. 2008). Thus it is important to understand physiological responses of
native grasses present under and near the canopy stands of acacia that can elucidate ecological
significance of this invasive specie. This can also provide basic information about the
management of A. melanoxylon.
Methods and Materials
Extraction of Phenolics from Flowers and Phyllodes of A. melanoxylon Fresh shoots of A.
melanoxylon were collected from a natural population in the area surrounding the Lagoas
Marcosende campus of University of Vigo, during flowering period. Phenolics were extracted
using the method proposed by Macheix et al. (1990) and optimized by Bolaño et al. (1997). The
fresh plant tissue (flowers or phyllodes) was cut into small pieces with scissors and placed in the
laboratory at room temperature (25 ºC) for air drying to avoid any degradation process due to
temperature. The dry flowers or phyllodes (1 g), were mixed with 50 mL of methanol/HCl
(1000:1, v/v) in a 100 mL Erlenmeyer flask and kept in darkness for 12 hrs with hand shaking
every 3 hr. The mixture was filtered by using a filter paper and a funnel. The filtrate was saved
in a refrigerator and the process was repeated with residual plant material for another 12 hr by
adding 50 mL of methanol/HCl. The mixture was filtered again and two methanolic fractions
were combined (approx. 100 mL). This mixture was dried in a rotary evaporator under vacuum.
The temperature of the rotary evaporator was maintained below 35 ºC. The remaining material
was redissolved in 40 mL of ethanol/water (2:8, v/v) and filtered. Afterwards, three sequential
extractions were carried out with 20 mL of diethyl ether. The mixture was extracted with an
extraction funnel by shaking vigorously for one min each time, waiting until complete
separation into two phases had been achieved: the aqueous one in the lower part and the organic
one, in the upper part of the funnel. The organic phase was removed and saved. We collected
three ethereal phases in an Erlenmeyer flask. After that, three sequential extractions were
carried out with 20 mL ethyl acetate on the aqueous phase, obtaining three new organic phases
that were collected and combined with the ethereal ones. The total organic fractions obtained in
this way were dehydrated with anhydrous sodium sulphate for 30 mints to remove minimum
residual water. Subsequently, they were filtered to withdraw sodium sulphate and evaporated to
dryness in a rotary evaporator. The final residue containing phenolics was redissolved in 1 mL
methanol/water (1:1, v/v) and filtered through a 0.45 µm pore size nylon membrane filter and
saved in a refrigerator at 4 ºC until HPLC analysis.
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Ecophysiological response of native herbs
Extraction of Flavonoids from Flowers and Phyllodes of A. melanoxylon The flavonoids were
extracted by the method of Markham (1989) with certain modifications. The fresh plant tissue
was cut into small pieces with scissors and placed in the laboratory at room temperature (25 ºC)
for air drying. The dried and powdered flowers or leaves (10 g) were macerated with 300 mL of
methanol/water (80:20) for 24 hr at room temperature with continuous shaking. The extract was
obtained through filtration by using a Buchner funnel with a filter paper. This mixture was dried
in a rotary evaporator under vacuum until the entire methanol had been removed. The remaining
material was redissolved in 40 mL of ethanol/water (2:8, v/v) and filtered. The resultant
aqueous material was extracted with petroleum ether in an extraction funnel to remove fats,
terpenes, chlorophylls and xanthophylls. This extraction was repeated three times. Afterwards
the mixture was extracted as described above (in phenolic extraction) i.e. three sequential
extractions with diethyl ether followed by three sequential extractions with ethyl acetate. The
total organic fractions obtained in this way were dehydrated with anhydrous sodium sulphate for
30 mints to remove minimum residual water. Subsequently, they were filtered to withdraw
sodium sulphate and evaporated to dryness in a rotary evaporator. The final residue was
redissolved in 2.5 mL methanol and filtered through a 0.45 µm pore size nylon membrane filter
and saved in a refrigerator at -20 ºC until HPLC analysis.
Chemical Analysis The Analysis was performed using an HPLC (Shimadzu chromatograph)
equipped with a UV-DIODE ARRAY detector to identify flavonoids and phenolics.
Identification of the compounds was achieved by using a reverse-phase Waters Nova-Pak C -18
(4.6 x 250 mm) column with a 4 µm particle size. For flavonoids, the extracts were analyzed
using two mobile phases: (A) methanol : phosphoric acid 999:l and (B) water: phosphoric acid
999:1. HPLC grade solvents were used in this study. Linear gradients starting with 20% (A) and
ending with 100% (A) were used over the first 40 min with an additional 5 min at 100% (A).
The flow rate of the mobile phase was 1 mL/min and the eluate was analyzed at 250-400 nm.
For the phenolics, extracts were analyzed using two mobile phases: (A) water: acetic acid (98:2)
and (B) water: methanol: acetic acid (68:30:2). Linear gradients starting with 100% (A) and
ending with 20% (A) were used over the first 59 min, with an additional 6 min at 20% (A). The
flow rate of the mobile phase was 0.8 mL/min and the eluate was analyzed at 210-400 nm.
Acacia melanoxylon Flowers and Phyllodes Aqueous Extract Preparation for Bioassays Fresh
shoots of A. melanoxylon were separated into flowers and phyllodes and soaked in distilled
water in the ratio of 1:1 (w/v) at room temperature and left in the laboratory for 24 hr. The
extract was collected, filtered through filter paper and described as 100%. Distilled water was
added to this solution to achieve different dilutions (75%, 50% and 25%) (Molina et al. 1991;
Lorenzo et al. 2008) We examined the effects of both phyllodes and flowers extracts made by
mixing plant material in water, as opposed to the often criticized method of tissue disruption
and organic solvent extraction that may yield artificially high levels of specific compounds
(Inderjit and Duke 2003).
Target Species and Growth Conditions The selection of a suitable target species in phytotoxic
research should be given importance (Sinkkonen 2006). Three grass species, (1) cocksfoot:
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Chapter V. Phytotoxic effects of A. melanoxylon
Dactylis glomerata L., cv Amba (Poaceae), (2) perennial ryegrass: Lolium perenne L. cv. Belida
(Poaceae), and (3) common sorrel (Rumex acetosa L. cv Belleville (Polygonaceae) were used as
representative grass species because they grow naturally in the Galician forest ecosystem, (north
western Spain) (Pasalodos-Tato et al. 2009). The Lettuce (Lactuca sativa L. [cv.] Great Lakes
California (Asteraceae) was selected as a general biotest species because it is frequently used as
a model species in allelopathic bioassays (Macías et al. 2000). The seeds of the test species were
purchased commercially from Semillas Fito (Barcelona, Spain).
Concentration-Dependent Phytotoxic Effects The seeds of three grass species and a crop species
were placed in plastic trays (32 x 20 x 6 cm) with a 5 cm deep layer of perlite (500 g/tray). The
trays were irrigated on alternate days with tap water until germination of seeds occurred and
thereafter with 500 mL 1:1 Hoagland solution/tray, twice a week. The germination conditions in
the growth chamber were as follows: L. perenne (day/night temperature 25/15 °C, 12/12 h of
photoperiod), L. sativa 18/8 °C (day/night temperature) and 12/12 h (light/darkness
photoperiod), R. acetosa (day/night temperature: 28/20 °C and 9/15 h of light/darkness) and D.
glomerata (day/night temperature: 25/20 °C and 14/10 h of light and darkness) and a relative
humidity of 80% (Hussain et al. 2008). One month old seedlings (when plants have three fully
expanded leaves), were transferred to pots (10 cm) containing perlite (70 g) to stimulate the
development of root system and shifted to a glasshouse with the same growing conditions and
nutrient solution (100 mL/pot). The glasshouse was ventilated with outside air to ensure steady
CO2. Every second day the pots were well watered with tap water through an automatic
irrigation system. One week later treatment solutions (100 mL/pot) were applied three times
(first, third, and fifth day) and measurements were taken. There were three replicates of each
concentration for each bioassay species and an experiment was laid out in Randomized
Complete Block Design (RCBD).
Chlorophyll Fluorescence Measurement Chlorophyll fluorescence was measured on fully
expanded exposed leaves (one leaf per plant) by means of a pulse-modulated instrument
fluorescence monitoring system (FMS) (Hansatech, Norfolk, England) using the method of
Weiss and Reigosa 2001. The parameters of chlorophyll fluorescence: initial fluorescence (F 0),
maximum fluorescence (Fm) and variable fluorescence Fv (Fm-F0), were measured after 20 min
in the dark adopted leaves using Walz leaf clips. The intensity of saturation pulses to determine
the maximum fluorescence emission in the presence (F′m) and absence (Fm) of quenching was
1800 µmol m-2 s-1 for 3 seconds. The steady state fluorescence (Fs), basic fluorescence after
light induction (F′0), maximum chlorophyll fluorescence (F′m) and variable fluorescence from
light adopted leaves (F′v) were also evaluated by attaching the optic fiber to a leaf clip holder.
The chlorophyll fluorescence ratios Fv/Fm were considered a useful measurement of
photosynthetic performance of plants and as stress indicators. The quantum efficiency of open
PSII reaction centers in the light-adapted state (ФPSII) was determined from F′m and Fs values
and the quantum efficiency of excitation energy trapping of PSII, (F′v/F′m), was also calculated
(Genty et al. 1989). After turning off the actinic light the leaves were illuminated with far-red
light (7 μmol m−2 s−1) to oxidize the PQ pool to determine the minimum fluorescence level of
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Ecophysiological response of native herbs
the light-adapted state (F′0). The fraction of open PSII reaction centers as qP = (F′m− Fs/F′m−
F′0). (F′0/Fs) were calculated using the ‗lake‘ model according to Kramer et al. (2004) and nonphotochemical quenching NPQ = (F′m−Fm)/(F′m) was calculated according to Bilger et al.
(1995).
Protein Content Determination Total protein was quantified by using the Spectrophotometric
Bradford (1976) assays. Target species leaves (200 mg fresh weight) from three replicates per
treatment were crushed in liquid nitrogen in a cooled mortar with 0.05 g PVPP, and the powder
was resuspended in 1 mL of Tris buffer containing 0.05 M Tris base, 0.1% w/v ascorbic acid,
0.1% w/v cysteine hydrochloride, 1% w/v PEG 4000, 0.15% w/v citric acid, and 0.008% v/v 2mercaptoethanol (Arulsekar and Parfitt 1986). Samples were centrifuged at 19000 gn and 4 ºC
for 20 min after resuspension. 0.1 mL supernatant was mixed for a protein dye-binding reaction
with 3 mL Bradford reactive containing 0.01% Coomassie® Brilliant Blue G-250, 4.7% v/v
ethanol 95%, and 8.5% v/v phosphoric acid 85%. Absorbance was recorded at 595 nm after 5
mins for quantification of the protein content. Commercial bovine seroalbumin (BSA) was used
as standard. Values were expressed per gram of dry weight.
Carbon Isotope Composition Analysis The leaf samples from four plant species were
immediately dried at 70 °C for 72 hr in a forced-air oven (Gallenkamp oven, Loughborough,
Leicestershire, UK) to constant weight and ground in Ball Mills (Retsch MM 2000, Haan,
Germany). Dry ground plant material was weighted (1700-2100 µg) with an analytical balance
(Metler Toledo GmbH: Greifensee Switzerland), and filled in tin capsules (5x3.5 mm,
Elemental Microanalysis Limited, U.K.). Each tin capsule was entered automatically in to a
combustion oven at 1600-1800 0C in the presence of oxygen and converted to CO2.
Subsequently isotope ratios were determined in an Isotopic Ratio Mass Spectrometer (Finnegan:
Thermo Fisher Scientific, model MAT-253, Swerte Germany) coupled with an Elemental
Analyzer (Flash EA-1112, Swerte Germany). The Isotopic ratio mass spectrometer has an
analytical precision better than 0.3‰ for 13C.
Carbon isotope compositions were calculated as:
δ (‰) = [(Rsample/Rstandard) —1)] x 1000
(1)
where Rsample is the ratio of 13C/12C and Rstandard is Vienna PeeDee Belemnite (VPDB),
standard for carbon. The standard carbon R is equal to 1.1237 x10-2. Pure CO2 (δ13C = -28.2 ±
0.1‰) gas calibrated against standard CO2 (-10.38‰) served as reference gas for δ13C. The
accuracy and reproducibility of the measurements of δ13C were checked with an internal
reference material (NBS 18 and IAEA-C6 for C), and Acetanilide for C %age ratios
respectively.
Carbon isotope discrimination is a measure of the carbon isotopic composition in plant
material relative to the value of the same ratio in the air on which plants feed:
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Chapter V. Phytotoxic effects of A. melanoxylon
Δ (‰) = [(δa – δp)/(1 + δp)] x 1000
(2)
where Δ represents carbon isotope discrimination, δa represents C isotope composition in the
source air, and δp represents C isotope composition in the plant tissue. Theory published by
Farquhar et al. (1989) and Farquhar and Richards (1984) indicates that carbon discrimination in
leaves of plants can be expressed in relationship to CO2 concentrations inside and outside of
leaves in its simplest form as:
Δ = a + (b - a) ci/ca
(3)
Δ = 4.4 + (27 - 4.4) ci/ca
where a is discrimination that occurs during diffusion of CO2 through the stomata
(4.4‰), b is discrimination by Rubisco (27‰), and ci/ca is the ratio of the leaf intercellular
CO2 concentration to that in the atmosphere. Equation [3] shows a direct and linear relationship
between Δ and ci/ca. Therefore, measurement of Δ gives an estimation of the assimilation-rate–
weighted value of ci/ca. A lower ci/ca ratio may result either from higher rates of photosynthetic
activity or from stomatal closure induced by environmental stress. Carbon isotope
measurements were performed in CACATI (Centro de Apoio Cientifico Tecnologico a la
Investigacion), University of Vigo, Spain.
Plant Growth Measurements Information about shoot length/ root length was obtained with a
ruler and values were expressed in cm. The fresh and oven dry plant leaf and root weights were
obtained by first weighting independently fresh leaves and roots then afterwards drying these
samples in a circulatory air oven at 70 °C for 72 hr. The samples were weighed again to get dry
weight of the plant.
Leaf Water Relations The leaf relative water content (RWC) is an appropriate measure of plant
water status in terms of physiological consequence of cellular water deficit. The RWC was
calculated by measuring fresh (Wf), saturated (Wt) and dry (Wd) weights of plant (Reigosa and
González 2001). Leaf plant pieces from three replicates per treatment were weighed in fresh
saturates at 4 °C for 3 hr, weighed again, oven dried at 70 °C for 72 hr, and finally weighed
once more to obtain the three weights required for the following equation:
RWC = Wf – Wd/ Wt – Wd x 100
(4)
Leaf Osmotic Potential For osmolality measurements (milliosmol kg-1) one leaf per replicate
was transferred into 10 mL syringes and kept frozen at -80 °C until further analysis (González
2001). In analysis, syringes were thawed until the sample reached room temperature and
osmotic potential was measured on the second drop from collected sap using a calibrated vapor
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Ecophysiological response of native herbs
pressure Osmometer (Automatic Cryoscopic Osmometer, Osmomat – 030, GmbH, Gonatec,
Berlin, Germany) according to the manufacturer‘s instructions.
Statistical Analysis Data were analysed with the statistical package SPSS® (version 15.00) for
Windows® (SPSS Inc., Chicago, IL, USA). Significant differences between means of treatment
were compared at 5% probability level. Multiple comparisons of means were performed using
the Dunnett test at 0.05 significance level (when variance was homogeneous) or the KruskalWallis test (when heterogeneous).
Results
Identification Of Phytotoxins From Extracted Tissues Qualitative analyses of chemical
components of flowers and phyllodes of A. melanoxylon confirmed the presence of numerous
phenolic compounds (benzene acetic, p-hydroxybenzoic, vanillic, p-coumaric, syringic acids)
and flavonoids (catechin, luteolin, rutin, and apigenin) (Table 1). Amounts are expressed as mg
per litre in the extract. The p-hydroxybenzoic acid (Phenolics) and rutin (flavonoids) were most
abundant in both extracts.
Effect On Plant Growth And Development The inhibitory effect was greatest on native grass
species, particularly on Lolium perenne (Table 2). The incorporation of flower extract at a
concentration of 100% inhibited the shoot length of Dactylis glomerata and Lolium perenne by
31 and 20% of control, respectively. The acacia phyllodes extract significantly reduced shoot
growth of two grass species at all four concentration and the inhibition was 22% (L. perenne)
and 29% (D. glomerata) at 100% phyllodes aqueous extract, respectively. A. melanoxylon
(flower and phyllodes extracts) significantly reduced the root growth of L. perenne and D.
glomerata and L. sativa. In R. acetosa, the inhibitory effect was only achieved at 100% and
75% concentration of acacia flowers extract (Table 3). Root growth was significantly decreased
in L. perenne as the concentrations of phyllodes extract increased in pots and the reduction was
two folds less as compared with control at 100% phyllodes extract (Table 3). The acacia flower
extract significantly reduced the root length of Dactylis glomerata and L. sativa at 100%, 75%,
50% respectively.
Leaf fresh weights of L. perenne, D. glomerata and L. sativa was significantly decreased when
grown in perlite having flower or phyllodes extract of A. melanoxylon at all concentrations but
maximum inhibition occurred at 100% extract (Fig. 1). A. melanoxylon (flower extract)
significantly reduced leaf dry weight of D. glomerata, L. perenne and L. sativa (Fig. 2 a). A.
melanoxylon (flower and phyllodes extract) significantly reduced root fresh weight of D.
glomerata, L. perenne and L. sativa at all concentrations (Fig. 3 a) but it did not significantly
affect root fresh weight of R. acetosa. Acacia phyllodes extract significantly reduced the root
dry weight in D. glomerata, L. perenne and L. sativa at 100%, 75%, 50% concentration (Fig. 4).
Leaf Water Status A. melanoxylon flower and phyllodes extract (100%, 75%, 50%) significantly
decreased relative water content (RWC) in leaves of D. glomerata and L. perenne (Fig. 5 a, b)
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Chapter V. Phytotoxic effects of A. melanoxylon
while highest concentration (100%) of acacia phyllodes extract reduced RWC 2-folds in grass
species (D. glomerata and L. perenne) (Fig. 6). There was non-significant effect of acacia
extracts on RWC in R. acetosa and L. sativa (data not shown). Acacia flower extract
significantly reduced leaf osmotic potential (LOP) as compared to control at all concentrations
in L. sativa (Fig. 6 a). Both flowers and phyllodes extract of acacia (100%, 75%, 50%)
significantly decreased LOP in L. perenne and D. glomerata as compared to control and the
lowest LOP was observed at 100% and 75% aqueous extract (Fig. 6 b, c).
Chlorophyll Fluorescence Measurements Damage caused by aqueous extract (100%) of flowers
and phyllodes of A. melanoxylon was evaluated on the basis of six days of treatment by
analyzing several fluorescence parameters determined under dark-adapted and steady state
conditions. In Lolium perenne, the quantum efficiency of open PSII reaction centre in the darkadapted state (Fv/Fm) was significantly decreased after treatment with 100% acacia flower or
phyllodes extract while strongest inhibition in Fv/Fm was observed during fifth day (Fig. 7). In
Dactylis glomerata, the Fv/Fm levels were significantly decreased by flower and phyllodes
extracts (100%) of acacia while there was more reduction in F v/Fm values during fifth and sixth
day (Fig. 7). The Fv/Fm level in L. sativa was significantly decreased after treatment with acacia
flower/phyllodes extract and this inhibition was stronger during last three days. In R. acetosa,
Fv/Fm level was significantly decreased after treatment with A. melanoxylon flower and
phyllodes extract during all days (Fig. 7). However, acacia phyllodes extract (100%) decreased
the Fv/Fm level in R. acetosa during fifth and sixth day (Fig. 7).
A. melanoxylon flower and phyllodes extract significantly decreased the level of quantum yield
(ΦPSII) of photosystem II in light conditions in Dactylis glomerata and L. perenne (Fig. 8). The
ΦPSII levels in L. sativa and R. acetosa were significantly decreased following exposure to
flower or phyllodes extracts (100%) of acacia (Fig. 8). A significant decrease in photochemical
fluorescence quenching (qP) was also observed in D. glomerata, L. perenne, R. acetosa and L.
sativa (Fig. 9). A. melanoxylon flower and phyllodes extract (100%) significantly decreased the
non-photochemical fluorescence quenching (NPQ) in D. glomerata, L. perenne, R. acetosa and
L. sativa (Fig. 10).
Carbon Isotope Discrimination (Δ13C) A. melanoxylon phyllodes aqueous extract (100%)
significantly reduced the carbon %age in the leaves of L. perenne, D. glomerata and R. acetosa
while acacia phyllodes extract significantly reduced the C% in L. sativa (Fig. 11). The carbon
isotope ratios (δ 13C) were significantly less negative in L. perenne, D. glomerata, R. acetosa
and L. sativa as compared to the control. In the L. perenne seedlings treated with A.
melanoxylon phyllodes aqueous extract (100%), the δ 13C ratio was significantly less negative (29.7) compared to the control (-30.6). The carbon isotope discrimination (Δ13C) values in L.
perenne, D. glomerata and L. sativa were significantly decreased by acacia flower and
phyllodes extract (100%) as compared to the control (Fig. 11).
95
Ecophysiological response of native herbs
Protein Contents Acacia flower and phyllodes aqueous extract (100%) was highly deleterious,
which significantly reduced leaf protein contents in D. glomerata, L. perenne and in L. sativa as
compared to the control. However, non-significant differences were observed in the leaf protein
content of R. acetosa at 100% concentration of A. melanoxylon flower and phyllodes extract
(Fig. 12).
Discussion
A number of plants have inhibitory effects on the growth of neighboring or successional plants
by releasing phytotoxic chemicals into the soil, either as exudates from living tissues or by
decomposition of plant residues (Korner and Nicklish 2002; Leu et al. 2002). The presence of
phytotoxins in flower and phyllodes of A. melanoxylon was confirmed by inhibitory effect on
native plants grown in perlite medium having aqueous extract at four different concentrations
(100, 75, 50, 25%). In all tested species shoot and root growth of three native grass weeds and
edible crops (lettuce) was strongly inhibited. The magnitude of inhibition increased with an
increase in concentrations of acacia flower and phyllode extract. The highest inhibition was
observed at 100% (w/v) (Table 2, 3). Native grasses, particularly L. perenne and D. glomerata
were the most sensitive to allelochemicals released from fresh flower and phyllode extracts. The
reduction in shoot and root growth ranged from 49-64% and 52-70% after treatment with acacia
flowers and phyllodes respectively. Meanwhile, a gradual decrease in fresh weights of the grass
species (L. perenne and D. glomerata) and the crop species, L. sativa, was observed at all
concentrations. The L. sativa experienced the highest leaf/root fresh weight loss. A.
melanoxylon (flower extract) significantly reduced root fresh weight of D. glomerata. Similarly,
Souto et al. 2001 concluded that the soils (up to 10 cm depth) infested with A. melanoxylon
phyllodes showed a maximum inhibitory activity on growth and germination of understory
plants, which leads to the assumption that leaching from intact phyllodes and decomposition of
phyllodes in soil, may contribute strongly to the allelopathic potential of A. melanoxylon. AlWakeel et al. (2007), in a greenhouse pot experiment reported that lower doses of Acacia
nilotica leaf residue (0.25 and 0.5%, w/w) stimulated the growth of pea shoot and root, but the
higher doses (0.75, 1.0, 1.5 and 2%, w/w) were inhibitory to seedling growth and the effect was
concentration dependent. A. melanoxylon flower and phyllode extract (100%, 75%) significantly
reduced the RWC and LOP in D. glomerata and L. perenne. The maximum amount of
phytotoxins was present in the flowers extract then in the phyllode extract (Table 1) and
maximum growth inhibition has also been reported in flower extract.
Fluorescence induction patterns and derived indices have been used as empirical
diagnostic tools in plant physiological studies (Strasser et al. 2000; Baker and Oxborough 2004;
Rosenqvist and van Kooten 2003). Recent advances in chlorophyll fluorescence analysis make
it easy to detect photoinhibition by measuring the maximum quantum efficiency of PSII
photochemistry (Fv/Fm) and are widely used as a measure of induced stress on photosynthetic
apparatus (Maxwell and Johnson 2000). The quantum efficiency of open PSII reaction centers
in the dark-adapted state (Fv/Fm) was significantly decreased in three grass species (L. perenne,
D. glomerata, R. acetosa) after treatment with A. melanoxylon flower or phyllode extract during
96
Chapter V. Phytotoxic effects of A. melanoxylon
six days. A sustained decrease of the Fv/Fm may indicate the occurrence of photoinhibitory
damage (Colom and Vazzana 2003). In the edible crop L. sativa the Fv/Fm level was
significantly reduced after treatment with A. melanoxylon flower or phyllodes extract. Barkosky
et al. (2000) reported that exposure to caffeic acid results in a significant increase in leaf
diffusive resistance and transpiration rate on the twelth day after the treatment and significant
decrease in Fv/Fm after the 28 day. Devi and Prasad (1996) observed that application of ferulic
acid to maize caused a decrease in chlorophyll fluorescence of both PSI and PSII. Similar
Barkosky et al. 1999 found that chronic reduction in available CO2 and water stress are the
possible causes for the reduction in Fv/Fm in leaf spurge after treatment with hydroxyquinone.
A. melanoxylon flower and phyllodes extract significantly decreased the quantum yield
(ΦPSII) of photosystem II in Dactylis glomerata, L. perenne, L. sativa and R. acetosa. The
reduced ΦPSII was a result of the decrease in the efficiency of excitation energy trapped by
PSII reaction centers. The quantum yield of electron transfer at PSII is a product of the
efficiency of the open reaction centers (Fv′/Fm′) and the photochemical quenching (qP) (Genty et
al. 1989; Lu and Zhang 1999; Sinsawat et al. 2004). Changes in the chlorophyll fluorescence
quenching parameters are good indicators of photosynthetic electron flow (Schreiber et al.
1994). There was a significant reduction in photochemical fluorescence quenching (qP) in D.
glomerata, L. perenne, R. acetosa and L. sativa after treatment with A. melanoxylon flower or
phyllode extract (100%). The decrease in qP level indicate that a larger percentage of the PSII
reaction centers was closed during this time and balance between excitation rate and electron
transfer rate had changed leading to a more reduced state of PSII reaction centers. A significant
decrease in non-photochemical fluorescence quenching (NPQ) was observed in all target
species after treatment with acacia flower and phyllode extract.
Stable isotope discrimination of several elements has been widely studied in plant
physiology (Griffiths 1991). The carbón isotope ratio was significantly less negative in all target
species as compared to the control. The carbon isotope discrimination (Δ 13C) values in leaves
of L. perenne, D. glomerata and L. sativa were significantly decreased by acacia flower and
phyllodes aqueous extract (100%) as compared to the control. Similarly, Barkosky et al. (2000),
concluded that dried leaf tissue from Leafy spurge (Euphorbia sula) treated with 0.25 mM
Caffeic acid had a less negative δ 13C compared to controls, indicating less discrimination
against 13C in these plants. Different types of biotic and abiotic stress can cause degradation of
proteins by senescence or by reduction in protein synthesis. El-Khawas and Shehata (2005)
concluded that maize samples treated with Acacia nilotica extracts show a significant increase
in levels of soluble proteins while there was a significant decrease in soluble proteins levels in
kidney beans.In this experiment, A. melanoxylon flower and phyllode aqueous extract (100%)
significantly reduced leaf protein contents in D. glomerata, L. perenne and L. sativa. SanchezMoreiras and Reigosa (2005), found that reduction of total soluble leaf proteins in BOAexposed plants were the consequence of the increase in lipid peroxidation, which has been
associated with high levels of denatured proteins. These results were confirmed by Duhan and
Lakshinarayana (1995), who revealed a drastic increase in the level of nucleic acids and
97
Ecophysiological response of native herbs
decrease in the level of soluble proteins in legume crops in response to Acacia nilotica extracts.
Baziramakenga et al. (1997) reported that many phenolic acids reduced the incorporation of
certain amino acids into proteins and thus reduce the rate of protein synthesis.
Allelochemicals could follow several paths to influence growth, photosynthesis,
stomatal control of CO2 supply, thylakoid electron transport, carbon reduction cycle and
changes in chlorophyll contents (Zhou and Yu 2006). The phytochemicals present in aqueous
extract of A. melanoxylon, caused a significant reduction in growth and ecophysiological
characteristics of L. perenne, D. glomerata, R. acetosa and a general biotest species, L. sativa.
Allelopathic activity depended on the concentration of the extracts, target species, and the plant
tissues from which the chemicals were extracted. Although flower aqueous extract of A.
melanoxylon appeared to have a more negative effect on the growth and ecophysiology of native
species than phyllode extract. This could be due to a greater concentration of diffusible active
compound in flowers than in the phyllodes or variation in chemical composition between the
tissues. We have identified several phenolics and flavonoids which are assumed to be watersoluble and diffusible from intact plant tissue. These may be reasonable candidates for the
active compounds in A. melanoxylon and phenolics are widely recognized for their allelopathic
potential in plants (Djurdjevic et al. 2004) and have potential as either herbicides or templates
for new herbicide classes (Dayan et al. 2009). Many other species of Genus Acacia
(Mimosaceae), such as Acacia dealbata Link (Carballeira and Reigosa 1999), Acacia confusa
Merr (Chou et al. 1998), Acacia auriculiformisA.Cunn. ex Benth (Hoque et al. 2003; Oyun
2006) and Acacia nilotica (El-Khawas and Shehata 2005; Al-Wakeel et al. 2007) are also
known to exhibit allelopathic activity.
In summary, this study demonstrates that water extracts of A. melanoxylon have the
potential to inhibit the growth and physiological characteristics of various grass and crop
species. A species-specific inhibitory effect was observed. More work clearly needs to be done
to address the ecological relevance of our findings, including bioassays in fields, on native grass
species (that co-occur with Acacia melanoxylon), where the influence of soil particles,
microbes, and other environmental factors on the putative allelopathic agents can be seen.
However, the phytotoxic effect of acacia, coupled with its dramatic effects on resource
availability, could contribute to the invasive success of this tree and its lasting impact in
restored sites.
Acknowledgements
We thank Dr. Aldo Barreiro, Carlos Bolaño, Alfredo Justo, Maite Ricart, and P. Lorenzo for
field and laboratory assistance. We are also grateful to Jesús Estévez Sío and Jorge Millos for
their technical assistance with isotope ratio mass spectroscopy. We also thank Dr. Nuria Pedrol
for her valuable help in leaf osmotic potential measurement and in statistical analysis.
98
Chapter V. Phytotoxic effects of A. melanoxylon
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Chapter V. Phytotoxic effects of A. melanoxylon
Table 1 Phenolics and flavonoids found in flower and phyllode extracts of Acacia melanoxylon R. Br.
RT: retention time (minutes) of compounds in column.
Sr. No.
Common Name
Scientific Name
Flowers mg/L
Phyllodes mg/L
RT
PHENOLICS
1
Gallic acid
3,4,5-trihydroxy benzoic acid
4.4
3.41
6.5
2
Protocatequic acid
3,4-dihydroxybenzoic acid
5.06
3
p-Hydroxybenzoic acid
4-hydroxybenzoic acid
12.33
1.46
21.9
4
p-Hydroxybenzaldehyde
0.91
0.16
28.6
5
Vanillic acid
4-hydroxy-3-methoxybenzoic acid
9.7
1.64
32.2
6
Syringic acid
4-hydroxy-3,5-dimethoxybenzoic acid
2.64
40.1
7
p-Comaric acid
4-hydroxycinnamic acid
3.19
3.6
49.7
8
Ferulic acid
4-hydroxy-3-methoxycinnamic acid
3.87
12.9
57.9
FLAVONOIDS
9
5342.39
Rutin
10
Quercetin
3,3',4',5,7-Pentahydroxyflavone
326.4
11
Luteolin
3',4',5,7-Tetrahydroxyflavone
388.89
12
Apigenin
13
Catechin
5032.87
20.4
25.2
706.89
26.2
4',5,7-Trihydroxyflavone
85.55
28.5
(±)-3,3',4',5,7-Flavanpentol
765.44
7.9
103
Ecophysiological response of native herbs
Table 2 Effect of aqueous extract of Acacia melanoxylon flowers and phyllodes on shoot growth (cm)
of test species
Flower Extract
Lolium
Dactylis
Rumex
Lactuca
0 (control)
55.89 ± 1.33
70.88 2.60
19.08 ± 0.70
15.30 ± 0.23
25
45.17 ± 1.59*
58.33 ± 4.41
13.00 ± 1. 15*
9.17 ± 0.92*
50
40.17 ± 1.59*
52.33 ± 1.33*
12.67 ± 0.72*
8.97 ± 0.89*
75
39.00 ± 3.60*
46.67 ± 7.164*
12.00 ± 0.76*
7.67 ± 0.33*
100
36.17 ± 3.42*
43.67 ± 4.34*
11.00 ± 0.577*
7.53 ± 0.29*
0 (control)
55.89 ± 1.33
70.88 2.60
19.08 ± 0.70
15.30 ± 0.23
25
41.33 ± 2.08*
48.9 ± 1.95*
13.33 ± 0.88*
11.31 ± 0.33*
50
40.17 ± 0.44*
48.4 ± 7.55*
13.67 ± 1.07*
10.53 ± 1.29*
75
39.83 ± 1.83*
48.17 ± 5.98*
12.83 ± 2.58*
8.75 ± 1.16*
100
39.67 ± 5.81*
40.7 ± 3.35*
11.77 ± 1.07*
8.00 ± 0.28*
(% age of 1:1 w/v)
Phyllode Extract
(% age of 1:1 w/v)
Each value represents the mean (± S.E.) of three replicates.
*Significant differences compared to the control for p < 0.05 according to Dunnett test.
104
Chapter V. Phytotoxic effects of A. melanoxylon
Table 3 Effect of aqueous extract of Acacia melanoxylon flowers and phyllodes on root growth (cm)
of test species
Flower Extract
Lolium
Dactylis
Rumex
Lactuca
0 (control)
33.00 ± 0.278
47.58± 1.48
16.38 ± 0.952
27.77 ± 0.22
25
31.00 ± 2.30
46.67 ± 2.96
16.00 ± 0.93
22.67 ± 1.45*
50
27.67± 1.76*
42.67 ± 2.18*
15.67 ± 0.88
21.33 ± 1.33*
75
26.67 ± 2.72*
37.00 ± 3.055*
14.50 ± 2.93*
21.50 ± 1.60*
100
25.67 ± 2.84*
32.67 ± 0.882*
13.83 ± 0.55*
19.00 ± 1.15*
0 (control)
33.00 ± 0.278
47.58 ± 1.48
16.38 ± 0.952
27.77 ± 0.22
48.67 ± 0.925
18.67 ± 1.667
26.82 ± 0.33
24.19 ± 1.29*
(% age of 1:1 w/v)
Phyllode Extract
(% age of 1:1 w/v)
25
20.00 ± 0.577*
50
19.33 ± 0.33*
42.67± 0.664*
18.33 ± 1.764
75
17.67 ± 0.667*
39.33 ± 2.93*
16.00 ± 1.764
20.37 ± 1.16*
100
15.00 ± 0.577*
38.00 ± 1.00*
15.00 ± 0.577*
18.27 ± 0.28*
Each value represents the mean (± S.E.) of three replicates.
*Significant differences compared to the control for p < 0.05 according to Dunnett test.
105
Ecophysiological response of native herbs
(a)
(b)
Fig. 1 Inhibitory effects of A. melanoxylon flowers (a) and phyllodes (b) on leaf
fresh weight (g) of native grasses and crop species at four different concentrations.
Each bars represents the mean (± S.E.) of three replicates.
*Significant differences compared to the control for p < 0.05 according to the
Dunnett test.
106
Chapter V. Phytotoxic effects of A. melanoxylon
a
b
Fig. 2 Inhibitory effects of A. melanoxylon flowers (a) and phyllodes (b)on leaf dry weight (g) of
of three native grasses and crop species at four different concentrations. Each bar represents
the mean (± S.E.) of three replicates. *Significant differences compared to the control for p < 0.05 according
to the Dunnett test.
107
Ecophysiological response of native herbs
a
b
Fig. 3 Inhibitory effects of A. melanoxylon flowers (a) and phyllodes (b)on root fresh weight (g)
of native grasses and crop species at four different concentrations. Each bar represents the
mean (± S.E.) of three replicates. *Significant differences compared to the control for p < 0.05
according to the Dunnett test.
108
Chapter V. Phytotoxic effects of A. melanoxylon
a
b
Fig. 4 Inhibitory effects of A. melanoxylon flowers (a) and phyllodes (b)on root dry weight (g)
of native grasses and crop species at four different concentrations. Each bar represents the
mean (± S.E.) of three replicates. *Significant differences compared to the control for p < 0.05
according to Dunnett test.
109
Ecophysiological response of native herbs
Acacia flowers
Acacia phyllodes
a
b
Fig. 5 Relative water content in leaves of Dactylis glomerata (a) and Lolium perenne (b) one week
after exposure to four concentrations (100%, 75%, 50%, 25%) of A. melanoxylon flowers and
phyllodes. Every bar in each graph represents the mean (± S.E.) of three replicates.
Asterisks indicate significant differences at level 0.05 with respect to the control.
110
Chapter V. Phytotoxic effects of A. melanoxylon
Fig. 6 Leaf osmotic potential (mmol kg-1) of (a) Lactuca sativa (b) and Lolium perenne (c) Dactylis glomerata
following exposure to four concentrations (100%, 75%, 50%, 25%) of A. melanoxylon (A) flowers and (B) phyllodes.
Every column in each graph represents the mean (± S.E.) of three replicates. Asterisks indicate significant
differences at level 0.05 with respect to the control.
111
Ecophysiological response of native herbs
Fig. 7 Changes in quantum efficiency (Fv/Fm) of open PSII reaction centers in the dark-adapted state
in leaves of L. perenne, D. glomerata, R. acetosa and L. sativa from days 1 to 6 under A. melanoxylon
flower and phyllode (100%) aqueous extracts. Every column in each graph represents the mean (± S.E.)
of three replicates. Asterisks indicate significant differences at level 0.05 with respect to the control.
112
Chapter V. Phytotoxic effects of A. melanoxylon
Fig. 8 Changes in quantum yield (ФPSII) of photosystem II in light condition in leaves of L. perenne,
D. glomerata, R. acetosa and L. sativa from days 1 to 6 under A. melanoxylon flower and phyllode
(100 %) aqueous extract. Every column in each graph represents the mean (± S.E.) of three replicates.
Asterisks indicate significant differences at level 0.05 with respect to the control.
113
qP
Ecophysiological response of native herbs
Fig. 9 Changes in photochemical fluorescence quenching (qP) in leaves of L. perenne,
D. glomerata, R. acetosa and L. sativa from days 1 to 6 under A. melanoxylon flower and phyllode
(100 %) aqueous extract. Every column in each graph represents the mean (± S.E.) of three replicates.
Asterisks indicate significant differences at level 0.05 with respect to the control.
114
Chapter V. Phytotoxic effects of A. melanoxylon
Fig. 10 Changes in non-photochemical fluorescence quenching (NPQ)in leaves of L. perenne,
D. glomerata, R. acetosa and L. sativa from days 1 to 6 under A. melanoxylon flower and phyllode
(100 %) aqueous extract. Every column in each graph represents the mean (± S.E.) of three replicates.
Asterisks indicate significant differences at level 0.05 with respect to the control
115
Ecophysiological response of native herbs
Fig. 11 Inhibitory effects of A. melanoxylon flowers and phyllodes on Carbon (C %), carbon
isotope ratios (δ13C12) and carbon isotope discrimination (Δ13C)of grass and crop species
at four different concentrations. Each bar represents the mean (± S.E.) of three replicates.
*Significant differences compared to the control for p < 0.05 according to the Dunnett test.
116
Chapter V. Phytotoxic effects of A. melanoxylon
Fig. 12 Inhibitory effects of A. melanoxylon flowers and phyllodes on leaf protein
contents of three native grasses and crop species at four different concentrations.
Each bar represents the mean (± S.E.) of three replicates.
*Significant differences compared to the control for p < 0.05 according to the Dunnett test.
117
Ecophysiological response of native herbs
118
Benzoxazolin-2(3H)-one
(BOA)
induced
changes
in
leaf
water
relations,
photosynthesis
and
carbon
isotope
discrimination in Lactuca sativa
Chapter VI
Chapter section submitted as an original article to SCI journal:
Hussain, M.I., González, L., Chiapusio, G., and Reigosa, M.J. 2010. Benzoxazolin2(3H)-one (BOA) induced changes in leaf water relations, photosynthesis and
carbon isotope discrimination in Lactuca sativa. Plant Physiology and Biochemistry
(accepted)
Physiological response of lettuce
Elsevier Editorial System(tm) for Plant Physiology and Biochemistry
Manuscript Number:
Title: Benzoxazolin-2(3H)-one (BOA) induced changes in leaf water relations,
photosynthesis and carbon isotope discrimination in Lactuca sativa
Article Type: Research Paper
Keywords: Allelochemicals; BOA, leaf/root growth; Chlorophyll fluorescence; leaf
water status; stable isotopes; Lettuce; protein contents
Corresponding Author: Dr. M. IFTIKHAR HUSSAIN, PhD in Plant Physiology
Corresponding Author's Institution: University of Vigo
First Author: M. IFTIKHAR HUSSAIN, PhD in Plant Physiology
Order of Authors: M. IFTIKHAR HUSSAIN, PhD in Plant Physiology; Genevieve
Chiapusio, PhD in Plant Biology; Luis Gonzalez, PhD in Plant Physiology; Manuel J.
Reigosa, PhD in Plant Physiology
Abstract: The effects are reported here of Benzoxazolin-2(3H)-one (BOA), an allelopathic compound, on
plant water relations, growth, components of chlorophyll fluorescence, and carbon isotope discrimination
in lettuce (Lactuca sativa L.). Lettuce seedlings were grown in 1:1 Hoagland solution in perlite culture
medium in environmentally controlled glasshouse. After 30 days, BOA was applied at concentration of
0.1, 0.5, 1.0 and 1.5 mM and distilled water (control). BOA, in the range (0.1-1.5 mM), decreased the
shoot length, root length, leaf and root fresh weight. Within this concentration range, BOA significantly
reduced relative water content while leaf osmotic potential remained unaltered. Stress response of lettuce
was evaluated on the basis of six days of treatment with 1.5 mM BOA by analyzing several chlorophyll
fluorescence parameters determined under dark-adapted and steady state conditions. There was no change
in initial fluorescence (F0) in response to BOA treatment while maximum chlorophyll fluorescence (Fm)
was significantly reduced. BOA treatment significantly reduced variable fluorescence (Fv) on second,
third, fourth, fifth and sixth day. Quantum efficiency of open reaction centers (Fv/Fm) in the dark-adapted
state was significantly reduced in response to BOA treatment. Quantum yield of photosystem II (ΦPSII)
electron transport was significantly reduced because of decrease in the efficiency of excitation energy
trapping of PSII reaction centers. Maximum fluorescence in light adapted leaves (F′m) was significantly
decreased but there was no change in initial fluorescence in light-adapted state (F′0) in response to 1.5
mM BOA treatment. BOA application significantly reduced photochemical fluorescence quenching (qP)
indicating that the balance between excitation rate and electron transfer rate has changed leading to a
more reduced state of PSII reaction centers. Non photochemical quenching (NPQ) was also significantly
reduced by BOA treatment on
third, fourth and fifth day. BOA had dominant effect on C isotope ratios (δ13C) that was significantly less
negative (-26.93) at 1.0 mM concentration as compared to control (-27.61). Carbon isotope discrimination
(Δ 13C) values were significantly less (19.45) as compared to control (20.17) at 1.0 mM. BOA also affect
ratio of intercellular to air CO2 concentration (ci/ca) that was significantly less (0.66) as compared to
control (0.69) when treated with 1.0 mM BOA. There was non-significant difference between control and
BOA treated plants regarding intrinsic water use efficiency (iWUE) while BOA 1.0
mM also stimulated iWUE. Protein content of lettuce leaf tissue was significantly decreased under BOA
stress at highest concentration (1.5 mM) as compared to control.
120
Chapter VI. Phytotoxic effects of BOA
Abstract
The effects are reported here of Benzoxazolin-2(3H)-one (BOA), an allelopathic
compound, on plant water relations, growth, components of chlorophyll fluorescence, and
carbon isotope discrimination in lettuce (Lactuca sativa L.). Lettuce seedlings were grown in
1:1 Hoagland solution in perlite culture medium in environmentally controlled glasshouse. After
30 days, BOA was applied at concentration of 0.1, 0.5, 1.0 and 1.5 mM and distilled water
(control). BOA, in the range (0.1–1.5 mM), decreased the shoot length, root length, leaf and
root fresh weight. Within this concentration range, BOA significantly reduced relative water
content while leaf osmotic potential remained unaltered. Stress response of lettuce was
evaluated on the basis of six days of treatment with 1.5 mM BOA by analyzing several
chlorophyll fluorescence parameters determined under dark-adapted and steady state conditions.
There was no change in initial fluorescence (F0) in response to BOA treatment while maximum
chlorophyll fluorescence (Fm) was significantly reduced. BOA treatment significantly reduced
variable fluorescence (Fv) on second, third, fourth, fifth and sixth day. Quantum efficiency of
open reaction centers (Fv/Fm) in the dark-adapted state was significantly reduced in response to
BOA treatment. Quantum yield of photosystem II (ΦPSII) electron transport was significantly
reduced because of decrease in the efficiency of excitation energy trapping of PSII reaction
centers. Maximum fluorescence in light adapted leaves (F′m) was significantly decreased but
there was no change in initial fluorescence in light-adapted state (F′0) in response to 1.5 mM
BOA treatment. BOA application significantly reduced photochemical fluorescence quenching
(qP) indicating that the balance between excitation rate and electron transfer rate has changed
leading to a more reduced state of PSII reaction centers. Non photochemical quenching (NPQ)
was also significantly reduced by BOA treatment on third, fourth and fifth day. BOA had
dominant effect on C isotope ratios (δ13C) that was significantly less negative (-26.93) at 1.0
mM concentration as compared to control (-27.61). Carbon isotope discrimination (Δ 13C)
values were significantly less (19.45) as compared to control (20.17) at 1.0 mM. BOA also
affect ratio of intercellular to air CO2 concentration (ci/ca) that was significantly less (0.66) as
compared to control (0.69) when treated with 1.0 mM BOA. There was non-significant
difference between control and BOA treated plants regarding intrinsic water use efficiency
(iWUE) while BOA 1.0 mM also stimulated iWUE. Protein content of lettuce leaf tissue was
significantly decreased under BOA stress at highest concentration (1.5 mM) as compared to
control.
Keywords: Allelochemicals; BOA, leaf/root growth; Chlorophyll fluorescence; leaf water
status; stable isotopes; Lettuce; protein contents
121
Physiological response of lettuce
Abbreviations: RWC, relative water content; LOP, leaf osmotic potential; Fo, initial
fluorescence level from dark adapted leaves; Fm, maximal fluorescence level from dark adapted
leaves; Fv, variable fluorescence level from dark adapted leaves; F′m, maximal fluorescence, F′o,
minimal fluorescence; F′v, variable fluorescence level from light adapted state; PSII (efficiency
of photosystem II photochemistry in the dark-adapted state (Fv/Fm); maximum quantum yield of
PSII (ΦPSII) electron transport; qP, photochemical quenching; NPQ, non-photochemical
quenching; N%, Nitrogen concentration; δ15N, Nitrogen isotope composition; Carbon%, Carbon
concentration; δ 13C, composition of carbon isotope ratios; Δ13C, carbon isotope discrimination;
ci/ca, ratio of intercellular CO2 concentration from leaf to air; iWUE, intrinsic water use
efficiency.
122
Chapter VI. Phytotoxic effects of BOA
1. Introduction
Allelopathy, the chemical inhibition of one plant species by another, represents a form
of chemical warfare between neighboring plants competing for limited light, water, and nutrient
resources [78]. Many plant species produce certain chemicals which released in the environment
by means of volatilization, leaching, decomposition of residues, root exudates and mediate plant
to plant interactions [38]. Allelochemicals produced by plants can induce both inhibitory and
stimulatory effects on organisms and may play important roles in shaping plant and microbial
communities [60]. Concerns about ecological, environmental and health problems possibly
associated with synthetic pesticides have increased interest in the development of new classes of
environmentally safe chemicals [21]. Most allelochemicals are produced as offshoots of the
primary metabolic pathways of the plant and some have been explored as natural substitutes for
commercial herbicides [23, 26].
Benzoxazolin-2(3H)-one (BOA) is an allelochemical most commonly associated with
monocot species, formed from the O-glucoside of 2,4-dihydroxy-2H-1,4- benzoxazin-3(4H)-one
by a two-step degradation process. BOA is common in both cultivated and wild Gramineae
plants [56, 66] particularly cereals such as wheat, rye and maize [32]. BOA was first discovered
by Virtanen et al. [75] as an anti-Fusarium secondary metabolite produced by some grasses.
Benzoxazinones are found in cereal seedlings and mature plants at millimolar levels; i.e. the
natural concentration of benzoxazinones in the vacuolar sap of corn seedlings is <10 mm [61].
BOA inhibits the growth of a number of plants such as Avena sativa [31], Cucumis sativus [18],
Lactuca sativa [43, 67], Lepidium sativum [7], Phaseolus aureus [72, 10] and Raphanus sativus
[19]. It is now being explored for herbicidal activity [16]. BOA inhibits plasma membrane
bound H ± ATPases in roots of Avena fatua [31], causes a number of ultrastructural changes in
the roots of C. sativus [16], and inhibits α-amylase activity in roots of L. sativa [43]. Recently,
Singh et al. [72] reported that BOA inhibits germination, early seedling growth and rhizogenesis
in the hypocotyls cuttings of mung bean (Phaseolus aureus). BOA occurs naturally in grass
species [69] and has been associated with dose-dependent germination inhibition and growth
reductions in crop and weed species [42]. Phytotoxic effects of BOA have been found on root
ultrastructural, where packed multiple columns of cells failed to lengthen at the root tip of
cucumber plants [15].
Baerson et al. [2] used whole genome oligonucleotide-based Gene Chip® arrays to
probe specific members of complex gene families likely to participate in xenobiotic
detoxification pathways in Arabidopsis exposed to BOA. They found that BOA induced many
genes involved in phases I, II and III detoxification processes; concluding that BOA elicits a
general response network of genes involved in chemical detoxification pathways, and providing
clear evidence of the phytotoxic capacity of BOA. Determining the mode of action of
allelochemicals is a difficult task owing to the multitude of potential target sites and the
difficulty in separating primary effects from secondary ones [20]. Recently, Sánchez-Moreiras
et al. [68] have pointed out that BOA has multiple modes of actions and causes alterations in
cell cycle and activity of ATPases and changes in water status. Degradation of BOA was
123
Physiological response of lettuce
recently studied in great detail [47]. One of the degradation products of BOA is APO (2aminophenoxazin-3-one), which is very stable and more phytotoxic than BOA.
Nevertheless, it remains necessary to incorporate allelopathy into the modern concept of
ecophysiology, as a means of resolving many unanswered questions [19]. Furthermore, little is
known about the molecular mode of action of allelochemicals and the plant defense response
against them (22, 23). The phytotoxic study of potential molecules offers useful clues in the
investigation of new models of natural herbicides that could be more specific and less harmful
than the synthetic substances used in agriculture. In the present work we have focused on the
analysis of the physiological response, protein profiles, leaf water relations and stable isotopic
fractionation responses in Lactuca sativa seedlings exposed to the model benzoxazolinone
allelochemical BOA. These studies using exogenously supplied BOA help us to better
understand its mode of action on new target sites and open the gate to the use of allelochemical
as natural herbicides.
2. Results
2.1. Effect on Plant growth and development
There was a significant (P < 0.05) reduction in root and shoot length of lettuce in
response to BOA exposure at all concentrations. In general, the reduction was more in root
length than shoot length. At 1.5 mM BOA, there was nearly 49.21% reduction in shoot length
compared to control, whereas in root length the reduction was 68.41%. Both root and shoot
lengths declined with increase in BOA concentration (Table 1). In response to 1.5 mM BOA
treatment, there was a significant reduction of leaf fresh weight while leaf dry weight reduced at
all concentration of BOA as compared to control but the results were not statistically significant.
In response to 0.1 mM BOA treatment, there was a reduction of root fresh weight of 46.90%.
These declined further and reached to nearly 70.68% reduction in root fresh weight at 1.5 mM
BOA (Table 1). The root dry weight was only significantly reduced at 1.5 mM BOA as
compared to control.
2.2. Leaf water relations
Leaf water relations measured in terms of relative water content (RWC) significantly
decreased in leaf tissue (except at 0.1 mM) in response to BOA treatment (Fig. 1). At highest
BOA concentration (1.5 mM), the RWC was nearly 0.5-folds less as compared to control,
whereas at lowest BOA concentration (0.1 mM), there was a slight decrease in RWC. Parallel
to RWC, leaf osmotic potential (LOP) significantly increased in response to lowest
concentration (0.1 mM) of BOA. There was non-significant difference in LOP in lettuce leaf
tissue among control and BOA treatment (Fig. 2) at all remaining concentration.
124
Chapter VI. Phytotoxic effects of BOA
2.3. Chlorophyll fluorescence measurements
Stress response of lettuce leaves to BOA was evaluated on the basis of six days of
treatment with 1.5 mM BOA by analyzing several chlorophyll fluorescence parameters
determined under dark-adapted and steady state conditions. There was no change in initial
fluorescence (F0) in response to BOA treatment while lettuce seedlings treated with 1.5 mM
BOA significantly stimulated F0 on second day (Fig. 3 A). There was significant reduction in
maximum chlorophyll fluorescence (Fm) in lettuce leaves when exposed to BOA during all six
days. (Fig. 3 B). BOA treatment significantly reduced variable fluorescence (Fv) during second,
third, fourth, fifth and sixth day (Fig. 4 A). In response to BOA (1.5 mM), the steady state
fluorescence (Fs) was significantly decreased during third and fourth day (Fig. 4 B), while BOA
also reduced Fs in all other days as compared to control but results were not statistically
significant. The quantum efficiency of open reaction centers in the dark-adapted state (Fv/Fm)
was significantly reduced in response to BOA treatment on all days except day first (Fig. 5 A).
In response to BOA (1.5 mM) treatment, quantum yield of photosystem II (ΦPSII) electron
transport in lettuce leaves was significantly reduced (Fig. 5 B). The reduced ΦPSII was a result
of the decrease in the efficiency of excitation energy trapping of PSII reaction centers, F′v/F′m.
As shown in table 2, the maximum fluorescence in light adapted leaves (F′m) in lettuce
leaves was significantly decreased when exposed to BOA treatment. There was no change in
initial fluorescence in light-adapted state (F′0) during first, third, fourth and fifth day while BOA
significantly reduced F′0 during second and sixth day (Table 2). BOA treatment significantly
reduced variable fluorescence (F′v) on second, third, fourth, and fifth day (Fig. 4 A). A
significant reduction in photochemical fluorescence quenching (qP) was observed after
treatment with 1.5 mM BOA (Fig. 6 A), indicating that the balance between excitation rate and
electron transfer rate has changed leading to a more reduced state of the PSII reaction centers.
Non photochemical quenching (NPQ) was also significantly reduced by BOA treatment on
third, fourth and fifth day while in all other days BOA also reduce NPQ but results were nonsignificant (Fig. 6 B).
2.4. Carbon and nitrogen stable isotope composition analysis
Up to certain extent BOA concentration (0.5 mM) increased the leaf nitrogen
concentration (N %) while non-significant differences were observed between control and BOA
at remaining concentrations. Similarly, BOA did not significantly affect nitrogen isotope ratios
(δ15N) (data not shown). Non-significant difference was observed between control and BOA
treated plants regarding carbon concentration (C %) in lettuce (Fig. 7 A). The BOA had
dominant effect on C isotope ratios (δ13C) that was significantly less negative (-26.93) at 1.0
mM concentration as compared to control (-27.61) (Fig. 7 B). Carbon isotope discrimination (Δ
13
C) values were significantly less (19.45) as compared to control (20.17) at 1.0 mM (Fig. 7 C).
The BOA had dominant effect on the ratio of intercellular to air CO2 concentration (ci/ca) that
was significantly less (0.66) as compared to control (0.69) when treated with 1.0 mM BOA( Fig.
125
Physiological response of lettuce
8 A). There was non-significant difference between control and BOA treated plants regarding
intrinsic water use efficiency (iWUE) while BOA 1.0 mM also stimulated iWUE (Fig. 8 B).
2.5. Protein contents
Protein content of lettuce leaf tissue was significantly decreased under BOA stress at
highest concentration (1.5 mM) as compared to control (Fig. 9).
3. Discussion
Plant phenolics, including phenolic acids, are ubiquitous secondary metabolites widely
involved in plant–plant interactions [76]. Here, the effect of a putative allelochemical, BOA on
growth, physiological and stable isotopic characteristics of 30-days old lettuce is reported. The
striking fact revealed in current study is that plants growing in the presence of 1.5 mM BOA
were significantly smaller, with less fresh biomass and shorter root length. In general, the
reduction was more in root length than shoot length and this increase with increase in
concentration from 0.1-1.5 mM BOA. In response to 0.1 mM BOA treatment, there was a
significant reduction in root fresh weight that declined further due to increase of BOA
concentration. Plants exposed to highest concentration (1.5 mM) of BOA were severely
affected, with more than 50% reduction in root/leaf biomass as compared to control. This
finding is of particular interest because the reduction in root growth has been considered one of
the first effects of allelochemicals associated with reduction in plant growth and development.
This data suggests that BOA act as stressor on plant metabolism. Similar results were reported
in literature [8, 19, 10, 67, 68, 72]. It affects mitotic activity in root tips [72], and causes a
number of ultra structural changes like increased cytoplasmic vacuolation, reduced ribosomal
density, dictyosomes and mitochondrial number, and decreased lipid catabolism [15]. The
greater phytotoxic effect on root is also reflected from higher levels of oxidative stress and
induction of antioxidant enzyme system in the roots compared to leaves.
The BOA interfered with the normal leaf water relations of lettuce and significantly
decreased the RWC of lettuce leaf tissue. At highest BOA concentration (1.5 mM), the RWC
was nearly 0.5-folds less as compared to control. Osmotic potential in the leaves of treated
plants remained unaltered and significantly stimulated after exposure to lowest concentration
(0.1 mM) of BOA. Contrary, other phenolic acids like p-coumaric, caffeic, ferulic, and salicylic
acid, are known to cause water stress in plants [24, 13, 5].
The measurement of chlorophyll fluorescence emitted by intact, attached leaves is
thought to be a reliable, non-invasive method for monitoring photosynthetic events and
physiological status of the plant. Fluorescence induction patterns and derived indices have been
used as empirical diagnostic tools in plant physiological studies [3, 44, 73]. In lettuce the
fluorescence quenching parameters are directly reflecting the photosynthetic activity during the
allelopathic stress. Chlorophyll fluorescence measurement reflects changes in thylakoid
membrane organization, function and inhibition of photosynthesis and oxygen evolution
126
Chapter VI. Phytotoxic effects of BOA
through interactions with components of photosystem II [24, 64]. The initial fluorescence (F0) in
the beginning when the primary quinine type electron acceptors of PSII (QA) were oxidized was
variable in control leaves during day first with a tendency of stimulation in lettuce treated with
BOA (1.5 mM) during all other days. F0 was usually higher in treatments and with the passage
of time increased over control values for BOA treated plants. An increase in F 0 may have a
variety of causes. It has been associated with a dissociation of light harvesting complex (LHC)
II from the reaction centers; it may due to the presence of photoinhibited reaction centers,
however also a more reduced PQ-pool in dark-adapted leaves will lead to an increase of the
measured F0. It is interesting that despite the measured strong decrease of the chlorophyll
content; no decrease of the F0 was observed. It suggests that the modulated measuring
equipment simply probes somewhat deeper in the stressed plants monitoring the same number
of reaction centers as in the control plants. There was significant reduction in maximum
chlorophyll fluorescence (Fm) in lettuce leaves when exposed to BOA treatment during all six
days. Similarly, Zlatev and Yordanov [80], reported that biotic stress (drought) induced in their
bean plants always an increase in F0 accompanied by a decrease in Fm. The variable
fluorescence (Fv) was significantly reduced during all days (except day first) after the
application of BOA. The allelopathic stress of BOA was very drastic that significantly reduced
Fv values during sixth day and BOA values becoming 65% of the control.
The application of chemicals tended to reduce the Fv/Fm and gives insight into the
ability of plant to tolerate against this chemical stress [52] and the extent to which that stress has
damaged the photosynthetic apparatus. The ratio Fv/Fm fell more than 15% during second day
when BOA was added as can be seen in Fig. 5 A. These declined further and Fv/Fm ratio reduced
more than 58% on the sixth day, indicating that continuous application of BOA caused a severe
damage to photosynthetic system of Lactuca sativa. Damage to the reaction centers embedded
in thylakoid membranes, especially those of PSII, and inhibition of inductive resonance energy
transfers from antenna molecules to reaction centers can lead to lower Fv/Fm ratios [46].
Similarly, Zhou and Yu [79], concluded that allelochemicals can significantly affect the
performance of the three main processes of photosynthesis: stomatal control of CO 2 supply,
thylakoid electron transport (light reaction), and carbon reduction cycle (dark reaction).
In response to BOA (1.5 mM) treatment, quantum yield of photosystem II (ΦPSII)
electron transport in lettuce leaves was significantly reduced. Nevertheless, the inhibition of
ΦPSII values could be a result of a weaker efficiency of PSII reaction centers, and it may
indicate an alteration of rate of linear electron transport [57], suggesting that the proportion of
photons absorbed by PSII and used for photosynthesis was not the same as in control. This
altered PSII efficiency, as well as the water unbalance observed on BOA-exposed plants, could
be explained through an alteration on membrane properties. As shown in table 2, the maximum
fluorescence in light adapted leaves (F′m) in lettuce leaves was significantly decreased when
exposed to BOA treatment in all days except on first and sixth day. There was no change in
initial fluorescence in light-adapted state (F′0) in response to 1.5 mM BOA treatment on first,
third, fourth and fifth day while BOA significantly reduced F′0 on second and sixth day when
lettuce seedlings were treated with 1.5 mM BOA. Changes in the chlorophyll fluorescence
127
Physiological response of lettuce
quenching parameters are good indicators of photosynthetic electron flow [71]. After the onset
of illumination, maximal chlorophyll fluorescence declines as the photosynthetic reaction
proceeds due to the photosynthetic electron flow, qP, and non-radiative energy dissipation, NPQ
[33]. A significant reduction in photochemical fluorescence quenching (qP) was observed after
treatment with 1.5 mM BOA, indicating that the balance between excitation rate and electron
transfer rate has changed leading to a more reduced state of the PSII reaction centers. A
decrease in qP induced by BOA indicates a higher proportion of closed PSII reaction centers,
which probably generates a decrease in the proportion of available excitation energy used for
photochemistry. Non photochemical quenching (NPQ) was also significantly reduced by BOA
treatment on third, fourth and fifth day (Fig. 6 B).
The significant changes in PSII photochemistry in the light adapted leaves, such as the
decreased qP and F′v/F′m, as well as the increased of NPQ, can be seen as the regulatory
response to down-regulate the quantum yield of PSII electron transport (ΦPSII) [33], that would
match with the high decrease of CO2 assimilation rate. It is suggested that the decay in ΦPSII of
the BOA stressed plants may be a mechanism to down-regulate the photosynthetic electron
transport so that production of ATP and NADPH would be in equilibrium with the decreased
CO2 assimilation capacity in stressed plants. The fraction of PSII reaction centers that is opened,
as indicated by the decrease of qP, is reduced in lettuce plant cultivated with nutrient solutions
added with 1.5 mM BOA.
Much less progress has been made on the relationship between stress and δ15N in the
plant, because the observed variation was often attributed to a shift in source δ15N, rather than
discrimination in the plant [41, 55]. In present research, BOA (1.5 mM) did not significantly
affect nitrogen isotope ratios (δ15N) at any concentration. Handley et al. [39], reported a
decrease in foliar δ15N because of moderate salt stress in a controlled experiment with barley
(Hordeum spp.). They reported that this effect would be consistent with a decrease in nitrate
reductase activity. Plant photosynthesis discriminates against the stable 13C isotope [27] when
atmospheric CO2 passes through stomata during CO2 carboxylation in RuBisCo. Carbon isotope
discrimination decreases with a decrease in intercellular CO2 concentration due to stomatal
closure, and consequently with water use efficiency. For example, δ13C data are used to study
plant water use efficiency [17], respiration and secondary fractionation processes [9], and to
partition net ecosystem CO2 fluxes between photosynthesis and respiration [81]. These
applications require robust estimates of net 13C discrimination (Δ13C) during photosynthesis. In
C3 species, leaf level 13C Δ during photosynthetic gas exchange primarily reflects the balance
between CO2 supply by diffusion through stomata and CO2 demand by biochemical reactions in
chloroplasts, most importantly catalysis by Rubisco [28, 29]. Both processes discriminate
against the heavier isotope, but the fractionation occurs during carboxylation by Rubisco [36,
58]. The carbon isotope fractionation patterns indicates that limitations to the diffusion of CO2
through the stomatal aperture can result in less negative δ13C values [59]. Our data suggest that
stomata‘s of lettuce plants treated with 1.5 mM BOA experienced some degree of closure,
which ultimately leads to less discrimination against the heavier isotope. Δ 13C values were
significantly less as compared to control at 1.5 mM. Previously, Barkosky and Einhellig [5],
128
Chapter VI. Phytotoxic effects of BOA
reported that carbon isotope ratio (δ 13C) in leaf tissue of soybean at the termination of the 28day experiment showed significantly less discrimination (less negative δ 13C) against 13C in
plants grown with 0.75 mM p-hydroxybenzoic acid. Similarly, Barkosky et al. [4], concluded
that dried leaf tissue from Leafy spurge (Euphorbia sula) treated with 0.25 mM Caffeic acid had
a less negative δ 13C compared to controls, indicating less discrimination against 13C in these
plants. The δ 13C value of Lactuca sativa was less negative at the higher pollutant level than at
the lower level and was statistically significantly different (P < 0.05) from that of control [49].
Different types of biotic and abiotic stress can cause degradation of proteins by
senescence or by reduction in protein synthesis. Baziramakenga et al. [11], reported that many
phenolic acids reduced the incorporation of certain amino acid into proteins and thus reduce the
rate of protein synthesis. In the present research, BOA also decreased leaf protein contents in
lettuce at 1.5 mM concentration. Similarly, Mersie and Singh [54], reported a degradation and
reduction in total protein contents after the application of p-hydroxybenzoic acid.
As regards the uptake, translocation and accumulation of BOA not much details are
available. The concentrations of the BOA (0.1, 0.5, 1.0, and 1.5 mM = 0.0946, 0.473, 0.946,
1.419 mg ml–1) used in the present study are ecologically relevant. BOA is a major metabolite
responsible for the weed suppressing ability of rye residues [7]. However, its phytotoxic effect
depends upon the amount of BOA available within the soil and the type of crop/weed growing
[16]. Burgos et al. [15], observed that residues from rye cultivars could release BOA at 1.17 mg
kg–1 soil. The realistic concentration of BOA present in the soil environment, however, depends
upon a number of factors including amount of residue and type of rye cultivar incorporated into
the soil and the microbial activity. Recently, Martyniuk et al. [51], reported that BOA in the
range of 0.125–4.0 mM inhibited the growth of several cereal pathogenic fungi including
Cephalosporium gramineum, Gaeumannomyces graminis var. graminis, and Fusarium
culmorum. The study concluded that BOA at concentration of 1% (approximately 74 mM)
could provide fungicidal activity similar to commercial fungicides. Glass and Dunlop [34],
reported that BOA depolarizes membranes and it is likely that membrane perturbations will
have multiple effects on metabolism and the efficiency of plant processes. In some cases,
highest concentrations of BOA were not lethal. This may be due to the glucosylation of BOA by
enzyme systems within the plant. Scholten et al. [70], found that cell cultures of Datura innoxia
and Scopolia carniolica are able to glucosylate hydroquinone, vanillin, and BOA acid by an
enzyme-mediated, concentration dependent, bioconversion. Tabata et al. [74], revealed that
certain plant cell cultures can glucosylate a wide variety of phenolics including BOA.
The results of this study show that BOA is a growth inhibitor of lettuce and mechanism
of inhibition is due to disruption of plant water balance. Furthermore, it significantly reduced
the shoot length, root length and leaf/root fresh weight of lettuce. Damage caused by BOA
application indicates a direct destruction in photosynthetic reaction center. These leads to
unbalance in process of photosynthesis which ultimately results in stunted leaf and root growth
with less plant biomass. Stable carbon isotope signatures were significantly less negative in
129
Physiological response of lettuce
BOA treated plants as compared to control. BOA caused denaturation of amino acids that
ultimately leads to reduction in protein synthesis.
4. Materials and Methods
4.1. Materials used
Lettuce (Lactuca sativa L. cv. Great Lakes California) was used for the present study
because of its fast germination and homogeneity [48]. Lettuce seeds were purchased
commercially from Semillas Fito (Barcelona, Spain). BOA (100% purity) was purchased from
Sigma Chemical Company, St. Louis, MO, USA. A stock solution (3 mM) of BOA was
prepared by dissolving requisite amount in Methanol: Water (20:80). The control was prepared
with distilled water and methanol. The methanol was evaporated in a rotary vaporizer. The stock
solution was further diluted with distilled water to get 0.1, 0.5, 1.0, 1.5 mM BOA solution and
adjusted to pH 6.0 with NaOH [49, 50]. The concentrations used in present study are
ecologically relevant and were selected based on previous studies reported in literature for
exploring mode of action of BOA (0.75–3.70 mM, Burgos and Talbert, [16]; 0.125–4.0 mM,
Martyniuk et al. [51]; 0.01–5 mM, Singh et al. [72]; 0.1- 5 mM, Batish et al. [10]; 0.01-0.5
mM, Baerson et al. [2]; 1 mM, Sánchez-Moreiras et al. [67]; 0.01- 6 mM, Sánchez-Moreiras et
al. [68].
4.2. Plant growth and BOA treatment
Seeds of lettuce were surface sterilized with sodium hypochlorite (0.1%, w/v)
followed by washing with distilled water three times. Seeds were placed in plastic trays
(32x20x6 cm) with a 5 cm deep layer (500 g/tray) of perlite in darkened at 20 0C temperatures
in environmentally controlled growth chamber. Seeds were irrigated on alternate days with tap
water until germination and thereafter with 500 ml 1:1 Hoagland solution/tray [40], twice in a
week. For seedling growth, the environmental conditions were as follows; temperature: 18/8 0C
(day/night) and 12/12 h (light/darkness) photoperiod, 80% relative humidity and 200 mol m–2
sec-1 irradiance. One month old seedlings (three fully expanded leaf stage), were transferred to
pots (10 cm) containing perlite (70 g) to stimulate the development of root system and shifted to
the glass house with same growing condition and nutrient solution. The glasshouse was
ventilated with outside air to ensure steady CO2. Test plants were selected randomly and
assigned one plant per pot. Three replicates were maintained for each treatment in a
randomized complete block manner. The treatments were applied three times (days 1, 3 and 5)
with 100 ml solution of four concentration (0.1, 0.5, 1.0, 1.5 mM) of BOA and control (distilled
water) were checked against the target specie.
4.3. Chlorophyll fluorescence measurement
Chlorophyll fluorescence measurements were performed in a glass house with a
portable, pulse-modulated instrument fluorescence monitoring system (FMS) (Hansatech,
130
Chapter VI. Phytotoxic effects of BOA
Norfolk, England) by the method of Weiss and Reigosa, [77], on randomly selected leaves of
lettuce. Following 20 min dark adaptation, the minimum chlorophyll fluorescence (F0) was
determined using a measuring beam of 0.2 μmol m−2 s−1 intensity. A saturating pulse (1 s white
light with a PPFD of 7500 μmol m−2 s−1) was used to obtain the maximum fluorescence (Fm) in
the dark-adapted state. The quantum efficiency of open PSII reaction centers in dark-adapted
plants (Fv/Fm) was calculated from (Fm− F0)/ Fm. Light-induced changes in chlorophyll
fluorescence following actinic illumination (300 μmol m−2 s−1) were recorded prior to
measurement of F′0 (minimum chlorophyll fluorescence in light saturated state) and F′m
(maximum fluorescence in light saturated stage). The quantum efficiency of open PSII reaction
centers in the light-adapted state, referred to as ФPSII (F′m−Fs/ F′m), was determined from F′m
and Fs values and also the quantum efficiency of excitation energy trapping of PSII,
(F′v/F′m),was calculated according to Genty et al. [33]. After turning off the actinic light the
leaves were illuminated with far-red light (7 μmol m−2 s−1) to oxidize the PQ pool to determine
the minimum fluorescence level of the light-adapted state (F′0). The fraction of open PSII
reaction centers as qP = (F′m− Fs/F′m− F′0). (F′0/Fs) were calculated using the ‗lake‘ model
according to Kramer et al. [45] and nonphotochemical quenching NPQ = (F′m−Fm)/(F′m) were
calculated according to Bilger et al., [12].
4.4. Determination of protein content
Total protein was quantified by using the Spectrophotometric Bradford assay [14].
Lettuce leaves (200 mg fresh weight) from three replicates per treatment were crushed in liquid
nitrogen in cooled mortar with 0.05 g PVPP, and the powder was resuspended in 1 mL of Tris
buffer containing 0.05 M Tris base, 0.1% w/v ascorbic acid, 0.1% w/v cysteine hydrochloride,
1% w/v PEG 4000, 0.15% w/v citric acid, and 0.008% v/v 2-mercaptoethanol [1]. Samples were
centrifuged at 19000 gn and 4 ºC for 20 min after resuspension. The supernatant (0.1 mL) was
mixed for protein dye-binding reaction with 3 mL Bradford reactive containing 0.01%
Coomassie® Brilliant Blue G-250, 4.7% v/v ethanol 95%, and 8.5% v/v phosphoric acid 85%.
Absorbance was recorded at 595 nm after 5 min for quantification of the protein content.
Commercial bovine seroalbumin (BSA) was used as standard.
4.5. Carbon and nitrogen isotope composition analysis
Collected plant leaf samples were immediately dried in a forced-air oven at 70°C
(Gallenkamp oven, Loughborough, Leicestershire, UK) to constant weight and ground in Ball
Mills (Retsch MM 2000, Haan, Germany). Dry ground plant material were weighed (1700-2100
µg) with weighing meter (Metler Toledo GmbH: Greifensee Switzerland), filled in tin capsules
(5x3.5 mm, Elemental Microanalysis Limited, U.K.). Each tin capsule was entered
automatically in combustion oven at 1600-1800 0C in the presence of oxygen and converted to
CO2 and N2. Subsequently isotope ratios were determined in an Isotopic Ratio Mass
Spectrometer (Finnegan: Thermo Fisher Scientific, model MAT-253, Swerte Germany) coupled
with an Elemental Analyzer (Flash EA-1112, Swerte Germany). The Isotopic ratio mass
spectrometer has an analytical precision better than 0.05‰ for 15N and 0.3‰ for 13C.
131
Physiological response of lettuce
Carbon and nitrogen isotope compositions were calculated as;
δ (‰) = [(Rsample/Rstandard) — 1)] x 1000
(1)
Where Rsample is the ratio of 13C/12C or 15N/14N, and Rstandard are standards used. Atmospheric N2
is the standard for nitrogen while Vienna PeeDee Belemnite (VPDB) is standard for carbon. The
accuracy and reproducibility of the measurements of δ13C and δ15N checked with an internal
reference material (NBS 18 and IAEA-C6 for C), and (IAEA-310A and IAEA-N1 for N), and
Acetanilide for C/N %age ratios respectively. Carbon isotope discrimination is a measure of the
C isotopic composition in plant material relative to the value of same ratio in the air on which
plants feed:
Δ (‰) = [(δa – δp)/(1 + δp)] x 1000
(2)
where Δ represents carbon isotope discrimination, δa represents C isotope composition in the
source air, and δp represents C isotope composition in the plant tissue. Theory published by
Farquhar et al. [27] and Farquhar and Richards [29] indicates that C discrimination in leaves of
plants can be expressed in relationship to CO2 concentrations inside and outside as:
Δ = a + (b - a) ci/ca
(3)
Δ = 4.4 + (27 - 4.4) ci/ca
where a is discrimination that occurs during diffusion of CO2 through the stomata
(4.4‰), b is discrimination by rubisco (27‰), and ci/ca is the ratio of the leaf intercellular CO2
concentration to that in the atmosphere. Equation [3] shows a direct and linear relationship
between Δ and ci/ca. Therefore, measurement of Δ gives an estimation of the assimilation-rate–
weighted value of ci/ca. A lower ci/ca ratio may result either from higher rates of photosynthetic
activity or from stomatal closure induced by water stress.
Intrinsic water use efficiency (iWUE) was calculated according to the following
equations (McCarroll and Loader, [53]:
iWUE = A/g = ca [1-(ci/ca)] x (0.625)
(4)
Where A is rate of CO2 assimilation and g is the stomatal conductance. Data of δ 13Cair, ci and ca
were obtained from McCarroll and Loader [53] which used the high precision records of
atmospheric Δ 13C from Antarctic ice cores [30], and the atmospheric CO2 concentration (ppm)
from Robertson et al. [65]. Carbon and nitrogen isotope measurements were performed in
CACATI (Centro de Apoio Cientifico Tecnologico a la Investigacion), University of Vigo,
Spain.
132
Chapter VI. Phytotoxic effects of BOA
4.6. Plant growth measurements
Information about plant height/ root length was obtained with a ruler and values were
expressed in cm. The fresh and oven dry plant leaves and roots weight were obtained by first
weighting independently fresh leaves and roots then after drying these samples in a circulatory
air oven at 70 0C for 72 h. The samples were weighed again to get dry weight of plant.
4.7. Determination of plant water content
The leaf relative water content (RWC) is an appropriate measure of plant water status in
terms of physiological consequence of cellular water deficit. The RWC was calculated by
measuring fresh (Wf), saturated (Wt) and dry (Wd) weights of plant [62]. Leaf plant pieces from
three replicates per treatment were weighed in fresh saturates at 4 oC for 3 h, weighed again,
oven dried at 70 oC for 72 h, and finally weighed once more for obtaining three necessary
weights required for the following equation:
RWC = Wf – Wd/ Wt – Wd x 100
(6)
To measure leaf osmotic potential (LOP) (milliosmol kg-1); one leaf per replicate was
transferred into 10 mL syringes and kept frozen at -80 ºC until further analysis [35]. During
analysis, syringes were thawed until sample reached at room temperature and osmotic potential
was measured on the second drop from collected sap by using a calibrated vapor pressure
Osmometer (Automatic Cryoscopic Osmometer, Osmomat–030, GmbH, Gonatec, Berlin,
Germany) according to manufacturer‘s instructions.
4.8. Statistical analysis
All experiments were performed in a randomized complete block manner with three
replicates. Data were analyzed with the statistical package SPSS® (version 15.00) for Windows®
(SPSS Inc., Chicago, IL, USA). In all cases, there was a comparison between control and
treatment using Student‘s t-test.
Acknowledgements
We thank Dr. Aldo Barreiro, Carlos Bolano, Alfredo Justo, Maite Ricart, and Paula Lorenzo
with field and laboratory assistance. We are also grateful to Jesús Estévez Sío and Jorge Millos
for their technical assistance with isotope ratio mass spectroscopy work.
133
Physiological response of lettuce
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Physiological response of lettuce
Table 1. Effect of BOA ( 0.1, 0.5, 1.0, 1.5 mM) on lettuce leaf and root growth and development after six days treatment.
Growth characteristics
BOA concentration (mM)
Control
0.1
0.5
1.0
1.5
Leaf fresh weight (g)
10.58±0.60
6.19±0.133*
6.15±1.09*
6.9±0.59*
4.93±1.69*
Leaf dry weight (g)
1.63±0.15
1.57±0.34
1.50±0.073
1.48±0.34
1.42±0.40
Root fresh weight (g)
6.31±0.34
4.46±1.26*
4.09±0.49*
2.99±0.90*
2.96±0.60*
Root dry weight (g)
0.49±0.08
0.42±0.057
0.42±0.062
0.413±0.031
0.23±0.078*
Shoot length (cm)
15.30±0.23
8.47±0.86*
8.10±0.10*
7.63±0.63*
7.53±0.50*
Root length (cm)
27.77±0.22
22.17±0.83*
20.77±1.49*
19.17±0.44*
19.00±0.57*
Each value represents the mean (± S.E.) of three replicates.
*Asterisk indicates significant differences as compared to control for p < 0.05 according according to Student's t-test.
138
Chapter VI. Phytotoxic effects of BOA
Table 2. Effect of BOA (1.5 mM) on F'm, F'o, and F'v in leaves of Lactuca sativa from days 1-6 after BOA treatment.
Chlorophyll
parameters
F'm
F'o
F'v
Days
Treatments
1
2
3
4
5
6
Control
2496.33 ± 201.79
3032.92±100.43
2528.67±188.10
1389.17±154.60
1191.75±101.39
868.67±114.38
BOA
2003.67 ± 800.48
2299.00±532.99*
835±28.18*
445.33±62.98*
463.67±33.22*
545.33±72.83
Control
75.78±8.70
75.40±2.59
66.74±7.75
33.5±2.04
76.4±4.47
84.25±5.35
BOA
66.96±2.35
115.80±5.18*
73.15±11.48
41.74±1.65
86.57±23.76
211.88±120.59*
Control
2420.54±203.13
2957.51±101.79
2461.92±192.04
1355.65±154.68
1115.34±99.04
784.4±116.97
BOA
1936.70±799.13
2183.19±528.38*
761.84±16.90*
403.58±64.64*
377.09±17.81*
333.45±163.63
Where F'm (maximum chlorophyll fluorescence), F'o (initial fluorescence), and F'v (variable fluorescence in light adapted state).
Each value represents the mean (± S.E.) of three replicates.
*Asteriks indicates significant differences as compared to control for p < 0.05 according to Student t-test.
139
Physiological response of lettuce
Fig. 1. Effect of BOA on RWC (%) in leaves of L. sativa determined 6-days after BOA treatment.
.Every column in each graph represents the mean (± S.E.) of three replicates.
*Asteriks indicates significant differences as compared to control for p < 0.05 according to Student t-test.
Fig. 2. Effect of BOA on LOP (milliosmol kg-1) in leaves of Lactuca sativa determined 6-days after BOA treatment.
Every column in each graph represents the mean (± S.E.) of three replicates.
*Asteriks indicates significant differences as compared to control for p < 0.05 according to Student t-test.
140
Chapter VI. Phytotoxic effects of BOA
(A)
(B)
Fig. 3. Effect of BOA on initial chlorophyll fluorescence in dark adopted states (Fo) (A) and maximum fluorescence (Fm) (B) in leaves
of L. sativa determined 6-days after BOA treatment. Every column in each graph represents the mean (± S.E.) of three replicates.
*Asteriks indicates significant differences as compared to control for p < 0.05 according to Student t-test.
141
Physiological response of lettuce
(A)
(B)
Fig. 4. Effect of BOA on variable chlorophyll fluorescence in dark adopted states (Fv) (A) and steady state fluorescence (Fs) (B) in leaves of lettuce
determined from 1- 6 days after BOA treatment. Every column in each graph represents the mean (± S.E.) of three replicates.
*Asteriks indicates significant differences as compared to control for p < 0.05 according to Student t-test.
142
Chapter VI. Phytotoxic effects of BOA
(A)
(B)
Fig. 5 Effect of BOA (1.5 mM) on quantum efficiency of open PSII reaction centers in the dark-adapted state (Fv/Fm) (A) and quantum
yield of PSII (ΦPSII) electron transport (B) in lettuce leaves from days 1 to 6 after treatment with BOA. Every column in each graph represents
the mean (± S.E.) of three replicates. Asterisks indicate significant differences at level 0.05 with respect to control.
143
Physiological response of lettuce
(A)
(B)
Fig. 6 Effect of BOA (1.5 mM) on chlorophyll flourescence (qP) (A) and non-chlorophyll fluorescence quenching (NPQ) (B) in lettuce
leaves from days 1 to 6 after treatment with BOA. Every column in each graph represents the mean (± S.E.) of three replicates
Asterisks indicate significant differences at level 0.05 with respect to control.
144
Chapter VI. Phytotoxic effects of BOA
(A)
(B)
( C)
Fig. 7. Effect of BOA (1.5, 1.0, 0.5 and 0.1 mM) on leaf carbon (C%) (A), carbon isotope ratios (δ13C)
(B) and carbon isotopes discrimination (Δ 13C) (C) in lettuce after treatment with BOA.
Every column in each graph represents the mean (± S.E.) of three replicates.
Asterisks indicate significant differences at level 0.05 with respect to control.
145
Physiological response of lettuce
(A)
Fig. 8 Effect of BOA (0.1 -1.5 mM) on ratio of intercellular to air CO2 concentration (ci/ca) (A) and
intrensic water use efficiency (iWUE) (B) after treatment with BOA.
Every column in each graph represents the mean (± S.E.) of three replicates.
* Asterisks indicate significant differences at level 0.05 with respect to control at Student t-test.
146
(B)
Chapter VI. Phytotoxic effects of BOA
Fig. 9. Effect of BOA on protein contents (mg g-1)in Lactuca sativa leaves determined 6-days after
BOA treatment. Every column in each graph represents the mean (± S.E.) of three replicates.
*Asteriks indicates significant differences as compared to control for p < 0.05 according to Student t-test.
147
Physiological response of lettuce
148
Cinnamic acid inhibited photosynthetic
efficiency, yield and non-photochemical
fluorescence quenching in leaves of Lactuca
sativa L.
Chapter VII
Chapter section submitted as an original article to SCI journal:
Hussain, M.I., and Reigosa, M.J. 2010. Cinnamic acid inhibited photosynthetic
efficiency, yield and non-photochemical fluorescence quenching in leaves of Lactuca
sativa L. Environmental and Experimental Botany (under review).
Photosynthetic response of lettuce
Elsevier Editorial System(tm) for Environmental and Experimental Botany
Manuscript Number: EEB-D-10-00040
Title: Cinnamic acid inhibited photosynthetic efficiency, yield and non-photochemical
fluorescence quenching in leaves of Lactuca sativa L.
Article Type: Research Paper
Keywords: Allelopathy, Plant water relation, Cinnamic acid, Chlorophyll fluorescence,
Carbon isotope composition, Lettuce, growth
Corresponding Author: Dr. Iftikhar Hussain, PhD in Plant Physiology
Corresponding Author's Institution: University of Vigo, SPAIN
First Author: Iftikhar Hussain, PhD in Plant Physiology
Order of Authors: Iftikhar Hussain, PhD in Plant Physiology; Manuel Joaquin Reigosa,
PhD in Plant Physiology
Abstract: ABSTRACT
This study investigated the effects of cinnamic acid (CA) on biochemical, physiological
and isotopic responses in leaves of lettuce. The results show that 1.5, 1.0 and 0.5 mM
CA treatment decreased lettuce leaf/root fresh weight, shoot/root length, but it did not
affect the relative water content and leaf osmotic potential. CA treatment (1.5 mM)
significantly reduced Fm, Fv, photochemical efficiency of PSII (Fv/Fm), quantum yield of
PSII (ΦPSII), photochemical fluorescence quenching (qP) and non-photochemical
quenching (NPQ). These suggest that the decrease in these chlorophyll fluorescence
components was a result to damage in photosynthetic apparatus and is at least partially
attributed to lowered photochemical efficiency of PSII in lettuce leaves following
exposure to 1.5 mM CA. The F′v/F′m and fraction of photon energy absorbed by PS II
antennae trapped by ―open PS II‖ reaction centers (P) values was reduced by CA (1.5
mm) while, portion of absorbed photon energy thermally dissipated (D) and photon
energy absorbed by PSII antennae and trapped by ―closed‖ PSII reaction centers (E)
was increased. CA (1.5 mm) treatment did not affect nitrogen percentage (N %),
nitrogen isotope ratios (δ 15N) and carbon percentage (C %) in lettuce. Carbon isotope
ratios (δ13C) was less negative (-27.10) in CA (1.5 mM) treated plants as compared to
control (-27.61) and carbon isotope discrimination (Δ 13C) were also less (19.63) in CA
treated plants as compared to control (20.18). The leaf protein contents of lettuce were
significantly decreased when treated with CA (1.5 mM) treatment.
150
Chapter VII. Phytotoxic effects of Cinnamic acid
ABSTRACT
This study investigated the effects of cinnamic acid (CA) on biochemical, physiological and
isotopic responses in leaves of lettuce. The results show that 1.5, 1.0 and 0.5 mM CA treatment
decreased lettuce leaf/root fresh weight, shoot/root length, but it did not affect the relative water
content and leaf osmotic potential. CA treatment (1.5 mM) significantly reduced Fm, Fv,
photochemical efficiency of PSII (Fv/Fm), quantum yield of PSII (ΦPSII), photochemical
fluorescence quenching (qP) and non-photochemical quenching (NPQ). These suggest that the
decrease in these chlorophyll fluorescence components was a result to damage in photosynthetic
apparatus and is at least partially attributed to lowered photochemical efficiency of PSII in
lettuce leaves following exposure to 1.5 mM CA. The F′v/F′m and fraction of photon energy
absorbed by PS II antennae trapped by ―open PS II‖ reaction centers (P) values was reduced by
CA (1.5 mm) while, portion of absorbed photon energy thermally dissipated (D) and photon
energy absorbed by PSII antennae and trapped by ―closed‖ PSII reaction centers (E) was
increased. CA (1.5 mm) treatment did not affect nitrogen percentage (N %), nitrogen isotope
ratios (δ 15N) and carbon percentage (C %) in lettuce. Carbon isotope ratios (δ13C) was less
negative (-27.10) in CA (1.5 mM) treated plants as compared to control (-27.61) and carbon
isotope discrimination (Δ 13C) were also less (19.63) in CA treated plants as compared to
control (20.18). The leaf protein contents of lettuce were significantly decreased when treated
with CA (1.5 mM) treatment.
Keywords: Allelopathy, Plant water relation, Cinnamic acid, Chlorophyll fluorescence, Carbon
isotope composition, Lettuce, growth
Abbreviations: RWC - relative water content, LOP - leaf osmotic potential, Fo - Initial
fluorescence from dark adapted leaves, Fm-maximal fluorescence from dark adapted leaves; Fvvariable fluorescence from dark-adapted leaves, F′m - maximal fluorescence from leaves in light,
F′0 - minimal fluorescence, F′v - variable fluorescence of leaves in light, Fv/Fm- quantum
efficiency of PSII photochemistry; qP- photochemical quenching; NPQ: non-photochemical
quenching; PSII- photosystem II; N% - Nitrogen concentration; δ15N -Nitrogen isotope
composition, C%- carbon concentration; δ13C- carbon isotope composition, Δ13C -Carbon
isotope discrimination, ci/ca - ratio of leaf intercellular CO2 concentration to that in air, iWUE –
intrinsic water use efficiency.
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Photosynthetic response of lettuce
1. Introduction
Plants synthesize an array of chemicals that are involved in a variety of plan-plant, plantmicrobe, and plant-herbivore interactions (Inderjit and Duke, 2003; Weir et al., 2004).
Allelochemicals that are responsible for plant-plant allelopathy are delivered into the
rhizosphere mainly through volatilization, leaching, decomposition of residues, root exudates
and mediate plant to plant interactions (Hadacek, 2002). The allelopathic interactions have been
proposed to have profound effects on the evolution of plant communities through the loss of
susceptible species via chemical interference and by imposing selective pressure favoring
individuals resistant to inhibition from a given allelochemical (Baerson et al., 2005).
Investigation of the chemical composition of allelopathic plants reveals that a great number of
secondary metabolites serve as allelochemicals. Among them, benzoic and cinnamic acid
derivatives are frequently identified from soils or root exudates (Yu and Matsui, 1994; Blum,
1996; Inderjit and Duke, 2003). Root growth inhibition by benzoic and cinnamic acids has been
widely observed (Chon et al., 2002; Iqbal et al., 2004; Hiradate et al., 2005; Rudrappa et
al.,2007; Batish et al., 2008). Furthermore, exogenous addition of allelochemicals can influence
physiological and biochemical reactions, such as photosynthesis, respiration, water and nutrient
uptake, and generation of reactive oxygen species (Ding et al., 2007), as well as induce changes
in gene expression (Golisz et al., 2008). All such physiological, biochemical, and transcriptional
changes are implicated directly or indirectly in plant growth inhibition.
Cinnamic acid (CA) is widely used in food, cosmetics, and pharmaceuticals because of
its reported antimicrobial, antifungal, and antioxidant properties (Chafer et al., 2009). CA is a
widespread phenolic acid released into soil by root exudates, leaf leachates, and decomposed
plant tissues of different plants e.g. quack grass (Elytrigia repens) (Baziramakenga et al., 1994
), cucumber (Yu and Matsui, 1994), alfalfa (Chon et al., 2002). The CA, and o-,m-, and pcoumaric acids inhibited the growth of etiolated seedlings of lettuce at concentrations higher
than 10–4 M and seed germination above 10–3 M (Li et al., 1993). Baziramakenga et al. (1994)
concluded that benzoic acid and trans-cinnamic acid were responsible for negative allelopathic
effects of quack grass on soybean by inhibiting root growth, reducing the root and shoot dry
mass, by altering ion uptake and transport, and by reducing chlorophyll content.
Although the mechanisms by which physiological and metabolic processes are affected
by allelochemical stress in higher plants are not fully understood, the photosynthetic process is
often the first to be inhibited by allelochemical stress (Hejl and Koster, 2004). In the
photosynthetic apparatus, light is absorbed by antenna pigments and excitation energy is
transferred to the reaction centers of the two photosystem (PSI and PSII). There, the energy
drives the primary photochemical reactions that initiate the photosynthetic energy conversion.
Under optimal conditions, the primary photochemistry occurs with high efficiency and the
dissipation of absorbed light energy by chlorophyll fluorescence is low and mainly emitted by
PSII associated chlorophyll molecules (Krause and Weis, 1991; Lichtenthaler, 1996). This
property of green plants is known to provide a simple and powerful non-intrusive means to
determine the health state of plants (Lichtenthaler, 1988). In toxicity investigations, the
saturation pulse method using pulse-amplitude-modulate (PAM) fluorometer is commonly used
152
Chapter VII. Phytotoxic effects of Cinnamic acid
to study photosynthesis and is able to provide different fluorescence parameters giving reliable
information of the effect of biotic and abiotic stress on plant physiology (Schreiber et al., 1994;
Juneau et al., 2002). Among these parameters, the maximum quantum efficiency of PSII
primary photochemistry (Fv/Fm) and, photochemical and non-photochemical quenching (qP and
NPQ) are very useful for laboratory and field studies (Conrad et al., 1993; Rascher et al., 2000).
The two types of quenching occur in chloroplast: the photochemical quenching (qP) directly
dependent to the electron transport, and the non-photochemical quenching (NPQ) representing
all non-radiative mechanisms involved in dissipation of excess absorbed light energy. NPQ is
composed of three different components according to their relaxation kinetics in darkness
following a period of illumination, as well as their response to specific inhibitors (Muller et al.,
2001).
Our laboratory is focusing research mainly on physiological mode of action of different
allelochemicals (Sánchez-Moreiras and Reigosa, 2005; Reigosa and Pazos-Malvido, 2007;
Sánchez-Moreiras et al., 2008; Sánchez-Moreiras et al., 2009). Previously we have
demonstrated that different allelochemicals including cinnamic acid significantly affect seed
germination and seedling growth of Lactuca sativa, Lolium perenne, Dactylis glomerata and
Rumex acetosa (Hussain et al., 2008), and it was subjected to further study. Since cinnamic acid
appeared to be a more potent inhibitor when used in combination with other allelochemicals, the
current study was performed. Here we report on plant growth inhibitory activity of cinnamic
acid, its interference with components of chlorophyll fluorescence in a general biotest specie
Lactuca sativa and speculate on the mechanism of this inhibition. Acquisition of such
knowledge may ultimately provide a rational and scientific basis for the design of safe and
effective herbicides.
2. Materials and methods
2.1. Growth Bioassays
Lettuce (Lactuca sativa L. cv. ―Great Lakes California‖) was used for this study. The
seeds were placed in plastic trays (32 x 20 x 6 cm) with a 5 cm deep layer of perlite (500 g/tray).
The trays were irrigated on alternate days with tap water until germination of seeds and
thereafter with 500 ml 1:1 Hoagland solution/tray, twice in a week. Seedlings were germinated
in darkened at 20 0C temperatures in environmentally controlled growth chamber. For seedling
growth, the environmental conditions were as follows; temperature: 18/8 0C (day/night) and
12/12 h (light/darkness) photoperiod, 80% relative humidity and 200 mol m–2 sec-1 irradiance.
One month old seedlings (when plants have three fully expanded leaves), were transferred to
pots (10 cm) containing perlite (70 g) to stimulate the development of root system and shifted
to the glass house with same growing condition and nutrient solution (100 ml/pot). The
cinnamic acid (CA), dissolved in methanol: water (20:80), methanol was evaporated in a rotary
vaporizer and solution was diluted to 1.5, 1.0, 0.5, 0.1 mM. The pH of these solutions was
adjusted to 6.0 with NaOH (Martin et al., 2002). When the lettuce seedlings were at three leaf
stage, CA was watered three times (days 1, 3 and 5) with 100 ml aqueous solution of four
concentration. The experiment was laid out in Randomized Complete Block Design (RCBD)
and replicated thrice.
153
Photosynthetic response of lettuce
2.2. Chlorophyll fluorescence measurement
Chlorophyll fluorescence was measured on fully expanded exposed leaves (one leaf per
plant) using a pulse-modulated instrument fluorescence monitoring system (FMS) (Hansatech,
Norfolk, England) by the method of Weiss and Reigosa (2001). The parameters of chlorophyll
fluorescence: initial fluorescence (F0), maximum fluorescence (Fm), variable fluorescence Fv
(Fm-F0), were measured after 20 min in the dark adopted leaves using Walz leaf clips. The
intensity of saturation pulses to determine the maximum fluorescence emission in the presence
(F′m) and absence (Fm) of quenching was 1800 µmol m-2 s-1 for 3 second. The steady state
fluorescence (Fs), basic fluorescence after light induction (F′0), maximum chlorophyll
fluorescence (F′m), variable fluorescence from light adopted leaves (F′v) and F (= Fm-Fs) were
also evaluated by attaching the optic fiber to a leaf clip holder. The chlorophyll fluorescence
ratios (Fv/Fm) was considered a useful measurement of photosynthetic performance of plants and
as stress indicators. Effective quantum yield of photosystem II (Ф PSII), photochemical
quenching (qP) and non- photochemical quenching (NPQ) were also recorded (Genty et al.
1989; Van Kooten and Snel 1990). The fraction of photon energy absorbed by PSII antennae
that was traped by open PSII reaction centers (centers with QA in the oxidized state) and utilized
in PSII photochemistry was estimated as P = F′v/F′m x qP. The portion of absorbed photon
energy that was thermally dissipated was calculated as the difference between the maximal
theoretical and actual levels of PSII efficiency (D = 1 – F′v/F′m). The fraction of absorbed
radiation not attributed to P or D (denoted as ―excess‖, E) and representing the photon energy
absorbed by PSII antennae and trapped by ―closed‖ PSII reaction centers was estimated using E
= 1- (D + P) = F′v/F′m x (1 – qP) (Demmig-Adams et al., 1996).
2.3. Plant growth measurements
Plant height/ root length was obtained with a ruler and values were expressed in cm.
The fresh and oven dry plant leaves and roots weight were obtained by first weighting
independently fresh leaves and roots then after drying these samples in a circulatory air oven at
70 0C for 72 h. The samples were weighed again to get dry weight of plant.
2.4. Plant leaf water relations
Relative Water Content (RWC) was calculated by measuring fresh (Wf), Saturated (Wt)
and Dry (Wd) weights of plant (Reigosa and Gonzalez, 2001). Leaf plant pieces from three
replicates per treatment were weighed in fresh, saturates at 4 oC for 3 hours, weighed again,
oven dried at 70 oC for 72 h, and finally weighed once more for obtaining three necessary
weights required for the following equation:
RWC = Wf – Wd/ Wt – Wd x 100
For osmolality measurements (milliosmol kg-1) one leaf per replicate was transferred
into 10 ml syringes and kept frozen at -80 0C until further analysis (González, 2001). In analysis
syringes were thawed until sample reached at room temperature and osmotic potential was
measured on the second drop from collected sap by using a calibrated vapor pressure
154
Chapter VII. Phytotoxic effects of Cinnamic acid
Osmometer (Automatic Cryoscopic Osmometer, Osmomat – 030, GmbH, Gonatec, Berlin,
Germany) according to manufacturer‘s instructions.
2.5. Protein Content determination
Total protein was quantified by using the Spectrophotometric Bradford assay (1976).
Lettuce leaves (200 mg fresh weight) from three replicates per treatment were crushed in liquid
nitrogen in cooled mortar with 0.05 g PVPP, and the powder was resuspended in 1 mL of Tris
buffer containing 0.05 M Tris base, 0.1% w/v ascorbic acid, 0.1% w/v cysteine hydrochloride,
1% w/v PEG 4000, 0.15% w/v citric acid, and 0.008% v/v 2-mercaptoethanol (Arulsekar and
Parfitt, 1986). Samples were centrifuged at 19000 gn and 4 ºC for 20 min after resuspension. 0.1
mL supernatant was mixed for protein dye-binding reaction with 3 mL Bradford reactive
containing 0.01% Coomassie® Brilliant Blue G-250, 4.7% v/v ethanol 95%, and 8.5% v/v
phosphoric acid 85%. Absorbance was recorded at 595 nm after 5 min for quantification of the
protein content. Commercial bovine seroalbumin (BSA) was used as standard. Values were
expressed per gram of dry weight.
2.6. Carbon and nitrogen isotope discrimination analysis
Lettuce leaf samples were dried at 70 °C for 72 h in a forced-air oven (Gallenkamp
oven, Loughborough, Leicestershire, UK) to constant weight and ground in Ball Mills (Retsch
MM 2000, Haan, Germany). Dry ground plant material was weighed (1700-2100 µg) with
weighing meter (Metler Toledo GmbH: Greifensee Switzerland), filled in tin capsules (5x3.5
mm, Elemental Microanalysis Limited, U.K.). Each tin capsule was entered automatically in
combustion oven at 1600-1800 °C in the presence of oxygen and converted into CO2 and N2.
Subsequently isotope ratios were determined in an Isotopic Ratio Mass Spectrometer (Finnegan:
Thermo Fisher Scientific, model MAT-253, Swerte Germany) coupled with an Elemental
Analyzer (Flash EA-1112, Swerte Germany). The isotopic ratio mass spectrometer has an
analytical precision better than 0.05‰ for 15N and 0.3‰ for 13C.
Carbon and nitrogen isotope compositions were calculated as;
δ (‰) = [(Rsample/Rstandard) —1)] x 1000
(1)
where Rsample is the ratio of 13C/12C or 15N/14N, and Rstandard are standards used. Atmospheric N2 is
the standard for nitrogen while Vienna PeeDee Belemnite (VPDB) is standard for carbon. The
standard carbon and nitrogen R equal 1.1237 x10-2 and 3.6764 x 10-3, respectfully. Pure CO2
(δ13C = -28.2 ± 0.1‰) and N2 (δ15N = - 2.1 ± 0.1‰) gases calibrated against standard CO2 (10.38‰) and N2 (-0.22‰) served as reference gases for δ13C and δ15N, respectively. The
accuracy and reproducibility of measurements of δ13C and δ15N was checked with an internal
reference material (NBS 18 and IAEA-C6 for C), and (IAEA-310A and IAEA-N1 for N), and
Acetanilide for C/N %age ratios, respectively.
Carbon isotope discrimination is a measure of the C isotopic composition in plant
material relative to the value of the same ratio in the air on which plants feed:
155
Photosynthetic response of lettuce
Δ (‰) = [(δa – δp)/(1 + δp)] x 1000
(2)
where Δ represents carbon isotope discrimination, δa represents C isotope composition in the
source air, and δp represents C isotope composition in the plant tissue. Theory published by
Farquhar et al. (1989) and Farquhar and Richards (1984) indicates that C discrimination in
leaves of plants can be expressed in relationship to CO2 concentrations inside and outside as:
Δ = a + (b - a) ci/ca
(3)
Δ = 4.4 + (27 - 4.4) ci/ca
where a is discrimination that occurs during diffusion of CO2 through the stomata (4.4‰), b is
discrimination by rubisco (27‰), and ci/ca is the ratio of the leaf intercellular CO2
concentration to that in the atmosphere. Equation [3] shows a direct and linear relationship
between Δ and ci/ca. Therefore, measurement of Δ gives an estimation of the assimilation-rate–
weighted value of ci/ca. A lower ci/ca ratio may result either from higher rates of photosynthetic
activity or from stomatal closure induced by stress.
Intrinsic water use efficiency (iWUE) was calculated according to the following
equations (McCarroll and Loader 2004):
iWUE = A/g = ca [1-(ci/ca)] x (0.625)
(4)
where A is the rate of CO2 assimilation and g is the stomatal conductance. Data of
δ Cair, ci/ca were obtained from McCarroll and Loader (2004) whom used the high precision
records of atmospheric Δ13C from Antarctic ice cores (Francey et al. 1999), and the atmospheric
CO2 concentration (ppm) from Robertson et al. (2001). Carbon and nitrogen isotope
measurements were performed in CACATI (Centro de Apoio Cientifico Tecnologico a la
Investigacion), University of Vigo, Spain.
13
2.7. Statistical analysis
Data were analysed with the statistical package SPSS® (version 15.00) for Windows®
(SPSS Inc., Chicago, IL, USA). The data were analyzed with a one-way analysis of variance
and differences between treatments were separated by the Student‘s t-test at the P <0.05 level.
3.0. Results
3.1. Plant growth
The inhibitory effect of CA on lettuce growth is concentration-dependent. Exposure to
1.5, 1.0 and 0.5 mM CA resulted in a significant inhibition on shoot/root growth and leaf
fresh/root fresh weight in lettuce. All concentration of CA decreased fresh biomass of the shoot
and root. CA, however, had little effect on the leaf and root dry weight of lettuce, even at the
highest concentration (Table 1).
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Chapter VII. Phytotoxic effects of Cinnamic acid
3.2. Effect on Plant leaf water relations
Non significant differences were observed in leaf relative water content as compared to
control at all four concentration of TCA; however, RWC was considerably less at highest
concentration of TCA as compared to control (Fig. 1 A). There was non-significant difference in
leaf osmotic potential in lettuce among control and TCA treatments and there was a tendency of
increase in LOP at lowest concentration (0.1 mM) of TCA (Figure 1 B).
3.3. Chlorophyll fluorescence measurements
The inhibitory effect of CA (1.5 mM) in lettuce was evaluated on the basis of six days
treatment by analyzing several fluorescence parameters determined under dark-adapted and
steady state conditions. After exposure to CA the Maximum (F m) and variable fluorescence (Fv)
was significantly reduced in lettuce on first, second, third and fourth day (Figure 2 B, 2 D).
Quantum efficiency of open PSII reaction centers in the dark-adapted state (Fv/Fm) was
significantly decreased on second, fourth and fifth day as due to CA (Figure 2 C). A significant
reduction in steady state fluorescence (Fs) in lettuce exposed to CA was observed on second,
third, fourth and fifth day while there was a tendency of increase in Fs on sixth day (Figure 3 A).
The reduction in maximum fluorescence in light adopted leaves (F′m) was observed in
all days except on first day and sixth day (Figure 3 B). Furthermore, CA significantly reduced
quantum yield of PSII (ΦPSII) in all days except day first (Figure 3 C). The ΔF was also
significantly reduced by CA on second, third, fourth and fifth day (Figure 3 D). The variable
fluorescence (F′v) in light state was significantly decreased on second, third, fourth and fifth day
(Figure 4 B). A significant decrease in photochemical fluorescence quenching (qP) was
observed on third, fourth, fifth and sixth day (Figure 4 C). However, a contrary trend was found
in the changes of nonphotochemical fluorescence quenching (NPQ), which increased on first
day and then gradually decreased in next five days. The efficiency of excitation by open
reaction centers (F′v/F′m) was significantly reduced by CA on second, third, fourth and fifth day
(Figure 5 A). Additionally, figure 5 B shows significantly gradual decrease in fractions of light
absorbed by PSII allocated to PSII photochemistry (P) values in lettuce. The portion of
absorbed photon energy that was thermally dissipated (difference between the maximal
theoretical and actual levels of PSII efficiency, D) and photon energy absorbed by PSII
antennae and trapped by ―closed‖ PSII reaction centers (E) was significantly increased after CA
exposure (Figure 5 C and 5 D).
3.4. Carbon ad nitrogen isotope ratios
Up to certain extent the CA (1.5, 1.0 mM) increased the leaf nitrogen concentration (N
%) in lettuce while CA did not significantly affect nitrogen isotope ratios (δ15N) at any
concentration (data not shown). CA did not significantly effect carbon concentration (%) in
lettuce leaves while carbon isotope ratios (δ13C) was less negative (-27.10) at 1.5 mM CA as
compared to control (-27.61) (Figure 6). Carbon isotope discrimination (Δ 13C) were less
(19.63) in CA (1.5 mM) treated plants as compared to control (20.18). However, there was nonsignificant difference regarding ratio of intercellular leaf to atmospheric CO 2 concentration
(ci/ca) and intrinsic water use efficiency (iWUE) (Figure 7 A and 7 B).
157
Photosynthetic response of lettuce
3.5. Protein contents
The CA (1.5 mM) significantly decreased leaf protein contents in lettuce (Figure 8).
4. Discussion
4.1. Plant growth
Allelochemicals appear to alter a variety of physiological processes in target plant
species and it is difficult to separate the primary from secondary effects. Some have been
explored as natural substitutes for commercial herbicides (Duke et al., 2000; 2005). A number
of studies have indicated that phenolic acids, including CA, affect membrane permeability and
thus affect plant growth (Doblinski et al., 2003; Ju et al., 2007). Baziramakenga et al. (1995)
demonstrated that benzoic and cinnamic acids induced reactive oxygen species generation,
which severely affected the integrity of the membrane. Doblinski et al. (2003) reported that pcoumaric and p-hydroxybenzoic acids (0.5–1.0 mM) caused lipid peroxidation in soybean roots,
resulting in growth inhibition. Cinnamic acid (0.5–0.25 mM) has recently been shown to cause
membrane peroxidation, increase H2O2 content and induces oxidative stress in cucumber roots
(Ju et al., 2007). Our observations are in agreement with earlier reports of the ability of
phytotoxins to inhibit root growth (Batish et al., 2006; Sánchez-Moreiras et al., 2005).
Baziramakenga et al. (1995) reported the effects of benzoic and cinnamic acids on the cell
plasma membrane in intact soybean (Glycine max L. cv. Maple Bell) seedlings. Treated intact
root systems rapidly increased electrolyte leakage and ultraviolet absorption of materials into
the surrounding solution. The two allelochemicals induced lipid peroxidation, which resulted
from free radical formation in plasma membranes, inhibition of catalase and peroxidase
activities, and sulfhydryl group depletion. Oxidation or cross-linking of plasma membrane
sulfhydryl groups is the first mode of action of both compounds. Fujita and Kubo, (2003)
reported that trans-cinnamic acid inhibited the root elongation of lettuce by 50% at 8.1 µM. Ju
et al. (2007) reported that CA significantly inhibited the growth of cucumber but did not inhibit
the growth of figleaf gourd.
4.2. Effect on plant leaf water relations
RWC and LOP did not significantly affected by CA at any concentration. However,
Sánchez-Moreiras and Reigosa, (2005) reported that a 10% decrease in relative water content
which was correlated with a decline in leaf water potential in BOA-exposed lettuce plants.
4.3. Chlorophyll fluorescence measurements
Several action modes of allelochemicals have been suggested, including direct
inhibition of PSII components and ion uptake, interruption of dark respiration, and ATP
synthesis and ROS-mediated allelopathic mechanisms (Inderjit and Duke, 2003; Belz and Hurle
2004).). Furthermore, chlorophyll fluorescence is a useful measurement to study several aspects
of photosynthesis because it reflects changes in thylakoid membrane organization and function
and inhibition of photosynthesis and oxygen evolution through interactions with components of
photosystem II (Einhelling 1995; Rimando et al. 1998). In the present study, CA induced strong
inhibition of efficiency of open PSII reaction centers (Fv/Fm). Damage to the reaction centers
158
Chapter VII. Phytotoxic effects of Cinnamic acid
embedded in thylakoid membranes, especially those of PSII, and inhibition of inductive
resonance energy transfers from antenna molecules to reaction centers can lead to lower F v/Fm
ratios (Krause and Wies, 1984). A sustained decrease of the Fv/Fm may indicate the occurrence
of photoinhibitory damage (Colom and Vazzana 2003). Ye et al. (2004) reported a decline in
Fv/Fm, ΦPSII after CA exposure in Cucumis sativus. Similarly, Zhou andYu, (2006), concluded
that allelochemicals can significantly affect the performance of the three main processes of
photosynthesis: stomatal control of CO2 supply, thylakoid electron transport (light reaction), and
carbon reduction cycle (dark reaction). The reduction in maximum fluorescence in light
adopted leaves (F′m) was observed. Furthermore, our results clearly show that CA significantly
inhibits the quantum yield of PSII electron transport (ΦPSII) in lettuce seedlings. We also
demonstrate that such a decrease in ΦPSII was associated to the alteration of qP and NPQ.
However, Huang and Bie, 2009 reported that 0.1mM cinnamic acid treatment decreased
photosynthetic rate (Pn) and RuBPC activity, but it did not affect the maximal photochemical
efficiency of PSII (Fv/Fm),the actual photochemical efficiency of PSII (ΦPSII),intercellular CO2
concentration (Ci), and relative chlorophyll content. A decrease in qP induced by CA indicates a
higher proportion of closed PSII reaction centers (Genty et al., 1989), which probably generates
a decrease in the proportion of available excitation energy used for photochemistry.
The significant changes in PSII photochemistry in the light adapted leaves, such as the
decreased qP and F′v/F′m, as well as the increased of NPQ, can be seen as the regulatory
response to down-regulate the quantum yield of PSII electron transport (ΦPSII) (Genty et al.,
1989), that would match with the high decrease of CO2 assimilation rate. It is suggested that the
decay in ΦPSII of the CA stressed plants may be a mechanism to down-regulate the
photosynthetic electron transport so that production of ATP and NADPH would be in
equilibrium with the decreased CO2 assimilation capacity in stressed plants. The fraction of PSII
reaction centers that is opened, as indicated by the decrease of qP, is reduced in lettuce plant
cultivated with nutrient solutions added with 1.5 mM CA.
In the present study, a gradual decrease in fractions of light absorbed by PSII allocated
to PSII photochemistry (P) values was observed. The portion of absorbed photon energy that
was thermally dissipated (D) and photon energy absorbed by PSII antennae and trapped by
―closed‖ PSII reaction centers (E) was significantly increased after CA exposure. It has been
shown in many studies that an increase in the thermal dissipation in the PSII antennae competes
with the excitation energy transfer from the PSII antennae to PSII reactions centers, thus
resulting in a decrease in the efficiency of excitation energy captured by open PSII reaction
centers F′v/F′m (Demmig-Adams et al., 1996).
3.5. Carbon and nitrogen isotope discrimination
Much less work has been done on relationship between stress and 15N in the plant,
mostly because the observed variation was often attributed to a shift in source 15N, rather than
discrimination in the plant (Nadelhoffer and Fry 1994; Högberg 1997). In the present study, CA
increased the leaf nitrogen concentration (N %) in lettuce and CA did not significantly affect
nitrogen isotope ratios (δ15N). However, Högberg, (1997) reported that foliar 15N discrimination
has been observed in plants under cold stress and in slow growing plants. Plant photosynthesis
159
Photosynthetic response of lettuce
discriminates against the stable 13C isotope (Farquhar et al., 1989) when atmospheric CO2
passes through stomata and during CO2 carboxylation in RuBisCo. 13C discrimination decreases
with a decrease in intercellular CO2 concentration due to stomatal closure, and consequently
with water use efficiency. Our results revealed that CA did not significantly effect carbon
concentration (%) while carbon isotope ratios (δ13C) was less negative at 1.5 mM CA. Carbon
isotope discrimination (Δ 13C) were less (19.63) in CA treated plants as compared to control
(20.18). The carbon isotope fractionation patterns indicates that limitations to the diffusion of
CO2 through the stomatal aperture can result in less negative δ13C values (O‘Leary, 1988). Our
data suggest that stomata‘s of lettuce plants treated with 1.5 mM TCA experienced some degree
of closure, which ultimately leads to less discrimination against the heavier isotope. Previously,
Barkosky and Einhellig (2003), reported that carbon isotope ratio (δ 13C) in leaf tissue of
soybean at the termination of the 28-day experiment showed significantly less discrimination
(less negative δ 13C) against 13C in plants grown with 0.75 mM p-hydroxybenzoic acid. The δ
13
C value of Lactuca sativa was less negative at the higher pollutant level than at the lower level
and was statistically significantly different (P < 0.05) from that of control (Martin et al., 1988).
3.6. Protein contents
Different types of biotic and abiotic stress can cause degradation of proteins by
senescence or by reduction in protein synthesis. Baziramakenga et al. (1997), reported that
many phenolic acids reduced the incorporation of certain amino acid into proteins and thus
reduce the rate of protein synthesis. In the present research, CA also decreased leaf protein
contents in lettuce at 1.5 mM concentration. Similarly, Mersie and Singh (1993), also reported
the degradation and reduction in total protein contents after the application of p-hydroxybenzoic
acid. The reduction in total soluble leaf proteins in BOA-exposed lettuce plants were also
reported by Sanchez-Moreiras and Reigosa (2005).
In conclusion, our results show that L. sativa plants are able to grow in CA amended
perlite culture but show a reduction in root/shoot length and a decrease in leaf and root fresh
weight. These are associated with a growth restriction induced by the unbalanced nutrient
uptake and a decreased upward translocation. We observed that CA had a significant effect on
the primary photochemistry of PSII and that photoinhibition occurred in leaves from 1.5 mM. A
modification in the PSII photochemistry apparatus leads to decreased requirements for ATP and
NADPH due to a decreased CO2 assimilation capacity induced by CA stress. These results
suggest that CA stress in plants is associated with the high accumulation of inactivated PSII
reaction centers and the higher fraction of reduction state of plastoquinone in plants.
Acknowledgements
We thank Dr. Aldo Barreiro, Carlos Bolano, Alfredo Justo, Maite Ricart, and P. Lorenzo with
field and laboratory assistance. We are also grateful to Jesús Estévez Sío and Jorge Millos for
their technical assistance with isotope ratio mass spectroscopy. We also thank Dr. Nuria Pedrol
for valuable help in leaf osmotic potential measurement and in statistical analysis.
160
Chapter VII. Phytotoxic effects of Cinnamic acid
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response and microtubule assembly disruption to execute rhizotoxicity. J. Chem. Ecol. 33,
1898–1918.
Sánchez-Moreiras, A. M., Reigosa, M.L., 2005. Whole plant response of lettuce after root exposure to
BOA (2(3H)-Benzoxazolinone). J. Chem. Ecol. 31, 2689-2703.
Sánchez-Moreiras, A.M., De La Peña, T.C., Reigosa, M. J., 2008. The natural compound benzoxazolin2(3H)-one selectively retards cell cycle in lettuce root meristems. Phytochemistry. 69, 2172–
2179.
Sánchez –Moreiras, A.M., Pedrol, N., González, L., Reigosa, M. J., 2009. 2-3H-Benzoxazolinone (BOA)
induces loss of salt tolerance in salt-adapted plants. Plant Biol. 11, 582-590.
Schreiber, U., Bilger, W., Neubauer, C., 1994. Chlorophyll fluorescence as a nonintrusive indicator for
rapid assessment of in vivo photosynthesis. In: Schulze, E.D., Caldwell, M.M. (Eds.),
Ecophysiology of Photosynthesis. Ecological Studies, vol. 3. Springer, Berlin, pp. 49-70.
Van Kooten, O., Snel, F.H., 1990. The use of chlorophyll fluorescence nomenclature in plant stress
physiology. Photosynth. Res. 25, 147–150.
Weiss, O., Reigosa, M. J., 2001. Modulated fluorescence, pp. 173-183, in M. J. Reigosa (ed.). Handbook
of Plant Ecophysiology Techniques. Kluwer Academic Publishers, Dordrecht.
Weir, T. L., Park, S.-W., Vivanco, J. M., 2004. Biochemical and physiological mechanisms mediated by
allelochemicals . Curr. Opin. Plant Biol. 7, 472- 479.
Ye, S.F.,Yu, J.Q., Peng, Y.H., Zheng, J.H., Zou, L.Y., 2004. Incidence of Fusarium wilt in Cucumis
sativus L. is promoted by cinnamic acid, an autotoxin in root exudates. Plant Soil. 263, 143–
150.
Yu, J.Q. Matsui, Y., 1994. Phytotoxic substances in the root exudates of cucumber (Cucumis sativus L.).
J. Chem. Ecol. 20, 21–31.
Zhou, Y.H., Yu, J.Q., 2006. Allelopathy and photosynthesis. In: Reigosa MJ, Pedrol N, González L,
editors. Allelopathy: a physiological process with ecological implications. Netherlands:
Springer, p. 127–139.
163
Photosynthetic response of lettuce
Table 1
Effect of different concentration of trans-cinnamic acid on lettuce shoot and root growth.
Growth characteristics
trans-cinnamic acid (mM)
Control
0.1
0.5
1.5
Leaf fresh weight (g)
10.58±0.60
7.29 ±1.00*
6.87 ±1.41*
6.03 ± 0.78*
6.18±1.03*
Leaf dry weight (g)
1.63±0.15
1.80± 0.20
1.39 ±0.32
1.12 ±0.38
1.04±0.28
Root fresh weight (g)
6.31±0.34
3.36 ±0.49*
3.27± 0.69*
3.20± 0.96*
2.95±0.40*
Root dry weight (g)
0.40±0.042
0.30 ±0.02
0.38 ±0.08
0.30 ±0.06
0.29±0.058
Shoot length (cm)
15.30±0.23
8.63 ±0.43*
8.60 ±0.70*
7.17± 0.52*
6.06±1.24*
Root lemgth (cm)
27.77±0.23
21.67± 2.20*
18.50± 1.25*
18.00± 0.57*
17.33±0.60*
Each value represents the mean (± S.E.) of three replicates.
*Significant differences as compared to control for p < 0.05 according to Student's t-test.
164
1.0
Chapter VII. Phytotoxic effects of Cinnamic acid
Fig. 1. Leaf relative water contents (%) (A) and leaf osmotic potential (mmol kg-1) (B) of
L. sativa following one week exposure to different concentrations (0, 0.1, 0.5, 1.0, 1.5 mM) of cinnamic acid (CA).
Every column in each graph represents the mean (± S.E.) of three replicates. Asterisks indicate significant differences at level 0.05
with respect to control.
165
Photosynthetic response of lettuce
(A)
(B)
(D)
(C)
Fig. 2 Changes in chlorophyll fluorescence parameters (A) Fo, (B) Fm, ( C ) Fv/Fm and (D) Fv in L. sativa leaves from days 1 to 6 following exposure to
cinnamic acid (CA: 1.5 mM) concentration. Every column in each graph represents the mean (± S.E.) of three replicates. Asterisks indicate significant
differences at level 0.05 with respect to control.
166
Chapter VII. Phytotoxic effects of Cinnamic acid
(B)
(A)
(C)
(D)
Fig. 3 Changes in chlorophyll fluorescence parameters (A)Fs, (B)Fm', (C)psII, and (D)ΔF in L. sativa
leaves from days 1 to 6 following esposure to cinnamic acid (CA: 1.5 mM) concentration.
Every column in each graph represents the mean (± S.E.) of three replicates.
Asterisks indicate significant differences at level 0.05 with respect to control.
167
Photosynthetic response of lettuce
(B)
(A)
(C )
(D)
Fig. 4 Changes in chlorophyll fluorescence parameters (A) Fo', (B) Fv', (C ) qP and (D) NPQ in L. sativa
leaves from days 1 to 6 following exposure to cinnamic acid (CA: 1.5 mM) concentration.
Every column in each graph represents the mean (± S.E.) of three replicates.
Asterisks indicate significant differences at level 0.05 with respect to control.
168
Chapter VII. Phytotoxic effects of Cinnamic acid
(A)
(B)
(C )
(D)
Fig. 5 Changes in chlorophyll fluorescence parameters (A) Fv'/Fm', (B) P, (C ) D and (D) E in L. sativa
leaves from days 1 to 6 following exposure to cinnamic acid (CA: 1.5 mM) concentration.
Every column in each graph represents the mean (± S.E.) of three replicates.
Asterisks indicate significant differences at level 0.05 with respect to control.
169
Photosynthetic response of lettuce
(C )
(B)
(A)
Fig. 6 Changes in leaf C% (A), δ13C (B), Δ13C ‰ (C ) of L. sativa following expopsure to
different concentration of cinnamic acid (CA).
Every column in each graph represents the mean value ± S.E. from three replicates.
Asterisks indicate significant differences at level 0.05 with respect to control.
170
Chapter VII. Phytotoxic effects of Cinnamic acid
(A)
(B)
Figure 7. Ratio of intercellular leaf to atmospheric CO2 concentration (ci/ca) (A) and intrinsic water use efficiency
(iWUE) (B) in lettuce following exposure to four concentrations (0.1, 0.5, 1.0, 1.5 mM) of cinnamic acid (CA).
Every column in each graph represents the mean (± S.E.) of three replicates.
Fig. 8. Protein contents in lettuce leaves after exposure to cinnamic acid (CA: 1.5 mM).
Every column in each graph represents the mean (± S.E.) of three replicates.
Asterisks indicate significant differences at level 0.05 with respect to control.
171
Photosynthetic response of lettuce
172
GENERAL DISCUSSION
Chapter VIII
General discussion
GENERAL DISCUSSION
The increased connectivity of the global human population has amplified the frequency and
effect of biological invasions and disease outbreaks. In addition, land-use and climate change
interact with human transportation networks to facilitate the spread of invasive species, vectors,
and pathogens from local to continental scales (Dukes and Mooney 1999; Sakai et al. 2001;
Benning et al. 2002; Patz et al. 2004; Smith et al. 2007). However, only a few introduced
species succeed in establishing in the recipient community (Holdgate 1986; Parker and Reichard
1998). Thus, invasive success of alien species depends on the biotic and environmental
characteristics of the recipient community as well as on biological attributes related to its
potential to colonize and expand (Lonsdale 1999). Many exotic plant species have
characteristics that seem to make them successful invaders such as a large production of viable
seeds which disperse widely, the ability to germinate and grow in a broad range of
environmental conditions, and being a good competitor (Baker 1965; Noble 1989; Roy 1990;
Gordon 1998). When an alien plant is introduced, competition for limited resources is one of the
first interactions the species has with the recipient community (Vilà and Weiner 2004). Field
observations and experiments have proved that the threat alien species pose to the persistence of
native species is usually driven by the competition effect of the alien species on natives (Parker
and Reichard 1998; Levine et al. 2003). Identifying mechanisms of invasion and impacts of
invasive plants in natural areas is important in order to determine their effect on biodiversity, to
predict rates of spread, and to inform control and restoration efforts. One mechanism of
invasion that may be employed by some invasive plants, and that has received renewed attention
in the literature in general, is allelopathy (Bais et al. 2003; Hierro and Callaway 2003; Callaway
et al. 2005).
Acacia species are distributed primarily in the dry tropics, as well as several Australian Acacia
species have become highly invasive weeds around the world (Blakesley et al. 2002). Acacia
melanoxylon R. Br. is an introduced species that has its origin in the temperate forests of southeast Australia and Tasmania. It is a versatile and highly adaptive tree species which has spread
all over the world (Knapic et al. 2006), and currently covers a considerable area in the coastal
area of the north-western Iberian Peninsula, both in monocultures and in mixed stands with
Eucalyptus globulus. Upon invasion, it establishes quickly in the alien environment, thereby
resulting in changes in the structure and dynamics of native ecosystems. Some Acacia species
affects crop growth through allelopathy, as their litter interferes with the establishment and
growth of adjoining crop plants (Kohli et al. 2006) due to the presence of numerous substances
including phenolic compounds in litter (Seigler 2003).
Only two studies have reported the allelopathic potential of Acacia melanoxylon leaf and bark
tissues or extracts Lonicera tissues or extracts. In one study, Soil from Acacia showed a strong
toxic effect on L. sativa and D. glomerata radicle growth, and on germination of D. glomerata,
an effect that may be related to several phenolic secondary metabolites found in the tissues
(Gonzalez et al. 1995). Souto et al. (2001) showed that macerated decomposing leaf extract
174
Chapter VIII. General discussion
showed strong inhibition on germination and growth of L. sativa. Only leaf tissues were
examined in these studies. In present study, we made several advances in exploring the
allelopathic potential of Acacia melanoxylon, with a focus on the inhibition of seed germination
and radicle growth by phyllodes and flowers tissue extracts. The selection of flower tissue was
based on a previous study which indicated that another Acacia species (A. dealbata Link)
releases several allelopathic compounds during flowering period (Carballeira and Reigosa 1999)
and its aqueous extract inhibited germination and growth of meadow species (Reigosa et al.
1984). Allelochemicals are found in different parts of a plant body; roots, stems, flowers, leaves
and likely enter the soil from foliar leaching in rainwater, exudation from roots into the soil
water, or after leaf fall and subsequent incorporation in the soil (Inderjit and Duke 2003). Thus,
both phyllodes and flowers may be sources of allelochemicals and extractions in water are most
ecologically relevant. Second, we examined effects of these extracts at four ecologically
relevant concentrations, relative to a control, which is important in determining causal
relationships and minimizes effects due simply to unnaturally high doses of putative
allelochemicals. Third, we examined the response of extracts on seeds of three native grass
species, Lolium perenne, Dactylis glomerata, Rumex acetosa, that vary in life history, seed
germination requirement than the donor species A. melanoxylon and including a general biotest
species, Lettuce (Lactuca sativa L.) because lettuce has been used extensively as a test species
in the allelopathic research (Macias et al. 2000). Fourth, we calculated four germination indices
(Chiapusio et al. 1997) in order to make comparisons between each of them. The bioassays were
undertaken in Petri dishes to avoid soil and microbial interactions and in a controlled
environment in order to decrease environmental variability (chapter III).
A. melanoxylon flowers (100%, 75%, and 50%) aqueous extract significantly inhibited seed
germination in D. glomerata, R. acetosa, L. perenne and in L. sativa while phyllodes (100%,
75%) extract completely inhibited germination of R. acetosa, L. perenne, and L. sativa during
different days. The mean LC50-value of A. melanoxylon flowers and phyllodes extract in the
germination inhibition of L. perenne was 43%, 41%, in R. acetosa (40%, 38%), and in L. sativa
was 53%, 41%. The LC50-value of A. melanoxylon phyllodes extract in the germination
inhibition of D. glomerata was 46%. All the four concentration of A. melanoxylon flowers
extract proved to be more toxic that significantly reduced the root length of D. glomerata, L.
perenne, R. acetosa and L. sativa while phyllodes extract decreased the root length of L.
perenne (83%) and R. acetosa (74%) at 100% concentration. Inhibition of seed germination of
target species showed a species-specific and dose-dependent response with highest inhibition
occurred at concentration of 100% and 75%. The possible use of secondary metabolites from
plants as herbicides has long been discussed. Although, the physiological and ecological actions
of allelochemicals are weaker than commercial herbicides. However, these secondary
metabolites have ecological advantages like, greater biodegradability. Allelochemicals affects
many physiological processes in target organisms, including inhibition of; seed germination,
growth of plant species, ion uptake, inhibition of electron transport in photosynthesis and
respiratory chain and involved in root growth inhibition.
175
General discussion
Laboratory bioassays were conducted to test the phytotoxicity of 6 allelochemicals (ferulic acid,
p-hydroxybenzoic acid, 2-benzoxazolinone, L-mimosine, juglone, trans-cinnamic acid), 6
allelochemicals mixture and two herbicides (pendimethalin and S-metolachlor) on germination
and seedling growth of four plants species (Rumex acetosa L., Lolium perenne L., Lactuca
sativa L. and Dactylis glomerata L.). Data obtained were used to compare four germination
indices viz., (total germination index (GT), speed of germination index (S), accumulated speed
of germination index (AS), and coefficient of rate of germination index (CRG) (Hussain et al.
2008). The allelochemicals 2-benzoxazolinone and trans-cinnamic acid inhibited the total
germination index of L. perenne at 1
allelochemicals mixture (1
M concentration. The L- mimosine and six
M concentration) inhibited GT, S, AS, in L. perenne and also
inhibited GT, S, AS in D. glomerata at 1 M and 0.1 µM concentrations. Juglone inhibited GT of
L. perenne at 1 M concentration and in D. glomerata at 1 M, 0.01 M and CRG in R. acetosa
at 0.01 M concentration. The application of both herbicides strongly inhibited GT, S, AS, CRG
in L. perenne L. and D. glomerata at concentration of 104 M. These results indicate that each
index led to a different interpretation of allelochemicals effect on germination.
Low-molecular-weight compounds are released through passive diffusion into soil solutions
(Neumann and Römheld 2001). The development of phytotoxicity of phyllodes from A.
melanoxylon R.Br. was reported by González et al. 1995, during their decomposition in four
different plots in which the dominant species of tree was Quercus robur L., A. melanoxylon,
Pinus radiata D. Don. and Eucalyptus globulus Labill. Soil from Acacia showed a strong toxic
effect and extremely inhibited germination, seedling growth and radicle length of Lactuca sativa
and Dactylis glomerata. Souto et al., 1994, concluded that Acacia melanoxylon L leaves has
shown strong toxic effects on germination and growth of Lactuca sativa. Allelochemical ferulic
acid (FA) reduced the quantum efficiency (Fv/Fm) of open PSII reaction centers in dark adapted
states. Reduction in ratio of Fv/Fm was stronger than both herbicides. Similarly, phydroxybenzoic acid (HBA) also reduced the quantum efficiency (Fv/Fm) in lettuce seedlings on
all days but the effect was stronger on day 5 and 6. Both herbicides (pendimethalin (PML) and
S-metolachlor (MTR) slightly reduced the Fv/Fm activity on day 2 and afterwards there was
Stimulation. Both allelochemicals significantly decreased the effective quantum yield (Ф PSII)
of photosystem II from 3-6 days. Contrarily there was no effect of herbicides on ФPSII levels.
The application of FA drastically decreased (3-folds) the photochemical fluorescence quenching
(qP) compared to control. The allelochemicals HBA also drastically reduced (3-folds) qP values
over control from 3-6 days than control. On the contrary, there was non-significant effect of
both herbicides on qP in lettuce. The application of FA also drastically decreased (3-folds) the
non-photochemical fluorescence quenching (NPQ) as compared to control on third day. The
allelochemical HBA also significantly reduced the NPQ values than control on 1-4 days but
there was non-significant effect of both herbicides on NPQ in lettuce on all days. However, on
third day, herbicide PML significantly stimulated the NPQ in lettuce. The FA, HBA, MTR and
PML treatments did not significantly affect the carbon %age. The lettuce seedlings treated with
HBA, carbon isotope ratio (δ13C) was significantly less negative (-26.93) than control (-27.61).
Similarly, lettuce plants treated with FA and MTR, the δ13C values were significantly less
176
Chapter VIII. General discussion
negative (-26.90) and (-26.88), respectively, as compared to control (-27.61). Carbon isotope
discrimination (Δ13C) values in lettuce leaves were significantly less after treatment with FA
(19.40) and HBA (19.25) at 1.5 mM concentration than control (20.17) (Hussain et al. 2010).
One of the principal criticisms of research on allelopathy is the frequent lack of correlation
between laboratory bioassays and field studies or observations (Wardle et al., 1996). Most of
researchers demonstrated the allelopathy and effect of allelochemicals on growth and
physiological characteristics of target plant species (Peng et al. 2004; Weir et al. 2004). One of
the best-characterized phytotoxic mechanisms induced by allelochemicals is the inhibition of
photosynthesis and oxygen evolution through interactions with components of photosystem II
(Einhelling 1995; Rimando et al. 1998) and changes in chlorophyll fluorescence emission from
photosynthetic organisms are frequently indications of changes in photosynthetic activity. The
measurement of chlorophyll fluorescence emitted by intact, attached leaves is thought to be a
reliable, non-invasive method for monitoring photosynthetic events and for judging the
physiological status of the plant. Recent advances in Chlorophyll fluorescence analysis make it
easy to detect photoinhibition by measuring the maximum quantum efficiency of PSII
photochemistry (Fv/Fm) and is widely used as a measure of induced stress on photosynthetic
apparatus (Maxwell and Johnson 2000). A decrease in this ratio is a good indicator of
photosynthetic damage caused by lights when plants were subject to stress. In our results
(Fv/Fm) was significantly decreased in response to allelopathic stress of A. melanoxylon flowers
on 2nd and 4th day. Similarly, Barkosky et al. (2000), reported that exposure to cafferic acid
result in a significant increase in leaf diffusive resistance and transpiration rate on 12 d of the
treatment and significant decrease in Fv/Fm on 28th day. The quantum yield of electron transfer
at PSII is the product of the efficiency of the open reaction centers (Fv′/Fm′) and the
photochemical quenching (qP) (Sinsawat et al. 2004). The extract of A. melanoxylon flowers
induced strong allelopathic stress which results in reduction in the quantum yield of PSII
electron transport (ΦPSII) in lettuce plants on first and 2nd day. A sustained decrease of the
(Fv/Fm) may indicate the occurrence of photoinhibitory damage (Colom and Vazzana 2003).
The lowered photosynthetic electrol transport (ΦPSII) were frequently observed in
allelochemicals treated plants such as cucumber seedlings. Changes in the Chl fluorescence
quenching parameters are good indicators of photosynthetic electron flow (Schreiber et al.
1994). After the onset of illumination, maximal Chl fluorescence declines as the photosynthetic
reaction proceeds due to the photosynthetic electron flow, qP, and nonradiative energy
dissipation, NPQ (Genty et al. 1989). A significant decrease in photochemical fluorescence
quenching (qP) was also observed on first and 4th day indicating that the balance between
excitation rate and electron transfer rate has changed leading to a more reduced state of the PSII
reaction centers. However, NPQ was significantly reduced under A. melanoxylon flowers
treatments. The quantum yield of electron transfer at PSII (ΦPSII) is the product of the
efficiency of the open reaction centers and the photochemical quenching (qP) (Sinsawat et al.
2004). In Lettuce a decrease of the qP was observed in response to the allelopathic stress
treatment, indicating that a larger percentage of the PSII reaction centers was closed at any time,
which in turn indicates that the balance between excitation rate and electron transfer rate had
changed. Stable isotope discrimination of several elements has been widely studied in plant
177
General discussion
physiology (Griffiths 1991). A. melanoxylon flowers aqueous had dominant effect on δ13C and
C isotope ratios (δ13C) varied significantly (P < 0.05) with respect to A. melanoxylon flowers
(100%, 75% and 50%) aqueous extract and varied by 1.00 ‰, ranging from - 27.11‰ to 26.72‰. Similarly, Handley et al. (1994), found that shoot δ13C was most negative in H.
spontaneum genotypes from sites receiving the least rainfall annually. A. melanoxylon flowers
(100%, 75% and 50%) aqueous extract had the dominant effect on Carbon isotope
discrimination of lettuce leaf (Δ 13C) and Δ 13C varied from – 0.731‰ – 0.727‰.
Plant phenolics and terpenoids are two major chemical groups implicated in allelopathy (Inderjit
and Mallik 2002). Plant phenolics originate from the shikimate pathway, and generally
terpenoids originate from the mevalonic acid pathway. In higher plants, there are two pathways
for the formation of C5 terpenoid monomers, isopentenyl diphosphate: (i) the glyceraldehyde-3phosphate/pyruvate pathway in the plastids, and (ii) the cytoplasmic acetate/mevalonate
pathway (Lichtenthaler et al. 1997). Biosynthesis of several mono-, sesqui- and diterpenes is
regulated at the transcriptional level (Steele et al. 1998; McConkey et al. 2000). Plants introduce
allelochemicals into the environment through foliar leaching, root exudation, residue
decomposition, volatilization and debris incorporation into soil (Inderjit and Keating 1999).
Precipitation passing through the canopy aids in leaching of chemicals from foliar parts of the
donor plant into the environment. We have reported earlier (chapter VI), that lettuce seedlings
treated with BOA (0.1, 0.5, 1.0 and 1.5 mM) and distilled water (control). BOA, in the range
(0.1–1.5 mM), decreased the shoot length, root length, leaf and root fresh weight. Quantum
efficiency of open reaction centers (Fv/Fm) in the dark-adapted state was significantly reduced in
response to BOA treatment. Quantum yield of photosystem II (ΦPSII) electron transport was
significantly reduced because of decrease in the efficiency of excitation energy trapping of PSII
reaction centers. BOA had dominant effect on C isotope ratios (δ13C) that was significantly less
negative (-26.93) at 1.0 mM concentration as compared to control (-27.61). Carbon isotope
discrimination (Δ 13C) values were significantly less (19.45) as compared to control (20.17) at
1.0 mM. BOA also affect ratio of intercellular to air CO2 concentration (ci/ca) that was
significantly less (0.66) as compared to control (0.69) when treated with 1.0 mM BOA. In
another experiment, (chapter VII), the effects of cinnamic acid (CA) (an allelopathic compound)
on biochemical, physiological and isotopic of lettuce were studied. The results show that 1.5,
1.0 and 0.5 mM CA treatment decreased lettuce leaf/root fresh weight, shoot/root length, but it
did not affect the relative water content and leaf osmotic potential. CA treatment (1.5 mM)
significantly reduced Fm, Fv, photochemical efficiency of PSII (Fv/Fm), quantum yield of PSII
(ΦPSII), photochemical fluorescence quenching (qP) and non-photochemical quenching (NPQ).
These suggest that the decrease in these chlorophyll fluorescence components was a result to
damage in photosynthetic apparatus and is at least partially attributed to lowered photochemical
efficiency of PSII in lettuce leaves following exposure to 1.5 mM CA. Carbon isotope
discrimination (Δ 13C) were also less (19.63) in CA treated plants as compared to control
(20.18). The leaf protein contents of lettuce were significantly decreased when treated with CA
(1.5 mM) treatment. Allelopathy has been well recognized in many natural and manipulated
ecosystems and is thought to be implicated in problems such as exotic plant invasion, replant
failure and soil sickness (Fitter 2003). Our results suggest that natural extracts of A.
178
Chapter VIII. General discussion
melanoxylon have the potential to inhibit the germination and/or growth of competing plants in
the field. This effect, coupled with its dramatic effects on resource availability, could contribute
to the invasive success of this tree. Because, it is very difficult (or even impossible, Inderjit and
Del Moral 1997) to separate allelopathy from other forms of compition, and allelopathy usually
is present altogether with resource competition (Thijs et al. 1994; Mallik 2000; Weih and
Karlsson 1999). More work clearly needs to be done to address the ecological relevance of our
findings, including bioassays in field where the influence of soil particles, microbes, and other
environmental factors on the putative allelopathic agents can be seen (Inderjit and Duke 2003).
Conclusions
The aqueous extracts of flowers and leaves of invasive Acacia melanoxylon suppressed seed
germination and radicle growth of L. perenne and L. sativa and inhibition effects increased with
increasing concentrations of extract. Our data suggest that allelopathy may contribute to the
success of A. melanoxylon as an invader of North Western Galician forests, but further work are
needed which could attempt to resolve interactions under field conditions to evaluate the effects
of allelochemicals in natural environment. The important implications for protection and
restoration of sites invaded by A. melanoxylon are extremely important. Chemically-mediated
effects on native flora and fauna are yet another reason to protect valuable natural areas from A.
melanoxylon invasion. Since these effects may persist for some time after removal of A.
melanoxylon it may be unwise to attempt to repopulate an area with native plants by spreading
seeds immediately after A. melanoxylon removal until enough time has elapsed for the
environmental breakdown of active compounds. Knowing the identity and half-life of
allelopathic compounds in the soil would help to guide restoration efforts. In addition to direct
effects on competing plants, A. melanoxylon may have chemically-mediated effects on soil
microbes, including mycorrhizae, which may also need to be remediated in restored sites. The
allelopathic properties of plants can be exploited successfully as a tool for pathogen and weed
reduction. Members of genus Acacia are widely distributed in tropical, subtropical and
temperate regions of the world. Some species are allelopathic and inhibits the crop growth and
development. Numerous growth inhibitors (allelochemicals) identified from these allelopathic
plants are responsible for their allelopathic properties and may be a useful source for the future
development of bio-herbicides and pesticides.
179
General discussion
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Chapter IX
Summary
SUMMARY
The experiment was conducted in growth chamber and glass house of Plant Ecophysiology
Laboratory, Faculty of Biology, University of Vigo, Spain during 2007-2009. The objectives of
present study were to check the Acacia melanoxylon (flowers and phyllodes aqueous extract),
different allelochemicals and two herbicides phytotoxic potential on germination, growth and
ecophysiological characteristics, Chlorophyll fluorescence mechanisms, carbon and nitrogen
isotopes discrimination, leaf water relations and leaf protein contents of four plant species to
understand the relationships between molecular mode of action of allelochemicals, herbicides
and phenolics compounds present in A. melanoxylon and their physiological effects.
In the laboratory bioassays, phytotoxicity of six allelochemicals (ferulic acid, phydroxybenzoic acid, 2-benzoxazolinone, L-mimosine, juglone, trans-cinnamic acid, six
allelochemical mixture and two herbicides (pendimethalin and S-metolachlor) were examined
on germination and seedling growth of four plants species (Rumex acetosa, Lolium perenne,
Lactuca sativa, and Dactylis glomerata). Data obtained were used to compare four germination
indices (GT, S, AS, and CRG) and root growth. Allelochemicals 2-benzoxazolinone and transcinnamic acid inhibited total germination index of L. perenne at 1 M concentration. The Lmimosine and six allelochemical mixture (1
M concentration) inhibited GT, S, AS, in L.
perenne and also inhibited GT, S, AS in D. glomerata at 1 M and 0.1 M concentration. Lmimosine also inhibited CRG in R. acetosa at 0.01 M and in D. glomerata at 0.001
M
concentration. The six allelochemical mixture (1 M concentration) inhibited S of D. glomerata
while trans-cinnamic acid has shown inhibition of S in R. acetosa at 0.001 M concentration.
Juglone inhibited GT of L. perenne at 1 M concentration and in D. glomerata at 1 M, 0.01 M
and CRG in R. acetosa at 0.01 M concentration. The application of both herbicides strongly
inhibited GT, S, AS, CRG in L. perenne and D. glomerata at concentration of 104 M. These
results indicate that each index led to a different interpretation of allelochemicals effect on
germination.
Plants may favourably or adversely affect other plants through allelochemicals which
may be released directly or indirectly into surrounding environment. Acacia melanoxylon R. Br.
is an invader in Galician forests with well-documented allelopathic tendencies that have
generally been ascribed to its most abundant secondary metabolites present in flowers and
phyllodes. Laboratory bioassays were conducted to test allelopathic potential of aqueous extract
of A. melanoxylon flowers and phyllodes on germination, seedling growth and radicle length of
two test species (Rumex acetosa and Dactylis glomerata). Inhibition of seed germination of
plant species showed a species-specific and dose-dependent response with highest inhibition
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Chapter IX. Summary
occurring at concentration of 100% flowers and phyllodes aqueous extract. D. glomerata seeds
are most sensitive, whereas those of R. acetosa were least sensitive. Aqueous extract of A.
melanoxylon flowers were more phytotoxic as compared to phyllodes even at lowest
concentration (25%). A. melanoxylon flowers significantly inhibited radicle length of both target
species at all concentration as compared to A. melanoxylon phyllodes and control. From present
study the use of multiple indices seems necessary to validate allelochemical activity of plant
extracts on germination behaviour.
Biological invasions are believed to be the second largest cause of current
biodiversity loss. Blackwood (Acacia melanoxylon) is an invasive, non-indigenous species
currently invading the understory of Galician woodlands where it is a serious threat to the native
flora. Allelopathy is believed to contribute to the invasiveness and impact of the exotic species.
Part of this success might be due to allelopathic interference by release of allelochemicals from
flowers and phyllodes of A. melanoxylon which fall down on ground. Two congeneric species,
the Lolium perenne, an under story species and Lactuca sativa, a general biotest species were
tested for allelopathic inhibition of germination and growth by A. melanoxylon. Seeds were
germinated on filter papers contaminated by aqueous extract of flowers and phyllodes of A.
melanoxylon. Extracts were selective in their allelopathic activity and A. melanoxylon flowers
significantly inhibited germination phenomena in both test species at all concentration as
compared to A. melanoxylon phyllodes. Inhibition of seed germination of target plant species
showed a species-specific and dose-dependent response with highest inhibition occurring at
100% and 75% A. melanoxylon flowers aqueous extract. L. perenne seeds were more sensitive
as compared to L. sativa. Aqueous extract of A. melanoxylon flowers were more phytotoxic as
compared to phyllodes even at lowest concentration (25%). A. melanoxylon flowers also
significantly inhibited radicle length of both species at all concentration. We counted number of
germinated seeds at each exposure time and total number of germinated seeds at the end of one
week. We also compared four germination indices (total germination index, speed of
germination index, accumulated speed of germination index, and coefficient of rate of
germination index) in order to discuss their physiological meaning.
Acacia melanoxylon is a naturalized Australian species that has invaded woodlands and
degraded natural habitats in the north western Iberian Peninsula (Galicia) in Spain. Several
phenolic (p-hydroxybenzoic, vanillic, p-coumaric, syringic, protocatequic, ferulic acids) and
flavonoides (catechin, luteolin, rutin, apigenin, quercetin) were isolated from methanol extracts
of flowers and phyllodes of A. melanoxylon by HPLC. A. melanoxylon flowers and phyllodes
aqueous extract was tested on growth and ecophysiological characteristics of lettuce because it
is one of the most allelopathic sensitive cash crop species, commonly used in allelopathic
studies. Leaf fresh weight, shoot length, root length and shoot/root length ratios were
185
Summary
significantly inhibited by A. melanoxylon flowers aqueous extract at all concentration. Non
significant differences were observed in leaf relative water content (RWC) at all four
concentration of phyllodes extract and lowest concentration (25%) of flowers stimulated RWC.
A. melanoxylon flowers and phyllodes extract was very lethal and significantly reduced leaf
osmotic potential at 100% and 75%. A. melanoxylon flowers and phyllodes extract (100%) were
checked on chlorophyll fluorescence characteristics. There was significant change in initial
fluorescence (Fo) under allelopathic stress of A. melanoxylon flowers on first, second and fifth
day but A. melanoxylon phyllodes extract significantly stimulated Fo in second day. A
significant reduction in Fm in lettuce exposed to A. melanoxylon phyllodes was observed only on
3rd day but there was no significant difference in F m between treatments and control in all other
days. Variable fluorescence (Fv) was significantly reduced on 3rd day by the application of A.
melanoxylon phyllodes extract. Quantum efficiency of open PSII reaction centers in the darkadapted leaves (Fv/Fm) was approximately 0.81–0.87 in control and decreased significantly in
response to A. melanoxylon flowers extract on 2nd and 4th day. With the passage of time, a full
recovery of the Fv/Fm value was observed on 6th day. A. melanoxylon flowers induced strong
allelopathic stress on Ist and 2nd day which results in reduction in the quantum yield of PSII
electron transport (ΦPSII) in lettuce. The reduced ΦPSII was a result of the decrease in the
efficiency of excitation energy trapping of PSII reaction centers. A significant decrease in
photochemical fluorescence quenching (qP) was also observed on first and 4th day indicating
that the balance between excitation rate and electron transfer rate has changed leading to a more
reduced state of the PSII reaction centers. NPQ were significantly reduced under A.
melanoxylon flowers extracts.
A. melanoxylon flowers aqueous had dominant effect on δ13C and significantly varied (P
< 0.05) with respect to A. melanoxylon flowers (100%, 75% and 50%) aqueous extract and
varied by 1.00 ‰, ranging from - 27.11‰ to -26.72‰. A. melanoxylon flowers (100%, 75% and
50%) aqueous extract had significant effect on Δ
C that varied from – 0.731‰ – 0.727‰.
13
Acacia melanoxylon flowers (100%) concentration also significantly inhibited protein contents
in L. sativa.
SUGGESTIONS FOR FUTURE RESEARCH
The acacia allelopathy can be exploited for managing weeds. Research is needed to
check allelopathic potential of different acacia species and selection of excellent ones.
Numerous phenolics and flavonoids compounds have been identified as causes of Acacia
allelopathy (Chapter III, table 1). Compounds present in plant tissues might possess allelopathic
potential, but they might not necessarily be leached or exuded from the plant into the
environment, causing seedling allelopathy under natural conditions. Future research needs to be
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Chapter IX. Summary
conducted in vitro to isolate, identify and quantify allelochemicals from living plant parts at
different growth stages.
Future research should focus on developing an assessment system of allelopathy as soon
as possible, in part, to ensure the protection of allelopathic effects of acacia. In natural and
artificial ecosystems, allelopathy is a critical factor influencing co evolution and other ecologic
processes. It is necessary to investigate acacia allelopathy in conjunction with global change,
biodiversity and biological invasion.
More research models on the allelopathic effects on A. melanoxylon should be built to
get a systemic understanding of the essence of allelopathy. In addition, with the determination
of proteins and genomes related to allelopathy in acacia, bio-information will come into being,
which will allow allelopathy in acacia, and other plants, to become prevalent, practical and
prosperous. Exposure to any stressful condition (such as allelochemicals presence in soil), over
a long period would permit the evolution of plant mechanisms by which to tolerate the stressful
factor (Reigosa et al., 2002). Given that natural communities have been evolving together for
centuries, Rabotnov (1974) proposed that the most important effects of llelochemicals might
appear in resource competition between species which did not share a common environment in
the past. This hypothesis is especially important in forestry, because allelopathy could be one of
the important factors influencing the successful introduction of tree species in new regions.
The possibility of plantation failure due to allelopathy should be analyzed before
introducing a new species into a forest ecosystem. Furthermore, not only this factor should be
taken into account for the success of introduced trees, but also in invasions or introductions of
exotic understory species that may be able to change the entire community. Recent studies have
pointed out that allelochemical production and tolerance could be a key factor in determining
success in exotic species invasion and in defining the new community resulting from the
invasion process.
RESUMEN
Se llevaron a cabo experimentos en cámaras de cultivo y en invernadero en el
laboratorio de Ecofisiología Vegetal, Facultad de Biología, Universidad de Vigo, España, entre
2007 y 2009. Los objetivos del presente estudio incluyeron comprobar la fitotoxicidad de
Acacia melanoxylon (extractos acuosos obtenidos a partir de flores y filodios), la de diferentes
aleloquímicos y de dos herbicidas a efectos comparativos sobre la germinación, el crecimiento,
diversas características ecofisiológicas, los mecanismos de fluorescencia de clorofila, la
discriminación isotópica del carbono y el nitrógeno, las relaciones hídricas y el contenido en
proteínas en hojas de cuatro especies, a fin de comprender las relaciones entre el modo de
187
Summary
acción molecular de los aleloquímicos, los dos herbicidas y diversos compuestos fenólicos
presentes en A. melanoxylon y sus efectos fisiológicos.
Se estudió mediante bioensayos de laboratorio la fitotoxicidad de seis aleloquímicos
(ácido ferúlico, ácido p-hidroxibenzoico, 2-benzoxazolinona, L-mimosina, juglona, ácido transcinámico, una mezcla de los seis y dos herbicidas (pendimethalin y S-metolachlor) sobre la
germinación y el crecimiento de plántulas de cuatro especies vegetales (Rumex acetosa L.,
Lolium perenne L., Lactuca sativa L. y Dactylis glomerata L.). Los datos obtenidos se
utilizaron para calcular cuatro índices de germinación (GT, S, AS, y CRG) y el crecimiento de
radículas. Los aleloquímicos 2-benzoxazolinona y ácido trans-cinámico inhibieron el índice de
germinación total de L. perenne a la concentración 1 M. La L- mimosina y la mezcla de seis
aleloquímicos (concentración 1 M) redujeron los índices GT, S y AS en L. perenne y también
GT, S y AS en D. glomerata a las concentraciones 1 M y 0,1 M. La L-mimosina también
inhibió los valores de CRG en R. acetosa a 0,01 M y en D. glomerata a la concentración de
0,001 M. La mezcla de seis aleloquímicos (concentración 1 M) inhibió el valor de S en D.
glomerata mientras que el ácido trans-cinámico también mostró inhibición de S en R. acetosa a
concentración 0,001 M. La juglona inhibió GT en L. perenne a concentración 1 M y en D.
glomerata a 1 M, 0,01 M y CRG en R. acetosa a concentración 0,01 M. Los dos herbicidas
ensayados inhibieron fuertemente GT, S, AS y CRG en L. perenne y D. glomerata a la
concentración de 104
M. Estos resultados indican que cada índice condujo a diferentes
interpretaciones de los efectos de los aleloquímicos sobre la germinación.
Las plantas pueden afectar a otras plantas de manera positiva o negativa a través de
aleloquímicos que pueden ser liberados directa o indirectamente al ambiente cercano. Acacia
melanoxylon R. Br. es una planta invasora en los bosques y plantaciones forestales gallegos,
mostrando capacidades alelopáticas que han sido previamente documentadas, generalmente
atribuidas a los abundantes metabolitos secundarios presentes en flores y filodios. Se llevaron a
cabo bioensayos de laboratorio para probar el potencial alelopático de extractos acuosos
obtenidos a partir de flores y filodios de A. melanoxylon sobre la germinación, el crecimiento de
plántulas y la longitud de raíces de dos especies (Rumex acetosa y Dactylis glomerata). La
inhibición de la germinación de granos de ambas especies mostró una respuesta variable
dependiendo de la dosis y de la especie ensayada, con los valores máximos de inhibición
correspondiéndose con la concentración máxima (100%) de extractos acuosos de flores y
filodios. Los granos de D. glomerata fueron más sensibles, mientras que los de R. acetosa
resultaron menos inhibidos. Los extractos acuosos de flores de A. melanoxylon fueron más
fitotóxicos que los obtenidos a partir de filodios incluso en las concentraciones más bajas
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Chapter IX. Summary
ensayadas (25%). Las flores de A. melanoxylon inhibieron significativamente la longitud de
radículas de ambas especies diana a todas las concentraciones ensayadas frente al efecto de los
filodios y frente al control. Del presente estudio se recomienda el uso de varios índices de
germinación; dicha utilización podría ser necesaria para validar la actividad alelopática de
extractos vegetales sobre la respuesta de la germinación.
Se cree que las invasiones biológicas son la segunda causa más importante de
pérdida de biodiversidad en la actualidad. La acacia de madera negra (Acacia melanoxylon) es
una especie alóctona que está invadiendo el sotobosque de bosques y plantaciones forestales en
Galicia, donde ha llegado a ser un problema serio que amenaza la flora autóctona. Se cree que
su capacidad alelopática contribuye a su capacidad invasora y al impacto sobre las especies
autóctonas. Parte de su éxito podría ser debido a la interferencia alelopática producida por la
liberación de aleloquímicos por sus flores y filodios que caen al suelo. Dos especies, Lolium
perenne, especie que se puede encontrar en el sotobosque, y Lactuca sativa, considerada una de
las especies de uso más frecuente en bioensayos de alelopatía, se utilizaron para medir la
inhibición alelopática de la germinación y el crecimiento producidos por A. melanoxylon. Los
granos se hicieron germinar sobre papel de filtro y se regaron con extractos acuosos de flores y
filodios de A. melanoxylon. Los extractos tuvieron actividad alelopática selectiva, con los
extractos obtenidos a partir de flores de A. melanoxylon inhibiendo significativamente la
germinación en ambas especies diana a todas las concentraciones con un efecto muy superior al
de los extractos obtenidos a partir de filodios. La inhibición de la germinación de los granos
mostró influencia de la especie y de la concentración, con máximas inhibiciones en las
concentraciones 100% y 75% de flores de A. melanoxylon. Los granos de L. perenne resultaron
más sensibles que los de L. sativa. Los extractos acuosos de flores de A. melanoxylon fueron
más fitotóxicos que los de filodios incluso a las concentraciones más bajas (25%). Los extractos
de flores de A. melanoxylon también inhibieron significativamente la elongación radicular de
ambas especies a todas las concentraciones ensayadas. Se contaron los granos germinados en
cada momento de exposición y el número total de granos germinados al final de una semana.
También se compararon cuatro índices de germinación (índice de germinación total, índice de
velocidad de germinación, índice de velocidad acumulada de germinación e índice ―coeficiente
tasa de germinación‖) antes de discutir el significado fisiológico del efecto encontrado.
Acacia melanoxylon es una especie australiana naturalizada que ha invadido hábitats
degradados, bosques y plantaciones forestales en la zona noroeste de la Península Ibérica
(Galicia) en España. Varios compuestos fenólicos (los ácidos p-hidroxibenzoico, vaníllico, pcumárico, siríngico, protocatéquico y ferúlico) y flavonoides (catequina, luteolina, rutina,
apigenina, quercetina) se aislaron a partir de extractos metabólicos de flores y filodios de A.
melanoxylon mediante HPLC. Los extractos acuosos obtenidos a partir de flores y filodios de A.
189
Summary
melanoxylon se ensayaron sobre el crecimiento y las características ecofisiológicas de lechuga
dada que se considera una de las especies de bioensayo estándar en alelopatía, siendo sensible y
de interés aplicado. El peso fresco de hojas, la longitud de partes aéreas y de radículas y la razón
parte aérea/raíz fueron inhibidos significativamente por los extractos acuosos de flores de A.
melanoxylon a todas las concentraciones ensayadas. Las diferencias encontradas en contenido
relativo hídrico no resultaron significativas en ninguna de las cuatro concentraciones ensayadas
de extractos de filodios y en la más baja de las concentraciones utilizadas de extractos acuosos
de flores (25%) se vio incrementado. Los extractos de flores y filodios de A. melanoxylon
fueron muy letales y redujeron significativamente el potencial osmótico de los filodios en las
dos concentraciones más elevadas (100% y 75%). Se midió el efecto de los extractos de flores y
filodios de A. melanoxylon sobre las características de la fluorescencia de clorofilas. Se
mostraron cambios significativos en fluorescencia inicial (Fo) bajo estrés alelopático producido
por extractos de flores de A. melanoxylon los días uno, dos y cinco tras la aplicación, mientras
que los extractos de filodios de A. melanoxylon estimularon significativamente Fo el segundo
día. Se observó una reducción estadísticamente significativa en Fm en lechuga sometida a
extractos de filodios de A. melanoxylon únicamente el tercer día, mientras que no existieron
diferencias significativas en Fm entre tratamientos y control en el resto de días. La fluorescencia
variable (Fv) se redujo significativamente el tercer día tras la aplicación de extracto de filodios
de A. melanoxylon. La eficiencia cuántica de los centros de reacción del fotosistema II en hojas
adaptadas a la oscuridad (Fv/Fm) varió entre 0,81 y 0,87 en el control y bajó significativamente
en respuesta a los extractos de flores de A. melanoxylon el día segundo y el cuarto. Con el paso
del tiempo, se observó la recuperación completa del valor de Fv/Fm a partir del sexto día. Las
flores de A. melanoxylon indujeron un fuerte estrés alelopático los días primero y Segundo
resultando en una reducción del rendimiento cuántico del transporte electrónico del PSII
(ΦPSII) en lechuga. Esta reducción en ΦPSII fue el resultado de la disminución en la eficiencia
de los centros de reacción del fotosistema II para atrapar energía de excitación. También se
observó una disminución significativa del atenuamiento fotoquímico de la fluorescencia (qP) en
los día primero y cuarto indicando que el equilibrio entre tasas de excitación y de transporte de
electrones ha cambiado conduciendo a un estado más reducido de los centros de reacción del
fotosistema II. NPQ también sufrió reducción significativa en respuesta a los extractos de flores
de A. melanoxylon.
Los extractos acuosos de flores de A. melanoxylon presentaron efectos dominantes
sobre δ C con diferencias significativas (P < 0,05) en lo tocante a las concentraciones de 100%,
13
75% y 50%, variando hasta 1,00 ‰, oscilando desde 27,11‰ hasta -26,72‰. Los extractos
acuosos de flores de A. melanoxylon (100%, 75% y 50%) produjeron efectos significativos
sobre Δ 13C, que varió desde – 0,731‰ hasta – 0,727‰. La concentración máxima ensayada de
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Chapter IX. Summary
extractos acuosos de flores de Acacia melanoxylon (100%) también inhibió significativamente
el contenido en proteínas en L. sativa.
Sugerencias para la investigación futura
La alelopatía de la acacia puede ser explotada para el manejo de malas hierbas.
Hace falta más investigación para comprobar el potencial alelopático de diferentes especies de
acacia y la selección de las más prometedoras. Numerosos compuestos fenólicos y flavonoides
se han identificado como causas de alelopatía en Acacia. Compuestos presentes en los tejidos de
las plantas podrían poseer potencial alelopático, pero que podría no ser necesariamente efectivo
si no se liberan al medio ambiente de manera natural sea por lixiviación de partes aéreas,
exudación radicular u otros medios. Debe llevarse a cabo investigación futura in vitro que
permita aislar, identificar y cuantificar aleloquímicos presentes en diferentes partes de la planta
que aparezcan en distintas etapas de crecimiento.
La investigación futura debería centrarse en el desarrollo de un sistema de evaluación
de la alelopatía tan pronto como sea posible, en parte, para garantizar la protección frente a los
efectos alelopáticos de la acacia de madera negra. En los ecosistemas naturales y artificiales, la
alelopatía es un factor crítico que influye en la co-evolución y en otros procesos ecológicos. Es
necesario investigar en la alelopatía de acacia en relación con el cambio global, la biodiversidad
y la invasión biológica.
Deben construirse más modelos teóricos sobre los efectos alelopáticos de A.
melanoxylon para tener una comprensión sistémica de la esencia de alelopatía. Además, con la
determinación de los genomas y proteínas relacionadas con la alelopatía en la acacia se
conseguirá información biológica valiosa, lo que permitirá que la alelopatía en la acacia y otras
plantas puedan producir efectos prácticos y beneficiosos.
La exposición a cualquier condición de estrés (como la presencia de aleloquímicos en el
suelo) durante un largo período permitiría la aparición evolutiva de los mecanismos por los
cuales las plantas sean capaces de tolerar el factor estresante (Reigosa et al. 2002). Dado que las
comunidades naturales han ido evolucionando juntas durante largos períodos de tiempo,
Rabotnov (1974) propone que los efectos más importantes producidos por los aleloquímicos
pueden aparecer en la competencia por los recursos entre especies que no comparten un entorno
común en el pasado. Esta hipótesis es especialmente importante en el sector forestal, porque la
alelopatía podría ser uno de los factores importantes que influyen en el éxito de la introducción
de especies de árboles en las nuevas regiones.
La posibilidad de fracaso en plantaciones debido a alelopatía debería analizarse
antes de introducir una nueva especie en un ecosistema forestal. Además, no sólo este factor
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Summary
debe tenerse en cuenta para el éxito de la introducción de los árboles, sino también en las
invasiones o introducciones de especies exóticas que puede ser capaz de cambiar toda la
comunidad de sotobosque y reducir la biodiversidad presente. Estudios recientes han señalado
que la producción y la tolerancia a los agentes alelopáticos podría ser un factor clave para
determinar el éxito en la invasión de especies exóticas y en la definición de la nueva comunidad
resultante del proceso invasivo.
References
Rabotnov T.A. 1974. On the allelopathy in the phytocenoses. Izo Akademie Nauk SSR Series Biology
6:811-820.
Reigosa M.J. Pedrol N. Sánchez-Moreiras A. González L. 2002. Stress and allelopathy. En: Reigosa,
M.J., Pedrol, N. (Eds.). Allelopathy from molecules to ecosystems, pp. 231-256. Science
Publishers, Enfield. New Hampshire, USA.
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