“Root morphology of rice through bipartite interaction
involving Trichoderma spp. influencing adaptation of plants
to abiotic stress and in-silico analysis of root related QTLs”
M. Sc. (Ag.) THESIS
by
SHINDE UMESH DNYANESHWAR
DEPARTMENT OF PLANT MOLECULAR BIOLOGY AND
BIOTECHNOLOGY
COLLEGE OF AGRICULTURE
INDIRA GANDHI KRISHI VISHWAVIDYALAYA
RAIPUR, (C.G.)
2014
“Root morphology of rice through bipartite interaction
involving Trichoderma spp. influencing adaptation of plants
to abiotic stress and in-silico analysis of root related QTLs”
Thesis
Submitted to the
Indira Gandhi Krishi Vishwavidyalaya, Raipur
by
SHINDE UMESH DNYANESHWAR
IN PARTIAL FULFILMENT OF THE
REQUIREMENTS FOR THE
DEGREE OF
Master of Science
in
Agriculture
(Plant Molecular Biology and Biotechnology)
Roll. No.15583
ID No. 120112023
JULY, 2014
List of Abbreviation
%
-
per cent
˚c
-
Degree centigrade
°C
-
degree Celsius
µl
-
Microlitre
ACC
-
1- aminocyclopropane-1-carboxylic acid
ADW
-
Autoclaved Distilled Water
AFLP
-
amplified fragment length polymorphism
AM
-
Association mapping
ANOVA
-
Analysis of variance
BAC
-
Bacterial artificial chromosome
BCA
-
Biocontrol agents
Bp
-
base pair
CAS
-
chromo azurol S solution
Cm
-
Centi meter
cM
-
centi morgan
CMA
-
chloromolybdic acid
CTAB
-
Cetryl trimethyl bromide
DNA
-
Deoxyribonucleicacid
dNTPs
-
deoxynucleotide triphosphates
DR
-
Deep rooting
EBA
-
Extraction buffer A
EBB
-
Extraction buffer B
EDTA
-
ethylenediamine tetra acetic acid
et al.
-
and others
gm
-
Gram
GGT
-
Graphical genotyping
H2O
-
Water
HCl
-
hydrochloric acid
HRPS
-
Higher root pulling strength
i.e.
-
that is
IAA
-
Indole Acetic Acid
KCl
-
potassium chloride
LWP
-
Leaf water potential
M
-
Molar
Mg
-
milligram
MgCl2
-
magnesium chloride
Ml
-
Milliliter
mM
-
milli molar
NaCl
-
sodium chloride
Ng
-
Nanogram
OD
-
Optical density
P
-
Phosphate
PAGE
-
polyacrylamide gel electrophoresis
PCR
-
polymerase chain reaction
PDA
-
Potato Dextrose Agar
PDB
-
Potato Dextrose Broth
PGPR
-
Plant growth promoting response
PRDW
-
Penetrated root dry weight
QTL
-
Quantitative trait loci
RFLP
-
restriction fragment length polymorphism
RL
-
Root length
Rpm
-
revolutions per minute
RPS
-
Root pulling strength
RV
-
Root volume
SL
-
Shoot length
SMA
-
Single marker analysis
SSR
-
Simple sequence repeats
TAE
-
tris acetic acid EDTA buffer
TE
-
tris EDTA buffer
TRN
-
Total root number
TSM
-
Trichoderma Selective Media
CHAPTER –I
INTRODUCTION
The need for increasing agricultural productivity and quality has led to an
excessive use of chemical fertilizers, creating serious environmental pollution. The
use of biofertilizers and biopesticides is an alternative for sustaining high production
with low ecological impact. Different soil-borne bacteria and fungi are able to
colonize plant roots and may have beneficial effects on the plant. Besides the classic
mycorrhizal
fungi
and
Rhizobium
bacteria,
other
plant-growth-promoting
rhizobacteria (PGPR) and fungi such as Trichoderma spp. and Piriformospora indica
can stimulate plant growth by suppressing plant diseases (Van Wees et al., 2008).
These micro-organisms can form endophytic associations and interact with other
microbes in the rhizosphere, thereby influencing disease protection, plant growth and
yield.
Every plant part have unique function as they performed in that the plant root
have a large range of functions, including acquisition of water and nutrients, as well
as structural support. Dissecting the genetic and molecular mechanisms controlling
rice root development is critical for the development of new rice ideotypes that are
better adapted to adverse conditions and for the production of sustainably achieved
rice yield potential. Most knowledge regarding the gene networks involved in root
development has been accumulated in the model dicotyledon plant species
Arabidopsis thaliana. Rice, the model monocotyledon species, presents several
singularities compared to Arabidopsis thaliana, including a root architecture
characterized by a fibrous root system comprising five types of embryonic and
postembryonic roots. For instance, a deep, thick, and branched root system is
correlated with better survival under adverse conditions, such as water or nutrients
1
deficits and this can be beneficial for development of sustainable agriculture. The
structure of root system is determined by an endogenous genetic programme as well
as by external environmental factors, including biotic and abiotic stresses. The high
adoptive plasticity of root development complicates the genetic dissection of gene
controlling root structure variation and represents bottleneck for efficient selection of
specific root ideotypes.
A significant progress in evaluating root morphology has been observed in the
last years. Despite, root research as a whole still remains challenging and costly.
Image analyses systems provide an opportunity to facilitate analyzing procedure.
They offer a rapid assessment of root characteristics like length and surface area,
diameter and tips, root branching patterns, etc. Root length and surface area are
important indicators for a potential uptake of water and nutrients. Root diameter
respective radius is one of the most important input parameters for rhizosphere
modeling. Various image analyses techniques are already used for the estimation of
root parameters. Early recognition of the importance of roots for drought resistance,
and the diversity in rice root architecture, provided a strong foundation for drought
research at the International Rice Research Institute (IRRI). IRRI was founded in
1960, and large efforts for research on root growth in response to drought were
ongoing by the mid-1970s, with an emphasis on deep root growth, formation of
coarse nodal roots, and the root pulling force method. In the 1980s, aeroponic studies
on root morphology and anatomy and line-source sprinkler field studies were
commonly conducted. The use of crosses to better understand the genetics of root
traits started in the 1980s. Further characterization of the genetics behind root traits
was conducted in the 1990s, specifically the use of molecular markers to select for
root trait QTLs. A shift toward rainfed lowland experiments in addition to upland
2
conditions began in the 1990s, with increased recognition of the different types of
drought stress environments and characterization of root water uptake. In the 2000s,
drought breeding efforts moved from selection of root traits to direct selection for
yield under drought. Today we have identified two major drought-yield QTLs to be
related to root traits, and phenotyping for association mapping of genes related to root
traits and functions. After direct selection for yield during the past decade that is now
approaching impact at the farm level, we are seeing that root traits are indeed
involved in improved yield under drought. Early reports indicated that droughttolerant upland varieties typically showed “long and thick” root systems (Chang and
Vergara 1975). This root ideotype of long/deep and thick (large diameter nodal) roots
remained the target drought/root phenotype for about the next four decades at IRRI.
However, it was also recognized that although upland varieties typically showed
deeper roots and could avoid drought, they were not as tolerant of stress as lowland
varieties. Of all the root techniques used at IRRI during the 1970s, perhaps the most
distinct was “root pulling force”, in which individual hills were vertically pulled out
of the soil, and the force required to do so was reported to be correlated with root
growth. Other root measurements in the field included core sampling coupled with the
use of photographs to estimate total root length. Both vertical and lateral root
distribution were assessed, and the need for uniformity of field soil was emphasized
for root studies.
The fungal pathogens play a major role in the development of diseases on
many important field and horticultural crops; resulting in severe plant yield losses.
Intensified use of fungicides has resulted in accumulation of toxic compounds
potentially hazardous to humans and environment and also in the buildup of resistance
of the pathogens. In order to tackle these national and global problems, effective
3
alternatives to chemical control are being employed (Rossman et. al., 1999).
Biological control is a nature friendly approach that uses specific microorganisms,
which interfere with plant pathogens and pests to overcome the problems caused by
chemical methods of plant protection. Commercial preparations of plant disease
biocontrol agents are based on the practical application of rhizosphere competent
species of bacteria or fungi. Fungi in the genus Trichoderma are among the most
promising biocontrol agents against plant pathogenic fungi. They show high level of
genetic diversity, and can be used to produce a wide range of products of commercial
and ecological interest. Trichoderma has gained immense importance since last few
decades due to its biological control ability against several plant pathogens.
Fungal species belonging to the genus Trichoderma are worldwide in
occurrence and easily isolated from soil, decaying wood, and other forms of plant
organic matter (Howell, 2003). They are classified as imperfect fungi, in that they
have no known sexual stage. Rapid growth rate in culture and the production of
numerous spores (conidia) that are varying shades of green characterize fungi in this
genus (Howell, 2003). The reverse side of colonies is often uncoloured, buff, yellow,
amber, or yellow-green, and many species produce prodigious quantities of thickwalled spores (chlamydospores) in submerged mycelium (Gams, and J. Bisset, 1998).
The role of microorganisms in plant growth promotion, nutrient management
and disease control is well known. These beneficial microorganisms colonize the
rhizosphere/ endorhizosphere of plants and promote growth of the plants through
various direct and indirect mechanisms (Saxena et al. 2005). However, of late, the
role of microbes in management of biotic and abiotic stresses is gaining importance.
The subject of PGPR elicited tolerance to abiotic stresses has been reviewed recently
(Venkateswarlu et al. 2008; Yang et al. 2009). The term Induced Systemic Tolerance
4
(IST) has been proposed for PGPR-induced physical and chemical changes that result
in enhanced tolerance to abiotic stress. Plant growth can also best imulated by PGPR
that produce1-aminocyclopropane-1-carboxylate (ACC) deaminase, which cleaves
ACC, the immediate precursor of the plant hormone ethylene, to produce αketobutyrate and ammonia (Todorovic & Glick, 2008). Ethylene is an important
signaling molecule in plants under pathogen attack or abiotic stresses and results in
plant growth inhibition (Abels et al., 1992). Inoculation of plants with PGPR
producing ACC deaminase (ACCD) lowers ethylene levels, which results in longer
roots and decreased inhibition of plant growth following environmental or pathogeninduced stress (Glick et al., 1998, 2007; Farwell et al., 2007). Microorganisms could
play a significant role in stress management, once their unique properties of tolerance
to extremities, their ubiquity, and genetic diversity are understood and methods for
their successful deployment in agriculture production are developed. The
development of molecular markers for the detection and exploitation of DNA
polymorphisms in plant systems is one of the most significant developments in the
field of molecular biology and biotechnology. Its high resolution is accounted for by
the historical recombination accumulated in natural populations and collections of
landraces, breeding materials and varieties.
The present investigation was conceived with the following major objectives:1) Analyzing root system morphology in response to candidate Trichoderma
isolates.
2) In-silico analysis of root related QTL’s
3) Molecular and biochemical characterization of Trichoderma isolates for ACC
deaminase (Tas-acdS) gene
5
CHAPTER- II
REVIEW OF LITERATURE
2.1 Rice
Microorganisms are used for eliminating problems associated with the use of
chemical fertilizers and pesticides; they are now widely applied in nature farming
and organic agriculture (Higa, 1991; Parr et al., 1994). Several microbes can
capable of mobilizing nutritive elements from non-usable form to usable form
through biological processes (Tiem et al., 1979). Either by living freely in the soil
or being associated symbiotically with plants (Chandraseker et al., 2005). Rice is
an economically important cereal grown in about one-third of the world’s total
cereal crop area, providing staple food and 35-60% of the calories consumed by
more than 2.7 billion people (Kush, 1997). Rice is life for human beings
especially in Asia, where 90 per cent of world’s rice is grown and consumed with
60 percent of population (Khush and Virk, 2000). Rice is grown over an area of
153.76 m. ha with production of 598.85 m.t. in the world (FAO, 2005). In India,
rice is cultivated on an area of 43.50 m. ha with production of 91.00 m.t. (DRR,
2007). Rice is grown under diverse growing conditions. Four major ecosystems
are generally recognized (Khush, 1984) as follows: (1) irrigated, (2) rainfed
lowland, (3) upland, and (4) floodprone. Approximately, 55% of the world rice
area planted to rice is irrigated and is the most productive rice growing system,
perhaps contributes 75% of the world rice production. Large areas of rice are
grown under lowland and upland rainfed conditions. Rice to be grown
successfully under a variety of climatic conditions across the globe; breeders
maintain rice at high genetic diversity. Second generation sequencing technologies
have enabled resequencing of a large number of genomes and have provided the
6
possibility of high-throughput genotyping and large scale genetic variation
surveys. Identification of allelic variations underpinning the phenotypic diversity
observed in rice will have enormous practical implications in rice breeding. The
International Rice Research Institute (IRRI) is a non-profit organization with a
mission to conduct scientific research to help rice farmers improve yields in a
range of agro ecosystems. For drought-prone environments, IRRI’s research
program has included plant breeding efforts to improve yield under drought as
well as the physiological investigation of the mechanisms behind drought
resistance, such as root traits. Root trait is an important parameter of study of
investigation to develop a variety resistance to biotic and abiotic stress which is
the requirement of sustainable agriculture to increase yield. It is important to study
phenotypic and molecular analysis of rice root system and identify the rice root
ideotype having denser, branched, thick, deep root system.
2.2 Genetics of rice root development
Plant root have a large range of functions, including acquisition of water and
nutrients, as well as structural support. The root grows in length due to the
proliferation of self-renewing stem cells in the RAM, which is found just behind
the root cap. Cells leaving the RAM are deposited in files (representing the radial
cell layers) and go through successive stages of elongation and differentiation.
The young root can thus be divided into four longitudinal zones: the cap, the
division zone, the elongation zone, and the differentiation zone. The RAM does
not produce lateral organs in the same way as the shoot meristem. Instead, lateral
roots may arise from the differentiated part of the main root. However, the RAM
does continuously supply cells to the root cap, which is worn away by abrasion as
the root pushes through the soil.
7
Dissecting the genetic and molecular mechanisms controlling rice root
development is critical for the development of new rice ideotypes that are better
adapted to adverse conditions and for the production of sustainably achieved rice
yield potential. Most knowledge regarding the gene networks involved in root
development has been accumulated in the model dicotyledon plant species
Arabidopsis thaliana. Rice, the model monocotyledon species, presents several
singularities compared to Arabidopsis thaliana, including a root architecture
characterized by a fibrous root system comprising five types of embryonic and
postembryonic roots. For instance, a deep, thick, and branched root system is
correlated with better survival under adverse conditions, such as drought or
nutrients deficits. The structure of root system is determined by an endogenous
genetic program as well as by external environmental factors, including biotic and
abiotic stresses. The high adoptive plasticity of root development complicates the
genetic dissection of gene controlling root structure variation and represents
bottleneck for efficient selection of specific root ideotypes. Genetic control of root
development has been studied mainly through quantitative trait loci analysis, and a
wide range of QTLs associated with small-medium effects on root biomass, root
length, root number has been identified in rice. Despite the availability of these
scientific resources, most of what we know about the genetic architecture of
complex traits in rice is based on quantitative trait locus (QTL) linkage mapping
using bi-parental populations. While providing valuable insights, the QTL
approach is clearly not scalable to investigate the genomic potential and
tremendous phenotypic variation. Thus for analysing or determining the genetic
basis of complex traits a new tool can be use called association mapping.
8
2.3 Specification of the Root Apical Meristem
The origins of the RAM can be traced back to the very first cell division in the
plant embryo, when the elongated zygote divides asymmetrically to produce a
small apical cell (which gives rise to most of the embryo) and a larger basal cell
(which forms the basal part of the embryo and the suspensor). The apical cell
undergoes a series of stereotyped divisions to produce the embryo, while the basal
cell divides several times in the same plane to produce the suspensor. The
embryonic root is derived from the lower tier of embryonic cells in the globular
stage embryo and the upper cell of the suspensor, which is called the hypophysis.
Axial patterning depends on the expression of a series of genes in zones along the
apical–basal axis (Costa S and Dolan L 2000). In the case of root development,
the most important genes are those that specify basal parts of the axis (Howell SA
(ed.) 1998). For example, the MONOPTEROUS (MP) gene is expressed in the
bottom tier of cells of the proembryo, and the mp mutant has no hypocotyl,
radicle, or RAM. Similarly, the HOBBIT (HBT) gene is required for the
formation of the hypophysis, and hbt mutants do not correctly form a RAM. The
product of the MP gene is a transcription factor that binds to auxin-responsive
elements. The role of auxin in root development has been revealed by the analysis
of genes that act upstream of MP. For example, the GNOM (GN) gene, which is
expressed just after fertilization, encodes a protein required for polarized auxin
transport. In normal embryos, auxin accumulates at the future basal pole because
auxin efflux pumps are localized specifically at the basal ends of each cell
(Paquette AJ and Benfey PN 2001). Once the RAM is specified, root growth is
dependent on its continuous proliferation. This does not occur in mutants lacking
functional ROOT MERISTEMLESS1 (RTM1 or RTM2) genes, while cell
9
proliferation in the shoot meristem is unaffected. Conversely, over proliferation of
cells is the defect in mass mutants, which are characterized by fat, barrel like roots
with extra cell layers.
2.4 Lateral Root Initiation
During embryogenesis, the first root in the embryo axis is called the radicle. When
it emerges during germination it becomes the primary root. Lateral roots
(sometimes called secondary roots) will then form the primary root. Roots that
develop on other parts of a plant, such as the stem, or leaves, are called
adventitious roots. Roots are heterotrophic organs, meaning that their nutrition
depends significantly on photosynthates produced by leaves. LRP initiation
usually starts in the primary root after seed germination. Lateral root cap cells
originate from the endodermis, while epidermis, ground tissues, columella, and
stele originate from the pericycle (Kawata S. and Shibayama H. 1965). However,
in some plants LRP initiation actually starts during the development of the radicle
in the embryo axis; cucumber (Cucumis sativum) is an example. Two kinds of
lateral roots can originate from primary roots: the large lateral roots and small
lateral roots. CRL1 encodes a lateral organ boundary (LOB) family transcription
factor, the expression of which is regulated by auxin through an auxin response
factor (ARF). Several LOB gene involved in lateral root formation (Okushima Y,
Overvoorde PJ, Arima K, Alonso JM, et. al.,). The plant specific LOB/ AS2
family contain 43 members in A. thaliana and 35 members in rice (Shuai B,
Reynaga-Pena CG).CRL1/ ARL1 is the master gene for crown root formation. The
CRL1 protein is a positive regulator of crown root and lateral root formation, and
its expression is regulated by an ARF via auxin pathway.
10
2.5 Root pulling force for estimating plant anchorage
Root length and diameter distribution are important characteristics to be
considered when describing and comparing root systems. Root pulling force was
first used in rice by O'Toole and Soemartono (1981) as an indirect estimate of root
related dehydration avoidance capacity. Ekanayake et. al., (1985) found a
significant positive correlation across diverse rice genetic materials between root
pulling force and dehydration avoidance as expressed in leaf water status
maintenance and visual scored of drought resistance under severe drought stress in
the field. Long fibrous roots have long been recognized as an important
dehydration avoidance mechanism in rice and such roots evidently also ascribe
stronger anchorage and greater resistance to pulling force. The method appear s
sensible for root depth phenotyping in rice, especially when other direct field
methods for root selection are not very forthcoming.
The root pulling strength (RPS) measurements showed a significant positive
correlation with maximum root length, root thickness, branching number, and root
dry weight. Rice genotypes that had a high RPS value were identified as having
longer, thicker, and denser root systems. When drought resistance is considered,
root function can be phenotype indirectly by assessing plant water status in a
drought managed breeding nursery rather than by accessing the root itself.
2.6 Drought and root traits
Drought is the most serious abiotic stress that limits crop production under
rainfed conditions. In particular, rice (Oryza sativa L.), which is generally grown
under flooded conditions, is susceptible to drought stress owing to its shallow root
distribution and limited capacity to extract water from deep soil layers (Kondo et.
al., 2000 and 2003). The global warming that has occurred in recent years has
11
caused serious drought damage in rice-growing areas that rely on rainwater and
that lack access to irrigation. Therefore, the enhancement of drought resistance in
rice is becoming an important strategy to stabilize rice production in areas with
rainfed agriculture. A deep root system is thought to enable plants to avoid
drought stress by absorbing water from deep soil layers (Yoshida and Hasegawa,
1982). Typical upland rice cultivars have deeper rooting than lowland cultivars
(O’Toole and Bland, 1987). The deep root system in upland rice may contribute
greatly to its drought resistance through enhanced water uptake (Price et. al.,
1999). Therefore, introducing the deep rooting characteristic of upland rice into
lowland rice cultivars may be one of ways to improve their drought resistance.
Deep rooting is a complex trait that combines the effects of the root growth
angle and root length in seminal and nodal roots of cereal crops (Araki et. al.,
2002). Neither growth angle nor length of roots alone determines the vertical root
distribution (Abe and Morita, 1994). Thus, a combination of a near-vertical
growth axis and increased root length along that axis is important for deep root
development. The different root parameters are involved in development of
drought resistance cultivars with the deep root system including lateral roots, sub
lateral root that affect the root volume. The more root volume, the higher will be
the water holding capacity.
2.7 Analyzing root morphology with Trichoderma seed treatment
Only the phenotypic and genotypic observations are not only enough to
identify the rice ideotype having deep, branched, thick root system so it is
necessary to scan the whole root of individual plants using root scanner machine
and then analyzed the image using various root analysis software’s e.g.
WinRHIZO. A significant progress in evaluating root morphology has been
12
observed in the Trichoderma treated roots and untreated roots. Despite, root
research as a whole still remains challenging and costly. Image analyses systems
provide an opportunity to facilitate analyzing procedure. They offer a rapid
assessment of root characteristics like length and surface area, diameter and tips,
root branching patterns, etc. Root length and surface area are important indicators
for a potential uptake of water and nutrients. Root diameter radius is one of the
most important input parameters for rhizosphere modeling. The complex and
dynamic interactions among microorganisms, roots, soil and water in the
rhizosphere induce changes in physicochemical and structural properties of the
soil (Haynes and Swift 1990). Microbial polysaccharides can bind soil particles to
form microaggregates and macroaggregates. Plant roots and fungal hyphae fit in
the pores between microaggregates and thus stabilize macroaggregates that
alleviate stress.
2.8 Characterization of ACC deaminase
Plant diseases are a major impediment to increasing yields of many crops, and
result in large economic losses. An environmentally safe strategy to control
diseases is biological control, which is based on natural antagonistic interactions
among microorganisms. Successful biocontrol agents (BCAs) colonize roots and
suppress pathogens by mechanisms that include niche exclusion and competition,
direct antagonism of pathogens by antibiosis, parasitism or predation and by
triggering systemic host plant defense responses (Chet&Chernin,2002; Harman et
al., 2004). Some BCAs are plant growth-promoting rhizobacteria (PGPR) and
fungi that also stimulate plant growth directly by altering plant hormone levels,
facilitating iron acquisition through siderophore production, fixing atmospheric
nitrogen and/or solubilising minerals (Lugtenberg&Kamilova, 2009). Plant
13
growth can also be stimulated by PGPR that produce1-aminocyclopropane-1carboxylate (ACC) deaminase, which cleaves ACC, the immediate precursor of
the plant hormone ethylene, to produce a-ketobutyrate and ammonia (Todorovic
& Glick, 2008). Ethylene is an important signalling molecule in plants under
pathogen attack or abiotic stresses and results in plant growth inhibition (Abels et
al., 1992). Inoculation of plants with PGPR producing ACC deaminase (ACCD)
lowers ethylene levels, which results in longer roots and decreased in hibition of
plant growth following environmental or pathogen-induced stress (Glicketal.,
1998, 2007; Farwell et al., 2007). Interestingly, it has been found that ACCD
activity is not unique to bacteria. ACCD activity was detected in Penicillium
citrinum (Jia et al., 2000). Two putative acdS genes were recently detected in the
genome of Arabidopsis thaliana and evidence was presented supporting the
hypothesis that these genes can act as regulators of ACC levels in A. thaliana and
also in tomato fruit development (McDonnell et al., 2009; Plett et al., 2009).
Certain Trichoderma spp. that has beneficial effects on plant growth and enhanced
resistance to both biotic and abiotic stresses (Harman et al., 2004) also possess
ACCD putative sequences (http://genome.jgi-psf.org/Trive1/Trive1.home.html).
However, the role of ACCD in beneficial fungi has not been investigated in depth.
2.9 Phosphate solubilisation
Phosphorus is abundant in soils in both organic and inorganic forms; nevertheless,
it is unavailable to plants. Accordingly, soil becomes phosphorus (P)-deficient,
making P one of the most important nutrient elements limiting crop productivity.
Phosphorus frequently is the least accessible macronuntrient in many ecosystems
and its low availability is often limiting to plant growth (Raghothama, 1999). To
circumvent the P deficiency, phosphate-solubilizing microorganisms could play
14
an important role in making P available for plants by dissolving insoluble P. Fungi
exhibit traits such as mineral solubilization, biological control, and production of
secondary metabolites. Altomare et. al., (1999) reported three possible
mechanisms by which Trichoderma might convert phosphate to a soluble form (i)
acidification, (ii) production of chelating metabolites, and (iii) redox activity,
concluding that chelation was the more likely mechanism for P solubilization by
Trichoderma. As such, their potential to enhance plant growth when present in
association with the roots is clear. The challenge is how to make use of such
biological resources to maintain soil health while increasing the crop productivity
by providing P to plants through the application of phosphate-solubilizing fungi
(Khan et. al., 2009). Khan et. al., (2009) focuses on the mechanisms of phosphate
solubilization, development and mode of fungal inoculants application and
mechanisms of growth promotion by phosphate-solubilizing fungi for crop
productivity under a wide range of agro-ecosystems, and the understanding and
management of P nutrition of plants through the application of phosphatesolubilizing fungi.
2.10 Production of different types of Siderophore
Siderophores, low molecular weight ligands produced by bacteria and fungi for
iron uptake, can be used to increase the potency of antibiotics. In addition to Fe
(III), siderophores can form complexes with Ga (III), Cr (III) and V (IV).
Siderophores are iron chelating ligands which can be beneficial to plants by
increasing the solubility of ferric iron (Fe III), which otherwise is unavailable for
plant nutrition (Renshaw et. al.,., 2002). This element is assimilated by root cells
in the reduced form (Fe II); however, especially in sufficiently aerated soils, the
oxidized state (Fe III) is predominant and needs to be reduced to be taken up by
15
plants. Trichoderma, can reduce Fe III through chelators such as siderophores, and
plants can take up chelated iron by reductases on the plasma membrane (Altomare
et. al.,., 1999; Jalal et. al.,., 1986 and 1987). Unlike bacterial siderophores, the
production of Trichoderma metabolites able to chelate iron is constitutive and
does not require Fe deficiency, these molecules bind Fe (III), and also other metals
including Pb (II), Cr (III), Al (III) and actinide ions ( Renshaw et. al., 2002).
Basically siderophores are considered to be of two different types, viz., secondary
hydroxamic acids and catechol type (Neilands, 1981). Using the method of
Alexander and Zuberer (1991), Hoyos-Carvajal et al. (2009) described the
production of ferric type of siderophores or other chelating compounds in 55 out
of 101 Trichoderma strains, based on the production of soluble ferrous iron
complexes indicated by colour changes in the media from blue to orange or pink.
Only 17 strains were Sid+ positive on 8-hydroxyquinoline media, in which Fe is
linked to the lipophylic 8-hydroxyquinoline. Hydroxyquinoline is used as a
complexing reagent which reacts with almost every metal in the periodic table to
form uncharged chelates. 8-HQ has a hydrogen atom that is replaceable by a
metal, and a heterocyclic nitrogen atom, which forms with this, metals a five
membered ring (Becher, 1966).The lipophylic chelant HQ can be toxic causing
lipid peroxidation and DNA breakage (Leanderson and Tagesson, 1996), possibly
explaining the relatively low number of positive tests on HQ media.
2.11 Production of Plant hormones and related compounds
Plant immunity and development are interconnected in a network of hormone
signalling pathways (Pieterse et. al., 2009). There is a cross-communication
between hormones salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), the
central players in defence, and the response pathways of other hormones such as
16
abscisic acid, commonly associated with plant development and abiotic stress,
IAA, related to plant growth and lateral root development, and gibberellins, which
control plant growth by regulating the degradation of growth-repressing DELLA
proteins (negative regulators of gibberellin (GA) signaling that act immediately
downstream
of
the
GA
receptor).
In
Trichoderma,
the
ACCD
(1-
aminocyclopropane1-carboxylic acid deaminase) activity reduces the availability
of ACC necessary for ET biosynthesis. Reductions in ET promote plant growth
via gibberellins signalling by increasing the degradation of DELLA proteins.
Moreover, gibberellins may control the onset of JA- and SA-dependent defence
responses of the plant through the regulation of DELLA protein degradation. (
Rosa Hermosa et. al., 2012).
In plants IAA plays a key role in root and shoots development. The hormone
moves from one part of the plant to another by specific transporter systems that
involve auxin importer (AUX1) and efflux (PIN1-7) proteins. IAA is a key
regulator of lateral root development and root hair development (Casimiro et. al.,
2001). Expression studies of the auxin-inducible marker DR5: uidA suggested that
T. virens inoculation increases the auxin response in Arabidopsis seedlings.
Several auxin-like secondary metabolites produced by Trichoderma strains were
able to induce plant growth and are required for development of lateral roots in
Arabidopsis (Contreras et. al., 2009; Vinale et. al., 2008). The observed effect of
Trichoderma in promoting lateral root development is similar to that described for
auxins in plants (Casimiro et. al.,., 2001). IAA is a molecule that is synthesized by
plants and a few microbes (Woodward and Bartel, 2005). IAA is also involved in
tomato fruit development, especially during fruit setting and in the final phase of
development (Srivastava and Handa, 2005). Indeed, IAA, including microbial, can
17
greatly influence the growth of the root system depending on the amount found in
the rhizosphere, through root elongation and the formation of lateral or
adventitious roots (Scott, 1972; Patten and Glick, 2002).
2.12 Cell-wall-degrading enzymes chitinases
The chitinolytic system of Trichoderma comprises many enzymes and the list
of its components is rapidly being updated as new enzymes and genes are
reported. Chitinases are divided into 1, 4-β-acetylglucosaminidases (GlcNAcases),
endochitinases and exochitinases. Two chitinolytic fungal strains, Trichoderma
aureoviride DY-59 and Rhizopus microsporus VS-9, were isolated from soil
samples of Korea and Vietnam, respectively. Enzymatic hydrolysis products from
crab swollen chitin were N-acetyl-β-D-glucosamine (GlcNAc) by DY-59
chitinase, and GlcNAc andN, N′-diacetylchitobiose (GlcNAc) 2 by VS-9 chitinases.
The chitinases degraded the cell wall of Fusarium solani hyphae to produce
oligosaccharides,
among
which
GlcNAc,
(GlcNAc) 2 ,
and
pentamer
(GlcNAc)5 were identified by high-pressure liquid chromatography. DY-59 and
VS-9 chitinases inhibited F. solani microconidial germination by more than 70%
and 60% at final protein concentrations of 5 and 27 μg mL −1 , respectively, at 30°C
for 20 h treatment (Nguyen et. al., 2008). Chitinolytic enzymes have been found
in Trichoderma harzianum, Trichoderma aureoviride, Trichoderma autroviride,
Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma viride,
Trichoderma
pseudokoningii,
Trichoderma
longipilis,
Trichoderma
minutisporum, Trichoderma hamatum and Trichoderma reesei, but the
chytinolytic systeme of Trichoderma harzianum is by far the most studied and
effective (Bruce et. al.,1995; de la Cruz et. al., 1993; Garcia et. al., 1994; Harman
18
et. al.,1993b; Lorito et. al., 1994a; Turoczi et. al., 1996; Ulhoa and Peberdy,
1991a,1992; Usui et. al., 1990).
Many GlcNAcases and their genes-exc1 (=nag1), exc2, tvnag1, and tvnag2 from
T. harzianum T25-1, T. atroviride P1 and T. virens Tv29-8 -have been described
(Harman et. al.,., 2004, Kim et. al., 2002). The 73-kDa Nag1 represents the main
GlcNAcase in T. atroviride. Nag1-disruption strain lacks chitinase activity and the
endochitinase chit42 mRNA is absent (Harman et. al.,., 2004).This indicates that
nag1 is essential for triggering chitinase gene expression. The pathogen cell wall
and chitin induce nag1, but it is only triggered when there is contact with the
pathogen (Carsolio et. al., 1999; Mach et. al., 1999) GlcNAcases CHIT73 and
CHIT102 were detected in T. harzianum TM and Trichoderma asperellum (Haran
et. al., 1996). CHIT102 triggers the expression of other chitinolytic enzymes
(Haran et. al., 1996). In addition, strain 2413 produces three extracellular
endochitinases whose genes, chit33, chit37 and chit42, have been cloned from this
strain. Other genes coding for Chit42 chitinase-ech42, cht42 and ThEn4-have also
been cloned from T. atroviride IMI206040 (Carsolio et. al., 1999), Gv2908
(Howell et. al., 2003) and T. atroviride P1 (Howell et. al., 2003), respectively.
Chit37 shows 89% similarity to Chit36 from T. harzianum TM at the amino-acid
level (Harman et. al., 2004). Chit36 inhibits B. cinerea spore germination and the
growth of both Sclerotium rolfsii and Fusarium oxysporum (Viterbo et. al., 2001).
Other genes homologous to chit36 have been cloned from T. harzianum TM, T.
atroviride P1 and T. asperellum T-203. Endochitinases are regulated by a variety
of mechanisms but induction by stress has been reported for chit33, chit36 and
chit42. Chitinase gene ech42 was obtained from Trichoderma aureoviride M. The
cloning vector pMD18-T and an E. coli DH5 alpha host were used to yield clones
19
as E. coli DH5alpha/ech42. The ech42 gene was integrated into the genomic DNA
of pYES2 by insertion into a single site for recombination, yielding the
recombinant pYES2/ech42. Chitinase expressed by pYES2/ech42 was induced by
galactose (maximal activity 0.50 units ml(-1)) and was produced in fermentation
liquid cultured for 36 h (Jinzhu s. et. al., 2005).
However, the induction under mycoparasitic conditions is not clear. Ech42 is
induced prior to any physical contact with R. solani (Kullnig et. al., 2000). Chit33
is expressed only during the contact phase and not before overgrowing R. solani
(Dana et. al., 2001); and chit36Y does not need the direct contact of the pathogen
to be expressed. chit33, chit42 and chit36 have been overexpressed in
Trichoderma spp. in order to test the role of these chitinases in mycoparasitism,
and the 42-kDa chitinase is believed to be a key enzyme (Howell et. al.,., 2003).
T. virens transformants overexpressing Chit 42 showed significantly enhanced
biocontrol activity compared with the wild-type when assayed against R. solani in
cotton seedlings experiments (Howell et. al., 2003). Other Trichoderma spp.
transformants overexpressing chit 42 resulted in better antagonism than obtained
with the wild-type (Carsolio et. al., 1999; Limon et. al., 2004). In greenhouse
biocontrol tests, however, the activity of chit 42 disruptants did not differ from
that of the wild-type (Harman et. al., 2004). T. harzianum transformants
overexpressing Chit33 chitinase constitutively inhibited the growth of R. solani
under both repressing and derepressing conditions; the antagonist tests
demonstrated that this chitinase also has an important role in mycoparasitism
(Limon et. al.,., 1999). T. harzianum Rifai TM transformants overexpressing
Chit36 chitinase inhibited F. oxysporum and S. rolfsii more strongly than the wildtype. Moreover, culture filtrates inhibited the germination of B. cinerea almost
20
completely (Viterbo et. al., 2001). The antagonism of Chit 33 and Chit 42
transformants has been improved by the addition of a cellulose-binding domain to
the chitinase genes. As a result, the strains producing the chimeric enzymes
increased their specific chitinase activity (Limon et. al., 2004). Trichoderma genes
can be expressed functionally in plants to confer beneficial features, mainly in the
control of plant diseases (Hermosa et. al., 2005). Co-expression of endo- (ech42)
and exo- (nag70) chitinases of T. atroviride in apple has been correlated with
increased resistance to V. inaequalis, and a negligible reduction in vigour (Bolar
et. al., 2001; Faize et. al., 2003). Carolina Carsolio et. al., 1994 described the
cloning and characterisation of an endochitinase gene of T.harzianum (ech42),
whose expression is strongly enhanced during mycoparasitism, as well as induced
by chitin and light-induced sporulation.
2.13 Quantitative trait loci (QTL) related to root traits
Genetic control of root development in rice is complex and the underlying
mechanisms (constitutive and adaptive) are poorly understood. Lowland and
upland varieties of indica and japonica rice with contrasting root development
characteristics have been crossed, mapping populations developed and a number
of QTLs in different chromosomes were identified. ‘QTL’ (Quantitative trait loci),
a term refers to “a region of the genome that is associated with an effect on a
quantitative trait” Conceptually, a QTL can be a single gene or it may be a cluster
of linked gene that affect trait. A wide range of QTLs associated with smallmedium effects on root biomass, root length, root number has been identified in
rice ( Kamoshita A, Wade J, Ali L, and et. al.,) has been identified in rice. As
21
these studies have used different sets of markers and many of the QTLs identified
are long, it is difficult to exploit the varietal difference for improved root traits by
marker assisted selection and for identification of concerned alleles. Intensive data
mining of literature resulted in the identification of 861 root development QTLs
and associated microsatellite markers located on different chromosomes. The QTL
and marker data generated and the genome sequence of rice were used for
construction of a relational database, Rootbrowse, using MySQL relational
database management system and Bio::DB::GFF schema. The data is viewed
using GBrowse visualization tool. It graphically displays a section of the genome
and all features annotated on it including the QTLs. The QTLs can be displayed
along with SSR markers, protein coding genes and/or known root development
genes for prediction of probable candidate genes.
22
CHAPTER-Ш
MATERIALS AND METHODS
The present investigation entitled “Root morphology of rice through
bipartite interaction involving Trichoderma spp. influencing adaptation of plants
to abiotic stress and in-silico analysis of root related QTLs” was conducted at the
Molecular Plant Pathology Laboratory, Department of Plant Molecular Biology and
Biotechnology, College of Agriculture, IGKV, Raipur (Chhattisgarh). During the
course of study, potato dextrose agar was used for maintaining the culture of different
isolates of Trichoderma spp, unless and until stated otherwise. Prior to the use
glassware’s were cleaned with labolin, and rinsed with tap water and / or distilled
water. Dried glassware’s were sterilized in hot air oven at 80˚C for two hours, while
forceps, inoculation needle and other metallic instruments were sterilized by dipping
them in alcohol followed by heating over the flame. Until and otherwise stated media
was sterilized by autoclaving at 121˚C at 15psi for 30 min.
3.1. Materials
During the course of investigation twenty genetically purified isolates of
Trichoderma spp. (Table 3.1) kindly provided by Dr. A. S. Kotasthane, Head of
Department, Department of Plant Pathology, College of Agriculture, IGKV, Raipur
were used. Rice lines IR 64 (Drought susceptible line), Sahbhagi Dhan (Drought
resistant line) and RRF 75 (Drought resistant line) was used. Indira barani dhan
(Drought resistant line)
23
Table 3.1:- Isolates of Trichoderma spp. used in the study
S.No.
1
2
3
4
5
6
7
8
9
10
Isolates/Source
IRRI-1 (IARI, NewDelhi)
IRRI-2(IARI, NewDelhi)
IRRI-3(IARI, NewDelhi)
IRRI-4(IARI, NewDelhi)
IRRI-5(IARI, NewDelhi)
IRRI-6(IARI, NewDelhi)
TH-3(IARI, NewDelhi)
TV12(IARI, NewDelhi)
94a (IGKV, Raipur)
T14(IGKV, Raipur)
ID
T1
T2
T10
T3
T4
T5
T6
T7
T8
T9
3.2. Methods
3.2.1 Study of Root characteristics:Root systems are responsible for the capture of below-ground resources such
as nutrients and water. As such, they are thought to play a central role in the yield
establishment of crop plants. The availability of a given resource for the plant can be
seen as the integration of soil and roots bio-physical constraints. Therefore, detailed
datasets containing root system architecture, root placement and soil resource
dynamics are required to improve our understanding of resource capture by plant
roots. The roots of Trichoderma treated plants were used for root scanning which
gives the detailed information about all root parameters. The root scanning was done
by using root scanner Epson Perfection V700/ V750 3.81 version and WinRhizo Reg
2009 (Lobet et. al., 2013) software. The data was recorded automatically in the
computer for different root parameters including root length, average root diameter,
root volume, number of tips, forks, surface area etc.
Following procedure was used for root scanning –
24
3.2.1.1 Acquiring Washed Roots
The first step is acquiring washed roots. This can be the most difficult and
laborious step in the experiment if plants are grown in a solid medium. The roots were
washed with tap water two times to remove soil completely and the roots were
preserved in 25% spirit in tarson tubes for root scanning. The procedure was
conducted cautiously to prevent supplementary root damage and losses.
3.2.1.2 Preparing Roots for Scanning
Roots are floated in water in acrylic trays on the scanner. This allows the roots
to be arranged to reduce overlap and crossing of roots. Plastic forceps were used as
tools. This is a delicate work; good lighting and steady hands are helpful.
3.2.1.3 Scanning Roots
For best results, WinRhizo is used with an approved scanner, which allows the
roots to be lit from above and below while being scanned. This is an important feature
(called "Dual Scan" in Regent's documentation), which reduces shadows on the root
image. Positioning System allows the trays to be consistently placed, thus obviating
the need to preview each scan. Optimum scanning resolution depends on the type of
samples. Lower resolution can speed up scanning significantly, especially if the
samples require the use of large trays. Root length analyses are carried out with
grayscale images; saving images in grayscale reduces the image file size substantially.
3.2.1.4. Threshold Parameters
Analysis results can be sensitive to the threshold parameters used. WinRhizo
can automatically set these, but one can also manually tweak them from time to time.
3.2.1.5 Analyzing Scanned Images
To analyze the image, selects the region(s) of interest and it is analyzed. When
scanned images are analyzed, the software uses thresholding to determine what is root
25
and what is not root (each pixel is classified as either root or not root based on its
grayscale value; this is why shadows in images are problematic). Portions of the
image can be excluded from analysis if necessary, and there are basic editing tools
if minor image editing is required.
3.2.2 Qualitative estimation of ACC Deaminase activity
1. Trichoderma isolates were grown in 5 ml of synthetic medium (SM; Yedida et
al., 1991) incubated at 28oC at120 rpm for 24 hrs.
2. The cells were harvested by centrifugation at 3000 g for 5 min and washed
twice with sterile 0.1 M Tris-HCl (pH 7.5) and resuspended in 1 ml of 0.1 M
Tris-HCl (pH 7.5) and spot inoculated on petri plates containing synthetic
medium (SM; Yedida et al., 1991) modified supplemented with 3 mM ACC as
sole nitrogen source.
3. Plates containing only synthetic medium without ACC served as negative
control and with (NH4)2 SO4 (0.2% w/v) as positive control.
4. The plates were incubated at 28°C for 72 h.
Growth of isolates on ACC supplemented plates was compared to negative
and positive controls and was selected based on growth by utilizing ACC as nitrogen
source.
3.2.2.1 Quantitative Estimation of ACC Deaminase activity
ACC deaminase activity was determined by measuring the production of αketobutyrate and ammonia generated by the cleavage of ACC by ACC deaminase
(Honma and Shimomura, 1978; Penrose and Glick, 2003). For determination of
ACCD activity in Trichoderma 20µl spore suspension was inoculated in 10ml of
synthetic medium (Yedida et al., 1991) and the culture was grown for 48 hrs. The
washed mycelia were then transferred to 5ml of SM without ammonium and with 0.326
3 mM ACC. At the end of the induction period, the culture were resuspended in half
volume of Tris buffer 0.1 M (pH 8.5) and homogenized. Toluene (25µl) was added to
a 200 µl aliquot and vortexed vigorously for 30s. ACC (20 µl of 0.5 M solution) was
added, and after an incubation period of 15 min at 30°C, 1 ml of 0.56N HCl was
added. The lysates were centrifuged (10000g, 10 min) and 1ml of the supernatant was
mixed with 800 µl of 0.56N HCl and 300 µl of 2,4-dinitrophenylhydrazine (0.2g in
100 ml of 2N HCl). The mixture was incubated for 30 min at 30°C, after which 2 ml
of 2N NaOH was added. The A 540nm was measured. ACCD activity was evaluated
quantitatively by measuring the amount of α-ketobutyrate produced by the
deamination of ACC. ACCD activity was expressed in µmol of α-ketobutyrate / mg
protein / hr.
3.2.3 Screening isolates of Trichoderma spp. for Chitinase activity:
3.2.3.1 Preparation of colloidal chitin:
Colloidal chitin was prepared from commercial chitin (Hi Media) and was
used for chitinase detection test.
3.2.3.2 Acid hydrolysis of chitin:To prepare colloidal chitin, 5.0 g of chitin was suspended in 60 ml conc. HCl
and was acid hydrolyzed by constant stirring using a magnetic stirrer at 4ºC
(refrigerator) overnight.
3.2.3.3. Extraction of colloidal chitin by ethanol neutralization:To the resulting slurry 2000 ml ice cold 95% ethanol was added and was kept
at 28±2°C for overnight to neutralize the acid hydrolyzed chitin, which was then
centrifuged at 3000 rpm for 20 min at 4ºC. Supernatant was discarded and the pellet
was washed with sterile distilled water by centrifugation at 3000 rpm for 5 min at
27
4ºC. The washing of pellets was repeated till the smell of alcohol vanished. Colloidal
chitin thus obtained was stored at 4ºC until further use.
3.2.4 Screening of isolates of Trichoderma for chitinase activity:
3.2.4.1 Qualitative estimation:
For estimation of chitinase activity a simple technique developed by Agrawal
and Kotasthane (2012) was followed. Colloidal chitin and a pH indicator was
supplemented to the minimal media (Table 3.2), 20 ml of the molten chitin
supplemented media was poured in pre sterilized petri dishes. After solidification, the
plates were inoculated with culture plugs containing young actively growing
mycelium of Trichoderma isolates (Table 3.1). The plates were then incubated at
28±2°C. Chitinase activity was identified by the formation of purple coloured zone.
Observations were recorded for the colour intensity and diameter of the purple
coloured zone until 5th day after inoculation.
3.2.4.2 Quantitative estimation:
Culture plugs containing young actively growing mycelium of trichoderma
isolates were inoculated in colloidal chitin supplemented broth and incubated at 28°C
for 5 days at 200 rpm. Culture filtrates were obtained by filtering through whatman
filter paper no. 1. The filtrate obtain were analyzed for chitinolytic and N-acetyl-β-Dglucosaminidase activity. Filtered supernatant were stored at -20°C until use.
Two different procedures were followed to analyze the culture filtrates for
chitinolytic and N-acetyl-β-D-glucosaminidase activity.
Procedure I
Chitinolytic activity was assayed by measuring the release of reducing
saccharides from colloidal chitin. A reduction mixture contain 5ml of culture
supernatant, 1.5ml of 1M sodium acetate buffer (SA-buffer) pH 4.6 and 1ml of
28
colloidal chitin was incubated at 40°C for 20 hrs and then centrifuged at 13,000rpm
for 5 min after centrifuge, an aliquot of 3ml of the supernatant, 1ml of 1% solution of
dinitrosalicylic acid in 0.7M NaOH and 0.4ml of 10M NaOH and heated at 100°C for
5min. Absorbance of the mixture at 582nm was measured after cooling. A calibration
curve with N-acetyl-β-D-glucosamine (NAGA) as a standard was used to determine
reducing saccharide concentration under the assay conditions described a linear
correlation interval of 40-800mg/ml NAGA. Chitinolytic activity was estimated in
terms of concentration (mg/ml) of NAGA released.
Procedure II
N-acetyl-β-D-glucosaminidase (exochitinase) activity was measured and
monitored spectrophotomerically as the release of p-nitrophenol (pNP) from pnitrophenyl-N-acetyl-β-D-glucosamide (pNPg). A mixture of 125µl of testing solution
(culture filtrate), 1ml of pNPg solution (1mg/ml), and 5ml of 0.1M SA buffer (pH4.6) was heated at 40°C for 20 hrs and then centrifuged at 13,000 rpm for 5min. An
aliquot of 1.2ml of 0.125M sodium tetraborate-NaOH buffer (pH-10.7) was added to
supernatant (2.4ml) obtained, absorbance at 400nm was measured immediately after
mixing and pNp concentration (in terms of volume activity) in the solution was
calculated using the pNp molar extinction (18.5mM -1CM-1) with the help of following
formula.
Vt
–
Total volume (3600µl)
Vs
–
Sample volume (125µl)
18.5
–
Milimolar extinction coefficient of p-nitrophenol under the assay
condition (cm2/micromole)
1.0
–
Light path length (cm)
t
–
Reaction time (20 hrs=1200 minutes)
df
–
Dilution factor (27.8)
29
3.2.5 Screening of Trichoderma isolates for siderophore production
3.2.5.1 The CAS agar plate test
It is the universal test for detection and determination of siderophores. Chrome
azurol S solution was prepared and added to melted Potato dextrose Agar medium in
the ratio 1:10 (1 part CAS solution and 10 parts PDA). A 5mm mycelial disc of
isolates of Trichoderma spp. was cut from the plates of actively growing colony and
placed in the centre of each Petri plate. Colonies exhibiting a pink halo after 5 days
incubation (28 ±2o C) were considered positive for siderophore production.
3.2.5.2 Hydroxyquinoline mediated siderophore test
For selection of fungal isolates with high ability to produce siderophore,
isolates were inoculated on PDA supplemented with a strong chelater 8 Hydroxyquinoline (50mg/L) (De Brito Alvarez and Gagne, 1995). The iron
availability of this medium is too low and only the isolates with high ability of
siderophore production can grow on this medium. Inoculated isolates were incubated
at 28±2o C for 48-72hrs.
3.2.5.3 Arnow’s assay
Arnow’s assay was used for quantification of catechole type of siderophore
production. For qualitative estimation of siderophore quantification, actively growing
cultures of Trichoderma spp. with four discs (3 mm diameter) was inoculated to 20 ml
PDB (Hi Media) in 50 ml Tarson tubes and incubated for 3 days at 28±2°C. The
mycelium growth was removed by centrifugation at 3000 rpm for 5 min. 3 ml of the
culture supernatant was then mixed with 0.3 ml 5 N HCl solution, 1.5ml Arnow’s
reagent (10g NaNO2, 10g Na2MoO4.2H2O dissolved in 50 ml distilled water) and
30
0.3ml 10 N NaOH. After 5-10 min the presence or absence of pink colour was
observed.
3.2.5.4 Tetrazolium test
This test is based on the capacity of hydroxamic acid to reduce tetrazolium salt
by hydrolysis of hydroxymate groups using a strong alkali. The reduction and release
of alkali shows red colour to a pinch of Tetrazolium salt when 1-2 drops of 2N NaOH
and 0.1 ml of test sample is added. Instant appearance of a deep red colour indicated
the presence of Hydroxamate siderophore.
3.2.5.5 FeCl3 test
One ml of the culture supernatant was mixed with freshly prepared 0.5 ml 2%
aqueous FeCl3 and observed for presence and absence of deep red colour.
3.2.5.6 Quantitative spectrophotometric assay for siderophore production (liquid
assay)
The chrome azurol sulfonate (CAS) assay (Schwyn and Neilands, 1987) was
used to evaluate the siderophore production. For siderophore quantification, actively
growing cultures of Trichoderma spp. with four discs (3 mm diameter) was inoculated
to 20 ml PDB (Hi Media) media in tarson tubes and incubated for 3 days at 28±2°C.
The mycelium growth was removed by centrifugation at 3000 rpm for 5 min. 0.5 ml
of the culture supernatant was then mixed with 0.5 ml CAS solution and 10µl
shuttling solution (sulfosalicyclic acid). After 20 min. of incubation, the colour
obtained was determined using the spectrophotometer at 630 nm. Only PDB was used
as blank while reference solution was prepared by adding CAS dye and shuttle
solution to PDB and absorbance was recorded. Values of siderophore released in PDB
was expressed in percent siderophore units and calculated using the formula:
31
Where,
Ar = OD of reference solution,
As = OD of sample
3.2.6 Screening the isolates of Trichoderma spp. for Plant growth promoting
traits
3.2.7 Estimation of inorganic phosphate for phosphate solubilisation ability of
Trichoderma spp.
3.2.7.1 Qualitative estimation in Agar medium
Isolates of Trichoderma were inoculated on Pikovskaya agar medium
(Himedia) for evaluating its ability to solubilize supplemented tri-calcium phosphate
in vitro. Pre-sterilized molten media was supplemented aseptically with BCP
(bromocresol purple 0.1 gL-1) as pH indicator (Vasquez et al., 2000) and poured in
petri dishe(s). Plates were inoculated with young actively growing cultures of
Trichoderma spp. and incubated at 28±2°C for 3 days. After incubation phosphate
solubilising positive isolates turned the media from purple to yellow in the zone of
acidification which indicated positive response for phosphate solubilising efficacy of
Trichoderma spp.
3.2.7.2 Quantitative estimation in Liquid medium
The quantitative procedure described by Nautiyal, (1999) was used to screen
phosphate solubilising efficacy of Trichoderma spp. with slight modification. Isolates
of Trichoderma spp. were inoculated on Pikovskaya broth medium (Pikovskaya,
1948) to evaluate its ability to solubilize supplemented phosphate in vitro. 30ml of
Pikovskaya broth was poured in 100ml conical flasks and sterilized. After sterilization
32
the media was cooled and inoculated with 1cm disc of different Trichoderma isolates.
The flasks were incubated at 28±2°C for 3-6 days on orbital shaker at 100rpm. After
incubation the whole culture was transferred to 50ml Tarson tube, centrifuged at
5000rpm for 10min. and the supernatant used for quantitative estimation of phosphate
solubilization. To 0.5ml of sample supernatant 5ml of chloromolybdic acid (CMA)
was added and the volume was made up to 20ml with distilled water. To the above
solution 125µl of chlorostannous acid (CSA) was added and final volume was made
to 25ml with DW. The tubes were incubated for 15min. at RT for the development of
blue color and the absorbance was measured at 610nm. Blank was prepared with DW
in place of sample supernatant, reference or control was prepared with un inoculated
broth supernatant instead of sample supernatant. The standard solution was prepared
with KH2PO 4 (10 mg/ml) in distilled water with the increase in conc. of stock 0µl,
1µl, 2.5µl, 5 µl, 10 µl, 20 µl, 30 µl, 40 µl, 50 µl, 60 µl, 70 µl, 80 µl. 90 µl and the
conc. of KH2PO4 (10 mg/ml) was 0µg, 10µg, 25 µg, 50 µg, 100 µg, 200 µg, 300 µg,
400 µg, 500 µg, 600 µg, 700 µg, 800 µg and 900 µg. Measurement of OD was taken
at 610nm.
3.2.8 Quantification of Indole acetic acid production by Trichoderma spp.
For the production of indolic compounds, an active cultures of Trichoderma
spp. with four discs (3 mm diameter) was inoculated to 20 ml DF salts minimal media
(Dworkin and Foster, 1958) in 100ml conical flasks and incubated for 3 days at
28±2°C. The medium was supplemented with L-tryptophan at a concentration of 1.02
g/L. After incubation for 72 hours, the mycelium was removed from the culture
medium by centrifugation at 5,000 rpm for 5 min. One ml aliquot of the supernatant
was mixed vigorously with 4 ml of Salkowski’s reagent (Gordon and Webber, 1951)
with blank as DF salts minimal media and allowed to stand at RT for 20 min. before
33
the absorbance at 535 nm was measured in colorimeter. The standard curve was
prepared from 2 mg/ml stock IAA and the concentration of IAA in each culture was
determined by comparison with standard curve.
3.2.9 Estimation of Hydrogen cynide (Volatile inhibitory compound)
The production of HCN compound was estimated by method of Wei et al.
(1991). The cultures was grown on PDA plates supplemented with 4.4g/L glycine as a
precursor molecule for hydrogen cynide production and the filter paper strips soaked
in saturated picric acid solution were exposed to the growing trichoderma isolates.
The plates were incubated for 7 days at 28±2°C and observations were recorded as
colour of filter paper turning to brown recorded as positive for HCN production.
3.2.10 Media preparation used in the present study
A.
Table 3.2 Composition of different media used in present investigation
Potato dextrose Agar (PDA) Medium.
1
Potato
250g
2
Dextrose
18g
3
Agar
15g
4
Distilled water
1000ml
B.
Chitin amended medium (pH4.7)
1
Colloidal chitin
4.5 g
2
MgSO4.7H2O
0.3 g
3
(NH4)2SO4
3g
4
KH2 PO4
2g
5
Citric acid monohydrate
1g
6
Tween 80
200µl
7
Bromo cresol purple
0.15 g
34
8
Agar
15 g
9
Distilled water
1000ml
D.
Pikovskaya agar medium
1
Yeast extract
0.50g
2
Dextrose
10g
3
Calcium phosphate
5g
4
Ammonium sulphate
0.5g
5
Pottasium chloride
0.2g
6
Magnesium sulphate
0.1g
7
Manganese sulphate
0.0001g
8
Ferrous sulphate
0.0001g
9
Agar
15 g
10
Distilled water
1000ml
E.
Pikovskaya broth medium
F.
In Pikovskaya broth medium all chemical compositions are same as Pikovskaya
Agar medium except absence of agar
Synthetic Medium
1
Glucose
15.0 g
2
MgSO4.7H2O
0.2g
3
K2HPO4
0.6 g
4
KCl
0.15 g
5
NH4NO3
1g
1 ml of trace elements (per liter of stock solution)
6
FeSO4·7H2O
0.005 g
7
MnSO4 ·H2O
0.006 g
35
8
ZnSO4 ·H2O
0.004 g
9
CoCl2
0.002 g
10
Distilled Water
1000ml
G.
DF Salts Minimal Medium (pH 7.2)
1
KH2 PO4
4g
2
Na2HPO4
6g
3
MgSO4.7H2O
0.2g
4
Glucose
2g
5
Gluconic acid (Sodium Gluconate)
2g
6
Citric acid
2g
7
FeSO4.7H2O
1mg
100µl of trace elements
8
H3BO3
10µg
9
MnSO4.H 2O
11.19µg
10
ZnSO4.7H2O
124.6µg
11
CuSO4.5H2O
78.22µg
12
MoO3
10µg
13
(NH4)2SO4
2g
3.2.11 Molecular Characterization of the isolates of Trichoderma
3.2.11.1 Fungal growth conditions and DNA extraction:
1. To obtain the vegetative growth of all the isolates of Trichoderma spp. 3 to 4
mycelial blocks from one week old culture (approx. 3mm × 3mm) were transferred
separately in 50 ml potato dextrose broth (HiMedia) medium contained in 100 ml
flasks. The flasks were incubated by shaking (100 rpm) at 28±2°C for 7 days.
36
2. Vegetative growth (mycelia) was harvested from the broth by suction filtration
through Whatman no. 1 filter paper using a Buchner filtration apparatus connected
to a vacuum pump. The mycelia mat was removed from the filter paper; blot dried
and was then used for DNA extraction.
3. Mycelia mat was grind to uniform consistency in 1000 µl resuspension buffer using
PowerlyzerTM 24.
4. The grinded sample (500 µl) were collected in a 1.5 ml eppendorf tube to which
150µl 5M potassium acetate buffer was added, vortexed briefly. Incubated in water
bath at 65°C for 10 min and then centrifuged at 13,000 rpm for 10 min.
5. To the supernatant added 600µl of chloroform : isoamyl alcohol (24:1), inverted the
tubes for 5 min. and centrifuged at the 12,000rpm for 10min. Supernatant was taken
carefully and transferred to another new eppendorf tubes.
6. Added equal volume of pre-chilled isopropanol and inverted tubes gently, kept the
tubes in deep freezer for half hour and then centrifuged again for 12,000 rpm for
10min. Supernatant was discarded and pellet was saved.
7. The pellets was washed with 300µl of 70% ethanol, air dried and resuspended in
50µl of TE buffer and left overnight.
8. 5µl RNase (10 mg/ml) was added to the samples and incubated at 37°C for 1 hour.
The samples were precipitated with double volume of pre chilled absolute alcohol
and incubated at -20°C deep freezer for 30 min. followed by spinning at 13000rpm
for 10 minutes.
9. Supernatant was discarded and the pellet was washed with 70% ethanol and then
air dried. The DNA pellet was dissolved in 100 µl of TE buffer and stored at -20°C
till further use.
3.2.12 Quantification of genomic DNA:-
37
The DNA samples were quantified using Nanodrop Spectrophotometer (ND
1000). One µl of isolated DNA was placed over lower pedestal of Nanodrop to record
absorbance at 260nm and concentration was estimated using TE buffer as blank
reference. After quantification, the DNA was diluted with sterile water to get a final
concentration 30ng /µl. The absorbance ratio (A260/A280) was recorded for each
sample to find out the purity of DNA. The acceptable absorbance ratio (A260/A280)
for pure DNA is 1.8.
3.2.13 Polymerase Chain Reaction (PCR) analysis of Isolates of Trichoderma spp.
A set of 9 primer pairs (Table 3.3) were used for PCR based DNA
fingerprinting for the analysis of 10 isolates of Trichoderma spp. The reaction mixture
and the temperature profiles used are summarized in Table 3.4 to Table 3.15
respectively. 5% non-denaturing PAGE was performed to separate the PCR amplified
products.
38
Table 3.3 List of DNA primers and their sequence were used in present investigation
S.
No
.
1
2
3
4
5
6
7
8
9
10
Primer
Sequence(5’-3’) and Tm value (oC)
Primer
Length
GAGTTCAAGGAGGCCTTCTCCC (68.3)
CATCTTTCTGGCCATCATGG (65.4)
ATGTGCAAGGCCGGTTTCGC (73.3)
TACGAGTCCTTCTGGCCCAT (65.8)
GGCAAGGTCGACATCTATGC (64.5)
GGCTTGCCATTCAGCTATG (63.3)
GAAATTCCATATGACGGTGCATAAAAAACAG (70.8)
22
20
20
20
20
19
31
CGGGATCCCTTGTCGCCTTGCTCT (77.1)
24
ATCGCCAGGGGCGGATGTGC (77.5)
ACGATGTGCTCGGCGTAC (65.4)
TGCGGCATGGGCGTGTGCCATTGCTGCCTGG (91.7)
CCGCTCTTGATCTGCAATTGCAGGCC (78.5)
GCTCCTCAGTGCTTCTTCC (61.8)
GGGAATGCCGACAAGAAGC (66.9)
GAACTGGAGGCTCATCTAC (56.2)
GATGATGTTGTCCATGTTG (57.1)
TGCGACCCGACCAAGAACTG ()
CAGATGATGGTGTCGAGGCTG ()
GCACGCTCTTCATTGACCAG (66)
CACAGTCATGCACATCAACCTG (66.6)
20
18
31
26
19
19
19
19
20
21
20
22
Reference
CAL-228F
CAL-737R
ACT-512F
ACT-782R
AcdS-F
AcdS-R
nitrilase P1-F
nitrilase
PfluHisB-R
hcnACa
hcnACb
hcnPM2
hcnPM7-26R
chit33-F
chit33-R
nag1RT-F
nag1RT-R
Nag-2RT-F
Nag-2RT-R
Nag-2F
Nag-2R
Chaverri et al (2003)
Carbone and
Kohn (1999)
Duan et al (2009)
Kiziak et al (2005)
Ramette et al (2003)
Svercel et al (2007)
Matarese et al(2010)
Seidl et al (2006)
Seidl et al (2006)
Seidl et al (2006)
39
Expected
Product size
500bp
1000bp
1052bp
587bp
571bp
130bp
146bp
1.8kb
Table 3.4 Reaction mixtures used for polymerase chain reaction (PCR) with gene
specific primers.
Sr. No. Component
Stock concentration Volume/reaction
1
DNA
40ng/ml
3µl
2
ADW
-
10.5 µl
3
10X PCR Buffer
10X
2 µl
4
dNTP
1mM
2 µl
5
Primer F
10µM
1 µl
6
Primer R
10µM
1 µl
7
Taq polymerase
1 U/µl
0.5 µl
Table 3.5 Thermal profile for amplification of primers Acc G
Steps
Activity
Temperature (°C)
Time (min.)
1 Initial Denaturation
95°C
3
2 Denaturation
95°C
1
3 Annealing
55°C
1
4 Extension
72°C
1
5 Final Extension
72°C
5
6 Storage
4°C
∞
Repeats
1
30
1
Table 3.6 Thermal profile for amplification of primers Actin
Steps
Activity
Temperature (°C)
Time (min.)
1 Initial Denaturation
94°C
5
1
2 Denaturation
94°C
1
30
3 Annealing
60°C
1
40
Repeats
4 Extension
72°C
1
5 Final Extension
72°C
7
6 Storage
4°C
∞
1
Table 3.7 Thermal profile for amplification of primers CAL
Steps
Activity
Temperature (°C)
Time (min.)
Repeats
1 Initial Denaturation
94°C
5
1
2 Denaturation
94°C
1
35
3 Annealing
58°C
1
4 Extension
72°C
45 sec
5 Final Extension
72°C
7
6 Storage
4°C
∞
1
Table 3.8 Thermal profile for amplification of primers Chitinase33
Steps
Activity
Temperature (°C)
Time (min.)
1 Initial Denaturation
94°C
3
1
2 Denaturation
94°C
1
25
3 Annealing
60°C
1
4 Extension
72°C
1
5 Final Extension
72°C
7
6 Storage
4°C
∞
41
Repeats
1
Table 3.9 Thermal profile for amplification of primers HCN AC and HCN
PM
Steps
Activity
Temperature (°C)
Time (min.)
Repeats
1 Initial Denaturation
94°C
02:30
1
2 Denaturation
94°C
30 sec
30
3 Annealing
63°C
30 sec
4 Extension
72°C
1
5 Final Extension
72°C
10
6 Storage
4°C
∞
1
Table 3.10 Thermal profile for amplification of primers NAG-1
Steps
Activity
Temperature (°C)
Time (min.)
Repeats
1 Initial Denaturation
94°C
5
1
2 Denaturation
94°C
1
30
3 Annealing
54°C
1
4 Extension
72°C
1
5 Final Extension
72°C
7
6 Storage
4°C
∞
1
Table 3.11 Thermal profile for amplification of primers NAG-2 RT
Steps
Activity
Temperature (°C)
Time (min.)
1 Initial Denaturation
95°C
5
1
2 Denaturation
95°C
1
30
3 Annealing
63°C
1
42
Repeats
4 Extension
72°C
1
5 Final Extension
72°C
7
6 Storage
4°C
∞
1
Table 3.12 Thermal profile for amplification of primers Nitrilase
Steps
Activity
Temperature (°C)
Time (min.)
Repeats
1 Initial Denaturation
94°C
3
1
2 Denaturation
92°C
1
30
3 Annealing
65°C
1
4 Extension
72°C
2
5 Final Extension
72°C
5
6 Storage
4°C
∞
1
3.2.14 Visualizing PCR products on native (non-denaturing) polyacrylamide gel
electrophoresis (PAGE)
1. The Biorad Sequencing gel apparatus was used for PAGE of PCR products.
The plates and spacers were cleaned thoroughly with three washes of distilled
water, followed by two washes with 75% alcohol and then with 100% alcohol
respectively.
2. 1ml bind silane (945µl Absolute ethanol, 45µl Autoclaved distilled water, 5µl
Glacial acetic acid, 5µl Bind solution (3-trimethoxysilyl)) solution was applied
on unnotched plated, uniformly spread by wiping with a tissue paper, and kept
for drying for 10 min.
3. 1 ml of sigma cote (sigma. cat. # SL-2) was applied to the notched plate,
uniformly spread with a tissue paper and kept for drying for 10 min.
43
4. Post-drying the gel plates were assembled as per manufacturer’s instructions
so that the processed surfaces of both plates face each other. The comb was
inserted in upside down orientation and the gel was allowed to polymerize for
at least an hour.
5. To 100 ml of the 5% Polyacrylamide gel solution, 650µl of freshly prepared
10% ammonium per sulphate (APS) solution and 65 µl TEMED were added
and swirled gently. The solution was gently poured in the gap between the two
glass plates by taking care to prevent the formation of air bubbles using
syringe.
6. The gel plates were fixed to the electrophoresis mold/gel tank and 1X TBE
(running buffer) was added to the buffer chamber of the mold.
7. After one hour, the comb was removed gently without disrupting the gel line
and the well was washed with TBE buffer by using a syringe.
8. The comb was inserted again to form the wells by ensuring that the teeth of
comb just touched the gel line uniformly.
9. The PCR reaction mixture from PCR was mixed with 3 µl of formamide dye
.3µl of each non-denatured sample was loaded and the samples were
electrophoresed at a constant 800 V for 30 min. After completion of the run,
the plates were allowed to cool to room temperature and then the bands were
visualized by silver staining.
3.2.14.1 Silver Staining
1. After electrophoresis, the power supply was disconnected, the comb was
removed and the notched plate was separated from the unnotched plate. The
gel adheres to the unnotched plate coated with bind silane.
44
2. The plate transferred to a tray containing 1 liter fix/stop solution and kept for
15 min at room temperature in the solution with shaking at 90 rpm. The gel
was then given a quick wash with distilled water for 30 s.
3. Then the gel plate was transferred to a tray containing 0.2% silver nitrate
solution, and kept for 25 min in dark with slow shaking, followed by a quick
wash with distilled water for 30 s.
4. The gel plate was then placed in the tray containing one liter developer
solution and kept for 5 to 10 min until bands began to appear. After bands with
detectable intensity appeared, the gel was transferred to a tray containing
distilled water and rinsed for few minutes.
5. The gel was kept for air-drying and subsequently the bands were captured by
scanning and/or photography
3.2.14.2 NATIVE PAGE / NON-DENATURING PAGE
CBS Scientific Dual vertical unit is used for preparation of PAGE
1. Clean the glass plates thoroughly with mild detergent and wipe it with alcohol.
After complete drying of plates apply gasket around the sides of rectangular
back plate starting from one end. Keep the flat side of gasket on bottom side of
the plate.
2. Align the notches of the gasket around the rounded corners of the glass plate.
Once the gasket is pushed over the bottom edge and corners, work it down the
remaining side.
3. Place the gasketed plate on flat surface with the tubing side up and place the
spacers on two sides of plate (rounded corners of spacers should face outside
bottom of the plate).
45
4. Place the notched plate on top of the bottom assembly and place two clamps
on bottom of the plate assembly.
5. Lift the assembly and stand it on the base of the clamps. Push the plates down
to level the assembly. Clamp the sides of the plates.
6. Apply PAGE solution to the gel plate sandwitch using notched beaker or
syringe.
7. Insert the comb and allow the gel to solidify.
8. After 20-30 minutes disassemble the clamps (only bottom clamps) and gasket.
9. Position the plate sets in the electrophoresis unit with notched plate facing the
upper buffer reservoir and clamp on sides. If running one side of dual plate
assembly, use unnotched back plate to seal the open side of upper reservoir.
10. Pour freshly prepared buffer in upper and lower chambers. Remove the combs
and load the gel samples mixed with dye.
11. Align the power leads, attach safety cover and connect to power supply. Run
at 150-200 volts for 20-30 minutes.
12. After the run completes, switch off the power supply and disassemble the
glass plates.
13. Remove the side clamps and pull notched plate outside taking into care that
the gel sticks to the plane glass plate.
14. Keep the gel for staining in 1X TBE buffer containing 4µl/100ml ethidium
bromide (10mg/ml stock). Stain for 5-10 minutes, rinse the gel with distilled
water and visualize the bands on UV transilluminator or gel documentation
system.
46
3.2.15 BUFFERs, SOLUTIONs AND REAGENTS
A. Chrome Azurol assay sulphonate (CAS) solution
1. 1M FeCl3.6H2O(stock solution)
Dissolved 2.703g of FeCl3.6H2O in 10ml of distilled water.
2. 1M HCl (stock)
Diluted 0.86ml of conc. HCl to 10ml with distilled water
3. Iron (Ш) solution
Chemicals
FeCl3.6H2O
Stock
Concentration
1M
Working
concentration
1mM
Volume required
per 1000ml
10ul
HCl
1M
1mM
100µl
Mixed both the components and made up the volume to 10ml with autoclaved
distilled water.
4. Chrome Azurol S solution
In a beaker 60.5mg of CAS was dissolved in 50ml autoclave distilled
water and mixed with 10ml Iron (Ш) solution (1mM FeCl3 .6H2O+10mM HCl)
stirred and to it HDTMA solution (72.9mg dissolved in 40ml of autoclave
distilled water) was added slowly. This resulted in a formation of a dark blue
liquid.
B. Arnow’s reagent
S. No.
Component
Amounts(g/L)
1
Sodium nitrite
10g
2
Sodium molybdate
10g
3
distilled water
50ml
47
C. Reagents used for quantitative estimation of inorganic phosphate1. Chloromolybdic acid
Dissolved 7.5g ammonium molybdate in 150ml autoclaved distilled water and
mixed into 162ml conc. HCl. Made up the final volume to 1000 ml with autoclaved
distilled water and stored in amber colour bottle at 4 oC.
2. Chlorostannous acid
Dissolved 25g stannous chloride in 100ml conc. HCl and made up the final
volume to 1000 ml with autoclaved distilled water and stored in amber colour bottle at
4 oC.
D. Reagents required for estimation of chitinase activity:
1. 10 M NaOH:Dissolved 40gm of NaOH pellets in 100ml autoclaved distilled water
2. 0.7 M NaOH:Dilute 7ml of 10 M NaOH to 100 ml with autoclaved distilled water
3. 1 M sodium acetate solution:Dissolved 13.61gm of trihydrated sodium acetate (or 8.204gm of anhydrous
sodium acetate) in 100ml of autoclaved distilled water.
4. 1M acetic acid solution:Dilute 6ml of glacial acetic acid to 100 ml with autoclaved distilled water.
5. 1 M sodium acetate-acetic acid buffer (pH-4.6):To prepare 100 ml of SA-AA buffer (pH-4.6) 48 ml of 1 M sodium acetate
solution was mixed with 52 ml of 1 M acetic acid solution.
6. 50 mM SA-AA solution:Dilute 5 ml of 1 M SA-AA solution to 100 ml with distilled water.
7. 1% colloidal chitin in 50 mM SA-AA solution:48
Dissolved 1gm of colloidal chitin in 100ml of 50 mM SA-AA solution by
continuous stirring in magnetic stirrer.
8. 1% DNSA solution in 0.7M NaOH:Dissolved 1gm of DNSA in 0.7M NaOH solution by continuous stirring in
magnetic stirrer.
9. 0.125M sodium tetraborate-NaOH buffer (pH-10.7)
Dissolved 4.768gm of sodium tetraborate in 80ml of autoclaved distilled water
with few NaOH pellets till the pH reached to 10.7. The final volume was made
upto 100ml with autoclaved distilled water.
10. p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNPg) solution (1mg/ml):Dissolved 50mg pNPg in 40 ml autoclaved distilled water by gentle warming
and then made upto 50ml by autoclaved distilled water.
11. 0.1M SA_AA solution:Dilute 10ml of 1M SA-AA solution to 100ml with autoclaved distilled water.
E. Genomic DNA extraction buffer:
1. 1M Tris-HCl solution (pH 8)
121.1gm Tris-HCl was dissolved in 800 ml distilled water. Volume was made
upto 1000 ml with distilled water and was sterilized by autoclaving after adjusting the
pH to 8 with HCl.
2. 0.5M EDTA solution (pH 8.0)
186.1 gm EDTA was dissolved in 800ml distilled water. The solution was
stirred vigorously on a magnetic stirrer and adjusted to pH to 8.0 by adding NaOH
pellets. The final volume was made up to 1000 ml with distilled water followed by
sterilizing in autoclaving.
3. Chloroform
49
4. Ethanol (100% & 70%)
5. Isoamyl alcohol.
6. 5M Potassium acetate-Acetic acid buffer.
S. No.
Chemicals
Volume required per 100ml
1
5M Potassium acetate
10 ml
2
Glacial acetic acid
11.5Ml
Mixed both the components and adjust the final volume 100ml with distilled
water. (5M potassium acetate was prepared by dissolving 49.07 g of potassium acetate
in 100ml autoclaved distilled water.)
7. Isopropanol.
8. DNA resuspension buffer(100ml)
Tris-HCl(pH 7.5)
Stock
Concentration
1M
Working
concentration
10mM
Volume required
per 1000ml
1ml
EDTA(pH 7.5)
0.5M
10mM
2ml
SDS
10%
1%
10ml
Chemicals
9. TE buffer (1 litre) (pH-8.0).
10 ml 1M Tris HCl and 2 ml 0.5 M EDTA were mixed and final volume was
made up to 1000ml with autoclaved distilled water.
10. Sodium acetate (3M pH 5.2)
3M Sodium acetate was prepared by dissolving 24.61g of unhydrous Sodium
acetate in 30ml autoclaved distilled water and adjusted pH 5.2 with glacial acetic acid.
The final volume was made up to 100ml with autoclaved distilled water.
50
11. 20% SDS:Dissolved 20gm SDS in 100ml autoclaved distilled water.( Do not autoclaved
SDS solution)
12. 5M NaCl:Dissolved 73.05gm NaCl in 200ml distilled water and made final volume upto
250ml with distilled water. (Warm water was used).
13. 1% NaCl:Dissolved 1gm of NaCl in 70ml autoclaved distilled water and made final
volume upto 100ml.
14. RNase Solution (10mg/ml):Dissolved 10g RNase powder in 1ml of TE buffer by boiling. Allowed to cool
at room temperature and final volume upto 250ml with distilled water.( Warm water
was used).
Stocks and solutions for PAGE and silver staining:1. 10X TBE:S.NO.
Components
Conc. required
Quantity
1
Tris Base
89mM
10.8g
2
Boric acid
89mM
5.5g
3
0.5M EDTA (pH 2mM
4ml
8.0)
Mixed all the components and adjusted the volume to 100ml with distilled
water.
2. 1X TBE:Diluted 10ml of 10X TBE to 100ml with distilled water.
51
3. Polyacrylamide Stock Solution (40%)
Dissolved 38g of Acrylamide and 2g Bis-acrylamide in 100ml of autoclaved
distilled water and filtered throughWatman filter paper no.1. Stored in brown
bottle in refrigerator.
4. 5% Polyacrylamide gel
S. NO.
Components
Volume per 100 ml
1
40% Polyacrylamide
12.5ml
2
10% APS
700µl
3
TEMED
70µl
Mixed all the components and adjusted the final volume to 100ml with 1X
TBE solution.
5. Formamide Dye was prepared by mixing following components
S. No
Stock
Final conc.
For 10ml
1
Formamide
98%
9.8ml
2
0.5 M EDTA
10mM
200µl
3
Xylene cyanol
0.1%
0.01g
4
Bromophenol blue
0.1%
0.01g
6. Fix/Stop solution was prepared by mixing following components
Sr. No.
Stock
For 1000ml
1
100% ethanol
100ml
2
Glacial acetic acid
5ml
3
Nanopure water
895ml
52
7. Staining Solution was prepared by mixing following components
Sr. No.
Stock
1
Silver nitrate
2
Nanopure water
Final
concentration
0.2%
For 1000ml
2g
1000ml
8. Developer was prepared by mixing following components and final
volume made up to 1000 ml with water
Sr. No.
Stock
Final
concentration
For 1000ml
1
Sodium hydroxide
3%
30g
2
Formaldehyde
0.5%
5ml
9. Ammonium persulphate (APS) – l0% solution was prepared by mixing
following components
Sr. No.
Component
Final concentration
1
Ammonium persulphate
1.0g
2
Distilled water
10ml
10. Bind silane solution
Bind Silane: (3- (Trimethoxysilyl) propyl methacrylate methacrylsaure-3
trimethoxysilyl propylester) was prepared by mixing following components.
S. No.
1
Stock
100% Ethanol
For 1 ml
945µl
2
Bind silane
5µl
3
Glacial acetic acid
5µl
4
Distilled water
45µl
53
CHAPTSER IV
RESULT AND DISCUSSION
4.1 Analyzing root system morphology against candidate Trichoderma isolates.
Ten isolates of trichoderma were evaluated in the present investigation to
study the effect on root morphology of the selected genotype/ rice lines. Differential
response of trichoderma isolates was observed against four selected rice genotypes
Like crops are affected by abiotic stresses, microbes are also known to be
affected by these conditions. However, successful deployment of these organisms in
stressed ecosystems depend on their ability to withstand and proliferate under adverse
environments such as drought, high temperature, salt stress, mineral deficiency,
chemical and heavy metal toxicity which are major problems in rainfed agroecosystems. Microorganisms could play a significant role in stress management, once
their unique properties of tolerance to extremities, their ubiquity and genetic diversity
are understood and methods for their successful deployment in agriculture production
are developed. These organisms also provide excellent models for understanding
stress tolerance mechanisms that can be subsequently engineered into crop plants.
Plant-associated microorganisms can play an important role in conferring resistance to
abiotic stresses. These organisms could include rhizoplane and endophytic bacteria
and symbiotic fungi and operate through a variety of mechanisms like triggering
osmotic response and induction of novel genes in plants. Increase in root length as
compared to control was observed in several different Trichoderma isolate vs. rice
genotype treatment combinations. Table 4.1 indicate a maximum increase in the root
and
shoot
length
(in
cm)
as
compared
54
to
untreated
control
plants
Table 4.1: Efficacy of Trichoderma isolates on different rice varieties for root and shoot length development
IR-64
Sahbhagi Dhan
RRF-75
IBD-1
Treatment
RL
SL
RL
SL
RL
SL
RL
SL
Control
30.50±3.96
22.08±2.53
34.50±2.87
22.33±3.58
32.67±3.09
24.33±4.84
36.80±1.06
18.60±2.43
IRRI-1
30.33±3.10
17.83±2.20
25.20±0.97
13.80±1.02
34.17±3.63
23.00±1.62
30.50±2.20
18.08±3.61
IRRI-2
33.50±2.45
23.08±1.98
27.83±4.37
19.75±4.11
27.33±2.85
19.83±2.83
33.67±4.00
22.50±2.60
IRRI-3
38.00±0.00
27.00±0.00
28.00±0.00
13.00±0.00
27.67±4.99
23.67±2.62
26.58±4.31
18.08±2.07
IRRI-4
33.60±5.79
19.90±3.82
35.75±1.50
29.08±3.71
35.83±2.36
23.00±2.08
35.50±4.83
28.40±1.93
IRRI-5
33.67±3.62
25.33±3.27
36.42±2.75
24.42±2.52
36.67±2.01
17.33±2.69
30.40±0.85
28.80±2.15
IRRI-6
33.83±1.62
26.67±1.61
26.00±1.67
26.00±2.05
29.00±2.72
35.17±2.61
31.90±2.03
19.50±2.18
TH3
28.00±2.90
19.00±2.44
33.25±3.17
23.00±2.31
27.38±1.28
12.13±0.67
23.83±4.42
14.25±1.03
TV12
33.58±3.098
26.17±2.40
33.20±1.90
26.20±3.50
29.00±1.66
15.00±0.77
37.00±3.05
26.80±1.59
94a
33.50±1.38
25.17±2.41
33.40±2.01
26.40±2.97
32.75±3.37
17.50±2.07
28.92±3.17
18.33±2.56
T14
40.00±1.91
24.00±1.53
34.67±2.56
23.83±1.83
39.33±2.51
22.83±0.91
32.92±1.07
19.83±2.70
Max.
40.00±1.91
26.17±2.40
36.42±2.75
29.08±3.71
39.33±2.51
35.17±2.61
37.00±3.05
28.80±2.15
Min.
28.00±2.90
17.83±2.20
25.20±0.97
13.00±0.00
27.33±2.85
12.13±0.67
23.83±4.42
14.25±1.03
55
Figures in parenthesis indicate the increase in root length over control. As observed
that trichoderma seed treatment contributes towards increased root length, (Plate no.
4.1, 4.2, 4.3) we therefore scanned the roots derived from all the treatment
combination using root scanner machine Epson Perfection V700/V750, 3.81 Version,
WinRhizo Reg 2009 and observations were recorded on several root parameters like
total length of root, total surface area, average diameter, root volume tips and forks
(Table no. 4.2 to 4.5). It shows maximum total length in IRRI-4 isolate treatment
while IRRI-3 shown the maximum surface area. Similarly T14 isolate gives maximum
root diameter, root volume has maximum in treatment with TV12 isolate in IR-64
rice line whereas in Sahbhagi dhan T14 and IRRI-4 shown highest total length and
surface area respectively while IRRI-6 and TV12 gives most diameter and root
volume. RRF-75 rice line when treated with different Trichoderma isolates they
shown differences in various parameters over control IRRI-4 shown the maximum
total length and surface area and root volume, while IRRI-6 shown the maximum
diameter. In Indira barani dhan rice line the most effective isolates are IRRI-4, IRRI3, T14 and IRRI-6 which gives maximum increase in total length, total surface area,
root diameter and root volume respectively. We hypothesized that upon trichoderma
treatment trichoderma may operate through a variety of mechanisms and induce of
novel genes in plants contributing toward enhanced root growth.
56
57
58
59
Table No. 4.2 Total phenotypic data for total length of root, total surface area,
average diameter, root volume, tips and forks of IR-64 rice line
Treatments
Control
IRRI-1
IRRI-2
IRRI-3
IRRI-4
IRRI-5
IRRI-6
TH3
TV12
T14
94a
Max
Min
Total
Length
379.26
362.11
449.04
452.23
457.98
431.77
450.50
419.70
434.77
426.07
433.39
457.98
362.11
Surface
Area
22.48
22.93
23.84
24.11
24.02
23.62
23.74
23.33
23.72
23.47
23.52
24.11
22.48
Average
Diameter
(mm)
0.18
0.19
0.18
0.19
0.16
0.18
0.18
0.18
0.20
0.21
0.20
0.21
0.16
Root
Volume
(cm3)
0.03
0.06
0.13
0.18
0.10
0.17
0.15
0.05
0.19
0.14
0.07
0.19
0.03
Tips
Forks
2068.00
2585.67
11186.00
9925.00
10543.50
11898.33
9411.33
3665.00
9583.67
6338.33
3738.33
11898.33
2068.00
1547.67
2298.67
9699.67
7802.00
7978.00
8514.33
5261.67
1700.00
5981.00
4355.67
2382.00
9699.67
1547.67
Table No. 4.3 Total phenotypic data for total length of root, total surface area,
average diameter, root volume, tips and forks of Sahbhagi dhan
Treatments
Control
IRRI-1
IRRI-2
IRRI-3
IRRI-4
IRRI-5
IRRI-6
TH3
TV12
T14
94a
Max.
Min.
Total
Length
423.80
433.74
426.87
439.17
439.28
432.97
428.78
430.92
434.32
440.26
435.57
440.26
423.80
Surface
Area
23.12
23.62
23.43
23.74
23.97
23.65
22.92
23.46
23.72
23.73
23.73
23.97
22.92
Average
Diameter
(mm)
0.19
0.21
0.21
0.18
0.20
0.17
0.23
0.21
0.21
0.19
0.22
0.23
0.17
60
Root
Volume
(cm3)
0.05
0.07
0.15
0.01
0.13
0.06
0.15
0.12
0.16
0.13
0.11
0.16
0.01
Tips
2725.00
3106.00
5693.00
984.00
7049.67
5150.33
4963.50
5013.00
7295.33
7559.67
4800.33
7559.67
984.00
Forks
1854.67
2313.50
3762.00
286.00
5491.33
2546.00
5903.50
3654.33
4535.00
4440.33
3063.33
5903.50
286.00
Table No. 4.4 Total phenotypic data for total length of root, total surface area,
average diameter, root volume, tips and forks of RRF-75
Treatments
Control
IRRI-1
IRRI-2
IRRI-3
IRRI-4
IRRI-5
IRRI-6
TH3
TV12
T14
94a
Max.
Min.
Total
Length
391.70
429.90
427.95
432.27
434.53
400.27
428.94
420.53
402.75
419.12
433.07
434.53
391.70
Surface
Area
22.55
23.45
23.32
23.53
23.68
23.27
23.23
23.62
23.39
23.39
23.57
23.68
22.55
Average
Diameter
(mm)
0.21
0.22
0.25
0.22
0.24
0.20
0.25
0.15
0.19
0.19
0.28
0.25
0.15
Root
Volume
(cm3)
0.06
0.16
0.23
0.31
0.31
0.05
0.26
0.03
0.05
0.06
0.30
0.31
0.05
Tips
Forks
2476.00 1630.00
8274.00 5264.00
8057.67 8514.00
12798.00 10903.00
8524.33 6826.33
2126.00 1278.33
8797.33 6506.00
2853.33 1323.33
3086.00 2035.00
2975.67 1875.67
5418.33 5055.00
12798.00 10903.00
2126.00 1278.33
Table No. 4.5 Total phenotypic data for total length of root, total surface area,
average diameter, root volume, tips and forks of Indira Barani Dhan
Treatments
Control
IRRI-1
IRRI-2
IRRI-3
IRRI-4
IRRI-5
IRRI-6
TH3
TV12
T14
94a
Max.
Min.
Total
Length
426.17
428.66
437.02
452.59
452.32
439.40
434.45
450.72
436.76
433.45
429.92
452.59
426.17
Surface
Area
23.26
23.41
23.52
24.04
23.77
23.81
23.56
23.85
23.70
23.41
23.45
24.04
23.26
Average
Diameter
(mm)
0.22
0.22
0.25
0.21
0.20
0.16
0.19
0.24
0.21
0.26
0.22
0.26
0.16
61
Root
Tips
Volume
(cm3)
0.04 2995.00
0.09 3581.67
0.22 6702.67
0.08 11998.50
0.19 11036.33
0.08 6609.67
0.40 1893.67
0.31 8627.50
0.19 9767.33
0.15 5389.00
0.13 4462.33
0.40 11998.50
0.04 2995.00
Forks
1112.67
2143.67
4382.00
9294.00
8558.33
3632.33
2317.50
8816.00
7692.33
3824.00
2606.00
9294.00
1112.67
4.1.1 Assessment of root pulling strength of elite rice lines
Root length and diameter distribution are important characteristics to be
considered when describing and comparing root systems. Root pulling force was first
used in rice by O'Toole and Soemartono (1981) as an indirect estimate of root related
dehydration avoidance capacity. Long fibrous roots have long been recognized as an
important dehydration avoidance mechanism in rice and such roots evidently also
ascribe stronger anchorage and greater resistance to pulling force. The method
appears sensible for root depth phenotyping in rice, especially when other direct field
methods for root selection are not very forthcoming.
In the present investigation we evaluated 4 rice line / genotypes for root
pulling strength. The observations of root pulling strength were recorded with the
puller machine by pulling the complete one hill of rice plant after 50 days and it was
observed that Root Pulling Strength (RPS) varied from 52.5±0.50 to 38.5±2.63 for
rice line IR-64. In Sahbhagi dhan rice line it was varied from 48.50±0.82 to
38.25±2.39. Rice line RRF-75 shown the root pulling strength between 50±0.82 to
37.25±2.87. Whereas Indirabarani dhan line shown 47±1.58 to 39.5±2.75 (Table 4.6).
Root pulling resistance is the vertical force required to pull a plant from the soil and
this technique has been widely used to assess the nature of root development in cereal
crops (Nass and Zuber, 1971). The method appears sensible for root depth
phenotyping in rice, especially when other direct field methods for root selection are
not very forthcoming. Several root characteristics in rice are associated with drought
tolerance and other biotic-abiotic stresses and avoidance capability of plants.
62
Table No. 4.6 Root Pulling Strength (RPS)
Treatment/Varities
IR-64
Sahbhagi Dhan
RRF-75
Control
39.75±2.21
38.25±2.39
37.25±2.87
IRRI-1
41±3.11
47±2.38
44±2.94
IRRI-2
52.5±0.50
47±1.29
45.25±2.29
IRRI-3
45.5±4.35
46.75±2.87
47.75±1.65
IRRI-4
50±0.82
44.5±3.86
38.5±2.75
IRRI-5
45±1.73
45±3.51
39.5±3.69
IRRI-6
38.5±2.63
41.5±4.03
39±2.38
TH3
42.5±2.63
39.5±2.06
36±1.83
TV12
46±2.94
45.5±0.96
45±2.52
T14
46±2.58
45±2.65
44±3.83
94a
46.5±1.26
48.50±0.82
50±0.82
Max
52.5±0.50
48.50±0.82
50±0.82
Min
38.5±2.63
38.25±2.39
37.25±2.87
IBD-1
39.5±2.75
46±4.08
44.5±1.50
47±1.58
40.5±2.75
44±1.83
41±2.38
41.5±1.26
42±3.74
43.5±2.87
46.75±1.70
47±1.58
39.5±2.75
4.2 Qualitative assay for ACC deaminase activity
Ali et. al. (2013) found that isolate SorgP4 utilized ACC as a sole source of
nitrogen by the production of ACC deaminase enzyme and it showed the greater
amount of ACC deaminase activity (3.71 ± 0.025 μM/mg protein/h of α-ketobutyrate)
under non-stress and 1.42 ± 0.039 μM /mg protein/h of α-ketobutyrate under drought
stress condition respectively. Similar results were obtained by Grichko & Glick, 2000
where strain produced significant amount of Acc deaminase enzyme (3.8 ±0.7 μM/mg
protein/h of α-ketobutyrate).
All the 10 isolates were screened for ACC deaminase based on the enrichment
method, where ACC was used as the sole nitrogen source. All the isolates were found
to be positive as they were able to grow on Petri plates containing Synthetic media
utilizing ACC as sole nitrogen source and on plates containing (NH 4 )2 SO4 which
served as positive control. The isolates could not grow on plates containing only
synthetic media without ACC which served as negative control (Plate no. 4.4)
63
64
4.2.1 Quantitative estimation of ACC deaminase
Since all the isolates were able to grow on petri plates containing synthetic
media supplemented with ACC, therefore all were selected for quantitative estimation
of enzyme activity. The ACC deaminase activity was assayed by quantifying the
amount of α-ketobutyrate produced during the deamination of ACC by the enzyme
ACC deaminase. Quantification of enzyme activity showed wide variations and
differed statistically from one isolate to the other both at 1% and 5% level of
significance. All the isolates showed activity in the range of 10.46±3.92 to 2.37±1.37
µmol α ketobutyrate/mg protein/h (Table 4.7, Figure 4.1). The isolates were placed in
high, medium and low ACC deaminase activity producing groups. Isolates IRRI-1,
IRRI-2, IRRI-3, and TV12 were placed in high enzyme activity group while isolates
IRRI-4 in medium enzyme activity group. Similarly, isolates IRRI-6, TH3, T14 and
94a were placed in low enzyme activity group. Isolate TV12 showed the highest
amount of ACC deaminase activity (10.46±3.92 µmol α ketobutyrate/mg protein /h)
while isolate T14 showed lowest (2.37±1.37 µmol α ketobutyrate/mg protein /h).
Table no. 4.7: Screening isolates of Trichoderma spp. for ACC deaminase activity
Isolates
IRRI-1
IRRI-2
IRRI-3
IRRI-4
IRRI-6
TH3
TV12
T14
94a
Conc.
5.75±0.37
5.86±0.20
9.10±1.28
4.25±0.45
2.66±0.13
2.92±0.39
10.46±2.77
2.37±0.97
2.84±0.96
65
µmol α ketobutyrate/mg protein/hr
14
12
10.46
10
9.1
8
6
5.75
5.86
4.25
4
2.66
2.92
2.37
2.8
T14
94a
2
0
IRRI-1 IRRI-2 IRRI-3 IRRI-4 IRRI-6
TH3
TV12
Treatments
Fig. No. 4.1 Estimation of ACC deaminase activity by different Isolates of
trichoderma
4.3 Screening of Isolates of Trichoderma spp. for chitinase activity
Ten isolates of Trichoderma spp. were screened for their ability to hydrolyse
chitin and were then grouped according to the diameter (mm) of the purple coloured
zone observed. (Plate No. 4.5) Isolates of trichoderma spp. used in the present
investigation showed variable responses for chitinase expression (Table 4.8, Table
4.9) for utilization of colloidal chitin supplemented as substrate in the agar medium.
Isolates with high chitinase activity are the isolates of choice for their application as
biological control agent and were also identified after screening on colloidal chitin
supplemented as substrate in the agar medium High chitinase activity observed in
#IRRI-1, #IRRI-2, #IRRI-3, #TV12, #T14 and #94a that was, 192.5 mg/ml, 176.25
mg/ml, 177.5 mg/ml, 187.5 mg/ml, 165.0 mg/ml, 141.25 mg/ml respectively. Medium
chitinase activity was observed for #IRRI-4, IRRI-6 and TH3 111.25 mg/ml, 75
mg/ml, 68.75 mg/ml respectively. No isolate was observed with low chitinase
activity. Trichoderma isolate #94a and IRRI-6 measured (70 mm radial diameter)
(Fig.4.2,4.3). Fungal Strains assigned to the genera trichoderma are well known
66
producers of chitinolytic enzymes and are used commercially as sources of these
proteins. Additional interest in these enzymes is stimulated by the fact that
chitinolytic strains of Trichoderma are among the most effective agents for biological
control of plant diseases and can be serious pathogens. (Chet, 1987; Harman, 1990;
Harman et al., 1993; Komatsu, 1976; Muthu meenakshi et al., 1994; Samuels, 1996).
Trichoderma directly attacks the plant pathogen by excreting lytic enzymes such as
chitinases, ß-1,3 glucanases and proteases (Haran et al., 1996). Because the skeleton
of filamentous fungi cell walls contains chitin, glucan and proteins, enzymes that
hydrolyze these components have to be present in a successful antagonist in order to
play a significant role in cell wall lysis of the pathogen (Lorito et al. 1994; Carsolio et
al. 1999). Several distinct chitinolytic enzymes have been reported in T. harzianum
(De la Cruz et al., 1992; Haran et al. 1996). These include endochitinases,
exochitinases and 1,4-ß-N-acetylglucosaminidases, which are induced during growth
of T. harzianum in liquid medium containing chitin as carbon source.
Table No. 4.8 Screening of Isolates of Trichoderma spp. for chitinase activity
Isolates
Chitinase activity
IRRI-1
192.5±22.50
IRRI-2
176.25±6.25
IRRI-3
177.5±10.001
IRRI-4
111.25±3.75
IRRI-6
75±2.50
TH3
68.75±8.75
TV12
187.5±12.50
T14
165±10.001
94a
141.25±1.25
67
250
200
192.5
176.25
187.5
177.5
165
141.25
µg/ml
150
111.25
100
75
68.75
50
0
IRRI-1 IRRI-2 IRRI-3 IRRI-4 IRRI-6
Isolates
TH3
TV12
T14
94a
Fig. No. 4.2 Quantitative estimation of Chitinase activity (Procedure I) (µg ml-1)
produced by different isolates of Trichoderma spp. in colorimeter (OD-582
nm)
Table no. 4.9 : Screening of Isolates of Trichoderma spp. for chitinase activity
Isolates
Chitinase activity
IRRI-1
0.0757±0.00033
IRRI-2
0.0810±0.00028
IRRI-3
0.0820±0.00033
IRRI-4
0.0858±0.00066
IRRI-6
0.0783±0.00609
TH3
0.0772±0.00143
TV12
0.0765±0.00094
T14
0.0400±0.00036
94a
0.0734±0.00005
68
69
0.1000
Chitinase activity in µg/ml
0.0900
0.0800
0.0810 0.0820
0.0858
0.0783 0.0772 0.0765
0.0757
0.0734
0.0700
0.0600
0.0500
0.0400
0.0400
0.0300
0.0200
0.0100
0.0000
IRRI-1 IRRI-2 IRRI-3 IRRI-4 IRRI-6
Isolates
TH3
TV12
T14
94a
Fig. No. 4.3 Quantitative estimation of Chitinase activity (Procedure II) (µg ml-1 )
produced by different isolates of Trichoderma spp. in colorimeter
(OD-400 nm)
4.4 Qualitative and quantitative assay for siderophore production
In soil, plant roots normally coexist with bacteria and fungi which may
produce siderophores capable of sequestering the available soluble iron and hence
interfere with plant growth and function. Siderophores are produced during extreme
iron-depleted conditions for the solubilization of extracellular ferric iron by most
bacteria and fungi.
In the present investigation, ten isolates of Trichoderma were screened by six
different siderophore assay viz., CAS assay, CAS agar medium, Hydroxyquinoline
test, Tetrazolium test, FeCl3 tests and Arnow’s assay. CAS assay is the universal
assay for detection of siderophores. The principle of this assay is based on a color
change of CAS from blue to orange resulting from siderophoral removal of Fe from
the chrome azurol dye (Guan et al., 2001). All the isolates showed positive reaction
on CAS agar plate. The diameter of orange halo due to production of siderophore by
70
the isolates ranged from 21 to 49 mm with maximum halo produced by TV12 (49
mm). The minimum halo was produced by isolate T14 (21 mm) (Table 4.10).
For selection of Trichoderma isolates with high ability to produce siderophore,
isolates were inoculated on PDA supplemented with a strong chelater 8 Hydroxyquinoline. All the isolates are not able to grow on this medium. Arnow’s
assay was performed to detect catechol type of siderophores. Slight colour change in
the media was observed by only by three isolates IRRI-2, IRRI-6 and 94a indicating
that they produced catechol type of siderophore. All isolates produced deep red colour
on addition of Tetrazolium salt and NaOH which used to test presence of
hydroxamate type of siderophore, indicating that the isolates have capacity to reduce
tetrazolium salt by hydrolysis of hydroxamate group in presence of strong alkali.
However, FeCl3 test was negative for all the isolates.
Carboxylate type of siderophore was determined by spectrophotometric test
and the percentage of siderophore unit ranged from1.83 to 24.58%. Among the ten
isolates, isolate 94a produced highest percent (24.58) of siderophore units. Minimum
siderophore unit was observed for isolate TH3 (1.83%) (Figure 4.4)
71
Table No. 4.10: Production of various siderophores by Trichoderma isolates
S.No
Isolates
% siderophore
units
CAS test
(diameter of
orange colormm)
Arnow’s test
(development
of pink color)
FeCl3 test
(instant
development of
deep red color)
Tetrazolium test
(instant development
of deep red color)
HQ test
1
IRRI-1
2.28
Negative
Slightly positive
Negative
positive
Negative
2
IRRI-2
18.95
3.5
Slightly positive
Negative
positive
Negative
3
IRRI-3
2.42
4.3
Negative
Negative
positive
Negative
4
IRRI-4
2.82
Negative
Slightly positive
Negative
positive
Negative
5
IRRI-5
Negative
Negative
positive
Negative
6
IRRI-6
2.59
Negative
Slightly positive
Negative
positive
Negative
7
TH3
1.83
Negative
Negative
Negative
positive
Negative
8
TV12
6.60
4.9
Negative
Negative
positive
Negative
9
94a
24.58
4.4
Slightly positive
Negative
positive
Negative
10
T14
14.71
2.1
Positive
Negative
positive
Negative
72
% Sidrophore production
30
24.58
25
18.95
20
14.71
15
10
5
6.60
2.28
2.42
2.82
2.59
1.83
0
IRRI-1 IRRI-2 IRRI-3 IRRI-4 IRRI-6 TH3 TV12 T14
Isolates
94a
Fig. No. 4.4 Quantitative estimation of % Sidrophore production by different
isolates of trichoderma spp.
4.5 Screening isolates of Trichoderma spp. for in vitro phosphate solubilization
ability
Phosphate solubilization efficacy of isolates of trichoderma spp. was
performed on Pikovaskaya broth medium. By preparing standard curve the amount of
inorganic phosphate solubilised by different Trichoderma isolates were estimated at
wavelength 610nm. Screening of isolates showed variation in their ability to utilized
calcium phosphate supplemented in different nutrient constituents. Quantitative
estimation of phosphate solubilization, carried out after incubation of 7 days, is
presented (Table 4.11, Figure 4.5). Some of the fungal isolates (#T14) showed
significantly good response to phosphate solubilization where as some was not good
solubilizer of phosphate. Qualitative estimation done on agar medium containing BCP
as a colour indicator it changes color purple to yellow (Plate no. 4.5). Phosphorus in
the soil is essentially unavailable to the plants and use of plant associated organisms
may help in solubilization of mineral Phosphate (P) for easy uptake by the plants.
Fungi are reported to solubilize P by production of organic acids and are known to
73
have a higher efficiency of solubilization than bacteria. It was observed that #T14
isolates showed higher ability to solublized the phosphate as they also exhibited good
responses to PGPR activity after direct seed treatments. Higher efficacy of
Trichoderma isolates to utilized calcium phosphate varied and was enumerated as
follows 1)
Trichoderma isolate (#T14) exhibited higher phosphate solubilising ability in
pikovaskaya broth media used for screening and therefore can be considered
as promising inducer of phosphate mobilization. The amount of inorganic
phosphate solubilised was 780 µg mL -1.
2)
Other Trichoderma isolates i.e #IRRI-1, #IRRI-2, #IRRI-3, #IRRI-6 and also a
good phosphate solubilizer that they solubilized 455µg mL-1 , 603 µg mL-1,
485 µg mL-1 , 542 µg mL-1 , respectively.
3)
Trichoderma isolate (#TH-3) showed good phosphate solubilising ability (403
µg mL-1) in pikovaskaya broth media as compare to control. Isolate #94a
produced (397 µg mL-1) and #Tv12 produced (451µg mL-1) phosphate in
media. µg mL-1.
Phosphatases play a key role in transforming organic forms of phosphorus into
plant available inorganic forms and are found to be active in microorganisms, plants
and roots. A significant correlation had been noticed between the depletion of organic
P and phosphatase activity in the rhizosphere soil of wheat (Tarafdar and Jungk,
1987). Simultaneous exudation of organic acids and phosphatases could increase both
P solubility, by releasing bound organic phosphates and its mineralization by
increasing the rate of hydrolytic cleavage (George et al., 2002). Satyavani and
Satyaprasad, (2009) demonstrated that two species of Trichoderma viz., T. harzianum
and T. aureoviride isolated from rhizosphere of pigeon pea promoted plant growth so
74
to understand the role of Trichoderma in growth promotion, the ability of
Trichoderma spp. in solubilizing rock phosphate was tested in vitro. Culture filtrates
of both species solubilized rock phosphate in which T.harzianum was more effective
than T.aureoviride in solubilizing phosphates and producing fungal biomass. For
example, Trichoderma harzianum was shown to solubilize phosphate and
micronutrients that could be made available to plant (Altomore et al., 1999).
Table 4.11 Screening isolates of Trichoderma spp. in vitro for phosphate
solubilizing ability
Isolates
IRRI-1
Inorganic phosphate solubilized
(ug/ml)
455
IRRI-2
603
4.540
IRRI-3
485
4.120
IRRI-4
42
4.060
IRRI-6
542
4.360
TH3
403
5.190
TV12
451
5.160
T14
780
3.940
94a
397
4.900
Control
42
4.700
75
pH of the culture media
4.810
900
780
800
700
603
µg/ml
600
500
455
542
485
403
400
451
397
300
200
100
42
42
0
Isolates
Fig. No. 4.5 Quantitative estimation of inorganic phosphate solubilization (µg/ml)
by different isolates of Trichoderma spp. in colorimeter (OD-610 nm)
4.6 Quantification of indole acetic acid (auxin) production by Trichoderma spp.
Laboratory studies have emphasized on use of (plant growth promoting fungi)
PGPF as biocontrol agents (Hossain et al., 2007) and the role of auxin (IAA)
production (Contreras-Cornejo et al., 2009) in plant growth promotion. Production of
IAA was evaluated for ten different isolates of Trichoderma spp. in DF salts culture
medium amended with 1.02 g/L from 5mM stock of L-tryptophan as precursor
molecule and without any IAA precursor as control. The mixture of culture
supernatant and salkowski’s reagent was incubated at room temperature for 20 min
and the absorbance was measured at 535 nm. The concentration of IAA compound
was evaluated by comparison with a standard curve prepared using serial dilutions (0 100 µg/ml) prepared from commercially available IAA. Interpolation of the
colorimeter readings with standard curve were used to quantify the amount of IAA
produced by different isolates of Trichoderma in the media which ranged from 0.36 to
29.09 µg/ml (Table 4.12, Figure 4.6). The highest IAA was produced by Trichoderma
isolate #T14 (29.09µg/ml) which is significant high where as isolate #TH3
(0.36µg/ml) was the lowest producer. Fungal isolate #IRRI-1, IRRI-2 and #94a were
76
also identified as promising producers of Indole acetic acid 8.36µg/ml, 18.81µg/ml
and 7.72µg/ml respectively. Isolate #IRRI-3, #TH3, and TV12 was produced Indole
acetic acid 0.54µg/ml, 0.36µg/ml, 1.09µg/ml respectively which is low than other
fungal isolates.
Microbial IAA could be involved in the growth stimulation observed in our
greenhouse assay. Production of plant growth regulators by the microorganisms is
another important mechanism often associated with growth stimulation (Vessey,
2003). The balance between vegetative and reproductive growth is controlled by
hormone signaling within the plant and can therefore be highly influenced by it (Taiz
and Zeiger, 1991). At relatively high concentrations, natural auxins, such as IAA,
stimulate shoot elongation and root induction while reducing root elongation
(Tanimoto, 2005). Trichoderma spp. have been also shown to exhibit plant growthpromoting activity on numerous cultivated plants (Kleifeld and Chet, 1992; Ousley et
al.,1994; Altomare et al., 1999; Harman, 2000; Yedidia et al.,2001). Gravel et al
(2007) in his results, showed that P. putida subgroup B strain 1 and T. atroviride are
able to synthesize IAA from different precursors in vitro, which supports the theory
that microbial IAA could be involved in the growth stimulation observed in our
greenhouse assay.
77
Table 4.12 Efficacy of different Trichoderma spp. for IAA production
Isolates
pH of the culture media IAA produced (ug/ml)
IRRI-1
6.92
8.36
IRRI-2
6.88
18.81
IRRI-3
6.51
0.54
IRRI-4
6.73
6.18
IRRI-6
8.15
3.54
TH3
7.94
0.36
TV12
6.44
1.09
T14
6.41
29.09
94a
6.91
7.72
Trichoderma inoculation affected root system architecture, which was related
to increased yield of plants. Reported effects include enhanced root biomass
production and increased root hair development (Bjorkman et al., 1998; Harman et
al., 2004) help the plants to grown in adverse condition such as drought stress. The
root system is important for plant fitness because it provides anchorage, contributes to
water-use efficiency and facilitates the acquisition of mineral nutrients from the soil
(Lopez-Bucio et al., 2005). Many lines of evidence strongly support a role for auxin
in the regulation of root system architecture. The production of plant growth
hormones or analogues is another mechanism by which strains of Trichoderma (T14,
IRRI-1, IRRI-4) can enhance plant growth. 162 species of fungi have been reported to
produce auxins, which are key hormones effecting plant growth and development that
can be produced by fungi in both symbiotic and pathogenic interactions with plants
(Gravel et al., 2007).
78
IAA production in ug/ml
35
29.09
30
25
18.81
20
15
10
8.36
7.72
6.18
3.54
5
0.36
1.09
IRRI-1 IRRI-2 IRRI-3 IRRI-4 IRRI-6 TH3
Isolates
TV12
0.54
0
T14
94a
Fig. No. 4.6 Quantitative estimation of IAA (µg ml-1) produced by different
isolates of Trichoderma spp. in colorimeter (OD-535 nm)
4.7 Screening of different isolates of Trichoderma spp. for hydrogen cyanide
production
In the present investigation twenty isolates of Trichoderma spp. were screened
for its ability to produce hydrogen cyanide. The production of hydrogen cyanide by
trichoderma spp. was screened using glycine as its precursor molecule. The plates
were incubated for 7 days at 28±2°C and observations were measured as colour of
filter paper turning to brown measured as positive for HCN production. Isolate #IRRI4, #IRRI-6, #IRRI-2, #94a were observed as positive for its ability to produce HCN
(Plate 4.6). Microbial cyanogenesis has been demonstrated in many species of fungi
(Hutchinson et al., 1973), but only in a few species of bacteria in the genera
Chromobacterium and Pseudomonas (Michaels and Corpe, 1965; Patty, 1921).
Glycine has usually been the indicated precursor of cyanide in fungi and bacteria
(Brysk et al., 1969; Ward et al., 1971; Wissing et al., 1974).
79
80
4.8 PCR amplification of Trichoderma isolates using different types of primers
Genomic DNA extracted from different isolates of Trichoderma were subjected to polymerase chain reaction using different types of
primers as detailed below.
Table No. 4.13: Details of PCR primers used in the present study
S. No.
Primers
Reference
Sequence(5’-3’) and Tm value (oC)
1
CAL-228F
CAL-737R
ACT-512F
ACT-782R
AcdS-F
AcdS-R
nitrilase P1-F
nitrilase PfluHisB-R
hcnACa
hcnACb
hcnPM2
hcnPM7-26R
chit33-F
chit33-R
nag1RT-F
nag1RT-R
Nag-2RT-F
Nag-2RT-R
Nag-2F
Nag-2R
Chaverri et al
(2003)
Carbone and
Kohn (1999)
Duan et al
(2009)
Kiziak et al
(2005)
Ramette et al
(2003a)
Svercel et al
(2007)
Matarese et al
(2010)
Seidl et al
(2006)
Seidl et al 2006
GAGTTCAAGGAGGCCTTCTCCC (68.3)
CATCTTTCTGGCCATCATGG (65.4)
ATGTGCAAGGCCGGTTTCGC (73.3)
TACGAGTCCTTCTGGCCCAT (65.8)
GGCAAGGTCGACATCTATGC (64.5)
GGCTTGCCATTCAGCTATG (63.3)
GAAATTCCATATGACGGTGCATAAAAAACAG(70.8)
CGGGATCCCTTGTCGCCTTGCTCT (77.1)
ATCGCCAGGGGCGGATGTGC (77.5)
ACGATGTGCTCGGCGTAC (65.4)
TGCGGCATGGGCGTGTGCCATTGCTGCCTGG (91.7)
CCGCTCTTGATCTGCAATTGCAGGCC (78.5)
GCTCCTCAGTGCTTCTTCC (61.8)
GGGAATGCCGACAAGAAGC (66.9)
GAACTGGAGGCTCATCTAC (56.2)
GATGATGTTGTCCATGTTG (57.1)
TGCGACCCGACCAAGAACTG ()
CAGATGATGGTGTCGAGGCTG ()
GCACGCTCTTCATTGACCAG (66)
CACAGTCATGCACATCAACCTG (66.6)
2
3
4
5
6
7
8
9
10
Seidl et al 2006
81
Primer
Length
22
20
20
20
20
19
31
24
20
18
31
26
19
19
19
19
20
21
20
Expected
Product size
500bp
1000bp
1052bp
587bp
571bp
130bp
146bp
1.8kb
22
4.9 Amplification of actin and calmodulin genes in Trichoderma isolates
Calmodulin (CaM) (an abbreviation for CALcium-MODULated proteIN) is
a calcium-binding messenger protein expressed in all eukaryotic cells. CaM is a
multifunctional intermediate messenger protein that transduces calcium signals by
binding calcium ions and then modifying its interactions with various target proteins
(Stevens
1983,
Chin
and
Means
2000).
Actin is
a globular multi-
functional protein that forms microfilaments. It is found in all eukaryotic cells. Actin
participates in many important cellular processes, including muscle contraction, cell
motility,
cell
division
and
cytokinesis, vesicle and organelle movement, cell
signalling, and the establishment and maintenance of cell junctions and cell shape.
Primer pairs CAL-228F & CAL-737R and ACT-512F & ACT-782R were
used to amplify a portion of the calmodulin (CAL) gene including two introns and a
portion of the actin (ACT) gene including one intron. Both the primers amplified
specific size fragments (~340bp actin and ~500bp calmodulin) (Plate no.4.7) in all the
isolates. Both the genes are of phylogenic and taxonomic importance and used for
sequencing and species identification in the way similar to internal transcribed spacers
ribosomal DNA (ITS rDNA) and
translation elongation factor-1a (EF-1a).These
genes serve as house-keeping genes and used during expression analysis such as that
of chitinases, cellulases, N-acetyl glucosaminases etc.
4.10 In vitro detection of ACC deaminase producing Trichoderma isolates using
gene-specific primers
Certain plant growth promoting rhizobacteria (PGPR) contain a vital enzyme, 1aminocyclopropane-1-carboxylic acid (ACC) deaminase (EC 4.1.99.4), which
regulates ethylene production by metabolizing ACC (an intermediate precursor of
82
83
ethylene biosynthesis in higher plants) into α- ketobutyrate and ammonia. This
pyridoxal phoshphate enzyme was first isolated in 1978 from pseudomonas sp. strain
ACP and from the yeast Hansenula satrunus (Honma and Shimonura 1978); since
then, it has been detected in fungi and in a number of other bacteria. When ACC
deaminase-containing plant growth-promoting bacteria are bound to a plant, they act
as a sink for ACC ensuring that plant ethylene levels do not become elevated to the
point. ACC deaminase-containing plant growth-promoting bacteria up-regulate genes
involved with plant growth and protein production while down-regulating plant genes
involved with ethylene stress and defence signaling pathways (Hontzeas et al. 2004).
This enzyme facilitates plant growth as a consequence of the fact that it
sequesters and cleaves plant produced ACC, thereby lowering the level of ethylene in
the plant. In turn, decreased ethylene levels allow the plant to be more resistant to a
wide variety of environmental stresses, all of which induce the plant to increase its
endogenous level of ethylene; stress ethylene exacerbates the effects of various
environmental stresses.
Enzymatic activity of ACC deaminase is assayed by monitoring the
production of either ammonia or α-ketobutyrate, the products of ACC hydrolysis.
ACC deaminase has been found only in microorganisms, and there are no
microorganisms that synthesize ethylene via ACC (Fukuda et al. 1993). Interestingly,
this enzyme is cytoplasmically localized so that the substrate ACC must be exuded by
plant tissues and subsequently taken up by an ACC deaminase-containing
microorganism before it is cleaved (Glick et al 1998).
Screening of isolates for the presence of acdS gene was done using primer pair
acdS. The results of the PCR analysis with primer pair AcdS-F & AcdS-R indicated
that a DNA fragment approximately 1500 bp in size was obtained in all Trichoderma
84
85
isolates. Amplification of acdS gene gives the proof for the presence of gene in the
form of 1.5 to 1.7 kb gel band. Additional band of 1270 bp was also observed in all
the isolates (Plate no.4.8). ACC deaminase producing microbes are known to facilitate
the growth of a variety of plants especially under stressful conditions such as
flooding, heavy metals, high salt and drought so, the acdS gene coding for enzyme
ACC deaminase can be a very useful candidate gene for the development of
transgenics for abiotic stress management in plants.
4.11 In vitro detection of Nitrilase producing Trichoderma isolates using genespecific primers
Nitrilase enzymes
(nitrile
aminohydrolase;
EC 3.5.5.1)
catalyse
the
hydrolysis of nitriles to carboxylic acids and ammonia, without the formation of
"free" amide intermediates. Nitrilases are involved in natural product biosynthesis and
post
translational
modifications
in
plants,
animals,
fungi
and
certain
prokaryotes. Nitrilase is also involved in synthesis of IAA (via an alternate pathway)
by hydrolysis of indole-3-acetonitrile (Mahadevan, 1963).
A large number of various nitrilases have been purified and characterized in
bacteria, but only a few in eukaryotic organisms (plants and filamentous fungi)
(O’Reilly and Turner 2003; Vejvoda et al, 2010).
The forward primer P1 and the reverse primer PfluHisB which were used to
amplify a 1052bp fragment containing the nitrilase gene in Pseudomonas fluorescens
EBC191 by Kiziak et al, (2005) was used to amplify nitrilase gene in Trichoderma
isolates. In the present investigation 1000, 900 and 800bp fragments were obtained in
all the isolates whereas additional bands of 400 and 280bp were also obtained in all
the isolates except IRRI-3 and T14. Intense bands below 100bp was observed in all
the isolates as well (Plate no.4.9)
86
In trichoderma sp. nitrilase serves defend against cyanide produced by R.
solani, but this has not yet been proven so far. Its expression may be a general
response of Trichoderma towards the presence of any host, but this clearly needs
further investigations (Atanasova et al, 2013).
4.12 In vitro detection of hydrogen cyanide producing Trichoderma isolates using
gene-specific primers
HCN production ability has been found to be associated with effective disease
suppression ability in different pathosystems. Prior studies have suggested that HCN
may be a distinctive marker for deleterious soil microorganisms (Schippers et al.,
1990; Paszkowski & Dwornikiewicz, 2003; Benizri et al., 2005), and, in fact, HCNproducing bacteria have been exploited for weed biocontrol (Kremer & Souissi, 2001;
Owen & Zdor, 2001). In fact, it is not uncommon for certain plant-beneficial traits to
be also found in deleterious bacteria (Blaha et al., 2006).
In the present investigation isolates of trichoderma were amplified with two
HCN specific primers of Pseudomonas sp. amplifying partial segments of HCN gene
(infact operon) Part (i.e. 587 bp) of the hcnBC genes should be amplified by PCR
using primers hcnAC-a & hcnAC-b as described by Ramette et al., 2003a whereas a
571 bp fragment of hcnAB was supposed to be amplified with primers hcnPM2 &
hcnPM7-26R (Svercel et al 2007). In the present investigation primer hcnAC-a &
hcnAC-b resulted in amplification of >1500bp fragment in isolates IRRI-2, IRRI-3
and Tv12 along with 120bp fragment in all the isolates of Trichoderma (Plate no.
4.10). However with primer hcnPM2 & hcnPM7-26R amplification was not observed
in any of the isolates.
87
88
4.13 Amplification of chitinase genes in Trichoderma isolates using gene-specific
primers
Cell wall degrading enzyme (CWDE) genes from biocontrol fungi belonging
to the genus trichoderma have been demonstrated to encode proteins with high
antifungal activity against a wide range of plant pathogenic fungi. The
characterisation of endochitinase genes of Trichoderma spp., whose expression is
strongly enhanced during mycoparasitism, as well as induced by chitin and lightinduced sporulation is performed using different primers. In the present investigation,
four different chitinase gene specific primers (Chit33, Nag1, Nag1RT, Nag2RT) were
used to analyse amplification of chitinase genes in Trichoderma isolates.
Primer Chit33 amplified fragments of 900 and 220 bp in all the isolates with
an additional small fragment of approx. 60bp size (Plate no.4.11). Nag-1 primer gave
products of different sizes ranging from 150 to 1700bp. Fragment size 1500 bp was
observed only in isolates IRRI-2, IRRI-3, Tv12 and T14 whereas fragments of 470,
300 and 220 bp was observed in all the isolates under investigation. Amplification
with primer Nag1-RT resulted in expected bands of 145bp in all the isolates (Plate no.
4.12) whereas an expected 150 bp fragment was amplified with primer Nag2-RT only
in isolates IRRI-2, IRRI-3, Th3, Tv12 and T14 (Plate no 4.13).
4.14 In-silico analysis of root related QTLs
Drought stress is the major constraint to rice (Oryza sativa) production and
yield stability in rainfed ecosystems. Identifying genomic regions contributing to
drought resistance will help to develop rice cultivars suitable for rainfed regions
through molecular marker-assisted breeding. One of the most exciting developments
in rice biotechnology is the advent of molecular markers.
89
90
91
Molecular markers help to track the genetic loci controlling drought-resistance
traits without having to measure the phenotype, thus reducing the need for extensive
field testing over space and time (Nguyen et al. 1997). Once the tightly linked
markers have been identified, the QTLs can be selected for in breeding programs. The
root trait was an important trait to resist plants in drought stress. It was noticed that
the deep, thick, branched root system was good to grown rice plants in adverse stress
conditions, so the identification of the genomic region for root traits was an important
to develop a variety resistance to drought stress. In the present investigation the in
silico analysis of root related QTLs was done for identification of chromosome region
responsible for different root parameters including root length (RL), root volume
(RV), total root numbers (TRN), penetrated root thickness (PRT), penetrated root dry
weight (PRDW), deep rooting (DR) using various bioinformatics tools. Using the
online Nipponbare BAC/PAC map (Oryza sativa L.) information, QTL regions were
identified. In rice, a dihaploid, RIL population derived from two cultivars provides a
useful references population for in silico analysis of QTLs.
Table 4.14 Details of in silico analysis of root related QTLs on chromosome
number #09 and #02
QTL trait
Root Length
(RL)
Deep Rooting
(DR)
Penetrate Root
Thickness
(PRT)
Root Length
(RL), Root
Volume (RV),
Total Root
Number(TRN)
9
Flanking
cM
North clone
South clone
marker
RZ20636-55.3 P0711F01
OJ1299_A11
RZ422
RM24393- 58.3-60.8 OJ1081_G10 OJ1123_B08
RM7424
9
ME9_6K985
Ch. #
9
63-69.5
B1045B05
Hemamalini
et al (2000)
Yusaku
Uga et al
(2011)
OSJNBa0087J09 Zang et al
(2001a)
S20944S- 57.9-77.8 OJ1288_D09 E0508B05
E30251S
2
92
Referances
Hemamalini
et al (2000)
93
94
95
96
Through in silico analysis five QTLs were identified on chromosome number #09 and
one on chromosome number #02. The three QTLs on chromosome #09 were
identified in silico between the flanking markers RZ206 and RZ422 covering a
distance 19.3 cM. The region of 36 cM to 55.3 cM on this chromosome govern by
Root Length (RL) having flanking markers RZ206 and RZ422 on P0711F01 and
OJ1299_A11 BAC/PAC clone respectively.
The QTL for penetrate root dry weight was identified by Nguyen et al (2004)
and for the same trait the QTL was identified in the Ct9993 rice population by Zhang
et al (2001). From this references the in silico analysis was done that showed the
region from 40.7 cM to 50.7 cM on chromosome number #02 having flanking
markers RM41 and ME2_10 govern by root parameter i.e. penetrate root dry weight.
The BAC/PAC clone on which flanking markers present was P0711A01 and
P043D11 (Plate 4.14).
The next QTL was identified on same chromosome (#09) for the root
parameter deep rooting (DR), this QTL region span the 2.5 cM distance with flanking
markers was RM24393 and RM7424 present on OJ1081_G10 and OJ1123_B08
respectively. This QTL region cover the distance from 58.3 cM to 60.8 cM which is
very short region govern by this trait (Plate 4.15). On the same chromosome, the
distances 6.5 cM govern by root trait Penetrate Root Thickness (PRT). The distance
from 63.0 cM to 69.5cM with flanking markers E61594S and C670SA on B1342C04
and OSJNBa0046G BAC/PAC clone respectively (Plate 4.16). The first time this
QTL region was identified by Zang et al, (2001) derived from CT9993 rice
population.
The QTL on chromosome #02 was first identified by Hemamalini et al in 2000
associated with number of root traits including Root Length (RL), Root Volume (RV),
97
Total Root Number (TRN) in IR64×Azucena derived population. In silico analysis of
chromosome number #02 revealed that the whole QTL region of 57.3 cM to 81.0 cM
with flanking markers S209445 and E302515 govern by above root parameters. This
major QTL region covered total 23.7 cM distance. The flanking marker for this QTL
was present on OJ1288_G09 (S209445) and P0508B05 (E30251S) (Figure 4.17).
98
CHAPTER V
SUMMARY, CONCLUSION AND SUGGESTION FOR
FUTURE RESEARCH WORK
Every plant part have unique function as they performed in that the plant root
have a large range of functions, including acquisition of water and nutrients, as well
as structural support. Dissecting the genetic and molecular mechanisms controlling
rice root development is critical for the development of new rice ideotypes that are
better adapted to adverse conditions and for the production of sustainably achieved
rice yield potential. Increased incidences of abiotic and biotic stresses impacting
productivity in principal crops are being witnessed all over the world.
Microorganisms could play a significant role in this respect, if we can exploit their
unique properties of tolerance to extremities, their ubiquity, genetic diversity, their
interaction with crop plants and develop methods for their successful deployment in
agriculture production. The fungus Trichoderma isolates have been identified as being
able to act as endophytic plant symbionts. The strains become endophytic in roots, but
the greatest changes in gene expression occur in shoots. These changes alter plant
physiology and may result in the improvement of abiotic stress resistance, nitrogen
fertilizer uptake, resistance to pathogens and photosynthetic efficiency.
5.1 Summary
5.1.1 Analyzing root system morphology against candidate Trichoderma isolates
1. Ten Trichoderma isolates were evaluated in the present investigation to study
the effect on root morphology of the selected genotype/ rice lines. It was
observed that Trichoderma seed treatment contributes towards increased root
length.
99
2. Seed treatment to IR-64 and RRF-75 by trichoderma isolate #T14 shows
maximum root length. In the same manner in IRRI-5 shows maximum root
length in sahbhagi dhan and TV12 shows in IBD-1
5.1.2 Screening of isolates of Trichoderma spp. for ACC deaminase activity
1. All the isolates showed activity in the range of 10.46±3.92 to 2.37±1.37 µmol
α ketobutyrate/mg protein /h.
2. Isolate TV12 showed the highest amount of ACC deaminase activity
(10.46±3.92 µmol α ketobutyrate/mg protein /h) while isolate T14 showed
lowest (2.37±1.37 µmol α ketobutyrate/mg protein /h).
5.1.3 Screening of isolates of Trichoderma spp. for chitinase and production
1. In this screening of ten trichoderma spp. to detect enzyme activity of chitinase
and, the isolates were grouped to high, medium and low according to the
radial diameter measured as the ability to produce chitinase and enzyme
activity.
2. Out of ten isolates of Trichoderma spp., six isolates (#IRRI-1, #IRRI-2,
#IRRI-3, #TV12, #T14 and #94a) showed the ability to produce high chitinase
activity and three isolates (#IRRI-4, #IRRI-6 and #TH3) showed the medium
chitinase producing ability where as low chitinase producing ability was not
shown by any of the isolates.
5.1.4 Screening isolates of Trichoderma spp. for siderophore and HCN
production
1. In the present investigation ten different species of Trichoderma were
screened for siderophore producing ability to chelate and solubilize iron to
make in available form to plants. Out of ten isolates screened three isolates
100
(IRRI-2, T14, 94a) of trichoderma spp. were found to be producing more than
10% siderophore production.
2. Screening of ten isolates of Trichoderma spp. carried for its ability to produce
volatile inhibitory compound hydrogen cyanide supplementing glycine as a
precursor molecule. Isolate #IRRI-2, #IRRI-4, IRRI-6 and isolate #94a
showed positive reaction for cyanide production.
5.1.5 Screening isolates of Trichoderma spp. for Phosphate solubilization and
IAA production
1. Trichoderma spp. has been known for plant growth promoting related activity
other than as biocontrol agent which enhances the root and shoot growth, seed
germination, solubilization and uptake of range of nutrients which ultimately
increases the crop productivity.
2. Screening of ten isolates of Trichoderma spp. for Phosphate solubilization
ability was carried out in two different types of media containing tricalcium
phosphate as a phosphate source. Isolates showed varying results of phosphate
solubilization carried out in three different types of media. Among twenty
isolates screened, isolate# T14 showed higher phosphate solubilization in all
three types of media.
3. In the present experiment, twenty isolates of Trichoderma spp. were evaluated
for the ability to produce IAA compounds from L-tryptophan as a precursor
molecule and compared with standard graph of IAA. Isolate # T14 showed
high IAA producing ability among the ten isolates.
5.1.6 Molecular characterization of ten isolates of Trichoderma spp.
The polymerase chain reaction (PCR) technique has created new ways of
revealing DNA polymorphism among closely related genotypes with high sensitivity
101
via a fast and easy to perform protocol. PCR based fingerprinting is widely applied
for the characterization of Trichoderma strains and species for various purposes.
1. The genetic relatedness among ten isolates of Trichoderma spp. were analyzed
by ten gene specific markers.
2. Primer pairs CAL-228F & CAL-737R and ACT-512F & ACT-782R were
used to amplify a portion of the calmodulin (CAL) gene including two introns
and a portion of the actin (ACT) gene including one intron. Both the primers
amplified specific size fragments (~340bp actin and ~500bp calmodulin) in all
the isolates
3. PCR analysis for presence of acdS gene with primer pair AcdS-F & AcdS-R
indicated that a DNA fragment approximately 1500 bp in size was obtained in
all Trichoderma isolates.
4. . In the present investigation for detection of Nitrilase producing gene used
primer pair of P1 and PfluHisB gives 1000, 900 and 800bp fragments were
obtained in all the isolates whereas additional bands of 400 and 280bp was
also obtained in all the isolates except IRRI-3 and T14. Intense bands below
100bp were observed in all the isolates as well.
5. In the present investigation isolates of Trichoderma were amplified with two
HCN specific primers of Pseudomonas sp. Primer hcnAC-a & hcnAC-b
resulted in amplification of >1500bp fragment in isolates IRRI-2, IRRI-3 and
Tv12 along with 120bp fragment in all the isolates of Trichoderma. However
with primer hcnPM2 & hcnPM7-26R amplification was not observed in any of
the isolates.
102
6. In the present investigation, four different chitinase gene specific primers
(Chit33, Nag1, Nag1RT, Nag2RT) were used to analyse amplification of
chitinase genes in Trichoderma isolates.
7. Primer Chit33 amplified fragments of 900 and 220 bp in all the isolates with
an additional small fragment of approx. 60bp size. Nag-1 primer gave
products of different sizes ranging from 150 to 1700bp. Fragment size 1500 bp
was observed only in isolates IRRI-2, IRRI-3, Tv12 and T14 whereas
fragments of 470, 300 and 220 bp was observed in all the isolates under
investigation. Amplification with primer Nag1-RT resulted in expected bands
of 145bp in all the isolates whereas an expected 150 bp fragment was
amplified with primer Nag2-RT only in isolates IRRI-2, IRRI-3, Th3, Tv12
and T14.
5.1.7 In-silico analysis of root related QTLs
1. Using the online Nipponbare BAC/PAC map (Oryza sativa L.) information,
QTL regions were identified in-silico. The region of 36 cM to 55.3 cM on
chromosome #09 govern by Root Length (RL) having flanking markers
RZ206 and RZ422 on P0711F01 and OJ1299_A11 BAC/PAC clone
respectively.
2. In-silico analysis was done that showed the region from 40.7 cM to 50.7 cM
on chromosome number #02 having flanking markers RM41 and ME2_10
govern by root parameter i.e penetrate root dry weight. The BAC/PAC clone
on which flanking marker anchored was P0711A01 and P043D11. The next
QTL was identified on same chromosome (#09) for the root parameter deep
rooting (DR), this QTL region span the 2.5 cM distance with flanking markers
was RM24393 and RM7424 present on OJ1081_G10 and OJ1123_B08
103
respectively. This QTL region cover the distance from 58.3 cM to 60.8 cM
which is very short region govern by this trait.
5.2 Conclusion
1. Treatment showed maximum root length, root thickness, root branching, root
number over the control in all the rice lines.
2. In-vitro screening for phosphate solubilizing activity and indol acetic acid
(IAA) producing assay showed that the fungal isolate T14 was identified as
potentially high responsive in both the screening assay. Iron chelating ability
observed maximum in isolate 94a.
3. These isolates were also screen for ACC deaminase activity experiment
showed that the isolate IRRI-1 has high activity.
4. PCR amplification with ACC primers showed homogeneity of all
Trichoderma isolates indicating that they all are able to produce ACC
deaminase.
5. Almost all the isolates showed the presence of endochitinase and nitrilase gene
fragments which proves their ability to produce chitinase production.
Homogenecity also observed in actin and calmodulin gene amplification.
5.3 Suggestion for future research work
1. Studying the relationship between plant growth regulators produced by
different Trichoderma spp. for increased growth response of plant specially
roots traits for alleviating stress conditions.
2. Those isolates which possessed high phosphate solubilizing ability, IAA
production, tolerance to fungicide and strong antagonism will be further
exploited for their above mentioned characteristics.
104
3. There will be the need to study the complex and dynamic interactions among
microorganisms, roots, soil and water in the rhizosphere induce changes in
physicochemical and structural properties of the soil.
4. QTL can help us to develop insight regarding the rice ideotype which have the
long, branched, deep root system.
105
106
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