2513
Advances in Environmental Biology, 6(9): 2513-2518, 2012
ISSN 1995-0756
This is a refereed journal and all articles are professionally screened and reviewed
ORIGINAL ARTICLE
Allelopathic Effects of Pine Needle Extracts on Germination and Seedling Growth of
Ryegrass and Kentucky Bluegrass
1
Ali Asghar Aliloo, 2Saleh Shahabivand, 1Leila Farjam and 1Somayeh Heravi
1
University of Maragheh, Faculty of Agriculture, Department of Agronomy and Plant Breeding, Maragheh,
Iran;
2
University of Maragheh, Faculty of Life Sciences, Department of Plant Biology, Maragheh, Iran
Ali Asghar Aliloo, Saleh Shahabivand, Leila Farjam and Somayeh Heravi: Allelopathic Effects of Pine
Needle Extracts on Germination and Seedling Growth of Ryegrass and Kentucky Bluegrass
ABSTRACT
The potential allelopathic effect of Pinus eldarica on seed germination and seedling growth was
investigated with two grasses: ryegrass (Lolium perenne) and kentucky bluegrass (Poa pratensis). Aqueous leaf
extracts significantly reduced both germination and seedling growth of grasses mostly starting from
concentrations of 2.5% to 10%. The effect of aqueous extracts on kentucky bluegrass was more than ryegrass.
Results also revealed that seeds with greater germination index had low inhibition rate for germination
percentage and seedling growth. The average inhibition rate on germination and seedling growth for grass
species was 13.6 and 25.9%, respectively. With increasing concentrations of the pine aqueous extract, shoot
length, root length, seedling length, root dry weight, shoot dry weight and seedling dry weight of both grasses
were significantly decreased and generally, high inhibitory effect was observed at high level of concentrations.
The sensitivity of the root part to the allelochemicals was more than the shoot part; thereby the root/shoot ratio
was decreased. The LD50 of pine aqueous extracts for germination inhibition rate and seedling inhibition rate
was 10.6% and 9.4%, respectively. Our finding suggested that the seed germination can be considered as a
relatively less sensitive period of the individual plant development, whereas the period of seedling growth is
suitable for potential testing of the allelopathic effect of the studied extracts under laboratory conditions. In
addition, the choice of vigorous seeds can be useful to alleviation of possible allelopathic risks.
Key words: allelopathy, kentucky bluegrass, ryegrass, pine, germination, seedling growth
Introduction
Conifers known as narrow-leaved or needled
evergreens are planted primarily for the
attractiveness of their evergreen foliage. The variety
of sizes, shapes, and colors available contributes to
their popularity. The species of Pinaceae are wide
spread in the landscapes and commonly are used
with grasses of Poaceae family. In North West of
Iran Pinus eldarica (as a decorative tree) is planted
with perennial ryegrass (Lolium perenne) and
kentucky bluegrass (Poa pratensis) in parks and
landscapes.
Seed dispersal and seed germination are critical
phases in the life-cycle of the plant, during these
phases the forces (biotic and abiotic stresses) have a
maximum opportunity to exert their influence. Seed
germination represents a risky transition from the
stage most tolerant to environmental conditions (i.e.,
resting seed) to the weakest and most vulnerable
stage in plant development, the seedling [9].
Different environmental factors may determine seed
germination, although the essentials are an
appropriate combination of temperature, moisture
and light [2]. In addition, the chemical environment
surrounding the seed must be suitable [14], and the
presence of allelochemicals inhibitors released by the
surrounding vegetation may also determine
germination success [5]. Allelopathy is “Any process
involving secondary metabolites produced by plants,
micro-organisms, viruses, and fungi that influence
the growth and development of agricultural and
biological systems (excluding animals), including
positive and negative effects” [17]. The most widely
used biological assays for allelochemicals are seed
germination and seedling growth studies. Allelopathy
in natural and agricultural ecosystems is receiving
increasing attention because allelochemicals
significantly reduce the growth of other plants and
the yields of crop plants [12].
Allelochemicals are found to be released to
environment in appreciable quantities via root
exudates, leaf leachates, roots and other degrading
plant residues, which include a wide range of
phenolic acids such as benzoic and cinnamic acids,
alkaloids, terpenoids and others [20]. These
Corresponding Author
Ali Asghar Aliloo, PhD.Department of Agronomy and Plant Breeding, Faculty of Agriculture,
E-mail: [email protected] University of Maragheh, Maragheh 55181-83111, Iran
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Adv. Environ. Biol., 6(9): 2513-2518, 2012
compounds are known to modify growth,
development of plants, including germination and
early seedling growth. The ‘visible’ physiological
effects from allelopathy interactions are frequently
observed as inhibited or delayed seed germination or
reduced seedling growth, which are secondary
expressions of primary effects on metabolic
processes as photosynthesis, respiration, cell
division, pigment synthesis, production of plant
hormones and their balance, membrane stability and
permeability, mineral uptake, movement of stomata
amino acid synthesis, nitrogen fixation, and specific
enzyme activities [15].
Information about allelopathic potential of pine
species may help landscape engineers to select a best
combination of species that they grow without or less
allelopathic effects on each other. For this reason, the
objective of the study was to distinguish allelopathic
impacts of Pinus eldarica on seed germination and
seedling growth of Lolium perenne and Poa
pratensis.
Material and Methods
A factorial experiment in completely
randomized block design was conducted under
laboratory conditions in the University of Maragheh
in Iran. The seeds of the tested grasses were
perennial ryegrass (Lolium perenne) and blue grass
(Poa pratensis). Needles of pine trees were collected
from nearby trees and dried. The dried plant material
was then ground to pass a 2-mm sieve. Aqueous
extracts (w/v) were prepared by extracting 100 g of
dried, ground plant samples with 1000 mL of
deionized water in a shaker for 24 h. The mixture
was then filtered using filter paper (Whatman no. 1)
to obtain 10% extracts. The extract was considered
as stock solution and a series of solution with
different concentrations (2.5,5 and 7.5%) were
prepared for each species by dilution and distilled
water was used as a control. The extracts were stored
at 4˚C until use within 24 h.
A number of 100 seeds of L. perenne and P.
pratensis were put in plastic containers between filter
paper. The seed surface was sterilized before use.
Each variant was laid out in four replications. The
samples were then placed in a germinator at a
temperature of 22˚С±2˚С for seven days. The
following
characteristics
were
determined:
percentage of germinated seeds (%), root length,
shoot length, root/shoot ratio, seedling length, root
dry weight, shoot dry weight, seedling dry weight,
percentage of abnormal seedling and number of
roots.
The inhibition rate on seed germination (GIR)
and seedling growth (SIR) were calculated using the
formula of Ahn and Chung [1]:
GIR or SIR =[(Control-Aqueous extract)/
Control] * 100 [1]
The estimates obtained by application of the
programme Probit were used to determine LC50 for
extracts (Hamilton et al., 1978).
The index of initial plant development=Index
Germinations(GI) was determined by the formula of
Gariglio et al., (2002):
GI = [G/G0 * L/L0 ]* 100 [2]
where: G –percentage of germinated seeds in the
studied variant; G0 – percentage of germinated seeds
in the control variant; L – average length (cm) of the
primary seedling in the studied variant transformed
into percentage as against the control variant; L0 –
average length (cm) of the primary seedling in the
control variant taken as 100%.
All experimental data were statistically
processed using the Statistical Analysis System
(SAS) program [22].
Results And Discussion
Results of variance analysis showed that
between two grass species were significantly
differences for germination percentage, seedling dry
weight, shoot dry weight and number of roots (table
1). The dissimilar results for grasses related to the
different growth potential of each species. Generally,
Lolium perenne have larger seed size than Poa
pratensis that produced larger seedling. Within a
seedlot, seeds with a greater seed weight have greater
storage reserves and thereby have increased seed
vigor [3]. Plants grown from smaller spring wheat
(Triticum aestivum L.) seeds emerged faster but
accumulated less shoot weight than plants grown
from large seeds. Seed size accounted for
approximately 50 percent of the variation in seedling
shoot dry weight [16]. Ellis et al., [4] reported that
seeds with high germination index have a high seed
vigor which could produce vigorous seedlings with a
high performance, thereby in this study Lolium
perenne with high initial vigor, showed high
germination index (table 2), results also revealed that
seeds with greater germination index had low
inhibition rate for germination percentage and
seedling growth (table 2). These finding can be
important for decision making in landscape
management, especially when there are not suitable
data about allelopathic potential of using species. So,
the choice of vigorous seeds that lead to high
germination index can be useful to alleviation of
possible risks.
With respect to table (1) different aqueous
extracts of pine needles had significantly effect on
number of roots(NR), shoot length(SL), root
length(RL),
shoot/root
ratio(S/R),
seedling
length(SeL), dry weight of root(RDW), dry weight of
shoot(SDW), dry weight of seedling(SeDW),
germination percentage(G), abnormal seedlings(AS),
germination inhibition rate(GIR), seedling inhibition
rate(SIR) and germination index(GI). The means of
traits that were affected by pine aqueous extracts
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Adv. Environ. Biol., 6(9): 2513-2518, 2012
(A.E.) have been shown in table (3). With increase of
extracts concentrations, the germination percentage
was dramatically decreased compared to the control.
Allelochemicals are found to be released to
environment in appreciable quantities via root
exudates, leaf leachates, roots and other degrading
plant residues, which include a wide range of
phenolic acids such as benzoic and cinnamic acids,
alkaloids, terpenoids and others [20]. These
compounds are known to modify growth,
development of plants, including germination and
early seedling growth.
Aqueous extracts of pine leaves applied to both
grasses decreased root length (RL), gradually.
Differences among first three concentrations (2.5, 5
and 7.5%) for root length were not significant,
however between these concentrations and
concentration of 10%, significant differences were
observed. The highest root growth inhibition belongs
to highest concentration. Jobidon [13] reported that
leaf leachates of Pinus divaricata (jack pine) and P.
resinosa (Norway pine) significantly decreased
germination and seedling growth of Phleum pratense
(timothy), Agropyron repens (couch grass), and
Epilobium angustifolium (fireweed). Aqueous
extracts of Artemisia vulgaris and Pinus roxburghii
were significantly inhibitory
effect on seed
germination of Lepidium virginicum [18].
The effects of pine needles extracts on shoot
length (SL) were more complex. With increase of
extracts to 5% the shoot length was significantly
increased and a stimulatory effect was observed in
low and medium concentrations, especially at 2.5%
(table 3) but with more increase of concentration to
the highest level, the inhibitory effect was seen and
shoots length decreased noticeably.
Results of this study revealed that with pine
aqueous extracts application, the Root/Shoot ratio
was significantly decreased as compared with control
(table 3), with increase of A.E. concentrations, the
ratio gradually decreased. But among different A.E.
differences were not significant. This is suggested
that allelopathic effect of pine A.E. on the root
growth is greater than the shoot growth. With regard
to pine needle biomass content in water extracts, it
was evident that only at high levels of pine needle
biomass content (7.5 and 10%), seedling length was
remarkably decreased (table 3), as compared to the
control variant. The mechanism of inhibition on the
seedling growth caused by allelochemicals can be the
result of reduced cell division and/or cell elongation
[11].
The obtained results are contradictory and
confirm to a different degree the inhibitory or
stimulatory effect of allelochemicals on the seed
germination and seedling growth. Few studies have
been reported that legumes crop residues have been
stimulatory effects on studied crops. For example,
chopped alfalfa added to soil stimulated the growth
of tomato, cucumber, lettuce and several other plants
[21]. Gill et al., [7] have also reported stimulatory
effect of mungbean, sesame and soybean residues on
wheat, chickpea and lentil at lower concentration.
Improvement in corn yields following legume crops
has also been reported [19,23].
Needle aqueous extracts of pine do not adversely
influenced root dry weight and shoot dry weight of
grasses in the 2.5 and 5% treatment compared to the
control (table 3). However, these traits significantly
affected at higher A.E. concentrations of 7.5 and
10%. Usually, with increase of allelochmical
intensity, the growth inhibition was increased.
The A.E. of pine affected the abnormal
seedlings, significantly (table 1). Highest abnormal
seedling percentage (47.23%) was associated with
highest A.E. (table 3). The rate of abnormality was
increased from 5% concentration and reached to
highest level at 10%.
According to the table (1) the influence of pine
needle A.E on root numbers was also significant, as
comparing to the control. Although, the decrease of
root numbers was continued with increasing of A.E.
concentrations, however, no statistically significant
differences were found among A.E for this trait
(table 3).
Table 1: Analysis of variance for Pine needles extracts effects on germination and seedling growth of two grass species
means of squares
Seedl
Sho
Roo
Seedl Seedli
Roo
Germin
ing
ot
Shoo
Germina
t dry
ng
Root/S ing
Germin
t
ation
dry
dry
t
tion
wei
Inhibi
lengt
ation
leng hoot
D
Inhibiti
weig
wei
lengt
Source
Percenta
ght
tion
h
Ratio
index
th
F
on
ht
ght
h
ge
(RD
Rate
(SeL
(GI)
(RL (R/S)
Rate
(SeD
(SD
(SL)
(G)%
W)
(IRS)
)
)
(IRG)
W)
W)
Replica
3
27.22
0.016
879.41
0.91
0.20 0.0029
1.93
0.080
1
0.01
1
tion
0*
Species
1
738.22*
0.064*
3226.6
1.06
1.44 0.0137
4.21
0.268
5*
0.15
7*
(S)
*
1**
7**
Extract
4
3161.83
0.375*
8131.8
25.2
5.47 0.0746
45.7
0.525
18*
0.43
24**
s(Ex)
**
*
4**
8**
**
**
0**
6**
*
**
S*Ex
4
241.66
0.033
226.91
0.68
1.25 0.0274
2.74
0.022
0.4
0.09
0.8
2
Error
2
91.48
0.013
323.46
0.62
0.57 0.0109
1.62
0.022
0.7
0.03
1
7
4
* Significant at the 0.05 level.
** Significant at the 0.01 level.
Abnor
mal
seedli
ng
(AS)
59.15
Root
Num
bers
(RN)
2839.
9**
28.93
0.099
**
0.096
*
0.201
**
0.031
98.51
0.017
3.15
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Adv. Environ. Biol., 6(9): 2513-2518, 2012
Table 2: Means comparison of Lolium perenne and Poa pratensis
Grass species
G%
GI%
GIR%
Lolium perenne
75.65
78.78
17.78
Poa pratensis
66.80
60.82
25.81
Data located at column have significant difference at p< 0.05.
SIR%
9.55
25.94
SDW(g)
0.0412
0.0326
Table 3: Comparison of means Pine needles extracts on grasses seed germination and seedling growth.
Treatmen
G
SL(cm
RL(cm
R/S
SeL(cm
SDW(g
RDW(g
SeDW(g
AS
t
%
)
)
)
)
)
)
%
Control
92
6.65 b
2.71 a
0.39
9.33 a
a
8a
2.5%
82
7.55 a
1.66 b
0.21
9.06 a
ab
8b
5%
77
6.88 ab 1.48 b
0.21
8.34 ab
b
7b
7.5%
65
6.20 b
1.05 bc 0.16
7.26 b
c
3b
10%
40
3.00 c
0.48 c
0.16
3.48 c
d
1b
Different letters indicating significant difference at p< 0.05.
0.0465
a
0.0422
a
0.0376
a
0.0276
b
0.0082
c
The results obtained for germination index was
similar to the results of germination percentage (table
3). with increasing concentration of A.E this index
significantly decreased and at severe concentrations
the suppression was intensive. According to
accumulation of various growth parameters in the
germination index [Eq. 1], their results are more
reliable than the germination percentage.
0.0068
a
0.0059
a
0.0059
a
0.0027
b
0.0014
b
0.0533 a
0.0481 a
0.0435 a
0.0300 b
0.0096 c
4.5
d
3.5
d
18.2
c
33.1
b
47.2
a
RN
0.26
0.16
RN
1.4
7a
1.1
9b
1.1
8b
1.1
2b
1.0
6b
GI
%
100
a
91
ab
80
b
58
c
20
d
GIR
%
SIR
%
0 d
0a
10 cd
0a
15 c
8 ab
29 b
20 b
56 a
61 c
The germination inhibition rate (GIR) and
seedling inhibition rate (SIR) were also affected by
pine aqueous extracts (p≤0.01). For both traits a
significantly
difference
was
obtained
at
concentrations above 2.5% as compared with control
(table 3). The increase in aqueous concentration was
concomitant with increase of inhibition rates and a
positive relationship was observed between
inhibition rates and A.E. concentrations (fig.1).
Fig. 1: The effects of different pine needls extracts on germination inhibition rate (series 1) and seedling
inhibition rate (series 2)
According to the figure (1) the effects of A.E. on
the germination inhibition rate (series 1) at 2.5, 5 and
7.5 concentrations is higher than the seedling
inhibition rate (series 2) and only at highest
concentration this position has changed. Therefore,
the first evaluation was that the inhibitory effect of
pine aqueous extracts on the germination stage is
greater than the seedling stage. In other words, the
seed germination stage in studied grasses is more
sensitive than the seedling stage. But the results of
probit analysis of these traits against concentration
showed that such evaluations are not accurate
because, according to the probit analysis the LD50 of
pine aqueous extracts for germination inhibition rate
and seedling inhibition rate was 10.6% and 9.4%,
respectively. Therefore, the Sensitivity of seedling
growth stage to the allelopathic substance was higher
than the germination stage.
Conclusions:
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Adv. Environ. Biol., 6(9): 2513-2518, 2012
The needle water extracts from dry biomass of
Pinus eldarica showed an inhibitory effect on the
seed germination and seedling growth of Lolium
perenne and Poa pratensis. The inhibitory effects on
seed germination and seedling growth of L.perenne
was more than the P.pratensis. our finding suggested
that the seed germination can be considered as a
relatively less sensitive period of the individual plant
development, whereas the period of seedling growth
is suitable for potential testing of the allelopathic
effect of the studied extracts under laboratory
conditions due to direct contact of the seedlings with
the extracts during the bioassays. Also the sensitivity
of seeds with high germination index under
allelopathic chemical treatment to seed germination
and seedling growth is probably low. Therefore,
these finding can be important for decision making in
landscape management, especially when there are not
suitable data about allelopathic potential of using
species. So the choice of vigorous seeds that lead to
high germination index can be useful to alleviation of
possible risks.
9.
10.
11.
12.
13.
14.
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