Allelopathic effects of water extracts of Sorghum halepense ( L . ) Pers . , Convolvulus arvensis L . and Cirsium arvense Scop . on early seedling growth of some leguminous crops

In order to study the allelopathic effect of aboveground dry biomass of Sorghum halepense, Convolvulus arvensis and Cirsium arvense on seed germination and early seedling growth of Pisum sativum (L.), varieties Mir (winter form) and Kerpo (spring form); Vicia sativa (L.), variety Tempo, and Medicago sativa (L.), variety Dara, a laboratory experiment was conducted at the Institute of Forage Crops Pleven. Four concentrations: 1.25, 2.5, 5.0 and 10.0% were applied to each weed biotype used to study allelopathic effects. The results showed that weed extracts significantly decreased germination percentage, shoot and root length (cm), shoot and root weight (g), and seed vigor index (SVI1 and SVI2) of the tested species. In general, the variable effects are related to the weed species and extract concentrations.


IntroductIon
One of the problems studied in organic farming is weed control and interspecies relationships between crops and weeds.It requires a selection of more competitive crops or an increase in crop competitiveness in order to reduce weed competition.
Allelopathy is a direct or indirect, beneficial or harmful effect of one plant on another through the production of secondary compounds (allelochemicals) and their release into the environment in sufficient quantity to cause recordable effects (Cheema et al., 1998;Khalid et al., 2002;Aleksieva and Serafimov, 2008).Allelochemicals are synthesized and accumulated in different plant parts (root, stem, leaves, flowers and fruits, etc.) and released into the environment by evaporation, decomposition and elution of weed residues which have an inhibitory effect on the germination, growth and development of a number of crops.In order to determine allelopathic relationships between weeds and crop plants, Moosavi et al. (2011) and Nouri et al. (2012) used plant extracts from dry weed biomass because such extracts had significantly higher concentrations than plants occurring in agrophytocenoses.The relatively low competitiveness of some legumes [Pisum sativum (L.) and Vicia sativa (L.)] during their early seedling growth, makes weeds a limiting factor in the production of grain and green biomass (Маринов-Серафимов and Димитрова, 2007).Holm et al. (1977) and Gustavsson (1997) described Sorghum halepense, Convolvulus arvensis and Cirsium arvense as species belonging to a group of "the world's worst weeds".
The purpose of this study was to determine the allelopathic effects of aboveground biomass of S. halepense, C. arvensis and C. arvense on seed germination and early seedling growth of Pisum sativum (L.), varieties Mir (winter form) and Kerpo (spring form); Vicia sativa (L.), variety Tempo, and Medicago sativa (L.), variety Dara.

mAterIALS And methodS
The experiment was conducted under laboratory conditions at the Institute of Forage Crops -Pleven during 2012 to study the allelopathic effects of S. halepense, C. arvense and С. аrvensis on the germination and early seedling growth of P. sativum, V. sativa and M. sativa.
Plant and seed material: Aboveground biomass (leaves and stems) of S. halepense, C. arvense and С. arvensis was collected at the stage BBCH 65 (Hess et al., 1997).All samples were cut to 0.5-3.0cm length and dried to constant dry weight at 60 ± 3 0 C and then ground.Seeds of P. sativum, varieties Мir (winter form) and Kerpo (spring form); V. sativa, variety Тempo, and M. sativa, variety Dara, were used in a germination test.The seeds were surface-sterilized for 20 minutes with 1% NaOCl (4% NaOCl commercial bleach), then rinsed three times with distilled water (Siddiqui et al., 2009).
Extraction procedure: Aqueous extract was prepared by soaking 100 grams of the powdered material of each weed (S. halepense, C. arvense and С. arvensis) in 1 L of distilled water at 24 ± 2 0 С for 24 h.After that, cold aqueous extract was filtered using Whatman filter paper.The stock solution (10% w/v) was diluted appropriately with distilled water to give the final concentrations of 1.25, 2.5, 5.0 and 10.0%.Thymol (C 10 H 14 O) was added to each extract as a preserving agent (Marinov-Serafimov et al., 2007a).
Bioassay for growth: Ten seeds (Hassan et al., 2012) of the test species (P. sativum, V. sativa and M. sativa) were placed between filter paper in 9.0 cm diameter Petri dishes.The Petri dishes were irrigated with extract solutions at ratios from 1:6 to 1:20 to the seed weight (Marinov-Serafimov et al., 2007b), and distilled water was used as control.Each treatment was replicated five times.Petri dishes were placed in a germinator at 22 0 C ± 2 0 C for seven days.A biochemical analysis of the aboveground biomass was performed to determine the contents of: cyanogenic glucosides (Ермаков et al., 1987), total phenols (Swain and Hillis, 1959) and condensed tannins (Terrill et al., 1992).The pH of aqueous extract was determined with pH meter.Distilled water was used for the control.
Data analysis.Seedling performance was assessed through relative seed germination (RSG), relative elongation of root + shoot (RERS) and relative weights of root + shoot (RWRS) by the formulas of Asgharipour and Armin (2010): RSG=(number of seeds germinated in extract/ number of seeds germinated in control)×100; RERS=(mean root+shoot elongation in extract/ mean root+shoot elongation in control)×100; RWRS=(mean root+shoot weight in extract/mean root+shoot weight in control)×100.
Mean time to germination (MTG) is an index of seed germination rate (Ellis and Roberts, 1981) and calculated by: MTG=(∑(Dn))/(n), where n is the number of seeds that had germinated on day D and D is the number of days counted from the beginning of germination.
Statistical analysis: The collected data were analyzed using the software Statgraphics Plus for Windows Ver.2.1.The percentage of germinated seeds in each variant was previously transformed by the formula: Y = arcsin √(x % /100) (Hinnkelmann and Kempthorne, 1994).To determine the LC 50 of weed extracts, the software Trimmed Spearman-Karber, Ver. 1.5, based on Hamilton et al. (1978), was used.

reSuLtS And dIScuSSIon
The 1% extracts of aboveground biomass of S. halepense, C. arvense and C. arvensis showed inhibitory effects on seed germination of P. sativum, V. sativa and M. sativa ranging from 42.0 to 87.5% for all treatments (Figure 1).With increasing concentrations of weed extract (from 1.25 to 10.00%), the percentage of germinated seeds decreased from 12.50 to 58.00% in all treatments, compared with the control.Based on the percentage of inhibition of seed germination of P. sativum, V. sativa and M. sativa (depending on extract), two groups were conditionally formed: Igroup from 27.89 to 31.12% (Kerpо, Mir and Tem-po), and II -group over 40.93% (Dara).The variety Dara was an exception to this as the extract of S. halepense caused its 100% inhibition at the highest concentration.Very small seeds make contact with the aqueous extract easily, so that even low concentrations can cause an immediate negative effect (Shang and Xu, 2012).
The LC 50 values were determined depending on the effect of concentrations of cold water extracts of S. halepense, C. arvensis and C. arvense on the germination of seeds of P. sativum, V. sativa and M. sativa (Table 1).The observed differences in test species can be attributed to genetic differences because comparison between them was performed at under identical conditions.Similar results had been reported by Einhellig (1996), Aleksieva and Serafimov (2008) and in both reports the cultivated plants and varieties showed different sensitivities to weed extracts, the allelopathic effect was species-specific and depended on concentrations.Germination of the seeds of test plants depended on concentration and pH of their extracts, ranging from 5.23 to 6.10 as shown in Figure 2.

Sorghum halepense Convolvulus arvensis Cirsium arvense
It is well-known that all parts of weed plants (leaf, stem, root and fruit) have different allelopathic potentials (Alam and Islam, 2002;Tinnin and Muller, 2006).Weeds also exert allelopathic effects on seed germination and growth of plants by releasing water-soluble compounds into the soil that are possible allelopathic components (Ashrafi et al., 2007;Batish et al., 2007).These are mostly сyanogenic glycosides, total phenols and condensed tannins (Fateh et al., 2012).The inhibitory effect of weed extracts on the germination and initial growth of the test plants can be attributed to the presence of allelochemicals (Table 2).Biochemical analysis revealed that the content of total phenols, condensed tannins and cyanogenic glycosides in aboveground biomass varied depending on weed species.ts depending on concentration and type of weed  The results confirm the findings of Agarwal et al. (2002), Iqbal et al. (2003), Fateh et al. (2012), Nouri et al. (2012) and Shang and Xu (2012), showing that allelochemicals have an inhibitory and/or lethal effects on seed germination, growth and development of crops.According to these authors, lower concentrations inhibit germination to different degrees, which is probably due to the lower contents of allelochemicals in them, while higher concentrations induce lethal effects on seed germination.
In addition, we detected a high negative correlation (r ranges from -0.914 to -0.995) between seed germination, and the concentration and pH of extracts (Table 3).The effects the extracts on seed germination of P. sativum, V. sativa and M. sativa can be expressed as a function: y = a -b.√x.
Weed еxtracts had negative effect on the growth of roots and shoots of the test plants (Tables 4, 5, 6 and 7).Generally, root and shoot length reacted with a significant difference at the 5% level.Exceptions were found for the lowest concentrations in the variants S. halepense -P.sativum var.Kerpo; S. halepense -P.sativum var.Mir; C. arvensis -V.sativa var.Tempo; C. ar-vense -V.sativa var.Tempo and C. arvense -M.sativa var.Dara, where the differences were nonsignificant.
It can be inferred from the screening results that the degree of inhibition of root and shoot growth depends on the type of extract used and the applied concentration.The most sensitive was M. sativa var.Dara (24.96-100.00%),followed by P. sativum var.Kerpo (14.76-93.78%),while relatively low sensitivity was seen in V. sativa var.Tempo (10.67-88.80%)and P. sativum var.Mir (9.24-84.46%).
There was a general trend of fresh biomass reduction in all studied variants, depending on the type and concentration of weed extracts.An exception to such dependence was observed at the lowest applied concentration in the variants P. sativum var.Kerpo -C.arvense and V. sativa var.Tempo -S.halepense, which exerted a small stimulatory effect.
The mean germination time of P. sativum, V. sativa and M. sativa significantly increased in all treatments compared to the control (Tables 4, 5, 6 and 7).The extract of S. halepense showed maximum MGT values in all crops.SG, SVI 1 and SVI 2 values decreased statistically with increased extract concentration.The results of the present study indicate allelopathic effects of P. sativum, V. sativa and M. sativa in several leguminous crops.The weed extracts contained water soluble compounds to varying degrees.Those compounds may be released by rain or irrigation and dissolve in wa-ter under field conditions.However, natural conditions in organic farming are, more complicated than laboratory bioassays (Mubeen et al., 2011).Therefore, field experiments are necessary before drawing final conclusions on the allelopathic effects of these weed species.

concLuSIonS
The tested extracts of S. halepense, C. arvensis and C. arvense demonstrated variable allelopathic effects on seed germination and early seedling growth of P. sativum, V. sativa and M. sativa.Such allelopathic effects depended both on the extract concentration and the weed from which that extract had been derived.Mean germination time of all test species significantly increased compared to the control, but the speed of germination and seedling vigour index (SVI 1 and SVI 2 ) significantly decreased.S. halepense, C. arvensis and C. arvense growing in farm fields should be controlled at an early stage to avoid their phytotoxic allelopathic effects.

Figure 2 .
Figure 2. pH of the extracts depending on concentration and type of weed

Table 2 .
Biochemical analysis of aboveground biomass of S. halepense, C. arvensis and C. arvense

Table 3 .
The effects of extracts on the germination of seeds of P. sativum, V. sativa and M. sativa

Table 4 .
The effects of aqueous extracts of S. halepense, C. arvensis and C. arvense on the germination of P. sativum var.Kerpo Means sharing the same letters in a column do not differ significantly at 0.05 probability level according to LSD test.

Table 5 .
The effects of aqueous extracts of S. halepense, C. arvensis and C. arvense on the germination of P. sativum var.Mir Means sharing the same letters in a column do not differ significantly at 0.05 probability level according to LSD test.

Table 6 .
The effects of aqueous extracts of S. halepense, C. arvensis and C. arvense on the germination of V. sativa var.Tempo

Table 7 .
The effects of aqueous extracts of S. halepense, C. arvensis and C. arvense on the germination of M. sativa var.Dara Means sharing the same letters in a column do not differ significantly at 0.05 probability level according to LSD test.