Allelopathic tolerance of alfalfa ( Medicago sativa L . ) varieties to dodder ( Cuscuta epithymum L . )

Allelopathic effects of cold water extracts of Cuscuta epithymum L. on seed germination and initial development of Medicago sativa L. varieties were investigated under laboratory conditions at the Institute of Forage Crops, Pleven, during 2016-2017. It was found that the water extracts from dry biomass of C. epithymum had a considerably stronger inhibitory effect on the studied M. sativa varieties (IR 32.7-100.0 %), as compared to the extracts of fresh biomass (IR 0.2-40.5%). Depending on the kind of C. epithymum extract (fresh or dry parasitic weed biomass), IRs for seed germination of the tested M. sativa varieties could be conventionally classified into five groups: 1) seed germination stimulation, -1.4% – extracts from fresh biomass affecting the variety „Multifoliolate“; 2) seed germination inhibition of 0.1-10% – extracts prepared from fresh biomass affecting the varieties „Prista 3“, „Pleven 6“, „Prista 5“ and „Obnova“; 3) seed germination inhibition of 11-20% – extracts from fresh biomass affecting the varieties „Roly“ and „Victoria“; 4) seed germination inhibition of 30-45% – extracts from dry biomass affecting the variety „Multifoliolate“; 5) seed germination inhibition of 46-60% – extracts from dry biomass affecting the varieties „Prista 5“, „Prista 3“, „Victoria“, „Roly“, „Dara“, „Pleven 6“ and „Obnova“. The varieties „Victoria“, „Prista 5“ and „Multifoliolate“ of M. sativa possess some allelopathic tolerance because their germination indexes (GI) range from 80.5 to 88.7 % for the extracts prepared from fresh weed biomass of C. epithymum, and from 47.1 to 48.6% for the extracts from dry weed biomass, compared to control treatment. These varieties can be used as components in future breeding programmes.


INTROdUCTION
In the last few decades, research work has been focused on studying allelopathic interrelations between cultivated plants and weed species with the purpose of finding varieties with high allelopathic potential (Masum et al., 2016;Trezzi et al., 2016).The discovery of varieties with high allelopathic potentials provides a possibility for decreasing inputs in agricultural crop growing and for producing organic foods (Bashir et al., 2011;Bashir et al., 2012).
Weed infestation of alfalfa crops in Bulgaria is of a mixed type with predominating early-and late-spring weeds, perennial mono-and dicotyledonous weeds and some parasitic weed species, which deteriorate the quality of the obtained produce and decrease fodder and seed production (Dimitrova, 2005;Dimitrova & Serafimov, 2007a;2007b;Dimitrova, 2008).
Literature data have revealed differences among the varieties of different crops (Avena sativa L., Glycine max (L.) Merr., Pisum sativum L., Sorghum sudanense Piper Stapf, Triticum spp., etc.) regarding their allelopathic tolerance to different weed species (Rice, 1995;Miralles et al., 2012;Fragasso et al., 2013;Cheng & Cheng, 2015).In Bulgaria, no detailed results have been published about allelopathic tolerance of Bulgarian alfalfa varieties to different weed species.The objective of this study was to determine allelopathic effects of cold aqueous extracts of fresh and dry biomass of dodder (Cuscuta epithymum L.) on seed germination, and growth and development of seedlings of different alfalfa (Medicago sativa L.) varieties, and to identify the varieties with allelopathic tolerance of that parasitic weed.

MATERIAL ANd METHOdS
The study was conducted under laboratory conditions at the Institute of Forage Crops in Pleven, Bulgaria, during 2015-2016.
The biomass of plant samples of C. epithymum was collected in a weed-infested natural environment in the environs of the Institute of Forage Crops, Pleven, at the growth stage BBCH -65-67 (Hess et al., 1997).

Plant extracts
Aboveground biomass of C. epithymum was chopped into 0.5-3.0cm long segments.Two kinds of weed extracts were prepared: А -from fresh weed biomass of C. epithymum, crushed with quartz sand, and В -from dry weed biomass after drying the samples to constant dry weight at 55 ± 2 0 С and grinding in Retsch SM-1 grinder to a sieve size of 1.0 mm.
A hundred grams of dry and the same amount of fresh biomass of C. epithymum were soaked in 1 l -1 distilled water.The samples were prepared in such a way that fresh or dry biomass of C. epithymum were cold extracted at a temperature of 24 ± 2 0 С for 24 h in a shuttle apparatus at 150/60 с -1 .The obtained extracts were decanted and filtered through filter paper.All available aqueous extracts were brought to weed biomass content of 25, 50 and 100 g biomass per litre of distilled water (presented hereinafter as g/l -1 ) (Faravani et al., 2008).Thymol (C 10 H 14 O) was added to each extract as a preserving agent (Marinov-Serafimov et al., 2007).

Bioassay
One hundred seeds of each alfalfa (M.sativa) variety ("Pleven 6", "Dara", "Obnova", "Victoria", "Prista 3", "Prista 5", "Roly" and "Multifoliolate") were put in Petri dishes (90 mm diameter) between filter papers.All prepared extracts, according to their parasitic weed biomass contents, were pipetted at a ratio of 1:20 as against the seed mass ( Marinov-Serafimov et al., 2007).Distilled water was used for control seeds.Each variant was laid out in ten replications.The samples were then placed in a thermostat-operated device at a temperature of 22 ± 2 0 С for seven days.

Effect assessment
For assessing the experimental results the following biometric parameters were examined: seedling length (mm), and fresh weight of seedlings (g).Seedling length was measured using graph paper, while weight was measured on an analytical balance.

Number of seeds germinated GS % = -
where Ccharacteristic in the control treatment; Т -characteristics in each treatment.
where G -germinated seeds in each treatment, %; G 0germinated seeds in the control treatment, %; L -average length (cm) of seedlings in treatment transformed into percentage as against the control treatment; L 0 -average length (cm) of the seedlings in the control treatment taken as 100%.
The percentage of seed germination in each treatment was previously transformed by Equation (4) (Hinkelman & Kempthorne, 1994) where x % -germinated seeds for each treatment (%).
The collected data were analyzed in Statgraphics Plus for Windows Ver.2.1 and STATISTICA Ver. 10.

RESULTS ANd dISCUSSION
The species and concentrations of the applied extracts of C. epithymum had a substantial influence on laboratory seed germination of the studied alfalfa varieties (Таble 1).With increasing concentrations there was a general tendency of germination decrease in all varieties, i.e. 0.5-59.6% on average, as against the control treatment.
The extract of C. epithymum dry biomass showed a considerably stronger inhibitory effect on the studied alfalfa varieties (IRs 32.7-100.0%)and the differences are statistically significant at P=0.05, as compared to the extract of fresh biomass (IRs 0.2-40.5%)(Таble 1).That was probably due to the considerably higher concentrations of that type of extract, compared to concentrations found in agrophytocenoses with falling off and decomposition of weed biomass.An exception was only the extract of 25 g l -1 parasitic fresh weed biomass of C. epithymum in the varieties "Pleven 6" and "Prista 3", which showed a weak stimulating effect (from 1.4 to 8.2%) but the differences were not statistically significant (Table 1).Seed germination was mostly unaffected by the tested fresh weed extract of C. epithymum only in the M. sativa variety "Multifoliolate", while nonsignificant stimulating effects were only found at the highest concentrations (50 and 100 g/l -1 ).Similar results were reported by Othman et al. (2012), showing that fresh plant extracts of C. campestris did not reduce seed germination of radish (R. sativus).
This relationship could be due to allelochemicals (terpenes, long-chain fatty acids, phenols, phenolic acids and lactone) that are present in the parasitic weed species C. campestris (Khanh et al., 2008, Smith et al., 2016).
Allelochemicals are known to be toxic and have lethal effect on seeds at higher concentrations, whereas their lower concentrations inhibit seed germination of test plants (Macías et al., 2007;Yu et al., 2011;Othman et al., 2012;Baličević et al., 2014).
Differences in inhibitory effects of the extracts made from fresh or dry weed biomass of C. epithymum on seed germination of the test plants can be attributed to diffusion of soluble allelochemicals during extraction of fresh and dry weed biomass (Dhima et al., 2009;Konstantinović et al., 2014;Ravlić et al., 2016).Similar results were reported by Sisodia & Siddiqui (2010), Mondal et al. (2015), Petrova et al. (2015) and Ravlić  Qasem (1995) indicated that dry biomass may exhibit lower inhibitory potential than fresh biomass since the harmful effect could be greatly reduced during the drying process.Similar results were obtained in our study determining the inhibition rate (IR) of seedling growth depending on the kind and content of weed biomass in water extracts (Table 2).
The rate of inhibition of seedling growth increased disproportionately with increase in weed biomass content in water extracts, from 0.2 tо 18.0% on average for extracts from fresh weed biomass, and from 25.0 tо 48.3% for extracts from dry weed biomass (Table 2).The mechanism of inhibition of seedling growth caused by allelochemicals can be the result of reduced cell division (Cheng & Cheng, 2015;Soltys et al., 2013).
There was a specific variety reaction with regard to the inhibitory effect of the extracts (fresh or dry parasitic weed biomass) on seedling growth of the studied varieties of alfalfa (Table 2).
The applied concentrations of the aqueous extracts from dry and fresh biomass of C. epithymum had no statistically significant inhibitory effect on seedling growth of the M. sativa variety "Victoria".A significant inhibitory effect of 14.1-40.8% of all applied concentrations (at P=0.05) was found only in the variety "Multifoliolate".
The extracts prepared from the fresh and dry parasitic weed biomass had various effects on the accumulation of fresh biomass in g per seedling of the test plants (Table 3).On average, the fresh biomass extracts of C. epithymum reduced accumulation of fresh biomass in g per seedling of alfalfa variety for up to 2.2%, while dry biomass extracts reduced the same parameter for over 50.0%, compared to controls.
The obtained experimental data confirmed the results of Seyyedi et al. (2013) and Nešić et al. (2016), according to which the impact of allelochemicals becomes evident already during seed germination but is even more pronounced during the subsequent growth of primary seedlings of plants.The index of germination (GI) depended on the same factors and followed the observed relationship pattern with regard to laboratory seed germination and growth of the seedlings of test plants (Table 4).The performed analyses showed that the studied extracts prepared from fresh and dry weed biomass provoked stimulating and/ or inhibitory effects on the initial plant development of alfalfa varieties.With increasing weed biomass content in the water extracts made from fresh biomass of С. epithymum, GIs decreased 6.8-26.3% on average, while the decrease rate reached 64.0% in the variant of dry biomass extracts, as compared to control treatment.Exceptions to the described relationship were found only at 25 g l -1 of extract from fresh biomass of C. epithymum in the alfalfa variety "Pleven 6" (117.0%) and the variety "Victoria" (101.7%).
Therefore, the observed differences among M. sativa varieties with regard to allelopathic effects of C. epithymum extracts may be attributed to the genetic differences of the examined alfalfa varieties because comparisons among them were performed at equal concentrations of extracts.Allelopathic tolerance was detected in the varieties "Victoria", "Prista 5" and "Multifoliolate".Similar results in radish (R. sativus) and lettuce (L.sativa) were reported by Othman et al. (2012).They found that the species of test plants and their varieties had different levels of susceptibility to allelopathic effects of plant extracts (from fresh or dry biomass) and that they depended on the applied concentrations.

CONCLUSION
The water extract prepared from dry biomass of C. epithymum showed a considerably stronger inhibitory effect on the studied M. sativa varieties (IR 32.7-100.0%)at P=0.05 statistical significance of difference, as compared to the extract of fresh biomass (IR from 0.2 to 40.5%).
Depending on the type of C. epithymum extract (fresh or dry parasitic weed biomass), the rates of inhibition of seed germination of the tested M. sativa varieties could be conventionally classified into five groups: the first group (seed germination stimulation -1.4%) includes extracts from fresh biomass as affecting the variety "Multifoliolate"; the second group (seed germination inhibition of 0.1-10%) includes extracts prepared from fresh biomass as affecting the varieties "Prista 3", "Pleven 6", "Prista 5" and "Obnova"; the third group (seed germination inhibition of 11-20%) includes extracts from fresh biomass as affecting the varieties "Roly" and "Victoria"; the fourth group (seed germination inhibition of 30-45%) includes extracts from dry biomass as affecting the variety "Multifoliolate"; and the fifth group (seed germination inhibition of 46-60%) includes extracts from dry biomass as affecting the varieties "Prista 5", "Prista 3", "Victoria", "Roly", "Dara", "Pleven 6" and "Obnova".
The studied alfalfa varieties showed different levels of susceptibility to the allelopathic effect of extracts prepared from fresh and dry biomass of C. epithymum, which was probably due to their genetic differences.The alfalfa varieties "Victoria", "Prista 5" and "Multifoliolate" possess some allelopathic tolerance because their index of germination (GI) ranged from 80.5 to 88.7% in extracts prepared from fresh weed biomass of C. epithymum, and from 47.1 to 48.6% in extracts from dry weed biomass, as compared to control treatments.These varieties can be used as components in future breeding programmes.

Table 1 .
Effects of different concentrations of aqueous extracts of C. epithymum on seed germination of M. sativa varieties, % a, b, c, d LSD at P=0.05 confidence interval, GS % -germination seeds, IR -Inhibition rate

Table 2 .
Effects of different concentrations of aqueous extracts of C. epithymum on early seedling growth of M. sativa varieties, mm

Table 3 .
Effects of different concentrations of aqueous extracts of C. epithymum on early accumulation of fresh biomass (g) of seedlings of M. sativa varieties a. b. c. d. e LSD at P=0.05 confidence interval, IR -Inhibition rate

Table 4 .
Effects of different concentrations of aqueous extracts of C. epithymum on the initial development (GI) of M. sativa varieties