Impact of Trichoderma spp . on Soybean Seed Germination and Potential Antagonistic Effect on Sclerotinia sclerotiorum

Trichoderma species have been registered as species with important plant growth promoting potential and antagonistic effect against various phytopathogens. Trichoderma isolates originating from different soil types from the Vojvodina region (Serbia) were screened using dual culture test for their antagonistic effect against the pathogen Sclerotinia sclerotiorum. All tested isolates had high radial growth inhibition (RGI) factors of the pathogen and high colonization percentage. Growth promoting activity of Trichoderma isolates on soybean seeds was tested under glasshouse conditions. Soybean seeds were coated with suspensions of different Trichoderma isolates and seed germination percentage, root and shoot length were measured. According to data analysed in Statistica 10, using Duncan’s test, there were no significant effects on shoot length among the tested isolates, compared to the control. The four tested Trichoderma isolates showed significant positive effects on germination, root length and vigour index, while two isolates exhibited no significant effect on any of the measured parameters.

As it was mentioned above, Trichoderma species are effective antagonists of the cosmopolite pathogen Sclerotinia sclerotiorum, which is found worldwide in many different soil types and environmental conditions and causes diseases of more than 400 plant species.This pathogen is common on various vegetable plants, sunflower, soybean, edible dry bean, chickpea, peanut, etc., causing white rot of plant roots, stems and fruits or pods, which ultimately ends in a complete plant wilting or rotting if infection had started early.In the case of early infection, the percentage of yield reduction is almost equal to the percentage of infected plants.In years with high amounts of rainfall during summer, white rot is the most dangerous soybean disease in Serbia and its infection intensity can exceed 50%, causing drastic reduction in soybean yields (Vidić and Jasnić, 2011).
Trichoderma species have attracted considerable scientific attention in the past few decades as potential fungal biocontrol agents against a vast number of plant pathogens, and are attractive in a wider research field.The aim of the present study was to assess the growth promoting activities of Trichoderma isolates on soybean seeds and antagonistic activities of those isolates against the common pathogen S. sclerotiorum.

MATERIAL AND METHODS
The tested Trichoderma isolates originated from different soil types and localities in Vojvodina (Serbia), while the S. sclerotiorum isolate originated from a sunflower crop grown at Rimski Šančevi (Novi Sad, Serbia).The isolates were refined to monospores for further research.
Dual culture test was used for screening nine Trichoderma isolates and their antagonistic effects against S. sclerotiorum in vitro.Each Trichoderma isolate plug of a 7-day old culture (5 mm 2 ) was confronted with identical plugs of S. sclerotiorum isolates in 90 mm Petri plates at 60 mm distance on PDA in four replicates.The antagonistic abilities of Trichoderma isolates were evaluated periodically, i.e. on the 7 th , 14 th , 21st and 28 th day of incubation in the dark at 25˚C.After 28 days of incubation, Radial Growth Inhibition (RGI) and Colonization percentages (C) were calculated according to Rodriguez et al. (2000) and Ibarra-Medina et al. (2010), respectively.
The growth promoting activity of the tested Trichoderma isolates on soybean seeds (Favorit variety) were tested under glasshouse conditions.One hundred of surface sterilized soybean seeds were coated with suspensions of each Trichoderma isolate according to a modified method proposed by Mukhtar et al. (2012), while sterile distilled water was used as a control.Spore suspension concentrations were adjusted to 1x10 6 conidia/ml by haemocytometer.Previously sterilized soybean seeds were dipped in seed coating suspensions for 30 minutes and then air dried on filter paper in Petri plates for 24 hours.Dry Trichoderma coated seeds were sown in four replications (25 seeds per pot sized 12.5 x 17 cm) in Classman substrate 2. Seed germination percentages and root and shoot lengths were measured 7 days after sawing.Vigour index was calculated according to formula (Asaduzzaman et al., 2010): Vigour index = [Mean of root length (cm) + Mean of shoot length (cm)] x percentages of seed germination.
All obtained data were analyzed in Statistica 10 using Duncan's test.

RESULTS AND DISCUSSION
According to the results obtained in dual culture tests in vitro, the best inhibition of S. sclerotiorum radial growth (RGI) was observed in interaction with the Trichoderma isolate K132 (52.2%), while Trichoderma isolate K174 achieved the best colonisation of the pathogen (100%).What is more, all tested Trichoderma isolates except K175 caused high colonisation of pathogen mycelia of more than 70% (Table 1).et al., 2010).Similar data showing Trichoderma isolates' antagonistic effect on other pathogen species have also been reported.Antagonism of Trichoderma isolates in dual culture tests was recorded on Pythium aphanidermatum by Mishra (2010) and maximal inhibition of pathogen micelial growth (RGI) was 72% in a dual culture test with a Trichoderma viride isolate.Nine Trichoderma spp.isolates originating from Mexican sandy soils have been found to reduce radial growth rate of Macrophomina phaseolina isolates by at least 50% using in vitro dual culture test (Larralde-Corona et al., 2008), while RGI of M. phaseolina mycelia by Trichoderma harzianum was 47% (Ilyas et al., 1985).Trichoderma isolates have also inhibited radial growth of Phytophtora palmivora mycelia by 33.29-97.86% (Mpica et al., 2009).
In glasshouse tests, there were no significant effects of any of the tested Trichoderma isolates on shoot length, compared to the control.In contrast, significant positive effects on both root length and germination were found in treatments with four Trichoderma isolates -K114, K132, K150 and K160.Vigour indexes of plants treated with those four Trichoderma isolates were significantly higher than the control as well (Table 1).Some tested Trichoderma isolates exhibited significant positive effect only on root length (K176) or germination (K178), or germination and vigour index (K175), while two isolates (K173, K174) showed no significant effect on any measured parameter, despite their high colonisation ability against the pathogen in dual culture test (Table 1).In their research of the influence of rhizosphere fungi on soybean, Maisura and Patel (2009) found that Trichoderma viride promoted an increase in root length, shoot length and seed germination (96.6%).Also, Mukhtar et al. (2012) reported enhanced germination of soybean seeds treated with 6 different Trichoderma species, with germination varying from 76-96%, depending on species, which was higher than the germination obtained in the control (76%).In our research, all tested Trichoderma isolates were also found to have similar growth promoting effect on other grown plants, such as sunflower, rice, maize, bean, safflower, papper, chilli, radish, cucumber, tomato, mustard etc. Ilyas et al. (1985) reported that T. harzianum isolates significantly increased plant height (16.2%) and dry stem weight (10.9%), compared to non-treated healthy sunflower plants in a pot experiment.The growth promoting effects of T. viride and T. harzianum isolates on safflower were tested in a pot experiment, and germination percentages (91.1% and 86.7% respectively), radicale and plumule lenghts were significantly higher than those of untreated seeds in a control (Singh et al., 2008).Joshi et al. (2010) found the maximal germination percent in chilli treated with Trichoderma isolates (79.49%) to be significantly higher than in a control, and enchanced root and shooth lenghts as well.To summarize, Trichoderma isolates tested in this research were equally effective as a growth promoting factor for soybean seeds as the Trichoderma strains tested so far on different other crops.

CONCLUSIONS
This research shows that eight out of nine Trichoderma isolates tested in vitro can be considered as effective antagonists against S. sclerotiorum based on their colonisation percentages that exceeded 70%.Also, four of those Trichoderma isolates had significant positive effects on germination, root length and vigour index of soybean seeds in the glasshouse experiment.Those isolates are promising antagonists and will be included in more comprehensive future research of their antagonistic effects against S. sclerotiorum.

Figure 1 .
Figure 1.Trichoderma isolates with the highest colonization percentages and RGI factors in dual culture tests with S. sclerotiorum

Table 1 .
Trichoderma isolates' growth promoting effect (root length, germination and vigour index) on soybean seeds and antagonistic effect against S. sclerotiorum in vitro Values in the columns followed by the same letters are not significantly different (p<0.05) by Duncan's test; values are average of four replicates *