Mycobiota of rape seeds in Romania . II . Evaluation of potential antagonistic fungi isolated from rape seeds against the main pathogens of rape crop

In vitro relationships between identified seedand soil-borne fungi from rape samples have been investigated in order to evaluate their antagonistic ability as potential biocontrol agents. The bioproduct obtained from the Trichoderma viride Pers. (strain Td50) has been tested in vivo against the main phytopathogens of rape: Sclerotinia sclerotiorum (Lib.) de Bary, Botrytis cinerea Pers., Alternaria spp. and Fusarium spp. in greenhouse at the Laboratory of Mycology and Plant Pathology, Biology Faculty, University of Bucharest – Romania and in the field at the Agricultural Experimental Research-Development Station Caracal (AERDS), Olt district. The T. viride (strain Td50) bioproduct formulated as a powder for the seed treatment has been effective in the protection of rape plantlets against the above mentioned phytopathogens.

The objective of this approach was the evaluation of potential antagonistic ability of some Trichoderma viride isolates against the main pathogens of rape crop: Sclerotinia sclerotiorum (Lib.) de Bary, Botrytis cinerea Pers., Alternaria spp., Fusarium spp.

MATERIAL AND METHODS
The investigations for evaluation of potential antagonistic ability of some T. viride isolates against the main rape phytopathogens have been conducted in vitro and in vivo (greenhouse, experimental field).
In vitro method of double cultures on the same medium in Petri plates it was used, after Jouan et al. (1964), presented in the Figure 1 the antagonistic fungal ability has been expressed by the coefficient x, calculated after the formula x = iA/iB x eB/eA.In this formula the coefficient x is the value of the quotient of inner radius (i) and outer radius (e) of the test-fungus (A) and the antagonistic fungus (B).In case of x = 1, no influence has been expressed between the two tested fungi; when x < 1, the antagonism is stronger when the coefficient x value is lower or close to 0 (zero); when x > 1, the tested isolates prove no antagonism against the checked phytopathogens.
For in vitro experiments, fungal isolates of Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium spp., Alternaria spp.and Trichoderma spp.In the field at AERDS Caracal, Olt district, the experimental variants were as follows: 1) seeds treated with Trichosemin 25 PTS -1 g/kg seed; 2) seeds treated with a chemical standard (procymidone 50) -1 g/kg seed and 3) untreated control.The Manitoba cv. was cultivated.

© The Author(s) 2014 Published by Polish Botanical Society
In this experiment, evaluation of number and percent of emerging healthy plantlets in comparaison with the same parameter in the standard variant (chemical fungicide procymidone 50) and in the untreated control was performed.

RESULTS AND DISCUSSION
In vitro.Among the tested isolates (Tab.1), the strain Td 35 proved the strongest antagonism against tested phytopathogens, the value of x coefficient ranging between 0.22 and 0.44 (Fig. 2).In the decreasing order, a good antagonistic ability have been proved for strains Td 50 (x = 0.30-0.70)and Td 49 (x = 0.30-0.89).The lowest antagonistic activity has been noticed for the isolates Td 45 , with x coefficient between 0.48 and 0.86 and Td 5 (control), with x coefficient between 0.45-0.89.
The antagonistic behaviour of the tested isolates is shown by the following decreasing order: Td 35 >Td 49 >Td 50 >Td 45 >Td 5.
In vivo.The experiments performed under greenhouse conditions (Tab.2, Fig. 3) proved that the percent of emerging healthy plantlets, in comparison with the untreated control, was higher by 47%.In the standard variant (procymidone 50), the value of the emerging and healthy rape seedlings was higher by 30% in comparaison with the untreated control.
Under the field conditions, in the variant with biological seed treatment, the percentage of emerging helthy plantlets was higher by 26% in comparaison with the untreated control and similar to the variant of the chemical standard (Tab.3).However, after 14 days (29th August 2008), no differences have been registered between the biological and chemical treatments.
These results obtained in the rape crop were similar to other experimental results obtained by us for other industrial oilcrops (sunflower, soybean) and annual pulses (bean, soybean, cickpea) (Baicu, Săvescu 1986;Şesan et al. 1997 a, b) and to our previous results in the rape crop (Galani et al. 2008).Also, these results confirm our tests in vivo proving the efficacy of our bioproduct with T. viride (Td 50 ), patented in 2012 (Şesan et al. 2012) for protecting oilseed plants among them rape (Şesan, Oancea 2010).
In order to obtain a healthy, non-polluted and productive rape crop, the instructions for the application of biological control of rape seeds with the bioproduct based on T. viride were prepared.These instructions consist of: 1) seed treatment: dry; 2) dose of treatment: 1-2 g/kg seeds; 3) time of treatment application: 1-2 days before sowing; 4) storage conditions for the bioproduct: in the dry, wellaired spaces, with a good ventilation, at low temperatures, in the shadow, avoiding 2. Bioproduct with Trichoderma viride -Td 50 strain -was efficient in the rape protection against the seed-and soil-borne pathogens (Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium spp., Alternaria spp.) in the greenhouse (Hydromel cv.) and in the field (Manitoba cv.), too.Applied as seed treatment at a rate of 1-2 g/kg seeds, T. viride bioproduct stimulated the emergence of plantlets and their health status.
3. The instructions for the application of biological control of rape seeds with the bioproduct based on T. viride were proposed: 1) seed treatment: dry; 2) dose of treatment: 1-2 g/kg seeds; 3) time of treatment application: 1-2 days before sowing; 4) storage conditions for the bioproduct: in the dry, well-aired spaces, at low temperatures, in the shadow, avoiding the direct; 5) proper conditions for bioproduct transport, protected against high temperatures and humidity.These instructions are very important for the agricultural practice for obtaining a healthy, productive, nonpolluted with chemicals rape crop.

T
from rape seeds have been used.As antagonistic fungi, 5 strains of Trichoderma viride (Td 5 , Td 35 , Td 45 ,Td 49 , Td 50 ), isolated by the author, have been tested.The bioproduct from Trichoderma viride -isolateTd 50 -(Şesan, Oancea 2010; Şesan et al. 2012) has been also, tested in greenhouse at the University of Bucharest, Laboratory of Mycology and Plant Pathology for the treatment of rape seeds in comparaison with an untreated control and with a chemical standard -procymidone 50.The experiment has been performed in sterile soil, in Petri plates of 12 cm diameter, each variant having 5 replicates.A number of 10 rape seeds Hydromel cv.(from AERDS Caracal, Olt district, 2007) have been used for each replicate.The application dose for biological seed treatment was 1g powder of T. viride bioproduct per kg seeds.

Table 2
Testing the Trichoderma viride (Td 50 ) bioproduct for protecting rape crop cv.Hydromel against soil-and seed-borne pathogenic fungi in greenhouse(2007 yield)