IN VITRO ANTAGONISM BETWEEN PHYTOPATHOGENIC FUNGI SCLEROTIUM ROLFSII AND TRICHODERMA STRAINS

Six Trichoderma strains (collected from IARI, New Delhi and MTCC, Chandigarh) were tested for their ability to inhibit soil born pathogen of groundnut mainly Sclerotium rolfsii (causing stem rot on groundnut). Morphological observations of Trichoderma strains as well as phytopathogenic fungi S. rolfsii were made from culture grown at 28 0C for about one week on PDA media. The radial growth, fastest growth and coiling of test fungus of various Trichoderma strains on plant pathogenic fungi S. rolfsii were examined under LEICA phase contrast binocular light microscope. In vitro percent growth inhibition of S.rolfsii by various Trichoderma strain was recorded at 5 days after inoculation at 28 C in the 90 cm petriplates. Results obtained from the antagonism study indicated that Trichoderma viride (NBAII Tv 23) inhibited 61% growth of phytopathogenic fungi S. rolfsii followed by T. harzianum (NBAII Th1) (55% growth inhibition of pathogen). This suggested that among different Trichoderma strains, T. viride was the best bio-control agent to inhibit in vitro growth of phytopathogen S.rolfsii which otherwise cause stem rot disease in groundnut.


Introduction
Groundnut (Arachis hypogaea L.) is the most important oil seed crop of India. Groundnut occupies the third place in regard to acreage and in production which provides a rich source of high-quality edible oil (45-50%), easily digestible protein (23-25%), minerals and vitamins. Plant disease cause losses of agricultural crops every year. The distribution of several phytopathogenic fungi such as Sclerotium rolfsii, Pythium, Phytophthora, Rizoctonia and Fusarium has spread during last few years due to changes introduced in farming, with detrimental effects on crops of economic importance. Chemical compounds have been used to control plant disease but it has adverse effect that creates health hazards for humans and other non-target organisms. The development of safer and environmentally feasible plant disease control alternative has become a top priority. In this context, biological control becomes an urgently needs for modern agriculture. Fungi of the genus Trichoderma are potential biocontrol agents of several soil born phytopathogens (Benitez et al., 2004;Hassan et al., 2014). Moreover, all Trichoderma isolates exhibited inhibition to the mycelial growth of all pathogens. This could be due to the production of diffusible components, such as lytic enzymes or water-soluble metabolites Anees et al. (2010). Trichoderma strains are free-living fungi that are common in soil and root ecosystems. They are highly interactive in root, soil and foliar environments. They produce or release a variety of compounds that induce localized or systemic resistance responses in plants. Among the plant pathogens, S. rolfsii were found to be cause for the stem rot disease in Groundnut. Trichoderma have been considered to play an important role in the biological control of stem rot disease. The main objective of this study was to evaluate the interaction between the antagonists Trichoderma strains and fungal pathogen S. rolfsii (Chet and Baker, 1981;Papavizas, 1985;Chet, 1987;Kumar and Mukerji, 1996;Hassan et al., 2013) and the possibility of mycoparasitism through examination of the coiling pattern during antagonism using light microscopy.

Materials and Methods
S. rolfsii was isolated from groundnut plants showing typical symptoms of stem rot were collected from university fields in Junagadh Agricultural University, Junagadh. Strain of T. harzianum (NBAII Th1), T. hamantum ( NBAII Tha -1), T. virens (NBAII Tvs 12) and T. viride was (NBAII Tv 23) collected from Indian Agriculture Research Institute (IARI), plant pathology department, New Delhi and two other strains T. koningii (MTCC 796) & T. pseudo koningii (MTCC 2048) was collected from MTCC, Chandigarh, India. Collected Trichoderma strains and isolated pathogen from stem rot of groundnut was sub-cultured on potato dextrose agar slants and kept at 28 ± 1 o C for seven to eight days for good growth. Such Research Article slants were preserved under refrigeration at 20 o C. This culture was sub-cultured at regular intervals and was used for further studies.
For each pathogen -Trichoderma interaction, a clean slide was placed in petri plates (9 cm) and sterilized. Then a small amount of autoclaved melted potato dextrose agar was spread over the slide to make a thin PDA film on the slide. The 5 mm discs of one week old growing colonies cut from the margin of each pathogen and Trichoderma strains were placed on the opposite sides of the slide 3 cm apart on the PDA surface. Then a few ml of double distilled water was added to the plate to prevent drying and then incubate at 25 ± 1 o C for a week. At the end of incubation period, meeting area of Trichodermapathogen hyphae was observed under a light microscope for the presence of coiling structures for wall disintegration (Hajieghrari et al., 2008).
Six strains of Trichoderma were screened against test pathogen in dual culture methods using potato dextrose agar (PDA) ( Dennis and Webster, 1971). The radial growth of the pathogen in dual culture and control plates was measured after seven day incubation at 28 ± 1 o C temperatures. The percent inhibition of mycelial growth of pathogen was calculated according to formula suggested by Watanabe (Watanabe, 1984).

% Growth inhibition = [(C-T)/C] X 100
Where, C = colony diameter of pathogen in control T = colony diameter of pathogen in inhibition plate

Morphological observation
Pigmentation of varying shades was recorded in some strains of Trichoderma. Generally white myceliya of Trichoderma had many aerial mycelia and its gradually turn to green colour. Yellow pigmentation was shown in T. koningii and dark green colours were observed in T. hamntum. Similar results were obtains with Trichoderma strains often can be readily identified to genus by a distinctive morphology that includes rapid growth, bright green or white conidial pigments and a repetitively branched, but otherwise poorly defined conidiophore structure (Bissett, 1991). Sclerotia begin to develop after 7-8 days mycelial growth of S .rolfsii. Initialy a felty white appearance, sclerotia quickly melanize to dark brown coloration. Narsimha rao also reported that, fungus Sclerotium rolfsii showed characteristics with white, radiating abundant mycelial growth on the affected portion of plant and tuber. Initially sclerotia exhibited white colour later turn to chocolate brown (Narasimha, 2000).

Evaluation of coiling pattern at 10 DAI
Trichoderma strains tested in this work had a marked significant variation in inhibition of pathogen growth. Maximum pathogen growth inhibition occurred in interacting with T. viride NBAII Tv 23 (T6 antagonist). At 10 DAI, T. viride NBAII Tv 23 completely destroyed the host and sporulated (Fig. 3). This process occurred at different intensities depending on the Trichoderma strains. suggesting that various antagonists have different mechanisms of host recognition (Silva et al., 2004). Microscopic study showed that T. viride NBAII Tv 23 was capable of overgrowing and degrading S. rolfsii mycelia, coiling around the hyphae with apressoria and hooklike structures (Fig. 2). Formation of apressoria-like structures enabled the hyphae of Trichoderma strains to firmly attach to the surface of its host mycelium. However, some antagonists (T. koningi MTCC 796, T. hamatum NABII Tha 1) used different mechanism against S. rolfsii just touched the hyphae without coiling, while, T. pseudo koningii showed spore around pathogen not attached to hyphae (Fig. 3). Variability in antagonistic potential among the different strains of Trichoderma against different pathogen have been reported (Bhagat and Pan, 2007). Science has reported that the Trichoderma strains inhibited mycelial growth of S. rolfsii to various degrees, with 52% of strains expressing an average inhibition, varying between 45% and 55%. So, Trichoderma strains are very useful as biocontrol agent (Khattabi et al., 2004). Efficient coiling process followed by a substantial production of hydrolytic enzymes was reported by dos Reis Almeida et al. (2007). The interaction between fifteen isolates of T. harzianum and the soil-borne plant pathogen, Rhizoctonia solani, was studied by light microscopy and transmission electron microscopy (TEM). Trichoderma attaches to the pathogen with cell-wall carbohydrates that bind to pathogen lectins (Howell, 2003;Hassan et al., 2014). Once Trichoderma is attached, it coils around the pathogen and forms the appresoria. The following step consists of the production of pathogenesis related enzymes and peptaibols. From above results, it can be concluded that the maximum (61%) growth inhibition of test pathogen with T. viride (NBAII Tv 23) followed by T. harzianum (NBAII Th1) (55%) was found. So, Trichoderma strains are very useful as biocontrol agent. It also can be concluded that the tested Trichoderma strains reduced the growth of all the three soil borne pathogens significantly and, therefore, can be incorporated for integrated disease management of soil borne plant pathogens. The degree of antagonism varied between and within strains of Trichoderma against the soil borne plant pathogen.