Production of Lytic Enzymes by Trichoderma Isolates during in vitro Antagonism with Aspergillus Niger, The Causal Agent of Collar ROT of Peanut

Twelve isolates of Trichoderma (six of T. harzianum, five of T. viride, one of T. virens), which reduced variably the incidence of collar rot disease caused in peanut by Aspergillus niger Van Tieghem, were evaluated for their potential to produce lytic enzymes during in vitro antagonism. T. viride 60 inhibited highest (86.2%) growth of test fungus followed by T. harzianum 2J (80.4%) at 6 days after inoculation (DAI) on PDA media. The specific activities of chitinase, β-1,3-glucanase and protease were 11, 3.46 and 9 folds higher in T6 antagonist (T. viride 60 and A. niger interactions) followed by 8.72, 2.85 and 9 folds in T8antagonist (T. harzianum 2J and A. niger interactions), respectively, compared to the activity produced by control petri plate T13 (A. niger alone) at 6 DAI. Activity of these lytic enzymes induced in antagonists’ plates comprises the growth of Trichoderma isolates. However, cellulase and poly galacturonase were found least amount in these antagonists treatment. A significant positive correlation (p=0.01) between percentage growth inhibition of test fungus and lytic enzymes – (chitinase, β-1,3-glucanase and protease) in the culture medium of antagonist treatment established a relationship to inhibit growth of fungal pathogen by increasing the levels of these enzymes. Among the Trichoderma isolates, T. viride 60 was found best strain to be used in biological control of plant pathogen A. niger.

. Management of collar rot disease can be achieved by use of tolerant varieties and adjustment of sowing dates (16).
However, several factors influence the efficacy of these management practices, including pathogenic variability in the fungus populations as well as abiotic factors such as temperature and moisture. Fungicides and organic compounds are widely used to control this disease. However, the degradation of fungicides and organic compounds are very difficult and it's accumulation in food chains is leading to higher toxic level in animals including humans (8, 33).
In this context, novel biocontrol agents have, therefore, been looked for (5) and Trichoderma have been the preferred choice. Besides other mode of action, lytic enzymes such as chitinases, -1,3 glucanases and proteases have been associated with the ability of Trichoderma spp to control plant pathogens (4). Three strains of Trichoderma (T. harzianum, T. viride, T. virens) were found to impair the progress of collar rot disease in field conditions caused in peanut by A. niger.
Trichoderma isolates (twelve isolates of three Trichoderma strains) could not work equally against specific phytopathogen. Hence, the objective of this study is to find out the best Trichoderma isolate as a biocontrol agent which effectively inhibits in vitro the growth of A. niger on solid PDA media. These Trichoderma isolates are correlated on the production of lytic enzymes (cellulase, poly galacturonase, chitinase, -1,3 glucanse and protease activities in the presence of corresponding substrate) during antagonism with A. niger.

Isolation and maintenance of microbes
Peanut seedlings which showed typical symptoms of collar rot were cut into small bits with the help of sterilized blade and the pure culture of pathogen (A. niger) was made by hyphal tip isolation method (45) on the solidified PDA medium in petri plates. A typical black mycelium (conidia) growth of A.
niger was observed after 72 h of incubation at 28 ± 1 0 C in an incubator. Virulence of isolated pathogen-A. niger was also tested on peanut seed.

Antagonistic capacity of Trichoderma isolates
The in vitro antagonism experiments were accomplished by the method described by Dennis and Webster (13). The isolates were cultivated in petri plates with PDA media for seven days. Disks of 5mm of diameter cut and removed from the growing borders of the colonies and transferred to another petri plate with PDA. Each plate received two disks, one with Trichoderma mycelium and another with A. niger, placed at a distance of 7 cm away from each other. The experiment was conducted in five replications for each antagonist. The plates were incubated at 28 ± 1 0 C temperatures and observed after six days for growth of antagonist and test fungus. Index of antagonism as percentage growth inhibition of A. niger was Production of lytic enzymes by Trichoderma determined by following the method of Watanabe (47).

Extraction of lytic enzymes
Crude enzyme extract was prepared from petri plates containing fungal pathogen and Trichoderma as well as the test fungus alone as a control (C) at 6 days after inoculation (DAI).
For that, 25 ml of 100mM phosphate buffer (pH-5.5) containing 50mM sodium chloride was added to each petri plate and transferred mycelia mat to conical flask. For enzyme induction, 1% of either carboxymethyl cellulose (CMC), sodium polypectate, chitin, laminarin or casein was added into culture medium of conical flask and pH adjusted to 5.5 (26, 35). Cultures were then shaken well in orbital shaker at 120 rpm at 28 0 C for about 6 hours (46). After that, it was transferred to 50 ml centrifuge tubes and centrifuged at 14,000 rpm for 10 min. Supernatant was collected and stored at -20 0 C until use for assay of enzymes activities (cellulase, poly galacturonase (PG), chitinase, -1, 3 glucanase, protease). The method of Folin-Lowry (32) was used to determine protein content in culture supernatant and to calculate the specific activity of the enzyme.

Enzymes assays
Cellulase activity (EC 3.2.1.21): Suitable aliquot (100 µl) of the culture supernatant was incubated with 400 µl of 100mM sodium citrate buffer (pH 5.2) containing 1 % CMC (12). After incubation at 55 0 C for 15 min., the glucose released was measured by the dinitrosalicylic acid (DNSA) method Then, 3 ml DMAB reagent (5.0 g DMAB dissolved in 500 ml of glacial acetic acid containing 12.5 % v/v 10 N HCl, stored at 20 0 C as a stock and prior to use, it was diluted with nine volume of glacial acetic acid) was added in each tubes and incubated at 38 0 C for 20 min. Tubes were then cooled and absorbance was measured at 544nm in spectrophotometer.
Standard N-acetylglucosamine was prepared in borate buffer and measured following the above procedure. The amount of N-acetylglucosamine was calculated and expressed as appropriate.

In vitro percentage growth inhibition of A. niger
Growth inhibition of A. niger during in vitro interaction with bio-control agents Trichoderma at 6 DAI was depicted in  solani.

Lytic enzymes
The results on in vitro production of various lytic enzymes such as cellulase, poly galacturonase (PG), chitinase, -1, 3 glucanase and protease during antagonism of Trichoderma isolates with test fungus A. niger in the culture medium at 6 DAI is presented below.
The activity of cellulase and PG in the culture medium reduced when the growth of Trichoderma isolates more during antagonism with test fungus in T 6 and T 8 . While, the same activities were higher in T 13  Inhibition of these CWDEs production leads to the reduction in virulence of fungal pathogens.

Pathogenesis related enzymes:
Chitinase activity was the highest (1.31 U.mg -1 protein) in the culture medium of T 6 and slightly reduced to 1.07 U.mg -1 protein in T 8, 0.90 U.mg -1 protein in T 3, 0.72 U.mg -1 proteins in T 1 (Fig. 3B). It was significantly reduced to 0.44 U.mg -1 protein in T 5 and T 9 antagonists followed by T 10 , T 4 , T 7, T 12 and T 13 antagonists.
The lowest chitinase activity (0.11 U.mg -1 protein) was recorded in T 13 (culture medium of test fungus as a control).
Chitinase activity was higher during antagonism when antagonist effectively inhibited the growth of test fungus (A. The specific activity of β-1,3-Glucanase recorded its maximum in T 6 (3.48 U.mg -1 protein) at 6 DAI (Fig. 3B) and it was significantly reduced to 3.01 U.mg -1 protein during T 8 antagonism followed by T 2 (2. 15 When expressing specific activity of protease in U.mg -1 protein (Fig. 3B), the highest activity was found in T 8 (5.63) followed by T 6 , T 11 , T 2 , T 12 , T 3 , T 1 , T 9 , T 5 , T 4 , T 10 , T 7 and T 13 antagonists in the culture medium at 6 DAI. The enzyme activity was more or less correlated with growth inhibition of test fungus except T 11 antagonist. The specific activity of protease attained highest in T 8 antagonism (T. harzianum 2J x test fungus A. niger) followed by T 6 (T. viride 60 x test fungus). However, maximum growth inhibition of pathogen A. niger was achieved by T 6 antagonist followed by T 8 .