Laboratory bioassays
Three Trichoderma species, i.e., T. viride (FCBP 644), T. harzianum (FCBP 1277), and T. hamatum (FCBP 907), were tested for their antagonism activity against M. phaseolina (FCBP 0751).
Antifungal activity of Trichoderma spp. by cell-free culture filtrates
Cell-free culture filtrates Trichoderma spp. were prepared in 2% ME (malt extract) broth medium (100 mL). After 20 days of inoculation, cell-free supernatants were collected after aseptic filtration through Whatman filter paper and centrifugation at 4000 rpm for 5 min, followed by re-filtration through Millipore filter paper (pore size 45 μm). Twenty-one different concentrations, ranging from 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,…, 100% (v/v) of each cell-free culture filtrate, were prepared by addition of 2% of ME. Flasks were inoculated with 5 mm disc of M. phaseolina and incubated at 28 ± 2 °C. After 7 days, mycelial mat was dried in oven at 45 °C for 24 h for measuring dry biomass.
Assessment of the antifungal potential of plant extract
Two hundred-gram powdered leaves of A. indica and M. azedarach were soaked in 2 L methanol separately for 12 days. Extracts were obtained from soaking materials by filtering and evaporating and finally drying. Original concentration was made by dissolving 9 g of extracting plant material in 5 ml of dimethyl sulphoxide (DMSO 99.5%) to prepare a final volume of 15 ml. Control solution was made by adding 5 ml of DMSO in 10 ml of sterilized distilled water. Six concentrations, i.e., 0, 1, 2, 3, 4, and 5%, were made by adding 0, 1, 2, 3, 4, and 5 ml of stock solution and 5, 4, 3, 2, 1, and 0 ml of control solution in 55 ml of each flask to make a final volume of medium 60 ml. Then, 60 ml of each treatment was equally divided into four 100-ml flasks to serve as replicates, where 0% was control treatment. Actively growing culture of M. phaseolina (5 mm disc) was inoculated in each flask and incubated at 28 ± 2 °C for 7 days. The fungal biomass was dried and weighed.
Pot bioassays
On the basis of the laboratory bioassays, two species of Trichoderma viz. T. harzianum and T. viride, and one member of Meliacaceae family, i.e., A. indica, were selected to conduct trials in pots (6 in. diameter × 10 in. height). Initially, presterilized potted soil (1 kg pot−1) was inoculated with cultural suspension (conidial count 4 × 106) of each of two Trichoderma spp. and left for 4 days for the establishment of the fungus in soil. Later dry leaves of A. indica were mixed at 1, 2, and 3% in 1 kg of soil and left for 7 days. Soil was inoculated with M. phaseolina (MP) and left for another 4 days for inoculum establishment. Finally, surface sterilized seeds of cowpea with 0.1% sodium hypochlorite solution were sown in each pot. The pots were arranged in a completely randomized design and were kept under natural environmental conditions having three replicates of each treatment. Experiment was comprised of 13 treatments including T
1
: negative control (without any inoculation or amendment); T
2
: positive control (inoculated with MP only); T
3
–T
5
: MP + 1% A. indica, MP + 2% A. indica and MP + 3% A. indica; T
6
: MP + T. harzianum; T
7
–T
9
: MP + T. harzianum + 1% A. indica, MP + T. harzianum + 2% A. indica, MP + T. harzianum + 3% A. indica; T
10
: MP + T. viride; T
11
–T
13
: MP + T. viride + 1% A. indica; MP + T. viride + 2% A. indica, and MP + T. viride + 3% A. indica.
Disease assessment
After 40 days of inoculation charcoal rot disease symptoms on cowpea plants, were appeared and were assessed, using disease rating scale, where 1: no symptoms on plants (highly resistant); 3: lesions are limited to cotyledonary tissues (resistant); 5: lesions have progressed from cotyledons to about 2 cm of stem tissues (tolerant); 7: lesions are extensive on stem and branches (susceptible); and 9: most of the stem and growing points are infected. A considerable amount of pycnidia and seclerotia are produced (highly susceptible). Disease incidence (DI) was determined, using the following formula:
$$ \mathrm{DI}\ \left(\%\right)=\frac{\mathrm{Number}\ \mathrm{of}\ \mathrm{infected}\ \mathrm{plants}}{\mathrm{Total}\ \mathrm{nutmber}\ \mathrm{of}\ \mathrm{plants}}\times 100 $$
Analysis of plant physiology
Physiological variations in different treatments were assessed in cowpea leaves after 40 days of seed sowing. Total chlorophyll content was quantitatively analyzed by taking absorbance properties for chlorophyll a (645 nm), chlorophyll b (663 nm), and carotenoid (270 nm), and the amount of pigment was calculated. Activity of catalase (CAT) was determined in the reaction mixture consisted enzyme extract (0.1 ml) that was added to 2.9 ml of H2O2 (20 mM) and sodium phosphate buffer (50 mol/L; pH 7.0) by monitoring the reduction in the absorbance at 240 nm (Maehly and Chance 1967). Activity of peroxidase (POX) was determined by taking 0.5 ml of enzyme extract in reaction mixture containing 2 ml of 0.1 mol/L phosphate buffer (pH 6.8) and 1 ml of pyrogallol. Solution was filled with 1 ml of 0.05 mol/L H2O2 (5:5 in H2O2 and distilled water), incubated at 25 °C, and reaction was stopped by adding 2.5 mol/L H2SO4 (24.5 ml of H2SO4 + 100 ml of distilled water). The amount of purpurogalline formed was determined by reading the absorbance at 430 nm against a blank prepared by adding the extract after the addition of 2.5 mol/L H2SO4 (Colville and Smirnoff 2008). Polyphenol oxidase activity (PPO) was assayed in a reaction mixture consisted of 0.1 ml enzyme extract and 1.5 ml of 0.1 mol/L sodium phosphate buffer (pH 7.0), 0.2 ml of 0.01 mol/L catechol. The changes in the absorbance were recorded at 30-s interval for 3 min at 495 nm (Mayer et al. 1965). For determination of phenylalanine ammonia-lyase (PAL) activity, reaction mixture [(0.4 ml of enzyme extract + 0.1 mol/L sodium borate buffer (pH 8.8) + 0.5 ml of 0.012 mol/L l-phenylalanine)] was incubated for 1 h in light at 25 °C and reaction was stopped by incubating at 47 °C for 10 min. The amount of trans-cinnamic acid formed was calculated after measuring absorbance of samples at 290 nm (Dickerson et al. 1984).
Harvesting and data collection
Plants were harvested after 65 days of sowing. Data regarding disease incidence, and plant height, shoot, root fresh, and dry weight were measured. Materials were dried at 70 °C, and dry weight was recorded on an electric balance.
Statistical analysis
Triplicate values reported are mean of ±SD. Kolmogorov-Smirnov test (normality test) and Levene’s test (variance test) were applied before any statistical analysis. When all the assumptions of ANOVA were satisfied, standard errors of all data were analyzed by analysis of variance (ANOVA), followed by LSD test, using computer software Statistix 8.1.
