Pot trial
For possible management of collar rot disease and subsequent improvement in growth and yield of chickpea, pot soil was amended with dry biomass of C. album and two antagonistic fungi, namely T. harzianum and T. viride, the protocol given by Javaid et al. (2017) was generally followed with some modifications.
Inoculum of S. rolfsii was prepared on pearl millet seeds. The seeds were moderately boiled and water was strained then seeds were air dried to remove moisture from the surface. Seeds were packed in polythene bags and autoclaved for 30 min at 121 °C. Afterward, seeds were cooled and inoculated with discs of S. rolfsii and incubated for 7 days at 28 °C.
In the pot trial, there were following 13 treatments:
T1 = Negative control
T2 = Positive control [Sclerotium rolfsii (SR)]
T3 = 1% C. album biomass + SR
T4 = 2% C. album biomass + SR
T5 = 3% C. album biomass + SR
T6 = T. harzianum (TH) + SR
T7 = T. viride (TV) + SR
T8 = 1% C. album biomass + TH + SR
T9 = 2% C. album biomass + TH + SR
T10 = 3% C. album biomass + TH + SR
T11 = 1% C. album biomass + TV + SR
T12 = 2% C. album biomass + TV + SR
T13 = 3% C. album biomass + TV + SR
Each treatment had five replicates and pots were arranged in a completely randomized design.
The soil used in the experiment was sandy loam in texture having pH 7.7, 95 mg kg−1 potassium, 6.3 mg kg−1 phosphorous, and 0.84% organic matter. Soil was fumigated with formylene-dipped cotton swabs and was covered with polythene sheet for 1 week to eliminate any kind of pathogens and insects. Thereafter, polythene sheet and cotton swabs were removed and soil was left for 1 week to evaporate any traces of the fumigant.
Earthen pots of 27 cm diameter were filled with 5 kg fumigated soil in each pot. Inoculum of S. rolfsii, prepared on pearl millet seeds, was thoroughly mixed in soil of each pot at 50 g per pot. After that, pots were irrigated and left for a week under natural environmental conditions for development of the pathogen in the soil. The pathogen was mixed in the soil of all the pots except those of negative control. In negative control, however, same amount of autoclaved pearl millet seeds was mixed in the pot soil. After 1 week, inocula of T. harzianum and T. viride were mixed in soil of the respective pots at 50 g per pot. In remaining pots, same amount of boiled pearl millet seeds was mixed. Pots were again left for 1 week after irrigation with a good quality tap water. After that, dry biomass of C. album was mixed at 1, 2, and 3% (w/w) in soil of respective pots, irrigated with tap water and left for 1 week for stabilizing the conditions.
Seeds of chickpea variety Noor 2009 were surface sterilized by 1% sodium hypochlorite solution for 3 min. After thorough washing with sterilized water, 20 seeds of each variety per pot were sown. Once seeds were germinated, thinning was done to sustain 10 uniform seedlings per pot. Pots were watered according to the requirement of the crop.
Plants were harvested at maturity. Data for total pod and seed weight, and root and shoot length were recorded. Plant materials were placed in an oven at 60 °C to constant weight and finally root and shoot biomasses were recorded in gram.
Physiological bioassays
Total chlorophyll and carotenoids extraction were carried out by mixing of 500 mg fresh leaf material (at flowering sage) in 80% of chilled ethanol (10 ml). Mixture was homogenate at 800 rpm for 15 min using centrifuge machine. The supernatant was evaluated for chlorophyll content. The absorbance was checked for chlorophyll a at 645 nm, for chlorophyll b at 663 nm, and for carotenoids at 470 nm by UV-spectrophotometer. The observed values were analyzed quantitatively, and the amount of chlorophyll was estimated by using the formula of Lichtenthaler and Buschmann (2001). The quantities of chlorophyll and carotenoid content were expressed as mg g−1 of fresh plant weight.
Leaves and root samples of each treatment were collected at flowering stage for enzyme assays. One gram of sample from each treatment was grinded in pre chilled liquid nitrogen with 0.1 M sodium phosphate buffer (4 ml) using pestle and mortar. Centrifugation of the mixture was done for 15 min at 1000 rpm. The supernatant was considered as crude enzyme extract for evaluating phenyl alanine ammonia lyase (PAL) and catalase (CAT) activities (Ramanujam et al. 2012). Estimation of phenyl PAL and CAT activities were carried following procedures of Dikerson et al. (1984) and Beers and Sizer (1952), respectively. The enzyme activity was expressed as U min−1 mg−1 of protein. For estimation of phenolic content, methodology of Malik and Singh (1980) was adopted and the quantity of total phenol was expressed in mg g−1 of fresh plant weight.
Statistical analysis
All data were subjected to analysis of variance (ANOVA), followed by Tukey’s HSD test to delineate the treatment means at p≤ 0.05 using computer software Statistix 8.1.