The present investigation consisted of two separate experiments with total 11 treatments & 3-replications, laid out in CRD (Completely Randomized Design) and RBD (Randomized Block Design) under laboratory (in vitro) of Department of Plant Pathology, and net house at the Research farm of Lovely Professional University, Punjab respectively, during the year, 2020-2021. The detailed description of treatments is presented below in Table 1.
Procurement, subculturing, confirmation & purification of pathogen
The mother culture of P. vexans was procured from Indian Type Culture Collection (ITCC), New Delhi and the other chemicals were collected from the agriculture store of Lovely professional university, Punjab. The confirmation of pathogen was done by transferring it (via hyphal tip method) in the petri dishes containing sterilized Potato Dextrose Agar (PDA) medium in the aseptic conditions, followed by microscopic observations (morphological/cultural characteristics). Subsequently, slants were prepared and kept under refrigeration for subculture. The purified culture was then maintained at 25±2°C in a biochemical oxygen demand (BOD) incubator.
In vitro evaluation
The poison food technique (PFT) was adopted to check the effects of treatments against pathogen. In this firstly the media was sterilized at 15lb psi pressure with maintaining 121.6°C temperature for 18 minutes into a vertical autoclave and the empty petri dishes were sterilized at 160°C for 120 minutes into a hot air oven. The biochar was admixed in the PDA before its sterilization and other chemicals were added just before the pouring of PDA into petri dishes. When the media got solidified, bits of 7-days old culture were inoculated at the center of the petri dishes using a cork borer and inoculated an inoculating needle. These inoculated petri dishes were wrapped with parafilm, marked and placed into a BOD incubator at 25±2°C for 7 days. After that, these petri dishes were regularly observed and growth of mycelium was measured on daily basis to calculate percent inhibition over control using the formula; [(C-T)/C×100] where ‘C’ was the ‘mycelium growth in control’ & ‘T’ was ‘mycelium growth in respective treatment’ as stated in Table 1.
Inoculation of pathogen in vivo
The inoculation of P. vexans was carried out using a previously prepared suspension with a conidial concentration of 1 x 106 per ml, applied to the vegetative parts of the host plant variety "Navkiran" (Mathur and Kongsdal 2003), 15 days after transplanting (DAT). These inoculated plants were subsequently covered with poly bags to create a suitable humid environment for 40 hours. Subsequently, at weekly intervals, disease symptoms were observed and, concurrently, pathogen confirmation was conducted through microscopic examination in the laboratory.
Soil pH and electrical conductivity (EC) evaluation
For analysing the soil pH and EC, total 5 random samples (500g each) were collected from the experimental site and brought to laboratory twice (first before the application of treatments and second after the completion of research). These samples were dried, grinded, diluted, and prepared for testing of pH and EC with the help of digital pH and EC meter respectively.
Biochemical analysis
In the biochemical analysis, estimation of total phenols (Bray and Thorpe 1954) and total soluble proteins (Lowry et al. 1951) were performed for the confirmation of development of disease management and resistance in brinjal. The evaluation for the measurement of total phenols and total soluble proteins was done twice (at 30 & 60 DAT) by spectrophotometric quantification.
Disease severity
The disease severity (leaf blight and fruit rot) was assessed by categorizing all the infected samples into different grades of infection (Percent disease index or PDI) using the grading scale as 0−5 (Hossain et al. 2010). The disease severity in terms of per cent disease index (PDI) was calculated by using the formula; [(Sum of numerical values of scale/total number of observations × maximum grade of scale) × (100)]. Based on PDI, the following grades (I = 0 = free from the disease; II = 0.1−5.0 = poorly affected; III = 5.1−20.0 = moderately affected; IV = 20.1−50.0 = severely affected & V = >50.1 = very seriously affected by disease) were classified and relevant observations are made.
Statistical analysis
The recorded data sets were analysed at 5% probability level of significance with using SPSS-22 software. The correlation and regression coefficient were established with total phenol and total soluble proteins in relation to disease severity to get the variability and efficiency of treatments.