Isolates of S. rolfsii
used in this investigation were isolated from the collar region of Lens culinaris
showing typical symptoms of collar rot disease caused by S. rolfsii
from different regions of Madhya Pradesh (Table 1). S. rolfsii
was confirmed in all isolates by comparing their morphology and further by ITS sequence information. The molecular work was carried out at Plant Molecular Biology Laboratory, Department of Plant Molecular Biology and Biotechnology, College of Agriculture, Rajmata Vijayaraje Scindia Agricultural University, Gwalior, India during the year 2021-22. For isolation of genomic DNA, tissue segments of 2-3 cm from collar rot disease infected plants were excised from rotten margins. The segments were sterilized with 1% sodium hypochlorite solution for 2 min followed by rinsing twice with sterilized double distilled water and placed onto Potato Dextrose Agar (PDA) medium. Then segments were transferred in Petri dishes and incubated at 25±2°C for 2 to 3 days and mycelia were then transferred and maintained on PDA for 15 days to allow sclerotia development. Twenty-five to thirty days old sclerotia (mature) were employed for DNA extraction.
Reagents and chemicals
Table 1: List Sclerotium rolfsii isolates collected from locations of Madhya Pradesh, India.
The following buffers and solutions were prepared for DNA extraction:[Extraction buffer (1M Tris-HCl (pH 8); 0.5 M EDTA (pH 8); % M NaCl; 2% CTAB (w/v); 1% PVP (Mr. 40,000); 2% β-mercaptoethanol (v/v)]; phenol: chloroform (24:1); wash solution (3M ammonium acetate in 70% (v/v) ethanol. The pH of DNA extraction buffer (pH-5.2) was adjusted and volume was made up to 100 ml with distilled water.
Genomic DNA extraction
About 150 mg of sclerotia was ground to a fine powder by using liquid nitrogen. The powder was directly added to 2 ml Eppendorf tube containing 500 μl of pre-warmed DNA extraction buffer (DEB). The tube was incubated for about 15 min at 55°C in water bath with frequent swirling. The samples were centrifuged @12000 rpm for 5 min and supernatant obtained was transferred into clean micro-centrifuge tube. To each tube 250 μl of chloroform: isoamyl alcohol (24:1) was added and solution was mixed by the gentle inversion. After mixing, the tube was spinned at 13000 rpm for 10 minutes. By using cut tips transfer the upper aqueous phase transferred to new 1.5 ml Eppendorf tube. To each tube 50 μl 3M sodium acetate was added tracked by 500 μl of ice-cold absolute ethanol. Tube was inverted several times to precipitate the DNA. These tubes were placed at -20°C for 1 hr after addition of ethanol to precipitate out the DNA. DNA was pelleted by centrifugation at 13000 rpm for 1 min and the supernatant was discarded. The DNA was washed twice by 70% ethanol and again centrifuged at 13000 rpm for a min. The supernatant was discarded and allowed the DNA to dry till the smell of ethanol gone away. The DNA was dissolved in nuclease free water (50 μl-100 μl). The dissolved DNA was stored at -20°C for further downstream reactions (PCR). The experiment was repeated thrice and result described as the mean of three independent experiments.
The quality of extracted DNA was checked by means of 0.8% gel electrophoresis tracked by ethidium bromide staining (0.5 mg ml-1
). The purity of DNA was estimated by 1.7 to 1.9 by calculating the A260
0 ratios and the yield was estimated by measuring absorbance at 260 nm.
To check the suitability of extracted DNA for downstream analysis, RAPD, ISSR and rDNA-ITS primers were employed for amplifications of extracted DNA samples. For this purpose, RAPD (OPA-8) primer, 5'-GTGACGTAGG-3' (Imperial Bio Medics, Coralville, USA), ISSR (ISSR-12) 5'- (GA)8T-3') and universal primers ITS-1 (5'-TCCGTAGGTGG ACCTGCGG-3') as forward primer and ITS-4 (5'-TCCTCCGCTTATTGATATGC-3') were used. Each PCR reaction mixture of 10 μl consisted of 200 ng genomic DNA, 1 μl of 6X green Taq
reaction buffer with MgCl2
(Thermofisher), 0.2 μl of 2.5 mM dNTPs, 100 ng primer. PCR amplification was performed in an Eppendorf thermal cycler (Bio-Rad).
The temperature profiles used for RAPD amplification were: an initial denaturation at 94°C for 5 min and then subjected to 30 cycles of (94°C for 1 min, 36°C for 2 min, 72°C for 2 min). After the last cycle, the final extension was carried out 72°C for 5 min. The temperature profiles employed for ISSR amplification were: an initial denaturation at 94°C for 5 min and then subjected to 30 cycles of (95°C for 30 sec, 48°C for 30 sec, 72°C for 1.5 min). After the last cycle, the final extension was carried out 72°C for 10 min. The amplified product was resolved on 1.5% gel comprehending 0.5 mg ml-1
ethidium bromide and visualized under UV light. Gel photographs were scanned through Gel Doc System (SYNGENE, USA). The temperature profiles used for rDNA-ITS region amplification were: an initial denaturation at 94°C for 4 min and then subjected to 35 cycles of (94°C for 1 min, 56°C for 1 min, 72°C for 1.5 min). Afterward the last cycle, the final extension was carried out at 72°C for 6 min. The amplified product was resolved on 1.8% gel containing 0.5 mg ml-1
ethidium bromide and visualized under UV light. Gel photographs were scanned through Gel Doc System (SYNGENE, USA).