Collection of Disease Sample Typical symptom of sheath rot disease was collected from greenhouse of Plant Breeding and Biotechnology Laboratory, Agrotechnology Discipline, Khulna University, Khulna, Bangladesh.
Isolation, Purification, Preservation and Identification of Fungus The fungus was isolated following standard procedures of Dhingra and Sinclair (1985). To obtain pure culture of S. oryzae, a hyphal tip from water agar was transferred aseptically to PDA petridish by using a sterile fine needle and then incubated at a temperature of 25±2 0C for 52 hours. Advanced hyphae were transferred aseptically into the test tube slant containing PDA with pH 6.5 and incubated at room temperature (25±2 0C) for 52 hours. After incubation, these slants were carefully checked for contamination and then preserved at 4 0C in a refrigerator for further use. Fungal isolates were identified based on the characteristics of hyphae and sclerotia (Lilly and Barnett, 1951).
Pathogenicity Test of S. oryzae Pathogenicity test was done on excised rice stem with sheath. Healthy stem of rice were cut into pieces (about 1 inch) and surface sterilized with 70% ethanol for 10 seconds. After that cut pieces were placed on sterilized blotting paper for the removal of excess ethanol present in the stem. Stem pieces were injured softly by flame sterilized pointed needles and then placed onto sterilized water agar. Advanced hyphae were cut from 30 hours old pure cultures aseptically with the help of cork borer and then placed at three injured places (both ends and centre) onto the stems (Dhingra and Sinclair, 1985). Then typical sheath rot symptoms were observed on sheath and reisolation was done following the protocol of Dhingra and Sinclair, 1985.
Preparation of Different Growth Media The various media composition (g L-1 ) shown below were tested. Potato dextrose agar (PDA) PDA was prepared following the standard procedure (Anonymous, 1968). Potato 200 g, dextrose 20 g, agar 15 g, distilled water 1L. Rice stem agar and Betel vine stem agar 20 g rice stem / betel vine stem was taken and pasted with the help of mortar-pestle by adding 5 ml distilled water for preparing the paste. After that 995 ml distilled water was added slowly in to the paste. It was then sieved in to a beaker; 20 g dextrose and 15 g agar added with it. Cornmeal agar 20 g cornmeal extract, 20 g dextrose, 15 g agar, 1L distilled water. Preparation of Different Carbon and Nitrogen Sources Media Czapek dox agar medium 2 g NaNO3 , 1 g K2HPO4 , 0.5 g MgSO4 , 0.5 g KCl, 0.01 g FeSO4, 30 g Sucrose and 15 g agar,1L distilled water.
Nitrogen sources Three carbon compounds viz; Peptone (2.5 g L-1 ), Sodium nitrate (8.5 g L-1 ) and potassium nitrate (10 g L-1 ) were tried individually as a constitute of nitrogen source in Czapek dox agar medium.
Preparation of Media at Different pH Five different pH levels namely 5.0, 6.0, 7.0, 8.0 and 9.0 were taken as treatments. Hundred (100) ml PDA was taken in 250 ml conical flask. Different pH were adjusted by adding either 0.1N HCl or 0.1N NaOH and measured by pH meter. All the media were sterilized at 1210C temperature, 15 PSI for 15 min. After cooling, the media were poured in to 90 mm petridishes.
Inoculation and Incubation All these treatments were replicated into five plates. Advanced hyphae of 3 days old culture was used for inoculation. A 5 mm block of the mycelium was cut with flame sterilized cork borer (5 mm). The mycelial blocks were taken from the edge of the colony. Each mycelial block was placed upside down at the centre of each petridish. All these operation were done under aseptic condition. The inoculated petridishes were kept in the growth chamber at (25±2 0C) until the mycelia touch the edge of petridishes.
Experimental Design and Data Analysis: The experiment was laid out under completely randomized design (CRD) with three replications. The data were analyzed statistically using STAR (statistical tools for agricultural research) program, version-02, IRRI, Los Baños, Philippines computer program.