Antibacterial mechanism of cow manure biogasslurry on Rhizoctonia solani

Abstract: In order to investigate the antibacterial mechanism of biogas slurry, Rhizoctonia solani was cultured at 28℃ for 96 h using potato dextrose agar medium in the center of Petri dishes that coated uniformly with 1 mL different treatments biogas slurry. 5 treatments of biogas slurry were applied as follows: 1) Biogas slurry without treatment; 2) Biogas slurry sterilization under 121℃ for 20 min; 3) Centrifugal supernatant at 12 000 r/min for 10 min; 4) Filtrate from centrifugal supernatant by 0.22 μm membrane; 5) Microbes solution, precipitate from high speed centrifugation of 4 mL biogas slurry washed 3 times with sterilized water to remove the chemical and bioactive substances and added with sterilized water to 4 mL. The colony diameters of Rhizoctonia solani were measured at 12 h intervals and the inhibitory rates were calculated. The inhibitory rate equals to the diameters of control colony subtracted the diameters of treated colony then divided by the diameters of control colony, at last multiply 100%. Origin8.1 and excel were used to analysis the data. Molecular biology technique was also used to identify the microbes which had inhibitory effect on Rhizoctonia solan,. The sequence analysis was based on the BLASTN of United States National Center for Biotechnology Information and software MEGA4.0. The results showed that all inhibitory rates of different treated biogas slurry rose quickly at the beginning, then reached a maximum value, and decreased slowly with the similar trend at last. Sterilization treatment got the lowest inhibitory rate, usually less than 13%, supernatant and membrane filtration treatments were below 20%, biogas slurry without treatment has the higher inhibitory rate of 53%, and the microbe solution treatment obtained the highest inhibitory rate of 74%. The microbe solution treatment also showed the most durable inhibitory effect which lasted for about 96 h. 16 kinds of microbe named as nfd-1 to nfd-16 were separated from biogas slurry by 10 time's dilution method. They were 8 kinds of fungi, 5 kinds of bacteria and 3 kinds of actinomyces, and their antagonism effects with Rhizoctonia solani were investigated respectively. The results showed that the fungi nfd-9 and nfd-16 had strong inhibitory effect on Rhizoctonia solani, however, the others had almost no inhibitory effect. Scanning electron microscopy was used to survey the interface of nfd-9 and nfd-16 with Rhizoctonia solani. It was found that nfd-9 and nfd-16 inhibit the growth of Rhizoctonia solani through the effects of competitions, antagonism, and hyperparasitism to prevent the crops diseases. Nfd-9 and nfd-16 were finally identified as Rhizopus oryzae and Trichoderma harzianum by molecular biology technique. The results can provide a theoretical reference for further understanding the fungal inhibitory mechanism of biogas slurry.