摘要
为探讨牛粪发酵沼液的抑菌机理,以立枯丝核菌为研究对象,进行了试验研究。采用平皿培养方法:培养基表面分别均匀涂布1mL5种不同处理的沼液,即不作处理沼液、沼液灭菌、离心上清液、无菌膜滤液和菌体溶液,中心接种立枯丝核菌并于生化培养箱中28℃培养96h,每12h测量立枯菌落的生长直径以计算抑制率,同时借助分子生物学手段鉴定了沼液中起抑制作用的微生物。试验结果表明菌体溶液和不作处理沼液对立枯的抑制效果较好,抑制率最高可达74%和53%,离心上清液的抑制效果次之,无菌膜滤液和沼液灭菌处理的抑制作用较弱,一般在20%以下;对沼液中的微生物菌群进行了分离、纯化与筛选,获16株菌种,并与立枯丝核菌进行对峙培养试验,其中nfd-9和nfd-16具有较强抑制作用,其他菌株几乎没有抑制作用。将nfd-9、nfd-16与立枯对峙交界面处进行了扫描电镜观察,结果显示:这2株微生物通过竞争、拮抗和重寄生等作用来抑制立枯丝核菌的生长,从而到达防止病害的目的。经分子生物学技术鉴定,确定了nfd-9和nfd-16分别为米根霉菌(Rhizopus oryzae)和哈茨木霉(Trichoderma harzianum)。研究结果为进一步了解沼液抑制真菌的机理提供一定的理论参考。
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.
出处
《农业工程学报》
EI
CAS
CSCD
北大核心
2013年第3期207-212,J0006,共7页
Transactions of the Chinese Society of Agricultural Engineering
基金
"十二五"国家支撑计划课题:寒冷地区干湿耦合发酵气
热
电联供技术集成与示范(2011BAD15B04)
黑龙江省教育厅科学技术研究项目:沼液特性数据库建立及抑菌机理研究
关键词
抑菌性
植物
疾病控制
牛粪发酵沼液
立枯丝核菌
米根霉
哈茨木霉
fungus resistance, plants, disease control, cow manure biogas slurry, Rhizoctonia solani, Rhizopus oryzae,Trichoderma harzianum
