解淀粉芽孢杆菌植物亚种CGMCC 11640对山核桃干腐病菌的抑制机制

Inhibitory mechanism of Bacillus amyloliquefaciens subsp. plantarum CGMCC 11640 against Botryosphaeria dothidea, the pathogen of canker disease of Carya cathayensis

葡萄座腔菌Botryosphaeria dothidea引起的山核桃Carya cathayensis干腐病是导致山核桃树发病甚至死亡的主要病害之一,对山核桃产业带来了严重威胁。通过平板对峙法成功筛选出1株对病原菌有明显抑制效果的解淀粉芽孢杆菌植物亚种Bacillus amyloliquefaciens subsp.plantarum(菌种保藏号:CGMCC 11640)。试图通过扫描电镜探究该菌株对山核桃干腐病菌的抑制机制。对峙培养4,6和7 d后,比较菌丝和孢子形态,发现病原菌菌丝出现萎缩干瘪现象,培养时间越长,菌丝萎缩干瘪程度越大;对峙培养4 d时病原菌孢子正常,而到第6天时部分病原菌孢子表现出凹陷、萎缩现象,第7天异常孢子数量增加。用CGMCC 11640发酵上清液与水分别按V(上清液)∶V(水)=1∶9,2∶8,5∶5的比例配制成的马铃薯葡萄糖琼脂(PDA)混合培养基培养病原菌,结果发现病原菌菌丝抑制率分别为75.81%,88.84%和94.30%,说明灭菌发酵上清液对病原菌有抑制作用。电镜观察发现,病原菌菌丝在含有灭菌发酵上清液的PDA混合培养基上出现萎缩干瘪现象。通过扫描电镜观察,推测CGMCC 11640菌株及其代谢物对病原菌的抑制作用主是通过破坏菌丝和孢子细胞壁和细胞膜,使细胞内原生质泄露,从而导致菌丝和孢子萎缩,最终杀死病原菌细胞。

The canker disease of Carya cathayensis caused by Botryosphaeria dothidea, is one of the diseases in China that has resulted in serious damage and even death for the tree. To overcome the serious disease threat to the C. cathayensis industry, Bacillus amyloliquefaciens subsp. plantarum CGMCC 11640, which has shown a strong inhibitive activity in vitro against Botryosphaeria dothidea, was obtained by screening with the dual-culture method and cultured in medium mixtures with ratios of sterilized fermentation supernate to PDA being 1 ：9, 2 ： 8, and 5 ： 5（volume ratio）. Then, the inhibitory mechanisms of strain CGMCC 11640 against Botryosphaeria dothidea were further explored by scanning electron microscopy（SEM）. Results revealed that the Botryosphaeria dothidea strain growth rate for the three volume ratio mediums were restrained by 75.8% for1 ： 9, 88.8% for 2 ： 8, and 94.3% for 5 ： 5. The SEM results showed that both bacterial cells and their sterilized fermentation supernate exhibited strong inhibition activity in vitro against Botryosphaeria dothidea and that the hyphae and spores of Botryosphaeria dothidea exhibited atrophy and introcession. Thus, CGMCC 11640 inhibited Botryosphaeria dothidea by destroying cell membranes（e.g. punching holes in them） and walls of hyphae and spores resulting in leakage of plasmatic material from the inside of the cells.