苹果树腐烂病拮抗菌株的分离、筛选和鉴定

Isolation,screening and identification of antagonistic strains against apple valsa canker

为筛选出对苹果树腐烂病有拮抗效果的生防菌,从内蒙古达茂草原采集土样,利用逐级稀释法分离菌株;以苹果树腐烂病病菌(Cytospora mali)QH2为供试病原菌,采用平板对峙、离体枝条法、抑菌谱,结合耐盐、耐紫外的稳定性测定筛选拮抗菌株,通过形态学、分子生物学和生理生化方法对拮抗菌株进行鉴定。结果表明,从6份土壤样品中分离纯化得到细菌288株、放线菌54株,经筛选得到DM5-29和DM3-18。DM5-29和DM3-18对C.mali QH2的皿内拮抗圈在1 cm以上,在金红和黄太平枝条上的防效分别在64%、77%以上,抑菌谱广,发酵液紫外照射120 min后仍对苹果树腐烂病具有一定的拮抗活性,可分别在5%NaCl与12%NaCl中生长,鉴定确定DM5-29为贝莱斯芽孢杆菌(Bacillus velezensis),DM3-18为萎缩芽孢杆菌(Bacillus atrophaeus),2个菌株对苹果树腐烂病均有良好的拮抗效果,抗逆性强、抑菌谱广,有应用潜力。

To screen out the resistance good effect antagonistic bacteria for apple valsa canker.Soil samples were collected from grassland in Darhan Muminggan Joint Banner,Inner Mongolia.The soil solution was then diluted step by step.Antagonistic bacteria were selected based on their antagonistic activity against C.mali QH2,plate confrontation and fresh apple branches and wide spectrum and tolerant to high NaCl content and UV light.Active strains were identified through morphological,molecular,as well as physiological and biochemical characteristics.A total of 288 bacteria and 54 actinomycetes were isolated from six soil samples.Bacteria DM5-29 and DM3-18 were selected based on their antagonistic activity against C.mali QH2.The inhibitory zone size of DM5-29 and DM3-18 against C.mali QH2 in confrontation tests were larger than 1 cm,and their inhibitory activity against C.mali QH2 on the‘Jinhong’and‘Huangtaiping’branches were higher than 60%and 75%,respectively.Both isolates caused significant hyphal malformation of C.mali QH2.DM5-29 grew normally in 5%NaCl,DM3-18 was toleramt to 12%NaCl,their spore free fermentation broth showed antagonistic activity against C.mali QH2 after treated with UV light for 2 h.Based on morphological,physiological and biochemical characteristics as well as 16S rRNA gene sequences,DM5-29 was identified as Bacillus velezensis and DM3-18 as Bacillus atrophaeus.B.velezensis DM5-29 and B.atrophaeus DM3-18 had effective and stable antagonistic activity against apple valsa canker,it induced malformation of the causal pathogen,and had wide antagonistic spectrum.B.velezensis DM5-29 and B.atrophaeus DM3-18 had potential for the effective and stable biological control of apple valsa canker in Inner Mongolia,China.