黄瓜青枯病内生拮抗菌株的分离及ARDRA分析

在黄瓜生长的不同阶段从根系分离内生细菌共469株。通过青枯菌平板拮抗试验,从中筛选到具明显拮抗作用的菌株59株。将内生拮抗菌纯培养物扩增近全长的16SrDNA并用限制性内切酶AluⅠ对PCR产物进行ARDRA(amplifiedrDNArestrictionanalysis)多态性分析,共得到5种不同的操作分类单元(OperationalTaxonomicUnit,OTU)。其中属于OTU1共有39株分离物,占内生拮抗菌总数的66%,为优势种群。进一步通过ERIC-PCR指纹图的方法在菌株水平上分析OTU1类群。结果表明,OTU1可分为12种不同的菌株,其中菌株HE-1和HE-2在黄瓜生长的5个不同阶段均可分离到。通过标记天然不具有利福平抗性的HE-1和HE-2菌株,获得抗利福平突变体菌株,回收检测结果表明,在栽培的不同时期,黄瓜植株根内均有HE-1和HE-2菌株的定殖。经防病效果的盆栽试验,发现HE-1和HE-2的浸种处理能有效降低黄瓜青枯病的病发率,与对照比较差异显著。因此确定HE-1和HE-2为黄瓜青枯病生物防治的优良菌株。

Isolation and ARDRA analysis of cucumber entophytic antagonists against Ralstonia solanacearum

Cucumber bacteria wilt, caused by Ralstonia solanacearum, is one of soilborne plant diseases of worldwide origin. Biological control is considered the most environment-safe and efficacious approaches to control this disease. In this study, a total of 469 entophytic bacteria were isolated from cucumber plant roots at different growth stages and 59 of these isolates were shown antagonistic against Ralstonia solanacearum. An analysis of the level of biodiversity of the isolates at the species level was performed by comparing the Alu I restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 59 isolates. This comparison was allowed to cluster the isolates into 5 operational taxonomic units (OTU), which were dominated by OTU1 group accounting for 39 isolates. This dominant OTU1 group was further investigated by a genomic fingerprinting technique, ERIC-PCR. The results indicated that there were 12 different ERIC-PCR types present among OTU1 groups. Isolates HE-1 and HE-2, which belonged to OTU1 and originated from all plant growth stages, were used to test the effect of biocontrol on the cucumber wilt caused be Ralstonia solanacearum. Results showed that while the growth of seedlings was promoted, the disease was suppressed too. As these strains are the same genotype, it can be hypothesized that if they were included in an inoculum, they would efficiently colonize the cucumber and protect the plant in whole growing season. Thus, the strains described here could be used in future screening programs for target biocontrol agents. The results of this study have practical importance in the context of using biocontrol agents to protect plants against rhizosphere pathogens.