蕙兰病株根部内生细菌种群变化

Population dynamics of endophytic bacteria isolated from the roots of infected Cymbidium faberi

为了研究褐斑病与蕙兰根部内生细菌群落结构和多样性的关联,从野生蕙兰健株和褐斑病株根部分离出内生细菌112株,采用核糖体DNA扩增片段限制性酶切分析(ARDRA),研究了健株和病株内生细菌多样性与群落结构。将内生细菌纯培养物扩增近全长的16S rDNA,并用ARDRA(Amplified Ribosomal DNA Restriction Analysis)对所分离的菌株进行分型,根据酶切图谱的差异,将健株中的内生细菌分成8个ARDRA型,病株分成13个ARDRA型。并选取代表性菌株进行16S rDNA序列测定。结果表明,健株分离出内生细菌6个属,优势菌群为Bacillus;病株分离出11个属,优势菌群为Mitsuaria和Flavobacterium。通过回接兰花植物和初步拮抗实验发现,从病株分离出的H5号菌株(Flavobacterium resistens)使兰花产生病症,而健株中的B02(Bacillus cereus)和B22号菌株(Burkholderia stabilis)对菌株H5有拮抗作用。

A study was conducted to investigate the community structure and the diversity of culturable endophytic bacteria in the root tissues of healthy and diseased plants of Cumbidium faberi Rolfe collected from Hubei Province of China.The endophytic bacteria were identified by characterization of cultural features,and some molecular features using methods of amplified ribosomal DNA restriction analysis（ARDRA） and sequence homology comparisons.Results showed that a total of 112 bacterial isolates were obtained,53 strains from healthy plants and 59 strains from diseased plants of C.faberi.The amount of bacteria reached 1.33×105 cfu/g in healthy plants,whereas was 1.21×104 cfu/g in the plants infected with brown spot disease.The number of endophytic bacteria decreased in the diseased plants.Comparison of the AluI-restriction patterns of the 16S rDNA sequence showed that 112 bacterial isolates from diseased plants could be grouped into 13 operational taxonomic units（OTUs）.The OTU10 and OTU11 were the major groups accounting for 13 and15 isolates,respectively.The bacterial isolates from healthy plants could be grouped into 8 OTUs and OTU1 was the major group accounting for 24 isolates.The representative strains for these OUTs were selected for characterization of the 16S rDNA sequences.Phylogenetic analysis on the basis of the 16S rDNA sequences from this study and from related bacterial species in the GenBank database showed that the representative bacterial isolates from the healthy plants of C.faberi belonged to six genera,Bacillus,Rhizobium,Burkholderia,Paenibacillus,Pseudomonas and Microbacterium.The genus Bacillus was the dominant bacteria,followed by Paenibacillus and Burkholderia.The bacterial isolates from diseased plants of C.faberi belonged to eleven genera,Mitsuaria,Flavobacterium,Bacillus,Enterobacter,Brevundimonas,Pedobacter,Burkholderia,Sphingomonas,Mucilaginibacter,Pseudomonas and Microbacterium.The dominant genera were Mitsuaria and Flavobacterium,followed by Burkholderia.The phylogenetic diversity of endophytic bacteria in diseased plants of C.faberi were different from that in healthy plants of C.faberi.Results also showed that the diversity of the endophytic bacterial community from diseased plants of C.faberi was higher than that in healthy plants of C.faberi.The dominant OTUs from diseased plants were suspected to be pathogens of C.faberi.Thus,representative bacterial isolates were selected to inoculate healthy plants of C.faberi,Ascocentrum ampullaceum and Cymbidium goeringii.Results showed that isolate H5 of Flavobacterium resistens from a diseased plant of C.faberi was pathogenic on leaves of C.faberi,Ascocentrum ampullaceum and Cymbidium goeringii.The endophytic bacterial isolates B02 of Bacillus cereus and B22 of Burkholderia stabilis from healthy plants were found to be antagonistic to the isolate H5 of F.resistens.These results indicate that the endophytic bacterial community in healthy plants and diseased plants of C.faberi differs greatly and the difference may be the cause responsible for brown spot disease in C.faberi.