Identification Of Endophytic Bacteria From Brassica Napus Using 16S RDNA Sequence Analysis

Endophytic bacteria reside in plant hosts without causing obvious diseases symptom. Although it is just several years since the definition of endophytic bacteria was introduced, it has been paid attention by researchers in many disciplines for their promoting plant growth and improving resistance to diseases and insects penetration. Oilseed rape (Brassica napus) is an important oleaginous crop. Kinds of diseases during its growth period are responsible for dramatic yield losses. The object of this study is to obtain some basic information for further studying diversity of endophytic population and utilizing effectively antifungal endophytes as biocontrol agents through identified partly bacterial endophytes isolated from oilseed rape and screened antagonistic isolates. In this study, many endophytic bacteria with distinctive colony morphological character were isolated from the different tissues of oilseed rape. It showed that many bacterial endophytes exist in the tissues of oilseed rape and have abundant groups. Total 122 entophytic bacterial strains were categorized preliminarily to 35 groups based on colony morphology. 16S rDNAs were amplified with universal primers of bacteria, all the strains could produce a single band about 1500bp. The PCR products were digested with restriction endoenzyme Haeâ…¢and Msp â… respectively, and the different patterns were obtained for each enzyme, by combining all the restriction patterns for the two enzymes, the 122 strains could be grouped into 39 16S rDNA genotypes. The results indicated that endophytic bacteria from oilseed rape were genetically diversity. By comparing two methods, colony morphology only could give us limited and preliminary information, but PCR-RFLP can be as a rapid and accurate tool for the diversity analysis of endophytic bacteria from oilseed rape. Antagonism of 122 endophytic bacteria isolated from healthy oilseed rape were evaluated against Alternaria brassicae,Sclerotinia sclerotiorum etc. ten pathogenic fungi,through dual culture in Petri dish, 22 isolates showed good antagonistic effect. 5 (G5,BS5,B5,J6,LT20) isolates among 22 have broad inhibitory spectrum and obvious inhibitory effect. The inhibition efficiency of culture filtrate of these 5 endophytes against ten pathogens were further tested, the result showed that these five bacterial endophytes had shown certain inhibition efficiency. This study indicated that antifungal bacteria can be screened from endophytic bacteria isolated from oilseed rape and they can produce antifungal metabolic substances, so these antifungal bacteria can be considered as the sources of screening of antifungal activity substances. These five strains were identified as Bacillus cereus,Acinetobacter calcoaceticu,Pantoea agglomrans,Pseudomonas putida and Pantoea ananas by 16S rDNA gene sequence phylogenetic analysis, respectively. These bacteria species is the first reported in oilseed rape rape. The main groups from PCR-RFLP analysis were identified by 16S rDNA gene sequence phylogenetic analysis.16 species were obtained, they were: Bacillus cereus,Mycoplana bullata ,Acinetobacter calcoaceticus ,Acinetobacter baumannii ,Chryseobacterium indolthetium,Acidovorax avenae,Pantoea ananas,Pantoea agglomerans,Pantoea dispersa,Pantoea spp.,Enterobacter cowanii,Sphingobacterium multivorum,Pseudomonas putida,Pseudomonas fulva,Pseudomonas parafulva and Pseudomonas spp., respectively. The results showed the population of endophytic bacteria from oilseed rape was very abundant.These bacteria species is the first reported in oilseed rape, and Mycoplana bullata,Acinetobacter calcoaceticus,Pantoea dispersa,Pantoea ananas,Enterobacter cowanii,Pseudomonas fulva,Pseudomonas parafulva are new record species of endophytic bactria..