Study On Diversity Of Endophytic Bacteria And A New Species Of Yeast In Rice Seeds

Diversity of endophytic bacterial communities in parental seeds of hybrid rice(Oryza sativa L.)at filling stage and maturity stage were analyzed by culture-dependent method.In addition diversity of endophytic bacterial communities in seeds of super hybrid rice “Shenliangyou 5814”(Oryza sativa L.,)were analyzed by the 16 S rRNA gene clone library technique and culture-dependent method.One of“Shenliangyou 5814” endophytic bacterialwas considered as the novel bacteria strains and identified according to the polyphasic taxonomic method.The main results were followed:The endophytic bacterial communities in the seeds of three rice(“Shen 08S”,“He 620S” and “16A007”)in filling stage and maturity stage were analyzed.Experimental results showed that the parental seeds of hybrid rice had diversity of the bacterial community structure.In filling stage and maturity stage the endophytic bacterial communities in “Shen 08S” included 6 Operational Taxonomic Units(OTUs)and 40 OTUs,respectively;they were belong to Proteobacteria,Bacteroidetes and Proteobacteria,Actinobacteria,Firmicutes,Bacteroidetes,respectively;they were 5and 22 genera.The endophytic bacterial communities in “He 620S” included 10 OTUs and 20 OTUs,respectively;they were belong to Proteobacteria,Actinobacteria and Proteobacteria,Actinobacteria,Firmicutes,respectively;they were 5 and 12 genera,respectively.The endophytic bacterial communities in “16A007” included 11 OTUs and 24 OTUs,respectively;they were belong to Proteobacteria and Proteobacteria,Actinobacteria,Firmicutes,respectively;they were 6 and 14 genera,respectively.From the results we found that the endophytic bacteria species in the seeds changed obviously with seed maturationand the seeds in maturity stage were richer in endophytes resources,we also found that different seeds were different in dominant species of endophytic bacteria.For “Shen 08S”,in illing stage the abundance of the first dominant bacterium Pantoea was 52.04%,the second and third dominant bacterium was Pseudomonas and Rhizobium,respectively;in maturity stage the abundance of the first dominant bacterium Rhizobium was 15.83%,the second dominant bacterium was Bacillus,the third dominant bacterium were Sphingomonas and Curtobacterium.For “He 620S”,in illing stage the abundance of the first dominant bacterium Pantoea was 69.02%,the second was Pseudomonas,the third dominant bacterium were Rhizobium and Sphingomonas;in maturity stage theabundance of the first dominant bacterium Curtobacterium was 25.33%,the second and third dominant bacterium was Microbacterium and Pantoea,respectively.For“16A007”,in illing stage the abudance of the first dominant bacterium Pseudomonas was 45.12%,the second and third dominant bacterium was Pantoea and Sphingomonas,respectively;in maturity stage the abudance of the first dominant bacterium Pantoea was 35.00%,the second and third dominant bacterium was Micrococcus and Pseudomonas,respectively.The 16 S rRNA gene libraries were established to make a survey of the endophytic bacterial community of “Shenliangyou 5814”.The results showed that180 positive clones belong to 13 OTUs,sequence analysis showed that the majority of clones were affiliated with Proteobacteria(94.44%).In addition,other clones belonged to Actinobacteria(0.56%).The first dominant bacterium Pantoea was89.44%,the second dominant bacterium were Flavobacterium and Methylobacterium,respectively;the dominant group was Proteobacteria,others was few.Culture-dependent method was used to further research the endophytic bacterial community of “Shenliangyou 5814”.Experiemental result showed that the endophytic bacterial communities in “Shenliangyou 5814” included 49 OTUs,they were belonged to Firmicutes,Proteobacteria and Actinobacteria.The abudance of the first dominant bacterium Bacillus was 37.5%,the second dominant bacterium was Fictibacillus,the third dominant bacteria were Pantoea and Klebsiella.From this study,we found that the seed of “Shenliangyou 5814” were rich in endophytes resources,and as far as we know this was the first plant material isolated lots of different kinds of bacterial species.Compared to 16 S rRNA gene libraries,the culture-dependent method had more bacterial species and was richer in endophytes community.There were some differences in the species and abundance of the dominant bacteria species obtained by the two methods.This was due to the different technical means between the traditional culture method based on microbial isolation and culture and the non-culture method based on molecular biology technology.The comprehensive analysis of the results of this study of the two methods will provide an objective and comprehensive analysis of the endophytic bacteria of "Shenliangyou5814".A novel species Z8 Tisolated from rice seeds was characterized by phenotypic characteristics,physiological and biochemical characteristics,genetic characteristics and chemical characteristics.The results were as follows: coloniesare brownish white,cells are ovoid or ellipsoidal,asexual reproduction is by budding,ascospores are not produced.Glucose,D-mannose,D-mannitol,D-maltose,D-cellobiose,D-fructose,D-arabinose,inulin,glycogen,potassium gluconate,2-ketogluconate,5-ketogluconate,soluble starch are assimilated.Nitrite and nitrate are utilized as nitrogen sources.Molecular phylogenetic analysis based on the D1/D2 domains of the 26 S rRNA gene and ITS regions revealed that isolate Z8 Twas located in the same clade with the type species(M.magnisporus)of the genus Microsporomyces.The DNA G+C content of strain Z8 Twas 56.7 mol %.The major ubiquinone of the strain is Q-10.These data suggest that strain Z8 T represents a novel species of the genus Microsporomyces.Strain Z8 T is considered to represent a novel species of Microsporomyces,for which the name Microsporomyces hainanensis sp.nov.is proposed.