| Plant micorbes exist in the whole process of plant growth.At present,most researches on microbes in plants mainly focus on roots,leaves and other parts.As an important propagation organ of plants,seeds are of great significance in crop production.A large number of previous studies have shown that the outer and inner surfaces of the seeds carry a rich microbial population.Seed endophytic bacteria have the potential to be selectively transferred to the next generation.They have a variety of beneficial functions for the host plant,and are essential for plant growth,health,and seed storage.In the early stage of this laboratory,two varieties of rice PA64(japonica rice)and Dular(indica rice)were bred in Jiangxi region as parent seeds and planted in four regions of Hainan,Yunnan,Jiangxi,and Hebei.Isolation culture and Illumina high-throughput sequencing methods were used to study the endophytic bacteria diversity and community composition in two generations of rice seeds.The common group of endophytic bacteria in rice seeds was initially defined as Xanthomonas sacchari,and 117 pure cultures were obtained.Although Xanthomonas spp.has been reported to be pathogenic in a variety of host plants,Xanthomonas sacchari in this genus has not been found pathogenic to date yet.In this study,this group was used as the research object.Through BOX-PCR and 16 S rRNA gene cluster analysis,combined with the source of the strains,25 strains were selected for genome analysis,plant growth promotion function verification,and colonization status was detected by GFP-labeled strains.Further to explore the interaction between endophytic bacteria and plants in rice.The following main results were obtained:1.The plant promoting functional characteristics such as IAA secretion,siderophoreACC deaminase and antagonistic phytopathogen were tested.All 25 strains have the function of secreting IAA,the amount of IAA secretion is between 7.65-135.08 mg/L;17 strains have the ability to produce siderophore,and the A/Ar ratio is between 0.41-1.19,of which strain JR3-14 has the strongest iron-producing ability,with an A/Ar ratio of 0.41;10 strains have ACC deaminase activity,and the enzyme specific activity is between 0.0402-0.1868 U/mg;2 strains and 14 strains have antagonistic effects for two pathogens,tomato wilt and the rice bacterial blight,respectively.In addition,it is determined that this group has no hemolytic properties,indicating that it has the basic property for the production of biological fertilizers.2.Bioassay experiments showed that the endophytic bacteria Xanthomonas sacchari of rice seeds could promote the growth of rice seedlings and Arabidopsis plants.SPSS analysis of variance revealed that LT5-3,JT6-9-1 and JT6-14 strains have significant effects on rice seedlings,JR3-14 and LT5-3strains have significant effects on the main root elongation of Arabidopsi.According to the pathogenicity test,this group has no pathogenicity to rice.3.Combining with genomic analysis,we knew that Xanthomonas sacchari is a group with high GC content(about 68%),with a genome of about 5.0 Mb and CDS of about 4000.Most of them have 5rRNAs and 55 tRNAs,Xanthomonas sacchari has no pathogenic gene clusters(T3SS,T6 SS and related effector proteins)compared to the known pathogenic strains of Xanthomonas spp.The cluster heat mapfound that different strains in the same area can better cluster on the same branch,and their core genome number is 2528,which indicated that there are more genes in the same group,and the genetic relationship is closer.4.JR3-14 strain was successfully labeled with GFP gene,and the colonization of the labeled strain in tissue culture seedling of rice plants was detected by microscopy.There were a small number of dynamic green fluorescent strains found inside the stems and leaves of the plant,showing that Xanthomonas sacchari might enter the rice inner through infection of stems and leaves.The specific colonization and transmission pathways need to be further studied. |
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