| Riportus pedestris is a kind of polyphagous agricultural pest,which harms broad bean,mung bean,soybean and other leguminous plants.It occurs in a wide range of areas,mainly in the Yellow River,Huaihe River,Tibet,and other regions of China,and can seriously lead to a crop failure in the bean field.Leguminous plants,especially soybeans,are one of China’s major food and economic crops.They not only play an important role in food crops,feed,etc.,but also have great scientific value in the research of the relationship with human physiological health.The research object of this study,R.pedestris,is deeply studied in Japan.As a secondary pest,coupled with the lack of systematic research in China,it has not been able to make people pay attention to.Insects and their living environment are accompanied by abundant bacterial communities,which have different functions and can play a key role in different stages of host development and in various organs.At present,most of the studies on the bugs focus on the functions of the insects and the specific strains in the bugs,but there are few studies on the bacteria that systematically describe the whole life cycle of R.pedestris.The purpose of this paper is to systematically explore the life history and associated bacteria in different organs of R.pedestris,and predict the potential function of associated bacteria,so as to enrich the bacterial resources of R.pedestris.The first scientific problem to be solved is what are these associated bacteria? What are the ecological changes of bacteria in the various stages of development and organs of R.pedestris? From this,we can infer its possible ecological function.The main content of this paper is to comprehensively explore the diversity and abundance changes of associated bacteria in the whole life cycle of R.pedestris by using traditional isolation and identification methods and Illumina high-throughput sequencing technology,combined with technical means such as diversity analysis,diversity analysis of bacterial community composition,diversity analysis of bacterial community,and microbial function prediction,It lays the foundation for the important topics such as how to help the growth and development of the bacteria of the bee edge bug in the follow-up research,and provides the possibility for the comprehensive control research,so as to effectively control its harm for a long time.The main results and conclusions are as follows: 1.Isolation by traditional culture method.The eggs were systematically separated,the antennae,heads,intestines and other tissues of five nymphs,and the antennae,heads,intestines and other tissues of female adults;The antennae,heads,intestines and other tissues of male adults were isolated and cultured from their associated bacteria.On the basis of obtaining the full length of the 16 SDNA gene fragment of the associated bacteria,they were finally identified by phylogenetic analysis.A total of 3223 strains of bacteria were purified in this experiment.After simple classification such as morphology,751 strains of bacteria were selected as representative strains.All of them were sequenced and identified.Finally,221 unique 16 S DNA genotypes were obtained.A total of221 species of bacteria were identified by the isolation method.The isolated bacteria belong to 4 phyla,29 families,66 genera,and 4 phyla are Proteobacteria,Actinobacteria,Firmicutes and Bacteroides.The main genera are Acinetobacter 、 Bacillus 、 Brevibacterium 、Enterobacter、Klebsiella、Mammaliicoccus、Pseudomonas、Staphylococcus,etc.2、High throughput sequencing results.A total of 1.74 G sequence data was obtained by sequencing,and the diversity data analysis of 91 samples was completed.A total of 4486445 optimized sequences were obtained,with an average sequence length of 376 bp.A total of3441 operational biological taxonomic units(OTUs)were obtained.The results of diversity analysis indicated that the species richness and diversity of the associated bacteria in the young nymph stage of R.pedestris were the highest,followed by the old nymph stage and adult stage,and the species richness and community diversity of the bacteria in the middle nymph stage were the lowest.From this,we can see that there is a dynamic change in the diversity of the associated bacteria of R.pedestris,which shows a trend of high-low-high.This change may be that R.pedestris gradually tend to have some important functions during their growth and development,and retain the associated bacteria with specific functions,thus changing the diversity of the flora.The analysis of the composition of associated bacteria flora shows that at the level of phylum,the dominant associated bacteria include Proteobacteria and Actinobacteria;At the generic level,the dominant bacteria include Pseudomonas,Methylbacillus,Stenotrophomonas,Microbacterium,etc.The prediction results of the microbial function of the associated bacteria in different developmental stages of R.pedestris showed that the functions of the microbial flora in nymph stage and adult stage were different.Associated bacteria in nymph stage are mainly related to growth and metabolism,while associated bacteria in adult stage are related to metabolism,degradation,translation,protein folding,etc.This indicates that associated bacteria enhance the adaptability of the host and have different functions at different developmental stages.During the growth and development of R.pedestris,these associated bacteria have some similarities,but also have great differences in each stage of their life cycle.In combination with other published studies on the associated bacteria of insects,some important bacteria groups also appear in the associated bacteria of R.pedestris,and the literature is summarized to speculate the possible ecological functions of the associated bacteria.To sum up,this study carried out systematic investigation and functional speculation analysis on the associated bacteria of R.pedestris through isolation,culture and microbial diversity.It has increased the understanding of the associated bacteria of Pteropterus punctatus,laid a good foundation for fully revealing the mechanism of Pteropterus punctatus invading host plants,and provided a new direction for the comprehensive control of Pteropterus punctatus in the future... |
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