| The root-knot nematode (RKN) belongs to the most damaging plant pathogens worldwide, is able to infect almost all cultivated plants, such as food, tobacco, vegetables, fruits, which was regarded as one major limiting factor in sustainable agriculture. RKN finish their major life history and infection cycle in the rhizosphere environment. In the same ecological niche, there is a tight relationship among RKN, rhizosphere microorganism and plant. In this study, the relationships among environmental factors, rhizosphere bacterial communities of RKN-infected and healthy tobacco in the field conditons were investigated. In addition, associated bacteria flora of different life stages of Meloidogyne incognita was also studied. The results are as follow:1. The rhizosphere bacterial community structure, diversity and predominant bacteria of tobacco at large-scale geographic environmentA total of65rhizosphere soil samples (28of RKN-infected tobacco,27of healthy tobacco and10of other plants) were collected from28geographical locations throughout the main tobacco growing areas of ten provinces acress China. The rhizosphere bacterial community and predominant taxa of RKN-infected and healthy tobacco were compared by sequencing the V1-V3region of16S rDNA using454GS FLX platform. A total of464879reads were preceded and analysed, the results showed that:(1) Tobacco rhizosphere bacteria were composed of23bacterial phyla and unclassified bacteria. Proteobacteria, Acidobacteria, Actinobacteria Bacteroidete, Firmicutes, candidate phyla WS3are the most abundant group in the tbacco rhizobacterial community.(2) Tobacco rhizobacaterial diversity are significant different among different region (alpha diversity) and are similar in the same region.(3) Rhizobium, Streptomyces, Sphingomonas, Singulisphaera and other13taxon are the predominant bacteria (bacterial taxa with realative aboundance above1%) in the tbacco rhizobacterial community.2. Enviroment and soil physicochemical propertises interact to sharpe the tobacco rhizobacterial community12soil physicochemical properties, such as nitrate, phosphorous, potassium and several other nutrients were analysis. We correlated each and all of physicochemical characteristics with geographic region and rhizobacterial community, the results showed that:(1) Tobacco rhizobacterial communitise from various locations have obvious differences and there is a good correlation between rhizobacterial community compositions and geographic origin using principal components analysis based on the spearman correlation coefficients.(2) Soil pH correlated with geographic origin and bacterial compositons. No significant realationship were observed between other soil physicochemical factors and geographic origin and bacterial compositons. These results indicate that environmental heterogeneity, such as pH, as well as geographic patterns, likely interact to shape the spatial scaling of the tobacco rhizosphere microbiota, and that the nature of these interactions may differ by environment.3. The influence of RKN infection on tobacco rhizobacteria communityThe rhizobacterial composition and diversity of RKN-infected and healthy tobacco have some differences by comparing alpha diversity, beta diversity and the distribution of predominant bacteria. The results showed that the RKN infection have effect on host plant rhizobacterial communities. Analysis of co-occurrence patterns between rhizobacterial communities of RKN-infected and healthy tobacco showed significant differences in the co-occurrence relationship.32bacterial clades belong to7phylum were significantly correlated to each other. The main co-occurrence clades in the rhizobacterial community of healthy tobacco were Proteobacteria (13clades)、 Actinobacteria (7clades)、Planctomycetes (4clades)'Firmicutes (3clades). Compare to the rhizobacteria taxon of healthy tobacco,42bacterial clades belong to10phylum were significantly correlated to each other in the rhizobacterial community of RKN-infected tobacco. There were22clades of Proteobacteria,6clades of Actinobacteria and lclade of Firmicutes and3clades of Bacteroidetes,1clade of Nitrospirae and Gemmatimonadetes were additionaly correlated with other bacterial clades in the the rhizobacterial community of RKN-infected tobacco. Different statistical testing methods were used to analysis the associated bacteria of tobacco RKN disease, the results showed that Rhizobium and Novosphingobium were significantly correlated with tobacco RKN disease.Correlation Analysis showed that single soil physicochemical properties had no correlation with tobacco RKN disease. We further constructed a generalized linear mixed models (RKN-infected and healthy as variables, geographic origin as random effect) to estimate the relationship between soil physicochemical properties and tobacco RKN disease. When using soil pH and major nutrient elements (N, P and K) as fixed effect, soil potassium content was significant negative correlated with tobacco RKN disease; When using trace nutrient elements (Copper, zinc, manganese, iron, magnesium) as fixed effect, soil Copper and manganese content was significantly negative correlated with tobacco RKN disease; When using oganic mixed nutrient elements (N、P、K、Na、 Ca) as fixed effect, potassium content was negative correlated with tobacco RKN disease. These results indicate that soil potassium content and the interaction with other biotic factors may have effect on the occurrence of tobacco RKN disease.4. Bacterial consortia associated with M. incognita at different developmental stages using pyrosequencing-based analysisIn this study, we explored the bacterial consortia associated with M. incognita at different developmental stages, including egg mass, adult female and second-stage juvenile using the454pyrosequencing approach. The results showed that:(1) Proteobacteria is the most abundant phylum associated with M. incognita, followed by Actinobacteria, Bacteriodetes, Firmicutes etc.(2) Egg mass, female and second-stage juvenile of M. incognita harbored a core microbiome with minor difference in communities and diversities, which could be due to differences in habitats and development of the nematode.(3) Several bacteria genera identified in M. incognita are recognized cellulosic microorganisms, pathogenic bacteria, nitrogen-fixing bacteria and antagonists to M. incognita. The potential biological control microorganisms, including the known bacterial pathogens and nematode antagonists, such as Actinomycetes and Pseudomonas, showed the largest diversity and proportion in egg mass, and dramatically decreased in second-stage juvenile and female of M. incognita. Some genera previously identified in other plant-parasitic nematodes were also found, such as the genera Pedobacter. The results indicated that M.incognita to be rich in a variety of bacteria, some of which may play important roles in the bioloy of RKN and have potential as biocontrol agents or target.The results of this study will help to elucidate the relationship of RKN-rhizobacteria-plant and provide reference data for recognizing the crop RKN-disease and may also open new avenues to RKN biocontrol. |
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