Investigation Of Bacterial Diversity In Root Nodules Of Three Woody Nodulation Plants

Nodule is a symbiosis established by the interaction between plant roots and some soil bacteria(especially nitrogen-fixing bacteria).It is the main place where plants and nitrogen-fixing bacteria symbiotic nitrogen fixation.Therefore,woody nodulation plants are important species for improving forest soil,greening wasteland,and promoting ecological balance.The specificity of nodules structure and metabolism(including nodules secretions)may alter the microbial community structure on the nodules surface and within the nodules and differentiate it from the soil and rhizosphere.At the same time,the diversity of bacteria not only affects the isolation of nitrogen-fixing bacteria,but also may affect the function of nitrogen-fixing bacteria.To investigate bacterial diversity of nodules of woody nodulation plants,the characteristics of bacterial community of nodules of Elaeagnus chinensis and Hippophae rhamnoides,Robinia pseudoacacia were analyzed using high-throughput sequencing technology and compared,in the meantime,the characteristics of bacterial community of nodules of H.rhamnoides in four different months were analyzed and compared.The result showed:1.The analysis of the characteristics of bacterial community of the nodules of E.chinense,H.rhamnoides and R.pseudoacacia collected on June 18,July 14 and May 20 respectively showed that: The bacterial community of the nodules of different types of woody nodular plants were rich and diverse.There were 8 phyla 13 classes 27 orders 40 families 50 genera,12 phyla 26 classes 54 orders 98 families 130 genera,10 phyla 19 classes41 orders 62 families 104 genera obtained from the nodules of E.chinensis,H.rhamnoides and R.pseudoacacia respectively.There are differences in the richness and diversity of the bacterial community of the nodules of different species.The community richness of the nodules of H.rhamnoides are significantly higher than E.chinensis and R.pseudoacacia,its diversity is also slightly higher than the latter two.At the level of phylum and class,11 classes of 7 phyla were the common bacteria.The bacteria of the nodules of E.chinensis and H.rhamnoides are dominated by the class Actinobacteria,the relative abundance is as high as 99.00% and 97.05%.The bacteria of the nodules of R.pseudoacacia were dominated by class Alphaproteobacteria in phylm Proteobacteria,with a relative abundance of 99.34%.At the level of order,the dominant bacteria of E.chinensis and H.rhamnoides were all order Frankiales,with a relative abundances of 98.87% and 95.48%,at the same time,both alsocontained a certain amount of Rhizobiales,the relative abundance were 0.13% and 0.77%.In the nodules of R.pseudoacacia,the domaint order was Rhizobiales,with a relative abundance of 99.18%,and only less than 0.01% of Frankiales.The genus Frankia was the dominant bacteria in the order Frankiales of the nodules of E.chinensis and H.rhamnoides,the relative abundance accounted for more than 99.90%.The genus Mesorhizobium in family Phyllobacteriaceae was the dominant bacteria in the order Rhizobiales of the nodules of R.pseudoacacia,the relative abundance accounted for 99.50% of Rhizobiales.2.The analysis of the characteristics of bacterial community of the nodules of H.rhamnoides collected in four different months show that: the diversity and the richness in March,new leaves and nodules have just begun to germinate(the nodules were formed in the previous year),were significantly higher than in May,July and August.The differences in May,July and August were not significant.The bacteria obtained from nodules in four sampling times can be classified into 21 phyla 36 classes 75 orders 145 families and 234 genera.Among them,17 classes of 8 phyla always present in all of the times.The class Actinobacteria was always dominant with a relative abundance of 65.30% in March,whereas above 96.05% in May,July and August.At the same time,many types of non-actinomycetes,such as the class Alphaproteobacteria,Betaproteobacteria and Gammaproteobacteria of the phylum Proteobacteria as well as the class Cytophagia and Sphingobacteriia of the phylum Bacteroidetes also present.In March,the relative abundance of the order Frankiales in the whole bacterial community was 48.60%,which was significantly lower than 93.54% in May,95.48% in July and 94.73% in August,correspondingly,the relative abundance of non-Frankiales in March was significantly higher than in other sampling times.A total of 10 operational taxonomic units(OTUs)belong to 6 genera of 5 families of Frankiales was obtained from the four sampling times,but OTU235 belongs to the genus Frankia always remained a relative abudance of about 99% in all of the sampling times.the diversity and the richness in March(new leaves develop and new root nodules germinate just)were significantly higher than in May,July and August.The bacteria obtained from nodules in four sampling times can be classified into 21 phyla 36 classes 75 orders 145 families and234 genera.Among them,17 classes of 8 phyla always present in all of the times.The class Actinobacteria was always dominant with a relative abundance of 65.30% in March,whereas above 96.00% in May,July and August.At the same time,many types of non-actinomycetes,such as the class Alphaproteobacteria,Betaproteobacteria and Gammaproteobacteria of the phylum Proteobacteria as well as the class Cytophagia and Sphingobacteriia of the phylum Bacteroidetes also present.In March,the relative abundance of the order Frankiales in thewhole bacterial community was 48.60%,which was significantly lower than 93.54% in May,95.48% in July and 94.73% in August,correspondingly,the relative abundance of non-Frankiales in March was significantly higher than in other sampling times.A total of 10 operational taxonomic units(OTUs)belong to 6 genera of 5 families of Frankiales was obtained from the four sampling times.Among them,5 OTUs belonged to the Frankia,among them,the relative abundance of OTU235 occupied an absolute dominant position at different sampling times.In summary,bacterial community in nodules of different types of woody nodulation plants was extremely rich and diverse.The domaint bacteria of nodules of actinorhizal plants were Frankia,and there are a small amount of nitrogen-fixing bacteria belonging to the order Rhizobiales.The domaint bacteria of nodules of R.pseudoacacia were Mesorhizobium.In addition,bacterial community in nodules of H.rhamnoides in different months was extremely rich and diverse,including many types of actinomycetes and non-actinomycetes.Frankia always was the dominate genus.And the bacterial community structure(especially the relative abundance of Frankia)of nodules changed significantly with the times.This change may be related to the phenology and growth habit of H.rhamnoides,and seasonal changes in the nitrogen fixation capacity of root nodules.