Microbial Diversity And Community Dynamic Structure Of Walnut Phyllosphere Based On High Throughput Sequencing

Walnut(Juglans regia L.)is an important "woody grain and oil" ecological tree species in China,known as "super food in the 21 st century".Its rich nutrients are beneficial to human health,with good economic and social benefits.With the continuous expansion of planting area,walnut disease is becoming more and more serious,among which leaf spot is one of the main diseases.At present,the development of disease has become a serious problem in the development of walnut industry.In this study,high-throughput sequencing technology was used to analyze the species diversity,richness and dynamic changes of the microbial populations of healthy leaves and diseased leaves.The difference of dominant groups between healthy leaves and diseased leaves was found.With the development of time,the dynamic development and change of the microbial community of diseased and healthy leaves laid the foundation for the prevention of leaf spot diseases such as anthracnose,black spot and brown spot of Walnut leaves by micro ecological control in the future.The main results are as follows:(1)The diversity index of infected leaves and healthy leaves showed dynamic changes.The diversity index of fungal and bacterial communities in healthy leaves was always lower than that in diseased leaves sampled at the same time,indicating that the diversity of fungal and bacterial communities in healthy leaves collected at each time was lower than that in diseased leaves sampled at the same time.The results showed that the occurrence and development of the disease played a key role in the diversity change of the microbial community,among which Vishnacozyma,Symmetrospora,Strelitziana,Colletrotrichum,Pseudomonas,Sphingomonas and other genera were the key ones(2)After ITS sequences and 16 Sr DNAsequences analysis,found the phyllosphere fungi belonging to three categories of fungi,bacteria,actinomyces,the results show that the Ascomycota,and Basidiomycota,Glomeromycota is the main walnut phyllosphere fungi phylum,Proteobacteria,Oxyphotobacteria,Bacteroidetes are major walnut phyllosphere bacteria phylum;Vishniacozyma,Cercospora and Ramularia are the main fungi genera of walnut phyllosphere,while Pseudomonas,unidentified?Oxyphotobacteria and Sphingomonas are the main bacteria genera of walnut leaves.This result fully shows that even though the physical environment of walnut leaves surface is bad,the microorganism still has the richness and diversity,and the utilization and development potential are high.(3)The results of dynamic analysis showed that the number of fungi and bacteria in different sampling time had obvious changes,and did not show a single downward and single upward trend.The trend of diversity index was that the Shannon index of fungi: HE4 > he5 >He2 > HE1 > He3;IN4 > IN5 > IN2 > IN3 > IN1.The Shannon index of bacteria: he1 >he2 > he3 > he5 > he4;in2 > in3 > in1 > in5 > in4.The diversity of fungi and bacterial communities is influenced by temperature,humidity and rainfall.(4)The experiments also showed that as the disease progresses,the rotting plants produced by the native colonies can produce metabolites,which can select later strains and cause changes in the fungal and bacterial communities.Among them,the fungi with large abundance change include Vishniacozyma,Cercospora,Ramularia,etc.,while the bacteria with large abundance change include Pseudomonas,Sphingomonas,Hymenobacter,etc.This suggests that the later composition of the microbial community depends on the order of the species inhabiting the plant.These data also reveal some complex ecological dynamics of microbial community in the growing season,and emphasize the importance of community succession.At the same time,there are a lot of nonpathogenic microbial communities in the phyllosphere.The colonization of different bacteria on the plant at the early stage of the disease can explain the change of microbial community caused by the same disease on the same plant.