Structural and Dynamic Analysis of Leaf-Associated Fungal Community of Walnut Leaves Infected by Leaf Spot Disease Based Illumina High-Throughput Sequencing Technology

Abstract

Leaf-associated microbiota is vital in plant-environment interactions and is the basis for micro-ecological regulation. However, there are no studies on the direct differences in microbial community composition between disease-susceptible and healthy walnut leaves. This study collected five samples of healthy and infected leaves (all leaves with abnormal spots were considered diseased leaves) from May to October 2018. Differences in fungal diversity (Chao1 index, Shannon index, and Simpson index) and community structure were observed by sequencing and analyzing diseased and healthy leaf microbial communities by Illumina HiSeq sequencing technology. The main fungal phyla of walnut leaf-associated were Ascomycota, Basidiomycota, and Glomeromycota. Diversity indices (Shannon and Chao1 index values) of healthy leaves differed significantly in the late stages of disease onset. The results showed that the fungal species that differed considerably between the healthy and infected groups differed, and the fungal species that differed significantly between the healthy and infected groups changed with the development of the leaf disease. Critical control time points were determined by analyzing the population dynamics of pathogenic fungi. Leaf-associated microorganisms are abundant and diverse, and fungal identification and diversity studies are helpful for developing more appropriate walnut management strategies

Profiling Walnut Fungal Pathobiome Associated with Walnut Dieback Using Community-Targeted DNA Metabarcoding

“…Finally, we evaluated the capacity of the combinations of the two selected primer pairs with and without ITS2 extraction to describe well the symptomatic environmental samples. All conditions tested successfully permitted the detection and amplification of the major fungal phytopathogens and endophytes or saprophytes associated with walnut dieback and trunk diseases worldwide, namely, Botryosphaeria , Colletotrichum , Diaporthe , Fusarium , Neofusicoccum , Juglanconis and Phaeoacremonium genera [ 5 , 10 , 25 , 70 , 71 , 72 ]. Alpha-diversity indices were not significantly different between the two primer pairs except for the number of detected genera associated with walnut husk samples, which was significantly higher with Kyo.…”

“…Metabarcoding has been extensively used to assess microbial diversity of various agroecosystems [ 21 , 22 , 23 , 24 ]. Recently, this method has been used to evaluate fungal diversity associated with leaves [ 25 ] and buds [ 5 ] of diseased walnut trees. Metabarcoding success and accuracy depend on the selection of the targeted region, the design of primer pairs, and the choice of adapted bioinformatic analysis pipelines [ 26 , 27 , 28 , 29 ].…”