Analysis of endophytic community diversity and functional taxa in walnut green husk tissues at the mature stage

【Objective】Walnut (Juglans regia L.) has both high edible and medical values, and is also one of the most widely cultivated economic forest species in temperate regions. China is currently the country with the largest walnut cultivation area and yield in the world, and North China is an importantwalnut producing area in China. Due to the high temperature and humidity climate in July and August, walnut fruit diseases are severe, resulting in a decline in yield and quality. Endophytes play an important role in host health, adaptive evolution and stress tolerance. However, the role of endophytes in fruit disease resistance is still unclear. This study aims to reveal the relationship between endophytic communities and walnut fruit diseases, and to identify beneficial functional microbiota. The findings will provide a theoretical basis for developing eco-friendly strategies to control fruit diseases.【Methods】In August 2022, six walnut varieties (lines) cultivated across three regions in Beijing were collected as experimental materials. Through high-throughput sequencing technology, the diversity and structural composition of endophytic communities in healthy/diseased walnut green husks of different resistant varieties (lines) were analyzed, the signature endophytes in healthy green husks were identified, and the metabolic functions of endophytic communities were predicted. Specifically, 16S rRNA and ITS1 gene sequencing were used to study endophytic bacterial and fungal communities, respectively.【Results】(1) The richness and diversity of endophytic communities in the green husks of resistant varieties (lines) were significantly higher than those of susceptible varieties (lines). In the same variety (line), the diversity of the endophytic fungal communities in the green husks increased significantly after the fruit became diseased, while there was no significant difference in the diversity of the endophytic bacterial communities. Beta diversity analysis revealed that differences in disease resistance significantly influenced the community structures of both endophytic bacteria (R2 =0.111, P=0.001) and endophytic fungi (R2 =0.179, P=0.001), with endophytic fungal communities exhibiting a more pronounced structural response to resistance variation. (2) The dominant endophytic bacterial phyla in each variety (line) of walnut were Proteobacteria, Firmicutes, Actinobacteriota, and Bacteroidota. Among these, Proteobacteria exhibited the highest relative abundance (11.19%-97.76% ), which increased significantly following fruit infection. Firmicutes was mainly distributed in the disease-resistant group, and their abundance decreased after fruit infection. At the genus level, Pseudomonas, Xanthomonas, Pantoea, and Sphingomonas were identified as predominant endophytic bacterial taxa. The dominant endophytic fungal phyla were Ascomycota and Basidiomycota, of which Ascomycota had the highest relative abundance (33.03%- 99.87%). Notably, the disease-resistant group exhibited higher relative abundance of Ascomycota compared to the susceptible group, and its abundance further increased following fruit infection. At the genus level, Colletotrichum, Talaromyces, Alternaria, and Nothophoma were identified as predominant endophytic fungal taxa. Among these, Colletotrichum displayed a significantly lower relative abundance in the resistant group than in the susceptible group, but its abundance markedly increased after fruit infection. In contrast, Talaromyces exhibited the opposite pattern: Its relative abundance was substantially higher in the resistant group and decreased significantly after fruit infection. (3) Pathogens had different preferences for different resistant varieties (lines), and different varieties (lines) also had different abilities to resist pathogens. Through LEfSe analysis, seventy-three signature endophytic bacterial taxa and ten signature endophytic fungal taxa were identified. In the healthy walnut green husks, genera such as Peptoclostridium, Cellulomonas, Sphingomonas, Methylobacterium- methylobacterium, Lactobacillus, Talaromyces, Apiosordaria and Coniosporium were enriched as signature taxa, while in the diseased walnut green husks, Pseudomonas, Paenibacillus, Stenotrophomonas, Saccharibacillus, Botryosphaeria and Microcyclosporella were enriched as signature taxa. (4) Functional prediction analysis revealed that the endophytic communities in walnut green husks had diverse metabolic functions. The endophytic bacterial communities included seven categories of primary functions and sixty categories of secondary functions, among which amino acid biosynthesis was identified as the predominant metabolic function.Meanwhile, endophytic fungal communities included five categories of primary functions and twentynine categories of secondary functions, among which nucleoside and nucleotide biosynthesis were identified as the predominant metabolic function. Further differential metabolic pathway analysis demonstrated that bacterial communities enhanced resistance against pathogens by upregulating the biosynthesis of amino acids and nucleotides, thereby improving fruit resistance to the disease.【Conclusion】The findings of this study revealed the structural differences in endophytic communities in walnut green husks of different resistant varieties (lines), as well as the impacts of pathogen infection. The richness and diversity of endophytic communities in walnut green husk of resistant varieties (lines) were significantly higher than those of susceptible varieties (lines), and the diversity of endophytic fungal communities in diseased fruits increased significantly. In addition, the healthy green husks of resistant varieties (lines) were enriched with beneficial endophytic taxa, which may have enhanced fruit disease resistance by upregulating amino acid and nucleotide biosynthesis. These results contribute to the understanding of the relationship between endophytic communities and fruit disease resistance. It can also lay a solid foundation for the subsequent development of beneficial functional endophytic resources, and provide a theoretical basis for developing eco-friendly strategies to control walnut fruit diseases.