Multi-Omics of Pine Wood Nematode Pathogenicity Associated With Culturable Associated Microbiota Through an Artificial Assembly Approach.

Shouping Cai,Xueyou He,Yu Fang,Xiang Lan,Chenyang He,Jiayu Jia,Guowen Qiu,Jun Su,Liqiong Zeng

doi:10.3389/fpls.2021.798539

Abstract

Pinewood nematode (PWN), the causal agent of pine wilt disease (PWD), causes massive global losses of Pinus species each year. Bacteria and fungi existing in symbiosis with PWN are closely linked with the pathogenesis of PWD, but the relationship between PWN pathogenicity and the associated microbiota is still ambiguous. This study explored the relationship between microbes and the pathogenicity of PWN by establishing a PWN-associated microbe library, and used this library to generate five artificial PWN–microbe symbiont (APMS) assemblies with gnotobiotic PWNs. The fungal and bacterial communities of different APMSs (the microbiome) were explored by next-generation sequencing. Furthermore, different APMSs were used to inoculate the same Masson pine (Pinus massoniana) cultivar, and multi-omics (metabolome, phenomics, and transcriptome) data were obtained to represent the pathogenicity of different APMSs at 14 days post-inoculation (dpi). Significant positive correlations were observed between microbiome and transcriptome or metabolome data, but microbiome data were negatively correlated with the reactive oxygen species (ROS) level in the host. Five response genes, four fungal genera, four bacterial genera, and nineteen induced metabolites were positively correlated with the ROS level, while seven induced metabolites were negatively correlated. To further explore the function of PWN-associated microbes, single genera of functional microbes (Mb1–Mb8) were reloaded onto gnotobiotic PWNs and used to inoculate pine tree seedlings. Three of the genera (Cladophialophora, Ochroconis, and Flavobacterium) decreased the ROS level of the host pine trees, while only one genus (Penicillium) significantly increased the ROS level of the host pine tree seedlings. These results demonstrate a clear relationship between associated microbes and the pathogenicity of PWN, and expand the knowledge on the interaction between PWD-induced forest decline and the PWN-associated microbiome.

Highlights

Pine wilt disease (PWD) is one of the most devastating forest diseases in the world
The current study aimed to explore the relationship between microbes and the pathogenicity of Pinewood nematode (PWN) by generating artificial assemblies of culturable PWN-associated microbiota from different geographic locations with the same PWN species population, and inoculating these assemblies onto the same Masson pine (Pinus massoniana) cultivar to normalize the background of the inoculation
There were 78, 27, 39, 51, and 30 specific operational taxonomic units (OTUs) in the fungal communities of different artificial PWN–microbe symbiont (APMS) that developed with microbes from FZ, NP, Xiamen (XM), LY, and Sanming (SM), 1http://www.kegg.jp 2http://www.metaboanalyst.ca/ 3https://www.genome.jp/kegg/pathway.html respectively (Figure 2E), while 119 OTUs were shared by all five geographical locations

Summary

Introduction

PWD has increased the number of its host species and its pandemic area rapidly in the last decade, and causes enormous economic losses annually (Proença et al, 2017b,a). The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causative agent of PWD and is transmitted by its insect vector, the Monochamus beetle (Zhao et al, 2014). Several critical pathogenic factors have been proposed [i.e., the abnormal regulation of reactive oxygen species (ROS) in the host, the genome of PWN, the regulation of metabolites, and the co-evolution of the PWN-insect vector], providing new insights into PWD management (Shinya et al, 2013; Zhao et al, 2016; Wen et al, 2021). It is well documented that abnormal regulation of ROS is the most important early symptom of PWD, which is highly correlated with the pathogenicity of PWN (Zhang et al, 2019)

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Results

Conclusion