The Changes of Microbial Communities and Key Metabolites after Early Bursaphelenchus xylophilus Invasion of Pinus massoniana

Abstract

Pine wood nematode, Bursaphelenchus xylophilus, is a worldwide pest of pine trees, spreading at an alarming rate and with great ecological adaptability. In the process of causing disease, the nematode causes metabolic disorders and changes in the endophytic microbial community of the pine tree. However, the changes at the pine nidus during early nematode invasion have not been well studied, especially the differential metabolites, in Pinus massoniana, the main host of B. xylophilus in China. In this study, we analyzed the endophytic bacterial and fungal communities associated with healthy and B. xylophilus-caused wilted pine trees. The results show that 1333 bacterial OTUs and 502 fungal OTUs were annotated from P. massoniana stem samples. The abundance of bacterial communities in pine trees varies more following infection by B. xylophilus, but the abundance changes of fungal communities are less visible. There were significant differences in endophytic microbial diversity between wilted and healthy P. massoniana. In wilted pine trees, Actinobacteria and Bacteroidia were differential indicators of bacterial communities, whereas, in healthy pine trees, Rhizobiales in the Proteobacteria phylum were the major markers of bacterial communities. Meanwhile, the differential markers of fungal communities in healthy pines are Malasseziales, Tremellales, Sordariales, and Fusarium, whereas Pleosporaceae is the key marker of fungal communities in wilted pines. Our study examines the effect of changes in the endophytic microbial community on the health of pine trees that may be caused by B. xylophilus infection. In parallel, a non-targeted metabolomic study based on liquid mass spectrometry (LC-MS) technology was conducted on pine trees inoculated with pine nematodes and healthy pine trees with a view to identifying key compounds affecting early pine lesions. Ultimately, 307 distinctly different metabolites were identified. Among them, the riboflavin metabolic pathway in pine trees may play a key role in the early pathogenesis of pine wood nematode disease.

Keywords: endophytic microbes; metabolite profiling; microbial community; pines; pinewood nematode.

Figure 1

Alpha Diversity Index of the microbial communities of healthy and B. xylophilus–infected P. massoniana. (A) Endophytic bacteria. (B) Endophytic fungi. Nematode-infected Wilted pines are designated as P. massoniana. EBH, healthy pine endophytic bacteria; EBW, wilted pine endophytic bacteria; EFH, healthy pine endophytic fungi; EFW, wilted pine endophytic fungi.

Figure 2

Clustering of the microbial communities of healthy and B. xylophilus−infected P. massoniana using PCoA and UPGMA (unweighted pair-group method with arithmetic means). (A) PCoA plots based on Bray–Curtis metrics for bacterial communities of healthy and B. xylophilus−infected P. massoniana. (B) UPGMA clustering of bacterial communities of healthy and B. xylophilus−infected P. massoniana. (C) PCoA plots based on unweighted Bray–Curtis metrics for fungal communities of healthy and B. xylophilus−infected P. massoniana. (D) UPGMA clustering of fungal communities of healthy and B. xylophilus−infected P. massoniana.

Figure 3

Venn diagrams of the unique and shared operational taxonomic units (OTUs) of sequenced samples and LEfSe diagrams between all the samples from healthy pines and wilted pines. (A) Bacterial data among all samples from healthy pines and wilted pines. (B) Fungal data from all the samples from healthy pines and wilted pines. (C) LEfSe analysis diagrams of bacteria data between all the samples from healthy pines and wilted pines. (D) LEfSe analysis diagrams of fungal data between all the samples from healthy pines and wilted pines.

Figure 4

PCA analysis of differential metabolites. The horizontal coordinate PC1 and the vertical coordinate PC2 indicate the scores of the first and second principal components, while different colors indicate samples from different treatments, and the confidence ellipse is 95%. (C,D) PLS-DA classification validation plots. The horizontal coordinates indicate the correlation between random group Y and original group Y, while the vertical coordinates indicate the scores of R2 and Q2. (A,C) positive model, (B,D) negative model.

Figure 5

Volcano plots and KEGG metabolic pathway enrichment map of differential metabolites. (A,C) Positive ion mode, (B,D) Negative ion mode; each point represents a metabolite: horizontal coordinates indicate different multiplicities of differential metabolites (log2 values), vertical coordinates indicate p-values (−log10 values), grey indicates metabolites with no significant differences (NoDiff), red indicates up–regulated metabolites (UP), green indicates down–regulated metabolites (DW), and the size of the points indicates VIP values. The abscissa in the figure is x/y (the number of differential metabolites in the corresponding metabolic pathway/the total number of metabolites identified in the pathway), and the larger the value, the higher the enrichment of differential metabolites in the pathway. The color of the point represents the p-value of the hypergeometric test. The smaller the value, the more reliable and statistically significant the test is. The size of the dots represents the number of differential metabolites in the corresponding pathway; the larger the number, the more differential metabolites in the pathway.

Figure 6

LIPID MAPS category annotation and heatmap analysis of differential metabolites of polyketides. (A) Positive ion mode, (B) Negative ion mode: the horizontal coordinate represents the number of metabolites, and the vertical coordinate represents the LIPID MAPS lipid categories annotated to; this figure shows the number of metabolites annotated to the Main_Class under the eight major lipid categories in LIPID MAPS; (C) Heatmap display of the different substances of polyketides.

Figure 6

LIPID MAPS category annotation and heatmap analysis of differential metabolites of polyketides. (A) Positive ion mode, (B) Negative ion mode: the horizontal coordinate represents the number of metabolites, and the vertical coordinate represents the LIPID MAPS lipid categories annotated to; this figure shows the number of metabolites annotated to the Main_Class under the eight major lipid categories in LIPID MAPS; (C) Heatmap display of the different substances of polyketides.