The novel nematicide chiricanine A suppresses Bursaphelenchus xylophilus pathogenicity in Pinus massoniana by inhibiting Aspergillus and its secondary metabolite, sterigmatocystin

Abstract

Introduction: Pine wilt disease (PWD) is responsible for extensive economic and ecological damage to Pinus spp. forests and plantations worldwide. PWD is caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus) and transmitted into pine trees by a vector insect, the Japanese pine sawyer (JPS, Monochamus alternatus). Host infection by PWN will attract JPS to spawn, which leads to the co-existence of PWN and JPS within the host tree, an essential precondition for PWD outbreaks. Through the action of their metabolites, microbes can manipulate the co-existence of PWN and JPS, but our understanding on how key microorganisms engage in this process remains limited, which severely hinders the exploration and utilization of promising microbial resources in the prevention and control of PWD.

Methods: In this study we investigated how the PWN-associated fungus Aspergillus promotes the co-existence of PWN and JPS in the host trees (Pinus massoniana) via its secondary metabolite, sterigmatocystin (ST), by taking a multi-omics approach (phenomics, transcriptomics, microbiome, and metabolomics).

Results: We found that Aspergillus was able to promote PWN invasion and pathogenicity by increasing ST biosynthesis in the host plant, mainly by suppressing the accumulation of ROS (reactive oxygen species) in plant tissues that could counter PWN. Further, ST accumulation triggered the biosynthesis of VOC (volatile organic compounds) that attracts JPS and drives the coexistence of PWN and JPS in the host plant, thereby encouraging the local transmission of PWD. Meanwhile, we show that application of an Aspergillus inhibitor (chiricanine A treatment) results in the absence of Aspergillus and decreases the in vivo ST amount, thereby sharply restricting the PWN development in host. This further proved that Aspergillus is vital and sufficient for promoting PWD transmission.

Discussion: Altogether, these results document, for the first time, how the function of Aspergillus and its metabolite ST is involved in the entire PWD transmission chain, in addition to providing a novel and long-term effective nematicide for better PWD control in the field.

Keywords: Monochamus alternatus; aspergillus; host performance; nematicide; pine wilt disease; pinewood nematode (Bursaphelenchus xylophilus); sterigmatocystin.

Figure 1

Sterigmatocystin (ST) increases Pinewood nematode (PWN) pathogenicity by suppressing ROS accumulation in Pinus massoniana. (A) Relative ST concentrations in the host plant P. massoniana quantified before (PWN(-)) and after (PWN(+)) invasion by PWN. The seedlings were inoculated with an equal amount of PWN (5000 individuals), followed by 0.1 mg/mL (ST_H), 0.01 mg/mL (ST_M), 0.001 mg/mL (ST_L) or a 0 mg/mL (ST(-)) of ST as the treatment, after which the (B) relative PWN amount, (C) death rate, (D) rate of ROS production, and (E) rate of H₂O₂ production were quantified at 14 days post-infection. Data shown is the mean ± standard deviation (SD). (F) Correlations between ST and their correlated ROS, transcripts, microbes, and metabolites. Metabolite pathways of P. massoniana as induced by ST with a highly correlated concentration. Different lowercase letters above bar columns show significant differences between treatments at P < 0.05, based on a one-way ANOVA, with multiple comparisons made using Tukey’s test. G1–G5 denote different transcripts (c60547.graph_c0, c82953.graph_c0, c64867.graph_c0, c68789.graph_c0, c81022.graph_c0); Mb1–Mb6 correspond to different microbial genera (Cladophialophora, Penicillium, Trichoderma, Achromobacter, Chitinophaga, and Flavobacterium); Mt1–Mt3 indicate different metabolites [maltotriose, 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine, and myo-inositol]. (G) KEGG enrichment map of metabolic pathways of metabolites found significantly related to ST. The abscissa represents the impact of each pathway and the ordinate represents the pathways’ name. Impact is expressed as the ratio of the number of differential metabolites to the number of metabolites annotated in a given pathway. The circle represents ST_H vs. ST(-), the square represents ST_M vs. ST(-), and the triangle represents ST_L vs. ST(-), whose sizes indicate the number of differentially expressed metabolites contained in that metabolic pathway. Coloring represents the P-values for the enrichment analysis.

Figure 2

Sterigmatocystin (ST) increases VOC accumulation in Pinus Massoniana to attract Monochamus alternatus (JPS). (A) VOC amounts in host plant P. massoniana quantified 14 days after treatment with 0.1 mg/mL (ST_H), 0.01 mg/ml (ST_M), 0.001 mg/mL (ST_L), or 0 mg/mL (ST(-)) of ST. (B) VOC compounds of P. massoniana induced by ST with a high concentration correlation. (C) Selective ratio of JPS to different plant samples were plotted, with the P-value for the t test between samples treated with (ST (+)) and without ST(ST(-)) presented in white. The (D) consumption area and (E) body weight of 3rd instar JPS larvae, and the (F) spawning rate, (G) EG (endo-1,4-β-D-glucanase) activity, (H) CBH (exo-β-1,4-D-glucanase) activity, and (I) β-GC (β-glucosidase) activity in the gut of the JPS adults that fed upon plant samples treated with (ST (+)) and without ST(ST(-)); data shown are the mean ± standard error (SE). The * and *** represents significant differences between treatments at P < 0.05 and P < 0.001, respectively, based on a one-way ANOVA, with multiple comparisons made using Tukey’s test. VOC1–VOC10 indicate different volatile organic compound (trans-anethole; acetophenone, 4’-hydroxy-; humulene; niacinamide; 4a(2H)-naphthalenol, octahydro-4,8a-dimethyl-,(4.alpha.,4a.alpha.,8a.beta.)-; 6-octen-1-ol,3,7-dimethyl-, (R)-; alpha-pinene; butanoic acid, 3-hydroxy-3-methyl-; phenol; beta-myrcene). The ** represents significant differences between treatments at P < 0.01.

Figure 3

Associated microbe Aspergillus arachidicola promotes PWN pathogenicity by increasing the sterigmatocystin (ST) accumulation in Pinus massoniana. (A) Richness of microbes belonging to the Aspergillus genus, (B) expression level of ST synthesis promoting gene aflR, (C) richness of A arachidicola and A sclerotioniger in plant samples before (PWN(-)) and after (PWN(+)), invasion by PWN invasion are plotted. Aspergillus arachidicola, A sclerotioniger, and inactivated A arachidicola (-) were co-incubated with sterilized PWN, then inoculated into P. massoniana and the (D) ST amount, (E) relative PWN amount, and (F) survival ratio of different host plants were measured. Data shown are the mean ± standard deviation (SD). The *** represents significant differences between treatments at P < 0.001, based on a one-way ANOVA, with multiple comparisons made using Tukey’s test; ‘n.s.’ denotes no significant differences found.

Figure 4

The fungal inhibitor chiricanine A can suppress the richness of Aspergillus fungi in Pinus massoniana to limit in vivo PWN population or pathogenicity, in both laboratory and field testing. Under laboratory conditions, 2-year-old P. massoniana seedlings were inoculated with PWN, and then inoculated with chiricanine A solution or methanol (control). For these samples, the (A) richness of Aspergillus fungi highly induced and (B) aflR expression level in response to PWN invasion were quantified. Relative PWN amount in PWN-carrying P. massoniana treated with (C) emamectin benzoate and chiricanine A, as well as those (D) injected with A arachidicola or (E) ST at 8 weeks post-inoculation. In the field trial, a solution of chiricanine A or methanol (control) was injected into PWN-carrying P. massoniana, and its (F) survival ratio, (G) relative PWN amount, and the (H) richness of Aspergillus fungi highly induced and (I) aflR expression level in response to PWN invasion were quantified after the 1st and 2nd year post-injection. Data shown are the mean ± standard deviation (SD). The *** represents significant differences between treatments at P < 0.001, based on a one-way ANOVA, with multiple comparisons made using Tukey’s test.

Figure 5

Schematic diagram of the pine wood nematode-Monochamus alternatus symbiosis promoted by Aspergillus fungi in Pinus massoniana host trees via sterigmatocystin (ST).