The nematicide emamectin benzoate increases ROS accumulation in Pinus massoniana and poison Monochamus alternatus

Abstract

Pine wilt disease (PWD) is caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus) and transmitted by a vector insect, the Monochamus alternatus. The PWN has caused much extensive damage to pine-dominated forest ecosystems. Trunk injection of emamectin benzoate (EB) has been found to be the most useful protective measure against the PWN, due to its low effective dose and long residence time in the field. However, the interactions between EB and the host or the environment remain largely unknown, which limits the efficacy and stability of EB in practical field settings. In this study, we investigated the impact on PWN from EB injection for both adult and young host plants (Pinus massoniana) by taking a multi-omics (phenomics, transcriptomics, microbiome, and metabolomics) approach. We found that EB injection can significantly reduce the amount of PWN in both living adult and young pine trees. Additionally, EB was able to activate the genetic response of P. massoniana against PWN, promotes P. massoniana growth and development and resistance to Pine wilt disease, which requires the presence of PWN. Further, the presence of EB greatly increased the accumulation of reactive oxygen species (ROS) in the host plant in a PWN-dependent manner, possibly by affecting ROS-related microbes and metabolites. Moreover, we uncovered the function of EB limiting the consumption of P. massoniana by the JPS. Based on biochemical and gut microbial data, we found that EB can significantly reduces cellulase activity in JPS, whose transcription factors, sugar metabolism, and the phosphotransferase system are also affected. These results document the impact of EB on the entire PWD transmission chain through multi-omics regarding the dominant pine (P. massoniana) in China and provide a novel perspective for controlling PWD outbreaks in the field.

Fig 1. Impact of EB (emamectin benzoate) injection on the amount of PWN (pine wilt nematode) in the host plant Pinus massoniana.

(a) The control efficacy of EB on the PWN in host trees carrying a high (PWN_H, PWN amount > 10000) and low (PWN_L, PWN amount < 6000) load of PWN in the field (a), and laboratory (b) were plotted according to the relative amount of PWN present. EB (+) and EB (-) represent injection with EB or a control chemical, respectively. The * represents a significant difference between samples, P ≤ 0.05, (a) with non-parametric tests (Kruskal-Wallis tests and Wilcoxon rank-sumtests), (b) based on a one-way ANOVA, with a multiple comparison analysis using Tukey’s test.

Fig 2. Injection of EB (emamectin benzoate) promotes ROS accumulation in the host plant Pinus massoniana.

The ROS level was quantified in field (a) and indoor seedling (b) samples. EB (+) and EB (-) respectively denote the injection of EB or a control chemical. PWN_H and PWN_L represent high and low amounts of PWN carriers in the field; PWN (+) or PWN (-) indicate whether the host plant carries PWN or not, respectively. The symbol * indicates a significant difference between samples at P < 0.05, the symbol ** indicates a highly significant difference between samples at P < 0.01, while “n.s.” indicates no significant difference between samples, with non-parametric tests (Kruskal-Wallis tests and Wilcoxon rank-sumtests).

Fig 3. Impact of EB (emamectin benzoate) injection on the defense genes of the host plant Pinus massoniana.

The PWN resistance genes of pine trees were quantified in the field (a) and seedlings (b); G1–G5 denotes gene c60547.graph_c0, c64867.graph_c0, c68789.graph_c0, c81022.graph_c0, and c82953.graph_c0, respectively. PWN (+) represents the host plants carrying the PWN, and PWN (-) those not.

Fig 4. Impact of EB (emamectin benzoate) injection on the symbiotic microbiota of the host plant Pinus massoniana.

The impact of EB on symbiotic microbiota of P. massoniana seedlings in terms of alpha diversity (a) and KEGG functional abundance of microbes present in differing proportions (b). Samples A, B, C, and D represent the seedlings containing PWN and EB, EB alone, PWN and a controlled chemical, and the controlled chemical alone, respectively, with non-parametric tests (Kruskal-Wallis tests and Wilcoxon rank-sum tests).

Fig 5. The impact of EB on symbiotic microbiota of P. massoniana seedlings in terms of the richness of ROS accumulation-related microbes in different seedlings plotted.

Samples A, B, C, and D represent the seedlings containing both PWN and EB, EB alone, PWN and a controlled chemical, and the controlled chemical alone, respectively, Mb1–Mb5 represent Cladophialophora, Penicillium, Trichoderma, Achromobacter, and Chitinophaga. The symbol “(+)” or “(-)” following the name of a microbe indicates its stimulation or suppression related to ROS accumulation.

Fig 6. Impact of EB (emamectin benzoate) injection on the metabolites of the host plant Pinus massoniana.

The impact of EB on the metabolites of P. massoniana seedlings in terms of the number of metabolites (a), the functions of differentially induced metabolites (b, c) and the richness of ROS accumulation-related metabolites (d) in different seedlings plotted. Samples A, B, C, and D represent those seedlings containing both PWN and EB, EB alone, PWN and a controlled chemical, and the controlled chemical alone, respectively, Mt1–Mt7 represent tyramine, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-octadecadienoyl-sn-glycero-3-phosphocholin, linalool oxide, 3alpha,6alpha-mannotriose, cellobiose, and maltotriose, respectively. The symbol “(+)” or “(-)” following the name of a metabolite indicates its stimulation or suppression related to ROS accumulation.

Fig 7. The impact of EB on the metabolites of P. massoniana seedlings in terms of the number of the richness of ROS accumulation-related metabolites in different seedlings plotted.

Samples A, B, C, and D represent those seedlings containing both PWN and EB, EB alone, PWN and a controlled chemical, and the controlled chemical alone, respectively, Mt1–Mt7 represent tyramine, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-octadecadienoyl-sn-glycero-3-phosphocholin, linalool oxide, 3alpha,6alpha-mannotriose, cellobiose, and maltotriose, respectively. The symbol “(+)” or “(-)” following the name of a metabolite indicates its stimulation or suppression related to ROS accumulation.

Fig 8. EB (emamectin benzoate) affects the diet of JPS (Japanese pine sawyer).

Adults of JPS fed on different seedlings, and the amount of seedling tissue consumed (a) was quantified, along with CBH (Exo-β-1,4-glucanase/cellobiose hydrolase, b), endo-β-1,4-glucanase (c), and β-glucosidase (d) activity of JPS. EB (+) or EB (-) denotes seedlings injected with EB or a controlled chemical, respectively. The symbols * and ** indicate significant differences between samples at P < 0.05 and P < 0.01, respectively, based on a one-way ANOVA, with a multiple comparison analysis using Tukey’s test.

Fig 9. EB (emamectin benzoate) affects the gut microbiota of JPS (Japanese pine sawyer) through its diet.

The gut microbiota of different JPS samples were compared in terms of their diversity (a) and function of differentially present microbes (b). The symbols “T” and “C” denote JPS feeding on seedlings containing EB or a controlled chemical, respectively. Significant differences between samples are based on a one-way ANOVA, with a multiple comparison analysis using Tukey’s test.