Mechanisms of Insecticidal Action of Metarhizium anisopliae on Adult Japanese Pine Sawyer Beetles (Monochamus alternatus)

Abstract

Pine wilt disease, caused by Bursaphelenchus xylophilus (pine wood nematode), leads to severe environmental and economic damage. Here, we report the results of experiments on the biological control of pine wilt disease through termination of the insect vector of the nematode and the mechanism of the insecticidal action of Metarhizium anisopliae JEF-279 against Monochamus alternatus (Japanese pine sawyer). A combined treatment with a fungal conidia suspension and a fungal protease-containing culture filtrate caused 75.8% mortality of the insect vector. Additionally, the presence of destruxins was confirmed in the dead Japanese pine sawyer adults, and half of the 10 protein spots in proteomic analysis were identified as an actin related to muscle contraction. Based on proteomic and microscopic analyses, the infection cycle of the Japanese pine sawyer by M. anisopliae JEF-279 was inferred to proceed in the following sequence: (1) host adhesion and germination, (2) epicuticle degradation, (3) growth as blastospore, (4) killing by various fungal toxins (insecticidal metabolites), (5) immune response as defense mechanism, and (6) hyphal extrusion and conidiation. Consequently, the combined fungal conidia suspension and protease-containing culture filtrate treatment may be applied as an insecticidal agent, and flaccid paralysis is likely a major mechanism underlying the insecticidal action of M. anisopliae JEF-279 on host insects.

Figure 1

(a) Insecticidal effects of fungal conidia suspension, protease-containing culture filtrate, and combined fungal conidia suspension and a protease-containing culture filtrate treatment with Metarhizium anisopliae JEF-279 on Japanese pine sawyer beetles. (b, c) Dorsal and (d) ventral views of nontreated JPS, (e, f) dorsal and (g) ventral views of FCS-treated JPS, and (h, i) dorsal and (j) ventral views of CFP-treated JPS. JPS, Japanese pine sawyer; FCS, fungal conidia suspension; and CFP, combined fungal conidia suspension and protease-containing culture filtrate. A total of 50 JPS adults were tested per treatment.

Figure 2

Analysis of two-dimensional gel electrophoresis of extracted protein from (a) nontreated JPS, (b) FCS-treated JPS, and (c) CFP-treated JPS adults. JPS, Japanese pine sawyer; FCS, fungal conidia suspension; and CFP, combined fungal conidia suspension and protease-containing culture filtrate. Spot 1, tropomyosin-2 isoform X12; spot 2, fructose-bisphosphate aldolase; spot 3, actin; spot 4, annexin-B9; spot 5, endocuticle structural glycoprotein SgAbd-2-like; spot 6, mitochondrial-processing peptidase subunit β; spot 7, actin; spot 8, actin; spot 9, actin; spot 10, actin; spot 11, actin; spot 12, ATP synthase subunit β; spot 13, probable isocitrate dehydrogenase; spot 14, heat shock protein 70; and spot 15, heat shock 70 protein.

Figure 3

Transmission electron microscopy images of the abdominal sternum of Japanese pine sawyer adults after the combined fungal conidia suspension and protease-containing culture filtrate treatment: (a) control, (b) 5 days, (c) 8 days, and (d) 13 days.