Associated bacteria of a pine sawyer beetle confer resistance to entomopathogenic fungi via fungal growth inhibition

Abstract

Background: The entomopathogenic Beauveria bassiana is a popular fungus used to control the Japanese pine sawyer, Monochamus alternatus Hope, the key vector of pine wood nematode (Bursaphelenchus xylophilus) that is the causal agent of pine wilt disease, resulting in devastating losses of pines in China and Portugal. However, recent studies have demonstrated that some insect-associated bacteria might decrease fungal toxicity and further undermine its biological control efficacy against M. alternatus. Thus, it is of great significance to uncover whether and how associated bacteria of M. alternatus become involved in the infection process of B. bassiana.

Results: Here, we show that axenic M. alternatus larvae died significantly faster than non-axenic larvae infected by four increasing concentrations of B. bassiana spores (Log-rank test, P < 0.001). The infection of B. bassiana significantly changed the richness and structure of the beetle-associated bacterial community both on the cuticle and in the guts of M. alternatus; meanwhile, the abundance of Pseudomonas and Serratia bacteria were significantly enriched as shown by qPCR. Furthermore, these two bacteria genera showed a strong inhibitory activity against B. bassiana (One-way ANOVA, P < 0.001) by reducing the fungal conidial germination and growth rather than regulating host immunity.

Conclusions: This study highlights the role of insect-associated bacteria in the interaction between pest insects and entomopathogenic fungi, which should be taken into consideration when developing microbial-based pest control strategies.

Keywords: Associated bacteria; Beauveria bassiana; Inhibition; Monochamus alternatus.

Fig. 1

Survival of axenic and nonaxenic Monochamus alternatus larvae (n = 25) following topical infection with different concentration Beauveria bassiana. a 1 × 10⁶ conidia mL⁻¹; b 1 × 10⁷ conidia mL⁻¹; c 1 × 10⁸ conidia mL⁻¹; d 1 × 10⁹ conidia mL⁻¹ (Statistically significant differences between axenic and nonaxenic larvae infected Bb3275 are indicated by the asterisks). The phenotypes of naturally infected M. alternatus larvae, uninfected larvae (e). In the early stage of infection, the larva surface produced black spots (f). The cuticle of the larvae were covered with mycelium (g)

Fig. 2

Principal coordinates analysis (PCoA) plots (a, c) and non-metric multidimensional scaling (NMDS) diagrams of associated bacteria structures (b, d) based on the OTU level used weighted UniFrac analysis. a and b Gut samples; c and d Cuticle samples

Fig. 3

Bacterial community structure of different groups. Taxonomic classification of highly abundant (> 1%) members of Monochamus alternatus larvae in the guts and on the cuticle under different concentrations of Beauveria bassiana. Taxa with a abundance lower (< 1%) were summarized as “other”. a Gut; b Cuticle

Fig. 4

Significance analysis of differences among the top 10 genera in relative abundance from infected larvae and Tween treated larvae. a Gut; b Cuticle. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Number of gut and cuticle microbiota in Monochamus alternatus larvae infected by Beauveria bassiana. qPCR analysis on M. alternatus larvae (gut, n = 5; cuticle, n = 3) using universal 16S rRNA gene primers (a, d), Pseudomonas specific primers (b, e) and Serratia specific primers (c, f). Bars with different letters are significantly different (P < 0.05)

Fig. 6

Germination of Beauveria bassiana was observed at × 400 magnification and in vitro assay of Enterobacter soli (b), Pseudomonas protegens (c) and Serratia marcescens (d) antifungal activity. Coculture bacteria of E. soli (f), P. protegens (g) and S. marcescens (h) and B. bassiana on PDA plates for 3 days. Inhibition was scored as the presence/absence of a distinct zone around inoculation hole. a and e showed the germination and culture of B. bassiana without bacteria. i Germination rate on PDA medium at 28 °C of B. bassiana germ tube under different bacteria coculture conditions and (j) bacteria antifungal units (= [inhibition diameter-5] × 10). Scale bars represent 20 μm (for a–d) and 10 mm (for e–h)

Fig. 7

Differential responses of immunity-related genes in Monochamus alternatus following Beauveria bassiana infection among different treatment groups. Hierarchical clustering analysis of the main immunity related genes in nonaxenic and axenic M. alternatus larvae treated with Tween-80 or B. bassiana. Data represent mean of the Log2 fold change in expression. The size of dot and color shade indicates the Log2FC values. Gene names are shown on the right side