Invasion History of the Pinewood Nematode Bursaphelenchus xylophilus Influences the Abundance of Serratia sp. in Pupal Chambers and Tracheae of Insect-Vector Monochamus alternatus

Abstract

Pine wilt disease (PWD) has caused extensive mortality in pine forests worldwide. This disease is a result of a multi-species interaction among an invasive pinewood nematode (PWN) Bursaphelenchus xylophilus, its vector Monochamus sp. beetle, and the host pine tree (Pinus sp.). In other systems, microbes have been shown to attenuate negative impacts on invasive species after the invasion has reached a certain time point. Despite that the role of PWD associated microbes involved in the PWD system has been widely studied, it is not known whether similar antagonistic "hidden microbial players" exist in this system due to the lack of knowledge about the potential temporal changes in the composition of associated microbiota. In this study, we investigated the bacteria-to-fungi ratio and isolated culturable bacterial isolates from pupal chambers and vector beetle tracheae across five sampling sites in China differing in the duration of PWN invasion. We also tested the pathogenicity of two candidate bacteria strains against the PWN-vector beetle complex. A total of 118 bacterial species belonging to 4 phyla, 30 families, and 54 genera were classified based on 16S sequencing. The relative abundance of the genus Serratia was lower in pupal chambers and tracheae in newly PWN invaded sites (<10 years) compared to the sites that had been invaded for more than 20 years. Serratia marcescens strain AHPC29 was widely distributed across all sites and showed nematicidal activity against PWN. The insecticidal activity of this strain was dependent on the life stage of the vector beetle Monochamus alternatus: no insecticidal activity was observed against final-instar larvae, whereas S. marcescens was highly virulent against pupae. Our findings improved the understanding of the temporal variation in the microbial community associated with the PWN-vector beetle complex and the progress of PWD and can therefore facilitate the development of biological control agents against PWN and its vector beetle.

Keywords: Monochamus alternatus; Serratia marcescens; bacteria isolation; invasion time; pathogenicity; pinewood nematode.

Figure 1

Comparison of pinewood nematode (PWN) fecundity collected from sites differing in the duration of PWN invasion. Fecundity of (A) wild population of PWN cultured in laboratory conditions for 7 days and (B) after the same populations were cultured in laboratory conditions for 30 days. A one-way ANOVA with Tukey's multiple comparison test, p < 0.05. Data are presented as means ± SEM. Different letters indicate significant differences. The numbers in the x-axis indicate the duration of PWN invasion in years (1–5: 1–5 years of invasion).

Figure 2

The relative bacterial and fungal abundance of pupal chambers and tracheae in sites differing in the duration of PWN invasion. The bacteria-to-fungi ratio of pupal chambers (A) and tracheae (B) samples in sites differing in the duration of PWN invasion (years of invasion in the X-axis). The total bacteria content of pupal chamber (C) and tracheae (D) samples from different sites differed in the duration of PWN Sinvasion. The abundance of bacterial and fungal biomass was estimated by RT-qPCR. Different letters indicate statistically significant differences from the one-way ANOVA with Tukey's multiple comparison test, p < 0.05. Data are presented as means ± SEM.

Figure 3

Phylogenetic tree showing the placement of 118 bacterial isolates with reference strains based on 16S rDNA sequence. The phylogenetic tree was produced by ML methods with 1,000 bootstrap replicates. Bootstrap values are represented by circles at each node. The letters in green: strains isolated from pupal chambers; letters in orange: strains isolated from tracheae. The type of strain indicated with “T”. LNPC67: bacteria strain isolated from pupal chambers in Liaoning province. AHTR10: bacteria strain isolated from tracheae in Anhui province (also see Supplementary Table 1).

Figure 4

The relative abundance of genus Serratia involved in pupal chambers (A) and tracheae (B) from sites differing in the duration of PWN invasion (years of invasion in the X-axis). Data are presented as means ± SEM, Kruskal-Wallis H-test, p < 0.05. Different letters indicate statistically significant differences.

Figure 5

Pathogenicity of Serratia marcescens strain to PWN and its vector beetle. (A) The mortality assay of PWN with two Serratia strains AHPC29 and ZJPC33. AHPC29: Serratia marcescens strain AHPC29; ZJC33: Serratia nematodiphila strain ZJPC33. Nematicidal activity of bacterial solution monitored after 24 h. Two-tailed paired t-test, ^***p < 0.001. (B,C) Meier survival curves of vector beetle last-instar larvae and pupae exposed to AHPC29 and ZJPC33. (D) Meier survival curves of vector beetle adults exposed to AHPC29. Survivorship was monitored daily for 10 days. The log-rank test was used for the comparison of survival curves.