Invasion History of the Pinewood Nematode Influences the Abundance of Sp. in Pupal Chambers and Tracheae of Insect-Vector

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) , its vector sp. beetle, and the host pine tree ( 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 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. 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 : no insecticidal activity was observed against final-instar larvae, whereas 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.