We provided an in-depth description of the microbial communities in the gut of the M. alternatus and its living environment. Using 16S rDNA gene amplicon sequencing, we explored the 60 different samples from M. alternatus, P. massoniana and soil. We examined the microbial diversity and investigated possible correlations between the M. alternatus, P. massoniana and its peripheral environment.
The number of OTUs showed that the microbial content in the root, rhizosphere soil, surface soil and peripheral environment of healthy and infected P. massoniana was abundant. This is consistent with Müller’s study, which found that the vertical microbial content of plants originated from the soil, so the soil microbial content is rich. Furthermore, many studies have suggested that rhizosphere soil is the main source of endophytic bacteria in plants [35, 36]. The gut and excretion of different instars of M. alternatus, showed a positive correlation between the microbial content of each instar larvae and their corresponding feeding plant tissues. Because pupae did not feed, there were fewer microbial species identified. The microbial species in infected wood tissues were higher in number than those in healthy wood tissues from the samples involved in the feeding process of II-III instars larvae and adults. This can be explained because P. massoniana is attacked by pine wood nematode and M. alternatus, resulting in a large number of wounds, followed by a large number of bacteria invading the plant through the wounds. The substances leaking from wounds provide nutrients for bacteria, thus promoting the colonization of a large number of endophytic bacteria [37]. Moreover, the intestinal excretion of larvae had a similar microorganism content than the corresponding infected wood tissues. Therefore, it can be inferred that food affects the intestinal microbial composition of M. alternatus as shown in previous studies that the effects of food on the intestinal microorganisms of insects are complex. Broderck et al. found that there are significant differences in the midgut microorganisms of Lymantria dispar larvae fed with different diets [38]. The intestinal microbial composition of lower and higher wood-eating termites is also affected by food. When the composition of food changes, the dominant bacteria changes accordingly [39].
Different organisms have different kinds of endophytic bacteria. Proteobacteria is the dominant bacteria in the gut and excretion of infected P. massoniana and its peripheral environment. Proteobacteria is classified into Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, and Gammaproteobacteria according to the diversity of morphology, physiology and metabolism. It is of great significance to the cycling of carbon, nitrogen and sulfur in living organisms [40]. Some studies have used 16S rRNA genes to isolate and identify microorganisms in the soil, and found that the dominant microflora is Proteobacteria [41, 42]. Besides, for insects, Proteobacteria is the resident bacteria in the gut of most insects [29, 43, 44]. Proteobacteria is the dominant intestinal bacteria of most insects, such as Helicoverpa armigera (Lepidoptera), Bombyx mori (Lepidoptera), locust (Orthoptera), and some species of termites (Isoptera) [38, 43, 45]. Previous studies have shown that the dominant bacteria of M. galloprovincialis and M. alternatus is Proteobacteria (mainly Gammaproteobacteria) [17, 44], and it has been found that most of the associated bacteria of pine wood nematode is Gammaproteobacteria [46].
Acidobacteria is one of the most dominant and abundant phyla in the soil [47]. In this study, Acidobacteria was the dominant bacteria in surface soil and rhizosphere soil, in which Gp1, Gp2, and Gp3 accounted for more than 50% of the total microbial abundance. Acidobacteria belongs to Acidophilus and studies have shown that Acidobacteria plays an important role in the ecosystem, and has a rich diversity of metabolic and genetic functions [48], as well as a great contribution to ecological stability [49]. Acidobacteria is the dominant bacteria in most soils because of its low pH value [50]. The soil in P. massoniana planting area is acidic. Different subgroups of Acidobacteria have different optimum pH values. For example, the subgroup Gp1 of Acidobacteria grows best in the soil environment with pH 4-5.5 [51, 52]. In addition, Cuie Shi et al. studied soil microbial diversity in P. massoniana forests which were infected and healthy by pine wilt disease. It was found that the infection of pine wood nematode causes changes in soil physical and chemical properties, bacterial community composition and diversity [18]. The important role of these microorganisms in the mechanism of pine wood nematode infection of P. massoniana needs further study.
Due to the infection with pine wood nematode, the dominant bacteria of infected and healthy P. massoniana changed significantly. The results indicated that feeding on P. massoniana could affect the distribution of these bacteria in different tissues and can be metabolized by M. alternatus larvae. According to the distribution of each sample, the transmission routes of these bacteria can be inferred as follows: Bradyrhizobium, Burkholderia, Dyella, Mycobacterium and Mucilaginibacter spread from the soil (healthy P. massoniana) to various tissues of infected P. massoniana. Rhizobium, Saccharibacteria, Terriglobus and Nocardioides spread from phloem of healthy P. massoniana to various tissues of infected P. massoniana. Granulicella and Sphingomonas spread from the bark of healthy P. massoniana to various tissues of infected P. massoniana. These microorganisms are related to plant growth. As plant growth-promoting rhizobacteria (PGPR), Rhizobium and Bradyrhizobium can colonize and survive in plant rhizosphere [53, 54], produce phytohormones and iron spores to promote plant growth and enhance the dissolution of inorganic phosphate [55]. Burkholderia occupies a rich niche in nature and has a wide range of functions, including controlling biological growth as a pathogen and promoting plant growth as PGPR [56, 57]. The phylum Candidate Saccharibacteria (former candidate division TM7) has been frequently detected in natural environments, the human oral cavities and activated sludge, moreover, the phylum was assigned the Saccharibacteria because of their sugar metabolisms [58-60]. Pseudoxanthomonas degrades complex organic compounds, so it is also used in environmental governance [61, 62]. Studies have shown that Cellulose-degrading bacteria of Dyella genus have been isolated from the gut of Cerambycidae insects [63]. Mycobacterium has been found in the gut of necrophagous beetles, suggesting that the bacteria can be transmitted through excretion by the digestive system of insects [64]. Some researchers had hypothesized that Nocardioides can degrade refractory organic compounds [65]. In this study, Gryllotalpicola was identified only in the tissues of infected P. massoniana, including bark, phloem, xylem and root, while Cellulomonas was present only in the phloem, and its content in infected wood was higher than that in healthy wood. These two strains can degrade cellulose [63]. The distribution of them in infected and healthy P. massoniana can be used as indicator of whether P. massoniana is infected with the pine wood nematode.
In a vital dynamic environment, the insect’s gut is associated with feeding, digestion, excretion and other important activities, which are related to the enteric microorganisms [66-69]. Enteric microorganisms are essential to insect growth and development, especially phytophagous insects [66, 70]. Insect intestinal microorganisms play an important role in nitrogen fixation, lignocellulose degradation, amino acid biosynthesis and uric acid degradation [70]. Therefore, the study of microorganisms in insect gut is of great significance to clarify the interaction between insects and plants. Many studies have been conducted on the diversity of enteric microorganisms in insects, such as Bombyx mori, Plutella xylostella, Helicoverpa armigera, Holotrichia parallela, etc [45, 71]. With the development of intestinal microbiology, it has been found that enteric microorganisms can be modified by genetic engineering to enrich and express insecticidal genes in the insect gut, which provides a feasible method for the use of microbes to prevent and control M. alternatus [72, 73]. The prevention and control of plant diseases and pests by pathogenic bacteria has become a popular research topic and new pathogenic bacteria have been isolated and found continuously, enriching the possibilities of biological control of plant diseases and pests [74-77]. The vector of pine wilt disease and its intestinal microorganisms have a very important influence on host selection and colonization. It can be seen from the results that in the gut and excretion of M. alternatus, the dominant microflora was Serratia and Stenotrophomonas in genus level. Serratia was not the dominant species in soil and plant tissues, but its content increased significantly in the gut of II instar larvae, and there was higher abundance of Serratia in the gut of different insect states after II instar, which proved that Serratia was likely to accumulate in the gut of II instar larvae through feeding pathway. A large number of Serratia were also isolated from indoor cultured pine wood nematode, infected P. massoniana and Monochamus galloprovincialis [46, 78]. Moreover, it has been proved that S. marcescens PWN146 can colonize on host plants [79]. S. marcescens has multiple roles after colonizing on plants. It can change from beneficial bacteria promoting plant growth to plant pathogenic bacteria under environmental stimulation [60]. Current studies have found that Serratia can secrete cellulase and other extracellular enzymes. This indicates that Serratia is ubiquitous in the gut of wood-eating insects like M. alternatus, and is closely related to the cellulose degradation function of the host. Serratia also has strong stability for rapid adaptation to the environment [80, 81]. If the role of Serratia in the mechanism of pine wilt disease transmission by M. alternatus can be clarified, a new method for the control of pine wilt disease can be provided.