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Characterization of bacterial communities associated with the pine wilt disease insect vectors Monochamus alternatus Hope and host trees Pinus massoniana CURRENT

Background Pine wilt disease (PWD) is a destructive disease caused by the pinewood nematode Bursaphelenchus xylophilus . Monochamus alternatus Hope is the main vector of this disease. The symbiotic microorganisms can play an important role in the transmission cycle mechanism. However, the role of bacterial microorganisms in the transmission of pine wood nematode by M. alternatus is not clear currently. The main purpose of this study is to reveal the composition and diversity of microbial flora in the gut of M. alternatus , as well as healthy and infected Pinus massoniana and its peripheral environment to discover the important microbial flora contributing to the transmission cycle. Methods In this study, total DNA was extracted from 60 samples, including 20 samples of M. alternatus gut from different larval instars, healthy P. massoniana , nematode-infected P. massoniana and their peripheral environment (needles, bark, phloem, xylem, root, surface soil and rhizosphere soil), by triplicate. Samples were used for 16S rDNA Amplicon sequencing to determine the composition and diversity of microbial flora in each sample. Results Infection of pinewood nematode resulted in an increase of the microbial community in the nematode-infected P. massoniana and its peripheral environment when compared with healthy P. massoniana , the microbial community in different tissues changed. Among them, Gryllotalpicola and Cellulomonas showed to be endemic microorganisms in nematode-infected P. massoniana , which can be used as indicators to detect the disease. Serratia was shown as an opportunistic pathogen, and was found to be enriched in M. alternatus gut and was also detected in the host plant tissues. Conclusions This study clarified the change of microbial community in the transmission of pine wilt disease by M. alternatus . An important theoretical basis for the prevention of pine wilt disease was structured by our research. plant P. massoniana and its peripheral environment. We analyzed the bacterial diversity in different M. alternatus larval instars and adults, as well as in the P. massoniana forest and peripheral soil, from nematode-infected and healthy sites. The samples (total 60 samples) collected from different tissues of nematode-infected and healthy P. massoniana and different larval instars of M. alternatus were sequenced and the dominant microflora of each sample and their occurrence were analyzed by 16S rDNA Amplicon. This study clarified the change of microbial community in the transmission of pine wilt disease by M. alternatus .

specific OTUs of infected pine xylems was 1328, which was far more than 237 of healthy pine xylems ( Fig. 1C). There were 84 mutual OTUs of the process of adult feeding on bark. The number of OTUs shared by adult gut and infected pine barks was 137. The number of specific OTUs in infected pine barks was about 2.5 times that in healthy pine barks (Fig. 1D). The results showed that the microbial species in infected wood tissues were higher in number than those in healthy wood tissues in the three stages of feeding by different larva instars especially III instar larvae feeding on xylem. This may be related to the decrease of self-protection ability of infected wood, which makes it is susceptible to various microorganisms from outside.

LEfSe (linear discriminant analysis effect size) analysis
The LEfSe analysis further identified the specific microbiota at the phylum, class, order, family and genus levels that were present or abundant in all samples (Fig. 2). The most significant differences were Proteobacteria in samples from M. alternatus and Chloroflexi in samples from healthy P.
massoniana. While Bacteroidetes, Armatimonadetes, Actinobacteria, Acidobacteria and Proteobacteria were mainly enriched in samples from infected P. massoniana. It was clear that the main bacterial species in samples from infected P. massoniana were highly similar in Proteobacteria and Acidobacteria to samples from M. alternatus and healthy P. massoniana respectively. It is possible that the ecological niche of various microbiota in P. massoniana may be changed due to PWN spread by M. alternatus. The microbiota in the gut of M. alternatus maybe also infected P. massoniana by feeding and even played an important role in P. massoniana. alternatus, the dominant bacteria were Proteobacteria (Additional file 1: Figure S3). Species distribution at the genus level indicated that in the peripheral environment of infected P. massoniana, Sphingomonas is the most abundant bacterial flora representing 7.66% of the abundance.

Microbial Community Composition in vectors
Burkholderia accounted for 6.51%, which is equal to Gp1. Among the microorganisms in the gut and excretion of M. alternatus, at the genus level, Serratia accounted for 25.25%, Enterobacter 12.42%, Halotalea 8.81% and Stenotrophomonas 6.68% respectively. The total proportion of Gp1, Gp2, and Gp3 in surface soil and rhizosphere soil exceeded 50% of their total microbial abundance respectively, with no differences between the peripheral environment of infected P. massoniana or healthy P. massoniana (Fig. 3). (Additional file 1: Figure S4, S5) The analysis of life history and coupling mechanism of M. alternatus show that the dominant bacteria in the excretion of II instars larvae was Saccharibacteria after feeding on the phloem of P. massoniana (Additional file 1: Figure S6). Burkholderia was the dominant bacteria in the excretion of III instars larvae after feeding on xylem (Additional file 1: Figure S7). Sphingomonas and Granulicella were the dominant bacteria in the process of adults feeding on the bark of P. massoniana (Additional file 1: Figure S8). It is not surprising to find that the bark, phloem, and xylem of infected P. massoniana contained more pathogenic bacteria than the corresponding tissues of healthy P. massoniana, and the pathogenic bacteria were mainly distributed between the Saccharibacteria, Burkholderia and Granulicella genus. Furthermore, it was found that the intestinal excretion of larvae of all instars has a similar bacterial content than the tissues of infected wood. Therefore, food might affect the intestinal microbial composition of M. alternatus. However, it can also be mentioned that there were significant differences in species composition between infected P. massoniana and healthy P. massoniana, and that the microbial composition of M. alternatus excretions was similar to infected P. massoniana Furthermore, all the samples in the three groups were classified by species. From the classification larvae (12.57%), followed by Burkholderia (11.68%). The abundance of Pseudoxanthomonas (5.31%) in the excretion of third instar larvae was higher than in the excretion of second instar larvae and the gut of different instar larvae (average 0.03%) (Fig. 5A). This may be related to the accumulation of multiple wounds, tree weakness and the invasion of pathogenic bacteria when third instar larvae feed on xylem.
Interestingly, Serratia was highly enriched in the insect samples, but less abundant in healthy or To confirm the relative concentration of Serratia in the gut of M. alternatus larvae from different larval instars, the intestinal enzyme solution of M. alternatus from reared indoors and wild selected separately were analyzed by flat colony counting. Despite the insects being collected in the field or reared indoors, the content of Serratia in the gut of second instar larvae were the highest, while the remaining instars had the lowest amount, and remained relatively stable in I and IV instars. However, the distribution of Serratia in the gut of V instarlarvae from indoor rearing was higher than the one from the field sampling, this suggests that the food has an effect on the distribution of Serratia in the gut of M. alternatus larvae, but its distribution pattern and whether it is related to the larval metabolic mechanism still needs further research ( Fig. 5C and D).
Also, the network map at the genus level showed that the abundance of Serratia was positively correlated with Lactococcus, Variovorax, Acinetobacter, Stenotrophomonas, and Achromobacter, and negatively correlated with Inquilinus and Burkholderia. Stenotrophomonas and Achromobacter had the same abundance trend as Serratia in the gut and excretion of M. alternatus (Additional file 1: Figure S9). Besides, Raoultella, which has ornithine-lysing activity in the samples, was abundant, especially in the gut of insects and sample grouping after II instar. It may be beneficial for M. alternatus to feed on crude fibrous tissues such as xylem and bark and promote related digestion and metabolism.

Discussion
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 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][67][68][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][75][76][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.

Conclusions
In this study, we analyzed the microbial diversity from each segment of the whole cycle infection mechanism and the dominant and endemic microflora from each part. Proteobacteria is the main microorganisms in the gut and peripheral environment of M. alternatus. Within the infection of pine wood nematode, the microbial community of infected P. massoniana was significantly higher than healthy P. massoniana. Rhizobium, Bradyrhizobium, Terriglobus, Granulicella, Sphingomonas, Dyella,

Burkholderia, Saccharibacteria, Pseudoxanthomonas, Mucilaginibacter, Mycobacterium, and
Nocardioides were dominant bacteria of infected P. massoniana. We also found that Gryllotalpicola and Cellulomonas mainly existed in infected P. massoniana but there were a little in healthy P.
Flat colony counting results showed that the content of Serratia in the gut of II instar larvae was the highest (80%), while that of III instar larvae was the lowest. The results indicated that Serratia could not only colonize in the intestinal tract but also enrich in the intestinal tract of M. alternatus. However, the role of these microorganisms in the whole cycle mechanism still needs to be further studied.
Clarifying the interaction among host, vector and microorganisms in the environment, we can provide a new strategy for the control of pine wilt disease.       massoniana. The bacteria labeled orange was identified only in the phloem and its content in infected wood was higher than that in healthy wood.

Supplementary Files
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