Investigation of Pine Wilt Disease in Chongqing: From Field Occurrence and Genetic Diversity to Endophytic Microbial Composition and Functional Analysis.

Pine wilt disease (PWD) caused by the pine wood nematode (PWN) is China’s most serious forest disease in recent decades. Its complex disease system with unclear pathogenesis and differentiated pathogenicity makes it challenging to manage and control. PWD has spread rapidly in Northeast and Northwest China in recent years, exceeding previous predictions and the traditional understanding of its potential distribution. This paper reviewed the comprehensive risk of PWD in Northeast and Northwest China through the occurrence of influencing factors. Regarding PWN’s temperature resistance, all regions of China are suitable for PWD. In terms of altitude, the PWD system is mainly distributed below 1000 m. In China, Pinus and Larix species are confirmed host trees of PWN, while Monochamus alternatus and M. saltuarius are confirmed vector insects of PWN. In this case, PWD may occur in all low-altitude pine forests in China. Northeast China has a higher risk than Northwest China due to its distribution of suitable host trees and vector beetles at low altitudes. To confront the threat, two scientific bottlenecks are required to be demonstrated. The first is the pathogenicity of Chinese PWN strains to Chinese Picea and Abies species. The other is the geographical distribution and highest altitude distribution of other Monochamus species in the Palaearctic region of China and their PWN transmission ability. We also suggest strengthening plant quarantining in Northeast China. In conclusion, this review comprehensively analyzes the risk of PWD in Northeast and Northwest China in terms of temperature, altitude, host trees, and vector insects. It provides a new perspective and reference for the management and research of PWD in China.

The pinewood nematode (PWN) Bursaphelenchus xylophilus, vectored primarily by the sawyer beetle, Monochamus alternatus, is an important invasive pest and causal agent of pine wilt disease of Chinese Masson pine, Pinus massoniana. Previous work demonstrated that the ratios and concentrations of α-pinene∶β-pinene differed between healthy trees and those trees containing blue-stain fungus (and M. alternatus pupae). However, the potential influence of the altered monoterpene ratios and concentrations on PWN and associated fungi remained unknown. Our current results show that low concentrations of the monoterpenes within petri dishes reduced PWN propagation, whereas the highest concentration of the monoterpenes increased PWN propagation. The propagation rate of PWN treated with the monoterpene ratio representative of blue-stain infected pine (α-pinene∶β-pinene = 1∶0.8, 137.6 mg/ml) was significantly higher than that (α-pinene∶β-pinene = 1∶0.1, 137.6 mg/ml) representative of healthy pines or those damaged by M. alternatus feeding, but without blue stain. Furthermore, inhibition of mycelial growth of associated fungi increased with the concentration of the monoterpenes α-pinene and β-pinene. Additionally, higher levels of β-pinene (α-pinene∶β-pinene = 1∶0.8) resulted in greater inhibition of the growth of the associated fungi Sporothrix sp.2 and Ophiostoma ips strains, but had no significant effects on the growth of Sporothrix sp.1, which is the best food resource for PWN. These results suggest that host monoterpenes generally reduce the reproduction of PWN. However, PWN utilizes high monoterpene concentrations and native blue-stain fungus Sporothrix sp.1 to improve its own propagation and overcome host resistance, which may provide clues to understanding the ecological mechanisms of PWN's successful invasion.

본 연구에서는 소나무재선충병 매개충의 소나무재선충 보유 정도와 산란 및 섭식행동을 통한 전파에 대하여 조사하였다. 첫째, 세 가지 종류의 벌채목으로부터 우화 탈출하는 솔수염하늘소의 소나무재선충 보유 정도를 조사하였다. (가)의 경우는 건전한 소나무를 벌채하여 감염림 내에 방치한 것이고, (나)의 경우는 감염목에서 우화 탈출한 솔수염하늘소를 건전한 벌채목에 산란시켰을 경우이며, (다)의 경우는 솔수염하늘소는 서식하고 있으나 소나무재선충은 보유하지 않은 소나무를 벌채하고 소나무재선충을 인공적으로 접종한 경우이다. 세 경우 모두 이듬해에 벌채목으로부터 우화탈출하는 솔수염하늘소의 소나무재선충 보유율과 보유수를 조사하였다. (가)와 (나)의 경우 소나무재선충 보유율은 각각 18.3%와 15.6%이었고, 보유한 소나무재선충 수는 각각 <TEX>$5,713.1{\pm}9,248.3$</TEX>마리와 <TEX>$2,034.1{\pm}4,746.8$</TEX>마리로서 차이가 없었다. 그러나 인공적으로 소나무재선충을 접종한 (다)의 경우에는 소나무재선충 보유율과 보유수가 각각 38.3%와 <TEX>$20,083.1{\pm}32,188.3$</TEX>마리로서 다른 두 경우에 비해 높은 경향이었다. 한편, 소나무재선충 보유수를 조사한 세 경우 전체 52마리의 솔수염하늘소 중에서 20마리(38.5%)가 5,000마리 이상의 소나무재선충을 보유하고 있었고, 이들 20마리가 보유한 소나무 재선충이 전체 소나무재선충의 97.9%를 차지하였다. 둘째, 야외의 소나무재선충 감염림에서 채취한 소나무로부터 우화한 솔수염하늘소와 북방수염하늘소의 섭식 과정 중에 이들로부터 이탈하는 소나무재선충의 수를 조사하였다. 소나무재선충이 솔수염하늘소와 북방수염하늘소 몸으로부터 이탈한 일수는 각각 <TEX>$34.9{\pm}12.4$</TEX>일과 <TEX>$23.9{\pm}16.2$</TEX>일이었고, 우화 후 2주째에 가장 많이 탈출하였다. 우화 후 2주 이내에 탈출한 소나무재선충의 비율은 솔수염하늘소의 경우는 44.5%, 북방수염하늘소의 경우에는 47.2%이었으며, 매개충 한 마리당 이탈하는 소나무재선충의 수는 각각 <TEX>$3,570.6{\pm}5,189.2$</TEX> 마리와 <TEX>$1,556.2{\pm}1,710.3$</TEX> 마리이었다. This study was performed to investigate the escape of pine wood nematode (PWN), Bursaphelenchus xylophilus, from two vector species (Monochamus alternatus and M. saltuarius) through oviposition and feeding behavior. First, we checked number of PWNs escaped from M. alternatus emerged from three different cases of pine logs. In case A, healthy pine trees were cut into logs and left in pine forest infected with PWN. In case B, healthy pine trees were cut into logs, left in large screen cage, and let them oviposited by M. alternatus emerged from pine trees infested with PWN. In case C, pine trees which were harboring M. alternatus were cut into logs, and PWN was inoculated artificially. The M. alternatus adults emerged from the above three cases of pine logs were checked in the next year to know how many PWN they were harboring in their bodies. The percentages of M. alternatus harboring PWN (18.3 and 15.6%, respectively) and number of nematodes per vector (<TEX>$5,713.1{\pm}9,248.3$</TEX> and <TEX>$2,034.1{\pm}4,746.8$</TEX> PWNs, respectively) in case A and B logs are similar to each other. However, the percentage and the number in case C (38.3% and <TEX>$20,083.1{\pm}32,188.3$</TEX> PWNs) were higher than those of case A and B. Among 52 M. alternatus adults harboring PWN from all the three cases, 20 adults (38.5%) were harboring more than 5,000 PWNs per beetle. And these 20 adults were harboring 97.9% of the total PWNs in 52 adults. Second, we checked the daily escape of PWNs from M. alternatus and M. saltuarius collected at pine forest infested with PWN. The PWN escaped from their vector body for <TEX>$34.9{\pm}12.4$</TEX> days for M. alternatus, and for <TEX>$23.9{\pm}16.2$</TEX> days for M. saltuarius, reaching at peak escape during the 2nd week of emergence of the two vector species. A 44.5 and 47.2% to the total PWNs escaped from vector body within 2 weeks of vector emergence for M. alternatus and M. saltuarius, respectively. The number of PWNs escaped from each vector was <TEX>$3,570.6{\pm}5,189.2$</TEX> and <TEX>$1,556.2{\pm}1,710.3$</TEX> for M. alternatus and M. saltuarius, respectively.

In order to study the key gene in internal causes of pinewood nematode (PWN), Bursaphelenchus xylophilus, a departure from its vector beetle, Monochamus alternatus, we collected PWNs extracted from newly emerged M. alternatus and beetles 7 days after emergence. The total RNAs of the two groups of PWNs were extracted, transcriptomes sequencing was performed, and gene expression differences between the two groups of PWN were analyzed. It was found that the expression of the choline-phosphate cytidylyltransferase gene (pcyt-1) was markedly up-regulated. After inhibition of pcyt-1 expression by RNA interference, the rate of lipid degradation in PWN decreased significantly, and the motility of PWN also decreased significantly. The analysis identified that phosphatidylcholine could promote the emulsification and degradation of neutral lipid granules in PWN, which provides sufficient energy for PWN departure from M. alternatus. The up-regulation of the gene pcyt-1 is an important internal factor for PWN departure from its vector.

I: Pine wilt disease: global issues, surveys, trade and economic impact. Session Summary. I.1. Eradication program for the pinewood nematode in Portugal. I.2. Incursion management in the face of multiple uncertainties: a case study of an unidentified nematode associated with dying pines near Melbourne, Australia. I.3. The risk of pine wilt disease to Australia and New Zealand.I.4. Potential threat and present status of survey of pine wood nematode in Turkey. I.5. Investigations on wood-inhabiting nematodes of the genus Bursaphelenchus in pine forests in the province Brandenburg, Germany. I.6. Official survey for Bursaphelenchus xylophilus carried out on the territory of the Republic of Poland. I.7. Bursaphelenchus spp. in wood packaging intercepted in China. II: Pine wood nematode: biology and microbial inter-relationships. Session Summary. II.1. Developmental biology and cytogenetics of B. xylophilus. II.2. The relationship between PWN and fungi cohabiting in pine trees inoculated with thee PWN. II.3. Influence of fungi on multiplication and distribution of the pinewood nematode. III: PWN taxonomy and detection methods. Session Summary. III.1. Taxonomic databases for Bursaphelenchus and other aphelenchoid nematodes. III.2. The enlargement of the xylophilus group in the genus Bursaphelenchus. III.3. Interspecific variation in ITS rDNA of Bursaphelenchus species of different groups. III.4. Molecular characterization of isolates of the Bursaphelenchus sexdentati group using ITS-RFLP and ribosomal DNA sequences. III.5. Analysis of Bursaphelenchus xylophilus provenances using ISSR and RAPD fingerprints. III.6. Satellite DNA as a versatile genetic marker for Bursaphelenchus xylophilus. III.7. An effective PCR-based diagnostic method for the detection of PWN in wood samples. IV: The insect vectors: biology, ecology and interaction with PWN. Session Summary IV.1. Biological studies relevant to the vector role of Monochamus species for pinewood nematode. IV.2. Potentialinsect vectors of Bursaphelenchus spp. in Spanish pine forests. IV.3. Genetic structure of Monochamus alternatus in Japan. IV.4. Distribution of nematodes (Bursaphelenchus xylophilus) in the beetle of Monochamus alternatus and its exiting transmission way. V: Ecology and modelling. Session Summary. V.1. Modelling PWN-induced wilt expression: a mechanistic approach. V.2. Field diagnosis of the asymptomatic carrier of pinewood nematode. VI: The tree: physiology, resistance and histopathology as a result of pine wilt disease. Session Summary.VI.1. Inoculation of pine trees with avirulent pinewood nematode under experimental conditions: risk-benefit analysis.VI.2. Rapidity of disease development seems to result in high mortality - insight from an inoculation test using hybridized populations between a virulent and an avirulent isolates of Bursaphelenchus xylophilus.VI.3. Defense systems of Pinus densiflora cultivars selected as resistant to pine wilt disease.VI.4. Histological observations of Bursaphelenchus xylophilus in symptomatic tissues of pinewood. VI.5. Development of external and internal symptoms in pine seedlings (Pinus sylvestris) due to inoculation with Bursaphelenchus vallesianus.VII: PWN and insect vector control methods. Session Summary. VII.1. Screening and isolation of antinematodal metabolites against Bursaphelenchus xylophilus produced by fungi and plant. VII.2. Microbial control of Bursaphelenchus xylophilus by fungi. VII.3. Attraction trap for monitoring Monochamus alternatus adults - its usefulness and limitations. VII.4. Studies on Scleroderma guani to control the pine sawyer, Monochamus alternatus. VII.5. Effect of aerial spraying insecticide as a control measure of pine wilt disease. VII. 6. Control program of pine wilt disease for landscape conservation - the case of Amanohashidate in Kyoto, Japan.

Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two nonidentical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to D-(+)-raffinose and N-acetyllactosamine were much higher than that to L-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

In Japan the pine wood nematode (PWN) is transmitted mainly by the Japanese pine sawyer, Monochamus alternatus, from wilt-killed to healthy pine trees (Mamiya and Enda 1972; Morimoto and Iwasaki 1972). The adult beetles of M. alternatus carry a great number of PWNs in their tracheae when they emerge from PWN-killed pines in early summer. Newly emerging adults fly to healthy trees and feed on the bark of young twigs for maturation of their reproductive organs (maturation feeding). At that time, the PWNs on the vector beetles are transmitted to healthy trees and invade them through the feeding wounds made by the beetles. A small number of PWNs disperse widely in the infected trees and causes cessation of oleoresin flow. Thereafter, PWNs propagate dramatically and the trees show wilting symptoms, releasing volatiles such as ethanol, and terpenes (reviewed by Kishi 1995). Mature beetles are attracted to these wilting trees and oviposit in them. The eggs hatch within a week and the larvae feed on the inner bark and outermost sapwood, and then in autumn bore into the sapwood to form pupal chambers (PCs). The number of PWNs reaches its maximum from autumn to winter, and then decreases gradually (Mamiya et al. 1973; Fukushige and Futai 1987). The PC of M. alternatus beetles is one of the most important places for PWNs, because as Mamiya (1972) reported numerous PWNs aggregated around the PCs of M. alternatus in wilt-killed pine trees and that the beetles emerging in the subsequent year harbored many nematodes on their bodies.

Antibacterial peptides from Monochamus alternatus induced oxidative stress and reproductive defects in pine wood nematode through the ERK/MAPK signaling pathway

Pine wilt disease (PWD) is responsible for extensive economic and ecological damage to Pinus spp. forests and plantations worldwide. PWD is caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus) and transmitted into pine trees by a vector insect, the Japanese pine sawyer (JPS, Monochamus alternatus). Host infection by PWN will attract JPS to spawn, which leads to the co-existence of PWN and JPS within the host tree, an essential precondition for PWD outbreaks. Through the action of their metabolites, microbes can manipulate the co-existence of PWN and JPS, but our understanding on how key microorganisms engage in this process remains limited, which severely hinders the exploration and utilization of promising microbial resources in the prevention and control of PWD. In this study we investigated how the PWN-associated fungus Aspergillus promotes the co-existence of PWN and JPS in the host trees (Pinus massoniana) via its secondary metabolite, sterigmatocystin (ST), by taking a multi-omics approach (phenomics, transcriptomics, microbiome, and metabolomics). We found that Aspergillus was able to promote PWN invasion and pathogenicity by increasing ST biosynthesis in the host plant, mainly by suppressing the accumulation of ROS (reactive oxygen species) in plant tissues that could counter PWN. Further, ST accumulation triggered the biosynthesis of VOC (volatile organic compounds) that attracts JPS and drives the coexistence of PWN and JPS in the host plant, thereby encouraging the local transmission of PWD. Meanwhile, we show that application of an Aspergillus inhibitor (chiricanine A treatment) results in the absence of Aspergillus and decreases the in vivo ST amount, thereby sharply restricting the PWN development in host. This further proved that Aspergillus is vital and sufficient for promoting PWD transmission. Altogether, these results document, for the first time, how the function of Aspergillus and its metabolite ST is involved in the entire PWD transmission chain, in addition to providing a novel and long-term effective nematicide for better PWD control in the field.

Study of the departure of pine wood nematode, Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) from Monochamus alternatus (Coleoptera: Cerambycidae)

Soil microbial carbon use efficiency (CUE) represents an important driver of soil organic C formation and turnover. The balance between anabolic and catabolic processes are known to regulate SOC formation through microbial growth and stabilization of microbial residues, and SOC mineralization, respectively. CUE is largely affected by environmental factors but also regulated by the availability of organic substrates and electron acceptors. This is particularly the case in soils that are temporarily subjected to shifts in redox conditions, as those that occur when rice paddy soils are flooded or drained. In such soils, microbial CUE may be affected by electron donor availability (e.g. presence or absence of labile crop residues) as well as electron acceptor availability as O2 becomes limiting and other oxidized species like nitrate and FeIII minerals are reduced. These changes in metabolic activities may also be accompanied by a change in microbial communities which are more adapted to changes in redox conditions and use their resources more efficiently affecting the overall community CUE.The aim of this work is to explore the effects of short-term changes in soil redox conditions (i.e. from aerobic to anaerobic) on the microbial physiology of a rice paddy soil, by unravelling the effects of management-related differences in electron donors and acceptors on microbial growth, respiration and CUE, as well as their dependence on changes in microbial community composition. For this we set up a microcosm experiment where a paddy soil was incubated for 17 d with a factorial combination of (i) redox conditions (oxic vs. anoxic), (ii) with or without rice straw, and (iii) with or without added nitrate. Soils were destructively sampled after 4, and were analysed for DOC, dissolved nitrate and FeII , and microbial biomass C (MBC). Soil aliquots were incubated with D2O for 48 h to measure rates of microbial respiration (CO2 and CH4) and growth by tracing isotope incorporation into phospholipid fatty acid (PLFA) biomarkers, to calculate CUE.&amp;#160;Our preliminary results showed that under anoxic conditions nitrate was rapidly consumed within 4 d while Fe(III) was reduced at a later stage particularly where easily degradable rice straw was added. This could mean that in the first few days the microorganisms are not facing C deficiencies, however, as the anoxic conditions persist the C input enhances microbial activity leading to an increase in Fe(II) in solution. This was also seen in the MBC, as the presence of rice straw enhanced MBC, irrespective of the other treatments (i.e. redox conditions and NO3- addition). Redox-driven changes in community level microbial physiology (growth, respiration and CUE) as well as changes in the active microbial community (PLFA-based) will provide further insights on the role of changing redox conditions and management on key parameters related to soil carbon accrual.

Sediment microorganisms help create and maintain mangrove ecosystems. Although the changes in vegetation during mangrove forest succession have been well studied, the changes in the sediment microbial community during mangrove succession are poorly understood. To investigate the changes in the sediment microbial community during succession of mangroves at Zhanjiang, South China, we used phospholipid fatty acid (PLFA) analysis and the following chronosequence from primary to climax community: unvegetated shoal; Avicennia marina community; Aegiceras corniculatum community; and Bruguiera gymnorrhiza + Rhizophora stylosa community. The PLFA concentrations of all sediment microbial groups (total microorganisms, fungi, gram-positive bacteria, gram-negative bacteria, and actinomycetes) increased significantly with each stage of mangrove succession. Microbial PLFA concentrations in the sediment were significantly lower in the wet season than in the dry season. Regression and ordination analyses indicated that the changes in the microbial community with mangrove succession were mainly associated with properties of the aboveground vegetation (mainly plant height) and the sediment (mainly sediment organic matter and total nitrogen). The changes in the sediment microbial community can probably be explained by increases in nutrients and microhabitat heterogeneity during mangrove succession.

Pine wood nematode (PWN) disease is a globally devastating forest disease caused by infestation with PWN, Bursaphelenchus xylophilus, which mainly occurs through the vector insect Japanese pine sawyer (JPS), Monochamus alternatus. PWN disease is notoriously difficult to manage effectively and is known as the "cancer of pine trees." In this study, dual enzyme-responsive nanopesticides (AVM@EC@Pectin) were prepared using nanocoating avermectin (AVM) after modification with natural polymers. The proposed treatment can respond to the cell wall-degrading enzymes secreted by PWNs and vector insects during pine tree infestation to intelligently release pesticides to cut off the transmission and infestation pathways and realize the integrated control of PWN disease. The LC50 value of AVM@EC@Pectin was 11.19 mg/L for PWN and 26.31 mg/L for JPS. The insecticidal activity of AVM@EC@Pectin was higher than that of the commercial emulsifiable concentrate (AVM-EC), and the photostability, adhesion, and target penetration were improved. The half-life (t1/2) of AVM@EC@Pectin was 133.7 min, which is approximately twice that of AVM-EC (68.2 min). Sprayed and injected applications showed that nanopesticides had superior bidirectional transportation, with five-times higher AVM contents detected in the roots relative to those of AVM-EC when sprayed at the top. The safety experiment showed that the proposed treatment had lower toxicity and higher safety for nontarget organisms in the application environment and human cells. This study presents a green, safe, and effective strategy for the integrated management of PWN disease.

Microbiomes play crucial roles in insect adaptation, especially under stress such as pathogen invasion. Yet, how beneficial microbiomes assemble remains unclear. The wood-boring beetle Monochamus alternatus, a major pest and vector of the pine wilt disease (PWD) nematode, offers a unique model. We conducted controlled experiments using amplicon sequencing (16S rRNA and ITS) within galleries where beetles and microbes interact. PWD significantly altered bacterial and fungal communities, suggesting distinct assembly processes. Deterministic factors like priority effects, host selection, and microbial interactions shaped microbiome composition, distinguishing healthy from PWN-infected galleries. Actinobacteria, Firmicutes, and Ophiostomataceae emerged as potentially beneficial, aiding beetle's development and pathogen resistance. This study unveils how nematode-induced changes in gallery microbiomes influence beetle's development, shedding light on microbiome assembly amid insect-pathogen interactions. Insights gleaned enhance understanding of PWD spread and suggest novel management strategies via microbiome manipulation.IMPORTANCEThis study explores the assembly process of gallery microbiomes associated with a wood-boring beetles, Monochamus alternatus, a vector of the pine wilt disease (PWD). By conducting controlled comparison experiments and employing amplicon approaches, the study reveals significant changes in taxonomic composition and functional adaptation of bacterial and fungal communities induced by PWD. It identifies deterministic processes, including priority effects, host selection, and microbial interactions, as major drivers in microbiome assembly. Additionally, the study highlights the presence of potentially beneficial microbes such as Actinobacteria, Firmicutes, and Ophiostomataceae, which could enhance beetle development and resistance to pathogens. These findings shed light on the intricate interplay among insects, microbiomes, and pathogens, contributing to a deeper understanding of PWD prevalence and suggesting innovative management strategies through microbiome manipulation.

Phyllosphere microbial communities have an important role in plant growth and resistance to pathogen infection and are partially influenced by leaf characteristics. Pinewood nematode, Bursaphelenchus xylophilus, is one of the greatest threats to pine trees and is spreading all over the world. However, studies on the resistance of plant–microbe interactions to pathogens during the nematode’s pathogenesis and the relationships of leaf chemical characteristics caused by pinewood nematode and phyllosphere microbial communities are limited. In this study, different stages of Pinus tabuliformis that were healthy or infected with B. xylophilus-associated leaf characteristics and phyllosphere bacterial and fungal communities were compared. These results demonstrated that soluble sugar and starch contents decreased based on the extent of infection. Phyllosphere microbial community changes potentially caused by B. xylophilus infection of P. tabuliformis and the fungal community compositions of healthy P. tabuliformis trees (Ya) were clearly different from diseased P. tabuliformis trees at an early stage of B. xylophilus infection (Yb) and P. tabuliformis trees in the last stage of B. xylophilus infection (Yc), particularly along the first coordinate axis. According to a linear discriminant effect size (LEfSe) analysis, the biomarker species in the phyllosphere of Yb were Acidobacteria, Deinococcus-Thermus, and Patescibacteria, while those in the phyllosphere of Ya were Proteobacteria, Aureobasidium, Dictyosporium, Alternariaster, Knufia, Microstroma, and Naganishia. Particularly at the end of PWD (pine wilt disease) infection, the majority of microbial taxa tended to co-exclude rather than co-occur with PWD infection. The result of a canonical correlation analysis (CCA) showed that the chemical properties of leaves, such as carbon and nitrogen, have significant impacts on phyllosphere microbial communities. These results expanded the possible connections between the phyllosphere communities and plant health.