Nematode Bursaphelenchus Xylophilus Research Articles

Population Density and Host Preference of the Japanese Pine Sawyer (Monochamus alternatus) in the Qinling-Daba Mountains of China.

Monochamus alternatus is a serious trunk-boring pest and is the most important and effective vector of the pine wood nematode Bursaphelenchus xylophilus, which causes pine wilt disease. The pine wilt disease poses a serious threat to forest vegetation and ecological security in the Qinling-Daba Mountains and their surrounding areas. In order to clarify whether the population density of M. alternatus larvae is related to the host preference of M. alternatus adults, we investigated the population density of M. alternatus overwintering larvae and explored the host preference of M. alternatus adults on Pinus tabuliformis, P. armandii, and P. massoniana. The results show that the population density of M. alternatus larvae was significantly higher on P. armandii than those on P. massoniana and P. tabuliformis. The development of M. alternatus larvae was continuous according to the measurements of the head capsule width and the pronotum width. Adults of M. alternatus preferred to oviposit on P. armandii rather than on P. massoniana and P. tabuliformis. Our results indicate that the difference in the population density of M. alternatus larvae between different host plants was due to the oviposition preference of M. alternatus adults. In addition, the instars of M. alternatus larvae could not be accurately determined, because Dyar's law is not suitable for continuously developing individuals. This study could provide theoretical basis for the comprehensive prevention and control of the pine wilt disease in this region and adjacent areas.

Teflon Coating and Anti-Escape Ring Improve Trapping Efficiency of the Longhorn Beetle, Monochamus alternatus

The longhorn beetle Monochamus alternatus is an important conifer stem-boring pest and the main vector in Asia of the invasive pine wood nematode Bursaphelenchus xylophilus, which causes a devastating pine disease. Field experiments were carried out to evaluate the effect of coating black panel traps with different concentrations of an anti-adhesive material, Teflon, on Monochamus captures. Tests were also conducted to look at the possible decrease in the Teflon effect with exposure duration as well as the effect of the implementation of an anti-escape ring on the trap. The three tested Teflon concentrations significantly improved the captures of M. alternatus with regard to those with uncoated traps. The beetle captures increased by 108.3% in traps coated with a 4x Teflon dilution, but no significant differences in captures were observed when the traps were coated with either a Teflon stock solution or a 8x dilution. The trapping efficiency gradually decreased with the duration of exposure of the coated traps. No significant difference was observed after one year of exposure, but the trapping efficiency significantly decreased by 36.6% after 2 years and by 72.0% after 3 years. Two-year old, coated traps equipped with an anti-escape ring were nearly three times more efficient than those without an anti-escape ring, but the captures were still lower than those obtained with newly coated traps. The present study provides valuable information for the future development of environmentally friendly prevention methods. Attractants combined with Teflon coating and an anti-escape ring could significantly improve the trapping efficiency of M. alternatus adults, which may allow for the reduction of the population density of M. alternatus and transmission probability of pine wilt disease.

Different responses of Monochamus galloprovincialis and three non-target species to trap type, colour, and lubricant treatment

With the increasing threat to forests in Europe from the invasive pine wood nematode (PWN) Bursaphelenchus xylophilus, effective methods are needed to monitor and reduce populations of its insect vector, the pine sawyer beetle Monochamus galloprovincialis. In the present study, we tested the effectiveness of different trap types (multiple-funnel, cross-vane, and triangular), colours (black, white and clear), and lubricant (polytetrafluoroethylene, PTFE) treatments (different PTFE formulations and timing of trap treatment) on the catches of M. galloprovincialis and three most commonly captured non-target beetle species (the xylophagous Spondylis buprestoides and two predators, Thanasimus formicarius and T. femoralis) in Poland. Of the traps not treated with PTFE, the white and black 6-funnel traps were most effective in trapping M. galloprovincialis beetles, while the catches in the cross-vane traps (both white and clear) were low. Trap treatment with PTFE significantly increased trap effectiveness, regardless of PTFE type and time of application. The catches of S. buprestoides were affected by trap type, while those of T. formicarius depended on trap colour and size. Both species seem to respond positively to ethanol and/or α-pinene in the lure composition. PTFE treatment had a significant effect on the catches of T. femoralis. In conclusion, for the monitoring of M. galloprovincialis, we recommend the white cross-vane traps treated with dry PTFE. They are less but still effective in catching the target species, while their use, together with lures containing no ethanol and α-pinene, greatly reduces the catches of non-target insects S. buprestoides and T. formicarius.

Discovery of entomopathogenic fungi across geographical regions in southern China on pine sawyer beetle Monochamus alternatus and implication for multi-pathogen vectoring potential of this beetle.

Entomopathogen-based biocontrol is crucial for blocking the transmission of vector-borne diseases; however, few cross-latitudinal investigations of entomopathogens have been reported for vectors transmitting woody plant diseases in forest ecosystems. The pine sawyer beetle Monochamus alternatus is an important wood borer and a major vector transmitting pine wilt disease, facilitating invasion of the pinewood nematode Bursaphelenchus xylophilus (PWN) in China. Due to the limited geographical breadth of sampling regions, species diversity of fungal associates (especially entomopathogenic fungi) on M. alternatus adults and their potential ecological functions have been markedly underestimated. In this study, through traditional fungal isolation with morphological and molecular identification, 640 fungal strains (affiliated with 15 genera and 39 species) were isolated from 81 beetle cadavers covered by mycelia or those symptomatically alive across five regional populations of this pest in southern China. Multivariate analyses revealed significant differences in the fungal community composition among geographical populations of M. alternatus, presenting regionalized characteristics, whereas no significant differences were found in fungal composition between beetle genders or among body positions. Four region-representative fungi, namely, Lecanicillium attenuatum (Zhejiang), Aspergillus austwickii (Sichuan), Scopulariopsis alboflavescens (Fujian), and A. ruber (Guangxi), as well as the three fungal species Beauveria bassiana, Penicillium citrinum, and Trichoderma dorotheae, showed significantly stronger entomopathogenic activities than other fungi. Additionally, insect-parasitic entomopathogenic fungi (A. austwickii, B. bassiana, L. attenuatum, and S. alboflavescens) exhibited less to no obvious phytopathogenic activities on the host pine Pinus massoniana, whereas P. citrinum, Purpureocillium lilacinum, and certain species of Fusarium spp.-isolated from M. alternatus body surfaces-exhibited remarkably higher phytopathogenicity. Our results provide a broader view of the entomopathogenic fungal community on the vector beetle M. alternatus, some of which are reported for the first time on Monochamus spp. in China. Moreover, this beetle might be more highly-risk in pine forests than previously considered, as a potential multi-pathogen vector of both PWN and phytopathogenic fungi.

Bursaphelenchus xylophilus detection and analysis system based on CRISPR - Cas12.

Pine wilt disease is caused by the pine wood nematode (Bursaphelenchus xylophilus) and leads to wilting and death of pines. It is one of the most damaging diseases of pines worldwide. Therefore, accurate and rapid detection methods are of great importance for the control of B. xylophilus. Traditional detection methods have some problems, such as being time-consuming and requiring expensive instruments. In this study, the loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) were used to establish a set of intelligent detection and analysis system for B. xylophilus, called LAMP-CRISPR/Cas12a analysis, which integrated field sampling, rapid detection and intelligent control analysis. The process can be completed within 1 hour, from sample pretreatment and detection to data analysis. Compared with the single LAMP method, the LAMP-CRISPR/Cas12a assay uses species-specific fluorescence cleavage to detect target amplicons. This process confirms the amplicon identity, thereby avoiding false-positive results from non-specific amplicons, and the large amounts of irrelevant background DNA do not interfere with the reaction. The LAMP-CRISPR/Cas12a assay was applied to 46 pine wood samples and the samples carrying B. xylophilus nematodes were successfully identified. To meet the needs of different environments, we designed three methods to interpret the data: 1) naked eye interpretation; 2) lateral flow biosensor assay; and 3) integrated molecular analysis system to standardize and intellectualize the detection process. Application of the B. xylophilus detection and analysis system will reduce the professional and technical requirements for the operating environment and operators and help to ensure the accuracy of the detection results, which is important in grass-root B. xylophilus detection institutions.

Comprehensive analysis of LRR-RLKs and key gene identification in Pinus massoniana resistant to pine wood nematode.

Pinus massoniana is a pioneer tree widely planted for afforestation on barren hills in southern China where the total planted area is 8.04 million ha. The invasive pine wood nematode (Bursaphelenchus xylophilus) poses a serious threat to the survival of P. massoniana. Plant resistance genes encoded by leucine-rich repeat-containing transmembrane-receptor proteins play important roles in plant defense. Leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest subfamily of the RLK protein family, play an important role in sensing stress signals in plants. However, the LRR-RLKs of P. massoniana have not been characterized previously, and their role in resistance to B. xylophilus is unknown. In this study, 185 members of the LRR-RLK subfamily were identified in P. massoniana and were categorized into 14 subgroups. Transcriptomic and quantitative real-time RT-PCR analyses showed that PmRLKs32 was highly expressed in the stem tissue after inoculation with B. xylophilus. The gene exhibited high homology with AtFLS2 of Arabidopsis thaliana. PmRLKs32 was localized to the plasma membrane and was significantly upregulated in nematode-resistant and nematode-susceptible individuals. The transient expression of PmRLKs32 resulted in a burst of reactive oxygen species production in P. massoniana and Nicotiana benthamiana seedlings. These results lay a foundation for further exploration of the regulatory mechanism of LRR-RLKs in response to biotic stress in P. massoniana.

In silico analysis of koranimine, a cyclic imine compound from Peribacillus frigoritolerans reveals potential nematicidal activity

Pine wilt disease (PWD) is a destructive vector-borne forest disease caused by the nematode Bursaphelenchus xylophilus. To date, several options are available for the management of pine wilt disease; however constant development and search for natural products with potential nematicidal activity are imperative to diversify management options and to cope with the possible future emergence of resistance in parasitic nematodes. Here, a combined metabolomics and genomics approach was employed to investigate the chemical repertoire and biosynthetic potential of the bacterial endophyte Peribacillus frigoritolerans BE93, previously characterized to exhibit nematicidal activity against B. xylophilus. Feature-based molecular networking revealed the presence of diverse secondary metabolites. A cyclic imine heptapeptide, koranimine, was found to be among the most abundant secondary metabolites produced. Genome mining displayed the presence of several putative biosynthetic gene clusters (BGCs), including a dedicated non-ribosomal peptide synthase (NRPS) BGC for koranimine. Given the non-ribosomal peptide nature of koranimine, in silico molecular docking analysis was conducted to investigate its potential nematicidal activity against the target receptor ivermectin-sensitive invertebrate α glutamate-gated chloride channel (GluCl). Results revealed the binding of koranimine at the allosteric site of the channel—the ivermectin binding site. Moreover, the ligand-receptor interactions observed were mostly shared between koranimine and ivermectin when bound to the α GluCl receptor thus, suggesting a possibly shared mechanism of potential nematicidal activity. This study highlights the efficiency of combined metabolomics and genomics approach in the identification of candidate compounds.

Screening, isolation and mechanism of a nematicidal extract from actinomycetes against the pine wood nematode Bursaphelenchus xylophilus

Actinomycetes are an important microbial resource closely related to human production and life. Approximately 70% of the antibiotics currently used are produced by various actinomycetes. Pine wilt disease (PWD) is caused by the pine wood nematode (PWN) (Bursaphelenchus xylophilus), which is a devastating forest pest that can cause the wilting and death of pine trees. The purpose of this work was to screen out, identify, and ferment active actinomycetes with high lethality to pine wood nematodes and study the active compounds in their secondary metabolites. A group of 227 original strains/isolates of actinomycetes was screened, and Streptomyces sp. TCS19-048 was selected for further analyses. The taxonomic characteristics and molecular analysis results indicated that the strain was a member of the genus Streptomyces and exhibited the highest similarity (99.77%) with the 16S rRNA gene sequences of Streptomyces lactacystinicus OM-6519. Furthermore, the active extract was isolated and had an inhibitory effect on the movement behaviour of B. xylophilus and was able to significantly increase the body fluid leakage of B. xylophilus. This study focused on isolating secondary metabolites from various soil actinomycetes to control PWD and identified an extract with nematicidal activity from a selected strain. The nematicidal effect of extract S-3-1 was confirmed, and the strain Streptomyces sp. TCS19-048 showed good inhibitory activity against B. xylophilus, which was reported for the first time at home and abroad.

Associated bacteria of a pine sawyer beetle confer resistance to entomopathogenic fungi via fungal growth inhibition

BackgroundThe entomopathogenic Beauveria bassiana is a popular fungus used to control the Japanese pine sawyer, Monochamus alternatus Hope, the key vector of pine wood nematode (Bursaphelenchus xylophilus) that is the causal agent of pine wilt disease, resulting in devastating losses of pines in China and Portugal. However, recent studies have demonstrated that some insect-associated bacteria might decrease fungal toxicity and further undermine its biological control efficacy against M. alternatus. Thus, it is of great significance to uncover whether and how associated bacteria of M. alternatus become involved in the infection process of B. bassiana.ResultsHere, we show that axenic M. alternatus larvae died significantly faster than non-axenic larvae infected by four increasing concentrations of B. bassiana spores (Log-rank test, P < 0.001). The infection of B. bassiana significantly changed the richness and structure of the beetle-associated bacterial community both on the cuticle and in the guts of M. alternatus; meanwhile, the abundance of Pseudomonas and Serratia bacteria were significantly enriched as shown by qPCR. Furthermore, these two bacteria genera showed a strong inhibitory activity against B. bassiana (One-way ANOVA, P < 0.001) by reducing the fungal conidial germination and growth rather than regulating host immunity.ConclusionsThis study highlights the role of insect-associated bacteria in the interaction between pest insects and entomopathogenic fungi, which should be taken into consideration when developing microbial-based pest control strategies.

Mathematical Modeling and Review of Pine Wilt Disease

Pine Wilt Disease (PWD), caused by the pinewood nematode (PWN) Bursaphelenchus xylophilus, causes significant losses in coniferous forests in eastern Asia, including Japan, China, and South Korea, as well as western Europe, including Portugal. The results of the research papers given at the International Symposium on Pine Wilt Disease (IUFRO Working Party Meeting 4.04.03) in Nanjing, China, in July 2009 are summarized in this article. The basic themes discussed included pine wilt disease (PWD), the pinewood nematode (PWN) Bursaphelenchus xylophilus, and other PWN-associated microorganisms that play a significant role in PWD, such as bacteria (e.g., Pseudomonas fluorescens). The majority of the papers are based on PWD-PWN research in East Asia and Russia. The following are some of the specific topics covered: 1) fundamental concepts of PWD development, 2) pathogenicity, 3) host-parasite relationships, including histopathology of diseased conifers and the role of toxins from bacteria-nematode ecto-symbionts, 4) PWN life cycle and transmission, 5) B. xylophilus dissemination models, 6) associations (with other nematodes), 7) diagnostics, 8) quarantine and control of the PWN and 9) biocontrol of the PWN.

Nematicidal activity of (Z)-ligustilide isolated from Angelica tenuissima Nakai root extract against the pine wood nematode Bursaphelenchus xylophilus

In this study, we investigated the nematicidal activities of the ethanol extracts of 49 medicinal plants against the pine wood nematode, Bursaphelenchus xylophilus, and isolated a main nematicidal constituent, (Z)-ligustilide, from Angelica tenuissima Nakai root extract. Among the 49 plant extracts, only the A. tenuissima root extract showed the strong nematicidal activity against the pine wood nematode, with a 92.3% mortality rate at a concentration of 2 mg/mL. Based on bioassay-guided isolation and gas chromatography-mass spectrometry (GC-MS) analysis, (Z)-ligustilide was identified as the active component of A. tenuissima root extract at 73.6% of the total content ratio. The LC50 value of (Z)-ligustilide against the pine wood nematode was 0.24 mg/mL. Our results indicated that (Z)-ligustilide as well as A. tenuissima root extract can be potential candidates for novel trunk injection agents against the pine wood nematode.

Analysis and numerical simulation of fractional order pine wilt disease model

Human survival is purely based on the environment. Through natural calamities or man-made depredation, the pleasant existence is disturbed. Trees provide not just foliage for the environment but also a pleasant atmosphere for the human population. One of the most serious threats to the forest and ecology is the pine wilt disease (PWD). It is a devastating disease that kills pine trees in a matter of weeks. The pine-wood nematode Bursaphelenchus Xylophilus causes PWD. This study emphasises on the infectious model of PWD engaging the [Formula: see text]-homotopy analysis transform method ([Formula: see text]-HATM) to a leading fractional operator Caputo–Fabrizio fractional time derivative to arrive for superior understanding. The denouements are presented in the forms of plots and tables. At last the paper supports to examine the real-world models and helps to forecast their behavior that communicate the instructions considered in the models.

The key molecular pattern BxCDP1 of Bursaphelenchus xylophilus induces plant immunity and enhances plant defense response via two small peptide regions.

The migratory plant-parasitic nematode Bursaphelenchus xylophilus is the pathogen of the pine wilt disease (PWD), causing serious damage to pine forests in China. During the process of plant resistance to multiple pathogens, plant immunity plays a key role. In this current study, the pathogen-associated molecular pattern (PAMP) BxCDP1 in B. xylophilus has been identified, but the host target protein of BxCDP1 and its key amino acid region inducing the plant immunity have yet to be elucidated. We found that BxCDP1 could trigger superoxide production, H2O2 production, and callose deposits. A RING-H2 finger protein 1 (RHF1) of Pinus thunbergii was screened and characterized as a target protein of BxCDP1 by yeast two-hybrid and co-immunoprecipitation (Co-IP). Moreover, two peptides (namely M9 and M16) proved to be key regions of BxCDP1 to induce PAMP-triggered immunity (PTI) in Nicotiana benthamiana, which also induced the expression of pathogenesis-related (PR) genes (PtPR-3, PtPR-4, and PtPR-5) in P. thunbergii and enhanced the resistance of the host to B. xylophilus. These results indicate that BxCDP1 plays a critical role in the interaction between B. xylophilus and P. thunbergii, and both peptides M9 and M16 have the potential to be developed and utilized as immune inducers of pines against B. xylophilus in future.

Cold Tolerance in Pinewood Nematode Bursaphelenchus xylophilus Promoted Multiple Invasion Events in Mid-Temperate Zone of China

Pinewood nematode (Bursaphelenchus xylophilus) is a highly destructive invasive species, causing extensive economic and ecological losses across Eurasia. It has recently invaded mid-temperate zone of northern China, threating large areas of coniferous forests. Herein, we evaluated the physiological and molecular basis of cold tolerance in pinewood nematode isolates from different temperature zones of China. After exposure to −5 °C and −10 °C, the survival rates of five pinewood nematode isolates from different temperature zones were 93.94%–94.77% and 43.26%–45.58% after 8 h, and 93.04%–94.85% and 9.93%–10.56% after 24 h, without significant differences among isolates. In a comparison of an isolate from a mid-temperate zone and an isolate from a subtropical zone under gradient cooling, the survival rates remained steady at nearly 95% when minimum temperature ranged from −5 °C to −15 °C, with no significant difference between isolates. In addition, phylogenetic and population structure analyses based on whole genome resequencing data suggested that isolates from mid-temperate and warm temperate zones are clustered with different isolates from subtropical zone, with no obvious geographic pattern. We did not detect significant variation in cold tolerance ability and selected gene among pinewood nematode isolates from different temperature zones. The recently invaded pinewood nematodes in the mid-temperate zone of northern China may spread by multiple invasion events from southern China, without adaptive revolution. Our research implies that it is important to reinforce quarantine inspection to control the rapid spread of pinewood nematode.

A GA and SVM Classification Model for Pine Wilt Disease Detection Using UAV-Based Hyperspectral Imagery

Pine wilt disease (PWD), caused by the pine wood nematode (Bursaphelenchus xylophilus), is a global destructive threat to forests and has led to serious economic losses all over the world. Therefore, it is necessary to establish a feasible and effective method to accurately monitor and estimate PWD infection. In this study, we used hyperspectral imagery (HI) collected by an unmanned airship with a hyperspectral imaging spectrometer to detect PWD in healthy, early, middle and serious infection stages. To avoid massive calculations on the full spectral dimensions of the HI, 16 spectral features were extracted from the HI, and a genetic algorithm (GA) was implemented to identify the optimal ones with the least fitness. Simultaneously, a support vector machine (SVM) classifier was established to predict the PWD infection stage for an individual pine tree. The following results were obtained: (1) the spectral characteristics for pine trees in different PWD infection stages were distinctive in the green region (510–580 nm), red edge (680–760 nm) and near-infrared (780–1000 nm) spectra; (2) the six optimal spectral features (Dgreen, SDgreen, Dred, DRE, DNIR, SDNIR) selected with the GA effectively distinguished the PWD infection stages of pine trees with a lower calculation cost; (3) compared with the traditional classifiers, such as k-nearest neighbor (KNN), random forest (RF) and single SVM, the proposed GA and SVM classifier achieved the highest overall accuracy (95.24%) and Kappa coefficient (0.9234). The approach could also be employed for monitoring and detecting other forest pests.

Drivers of Forest Pathogen Invasions

In the past 25 to 30 years, a marked increase has occurred in numbers of invasive alien pests and pathogens recorded, damaging agriculture, horticulture and forest environments. The reasons for this upsurge are clear: all involve human-driven factors, with, arguably, global trade as the most important component in the complex. Climate change is another major factor. Each plant transported through trade could carry cryptic pests or pathogens, which may be introduced into previously naïve environments and lead to major health issues on previously unaffected plants. Global trade in plants is a complex system, driven by a desire to use large plants in landscapes and demand for varied species for gardens. Examples of invasive alien pathogens causing wide-spread problems on a global scale currently include many Phytophthora species, Dothistroma needle blight of pines, and Xylella fastidiosa on a wide range of trees. A striking example recently was the spread of the Dutch elm disease pathogen, Ophiostoma novo-ulmi, in North America and Western Europe. Many invasive pathogens cause problems in localized regions, including Ceratocystis platani, killing Platanus in some European countries. Other examples include the threat of pine wilt nematode (Bursaphelenchus xylophilus), damage to Cupressus in Mediterranean landscapes caused by Seiridium cardinale, dieback of sweet chestnut (Castanea) caused by the canker pathogen Cryphonectria parasitica, and many others. In addition to global trade, climate change appears to be exacerbating the problems, with major influences on establishment and spread of alien invasive species. Hope lies in the enormous genetic ‘power’ of plants: humans could establish selection and breeding programmes that will ultimately enable us to plant trees with greater tolerance of changing climate and pests and pathogens, whether native or invasive.

Selenium nanoparticles are effective in penetrating pine and causing high oxidative damage to Bursaphelenchus xylophilus in pine wilt disease control.

Research on selenium nanoparticles (SeNPs) in chemical defense and chemotherapy of plants has developed rapidly owing to their high microbial toxicity, environmental safety, and degradability. Pine wilt disease (PWD) threatens pine forests worldwide; however, it is difficult to kill the nematodes (Bursaphelenchus xylophilus) inside the tree that cause PWD using traditional pesticide formulations. SeNPs could be the key to controlling PWD. In this study, approximately 50 nm SeNPs were prepared using a simple and green method, and chitosan was used to increase their biocompatibility and stability. The preparation and characterization results showed that the prepared SeNPs coated with chitosan (SeNPs@CS) were spherical and evenly dispersed. The bioassay results showed that SeNPs@CS had an LC50 of 15.627 mg L-1 against B. xylophilus. In addition, the killing mechanism of SeNPs@CS against B. xylophilus was studied. Confocal microscopy and transmission electron microscopy demonstrated that B. xylophilus were killed by reactive oxygen species, and the penetration of nano-form materials to B. xylophilus was higher than that of non-nano-form materials. To verify the effective penetration of SeNPs in pine tissues, Cy5-labeled SeNPs@CS was observed inside pine needles and branches using frozen sections and confocal microscopy. In addition, the cytotoxicity of SeO2 and SeNPs@CS was tested, and the results showed that the cytotoxicity of SeNPs@CS to MC3T3-E1 cells was reduced. These results show that SeNPs are expected to be used as a new strategy for the control of PWD with oxidative damage and high penetration to B. xylophilus and effective target penetration and biosafety. © 2022 Society of Chemical Industry.

UGT440A1 Is Associated With Motility, Reproduction, and Pathogenicity of the Plant-Parasitic Nematode Bursaphelenchus xylophilus.

Pine wilt disease (PWD) caused by Bursaphelenchus xylophilus is considered a major threat to pine forests worldwide. Uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyze the conjugation of small lipophilic compounds with sugars and play crucial roles in the detoxification and homeostatic processes in all living organisms. We investigated the molecular characteristics and biological functions of the gene UGT440A1 that encodes UGTs in B. xylophilus. The in situ hybridization results indicated that UGT440A1 is expressed in all developmental stages of B. xylophilus, particularly in the head, intestine, and hypodermis of the second-stage of juveniles (J2), third-stage of juveniles (J3) and fourth-stage of juveniles (J4) females and in almost the whole body of J4 males and adults. Recombinant UGT440A1 was observed mainly in the inclusion bodies, and the enzyme activity assay revealed that UGT440A1 could catalyze the glycosylation reaction of two types of flavonols (kaempferol and quercetin). RNA interference (RNAi) of UGT440A1 suppressed motility, feeding, and reproduction of B. xylophilus. Furthermore, UGT440A1 knockdown caused a delay in the development of PWD symptoms in the pine seedlings inoculated with the nematodes. These results suggest that UGT440A1 is involved in the pathogenic process of B. xylophilus and the information may facilitate a better understanding of the molecular mechanism of PWD.

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

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.

Genes Encoding Potential Molecular Mimicry Proteins as the Specific Targets for Detecting Bursaphelenchus xylophilus in PCR and Loop-Mediated Isothermal Amplification Assays.

The introduction of the pine wood nematode (Bursaphelenchus xylophilus) to new areas has affected the international forestry industry because this pathogen causes pine wilt disease (PWD). Therefore, methods for the accurate and reliable detection of B. xylophilus are essential for controlling and managing this pest. The PCR and Loop-Mediated Isothermal Amplification (LAMP) techniques developed in this study involve species-specific primer sets targeting B. xylophilus genes encoding potential molecular mimicry proteins (Bx-tlp-1, Bx-tlp-2, and Bx-cpi), which are associated with pathogenicity. The PCR and LAMP results revealed that the primers were specific for B. xylophilus Bx-tlp-1, Bx-tlp-2, and Bx-cpi. Moreover, our LAMP assay targeting Bx-tlp-1 conducted at 63°C detected B. xylophilus within 20 min and B. xylophilus from Monochamus alternatus or M. saltuarius within 30 min. The lower limits of detection for the LAMP and PCR assays were 10 pg and 10 ng genomic DNA, respectively, implying these assays may be useful for the rapid detection of B. xylophilus in pine forests. Designing primers specific for Bx-tlp-1, Bx-tlp-2, and Bx-cpi enabled the relatively rapid detection of B. xylophilus isolates as well as M. alternatus or M. saltuarius carrying B. xylophilus. These primers, which were designed following a thorough functional analysis of key B. xylophilus pathogenicity-related genes, may be useful for developing improved assays for the early diagnosis and prevention of PWD.