Pine Wood Nematode Bursaphelenchus Xylophilus Research Articles

Nematotoxicity of a Cyt-like protein toxin from Conidiobolus obscurus (Entomophthoromycotina) on the pine wood nematode Bursaphelenchus xylophilus.

The pine wood nematode Bursaphelenchus xylophilus is a destructive pest on Pinus trees and lacks effective control measures. The present study identified a novel nematotoxic cytolytic (Cyt)-like protein originating from the entomopathogenic fungus Conidiobolus obscurus. The protein was successfully purified using heterologous expression in Escherichia coli and affinity chromatography. N-hydroxysuccinimide-rhodamine-labeled Cyt-like protein was used to establish the route of toxin uptake, and revealed that the toxin can enter the nematode via the stylet. In bioassays, the purified protein had high nematicide activity against B. xylophilus, with a median lethal concentration at 24 h of 15.8 and 29.4 μg mL-1 for juveniles and adults, respectively. Compared with the deionized water control, fecundity, thrashing, and egg hatching were significantly reduced by 97%, 98%, and 83%, respectively, with 40 μg mL-1 Cyt-like protein at 24-36 h. Staining with Oil-Red-O showed a decrease in large lipid droplet formation in the protein-treated adult nematodes. The Cyt-like protein toxin possesses high nematicide activity against B. xylophilus with effects on nematode vitality and fecundity. The potential exists to use the Cyt-like protein for the control of B. xylophilus.

Effects of Temperature Factors on Resistance against Pine Wood Nematodes in Pinus thunbergii, Based on Multiple Location Sites Nematode Inoculation Tests

Pine wilt disease (PWD) caused by the pinewood nematode (PWN) (Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle) is a worldwide issue. Infection is considered to be promoted mainly by the increased air temperature, but it is important to investigate whether the effect of high temperature similarly influences the different ranks of resistant clone. In the present study, we conducted PWN inoculation tests using six common open-pollinated families of resistant Pinus thunbergii Parl. The tests were conducted at nurseries of five test sites across Japanese archipelago between 2015 and 2017. Our analysis focused specifically on temperature. Firstly, we examined the effects of test sites, inoculation year, and their interaction on unaffected seedling rate and found that the unaffected seedling rate of all tested pine families decreased as the cumulative temperature increased. We found that the unaffected seedling rate decreased as the cumulative temperature increased for all tested pine families. In general, higher cumulative temperatures were required for having an effect on the unaffected seedling rates of higher PWN-resistant families. Typically, early cumulative temperatures, i.e., 19 days after inoculation, had the greatest effect on the unaffected seedling rates of PWN-resistant pines. However, the relationship between cumulative temperature and predicted unaffected seedling rate follow similar rate for all families. Thus, the order of resistance level is maintained in terms of the cumulative temperature required for having an effect.

Adaptation of pine wood nematode Bursaphelenchus xylophilus to \u03b2-pinene stress

BackgroundThe pine wood nematode (PWN; Bursaphelenchus xylophilus) is the most damaging biological pest in pine forest ecosystems in China. However, the pathogenic mechanism remains unclear. Tracheid cavitation induced by excess metabolism of volatile terpenes is a typical characteristic of pine trees infected by B. xylophilus. β-pinene, one of the main volatile terpenes, influences PWN colonization and reproduction, stimulating pathogenicity during the early stages of infection. To elucidate the response mechanism of PWN to β-pinene, pathogenesis, mortality, and reproduction rate were investigated under different concentrations of β-pinene using a transcriptomics approach.ResultsA low concentration of β-pinene (BL, C < 25.74 mg/ml) inhibited PWN reproduction, whereas a high concentration (BH, C > 128.7 mg/ml) promoted reproduction. Comparison of PWN expression profiles under low (BL, 21.66 mg/ml) and high (BH, 214.5 mg/ml) β-pinene concentrations at 48 h identified 659 and 418 differentially expressed genes (DEGs), respectively, compared with controls. Some key DEGs are potential regulators of β-pinene via detoxification metabolism (cytochrome P450, UDP-glucuronosyltransferases and short-chain dehydrogenases), ion channel/transporter activity (unc and ATP-binding cassette families), and nuclear receptor -related genes. Gene Ontology enrichment analysis of DEGs revealed metabolic processes as the most significant biological processes, and catalytic activity as the most significant molecular function for both BL and BH samples. Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO) analysis showed that xenobiotics biodegradation and metabolism, carbohydrate metabolism, lipid metabolism, amino acid metabolism, metabolism of cofactors and vitamins, and transport and catabolism were the dominant terms in metabolism categories.ConclusionIn addition to detoxification via reduction/oxidation (redox) activity, PWN responds to β-pinene through amino acid metabolism, carbohydrate metabolism, and other pathways including growth regulation and epidermal protein changes to overcome β-pinene stress. This study lays a foundation for further exploring the pathogenic mechanism of PWN.

A Diketopiperazine, Cyclo-(L-Pro-L-Ile), Derived From Bacillus thuringiensis JCK-1233 Controls Pine Wilt Disease by Elicitation of Moderate Hypersensitive Reaction.

Pine wilt disease (PWD) caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus is one of the devastating diseases affecting pine forests worldwide. Although effective control measurements are still missing, induction of resistance could represent a possible eco-friendly alternative. In this study, induced resistance-based in vitro and in vivo screening tests were carried out for selection of bacteria with the ability to suppress PWD. Out of 504 isolated bacteria, Bacillus thuringiensis JCK-1233 was selected for its ability to boost pathogenesis-related 1 (PR1) gene expression, a marker of systemic acquired resistance. Moreover, treatment of pine seedlings with B. thuringiensis JCK-1233 resulted in increased expression of other defense-related genes, and significantly inhibited PWD development under greenhouse conditions. However, B. thuringiensis JCK-1233 showed no direct nematicidal activity against B. xylophilus. To identify the effective compound responsible for the induction of resistance in B. thuringiensis JCK-1233, several diketopiperazines (DPKs) including cyclo-(D-Pro-L-Val), cyclo-(L-Pro-L-Ile), cyclo-(L-Pro-L-Phe), and cyclo-(L-Leu-L-Val) were isolated and tested. Foliar treatment of pine seedlings with Cyclo-(L-Pro-L-Ile) resulted in suppression of PWD severity and increased the expression of defense-related genes similarly to B. thuringiensis JCK-1233 treatment. Interestingly, treatment with B. thuringiensis JCK-1233 or cyclo-(L-Pro-L-Ile) showed moderately enhanced expression of PR-1, PR-2, PR-3, PR-4, PR-5, and PR-9 genes following inoculation with PWN compared to that in the untreated control, indicating that they mitigated the burst of hypersensitive reaction in susceptible pine seedlings. In contrast, they significantly increased the expression levels of PR-6 and PR-10 before PWN inoculation. In conclusion, foliar spraying with either B. thuringiensis JCK-1233 culture suspension or DPKs could induce resistance in pine seedlings, thereby alleviating the serious damage by PWD. Taken together, this study supports aerial spraying with eco-friendly biotic or abiotic agents as a valuable strategy that may mark an epoch for the control of PWD in pine forests.

Mating and reproductive characteristics of the pathogenic nematode Bursaphelenchus xylophilus

Pine wood nematode (Bursaphelenchus xylophilus), one of the most destructive invasive species, has caused extremely serious economic, ecological and social losses in many countries throughout the world. Since the high reproductive rate of B. xylophilus PWN is the main cause of rapid death of its pine hosts (Pinus spp.), understanding the reproductive and population biology and the ecology of this nmatode are of great importance. This study mainly focused on analyzing the mating process and population structure under different combinations of sex ratios for mating. Reproductive efficiency of B. xylophilus peaked when the sex ratio (female to male) was 3.4:1. Phases of the mating process for the different sex-ratio combinations indicated that B. xylophilus had evolved alternative reproductive strategies to cope with complex copulating conditions to obtain a suitable population structure for further propagation. This research provides fundamental information on the mechanism that is responsible for the rapid population growth of B. xylophilus.

Inexpensive Screening Method to Validate the Efficacy of Ethanedinitrile Fumigant on the Forest Invasive Nematode Pest Bursaphelenchus xylophilus

At a global scale, the sustainability of forests is endangered by multiple invasive species, including the pine wood nematode (Bursaphelenchus xylophilus), a quarantine pest. International laws and standards require that all exported wood coming from countries in which B. xylophilus is present be chemically or physically treated. Since a major fumigant, methyl bromide, was banned, there has been a need to generate data for alternative fumigants, such as ethanedinitrile (EDN), for this purpose. Since the field screening of fumigants (i.e., the application of various doses to and exposure times of naturally infested wood logs) is prohibitively expensive, the aim of this study was to develop a quick and inexpensive laboratory method. Here, we suggest and describe an innovative method based on sawdust cultures for EDN efficacy screening. In the validation part of this study, we demonstrated (i) the high survival of the nematodes in the sawdust and (ii) the high efficacy of EDN against this pest under in vitro conditions; 100% mortality was observed after 6 h of EDN exposure to a dose of 25 g/m3. In particular, our newly developed model system could be used for the initial screening of various doses of and exposure protocols for EDN and similar types of fumigants developed with the intention of regulating B. xylophilus occurrence in exported wood. It is believed that the validated method may help to develop new and effective EDN fumigation procedures and thereby contribute to the long-term protection of forests worldwide.

Characterization of bacterial communities associated with the pinewood nematode insect vector Monochamus alternatus Hope and the host tree Pinus massoniana

BackgroundMonochamus alternatus Hope is one of the insect vectors of pinewood nematode (Bursaphelenchus xylophilus), which causes the destructive pine wilt disease. The microorganisms within the ecosystem, comprising plants, their environment, and insect vectors, form complex networks. This study presents a systematic analysis of the bacterial microbiota in the M. alternatus midgut and its habitat niche.MethodsTotal DNA was extracted from 20 types of samples (with three replicates each) from M. alternatus and various tissues of healthy and infected P. massoniana (pines). 16S rDNA amplicon sequencing was conducted to determine the composition and diversity of the bacterial microbiota in each sample. Moreover, the relative abundances of bacteria in the midgut of M. alternatus larvae were verified by counting the colony-forming units.ResultsPinewood nematode infection increased the microbial diversity in pines. Bradyrhizobium, Burkholderia, Dyella, Mycobacterium, and Mucilaginibacter were the dominant bacterial genera in the soil and infected pines. These results indicate that the bacterial community in infected pines may be associated with the soil microbiota. Interestingly, the abundance of the genus Gryllotalpicola was highest in the bark of infected pines. The genus Cellulomonas was not found in the midgut of M. alternatus, but it peaked in the phloem of infected pines, followed by the phloem of heathy pines. Moreover, the genus Serratia was not only present in the habitat niche, but it was also enriched in the M. alternatus midgut. The colony-forming unit assays showed that the relative abundance of Serratia sp. peaked in the midgut of instar II larvae (81%).ConclusionsOverall, the results indicate that the bacterial microbiota in the soil and in infected pines are correlated. The Gryllotalpicola sp. and Cellulomonas sp. are potential microbial markers of pine wilt disease. Additionally, Serratia sp. could be an ideal agent for expressing insecticidal protein in the insect midgut by genetic engineering, which represents a new use of microbes to control M. alternatus.

Diversity and Function of Endo-Bacteria in Bursaphelenchus xylophilus from Pinus massoniana Lamb. in Different Regions

The pine wood nematode (PWN) Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease (PWD), a devastating forest disease. PWN-associated bacteria may play a role in PWD. However, little is known about the endo-bacteria in PWN. We analyzed the diversity of endo-bacteria in nine isolates of PWNs from Pinus massoniana Lamb. in nine epidemic areas from three Chinese provinces by high-throughput sequencing of 16S rDNA and isolated and identified culturable endo-bacteria through construction of a 16S rDNA phylogenetic tree and Biolog microbial identification. We also examined the effects of endo-bacteria on PWN fecundity, antioxidant capacity, and virulence using sterile nematodes as a control. While the dominant endo-bacteria in PWNs from different regions exhibited no significant difference in the classification levels of class and genus, their proportions differed. Pseudomonas and Stenotrophomonas were highly abundant in all PWN isolates. A total of 15 endo-bacterial strains were successfully isolated and identified as six species: Stenotrophomonas maltophilia, Pseudomonas fluorescens, Kocuria palustris, Microbacterium testaceum, Rhizobium radiobacter, and Leifsonia aquatica. We also found that P. fluorescens significantly increased the egg production of PWN, and that both P. fluorescens and S. maltophilia enhanced the mobility of PWN under oxidative stress and reduced the content of reactive oxygen species by increasing antioxidant enzyme activity in PWN. These strains also accelerated the development of PWD, and P. fluorescens had a more beneficial effect on PWN than S. maltophilia. Diversity exists among the endo-bacteria in PWNs from different regions, and some endo-bacteria can promote PWN infestation by enhancing the fecundity and antioxidant capacity of the nematode. Our study contributes to clarifying the interaction between endo-bacteria and PWN.

Comparison of methods to obtain and maintain cultures of the pinewood nematode, Bursaphelenchus xylophilus

ABSTRACTThe pinewood nematode Bursaphelenchus xylophilus, native from North America, is the causal agent of the pine wilt disease, being considered one of the most important invasive pests in Eurasian pine forests. The establishment of laboratory isolates of B. xylophilus has been crucial to research on this important pathogen of pine trees. However, studies on the effects of in vitro serial passage on the pathogen evolution, and suitability of standard culture media to the wild populations have been surprisingly few. In the present work, we tested the suitability of different culture media for obtaining B. xylophilus lab populations from dauer larvae that have been isolated from pine sawyer beetles (Monochamus spp.), which serve as their vector. We conducted studies in North America and Portugal. Results showed that adding groundwood (xylem from pine trees) to standard agar medium dramatically improves the survival and growth of wild populations of B. xylophilus in the laboratory. Further, malt extract agar proved to be more suitable than potato dextrose agar. Once in culture, B. xylophilus grew well feeding on either of two fungus species: Botrytis cinerea or Ophiostoma minus. Growth rates were highest on B. cinerea, the most widely used fungus to maintain lab cultures of B. xylophilus, but O. minus has the advantage of being a natural associate and putative diet of wild populations. There remains a need for better understanding of how to obtain and maintain laboratory populations of this pathogen such that their natural characteristics are maintained.

α-l-Fucosidases from Bursaphelenchus xylophilus Secretome—Molecular Characterization and Their Possible Role in Breaking Down Plant Cell Walls

The pinewood nematode (PWN) Bursaphelenchus xylophilus, the causal agent of the pine wilt disease (PWD), enters above-ground parts of the tree, migrates through the resin canals and feeds on plant cells causing extensive damage. In order to penetrate the cell wall and establish a parasitic relationship with host trees, the PWN needs to secretea mixture of active cell wall degrading enzymes. In maritime pine, Pinus pinaster, which is high susceptible to PWN, xyloglucan is the major hemicellulosic polysaccharide in primary cells. The xyloglucan backbone is susceptible to hydrolysis by numerous endoglucanases, some of them specific to xyloglucan. However, to completely degrade xyloglucan, all substitutions on the glucan backbones must be released, and l-fucose residues in xyloglucan branches are released by α-l-fucosidases. In the present study, the molecular characterization of two α-l-fucosidases found in PWN secretome was performed. Moreover, a novel α-l-fucosidase was identified and its cDNA and gene sequence were determined. The three-dimensional structures of these α-l-fucosidases were predicted and the transcript levels were analyzed, thus providing new insights into fundamental PWN biology and the possible role of these proteins as cell wall degrading enzymes.

First detection of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) on Monochamus sutor (Coleoptera: Cerambycidae) in Romania

AbstractAs a result of the detection of the pinewood nematode Bursaphelenchus xylophilus in Portugal, and its subsequent spread to Spain, intense surveys were conducted to screen for the presence of Bursaphelenchus species in Romania. Herein, we report recent surveys of insects potentially vectoring Bursaphelenchus species collected using trap trees or pheromone‐baited traps placed in the forest. Trap felled spruce trees (Picea abies) and pheromone‐baited traps were installed in six different counties in Romania (Brașov, Sibiu, Suceava, Hunedoara, Timiș and Dâmbovița). Ten different species of insects distributed among Curculionidae and Cerambycidae were obtained. Nematodes were extracted from insects and observed to validate the presence of Bursaphelenchus specimens. One female identified as Monochamus sutor was the only specimen carrying nematodes in the genus Bursaphelenchus. Nematodes were identified as B. mucronatus based on morphological and molecular features. This is the first detection and report of natural spread of B. mucronatus in Romania. The absence of B. xylophilus was confirmed in the areas of Romania surveyed in this work.

Effectiveness of clear‐cuttings in non‐fragmented pine forests in relation to EU regulations for the eradication of the pine wood nematode

Abstract The invasive pine wood nematode (PWN) Bursaphelenchus xylophilus is one of the most serious threats to pine forests across the world. Detected in Europe in 1999, it has largely spread despite containment measures. Following the European Union regulations, the requested eradication measure is to fell, remove and dispose of all susceptible plants within a clear‐cut zone (CCZ) of a radius of 500 m around any infected tree. This measure is controversial since its effectiveness is questioned. An individual‐based model, describing the dispersal of the nematode vector and the nematode transmission, was used to estimate the relationship between the radius and the effectiveness of the CCZ at eradicating the PWN. Clear‐cutting of a 500‐m radius is poorly effective in non‐fragmented pine forests since it reduces the number of PWN transmissions by only 0.6%–11.5%. To significantly reduce the number of transmissions, the radius should be between 14 and 38 km, which is not technically nor ethically feasible. Policy implications. Our results, based on model simulations at a fine spatial scale, prove that clear‐cutting susceptible trees 500 m around any infested tree—as requested by EU regulation to eradicate the PWN—is not effective in large and continuous pine forests. Instead, strengthened surveillance and sanitation felling could be explored.

Bioinformatics analysis of structure and function in the MRP gene family and its expression in response to various drugs in Bursaphelenchus xylophilus

Genes homologous to members of the MRP gene family in Caenorhabditis elegans are important in drug resistance. To further explore the molecular mechanism of drug resistance in pine wood nematode (Bursaphelenchus xylophilus), we used bioinformatics approaches to analyze genomic data for B. xylophilus and identified Bx-MRP genes. We predicted the structure and function of the genes and encoded proteins. Using bioinformatics programs to predict and analyze various properties of the predicted proteins, including hydrophobicity, transmembrane regions, phosphorylation sites, and topologically isomeric structures, of these Bx-MRP genes, we determined that they function in transmembrane transport. From the results of RT-qPCR, the Bx-MRP family members confer significant differential resistance to different drug treatments. After treatment with different concentrations of emamectin benzoate, avermectin and matrine, the expression of each gene increased with increasing drug concentrations, indicating that the family members play a positive role in the regulation of multidrug resistance.

Bidens pilosa Extract Effects on Pine Wilt: Causal Agents and Their Natural Enemies

Abstract We investigated the nematicidal and insecticidal activities of the aqueous extract from Bidens pilosa var. radiata on pine-wood nematode (Bursaphelenchus xylophilus) and Japanese pine sawyer (Monochamus alternatus) larvae and adults, the causal agent and vector of pine wilt, respectively. The aqueous extract killed the pine-wood nematode, adults, and larvae of the vector in vitro at all concentrations tested, and the effect decreased significantly with increasing extract dilution. Repellent activity was observed on the nematode and the vector adults as well, although the activity decreased with time in case of the vector. Furthermore, the aqueous extract of B. pilosa effectively promoted approximately 3.0–9.0 mm of hyphal growth in Beauveria spp., when compared with the control treatment. A minor insecticidal effect was also observed on two species of click beetle (Cryptalaus larvatus pini and Paracalais berus), which are natural predators of the Japanese pine-sawyer larvae. Pesticidal and repellent activities of the aqueous extract observed on several organisms related to pine wilt suggest that a multifactorial approach may effectively control this devastating disease.

Influence of temperature on pine wilt disease progression in Pinus thunbergii seedlings

Pine wilt disease (PWD) affects forests globally and is caused by the pinewood nematode (PWN) Bursaphelenchus xylophilus. It has been proposed that PWD development has a strong association with air temperature. Because symptom development is related to the number of PWNs and they are poikilothermic animals whose development and multiplication are affected by temperature; it appears that temperature conditions affect the PWN multiplication rate, which leads to difference in disease progression. However, limited information is available about the temperatures inside pine trees, and the understanding of how temperature affects PWN multiplication and PWD progression remains incomplete. Therefore, in the present study, we evaluated the temperature, number of PWNs, and external symptoms of PWD in Pinus thunbergii seedlings infected with PWNs at air temperatures of 20 °C, 25 °C, and 30 °C over time. We found that the seedling temperatures were slightly higher than the air temperature under each temperature condition and that the effective accumulated temperature calculated using a base temperature of 10 °C was related to the number of PWNs and symptom development in all temperature test groups. However, at 20 °C, there were very few PWNs in some seedlings and none of the seedlings showed partial or full wilting. These findings suggest that PWD progression is greatly affected by ambient temperature and facilitated by increase in PWN populations with effective accumulated temperature especially in the range of 25 °C–30 °C.

Construction of genetic linkage map and identification of a novel major locus for resistance to pine wood nematode in Japanese black pine (Pinus thunbergii)

BackgroundPine wilt disease (PWD), which is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, is currently the greatest threat to pine forests in Europe and East Asian countries including Japan. Constructing a detailed linkage map of DNA markers and identifying PWD resistance genes/loci lead to improved resistance in Pinus thunbergii, as well as other Pinus species that are also susceptible to PWD.ResultsA total F1 mapping population of 188 individuals derived from a cross between the PWD-resistant P. thunbergii varieties ‘Tanabe 54’ (resistant rank 2 to PWD) and ‘Tosashimizu 63’ (resistant rank 4 to PWD) was inoculated with PWN, and was evaluated for disease symptoms. To perform linkage analysis for PWN resistance, a set of three maps was constructed; two parental maps generated using the integrated two-way pseudo-testcross method, and a consensus map with population-type cross-pollination. The linkage map of ‘Tanabe 54’ consisted of 167 loci, and covered 14 linkage groups (LGs), with a total genetic distance of 1214.6 cM. The linkage map of ‘Tosashimizu 63’ consisted of 252 loci, and covered 14 LGs, with a total genetic distance of 1422.1 cM. The integrated consensus map comprised 12 LGs with the basic chromosome number of P. thunbergii, and a total genetic distance of 1403.6 cM. Results from quantitative trait loci (QTL) analysis using phenotype data and linkage maps indicated that PWN resistance is controlled by a single dominant allele, which was derived from the ‘Tanabe 54’ female parent. This major QTL was located on linkage group 3 and was designated PWD1 for PINE WILT DISEASE 1.ConclusionsThe PWD1 locus is a major resistance QTL located on the Pinus consensus LG03 that acts in a dominant manner to confer pine wood nematode resistance. Information from the present study will be useful for P. thunbergii breeding programs to improve resistance to PWD, and also to help identify susceptibility genes in Pinus species.

Thaumatin-Like Protein-1 Gene (Bx-tlp-1) Is Associated with the Pathogenicity of Bursaphelenchus xylophilus.

The pine wood nematode Bursaphelenchus xylophilus is a destructive species affecting pine trees worldwide; however, the underlying mechanism leading to pathogenesis remains unclear. In this study, a B. xylophilus gene encoding thaumatin-like protein-1 (Bx-tlp-1) was silenced by RNA interference to clarify the relationship between the Bx-tlp-1 gene and pathogenicity. The in vitro knockdown of Bx-tlp-1 with double-stranded RNA (dsRNA) decreased B. xylophilus reproduction and pathogenicity. Treatments with dsRNA targeting Bx-tlp-1 decreased expression by 90%, with the silencing effect maintained even in the F3 offspring. Pine trees inoculated with B. xylophilus treated with Bx-tlp-1 dsRNA decreased the symptom of wilting, and the disease severity index was 56.7 at 30 days after inoculation. Additionally, analyses of the cavitation of intact pine stem samples by X-ray microtomography revealed that the xylem cavitation area of pine trees inoculated with B. xylophilus treated with Bx-tlp-1 dsRNA was 0.46 mm2 at 30 days after inoculation. Results from this study indicated that the silencing of Bx-tlp-1 has effects on B. xylophilus fitness. The data presented here provide the foundation for future analyses of Bx-tlp-1 functions related to B. xylophilus pathogenicity.

Relationship between Pine Wilt Disease Outbreaks and Climatic Variables in the Three Gorges Reservoir Region

Outbreaks of pine wilt disease (PWD, caused by the pinewood nematode Bursaphelenchus xylophilus), have caused mass mortality of the genus Pinus in Eurasia. Climate change may greatly influence the distribution and population dynamics of longhorn beetles of the genus Monochamus (the main vector of B. xylophilus), the survival and development of B. xylophilus, and the resistance of pines. The aim of this study was to investigate the effect of climatic variables associated with extensive PWD outbreaks in Masson pine (Pinus massoniana Lamb.) forest across the eastern part of the Three Gorges Reservoir region. Since its discovery in 2006, the most serious PWD outbreak occurred from 2014 to 2018; the most striking characteristic of this outbreak is the consistent increase in Masson pine mortality and extent of the affected areas. Moreover, 28 out of 46 PWD biological relevant climatic variables were selected and used for redundancy analysis. The ordination biplots reflect the complicated quantitative relationship between the PWD epidemic variables and the biologically relevant climatic variables of temperature, precipitation, relative humidity, and wind speed. The results will be useful for understanding the role climatic variables play in PWD outbreaks, for predicting the spread and pattern of PWD outbreaks, and for the advance preparation of management strategies with the purpose of preventing future PWD outbreaks.

Identification of a novel effector BxSapB3 that enhances the virulence of pine wood nematode &amp;lt;italic&amp;gt;Bursaphelenchus xylophilus&amp;lt;/italic&amp;gt;

Pine wilt disease, caused by the pine wood nematode Bursaphelenchus xylophilus, leads to severe damage to pine forests in China. In our previous study, effectors secreted by this pathogen were shown to play roles in the different infection stages of pine wilt disease, and a series of candidate effectors were predicted by transcriptome sequencing. This study identified and characterized a novel effector, BxSapB3, which was among these candidate effectors. Agrobacterium-mediated transient expression was used to identify BxSapB3. BxSapB3 was secreted by B. xylophilus and found to be capable of inducing cell death in Nicotiana benthamiana. Quantitative real-time PCR (qRT-PCR) analysis revealed that BxSapB3 was upregulated in a highly virulent strain of B. xylophilus and expressed at lower levels in a weakly virulent strain at the early stages of infection. When BxSapB3 was silenced in B. xylophilus, the process of infection was delayed. These results indicate that BxSapB3 acts as an effector and contributes to virulence at the early stages of B. xylophilus infection.

Phylogenetic characterization of bacterial endophytes from four Pinus species and their nematicidal activity against the pine wood nematode

Recently, bacterial endophytes (BEs) have gained importance in the agricultural sector for their use as biocontrol agents to manage plant pathogens. Outbreak of the pine wilt disease (PWD) in Korea has led researchers to test the feasibility of BEs in controlling the pine wood nematode (PWN) Bursaphelenchus xylophilus. In this study, we have reported the diversity and biocontrol activity of BEs against the PWN. By employing a culture-dependent approach, 1,622 BEs were isolated from the needle, stem, and root tissues of P. densiflora, P. rigida, P. thunbergii, and P. koraiensis across 18 sampling sites in Korea. We classified 389 members based on 16S rDNA analysis and taxonomic binning, of which, 215 operational taxonomic units (OTUs) were determined. Using Shannon’s indices, diversity across the Pinus species and tissues was estimated to reveal the composition of BEs and their tissue-specific preferences. When their ethyl acetate crude extracts were analysed for biocontrol activity, 44 candidates with nematicidal activity were obtained. Among these, Stenotrophomonas and Bacillus sp. exhibited significant inhibitory activity against PWN during their developmental stages. Altogether, our study furnishes a basic comprehension of bacterial communities found in the Pinus species and highlights the potential of BEs as biocontrol agents to combat PWD.