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Background: Global climate change is causing an increase in extreme high temperatures (EHTs), which subject insects to unprecedented stress. Behavior plasticity in response to EHTs, particularly oviposition behavior, is important for the persistence and outbreak of insect populations. Investigating the plasticity of oviposition behavior and its underlying mechanisms has theoretical importance to pest management, but knowledge gaps still remain. Results: Herein, we characterized the reproductive traits of Monochamus alternatus, a dominant insect vector of the destructive pine wilt disease, including oviposition behavioral patterns, fecundity, offspring fitness and sperm viability, under simulated heatwave conditions in the laboratory. The results showed that (i) EHTs induced a novel oviposition behavior, whereby females deposited multiple eggs into a single groove rather than laying one egg per groove under normal condition; (ii) EHTs exerted stage‐ and sex‐specific effects on fecundity, offspring fitness and sperm viability; and (iii) there was a significant correlation between frequency of the novel oviposition strategy and sperm viability. Conclusion: We hypothesized that this beetle pest has the ability to flexibly shift towards a low‐cost oviposition strategy to counteract the fitness costs caused by heat stress. Taken together, these findings provide a theoretical foundation for personalized pest management strategies in the context of climate change. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

In this study, we focused on evaluating the impact of Pseudomonas abietaniphila BHJ04 on the growth of Pinus massoniana seedlings and its biocontrol efficacy against pine wilt disease (PWD). Additionally, the colonization dynamics of P. abietaniphila BHJ04 on P. massoniana were examined. The growth promotion experiment showed that P. abietaniphila BHJ04 significantly promoted the growth of the branches and roots of P. massoniana. Pot control experiments indicated that strain BHJ04 significantly inhibited the spread of PWD. There were significant changes in the expression of several genes related to pine wood nematode defense in P. massoniana, including chitinase, nicotinamide synthetase, and triangular tetrapeptide-like superfamily protein isoform 9. Furthermore, our results revealed significant upregulation of genes associated with the water stress response (dehydration-responsive proteins), genetic material replication (DNA/RNA polymerase superfamily proteins), cell wall hydrolase, and detoxification (cytochrome P450 and cytochrome P450 monooxygenase superfamily genes) in the self-regulation of P. massoniana. Colonization experiments demonstrated that strain BHJ04 can colonize the roots, shoots, and leaves of P. massoniana, and the colonization amount on the leaves was the greatest, reaching 160,000 on the 15th day. However, colonization of the stems lasted longer, with the highest level of colonization observed after 45 d. This study provides a preliminary exploration of the growth-promoting and disease-preventing mechanisms of P. abietaniphila BHJ04 and its ability to colonize pines, thus providing a new biocontrol microbial resource for the biological control of plant diseases. [ABSTRACT FROM AUTHOR]

Pinus thunbergii Parl. is an economically and medicinally important plant, as well as a world-renowned horticultural species of the Pinus genus. Pine wilt disease is a dangerous condition that affects P. thunbergii. However, understanding of the genetics underlying resistance to this disease is poor. Our findings reveal that P. thunbergii's resistance mechanism is based on differential transcriptome responses generated by the early presence of the pathogen Bursaphelenchus xylophilus, also known as the pine wood nematode. A transcriptome analysis (RNA-seq) was performed to examine gene expression in shoot tissues from resistant and susceptible P. thunbergii trees. RNA samples were collected from the shoots of inoculated pines throughout the infection phases by the virulent Bursaphelenchus xylophilus AMA3 strain. The photosynthesis and plant–pathogen interaction pathways were significantly enriched in the first and third days after infection. Flavonoid biosynthesis was induced in response to late infestation (7 and 14 days post-infestation). Calmodulin, RBOH, HLC protein, RPS, PR1, and genes implicated in phytohormone crosstalk (e.g., SGT1, MYC2, PP2C, and ERF1) showed significant alterations between resistant and susceptible trees. Furthermore, salicylic acid was found to aid pine wood nematodes tolerate adverse conditions and boost reproduction, which may be significant for pine wood nematode colonization within pines. These findings provide new insights into how host defenses overcame pine wood nematode infection in the early stage, which could potentially contribute to the development of novel strategies for the control of pine wilt disease. [ABSTRACT FROM AUTHOR]

Pine wilt disease (PWD) is a devastating disease that threatens pine forests worldwide, and breeding resistant pines is an important management strategy used to reduce its impact. A batch of resistant seeds of P. thunbergii was introduced from Japan. Based on the resistant materials, we obtained somatic plants through somatic embryogenesis. In this study, we performed transcriptome analysis to further understand the defense response of resistant somatic plants of P. thunbergii to PWD. The results showed that, after pine wood nematode (PWN) infection, resistant P. thunbergii stimulated more differential expression genes (DEGs) and involved more regulatory pathways than did susceptible P. thunbergii. For the first time, the alpha-linolenic acid metabolism and linoleic acid metabolism were intensively observed in pines resisting PWN infection. The related genes disease resistance protein RPS2 (SUMM2) and pathogenesis-related genes (PR1), as well as reactive oxygen species (ROS)-related genes were significantly up-expressed in order to contribute to protection against PWN inoculation in P. thunbergii. In addition, the diterpenoid biosynthesis pathway was significantly enriched only in resistant P. thunbergii. These findings provided valuable genetic information for future breeding of resistant conifers, and could contribute to the development of new diagnostic tools for early screening of resistant pine seedlings based on specific PWN-tolerance-related markers. [ABSTRACT FROM AUTHOR]

Introduction: Pine wilt disease (Bursaphelenchus xylophilus) was recently detected in Liaoning Province, which was previously considered an unfavourable area for B. xylophilus due to its lowtemperatures. This studyaims tocomparethe reproductivity and genetic variations of B. xylophilus isolates from Liaoning Province and other parts of China to explore their phenotypic and genomic differences. Methods: The samples from Liaoning, Anhui, Hubei, Henan, Zhejiang and Jiangsu were isolated and purified to obtain the strains. The reproductivity of the strains was determined at 15 °C. The genetic structure was analyzed by using SNP molecular markers, and the whole genome association analysis was carried out by integrating SNP information and feculence traits. Results: A reproductivity experiment showed that Liaoning isolates have higher reproductive ability at 15 °C. Subsequent SNP profiling and population differentiation analysis revealed obvious genetic differentiation of Liaoning isolates from other isolates. A genome-wide association study showed that SNPs closely related to low-temperature tolerance were mainly located in GPCR, Acyl-CoA, and Cpn10, which are responsible for adaptation to environmental factors, such as temperature change. Discussion: Pine wood nematodes likely adapted to the climate in Liaoning and maintained a certain reproductive capacity at low temperature via variants of adaptation-related genes. This study provides a theoretical basis for elucidating the prevalence and diffusion status of B. xylophilus in China. [ABSTRACT FROM AUTHOR]

(1) Pine wilt disease (PWD) is a devastating disease of pine forests caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus. Control of the disease is a worldwide problem due to the impossibility of using chemical nematicides on a large scale and for long periods. (2) Based on preliminary tests of microemulsion quality and stability, the optimum formulation was selected from 14 formulated microemulsions. The median lethal concentration (LC50) of the selected formulation at 48 h after treatment of B. xylophilus was 31.45 μg/mL of emamectin benzoate. The active ingredient reached the apical branches of Pinus thunbergii within 90 days of injection. (3) P. thunbergii was inoculated with B. xylophilus at 100 days post-injection, and the trees treated with the formulation remained uninfected for 450 days. Trunk injection exerted substantial control over PWD. (4) These results indicate that this formulation has the advantages of good transportability and long persistence in pine trees after injection and that it effectively prevents PWD. Therefore, this emamectin benzoate formulation can effectively reduce PWD occurrence in pine forests. [ABSTRACT FROM AUTHOR]

Hubei, Hunan and Henan Provinces are located in Central China, a region with extensive transport networks and trade. The pine wilt nematode (PWN), Bursaphelenchus xylophilus, the causative agent of pine wilt disease, is spread mainly through human activities. To further understand the genetic structure of PWN in Central China, we studied the genetic information of PWN populations in this region and compared the genetic relationship with strains from Guangdong and Jiangsu provinces. We found that the HB (Hubei) 15, HEN (Henan) 20, HN (Hunan) 07, HN08 and HN10 had significantly more SNPs and homozygotes than other strains from Central China, and their most frequent mutant genotypes also differed from other strains. The clustering results indicated that HB15, HEN 20, HN07, HN08 and HN10 were genetically distinct from other strains and closely related to Guangdong strains. We also observed significant genetic variation among strains in Henan province, suggesting that some of them might have different transmission sources than those from Hubei and Hunan provinces. Introgression analysis identified three possible pathways: (1) Guangdong to Henan; (2) Guangdong to Hunan; and (3) Jiangsu to Hubei. The results provide a basis for tracing the origin and spread of pine wood disease in China. [ABSTRACT FROM AUTHOR]

In previous research, the pine wood nematode Bxadh2 gene significantly increased its expression in pine seedlings inoculated with endophytic Bacillus cereus GD2 and pine wood nematode. To explore pine wood nematode Bxadh2 gene function, we cloned and analyzed its biological information, and we preliminarily studied its physiological function through RNA interference. We found that the Bxadh2 gene's full CDS length is 1269 bp, which encodes 422 amino acids, and presents a relatively stable hydrophobic protein. The protein encoded by the Bxadh2 gene has no signal peptide or transmembrane structure, and it is an intracellular protein that does not participate in transmembrane movement. The RNAi interference results showed that when the pine wood nematode's Bxadh2 gene was suppressed, its survival rate and fecundity significantly decreased, indicating that the expression of the Bxadh2 gene was related to the growth and development of pine wood nematodes. [ABSTRACT FROM AUTHOR]

Objective: There is a growing need for nematode resistant Pinaceae species plantlets to cope with the global scale degradation of coniferous forests, due to the prevalence of pine wilt disease. One of the bottlenecks that limits the commercialization of Pinaceae species plantlets is regeneration following their transfer from controlled sterile environments to the field while maintaining high survival rates. Methods: The growth factors of somatic plantlets (SPs), such as sucrose, media, culture substrate, brassinolide and spectrum were investigated to promote the application of somatic nematode-resistant P. thunbergii plants in afforestation. Results: The 1/2 WPM liquid medium, culture substrate (perlite and vermiculite =1:1), and carbohydrate (20 g/L sucrose) were effective in stimulating the growth of rooted SPs. While for unrooted SPs, 1 ug/L of brassinolide enhanced plantlet growth and rooting. And blue light (B) significantly promoted the longitudinal growth of shoots, while red light (R) was beneficial for root growth during the laboratory domestication stage. High quality SPs were obtained at a R/B ratio of 8:2. Following this acclimatization protocol, the P. thunbergii SPs could be directly transplanted to the field with a higher survival rate (85.20 %) in a forcing house. Conclusion: this acclimatization protocol extremely improved the survival rate of P. thunbergii SPs. Moreover, this work will contribute to enhancing the possibilities for somatic plant afforestation with Pinus species. [ABSTRACT FROM AUTHOR]

Pine wilt disease (PWD) is greatly serious to Pinus, and there are still no effective therapeutic measures at present. It is necessary to explore a method of early diagnosis of PWD and to study rehabilitation treatment technology for diseased plants diagnosed early. This paper searched for infected pine trees in natural pine forests according to various subtle symptoms and divided the disease development stages. Different doses of 20% emamectin benzoate were injected at different stages, and the symptom development of pine trees was observed after injection. According to different external symptoms, the stage after being infected by PWD was divided into early stage I and II, middle stage I and II, late stage. It was shown by the results that the diagnostic rate of initial diagnosis based on the symptoms of early stage II was as high as 80%. Additionally, for early stage infected pine trees, an injection of 5–10 mL of 20% emamectin benzoate can inhibit the expansion of symptoms of PWD. One year after injection of 20 mL and 10 mL of 20% emamectin benzoate, the residues measured in the lateral branches 4 m above the injection point were 0.18 mg kg−1 and 0.06 mg kg−1, respectively. In summary, the characteristics of early stage II are ideal for identifying early infection, it has a certain therapeutic effect on early infected pine trees by injection of emamectin benzoate. [ABSTRACT FROM AUTHOR]

As the most severe forestry quarantine disease in several countries, pine wilt disease (PWD) causes substantial economic losses and poses a significant threat to the forest ecosystem. It is necessary to find a rapid and sensitive method for the early diagnosis of the disease to control the development of the disease effectively. This paper investigated the effect of Bursaphelenchus xylophilus (the pinewood nematode; PWN) on the chlorophyll fluorescence kinetic curve (OJIP curve) and the parameters of needles using four-year-old Pinus thunbergii as experimental materials and chlorophyll fluorescence analysis as a technical tool. It was shown by the results in the OJIP curve that the fluorescence intensity of the inoculated plants was significantly increased at points O and I. Additionally, the relative variable fluorescence intensity at points K and J was comparable to that of the control plants. Several chlorophyll fluorescence parameters of the treatment significantly increased or decreased with disease progression. At the same time, the control group had no significant changes in each parameter. Therefore, chlorophyll fluorescence parameters can be used as indicators for the early diagnosis of PWD, among which the DIo/RC parameter was the best. In summary, PWN invasion will produce fluorescence on the PSII of P. thunbergii, and its chlorophyll fluorescence parameters are expected to achieve early PWD diagnosis. [ABSTRACT FROM AUTHOR]

Bursaphelenchus xylophilus is considered the most dangerous quarantine pest in China. It causes enormous economic and ecological losses in many countries from Asia and Europe. The glycoside hydrolase 45 gene family has been demonstrated in early studies to contribute to the cell wall degradation ability of B. xylophilus during its infection. However, the copy number variation (CNV) of the GH45 gene and its association with B. xylophilus pathogenicity were not fully elucidated. In this study, we found that the GH45 gene with two copies is the most predominant type among 259 B. xylophilus strains collected from China and Japan. Additionally, 18 strains are identified as GH45 genes with a single copy, and only two strains are verified to have three copies. Subsequent expression analysis and inoculation test suggest that the copy numbers of the GH45 gene are correlated with gene expression as well as the B. xylophilus pathogenicity. B. xylophilus strains with more copies of the GH45 gene usually exhibit more abundant expression and cause more severe wilt symptoms on pine trees. The aforementioned results indicated the potential regulatory effects of CNV in B. xylophilus and provided novel information to better understand the molecular pathogenesis of this devastating pest. [ABSTRACT FROM AUTHOR]

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The pine wilt disease (PWD) caused by pine wood nematode (PWN), Bursaphelenchus xylophilus , is a destructive forest disease and causes global economic losses. Effective and affordable detection of PWN is critical for the management of this pest. In this study, we developed a rapid diagnostic assay using recombinase polymerase amplification (RPA) combined with the CRISPR/Cas12a system to detect PWN. The assay was validated as being fast, accurate, and reliable based on detection of PWN from pine wood samples. Test results for 33 PWN were positive, but negative for 11 isolates Bursaphelenchus species, 10 other nematodes, 20 isolates of 15 fungi species, and 6 isolates of Phytophthora species. This method allow us to have the results naked-eye directly. Results from this study indicated that this sensitive, specific, and rapid RPA-CRISPR/Cas12a assay has potentially significant applications to detect PWN, especially under time- and resource limited conditions. • The first combination of RPA and CRISPR/Cas12a was utilized for identification of B. xylophilus. • RPA-CRISPR/Cas12a can complete the rapid detection of B. xylophilus at 37 °C for 30 min. • The accuracy of RPA-CRISPR/Cas12a was verified by collecting 100 suspected B. xylophilus pine samples in China. • RPA-CRISPR/Cas12a assay has potentially significant applications to detect PWN. [ABSTRACT FROM AUTHOR]

The pine wood nematode Bursaphelenchus xylophilus parasitizes millions of pine trees worldwide each year, causing severe wilt and the death of host trees. Glycoside hydrolase 45 genes of B. xylophilus are reported to have been acquired by horizontal gene transfer from fungi and are responsible for cell wall degradation during nematode infection. Previous studies ignored the possibility of copy number variations of such genes. In this study, we determined that two of the glycoside hydrolase 45 genes evolved to maintain multiple copies with distinct expression levels, enabling the nematode to infect a variety of pine hosts. Additionally, tandem repeat variations within coding regions were also detected between different copies of glycoside hydrolase 45 genes that could result in changes in protein sequences and serve as an effective biological marker to detect copy number variations among different B. xylophilus populations. Consequently, we were able to further identify the copy number variations of glycoside hydrolase 45 genes among B. xylophilus strains with different virulence. Our results provide new insights into the pathogenicity of B. xylophilus, provide a practical marker to genotype copy number variations and may aid in population classification. [ABSTRACT FROM AUTHOR]

Cytochrome P450 genes are very important for plant-parasitic nematodes to reproduce and to metabolize xenobiotic compounds generated by their host plants. The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes very high annual economic losses by killing large numbers of pine trees across Asia and into Europe. In this study, we used RNA interference (RNAi) to analyze the function of the cyp-33C9 gene of PWN. Our results showed that expression of the cyp-33C9 gene was suppressed successfully after soaking nematodes for 24 h in cyp-33C9 double-stranded RNA (dsRNA). The silencing of the cyp-33C9 gene significantly decreased the feeding, reproduction, oviposition and egg hatch of B. xylophilus. Meanwhile, the migration speed of B. xylophilus in Pinus thunbergii was reduced in the early stages when the cyp-33C9 gene was silenced in the nematodes. Moreover, knockdown of the cyp-33C9 gene in B. xylophilus caused a decrease in pathogenicity to pine trees. These results suggest that the cyp-33C9 gene plays an important role in the reproduction and pathogenicity of B. xylophilus. This discovery identified several functions of the cyp-33C9 gene in B. xylophilus and provided useful information for understanding the molecular mechanism behind pine wilt disease caused by PWN. [ABSTRACT FROM AUTHOR]

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. However, the pathogenesis-related genes of B. xylophilus are not well characterized. Thus, DNA microarrays were used to investigate differential gene expression in PWN where Pinus thunbergii was inoculated with nematodes, compared with those cultured on Botrytis cinerea. The microarrays comprised 31121 probes, 1310 (4.2%) of which were differentially regulated (changes of >2-fold, P < 0.01) in the two growth conditions. Of these 1310 genes, 633 genes were upregulated, whereas 677 genes were downregulated. Gene Ontology (GO) categories were assigned to the classes Cellular Component, Molecular Function, and Biological Process. The comparative gene expression analysis showed that a large number of the pathogenesis-related genes of B. xylophilus, such as pectate lyase genes, cytochrome P450s, UGTs, and ABC transporter genes, were highly expressed when B. xylophilus infected P. thunbergii. Annotation analysis indicated that these genes contributed to cell wall degradation, detoxification, and the reproduction process. The microarray results were validated using quantitative RT-PCR (qRT-PCR). The microarray data confirmed the specific expression of B. xylophilus genes during infection of P. thunbergii, which provides basic information that facilitates a better understanding of the molecular mechanism of PWD. [ABSTRACT FROM AUTHOR]

• Bxy-octr-1 expression throughout the whole life cycle of B. xylophilus. • Bxy-octr-1 plays key roles in early ontogenesis of B. xylophilus. • Bxy-octr-1 is indispensable for mating behavior of B. xylophilus. Bursaphelenchus xylophilus is an invasive plant-parasitic nematode causing the notorious pine wilt disease (PWD) worldwide, which results in huge economic losses. G protein-coupled receptors (GPCRs) play an essential role in mating and reproduction behavior of animals. As a unique biogenic amine in invertebrates, octopamine (OA) can regulate a variety of physiological and behavioral responses by binding specific GPCRs. These specific GPCRs are also called octopamine receptors (OARs), and octr-1 is one of them. However, Bxy-octr-1 is unknown in B. xylophilus. Therefore, we investigated the expression pattern and biological function of Bxy-octr-1. Bioinformatics analysis indicated that Bxy-octr-1 was evolutionarily conserved. The real-time quantitative PCR data revealed that Bxy-octr-1 expression was required throughout the entire life of B. xylophilus. mRNA in situ hybridization showed that Bxy-octr-1 was mainly located in the cephalopharynx, body wall muscle, intestine, and gonadal organs of B. xylophilus. RNA interference (RNAi) showed that embryo hatching rates and locomotion speeds were both dramatically decreased. Obvious abnormal phenotypes were observed in the second-stage of juveniles after RNAi treated. Furthermore, its ontogenesis was stunting. Lack of Bxy-octr-1 reduced fecundity of females, of which 31.25% of them could not successfully ovulate. In addition, the error positioning ratio of the nematode was significantly increased. Our study suggests that Bxy-octr-1 is indispensable for locomotion, early ontogenesis and mating behavior in B. xylophilus. [ABSTRACT FROM AUTHOR]