Cloning of sft-4 and its influence on vitality and virulence of pine wood nematode,Bursaphelenchus xylophilus

In our previous screening of the transcriptome of the causal agent of the devastating pine wilt disease,pine wood nematode(PWN,Bursaphelenchus xylophilus),after treatment with the nematicide fomepizole,Surfeit locus gene sft-4,which encodes a regulatory factor,was found to be downregulated.In situ hybridization results showed that the sft-4 was continuously expressed from egg to adult and was especially high in the reproductive system.Here in a study of the effect of RNA interference(RNAi)of sft-4 and recombinant SFT-4 on PWN activity,treatment with sft-4 dsRNA inhibited feeding,reproduction,oviposition and egg hatching of PWN with the greatest inhibition on reproduction and oviposition,whereas recombinant SFT-4 had the opposite effect.In addition,RNAi of sft-4 changed the female–male ratio and lifespan of PWN.In bioassays of PWNs,with RNAi of sft-4 on seedlings and 2-year-old Pinus thunbergii trees,none of the treated plants developed symp-toms during the monitoring period,indicating that virulence of PWNs was either significantly weakened.These results indicate that the influence of sft-4 on PWN pathogenicity may be mainly through regulating reproductive function of PWN and its lifespan.

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.

Molecular characterization and functional analysis of daf-8 in the pinewood nematode,Bursaphelenchus xylophilus

Bursaphelenchus xylophilus,causal agent of pine wilt disease,causes extensive damage worldwide.Strate-gies are needed to inhibit population growth or block the spread of the invasive nematode to control pine wilt dis-ease.The gene daf-8 plays crucial roles in larval develop-ment and dauer formation in Caenorhabditis elegans,but little is known about its orthologue in B.xylophilus.In the present molecular characterization and functional analysis of daf-8 in B.xylophilus(Bx-daf-8),RT-qPCR showed that the expression of Bx-daf-8 gradually increased during the embryonic stage,peaked in the second-stage juvenile(J2),then dramatically dropped in the J3,and remained at that low level for the rest of its life.Bx-daf-8-transgenic C.elegans was employed to mimic the spatiotemporal expression of Bx-daf-8,which was expressed close to the pharynx during early embryogenesis and weakly throughout the whole body during late embryogenesis.It was observed in head neurons and tail ganglions throughout all postembryonic stages.B.xylophilus embryos were severely abnormal,and hatching rate decreased sharply after Bx-daf-8 knockdown.daf-16-1 and da-f16-2,downstream genes in the IIS pathway,also dropped sharply after Bx-daf-8 knockdown.We propose that TGFβmay crosstalk with the IIS pathway upstream of Bx-daf-16 and that daf-8 may act as a master regulator of daf-16 in B.xylophilus.However,knockdown of Bx-daf-8 did not lead to constitutive developmental arrest at the dauer larval stage,indicating that dauer entry in B.xylophilus might be controlled by several genes and is more complicated than in C.elegans.Bx-daf-8 alone did not control the dauer entry in B.xylophilus,but it was indispensable for embryogen-esis,providing a potential target for suppressing population growth of B.xylophilus.

Comparative study on the mRNA expression of Pinus massoniana infected by Bursaphelenchus xylophilus

Pine wilt disease(PWD)is a devastating disease affecting the growth of Pinus massoniana,often leading to withering and death.To reveal the changes involved during disease progression,we investigated the mRNA expression profile of P.massoniana infested by Bursaphelenchus xylophilus.The infestation resulted in the downregulation of genes involved in interactions with pathogenic pathways such as disease resistance gene,CC-NBS-LRR resistancelike protein,and the gene encoding a putative nematode resistance protein.Increased infestation pressure(number of nematodes inoculated)caused a continuous decline in the gene expression of stem samples.An infestation of P.massoniana also resulted in a pathway enrichment of genes involved in phenylpropanoid metabolism and flavonoid biosynthesis,which in turn reduced the levels of total phenols and total flavonoids.A downregulation of auxin responsive family protein was observed in infested samples,which resulted in a suppression of plant growth.Thus,upon B.xylophilus infestation,a downregulation of genes associated with the recognition of pathogens,PWD resistance,and growth regulation was observed in P.massoniana,together with a decrease in the levels of phytoalexinlike secondary substances,all of which resulted in withering and ultimately death of P.massoniana.

Suggestions on management measures of pine forest ecosystems invaded by Bursaphelenchus xylophilus

Pine wood nematode （PWN）, Bursaphelenchus xylophilus （Steiner et Buhrer） Nickle is an important invasive alien species in forests of China and has become one of the most destructive forest diseases. In order to improve the resistance and resilience of pine forest ecosystems against B. xylophilus invasion and make the pine forest ecosystem more timely responsive to PWN invasion, we made some recommendations based on five years of intensive observations. We advocate a set of management measures with the theme “Prevention is priority, but integrated with curative techniques and ecological resilience” on the pine forest ecosystem invaded by B. xylophilus; details of accurate measures are proposed. The aim is to discover the underlying problems of present pine forest ecosys-tems and to take, correspondingly, administrative measures and strategies, which will encourage the pine forest ecosystem to develop in a benign way.

Transcriptomic response of Pinus massoniana to infection stress from the pine wood nematode Bursaphelenchus xylophilus

The pinewood nematode(PWN)Bursaphelenchus xylophilus is a forestry quarantine pest and causes an extremely dangerous forest disease that is spreading worldwide.Due to the complex pathogenic factors of pine wood nematode disease,the pathogenesis is still unknown.B.xylophilus ultimately invades a host and causes death.However,little is known about the defence-regulating process of host pine after infection by B.xylophilus at the molecular level.Therefore,we wanted to understand how Pinus massoniana regulates its response to invasion by B.xylophilus.P.massoniana were artificially inoculated with B.xylophilus solution,while those without B.xylophilus solution were used as controls.P.massoniana inoculated with B.xylophilus solution for 0 h,6 h,24 h,and 120 h was subjected to highthroughput sequencing to obtain transcriptome data.At various time points(0 h,6 h,24 h,120 h),gene transcription was measured in P.massoniana inoculated with PWN.At different time points,P.massoniana gene transcription differed significantly,with a response to early invasion by PWN.According to Gene Ontology(GO)classification and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis,P.massoniana response to PWN invasion involves a wide range of genes,including plant hormone signal transformation,flavonoid biosynthesis,amino sugar and nucleoside sugar metabolism,and MAPK signalling pathways.Among them,inoculation for 120 hours had the greatest impact on differential genes.Subsequently,weighted gene coexpression network analysis(WGCNA)was used to analyse transcriptional regulation of P.massoniana after PWN infection.The results showed that the core gene module of P.massoniana responding to PWN was“MEmagenta”,enriched in oxidative phosphorylation,amino sugar and nucleotide sugar metabolism,and the MAPK signalling pathway.MYB family transcription factors with the highest number of changes between infected and healthy pine trees accounted for 20.4% of the total differentially expressed transcription factors.To conclude,this study contributes to our understanding of the molecular mechanism of initial PWN infection of P.massoniana.Moreover,it provides some important background information on PWN pathogenic mechanisms.

基于灰色关联度分析气候因素对江西省松材线虫病的影响

为分析不同气候因素对松材线虫Bursaphelenchus xylophilus引起的松材线虫病的影响程度,本文以江西省全南县(代表赣南,松墨天牛一年2代)和德兴市(代表赣北,松墨天牛一年1代)遭受松材线虫病危害的马尾松林生态系统为研究对象,获取2018年-2021年松材线虫病普查数据和1990年-2021年的主要气象因子数据,结合滑动平均法和灰色关联度分析法比较分析两地的气候差异和疫情年间气候变化对松材线虫病发病面积的影响。结果显示:除德兴市的年均相对湿度和年降水量与历年平均值相比有所降低外,两地其他气象因子均呈上升趋势;灰色关联度分析表明,年均最高气温和年均风速是影响赣南松材线虫疫情的重要气象因子,年均最高气温和年降水量是影响赣北松材线虫疫情的重要气象因子;气温升高促进松材线虫和其媒介昆虫的生长繁殖与活动,风速升高可使媒介昆虫远距离传播松材线虫,降水量减少可降低松材线虫和媒介昆虫染病死亡几率。赣南赣北气候的整体变化趋势都有利于松材线虫病的扩散蔓延。

New insights into the life history of Monochamus saltuarius(Cerambycidae:Coleoptera)can enhance surveillance strategies for pine wilt disease

Pine wilt disease was first discovered in Dongtang town,Liaoning Province,China,in 2017.Monochamus saltuarius Gebler is a new vector of pinewood nematode and the only known vector in Liaoning Province.The biology of this pest has not been reported thus far;therefore,it is necessary to study its life history.During 2018 and 2019,we collected 138 and 491 adult M.saltuarius beetles,respectively,to analyze their eclosion from larva to adult stage.In mid-March,overwintering larvae began to feed(on xylem)and seek nutrition in preparation for pupation and eclosion.The adults began to appear in mid-April,and the population reached its peak in late May.The life span of the adults was 28-76 days.After approximately 1 week of supplemental nutrition(feeding on twigs),adults began to mate and lay eggs.The egg stage of M.saltuarius lasted 4-8 days.The larvae in Dongtang town have 4 instars and overwinter in tunnels as 3rd-4th instars.The 1st-instar stage lasted 3-9 days,the 2nd-instar stage lasted 11-23 days,the 3rd-instar stage lasted 30-130 days,and the 4th-instar stage lasted 44-180 days.The pupal stage lasted 7-12 days,and the life span of the adults was 28-76 days.In this study we systematically monitored the life history of M.saltuarius for the first time.Our objective was to lay a foundation for improving control of this pinewood nematode vector.

Classification of dendrocola nematode-trapping fungi

Pine wilt disease,caused by Bursaphelenchus xylophilus and Bursaphelenchus mucnatus,is a serious quarantined disease.Arboreal nematode-trapping fungi of P inus spp.are effective predators on nematodes and have strong host adaptability.The development of these fungal resources may be an effective way to control pine wood nematodes.We collected 515 samples of pine wilt disease from the areas of Ninghai City(Zhejiang province),Shuangbai County(Yunnan province),and Daxing'anling(Heilongjiang province),China.Through isolation,culture and identification,6 species of nematode-trapping fungi(A rthrobotrys cladodesr,A.oligospora,A.musiformis,A.dendroides,Dactylellina ellipsospora,Monacrosporium thaumasium)were identified for predation against B.xylophilus,and 9 species(Arthrobotrys dactyloides,A.cladodes r A.oligospora A.dendroides,Dactylellina ellipsospora,Dactylella asthenopaga,D.leptospora,Arthrobotrys superba,Monacrosporium drechseri)were identified for predation against B.mucnatus.This study provides information in the classification of arboreal predator nematodes and provides an important basis for future biological control of pine wood nematodes.

Predatory behavior of Tetrigus lewisi on Monochamus alternatus

Pine wilt is a serious and fatal disease in pine forests. It is caused by the pine wood nematode, Bursaphelenchus xylophilus, transmitted by the Japanese saw beetle Monochamus alternatus. Control of this disease depends on reducing its vector population. The public is demanding environmentally friendly control methods and biological control has become increasingly seen as an impor-tant, safe and effective approach. Through our investigations both in the field and by breeding in our laboratory, a new predator of M. alternatus, Tetrigus lewisi （Coleoptera： Elateridae）, was found for the first time in China. Given our continuous observations, we are assured that the predatory behavior of the elater can be separated into four parts, i.e., foraging and sensing, killing, eating and excre-tion. They always took several hours to eat the preys that have been killed momentarily. We finally found that most of the elaters preferably attacked in the stomach and abdomen of the larva of M. alternatus.

Research Advances in Interplay of Bursaphelenchus xylophilus and Other Key Biological Factors Related to Pine Wilt Disease

The paper separately discusses interplay of pine wood nematode and several key biological factors related to pine wilt disease, such as host trees, insect vectors, fungi, symbiotic bacteria, and natural enemy acarid, etc. By virtue of so much complicated interplay among the biological factors related to pine wilt disease which is different from other forest disease, it also points out that the further researches about pine wilt disease should focus on interplay mechanism of key biological factors to discover pathogenic mechanism, and simple and quick inspection and quarantine methods.

Differences in arthropod communities between island and inland Masson pine forests infested by pine wilt disease in Zhejiang Province

The invasion of Bursaphelenchus xylophilus （pine wood nematode, PWN） carried by Monochamus alternatus predominately attacks Masson pine （Pinus massoniana） forests and causes great economic losses in China. In this study, we assessed whether the effect of the invasion of PWN is different between island and inland forests. Arthropods were sampled in Fuyang （inland） and Zhoushan （island） counties in Zhejiang Province with sweep netting and light traps at four plots. During two field periods （May to June 2004 and September to October 2005） a total of 21,916 insects, representing 384 species belonging to 99 families and 15 orders, were collected in the sample plots from the island, whereas, from the inland forest a total of 29,262 insects, representing 308 species belonging to 96 families and 13 orders, were found. A hierarchical cluster analysis （HCA） and one-way ANOVA, based on the composition of different arthropod guilds, were performed. The results showed that there was no significant difference in the composition of arthropod communities at the family level between inland and island. But these two habitats had a significant effect on the composition of species, individuals, sub-communities and energy class levels. Statistically, the composition of the two orders, Lepidoptera and Diptera, in the two habitats were significantly different.

Monitoring of Pinus massoniana spatial pattern changes based on RS and GIS techniques

Our research focused on Pinus massoniana information extracted from remote sensing images based on the knowledge detection and decision tree algorithm and established a spatial pattern model, combining quantitative theoretical ecology with remote sensing （RS） and geometric information system （GIS） techniques. Applying information extraction methods and a spatial pattern model, we studied P. massoniana spatial patterns changes before and after the invasion by pine wood nematode （Bursaphelenchus xylophilus） in Fuyang and Zhoushan counties, Zhejiang Province, east China. The P. massoniana spatial patterns are clustering, whether the invasion happened or not. But the degree of clustering is different. Our results show good agreement with field data. Applying the results, we analyzed the relationship between spatial patterns and the invasion level. Then we drew the elementary conclusion that there are two kinds of patterns for pine wood nematode to spread： continuous and discontinuous diffusion. This approach can help monitor and evaluate the changes in ecological systems.

马尾松打孔注射20%甲维盐可溶液剂防治松材线虫病的林间药效试验

本试验在27~33℃高温下,利用木材麻花钻给松树打孔注射新型打孔注药剂20%甲氨基阿维菌素可溶液剂,于注药后2h、1d、2d观察注药松树对药剂的吸收效果,注药后50d检测注药松树中松材线虫(Bursaphelenchus xylophilus)数量,并计算虫口减退率及防治效果。结果显示20%甲维盐可溶液剂在注药后2h吸收量达到80%以上,在注药1d后全部被吸收。对照组3%甲维盐微乳剂在注药后2h吸收量仅11.25%,注药后2d仅吸收21.25%,且被大量松脂堵塞注药口;20%甲维盐可溶液剂有效成分用量60~120 mg/cm胸径处理药后50d虫口减退率在88.69%~96.40%,防效在94.82%~98.35%;对照组3%甲维盐微乳剂有效成分用量90 mg/cm胸径处理后虫口减退率为59.52%,防效为81.47%,与试验组差异极显著(P<0.01)。经调查发现,该药剂对松树以及周围非靶标生物均无明显影响。由此说明20%甲维盐可溶液剂在27~33℃高温下吸收良好,在60~120mg/cm胸径浓度范围内对松材线虫防效好,且对松树及周围环境安全。

3种松墨天牛引诱剂诱捕效果比较

比较了3种松墨天牛引诱剂NL-01型、F2型、APF-1型对松墨天牛(Monochamus alternatus)成虫的诱捕效果。结果显示:试验组3种引诱剂诱捕松墨天牛成虫的数量相比对照组存在显著差异(P<0.05)。不同引诱剂诱捕松墨天牛成虫数量存在一定差异,总体趋势为APF-1>F2>NL-01,三者之间差异不显著。试验组3种引诱剂诱捕松墨天牛雌成虫和雄成虫的数量相比对照组存在显著差异(P<0.05),试验组之间差异不显著。试验结果表明,基于植物源和性信息素组合使用的引诱剂具有高效的诱捕效率,可以用于松墨天牛的诱杀和防治,从而降低松材线虫病(Bursaphelenchus xylophilus)的传播速度。

松材线虫的伴生微生物及其致病性研究概述

松材线虫(Bursaphelenchus xylophilus Nickle)是全球首要检疫性有害生物,在松材线虫的生长、繁殖、侵染和致病过程中,有多种生物因子参与其中,形成复杂的微生态系统,特别是松材线虫与其体表携带微生物形成了密切的伴生关系。本文综述了松材线虫伴生微生物种类的多样性,并探讨了松材线虫与其伴生微生物的相互关系及其致病性。伴生细菌以松材线虫为载体(附着于松材线虫体表)一同侵入松树寄主,松材线虫的存在有利于伴生细菌的繁殖和产毒,反过来,伴生细菌也可为松材线虫的生长发育、扩散、繁殖提供某些营养和有利的环境条件,促进其生长和繁殖,并有助于松材线虫降解松萜类物质,从而提高了松材线虫的适应性和致病性;伴生真菌能为松材线虫提供食物,维持松材线虫种群繁衍,促进扩散型松材线虫种群数量增加,提高松材线虫侵染、扩散机率。文中还结合伴生细菌在松材线虫病生物防治中的应用,提出了今后研究的方向。

Traits of Masson Pine Affecting Attack of Pine Wood Nematode

Masson pine characteristics were analyzed in five sample plots in Zhejiang Province, China. Bursaphelenchus xylophilus （Steiner et Buhrer） Nickle （pine wood nematode, PWN） carried by Monochamus alternatus predominately attacked Masson pines in the lower diameter classes. Among the 10 tree characteristics examined, mean crown width, percentage of bole with crown, 5-year cumulative diameter growth, and resin amount showed significant variation between successfully attacked and unattacked trees. The attacked trees had a lower percentage of the bole covered with tree crown, lower crown width, lower radial growth in the last 5 years, and produced less induced resinosis than unattacked trees. Results allowed for effective ranking of the pine forest based on individual tree resistance to PWN. This index of resistance should be considered throughout the development of an ＂Evaluation Criterion and Indicator System＂. The preceding ranking can be used to evaluate the resistance and resiliency of the pine forest ecosystem to PWN＇s invasion, which is similar to Pest Risk Analysis （PRA）.

松材线虫病除治迹地补植树种选择及其对立地条件的响应

为研究适宜松材线虫病(Bursaphelenchus xylophilus)除治迹地恢复的补植树种选择及其栽培技术,对浙江临海市的3种土层厚度的地类开展林地更新补植造林试验。经调查分析7个树种15年的树高、胸径和冠幅生长结果表明,3种地类间不同树种生长存在极显著差异,其中木荷、樟树和枫香表现出对优良立地条件的需求特性,而青冈、黄檀、白栎则表现出较好的耐瘠薄特性。分析木荷单株树高和胸径的频率分布结果显示,3种地类的树高和胸径均呈类正态分布,且在土层厚度小于40 cm的地类Ⅱ、Ⅲ中仍然存在0.84%~12.8%(树高)和10.4%~32.4%(胸径)的优良植株,其树高和胸径高于地类Ⅰ的平均水平,预示在南方相对瘠薄山地内存在优良的微立地环境,可通过选择微立地补植珍贵彩色树种实现瘠薄林地的质量精准提升。基于本研究结果,综合考虑具有相似生长习性的树种,提出适于浙江沿海松材线虫病除治迹地更新补植造林树种及其人工补植技术。

Comparative analysis of the Monochamus alternatus immune system

The pine sawyer beetle, Monochamus alternatus, is regarded as a notorious for- est pest in Asia, vectoring an invasive pathogenic nematode, Bursaphelenchus xylophilus, which is known to cause pine wilt disease. However, little sequence information is available for this vector beetle. This hampered the research on its immune system. Based on the transcriptome of M. alternatus, we have identified and characterized 194 immunity-related genes in M. alternatus, and compared them with homologues molecules from other species known to exhibit immune responses against invading microbes. The lower number of puta- tive immunity-related genes in M. alternatus were attributed to fewer C-type lectin, serine protease （SP） and anti-microbial peptide （AMP） genes. Phylogenetic analysis revealed that M. alternatus had a unique recognition gene, galectin3, orthologues of which were not identified in Tribolium castaneum, Drosophila melanogastor, Anopheles gambiae and Apis mellifera. This suggested a lineage-specific gene evolution for coleopteran insects. Our study provides the comprehensive sequence resources of the immunity-related genes ofM. alternatus, presenting valuable information for better understanding of the molecular mechanism of innate immunity processes in M. alternatus against B. xylophilus.

Major ascaroside pheromone component asc-C5 influences reproductive plasticity among isolates of the invasive species pinewood nematode

Pheromones are communication chemicals and regulatory signals used by animals and represent unique tools for organisms to mediate behaviors and make“decisions”to maximize their fitness.Phenotypic plasticity refers to the innate capacity of a species to tolerate a greater breadth of environmental conditions across which it adapts to improve its survival,reproduction,and fitness.The pinewood nematode,Bursaphelenchus xylophilus,an invasive nematode species,was accidentally introduced from North America into Japan,China,and Europe;however,few studies have investigated its pheromones and phenotypic plasticity as a natural model.Here,we demonstrated a novel phenomenon,in which nematodes under the condition of pheromone presence triggered increased reproduction in invasive strains(JP1,JP2,CN1,CN2,EU1,and EU2),while it simultaneously decreased reproduction in native strains(US1 and US2).The bidirectional effect on fecundity,mediated by presence/absence of pheromones,is henceforth termed pheromone-regulative reproductive plasticity(PRRP).We further found that synthetic ascaroside asc-C5(ascr#9),the major pheromone component,plays a leading role in PRRP and identified 2 candidate receptor genes,Bxydaf-38 and Bxysrd-10,involved in perceiving asc-C5.These results suggest that plasticity of reproductive responses to pheromones in pinewood nematode may increase its fitness in novel environments following introduction.This opens up a new perspective for invasion biology and presents a novel strategy of invasion,suggesting that pheromones,in addition to their traditional roles in chemical signaling,can influence the reproductive phenotype among native and invasive isolates.In addition,this novel mechanism could broadly explain,through comparative studies of native and invasive populations of animals,a potential underlying factor behind of the success of other biological invasions.