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.

松材线虫(Bursaphelenchus xylophilus)入侵对马尾松(Pinus massoniana)林分生长的影响及相关生长模型(英文)

松材线虫(Bursaphelenchus xylophilus)是一种松树上发生严重的有害生物,它不仅改变了生态系统的结构和功能,而且改变了系统内生物的原有特性和地理分布。松材线虫及其引起的松树萎蔫病已对中国马尾松林(Pinus massoniana)的树木成长产生了巨大影响。基于此,使用"每木调查法"和"样方法",对松材线虫入侵后的马尾松林内松树的各项生长指标因子进行了调查分析,其结果表明:自松材线虫1996年入侵所调查地区的松林后,对于受害松树不管是伐倒木(被伐倒)还是倒木(自然倒地),其对周围马尾松胸径生长的影响是显著的,而对树高生长的影响不显著。最后建立了一系列的灰色和灰色-马尔可夫链数学模型,其预测结果精度高,可用于今后受害和未受害区马尾松林分因子的生长预测。

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.

Interspecific hybridization between Bursaphelenchus xylophilus and Bursaphelenchus mucronatus

Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease, which has greatly damaged forests and ecosystems in countries of East Asia and Europe.Bursaphelenchus mucronatus is closely related to B. xylophilus in morphology and host plant specificity. A longrunning debate has existed regarding whether these two species can successfully produce hybrid offspring. In the present study, we performed in the laboratory, hybridization of two B. xylophilus nematode isolates from China and Japan and three B. mucronatus isolates from China, Japan and France. Nematode isolates of B. xylophilus were successfully crossed with B. mucronatus isolates, and the rate of hybridization was relatively high; however, some hybrid offspring died. Successful hybridization occurred between B. xylophilus and B. mucronatus isolates from China, and 22 generations of hybrids were produced. All F1 hybrids could be backcrossed with their parents and produce offspring. Variation in mucro length among the hybrid offspring and their parents was observed. The hybrid offspring and their parents were inoculated into 3-months-old black pine(Pinus tunbergii) seedlings. Weaker pathogenicity of hybrid offspring was observed compared with that of their parents, and significantly fewer offspring nematodes than parents were reisolated from pines. Therefore, the offspring of B. xylophilus and B. mucronatus may exist in the forest and could influence disease epidemics.

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.

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.

SCAR Marker and Detection Technique of Bursaphelenchus xylophilus

OPM05-M2100,the specific RAPD fragment of Bursaphelenchus xylophilus,was collected from agarose gels and purified.Then,the purified fragment was inserted into the pGEMR -T Vector that was transformed into Escherichia.coli and cloned and sequenced.Based on the sequence of RAPD marker,the sequences characterized amplified region（SCAR） primers were designed by the aid of the software Oligo5.0.The forward primer is M05F2 （5’-CGGGT CATGG CTGGA GGTAT CGT-3’）,and the backward primer is M05R1(5’-TGGCT CAATG GCAAA TCCTT CGTA-3’.The specific fragment (OPM05-M2100) was successfully converted to SCAR marker(SCAR-M05-X600) by using M05 F2/R1,which was the specific markers of B.xylophilus.Then, the DNA of 92 isolates of Bursaphelenchus,B. mucronatus,B.hofmanni,Aphelenchoides macronucleatus and Seinura sp.which were isolated from dead pines,were marked,and the DNA of a single nematode extracted with a simple method was detected using this set of specific primers.The results indicated that the PCR product of all 81 isolates of B.xylophilus was a clear and bright fragment about 600 bp with M05 F2/R1.But eight isolates of B. mucronatus,one B.hofmanni,one A.macronucleatus and one Seinura sp.had no any fragments.Assay M05 F2/R1 also successfully detected single pinewood nematode.Therefore,the specific pairwises would be used for constructing identification kits of B. xylophilus,implementing the aim of quick detection, and achieving the purpose of identify juvenile successfully.

松材线虫侵袭引发的植被演替对鸟类群落的影响

松材线虫(Bursaphelenchusxylophilus)是引起严重森林病害的外来物种,可能导致森林生态系统急剧退化。为了探讨在松材线虫侵袭所引起的森林演替过程中植被结构变化对鸟类群落的影响,我们研究了这些常绿阔叶演替林中的鸟类群落结构。2004年5–6月,我们在浙江省宁波市选择了分别处于3个不同演替阶段的7个样地:包括象山县被松材线虫侵袭5年后的常绿阔叶灌丛和侵袭12年后的常绿阔叶林样地各3个,同时在天童国家级森林公园选择了约40龄的常绿阔叶林样地1个。结果显示,在常绿阔叶灌丛和松材线虫侵袭12年后的常绿阔叶林中,鸟类物种多样性和丰富度均显著高于约40龄常绿阔叶林;3类栖息地的鸟类多度、均匀度均无显著差异;在侵袭12年后的常绿阔叶林中鸟类群落最丰富。多元回归分析结果显示,立地年龄是影响鸟类群落组成的主要因素。

防御酶与不同地理种源马尾松松材线虫病抗性的关系

采用比色法及聚丙烯酰胺凝胶电泳法(PAGE)测定了一年生马尾松(Pinus massoniana)抗性种源衡阳1号和敏感种源广西1号接虫松材线虫(Bursaphelenchus xylophilus)后其过氧化物酶(POD)、多酚氧化酶(PPO)活性及其同工酶的动态变化。结果表明,在抗性种源中,POD活性有2个峰值,PPO活性有1个峰值,且两种酶活性的峰值明显高于敏感种源。同工酶活性电泳图谱显示,尽管在两种种源的POD、PPO同工酶谱中均未见有新的酶带产生,但抗性种源中活性增强的POD同工酶酶带数量要多于敏感种源,且一些PPO同工酶酶带也能对松材线虫侵染作出较快反应。因此,POD和PPO在马尾松抗松材线虫病中发挥重要作用。

利用荧光PCR快速检测松材线虫

利用荧光PCR从美国进境的木质包装材料中成功检出松材线虫(Bursaphelenchus xylophilus).结果表明,探针检测松材线虫样品时,可产生明显的荧光信号,但空白水对照、小杆线虫(Rhabditida)、拟松材线虫(B.mucronatus)无荧光信号,表明探针有较强的特异性,且具有简单、灵敏、快速等特点.

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。

Metabolites from Resistant and Susceptible <i>Pinus thunbergii</i>after Inoculation with Pine Wood Nematode

Pine wilt disease (PWD), which is caused by pine wood nematodes (PWN), is one of the most serious forest diseases worldwide. To clarify the mechanism of resistance to PWD, we compared metabolites from resistant and susceptible Japanese black pine (Pinus thunbergii) families after inoculation with PWN. After 2 weeks to 1 month post inoculation, the number of PWN dramatically increased in susceptible plants, but not in resistant plants. At this PWN-proliferation phase, ethyl acetate soluble fractions extracted from PWN-inoculated plants were analyzed by gas chromatogramphy-mass spectrometry (GC-MS). Although most compounds were qualitatively and quantitatively similar between resistant and susceptible plants, resistant plants accumulated 2.0-fold more linoleic acid (LA) than susceptible plants. On the other hand, benzoic acid (BA) was barely detected in resistant plants, but it accumulated in susceptible plants as the number of PWN increased. Susceptible plants contained greater levels of the nematicidal compounds pinosylvin and pinosylvin monomethyl ether, compared with resistant plants. These results suggested that LA is involved in the resistance reaction against PWN-proliferation, and that BA could be a good biomarker for PWD.

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.

Micropropagation of Pinus densiflora and the evaluation of nematode resistance of regenerated microshoots in vitro

To accelerate the breeding and selection of Pinus densiflora Siebold and Zucc. resistance to pine wilt disease, a micropropagation system was established and nematode resistance evaluated in vitro. Cotyledon-hypocotyl explants from 28-day-old seedlings were first cultured on Gresshoff and Doy medium supplemented with 4.0 mg L^(-1) 6-benzyladenine and 0.2 mg L^(-1) a-naphthaleneacetic acid(NAA) to stimulate the formation of buds. Induced buds were subsequently subcultured on Gupta and Durzan medium supplemented with 0.1%(w/v)activated charcoal for elongation. Stem sections derived from shoots were used as explants for the further multiplication. Roots were formed from shoots transferred to woody plant medium containing 0.2 mg L^(-1) NAA for4 weeks. The nematode resistance test showed that symptoms in micropropagated shoots after infection with pine wood nematode(PWN) were similar to those in plants infected in the field. The wilting rate varied from 20 to100% among different clones 18 days after inoculation.The most susceptible clone was Clone 6-4 with a 100%wilting rate, while Clone 8-4 showed a relatively high resistance with a 20% wilting rate. The number of nematodes recovered from Clone 8-4 shoots was significantly lower(P = 0.05) than from Clones 5-10 and 16-4. This work contributes to the breeding of PWN resistance in P.densiflora.

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.

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.