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。

关于Bursaphelenchus中译名的商榷

鉴于在国内Bursaphelenchus学名的中文名被写成"伞滑刃线虫",作者指出其确切的中文名应当是伞真滑刃线虫,同时,指出其所隶属的Bursaphelenchinae应叫做伞真滑刃亚科,而不应叫做"伞滑刃亚科".此外,该亚科下的另外2个属,Parasitaphelenchus应叫做寄生真滑刃属,而不应叫做"寄生滑刃属",Rhadinaphelenchus应叫做细杆真滑刃属,而不应叫做"细杆滑刃属".

Risk Assessment and Analysis of Bursaphelenchus Species in the Coniferous Forest in Yuyao, Ningbo

The method for pest risk analysis was used to make the potential risk analysis and assessment to the 8 Bursaphelenchus species found in the pines in Yuyao, Ningbao, and the corresponding risk management strategies were put forward.

First record of Bursaphelenchus rainulfi on pine trees from eastern China and its phylogenetic relationship with intro-genus species

Bursaphelenchus rainulfi isolated from dead pine trees in Zbejiang, China, is described and illustrated. It also provided some molecular characters of the Chinese population, including the PCR-RFLP and sequences of ITS region and D2-D3 expansion region of the large subunit （LSU） rRNA gene. Both the morphological characters and ITS-RFLP patterns match with the original description. The phylogenetic trees based on the 13 sequences of D2-D3 expansion region of the LSU rRNA gene and ITS region of Bursaphelenchus species were constructed, respectively, with the results showing the similar clades. The phylogenetic relationship based on the molecular data is similar to that with morphological characters. This is the first report of the species on pine wood in eastern China.

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.

Recent Advances in Classification and Molecular Biology of Bursaphelenchus Species

This paper mainly summarizes recent advances in the classification of Bursaphelenchus species, introduces several common methods of molecular biologi- cal detection and identification methods, and analyzes the advantages and disad- vantages of these methods.

Growth and decline of arboreal fungi that prey on Bursaphelenchus xylophilus and their predation rate

Pine wilt disease caused by pine wood nema-todes is a deadly disease of the genus Pinus requiring strong quarantine measures.Since its discovery,it has been widely distributed throughout the world.China is one of the coun-tries with a severe rate of infections due to its abundant pine resources.In this study,nematode-trapping fungi were collected from pine trees in Ninghai City,Zhejiang Prov-ince,which is the key area of pine wilt control in Febru-ary,May,September,October and November.The results showed that nematode-trapping fungi of pine are abundant,especially the number and species detected in each month and are quite different;species of fungi in July,Septem-ber and November were more numerous and had higher separation rates.The dominant species in November was Arthrobotrys oligospora followed by Arthrobotrys cladodes;July and September it was A.cladodes.In each part of the tree,A.cladodes was the most widely distributed with a high separation rate.Therefore,it is the dominant fungal species in the area.Arboreal Bursaphelenchus xylophilus-trapping fungi are dependent on pine wood nematodes,and none were isolated from healthy pine trees.More arboreal B.xylophilus-trapping fungi were found under the bark than in the xylem.There is a close relationship between arboreal B.xylophilus-trapping fungi and pine wood nematodes.How to make use of these resources to reduce the damage of pine wood nematode fusarium is the focus of future research.

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.

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.

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.

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。

福建省松树线虫的研究 I.松干伞真滑刃线虫的种类

自2001年7月至2002年12月在福建省各地共采集松树木质部样品256份,分离发现88份(34%)样品中含有伞真滑刃属Bursaphelenchus的线虫8种,本文先报道其中的4种:拟松材线虫Bursaphelenchus mucronatusMamiya&En-da,1979、赫列尼克斯伞真滑刃线虫B.hellenicus SkarmoutsosG.et al.,1998、霍夫曼尼伞真滑刃线虫B.hofmanniBra-asch,H.,1998、奇异伞真滑刃线虫B.aberransFang,et al.,2002.

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.

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。