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。

上海佘山发现黑松松材线虫（Bursaphelenchus xylophilus）M型株系

１９８９～１９９１年对上海佘山濒死黑松调查中发现松材线虫（Ｂｕｒｓａｐｈｅｌｅｎｃｈｕｓｘｙｌｏｐｈｉｌｕｓ）的“Ｍ”型株系。该线虫形态学上带有某些拟松材线虫（Ｂ．ｍｕｃｒｏｎａｔｕｓ）特征，但它发现于尚未见枯萎症的染病松材中，并随着线虫密度增加病树进入枯萎和死亡。该株系与江苏六合群体（国内典型钝尾高毒力株系）在灭菌黑松木材上的繁殖系数相近，分别达３．３２和４．９６，这两个株系间的配对杂交产生了有尾突后代。

松材线虫(Bursaphelenchus xylophilus)耐久型幼虫(L_Ⅳ)携带的香茅醇假单胞杆菌(Pseudomonas citronellolis)分离与鉴定

[目的]目前有关松材线虫与伴生细菌的关系及伴生细菌的病原作用是松树枯萎病研究的重点。为了揭示松材线虫与伴生细菌之间存在的密切关系,作者对松材线虫L_Ⅳ幼虫携带的细菌进行了分离鉴定。[方法]根据培养性状和16S rDNA序列同源性以及系统发育学等方面进行分析鉴定。[结果]确定L_Ⅳ幼虫携带的是香茅醇假单胞杆菌(Pseudomonas citronellolis),携带率为100%;每条L_Ⅳ幼虫携带量在1.4×10~5~4.5×10~5。L_Ⅳ幼虫生活在松褐天牛体内,是引起松材线虫病侵染流行的唯一虫态;新发现的香茅醇假单胞杆菌能分解纤维素及降解或合成萜烯和酚类化合物。[结论]L_Ⅳ幼虫携带香茅醇假单胞杆菌的发现,揭示了松树、松褐天牛、松材线虫、细菌同为一体的紧密关系,并为揭示松树枯萎病机制提供了一种新病原和重要的研究思路。

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.

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.

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.

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.

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.

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".

中国新纪录种——东京伞滑刃线虫(Bursaphelenchus tokyoensis)

松材线虫是危害松树的重要病原线虫,在调查松材线虫时经常发现其他种类的伞滑刃线虫,明确中国的伞滑刃线虫种类有助于松材线虫的准确鉴定。2015年在广东省的黑松(Pinus thunbergii)上分离到的一种伞滑刃线虫属线虫,除体长比东京伞滑刃线虫(Bursaphelenchus tokyoensis)短及交合伞形状不同外,其余形态特征和测量值基本与东京伞滑刃线虫一致。此外,r DNA-ITS序列与东京伞滑刃线虫的r DNA-ITS序列一致性高达98.2%,基于r DNA-ITS序列的贝叶斯系统发育树表明,该线虫与东京伞滑刃线虫以100%的置信度聚在一起,且遗传距离小。形态学和分子生物学结果确定该线虫为东京伞滑刃线虫。东京伞滑刃线虫是中国新纪录种,黑松是该线虫的新寄主,同时该线虫也是继原始报道后的再次发现。

宁波地区马尾松中湖南伞滑刃线虫Bursaphelenchus hunanensis的鉴定

在宁波地区采集的4份马尾松中截获同一种伞滑刃线虫,经形态学观察、测量,鉴定为湖南伞滑刃线虫(Bur-saphelenchus hunanensisY in,Fang&Tarjan,1988),目前除澳大利亚和我国湖南省外,尚未有该线虫分布的报道。

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.

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.

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RPA等温扩增的松材线虫快速检测技术体系建立

利用松材线虫Bursaphelenchus xylophilus基因组分析和等温扩增技术,设计生产松材线虫野外快速检测技术产品并测试其效果。结果表明:比较分析松材线虫和拟松材线虫Bursaphelenchus mucronatus基因组发现松材线虫特异性基因Bx-gene12,以此为基础设计特异性扩增引物和探针,对采集自不同地区的松材线虫、拟松材线虫和松树内其他线虫株系采用研磨颗粒以1500 r/min转速震荡5 min的破碎方法有效释放线虫DNA,40℃等温扩增15 min反应条件下可有效区分松材线虫和其他线虫,检测灵敏度达到100 fg或1条线虫;该快速检测方法在25 min内有效检测出染病黑松Pinus thunbergii和赤松Pinus densiflora松木内的松材线虫。利用RPA等温扩增技术实现25 min内对野外环境下松材线虫的快速检测,可为松材线虫病及时有效防控提供支持。

松墨天牛监测及诱捕效果评价

为更好地对松墨天牛进行监测和诱杀,研究使用标记重捕法进行松墨天牛监测并选取3种引诱剂在林间诱捕,结果表明:在羽化初期诱集到的雄虫数量高于雌虫;添加天牛信息素可以有效提高引诱剂的效果,引诱到的雌虫数量高于雄虫;试验地内标记回捕率11.56%,占总诱捕量的6.45%,估算试验地内松墨天牛的种群数量为9 330头,雌虫4 582头,雄虫4 748头;北京中捷四方引诱剂的诱集数量、诱集纯度、诱集时效在3种引诱剂中最优,但对羽化1—20 d天牛的诱集效果不如杭州费洛蒙。以松墨天牛监测为目标时可以使用北京中捷四方引诱剂,以松墨天牛诱杀为目标时建议使用杭州费洛蒙引诱剂。

冠层尺度的无人机高光谱染病单木识别

基于马尾松感染松材线虫病后的光谱与纹理特征,探索机载高光谱影像在松材线虫病单木识别的应用效果与前景。本文分析了机载高光谱影像上不同程度染病与健康马尾松冠层的光谱特性,根据不同染病程度的光谱与纹理特征建立机载高光谱监督分类识别模型,并对不同监督分类方法识别结果进行分析与评价,提出适用于广东省的机载高光谱影像松材线虫病识别分类模型。研究表明,基于机载高光谱影像的松树冠层光谱曲线与地面非成像高光谱变化基本一致,这说明基于机载高光谱影像分析松树冠层光谱特性是可行的。同时,相较于光谱指数分析方法及高分辨率影像数据,基于高光谱影像的监督分类方法识别枯死松树可最大限度剔除裸土和阴影的干扰,但针对裸土边缘的干扰仍较难剔除。