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。

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.

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.

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.

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。

湖南省松材线虫病防控现状与对策

松材线虫是极具危险性的外来入侵物种,是重大植物疫情来源之一,严重威胁生态安全、生物安全和经济发展。为适应新形势下松材线虫病疫情防控工作,开创防控工作新局面,笔者选取32个松材线虫病重型疫区、18个轻型疫区和9个预防区开展实地调查,广泛征求地方政府、林业主管部门、乡镇林业站、林农和相关防控企业的意见建议,采取查看现场、走访询问、问卷调查、座谈研讨等多种方式,对当前防控现状、存在的问题开展深入调查,针对性地提出加强我省松材线虫病防控工作的对策。

灰葡萄孢培养基种类和继代培养对松材线虫致病力的影响

本试验在8种培养基上培养灰葡萄孢Botrytis cinerea,以灰葡萄孢喂饲松材线虫Bursaphelenchus xylophilus,采用3年生黑松Pinus thunbergii作为接种材料,研究松材线虫繁殖力和致病力的变化,同时探究了在PDA-灰葡萄孢上继代培养84代的松材线虫致病力的变化。结果表明,以玉米、小麦和PDA 3种培养基培养的灰葡萄孢喂饲松材线虫,其繁殖力显著高于CDA和4种合成培养基,其中PDA-灰葡萄孢上的松材线虫数量最多,比合成培养基C或D培养的灰葡萄孢上的线虫数量提升了48.94%;接种PDA-灰葡萄孢组松材线虫的黑松病情指数最高,较最低的合成培养基D-灰葡萄孢组高了47.87%。松材线虫在PDA-灰葡萄孢上继代培养至第14代后,继代次数的增加会导致其致病力下降,接种第79代松材线虫后黑松病情指数已为0。因此,为保证松材线虫的致病力不衰退,应使用PDA-灰葡萄孢培养基喂饲松材线虫,且松材线虫继代培养应控制在14代以内。研究结果将为松材线虫的繁殖技术与致病相关机理分析等提供参考。

松材线虫潜育期延长导致红松越年枯死

【目的】探究红松越年枯死的病理学过程和产生原因,以期为有效除治无松材线虫病症状的携带者松树提供理论依据。【方法】统计抚顺市大伙房实验林场自然侵染红松越年枯死比例,观测自然侵染和人工接种红松病症病状,测定红松当年显症的松材线虫数量阈值。实验室内不同温度下测定松材线虫虫口增长情况,以及媒介昆虫羽化后适宜松材线虫虫口有效增长的时期(虫口增长期),计算从寄生关系的建立到症状开始出现的时间(潜育期),并揭示红松感染松材线虫后越年枯死的原因。【结果】截至6月,自然侵染红松的越年枯死比例为41.4%。观测的自然侵染和低剂量人工接种红松在被侵染或接种当年均未出现显著外部、内部症状变化,而是到翌年松脂分泌停止,树干水分低,针叶变红褐色,只有进行高剂量饱和接种才出现当年半数显症。病症表现为松材线虫能够正常侵染红松,可有效繁殖但在低温时繁殖受阻,可成功越冬但越冬后虫口数量骤减。红松显症的全株阈值St1=(163±22)条·g^(-1),局部阈值St2=(621±20)条·g^(-1)。以每株50000条的接种量接种38年生红松,在当年虫口增长期内全株松材线虫数量仅为(90±8)条·g^(-1),低于St1、局部松材线虫数量最高为(350±18)条·g^(-1),也低于St2。抚顺的虫口增长期(141天)比大连(154天)和南京(173天)短。经计算,抚顺市松材线虫的潜育期为146天,长于虫口增长期。【结论】云杉花墨天牛携带的松材线虫数量相对较低,导致初始侵染量低。松材线虫生命周期受温度特性的影响,表现为非增长期长、虫口增长期短,最终导致在虫口增长期内未能达到红松显症的松材线虫数量阈值。这些综合作用导致松材线虫潜育期延长,其中41.4%的死亡红松表现出越年枯死现象。

温度调控ALP1表达对叶状枝节丛孢捕食松材线虫效率的影响

转录组测序筛选并克隆了叶状枝节丛孢丝氨酸蛋白酶基因ALP1,并通过生物信息学分析揭示了其对松材线虫捕食效率的影响。结果表明:ALP1编码的蛋白质是一个409个氨基酸残基的分泌性蛋白,分子质量为41.89 ku,等电点为6.45,表现出稳定且亲水的蛋白质特性。ALP1蛋白无跨膜螺旋,含有信号肽,预测的切割位点位于第21至22个氨基酸残基间。ALP1蛋白含有丝氨酸蛋白酶家族的保守基序,其二级结构主要由α螺旋、无规卷曲和β-折叠组成,与Drechslerella coelobrocha丝氨酸蛋白酶基因具有高度同源性。实时定量PCR(RT-qPCR)结果表明,ALP1基因表达水平与叶状枝节丛孢的捕食效率呈正相关,尤其在28℃时表达显著上调。在18~32℃梯度范围内,以28℃为叶状枝节丛孢捕食效率最优温度条件。GO分类和通路富集分析进一步确认了ALP1在叶状枝节丛孢适应环境和捕食过程中的核心作用。ALP1基因在叶状枝节丛孢适应温度变化和提高捕食效率中起到关键调控功能,还为松材线虫病的生物防治提供了新的策略和基因资源。

松材线虫侵染对油松的生理生化影响

为研究油松Pinus tabuliformis感染松材线虫Bursaphelenchus xylophilus后针叶内一些生理生化物质的变化规律,以5 a生油松盆栽苗为对象,在接种松材线虫后0,5,10,20,30 d测定油松针叶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)活性及渗透调节物质和次生代谢物质的含量。结果表明:接种松材线虫后,油松针叶POD活性整体表现为较低水平,接种后5,20 d处理组POD活性均显著低于对照组;CAT活性在接种后30 d显著高于对照组;SOD活性呈现先小幅下降后逐渐升高的趋势,接种后30 d达到峰值,显著高于对照组;PAL活性呈现出持续升高趋势,且在接种后20 d和30 d与对照组相比达到极显著差异水平;可溶性蛋白含量表现为先升高后降低,在接种后10 d达到峰值,在接种后10 d和20 d时显著高于对照组;甜菜碱含量总体呈现较高水平,接种后5 d,处理组甜菜碱含量达到峰值,显著高于对照组;黄酮含量呈现出先升高后降低的变化趋势,且在接种后20 d和30 d,处理组黄酮含量极显著高于对照组。油松幼苗被松材线虫侵染时多种防御酶活性及生理生化物质会发生动态变化,诱导植株产生防御反应。

粘质沙雷氏菌BRC-CXG2对松材线虫的防治效果及全基因组分析

为验证粘质沙雷氏菌Serratia marcescens BRC-CXG2对松材线虫Bursaphelenchus xylophilus的杀线活性,挖掘有效的杀线代谢产物,明确菌株生防机制,利用生物学测定验证粘质沙雷氏菌BRC-CXG2菌株杀线活性,基于Nanopore测序平台对菌株进行全基因组测序,采用生物信息学技术进行预测和功能注释,并通过生测检验从粘质沙雷氏菌BRC-CXG2菌株分离出的灵菌红素对松材线虫的毒力。结果表明:粘质沙雷氏菌BRC-CXG2对松材线虫具有较强的毒力,处理48 h的杀线LC_(50)为4.36×10^(4)CFU/mL。全基因组长5462607 bp,GC含量为59.36%,预测编码基因5087个,长4767972 bp。基于Nr数据库共注释基因5062个,其中包含灵菌红素、几丁质酶等杀线代谢物的相关基因。灵菌红素同样具有良好的杀线活性,处理48 h的杀线LC_(50)为20.17 mg/L。该结果可为该菌株的研究和开发利用提供参考。

蜡样芽孢杆菌NJSZ-13中蛋白酶ATP-α与ClpX的杀线虫活性研究

【目的】阐明蜡样芽孢杆菌NJSZ-13中杀线蛋白酶ATP-α与ClpX的杀线分子机制,构建杀线蛋白酶ATP-α与ClpX的原核表达载体。【方法】克隆杀线蛋白酶编码基因atpA与clpX,利用基因工程手段对杀线蛋白酶编码基因进行PCR扩增,并与载体pET-21b连接构建重组载体,提取重组质粒转入蛋白表达载体大肠杆菌BL21(DE3)。利用菌落PCR和测序验证转化效果。加入IPTG诱导蛋白表达,使用Ni-NTA柱进行重组蛋白的纯化,利用SDS-PAGE与Western blot验证ATP-α与ClpX纯化效果并进行杀线活性测定。【结果】菌落PCR验证和测序表明,重组载体中含有蛋白酶编码基因atpA与clpX,且基因序列与参考序列一致,证明目的基因已经成功插入BL21(DE3)表达载体中。SDSPAGE与Western blot验证表明,重组蛋白得到正确诱导与纯化,成功构建杀线蛋白酶ATP-α与ClpX的原核表达载体。杀线活性测定结果表明ATP-α与ClpX均具有较强的杀线效果。ATP-α在72 h达到66.75%的杀线率,ClpX在72 h达到75.46%的杀线率。同时,两个蛋白酶共同作用杀线能力显著增强,24 h便达到66.32%的杀线率,72 h杀线率达到91.01%。【结论】ATP-α与ClpX经原核表达及纯化后具有较高的杀线活性,且两者共同处理松材线虫,杀线效果更强,证明蛋白酶ATP-α与ClpX是重要的杀线因子,为设计和筛选杀线药物提供了新的线索和依据。

4种烟碱受体激动剂对松材线虫的毒杀活性

【目的】探究4种烟碱受体激动剂对松材线虫的杀虫作用。【方法】采用浸渍法以及马尾松松枝水培试验分别进行杀线虫活性测定,并测定4种烟碱受体激动剂水溶液对松材线虫体内三磷酸腺苷酶(ATP)、过氧化氢酶(CAT)、乙酰胆碱酯酶(AChE)、超氧化物歧化酶(SOD)的影响。【结果】盐酸左旋咪唑、盐酸四咪唑、烟碱、酒石酸噻吩嘧啶4种烟碱受体激动剂对松材线虫有良好的毒杀活性,4种药物水溶液浸渍处理松材线虫48 h,半致死浓度LC_(50)值分别为0.09、0.31、0.08、0.14 mg·mL^(-1);马尾松水培试验处理12 d后结果显示,盐酸左旋咪唑、盐酸四咪唑、烟碱、CK处理组松枝均不发病,病情指数为0,酒石酸噻吩嘧啶处理组部分枝叶变黄,病情指数为31%,“CK+线虫”松枝枝叶完全变黄萎蔫,病情指数为100%;4种烟碱受体激动剂水溶液浸渍处理48 h后松材线虫体内ATP、CAT、AChE、SOD酶活性均低于对照组(P<0.05)。【结论】盐酸左旋咪唑、盐酸四咪唑、烟碱、酒石酸噻吩嘧啶对松材线虫有良好的防治潜力,具有进一步深入研究和开发利用的前景。