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。

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。

防治松材线虫病的一种新型复配注干剂增效配比筛选

为了筛选出一种新型的防治松材线虫病复配药剂增效配比,将氟吡菌酰胺和甲维盐按照不同的比例进行复配,测定了两种药剂在不同配比时的增效情况,以及对松材线虫产卵、卵孵化的影响。结果表明:将氟吡菌酰胺和甲维盐按照不同的比例进行复配,对防治松材线虫病具有一定的增效效果,氟吡菌酰胺与甲维盐质量比为3∶5时的共毒系数为334.673 1,在7组不同配比中达到最高,在质量比为5∶3、2∶6、4∶4,1∶7时,共毒系数分别为159.768 2,215.704 1,126.850 2,122.429 5,均表现出较高的增效作用。并且在质量比为3∶5、5∶3、2∶6时,松材线虫的产卵量和卵孵化率都低于两种药剂单独使用时的结果。

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。

松材线虫泛素结合酶基因家族生物信息学与表达分析

松材线虫引起的松材线虫病对东亚、欧洲等多个国家和地区的松科植物破坏严重,威胁着世界森林生态系统的稳定发展。泛素结合酶(ubiquitin conjugating enzyme,UBCs)通过蛋白酶体降解靶蛋白,在生物体生长发育及逆境胁迫应答等方面发挥着重要作用。通过松材线虫基因组分析获得了20个泛素结合酶家族成员,对不同发育状态的松材线虫泛素结合酶家族基因进行理化性质、蛋白结构及在松材线虫发育过程中表达水平分析。结果表明,基因染色体定位结果显示,泛素结合酶基因均匀分布于除第5条染色体外的其余染色体上。基因拓扑分析显示大部分泛素结合酶基因位于细胞内,基因结构分析显示泛素结合酶基因主要由2~8个外显子组成。外显子-内含子结构在进化上高度保守,与氨基酸序列的多态性一致,且在外显子上存在密码子偏好性。基因表达数据显示,泛素结合酶家族成员在不同生长发育状态中存在差异性表达。通过生物信息学和基因表达分析方法初步了解泛素结合酶在松材线虫的生长发育过程中所扮演的角色,对于了解松材线虫发育机制和构建分子防治靶标具有重要的指导意义和理论价值。

四类重大林业有害生物的防控有效性分析

利用普通最小二乘法的稳健标准误模型及似不相关回归模型,定量分析松材线虫Bursaphelenchus xylophilus、美国白蛾Hyphantria cunea、薇甘菊Mikania micrantha及林业害鼠(兔)4类重大林业有害生物的防控有效性。结果显示:预防措施可以显著使松材线虫、美国白蛾及薇甘菊的发生面积呈递减趋势,防治资金的使用效率为抑制薇甘菊扩散起到显著作用。对于松材线虫病的防控,应加强生物化学农药在媒介昆虫防治中的应用,提升防控资金的使用效果;轻度发生的美国白蛾可采用综合防治措施,中度及重度发生首选生物化学农药防治;薇甘菊主要采用化学及营林防治措施,轻度发生时可结合采用人工物理防治;轻度发生的林业害鼠(兔)可采用综合防治措施,中度及重度发生采用生物、化学及人工物理防治,当重度发生时,营林措施控制灾害扩散的效果最好。

松材线虫病监测普查与防治技术

为了有效控制松材线虫病疫情的发生,对松材线虫病监测普查与防治技术进行了探讨。提出应根据松材线虫病的发病特点,先从监测范围、监测时段、监测的内容、监测方法等方面制定监测普查方案,再从取样方法、分离鉴定等方面确认疫情和疫情详查,最后根据疫情情况从检疫检验、物理防治、化学防治、生物防治4方面进行防治,从根本上控制松材线虫病的扩散。

秋冬季松材线虫在马尾松中的分布特征

秋冬季是松材线虫病普查及枯死松树采伐的重要时期,探究该时期松材线虫在马尾松中的空间分布特征,可有效提高松材线虫检测效率。通过松材线虫分离和形态学观察,研究秋冬季松材线虫在马尾松不同部位的分布特征。结果表明,秋季松材线虫在不同部位的检出率和数量分别为66.67%~78.00%和321.83~429.56条·g-1,显著高于冬季的33.33%~52.67%和33.00~52.82条·g-1。同时,秋季松材线虫在马尾松主干内的分布为8~11 m数量较多,尤其是距离顶梢1 m的位置,虫口密度达到最高的1349.50条·g-1。说明秋季更适合开展松材线虫病普查,在这一时期,马尾松主干的一级侧枝和主干上部是重点取样检测部位,利于提高松材线虫检出率和检测效率,缩短检测鉴定时间,降低调查成本。

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.

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.

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.

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.

砧木及抗性马尾松松脂组分对松材线虫的响应

[目的]通过对马尾松接种松材线虫,分析接种前后松脂化学组分的变化,为马尾松抗松材线虫病的研究提供理论基础。[方法]以浙江省临海市5年生马尾松无性系为研究对象,提取接种前和接种松材线虫1、7、15 d的不同砧木(马尾松砧木和湿地松砧木)、不同抗性(高抗和易感)马尾松的松脂组分,分析各松脂组分含量及动态变化;利用松脂内所得有效萜类对松材线虫实施外源处理,测定松材线虫存活率。[结果]从不同砧木(马尾松砧木和湿地松砧木)、不同抗性(高抗和易感)马尾松中检出19种主要化学组分,其中,α-蒎烯、β-蒎烯、水芹烯、海松酸、山达海松酸、长叶松酸/左旋海松酸、去氢枞酸、枞酸和新枞酸含量较高。接种松材线虫1 d时马尾松砧木中β-月桂烯含量显著高于湿地松砧木(p<0.05);接种松材线虫7 d与15 d时上述组分含量在不同砧木间无显著差异。不同抗性马尾松中α-蒎烯、β-蒎烯、β-月桂烯、柠檬烯、龙脑、长叶烯、α-石竹烯、反式-β-金合欢烯和新枞酸含量在接种松材线虫后变化规律不同,接种松材线虫1 d时高抗马尾松中α-蒎烯和β-蒎烯含量显著高于易感马尾松;接种松材线虫7 d时高抗马尾松中α-蒎烯、β-蒎烯、柠檬烯和长叶烯含量显著高于易感马尾松(p<0.05);接种松材线虫15 d时高抗马尾松中α-蒎烯、β-蒎烯、柠檬烯和长叶烯含量显著高于易感马尾松(p<0.05)。对上述萜类组分进行梯度浓度松材线虫外源试验:以150 mg·g^(−1)浓度α-蒎烯溶液处理0.5 h时,松材线虫存活率达20%;不同浓度β-蒎烯溶液对松材线虫抑制作用相同,1.20 mg·g^(−1)浓度处理0.5 h时松材线虫存活率达50%左右;以10 mg·g^(−1)浓度柠檬烯溶液处理0.5 h时,松材线虫存活率趋于零;以10 mg·g^(−1)浓度长叶烯溶液处理0.5 h时,松材线虫存活率达18%。[结论]不同砧木对马尾松抗性无明显作用;接种松材线虫后,α-蒎烯、β-蒎烯、柠檬烯和长叶烯含量存在显著差异,且均为高抗马尾松显著高于易感马尾松。α-蒎烯、β-蒎烯、柠檬烯和长叶烯低浓度下抑制松材线虫活性,与马尾松抗性有关。

松材线虫黄素单加氧酶家族基因结构与表达模式分析

黄素单加氧酶(flavin-containing monooxygenases,FMOs)是一类广泛存在于植物、动物、微生物中,主要参与调控生物体对内源性及外源性物质代谢的生物酶家族。为探究松材线虫FMOs(Bursaphelenchus xylophilus FMOs,BxFMOs)在宿主中定殖和响应杀线剂胁迫过程中所发挥的功能,本研究首先通过生物信息学手段从松材线虫基因组中筛选并鉴定了13个BxFMOs家族基因,随后对获得的BxFMOs家族基因进行理化特性、进化发育、蛋白结构及基因表达模式分析。结果表明:BxFMOs家族成员分布于5条染色体上,氨基酸数目为432~572个,分子量为49.77~66.09 kDa,等电点为6.26~9.27,其结构较为保守。基因表达模式分析揭示,BxFMOs家族对松材线虫定殖和响应杀线剂胁迫具有显著影响。本研究对探索松材线虫在宿主中的定殖机制和寻求防治松材线虫分子靶标具有重要的指导意义和理论价值。

花绒寄甲与松褐天牛及松材线虫病三者间长效关系研究

利用天敌昆虫开展生物防治是一条优良的、长效的措施,花绒寄甲是控制松褐天牛防治松材线虫病的重要优势天敌。为评价花绒寄甲林间应用效果,于2020—2022年在江西部分疫区开展了“疫木不砍伐或不清除,只释放花绒寄甲”试验,通过对林间松褐天牛种群密度监测、花绒寄甲寄生率及病死树下降率调查,研究了花绒寄甲林间种群增加量、松褐天牛种群下降率与病死树减退率的关系。结果显示:花绒寄甲对松褐天牛的最高寄生率达94.19%,平均寄生率由2020年的13.19%增加到2022年的72.44%,提高5.49倍;病死株数量在前2 a下降不明显,直至第3 a之后,病死树数量才急剧下降,病死树校正减退率平均为68.00%,其中马尾松纯林的校正减退率为72.37%,显著高于松阔混交林的校正减退率(59.26%);松材线虫病的病株率随松褐天牛种群密度降低而降低,呈显著正相关;与防治前(2019年)相比,2022年的松褐天牛种群密度下降58.43%~98.67%,平均下降了89.43%,而花绒寄甲的种群数量却呈现急剧增加,3 a平均上升221.03%,这说明,在疫木不清除条件下,通过3 a的天敌释放,能大幅度提高林间自然花绒寄甲虫口密度,同时显著降低害虫天牛的虫口密度,显著降低松材线虫病的病死树数量,从而达到持续控制松材线虫病的目标。

沈阳市油松松材线虫病发病规律观察

为了明确自然状态下油松感染松材线虫后的发病规律,采用直接观察法、流脂观察法、松材线虫分离鉴定等方法对沈阳市东陵公园内约70 a生油松大树发病过程及症状特征进行了持续性观察。结果表明:感病油松表现为当年、越1年和越2年枯死,以越1年枯死类型为主。油松受害初期,不同小枝的同一时间受害症状不同,枯死针叶在树冠上无规则星状分布,受害后期,树冠下小枝发病过程整齐统一。油松枯死5~6个月后针叶开始脱落。枯死油松胸径以下树皮上多有天牛危害痕迹,无流脂现象。该结果可为东北地区松材线虫病的防治提供依据。