Pathogenicity studies with Bursaphelenchus mucronatus in Scots pine in Finland

The objective of the study was to determine the role of Finnish Bursaphelenchus mucronatus as a possible pathogen in Scots pine, Pinus sylvestris, under Finnish ambient weather conditions. Small seedlings and young field grown trees were used as inoculation targets. The results suggested the nematode being of minor importance as a tree pathogen in Finland.     

Pathogenicity of Bursaphelenchus sexdentati,Bursaphelenchus leoni and Bursaphelenchus hellenicus on European pine seedlings

In Greece extensive pine wilting is associated with the presence of nematodes of the genus Bursaphelenchus. In order to check the pathogenicity of Bursaphelenchus sexdentati, Bursaphelenchus leoni and Bursaphelenchus hellenicus, inoculation tests were carried out on 3‐year‐old seedlings of Pinus brutia, Pinus halepensis, Pinus nigra, Pinus pinaster and Pinus sylvestris. For inoculations, 6000 nematodes in 0.5 ml of water were used per seedling. Bursaphelenchus sexdentati proved to be highly virulent causing mortality of up to 100%, followed by B. leoni whereas B. hellenicus was nonpathogenic.     

不同寄主拟松材线虫雌虫虫株间的形态变异

从5种不同松树上分离到拟松材线虫24个虫株,并在高倍显微镜下对其雌成虫的12个形态指标进行了测计,并以这些形态指标值作为形态学变异研究的依据。结果表明,拟松材线虫雌成虫的平均体长约为954.41μm,最长的能达1 312.50μm,最短为527.80μm;24个不同虫株的拟松材线虫雌成虫同一形态指标的变异系数在不同的虫株间各不相同,这反映了拟松材线虫形态多样性是很丰富的。但雌成虫的生殖系统指标(V’、V)和消化系统口针长度变异度较小,可以作为拟松材线虫的重要鉴定特征。不同寄主树种上的拟松材线虫雌成虫体长的均值大小依次为黑松、华山松、湿地松、马尾松、火炬松。借助于SPSS 13.0方差分析表明,不同寄主树种上的拟松材线虫雌成虫绝大部分的形态指标在0.01水平上差异显著,也就说寄主树种因素对拟松材线虫形态的分化有显著性影响。     

拟松材线虫研究进展

松材线虫和拟松材线虫共同构成松材线虫复合群体,为松材线虫病的病原物。拟松材线虫危害过去没有引起足够重视,相关研究工作不如松材线虫细致。近年来,随着拟松材线虫致病性增强现象不断被报道,人们逐步加强了拟松材线虫研究。本文从拟松材线虫分类学特征、致病性研究及与松材线虫关系等角度对拟松材线虫研究进展进行综述,并展望未来可能的研究热点。     

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.     

松材线虫与拟松材线虫的几种鉴别方法

在形态、生化与分子以及致病性3个方面综述了松材线虫和拟松材线虫的鉴别方法,旨在为松材线虫病的防治和进出口检疫提供更多更安全的检疫方法,同时为更好的区别两种线虫提供理论依据。     

Reproductive success of Bursaphelenchus xylophilus and B. mucronatus under Finnish summer temperature conditions

The objectives of this study were to determine the reproductive potential and the time required for completion of the life cycle of Bursaphelenchus xylophilus under Finnish summer temperature conditions. The reproductive success of B. xylophilus was compared with the potential of a Finnish isolate of B. mucronatus. The latter appeared more successful in reproduction particularly when cultured on Finns syhestris wood.     

松材线虫病致死松树上的松褐天牛季节性分布规律

连续两年定期监测、调查采样和鉴定证明：每年６～１０月出现的松材线虫病Ｂｕｒｓａｐｈｅｌｅｎｃｈｕｓｘｙｌｏｐｈｉｌｕｓ致死松树上，松褐天牛Ｍｏｎｏｃｈａｍｕｓａｌｔｅｒｎａｔｕｓ虫株率为１００％，而１１月至次年５月间出现的病死株上，虫株率为零。对前者应集中砍除，并加以严格的除害处理；后者可经剥皮处理后在疫情发生区内加工利用，部分可留在林间作为天牛产卵的饵木     

宁波市东部用二阶原虫除害法防治松材线虫病效果评价

本文认为用二阶原点除害法清理松材虫病死树能取得次年病死树防治效果（减退率下降５３．２％）,部分林地防治无效是由于防治质量差,松林火灾、人为传播和自然传播因子所致。     

松树抗松材线虫病机理的研究进展

松材线虫病 [Bursaphelenchusxylophilus(Steiner&Buhrer)Nickle]是一种毁灭性的森林病害。不同松种和同一松种不同种源的抗病性存在明显差异 ;松树通过固有或诱导的防卫系统抵抗松材线虫的扩散和繁殖。本文就国内外关于松树抗松材线虫病机理方面的研究进行综述     

贵州省拟松材线虫病的风险性分析

采用多指标综合评价方法对贵州省拟松材线虫病的危险性进行了分析。通过风险性分析和计算,得出拟松材线虫是松树的重要病害种类,其R值为1.74,属于中度危险的林业有害生物。在此基础上,探讨了贵州省拟松材线虫病的风险管理方法。     

Genetic diversity of pine‐parasitic nematodes Bursaphelenchus xylophilus and Bursaphelenchus mucronatus in China

The internal transcribed spacer (ITS) regions of rDNA have been routinely employed for identification and phylogenetic analysis of many nematode species. In this study, the intra‐ and interspecies ITS genetic diversity of Bursaphelenchus xylophilus and Bursaphelenchus mucronatus was evaluated. Ninety‐one isolates of the two nematode species collected from 14 Chinese provinces, Japan and Korea were used for ITS‐PCR and sequencing. An unweighted pair group cluster analysis dendrogram clustered them as two B. mucronatus and one B. xylophilus independent clades. Principal component analysis showed the phylogenetic relationship of the two nematode species more clearly; B. mucronatus isolates were separated into more than four groups, whereas B. xylophilus isolates still clustered into a group. The results of the Mantel test indicated the correlation of genetic distance matrices and geographic distance matrices was significant for both nematode species. The genetic differentiation coefficient (G_st) and gene flow (N_m) of B. mucronatus were 0.341 and 1.091, respectively, suggesting the importance of landscape heterogeneity and considerable obstacles for genetic exchange among B. mucronatus isolates in China. However, G_st and N_m of B. xylophilus were 0.188 and 2.151, respectively, very different compared to B. mucronatus. This could be owing to the short‐term introduction of B. xylophilus into China and a rapid spread through anthropogenic pathways. Our work adds to the understanding of the genetic diversity and genetic relationship of the two pine‐parasitic nematode species, and will aid in controlling them in the future.     

Discrimination of Bursaphelenchus xylophilus and Bursaphelencus mucronatus by PCR-RFLP technique

A polymerase chain reaction-restriction fragment length polymorphism analysis was used to discriminate isolates of Bursaphelenchus xylophilus and B. mucronatus. The amplifications of B. xylophilus isolates yielded one fragment of approximately 890 bp and that of B. mucronatus was about 930 bp. Digestion of amplified products of each nematode isolate with five restriction endonucleases revealed the following results: 1) Dra I digestion of the internal transcribed spacer (ITS) products of B. xylophilus populations yielded two fragments of 510 and 380 bp. Dra I could not digest the ITS products of B. mucronatus populations; 2) Sal I could not digest the ITS products of all B. xylophilus populations, but it could digest those of B. mucronatus populations into two fragments, which were 720 and 220 bp; 3) digested products of four B. xylophilus populations by Msp I yielded two fragments of 530 and 360 bp, except GZ02, which could not be digested. B. mucronatus populations yielded three fragments: 340, 290, and 180 bp; 4) all populations of B. xylophilus and B. mucronatus could not be digested by Apa I; 5) digestion of the ITS products of B. xylophilus and B. mucronatus yielded two fragments of 520 and 370 bp, and 530 and 400 bp respectively. The restriction endonucleases Dra I and Sal I could be used to identify B. xylophilus and B. mucronatus. Because the results of digestion of B. xylophilus and B. mucronatus were markedly different, they were very easy to be identified and applied; Msp I and Xho I were not suitable for identification of B. xylophilus and B. mucronatus and Apa I could not identify and distinguish between B. xylophilus and B. mucronatus. __________ Translated from Journal of Nanjing Forestry University, 2005, 30(4): 5–9 [译自: 南京林业大学学报]     

松材线虫与拟松材线虫迁移能力的比较

松材线虫病,又称松树萎蔫病、松树枯萎病,是由松材线虫(Bursaphelenchus xylophilus)引起的一种毁灭性森林病害。松材线虫原来广泛分布于北美地区,但在原产地并未造成严重危害。1905     

松材线虫在致病松树上的分布与采样的关系

通过对松村线虫在致病松树上的分布调查，在马尾松病树上的垂直分布，随着感病程度加深，在老发生区，小枝上的分布逐渐减少，倒枝、胸部和基部树干上的分布逐渐增多；在新发生区，树干胸部和基部的分布无明显差异，但以胸部为多，侧枝、小枝上的分布逐渐增多。在黑松病树上，普遍分布在新、老发生区中不同症状类型病树的各个部位。松材线虫虫量在黑松中明显高于马尾松，尤其在枝条上。在马尾松和黑松树干胸高处的水平分布，一般内材虫量多于外边材。为此，建议对该2个树种采样，样树应选择感病优势木、林缘木；时间以症状前期为主，分批采样；部位上级树干胸高处为主，多点采样，特别是马尾松。     

不同株系拟松材线虫对黄山松和黑松苗木的致病性

采用23个不同来源的拟松材线虫株系对2年生黄山松和2年生黑松进行人工接种试验.结果表明:1)对黄山松苗具有致病作用的拟松材线虫有4个株系,分别为CFS4,SLD3,ZJ1和KOR1.其中接种ZJ1,SLD3 2个株系的黄山松苗的死亡率均为16.7%.2)对黑松具有致病作用的拟松材线虫有9个株系,分别是CFL1,SFS1,SFS4,SLD9,SDZ1,ANL5,ANL7,AHS9和HYC1.感病指数最高的ANL5达到83.3.其次是AL7,感病指数为54.2.发病率最高的拟松材线虫株系同样是ANL5,发病率达100%.死亡率最高的拟松材线虫系是ANL5和ANL7,达50%.3)根据拟松材线虫对2年生黑松苗的感病指数、发病率、死亡率等指标,将供试的23个拟松材线虫株系划分为3个致病等级:Ⅰ级(强致病性):ANL5和ANL7个株系;Ⅱ级(弱致病性):CKL1,SFS1,SFS4,SLD9,SDZ1,AHS9和HYC1 7个株系;Ⅲ级(无致病性):ANL1,JLH1,JLH10,JXY7,JYX31,CFL4,SLD3,YWF1,GBL3,ZJ1,HN1,TW1,JAP1和KOR1 14个株系.4)来自安徽的4个拟松材线虫株系,有3个株系对2年生黑松苗具有致病作用,其中2个株系(ANL5,ANL7)具有强致病作用;来自四川的5个拟松材线虫株系,有4个株系(SFS1,SFS4,SLD9,SDZ1)对2年生黑松苗具有中等强度的致病作用,而另外1个株系(SLD3)对2年生的黄山松苗具有致病作用;来自江苏的4个拟松材线虫株系(JLH1,JLH10,JXY7,JYX3)对2年生黄山松苗、2年生黑松苗均无致病性.     

A new on‐site detection method for Bursaphelenchus xylophilus in infected pine trees

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease (PWD), which is a major problem in East Asia and West Europe. Quick identification of PWN is needed to prevent the dispersal of PWD to healthy forests. Various detection methods of PWN have been developed using anatomical characters and molecular markers. These methods are not suitable for rapid diagnosis because it is difficult to distinguish B. xylophilus from the non‐pathogenic species Bursaphelenchus mucronatus based on morphological characters without expertise in nematode taxonomy and most PCR or isothermal amplification detection methods require time‐consuming processes. In this study, we developed an on‐site PWN detection method using a recombinase polymerase amplification (RPA) assay with a novel extraction buffer (DAP buffer). This new PWN detection method is able to extract genomic DNA from PWN in pinewood by simple buffer consisting of sodium hydrate, polyethylene glycol 200 and dimethyl sulfoxide in 10 min without using the experimental devices and able to distinguish between B. xylophilus and other Bursaphelenchus spp. by amplifying the species‐specific 5S rDNA fragment of B. xylophilus in 10 min. Taken together, our protocol can obtain the result for the detection of PWN in pine tree samples within 30 min. This result suggests that RPA/DAP assay is much faster, easier and cheaper than the conventional methods for detecting PWN.     

Pathogenicity testing of four Bursaphelenchus species on conifer seedlings under greenhouse conditions

The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer, J. Agric Res. 48, 1934, 949), Nickle (J. Nematol. 2, 1970, 375), is the causative agent of the pine wilt disease and causes serious damage to pine forests around the world. During a survey for the pinewood nematode, four other Bursaphelenchus species (Bursaphelenchus mucronatus, B. sexdentati, B. anamurius and B. vallesianus) were isolated from wilted pine trees in Turkey. To understand the effects of these Bursaphelenchus species on wilting of pine trees, a study was conducted under greenhouse conditions. Two‐year‐old seedlings of three pine species (Pinus nigra, P. brutia and P. pinea) and one cedar species (Cedrus libani) were used. Fifteen seedlings of each species were inoculated with nematodes and 10 seedlings of each species served as controls. The inoculum densities used for each seedling contained approximately 1000 (±100) nematodes of all life stages in 0.25 ml of distilled water. The first wilting symptoms were observed in the fifth week in all pine species but not in the cedar seedlings. All seeding mortality occurred between the 5th and 13th weeks of the study; no mortality was observed outside of this period. The most pathogenic nematode species was B. mucronatus, closely followed by the other species. The most susceptible seedling species was P. nigra, and C. libani was the most resistant species.     

松材线虫与拟松材线虫杂交及后代的致病性

收集松材线虫5虫株与拟松材线虫1虫株进行生物学杂交,结果显示:松材线虫各虫株均能与拟松材线虫杂交且杂交率均较高,但杂交后代中普遍存在线虫死亡率较高的现象.用杂交后代线虫与未杂交亲本线虫分别接种2年生马尾松苗,结果表明杂交后代线虫对接种松苗的致病性小于其未杂交亲本线虫,从接种后萎蔫的松苗中再分离线虫,杂交后代线虫再分离数远少于亲本线虫数.     

松材线虫和拟松材线虫繁殖能力差异研究

松材线虫作为外来入侵种,逐步取代拟松材线虫占据生态位。初步研究表明这可能与两种线虫的繁殖能力差异关系密切。本研究采取了两种方法,即离体条件竞争试验和体内竞争试验比较二者繁殖力。结果表明,松材线虫的繁殖水平明显高于拟松材线虫。松材线虫和拟松材线虫混合物长时间培养后,松材线虫逐步竞争淘汰拟松材线虫。除此以外,离体培养和体内竞争结果均表明,竞争条件下松材线虫的繁殖力高于非竞争条件。