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.     

A key effector,BxSapB2, plays a role in the pathogenicity of the pine wood nematode Bursaphelenchus xylophilus

The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses in pine forests. The plant‐parasitic nematodes have a complex life cycle that includes the secretion of effector proteins through a stylet into the host cell to promote parasitism. In this study, SignalP 4.1 and TMHMM 2.0 were used in preliminary screens for candidate effectors and were expressed in Nicotiana benthamiana through the PVX virus expression vector. The yeast signal sequence trap system was used to further study the function of the signal peptide of an effector, BxSapB2. In situ hybridization was conducted to investigate the localization of BxSapB2, followed by RNA interference technology (RNAi) to assess the functions of BxSapB2. The results demonstrate that BxSapB2 is a secreted protein that induces cell death in N. benthamiana and is highly expressed in esophageal gland cells and amphids of B. xylophilus. BxSapB2 was determined to be related to the pathogenicity of B. xylophilus. The results of this work indicate that BxSapB2 plays an important role in the interactions between B. xylophilus and the hosts.     

国内部分地区松材线虫和拟松材线虫基因多态性的RAPD分析

采用SDS法提取线虫基因组DNA,分别选取了6种随机引物对部分地区的10株松材线虫和10株拟松材线虫进行随机扩增和亲缘关系比较。RAPD的结果可以明显区分松材线虫和拟松材线虫,与形态学分类相吻合。聚类分析的结果也表明,线虫种内和种间均存在差异,但种间差异明显大于种内差异,同种线虫在同一地区或临近地区的同源性明显高于其它地区,同一地区内寄主对线虫种类分布的影响较小。该方法具有所需样品量少、检测灵敏度高、结果准确等优点,为松材线虫种群的亲缘关系、传播途径及其检测防疫等方面提供一种灵敏、可靠的研究方法。     

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]     

松材线虫病在我国适生性分布的定量估计

在前人对影响松材线虫以及媒介昆虫生长发育因素研究的基础上,应用模糊综合评判、地理信息系统、地统计学等理论与方法,综合分析影响松材线虫病定殖的4个基本条件(即寄主植物、松材线虫病原、媒介昆虫和环境条件),直观、定量和较为准确地获得松材线虫病在我国的潜在适生分布范围以及在云南省具体的潜在分布区域.研究可为我国松材线虫病检疫及其防治决策的制定提供科学依据.     

First detection of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) on Monochamus sutor (Coleoptera: Cerambycidae) in Romania

As a result of the detection of the pinewood nematode Bursaphelenchus xylophilus in Portugal, and its subsequent spread to Spain, intense surveys were conducted to screen for the presence of Bursaphelenchus species in Romania. Herein, we report recent surveys of insects potentially vectoring Bursaphelenchus species collected using trap trees or pheromone‐baited traps placed in the forest. Trap felled spruce trees (Picea abies) and pheromone‐baited traps were installed in six different counties in Romania (Bra?ov, Sibiu, Suceava, Hunedoara, Timi? and Dâmbovi?a). Ten different species of insects distributed among Curculionidae and Cerambycidae were obtained. Nematodes were extracted from insects and observed to validate the presence of Bursaphelenchus specimens. One female identified as Monochamus sutor was the only specimen carrying nematodes in the genus Bursaphelenchus. Nematodes were identified as B. mucronatus based on morphological and molecular features. This is the first detection and report of natural spread of B. mucronatus in Romania. The absence of B. xylophilus was confirmed in the areas of Romania surveyed in this work.     

Genetic diversity and differentiation of fall webworm (Hyphantria cunea Drury) populations

The molecular variation and genetic relationships among five populations of the fall webworm (Hyphantria cunea Drury) in China were assessed using microsatellite technology. Ten microsatellite primers, producing polymorphic bands, were used across 300 samples. The percentage of polymorphic loci (PPB) was 98.36%; the percentage of polymorphic loci in five populations ranged from high to low in the following order: Cangzhou population, Yantai population, Qinhuangdao population, Dandong population, and Shijiaz...     

不同基质对抗松材线虫病黑松“虹青”容器苗生长的影响

以采自日本不同地区的抗松材线虫病黑松品种"虹青"(Pinus thunbergii'Hongqing')种子为材料,研究不同基质处理下的种子出苗率、成苗率及幼苗生长的差异.结果表明:以蛭石为黑松容器育苗基质,除东京都町田市种子的成苗率低于净营养基质处理外,其出苗率、以及静岡县御殿场市、九州种子的出苗率和成苗率均高于净营养基质和河沙处理,其中九州种子的出苗率及成苗率最高,分别为62.7%和52.1%;河沙处理的出苗率和成苗率均最低;九州的种子质量最佳,播种于蛭石中的7个月幼苗平均高8.85 cm,地径为5.1 mm;东京都町田市的种子质量最差.采用蛭石为育苗基质,对立枯病进行早防早治,能大幅度提高黑松出苗率及成苗率.     

斑背大尾莺汉口亚种(Locustella pryeri sinensis)遗传多样性及种群结构研究

采用线粒体控制区（807bp）序列分析对中国境内斑背大尾驾3个繁殖种群及1个越冬种群的遗传多样性及种群遗传结构,结果表明：斑背大尾莺的单倍型歧义度（Hd）为0.759±0.056,核苷酸多样性较（π）为0.002。三个地理种群的FST值以及繁殖种群同越冬种群之间的ΦST值表明不同地理单元之间无显著遗传分化。对不同单倍型的聚类分析（UPGMA）结果以及网络图（Network picture）结果也支持不同种群之间无显著分化。分子变异分析 （AMOVA）显示斑背大尾莺汉口亚种不同地理种群间遗传差异不大,98.5%的差异源自种群内部,仅1.5%源自种群间。中性检验结果Fu’sFS值为负值,错配分布分析结果呈单峰,表明斑背大尾驾在我国的进化史经历了种群扩张。这一假设也得到Tajima’D检验和Fu’s检验结果的支持（D=-1.80,p=0.02;Fs=-22.11,p=0.001）,该扩张大约发生在28,700年前。     

Genetic diversity and differentiation of fall webworm (Hyphantria cunea Drury) populations

The molecular variation and genetic relationships among five populations of the fall webworm (Hyphantria cunea Drury) in China were assessed using microsatellite technology. Ten microsatellite primers, producing polymorphic bands, were used across 300 samples. The percentage of polymorphic loci (PPB) was 98.36%; the percentage of polymorphic loci in five populations ranged from high to low in the following order: Cangzhou population, Yantai population, Qinhuangdao population, Dandong population, and Shijiazhuang population. The results showed that 34.38% of the total genetic variation of the fall webworm (GST) occurs among populations, while most variation (65.62%) exists within populations. Nei’s genetic distances ranged from 0.1386 to 0.3224. Using the unweighted pair group method with arithmetic mean (UPGMA), Nei’s genetic distances were analyzed by a clustering technique and the dendrogram shows that population differentiation is closely related to the time and geographic origin of the invasion. The major factors impacting genetic diversity of fall webworm populations are longitude, the plain area ratio, annual precipitation, latitude and time of invasion. The formation of genetic structure is correlated with characteristics of the life history and invasion ecology of the species.     

木荷1代育种群体遗传多样性分析

木荷为我国亚热带地区主要的珍贵优质阔叶用材树种和生态防护树种.利用筛选的14对多态性强的SSR引物,对木荷1代育种群体中来自15个产地133个亲本进行遗传多样性分析,为其优异种质资源保存、杂交亲本选配及新种质创制提供科学依据.结果表明:14对引物共扩增86个位点,每对引物检测到的等位基因数(Na)变异范围为2～11个,平均等位基因数(Na)为6.14个,平均有效等位基因数(Ne)为3.23个,平均观察杂合度(Ho)为0.572 0,平均Shannon信息指数(I)和平均Nei's基因多样性指数(Nei)分别为1.224 7和0.599 0,说明木荷1代育种群体具有丰富的遗传多样性,其中,福建建瓯产地遗传多样性最高,浙江遂昌产地遗传多样性最低.木荷1代育种群体中成对亲本间遗传距离为0.023 3～1.633 8,平均为0.6067.不同产地遗传多样性与纬度呈显著的负相关关系(r=-0.5162,p=0.048 9).通过UPGMA聚类,可将133个育种亲本分成3个类群,其中,类群3又分为4个亚类群.木荷亲本选配时,应充分考虑优树的产地来源.     

Genetic diversity of apple‐ and crabapple‐infecting isolates of Venturia inaequalis in Pennsylvania,the United States,determined by microsatellite markers

Apple scab caused by Venturia inaequalis is the most destructive disease of apple worldwide. In this study, genetic diversity of 101 V. inaequalis isolates from cultivated apples and ornamental crabapples in Pennsylvania (PA, USA) was characterized using 14 microsatellite markers. A total of 157 alleles ranging from 5 (Vitg9/99) to 26 (Vica10/154) per locus were detected. Regardless of the host of origin, isolates were grouped into five clusters, which were largely supported by STRUCTURE and principal coordinate analysis. Cluster analyses based on genetic distances and population structure analysis suggest very small differentiation (PhiPT ranging from 0.016 to 0.103, depending on the population comparison) between apple and crabapple isolates of V. inaequalis. Pairwise comparisons among populations from different locations showed very low differentiation, and POPGENE analysis indicated frequent migration of alleles (Nm = 1.47). In pathogenicity tests using a detached leaf assay, isolates of V. inaequalis from crabapple caused characteristic scab symptoms on apple and were highly virulent. Results of the study indicate that scab lesions in crabapple trees in close vicinity to apple orchards could serve as reservoirs for spread of the pathogen. Movement of inoculum among locations and between hosts may be responsible for the limited population structure observed. Understanding the population structure of V. inaequalis isolates is significant for apple scab management as crabapples are often used as pollinizers and rootstock in apple orchards, and as ornamental trees.     

Genetic diversity of natural Hepatacodium miconioides populations in Zhejiang Province

Hepatacodium miconioides is the Class II protected plant species in China. This paper studies the genetic diversity and differentiation of its nine natural populations in Zhejiang Province by using random amplified polymorphic DNA (RAPD) technique. Twelve random primers were selected in the amplification, and 164 repetitive loci were produced. The percentage of polymorphic loci in each H. miconioides population ranged from 14.60% to 27.44%, with an average of 20.73%. Among the test populations, Kuochangshan had the highest percentage of polymorphic loci, Simingshan took the second place, and Guanyinping had the lowest percentage. As estimated by Shannon index, the genetic diversity within H. miconioides populations accounted for 27.28% of the total genetic diversity, while that among H. miconioides populations accounted for 72.72%. The genetic differentiation among H. miconioides populations as estimated by Nei index was 0.715,7. This figure was generally consistent with that estimated by Shannon index, i.e., the genetic differentiation among populations was relatively high, but that within populations was relatively low. The gene flow among H. miconioides populations was relatively low (0.198,7), and the genetic similarity ranged from 0.655,7 to 0.811,9, with an average of 0.730,6. The highest genetic distance among populations was 0.422,9, while the lowest was 0.208,3. All the results showed that there was a distinct genetic differentiation among H. miconioides populations. The genetic distance matrix of nine test populations was calculated using this method, and the clustering analysis was made using the unweighted pair group method with arithmetic mean (UPGMA). The cluster analysis suggested that the nine populations of H. miconioides in Zhejiang Province could be divided into two groups, the eastern Zhejiang group and the western Zhejiang group. __________ Translated from Chinese Journal of Applied Ecology, 2005, 16(5): 795–800 [译自: 应用生态学报, 2005, 16(5): 795–800]     

燕山山脉西伯利亚杏的遗传多样性和遗传结构

以西伯利亚杏分布的核心区域燕山山脉地区的17个群体为材料,利用9对微卫星标记进行遗传多样性和遗传结构的分析。在533个个体扩增得到203个等位基因,每位点平均等位基因数为22.556个。分析表明燕山山脉西伯利亚杏群体具有较高的遗传多样性,每位点平均有效等位基因数(Ne)为5.714,多态位点百分率(P)为100%,期望杂合度(He)为0.788。根据有效等位基因数(Ne)、期望杂合度(He)和Shannon信息指数(I)3个遗传多样性参数,遗传多样性最高的群体为北京八达岭,其次为平泉榆树林子,而最低的群体为崇礼驿马图。群体间总的遗传分化系数FST为0.065,总的基因流Nm为3.836。分子方差分析(AMOVA)结果显示燕山山脉地区西伯利亚杏群体的遗传变异主要存在于群体内(95.62%)。Mantel检验发现遗传距离与地理距离呈显著相关性(r=0.5894,P<0.0001)。UPGMA聚类结果显示,地理距离接近的群体聚在一起,进一步验证了Mantel检验结果。基于上述分析结果,提出西伯利亚杏的种质收集策略。研究结果为西伯利亚杏可持续利用与保护提供一定的理论依据。     

广西火桐自然种群和迁地保护种群的遗传多样性比较

采用ISSR标记方法,对广西火桐Erythropsis kwangsiensis 10个自然种群和1个迁地保护种群的遗传多样性和遗传结构进行了分析和比较。结果表明:用10个引物从广西火桐总DNA中共扩增出60条带,其中多态性条带51条。自然种群中以维新种群的遗传多样性最高,而三叠岭瀑布种群最低。自然种群的总遗传变异Ht为0.281 5,种群内遗传变异HS为0.076 4,种群间遗传变异为0.205 1,基因流为0.183 2。迁地保护种群的HEt和HES均为0.118 8。自然种群间存在一定程度的遗传分化(Gst=0.731 9),种群间变异占主导地位。合并后的自然种群的多态性条带百分率(RPPB)、Nei’s多样性指数(H)和Shannon信息指数(I)均高于迁地保护种群,分别为85.00%和31.67%、0.284 1和0.118 8、0.428 5和0.175 7。研究结果表明:广西火桐迁地保护种群的遗传多样性较低,未能涵盖整个种群。建议从多个自然种群中收集种子,尽可能多的采集样本,分区育苗种植,以增加遗传多样性水平。     

A multiplex one‐step PCR method for the simultaneous identification of Bursaphelenchus xylophilus,B. mucronatus and B. doui– three species within the xylophilus group

The pine wood nematode, Bursaphelenchus xylophilus, causes severe damage to pines in Eastern Asia. Bursaphelenchus mucronatus and B. doui resemble closely B. xylophilus morphologically, moreover they were found frequently in this area recently. It is necessary to identify the three species precisely and rapidly. In this study, we report the results of a multiplex one‐step polymerase chain reaction (PCR) utilizing five primers to identify and discriminate the three Bursaphelenchus species simultaneously. The multiplex one‐step PCR yielded one fragment of about 1000 bp for all Bursaphelenchus populations tested. Futhermore, B. xylophilus, B. mucronatus and B. doui produced another fragment of about 100, 350 and 600 bp respectively. This approach is simple and reliable to simultaneously identify the above three species within the xylophilus group usually encountered together in a nematode assay.