松材线虫检测鉴定技术研究进展

松材线虫是全球十分关注的检疫性线虫，对松林危害巨大。该文介绍了松材线虫的研究背景，以及形态学和分子生物学检测鉴定技术的最新进展，并对口岸松材线虫鉴定流程提出建议。未来，快速、便捷的线虫智能识别和等温扩增技术将取得广泛应用。     

松材线虫rDNA-ITS1区分子检测与鉴定

利用两对松材线虫特异性引物,分别对来自日本、美国和葡萄牙的松材线虫虫样进行PCR检测,成功扩增出330 bp和220 bp rDNA-ITS1区基因片段,该方法对松材线虫成虫或幼虫均能作出准确鉴定.     

松材线虫PCR快速检测方法研究

松材线虫是许多国家的检疫性有害生物 ,其形态与拟松材线虫非常相似。为克服传统的形态学鉴定的不足 ,本研究依据松材线虫rDNA基因部分序列 ,通过单个碱基的故意错配 ,设计了一对特异性引物 ,运用PCR方法 ,最终实现了对单条松材线虫的快速检测。该检测方法具有很好的实用性和稳定性     

日本松材线虫和南京松材线虫对雪松和马尾松的致病性研究

用来自日本的木质包装材料上截获的松材线虫和南京松材线虫进行对雪松Ｃｅｄｒｕｓｄｅｏｄａｒａ和马尾松Ｐｉｎｕｓｍａｓｏｎｉａｎａ的致病力试验，发现日本松材线虫能使４～５年生雪松苗２０％发病，１０％左右枯死，而南京松材线虫则没有致病力；１０年生雪松对两种松材线虫具有高度抗性。这两种松材线虫都能使马尾松中度感病和１０％左右的枯死     

木质包装中松材线虫的实时荧光PCR检测

松材线虫是进境木质包装中的重要检疫对象之一。本研究以进境木质包装中截获的12种伞滑刃属线虫,包括4种不同来源的松材线虫、3种不同来源的拟松材线虫、豆伞滑刃线虫、大尖尾伞滑刃线虫、阿苏里伞滑刃线虫、伯氏伞滑刃线虫及拟小刺伞滑刃线虫为材料,进行实时荧光PCR检测。结果显示,仅松材线虫可观察到明显的荧光强度变化,而其他线虫的荧光强度没有变化。通过研磨样品的方法,提取单条松材线虫的DNA,进行实时荧光PCR试验,检测成功率为100%,且不论是雄虫、雌虫或幼虫均能检测出来。该研究对于我国口岸进境木质包装中松材线虫的检测具有一定的实际意义。     

松材线虫病取样部位及检测技术初探

本文介绍了从松材线虫病疫木的不同高度进行取样和分离,通过传统病原形态学方法进行检测和计数的技术,探讨松材线虫在松树体内的数量分布情况,从而提高松材线虫的检出率和缩短检疫时间.     

天津局从船舶垫舱木料中截获松材线虫

天津局从船舶垫舱木料中截获松材线虫１９９５年１２月２５日，天津动植物检疫局在对“海桥”轮上来自日本的垫舱木料实施检疫时，截获了大量活体线虫。经初步鉴定为松材线虫Ｂｕｒｓａｐｈｅｌｅｎｃｈｕｓｘｙｌｏｐｈｉｌｕｓ和拟松材线虫Ｂｕｒｓａｐｈｅｌｅｎｃｈｕ...     

应用TaqMan-MGB探针进行松材线虫的实时荧光定量检测技术研究

 松材线虫是国际公认的最重要的检疫性有害生物之一,也是我国2类检疫危险性有害生物,我国口岸多次从货物的木质包装中截获该线虫。由于松材线虫与拟松材线虫在形态上极其相似,难以区分,幼虫更无法用于鉴定。传统的形态学鉴定、生化以及其他分子技术等方法存在费时、准确度不高、灵敏度低等缺点,不易形成标准。我们设计筛选一对引物以及一条MGB探针,对松材线虫进行实时荧光PCR检测。建立了一条从1 pg到10⁴pg标准曲线,相关系数r=0.965。该检测方法省时、准确、快速、无污染。     

利用RAPD研究松材线虫和拟松材线虫亲缘关系

 用RAPD技术对18个松材线虫株系、8个拟松材线虫株系基因组DNA多态性进行研究。在事先优化的条件下,用15个随机引物进行PCR扩增,一共扩增出165条谱带,多态性为91.5%。通过聚类分析,结果表明:松材线虫和拟松材线虫种间和种内都表现出很大的差异性,但种间差异大于种内差异,因而松材线虫和拟松材线虫应为2个独立的种。     

Duplex PCR 快速检测松材线虫

利用duplex PCR技术对松材线虫(Bursaphelenchus xylophilus)与拟松材线虫(B. mucronatus)的rDNA部分核苷酸序列扩增.根据松材线虫与拟松材线虫的ITS1序列区别,设计出特异性引物,检测松材线虫的存在;在5.8S,28S保守序列区设计通用引物,检测伞滑刃线虫的存在.     

Short communication: Satellite DNA-based species-specific identification of single individuals of the pinewood nematode <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis> (Nematoda: Aphelenchoididae)

The pinewood nematode Bursaphelenchus xylophilus is a severe pest of coniferous trees, and has been designated as a quarantine organism in the European Union. From the sequence of a satellite DNA family characterized in the genome of this nematode, we developed a PCR procedure that allowed the specific discrimination of this species from closely related Bursaphelenchus species found on coniferous trees. Moreover, because of the repetitive nature of satellite DNA, positive amplification was achieved from B. xylophilus single individuals, which should contribute to an easy diagnostic procedure for assisting in the management of this major pest of conifer forests.     

Detection of the pine wood nematode using a real-time PCR assay to target the DNA topoisomerase I gene

A species-specific real-time PCR assay targeting the DNA topoisomerase I gene has been developed to detect Bursaphelenchus xylophilus. The specificity of the assay was confirmed by the lack of amplification of genomic DNA from other Bursaphelenchus or Seinura species. The sensitivity test showed that the limit of the reaction was 0.01 ng of genomic DNA or one individual nematode, as small as an egg. The validity of the real-time PCR assay was evaluated by analyzing mixed nematode samples extracted from diseased pine trees in which B. xylophilus was associated with several closely related species, B. mucronatus, B. hofmanni, Aphelenchoides macronucleatus, S. lii and S. wuae. These results demonstrate the potential of the assay to provide rapid, specific and sensitive molecular identification of B. xylophilus for use in pest risk assessment and quarantine regulations.     

A nested PCR assay targeting the DNA topoisomerase I gene to detect the pine wood nematode, <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis>

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a disastrous pathogen of the pine forests in East Asia and Europe. Plant quarantine is one of the most important ways to prevent its infection in current situation. A nested polymerase chain reaction (PCR) assay targeting the topoisomerse I gene has been developed to detect PWN in this study. To assess the specificity of the assay, 44 morphologically characterized nematode isolates including B. xylophilus, B. mucronatus, B. hofmanni, Seinura wuae, S. lii and Aphelenchoides macronucleatus were tested. Positive reactions characterized by amplification product of 509 bp were shown from all isolates of PWN. The nested PCR assay can detect 50 femtogram (fg) of template DNA or one individual nematode, as small as an egg. The validity was evaluated by analyzing the nematode samples extracted from the nematode-infested wood in the field. These results show that the assay is a specific, sensitive method for detection of PWN with the potential in relation to the pest risk assessment and quarantine regulations.     

Direct molecular detection of the pinewood nematode, <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis>, from pine wood,bark and insect vector

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. The international economic impact of the introduction of the PWN into new areas has highlighted the need for the development of accurate and reliable detection methods of B. xylophilus, which are essential to define aspects of its control and management. In the present study, a methodology was developed for the direct detection of PWN by conventional PCR assay, with a species specific set of primers based on PWN satellite DNA, using total DNA extracted directly from maritime pine, Pinus pinaster, wood and bark samples, and from the insect vector, Monochamus galloprovincialis. This methodology involves homogenisation of wood, bark and insects using liquid nitrogen, DNA extraction and one or two PCR amplification steps, which permit the rapid and direct detection of one single nematode present in 100 mg of wood and bark and in one entire insect without the preliminary steps of nematode extraction.     

Recent surveys for Bursaphelenchus xylophilus in the Czech Republic1

The State Phytosanitary Administration of the Czech Republic (SPA) conducted a survey of Bursaphelenchus xylophilus in 2004 and 2005. In 2004 97 locations were surveyed and 60 samples were taken. B. xylophilus was not found in any of the samples. In 2005, the SPA performed 120 phytosanitary inspections on solitary conifers, and in forest stands and clumps of trees. Checks were performed on Pinus sylvestris and Pinus nigra trees and in coniferous woodland mainly in areas at risk of invasion from B. xylophilus. Of 70 phytosanitary inspections performed in at risk areas, 13 samples were taken for laboratory processing and analysis. Fifty phytosanitary inspections were carried out outside risk areas, mainly in forests and parks, and 28 samples were taken. The Czech University of Agriculture in Prague together with the diagnostic laboratory of the SPA carried out a second survey in 2005. Fifty‐eight samples were taken from 58 surveyed locations in forests where coniferous trees with symptoms had been found. Laboratories performed a survey of B. xylophilus vectors to find only one vector species, Monochamus galloprovincialis subsp. pistor. In total in 2005, 178 phytosanitary inspections were carried out in the Czech Republic. B. xylophilus was not detected in either samples or vectors. Thus the results of the surveying were negative in both 2004 and 2005. The status of Bursaphelenchus xylophilus in the Czech Republic is: absent, confirmed by survey.     

Assessing risks of pine wood nematode Bursaphelenchus xylophilus transfer between wood packaging by simulating assembled pallets in service

The risks of Bursaphelenchus xylophilus (pine wood nematode) transfer in relation to wood material were assessed. Combinations of infested and non‐infested adjacent boards, long‐blocks and blocks of Pinus pinaster, simulating assembled pallets, were assessed. For the recipient wood, pieces with natural moisture content (MC), heat‐treatment (56°C for 30 min in the core) and kiln‐drying to <20% MC were tested, along with in‐service boards from pallets. Donor and recipient wood materials were kept in direct contact at 25°C or 10°C, with nine replicates per treatment. Bursaphelenchus xylophilus was found to transfer rapidly at 25°C when the wood had an MC above fibre‐saturation point (>30%). Nematode reproduction was rapid and sustained, gradually declining to zero at 40 weeks. Bursaphelenchus xylophilus did not transfer to kiln‐dried or to in‐service wood with an MC below fibre‐saturation point, or to wood at 10°C. The key factors determining nematode transfer were the ambient temperature, the nematode load of the donor wood and the MC of the recipient wood, with a ‘barrier’ of 20% MC below which it becomes unsuitable for nematode transfer. This finding indicates that there is a limited risk of spread of B. xylophilus in treated and untreated solid wood packaging materials.     

Experiences with Bursaphelenchus xylophilus in Finland1

In 1984 a heavy Bursaphelenchus xylophilus infestation was observed in pinewood chips imported from USA and Canada into Finland. Because there are good reasons to suppose that B. xylophilus, if spread to pine forests in North Europe, can cause significant damage, the import of coniferous wood into Finland has been prohibited since autumn 1984. In surveys B. xylophilus has not been found in roundwood or in chips imported from European countries into Finland. Neither has B. xylophilus been detected in Finnish pine trees around harbours nor in paper mills handling North American wood.     

Survival of Bursaphelenchus xylophilus in wood chips1

Preliminary results are presented on elimination of Bursaphelenchus xylophilus from pine wood chips by dry heat, hot water treatment and immersion in dithiocarbamate solution.     

Report on the survey for Bursaphelenchus xylophilus and the occurrence of other Bursaphelenchus species in Hungarian coniferous forests

An ongoing official survey to detect the pine wood nematode Bursaphelenchus xylophilus, a quarantine pest, started in 2003 in coniferous forests in Hungary. Based on the results of the study from 2003–11, B. xylophilus has not yet been detected in Hungary. Two other Bursaphelenchus species (B. mucronatus and B. vallesianus) were identified in samples in 2009. Details of the survey and the measurements of B. mucronatus and B. vallesianus are provided.     

高效新型杀松材线虫剂的筛选及其林间防效

为筛选出新型的高效松材线虫Bursaphelenchus xylophilus防治药剂,采用浸渍法室内测定了氯氟氰虫酰胺、阿维菌素、甲维盐、灭多威、特丁硫磷、甲基异柳磷和吡虫啉7种药剂对松材线虫的毒杀作用和对其繁殖能力的影响,采用高效液相色谱仪测定氯氟氰虫酰胺在马尾松体内的输导分布,并在林间开展树干注射氯氟氰虫酰胺抗松材线虫病试验。结果表明,7种药剂中氯氟氰虫酰胺对松材线虫的毒力最强,LC_(50)为0.019 mg/L,阿维菌素和甲维盐次之,LC_(50)分别为0.0213 mg/L和0.0214 mg/L。氯氟氰虫酰胺树干注射3个月内能迅速从马尾松基部传导至树梢顶部,树干边材东、南面的药剂含量显著高于西、北面,且树干不同高度的氯氟氰虫酰胺含量均高于其LC50;氯氟氰虫酰胺制剂在林间注干后,对人工接种病原松材线虫松树的防治效果为73.33%,自然感染松材线虫病松树的死亡率降至2.00%,而对照试验中人工接种松材线虫松树和自然感染松材线虫病松树的死亡率分别为93.33%和19.67%。表明氯氟氰虫酰胺制剂能有效预防松树个体的松材线虫病发生与为害,可以推广使用。