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.     

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.     

Pinewood nematode population growth in relation to pine phloem chemical composition

The pinewood nematode, Bursaphelenchus xylophilus, native to North America (NA), is now a major invasive pine pest in Eurasia. Pine species differ greatly in their susceptibility to B. xylophilus, and differences in susceptibility might be related to differences in phytochemistry. This study addressed two major questions: (i) which plant chemical profiles (including compounds that act as plant defences and those important for nutrition) favour pinewood nematode development; and (ii) are NA and Portuguese pinewood nematodes different in their capacity to subsist on plant tissues? For this, B. xylophilus isolates from NA and Portugal were tested on pine phloem and xylem of different pine species. It was found that unaltered pine phloem can sustain the population growth of B. xylophilus, but only in a few cases, and this was clearly related to distinct chemical profiles in some pine species. No evidence was found of evolutionary divergence between B. xylophilus populations in Portugal and NA. The pine species with phloem on which pinewood nematodes could grow tended to have low levels of total phenols, condensed tannins, flavonoids and lignin. Evidence was also found that nutrients such as N may be important.     

松材线虫rNDA-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330bp和220bprDNA-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。     

Differences in constitutive and inducible defences in pine species determining susceptibility to pinewood nematode

The pinewood nematode, Bursaphelenchus xylophilus, originating from North America (NA), is a major invasive pine pest in Eurasia. It was first detected in Portugal in 1999 associated with maritime pine, Pinus pinaster, and has been differently affecting the main local pine species, P. pinaster and P. pinea. Field studies and direct inoculation experiments in Pinus spp. seedlings, under controlled conditions, were performed to assess whether the differences in constitutive and inducible defences are determining the different susceptibility of pine host species to B. xylophilus. Host co‐evolution with the pathogen was also assessed, including the NA P. radiata, widely used in forestry in the northeast of the Iberian peninsula. Pine mortality in the field was positively related with the abundance of B. xylophilus, and concentration of phenolics and condensed tannins in pines. In the greenhouse assay, seedling tissues were analysed for constitutive investment in defences, as well as the potential inducibility of those defences as driven by B. xylophilus inoculation. Slower growing P. pinea presented higher levels of constitutive defences than faster growing P. pinaster, with only P. pinaster being affected by B. xylophilus. Furthermore, co‐evolution with the pathogen is important, with the fast‐growing NA P. radiata presenting an inducible and effective response to B. xylophilus. Results point to the importance of integrating data on pine life history traits, including growth rate, and production of constitutive and inducible defences, into predictive models for this invasive forest pest.     

Improved morphological key to the species of the xylophilus group of the genus Bursaphelenchus Fuchs, 1937

Correct identification of the pine wood nematode Bursaphelenchus xylophilus becomes more difficult, as more related or similar species are described. Spread of the nematode is being monitored worldwide, and in view of the fact that several new Bursaphelenchus species have been described, identification keys should be adjusted accordingly. Based on the identification key of the xylophilus group published by Braasch (2008) and the revised intrageneric grouping of Bursaphelenchus by Braasch et al. (2009), an improved dichotome morphological key including newly described species (B. paraluxuriosae, B. populi, B. macromucronatus, B. firmae, B. koreanus and B. gillanii) is presented. The recognition of the quarantine pest B. xylophilus using morphological characteristics in laboratories of the National Plant Protection Services is facilitated by a simplified key.     

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.     

Survey of the genus Bursaphelenchus Fuchs, 1937 (Nematoda: Aphelenchoididae) in Romania

An overview of the occurrence of species of the genus Bursaphelenchus in Romania is presented. The data is based on recent surveys conducted for the first time throughout the country, to monitor and evaluate the potential entry of the pine wood nematode, Bursaphelenchus xylophilus. Wood samples were collected from declining trees, wood‐processing companies and imported wood packaging material. Of the 895 wood samples examined, 11 contained Bursaphelenchus specimens. Morphological and molecular analyses were carried out to characterize the species detected. With respect to the possible presence of B. xylophilus, all samples were negative, confirming the absence of this quarantine pest in Romania. Nevertheless, five Bursaphelenchus species were found: B. hofmanni, B. poligraphi, B. vallesianus, B. willibaldi, and one putative new species belonging to the sexdentati group, classified here as Bursaphelenchus sp. NR512. These results constitute the first report of the genus Bursaphelenchus in Romania.     

Identification of Meloidogyne chitwoodi, M. fallax and M. hapla Based on SCAR-PCR: A Powerful Way of Enabling Reliable Identification of Populations or Individuals that Share Common Traits

This study describes the development of species-specific pairs of PCR primers for the root-knot nematodes Meloidogyne chitwoodi, M. fallax and M. hapla that amplify species-specific RAPD fragments. After sequencing the fragments, longer primers were designed to complement the terminal sequences of the polymorphic DNA fragments. The resulting pairs of primers were used to generate the sequence-characterized amplified regions (SCARs). Using the developed pairs of SCAR primers, SCAR fragments of M. chitwoodi, M. fallax or M. hapla were easily amplified from DNA extracts from juveniles, egg masses, females of the particular nematode species investigated, either present alone, in a mixture with other nematode species or in infested plant material. A specially designed multiplex assay using three pairs of SCAR primers enabled the identification of multiple species in a mixture in a single PCR step. Single juveniles were easily identified by applying this multiplex assay followed by a subsequent multiplex PCR using three pairs of nested primers. The SCAR-PCR-based assays described have potential to be optimized for routine practical diagnostic tests. The usefulness of converting RAPD markers into SCAR markers is discussed.     

Sublethal infection of different pine species by the pinewood nematode

Bursaphelenchus xylophilus, the pinewood nematode and causal agent of pine wilt disease (PWD), is a globally destructive pine pest. PWD is favoured by high temperatures and drought, but B. xylophilus can also persist in symptomless hosts in areas not prone to PWD. We compared seedlings of different Pinus species that were inoculated with B. xylophilus when well-watered and under moderate temperatures, conditions that favour sublethal infections. We compared isolates of B. xylophilus in south-west Europe (Portugal) and south-east USA (Louisiana). P. pinea, P. pinaster, and P. radiata were challenged in both areas with inoculations of local B. xylophilus. P. sylvestris in Portugal and P. palustris in Louisiana were also tested. Seedling mortality was low, as expected, but nematode establishment was common. There were higher nematode densities and more damage in seedlings of P. sylvestris and P. radiata, followed by P. pinaster, with P. pinea and P. palustris proving to be more tolerant. Life history of hosts was a better predictor of resistance to B. xylophilus than geographical origin, even under sublethal conditions. Fast-growing species (P. radiata and P. pinaster) appeared most likely to be symptomless carriers in areas not prone to the development of PWD. Cold-adapted species (P. sylvestris) may still be suitable hosts for the pathogen, even at the sublethal conditions of their distribution areas. There were more nematodes per gram of host using nematode isolates from Portugal. Nonetheless, our results opposed the hypothesis that North American pinewood nematodes have such low virulence that they are obligatorily saprophytic.     

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for <Emphasis Type="Italic">Xiphinema diversicaudatum</Emphasis>, <Emphasis Type="Italic">X. index</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis>

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.     

Genetic diversity of the root‐knot nematode Meloidogyne ethiopica and development of a species‐specific SCAR marker for its diagnosis

Meloidogyne ethiopica is an important nematode pathogen causing serious economic damage to grapevine in Chile. In Brazil, M. ethiopica has been detected with low frequency in kiwifruit and other crops. The objectives of this study were to evaluate the intraspecific genetic variability of M. ethiopica isolates from Brazil and Chile using AFLP and RAPD markers and to develop a species‐specific SCAR‐PCR assay for its diagnosis. Fourteen isolates were obtained from different geographic regions or host plants. Three isolates of an undescribed Meloidogyne species and one isolate of M. ethiopica from Kenya were included in the analysis. The results showed a low level of diversity among the M. ethiopica isolates, regardless of their geographical distribution or host plant origin. The three isolates of Meloidogyne sp. showed a high homogeneity and clustered separately from M. ethiopica (100% bootstrap). RAPD screenings of M. ethiopica allowed the identification of a differential DNA fragment that was converted into a SCAR marker. Using genomic DNA from pooled nematodes as a template, PCR amplification with primers designed from this species‐specific SCAR produced a fragment of 350 bp in all 14 isolates of M. ethiopica tested, in contrast with other species tested. This primer pair also allowed successful amplification of DNA from single nematodes, either juveniles or females and when used in multiplex PCR reactions containing mixtures of other root‐knot nematode species, thus showing the sensitivity of the assay. Therefore, the method developed here has potential for application in routine diagnostic procedures.     

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.     

Development of PCR-based Detection Methods for the Quarantine Phytopathogen <Emphasis Type="Italic">Synchytrium endobioticum</Emphasis>, Causal Agent of Potato Wart Disease

PCR-based methods were developed for the detection and quantification of the potato pathogen Synchytrium endobioticum in soil extracts and in planta. PCR primers, based on the internal transcribed spacer region of the multi-copy gene rDNA were tested for specificity, sensitivity and reproducibility in conventional and real-time PCR assays. Soil extraction procedures compared included the Hendrickx centrifugation (HC) procedure, nested wet sieving (NWS) and a method used by the Plant Protection Service (PPS). The primers amplified a 472 bp product from S. endobioticum DNA, but did not amplify DNA from other potato pathogens, other plant pathogens, and related species. Standard cell disruption and DNA extraction and purification methods were optimized for amplification of S. endobioticum DNA from resting sporangia. DNA was successfully amplified from a single sporangium and equivalent DNA preparations from soil extracts. Low levels of target DNA in water did not amplify, possibly due to DNA loss during final purification steps. A real-time PCR assay, developed for soil-based extracts using primers and probe based on the rDNA gene sequences, involved co-amplification of target DNA along with an internal DNA fragment. Both conventional and real-time PCR methods performed well with HC- and NWS-extracts having a threshold sensitivity of 10 sporangia per PCR assay. Of the three soil extraction methods, only with the HC method could 100 g soil samples be efficiently processed in one single PCR assay. Such a high capacity assay could be useful for routine soil analysis in respect to disease risk assessments and to secure de-scheduling according to EPPO guidelines.     

Pine Wilt Disease Caused by the Pine Wood Nematode: The Induced Resistance of Pine Trees by the Avirulent Isolates of Nematode

Since the beginning of the 20th century, pine trees in Japan have been seriously damaged by the pine wilt disease. This disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which is transmitted by the Japanese pine sawyer, Monochamus alternatus. The control of disease depends to a large extent on chemicals, but the public is now demanding environmentally friendly control methods. The virulence of B. xylophilus varies very widely. Pre-inoculation of young pine trees in a nursery with avirulent B. xylophilus has induced systemic resistance of trees against a subsequent inoculation with virulent B. xylophilus. This induced resistance was considered a hopeful means for developing a biological control for the disease. The induced resistance by the avirulent nematodes was also expressed in mature pine trees in a forest where the disease was naturally epidemic. However, the effects of induced resistance were not satisfactory for practical biological control. Since the inoculation with higher concentrations of the avirulent B. xylophilus induced the resistance more effectively, the pre-inoculation method will need to be improved to develop the biological control. The induced resistance of pine trees by avirulent B. xylophilus should be one of the candidate biological control methods against pine wilt disease. This induced resistance also provides an experimental system to clarify physiological interactions between the nematodes and pine trees.     

Multiplex real-time PCR assays for the detection and identification of Heterobasidion species attacking conifers in Europe

Four species of the destructive forest pathogen Heterobasidion annosum sensu lato (s.l.) are present in Europe: H. annosum sensu stricto (s.s.), H. abietinum and H. parviporum are native species, while H. irregulare is a non-native invasive species currently reported only in Italy, yet recommended for regulation throughout Europe. In this study, real-time PCR detection tests were developed for each of the four species, which can be used simultaneously or individually thanks to probes labelled with species-specific fluorescent dyes. The different performance criteria of each assay were evaluated, and it was determined that they were theoretically capable of detecting amounts of DNA corresponding to 311, 29 and 29 cell nuclei in H. annosum s.s., H. irregulare and H. parviporum, respectively. The specificity of each assay was assessed with a wide set of strains. Real-time PCR tests successfully detected Heterobasidion species from 36 fruiting bodies taken from the forest, as well as from artificially inoculated or naturally infected wood samples. The multiplex real-time PCR assays developed in this study could have practical applications both in forest management and in phytosanitary monitoring.     

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。     

Occurrence and ITS diversity of wood‐associated Bursaphelenchus nematodes in Scots pine forests in Switzerland

The quarantine pathogen Bursaphelenchus xylophilus (pine wood nematode, PWN) represents a serious threat for Pinus species in Europe. To exclude its presence in Switzerland, in 2010 and 2011 a countrywide survey was conducted in 102 Pinus sylvestris stands, chosen according to whether they contained dying or dead trees or were located in areas at risk of PWN introduction. In total, 285 trees (1–5 per site) were sampled. Nematodes were extracted from wood chips using a standard procedure, and identified to species by internal transcribed spacer (ITS) sequencing. Bursaphelenchus species were present in 34% of the trees, but no B. xylophilus was identified, i.e. PWN is still not present in Switzerland. The nematodes found belonged to seven different species, with B. vallesianus the most frequent species, followed by B. sexdentati, B. mucronatus kolymensis and B. eggersi. Three other species (B. borealis, B. pinophilus, B. poligraphi) were each only present in one or two trees. Three groups of sequences could not be assigned to a species because of the lack of matching reference sequences. The species composition found in Switzerland suggests co‐existence of southern and central European Bursaphelenchus species. Intraspecific ITS variability differed considerably among the four most common species. Bursaphelenchus eggersi, B. mucronatus kolymensis and B. sexdentati had several variable sites in the ITS region, resulting in multiple ITS genotypes in each species. In contrast, all 99 B. vallesianus isolates had an identical ITS region. This could indicate a founder effect, and possibly that B. vallesianus is not native to the Alpine region.     

Potential for establishment of Bursaphelenchus xylophilus and the pine wilt disease under Nordic conditions1

Serious concern about the pinewood nematode, Bursaphelenchus xylophilus, and the pine wilt disease arose in 1984 when the nematode was discovered in wood chips imported for the Finnish pulp industry. It has been questioned whether this nematode could establish itself and actually damage pine trees under Nordic conditions. It seems likely that B. xylophilus could reproduce in Nordic forest lands. It is also likely that potential vector insects are present and could be functional in the establishment and epidemic development of the disease. Although temperatures in Nordic areas hardly seem conducive to pine wilt disease, other factors may contribute to disease expression. Research is needed on interactions of stress factors, particularly temperature and soil moisture, in the expression of pine wilt disease. A better understanding of these interactions would aid in evaluating the risk connected with an accidental introduction of the nematode into Nordic countries and provide a sound basis for trade regulations. At present, there are reasons to be most restrictive concerning any possible means of introduction of B. xylophilus.