Direct PCR‐based method for detecting Bursaphelenchus xylophilus,the pine wood nematode in wood tissue of Pinus massoniana

Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, leads to serious losses to pine forestry around the world. Pinus massoniana, which is vulnerable to be attacked by the PWN, is the dominant species used in pine forestry in China. The objective of this study is to develop a direct PCR‐based method for detecting B. xylophilus in the wood of P. massoniana without a separate nematode extraction step. A simple procedure was first developed for isolating B. xylophilus DNA in 5 mg pine wood tissue samples harbouring PWN for detection by PCR amplification. A B. xylophilus‐specific amplicon of 403 bp (DQ855275) was generated by PCR from the infested wood tissue. The entire procedure can be completed within 5 h with one pair of primers. This assay can serve as a rapid, cheap and environmentally friendly method to detect B. xylophilus in samples of P. massoniana.     

Aseptic Bursaphelenchus xylophilus does not reduce the mortality of young pine tree

To assess the role of bacteria in pine wilt disease (PWD), aseptic M form (with a mucronated tail) and R form (with a round tail) of Bursaphelenchus xylophilus and B. mucronatus were obtained and compared, in terms of reproduction and pathogenicity, with non‐aseptic nematode. In addition, bacteria isolated from non‐aseptic nematodes and pine trees inoculated with non‐aseptic nematodes were identified. The results indicated that the bacteria associated with nematodes significantly lowered the reproduction of R form of B. xylophilus and B. mucronatus. Both the non‐aseptic and aseptic R forms of B. xylophilus induced death in all infected 7‐ to 8‐year‐old pine trees, while the non‐aseptic and aseptic M forms of B. xylophilus and B. mucronatus caused almost no plant mortality. High numbers of the non‐aseptic and aseptic R forms of B. xylophilus were distributed throughout the inoculated trees, while B. mucronatus and M form of B. xylophilus nematodes were lower in number and their distribution in stems limited within the inoculation site. Bacteria isolated from non‐aseptic nematodes were not recovered from the pine trees inoculated with these same kinds of nematodes. Two species of bacteria were both isolated from non‐aseptic B. mucronatus and from R form of B. xylophilus. Microbacterium trichotecenolyticum was common to both the control and inoculated pine trees. These results suggest that R form of B. xylophilus is the causal agent of PWD and that bacteria cannot increase the virulence of B. xylophilus and B. mucronatus.     

Development of two reverse transcription‐PCR methods to detect living pinewood nematode,Bursaphelenchus xylophilus,in wood

Pinewood nematode, Bursaphelenchus xylophilus, is an inhabitant of native pine species of North America, where its presence in trees is non‐pathogenic. By contrast, the introduction of this nematode to forests overseas has devastated some pine stands and is recognized as a pest of phytosanitary concern by some countries' National Plant Protection Organizations. The ability to detect B. xylophilus in internationally traded wood products is crucial to reduce the spread of this organism. Current molecular techniques for the detection of B. xylophilus rely on the presence of genomic DNA and thus will detect both living and dead nematodes without differentiation. The detection of dead nematodes could lead to unnecessary trade disruption. Therefore, accurate techniques for the detection of and differentiation between live and dead B. xylophilus are critical. We have developed an endpoint RT‐PCR assay and a SYBR Green 1 real‐time RT‐PCR assay, both of which selectively identify living pinewood nematode by detecting the presence of Hsp70 mRNA as a viability marker. Both of these assays may help overcome or resolve disputes involving the detection of pinewood nematode at the port of entry and can also be used to evaluate the efficiency of wood treatment procedures.     

Detection of Bursaphelenchus xylophilus from old discs of dead Pinus armandii var. amamiana trees using a new detection kit

We examined the effectiveness of a new Bursaphelenchus xylophilus detection kit, based on loop‐mediated isothermal amplification (LAMP), in old discs taken from the stem base of B. xylophilus‐infested dead trees of Pinus armandii var. amamiana (PAAm) occurring in their natural habitats. LAMP products, representing a past B. xylophilus infection, were detected in two consecutive trials from 16 of 20 discs collected from PAAm trees that died between 2003 and 2006. Bursaphelenchus xylophilus were more frequently detected using LAMP in wood samples taken from sapwood than from heartwood. No significant differences in the detection of B. xylophilus using LAMP were observed in relation to the disc collection time (from 3 to 6 years before the analysis). Bursaphelenchus xylophilus were not detected using LAMP in four discs, although a B. xylophilus infection had been confirmed for the original PAAm trees at the time they were found dead. This may have resulted from the small amount of wood chips needed for the LAMP test or the reduced number and uneven distribution of the nematode in the old dead trees. The results indicate that the new B. xylophilus detection kit will be a very efficient tool for conducting retrospective analysis of PAAm mortality factors.     

Inoculation of several Bursaphelenchus xylophilus group nematodes into adult trees of Pinus thunbergii and their survival in the trees

To clarify the pathogenicity of Bursaphelenchus nematodes to adult pine trees, inoculation experiments using six species of B. xylophilus group nematodes and ca. 10‐year‐old trees of Pinus thunbergii were conducted. Trees inoculated with an avirulent isolate (C14‐5) of B. xylophilus did not die during the survey, but showed a decline in oleoresin exudation compared with the controls. Fifteen months after the inoculation, a small number of B. xylophilus survived in a tree inoculated with B. xylophilus C14‐5. Trees inoculated with B. mucronatus, B. doui, B. luxuriosae, B. conicaudatus and Bursaphelenchus sp. NK224 (undescribed) showed no decline in oleoresin exudation and no external symptoms of wilt. However, 9 months after the inoculation, a small number of B. luxuriosae survived in a tree inoculated with the nematodes, although four other nematode species were not isolated from trees inoculated with them. These results were approximately consistent with our previous results (Kanzaki, N.; Aikawa, T.; Maehara, N.; Ichihara, Y., 2010, J. For. Res.; in press), in which an avirulent isolate (OKD‐1) of B. xylophilus and B. luxuriosae caused water flow inhibition without external symptoms in 3‐year‐old seedlings. Therefore, to examine the pathogenicity of the nematodes to pines, it is useful to use 3‐year‐old seedlings in inoculation experiments when adult trees cannot be used.     

Virulence of Bursaphelenchus mucronatus to pine seedlings and trees under field cownditions

Bursaphelenchus mucronatus is a parasitic nematode of pine that is widely distributed in the natural pine forests of Asia and Europe. It has a very similar morphology and biology to that of Bursaphelenchus xylophilus, the causal agent of pine wilt disease, but has generally been considered to be non‐pathogenic to pine. However, in some provinces of China, B. mucronatus has been isolated from dead pine trees rather than B. xylophilus. Previous studies have shown that B. mucronatus can induce the death of pine seedlings under glasshouse conditions. To investigate the virulence of B. mucronatus, 2‐year‐old seedlings of Pinus massoniana and Pinus elliottii were inoculated with one of six isolates of B. mucronatus under field conditions in April 2014 and their condition was monitored over a year. The virulence of the six B. mucronatus isolates differed on the three host species: P. elliottii seedling mortality ranged from zero to six of the 18 inoculated seedlings, whereas P. massoniana seedling mortality ranged from four to 12 of the 18 inoculated seedlings. Three B. mucronatus isolates that appeared to cause different levels of mortality among the seedlings were used to inoculate 12‐year‐old Pinus thunbergii trees in August 2014. The trees were monitored for a year, during which time between 4 and 12 of the 18 inoculated trees in each treatment wilted and died. The average monthly temperature during the test period appeared to be similar to that of the historical average in the test areas; however, both study sites experienced above‐average rainfall. This study demonstrated that B. mucronatus has potential virulence on pine trees and provided experimental evidence that high temperatures or drought stress is not essential for the virulence of B. mucronatus.     

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.     

Records of Bursaphelenchus spp. intercepted in imported packaging wood at Ningbo,China

From January 2003 to June 2005, samples from 3416 batches of wooden packaging material were inspected for the presence of nematodes in the Ningbo Entry–Exit Inspection and Quarantine Bureau, China. Bursaphelenchus spp. were detected in 202 batches from 25 different countries. The following species were identified on the basis of their morphology and their intergenic transcribed spacer‐restriction fragment length polymorphism (ITS‐RFLP) patterns: B. xylophilus, B. fungivorus, B. rainulfi, B. hylobianum, B. thailandae, B. mucronatus, B. aberrans, B. lini, B. singaporensis, B. doui, B. conicaudatus, B. vallesianus, B. pinasteri, B. hofmanni and B. arthuri. The most frequently found species were B. mucronatus (57 batches), B. xylophilus (40 batches), B. fungivorus (21 batches), B. rainulfi (19 batches) and B. thailandae (nine batches). The pine wood nematode B. xylophilus was not only found in packaging wood imported from areas where it is known to occur (i.e. The United States of America, Japan, Korea, Hong Kong and Taiwan), but also from countries considered to be free of this dangerous pest (i.e. Brazil, Thailand, Belgium, The Netherlands, Italy and Spain). The occurrence of B. xylophilus in packaging wood from countries regarded as being free of the nematode can most likely be explained by the global circulation of wooden packaging material among infested and non‐infested countries. Our findings emphasize the need to fully implement international standards on phytosanitary treatment of packaging wood, in order to prevent further spread of the pine wood nematode, and the need for careful re‐examination of the current heat treatment measures.     

A simple,polymerase chain reaction‐restriction fragment length polymorphism‐aided diagnosis method for pine wilt disease

For diagnosis of pine wilt disease, a simple PCR‐RFLP method was developed to identify and to differentiate two similar nematode species, based on a living or preserved single specimen. Pinewood nematodes, Bursaphelenchus xylophilus, and Bursaphelenchus mucronatus were examined. A single nematode in 1 µl of distilled water was put on a glass slide. When the water had almost dried the nematode was crushed with a filter paper chip, 1.5 mm × 1.5 mm, with the aid of forceps. The filter paper chip containing nematode remains was immediately placed into PCR buffer as the DNA template. The primer set used was to amplify ribosomal DNA containing the inter‐transcribed spacer (ITS) 1, 5.8S and ITS2 regions. The PCR product was consistently obtained from a single nematode, and digesting the product with restriction endonuclease, Hinf I, enabled discrimination between B. xylophilus and B. mucronatus. This method was simple, convenient and definitive, and could successfully determine the pathogen in the diagnosis of pine wilt disease. This method was applicable also to nematode specimens preserved under various conditions except in the case of those preserved in aldehyde‐containing fixatives.     

A SCAR molecular marker to distinguish Bursaphelenchus mucronatus from the pinewood nematode,B. xylophilus

Bursaphelenchus mucronatus is closely related to the pinewood nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease. B. xylophilus became a devastating pest when it was introduced in the Far East; however, B. mucronatus is considered to have low virulence. Morphological similarities between B. xylophilus and B. mucronatus make the accurate morphological identification of both species difficult. Thus, it has become important to pay attention towards B. mucronatus impact and the need of discrimination of these two species. To distinguish among the two species, a B. mucronatus‐specific sequence‐characterized amplified region (SCAR) marker has been developed. The specific Random amplified polymorphic DNA (RAPD) fragment of B. mucronatus, OPY₀₁‐M₈₅₀ was excised from agarose gels and purified. The gel‐purified fragment was cloned into the pGEM^®‐T Vector and subjected to sequencing. Based on the sequenced RAPD fragments, a number of SCAR primers were designed. It is demonstrated that OPY₀₁‐M₈₅₀ through primers Y₀₁F/R can be transformed into a B. mucronatus‐specific SCAR‐Y₀₁‐M₆₀₉ marker. Primers set Y₀₁F/R had high specificity that could be used for the discriminative identification of B. mucronatus versus B. xylophilus.     

Comparison of the gene expression profiles of resistant and non‐resistant Japanese black pine inoculated with pine wood nematode using a modified LongSAGE technique

The Japanese black pine (Pinus thunbergii) is highly susceptible to pine wilt disease caused by the pine wood nematode (PWN; Bursaphelenchus xylophilus). To cope with this disease, researchers and tree breeders selected PWN‐resistant individuals in a previous breeding program. In an attempt to understand the mechanisms of resistance in the Japanese black pine, we created four LongSAGE (serial analysis of gene expression) libraries. A total of 20 818 tags were studied, including 5194 tags from a PWN inoculated resistant pine, 5218 a non‐inoculated resistant pine, 5194 an inoculated non‐resistant pine, and 5212 a non‐inoculated non‐resistant pine. The analysis of the libraries indicated that 14 tag species were significantly up‐regulated (e.g., pathogenesis‐related proteins 2 and 4, osmotin, lipoxygenase, and chalcone synthase), and nine were down‐regulated (eukaryotic translation initiation factor SUI1, translationally controlled tumor protein, and xyloglucan endotransglycosylase) by the PWN inoculation in both the resistant and non‐resistant pines. On the other hand, 38 tag species were significantly expressed at a higher level only in the resistant pine (catalase, dienelactone hydrolase family protein) and 25 were expressed at a higher level in the non‐resistant pine (pathogenesis‐related proteins 1, 2, and 3, and leucoanthocyanidin dioxygenase). These differentially expressed genes are presumed to reflect some of the differences between the resistant and non‐resistant pines. Our results provide valuable information on the complex responses induced in the resistant and non‐resistant pine trees in response to PWN invasion.     

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.     

Development of two alternative Loop‐mediated isothermal amplification tools for detecting pathogenic pine wood nematodes

Loop‐mediated isothermal amplification (LAMP) detection tools have great potential for diagnosing the causal agents of infectious diseases in clinics and in agriculture. In this work, we developed two alternative LAMP protocols for detecting the pathogenic nematode Bursaphelenchus xylophilus, causal agent of pine wilt disease. We first identified a pectate lyase 3 gene as a biomarker for developing a LAMP Detection Kit, as there was no homologue in non‐pathogenic nematodes that live in pine timber or bark and show structural similarities to B. xylophilus. The first LAMP protocol used the Genie II equipment and an isothermal master mix containing Geobacillus sp. M 2.0 large fragment DNA polymerase showed approximately 10 times greater sensitivity with a shorter incubation period compared with that of the second LAMP protocol, which utilized a fluorescence metal indicator, calcein and an engineered Bacillus stearothermophilus DNA polymerase I, large fragment (Bst 2.0 DNA polymerase). However, the LAMP reactions with calcein and Bst 2.0 polymerase were the cost‐effective method because the reaction could be performed using a simple isothermal block and relatively inexpensive calcein as a fluorescence indicator visible to the naked eye.     

Pine wilt disease: detection of the pinewood nematode (Bursaphelenchus xylophilus) as a tool for a pine breeding programme

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a serious quarantine pest first detected in Portugal and Europe in 1999. It is the causal agent of pine wilt disease (PWD). A resistance breeding programme has been initiated to contribute to control the evolution of the disease. Five hundred and four adult maritime pine, Pinus pinaster, trees were phenotypically selected as candidate trees for this programme from an area affected by PWD. To identify tolerance to the nematode, the selected trees were monitored monthly. Over the course of 1 year, 57 candidate trees died and were tested for the presence/absence of the PWN. As accuracy of detection is of major importance, an ITS‐PCR‐based method applied directly to wood from adult maritime pine trees was tested and compared with a standard morphological identification method. The results showed that the use of PCR to detect the pathogen provided more rapid and accurate results in comparison with the standard morphological identification. Thus, this method is suitable to be used in the survey of the breeding population for resistance/tolerance to PWD.     

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.     

Pathogenitätstests mit Bursaphelenchus mucronatus an Kiefern und Fichtensämlingen in Deutschland

Pathogenicity tests with Bursaphelenchus mucronatus on pine and spruce seedlings in Germany Inoculation tests on Pinus sylvestris seedlings with a German, a Siberian and a French isolate of Bursaphelenchus mucronatus and on Picea abies seedlings using only the German isolate have clearly shown, under German field conditions, that the nematode can cause wilt symptoms on the apex of about 60% of the inoculated P. sylvestris plants. The nematodes in all these tests mostly remained near the inoculation site. However, further spread of the nematodes and wilt symptoms occured when a climate chamber, at 25°C, was used or when the French isolate was used outdoors. At 25°C, 10% of the inoculated plants with at least 1000 nematodes per seedling died. Seedlings' apices wilted if more than 50 nematodes per trunk were present, whereas smaller numbers of nematodes could cause partial wilting. Nevertheless, a few pine seedlings did tolerate high nematode-population densities, up to 966 per plant. No further progress of wilt of plants with symptoms was observed the following year. Inoculations of P. abies with a German isolate resulted in population development near the site of inoculation. Spruces largely tolerated nematode infestations without any development of wilt symptoms.     

Localized within‐ and between‐tree variation in nematode distribution during latent state of pine wilt disease makes the disease status cryptic

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease, an epidemic disease that has severely damaged pine forests in East Asia. The disease has spread to northern areas in Asia and parts of Europe. To prevent disease spread as the forefront of damage prevention, a better understanding of infection status is highly important. Not all infected trees show disease symptoms, and such asymptomatic PWN‐carrying trees are likely to be overlooked and can become a pathogen reservoir. To elucidate PWN infection status in asymptomatic trees, we performed PWN inspection of branches and trunks in 21 test trees in two different conditions: trees that had experienced PWN inoculation and those with suspected PWN infection that had experienced transient foliage discoloration. We detected PWNs in eight test trees (38%) and in 13 (1.5%) of a total of 843 samples. The difference in these percentages suggests that nematode inhabitation was highly localized within the trees, possibly owing to the restricted migration of PWNs. Our data demonstrated that trees that were once weakened but recovered their vigour can persist, as the asymptomatic carriers, in the forest. The implications for disease control are also discussed.