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.     

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.     

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.     

Long‐term survival and non‐vector spread of the pinewood nematode,Bursaphelenchus xylophilus,via wood chips

The pinewood nematode, Bursaphelenchus xylophilus, is the causal agent of pine wilt disease and is transmitted to new host trees by beetles of the genus Monochamus. The increasing interest in imported wood chips from North America for paper production and energy purposes and the corresponding phytosanitary risk of non‐vector transmission of B. xylophilus has been discussed since 1984, the year of the first interception of B. xylophilus in wood chips in the European Union. The long‐term survival of B. xylophilus in wood chips and its non‐vector spread from infested wood chips to non‐infested trees were studied. Pinus sylvestris logs were inoculated with a suspension of B. xylophilus to produce infested wood chips. During the long‐term storage test, B. xylophilus in P. sylvestris wood chips were examined. Four variants, including sealed and openly stored wood chips at both 15°C and 25°C, were studied. For the test of non‐vector spread, B. xylophilus ‐infested wood chips were placed on three‐ to four‐year‐old P. sylvestris saplings under different conditions. Bursaphelenchus xylophilus survived for more than 1 year at both temperatures in the sealed wood chips, which was significantly longer than for the openly stored variant at 25°C. Temperature, tree condition and wood chip location all influenced non‐vector spread through wood chips. Of the 480 trees that were in contact with infested wood chips and showed clear symptoms of pine wilt disease, B. xylophilus were extracted from 42 pines at 25°C and one pine at 15°C. The highest B. xylophilus infestation rates resulting in clear pine wilt disease symptoms (75%) were found in infested wood chips directly attached to stem‐wounded trees at 25°C. However, more variants exhibited B. xylophilus infestation at this temperature; trees with stem or root injuries plus direct contact with infested wood chips to the wounded part were primarily affected. Moreover, non‐vector spread was also detected in stem‐ and root‐injured pines without any direct contact with infested wood chips. Our results confirmed that B. xylophilus can survive for long periods in wood chips and can be transmitted from infested wood chips to damaged trees, but the likelihood of such PWN establishment should be low compared to spread through vectors. These findings must be considered in the pest risk analysis of B. xylophilus, and studies using outdoor trials should be carried out to complete this pest risk analysis.     

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.     

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.     

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.     

Population structure of Bursaphelenchus xylophilus within single Pinus thunbergii trees inoculated with two nematode isolates

A study was performed to clarify the population structure of the pinewood nematode, Bursaphelenchus xylophilus, within single Pinus thunbergii trees after double infection of nematode populations using the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method. Two nematode isolates, which had different levels or the same level of virulence, were inoculated into 6‐year‐old trees at the same or different times and then the propagated nematodes were collected from the trees after 1, 6 and 9 months. When a virulent and an avirulent isolate were inoculated into a single tree, an overwhelming propagation of the virulent isolate was observed there irrespective of the inoculation order of isolates or collection time of nematodes. However, when two virulent isolates were inoculated, propagation through the interbreeding between the two isolates was observed. In the case of the staggered inoculations with two virulent isolates, the frequency of nematodes with a PCR‐RFLP pattern of the primarily inoculated isolate increased with the time after nematode inoculations. This suggested that the population structure of B. xylophilus within a single tree varied by the virulence level of nematode populations transmitted and their transmission order.     

Molecular characteristics and functional analysis of the β‐1,4‐endoglucanase Bm‐eng‐1 gene of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae)

The pine nematode Bursaphelenchus mucronatus has been associated with pine wilt in China. This work was intended to investigate the role of the pathogenicity‐related β‐1,4‐endoglucanase gene of B. mucronatus in the infection of pines. In this study, the full‐length cDNA of the Bm‐eng‐1 gene was cloned and characterized from a B. mucronatus isolate. Phylogenetic analysis suggested that the Bm‐eng‐1 gene of B. mucronatus may be acquired from fungi through horizontal gene transfer. The function of the gene was demonstrated using RNA interference. RNA interference indicated that Bm‐eng‐1 was involved in the dispersal, reproductive ability and pathogenicity of B. mucronatus. In situ hybridization showed that Bm‐eng‐1 was specifically expressed in the oesophageal gland of B. mucronatus. Furthermore, to investigate the relation between β‐1,4‐endoglucanase activities and virulence of different isolates, the β‐1,4‐endoglucanase activities and the expression levels of Bm‐eng‐1 were detected in six B. mucronatus isolates with different virulence. β‐1,4‐Endoglucanase activity was generally higher in pathogenic isolates than in non‐pathogenic isolates, and the Bm‐eng‐1 expression levels in different isolates were positively correlated with the β‐1,4‐endoglucanase activity. These findings suggested that β‐1,4‐endoglucanase plays important roles in the pathogenic process of B. mucronatus, and the differential expression of the gene may underlie the different activity levels of β‐1,4‐endoglucanase, subsequently influencing variations in virulence of B. mucronatus isolates.     

Records of Bursaphelenchus spp. (Nematoda,Parasitaphelenchidae) in coniferous timber imported from the Asian part of Russia

Coniferous wood imported from the Asian part of Russia was surveyed in Germany (Mukran ferry terminal, highway and railway border station in Frankfurt/Oder) and Austria (railway in Marchegg, Retz and Wr. Neustadt, Lower Austria). The consignments consisted of mixed timber of Pinus/Picea or Pinus/Larix. Out of 625 samples investigated, 51 samples (8.5%) yielded eight Bursaphelenchus species. Bursaphelenchus mucronatus was found 42 times in Pinus, Picea and Larix wood, Bursaphelenchus hylobianum and Bursaphelenchus fraudulentus were detected twice, in Pinus/Larix and in Larix, respectively. The following species were each found once: Bursaphelenchus leoni in mixed timber of Pinus/Picea, Bursaphelenchus ‘borealis’ in Pinus, Bursaphelenchus hellenicus and Bursaphelenchus paracorneolus in Larix. One sample of Larix wood from Krasnoyarsk region contained a few specimens corresponding to Bursaphelenchus xylophilus. Bursaphelenchus mucronatus was present in about 30% of the samples showing signs of insect attack. A Monochamus species was found in a sample from Irkutsk. Most of the B. mucronatus isolates found belonged to the European genotype, whereas the East Asian genotype was found in three instances. This is the first report of B. fraudulentus, B. hellenicus, B. leoni, B. paracorneolus and the East Asian genotype of B. mucronatus in Russia. Bursaphelenchus hylobianum is the only species found in Russian wood and not in Europe so far. The species were identified morphologically and by internal transcribed spacer (ITS)‐restriction fragment length polymorphism (RFLP) technique. Species‐specific ITS‐RFLP patterns were established for B. hylobianum. In the case of the isolate morphologically corresponding to B. xylophilus, DNA extraction from the available low number of specimens failed to yield sufficient rDNA for ITS‐RFLP analysis.     

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.     

Correlation of seedling size and branch number with disease resistance of Pinus thunbergii seedlings to Bursaphelenchus xylophilus

Japanese black pine (Pinus thunbergii) seedlings resistant to pine wood nematode (PWN; Bursaphelenchus xylophilus) are routinely selected in Japanese field inoculation trials. Correlations between morphological factors (such as height, stem diameter at ground level and number of branches on seedlings) and disease resistance were examined to improve the production efficiency of 1‐year‐old black pine seedlings for inoculation. Family relatedness and environmental conditions strongly affected seedling resistance; accordingly, logistic regression analysis was used to separate effects of these two variables. Height and stem diameter at ground level significantly correlated with disease resistance in seedlings inoculated with PWN. Because (a) interactions between stem diameter at ground level and environmental condition were significant and (b) height did not interact with any other factor, it was concluded that height of 1‐year‐old Japanese black pine seedlings independently correlated with PWN resistance. Thus, field inoculation tests should use tall seedlings to achieve enhanced survival rates.     

Avirulent isolate of the pinewood nematode Bursaphelenchus xylophilus,survives 7 months in asymptomatic host seedlings

An avirulent isolate (C14‐5) of the pinewood nematode, Bursaphelenchus xylophilus, was inoculated onto 45 seedlings of the susceptible host Japanese black pine (Pinus thunbergii) and its viability was investigated. The nematode survived inside host seedlings for approximately 7 months even when the host seemed to overcome the infection based on lack of foliar wilting and the observation of normal oleoresin flow.     

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.     

Suppression of ectomycorrhizal development in young Pinus thunbergii trees inoculated with Bursaphelenchus xylophilus

In order to study the changes in ectomycorrhizal development during symptom expression of pine wilt disease, root window observations were conducted concurrent with measurements of leaf water potential as well as photosynthetic and transpiration rates of 5‐year‐old Pinus thunbergii trees that were inoculated with the pinewood nematode (PWN) Bursaphelenchus xylophilus. Infected trees were compared with girdled and uninfected control trees. Ectomycorrhizas developed constantly during the experimental period in control trees but did not develop in the girdled trees. Ectomycorrhizal development ceased within 2 weeks in those trees that finally died after PWN infection. In the trees that survived PWN infection, ectomycorrhizal development ceased within 1–4 weeks of inoculation but was resumed thereafter within 3–6 weeks. Ectomycorrhizal development ceased prior to a decrease in both photosynthetic rate and leaf water potential in the inoculated trees.     

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.     

Susceptibility of six pine species to Gremmeniella abietina isolates from Spain

Inoculation experiments were performed in order to evaluate the virulence of Gremmeniella abietina isolates from Spain on the main pine species planted in the Iberian Peninsula, as well as the influence of seedling age on this virulence. Two different experiments were carried out with four isolates of G. abietina from Spain. The greenhouse experiments consisted of seedling inoculations. One‐ and 2‐year‐old seedlings of the following five pine species were used: Pinus halepensis, Pinus pinea, Pinus pinaster, Pinus sylvestris and Pinus uncinata; also, 1‐year‐old seedlings of P. nigra were inoculated. The relative necrosis length (RNL) caused by the pathogen after 130 days was used as a response variable. The laboratory experiments were performed on 2‐ to 6‐year‐old internodes of the above pine species excluding P. uncinata. The necrosis length after 6 weeks of incubation was measured. The results have shown that all G. abietina isolates were pathogens on seedlings of these six pine species and seedlings of P. halepensis were consistently the most susceptible ones, although it is important to take into account that all the isolates used in the present work were isolated from P. halepensis, the only pine species in Spain where G. abietina has been recovered up to now. The susceptibility of the other pine species depended on the age of the seedlings.     

Occurrence of Bursaphelenchus species in declining Pinus sylvestris in a dry Alpine valley in Switzerland

We determined the incidence of Bursaphelenchus species in declining Pinus sylvestris stands in the Swiss Alps. A total of 217 trees showing different degree of decline were sampled from various locations in Valais between 2001 and 2004. Bursaphelenchus species were only found in recently dead or dying trees with an overall incidence of 40%. There was a positive relationship between Bursaphelenchus infestation and blue stain in these trees. Five Bursaphelenchus species were identified: Bursaphelenchus vallesianus, Bursaphelenchus mucronatus, Bursaphelenchus sexdentati, Bursaphelenchus leoni and Bursaphelenchus silvestris. The most frequent species were B. vallesianus detected in 75% and B. mucronatus in 20% of all Bursaphelenchus‐positive trees. Bursaphelenchus vallesianus is a recently described species within the B. sexdentati group, and could be a contributing factor in the observed pine decline in Valais.     

The effects of long‐term exposure to simulated acid rain on the development of pine wilt disease caused by Bursaphelenchus xylophilus

To elucidate the synergetic effects of acid rain on the development of pine wilt disease, we measured the sap flow rate in the stems and the chlorophyll content in the needles of 10‐year‐old Japanese black pine trees, Pinus thunbergii and 12‐year‐old Japanese red pine trees, Pinus densiflora, after exposure to simulated acid rain (SAR, pH 3) or tap water (TW, pH 6.3) as a control. The heat pulse method was used for the estimation of the sap flow rate. No apparent difference was found in the sap flow rate between the trees exposed to SAR and TW, but the chlorophyll content of needles at the end of the treatment was significantly higher in the trees exposed to SAR than in those exposed to TW. When the pinewood nematode, Bursaphelenchus xylophilus, the causal agent of pine wilt disease, was inoculated onto the Japanese black pines that had been exposed to SAR repeatedly for 1 year, the period to death was shortened. Japanese red pines that had been exposed to SAR for 2 years, however, did not show any development of symptoms after the nematode inoculation, suggesting that acid rain only affects pine wilt disease slightly, if at all.     

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.