On the genus Bursaphelenchus Fuchs, 1937 (Nematoda: Parasitaphelenchinae) associated with wood and insects from declining forest trees in the Czech Republic

An overview of the genus Bursaphelenchus in the Czech Republic is presented, based on a recent survey for monitoring the presence of the pinewood nematode, Bursaphelenchus xylophilus, as well as on previous reports of this genus in the country. In addition, we provide a morphological and molecular characterization of four Bursaphelenchus species (B. eremus, B. pinophilus, B. vallesianus and B. borealis) found during the monitoring programme for forest pests, conducted during 2006–2010, within the Moravian and Bohemian regions. Nematodes were extracted from over 1917 insects and 1493 wood samples collected from deciduous and coniferous trees exhibiting wilting and declining symptoms. Bursaphelenchus species were found only in 0.73% of insects and 0.47% of the total number of wood samples. Bursaphelenchus borealis and B. pinophilus dauer juveniles were found associated with the insect vectors Dryocetes autographus and Pityogenes bidentatus, respectively. While a total of seven Bursaphelenchus species are now reported from the Czech Republic, the status of B. xylophilus remains as absent.     

Occurrence of <Emphasis Type="Italic">Bursaphelenchus mucronatus</Emphasis> (Nematoda; Aphelenchoididae) in France and association with <Emphasis Type="Italic">Monochamus galloprovincialis</Emphasis> (Coleoptera: Cerambycidae)

As a consequence of the recent introduction of the pine wood nematode Bursaphelenchus xylophilus in Portugal, nematodes of the genus Bursaphelenchus were looked for in various French pine forests, in trees attacked by Monochamus galloprovincialis, the vector insect of B. xylophilus, and in the insects themselves. Trap trees were felled in 12 localities distributed all over the country. Nematodes were extracted from transversal stem discs; insects emerging from the trap trees were studied. B. hellenicus, B. leoni, B. mucronatus and B. sexdentati were isolated, but not B. xylophilus. The presence of B. mucronatus and the absence of B. xylophilus were confirmed by molecular markers. B. mucronatus was isolated from several regions with an average prevalence of 19%. The infestation of M. galloprovincialis by B. mucronatus reached 26.7%. The wide distribution of B. mucronatus in France could have an effect on the extension of B. xylophilus in a case of an introduction.     

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.     

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.     

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.     

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 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.     

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.     

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.     

New reports of Bursaphelenchus species associated with conifer trees in Romania

The 2013 annual monitoring programme for the pinewood nematode, Bursaphelenchus xylophilus, analysed a total of 267 wood samples collected from declining or symptomatic coniferous trees distributed among national forests, gardens, public parks, distribution centres, wood‐processing industries, as well as 104 samples collected from wood packing material originated from several other countries. From a total of eight species found, five have been previously reported from Romania, while three other species (Bursaphelenchus abietinus, Bursaphelenchus fraudulentus and Bursaphelenchus fuchsi) represent new findings for Romania. In addition, B. mucronatus was found in packing wood originated from Russia. Herein, we provide a morphological, morphometric and molecular characterization for the new species found for Romania. Bursaphelenchus xylophilus was not detected.     

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 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.     

Effects of accumulate dead and alive insects in trap on the capture ofTribolium castaneum (herbst)

In the trapping ofTribolium castaneum (Herbst), the activity of traps baited or not baited with aggregation pheromone 4,8-dimethyldecanal is influenced by the presence in the traps of accumulate specimens trapped. This fact has been observed both in trap containing alive or dead specimens. Interactions in the different substances produced by adults of Red Flour Beetle during their communication relationship are involved in this behaviour.The results obtained in the trials suggests that the presence of alive insects in traps is attractive for other free specimens, but under conditions of overcrowding quinone produced by them regulate population density by counteracting the effect of the aggregation pheromone, causing the insects to disperse or suppressing aggregation behaviour.The presence of dead insects in the traps, baited or not baited with pheromone, can be repellent for other free specimens ofT. castaneum, probably related to the presence of a residual alarm pheromone produced by the specimens before of death.With 10 figures and 3 tables     

First record of aBursaphelenchus-species (Nematoda) inMonochamus beetles in Scandinavia

A national research program was initiated in 1988 in Sweden to provide more information on the distribution and bionomics ofBursaphelenchus species. As a result of the intimate association known to occur between the pinewood nematodeB. xylophilus (Steiner andBuhrer) Nickle and its insect vectors a survey was directed to Scots pine wood (Pinus sylvestris L.) in whichMonochamus species were breeding. A total of 59M. sutor L. and twoM. galloprovincialis Ol. emerged from the collected material. Fourteen specimens of theM. sutor and bothM. galloprovincialis specimens containedBursaphelenchus nematodes which resembled the speciesB. mucronatus Mamiya andEnda. To our knowledge this is the first record of theB. mucronatus-type nematodes inM. galloprovincialis and the first finding of such nematodes inM. sutor in Europe.     

An inoculation experiment of Japanese <Emphasis Type="Italic">Bursaphelenchus</Emphasis> nematodes on Japanese black and red pine, <Emphasis Type="Italic">Pinus thunbergii</Emphasis> and <Emphasis Type="Italic">P. densiflora</Emphasis>

The pinewood nematode (PWN) Bursaphelenchus xylophilus is an invasive pathogen that was introduced from North America to Asian countries and Portugal and is devastating native pine forests. Some native European and Asian Bursaphelenchus nematodes also have weak to moderate pathogenicity to native pine species. To evaluate the potential risk of native Bursaphelenchus species, we inoculated ten Japanese Bursaphelenchus species into native pine species (the dominant forest species) in Japan, and evaluated their pathogenicity using mortality and tracheal tissue damage as indices. Inoculation was conducted on August 3, 2007, and the symptoms were observed every 2 weeks until February 1, 2008. None of the inoculated trees, excluding the pathogenic PWN inoculated control, showed external disease symptoms; however, four species [a less pathogenic PWN isolate, B. luxuriosae, Bursaphelenchus sp. NK215 (undescribed), and NK224 (undescribed)] caused tracheal tissue damage in inoculated seedlings and showed weak pathogenicity. Therefore, we conclude that there are some potentially pathogenic native species of nematodes distributed in Japan. Interestingly, two of these weakly pathogenic species, B. luxuriosae and NK215, are not associated with Pinaceae trees, suggesting that nematode pathogenicity may be a pre-adaptive character. More experimental studies under different conditions are necessary to accurately evaluate the potential risk of these pathogens.     

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.     

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]     

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.     

Non-target beetles (Coleoptera) in Trypodendron pheromone traps in Finland

Non-target beetles were surveyed in Trypodendron pheromone traps in 3 localities in Finland. Two window traps baited with Trypolure, containing lineatin dissolved in ethanol, and one control trap without attractant were used in each locality. A total of 1648 specimens belonging to 223 non-target species were recorded. The species were divided into five ecological groups to be treated separately. Hylurgops palliatus and Hylastes cunicularius were abundant in baited traps, whereas other scolytids as a group did not show clear attraction. Other species living in recently dead trees, including bark- and wood-boring species and associated species (predators, scavengers, fungivores, etc.), were clearly attracted by Trypolure. The most abundant ones were Hylecoetes flabellicornis, Thanasimus formicarius, Rhizophagus dispar, and Rhizophagus nitidulus. Although numerous in the samples, saproxylic species inhabiting more decayed wood and non-saproxylic species did not seem to be attracted. Grouping of species into ecological groups was helpful in the search for attraction patterns among species that usually are scarce in pheromone traps.