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.     

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.     

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.     

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.     

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.     

Chitosan as a biocontrol agent against the pinewood nematode (Bursaphelenchus xylophilus)

The pine wilt disease (PWD) is caused by Bursaphelenchus xylophilus and poses great environmental and economic challenges. Thus, the development of sustainable techniques for the control of this epidemic disease is of major importance. This work aimed at evaluating if the application of different molecular weight (MW) chitosans as a soil amendment could be used to control the PWD in maritime pine (Pinus pinaster, very susceptible to the disease) and stone pine (Pinus pinea, less susceptible). At the end of the experimental period (24 days after inoculation), P. pinaster and P. pinea untreated plants presented ca. 3825 ± 100 and 70 ± 47 nematodes, respectively. In P. pinaster, the high‐MW chitosan prompted the most drastic results, inducing a 21.9‐fold reduction in nematodes numbers, whereas in P. pinea, the most effective was the low MW chitosan, which reduced nematodes numbers up to 7‐fold, compared with untreated plants. P. pinea seems to be highly resistant to the disease, presenting nematode numbers up to 54.6‐fold lower than P. pinaster and less severe chlorophyll loss (ca. 2‐fold).     

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.     

寄生加勒比松松材线虫的鉴定及其致病性测定*

运用线虫比较形态学和rDNA—ITS—PCR-RFLP方法对采自广东省广州市白云山加勒比松上的伞滑刃线虫进行鉴定,结果确定该线虫为松材线虫。致病性测定表明该松材线虫种群对加勒比松苗有较强的致病力,接种的松苗均出现不同程度的枯萎,而且从枯死木中可重新分离到该线虫。     

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.     

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.     

A rapid staining‐assisted wood sampling method for PCR‐based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR‐based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining‐assisted wood sampling method aiding PCR‐based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5‐mg wood cross‐sections, of 0.5 × 0.5 × 0.015 cm dimension, that were cut from a wood sample of 0.5 × 0.5 × 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5‐mg wood cross‐sections (that adjoined the three 5‐mg wood cross‐sections, mentioned above) were cut. Nematode‐staining‐spots (NSSs) in each of the two stained sections were counted under a microscope at 100× magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus– specific amplicon of 403 bp ( DQ855275 ) was generated by PCR assay from 100.00% of 5‐mg wood cross‐sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx‐detection methods such as PCR assay.     

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.     

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.     

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.     

Bursaphelenchus fungivorus (Nematoda: Aphelenchida) associated with Orthotomicus erosus (Coleoptera: Scolitydae) in Spain

A survey of the xylophagous insects in the conifer forests, mainly Pinus pinaster woodlands, in Spain was carried out in order to study the phoretic vectors of nematodes of the genus Bursaphelenchus. Fifteen environmental sites were characterized on the bases of altitude, rainfall, temperature and conifer species. Specimens of Orthotomicus erosus (n = 3476) were captured of which 59% carried nematodes under their elytrous, most of them were aphelenchid fungal‐feeding nematodes, but B. fungivorus was found on the beetles from Las Cumbres, Villa Blanca, (Huelva), southern Spain, which represent the first record of O. erosus as vector of a Bursaphelenchus sp. The nematode is characterized by morphometric and biomolecular techniques and distribution and ecology of the insect is discussed.     

Canadian conifers as hosts of the pinewood nematode (Bursaphelenchus xylophilus): Results of seedling inoculations

Seedlings of 22 species of conifers from across Canada were inoculated with m and r form isolates of the pinewood nematode (Bursaphelenchus xylophilus). In an experiment made under ambient (summer‐fall) temperatures in a shadehouse at Victoria, British Columbia, Canada, 8 of the 22 conifer species were killed by the nematodes, but mortality was low, i.e. 4–30%. Pines (eastern white, Jack and red) were more susceptible than other conifers. Yellow cypress, eastern white cedar, western red cedar and western hemlock were not killed by the nematodes. In a second experiment made at elevated temperatures (30°C‐16h long days, 25°C‐8 h long nights) in a greenhouse, 18 of the 22 conifer species died following nematode inoculation. Again, pines (lodgepole, eastern white, western white and red) were among the most susceptible tree species and the four conifers that were unaffected in the first experiment were not killed. Tamarack and western larch, both immune at ambient temperatures, were the two most susceptible conifers at elevated temperatures. Compared to ambient temperatures, seedlings at elevated temperatures died quicker and contained more nematodes. M and r form nematodes were equally pathogenic in both experiments.     

Pathogenicity of canadian isolates of the Bursaphelenchus xylophilus (pinewood nematode) to provenances of Pinus sylvestris and Pinus contorta as grown in Finland: A greenhouse study

Seedlings of Scots pine (Pinus sylvestris L.) and lodgepole pine (P. contorta Dougl.) provenances, as grown in Finland, were inoculated with “m”; and “r”; “forms”; of the pinewood nematode (Bursaphelenchus xylophilus) from Canada, an m form isolate from France and an r form isolate from Missouri, USA. Scots pine was highly susceptible to an Alberta r form and a British Columbia m form isolate and moderately susceptible to two Quebec m form isolates. Lodgepole pine was higly susceptible to the two r form (Alberta and Missouri) isolates and moderately susceptible to the British Columbia m form and the two Quebec m form isolates. Mortality of both pines after inoculation with the French isolate was inconsistent. Mortality of both pines occurred more rapidly following inoculation with r form than with m form nematodes. Large numbers of nematodes were generally found in the tissues of both pines. Our results with seedlings need to be corroborated by inoculating larger field‐grown trees.