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.     

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.     

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.     

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.     

Pathogenicity of Bursaphelenchus sexdentati,Bursaphelenchus leoni and Bursaphelenchus hellenicus on European pine seedlings

In Greece extensive pine wilting is associated with the presence of nematodes of the genus Bursaphelenchus. In order to check the pathogenicity of Bursaphelenchus sexdentati, Bursaphelenchus leoni and Bursaphelenchus hellenicus, inoculation tests were carried out on 3‐year‐old seedlings of Pinus brutia, Pinus halepensis, Pinus nigra, Pinus pinaster and Pinus sylvestris. For inoculations, 6000 nematodes in 0.5 ml of water were used per seedling. Bursaphelenchus sexdentati proved to be highly virulent causing mortality of up to 100%, followed by B. leoni whereas B. hellenicus was nonpathogenic.     

Promotion of the population growth of pinewood nematode in 4-month-old Japanese black pine seedlings by pretreatment with simulated acid rain

Simulated acid rain (SAR) at three pH levels (pH 4, 3, 2) was applied to only the top or both the top and roots of 4-month-old Japanese black pine seedlings repeatedly for two months. Then the seedlings were inoculated with a virulent isolate (S10) of pinewood nematode,Bursaphelenchus xylophilus. The exposure to SAR at any level did not kill the seedlings. When inoculated with nematodes, however, the nematode population build-up was significantly higher in the seedlings pretreated with SAR at any level than in the control at the 7th and 17th day after inoculation, and subsequent disease symptom development was also significantly accelerated by the exposure to SAR at pH 2 and 3. This result indicates that even acid rain at pH 4 has the potential for promoting population growth of pinewood nematodes in 4-month-old Japanese black pine seedlings.     

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.     

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.     

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.     

Fluorescein-labeled wheat germ agglutinin stains the pine wood nematode, <Emphasis Type="Italic">Bursaphelenchus </Emphasis><Emphasis Type="Italic">xylophilus</Emphasis>

Pine wilt disease, caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is a major threat to pine forests throughout East Asia. Nonetheless, its mechanism of invasion has not yet been described in detail. To better understand the pathology of this disease, it is important to examine the distribution of PWNs within pine tissue during the course of disease development. We attempted to stain nematodes with fluorescein-conjugated wheat germ agglutinin (F-WGA) as a means to locate and track the spread of PWNs. Although PWNs proliferated on Botrytis cinerea fungus were successfully stained only on their vulvas and spicule holes, PWNs extracted from inoculated Pinus thunbergii seedlings were stained on their surface. Stainability, or the percentage of PWNs stained with F-WGA over more than half of their surface, was about 20% by 1 day after inoculation, but increased to 80% at 10 days. The stainability of PWNs extracted from a 5-cm main stem segment that included the inoculation site was less than that of PWNs extracted from other parts of the main stem farther away (i.e., those that had dispersed). These results suggest that stainability is related to dispersal activity in time. Thus, to raise the stainability of PWNs at shorter timeframes after inoculation, PWNs with more than 80% stainability were re-inoculated into pine seedlings. This resulted in more than 70% stainability from 1 to 6 days after inoculation. In F-WGA stained thin paraffin sections of pine tissue of re-inoculated seedlings, PWNs brightly fluoresced under epifluorescence and were easily detected against the dark background of pine tissue. This staining technique with F-WGA is an excellent tool for detecting PWNs in pine tissue.     

Susceptibility of adult trees of the endangered species Pinus armandii var. amamiana to pine wilt disease in the field

Adult trees of Pinus armandii var. amamiana (PAAm) and P. thunbergii grown in the field were inoculated with 100000 or 1000 of the nematode Bursaphelenchus xylophilus to evaluate their susceptibility to pine wilt disease. PAAm trees inoculated with 100000 nematodes started to show disease symptoms 2 weeks after inoculation, and all died within 29 weeks. Although the PAAm trees inoculated with 1000 nematodes tended to show delayed disease symptoms compared with those inoculated with 100000 nematodes, all of them died within 33 weeks after inoculation. All P. thunbergii trees inoculated with 1000 nematodes had died 6 weeks after inoculation. In the nematode-inoculated PAAm trees, death of branches distal to the nematode inoculation site was the first visible symptom, followed by the systemic discoloration of needles, whereas the whole tree wilted simultaneously in P. thunbergii trees. In nematode-inoculated PAAm trees, the period from inoculation to death was longer than that in P. thunbergii. These results suggest that adult PAAm trees are susceptible to pine wilt disease, but are less vulnerable than P. thunbergii.     

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.     

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.     

Physiological process of the symptom development and resistance mechanism in pine wilt disease

Pathophysiological changes during the symptom development of pine wilt disease are reconsidered from recent investigations. The symptom development is divided into two stages: the early and the advanced stages. In the early stage, small number of nematodes migrate in cortex, then in xylem of the stem, and induce denaturation and necrosis of parenchyma cells. These changes in parenchyma are regarded as defense reactions of pines which result in terpene synthesis in xylem cells and embolism in tracheids. Such changes in the early stage can be induced in both susceptible and resistant pine species by either virulent or avirulent isolates of pinewood nematode (Bursaphelenchus xylophilus), or byB. mucronatus. No change occur in physiological status of leaves, and nematode reproduction is suppressed during this stage. Pine trees can survive if symptom does not progress from this stage. The symptoms of the advanced stage usually occur only in susceptible pines infected by virulent nematode isolates. At the beginning of the advanced stage, enhanced ethylene production by stem which coincides with cambial destruction occurs, and results in embolism of the outermost xylem in the portion. The embolism causes decrease in leaf water potential and cessation of photosynthesis. After cessation of photosynthesis, symptoms develop drastically with a burst of nematode population. There seems to be some unknown mechanism which suppress nematode reproduction and invasion to the cambial zone. This mechanism is thought to be photosynthesis-dependent, so that in photosynthesis-decrased conditions, even avirulent nematodes can multiply and invade cambium to induce tree death. Water stress in hot and dry summer should accelerates symptom development from the early to the advanced stage through such decrease of photosynthesis-dependent “cambial resistance”.     

Different development of pine wilt disease in artificially infectedPinus thunbergii seedlings potted together with different tree species

To evaluate the effect of adjacent tree species on the susceptibility of Japanese black pine (Pinus thunbergii) to pine wilt disease, an inoculation experiment was conducted usingP. thunbergii seedlings potted with seedlings of six tree species,i. e. Alnus sieboldiana, Eurya japonica, Lespedeza bicolor formacutifolia, Pinus thumbergii, Robinia pseudo-acacia andSarothamus scoparius. About ten months after planting, they were inoculated with the pinewood nematode (Bursaphelenchus xylophilus) in early July 1992. After that, the proportion of pine seedlings with completely discolored foliage increased more quickly when the seedlings were potted withR. pseudo-acacia, S. scoparius orA. sieboldiana than when potted withP. thunbergii, L. bicolor orE. japonica. At the end of the study period, 17 weeks after inoculation, it reached 90.6%, 90.0%, 87.5%, 72.7%, 63.3%, and 59.4% when the pine seedlings were potted withR. pseudo-acacia, S. scoparius, A. sieboldiana, P. thunbergii, L. bicolor andE. japonica, respectively. This indicated that the susceptibility ofP. thunbergii seedlings to pine wilt disease was influence by the species of adjacent trees.     

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.     

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.     

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.     

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 of the pine wood nematode at different developmental stages

The ability of the pine wood nematode,Bursaphelenchus xylophilus, a pathogen that causes pine wilt disease, to kill cortical cells of Japanese black pine,Pinus thunbergii, during early development of the disease was conjectured to be a function of nematode developmental stage. A tangential segment of bark was removed from a 2-cm-long current-year stem. The cortex-exposed segments with cut cortical resin canals were designated as + RC-segments and those without them as − RC-segments. When a nematode population containing many older juveniles and adults (NL) was inoculated onto the cut surface, the − RC-segments were still alive 4 d after inoculation, as were non-inoculated control segments. When cortex-exposed segments were inoculated with either a nematode population containing many younger nematodes (NS) or with nematodes isolated from inoculated pine cuttings that also contained many younger juveniles, most tissue cells in − RC-segments died 4 d after inoculation, suggesting that younger juveniles killed pine cells directly, in contrast with older juveniles and adults. When nematodes were inoculated onto + RC-segments in which they could easily enter resin canals, both NL and NS killed the segment tissues. This suggests that NL is pathogenic to pine cells while living in resin canals. Such differences in the pathogenicity of NL and NS to pine parenchymatous cells were also demonstrated in a pathogenicity assay system using bark peelings, which allowed an estimate of direct attack on the cambial cells by nematodes. Based on these results, we hypothesize that younger juveniles are pathogenic to pine parenchymatous cells, while adults and older juveniles are not pathogenic. This work was supported in part by Grants-in-Aid for Scientific Research (No.01440012 and 06454088) and for Young Scientists (to K.I.) from Ministry of Education, Science, Culture, and Sports of Japan, and by a grant from PROBRAIN.