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.     

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.     

Migration of pine wood nematodes in the tissues of <Emphasis Type="Italic">Pinus thunbergii</Emphasis>

The distribution of pine wood nematodes (Bursaphelenchus xylophilus, PWNs) in Japanese black pine (Pinus thunbergii) tissues was investigated by staining with fluorescein isothiocyanate-conjugated wheat germ agglutinin. After PWNs were inoculated to current-year stems of pine seedlings, their distribution at about 5 cm below the inoculation site was confined only to cortical resin canals 1 day after inoculation, and then spread to other tissues, including resin canals of short branches. When PWNs were inoculated onto cross or tangentially cut surfaces of stem segments, maximal PWN migration speed was estimated to be faster through cortical resin canals and xylem axial resin canals vertically (>6.7 and <2.3 mm/h, respectively) than through cortical tissues both vertically and horizontally (<1.2 and <0.2 mm/h). To examine whether PWNs in cortical resin canals could invade surrounding tissues, segments in which PWNs resided only in cortical resin canals were prepared by removing the top portion 6 h after inoculation. Additional incubation of such segments caused extended PWN distribution to xylem axial resin canals and then to other tissues. A similar experiment with top portions of girdled segments removed 12 h after inoculation also showed extended PWN distribution from xylem axial resin canals and pith to cortical resin canals and then to other tissues. These results provided direct evidence that PWNs have the ability to migrate from cortical resin canals and xylem axial resin canals to other tissues.     

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.     

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

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

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

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

开展松材线虫病抗性选育 保护我国松林资源

松材线虫病是我国松林毁灭性病害,已造成数千万松株死亡,发生区经济损失巨大,目前此病害正威胁着南方重点林区及重要风景名胜区松林的安全。我国松属资源丰富,科技人员队伍组织体系完备,抗性选育研究已有较好基础。因此尽快开展抗性选育有利于发生区森林恢复、非发生区林分改造和结构调整,有利于保护我国森林资源。本文对开展抗性选育提出了具体建议。     

Migration of Bursaphelenchus xylophilus in cortical and xylem axial resin canals of resistant pines

Migration of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in susceptible and resistant pines was investigated at the tissue level. PWN was inoculated onto the top cross‐cut surface of 20‐cm stem cuttings of susceptible Pinus thunbergii and resistant pines (P. strobus, P. rigida and P. thunbergii of a resistant family Namikata‐(t)‐73 (half‐sib)). PWNs were mainly distributed in cortical resin canals of susceptible P. thunbergii down to 15 cm from the inoculated surface by 6 h after inoculation (HAI) and all tissues (including cortical and xylem resin canals) down to the bottom at 192 HAI. In P. strobus, P. rigida and P. thunbergii family Namikata‐(t)‐73 (half‐sib), PWN was distributed in cortical resin canals down to 5 cm by 6 HAI and down to the base at 192 HAI. However, the distribution of PWN in xylem resin canals of the resistant pines was restricted near inoculated surfaces down to 5 cm, even at 192 HAI. These results demonstrated that migration of PWN in resistant pines was slowed in cortical resin canals and restricted in xylem axial resin canals, features which may be associated with the resistance.     

Tree chitinases – stress‐ and developmental‐driven gene regulation

In recent years, a considerable number of studies have harnessed the power of genomics to decipher the role of pathogenesis‐related (PR) proteins in plant defence against various biotic and abiotic stresses. Chitinases are PR antifungal proteins expressed constitutively at low levels in plants and induced during biotic pressures and are demonstrated to be involved in the plant defence responses. Remarkable induction of chitinase enzymes by various abiotic agents (salicylic acid, jasmonic acid, ethylene and ozone) and biotic components (pathogens, insect pest, fungal cell wall components and oligosaccharides) is well demonstrated in plants. Several reviews on plant chitinase expression during host–pathogen interaction are available for annual species, whilst reports of their expression in tree species are limited to a few woody perennials: Populus, Pinus, Picea, Eucalyptus, Castanea and Pseudotsuga. The aim of this paper is to review the induction of chitinase during various stresses and developmental processes in forest tree species.     

Comparison of histological responses and tissue damage expansion between resistant and susceptible Pinus thunbergii infected with pine wood nematode Bursaphelenchus xylophilus

Pine wilt disease caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has been epidemic and has had disastrous impacts on pine forests and forest ecosystems in eastern Asia. Many pine species in this area are susceptible to this disease. Pinus thunbergii is particularly susceptible. In Japan, tree breeders have selected surviving trees from severely damaged forests as resistant candidates, and have finally established several resistant varieties of P. thunbergii. However, this breeding procedure requires much time and effort due to the lack of physiological and phenotypical information about resistance. To investigate the resistance mechanisms of selected P. thunbergii, we compared histochemical responses, tissue damage expansion, and PWN distribution in resistant and susceptible clones of P. thunbergii after PWN inoculation. The results suggested that the mechanisms of resistance are as follows: damage expansion in the cortex, cambium, and xylem axial resin canals are retarded in resistant trees soon after inoculation, probably due to the induction of wall protein-based defenses. Suppression of PWN reproduction was particularly caused by inhibition of damage expansion in the cambium. The slow expansion of damage in each tissue provides time for the host to complete the biosynthesis of lignin in the walls of cells that surround the damaged regions. This lignification of cell walls is assumed to effectively inhibit the migration and reproduction of the PWNs. The mechanism of initial damage retardation is presumed to be a key for resistance.     

Comparison of the potential spread of pinewood nematode (Bursaphelenchus xylophilus) in Finland and Iberia simulated with a cellular automaton model

Pinewood nematode (PWN) is one of the most threatening invasive pests in the pine forests of Europe, and it has recently spread to the Iberian Peninsula via import of timber and wooden packaging material from East Asia. A cellular automaton (CA) model was developed to simulate and compare the potential spread of PWN by transportation and its vectors, Monochamus beetles in the pine forests of Finland and Iberian Peninsula. The model assumes that all pines are equally sensitive to PWN. The CA is a spatio‐temporal grid‐based model, which can easily be applied on different geographical scales. The effects of climate warming and number of entries from ports on the spread of PWN were studied. A sensitivity analysis was conducted on the most uncertain model parameters. Twenty years after hypothetical entries, the predicted area of symptomatic PWN infection (pine wilt disease, PWD) was very low in Finland compared to Iberia. This was because of the low probability of warm July in Finland. The increase in the mean July temperature increased the area of PWD‐infected pine forest relatively more in Finland than in Iberia. An increase in the number of entries also increased the area of PWD‐infected pine forest relatively more in Finland than in Iberia. The probability of PWD infection was the highest in pine forests that were close to entry points and in areas with low elevation and high human population density.     

Abundance-dependent transmission of the pinewood nematode, <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis> (Nematoda: Aphelenchoididae), to the Japanese pine sawyer, <Emphasis Type="Italic">Monochamus alternatus</Emphasis> (Coleoptera: Cerambycidae), adult in its pupal chamber

The transmission ratio of the pinewood nematode (PWN), Bursaphelenchus xylophilus, to the emerging adult Japanese pine sawyer (JPS), Monochamus alternatus, in its pupal chamber is a determinant of the number of the nematodes carried by JPS beetles. To investigate the factors affecting the transmission ratio, we counted the number of the nematodes carried by 36 newly emerged JPS beetles and the number remaining in and around their pupal chambers, and then estimated the transmission ratio (the number of nematodes carried by a JPS adult as a percentage of the total number of nematodes aggregating in and around its pupal chamber). The total number of nematodes aggregating in and around a pupal chamber ranged from 0 to 19,041, and the number of nematodes carried by a beetle ranged from 0 to 18,920. The transmission ratio correlated with neither the water content of the wood around the pupal chamber nor the degree of wood discoloration caused by blue-stain fungus. The transmission ratio varied with the abundance of the nematodes aggregating in and around the pupal chamber. In pupal chambers with more than 1,000 nematodes, almost all the nematodes were transmitted to the beetle. However, in pupal chambers with fewer than 1,000 nematodes, the transmission ratio varied greatly, from 0 to 100%. These results suggest that aggregation of many PWNs in the pupal chamber might stimulate transmission of the PWNs to the JPS adult and that this abundance-related transmission might contribute to the large variation in the number of PWNs carried by the JPS beetle.     

A key effector,BxSapB2, plays a role in the pathogenicity of the pine wood nematode Bursaphelenchus xylophilus

The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses in pine forests. The plant‐parasitic nematodes have a complex life cycle that includes the secretion of effector proteins through a stylet into the host cell to promote parasitism. In this study, SignalP 4.1 and TMHMM 2.0 were used in preliminary screens for candidate effectors and were expressed in Nicotiana benthamiana through the PVX virus expression vector. The yeast signal sequence trap system was used to further study the function of the signal peptide of an effector, BxSapB2. In situ hybridization was conducted to investigate the localization of BxSapB2, followed by RNA interference technology (RNAi) to assess the functions of BxSapB2. The results demonstrate that BxSapB2 is a secreted protein that induces cell death in N. benthamiana and is highly expressed in esophageal gland cells and amphids of B. xylophilus. BxSapB2 was determined to be related to the pathogenicity of B. xylophilus. The results of this work indicate that BxSapB2 plays an important role in the interactions between B. xylophilus and the hosts.     

Identification and genetic analysis of the pinewood nematode Bursaphelenchus xylophilus from Pinus yunnanensis

In China, pinewood nematode (PWN), Bursaphelenchus xylophilus, was first discovered from Pinus thunbergii in 1982. Thus far, 14 species in the genus Pinus have been reported to be infected by PWN under natural conditions. Pinus yunnanensis, a pine species native to south‐western China, is considered a pioneer tree for barren hill afforestation in areas undergoing rocky desertification. In this study, we detected PWN in dead P. yunnanensis trees in Anlong County, Guizhou Province, China, using both morphological and molecular methods. To our knowledge, this is the first report of PWN from P. yunnanensis in China. To investigate the possible origin of this new outbreak, mitochondrial cytochrome oxidase gene subunit I and cellulase gene sequences were used to evaluate genetic relationships among worldwide PWN isolates. Phylogenetic tree and haplotype networks revealed that the Anlong isolate (BxChQAL008) sequence was identical to those of seven Chinese isolates collected from Sichuan, Chongqing, Zhejiang, Anhui and Shandong (372–1500 km from Anlong County), but different from the isolate BxChQZY030 collected from the same province (330 km from Anlong County). It is suggested, therefore, that more than one introduction of PWN into Guizhou Province has taken place. The Anlong isolate was likely introduced from neighbouring or more distant provinces rather than from outside China. Moreover, the absence of a correlation between geographic and genetic distance was observed using Mantel test analysis, providing evidence that human‐induced dispersal plays a fundamental role in the spread of the PWN in this region.     

Mutualistic symbiosis between Bursaphelenchus xylophilus and bacteria of the genus Pseudomonas

Interactions between the pine wood nematode (PWN), Bursaphelenchus xylophilus, and bacteria of the genus Pseudomonas were examined by cultivating axenic PWN and bacterial strains using callus of Pinus thunbergii. Ten (Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia and Pseudomonas spp.) of the 29 bacterial strains tested, significantly increased the reproduction of PWN. The rest of the bacteria (19 strains of 10 species) inhibited the reproduction of PWN completely. The growth of 18 of the 29 bacterial strains tested, including the 10 strains promoting PWN reproduction, was significantly increased by the presence of PWN. It indicated a mutualistic symbiotic relationship between PWN and the 10 bacterial strains in the genus Pseudomonas. The bacterial mutualistic symbionts are organisms, which may have co‐evolved with PWN rather than being accidentally associated. The finding provides further evidence for our hypothesis that pine wilt disease is complex, induced by both PWN and associated phytotoxin‐producing bacteria.     

Pine wilt disease: a short review of worldwide research

This article summarizes the results of the research papers presented at the International Symposium on pine wilt disease (IUFRO Working Party Meeting 4.04.03) held in July 2009, at Nanjing, China. The general topics covered were on pine wilt disease (PWD), its causal organism, the pinewood nematode (PWN) Bursaphelenchus xylophilus, plus other PWN-associated microorganisms that play a significant role in PWD such as bacteria (e.g. Pseudomonas fluorescens). Most of the papers that are reviewed are based on work on PWD-PWN in East Asia and Russia. Specific topics covered include: 1) the fundamental conceptions of PWD development, 2) pathogenicity, 3) host-parasite relationships including the histopathology of diseased conifers and the role of toxins from bacteria-nematode ecto-symbionts, 4) PWN life cycle and transmission, 5) B. xylophilus dissemination models, 6) associations (with other nematodes), 7) diagnostics, 8) quarantine and control of the PWN and 9) biocontrol of the PWN.     

First report of Fusarium solani causing wilt of Melia dubia

Melia dubia, a multipurpose tree species, is gaining importance to meet the demand supply gap of timber, plywood and pulpwood . In June 2016, a serious outbreak of wilt disease was observed in M. dubia seedlings planted in the Central Nursery of Forest Research Institute (FRI), Dehradun, India. The disease led to the destruction of one hundred thousand (100,000) seedlings. Earlier in June 2012, serious wilting of M. dubia seedlings was observed in Haryana, India. The pathogen was identified as Fusarium solani following standard laboratory procedures and sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA). The pathogenicity of three isolates has been proved under greenhouse conditions. This is the first report of F. solani causing wilt of M. dubia.