Virulence of Bursaphelenchus xylophilus Isolated from Naturally Infested Pine Forests to Five Resistant Families of Pinus thunbergii

Pine wilt disease is one of the most serious epidemic tree diseases in Japan, and resistant pine trees have been developed through a breeding program. To evaluate resistance of resistant families of Japanese black pine, Pinus thunbergii, to the pinewood nematode, Bursaphelenchus xylophilus, isolated from the field, and to determine whether differentiation of pathogenicity to resistant pine families appears in the nematode isolates, seedlings of five resistant pine families were inoculated with 25 nematode isolates. Disease incidence 18 weeks after inoculation was significantly different among nematode isolates and among pine families but there was no interaction effect between nematode isolate and pine family. This indicates that nematode isolates did not have differential host specificity to resistant families of P. thunbergii. Isolate Shimabara, a test isolate of the breeding program, showed the same degree of virulence as the highly virulent isolates frequently used in experiments. However, more virulent isolates than Shimabara were found among the isolates collected from natural pine forest. This indicated that B. xylophilus populations with higher virulence than Shimabara exist in the natural population. These findings are important in development of more efficient breeding procedures for resistant pine trees.

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Study of symptoms and gene expression in four Pinus species after pinewood nematode infection

Plant Genetic Resources ◽

10.1017/s1479262111000062 ◽

2011 ◽

Vol 9 (2) ◽

pp. 272-275 ◽

Cited By ~ 5

Author(s):

Albina R. Franco ◽

Carla Santos ◽

Mariana Roriz ◽

Rui Rodrigues ◽

Marta R. M. Lima ◽

...

Keyword(s):

Virulent Strain ◽

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Wilt Disease ◽

Pinewood Nematode ◽

Pine Trees ◽

Severe Infections ◽

Genomic Results ◽

Avirulent Isolate

Pine wilt disease, caused by the pinewood nematode Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, is originating severe infections in pine trees. The disease is detected when external symptoms appear (e.g. needle chlorosis), but trees could remain asymptomatic for long periods and serve as a long-term host. The primary goal of this study was to assess the effect of inoculation with an avirulent isolate of B. xylophilus (C14-5) on different Pinus spp. seedlings (P. sylvestris, P. nigra, P. pinea and P. pinaster). At the same time, seedlings were also inoculated with a virulent strain, HF, in order to compare the phenotypic and genomic results of the two types of inoculations. The effect of inoculation was determined in terms of expression of various Pinus genes potentially involved in the response to the disease.The results suggest that P. pinea and P. nigra are more resistant to infection by the nematode than P. sylvestris and P. pinaster. The phenotypic and genetic differences were more marked among P. pinea and P. pinaster.

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Pine chemical volatiles promote dauer recovery of a pine parasitic nematode, Bursaphelenchus xylophilus

Parasitology ◽

10.1017/s0031182019001264 ◽

2019 ◽

Vol 147 (1) ◽

pp. 50-57 ◽

Cited By ~ 2

Author(s):

Wei Zhang ◽

Yongxia Li ◽

Long Pan ◽

Xuan Wang ◽

Yuqian Feng ◽

...

Keyword(s):

Parasitic Nematode ◽

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Wilt Disease ◽

Pine Forests ◽

Pinewood Nematode ◽

Dispersal Stage ◽

Pine Trees ◽

The Common ◽

Gradient Effects

AbstractPinewood nematode, Bursaphelenchus xylophilus, a pine parasitic nematode, poses a serious threat to its host pine forests globally. When dispersal-stage larvae 4 (dauer, DL4) of B. xylophilus enters the new pine, it moults into propagative adult (dauer recovery) and reproduces quickly to kill the host pine. Here, we found pine chemical volatiles, rather than the common dauer recovery factors of nematodes (e.g. suitable temperatures, nutrient availability or density), promote B. xylophilus dauer recovery. The results showed that volatilization of chemicals in host pines could attract DL4 and promote DL4 recovery. To identify which chemicals promote this process, we determined the stimulated activity of the main volatiles of pines including six monoterpenes and two sesquiterpenes. Results showed that all the six monoterpenes promoted dauer recovery, especially β-pinene and β-myrcene, but the two sesquiterpenes have no effect on the transformation. Furthermore, β-pinene performed gradient effects on dauer recovery. We hypothesized that when DL4 infect pine trees, the pine volatiles released from the feeding wounds are used as chemical signals for DL4 transformation to adult to reproduce and rapidly kill the pines. Our study identified the B. xylophilus dauer recovery chemical signal and may contribute to preventing pine wilt disease.

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The specialization of Esteya vermicola hyphae in infection to Bursaphelenchus xylophilus and its colonization of pine tree

10.1101/2020.08.21.260729 ◽

2020 ◽

Author(s):

Hai-Hua Wang ◽

Can Yin ◽

Jie Gao ◽

Ran Tao ◽

Piao-Piao Dai ◽

...

Keyword(s):

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Wilt Disease ◽

Fungal Colonization ◽

Pinewood Nematode ◽

Pine Tree ◽

Pine Wilt ◽

Pine Trees ◽

Colonization Patterns ◽

Esteya Vermicola

AbstractPine wilt disease (PWD) caused by the nematode Bursaphelenchus xylophilus is a serious problem on pines, and there is currently no effective control strategy for this disease. Although the endoparasitic fungus Esteya vermicola showed great effectiveness in controlling pine wilt disease, the colonization patterns of the host pine tree xylem by this fungus are unknown. To investigate the colonization patterns of pine xylem by this fungus, the species Pinus koraiensis grown in a greenhouse was used as an experimental host tree. The fungal colonization of healthy and wilting pine trees by E. vermicola was quantified using PCR with a TaqMan probe, and a green fluorescence protein (GFP) transformant was used for visualization. The results reported a specific infection approach used by E. vermicola to infect B. xylophilus and specialized fungal parasitic cells in PWN infection. In addition, the inoculated blastospores of E. vermicola germinated and grew inside of healthy pine xylem, although the growth rate was slow. Moreover, E. vermicola extended into the pine xylem following spray inoculation of wounded pine seedling stems, and a significant increase in fungal quantity was observed in response to B. xylophilus invasion. An accelerated extension of E. vermicola colonization was shown in PWN-infected wilting pine trees, due to the immigration of fungal-infected PWNs. Our results provide helpful knowledge about the extension rate of this fungus in healthy and wilting PWN-susceptible pine trees in the biological control of PWD and will contribute to the development of a management method for PWD control in the field.Author summaryPine wilt disease, caused by Bursaphelenchus xylophilus, has infected most pine forests in Asian and European forests and led to enormous losses of forest ecosystem and economy. Esteya vermicola is a bio-control fungus against pinewood nematode, showed excellent control efficient to pine wilt disease in both of greenhouse experiments and field tests. Although this bio-control agent was well known for the management of pine wilt disease, the infection mechanism of fungal infection and colonization of host pine tree are less understand. Here, we use GFP-tagged mutant to investigate the fungal infection to pinewood nematode; additionally, the temporal and spatial dynamics of E. vermicola colonize to pine tree were determined by the TaqMan real-time PCR quantification, as well as the response to pinewood nematode invasion. We found a specific infection approach used by E. vermicola to infect B. xylophilus and specialized fungal parasitic cells in PWN infection. In addition, the fungal germination and extension inside of pine tree xylem after inoculation were revealed. In addition, the quantity of E. vermicola increased as response to pinewood nematode invasion was reported. Our study provides two novel technologies for the visualization and detection of E. vermicola for the future investigations of fungal colonization and its parasitism against pinewood nematode, and the mechanisms of the bio-control process.

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Effects of Temperature Factors on Resistance against Pine Wood Nematodes in Pinus thunbergii, Based on Multiple Location Sites Nematode Inoculation Tests

Forests ◽

10.3390/f11090922 ◽

2020 ◽

Vol 11 (9) ◽

pp. 922

Author(s):

Taiichi Iki ◽

Koji Matsunaga ◽

Tomonori Hirao ◽

Mineko Ohira ◽

Taro Yamanobe ◽

...

Keyword(s):

Pine Wilt Disease ◽

Similar Rate ◽

Bursaphelenchus Xylophilus ◽

Pinus Thunbergii ◽

Pinewood Nematode ◽

Resistance Level ◽

Japanese Archipelago ◽

Effects Of Temperature ◽

The Relationship ◽

Temperature Factors

Pine wilt disease (PWD) caused by the pinewood nematode (PWN) (Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle) is a worldwide issue. Infection is considered to be promoted mainly by the increased air temperature, but it is important to investigate whether the effect of high temperature similarly influences the different ranks of resistant clone. In the present study, we conducted PWN inoculation tests using six common open-pollinated families of resistant Pinus thunbergii Parl. The tests were conducted at nurseries of five test sites across Japanese archipelago between 2015 and 2017. Our analysis focused specifically on temperature. Firstly, we examined the effects of test sites, inoculation year, and their interaction on unaffected seedling rate and found that the unaffected seedling rate of all tested pine families decreased as the cumulative temperature increased. We found that the unaffected seedling rate decreased as the cumulative temperature increased for all tested pine families. In general, higher cumulative temperatures were required for having an effect on the unaffected seedling rates of higher PWN-resistant families. Typically, early cumulative temperatures, i.e., 19 days after inoculation, had the greatest effect on the unaffected seedling rates of PWN-resistant pines. However, the relationship between cumulative temperature and predicted unaffected seedling rate follow similar rate for all families. Thus, the order of resistance level is maintained in terms of the cumulative temperature required for having an effect.

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Monochamus Saltuarius Endangers Pinus tabuliformis Carr. and Carries Bursaphelenchus xylophilus (Steiner and Buhrer) in China

Forests ◽

10.3390/f11101051 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1051

Author(s):

Long Pan ◽

Yongxia Li ◽

Rong Cui ◽

Zhenkai Liu ◽

Xingyao Zhang

Keyword(s):

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Pinewood Nematode ◽

Vector Species ◽

Pinus Tabuliformis ◽

Longhorn Beetles ◽

Average Annual Temperature ◽

Pine Trees ◽

Pinewood Nematodes ◽

And Control

The pinewood nematode (Bursaphelenchus xylophilus) can cause fatal damage to trees and is transmitted by the vector species of the Monochamus genus. In December 2017, pinewood nematodes were found to be harming a large plot of pine trees, Pinus tabuliformis, in a new region with an average annual temperature of 6.7 °C in China. However, the vector insects were unknown and urgently needed to be identified. Hence, in April 2018, we collected wood sections of P. tabuliformis trees that had died from pine wilt disease. All 127 longhorn beetles that emerged from the P. tabuliformis samples were identified as Monochamus saltuarius, and the nematodes they carried were fourth-stage dispersal juveniles of the pinewood nematode. The carrier rate of pinewood nematodes in the M. saltuarius population was 58.3%, and the average carrying capacity was 642.4 ± 89.3. In the same P. tabuliformis plot, 742 M. saltuarius beetles were collected from hormone traps. Our results revealed that M. saltuarius can damage P. tabuliformis and is a new vector species of the pinewood nematode in China. These findings can inform the prevention and control of pinewood nematode damage to pine forests.

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Comparative Secretome and Functional Analyses Reveal Glycoside Hydrolase Family 30 and Cysteine Peptidase as Virulence Determinants in the Pinewood Nematode Bursaphelenchus xylophilus

Frontiers in Plant Science ◽

10.3389/fpls.2021.640459 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ryoji Shinya ◽

Haru Kirino ◽

Hironobu Morisaka ◽

Yuko Takeuchi-Kaneko ◽

Kazuyoshi Futai ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Wilt Disease ◽

Glycoside Hydrolase Family ◽

Pinewood Nematode ◽

Virulence Determinants ◽

Pine Wilt ◽

Pine Trees ◽

Hydrolase Family

Pine wilt disease, caused by the pinewood nematode, Bursaphelenchus xylophilus, is one of the world’s most serious tree diseases. Although the B. xylophilus whole-genome sequence and comprehensive secretome profile have been determined over the past decade, it remains unclear what molecules are critical in pine wilt disease and govern B. xylophilus virulence in host pine trees. Here, a comparative secretome analysis among four isolates of B. xylophilus with distinct virulence levels was performed to identify virulence determinants. The four candidate virulence determinants of B. xylophilus highly secreted in virulent isolates included lipase (Bx-lip1), glycoside hydrolase family 30 (Bx-GH30), and two C1A family cysteine peptidases (Bx-CAT1 and Bx-CAT2). To validate the quantitative differences in the four potential virulence determinants among virulence groups at the protein level, we used real-time reverse-transcription polymerase chain reaction analysis to investigate these determinants at the transcript level at three time points: pre-inoculation, 3 days after inoculation (dai), and 7 dai into pine seedlings. The transcript levels of Bx-CAT1, Bx-CAT2, and Bx-GH30 were significantly higher in virulent isolates than in avirulent isolates at pre-inoculation and 3 dai. A subsequent leaf-disk assay based on transient overexpression in Nicotiana benthamiana revealed that the GH30 candidate virulent factor caused cell death in the plant. Furthermore, we demonstrated that Bx-CAT2 was involved in nutrient uptake for fungal feeding via soaking-mediated RNA interference. These findings indicate that the secreted proteins Bx-GH30 and Bx-CAT2 contribute to B. xylophilus virulence in host pine trees and may be involved in pine wilt disease.

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Chitosan increases Pinus pinaster tolerance to the pinewood nematode (Bursaphelenchus xylophilus) by promoting plant antioxidative metabolism

Scientific Reports ◽

10.1038/s41598-021-83445-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marta Nunes da Silva ◽

Carla S. Santos ◽

Ana Cruz ◽

Adrián López-Villamor ◽

Marta W. Vasconcelos

Keyword(s):

Phenolic Compounds ◽

Pinus Pinaster ◽

Plant Defence ◽

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Nematode Population ◽

Post Inoculation ◽

Pinewood Nematode ◽

Plant Tolerance ◽

The Moment

AbstractThe pine wilt disease (PWD), for which no effective treatment is available at the moment, is a constant threat to Pinus spp. plantations worldwide, being responsible for significant economic and environmental losses every year. It has been demonstrated that elicitation with chitosan increases plant tolerance to the pinewood nematode (PWN) Bursaphelenchus xylophilus, the causal agent of the PWD, but the biochemical and genetic aspects underlying this response have not been explored. To understand the influence of chitosan in Pinus pinaster tolerance against PWN, a low-molecular-weight (327 kDa) chitosan was applied to mock- and PWN-inoculated plants. Nematode population, malondialdehyde (MDA), catalase, carotenoids, anthocyanins, phenolic compounds, lignin and gene expression related to oxidative stress (thioredoxin 1, TRX) and plant defence (defensin, DEF, and a-farnesene synthase, AFS), were analysed at 1, 7, 14, 21 and 28 days post-inoculation (dpi). At 28 dpi, PWN-infected plants elicited with chitosan showed a sixfold lower nematode population when compared to non-elicited plants. Higher levels of MDA, catalase, carotenoids, anthocyanins, phenolic compounds, and lignin were detected in chitosan-elicited plants following infection. The expression levels of DEF gene were higher in elicited plants, while TRX and AFS expression was lower, possibly due to the disease containment-effect of chitosan. Combined, we conclude that chitosan induces pine defences against PWD via modulation of metabolic and transcriptomic mechanisms related with plant antioxidant system.

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In Silico Screening of Agonist and Antagonist Natural Compounds from Reported Essential Oils against Bursaphelenchus xylophilus

Chemistry Proceedings ◽

10.3390/ecsoc-24-08386 ◽

2020 ◽

Vol 3 (1) ◽

pp. 31

Author(s):

Jorge M. S. Faria ◽

Ana Margarida Rodrigues ◽

Pedro Barbosa ◽

Manuel Mota

Keyword(s):

Essential Oils ◽

Pine Wilt Disease ◽

Natural Compounds ◽

Bursaphelenchus Xylophilus ◽

Pinewood Nematode ◽

Highly Active ◽

Bibliographic Data ◽

Agonist And Antagonist ◽

Containment Strategy ◽

Detailed Chemical

Chemical control has been the most effective and reliable containment strategy in integrated pest management of pine wilt disease (PWD), caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus. Yet, large spectrum nematicides can be dangerous to human health and the environment. Essential oils (EOs) are safer sustainable alternatives, being composed of highly active natural compounds. A survey of bibliographic data on the detailed chemical composition and activity of the EOs used against the PWN allowed pinpointing monoterpenes as the main source of structures with agonist or antagonist properties. Transversal EO data treatment can identify potential highly active anti-PWN compounds.

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α-l-Fucosidases from Bursaphelenchus xylophilus Secretome—Molecular Characterization and Their Possible Role in Breaking Down Plant Cell Walls

Forests ◽

10.3390/f11030265 ◽

2020 ◽

Vol 11 (3) ◽

pp. 265

Author(s):

Joana M.S. Cardoso ◽

Luís Fonseca ◽

Isabel Abrantes

Keyword(s):

Cell Wall ◽

Molecular Characterization ◽

Three Dimensional ◽

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Pinewood Nematode ◽

Cell Wall Degrading Enzymes ◽

Degrading Enzymes ◽

Host Trees ◽

Resin Canals

The pinewood nematode (PWN) Bursaphelenchus xylophilus, the causal agent of the pine wilt disease (PWD), enters above-ground parts of the tree, migrates through the resin canals and feeds on plant cells causing extensive damage. In order to penetrate the cell wall and establish a parasitic relationship with host trees, the PWN needs to secretea mixture of active cell wall degrading enzymes. In maritime pine, Pinus pinaster, which is high susceptible to PWN, xyloglucan is the major hemicellulosic polysaccharide in primary cells. The xyloglucan backbone is susceptible to hydrolysis by numerous endoglucanases, some of them specific to xyloglucan. However, to completely degrade xyloglucan, all substitutions on the glucan backbones must be released, and l-fucose residues in xyloglucan branches are released by α-l-fucosidases. In the present study, the molecular characterization of two α-l-fucosidases found in PWN secretome was performed. Moreover, a novel α-l-fucosidase was identified and its cDNA and gene sequence were determined. The three-dimensional structures of these α-l-fucosidases were predicted and the transcript levels were analyzed, thus providing new insights into fundamental PWN biology and the possible role of these proteins as cell wall degrading enzymes.

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Volatile compounds in pine stands suffering from pine wilt disease: qualitative and quantitative evaluation

Nematology ◽

10.1163/156854106779799295 ◽

2006 ◽

Vol 8 (6) ◽

pp. 869-879 ◽

Cited By ~ 10

Author(s):

Kazuyoshi Futai ◽

Natsumi Kanzaki ◽

Yuko Takeuchi

Keyword(s):

Volatile Compounds ◽

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Wilt Disease ◽

Economic Damage ◽

Qualitative And Quantitative ◽

Pine Wilt ◽

Pine Trees ◽

Quantitative Properties ◽

Pine Stands

AbstractPine wilt disease causes ecological and economic damage in Japanese pine forests in spite of intensive effort to protect them from the pine wood nematode, Bursaphelenchus xylophilus. Pine trees infected with B. xylophilus emit a characteristic bouquet of volatile compounds bioactive to the vector beetle of the nematode, Monochamus alternatus, and potentially affecting symptom development inside the trees. To investigate the qualitative and quantitative properties of volatile compounds in the field, we profiled the volatile emissions in two Japanese black pine stands, one naturally suffering from pine wilt disease and the other artificially inoculated with B. xylophilus. In both pine stands, the emission of some terpenoids from the infected trees such as (−)-α-pinene, began to increase in summer, overlapping the oviposition season of the vector beetle, but peaked in the summer and autumn. These data suggest that the beetles may not necessarily depend on the tremendous quantity of volatiles alone when they search for suitable trees on which to oviposit.

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