Copy Number Variations of Glycoside Hydrolase 45 Genes in Bursaphelenchus xylophilus and Their Impact on the Pathogenesis of Pine Wilt Disease

The pine wood nematode Bursaphelenchus xylophilus parasitizes millions of pine trees worldwide each year, causing severe wilt and the death of host trees. Glycoside hydrolase 45 genes of B. xylophilus are reported to have been acquired by horizontal gene transfer from fungi and are responsible for cell wall degradation during nematode infection. Previous studies ignored the possibility of copy number variations of such genes. In this study, we determined that two of the glycoside hydrolase 45 genes evolved to maintain multiple copies with distinct expression levels, enabling the nematode to infect a variety of pine hosts. Additionally, tandem repeat variations within coding regions were also detected between different copies of glycoside hydrolase 45 genes that could result in changes in protein sequences and serve as an effective biological marker to detect copy number variations among different B. xylophilus populations. Consequently, we were able to further identify the copy number variations of glycoside hydrolase 45 genes among B. xylophilus strains with different virulence. Our results provide new insights into the pathogenicity of B. xylophilus, provide a practical marker to genotype copy number variations and may aid in population classification.

Download Full-text

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.

Download Full-text

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.

Download Full-text

“Abnormal vertebral patterns in genetically heterogeneous deceased fetuses and neonates: evidence of selection against variations”

10.1101/784926 ◽

2019 ◽

Author(s):

Pauline C. Schut ◽

Erwin Brosens ◽

Frietson Galis ◽

Clara M. A. Ten Broek ◽

Inge M.M. Baijens ◽

...

Keyword(s):

Copy Number ◽

Single Nucleotide Polymorphism Array ◽

Copy Number Variations ◽

Cervical Region ◽

Nucleotide Polymorphism ◽

Rare Cnvs ◽

Single Nucleotide ◽

Neonatal Deaths ◽

Coding Regions ◽

Cervical Ribs

AbstractObjectiveTo assess the vertebral pattern in a cohort of deceased fetuses and neonates, and to study the possible impact of DNA Copy Number Variations (CNVs) in coding regions and/or disturbing enhancers on the development of the vertebral pattern.MethodRadiographs of 445 fetuses and infants, deceased between 2009 and 2015, were assessed. Terminations of pregnancies, stillbirths and neonatal deaths were included. Patients were excluded if the vertebral pattern could not be determined. Copy number profiles of 265 patients were determined using single nucleotide polymorphism array.Results274/374 patients (73.3%) had an abnormal vertebral pattern. Cervical ribs were present in 188/374 (50.3%) and were significantly more common in stillbirths (69/128 (53.9%)) and terminations of pregnancies (101/188 (53.7%)), compared to live births (18/58, 31.0%, p = 0.006). None of the rare CNVs were recurrent or overlapped candidate genes for vertebral patterning.ConclusionThe presence of an abnormal vertebral pattern, particularly in the cervical region, could be a sign of disruption at critical, highly interactive and conserved stages of embryogenesis. The vertebral pattern might provide valuable information regarding fetal and neonatal outcome. CNV analyses did not identify a mutual genetic cause for the occurrence of vertebral patterning abnormalities, indicating genetic heterogeneity.

Download Full-text

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.

Download Full-text

Boarding abilities of Bursaphelenchus mucronatus and B. xylophilus (Nematoda: Aphelenchoididae) on Monochamus alternatus (Coleoptera: Cerambycidae)

Nematology ◽

10.1163/156854103773040745 ◽

2003 ◽

Vol 5 (6) ◽

pp. 843-849 ◽

Cited By ~ 12

Author(s):

Shota Jikumaru ◽

Katsumi Togashi

Keyword(s):

Pine Wilt Disease ◽

Nematode Species ◽

Bursaphelenchus Xylophilus ◽

Insect Vector ◽

Species Replacement ◽

Monochamus Alternatus ◽

Bursaphelenchus Mucronatus ◽

Initial Load ◽

Pine Trees ◽

The Difference

Abstract Bursaphelenchus mucronatus is closely related to Bursaphelenchus xylophilus, the causative agent of pine wilt disease. Both nematodes are transmitted between host pine trees as the fourth-stage dispersal juveniles (JIV) by insect vectors. After the invasion of Japan by B. xylophilus, B. mucronatus, native to Japan, appears to have been replaced in the pine forests during the spread of the disease. To help understand this species replacement, the number of JIV carried by an insect vector (the initial nematode load) was compared between the two nematode species by using the beetle, Monochamus alternatus, in the laboratory. The initial load of B. mucronatus was significantly smaller than that of B. xylophilus although the number of third-stage dispersal juveniles (JIII) concentrated at the pupal chambers did not differ. Statistical analysis showed that the proportion of JIII moulting to JIV was the most important among three components explaining the difference in the initial load of B. mucronatus while the number of JIII concentrated at the pupal chamber was the most important for B. xylophilus. The phoretic affinity between the nematode and its vector is discussed in relation to its role in the species replacement.

Download Full-text

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.

Download Full-text

Molecular Characterization and Functional Analysis of Three Autophagy Genes, BxATG5, BxATG9, and BxATG16, in Bursaphelenchus xylophilus

International Journal of Molecular Sciences ◽

10.3390/ijms20153769 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3769 ◽

Cited By ~ 2

Author(s):

Hong-Bin Liu ◽

Lin Rui ◽

Ya-Qi Feng ◽

Xiao-Qin Wu

Keyword(s):

Regulatory Mechanism ◽

Expression Patterns ◽

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Wilt Disease ◽

Molecular Characteristics ◽

Chain Reaction ◽

Forest Disease ◽

Pine Trees ◽

Polymerase Chain

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen responsible for pine wilt disease (PWD), a devastating forest disease with a pathogenic mechanism that remains unclear. Autophagy plays a crucial role in physiological and pathological processes in eukaryotes, but its regulatory mechanism and significance in PWN are unknown. Therefore, we cloned and characterized three autophagy genes, BxATG5, BxATG9, and BxATG16, in PWN. BxATG9 and BxATG16 were efficiently silenced through RNA interference, and we found that BxATG16 positively regulated the expression of BxATG5. Silencing BxATG9 and BxATG16 severely inhibited feeding and reproduction in PWN, indicating that autophagy is essential for these processes. We then examined the expression patterns of these three autophagy genes in PWN under the stresses of α-pinene and H2O2, the main defense substances of pine trees, and during the development of PWD using quantitative reverse transcription polymerase chain reaction. The expression levels of BxATG5, BxATG9, and BxATG16 all significantly increased after nematodes were stressed with α-pinene and H2O2 and inoculated into pine trees, suggesting that autophagy plays an important role in the defense and pathogenesis of PWN. In this study, the molecular characteristics and functions of the autophagy genes BxATG5, BxATG9, and BxATG16 in PWN were elucidated.

Download Full-text

Resistance of an indigenous biological system against expansion of the invasive nematode, Bursaphelenchus xylophilus, in cool areas of Japan

Nematology ◽

10.1163/156854108785787172 ◽

2008 ◽

Vol 10 (5) ◽

pp. 679-687 ◽

Cited By ~ 2

Author(s):

Shota Jikumaru ◽

Katsumi Togashi

Keyword(s):

Air Temperature ◽

Pinus Densiflora ◽

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Vector System ◽

Insect Vector ◽

Disease Development ◽

Biotic Factors ◽

Pine Tree ◽

Pine Trees

Abstract Bursaphelenchus xylophilus is the pathogen associated with pine wilt disease (PWD), an infectious disease of pine trees transmitted by cerambycid beetles of the genus Monochamus. Bursaphelenchus xylophilus is an invasive species, whilst B. mucronatus is a native congener and non-pathogenic to pine trees in Japan. To provide experiment evidence of the biotic and/or abiotic resistance to PWD expansion in a cool area of Japan, we inoculated B. xylophilus into healthy pine trees in a Japanese red pine (Pinus densiflora) stand with the indigenous pine tree-B. mucronatus-insect vector system before invasion of B. xylophilus in 1993 and 1994. Extremely cool air temperature with high precipitation and extremely high air temperature with low precipitation were observed in the summers of the 2 years of inoculation, respectively. The cool summer induced a low incidence of PWD and delayed disease development, resulting in the replacement of B. xylophilus by B. mucronatus within diseased trees and the emergence of Monochamus saltuarius beetles carrying B. mucronatus from the trees 2 years after the inoculation. The hot summer induced disease development in trees in the year of inoculation, but such diseased trees did not become infection sources because of the lack of M. alternatus, whose oviposition was synchronised with the period of disease development in pine trees. This study indicated that biotic factors were important in the inhibitory mechanism in a pine forest against the spread of PWD in the stand. Relevant biotic factors were the lack of M. alternatus and the presence of M. saltuarius carrying B. mucronatus.

Download Full-text

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

Plant Disease ◽

10.1094/pdis-12-10-0910 ◽

2012 ◽

Vol 96 (2) ◽

pp. 249-252 ◽

Cited By ~ 10

Author(s):

Mitsuteru Akiba ◽

Makoto Ishihara ◽

Norio Sahashi ◽

Katsunori Nakamura ◽

Mineko Ohira ◽

...

Keyword(s):

Disease Incidence ◽

Pine Wilt Disease ◽

Bursaphelenchus Xylophilus ◽

Pinus Thunbergii ◽

Breeding Program ◽

Pinewood Nematode ◽

Differential Host ◽

Pine Trees ◽

Efficient Breeding ◽

Highly Virulent

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.

Download Full-text

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.

Download Full-text