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

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.

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

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.

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

2011 ◽  
Vol 9 (2) ◽  
pp. 272-275 ◽  
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.

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

Nematology ◽  
2006 ◽  
Vol 8 (6) ◽  
pp. 869-879 ◽  
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.

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

Parasitology ◽  
2019 ◽  
Vol 147 (1) ◽  
pp. 50-57 ◽  
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.

scholarly journals Prevention of Pine Wilt of Scots Pine (Pinus sylvestris) with Systemic Abamectin Injections

2006 ◽  
Vol 32 (5) ◽  
pp. 195-201
Author(s):  
Randall James ◽  
Ned Tisserat ◽  
Tim Todd
Keyword(s):  
Pinus Sylvestris ◽  
Scots Pine ◽  
Pine Wilt Disease ◽  
Bursaphelenchus Xylophilus ◽  
Wilt Disease ◽  
Pinewood Nematode ◽  
Commercial Formulation ◽  
Breast Height ◽  
Pine Wilt ◽  
Trunk Injection

We examined the efficacy of the insecticide/nematicide abamectin to prevent pine wilt disease caused by the pinewood nematode (Bursaphelenchus xylophilus) in Scots pine (Pinus sylvestris). Pinewood nematode movement was inhibited (>80% death or paralysis) following a 48 hr exposure to abamectin concentrations as low as 0.1 μL a.i. per L (100 ppb). A commercial formulation of abamectin (Avid™) was injected into Scots pine using a pressurized systemic trunk injection tube (STIT) technique. Fifteen to 30 mL (0.45 to 0.90 fl oz) of Avid per STIT could be injected into the trees in less than 1 hr. Trees were successfully injected throughout February, March, and April at temperatures above 4.4°C (40°F). Survival after 1 year of 10 cm diameter (4 in) at breast height (dbh) Scots pines injected with Avid and subsequently inoculated with pinewood nematode was higher (75%) than in pines injected with water (42%). Similarly, survival after 3 years of large Scots pines (30 to 60 cm [12 to 24 in] dbh)] injected with Avid and exposed to a natural epidemic of pine wilt was higher (96%) than in noninjected pines (33%) or those injected with water (71%). These results indicate that preventive injections of Scots pine with Avid are effective in protecting against pine wilt disease.

scholarly journals Assessment of the PCR technique for identification of Bursaphelenchus mucronatus Mamiya & Enda 1979, B. xylophilus Steiner & Buhrer 1934 (Nickle 1970) and B. fraudulentus Rühm 1956 (Nematoda, Aphelenchoididae) in crude extract of nematodes isolated from wood

2021 ◽  
Vol 61 (4) ◽  
pp. 346-351
Keyword(s):  
Pine Wilt Disease ◽  
Bursaphelenchus Xylophilus ◽  
Wilt Disease ◽  
Coniferous Forests ◽  
Pine Wood Nematode ◽  
Direct Examination ◽  
Pine Wood ◽  
Pine Wilt ◽  
Detection And Identification ◽  
Pine Trees

The pine wood nematode, Bursaphelenchus xylophilus Steiner & Buhrer 1934 (Nickle 1970) is the major causative agent of the pine wilt disease which has become devastating to Asian and European coniferous forests. These regions are also naturally occupied by two other native but nonpathogenic species, i.e. B. mucronatus Mamiya & Enda 1979 and B. fraudulentus Rühm 1956 which are closely related to the invasive B. xylophilus. Moreover, all these three species can colonize pine trees, and potentially be extracted from the same wood samples. Due to the cosmopolitan character and wide genetic variation within- and between existing populations the taxonomic distinction of these species based exclusively on their morphology is difficult or, almost impossible. The present quarantine regulations related to B. xylophilus require the most credible and simple methods which could allow for a possibly earliest detection and precise identification of this species in wood shipments and conifer forests stands. The main objectives of the presently reported research were to simplify the presently available procedures for possibly fast and precise detection and identification of B. xylophilus examined in the background of the remaining Bursaphelenchus species of the xylophilus group and other bacterio- and mycetophagous nematodes naturally present in the pine wood samples. The developed method is based on a direct examination of the crude nematode extract from wood samples and subsequent use of PCR technique with earlier designed specific reaction starters amplifying ITS1–28S rDNA regions.

scholarly journals Use of Magnetic Resonance Microscopy for the Nondestructive Observation of Xylem Cavitation Caused by Pine Wilt Disease

2005 ◽  
Vol 95 (7) ◽  
pp. 737-743 ◽  
Author(s):  
Shin Utsuzawa ◽  
Kenji Fukuda ◽  
Daisuke Sakaue
Keyword(s):  
Magnetic Resonance ◽  
Spin Echo ◽  
Pine Wilt Disease ◽  
Bursaphelenchus Xylophilus ◽  
Wilt Disease ◽  
Pinewood Nematode ◽  
Xylem Cavitation ◽  
Mr Microscopy ◽  
Pine Wilt ◽  
Spin Echo Sequence

The development of xylem cavitation caused by pine wilt disease was visualized nondestructively with a compact magnetic resonance (MR) microscope system. A T1-weighted spin-echo sequence clearly visualized the water-filled xylem of Japanese black pine (Pinus thunbergii) as white zones, whereas cavitated xylem was represented as dark areas. Cavitated areas in the xylem were first observed 6 to 9 days after inoculation with the pinewood nematode (Bursaphelenchus xylophilus), and enlarged gradually over several days. After 11 to 18 days, cavitated areas rapidly increased in size, fused, and reached the cambium. This drastic expansion in cavitation coincided with and appeared to explain the sudden wilting of the seedlings. The development of cavitation observed through MR microscopy corresponded well with previous descriptions of disease progression.

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