scholarly journals Silencing of cyp-33C9 Gene Affects the Reproduction and Pathogenicity of the Pine Wood Nematode, Bursaphelenchus xylophilus

2019 ◽  
Vol 20 (18) ◽  
pp. 4520 ◽  
Author(s):  
Xiuwen Qiu ◽  
Lili Yang ◽  
Jianren Ye ◽  
Wei Wang ◽  
Tiantian Zhao ◽  
...  
Keyword(s):  
Pine Wilt Disease ◽  
Bursaphelenchus Xylophilus ◽  
Economic Losses ◽  
Parasitic Nematodes ◽  
Pine Wood Nematode ◽  
Egg Hatch ◽  
Pine Wood ◽  
Large Numbers ◽  
Cytochrome P450 Genes ◽  
Pine Trees

Cytochrome P450 genes are very important for plant-parasitic nematodes to reproduce and to metabolize xenobiotic compounds generated by their host plants. The pine wood nematode (PWN), Bursaphelenchus xylophilus, causes very high annual economic losses by killing large numbers of pine trees across Asia and into Europe. In this study, we used RNA interference (RNAi) to analyze the function of the cyp-33C9 gene of PWN. Our results showed that expression of the cyp-33C9 gene was suppressed successfully after soaking nematodes for 24 h in cyp-33C9 double-stranded RNA (dsRNA). The silencing of the cyp-33C9 gene significantly decreased the feeding, reproduction, oviposition and egg hatch of B. xylophilus. Meanwhile, the migration speed of B. xylophilus in Pinus thunbergii was reduced in the early stages when the cyp-33C9 gene was silenced in the nematodes. Moreover, knockdown of the cyp-33C9 gene in B. xylophilus caused a decrease in pathogenicity to pine trees. These results suggest that the cyp-33C9 gene plays an important role in the reproduction and pathogenicity of B. xylophilus. This discovery identified several functions of the cyp-33C9 gene in B. xylophilus and provided useful information for understanding the molecular mechanism behind pine wilt disease caused by PWN.

scholarly journals Cathepsin L-like Cysteine Proteinase Genes Are Associated with the Development and Pathogenicity of Pine Wood Nematode, Bursaphelenchus xylophilus

2019 ◽  
Vol 20 (1) ◽  
pp. 215 ◽  
Author(s):  
Qi Xue ◽  
Xiao-Qin Wu ◽  
Wan-Jun Zhang ◽  
Li-Na Deng ◽  
Miao-Miao Wu
Keyword(s):  
Developmental Stages ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Pine Wilt Disease ◽  
Bursaphelenchus Xylophilus ◽  
Parasitic Nematodes ◽  
Pine Wood Nematode ◽  
Pine Wood ◽  
Real Time Quantitative Pcr ◽  
Rapid Amplification

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (PWD), resulting in huge losses in pine forests. However, its pathogenic mechanism remains unclear. The cathepsin L-like cysteine proteinase (CPL) genes are multifunctional genes related to the parasitic abilities of plant-parasitic nematodes, but their functions in PWN remain unclear. We cloned three cpl genes of PWN (Bx-cpls) by rapid amplification of cDNA ends (RACE) and analyzed their characteristics using bioinformatic methods. The tissue specificity of cpl gene of PWN (Bx-cpl) was studied using in situ mRNA hybridization (ISH). The functions of Bx-cpls in development and pathogenicity were investigated using real-time quantitative PCR (qPCR) and RNA interference (RNAi). The results showed that the full-length cDNAs of Bx-cpl-1, Bx-cpl-2, and Bx-cpl-3 were 1163 bp, 1305 bp, and 1302 bp, respectively. Bx-cpls could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpls in the egg stage was highest. After infection, the expression levels of Bx-cpls increased and reached their highest at the initial stage of PWD, then declined gradually. The silencing of Bx-cpl could reduce the feeding, reproduction, and pathogenicity of PWN. These results revealed that Bx-cpls play multiple roles in the development and pathogenic processes of PWN.

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.

A rapid in vitro bioassay system for testing resistance factors of pine trees to Bursaphelenchus xylophilus

Nematology ◽  
2013 ◽  
Vol 15 (6) ◽  
pp. 665-670
Author(s):  
Zhang Fujia ◽  
Zhang Fujia ◽  
Yasuhiro Mori ◽  
Zhang Fujia ◽  
Yasuhiro Mori ◽  
...  
Keyword(s):  
Pine Wilt Disease ◽  
Bursaphelenchus Xylophilus ◽  
Rapid Screening ◽  
Pine Wood Nematode ◽  
Pine Wood ◽  
Resistance Factors ◽  
Methanol Extracts ◽  
Bioassay System ◽  
Pine Trees

Understanding resistance mechanisms to pine wilt disease is essential to a successful breeding programme because plant selection cannot always guarantee absolute resistance against every isolate of the pine wood nematode, Bursaphelenchus xylophilus. To examine resistance factors in Japanese black pine, Pinus thunbergii, we devised a novel in vitro bioassay system in which we tested proliferation of pine wood nematodes co-cultured with wood slices or methanol extracts from pines. Proliferation of pine wood nematodes was inhibited in assays with fresh wood slices from resistant Japanese black pines but not with susceptible pine or without wood slices (control). When resistant wood slices were extracted by methanol, the inhibition effect of proliferation of pine wood nematodes was diminished, whereas methanol extraction from susceptible wood slices did not affect the proliferation. To verify whether nematode proliferation was inhibited by pine extracts, methanol extracts were loaded on paper disks and used in the assay. Populations of pine wood nematodes were significantly suppressed when methanol extracts originated from a resistant pine, but not from a susceptible pine. These results strongly suggest that methanol extracts from resistant pine trees contain the inhibitors of pine wood nematode proliferation. This bioassay system is available not only for identifying inhibitors of pine wood nematode proliferation but also for rapid screening of resistant pines.

How different is induced host resistance against the pine wood nematode, Bursaphelenchus xylophilus, by two avirulent microbes?

Nematology ◽  
2006 ◽  
Vol 8 (3) ◽  
pp. 435-442 ◽  
Author(s):  
Yuko Takeuchi ◽  
Natsumi Kanzaki ◽  
Kazuyoshi Futai
Keyword(s):  
Induced Resistance ◽  
Host Resistance ◽  
Pine Wilt Disease ◽  
Bursaphelenchus Xylophilus ◽  
Wilt Disease ◽  
Pine Wood Nematode ◽  
Pine Wood ◽  
Pine Wilt ◽  
Pine Trees ◽  
Induce Resistance

AbstractPine wilt disease, caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has been devastating pine forests in Japan for a century but an effective method for controlling the disease is still needed. Pre-inoculation of the host plant with an avirulent pathogen can induce resistance against subsequent infection with virulent pathogens; this is called 'induced resistance'. Host pine trees have this type of resistance against pine wilt disease but the detailed mechanism is unknown. In this study, 1-year-old potted seedlings of the susceptible Japanese black pine, Pinus thunbergii, were pre-inoculated with an avirulent isolate of PWN (C14-5), as well as an avirulent fungus, Botrytis cinerea, to induce host resistance against PWN. One, 2, 3, 4 and 5 weeks after inoculation, the seedlings were challenged with a virulent isolate of PWN (S10), and the survival of these seedlings was estimated by their external symptoms. Pre-inoculation with either C14-5 or Botrytis delayed the symptom development caused by subsequent inoculation with S10, suggesting that pre-inoculation induced host resistance against PWN. The resistance induced by pre-inoculation with C14-5 lasted for at least 5 weeks, while that induced by Botrytis weakened with time. It has been reported that the PWN survives inside the host seedlings for a long period without causing any symptoms under certain conditions, but Botrytis survives only for a while in the seedlings. Thus, the duration of host resistance induced by pre-inoculation may vary with the viability of the inoculum used for pre-inoculation. In order to obtain durable induced resistance, it may be necessary to repeat inoculations of avirulent microbes to be able to control pine wilt disease.

Identification of a novel effector BxSapB3 that enhances the virulence of pine wood nematode Bursaphelenchus xylophilus

2019 ◽  
Vol 51 (10) ◽  
pp. 1071-1078
Author(s):  
Xin Huang ◽  
Longjiao Hu ◽  
Xiaoqin Wu
Keyword(s):  
Virulent Strain ◽  
Pine Wilt Disease ◽  
Bursaphelenchus Xylophilus ◽  
Wilt Disease ◽  
Pcr Analysis ◽  
Pine Wood Nematode ◽  
Pine Wood ◽  
Early Stages ◽  
Pine Wilt ◽  
Weakly Virulent

Abstract Pine wilt disease, caused by the pine wood nematode Bursaphelenchus xylophilus, leads to severe damage to pine forests in China. In our previous study, effectors secreted by this pathogen were shown to play roles in the different infection stages of pine wilt disease, and a series of candidate effectors were predicted by transcriptome sequencing. This study identified and characterized a novel effector, BxSapB3, which was among these candidate effectors. Agrobacterium-mediated transient expression was used to identify BxSapB3. BxSapB3 was secreted by B. xylophilus and found to be capable of inducing cell death in Nicotiana benthamiana. Quantitative real-time PCR (qRT-PCR) analysis revealed that BxSapB3 was upregulated in a highly virulent strain of B. xylophilus and expressed at lower levels in a weakly virulent strain at the early stages of infection. When BxSapB3 was silenced in B. xylophilus, the process of infection was delayed. These results indicate that BxSapB3 acts as an effector and contributes to virulence at the early stages of B. xylophilus infection.

scholarly journals Sampling design for efficient detection of pine wood nematode, Bursaphelenchus xylophilus, in diseased trees using a DNA detection kit: variation across branch, trunk and tree

Nematology ◽  
2018 ◽  
Vol 20 (7) ◽  
pp. 641-652 ◽  
Author(s):  
Yuuki Nakabayashi ◽  
Takuya Aikawa ◽  
Michinari Matsushita ◽  
Kazuhiko Hoshizaki
Keyword(s):  
Sampling Design ◽  
Dna Detection ◽  
Pine Wilt Disease ◽  
Bursaphelenchus Xylophilus ◽  
Molecular Techniques ◽  
Effective Control ◽  
Pine Wood Nematode ◽  
Pine Wood ◽  
Efficient Detection ◽  
Minimum Number

Summary Detection of pine wood nematode, Bursaphelenchus xylophilus, is fundamental for effective control of pine wilt disease. Recent molecular techniques, such as DNA detection, have enhanced detectability of the nematodes whereas appropriate field sampling has received less attention. In order to elucidate a sampling design that most efficiently detects B. xylophilus using a commercially-distributed DNA detection kit, we compared detection levels of B. xylophilus using wood chips taken from various positions on dead trees. Results showed that the DNA kit had a higher detection level than the conventional method, and that trunk samples had higher levels than branch samples. Statistical model revealed that among-tree variation influenced the detectability more strongly than within-tree variation. Our results suggest that, in practice, with limited resources for control, it is more efficient to take samples from many trees with a minimum number from each tree, rather than taking many samples from a small number of trees.

Influence of cellulase gene knockdown by dsRNA interference on the development and reproduction of the pine wood nematode, Bursaphelenchus xylophilus

Nematology ◽  
2010 ◽  
Vol 12 (2) ◽  
pp. 225-233 ◽  
Author(s):  
Xin-Yue Cheng ◽  
Su-Ming Dai ◽  
Luo Xiao ◽  
Bing-Yan Xie
Keyword(s):  
Host Plants ◽  
Plant Cell Wall ◽  
Biological Effects ◽  
Cellulase Activity ◽  
Bursaphelenchus Xylophilus ◽  
Parasitic Nematodes ◽  
Transcriptional Gene Silencing ◽  
Pine Wood Nematode ◽  
Cellulase Gene ◽  
Pine Wood

Abstract Cellulase genes are very important for plant-parasitic nematodes to move and feed within their host plants. The pine wood nematode, Bursaphelenchus xylophilus, causes destructive damage by killing pine trees. In this study, by employing dsRNA interference technology, knockdown of a cellulase gene (Bx-eng-1) of B. xylophilus was achieved and the biological effects of RNAi on the nematode were observed. The result showed that, after 24 h soaking, dsRNA of the Bx-eng-1 gene was effectively delivered into the nematode causing a post-transcriptional gene silencing and decrease in cellulase activity. Moreover, the number of F1 generation offspring was reduced significantly when the dsRNA-treated nematodes were cultured on fungal mats. We consider that cellulase is important to B. xylophilus because it not only hydrolyses cellulose of plant cell wall for its parasitism and penetration in host plants, but also influences its feeding, development and propagation on fungal mats.

scholarly journals Ophiostomatoid fungi associated with pines infected by Bursaphelenchus xylophilus and Monochamus alternatus in China, including three new species

MycoKeys ◽  
2018 ◽  
Vol 39 ◽  
pp. 1-27
Author(s):  
HuiMin Wang ◽  
YingYing Lun ◽  
Quan Lu ◽  
HuiXiang Liu ◽  
Cony Decock ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pine Wilt Disease ◽  
Molecular Data ◽  
Bursaphelenchus Xylophilus ◽  
Its2 Region ◽  
Ophiostomatoid Fungi ◽  
Pine Wood Nematode ◽  
Pine Wood ◽  
Bark And Ambrosia Beetles ◽  
Japanese Pine Sawyer

The activity of the pine wood nematodeBursaphelenchusxylophilusleads to extremely serious economic, ecological and social losses in East Asia. The nematode causes pine wilt disease, which is currently regarded as the most important forest disease in China. The pathogenic nematode feeds on dendrocola fungi to complete its cycle of infection. As the vector of the nematode, the Japanese pine sawyer (Monochamusalternatus) also carries dendrocola fungi. Pine woods, infected byB.xylophilusand tunnelled byM.alternatus, are also inhabited by ophiostomatoid fungi. These fungi are well known for their association with many bark and ambrosia beetles. They can cause sapstain and other serious tree diseases. The aims of our study were to investigate and identify the ophiostomatoid communities associated with the epidemic pine wood nematode and the pine sawyer inPinusmassonianaandP.thunbergiiforests, which are the main hosts of the pine wood nematode in China. Two hundred and forty strains of ophiostomatoid fungi were isolated from nematode and sawyer–infected trees in the coastal Shandong and Zhejiang Provinces, representing newly and historically infected areas, respectively. Six ophiostomatoid species were identified on the basis of morphological, physiological and molecular data. For the latter, DNA sequences of the internal transcribed spacer (ITS1–5.8S–ITS2) region and partial b-tubulin gene were examined. The ophiostomatoid species included one known species,Ophiostomaips, three novel species, viz.Ophiostomaalbumsp. nov.,Ophiostomamassonianasp. nov.andSporothrixzhejiangensissp. nov.and two species whose identities are still uncertain, Ophiostomacf.deltoideosporum and Graphilbumcf.rectangulosporium, due to the paucity of the materials obtained. The ophiostomatoid community was dominated byO.ips. This study revealed that a relatively high species diversity of ophiostomatoid fungi are associated with pine infected byB.xylophilusandM.alternatusin China.

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