Isolates Of Pinewood Nematode Research Articles

Pathogenicity of Three Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle. Isolates in Pinus koraiensis (Siebold & Zucc.) Seedlings

The pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle., is one of the most dangerous invasive species in the world, causing devastating pine wilt disease (PWD) in pine trees from many countries. The PWN is now established in 18 provinces in China from the south to north, and it has expanded to some areas of Liaoning Province with temperatures that are beyond the ideal range. It has been reported that Pinus koraiensis Siebold & Zucc., one of the representative pine trees of Liaoning Province, has been infected by PWNs. To investigate the pathogenicity of the PWN in P. koraiensis, the reproductive ability of PWNs on fungal culture was compared among three isolates: QH-1, NM-1, and CM-1 (QH-1 from Liaoning Province, NM-1 from Nanjing Province, and CM-1 from Chongqing Municipality). Four-year-old P. koraiensis seedlings were inoculated with QH-1, NM-1, and CM-1 at a rate of 2000 per seedling. Pathogenicity, external symptoms, and nematode migration were all monitored on a daily basis over the next few days. The results from the experiment showed that all three PWN isolates caused wilt in P. koraiensis seedlings, with QH-1 being more virulent than NM-1 and CM-1. In addition, QH-1 exhibited greater reproductive and migration abilities in the seedlings than NM-1 and CM-1. These results indicate that the virulence of the B. xylophilus isolates QH-1, NM-1, and CM-1 can differ in terms of seedling mortality, migration ability, and reproductive ability (in trees).

Cold Tolerance in Pinewood Nematode Bursaphelenchus xylophilus Promoted Multiple Invasion Events in Mid-Temperate Zone of China

Pinewood nematode (Bursaphelenchus xylophilus) is a highly destructive invasive species, causing extensive economic and ecological losses across Eurasia. It has recently invaded mid-temperate zone of northern China, threating large areas of coniferous forests. Herein, we evaluated the physiological and molecular basis of cold tolerance in pinewood nematode isolates from different temperature zones of China. After exposure to −5 °C and −10 °C, the survival rates of five pinewood nematode isolates from different temperature zones were 93.94%–94.77% and 43.26%–45.58% after 8 h, and 93.04%–94.85% and 9.93%–10.56% after 24 h, without significant differences among isolates. In a comparison of an isolate from a mid-temperate zone and an isolate from a subtropical zone under gradient cooling, the survival rates remained steady at nearly 95% when minimum temperature ranged from −5 °C to −15 °C, with no significant difference between isolates. In addition, phylogenetic and population structure analyses based on whole genome resequencing data suggested that isolates from mid-temperate and warm temperate zones are clustered with different isolates from subtropical zone, with no obvious geographic pattern. We did not detect significant variation in cold tolerance ability and selected gene among pinewood nematode isolates from different temperature zones. The recently invaded pinewood nematodes in the mid-temperate zone of northern China may spread by multiple invasion events from southern China, without adaptive revolution. Our research implies that it is important to reinforce quarantine inspection to control the rapid spread of pinewood nematode.

Diversity and Function of Endo-Bacteria in Bursaphelenchus xylophilus from Pinus massoniana Lamb. in Different Regions

The pine wood nematode (PWN) Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease (PWD), a devastating forest disease. PWN-associated bacteria may play a role in PWD. However, little is known about the endo-bacteria in PWN. We analyzed the diversity of endo-bacteria in nine isolates of PWNs from Pinus massoniana Lamb. in nine epidemic areas from three Chinese provinces by high-throughput sequencing of 16S rDNA and isolated and identified culturable endo-bacteria through construction of a 16S rDNA phylogenetic tree and Biolog microbial identification. We also examined the effects of endo-bacteria on PWN fecundity, antioxidant capacity, and virulence using sterile nematodes as a control. While the dominant endo-bacteria in PWNs from different regions exhibited no significant difference in the classification levels of class and genus, their proportions differed. Pseudomonas and Stenotrophomonas were highly abundant in all PWN isolates. A total of 15 endo-bacterial strains were successfully isolated and identified as six species: Stenotrophomonas maltophilia, Pseudomonas fluorescens, Kocuria palustris, Microbacterium testaceum, Rhizobium radiobacter, and Leifsonia aquatica. We also found that P. fluorescens significantly increased the egg production of PWN, and that both P. fluorescens and S. maltophilia enhanced the mobility of PWN under oxidative stress and reduced the content of reactive oxygen species by increasing antioxidant enzyme activity in PWN. These strains also accelerated the development of PWD, and P. fluorescens had a more beneficial effect on PWN than S. maltophilia. Diversity exists among the endo-bacteria in PWNs from different regions, and some endo-bacteria can promote PWN infestation by enhancing the fecundity and antioxidant capacity of the nematode. Our study contributes to clarifying the interaction between endo-bacteria and PWN.

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

SummaryIn 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.

국내 소나무재선충에서 분리한 세균의 특성

국내 20개 지역의 소나무재선충병에 감염된 소나무와 잣나무에서 분리된 소나무재선충으로부터 총 110개의 세균을 분리하여 동정한 결과 Cedecea neteri(64균주)가 가장 높은 빈도로 검출되었으며, 다음으로 Ewingella americana(21균주), Pseudomonas sp.(15균주), Flavobacterium sp.(8균주), Rahnella aquatilis (2균주) 순으로 분리되었다. 분리된 세균의 phytotoxin 생성능을 조사한 결과 Pseudomonas sp.(Ba2) 세균은 해송 유묘에 갈변과 고사를 유발하였다. 21개의 항생물질을 이용하여 분리된 3개의 세균, Pseudomonas sp.(Ba2), E. ameircana(Ba4), C. neteri(Ba10)에 대한 감수성 조사 결과 carbenicillin, doxcycline, tetracycline에서 생장억제 효과가 높게 나타났다. A survey of bacterial species associated with Korean isolates of pine wood nematode (PWN) was performed. A total of 110 bacterial isolates were obtained from the PWN isolates that were previously isolated from Pinus densiflora and P. koraiensis. Among the bacterial isolates, Cedecea neteri was most frequent (64 isolates) followed by Ewingella americana (21 isolates), Pseudomonas sp. (15 isolates), Flavobacterium sp. (8 isolates) and Rahnella aquatilis (2 isolates). Both E. americana and Pseudomonas sp. which are assumed to be closely associated with PWN were examined for their phytotoxicity to P. thunbergii seedlings. Ethyl acetate extracts of Psuedomonas sp. (Ba2 strain) cultures were found to induce wilting and mortality in the tested seedlings. The three bacterial species, Pseudomonas sp. (Ba2 strain), E. ameircana (Ba4 strain) and C. neteri (Ba10 strain) were examined in vitro for their sensitivity to 21 kinds of antibiotics. All of the strains were highly susceptible to carbenicillin, doxcycline and tetracycline.

Genetic diversity of ITS sequences of Bursaphelenchus xylophilus

The sequence variation of internal transcribed spacer (ITS) regions of ribosomal DNA has been routinely used for species identification and species-level phylogeny of the pinewood nematode, Bursaphelenchus xylophilus. In this study, the intraspecies ITS genetic diversity of B. xylophilus was evaluated. Three pinewood nematode isolates from the United States, Japan, and Portugal were used for polymerase chain reaction (PCR) ITS region amplification and sequencing. Multiple peaks were observed in sequencing chromatograms from ITS regions of American and Japanese isolates, suggesting the presence of more than one ribosomal sequence for each isolate. PCR products were further cloned and 10 clones of each isolate were subsequently sequenced. Additionally, the ITS regions of individual nematodes from each isolate were amplified, cloned and sequenced. Among the 3 B. xylophilus isolates analyzed, an intraspecific and intra-isolate molecular variability was found. The intra-isolate ITS molecular diversity in the American isolate was higher than that in the Japanese and Portuguese isolates. However, the level of sequence variation observed within isolates was about the same as that described among ITS repeats within individuals.

A nested PCR assay targeting the DNA topoisomerase I gene to detect the pine wood nematode, Bursaphelenchus xylophilus

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a disastrous pathogen of the pine forests in East Asia and Europe. Plant quarantine is one of the most important ways to prevent its infection in current situation. A nested polymerase chain reaction (PCR) assay targeting the topoisomerse I gene has been developed to detect PWN in this study. To assess the specificity of the assay, 44 morphologically characterized nematode isolates including B. xylophilus, B. mucronatus, B. hofmanni, Seinura wuae, S. lii and Aphelenchoides macronucleatus were tested. Positive reactions characterized by amplification product of 509 bp were shown from all isolates of PWN. The nested PCR assay can detect 50 femtogram (fg) of template DNA or one individual nematode, as small as an egg. The validity was evaluated by analyzing the nematode samples extracted from the nematode-infested wood in the field. These results show that the assay is a specific, sensitive method for detection of PWN with the potential in relation to the pest risk assessment and quarantine regulations.

Surface coat proteins of the pine wood nematode, Bursaphelenchus xylophilus: profiles of stage- and isolate-specific characters

AbstractThe present study was made to determine the binding patterns of several lectins to the surface coat (SC) proteins of various isolates and developmental stages of the pine wood nematode (PWN), Bursaphelenchus xylophilus. Also, the detailed characteristics of the SC proteins were profiled by using molecular techniques. The lectin-binding study demonstrated the stage-specific characters of SC in binding to the lectin, wheat germ agglutinin (WGA). WGA binding was observed only to the outer surfaces of third-stage propagative juveniles and to the egg shells, and this occurred more frequently in virulent than in avirulent PWN isolates. A greater variety of lectins bound to eggs than to any other life stage. For characterisation, the SC proteins extracted were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and analysed by lectin blotting. The results showed that the carbohydrate and protein patterns of the SC of the PWN changed during nematode development.

Satellite DNA as a target for TaqMan real‐time PCR detection of the pinewood nematode, Bursaphelenchus xylophilus

SUMMARY The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a major pathogen of conifers, which impacts on forest health, natural ecosystem stability and international trade. As a consequence, it has been listed as a quarantine organism in Europe. A real-time PCR approach based on TaqMan chemistry was developed to detect this organism. Specific probe and primers were designed based on the sequence of the MspI satellite DNA family previously characterized in the genome of the nematode. The method proved to be specific in tests with target DNA from PWN isolates from worldwide origin. From a practical point of view, detection limit was 1 pg of target DNA or one individual nematode. In addition, PWN genomic DNA or single individuals were positively detected in mixed samples in which B. xylophilius was associated with the closely related non-pathogenic species B. mucronatus, up to the limit of 0.01% or 1% of the mixture, respectively. The real-time PCR assay was also used in conjunction with a simple DNA extraction method to detect PWN directly in artificially infested wood samples. These results demonstrate the potential of this assay to provide rapid, accurate and sensitive molecular identification of the PWN in relation to pest risk assessment in the field and quarantine regulation.

Early Symptom Development and Histological Changes Associated with Migration of Bursaphelenchus xylophilus in Seedling Tissues of Pinus thunbergii.

In order to clarify the mechanism of pine wilt caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus, nematode migration in tissues and disease symptoms in Pinus thunbergii seedlings were investigated. One-year-old seedlings were inoculated with different pathogenic isolates of PWN under two different temperatures. At an early stage of symptom development, a virulent isolate of PWN multiplied in both bark and xylem and was distributed in cortical resin canals, cortical tissue, and xylem resin canals at 30°C. Cell death and disease symptoms developed in both bark and xylem. The virulent isolate of PWN at 25°C and the avirulent isolate of PWN at 30°C were distributed mainly in cortical resin canals, but rarely in xylem resin canals and cortical tissue. Disease symptoms and cell death occurred in cortical resin canals and rarely occurred in other tissues. These results demonstrated that the virulent isolate of PWN at low temperature and avirulent nematodes could not easily migrate to xylem resin canals and cortical tissue. It was shown that cell death and early symptom development coincided with PWN migration and, therefore, PWN invasion induces cell death and early symptom development.