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Chen F.-M.,Nanjing Forestry University | Chen F.-M.,Jiangsu Key Laboratory for Prevention and Management of Invasive Species | Negi S.,Nanjing Forestry University | Negi S.,Jiangsu Key Laboratory for Prevention and Management of Invasive Species | And 2 more authors.
Forest Pathology | Year: 2011

Bursaphelenchus mucronatus is closely related to the pinewood nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease. B. xylophilus became a devastating pest when it was introduced in the Far East; however, B. mucronatus is considered to have low virulence. Morphological similarities between B. xylophilus and B. mucronatus make the accurate morphological identification of both species difficult. Thus, it has become important to pay attention towards B. mucronatus impact and the need of discrimination of these two species. To distinguish among the two species, a B. mucronatus-specific sequence-characterized amplified region (SCAR) marker has been developed. The specific Random amplified polymorphic DNA (RAPD) fragment of B. mucronatus, OPY 01-M 850 was excised from agarose gels and purified. The gel-purified fragment was cloned into the pGEM ®-T Vector and subjected to sequencing. Based on the sequenced RAPD fragments, a number of SCAR primers were designed. It is demonstrated that OPY 01-M 850 through primers Y 01F/R can be transformed into a B. mucronatus-specific SCAR-Y 01-M 609 marker. Primers set Y 01F/R had high specificity that could be used for the discriminative identification of B. mucronatus versus B. xylophilus. © 2010 Blackwell Verlag GmbH.


Ding X.,Nanjing Forestry University | Ding X.,Jiangsu Key Laboratory for Prevention and Management of Invasive Species | Ye J.,Nanjing Forestry University | Ye J.,Jiangsu Key Laboratory for Prevention and Management of Invasive Species | And 8 more authors.
Gene | Year: 2015

Bursaphelenchus xylophilus is known as the causative agent of pine wilt disease with complex life cycles. In this research, four small RNA libraries derived from different infection stages of pine wilt disease were constructed and sequenced. Consequently, we obtained hundreds of evolutionarily conserved miRNAs and novel miRNA candidates. The analysis of miRNA expression patterns showed that most miRNAs were expressed at extraordinarily high levels during the middle stage of pine wilt disease. Functional analysis revealed that expression levels of miR-73 and miR-239 were mutually exclusive with their target GH45 cellulase genes. In addition, another set of atypical miRNAs, termed mirtrons, was also identified in this study. Thus, our research has provided detailed characterization of B. xylophilus miRNA expression patterns during the pathological process of pine wilt disease. These findings would contribute to more in-depth understanding of this devastating plant disease. © 2014 Elsevier B.V.


Ding X.,Nanjing Forestry University | Ding X.,Jiangsu Key Laboratory for Prevention and Management of Invasive Species | Ye J.,Nanjing Forestry University | Ye J.,Jiangsu Key Laboratory for Prevention and Management of Invasive Species | And 8 more authors.
PLoS ONE | Year: 2016

Bursaphelenchus xylophilus is the causative agent of pine wilt disease which has caused huge economic losses in many countries. It has been reported that two forms of pine wood nematodes existed in its native region, i.e., with strong virulence and weak virulence. However, little is known about the molecular differences between the two forms. To better understand their molecular variations, transcriptome and genome sequences of three strongly virulent and one weakly virulent strains were analyzed. We found 238 transcripts and 84 exons which showed notable changes between the two virulent forms. Functional analyses of both differentially expressed transcripts and exons indicated that different virulence strains showed dissimilar nematode growth, reproduction, and oxidoreductase activities. In addition, we also detected a small number of exon-skipping events in B. xylophilus. Meanwhile, 17 SNPs were identified as potential genetic markers in distinguishing the two forms. Four of them were further proved to have undergone allele specific expressions and possibly interrupted the target site of evolutionary conserved B. xylophilus miR-47. These particular SNPs were experimentally verified by including eight additional strains to ensure the validity of our sequencing results. These results could help researchers to better diagnose nematode species with different virulence and facilitate the control of pine wilt disease. © 2016 Ding et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Qiu X.,Nanjing Forestry University | Qiu X.,Jiangsu Key Laboratory for Prevention and Management of Invasive Species | Wu X.,Nanjing Forestry University | Wu X.,Jiangsu Key Laboratory for Prevention and Management of Invasive Species | And 6 more authors.
PLoS ONE | Year: 2013

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. However, the pathogenesis-related genes of B. xylophilus are not well characterized. Thus, DNA microarrays were used to investigate differential gene expression in PWN where Pinus thunbergii was inoculated with nematodes, compared with those cultured on Botrytis cinerea. The microarrays comprised 31121 probes, 1310 (4.2%) of which were differentially regulated (changes of >2-fold, P < 0.01) in the two growth conditions. Of these 1310 genes, 633 genes were upregulated, whereas 677 genes were downregulated. Gene Ontology (GO) categories were assigned to the classes Cellular Component, Molecular Function, and Biological Process. The comparative gene expression analysis showed that a large number of the pathogenesis-related genes of B. xylophilus, such as pectate lyase genes, cytochrome P450s, UGTs, and ABC transporter genes, were highly expressed when B. xylophilus infected P. thunbergii. Annotation analysis indicated that these genes contributed to cell wall degradation, detoxification, and the reproduction process. The microarray results were validated using quantitative RT-PCR (qRT-PCR). The microarray data confirmed the specific expression of B. xylophilus genes during infection of P. thunbergii, which provides basic information that facilitates a better understanding of the molecular mechanism of PWD. © 2013 Ye et al.


Li G.,Nanjing Forestry University | Li G.,Lianyungang Municipal Academy of Agricultural science | Li G.,Jiangsu Key Laboratory for Prevention and Management of Invasive Species | Wu X.,Nanjing Forestry University | And 3 more authors.
Shengtai Xuebao/ Acta Ecologica Sinica | Year: 2013

Long-term or inappropriate use of glyphosate may damage the non-target organisms and environment. Microbes are important biological resources for bioremediation. In recent years, the method of using microbes and its catabolic enzymes to remediate organophosphorus pesticides in the environment has shown a promising potential in application. It is the main direction of research on organophosphorus pesticide degradation. This study aimed to investigate the degradation of glyphosate by an efficient phosphate solubilizing bacterium Burkholderia. multivorans WS-FJ9 which was isolated from pine rhizosphere. Strain WS-FJ9 was grown on the plates of nutrient agar (NA) with different concentrations of glyphosate to determine the tolerance of strain WS-F19 to glyphosate. Glyphosate can be used by strain WS-FJ9 as the sole sources of carbon, nitrogen and phosphorus, respectively. The degradation dynamical parameters of strain WS-FJ9 on glyphosate were determined using low flooding quantity batch reactor method (FBR). Plackett-Burman (PB) design, central composite design (CCD), and Response Surface Methodology (RSM) were employed to screen and optimize the main factors of strain WS-FJ9 degrading glyphosate. The maximum concentration of tolerance to glyphosate for strain WS-FJ9 to remain efficiently degrading glyphosate was 0.4%. The affinity constant (Ks) of strain WS-FJ9 to glyphosate was 65 μL/mL and the minimal concentration (Smin) of glyphosate degraded by WS-FJ9 was 21.9 μL/mL. Three key factors (cultural temperature,glucose and ammonium sulfate) for the glyphosate degradation by strain WS-FJ9 were selected using PB design. The quadratic model for the three significant factors was established using CCD design and RSM with glyphosate degradation rate as the target response. Under the optimal degradation conditions, the incubation temperature was 27.7°C, and the amount of glucose and ammonium sulfate supplemented were 9.67 g/L and 0.50 g/L, respectively. The degradation rate of glyphosate by strain WS-FJ9 reached 72.83%.


Zhu L.-h.,Nanjing Forestry University | Zhu L.-h.,Jiangsu Key Laboratory for Prevention and Management of Invasive Species | Ye J.,Nanjing Forestry University | Ye J.,Jiangsu Key Laboratory for Prevention and Management of Invasive Species | And 8 more authors.
PLoS ONE | Year: 2012

Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus. However, the pathogenic mechanism of pine wilt disease (PWD) remains unclear. Although the PWN was thought to be the only pathogenic agent associated with this disease, a potential role for bacterial symbionts in the disease process was recently proposed. Studies have indicated that aseptic PWNs do not cause PWD in aseptic pine trees, while PWNs associated with bacteria cause wilting symptoms. To investigate the pathogenicity of the PWN and its associated bacteria, 3-month-old microcuttings derived from certain clones of Pinus densiflora Siebold & Zucc. produced in vitro were inoculated under aseptic conditions with aseptic PWNs, non-aseptic PWNs and bacteria isolated from the nematodes. Six-month-old aseptic P. densiflora microcuttings and 7-month-old P. massoniana seedlings were also inoculated under aseptic conditions with aseptic PWNs and non-aseptic PWNs. The results showed that the aseptic microcuttings and seedlings inoculated with aseptic PWNs or non-aseptic PWNs wilted, while those inoculated with bacterial isolates did not wilt. Nematodes were recovered from wilted microcuttings and seedlings inoculated with aseptic PWNs and non-aseptic PWNs, and the asepsis of nematodes recovered from aseptic PWN-inoculated microcuttings and seedlings was reconfirmed by culturing them in NB liquid medium at 30°C for more than 7 days. Taken together, the results indicate that the asepsis of PWN did not cause the loss of pathogenicity. © 2012 Zhu et al.

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