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Development of a specific molecular tool for the detection of epidemiologically active mulberry causing-disease strains of Ralstonia solanacearum phylotype I (historically race 5-biovar 5) in China

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Abstract

Ralstonia solanacearum causes bacterial wilt disease in many plant species, including mulberry. Here, we used a suppression subtractive hybridization (SSH) approach to identify specific DNA fragments in R. solanacearum race 5-biovar 5. The genome of the R. solanacearum M7 strain was subtracted from that of the GMI1000 strain, resulting in the identification of 85 subtracted fragments. The primer set MG67-F/R for identification of Ralstonia solanacearum race 5-biovar 5 strains was designed on the basis of the clone MG67 sequence. Furthermore, a multiplex PCR was developed by using the primer set MG67-F/MG67R in combination with the species-specific primer pair 759/760. A 156 bp r5-bv5-specific fragment, together with a 282 bp species-specific fragment, was amplified from all tested R. solanacearum r5-bv5 strains. The sensitivity of the multiplex PCR made it possible to detect concentrations as low as 102 CFU ml−1 of pure culture. Moreover, the r5-bv5-specific multiplex PCR was successfully applied to detect Ralstonia solanacearum race 5-biovar 5 strains in diseased mulberry samples. Therefore, the multiplex PCR assay can be used as a reliable diagnostic technique to enable researchers to rapidly identify isolates of R. solanacearum race 5-biovar 5.

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Abbreviations

r:

Race

bv:

Biovar

IS:

Insertion sequences

SSH:

Suppression subtractive hybridization

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Acknowledgments

We thank Guan-Lin Xie, Institute of Biotechnology, Zhejiang University, for providing Enterobacter mori Rs18-2 strain. This work was supported by grants from the National Basic Research and Development Program (973 Program) (No. 2009CB119200), the National High Technology Research and Development Program (863 Program) (No. 2012AA101501), Special Fund for Agro-scientific Research in the Public Interest (201303015, 201303129 & 201303018), the National Natural Science Foundation (No. 31272008), Yunnan Natural Science Foundation (No. 2013FZ152), Yunnan Science and Technology Innovation Platform Construction Program (No. 2011DH018) and Yunnan High-Level Talent Program (No. 2011CI133).

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Authors and Affiliations

  1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 West Yuanmingyuan Road, Beijing, 100193, People’s Republic of China

    Z. C. Pan, J. Xu, J. S. Xu, H. Zhang, K. Y. Chen, Q. Tian, L. Q. Zhang, L. Liu, L. Y. He & J. Feng

  2. CIRAD/INRA, UMRC53, Plant Communities and Biological Invaders in Tropical Climates (PVBMT), 97410, Saint-Pierre, Réunion, France

    P. Prior

  3. National Potato Improvement Center Yunnan Branch, Yunnan Potato Engineering and Technology Research Center, Industrial Crop Research Institute, Yunnan Academy of Agricultural Sciences, Beijing Road 2238, Kunming, Yunnan, 650200, People’s Republic of China

    Z. C. Pan

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  1. Z. C. Pan
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  2. J. Xu
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  3. P. Prior
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  4. J. S. Xu
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  9. L. Liu
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  10. L. Y. He
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  11. J. Feng
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Correspondence to J. Feng.

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Z. C. Pan J. Xu and P. Prior contributed equally to this paper.

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Pan, Z.C., Xu, J., Prior, P. et al. Development of a specific molecular tool for the detection of epidemiologically active mulberry causing-disease strains of Ralstonia solanacearum phylotype I (historically race 5-biovar 5) in China. Eur J Plant Pathol 137, 377–391 (2013). https://doi.org/10.1007/s10658-013-0249-9

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