The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

  • Qiao, Tian-Min (College of Forestry, Sichuan Agricultural University) ;
  • Zhang, Jing (College of Forestry, Sichuan Agricultural University) ;
  • Li, Shu-Jiang (College of Forestry, Sichuan Agricultural University) ;
  • Han, Shan (College of Forestry, Sichuan Agricultural University) ;
  • Zhu, Tian-Hui (College of Forestry, Sichuan Agricultural University)
  • Received : 2016.03.17
  • Accepted : 2016.07.06
  • Published : 2016.10.01
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Abstract

Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

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References

  1. Anonymus. 1984. Nursery diseases of Eucalyptus in Kerala and their possible control measures. Kerala For. Res. Inst. Inf. Bull. 6:16.
  2. Bonants, P., Hagenaar-de Weerdt, M., van Gent-Pelzer, M., Lacourt, I., Cooke, D. and Duncan, J. 1997. Detection and identification of Phytophthora fragariae Hickman by the polymerase chain reaction. Eur. J. Plant Pathol. 103:345-355. https://doi.org/10.1023/A:1008640227432
  3. Crous, P. W., Phillps, A. J. L. and Wingffield, M. J. 1993. New records of Cylindrocladium and Cylindrocladiella spp. in South Africa. Plant Pathol. 42:302-305. https://doi.org/10.1111/j.1365-3059.1993.tb01504.x
  4. Crous, P. W. and Wingfield, M. J. 1994. A monograph of Cylindrocladium, including anamorphs of Calonectria. Mycotaxon 51:341-435.
  5. Feng, X., Zhang, X. and Yan, D. 2012. Detection to pathogens of poplar cankers by the multiplex PCR technique. Scientia Silvae Sinicae 48:72-77 (in Chinese).
  6. Glass, N. L. and Donaldson, G. C. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 61:1323-1330.
  7. Grote, D., Olmos, A., Kofoet, A., Tuset, J. J., Bertolini, E. and Cambra, M. 2002. Specific and sensitive detection of Phytophthora nicotaianae by simple and nested-PCR. Eur. J. Plant Pathol. 108:197-207. https://doi.org/10.1023/A:1015139410793
  8. Guglielmo, F., Bergemann, S. E., Gonthier, P., Nicolotti, G. and Garbelotto, M. 2007. A multiplex PCR-based method for the detection and early identification of wood rotting fungi in standing trees. J. Appl. Microbiol. 103:1490-1507. https://doi.org/10.1111/j.1365-2672.2007.03378.x
  9. Hagemann, G. D. and Rose, P. D. 1988. Leaf spot and blight on Acacia longifolia caused by Cylindrocladium scoparium. Phytophylactica 20:311-316.
  10. Hansen, Z. R., Knaus, B. J., Tabima, J. F., Press, C. M., Judelson, H. S., Grunwald, N. J. and Smart, C. D. 2016. Loopmediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans. J. Appl. Microbiol. 120:1010-1020. https://doi.org/10.1111/jam.13079
  11. Henson, J. M. and French, R. 1993. The polymerase chain reaction and plant disease diagnosis. Annu. Rev. Phytopathol. 31:81-109. https://doi.org/10.1146/annurev.py.31.090193.000501
  12. Huang, J., Song, D., Flores, A., Zhao, Q., Mooney, S. M., Shaw, L. M. and Lee, F. S. 2007. IOP1, a novel hydrogenase-like protein that modulates hypoxia-inducible factor-1alpha activity. Biochem. J. 401:341-352. https://doi.org/10.1042/BJ20060635
  13. Li, G., Wu, Z., Xie, Y., Li, T., Chang, R., Wang, Y. and Zhou, X. 2013. Research progress of Eucalyptus dieback in China and abroad. Eucalypt Sci. Technol. 1:45-50 (in Chinese).
  14. Li, W., Li, D., Twieg, E., Hartung, J. S. and Levy, L. 2008. Optimized quantification of unculturable Candidatus liberibacter spp. in host plants using real-time PCR. Plant Dis. 92:854-861. https://doi.org/10.1094/PDIS-92-6-0854
  15. Mori, Y. and Notomi, T. 2009. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J. Infect. Chemother. 15:62-69. https://doi.org/10.1007/s10156-009-0669-9
  16. O'Donnell, K. and Cigelnik, E. 1997. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol. Phylogenet. Evol. 7:103-116. https://doi.org/10.1006/mpev.1996.0376
  17. Pang, L. D., Pang, W. W., Zeng, W. Q., Liu, Z. C., Wei, Z. Z. and Tan, G. B. 2001. Chemical control of Eucalyptus dieback. Plant Quar. 15:273-275 (in Chinese).
  18. Rees-George, J., Vanneste, J. L., Cornish, D. A., Pushparajah, I. P. S., Yu, J., Templeton, M. D. and Everett, K. R. 2010. Detection of Pseudomonas syringae pv. actinidiae using polymerase chain reaction (PCR) primers based on the 16S-23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions. Plant Pathol. 59:453-464. https://doi.org/10.1111/j.1365-3059.2010.02259.x
  19. Sharma, J. K. and Mohanan, C. 1982. Cylindrocladium spp. associated with various diseases of Eucalyptus in Kerala. Eur. J. For. Pathol. 12:129-136. https://doi.org/10.1111/j.1439-0329.1982.tb01385.x
  20. Sipos, R., Szekely, A. J., Palatinszky, M., Revesz, S., Marialigeti, K. and Nikolausz, M. 2007. Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis. FEMS Microbiol. Ecol. 60:341-350. https://doi.org/10.1111/j.1574-6941.2007.00283.x
  21. Tan, Z., Tong, X., Fang, C., Jiang, C. and Chen, C. 2009. Prokaryotic expression of ${\alpha}$-tubulin gene of Camellia sinensis and preparation of ${\alpha}$-tubulin polyclonal antibody. J. Tea Sci. 29:336-340 (in Chinese).
  22. Tomlinson, J. A., Dickinson, M. J. and Boonham, N. 2010. Detection of Botrytis cinerea by loop-mediated isothermal amplification. Lett. Appl. Microbiol. 51:650-657. https://doi.org/10.1111/j.1472-765X.2010.02949.x
  23. Wang, Z., Yang, M., Yang, Y. Q., Peng, Z. K. and Zhou, E. X. 2010. Morphological and molecular identification of citrus anthracnose pathogen from Guangdong province. Mycosystema 29:488-493 (in Chinese).
  24. White, T. J., Bruns, T., Lee, S. and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR protocols: a guide to methods and applications, eds. by M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White, pp. 315-322. Academic Press, New York, NY, USA.
  25. Zhu, J. H., Chen, H. M., Wu, J. Q., Chen, Q. Z., Feng, L. Z., Meng, X. M. and Guo, W. S. 2011a. Loss estimation of eucalyptus growth caused by of eucalyptus dieback. For. Pest Dis. 30:6-10 (in Chinese).
  26. Zhu, J. H., Chen, H. M., Wu, J. Q., Chen, Q. Z., Feng, L. Z., Meng, X. M. and Guo, W. S. 2011b. Study on the control technology of Clindrocladium quinqueseptatum. J. Fujian For. Sci. Technol. 38:41-47 (in Chinese).