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Development of a molecular marker for specific detection of Fusarium oxysporum f. sp. cubense race 4

  • Ying-Hong Lin
  • Jing-Yi Chang
  • En-Tzu Liu
  • Chih-Ping Chao
  • Jenn-Wen Huang
  • Pi-Fang Linda Chang
Article

Abstract

Fusarium oxysporum f. sp. cubense is the causal agent of Panama disease of banana. A rapid and reliable diagnosis is the foundation of integrated disease management practices in commodity crops. For this diagnostic purpose, we have developed a reliable molecular method to detect Foc race 4 isolates in Taiwan. By PCR amplification, the primer set Foc-1/Foc-2 derived from the sequence of a random primer OP-A02 amplified fragment produced a 242 bp size DNA fragment which was specific to Foc race 4. With the optimized PCR parameters, the molecular method was sensitive and could detect small quantities of Foc DNA as low as 10 pg in 50 to 2,000 ng host genomic DNA with high efficiency. We also demonstrated that by using our PCR assay with Foc-1/Foc-2 primer set, Foc race 4 could be easily distinguished from other Foc races 1 and 2, and separated other formae speciales of F. oxysporum.

Keywords

Molecular detection Panama disease PCR RAPD Reliable diagnosis 

Notes

Acknowledgements

We thank Dr. Peter P. Ueng of USDA-ARS for providing DNA samples of reference isolates of Foc race 1, 2, and 4, and his critical review of this manuscript. We are grateful to Drs. Y.-S. Lin (NCHU), K.-S. Chen (FTHEB, ARI), S.-P.-Y. Hsieh (NCHU), S.-C. Hwang (TBRI), Miss H.-L. Lee (TDARES), WVC/AVRDC, and ARI for providing the tested microorganisms, and to Dr. W.-H. Ko for critical reading and useful suggestions for this manuscript. We also thank Miss L.J. Smith for VCG identification, Dr. R.C. Ploetz for information about Foc race 4, Miss Y.-L. Wan and Mr. C.-C. Su for technical assistance. This research was supported in part by Bureau of Animal and Plant Health Inspection and Quarantine, and Department of International Affairs, Council of Agriculture, Executive Yuan, Taiwan, R.O.C. under grant numbers 89ST-6.2-BQ-65(06), 91AS-7.3.1-BQ-B2(3), 93AS-1.9.2-BQ-B1, 96AS-4.1.2-IC-I1(2), and 97AS-4.1.2-IC-I1(2); by the Ministry of Education, Taiwan, R.O.C. under the ATU plan; and also by National Chung Hsing University, Taiwan, R.O.C.

Supplementary material

10658_2008_9372_MOESM1_ESM.doc (3.3 mb)
ESM 1 Amplification of PCR products using the primer set Foc-1/Foc-2 specific to Fusarium oxysporum f. sp. cubense (Foc) race 4 isolates. The fungal isolates used for extracting genomic DNA (gDNA) as PCR templates (50 ng) were as listed in Table 1. The DNA gels were subjected to Southern hybridisation (shown as the panel below each ethidium bromide-stained DNA gel pattern with light background) using the Biotin-labelled OPA02404 as a probe. The location of a 242-bp DNA band specific to F. oxysporum f. sp. cubense race 4 isolates (labeled as Foc) is indicated on the left. Fon-H0103 is the Fusarium wilt pathogen of watermelon to serve as a negative control here. N = negative control using sterile dH2O as the PCR template. M = molecular markers of Gen-100 DNA ladder (DOC 3.34 MB).

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Copyright information

© KNPV 2008

Authors and Affiliations

  • Ying-Hong Lin
    • 1
  • Jing-Yi Chang
    • 1
  • En-Tzu Liu
    • 1
  • Chih-Ping Chao
    • 2
  • Jenn-Wen Huang
    • 1
  • Pi-Fang Linda Chang
    • 1
  1. 1.Department of Plant PathologyNational Chung Hsing UniversityTaichungRepublic of China
  2. 2.Taiwan Banana Research InstitutePingtungRepublic of China

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