Enhancement of a Co-PCR Technology for Detection of Bacterial Fruit Blotch Pathogen, Acidovorax citrulli, in Watermelon Seed

Authors

  • Jutatape Watcharachaiyakup Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus
  • Supananya Chansri Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus
  • Thanyaluk Thaitae Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus
  • Kanchanaphorn Sawangchaitham Center for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus

DOI:

https://doi.org/10.14456/thaidoa-agres.2023.2

Keywords:

Seed certification, Watermelon, Cucubitae, Bacterial fruit blotch

Abstract

Enhancement of Co-PCR technology for the detection of Acidovorax citrulli (Ac) using the 521 primer set was developed in this study. The four primers comprised AC-ORF12F and AC-ORF1F4 at 0.4 µM, and primers AC-ORF13R and AC-ORF3F3 at 0.2 µM with the specificity of annealing temperature at 72 o C were employed. Results revealed that detection limit of the target DNA detection was 1 pg. The detection limit of this method in the 0.01% contamination seed lot was 37 colony forming unit (CFU). By comparison, the standard ISHI-Veg protocol was unable to detect Ac at the same level of contamination.

References

Dutta, B., H. Scherm, R. D. Gitaitis and R. R. Walcott. 2012. Acidovorax citrulli seed inoculum load affects seedling transmission and spread of bacterial fruit blotch of watermelon under greenhouse conditions. Plant Dis. 96(5): 705-711.

EPPO. 2021. EPPO A1 List of pests recommended for regulation as quarantine pests version 2021-09. Available at: https://www.eppo.int/ACTIVITIES/plant_quarantine/A1_list. Accessed: 14 November 2021.

Giovanardi, D., S. A. Sutton, E. Stefani, and R. R. Walcott. 2018. Factors influencing the detection of Acidovorax citrulli in naturally contaminated cucurbitaceous seeds by PCR-based assays. Seed Sci. Technol. 46(1): 93-106.

ISHI-Veg. 2021. Detection of Acidovorax citrulli in Cucubit Seeds by SE-qPCR. International Seed Federation (ISHI), Nyon, Switzerland.

Kim, Y. T., K. S. Park, H. S. Kim, H. I. Lee and J. S. Cha. 2015. Development of nested-PCR assay to detect Acidovorax citrulli, a causal agent of bacterial fruit blotch at Cucurbitaceae. Res. Plant Dis. 21: 74-81.

Korakngam, C., J. Watcharachaiyakup, S.Patarapuwadol, W. Kositratana. 2018. Detection of Acidovorax citrulli by co-operational polymerase chain reaction for seed health testing of watermelon seed. Thai J. Agric. Sci 36(3): 280-292.

Kositcharoenkul, N., O. Chatchawankanphanich, A. Bhunchoth and W. Kositratana. 2011. Detection of Xanthomonas citri subsp. citri from field samples using single‐tube nested PCR. Plant Pathol. 60(3): 436-442.

Lu, Y., H. J. Zhang, Z. J. Zhao, C. L. Wen, W. U. Ping, S. H. Song, S. C. Yu, L. X. Luo and X. L. Xu. 2020. Application of droplet digital PCR in detection of seed-transmitted pathogen Acidovorax citrulli. J Integr. Agric. 19(2): 561-569.

Olmos, A., E. Bertolini and M. Cambra. 2002. Simultaneous and co-operational amplification (Co-PCR): a new concept for detection of plant viruses. J. Virol. Methods 106: 51–59.

Öztürk, N. and H. Basim. 2022. A real-time PCR assay using locked nucleic acid probe for detection of Acidovorax citrulli. J Plant Dis. Prot. 129(2): 395-409.

Weisburg, W. G., S. M. Barns, D. A. Pelletier and D. J. Lane. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173(2): 697-703.

Downloads

Published

2023-05-25

How to Cite

Watcharachaiyakup, J., Chansri, S. ., Thaitae, T., & Sawangchaitham, K. (2023). Enhancement of a Co-PCR Technology for Detection of Bacterial Fruit Blotch Pathogen, Acidovorax citrulli, in Watermelon Seed . Thai Agricultural Research Journal, 41(1), 14–27. https://doi.org/10.14456/thaidoa-agres.2023.2

Issue

Vol. 41 No. 1 (2023): January - April

Section

Technical or research paper

License

Copyright (c) 2023 Thai Agricultural Research Journal

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Thai Agricultural Research Journal