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Universal, Rapid, and Visual Detection Methods for Phytoplasmas Associated with Coconut Lethal Yellowing Diseases
Shao-Shuai Yu1, Ying-Wen Pan2, Hui Zhu1
1Coconut Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wenchang 571339, Hainan Province, China.
Abstract:
Coconut lethal yellowing (LY) diseases caused by phytoplasmas are devastating diseases for coconut cultivation and seriously threaten the coconut industry around world. The phytoplasmas associated with the LY diseases belonged to six 16Sr groups containing 16SrI, 16SrIV, 16SrXI, 16SrXIV, 16SrXXII, and 16SrXXXII with comparatively higher variable levels. Conserved regions of the 16S rRNA genes of LY phytoplasmas belonging to the six 16Sr groups were obtained in the study. Based on the conserved region sequences of 16S rRNA genes, two sets of LAMP primers, Co-4 and Co-6, were designed and screened, and the rapid and visual detection methods universal for different groups LY phytoplasmas were established. The entire detection reactions of the universal detection methods could be completed with only 30 to 40 min of constant temperature amplification at 64°C, and the detection results were judged by the color changes of the reaction systems, which are convenient and quick. For the six groups of phytoplasmas, the estimated minimum detection limit range of the universal detection primers Co-4 and Co-6 were identical: 4.8 × 101 to 4.8 × 107 copies per 200 μl. The universal detection methods for the LY phytoplasmas established in the study are of great significance for the rapid diagnosis and identification and the efficient monitoring and early warning as well as the port inspection and quarantine of the LY phytoplasmas and their related diseases.
Insights
New LAMP primers, Co-4 and Co-6, enable rapid, visual detection of lethal yellowing (LY) phytoplasmas in 30-40 minutes. This breakthrough aids in early diagnosis and monitoring of devastating coconut diseases worldwide.
Area of Science:
- Plant Pathology
- Microbiology
- Molecular Diagnostics
Background:
- Coconut lethal yellowing (LY) diseases, caused by diverse phytoplasmas, pose a significant threat to global coconut cultivation.
- Phytoplasmas associated with LY belong to six distinct 16Sr groups, exhibiting high variability.
- Accurate and rapid detection methods are crucial for managing these devastating plant diseases.
Purpose of the Study:
- To develop a universal, rapid, and visual detection method for phytoplasmas causing coconut lethal yellowing.
- To design and screen novel LAMP primers targeting conserved regions of LY phytoplasma 16S rRNA genes.
- To establish efficient diagnostic tools for early warning and quarantine of LY diseases.
Main Methods:
- Sequencing conserved regions of 16S rRNA genes from six 16Sr groups of LY phytoplasmas.
- Designing and screening two sets of LAMP primers (Co-4 and Co-6) based on conserved sequences.
- Establishing a rapid, visual detection assay using constant temperature amplification (64°C) and colorimetric detection.
Main Results:
- Two universal LAMP primer sets, Co-4 and Co-6, were successfully designed and validated.
- A rapid detection method yielding results within 30-40 minutes via visual color change was established.
- The detection limit for both primer sets ranged from 4.8 × 10^1 to 4.8 × 10^7 copies per 200 μl for all six phytoplasma groups.
Conclusions:
- The developed universal LAMP methods provide a quick, convenient, and accurate tool for detecting diverse LY phytoplasmas.
- These methods are significant for rapid diagnosis, identification, monitoring, and quarantine of lethal yellowing diseases.
- The findings contribute to safeguarding the global coconut industry through effective disease management strategies.
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