A long-amplicon viability-qPCR test for quantifying living pathogens of bacterial spot in tomato seed

Authors: WANG, HEHE - Clemson University; WAGNON, RIEANNA - Clemson University; MORENO, DANIELA - Clemson University; TIMILSINA, SUJAN - University Of Florida; JONES, JEFF - University Of Florida; VALLAD, GARY - University Of Florida; Turechek, William

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/7/2021
Publication Date: 4/6/2022
Citation: Wang, H., Wagnon, R., Moreno, D., Timilsina, S., Jones, J., Vallad, G., Turechek, W. 2022. A long-amplicon viability-qPCR test for quantifying living pathogens of bacterial spot in tomato seed. Plant Disease. 106:1474-1485. https://doi.org/10.1094/PDIS-11-21-2509-RE.
DOI: https://doi.org/10.1094/PDIS-11-21-2509-RE

Interpretive Summary: Bacterial spot is a serious diseases of tomato caused by any of four different species of Xanthomonas. Contaminated and/or infected seed serve as a major inoculum source for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. However, current seed certification protocols rely mainly on culturing and conventional PCR, and both methods have limited capability to accurately quantify viable pathogen cells. To improve the method for seed certification, a long-amplicon PMA-qPCR assay was developed to selectively quantify the four Xanthomonas pathogen species in tomato seed. Tthe PMA-qPCR assay was able to detect targets in seed samples with higher accuracy than any previous method. Being much faster and more sensitive, PMA-qPCR has a promising potential to serve as a standalone tool or used in combination with culturing to improve tomato seed certification and advance the production of clean seed.

Technical Abstract: Bacterial spot is one of the most serious diseases of tomato. It is caused by four different species of Xanthomonas: X. euvesicatoria, X. gardneri, X. perforans, and X. vesicatoria. Contaminated and/or infected seed serve as a major inoculum source for this disease. The use of certified pathogen-free seed is one of the primary management practices to reduce the inoculum load in commercial production. However, current seed certification protocols rely mainly on culturing and conventional PCR, and both methods have limited capability to accurately quantify viable pathogen cells. To improve the sensitivity and specificity of the seed certification method, a long-amplicon PMA-qPCR assay was developed to selectively quantify the four Xanthomonas pathogen species in tomato seed. The optimized PMA-qPCR procedure was evaluated on pure bacterial suspensions, bacteria-spiked seed suspensions, as well as inoculated and naturally-infected seed. A crude DNA extraction protocol was also developed and it provided comparable detection as the purified DNA in non-concentrated seed suspensions. PMA-qPCR with crude DNA extract was able to accurately quantify 10^4-10^8 CFU/ml of viable bacteria in the mixture with dead cells as high as 10^7 CFU/ml in the seed suspensions. With purified DNA from concentrated seed suspensions, the PMA-qPCR assay was able to detect targets in seed samples spiked with =75 CFU/ml (~0.5 CFU/seed) of the viable pathogens. Latent class analysis of the inoculated and naturally-infected seed samples showed that PMA-qPCR had similar specificity but much higher sensitivity than the colony counts on the semi-selective MTMB and CKTM media across all target species. Being much faster and more sensitive, PMA-qPCR has a promising potential to serve as a standalone tool or used in combination with semi-selective culturing to improve tomato seed certification and advance the production of clean seed.