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ARS Home » Pacific West Area » Corvallis, Oregon » Horticultural Crops Research Unit » Research » Publications at this Location » Publication #322737
Title: Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans

Author
item HANSEN, Z - Cornell University
item Knaus, Brian
item TABIMA, J - Oregon State University
item JUDELSON, H - University Of California
item Grunwald, Niklaus - Nik
item SMART, C - Cornell University

Submitted to: Journal of Applied Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/21/2016
Publication Date: 3/7/2016
Citation: Hansen, Z.R., Knaus, B.J., Tabima, J.F., Judelson, H.S., Grunwald, N.J., Smart, C.D. 2016. Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans. Journal of Applied Microbiology. 120(4):1010–1020. doi: 10.1111/jam.13079.

Interpretive Summary: Late blight, caused by the oomycete Phytophthora infestans, continues to be an important disease of potato and tomato. We designed and validated a detection assay for rapid detection of P. infestans DNA. A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of P. infestans. Two sets of LAMP primers, targeting two different genomic regions, were designed and evaluated for their sensitivity and specificity for P. infestans. Primers were tested against the pathogens in culture as well as against a range of potato and tomato pathogens and were specific and able to detect infection of P. infestans on tomato or potato.

Technical Abstract: Aims: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of P. infestans DNA. Methods and Results: Two sets of LAMP primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the internal transcribed spacer region of ribosomal DNA. These primers had a limit of detection of 2 pg P. infestans DNA and cross reacted with the closely related species P. nicotianae. Rgn86_2 primers, designed to improve assay specificity, targeted a portion of a conserved hypothetical protein. These primers had a limit of detection of 200 pg P. infestans DNA and did not cross react with P. nicotianae. The specificity of the Rgn86_2 assay was tested further using the closely related species P. andina, P. ipomoeae, P. mirabilis, and P. phaseoli. Cross reactions occurred with P. andina and P. mirabilis, but neither species occurs on tomato or potato. Both primer sets were able to detect P. infestans DNA extracted from tomato late blight leaf lesions. Conclusions: Two colorimetric LAMP assays detected P. infestans DNA from pure cultures as well as infected leaf tissue. The ITSII primers had higher sensitivity, and the Rgn86_2 primers had higher specificity.