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Title:   Author  item
                                            HANSEN, Z - Cornell Universityitem
                                            Knaus, Brianitem
                                            TABIMA, J - Oregon State Universityitem
                                            JUDELSON, H - University Of Californiaitem
                                            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.


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