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Title: Multiplex real-time PCR for detection, identification and quantification of "Candidatus Liberibacter solanacearum" in potato plants with zebra chip

Author
item Li, Wenbin
item ABAD, JORGE - USDA APHIS BELTSVILLE MD
item FRENCH-MONAR, RONALD - TEXAS A&M AMARILLO TX
item RASCOE, JOHN - USDA APHIS BELTSVILLE MD
item WEN, AIMIN - ND STATE UNIV FARGO ND
item GUDMESTAD, GARY - ND STATE UNIV FARGO ND
item SECOR, GARY - ND STATE UNIV FARGO ND
item Lee, Ing Ming
item LEVY, LAURENE - USDA APHIS BELTSVILLE MD

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/17/2009
Publication Date: 5/2/2009
Citation: Li, W.N., Abad, J.A., French-Monar, R.D., Rascoe, J., Wen, A., Gudmestad, G., Secor, G.A., Lee, I., Levy, L. 2009. Multiplex real-time PCR for detection, identification and quantification of "Candidatus Liberibacter solanacearum" in potato plants with zebra chip. Phytopathology. 78:59-65.

Interpretive Summary: Potato purple top wilt (PPT) and similar diseases have caused tremendous damage to potato tuber production in South America, Mexico, and the U.S. In 2002 and 2003, a major epidemic of PPT occurred in Washington and Oregon, causing great economic damage to the potato industry. A phytoplasma, a small cell wall-less bacterium, belonging to the clover proliferation phytoplasma group was the causal agent. In 2004 and 2005, a new potato disease with symptoms similar to those of PPT occurred in Texas and Nebraska, causing patchy brown discoloration of chips produced from commercial processing potatoes. This new disease is termed potato zebra chip (ZC) disease. Earlier, we identified two phytoplasmas that were associated with this new PPT disease (ZC) in northern regions of Texas and in Nebraska. However, in Mexico and southern regions of Texas, the cause of ZC is still unknown. Recently, a new uncultured bacterium, ‘Candidatus Liberibacter psyllaurous’, was detected and found to be associated with ZC-affected potato plants collected in southern Texas. However, because of low concentration of this probable pathogen in potato tissues, conventional polymerase chain reaction (PCR) procedures often fail to detect this pathogen. In this study, in collaboration with scientists from the National Plant Germplasm and Biotechnology Laboratory, USDA-APHIS-PPQ-CPHST, Beltsville, MD, we developed a highly sensitive real-time PCR procedure that enable us to readily detect and quantify the pathogen. The information will aid implementation of quarantine regulations and help extension workers and plant diagnosticians to determine how to combat the disease.

Technical Abstract: A new ‘Candidatus Liberibacter species’, Ca Liberibacter psyllarous’ (Lps) was found to be consistently associated with a newly emerging potato zebra chip (ZC) disease in Texas and other southwestern states in the U.S. This species is nearly identical on the basis of 16S rDNA sequences to that reported to be associated with potato/tomato psyllid-transmitted diseases in tomato and capsicum in New Zealand and in Texas. A species-specific primer LpsF was developed for both quantitative real-time PCR (qPCR) and conventional PCR (cPCR) to detect and quantify Lps in potato and tomato plants affected with ZC. In multiplex qPCR, a plant cytochrome oxidase (COX)-based probe-primer set was used as a positive internal control for host plant DNA, which could be used to reliably access the DNA quality and to normalize qPCR data for accurate quantification of the bacterial populations in environment samples. Neither qPCR nor cPCR reacted with other ‘Ca.Liberibacter species’ of citrus or other potato pathogens. The low detection limit of the multiplex qPCR was about 20 copies of the target DNA template per reaction for field-collected samples. Lps was readily detected and quantified in various tissues of potato plants affected by ZC collected from fields in Texas. A thorough but uneven colonization of Lps was revealed in various organs of the potato plant. The highest Lps populations were about 3 x 108 genomes/g tissue in the root, which were 3-orders higher than those in the above-ground tissues of potato plants. The Lps bacterial population dynamics was normally distributed across the ZC-infected potato plants collected from potato fields in Texas, with 60% of ZC-affected potato plants harboring an average Lps population from 105 to 106 genomes/g, 4% of plants hosting above 107 Lps genomes/g, and 7% of plants holding bellow 104 Lps genomes/g. Lps was not detected in field-grown citrus plants in Texas. The rapid, sensitive, specific and reliable multiplex qPCR has potential to become a powerful tool for early detection and quantification of the new ‘Ca Liberibacter psyllaurous’ in ZC-diseased potato plants, and will be very useful for Potato Quarantine Program and seed potato certification programs, to ensure the availability of clean seed potato stocks, and also for epidemiologic studies on the disease.