Oral: Pathogen Detection
An inexpensive visual PCR method for field detection of Candidatus Liberibacter asiaticus associated with citrus huanglongbing.
M. KEREMANE (1), C. Ramadugu (2), D. Hall (3) (1) USDA ARS, U.S.A.; (2) University of California Riverside, U.S.A.; (3) US Horticultural Research Laboratory, U.S.A.
Huanglongbing (HLB) is threatening citrus cultivation in California and Texas. Testing for Liberibacter (Las, the HLB pathogen) by qPCR is labor-intensive and requires expensive instrumentation. The key to HLB management is large scale testing by many interested personnel. Development of on-site testing methods will increase the chance of pathogen detection by growers, homeowners and extension agents and enables timely implementation of disease management strategies. We describe an easy method to test psyllids (HLB vectors) for the presence of the Las pathogen. The initial equipment needed includes a heating block, table top centrifuge, tubes and reaction reagents. The cost for all of the equipment needed for testing by this method will be under $300. Psyllids are captured, stored in ethanol, air-dried, and DNA extracted by heating at 80 °C in extraction buffer. Pathogen DNA is amplified using a loop-mediated amplification method at ambient temperatures (65 °C) for 30 minutes. Samples positive for Las will appear yellow after the reaction and negative samples will remain pink. The whole reaction can be performed within an hour in a farmhouse by non-scientific personnel with very little training. Availability of methods for large scale testing will facilitate better disease management.