Increased efficiency and sensitivity for identifying citrus greening and citrus tristeza virus using real-time PCR testing

Abstract

All certified citrus nursery trees can trace their origins to fully tested scion and foundation source trees that remain clean and disease-free in screen houses. Implementation of real-time polymerase chain reaction (PCR) for pathogen testing has lead to an increased requirement for both the testing efficiency and sensitivity in the Florida Bureau of Citrus Budwood Registration testing program. Budwood source citrus trees are tested annually for the vectored diseases Citrus tristeza virus (CTV) and citrus greening (HLB) and once every 6 years for other graft-transmissible diseases. To test the current number (more than 6000) of source trees annually, streamlining of collection and extraction of samples was necessary. Advance preparation allowed greater efficiency during the late summer to early fall months that samples are collected, extracted, and tested for HLB. Specialized equipment, including a new type of grinding apparatus, reduced costs and the time needed for processing. The second yearly collection from the same trees had been carried out during Spring 2008 for CTV ELISA testing. Two real-time CTV PCR tests, used in conjunction from a single extraction, have replaced the second collection and CTV enzyme-linked immunosorbent assay (ELISA) test. After extraction, testing for each pathogen can be completed in one month, previously an impossible task by conventional PCR. Citrus leaf blotch virus was the fi rst pathogen tested using the same extraction as for HLB. One collection, one sample, and one extraction save money and allow for thorough, sensitive budwood testing for multiple citrus pathogens.