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United States Department of Agriculture

Agricultural Research Service

Title: Sensitivity of Pcr-Based Methods for Detection of Asiatic Strain of the Huanglongbing Bacterium in Symptomatic and Asymptomatic Citrus Tissues

Authors
item Irey, Michael
item Gast, Tim - SOUTHERN GARDENS CITRUS
item Gottwald, Timothy

Submitted to: Meeting Abstract
Publication Type: Proceedings
Publication Acceptance Date: July 14, 2006
Publication Date: July 22, 2006
Citation: Irey, M.S., Gast, T., Gottwald, T.R. 2006. Sensitivity of pcr-based methods for detection of asiatic strain of the huanglongbing bacterium in symptomatic and asymptomatic citrus tissues. Meeting Abstract. Proceedings of the Huanglongbing-greening International Workshop, July 14-21, 2006, Ribeiro Preto, Brazil. P. 19-20.

Interpretive Summary: Citrus Huanglongbing (HLB) (syn = citrus greening) was first confirmed in the US in September, 2005 in Dade County, FL. Since then it has been found in 12 counties in southern Florida and survey efforts to delimit the areas of infection are continuing. The survey process consists of visual inspections using trained inspectors followed by confirmatory laboratory testing using one of several PCR procedures. PCR testing has been limited only to the confirmation of suspect samples due to the perception that PCR testing was only able to reliably detect the pathogen that causes HLB in symptomatic tissue. Although there are many publications describing multiple PCR procedures to detect the three species of Candidatus Liberibacter that are known to cause citrus greening, there are few direct comparisons of the procedures and little data are available in the literature describing how and when samples should be collected for subsequent PCR testing. In the months since HLB was first found in Florida, samples from naturally infected trees in commercial groves have been used to evaluate the sensitivity of three PCR protocols on leaves from symptomatic tissue. In addition, the same three procedures were evaluated on asymptomatic tissue of six different tissue types over a five month period. Although the study is still in progress, marked differences in sensitivity were found between the three methods when applied to both the asymptomatic and the symptomatic tissue. Differences were also found within a method and between methods depending on the type of tissue sampled. Similarly, temporal differences were also observed over the five months of the study. All combined, the differences were substantial enough that if the wrong method was used to sample from a less than optimum tissue type during a less than optimum time of the year, it is possible or maybe even probable that the infection would not be detected and the sample would lead to a false negative conclusion.

Technical Abstract: Citrus Huanglongbing (HLB) (syn = citrus greening) was first confirmed in the US in September, 2005 in Dade County, FL. Since then it has been found in 12 counties in southern Florida and survey efforts to delimit the areas of infection are continuing. The survey process consists of visual inspections using trained inspectors followed by confirmatory laboratory testing using one of several PCR procedures. PCR testing has been limited only to the confirmation of suspect samples due to the perception that PCR testing was only able to reliably detect the pathogen that causes HLB in symptomatic tissue. Although there are many publications describing multiple PCR procedures to detect the three species of Candidatus Liberibacter that are known to cause citrus greening, there are few direct comparisons of the procedures and little data are available in the literature describing how and when samples should be collected for subsequent PCR testing. In the months since HLB was first found in Florida, samples from naturally infected trees in commercial groves have been used to evaluate the sensitivity of three PCR protocols on leaves from symptomatic tissue. In addition, the same three procedures were evaluated on asymptomatic tissue of six different tissue types over a five month period. Although the study is still in progress, marked differences in sensitivity were found between the three methods when applied to both the asymptomatic and the symptomatic tissue. Differences were also found within a method and between methods depending on the type of tissue sampled. Similarly, temporal differences were also observed over the five months of the study. All combined, the differences were substantial enough that if the wrong method was used to sample from a less than optimum tissue type during a less than optimum time of the year, it is possible or maybe even probable that the infection would not be detected and the sample would lead to a false negative conclusion. To test the utility of PCR testing of asymptomatic trees in a grove situation, the most sensitive of the three PCR protocols evaluated was used to test trees in five 14 by 14 tree plots of commercial trees in an area with known infection with Candidatus Liberibacter asiaticus (CLa). Prior to PCR testing, the trees were inspected by trained scouts to determine the location of CLa symptomatic trees. Leaves from each tree were then collected randomly from 3 of the 4 sides of the trees and tested by PCR. In all five grids, asymptomatic but CLa+ trees were identified by the testing procedure. On average, approximately a similar number of asymptomatic but CLa+ trees were identified as there were symptomatic trees. A high concentration of asymptomatic but CLa-infected trees were often located in close proximity to the CLa symptomatic trees indicating that more recent infections may have occurred within a short distance of known CLa-infected trees. Based on these early results, it appears that with proper refinement in the sampling, extraction, and testing protocols, PCR testing will be useful in budwood testing, epidemiological studies, survey procedures, and other procedures requiring the testing of asymptomatic trees.

Last Modified: 11/26/2014