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United States Department of Agriculture

Agricultural Research Service

Title: Using Pcr for Detection and Quantification of Xanthomonas Axonopodis Pv Citri in Wind Driven Splash

Authors
item Bock, C. H. - UNIV. OF FLORIDA
item Parker, P. E. - USDA-APHIS
item Gottwald, Timothy
item Mavrodieva, V. A. - USDA-APHIS
item Levy, L. - USDA-APHIS

Submitted to: Meeting Abstract
Publication Type: Proceedings
Publication Acceptance Date: September 30, 2005
Publication Date: November 7, 2005
Citation: Bock, C., Parker, P., Gottwald, T.R., Mavrodieva, V., Levy, L. 2005. Using pcr for detection and quantification of xanthomonas axonopodis pv citri in wind driven splash. Second International Citrus Canker and Huanglongbing Workshop, Orlando, FL, November 7-11, 2005. C23, p.33.

Interpretive Summary: An eradication program has been developed to remove citrus canker (caused by Xanthomonas axonopodis pv citri, Xac) from Florida. The eradication program is based on knowledge of the disease epidemiology. Additional information on the spread of Xac bacteria in wind blown splash will provide further insights into pathogen dynamics, and ways of detecting and quantifying epidemiologically significant (living) bacteria of Xac. The objective here was to assess a molecular method (PCR) and dilution plating for detecting and quantifying the Xac bacteria in wind-driven splash. Samples were collected down wind from canker-infected trees and quantified by the two systems. Detection agreed between the two systems on 75% of the samples tested. However, quantification suggested that at low concentrations PCR was not reliable as there were insufficient bacteria, and at higher concentrations despite good agreement on detection, quantiofication by PCR was unreliable. The results confirm previous observations on the sensitivity of real-time PCR for detecting citrus canker bacteria (down to 103/ml). For epidemiological studies a more sensitive PCR protocol is desirable, particularly one that can quantify epidemiologically active bacteria. Development of such a system is currently underway.

Technical Abstract: An eradication program has been developed to remove citrus canker (caused by Xanthomonas axonopodis pv citri, Xac) from Florida (1,4). The eradication program is based on knowledge of the disease epidemiology. Additional information on the spread of Xac bacteria in wind blown splash will provide further insights into pathogen dynamics, and ways of detecting and quantifying epidemiologically significant (living) bacteria of Xac. The objective here was to assess a molecular method (PCR) and dilution plating for detecting and quantifying the pathogen in wind-driven splash. Samples of wind-driven splash (a total of 314) were collected down wind from canker-infected trees. The systems used to simulate rain splash, and the sampling procedures, have been described previously (3). Control samples comprised water and known positive samples of Xac. The samples were subject to dilution plating on nutrient agar and the colonies counted after 5 days and compared to that estimated using an established citrus canker SYBR Green real-time PCR method for detection from symptomatic tissues using universal primers VM3+4 (2). Detection agreed between the two systems on 75% of the samples tested (235 of 314 samples). However, false negatives using PCR were found in 75 (24%) of samples, all of which contained 3 to 863 bacteria/per ml as detected by dilution plating, but only 4 samples (1%) were positive using PCR, but not by dilution plating. However, at PCR scores of 0.5 and above, there was excellent agreement on detection of the bacteria between the two methods. There was a positive linear relationship between the real-time PCR score and log number of living bacteria collected/ml (Table 1), although a large range of living bacteria/ml existed within each PCR category, implying some discrepancy in quantification of bacteria between the two methods. No modification was made to the PCR protocol and no splash-sample concentrating procedures were performed. The results confirm previous observations on the sensitivity of real-time PCR for detecting citrus canker bacteria (down to 103/ml, 2). Differences existed in quantification of bacteria between the two methods. For epidemiological studies a more sensitive PCR protocol is desirable, particularly one that can quantify epidemiologically active bacteria. Development of such a system is currently underway.

Last Modified: 12/20/2014
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