Diagnosis of Xanthomonas axonopodis pv. citri, causal agent of citrus canker, in commercial fruits by isolation and PCR-based methods

Abstract

Aims: To show the results of the detection of an EU quarantine organism, Xanthomonas axonopodis pv. citri (Xac), in citrus fruits imported from countries where this bacterium is present, using an integrated approach that includes isolation, pathogenicity assays and molecular techniques. Methods and Results: Citrus fruits with canker‐like symptoms, exported to Spain from South American countries were analysed by several methods. Bacterial isolation, three conventional polymerase chain reaction (PCR) protocols, and real‐time PCR with SYBR Green or a TaqMan probe, were compared. Canker‐like lesions were disrupted in PBS buffer, and the extract used for bacterial isolation and DNA extraction followed by PCR amplification. Canker lesions, identified by PCR, showed viable bacteria in eleven of fifteen fruit samples. In 16 out of 130 lesions analysed from these samples, Xac was isolated, and pathogenicity on grapefruit leaves confirmed. By real‐time PCR, using SYBR green or a Taqman probe, Xac was detected in 58 and 80 lesions respectively. By conventional PCR the bacterium was detected in 39–52 lesions depending on the protocol employed. Conclusions: An integrated approach for reliable detection of Xac in lesions of fruit samples, employing several techniques and with real‐time PCR using a TaqMan probe as the fastest and most sensitive screening method, has been established and validated and is proposed as a useful tool for the analysis of Xac on fresh fruits. Significance and Impact of the Study: This work faces up to the real threat of the importation of citrus fruits that can harbour quarantine bacteria and will be useful in diagnostic laboratories for the analysis of commercial fresh fruits from countries where citrus canker is present.

Comparison between Serological and Molecular Detection of Citrus Canker Pathogen (<i>Xanthomonas axonopodis</i> pv. <i>citri</i>)

“…The results showed that, canker pathogen were detected by these specific primers on the first day of inoculation while ELISA was unable (Table 4). The amplification assay using specific primers have been reported to detect at low pathogen population (10 2~1 0 3 CFU/mL) (Cubero et al, 2001, Cubero and Graham, 2002, Coletla-Filho 2006, Park et al, 2006, Golmohammadi et al, 2007, de Leon et al, 2008. The sensitivity of PCR amplification of this study showed very high sensitivity at 10 3 CFU/mL or 1 cell in the PCR reaction (Figure 3).…”

Statement in response to the comments submitted by the US phytosanitary authorities on a previous EFSA opinion (EFSA Journal 2011;9(12):2461) regarding the export of Florida citrus fruit to the EU

“…The amount of Taq polymerase utilised in the amplification protocol (0.5 U Ampli Taq per reaction mixture) was half of that used by Golmohammadi et al (2007) and this could also have played a role in obtaining a lower sensitivity in the PCR reactions. At that time (i.e.…”

“…This PCR (primer pair 2-3) produced a sensitivity of 10 3 cfu mL -1 (Hartung et al 1993) to 10 2 cfu mL -1 from pure cultures (Golmohammadi et al, 2007). To obtain similar sensitivity in citrus fruit, Golmohammadi et al (2007) indicated that a DNA extraction was required before amplification but no indication of this step is given by Shiotani et al (2009).…”

“…citri was identified even though these fruit received a post-harvest treatment (as indicated by their certificates). According to Golmohammadi et al(2007), 16 bacterial isolates from 11 different samples were found to contain viable Xanthomonas citri subsp. citri.…”

“…2006), real-time PCR procedures had already been developed to detect Xanthomonas citri subsp. citri and were shown to be more sensitive (Mavrodieva et al, 2004;Golmohammadi et al, 2007). Those procedures were not used in this study even though the detection of the pathogen was the key point in answering the objectives.‖ Response from Dr Shiotani: -We used the PCR procedure developed by Hartung et al (1993).…”