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.

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The purpose of this study was to assess the stability of mRNA and rRNA for evaluation of viability for Xanthomonas citri subsp. citri (Xcc). Total RNA from Xcc suspensions subjected to different stress treatments (high temperature or chemical treatment with sodium orthophenylphenate at different concentrations) was extracted at different time periods post-treatment (0, 3, 24 and 48 h) and analysed by quantitative real-time reverse transcription PCR (Q-RT-PCR). Primers were designed from selected fragments of rRNA and mRNA from genes involved in bacterial fitness, virulence or general metabolic mechanisms (gumD, rpfB, avrBs2 and gyrB). After stress treatment, only a 445-bp fragment from the gumD mRNA was detected in live Xcc cells specifically, whereas other RNA fragments, as well as DNA targets, were detected in both viable and nonviable cells. Statistical analyses demonstrated that the amount of some transcripts from genes involved in xanthan synthesis, pathogenicity factor regulation and DNA processing was significantly reduced after lethal treatments. The amplification of the 445-bp product from gumD mRNA was demonstrated to be useful for the detection of viable Xcc; the product was detected specifically from viable bacteria on leaf and citrus fruit surfaces and in citrus canker lesions. Instability of long RNA fragments can be used as a practical tool for the study of survival of citrus canker bacteria or for diagnostic purposes when the presence of viable bacteria needs to be confirmed.

Nucleic acid sequence based amplification (NASBA) is a method of amplifying RNA, for the detection of RNA viruses and human pathogenic bacteria. Recently, NASBA has also been employed for the detection of plant diseases caused by viruses and quarantine bacteria. A major citrus pathogen, Xanthomonas citri subsp. citri (Xcc), causal agent of citrus bacterial canker, is being studied in depth due to its economic importance, with recent focus concentrating on its viability and survival under different stress conditions and control treatments. In this work, a NASBA protocol using primers for gumD mRNA has been developed to assess the viability of this pathogen under different bacteriocidal treatments. This method is rapid, specific and sensitive, and is able to detect viable bacterial cells, using a hybridization device which allows the visualization of the results in only 30 min. The usefulness of the method has been confirmed with bacterial suspensions subjected to different heat treatments and to sodium orthophenylphenate.

Citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri (Xcc), is the most devastating of the citrus diseases worldwide. During our study, we found that Essential oils (EOs) of some citrus cultivars are effective on Xcc. Therefore, it prompted us to determine the plant metabolites responsible for the antibacterial properties. We obtained EOs from some locally cultivated citrus by using a Clevenger apparatus and their major constituents were identified by gas chromatography/mass spectrometry (GC-MS). The effect of Citrus aurantium, C. aurantifolia, Fortunella sp. EOs and their major constituents were evaluated against Xcc-KVXCC1 using a disk diffusion assay. Minimal inhibitory and bactericidal concentration of the EOs and their constituents were determined using the broth microdilution method. C. aurantium, C. aurantifolia Eos, and their major constituents including citral, linalool, citronellal, geraniol, α-terpineol, and linalyl acetate indicated antibacterial effects against Xcc. The C. aurantifolia EO and citral showed the highest antibacterial activity among the tested EOs and constituents with inhibition zones of 15 ± 0.33 mm and 16.67 ± 0.88 mm, respectively. Synergistic effects of the constituents were observed between α-terpineol-citral, citral-citronellal, citral-geraniol, and citronellal-geraniol by using a microdilution checkerboard assay. Transmission electron microscopy revealed that exposure of Xcc cells to citral caused cell wall damage and altered cytoplasmic density. We introduced C. aurantifolia and C. aurantium EOs, and their constituents citral, α-terpineol, citronellal, geraniol, and linalool as possible control agents for CBC.

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