Development of PCR-Based Assays for Detecting <i>Xanthomonas campestris</i> pv. <i>carotae</i>, the Carrot Bacterial Leaf Blight Pathogen, from Different Substrates

Meng, Xianglong; Umesh, Kodira C.; Davis, R. M.; Gilbertson, R. L.

doi:10.1094/pdis.2004.88.11.1226

Cited by 32 publications

(48 citation statements)

References 18 publications

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“…Due to the high genetic diversity of this pathogen (11,12,33,41), two PCR-based sequence-characterized amplified regions (SCARs) identified in RAPD and AFLP analyses were required to detect all isolates of this pathovar. The RAPD technique has been widely used for SCAR development to obtain PCR diagnostic tools for bacterial plant pathogens (23,38,54). Examples of SCARs derived from AFLP analysis are less common in molecular phytodiagnostics (10,36), whereas this technique is capable of generating a large number of markers over a relatively short time without any prior sequence knowledge (48).…”

Section: Discussionmentioning

confidence: 99%

“…Alternatively, it is possible that our PCR-based method detects free DNA from nonviable bacteria, as demonstrated for PCR detection of X. campestris pv. carotae (23), and thus results in false-positive responses. Nevertheless, detection of free target DNA in a seed lot represents a history of contamination, which indicates that there is a need for further analysis.…”

Section: Discussionmentioning

confidence: 99%

“…PCR-based techniques have been reported to be highly efficient for detecting and identifying xanthomonads from seeds, such as X. campestris pv. carotae (23), X. oryzae pv. oryzae (46), Xanthomonas pathogens that cause cereal leaf streak (21), and X. axonopodis pv.…”

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confidence: 99%

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Multiplex Nested PCR for Detection ofXanthomonas axonopodispv. allii from Onion Seeds

Robène-Soustrade

Legrand

Gagnevin

et al. 2010

Appl Environ Microbiol

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Bacterial blight of onion (BBO) is an emerging disease that is present in many onion-producing areas. The causal agent, Xanthomonas axonopodis pv. allii, is seed transmitted. A reliable and sensitive diagnostic tool for testing seed health is needed. Detection of X. axonopodis pv. allii was achieved using a multiplex nested PCR assay developed using two randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) sequences corresponding to pilus assembly genes (pilW and pilX) and the avrRxv gene, respectively. The multiplex nested PCR was used with a large collection of X. axonopodis pv. allii strains pathogenic to onion and/or other Allium species isolated in different regions of the world. The internal primers used in the multiplex PCR assay directed amplification for all 86 X. axonopodis pv. allii strains tested, resulting in a 401-bp amplicon, a 444-to 447-bp amplicon, or both amplicons, depending on the strain. No amplification was obtained for 41 unrelated phytopathogenic bacteria and for 14 saprophytic bacteria commonly isolated from onion leaves and seeds. Most Xanthomonas strains also did not produce amplicons, except for nine strains classified in X. axonopodis genetic subgroup 9.1 or 9.2 and not pathogenic to onion. Nevertheless, sequence signatures distinguished most of these strains from X. axonopodis pv. allii. The assay detected X. axonopodis pv. allii in seed lots with contamination levels of 5 ؋ 10 2 CFU g ؊1 or higher. The sensitivity threshold of the multiplex nested PCR assay was found to be 1 infected seed in 27,340 seeds. This PCR-based assay should be useful for certifying that commercial seed lots are free of this important seed-borne pathogen.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Multiplex Nested PCR for Detection ofXanthomonas axonopodispv. allii from Onion Seeds

Robène-Soustrade

Legrand

Gagnevin

et al. 2010

Appl Environ Microbiol

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“…Therefore, planting seed free of bacterial contamination is an important disease management strategy. Several molecular and immunological methods, such as PCR (3,4,5,13,17,30), enzymelinked immunosorbent assay (1,35), and flow cytometry (7), were developed to test seeds for the presence of these pathogens. However, these methods cannot directly distinguish dead and viable cells and may vary in specificity and sensitivity according to seed and other sample types (plant tissue, soil, and water).…”

mentioning

confidence: 99%

Combination of Chromogenic Differential Medium and estA -Specific PCR for Isolation and Detection of Phytopathogenic Xanthomonas spp

Lee

Sung

Liu

et al. 2009

Appl Environ Microbiol

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A xanthomonad differential medium (designated Xan-D medium) was developed, on which streaks and colonies of xanthomonads, including 13 species of the genus Xanthomonas, turned wet-shining yellow-green and were surrounded with a smaller milky zone and a bigger clear zone in 3 to 4 days. The characteristics could easily be differentiated from those of yellow nonxanthomonads and other bacteria. The mechanism of color change and formation of a milky zone on the medium are mainly due to the Tween 80 hydrolytic capacity of xanthomonads. The gene, estA, responsible for Tween 80 hydrolysis was cloned and expressed in Escherichia coli, which acquired a capacity to hydrolyze Tween 80 and could turn green and form a milky zone on the Xan-D medium. The nucleotide sequence of estA is highly conserved in the xanthomonads, and the sequence was used to design a specific PCR primer set. The PCR amplification using the primer set amplified a 777-bp specific DNA fragment for all xanthomonad strains tested. The Xan-D medium was used to isolate and differentiate Xanthomonas campestris pv. campestris from naturally infected cabbages with black rot symptoms for a rapid diagnosis. All isolated X. campestris pv. campestris strains developed characteristic colonies and were positive in the PCR with the estA primer set. The Xan-D medium was further amended with antibiotics and successfully used for the detection of viable X. campestris pv. campestris cells from plant seeds. Although some yellow nonxanthomonads and other saprophytic bacteria from plant seeds could still grow on the medium, they did not interfere with the color development of X. campestris pv. campestris. However, Stenotrophomonas maltophilia, which is closely related to xanthomonads, existing in a seed lot could also develop yellow-green color but had different colony morphology and was negative in the PCR with the estA primer set. Accordingly, the combination of the Xan-D medium with the estA-specific PCR is a useful and reliable method for the isolation and detection of viable xanthomonad cells from plant materials.

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“…PCR along with primer pairs developed from sequences of cloned random amplifi ed polymorphic DNA (RAPD) fragments could be able to detect X. campestris pv. carotae (Xcc), causing bacterial leaf blight disease in carrot seeds, leaves, and stem tissues (Meng et al 2004 ).…”

Section: Molecular Techniquementioning

confidence: 99%

Diagnostics of Seed-Borne Plant Pathogens for Safe Introduction and Healthy Conservation of Plant Genetic Resources

et al. 2016

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Development of PCR-Based Assays for Detecting Xanthomonas campestris pv. carotae, the Carrot Bacterial Leaf Blight Pathogen, from Different Substrates

Cited by 32 publications

References 18 publications

Multiplex Nested PCR for Detection ofXanthomonas axonopodispv. allii from Onion Seeds

Multiplex Nested PCR for Detection ofXanthomonas axonopodispv. allii from Onion Seeds

Combination of Chromogenic Differential Medium and estA -Specific PCR for Isolation and Detection of Phytopathogenic Xanthomonas spp

Diagnostics of Seed-Borne Plant Pathogens for Safe Introduction and Healthy Conservation of Plant Genetic Resources

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