Simultaneous PCR Detection of the Two Major Bacterial Pathogens of Geranium

Glick, David L.; Coffey, Caroline M.; Sulzinski, Michael A.

doi:10.1046/j.1439-0434.2002.00716.x

Cited by 17 publications

(6 citation statements)

References 9 publications

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“…manihotis, a nested PCR (N-PCR) protocol was used to enhance the sensitivity and specificity of detection. In addition, multiplex PCR protocols have been developed in order to detect several pathogens (13) or genetically heterogeneous strains of a single pathovar (24,56) simultaneously.…”

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confidence: 99%

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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confidence: 99%

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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“…In addition, this study demonstrated the feasibility of multiplexing the GspDm PCR with primers amplifying either a general prokaryotic 16S rDNA target or a general plant mitochondrial ribosomal large subunit target. Multiplex PCR approaches with internal controls have effectively been used in diagnosis of various xanthomonads (Glick et al. , 2002; Berg et al.…”

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confidence: 99%

Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

et al. 2011

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The present study developed a pathovar-specific PCR for the detection of Xanthomonas campestris pv. musacearum (Xcm), the cause of banana xanthomonas wilt, by amplification of a 265-bp region of the gene encoding the general secretion pathway protein D (GspD). A distinct DNA fragment of the expected size was amplified from genomic DNA from all of 12 Xcm isolates tested and no amplification of DNA was observed from other xanthomonads or plant-associated bacteria, including the two closely related species Xanthomonas vasicola pv. holcicola and Xanthomonas axonopodis pv. vasculorum. The Xcm-specific PCR was successfully multiplexed with internal control primers targeting 16S rDNA for application on DNA from bacterial cultures and with primers targeting plant mitochondrial 26S rDNA for application on DNA extracted from plant material. Diagnostic discrimination of healthy and infected plants was subsequently demonstrated in tests on artificially inoculated screenhouse cultivars of banana and field bananas with and without symptoms sampled from different parts of Uganda. This study therefore demonstrated a robust and specific Xcm diagnostic tool with the added advantage of applying internal PCR controls for direct quality assessment of results.

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“…The best result for the triple primer pair multiplex-PCR designed in this study was achieved with optimization of primer concentration and annealing temperature which allows detection of either or both of two groups of the pathogen simultaneously. Multiplex-PCR has also been used by Fegan et al (1998) and Glick et al (2002) for concurrent detection of two phytopathogenic bacteria in other studies.…”

Section: Discussionmentioning

confidence: 99%

The Use of ARMS PCR in Detection and Identification of Xanthomonads Associated with Pistachio Dieback in Australia

et al. 2006

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Pistachio dieback occurs in the main pistachio growing areas of Australia. Xanthomonas strains belonging to the translucens group have been identified as the causal agent of the disease and two distinct groups, A and B, have been recognised within the pathogen population. In this study, specific primers for amplification of DNA of the pathogen were developed by sequencing the Internal Transcribed Spacer (ITS) region of rDNA from strains representing groups A and B, as well as from X. translucens isolated from wheat in Australia and one Xanthomonas translucens strain from orchard floor grasses. Primers were designed for amplification of DNA sequences specific to each group and a multiplex PCR test was developed that identified and differentiated strains of each group in a single PCR assay. To determine the specificity of the primers, PCR was carried out with DNA from 65 strains of the pistachio pathogen, 31 type and reference strains of Xanthomonas, and from 191 phytobacteria commonly found in and around pistachio orchards. In the multiplex PCR, a 331 bp fragment was amplified from all strains belonging to group A and a 120 bp fragment from all strains in group B. No PCR products were obtained from the other bacteria tested except for the type strain of X. translucens pv. cerealis, which has not been found in Australia. The assay was used to detect strains from both groups of the pathogen in pistachio plant material.

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Simultaneous PCR Detection of the Two Major Bacterial Pathogens of Geranium

Cited by 17 publications

References 9 publications

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

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

Multiplex PCR for specific and robust detection of Xanthomonas campestris pv. musacearum in pure culture and infected plant material

The Use of ARMS PCR in Detection and Identification of Xanthomonads Associated with Pistachio Dieback in Australia

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