Detection of Xanthomonas campestris pv. campestris through a real-time PCR assay targeting the Zur gene and comparison with detection targeting the hrpF gene

Eichmeier, Aleš; Peňázová, Eliška; Pokluda, Robert; Vicente, Joana G.

doi:10.1007/s10658-019-01820-0

Cited by 11 publications

(15 citation statements)

References 31 publications

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“…Primer pair DLH 120 and DLH 125 ( Table 1 ) designed by Berg et al [ 17 ] were used to amplify a 619 bp fragment of hrpF gene region. For specific amplification of the hrpF region, a GoTaq ® G2 Flexi kit (Promega, Madison, WI, USA) was used as described in Eichmeier et al [ 18 ]. The reaction mixture contained 10.5 µL of nuclease-free water (Ambion, Foster City, CA, USA), 4 µL 5× for polymerase, 0.7 µL 5× Green GoTaq ® G2 Flexi Buffer (Promega, Madison, WI, USA), 0.2 µL GoTaq ® G2 Flexi DNA polymerase (5 U·µL −1 ) (Promega, Madison, WI, USA), 1.2 µL MgCl 2 (25 mM) (Promega, Madison, WI, USA), 0.2 µL of dNTPs mix (10 mM) (MCLAB, San Francisco, SF, USA), 1 µL of each primer (10 µM) and 2 µL of template DNA, total volume 20.8 µL.…”

Section: Methodsmentioning

confidence: 99%

“…Another recently developed method for detection of Xcc in cabbage tissues and seeds is a nested real-time PCR assay targeting Zur ( Zinc uptake regulator ) gene [ 18 ]. A pair of outer primers amplify a 305 bp product in an initial reaction and an internal region is then amplified in real-time PCR using a second pair of primers.…”

Section: Introductionmentioning

confidence: 99%

“…In this paper we monitor the persistence of Xcc in a field soil using two different assays, the conventional PCR assay using hrpF primers [ 17 ] and the recently developed nested real-time PCR assay [ 18 ] for detection of Xcc DNA in a field soil under natural conditions for two years following a black rot infected cabbage crop in the Czech Republic.…”

Section: Introductionmentioning

confidence: 99%

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Persistence of Xanthomonas campestris pv. campestris in Field Soil in Central Europe

Gazdík

Magnus

Roberts³

et al. 2021

Microorganisms

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Xanthomonas campestris pv. campestris (Xcc) is a bacterium that causes black rot of crucifers. The greatest losses of brassica crop production usually result from seed-borne infection, but carry-over of inoculum in field soil may also be possible. The aim of this study was to monitor persistence of Xcc in field soil in central Europe using a conventional PCR assay with hrpF primers and a two-step nested real-time PCR assay using Zur primers. The work has demonstrated that nested real-time PCR can be used to improve the analytical sensitivity for detection of Xcc in soil compared to conventional PCR, and that Xcc may persist in soil for up to two years following an infected brassica crop in central European climatic conditions.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Persistence of Xanthomonas campestris pv. campestris in Field Soil in Central Europe

Gazdík

Magnus

Roberts³

et al. 2021

Microorganisms

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“…Approximately 0.2 g cabbage seeds were artificially inoculated with Xcc (isolate HRIW 3811, UK). The inoculation was performed by soaking seeds in a bacterial suspension containing approximately 10 8 CFU/mL for two hours at room temperature, followed by drying seeds on the filter paper in a ventilation hood [29], as proposed previously [30]. In total, 60 seedlings, 30 Xcc-inoculated and 30 non-inoculated plants, were grown under controlled conditions in the growth chamber.…”

Section: Plant Materials and Samplingmentioning

confidence: 99%

The Change of Bacterial Spectrum after Storage of X. campestris pv. campestris Inoculated Cabbage Heads (Brassica oleracea var. capitata L.)

et al. 2020

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Changes in the bacterial spectrum of cabbage heads after storage under commonly used storage conditions were examined in this study. Cabbage seeds (Brassica oleracea var. capitata L.) were artificially inoculated with X. campestris pv. campestris (Xcc), a serious pathogen of cruciferous plants causing black rot. Isolation of bacterial cultures from Xcc-inoculated and non-inoculated cabbage heads were carried out in two time points—at the day of harvest and after four months of storage. According to our previous research and literature reports, the most frequent genera of bacteria were chosen for PCR testing, i.e., Bacillus cereus group, Bacillus subtilis group, Pseudomonas sp., and X. campestris pv. campestris. A few of the obtained bacterial cultures were negative for the four above-mentioned species. In those, other bacteria were identified by 16S rRNA sequencing. In both Xcc-inoculated and non-inoculated cabbage heads, changes of the bacterial spectrum over time were observed. The severity of Xcc infection of heads increased after four months of storage. Bacillus species represented the most frequently occurring bacterial genus. The presence of the Bacillus subtilis group increased significantly after storage in non-inoculated cabbage heads. The minor part of the other genera identified by sequencing in the first sampling were not detected in the stored cabbage heads. This was associated with a possible antagonistic behavior of Pseudomonas sp. and Bacillus sp.

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“…Considering the great yield loss rate and a resulting need for fast detection and suppression of Xcc, many molecular, PCR-based techniques for fast and sensitive detection of this pathogen in seeds and plants have been developed. Pathovar-specific primers are mainly designed to target genes involved in pathogen virulence, such as zinc uptake regulator (Zur) and genes belonging to the hypersensitive reaction and pathogenicity (hrpF) cluster (Eichmeier et al, 2019;Rubel et al, 2019). The multilocus sequence typing (MLST) approach, based on PCR amplifications of various housekeeping genes is becoming one of the main techniques for more precise identification and characterization (Young et al, 2008;Almeida et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Allelic profile of Serbian Xanthomonas campestris pv. campestris isolates from cabbage

et al. 2020

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Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot disease of cabbage (Brassica oleracea var. capitata L.), is one of the most important bacteria which affect proper cabbage growth, leading to head weight and quality losses and thereby drastically reducing its marketing value. The pathogen is genetically diverse, which is evident from the presence of eleven races worldwide and more than thirty combinations of allelic profiles. Therefore, this study aimed to determine the allelic profiles of Serbian cabbage Xcc strains obtained in 2014. The analysis was done on three selected Xcc strains whose DNA was first amplified using polymerase chain reaction (PCR) with four housekeeping genes - P-XdnaK, fyuA, gyrB, and rpoD, then sequenced, and the obtained sequences were finally used to determine allelic profiles. Allelic profiles were determined by comparison with 33 Xcc strains obtained from different hosts and regions, whose allelic profiles had been determined previously. A non-redundant database (NRDB) from the pubMLST was used for allelic profile determination and Phyloviz software for constructing a minimum spanning tree. The obtained allelic profile of all Serbian Xcc cabbage strains was 1, 3, 1, 1 for the P-X-dnaK, fyuA, gyrB and rpoD genes, respectively. This profile is assigned as sequence type 2 (ST2) and it coincides with a Portuguese B. oleracea Xcc strain, CPBF 213, originating from B. oleracea var. costata. No connection between sequence type (ST) and the host was detected. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 451-03-68/2020- 14/200010 and Grant no. 451-03-68/2020-14/200053]

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Detection of Xanthomonas campestris pv. campestris through a real-time PCR assay targeting the Zur gene and comparison with detection targeting the hrpF gene

Cited by 11 publications

References 31 publications

Persistence of Xanthomonas campestris pv. campestris in Field Soil in Central Europe

Persistence of Xanthomonas campestris pv. campestris in Field Soil in Central Europe

The Change of Bacterial Spectrum after Storage of X. campestris pv. campestris Inoculated Cabbage Heads (Brassica oleracea var. capitata L.)

Allelic profile of Serbian Xanthomonas campestris pv. campestris isolates from cabbage

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