Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

Tebaldi, Nilvanira Donizete; Peters, Jeroen; Souza, Rosemeri Melo e; Chitarra, L. G.; Zouwen, Patricia van der; Bergervoet, J.H.W.; Wolf, Jan van der

doi:10.1590/s1982-56762010000400002

Cited by 14 publications

(7 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Surprisingly, in none of these studies, the NADS protocol was used in combination with taxa-or species-specific fluorescent labeling techniques to resolve the viability of individual species in mixed communities. In general, only few reports of viability studies dealing with species discrimination are available (12,25,26).…”

mentioning

confidence: 99%

A flow cytometric method for viability assessment of Staphylococcus aureus and Burkholderia cepacia in mixed culture

et al. 2012

View full text Add to dashboard Cite

Mixed bacterial communities are commonly encountered in microbial infections of humans. Knowledge on the composition of species and viability of each species in these communities allows for a detailed description of the complexity of interspecies dynamics and contributes to the assessment of the severity of infections. Several assays exist for quantification of specific species in mixed communities, including analysis of quantitative terminal restriction fragment length polymorphisms. While this method allows for species-specific cell enumeration, it cannot provide viability data. In this study, flow cytometry was applied to assess the viability of Staphylococcus aureus and Burkholderia cepacia in mixed culture by membrane integrity analysis using SYBR® Green I and propidium iodide staining. Both bacteria are relevant to pulmonary infections of cystic fibrosis patients. Fluorescence staining was optimized separately for each species in pure culture due to differences between species in cell wall structure and metabolic capabilities. To determine viability of species in mixed culture, a protocol was established as a compromise between optimum conditions determined before for pure cultures. This protocol allowed the detection of viable and dead cells of both species, exhibiting an intact and a permeabilized membrane, respectively. To discriminate between S. aureus and B. cepacia, the protocol was combined with Gram-specific fluorescent staining using wheat germ agglutinin. The established three-color staining method was successfully tested for viability determination of S. aureus and B. cepacia in mixed culture cultivations. In addition, growth of both species was monitored by quantitative terminal restriction fragment length polymorphisms. The obtained data revealed alterations in viability during cultivations for different growth phases and suggest interspecies effects in mixed culture. Overall, this method allows for rapid simultaneous Gram-differentiation and viability assessment of bacterial mixed cultures and is therefore suitable for the analysis of dynamics of mixed communities of medical, environmental, and biotechnological relevance.

show abstract

mentioning

confidence: 99%

A flow cytometric method for viability assessment of Staphylococcus aureus and Burkholderia cepacia in mixed culture

et al. 2012

View full text Add to dashboard Cite

show abstract

“…At the moment, the limited number of specific primers described for X. fragariae (29,31,45), for X. axonopodis pv. phaseoli by Audy et al (5) and other works that followed based on the same set of primers (19,26,30,37), and for X. fuscans subsp. fuscans (38) have been hampering the implementation of DNA-based detection protocols as trustworthy alternatives to isolation in pure culture.…”

Section: Vol 77 2011 Novel Markers For Identification Of Xanthomonamentioning

confidence: 99%

Identification of Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans with Novel Markers and Using a Dot Blot Platform Coupled with Automatic Data Analysis

Albuquerque

Caridade

Marçal

et al. 2011

Appl Environ Microbiol

View full text Add to dashboard Cite

Phytosanitary regulations and the provision of plant health certificates still rely mainly on long and laborious culture-based methods of diagnosis, which are frequently inconclusive. DNA-based methods of detection can circumvent many of the limitations of currently used screening methods, allowing a fast and accurate monitoring of samples. The genus Xanthomonas includes 13 phytopathogenic quarantine organisms for which improved methods of diagnosis are needed. In this work, we propose 21 new Xanthomonas-specific molecular markers, within loci coding for Xanthomonas-specific protein domains, useful for DNA-based methods of identification of xanthomonads. The specificity of these markers was assessed by a dot blot hybridization array using 23 non-Xanthomonas species, mostly soil dwelling and/or phytopathogens for the same host plants. In addition, the validation of these markers on 15 Xanthomonas spp. suggested species-specific hybridization patterns, which allowed discrimination among the different Xanthomonas species. Having in mind that DNAbased methods of diagnosis are particularly hampered for unsequenced species, namely, Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans, for which comparative genomics tools to search for DNA signatures are not yet applicable, emphasis was given to the selection of informative markers able to identify X. fragariae, X. axonopodis pv. phaseoli, and X. fuscans subsp. fuscans strains. In order to avoid inconsistencies due to operator-dependent interpretation of dot blot data, an image-processing algorithm was developed to analyze automatically the dot blot patterns. Ultimately, the proposed markers and the dot blot platform, coupled with automatic data analyses, have the potential to foster a thorough monitoring of phytopathogenic xanthomonads.Xanthomonas is a genus of Gammaproteobacteria that includes numerous phytopathogenic species, each characterized by a narrow host range. However, as a whole, the genus members are able to infect a broad range of plants, distributed over 124 monocotyledonous and 268 dicotyledonous plant species (15). The nomenclature of this complex genus is still under debate, and the taxonomic rank of many previously described pathovars has been revised (28,41,48). At the moment, the European and Mediterranean Plant Protection Organization (EPPO) recommends that 13 members of the genus Xanthomonas be considered quarantine pests. Therefore, reliable, fast, and technically and commercially accessible screening methods of detection and identification are needed to allow the survey of a large number of samples. This would ensure the phytosanitary certification of plants, prevent the spread of contaminated plant material, and facilitate the implementation of timely phytosanitation and quarantine measures (4).The current certified methods of bacterial detection rely mainly on culture-based approaches and plant bioassays (35). While these methods allow for a presumptive identification, they lack resolution of dete...

show abstract

“…, 2007), PCR (Audy et al. , 1994), and serology such as indirect immunofluorescence microscopy and ELISA (Wong, 1991) or immunostaining and flow cytometry (Tebaldi et al. , 2010).…”

Section: Introductionmentioning

confidence: 99%

“…Several different approaches have been used for detecting Xap in seeds, including a dome test (Venette et al, 1987), phage test (Kahveci & Maden, 1994), selective media (Mabagala, 1992;Remeeus & Sheppard, 2006;Sheppard et al, 2007), PCR (Audy et al, 1994), and serology such as indirect immunofluorescence microscopy and ELISA (Wong, 1991) or immunostaining and flow cytometry (Tebaldi et al, 2010). Only a few of the published methods have been accepted as routine diagnostic tools.…”

Section: Introductionmentioning

confidence: 99%

Comparison of extraction procedures for detection of Xanthomonas axonopodis pv. phaseoli in common bean seed

Munkvold

2012

Plant Pathology

View full text Add to dashboard Cite

Xanthomonas axonopodis pv. phaseoli (Xap) is an important seedborne pathogen of Phaseolus vulgaris. Accurate seed health testing methods are critical to protect seed quality and meet phytosanitary requirements. Currently employed selective media-based methods include several variations in extraction procedures. In order to optimize pathogen extraction from seeds, the influence of different extraction steps on the sensitivity of Xap detection was assessed. Seeds were inoculated by vacuum infiltration with Xap to achieve inoculum levels from 10 1 to 10 5 CFU per seed; one contaminated seed was mixed into 1000-seed subsamples of uncontaminated P. vulgaris seeds. Thirty subsamples of 1000 seeds were tested using each different extraction procedure. These included soaking whole seeds in sterilized saline phosphate buffer, either overnight at 4°C or for 3 h at room temperature, with or without vacuum extraction, and either with or without concentrating the seed extract by centrifuging. Seed extract dilutions were cultured on semiselective agar media MT and XCP1. The percentages of positive subsamples were compared to measure the effects of each extraction step on detection sensitivity. Vacuum extraction and centrifugation of seed extracts increased sensitivity; the highest sensitivity was obtained with the 3 h vacuum extraction followed by centrifugation. These results were confirmed with naturally infested seeds; Xap was detected in 48 of 70 samples using the 3 h vacuum extraction with centrifugation, whereas only 35 of 70 field samples tested positive using overnight soaking, a significant difference. The results suggest that these steps would be valuable modifications to the current method approved by the International Seed Testing Association (ISTA).

show abstract

Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

Cited by 14 publications

References 19 publications

A flow cytometric method for viability assessment of Staphylococcus aureus and Burkholderia cepacia in mixed culture

A flow cytometric method for viability assessment of Staphylococcus aureus and Burkholderia cepacia in mixed culture

Identification of Xanthomonas fragariae, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans with Novel Markers and Using a Dot Blot Platform Coupled with Automatic Data Analysis

Comparison of extraction procedures for detection of Xanthomonas axonopodis pv. phaseoli in common bean seed

Contact Info

Product

Resources

About