A single loop-mediated isothermal amplification (LAMP) assay was developed for specific detection of both pathogens that cause bacterial blight in common bean, Xanthomonas phaseoli pv. phaseoli (Xpp) and Xanthomonas citri pv. fuscans (Xcf). The objective was to provide a simple, easy-to-use, specific, and sensitive method to investigate the presence of one or both pathogens in plant material and seeds for routine diagnosis. The detection limits for both pathogens were 10 CFU/ml for cell suspensions and 1 fg of DNA, whereas in conventional PCR, the primers detected up to 105 CFU/ml and 1 ng of DNA. Specificity was confirmed by testing DNA from bean leaves, other Xanthomonas species, common fungal and bacterial bean pathogens, and bacteria from the leaf microbiota. The method was tested with bean leaves inoculated with Xpp, and the pathogen could be detected from 4 h up to 15 days postinoculation, even before disease symptoms were visible. When the method was applied to bacterium detection (Xpp or Xcf) in seed lots from infected plants, the bacterium detection rate was 100% (24 of 24). The pathogens were detected in seeds incubated for just 1 h in saline solution (0.85%), reducing the time needed for bacterium detection. The LAMP assay could be useful as a tool in bean bacterial blight management. Rapid and sensitive detection of bacteria in bean seed lots would reduce the risks of planting highly contaminated seeds in environments favorable to blight multiplication.
A molecular tool was adapted for practical identification of the world's first transgenic event (Embrapa 5.1) in common bean (Phaseolus vulgaris L.). Based on the technology of RNA interference and on the transformation of plants via the Biobalistic method, a transgenic bean was created with effective resistance to Bean golden mosaic virus, its principal virus. To support the monitoring of this technology, the development of new cultivars, and the process of producing, benefiting, and distributing of seeds and grains, a molecular detection tool based on loop‐mediated isothermal amplification (LAMP) was developed. This tool can facilitating diagnosis, making it fast, specific, low‐cost, and easily executed. Based on the gene Ahas (GenBank M88686) of the Embrapa 5.1 event, sets of initiators were planned (forward outer primer–backward outer primer and forward inner primer–backward inner primer) that are characteristic of the LAMP method. These were evaluated for their sensitivity and specificity in detecting their target. The LAMP method allows direct visual interpretation. The Neutral Red (NR) pH indicator was adopted to facilitate molecular diagnosis in moderately well equipped environments, thus reducing the risk of contamination and the need for skilled labor. This agility in diagnosis occurs because several phases commonly used in molecular techniques, such as polymerase chain reaction, can be excluded. The LAMP method, combined with a rapid DNA extraction method (modified NaOH) and the addition of NR, provides technological support for addressing issues of biosafety, traceability, and identification of the Embrapa 5.1 event in beans in the field and in industry.
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