A simple method for extracting DNA from rhododendron plants infected with Phytophthora spp. for use in PCR

Abstract

Among the numerous protocols that describe the extraction of DNA, those relating to the isolation of DNA from infected plants, are rare. This study describes a rapid and reliable method of extracting a high quality and quantity of DNA from rhododendron leaves artificially infected with Phytophthora cactorum, P. cambivora, P. cinnamomi, P. citrophthora, and P. plurivora. The use of the modified Doyle and Doyle protocol (1987) allowed us to obtain high quantity and quality DNA (18.26 µg from 100 mg of the fresh weight of infected leaves at the ratios of A 260/280 and A 260/230 -1.83 and 1.72, respectively), suitable for conventional polymerase chain reaction (PCR) and real-time PCR amplifications.

Protocol for detecting Curtobacterium flaccumfaciens pv. flaccumfaciens in the leaves of infected bean plants (Phaseolus vulgaris L.)

“…Most bacterial detection methods are designed for gram-negative bacteria, as most phytobacteria belong to this category (Salton, 1953), while protocols for isolating DNA of gram-positive bacteria from diseased plant tissues are scarce (Trzewik, Nowak, & Orlikowska, 2016). Hence, there is a need for methodologies that can be used to extract DNA from gram-positive bacteria to enable their rapid detection.…”

“…Likewise, although it speeds up the processing of samples and generates a high yield, the use of commercial DNA extraction kits increases the cost per sample (Junqueira & Carneiro, 2012). Moreover, such kits do not always produce a satisfactory quantity of DNA or high-quality DNA, which is an essential requirement for PCR analysis (Trzewik et al, 2016).…”

Rapid and Simple Method for the Extraction of Genomic DNA from Tobacco (Nicotiana tabacum L.) Seedlings

“…The presence of NaCl in the EB allows the removal of the polysaccharides and proteins that are bound to the DNA and increases the solubility of these two biomolecules (have a solubility similar to that of DNA) in isopropanol or ethanol, which reduces their co-precipitation with the DNA in the polar solvents [6,9,12]. On the other hand, EDTA, a chelating agent, traps divalent cations such as Mg 2+ and Ca 2+ and inhibits the action of nucleases that require these ions for their enzymatic activity [13,14]. SDS detergent is able to dissolve the lipids of cell membranes and thus releasing cytoplasmic and nuclear contents [15].…”

Especies de Phytophthora asociadas a agua de irrigación en el Valle de Culiacán

“…Así, Álvarez et al (2016) lograron aislar e identificar las especies P. hydropathica y P. drechsleri de distintos canales de agua de uso agrícola en Culiacán, Sinaloa, México. Por otro lado, comúnmente la identificación de géneros y especies de oomycetes se basaba en sus características morfológicas y culturales; sin embargo, fueron frecuentes identificaciones erróneas, debido a que algunas especies comparten dichas características; actualmente, esas prácticas son complementadas mediante técnicas biotecnológicas basadas en la extracción y secuenciación de ácidos nucleicos, lo cual permite la identificación de organismos de forma eficaz y concreta (Trzewik et al, 2016). Por lo anterior, el objetivo del presente estudio fue determinar la incidencia de especies del género Phytophthora en aguas superficiales de uso agrícola en Culiacán, Sinaloa y determinar su potencial patogénico en plantas cultivadas en México.…”