Abstract
The most fungal DNA isolation protocols are designed to obtain high amounts of very pure DNA, requiring large fungal cultures and extraction procedures with many purification steps. Since the PCR does not require high purity DNA, the aim of this investigation was to evaluate three fast and simple fungal DNA isolation protocols for further use in Cercospora PCR based research. The purity and quantity of isolated DNAs were determined spectrophotometrically, electrophoretically and by PCR reaction with universal primers. The amounts of DNA evaluated on agarose gels, isolated by protocols A and C, did not correspond to the spectrophotometrical values, probably due to RNA impurities. In samples isolated by protocol B these impurities were not detected and the DNA concentrations were more similar. Neither protocol eliminated impurities such as carbohydrates and phenol. The average DNA yield of protocol A was 1.04 μg/μl, protocol B 0.88 μg/μl, and protocol C 0.55 μg/μl. The DNA quality most suitable for PCR analysis was obtained by protocol A, where amplification product with universal primers was detected in all DNA samples. The amplification product was detected in 87% of samples isolated by protocol C and in only 60% of samples isolated by protocol B. Although DNA obtained by protocol A had the highest yield and best quality, the isolation protocol C should be also recommended, for it does not require phenol, chlorophorm or liquid nitrogen.
Highlights
Cercospora leaf spot of sugar beet (Beta vulgaris L.) caused by Cercospora beticola Sacc. is the most important foliar disease of sugar beet in warm and humid agroecological conditions
Sugar beet leaves with typical symptoms of Cercospora leaf spot were collected from sugar CFFUđFMETJO7PKWPEJOB 4FSCJB"ĕFSNPOPTQPSJBM isolations from collected leaves, the obtained isolates were transferred on potato dextrose agar
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Summary
Cercospora leaf spot of sugar beet (Beta vulgaris L.) caused by Cercospora beticola Sacc. is the most important foliar disease of sugar beet in warm and humid agroecological conditions. The most fungal DNA isolation protocols require relatively large fungal cultures and long extraction procedures for obtaining high amounts of very pure DNA. These protocols are not necessary for experiments with polymerase chain reaction (PCR), since it does not require use of high purity DNA. Having in mind that PCR can amplify small amounts of low purity DNA, the aim of our research was to evaluate three fast and simple fungal DNA isolation protocols and determine which could be the most suitable for use in further investigation of Cercospora genome. Sugar beet leaves with typical symptoms of Cercospora leaf spot were collected from sugar CFFUđFMETJO7PKWPEJOB 4FSCJB"ĕFSNPOPTQPSJBM isolations from collected leaves, the obtained isolates were transferred on potato dextrose agar. Supported by Ministry of Education and Science of the Republic of 4FSCJB 1SPKFDU/P53
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