Anthracnose caused by Colletotrichum species is one of the most destructive fungal diseases of sorghum with annual yield losses of up to 100%. Although the resistance to anthracnose has been identified elsewhere, the usefulness of the resistance loci differs depending on the pathogen species and pathotypes. Accurate species identification of the disease-causing fungal pathogens is essential for developing and implementing suitable management strategies. The use of host resistance is the most effective strategy of anthracnose management and therefore identification of sources for resistance against unique pathogen pathotypes is fundamental. The aims of this study were to identify and characterize Colletotrichum species associated with sorghum anthracnose and screen sorghum germplasm for resistance to anthracnose. Symptomatic sorghum leaf samples were collected from smallholder farmers in lower eastern Kenya and used for the isolation, identification and characterization of Colletotrichum species using morpho-cultural and phylogenetic analyses with the sequences of the rDNA internal transcribed spacer (ITS) region. Pathogenicity tests of the seven fungal isolates showed that there were no significant differences in the pathogenicity on host plants. The fungal isolates were variable in cultural and morphological characters such as colony type and color, colony diameter, mycelia growth and hyaline. The phenotypic characters observed were useful in the identification of the genus Colletotrichum and not the species. Based on the sequence and phylogenetic analysis of ITS, Colletotrichum sublineola was revealed to be associated with anthracnose on sorghum. Germplasm screening for resistance to anthracnose showed differential reactions of sorghum genotypes to anthracnose under greenhouse and field conditions. The results revealed four resistant genotypes and ten susceptible genotypes against Colletotrichum sublineola. Significant (p ≤ 0.05) differences were observed in grain weight, grain yield, weight of 100 seeds and harvest index among the tested sorghum genotypes. The present study indicated that the Kenyan accessions could be an important source of resistance to anthracnose. The findings from this study provide a platform towards devising efficient disease control strategies and resistance breeding.
Ribonucleic acid (RNA) integrity, quality and quantity are critical in most plant molecular studies. Extracting high quality RNA from cassava leaves and other recalcitrant plant tissues are difficult due to the presence of polysaccharides, polyphenols and other secondary metabolites that often co-precipitate with the final RNA extract. This is an optimised a CTAB-based method that suitably extracts RNA from the polysaccharide-rich cassava leaves. The modifications were introduced into a version of the CTAB protocol as described by Gasic and colleagues [1]. The changes included an increased rate or use of Extraction Buffer (EB) for every gram ground leaf tissue (20 ml EB per 1 gram tissue), incubation of the Tissue-EB and Chloroform: Isoamyl alcohol (24:1) mixture at a lower water-bath temperature of 50°C and all centrifugation steps carried out at 4°C. In addition, the EB contained a higher concentration of soluble polyvinylpyrrolidone (PVP-K-30). The pH of sodium acetate was lowered to 5.2 and a final two-step high molarity (10M) Lithium Chloride (LiCl) precipitation was applied. Ethyl alcohol concentration was raised to 100%. The modified CTAB method produced RNA of high concentration (>1.0 μg), high A260:A280 and A260:A230 ratios
Study Protocol
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