Evaluation of the production of antifungal metabolites against Colletotrichum gloeosporioides in Streptomyces 5.1 by random mutagenesis

Streptomyces 5.1 is a bacterium isolated from rice soils in the south of the Tolima department (Colombia). This microorganism is characterized by its antagonistic activity against rubber tree phytopathogens like Colletotrichum gloeosporioides, the causal agent of leaf anthracnose. The antifungal activity of this Streptomyces isolate has been associated with secondary metabolites production. However, the identity of those metabolites is unknown because its purification and identification have not been possible through classic chemical studies. Therefore, aiming to contribute in the study of the secondary metabolites produced by 5.1 from a molecular approach, this research seeks to identify -preliminarily- the genomic fingerprint changes associated with the production of antifungal secondary metabolites produced by Streptomyces 5.1 through the evaluation of a mutant library of 5.1 obtained by random mutagenesis using controlled ultraviolet light exposure. The antifungal activity of obtained mutants was evaluated using Colletotrichum gloeosporioides (C1) fungus as a biosensor, isolated by the Biotechnology Institute of Universidad Nacional de Colombia. In this way, the library of mutants of 5.1, initially formed by 300 isolations, was classified into two phenotypic groups of interest: enhanced mutants (1 isolate) and null mutants (11 isolates) of secondary metabolites. The genomic changes in both groups were analyzed by obtaining the genomic profile of the isolates using Repetitive Extragenic Palindromic (Rep-PCR). The obtained profiles evidenced the presence of one additional band in the enhanced mutant, and the absence of a specific band in the non-producing mutants, both in comparison with the original strain. These bands are proposed for a future sequencing study which will define their role in the production process of metabolites with antifungal activity in Streptomyces 5.1.

RAPD based DNA markers linked to anthracnose disease resistance in Sorghum bicolor ( L.) Moench.

Anthracnose caused by Colletotrichum graminicola is one of the major diseases of sorghum. The locus for disease resistance in sorghum [Sorghum biocolor (L.) Moench] accession G73 was found to segregate as a simple recessive trait in a cross to susceptible cultivar HC136. In order to identify molecular markers linked to the locus for disease resistance, random amplified polymorphic DNA (RAPD) analysis was coupled with bulk segregant analysis. DNA from the parental cultivars and the bulks were, screened by PCR amplification with 114 RAPD primers. Three RAPD primers amplified a sequence that consegregated with the recessive resistance allele, while another three amplified a band linked to the susceptible allele. The six disease linked markers were screened with individual resistant and susceptible genotypes to observe degree of linkage of identified RAPD markers with the gene for resistance. Two primer sequences (OPI 16 and OPD 12) were found to be closely linked to the locus for disease resistance.