A Pipeline For The Genetic Improvement Of A Biological Control Agent Enhances Its Potential For Controlling Soil-Borne Plant Pathogens

Use of biopesticides results in a reduction of the worldwide dependence on chemical pesticides, ending in a more sustainable crop protection. For this reason, biological control industry invests in continued research for more effective biocontrol agents (BCAs) using basically two strategies: isolation of new strains from nature or conventional genetic improvement. However, both are highly onerous and time-consuming. Here we show a pipeline for the genetic improvement of fungal BCAs, based on genome shuffling, the most recent and promising non-recombinant DNA technology for the rapid phenotype improvement of microbial strains. The method consisted of the construction of a parent library using mutagenic agents, followed by genome shuffling and high throughput screening. Sarocladium oryzae BRM 6461, a known cerulenin antifungal producer strain and high biocontrol potential, served as the model fungal BCA. The pipeline aimed to select mutant enhanced strains at least one of these three desirable characteristics: antagonism, UV-B irradiation tolerance and high temperature tolerance. The experiments were conducted in laboratory and green house. After four cycles of genome shuffling we selected the superior S. oryzae GS4-03 strain, showing cerulenin production of 203.3 +/- 1.4 mu g/mL, 42% higher than the wild-type strain BRM 6461. The GS4-03 strain exhibited good genetic stability for at least five successive subcultivation assays. Antagonism assay showed an increased micelial inhibition of Rhizoctonia solani and Sclerotinia sclerotiorum when using GS4-03 strain compared to the BRM 6461 strain. The bioassays, showed that the superior strain GS4-03 have an increased ability to control root rot (23.4%) and white mold (8.1 times) disease compared to the wild type strain BRM 6461. Assessment of virulence of the superior strain GS4-03 against rice plants showed that improvement procedures did not change the original strain behavior in this subject. Hydrolytic activity of enzymes related to the biocontrol action as chitinase, protease and beta-1,3-glucanase did not present statistical difference between GS4-03 and BRM 6461 strains. Results of dual assay culture between GS4-03 and BRM 6461 strains showed that both have different genetic backgrounds, although RAPD and ITS-rDNA tests were not fully efficient to distinguish genetically the strains. Moreover, mutations resulted in S. oryzae strains more tolerant to UV-B irradiation, but with low genetic stability. Our results report for the first time an efficient pipeline for the genetic improvement of fungal BCAs based on genome shuffling.