Biological Control Agent for Rice Weeds from Protoplast Fusion betweenCurvularia lunataandHelminthosporium gramineum

Abstract

The advancement of biological weed control is limited by the slow development of effective, broader-spectrum biological control agents. Protoplast fusion was carried out between theHelminthosporium gramineumsubsp.echinochloae(HGE) strain, HM1, andCurvularia lunata(CL) to breed new strains with improved biocontrol efficiency. The HM1 strain was derived from HGE by ultraviolet (UV) treatment. Conditions for protoplast fusion were optimized, including lytic enzyme mixtures and incubation time. The efficacy of lytic enzyme mixtures on cell wall digestion was also compared. The most effective lytic enzyme mixture for CL was 2% lywallzyme plus 2% snailase and for HM1, 2% cellulase plus 2% snailase. The optimum incubation time was 16 h for CL and 24 h for HM1. All fusant strains exhibited similar morphological and conidial properties to the HM1 parent. A total of 1,360 fusant strains were produced, 136 of which were randomly selected for characterization. Seven fusant strains showed improved spore productivity and four fusant strains had increased production of the phytotoxin ophiobolin A, compared with HM1. Random amplified polymorphic DNA (RAPD) analysis of the fusant strains showed that the seven phytoactive fusant strains had a high similarity index (95 to 99%) to the parent HM1, but low similarity (22 to 26%) to the parent CL. Ethyl acetate extract of the culture broth and mycelia effectively controlled major weeds in rice production.