Novel point mutations in β-tubulin gene for carbendazim resistance maintaining nematode pathogenicity of Paecilomyces lilacinus

The application of fungicides is so critical, especially in greenhouses, to avoid fungal infections. Carbendazim, an inhibitor of tubulin biosynthesis, is the most widely known broad-spectrum benzimidazole fungicide. The application of carbendazim affects other beneficial fungi as well. Paecilomyces lilacinus 36-1 (Pl36-1) is a beneficial fungus used for biological control, and the most effective biocontrol agents of nematode eggs. The Pl36-1 is sensitive to carbendazim (0.3 mu g/ml). There is a general consensus that the mechanisms of resistance to carbendazim in the beta-tubulin gene have been analyzed in detail. However, no studies were conducted on P. lilacinus strains. In the present study, two carbendazim-resistant mutants of Pl36-1, P50 and P100, were obtained from UV exposure and tested. The beta-tubulin gene fragments were cloned and sequenced in the three strains, Pl36-1, P50 and P100. The resistance to carbendazim was developed when amino acid substitutions occurred at beta-tubulin gene positions of S145A, T185A and F200Y. The beta-tubulin gene was overexpressed in Pl36-1 strains. The beta-tubulin expression level of the overexpressed mutant (PL3), quantified by qRT-PCR, was increased 4-folds over its normal level in Pl36-1. In vitro, the PL3 was resistant to carbendazim with maintaining growth, sporulation and pathogenicity rates. Three-year field trial demonstrated that P100 and PL3 strains exhibited carbendazim resistance combined with high nematode reduction and yield improvement.