Establishment of polyethylene-glycol-mediated protoplast transformation for Lecanicillium lecanii and development of virulence-enhanced strains against Aphis gossypii.

BACKGROUND: Lecanicillium lecanii has been developed as a biopesticide and used in biological control of several agricultural insects. To improve fungal virulence, an optimised polyethylene glycol (PEG)-mediated protoplast transformation system was established for L. lecanii. Pr1A-like cuticle-degrading protease gene (Cdep1) from Beauveria bassiana was transferred into L. lecanii, and its resulting activity against Aphis gossypii was assessed. RESULTS: The optimised protoplast generation yielded 2.5 × 10(8) protoplasts g(-1) wet mycelium of fungi, and gave nearly 98% viability and 80% regeneration on plates. Protease activities were increased about fivefold in transformants expressing CDEP1. The median lethal concentration (LC50 ) for transformants expressing CDEP1 was twofold lower than that for the wild type (WT). The median survival time (LT50 ) for transformants expressing CDEP1 was also 14.2% shorter than that for WT, though no significant difference. There were no significant differences in conidial germination as colony growth and conidial yield on plates between transformants expressing CDEP1 and WT. The transformants expressing CDEP1 grew significantly quicker than WT in insects. The transformants expressing CDEP1 were lower in conidial yields on insect cadavers, but insignificantly different from WT. CONCLUSION: The PEG-mediated protoplast transformation system was effective for L. lecanii, and the expression of CDEP1 significantly enhanced fungal virulence against cotton aphids. © 2016 Society of Chemical Industry.

Persistence and Viability of Lecanicillium lecanii in Chinese Agricultural Soil.

The entomopathogenic fungus L. lecanii has been developed as biopesticides and used widely for biological control of several insects in agricultural practice. Due to the lack of isolation/count methods for L. lecanii in soil, the persistence of this fungus in soil appears to have attracted no attention. A selective medium and count method for L. lecanii in soil based on cetyl trimethyl ammonium bromide (CTAB) was developed, and then the persistence and viability of this fungus in soil were investigated under field conditions between 2012 and 2014. The results showed that the rate of recovery for L. lecanii in soil on the selective CTAB medium was satisfactory. The minimum CFUs for L. lecanii on the selective medium (0.5 g/L CTAB) was about 102 conidia/g soil. The L. lecanii density in soil declined quickly in the first month after inoculation with fungal conidia, kept stable for 6 to 10 months, and then decreased gradually until undetectable. L. lecanii could persist for at least 14 months in the agricultural soil of northern China. The colony growth, conidia yield and germination rate on plates, as well as the median lethal concentration or times (LC50 or LT50) to aphids, mycelium growth in aphids and sporulation on aphids of L. lecanii did not change significantly during the persistence in soil. In general, the count method developed here was a very useful tool for monitoring the dynamics of natural or introduced L. lecanii populations in soil, and the data on the persistence of L. lecanii in soil reported here were helpful for biological control and environmental risk assessment.