Ming Xie

Agrobacterium tumefaciens-mediated transformation in the entomopathogenic fungus Lecanicillium lecanii and development of benzimidazole fungicide resistant strains.

Lecanicillium lecanii has been used in the biological control of several insects in agricultural practice. Since the gene manipulation tools for this entomopathogenic fungus have not been sufficiently developed, Agrobacterium tumefaciens-mediated transformation (ATMT) in L. lecanii was investigated in this study, using the wild-type isolate FZ9906 as a progenitor strain and the hygromycin B resistance (hph) gene as a selection marker. Furthermore, a field carbendazim-resistant (mrt) gene from Botrytis cinerea was expressed in L. lecanii FZ9906 via the ATMT system. The results revealed that the frequency of transformation surpassed 25transformants/10(6) conidia, most of the putative transformants contained a single copy of T-DNA, and the T-DNA inserts were stably inherited after five generations. All putative transformants had indistinguishable biological characteristics relative to the wild-type strain, excepting two transformants with altered growth habits or virulence. Moreover, the resistance of the putative transformants to carbendazim (MBC) was improved, and the highest one was 380-fold higher than the wild-type strain. In conclusion, ATMT is an effective and suitable system for L. lecanii transformation, and will be a useful tool for the basic and application research of gene functions and gene modifications of this strain.

Expression of a scorpion toxin gene BmKit enhances the virulence of Lecanicillium lecanii against aphids

Lecanicillium lecanii has been developed as biopesticides and widely used in the biological control of several insects in agricultural practice. However, the poor efficacy has blocked its application. The genetic manipulation has been proved as a useful tool to improve the virulence of entomopathogenic fungus. In this study, a scorpion toxin gene (BmKit) from Buthus martensi was cloned and transferred into L. lecanii and then its resulting activity against cotton aphids (Aphis gossypii) was assessed. The results showed that the engineered strain BmKit-12 grew significantly quicker than WT in host insects. The median lethal concentration (LC50) for BmKit-12 was 7.1-fold lower than that for WT, and the median survival time (LT50) for BmKit-12 was reduced by 26.5xa0% compared with that for WT. Although the conidial yield was reduced by 31.4xa0% on insect cadavers, the mycelia growth, sporulation, and conidia germination of BmKit-12 on plates were not significantly different from WT. Considering the lower amount of mycelia for BmKit-12 in host insects in late growth stage (fungal sporulation stage), we think the expression of BmKit may not affect the capability of sporulation. In conclusion, the expression of BmKit gene can significantly enhance the pathogenicity of L. lecanii against cotton aphids in a relatively cost-effective way.

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

BACKGROUNDnLecanicillium 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.nnnRESULTSnThe 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.nnnCONCLUSIONnThe PEG-mediated protoplast transformation system was effective for L. lecanii, and the expression of CDEP1 significantly enhanced fungal virulence against cotton aphids.

Genetic improvement of the nematicidal fungus Lecanicillium attenuatum against Heterodera glycines by expression of the Beauveria bassiana Cdep1 protease gene.

Lecanicillium attenuatum is an important nematophagous fungus with potential as a biopesticide against plant-parasitic nematodes. The Pr1A-like cuticle-degrading protease (Cdep1) gene originating from the entomopathogenic fungus Beauveria bassiana was transformed into the nematophagous fungus L. attenuatum using a polyethylene-glycol mediated protoplast-based transformation system. Protease activity was increased 0.64- to 1.63-fold 2-10d after growth in the transformed L. attenuatum. Inhibition of egg-hatching and J2 motility of soybean cyst nematodes (Heterodera glycines) by cell-free fungal culture filtrates were enhanced by 17-76% 2-14d and 43-152% 1-13d after incubation, respectively.

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.

Effects of a NTG-based chemical mutagenesis on the propamocarb-tolerance of the nematophagous fungus Lecanicillium attenuatum

Lecanicillium attenuatum is an important nematophagous fungus with potential as a biopesticide for control of plant-pathogenic nematodes. However, relatively low fungicide-tolerance limits its application in the field. To improve the propamocarb-tolerance of L. attenuatum, a NTG-based mutagenesis system was established. Among different combinations of NTG concentration and treatment time in the first-round NTG treatment, the treatment of 1.0mg/ml NTG for 60min gave a proper conidial lethality rate of 84.6% and the highest positive mutation rate of 7.7%, and then produced the highest propamocarb-tolerant mutant LA-C-R1-T4-M whose EC50 value reached to 1050.0μg/ml. The positive mutation range was 105.1% in the first-round NTG treatment. Multiple-round NTG treatment was further employed to enhance the propamocarb tolerance of L. attenuatum. The positive mutation range was significantly accumulated to 179.3% on the third-round NTG treatment, and then appeared to level-off and remained constant. These results indicated that multiple-round NTG treatment had a significant accumulative effect on fungal tolerance to propamocarb. Among all chemical-mutants, the LA-C-R3-M was the highest tolerant to propamocarb, whose EC50 value was increased 2.79-fold compared to the wild-type strain, and it was mitotic stable after 20 passages on PDA medium. Colony growth, conidia yield and conidial germination on plates, and parasitism of nematode eggs of M. incognita and H. glycines were not significantly changed by the NTG-based mutagenesis compared to the wild-type strain in either single- or multiple-round NTG treatment. In conclusion, we succeeded in improving the propamocarb tolerance of L. attenuatum via the optimized NTG-based mutagenesis system. The improved strain LA-C-R3-M could be potentially applied with propamocarb in the field.