Studies On Multidrug Resistance Of Fungal Biocontrol Agents And A Strategy For Screening Candidate Strains Highly Virulent To Rice Planthoppers

Entomopathogenic fungi are important biocontrol agents of various insect pests in agriculture and forestry. The field efficacies of fungal formulations against the pests are not only dependent on the generic traits of fungal candidate strains but also affected by environmental stressful factors, such as high temperature, solar UV irradiation and chemical pesticides. Of those, fungicides applied for plant disease control are most stressful (harmful) towards the fungal agents simultaneously applied for insect control in the field. Therefore, probing possible mechanisms involved in fungal resistance to fungicides and means to engineering the fungal agents for improved fungicide resistance is of special value for developing more field-persistent mycoinsecticdes. ATP-binding cassette (ABC) transporter proteins existent in eukaryotic species are involved in cell protection from fungicide damage. However, such an important antidrug mechanism is unknown from the fungal biocontrol agents.This study began from evaluating the resistance of Isaria fumosorosea (previously Paecilomyces fumosoroseus) strains to carbendazim (methyl2-benzimidazole carbamate; MBC) and analyzing the site muations of genes in highly MBC-resistant mutants, which resulted from chemical mutagenesis and showed multidrug resistance (MDR) and reduced thermotolerance. These mutants differed form previously constructed Beauveria bassiana mutants, whose high MBC resistance was primarily associated with a few crucial site mutations of (3-tubulin gene. Thus, a hypothesis that an antidrug mechanism other than the β-tubulin mutation may exist in the fungal entomopathogens was proposed and testified by gene disruption and phenotypic analysis. The main results are summarized below.In vivo passages of heterologous Beauveria bassiana isolates for improved virulence to Nilaparvata lugens. Since desired candidates against brown planthopper, Nilaparvata lugens, are lacking,21Beauveria bassiana isolates from heterologous host insects were bioassayed on third-instar nymphs at the high concentration of～1000conidia/mm2, causing only2-23%mortalities. Of those, three isolates killed significantly more nymphs (up to45-62%) after two in vivo passages but no more after further passage. Conidial hydrophobicity rates (Hr), zeta potentials (Pz), and subtilisin-like protease (Prl) activities (Ap) of these isolates showed the same trends in the three host passages (N:0-3). In multivariate correlation, the variables N, Hr and Pz were found contributing89%to the mortality variation (r2=0.89). Significant positive correlations were also found between Hr and N (r2=0.64), Pz and N (r2=0.52), Ap and N (r2=0.51), HT and Ap (r2=0.45), and Pz and Ap (r2=0.57), respectively. However, irregular changes of Hr and Pz occurred in four other isolates, whose pathogenicity to N. lugens was not enhanced by repeated host passages, resulting in no correlation between the variables. Our data indicate that the conidial surface properties Hr and Pz associated with cuticle adhesion reflect the heterologous host-induced adaptation and help to select fungal candidates against N. lugens from repeated in vivo passages.Analysis of differentially expressed genes in MBC-resistant B. bassiana strains. Two MBC-resistant mutants (2860-M2-6and2860-M2-11) previously obtained by chemical mutagenesis of the wild-type strain B. bassiana2860(Bb2860) were grown on SDAY plates including MBC or not. Four RNA samples from the cultures were subjected to Digital Gene Expression Profiling (DEG) analysis, resulting in144up-regulated genes under the MBC-inducive conditions. Using R statistical programming language to do cluster analysis and online KEGG (Kyoto Encyclopedia of Genes and Genomes) classified indicated that those genes were likely involved in the processes of metabolism, Genetic Information Processing (GIP), Environmental Information Processing (ETP), Cellular Processes(CP) and Others(function unknow). Of those, the genes encoding five TFs, five signal transduction-related kinases and two transporters were associated with MBC resistance based on their expression levels in real-time PCR.Disruption and phenotypic analysis of five ABC transporter coding genes in B. bassiana. Twelve genes encoding drug transporters found from B. bassiana genome were classified to three subfamilies, including six PDR genes, five MDR genes and one MRP gene. Of those, two PDR genes and one MDR gene were up-regulated by MBC, azoxysyrobin (ASB) and phosphinothricin (PPT) included in growth medium. The three genes (BbABC1, BbABC2and BbMDRl) and two other ABC transporter genes (BbABC5and BbMRP) identified via DEG analysis were disrupted from Bb2860, respectively. The knockout mutants ΔBbABC1, ΔBbABC2, ΔBbABC5, ΔBbMDR1and ΔBMRP differed significantly in sensitivity to nine structure-different drugs (fungcides and Congo red), mostly down-regulated at varying degree. Their tolerances to the oxidative stresses induced by H2O2and menadione were also significantly reduced. Moreover, ΔBbABCl and ΔBbMDRl showed decreced virulence to the second-instar larvae of Spodoptera litura. The results indicate that the expression levels of drug transporter proteins to large extent determine B. bassiana multidrug resistance and that at least some of them are virulence associated factors.Characterization of B. bassiana transcripotion activitor Bbgal. In eukaryotic cells, PXR or Gal11is a transcription activator to activate some genes encoding drug efflux pumps via binding xenobiotics. A similar gene, Bbgal, was cloned from Bb2860genome. Real-time PCR indicated that the gene Bbgal was up-regulated by including MBC, ASB and PPT in growth medium. Disrupting this gene from Bb2860resulted in significant diference in in sensitivity to eight fungcides and Congo red but little change in sensitivity to ASB. Moreover, ΔBbgal showed increased tolerance to H2O2-induced oxidative stress but decreased tolerance to menadione-induced oxidative stress. The gene disruption also resulted in decreced virulence to the second-instar larvae of S. litura. The results suggest that Bbgal be a gene encoding the B. bassiana transcription activator like PXR and Gal11found in human and yeast cells, respectively and thus bind xenobiotic drugs to activate some drug transportanter proteins.Genetic analysis for MBC resistance of I. fumosorosea. A highly MBC-sensitive wild-type strain (EC50:1.67μg/ml) of I. fumosorosea (If116) was subjected to chemical mutagenesis, yielding nine mutants whose MBC resistance was drastically elevated with EC50ranging from99.4to1000μg/ml, accompanied by decreased thermotolerance. Sequence comparisons of α-and β-tubulin genes cloned from the wild-type and mutant strains revealed no site mutations associated with the increased MBC resistance of the mutant strains. These mutants also displayed higher resistance to other antifungal drugs than the wild-type strain, including tricyclazole, cymoxanil, thiophanate-methyl, azoxystrobin and iprodione. The fluorescence assay of If116blastospores stained with rhodamine indicated that MBC competed with the stain in the cell efflux, suggesting the possible involvement of a pleiotropic drug resistance (PDR) transporter in the fungal MBC resistance. This was partially confirmed with less accumulation of the fluorescence stain in the cells of all mutant strains with much higher MBC resistance. A gene encoding the ABC transporter protein of I. fumosorosea (ifTl) was cloned separately from the wild-type and mutant strains. Sequence alignments revealed no site mutations in the coding areas of all clones. However, three common site mutations were located at the biding sites of three transcription factors (TFs) upstream of the coding areas, i.e.,-2202G→A,-836C→T and -300C→T. Based on real-time PCR, the transcription level of the ifTl gene in the wild-type cells increased with the MBC concentration including in growth medium. Moreover, the gene expression level in the cells of all mutants was enhanced by17-137fold compared with that in the wild-type cells. Conclusively, the overexpression of the MBC transporter coding gene ifTl regulated by the mutated TFs was likely responsible for the high MBC resistance of all mutant strains.