Characterization Of The Resistance Of Metarhizium To Carbendazim And Of The Biological Functions Of Histidine Kinase And Adenylate Cvclase In M. Robberstii

Metarhizium-based mycoinsecticides have been widely applied in the control of insect pests, such as locusts and grasshoppers. As the active ingredients of fungal formulations, formulated fungal cells, such as conidia, are often exposed to environmental stresses including high temperatures, solar UV irradiation and fungicides applied for plant disease control in the field, suffering from some degree of viability loss that affects the field persistence and efficacy of a fungal application. This study started from evaluating the resistance of30wild-type Metarhizium strains to carbendazim (benzimidazole fungicide), followed by examining common site mutations in the tubulins of carbendazim-resistant mutants from chemical mutagenesis in order to probe the cause of the resistance, and constructing the knockout and complement mutants of Metarhizium robertsii histidine kinase (mhkl) and adenylate cyclase (macy) for various phenotypic analyses in order to reveal the roles of the two enzymes in regulating growth, development, sproulation, multi-stress responses and infectivity, respectively. The results are summarized below.Metarhizium resistance to carbendazim and associated changes in response to multiple stresses. Thirty wild-type strains consisting of21Metarhizium anisopliae, eight M. acridum and one M. anisopliae var. majus with different host and geographic origins were assayed for their sensitivities to carbendazim (methyl2-benzimidazole carbamate, MBC herein) using a gradient-concentration system. Of those, only one M. anisopliae strain (Man456) showed low resistance due to the minimal inhibition concentration (MIC) of5.76μg/ml for99%inhibition of colony formation while all other29strains were sensitive to the fungicide with the mean MIC of2.47(1.14-4.98)μg/ml. The selected strain, Man456, was subjected to NaNO2-induced mutagenesis, generating11mutants highly resistant to MBC. The MICs of all mutants exceeded1000μg/ml. Five of mutants even had the EC50(an MBC concentration suppressing50%colony fomation) of>1000μg/ml and the rest mutants had the EC50from24to447μg/ml. Accompanied by the enhanced MBC resistance, conidial tolerance to wet-heat stress at48℃was reduced for all the mutants and their UV-B resistance also changed. Two genes encoding a-tubulin and (3-tubulin were cloned separately from the wild-type strain and the MBC-resistant mutants by means of the technique of splicing-by-overlap extension (SOE), followed by online analysis of their protein sequences. As a result, the common site mutations S231L and S144G were found in the a-tubulin and β-tubulin sequences of all mutants, respectively. The same site-directed mutations were constructed in the wild-type strain, yielding mutants αT-S231L and βT-S144G. Both mutants showed higher resistance to MBC than wild-type, lower tolerance to the wet-heat stress and altered UV-B resistance. However, the changes in the MBC resistance of the two site-directed mutants are not enough to elucidate the extraordinarily high MBC resistance in the mutants from chemical mutagenesis. This suggests other mechanism(s) likely involved in the high MBC resistance of M. anisopliae.Characterized functions of M. roberstii histidine kinase (mhkl). The role of M. robertsii Group III histidine kinase(mhkl) in regulating various phenotypes of the fungal entomopathogen and the transcripts of the genes encoding three mitogen-activated protein (MAP) kinases in HOG-pathway and13proteins possibly associated with the phenotypes were probed by constructing Amhkl and Amhkl/mhkl mutants via Agrobacterium-mediated transformation. All examined Amhkl phenotypes except no change in fungicide (dimethachlon) sensitivity differed significantly from those of wild-type and Amhkl/mhkl, which were similar or close to each other in all phenotypic parameters. Significant main phenotypic changes in Amhkl included50-67%increases in conidial yield on two normal media,13-46%increases in osmotolerance to salts and sugars,27-37%increases in conidial thermotolerance at48℃,41-51%increases in formic-acid-extractable protein content relating to the thermotolerance,～6%reduction in conidial UV-B resistance, and-25%decrease in virulence to Tenebrio molitor larvae. Quantitative real-time RT-PCR (qRT-PCR) indicated that the mhkl disruption caused3.7-8.5-fold up-regulation of three MAP kinase genes(mapkkk, mapkk and mapk),1.6-4.7-fold up-regulation of two conidiation-related genes (flbC and hymA),1.6-15.1-fold up-regulation of three catalase (CAT) genes, and67-89%down-regulation of three superoxide dismultase (SOD) genes, but little change in the transcriptional expression of other CAT and SOD genes examined. The results provide new insights into the crucial role of mhkl in regulating the downstream genes and associated phenotypes important for the fungal biocontrol potential.Characterized functions of M. roberstii adenylate cyclase. Fugnal adenylate cyclase (ACY) enables to catalyze ATP into cAMP to activate cAMP-dependent protein kinase (PKA), thereby acting as a crucial enzyme in the pathway of cAMP-PKA signaling transduction. The sequence of M. roberstii ACY (macy) showed23-94%identity to98ACY sequences of other fungi. The macy gene was successfully disrupted and complemented via Agrobacterium-mediated transformation. Compared to Δmacy/macy, and WT, the disruptant Δmacy had significant phenotypic changes. These included:(1) reduced colony growth and greatly decreased sporulation capacity under normal conditions;(2) colony growth less suppressed by the osmotic stress of KC1or NaCl but facilitated by the osmotic stress of glucose or sucrose;(3) colony growth inhibited by10mM menadione but greatly facilitated by100mM H2O2;(4) colony growth facilitated by Fe3+but suppressed by Cu2+, Mg2+, Mn2+and Ca2+in medium;(5) colony growth less sppressed by sucrose than galactose as a mere carbon source but greatly facilitated by NH4CI or NaNO2as a mere nitrogen source;(6) significant dicreases in conidial thermotolerance, formic-acid-extractable conidial protein content and conidial UV-B tolerance;(7)22-29%reduction in conidial infectivity to T. molitor larvae. Exceptionally, the Amacy sensitivity to dimethachlon had little change at the concentration of200μg/ml. The qRT-PCR analyses indicated that the macy knockout resulted in3.93fold up-regulation of pka gene in the cAMP-PKA pathway,82%down-regulation of a gene (mmc) associated with microcycle conidiation,4.2and11.6fold up-regulation of two CAT genes (catl and cat3), and87-89%down-regulation of three SOD genes (sod4, sod5and sod6) but little change in the transcript levels of other10genes, including flbC, hymA, five CAT genes and three SOD genes. The results provided the first overview to the crucial role of macy in regulating the growth, development, sporulation, multi-stress responses and infectivity of M. robertsii.