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Cao Y.,Chongqing University | Cao Y.,Chongqing Engineering Research Center for Fungal Insecticides | Cao Y.,Key Laboratory of Functional Gene and Regulation Technology Under Chongqing Municipal Education Commission | Du M.,Chongqing University | And 8 more authors.
Applied Microbiology and Biotechnology | Year: 2014

Calcineurin is highly conserved and regulates growth, conidiation, stress response, and pathogenicity in fungi. However, the functions of calcineurin and its regulatory network in entomopathogenic fungi are not clear. In this study, calcineurin was functionally analyzed by deleting the catalytic subunit MaCnA from the entomopathogenic fungus Metarhizium acridum. The ΔMaCnA mutant had aberrant, compact colonies and blunt, shortened hyphae. Conidia production was reduced, and phialide differentiation into conidiogenous cells was impaired in the ΔMaCnA mutant. ΔMaCnA had thinner cell walls and greatly reduced chitin and β-1,3-glucan content compared to the wild type. The ΔMaCnA mutant was more tolerant to cell wall-perturbing agents and elevated or decreased exogenous calcium but less tolerant to heat, ultraviolet irradiation, and caspofungin than the wild type. Bioassays showed that ΔMaCnA had decreased virulence. Digital gene expression profiling revealed that genes involved in cell wall construction, conidiation, stress tolerance, cell cycle control, and calcium transport were downregulated in ΔMaCnA. Calcineurin affected some components of small G proteins, mitogen-activated protein kinase, and cyclic AMP (cAMP)-protein kinase A signaling pathways in M. acridum. In conclusion, our results gave a global survey of the genes downstream of calcineurin in M. acridum, providing molecular explanations for the changes in phenotypes observed when calcineurin was deleted. © 2014, Springer-Verlag Berlin Heidelberg.


Yu Y.,Chongqing University of Technology | Cao Y.,Chongqing University | Cao Y.,Chongqing Engineering Research Center for Fungal Insecticides | Cao Y.,Key Laboratory of Functional Gene and Regulation Technology Under Chongqing Municipal Education Commission | And 4 more authors.
Journal of Invertebrate Pathology | Year: 2016

Hemocytes are the first line of defense in the invertebrate immune system. Understanding their roles in cellular immunity is important for developing more efficient mycoinsecticides. However, the exact classification of hemocytes has been inconsistent and the various types of phagocytes in Locusta migratoria are poorly defined. Herein, the Wright–Giemsa staining method and microscopy were employed to characterize the hemocytes of L. migratoria following infection by Metarhizium acridum. Hemocytes were classified into four types, including granulocytes, plasmatocytes, prohemocytes, and oenocytoids, based on size, morphology, and dye-staining properties. Each type of hemocyte was classified into several subtypes according to different ultrastructural features. At least four subtypes of granulocytes or plasmatocytes, including small-nucleus plasmatocytes, basophil vacuolated plasmatocytes, homogeneous plasmatocytes, and eosinophilic granulocytes, carried out phagocytosis. The percentage of total phagocytes increased two days after infection by M. acridum, then gradually declined during the next two days, and then increased sharply again at the fifth day. Our data suggested that plasmatocytes and granulocytes may be the major phagocytes that protect against invasion by a fungal pathogen in L. migratoria. Total hemocytes in locusts significantly increased in the initial days after infection and decreased in the late period of infection compared to controls. In the hemocoel, hyphal bodies were recognized, enwrapped, and digested by the phagocytes. Then, the broken hyphal pieces were packaged as vesicles to be secreted from the cell. Moreover, locusts might have a sensitive and efficient cellular immune system that can regulate phagocyte differentiation and proliferation before fungi colonize the host hemolymph. © 2016 Elsevier Inc.


Liu S.,Chongqing University | Liu S.,Chongqing Engineering Research Center for Fungal Insecticides | Liu S.,Chongqing Municipal Education Commission | Peng G.,Chongqing University | And 5 more authors.
BMC Microbiology | Year: 2012

Background: The efficacy of entomopathogenic fungi in pest control is mainly affected by various adverse environmental factors, such as heat shock and UV-B radiation, and by responses of the host insect, such as oxidative stress, osmotic stress and fever. In this study, an adenylate cyclase gene (MaAC) was cloned from the locust-specific entomopathogenic fungus, Metarhizium acridum, which is homologous to various fungal adenylate cyclase genes. RNA silencing was adapted to analyze the role of MaAC in virulence and tolerance to adverse environmental and host insect factors. Results: Compared with the wild type, the vegetative growth of the RNAi mutant was decreased in PD (potato dextrose medium), Czapek-dox and PDA plates, respectively, demonstrating that MaAC affected vegetative growth. The cAMP levels were also reduced in PD liquid culture, and exogenous cAMP restored the growth of RNAi mutants. These findings suggested that MaAC is involved in cAMP synthesis. The knockdown of MaAC by RNAi led to a reduction in virulence after injection or topical inoculation. Furthermore, the RNAi mutant grew much slower than the wild type in the haemolymph of locust in vitro and in vivo, thus demonstrating that MaAC affects the virulence of M. acridum via fungal growth inside the host locust. A plate assay indicated that the tolerances of the MaAC RNAi mutant under oxidative stress, osmotic stress, heat shock and UV-B radiation was decreased compared with the wild type. Conclusion: MaAC is required for virulence and tolerance to oxidative stress, osmotic stress, heat shock and UV-B radiation. MaAC affects fungal virulence via vegetative growth inside the insect and tolerance against oxidative stress, osmotic stress and locust fever. © 2012 Liu et al.; licensee BioMed Central Ltd.


Tang L.,Chongqing University | Tang L.,Chongqing Engineering Research Center for Fungal Insecticides | Tang L.,Chongqing Municipal Education Commission | Wei W.,Chongqing University | And 11 more authors.
Indian Journal of Biochemistry and Biophysics | Year: 2010

A strain F1 with high cellulase activity obtained from the deadwood stack was characterized as Ceriporia lacerate by examination of the general taxonomical characteristics and phylogenetic sequence analysis of rDNA ITS gene. The endoglucanase (EG) and filter paper cellulase (FPase) activities of the strain showed remarkable stability in the pH range of 4.0-7.0, and maintained about their maximal value of 76% and 50% after incubation at 70°C for 6 h respectively. The strain grew particularly well with CMC-Na (1.0%) and yeast extract (0.4%) at 28°C (pH 6.0) in flasks stirred at 150 × g for 6 days. Based on the thermostability and pH stability of cellulase, the strain appears to have potential in industrial applications and bioresource utilization.


Tang L.,Chongqing University | Tang L.,Chongqing Engineering Research Center for Fungal Insecticides | Tang L.,Key Laboratory of Functional Gene and New Regulation Technologies under Chongqing Municipal Education Commission | Wei W.,Chongqing University | And 11 more authors.
Indian Journal of Geo-Marine Sciences | Year: 2010

A strain F1 with high cellulase activity obtained from the deadwood stack was characterized as Ceriporia lacerate by examination of the general taxonomical characteristics and phylogenetic sequence analysis of rDNA ITS gene. The endoglucanase (EG) and filter paper cellulase (FPase) activities of the strain showed remarkable stability in the pH range of 4.0-7.0, and maintained about their maximal value of 76% and 50% after incubation at 70°C for 6 h respectively. The strain grew particularly well with CMC-Na (1.0%) and yeast extract (0.4%) at 28°C (pH 6.0) in flasks stirred at 150 × g for 6 days. Based on the thermostability and pH stability of cellulase, the strain appears to have potential in industrial applications and bioresource utilization. © 2010, National Institute of Science Communication and Information Resources (NISCAIR). All rights reserved.


Cao Y.,Chongqing University | Cao Y.,Chongqing Engineering Research Center for Fungal Insecticides | Cao Y.,Municipal Education Commission | Li K.,Chongqing University | And 5 more authors.
Research in Microbiology | Year: 2011

Mrd1 is one of the trans-acting proteins and plays an important role in precursor ribosomal RNA processing. Here we characterized the Mamrd1 gene from Metarhizium acridum and studied its function in growth, conidiation and virulence using RNA interference. The Mamrd1 gene was identified as participating in the processing of pre-rRNA in M. acridum and was highly upregulated during the infection process. A Mamrd1-RNAi strain exhibited an appearance of fluffy mycelia, a defective branching pattern and delayed conidiation compared to the wild-type strain. Downregulation of Mamrd1 in M. acridum suppressed growth both on artificial medium and inside the insect, and significantly reduced hyphal biomass, conidium production and virulence against Locusta migratoria manilensis. These results demonstrated that Mamrd1 plays an important role in growth, conidiation and virulence in M. acridum. © 2011 Institut Pasteur.


Tang L.,Chongqing University | Tang L.,Chongqing Engineering Research Center for Fungal Insecticides | Li J.,Chongqing University | Li J.,Chongqing Engineering Research Center for Fungal Insecticides | And 10 more authors.
Plant Growth Regulation | Year: 2012

Although irregular monoterpenes are important and common in the Asteraceae family, little is known about their biosynthesis at the genetic level via the MEP pathway. Chrysanthemyl diphosphate (CPP) is an intermediate in the biosynthesis of pyrethrins which are irregular monoterpenes with excellent insecticidal activity in Tanacetum cinerariaefolium (T. cinerariaefolium). In this study, a chrysanthemyl diphosphate synthase (CDS) gene named CDS_CCI2 (GenBank accession no. HQ235057) was isolated from T. cinerariaefolium. It was homologous to T. cinerariaefolium CPP gene family, and proved to be located in the plastid by the in situ subcellular localization. CDS_CCI2 was found to be expressed in roots, stems, leaves, buds and flowers. Moreover, the expression of CDS_CCI2 can be up-regulated by abscisic acid (ABA), methyl jasmonate and ethrel treatment. Phenotypic and molecular analysis showed that overexpression of CDS_CCI2 in micro-tom tomato resulted in dwarf phenotypes characterized with infertile flowers and seedless fruits. Furthermore, overexpression of CDS_CCI2 altered the production of endogenous secondary metabolites. Our data indicate that CPP affects the synthesis of gibberellic acid (GA) and ABA. © 2012 Springer Science+Business Media B.V.


Leng Y.,Chongqing University | Peng G.,Chongqing University | Peng G.,Chongqing Engineering Research Center for Fungal Insecticides | Peng G.,Regulation Technologies under Chongqing Municipal Education Commission | And 6 more authors.
BMC Microbiology | Year: 2011

Abstract. Background: The entomopathogenic fungus Metarhizium acridum has been used as an important biocontrol agent instead of insecticides for controlling crop pests throughout the world. However, its virulence varies with environmental factors, especially temperature. Neutral trehalase (Ntl) hydrolyzes trehalose, which plays a role in environmental stress response in many organisms, including M. acridum. Demonstration of a relationship between Ntl and thermotolerance or virulence may offer a new strategy for enhancing conidiospore thermotolerance of entomopathogenic fungi through genetic engineering. Results: We selected four Ntl over-expression and four Ntl RNA interference (RNAi) transformations in which Ntl expression is different. Compared to the wild-type, Ntl mRNA expression was reduced to 35-66% in the RNAi mutants and increased by 2.5-3.5-fold in the over-expression mutants. The RNAi conidiospores exhibited less trehalase activity, accumulated more trehalose, and were much more tolerant of heat stress than the wild-type. The opposite effects were found in conidiospores of over-expression mutants compared to RNAi mutants. Furthermore, virulence was not altered in the two types of mutants compared to the wild type. Conclusions: Ntl controlled trehalose accumulation in M. acridum by degrading trehalose, and thus affected conidiospore thermotolerance. These results offer a new strategy for enhancing conidiospore thermotolerance of entomopathogenic fungi without affecting virulence. © 2011 Leng et al; licensee BioMed Central Ltd.


Peng G.,Chongqing University | Peng G.,Chongqing Engineering Research Center for Fungal Insecticides | Xia Y.,Chongqing University | Xia Y.,Chongqing Engineering Research Center for Fungal Insecticides
Pest Management Science | Year: 2015

BACKGROUND: Entomopathogenic fungi have been developed as biopesticides, but poor efficacy has blocked their application. One approach to improving virulence is by genetic manipulation. BjαIT from the venom of Buthotus judaicus is an insect-selective neurotoxin. To clarify the insecticidal potency of BjαIT as a virulence candidate in microbial biocontrol agents, the entomopathogenic fungus Metarhizium acridum was genetically modified with BjαIT, and its resulting activity against locusts (Locusta migratoria manilensis) was assessed. RESULT: In comparison with the wild-type strain, the engineered isolate BjαIT-102 grew significantly quicker in locust haemolymph. Correspondingly, the median lethal dose (LC50) for BjαIT-102 was 18.2-fold lower, and the median lethal times (LT50) for BjαIT-102 were reduced by 28.1 and 30.4%, respectively, after topical inoculation and injection. BjαIT-102 formed conidia on dead locusts, although the conidial yield was reduced 1.58-fold. Moreover, there were no significant differences in germination and appressorium formation between the BjαIT-102 and wild-type strains. CONCLUSION: Expression of BjαIT in M. acridum significantly increased virulence against locusts by shortening the in vivo infection period without affecting conidium formation on the carcasses. This study demonstrated that engineering entomopathogenic fungi to incorporate BjαIT offers great potential for increasing their virulence. © 2014 Society of Chemical Industry.


Peng G.,Chongqing University | Peng G.,Chongqing Engineering Research Center for Fungal Insecticides | Xia Y.,Chongqing University | Xia Y.,Chongqing Engineering Research Center for Fungal Insecticides
Journal of Industrial Microbiology and Biotechnology | Year: 2014

LqhIT2 is an insect-specific neurotoxin from the venom of scorpion. In this study, the LqhIT2 gene was introduced into the entomopathogenic fungus, Metarhizium acridum. The virulence of the genetically modified strain MaLqhIT2 was then evaluated against locusts (Locusta migratoria manilensis). Compared with the wild-type strain, the median lethal cell density (LC50) for MaLqhIT2 was a 22.6-fold lower, and the median times to death (LT50) for MaLqhIT2 were reduced by 30.3 and 29.6 %, respectively, after topical inoculation and injection. MaLqhIT2 also grew significantly faster in the hemolymph than wild-type strain. There were no significant differences in germination, appressorium formation and sporulation in locust carcasses between the MaLqhIT2 and wild-type strain. These results indicate that LqhIT2 increased the virulence of M. acridum towards locusts by shortening the in vivo infection period, without affecting cuticle penetration or conidia formation in the carcasses. LqhIT2 thus shows considerable potential for increasing fungal virulence against locusts. © 2014, Society for Industrial Microbiology and Biotechnology.

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