Chongqing Engineering Research Center for Fungal Insecticide

Chongqing, China

Chongqing Engineering Research Center for Fungal Insecticide

Chongqing, China
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Jin K.,Chongqing University | Jin K.,Chongqing Engineering Research Center for Fungal Insecticide | Jin K.,Regulation Technologies under Chongqing Municipal Education Commission | Ming Y.,Chongqing University | And 5 more authors.
Microbiology (United Kingdom) | Year: 2012

Fungal biocontrol agents have great potential in integrated pest management. However, poor efficacy and sensitivity to various adverse factors have hampered their wide application. In eukaryotic cells, Hog1 kinase plays a critical role in stress responses. In this study, MaHog1 (GenBank accession no. EFY85878), encoding a member of the Hog1/Sty1/p38 mitogenactivated protein kinase family in Metarhizium (Me.) acridum, was identified. Targeted gene disruption was used to analyse the role of MaHog1 in virulence and tolerance of adverse factors. Mutants with MaHog1 depletion showed increased sensitivity to high osmotic stress, high temperature and oxidative stress, and exhibited remarkable resistance to cell wall-disturbing agents. These results suggest that Hog1 kinase has a conserved function in regulating multistress responses among fungi, and that MaHog1 might influence cell wall biogenesis in Me. acridum. Bioassays conducted with topical inoculation and intrahaemocoel injection revealed that MaHog1 is required for both penetration and postpenetration development of Me. acridum. MaHog1 disruption resulted in a significant reduction in virulence, likely due to the combination of a decrease in conidial germination, a reduction in appressorium formation and a decline in growth rate in insect haemolymph, which might be caused by impairing fungal tolerance of various stresses during infection. © 2012 SGM.

Hong M.,Chongqing University | Hong M.,Chongqing Engineering Research Center for Fungal Insecticide | Hong M.,Key Laboratory of Gene Function and Regulation Technologies under Chongqing Municipal Education Commission | Peng G.,Chongqing University | And 7 more authors.
Applied Microbiology and Biotechnology | Year: 2017

Microbial pesticides form critical components of integrated pest management (IPM) practices. Little, however, is known regarding the impacts of these organisms on the indigenous microbial community. We show that Metarhizium anisopliae strain CQMa421 was highly effective in controlling the rice leafroller, Cnaphalocrocis medinalis Guenee. In addition, M. anisopliae distribution and its effects on phyllosphere microbial diversity after application in field trials were investigated. Phylloplane specific distribution of the fungus was observed over time, with more rapid declines of M. anisopliae CFUs (colony-forming units) seen in the top leaf layer as compared to lower layers. Application of the fungus resulted in transient changes in the endogenous microbial diversity with variations seen in the bacterial observed species and Shannon index. Notable increases in both parameters were seen at 6-day post-application of M. anisopliae, although significant variation within sample replicates for bacteria and fungi were noted. Application of M. anisopliae increased the relative distribution of bacterial species implicated in plant growth promotion and organic pollutant degradation, e.g., Methylobacterium, Sphingobium, and Deinococcus. These data show minimal impact of M. anisopliae on endogenous microbial diversity with transient changes in bacterial abundance/diversity that may result in added benefits to crops. © 2017 Springer-Verlag GmbH Germany

Zhang W.,Chongqing University | Chen J.,Chongqing University | Keyhani N.O.,University of Florida | Zhang Z.,Chongqing University | And 4 more authors.
BMC Genomics | Year: 2015

Background: The migratory locust, Locusta migratoria manilensis, is an immensely destructive agricultural pest that forms a devastating and voracious gregarious phase. The fungal insect pathogen, Metarhizium acridum, is a specialized locust pathogen that has been used as a potent mycoinsecticide for locust control. Little, however, is known about locust immune tissue, i.e. fat body and hemocyte, responses to challenge by this fungus. Methods: RNA-seq (RNA sequencing) technology were applied to comparatively examine the different roles of locust fat body and hemocytes, the two major contributors to the insect immune response, in defense against M. acridum. According to the sequence identity to homologies of other species explored immune response genes, immune related unigenes were screened in all transcriptome wide range from locust and the differential expressed genes were identified in these two tissues, respectively. Results: Analysis of differentially expressed locust genes revealed 4660 and 138 up-regulated, and 1647 and 23 down-regulated transcripts in the fat body and hemocytes, respectively after inoculation with M. acridum spores. GO (Gene Ontology) enrichment analysis showed membrane biogenesis related proteins and effector proteins significantly differentially expressed in hemocytes, while the expression of energy metabolism and development related transcripts were enriched in the fat body after fungal infection. A total of 470 immune related unigenes were identified, including members of the three major insect immune pathways, i.e. Toll, Imd (immune deficiency) and JAK/STAT (janus kinase/signal transduction and activator of transcription). Of these, 58 and three were differentially expressed in the insect fat body or hemocytes after infection, respectively. Of differential expressed transcripts post challenge, 43 were found in both the fat body and hemocytes, including the LmLys4 lysozyme, representing a microbial cell wall targeting enzyme. Conclusions: These data indicate that locust fat body and hemocytes adopt different strategies in response to M. acridum infection. Fat body gene expression after M. acridum challenge appears to function mainly through activation of innate immune related genes, energy metabolism and development related genes. Hemocyte responses attempt to limit fungal infection primarily through regulation of membrane related genes and activation of cellular immune responses and release of humoral immune factors. © 2015 Zhang et al.

Ming Y.,Chongqing University | Ming Y.,Chongqing Engineering Research Center for Fungal Insecticide | Ming Y.,Key Laboratory of Gene Function and Regulation Technologies under Chongqing Municipal Education Commission | Wei Q.,Chongqing University | And 8 more authors.
Applied Microbiology and Biotechnology | Year: 2014

The protein kinase sucrose non-fermenting-1(Snf1) regulates the derepression of glucose-repressible genes and plays a major role in carbon source utilization. In this study, MaSnf1, a sucrose non-fermenting protein kinase gene, has been identified from the entomopathogenic fungus Metarhizium acridum, which has a great potential as a biocontrol agent. The functions of MaSnf1 were characterized using gene disruption and complementation strategies. Disruption of MaSnf1 reduced the conidial yield and delayed the conidial germination on potato dextrose agar (PDA) medium. MaSnf1 is also important for response to ultraviolet radiation and heat shock stress and carbon source utilization in M. acridum. Bioassays by topical inoculation and intrahemocoel injection showed that the MaSnf1 deletion mutant exhibited greatly reduced pathogenicity. The reduced expression level of chitinase gene (Chi) and protease gene (Pr1A) in MaSnf1-disruption transformant (ΔMaSnf1) most likely affects the initial penetration into its host. Additionally, the reduced expression level of acidic trehalase gene (ATM1) probably causes a decline in growth rate in insect hemolymph. Inactivation of MaSnf1 led to a significant decrease in virulence, probably owing to reduction in conidial germination, and appressorium formation, impairment in penetration, and decrease in growth rate in insect hemolymph. © 2014, Springer-Verlag Berlin Heidelberg.

Jin K.,Chongqing University | Jin K.,Chongqing Engineering Research Center for Fungal Insecticide | Jin K.,Chongqing Municipal Education Commission | Han L.,Northwest University, China | And 3 more authors.
Journal of Invertebrate Pathology | Year: 2014

Entomopathogenic fungi have great potential for development as insecticides. However, large-scale use of mycoinsecticides is partially limited by poor efficiency. In many fungal pathogens, the yeast and fungal extracellular signal-regulated kinase (YERK1) subfamily is crucial to the fungal pathogenicity. In this study, a Fus3/Kss1-type mitogen-activated protein kinase (MAPK) gene MaMk1 (GenBank accession No. EFY93607) was identified in Metarhizium acridum, which encodes a member of the YERK1 subfamily. Targeted gene disruption was used to analyze the function of MaMk1 in fungal growth, conidial yield and virulence. Growth assays showed that MaMk1 disruption did not affect fungal growth and conidial yield on potato dextrose agar (PDA) plates. Bioassays by topical inoculation showed that a MaMk1-disruption mutant entirely lost its pathogenicity for the locusts, likely because of failure to penetrate the insect cuticle, which might have been caused by inability to form appressoria during infection. However, bioassays by injection showed no significant difference in virulence among the wild type (WT), δMaMk1 mutant and complementary transformant. δMaMk1 mutant failed to penetrate the cuticle outwards and sporulate on the locust cadaver. These results suggest that MaMk1 is required for penetration of the insect cuticle both into the hemocele and outside from the hemocele, but is dispensable for fungal growth in insect hemolymph. Gene expression pattern analysis showed that MaMk1 disruption downregulated expression of Mad1 and Mpl1, but did not reduce expression of Pr1 in M. acridum. © 2013 Elsevier Inc.

Jin K.,Chongqing University | Jin K.,Chongqing Engineering Research Center for Fungal Insecticide | Jin K.,Key Laboratory of Gene Function and Regulation Technology under Chongqing Municipal Education Commission | Peng G.,Chongqing University | And 7 more authors.
Fungal Genetics and Biology | Year: 2015

For pathogens, the ability to acquire available nutrients in a host is a key to their survival and replication. Entomopathogenic fungi of the genus Metarhizium secrete trehalase, which enables them to use trehalose, the predominant sugar in insects. Here, the roles of the acid trehalase gene (ATM1) in the in vivo growth and virulence of Metarhizium acridum were investigated. Phenotypic analysis showed that disruption of ATM1 severely reduced fungal growth on exogenous trehalose as the sole carbon source. Bioassays showed that ATM1 disruption impaired the virulence of M. acridum against the host insect Locusta migratoria. The ATM1-disruption strain (δ. ATM1) grown more slowly than the wild-type strain (WT) and complemented transformant (CP) in locust blood, which was consistent with the activity of acid trehalase in the hemolymph of infected locusts. Correspondingly, the trehalose concentration in locusts infected by δ. ATM1 was significantly higher than in those infected by WT or CP. Thus, ATM1 disruption led to a significant reduction in virulence by adversely affecting the fungal growth in insect hemolymph, which resulted from the inability of the mutant strain to use trehalose. © 2015 Elsevier Inc.

Jin K.,Chongqing University | Jin K.,Chongqing Engineering Research Center for Fungal Insecticide | Jin K.,Key Laboratory Of Gene Function Regulation Technologies Under Chongqing Municipal Edu Commission | Zheng X.,Chongqing University | And 5 more authors.
PLoS ONE | Year: 2011

We established a novel method, Gene Expression Profiling via Multigene Concatemers (MgC-GEP), to study multigene expression patterns simultaneously. This method consists of the following steps: (1) cDNA was obtained using specific reverse primers containing an adaptor. (2) During the initial 1-3 cycles of polymerase chain reaction (PCR), the products containing universal adaptors with digestion sites at both termini were amplified using specific forward and reverse primers containing the adaptors. (3) In the subsequent 4-28 cycles, the universal adaptors were used as primers to yield products. (4) The products were digested and ligated to produce concatemers. (5) The concatemers were cloned into the vector and sequenced. Then, the occurrence of each gene tag was determined. To validate MgC-GEP, we analyzed 20 genes in Saccharomyces cerevisiae induced by weak acid using MgC-GEP combined with real-time reverse transcription (RT)-PCR. Compared with the results of real-time RT-PCR and the previous reports of microarray analysis, MgC-GEP can precisely determine the transcript levels of multigenes simultaneously. Importantly, MgC-GEP is a cost effective strategy that can be widely used in most laboratories without specific equipment. MgC-GEP is a potentially powerful tool for multigene expression profiling, particularly for moderate-throughput analysis. © 2011 Jin et al.

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