The adenylate cyclase gene MaAC is required for virulence and multi-stress tolerance of Metarhizium acridum
© Liu et al.; licensee BioMed Central Ltd. 2012
Received: 14 February 2012
Accepted: 26 July 2012
Published: 1 August 2012
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.
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.
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.
KeywordsBiocontrol agents Entomopathogenic fungi Conidia Virulence Environmental stress
Fungal biocontrol agents, which are widespread and environmentally safe, have great potential in integrated pest management. However, the application of entomopathogenic fungi such as Metarhizium acridum in the field has been held back owing to their poor efficacy . During the infection process of entomopathogenic fungi, germ tubes are produced after the fungal conidia attach to the insect cuticle, and then differentiate into swollen infection structures called appressoria. The appressoria produce penetration pegs, which penetrate the host cuticle via a combination of mechanical pressure and cuticle degrading enzymes, before piercing the surface of the host into the blood cavity. They produce a large number of hyphae through budding, thereby exhausting the nutrition of the insect host . During the course of fungal infection, pathogenic fungi encounter various adverse factors from the host insect, such as antifungal substances on the cuticle , oxidative stress during infection , osmotic stress inside the host body , behavioral changes, such as locust fever during the early infection stage [6, 7], and environmental stresses, including heat shock and UV radiation . Research on the regulatory processes involved in response to adverse factors from the host and environment is essential for the commercial development and improvement of fungi as biocontrol agents.
As major regulators of virulence determinants, the signal transduction pathways of fungal pathogens have been extensively researched. In fungi and yeasts, the cAMP (adenosine 3′, 5′-cyclic monophosphate) signaling cascade has been co-opted for a multitude of cellular processes and development. cAMP regulates morphogenesis and virulence in a variety of fungi . Adenylyl cyclase anchored in membrane is responsible for catalyzing the conversion of ATP to cAMP . Recent studies indicate that adenylate cyclase is required for normal vegetative growth, infection structure formation and virulence in phytopathogenic fungi. The role of adenylate cyclase enzymes has been investigated in several fungal species [10–12]. Magnaporthe oryzae depleted of adenylate cyclase (MAC1) was incapable of penetrating the surface of susceptible rice leaves because it could not form appressoria . In the post-harvest necrotrophic fungus Botrytis cinerea, the deletion of the gene encoding adenylate cyclase reduced intracellular cAMP levels, causing delayed vegetative growth, lesion development and in planta sporulation . An adenylate cyclase (SAC-1) deletion mutant in Sclerotinia sclerotiorum exhibited aberrations in sclerotial initiation, possessed altered oxalate levels, and showed reduced virulence due to the lack of infection cushion formation . Targeted disruption of the adenylate cyclase-coding gene in Fusarium proliferatum retarded vegetative growth, increased conidiation and delayed conidial germination . Although adenylate cyclase plays various roles in a number of fungi, the function of adenylate cyclase in entomopathogenic fungi has not been explored up to date.
In this study, we cloned the full-length cDNA of adenylate cyclase from the locust-specific M. acridum strain, CQMa 102, designated MaAC. The MaAC transcript level of M. acridum was knocked-down by RNAi and the roles of MaAC in pathogenicity and tolerance to stresses were analyzed. Our results showed that MaAC contributed to vegetative growth, virulence and tolerance to various adverse host insect and environmental factors. The results demonstrated that impairment in the virulence of the MaAC RNAi mutant was caused by decreased vegetative growth and tolerance to adverse conditions encountered during host infection.
Isolation and characteristics of MaAC
Knocked-down MaAC transcription by RNAi
MaAC affects growth in vitro
Vegetative growth in vitro was further quantified by assaying the living cells in PD liquid culture by CellTiter 96® AQueous One Solution Assay (Figure 3B). In contrast to the wild type, the growth rate of the AC-RNAi-1 mutant was similar to the wild type, while the other four RNA mutants grew conspicuously slowly (p <0.01). These results indicated that MaAC affects growth in vitro. The correlation coefficient of the relative expression rate and the growth rate was 0.94, which was highly significant (p <0.01). These result showed that the growth rate is related to the relative expression rate of MaAC.
MaAC regulates intracellular cAMP levels in M. acridum
MaAC is required for in vivo virulence and growth
To confirm the effect of MaAC on virulence, fungal growth in vivo was observed by photomicroscopy and quantified by real-time PCR. The M. acridum mutant grew significantly more slowly than the wild type (Figure 6E), which was further confirmed by a quantitative assay. Here, the fungal DNA of the wild type was conspicuously higher (~4 times) than that of the RNAi mutant (Figure 6D). Fungal growth cultured in the haemolymph of the locusta in vitro was also observed by photomicroscopy, which showed that the RNAi mutant grew evidently more slowly than the wild type (Figure 6F). Taken together, these results demonstrate that MaAC affects fungal growth both in vivo and in vitro.
MaAC is involved in the tolerance of M. acridum to oxidative stress and osmotic stress
MaAC affects the tolerance to heat and UV light
Adenylate cyclase regulates a variety of physiological processes in phytopathogenic fungi, including conidiation, conidial germination, vegetative growth, appressoria formation and virulence. In this study, an adenylate cyclase gene, MaAC, was identified in a locust-specific entomopathogenic fungus, M. acridum. Bioinformatic analysis showed that the cloned MaAC had significant similarity to its homolog from M. oryzae and to many other fungal adenylate cyclase genes; the highest degree of similarity was found with the adenylate cyclase of M. anisopliae (98% identity). The cAMP level of the MaAC RNAi mutant was significantly reduced, and the exogenous addition of cAMP could restore the growth of the RNAi mutant, thus confirming that the MaAC gene encodes adenylate cyclase in M. acridum. These results were similar to previous studies on other fungi [10, 12, 14]. Following the deletion of the entire SAC1 coding sequence of S. sclerotiorum, cAMP underwent a four-fold reduction in the SAC1 deletion strain compared to the wild type. In BAC1- and UAC1-defective mutants, intracellular cAMP was detected, which contrasted with the wild type [13, 15].
In this report, the downregulation of MaAC led to inhibited growth on in vitro media, including PDA and Czapek-dox medium. In PD liquid culture, it caused similar effects to previously described adenylate cyclase mutants, such as the SAC1 mutant in S. sclerotiorum and the BAC1 mutant in B. cinerea. Furthermore, MaAC is also involved in the growth of M. acridum inside locusts. The virulence of the MaAC mutant was also significantly reduced, thus indicating that MaAC is required for M. acridum virulence. This finding is consistent with the role of adenylate cyclase in the virulence of other fungi, including M. oryzae, B. cinerea and U. maydis.
Previous research has demonstrated that the tolerance of fungi to stresses such as high temperature , UV-B radiation [8, 16], oxidative  and osmotic stress [4, 5, 17] is a factor that limits their widespread use. The elevated thermo- and H2O2-tolerance of the ΔFpacy1 mutants indicated that the adenylate cyclase may have negative regulatory roles on the stress response mechanisms of fungal cells . However, the tolerance of the RNAi mutant to the osmotic-, H2O2-, UV-B and thermal stress was reduced in this study, thus indicating that MaAC may affect the tolerance to multiple stresses through similar regulatory mechanisms in fungal cells.
Germination and appressoria formation on locust wings
Appressorium formation rateb(%)
33.3 ± 4.7
25.0 ± 5.6
55.7 ± 4.0
40.3 ± 1.5
80.6 ± 6.1*
66.3 ± 6.5*
53.7 ± 5
48.3 ± 3
99.3 ± 1.7
98.0 ± 2.9
79.6 ± 5
77.6 ± 4
An adenylate cyclase encoding gene (MaAC) was cloned from the locust-specific entomopathogenic fungus, M. acridum. MaAC affects virulence and fungal growth inside the insect, and is required for its tolerance to oxidative stress, osmotic stress, heat shock and UV-B radiation. MaAC affects fungal virulence via vegetative growth and tolerance to oxidative stress, osmotic stress and locust fever.
Strain and culture conditions
M. acridum strain CQMa102 was isolated from infected yellow-spined bamboo locusts ( Ceracris kiangsu Tsai) and was used to derive all strains in this study . The conidia were collected after the fungus was cultured on 1/4 strength Sabouraud’s dextrose agar yeast medium (1/4 SDAY; 1% dextrose, 0.25% mycological peptone, 2% agar and 0.5% yeast extract, w/v) at 28°C for 15 d. The medium used for growing mycelia was PD (potato dextrose medium) liquid culture. Czapek-dox medium (3% saccharose, 0.2% NaNO3, 0.1% K2HPO4, 0.05% KCl, 0.05% MgSO4, 0.001% FeSO4) and potato medium (PDA, 20% potato, 2% sucrose, 2% agar) were used for colony phenotype testing.
Gene cloning, phylogenetic analysis and construction of the MaAC RNAi vector
Genomic DNA of M. acidum was extracted as previously described . The genomic DNA sequence was acquired by PCR with the primer pairs (5′-TTCCACGCCAAACCTCAA -3′) and MaAC-R (5′-AGCCAAGTTGTTTCGGTA -3′) from the whole genomic sequence of M. acidum. A full-length cDNA clone of MaAC was amplified using Pyrobest DNA polymerase (TaKaRa, Japan) from a cDNA library of M. acridum established in our laboratory  with gene-specific primers MaAC-F (5′- TTCCACGCCAAACCTCAA -3′) and MaAC-R (5′- AGCCAAGTTGTTTCGGTA -3′). The resulting PCR product was subcloned into the pMD19-T vector, and transformed into E. coli XL-Blue for determination by GenScript (Nanjing, China). To study the function of MaAC, an RNA interference (RNAi) vector was constructed. The partial sequence of MaAC (500 bp) was amplified by MaAC-F (5′- GCGATACACGCCACAAGGACAAAGA-3′) and MaAC-R (5′-CCCAAGCTTACTACCAATCTCATCCACCTC-3′) from M. acridum MaAC cDNA. The resulting PCR product was cloned into pMD19-T (Takara, China) to form pMD19-MaAC. A fragment of MaAC was recovered from pMD19-MaAC by digestion with EcoRI and EcoRV and inserted into the vector pDPB . The fragment PgpdA-MaAC-PtrpC from pDPB was inserted at the site between HindIII and XbaI of pPK2-pB [23, 24] to form pPK2-pB-MaAC-RNAi. Transformation of M. acridum was mediated by Agrobacterium tumefaciens according to the procedure described previously . Transformants were screened on Czapek-dox medium containing 80 μg/mL phosphinothricin (PPT) and incubated at 27°C for 8 d. Transformants were confirmed by PCR amplification of the RNAi cassette.
Real-time quantitative reverse transcript (qRT-PCR) analysis
To confirm the expression levels of MaAC, the wild type and MaAC-RNAi transformants were grown in PD liquid culture for 2 d and the mycelia were collected and washed with sterile water. Total RNA was isolated using the SV Total RNA Isolation System (Promega, USA). The synthesis of cDNA and real-time RT-PCR were performed using the method described by Leng et al. . Primers of MaAC-F (5′- GGACGAAGGACTTGACAGACC-3′) and MaAC-R (5′-CACAGCATCTCCAGACGAGG-3′) were used to detect MaAC expression levels.
Determination of fungal growth
To characterize the role of MaAC in vegetative growth, the growth rate of the wild type and the RNAi mutants were analyzed using CellTiter 96® AQueous One Solution Assay (Promega, USA). In this study, the wild type or RNAi mutants were inoculated in PD liquid culture for 24, 30, 36, 48, 54, 60 and 72 h, respectively. CellTiter 96® AQueous One Solution Reagent (20 μL) and 100 μL culture fluid were directly added to the culture wells, the mixture was incubated for 2 h at 37°C, and then the absorbance was recorded at 490 nm with a 96-well plate reader.
The MaAC mutant and the wild type were cultured in PD liquid culture for 36 h. After harvesting, 20 mg mycelia were collected and washed three times with sterile water, followed by treatment with 2 mL 0.01 M PBS. Samples were then lyophilized and dissolved in the mixture. They were centrifuged at 4,000 rpm for 20 min and the supernatant was collected for the test. The cAMP levels were measured with cAMP Enzyme Immunoassay Kit (Sigma, USA), according to the manufacturer’s instructions. In total, each assay was repeated three times independently with three biological replicates for every strain.
To test whether exogenous cAMP could restore the growth of RNAi mutant, the cAMP analog, 8-Br-cAMP (Sigma, USA) was added to PDA at a final concentration of 5 mM. 8-Br-cAMP (a membrane permeable variant of cAMP) has been extensively used in various studies to artificially cause the enhancement of endogenous cAMP levels [27–29].
Biomass assay and fungal growth in the haemolymph of locust in vivo and in vitro
The virulence of the RNAi mutant and the wild type was tested by topical inoculation and injection into Locusta migratoria adults reared under crowded conditions as previously described by He et al. . The Locusta migratoria used were all male adult 3 days post-molt. Wild type and RNAi mutants were incubated at 28°C on 1/4 SDAY plates for 15 d. Aliquots of 5 μL solution of 107 conidia/mL of either wild type M. acridum or RNAi mutant in cottonseed oil were inoculated on the pronotum. Aliquots of 5 μL suspensions (2 × 106 conidia/mL) in sterile water were injected into the hemocoel. Both experiments were repeated five times with 30 insects per replicate. Mortality was recorded every 12 h after topical inoculation and injection. Mortality was then recorded daily, and lethal time values for 50% mortality (LT50) values were used to estimate the infectivity of M. acridum by DPS software . The growth of M. acridum in the host locust was quantified by the detection of fungal rDNA in the infected locust using real-time PCR . After the extraction of M. acridum DNA and fungal DNA from the infected locust, fungal DNA was detected by an Icycler iQ Quantitative PCR was performed using specific primers of M. acridum: CQMaP-F1: 5′-TGGCATCTTCTGAGTGGTG-3′and CQMaP-R1: 5′-CCCGTTGCGAGTGAGTTA- 3′. To test the fungal growth in the haemolymph of locust in vitro, 50 μL of a conidial suspension (1 × 107 conidia/mL) was inoculated into 950 μL of locust haemolymph, and the growth of the wild type and mutant was detected 24 h post inoculation.
Germination and appressoria formation against insect cuticles
The percentage of germination of wild type and RNAi mutant were measured as described by Liu et al.. The appressorium formation rates were determined from 300 conidia after an 18 h induction on locust hind wings according to He and Xia . The assay was replicated at least three times.
Oxidative stress, osmotic stress, heat shock and UV-B treatment test
Growth characterization of the wild type and RNAi mutants were carried out on 1/4 SDAY supplemented with H2O2 (6 mM) or KCl (1 M). Samples of conidial suspensions (2 μL; 5 × 105 conidia/mL) were spotted on each Petri dish and the plates were incubated at 28°C for 10 d. The tolerance to wet-heat shock and UV-B treatment of the RNAi mutant and wild type was analyzed as described by Liu et al. .
1/4 strength Sabouraud’s dextrose agar medium
Potato dextrose medium
Potato dextrose agar medium
Phosphate Buffered Saline
Lethal time of 50%
Data Processing Standards.
This work was supported by a grant from the National Natural Science Foundation of China (No. 30771446) and High Technology Research and Development Program (863) of China (No. 2011AA10A204).
- St Leger RJ, Joshi L, Bidochka MJ, Roberts DW: Construction of an improved mycoinsecticide overexpressing a toxic protease. Proc Natl Acad Sci. 1996, 93: 6349-6354. 10.1073/pnas.93.13.6349.PubMedPubMed CentralView Article
- Weiguo F, Monica P, Sibao W, St Leger R: Protein kinase A regulates production of pathogenicity determinants by the entomopathogenic fungus, Metarhizium anisopliae. Fungal Genet Biol. 2009, 46: 277-285. 10.1016/j.fgb.2008.12.001.View Article
- Charnley AK, St Leger RJ: The role of cuticle-degrading enzymes in fungal pathogenesis in insects. 1991, Plenum Press, New York, 267-287.
- Yueqing C, Min L, Yuxian X: Mapmi gene contributes to stress tolerance and pathogenicity of the entomopathogenic fungus, Metarhizium acridum. J Invertebr Pathol. 2011, 108: 7-12. 10.1016/j.jip.2011.06.002.View Article
- Wang CS, Duan ZB, St Leger RJ: MOS1 osmosensor of Metarhizium anisopliae is required for adaptation to insect host hemolymph. Eukaryot Cell. 2008, 7: 302-309. 10.1128/EC.00310-07.PubMedPubMed CentralView Article
- Inglis GD, Johnson DL, Goettel MS: Effects of temperature and thermoregulation on mycosis by Beauveria bassiana in grasshoppers. Biol Contr. 1996, 7: 131-139. 10.1006/bcon.1996.0076.View Article
- Lock GD, Pickering SG, Charnley AK: Application of infrared thermography to the study of behavioural fever in the desert locust. J Therm Biol. 2011, 36: 443-451. 10.1016/j.jtherbio.2011.07.008.View Article
- Rangel DEN, Braga GUL, Flint SD, Anderson AJ, Roberts DW: Variations in UV-B tolerance and germination speed of Metarhizium anisopliae conidia produced on insects and artificial substrates. J Invertebr Pathol. 2004, 87: 77-83. 10.1016/j.jip.2004.06.007.PubMedView Article
- Lee N, D’Souza CA, Kronstadt JW: Of smuts, mildews, and blights: cAMP signaling in phytopathogenic fungi. Annu Rev Phytopathol. 2003, 41: 399-427. 10.1146/annurev.phyto.41.052002.095728.PubMedView Article
- Wayne MJ, JeVrey AR: Deletion of the adenylate cyclase (sac1) gene aVects multiple developmental pathways and pathogenicity in Sclerotinia sclerotiorum. Fungal Genet Biol. 2007, 44: 521-530. 10.1016/j.fgb.2006.11.005.View Article
- Choi W, Dean RA: The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development. Plant Cell. 1997, 9: 1973-83.PubMedPubMed CentralView Article
- Klimpel A, Gronover CS, Williamson B, Stewart JA, Tudzinski B: The adenylate cyclase (BAC) in Botrytis cinerea is required for full pathogenicity. Mol Plant Pathol. 2002, 3: 439-450. 10.1046/j.1364-3703.2002.00137.x.PubMedView Article
- Gábor K, Brigitta O, Attila LÁ, García-Martínez J, Hornok L: Adenylyl cyclase regulates heavy metal sensitivity, bikaverin production and plant tissue colonization in Fusarium proliferatum. J Basic Microbiol. 2010, 50: 59-71. 10.1002/jobm.200900113.View Article
- Gold SE, Duncan GA, Barret KJ, Kronstad JW: cAMP regulates morphogenesis in the fungal pathogen Ustilago maydis. Genes Dev. 1994, 8: 2805-2816. 10.1101/gad.8.23.2805.PubMedView Article
- Alfredo DM, Jose R, Claudia GL, Scott EG: MAP Kinase and cAMP Signaling Pathways Modulate the pH-Induced Yeast-to-Mycelium Dimorphic Transition in the Corn Smut Fungus Ustilago maydis. Curr Microbiol. 2004, 49: 274-281. 10.1007/s00284-004-4315-6.View Article
- Braga GUL, Flint SD, Messias CL, Anderson AJ, Roberts DW: Effect of UV-B on conidia and germlings of the entomopathogenic hyphomycete Metarhizium anisopliae. Mycol Res. 2001, 105: 874-882. 10.1017/S0953756201004270.View Article
- Hallsworth JE, Magan N: Effects of KCl concentration on accumulation of acyclic sugar alcohols and trehalose in conidia of three entomopathogenic fungi. Lett Appl Microbiol. 1994, 18: 8-11. 10.1111/j.1472-765X.1994.tb00785.x.View Article
- Peng G, Wang Z, Yin Y, Zeng D, Xia Y: Field trials of Metarhizium anisopliae var. acridum (Ascomycota: Hypocreales) against oriental migratory locusts, Locusta migratoria manilensis (Meyen) in Northern China. Crop Prot. 2008, 27: 1244-1250. 10.1016/j.cropro.2008.03.007.View Article
- Reader U, Broda P: Rapid preparation of DNA from filamentous fungi. Lett Appl Microbiol. 1985, 1: 17-20. 10.1111/j.1472-765X.1985.tb01479.x.View Article
- Qiang G, Kai J, Sheng-Hua Y, Yongjun Z, Guohua X, Yanfang S, Zhibing D, Xiao H, Xue-Qin X, Gang Z, Guoxiong P, Zhibing L, Wei H, Bing W, Weiguo F, Sibao W, Yi Z, Li-Jun M, Raymond J, St L, Guo-Ping Z, Yan P, Ming-Guang F, Yuxian X, Chengshu W: Genome Sequencing and Comparative Transcriptomics of the Model Entomopathogenic Fungi Metarhizium anisopliae and M. acridum. PLoS Genet. 2011, 7: 1-
- Zhang C, Xia Y: Identification of genes differentially expressed in vivo by Metarhizium anisopliae in the hemolymph of Locusta migratoria using suppression subtractive hybridization. Curr Genet. 2009, 55: 399-407. 10.1007/s00294-009-0254-x.PubMedView Article
- Liu J, Cao Y, Xia Y: Mmc, a gene involved in microcycle conidiation of the entomopathogenic fungus Metarhizium anisopliae. J Invertebr Pathol. 2010, 105: 132-138. 10.1016/j.jip.2010.05.012.PubMedView Article
- Hervás-Aguilar A, Rodríguez JM, Tilburn J, Arst HN, Peñalva MA: Evidence for the direct involvement of the proteasome in the proteolytic processing of the Aspergillus nidulans zinc finger transcription factor PacC. J Biol Chem. 2007, 282: 34735-34747. 10.1074/jbc.M706723200.PubMedView Article
- Lodi T, Fontanesi F, Guiard B: Co-ordinate regulation of lactate metabolism genes in yeast: the role of the lactate permease gene JEN1. Mol Genet Genomics. 2002, 266: 838-847. 10.1007/s00438-001-0604-y.PubMedView Article
- Fang W, Zhang Y, Yang X, Zheng X, Duan H, Li Y, Pei Y: Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker. J Invertebr Pathol. 2004, 85: 18-24. 10.1016/j.jip.2003.12.003.PubMedView Article
- Leng Y, Peng G, Cao Y, Xia Y: Genetically altering the expression of neutral trehalase gene affects conidiospore thermotolerance of the entomopathogenic fungus Metarhizium acridum. BMC Microbiol. 2011, 11: 32-10.1186/1471-2180-11-32.PubMedPubMed CentralView Article
- Hao L, Angayarkanni S, Willard FS, Siderovski DP, Shen L, Naqvi NI: Rgs1 regulates multiple Galpha subunits in Magnaporthe pathogenesis, asexual growth and thigmotropism. EMBO J. 2007, 26: 690-700. 10.1038/sj.emboj.7601536.View Article
- Skamnioti P, Gurr SJ: Magnaporthe grisea cutinase2 mediates appressorium differentiation and host penetration and is required for full virulence. Plant Cell. 2007, 19: 2674-2689. 10.1105/tpc.107.051219.PubMedPubMed CentralView Article
- Ravikrishna R, Naqvi NI: PdeH, a High-Affinity cAMP Phosphodiesterase, Is a Key Regulator of Asexual and Pathogenic Differentiation in Magnaporthe oryzae. PLoS Pathog. 2010, 6: 5-
- He ZB, Cao YQ, Yin YP, Wang ZK, Chen B, Peng GX, Xia YX: Role of hunchback in segment patterning of Locusta migratoria manilensis revealed by parental RNAi. Dev Growth Differ. 2006, 48: 439-445. 10.1111/j.1440-169X.2006.00881.x.PubMedView Article
- Tang QY, Feng MG: DPS Data Processing System for Practical Analysis. 2002, Science Press, Beijing, 1-648.
- Peng G, Xia Y: The mechanism of the mycoinsecticide diluents on the efficacy of the oil formulation of insecticidal fungus. BioControl. 2011, 56: 893-902. 10.1007/s10526-011-9360-z.View Article
- He M, Xia Y: Construction and analysis of a normalized cDNA library from Metarhizium anisopliae var. acridum germinating and differentiating on Locusta migratoria wings. FEMS Microbiol Lett. 2009, 291: 127-135. 10.1111/j.1574-6968.2008.01447.x.PubMedView Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.