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Genetic improvement of the nematicidal fungus Lecanicillium attenuatum against Heterodera glycines by expression of the Beauveria bassiana Cdep1 protease gene
Journal of Invertebrate Pathology 138 (2016) 86–88
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Genetic improvement of the nematicidal fungus Lecanicillium attenuatum against Heterodera glycines by expression of the Beauveria bassiana Cdep1 protease gene Ming Xie, Yan-Jun Zhang ⇑, Xiao-Lin Zhang, De-Liang Peng, Wen-Bin Yu, Qian Li State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 4 May 2016 Revised 17 June 2016 Accepted 20 June 2016 Available online 21 June 2016 Keywords: Nematophagous fungi Genetic engineering Protease Soybean cyst nematode
a b s t r a c t Lecanicillium attenuatum is an important nematophagous fungus with potential as a biopesticide against plant-parasitic nematodes. The Pr1A-like cuticle-degrading protease (Cdep1) gene originating from the entomopathogenic fungus Beauveria bassiana was transformed into the nematophagous fungus L. attenuatum using a polyethylene-glycol mediated protoplast-based transformation system. Protease activity was increased 0.64- to 1.63-fold 2–10 d after growth in the transformed L. attenuatum. Inhibition of egg-hatching and J2 motility of soybean cyst nematodes (Heterodera glycines) by cell-free fungal culture filtrates were enhanced by 17–76% 2–14 d and 43–152% 1–13 d after incubation, respectively. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Soybean cyst nematodes (SCN, Heterodera glycines Ichinohe) are amongst the most destructive soybean-parasitic nematodes in the world. Due to environmental concerns with respect to the largescale use of chemical nematicides and severe limitations on rotated crops, nematophagous fungi are considered as effective biological agents for control of plant pathogenic nematodes (Yang and Zhang, 2014). However, the low nematicidal activity and instability in the field have hampered the application of nematophagous fungi. Proteases are important in nematode-cuticle penetration and host-cell digestion (Åhman et al., 2002; Yang et al., 2007). Previous studies have shown that biological control potential of nematophagous fungi can be improved by increasing fungal pathogenicity through genetic engineering (Åhman et al., 2002; Yang et al., 2011), e.g., Arthrobotrys oligospora or Paecilomyces lilacinus were transformed with the PII or Ver112 protease gene respectively. These enzymes originated from the nematophagous fungi A. oligospora and L. psalliotae respectively, and the transformed strains had higher protease and nematicidal activities. Furthermore, expression of the Beauveria bassiana Pr1A-like ⇑ Corresponding author at: Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 12, Zhong-Guan-Cun Nan-Da-Jie, Haidian, Beijing 100081, People’s Republic of China. E-mail address: [email protected] (Y.-J. Zhang). http://dx.doi.org/10.1016/j.jip.2016.06.008 0022-2011/Ó 2016 Elsevier Inc. All rights reserved.
cuticle-degrading protease (Cdep1) gene in Lecanicillium lecanii resulted in greater virulence to insects (Zhang et al., 2016). Thus, genetic engineering has proved to be a powerful tool with which to manipulate fungal virulence particularly of insect and nematode pathogens to improve their efficacy (Fan et al., 2012; Ortiz-Urquiza et al., 2015). Lecanicillium attenuatum is an important nematophagous fungus with potential as a biopesticide against SCN (Chen and Chen, 2003). Our objective was to transfer the Cdep1 gene from B. bassiana into L. attenuatum, and determine whether the pathogenicity of L. attenuatum against SCN can be enhanced by heterologous expression of the Cdep1 protease gene. 2. Materials and methods 2.1. Microbial strains L. attenuatum and B. bassiana were deposited in the China General Microbiological Culture Collection Center (CGMCC) as the strain code CGMCC No. 9220 and No. 8461 respectively. 2.2. Nucleic acid extraction and plasmid construction DNA and RNA were extracted from fungal mycelia as described previously (Zhang et al., 2016). The gdpA promoter (PgdpA) and the fungal trpC terminator (TtrpC) were amplified from the pAN7-1 plasmid, the ORF of the Cdep1 gene was amplified from
M. Xie et al. / Journal of Invertebrate Pathology 138 (2016) 86–88
B. bassiana, and these three fragments were fused to form the PgdpA-Cdep-TtrpC (PCT), and then the expression vector of pBHt2PCT was produced as described previously (Zhang et al., 2016).
87
2.3. Protoplast preparation and transformation, and verification of transformants Protoplast preparation of L. attenuatum was performed as described for L. lecanii (Zhang et al., 2016) with some modifications; mycelia were used 14 h after inoculation of conidia and an enzymatic digestion time of 6 h was used. Transformation and verification of transformants were performed as described for L. lecanii (Zhang et al., 2016). 2.4. Biological characteristics, protease and nematicidal activities of transformants
Fig. 1. Molecular verification of transformants. PCR analysis (A). RT-PCR analysis (B). Western blotting analysis (C).
Colony growth, conidial yield and germination rates on PDA plates were tested as described for L. lecanii (Xie et al., 2015). Fungal conidia were incubated in protease-inducing (PI) liquid medium and protease activity was measured as described previously (Yang et al., 2007). Soybean cyst nematodes (SCN, H. glycines) were kept on the soybean cultivar of ‘Heihe-13’ on the Langfang
Fig. 2. Biological characteristics, protease and nematicidal activities of L. attenuatum. Colony growth (A). Conidia yield (B). Conidia germination (C). Protease activities in culture filtrates at various time points (D). Inhibition of hatching of SCN mature eggs by cell-free culture filtrates (E). Inhibition of SCN J2 motility by cell-free culture filtrates (F). The values were means ± standard deviation of five replicates. Different letters on error bars indicate significant difference (Tukey’s HSD, P < 0.05).
88
M. Xie et al. / Journal of Invertebrate Pathology 138 (2016) 86–88
Experimental Farm (in Hebei Province). SCN cysts were collected from the infested soils, and inhibition of hatching of mature eggs by cell-free fungal culture filtrates (i.e. without mycelia or conidia) and inhibition of the motility of second stage juveniles (J2) of SCN were determined as described previously (Shinya et al., 2008).
provide an example of increasing the nematicidal activity of L. attenuatum via expression of a protease implicated in insect virulence from B. bassiana (Zhang et al., 2008; Fan et al., 2010), paving the way for future studies improving the efficiency of using fungi for nematode control.
2.5. Data analysis
Acknowledgments
Data were subjected to the analyses of variance (ANOVA). The means were compared by the Tukey’s HSD test when the F-tests were statistically significant (P = 0.05). All analyses were run in the SPSS 19.0 software (SPSS Inc., Chicago, USA).
We thank Dr. Nan-Jun Wu (Vanderbilt University) for the critical revision of this manuscript. We also acknowledge supports from the Special Fund for Agri-scientific Research in the Public Interest of China (201503114), National Natural Science Foundation of China (31201571), Ministry of Science and Technology of China (2011AA10A203/204), National Key Technology Support Program (2012BAD15B03), International Cooperation Project of China (S2016G5103), National Special Program for Basic Work of Science and Technology (2006FY111000) and Agricultural Science and Technology Innovation Program (ASTIP).
3. Results and discussion The Cdep1 gene was successfully integrated into the L. attenuatum fungal genome (Fig. 1A), and the gene was transcribed (Fig. 1B) and expressed in the transformant (Fig. 1C). Colony growth, conidia yield and germination of the transformant on PDA plates were not significantly different from the wild-type strain (Fig. 2A–C). Serine proteases, secreted by nematophagous fungi, can degrade proteins in nematode cuticles facilitating fungal penetration into the nematode body (Yang and Zhang, 2014). Overexpression of the PII and Ver112 protease gene increased nematicidal activities of A. oligospora or P. lilacinus (Åhman et al., 2002; Yang et al., 2011). Here, we demonstrate that expression of the Cdep1 gene from the entomopathogenic fungus B. bassiana improved fungal virulence of the nematophagous fungus L. attenuatum to SCN. Overall protease activity was 2–3 fold higher in the transformant as compared to the wild type parent and reached the highest in PI medium on the fifth day of growth after incubation in the transformant. Onset of protease activity was earlier (at 3 d, i.e. 1 d earlier) in the transformant compared to the wild-type strain (Fig. 2D). Protease activity appeared to level-off and remained essentially constant over the time course of the experiment for both strains. A similar dynamics of protease activity was reported by Yang et al. (2011), in which the highest protease activity occurred on the sixth day of growth after incubation in a P. lilacinus strain expressing the Ver112 protease gene, while the wild-type strain showed the highest activity levels on the seventh day of growth after incubation. In order to examine whether cell-free culture supernatants could display biological activity against nematodes, culture supernatants derived from cells grown in PI medium for 6 d were prepared and assayed for their ability to inhibit nematode egg-hatching and to block the motility of nematode juveniles. Cell-free fungal culture filtrates derived from the Cdep1expressing L. attenuatum strain showed 17–76% increased inhibition of nematode egg-hatching activity 2–14 d after incubation as compared to the wild-type strain. In addition, inhibition of the motility of nematode J2 juveniles was increased by 43–152% 1–13 d after incubation (Fig. 2E and F). These data
References Åhman, J., Johanson, T., Olsson, M., Punt, P.J., van den Hondel, C.A.M.J.J., Tunlid, A.S., 2002. Improving the pathogenicity of a nematodetrapping fungus by genetic engineering of a subtilisin with nematotoxic activity. Appl. Environ. Microbiol. 689, 3408–3415. Chen, S.Y., Chen, F.J., 2003. Fungal parasitism of Heterodera glycines eggs as influenced by egg age and pre-colonization of cysts by other fungi. J. Nematol. 35, 271–277. Fan, Y.H., Borovsky, D., Hawkings, C., Ortiz-Urquiza, A., Keyhani, N.O., 2012. Exploiting host molecules to augment mycoinsecticide virulence. Nat. Biotechnol. 30, 35–37. Fan, Y.H., Pei, X.Q., Guo, S.J., Zhang, Y.J., Luo, Z.B., Liu, X.G., Pei, Y., 2010. Increased virulence using engineered protease-chitin binding domain hybrid expressed in the entomopathogenic fungus Beauveria bassiana. Microbial Pathogen. 49, 376– 380. Ortiz-Urquiza, A., Luo, Z.B., Keyhani, N.O., 2015. Improving mycoinsecticides for insect biological control. Appl. Microbiol. Biotechnol. 99, 1057–1068. Shinya, R., Aiuchi, D., Kushida, A., Tani, M., Kuramochi, K., Koike, M., 2008. Effects of fungal culture filtrates of Verticillium lecanii (Lecanicillium spp.) hybrid strains on Heterodera glycines eggs and juveniles. J. Invertebr. Pathol. 97, 291–297. Xie, M., Zhang, Y.J., Peng, D.L., Zhou, J., Zhang, X.L., Zhang, Z.R., Zhao, J.J., Wu, Y.H., 2015. Persistence and viability of Lecanicillium lecanii in Chinese agricultural soil. PLoS ONE 10, e0138337. Yang, J.K., Zhang, K.Q., 2014. Biological control of plant-parasitic nematodes by nematophagous fungi. In: Zhang, K.Q., Hyde, K.D. (Eds.), Nematode-Trapping Fungi. Springer, Dordrecht, the Netherlands, pp. 231–262. Yang, J.K., Tian, B.Y., Liang, L.M., Zhang, K.Q., 2007. Extracellular enzymes and the pathogenesis of nematophagous fungi. Appl. Microbiol. Biotechnol. 75, 21–31. Yang, J.K., Zhao, X.N., Liang, L.M., Xia, Z.Y., Lei, L.P., Niu, X.M., Zou, C.G., Zhang, K.Q., 2011. Overexpression of a cuticle-degrading protease Ver112 increases the nematicidal activity of Paecilomyces lilacinus. Appl. Microbiol. Biotechnol. 89, 1895–1903. Zhang, Y.J., Feng, M.G., Fan, Y.H., Luo, Z.B., Yang, X.Y., Wu, D., Pei, Y., 2008. A cuticledegrading protease (CDEP-1) of Beauveria bassiana enhances virulence. Biocontr. Sci. Technol. 18, 543–555. Zhang, Y.J., Xie, M., Zhao, X.L., Peng, D.L., Yu, W.B., Li, Q., Li, Q., Zhao, J.J., Zhang, Z.R., 2016. Establishment of polyethylene-glycol mediated protoplast transformation for Lecanicillium lecanii and development of virulenceenhanced strains against Aphis gossypii. Pest Manag. Sci. 2016. http://dx.doi. org/10.1002/ps.4236.
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Genetic improvement of the nematicidal fungus Lecanicillium attenuatum against Heterodera glycines by expression of the Beauveria bassiana Cdep1 protease gene Ming Xie, Yan-Jun Zhang ⇑, Xiao-Lin Zhang, De-Liang Peng, Wen-Bin Yu, Qian Li State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 4 May 2016 Revised 17 June 2016 Accepted 20 June 2016 Available online 21 June 2016 Keywords: Nematophagous fungi Genetic engineering Protease Soybean cyst nematode
a b s t r a c t Lecanicillium attenuatum is an important nematophagous fungus with potential as a biopesticide against plant-parasitic nematodes. The Pr1A-like cuticle-degrading protease (Cdep1) gene originating from the entomopathogenic fungus Beauveria bassiana was transformed into the nematophagous fungus L. attenuatum using a polyethylene-glycol mediated protoplast-based transformation system. Protease activity was increased 0.64- to 1.63-fold 2–10 d after growth in the transformed L. attenuatum. Inhibition of egg-hatching and J2 motility of soybean cyst nematodes (Heterodera glycines) by cell-free fungal culture filtrates were enhanced by 17–76% 2–14 d and 43–152% 1–13 d after incubation, respectively. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Soybean cyst nematodes (SCN, Heterodera glycines Ichinohe) are amongst the most destructive soybean-parasitic nematodes in the world. Due to environmental concerns with respect to the largescale use of chemical nematicides and severe limitations on rotated crops, nematophagous fungi are considered as effective biological agents for control of plant pathogenic nematodes (Yang and Zhang, 2014). However, the low nematicidal activity and instability in the field have hampered the application of nematophagous fungi. Proteases are important in nematode-cuticle penetration and host-cell digestion (Åhman et al., 2002; Yang et al., 2007). Previous studies have shown that biological control potential of nematophagous fungi can be improved by increasing fungal pathogenicity through genetic engineering (Åhman et al., 2002; Yang et al., 2011), e.g., Arthrobotrys oligospora or Paecilomyces lilacinus were transformed with the PII or Ver112 protease gene respectively. These enzymes originated from the nematophagous fungi A. oligospora and L. psalliotae respectively, and the transformed strains had higher protease and nematicidal activities. Furthermore, expression of the Beauveria bassiana Pr1A-like ⇑ Corresponding author at: Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 12, Zhong-Guan-Cun Nan-Da-Jie, Haidian, Beijing 100081, People’s Republic of China. E-mail address: [email protected] (Y.-J. Zhang). http://dx.doi.org/10.1016/j.jip.2016.06.008 0022-2011/Ó 2016 Elsevier Inc. All rights reserved.
cuticle-degrading protease (Cdep1) gene in Lecanicillium lecanii resulted in greater virulence to insects (Zhang et al., 2016). Thus, genetic engineering has proved to be a powerful tool with which to manipulate fungal virulence particularly of insect and nematode pathogens to improve their efficacy (Fan et al., 2012; Ortiz-Urquiza et al., 2015). Lecanicillium attenuatum is an important nematophagous fungus with potential as a biopesticide against SCN (Chen and Chen, 2003). Our objective was to transfer the Cdep1 gene from B. bassiana into L. attenuatum, and determine whether the pathogenicity of L. attenuatum against SCN can be enhanced by heterologous expression of the Cdep1 protease gene. 2. Materials and methods 2.1. Microbial strains L. attenuatum and B. bassiana were deposited in the China General Microbiological Culture Collection Center (CGMCC) as the strain code CGMCC No. 9220 and No. 8461 respectively. 2.2. Nucleic acid extraction and plasmid construction DNA and RNA were extracted from fungal mycelia as described previously (Zhang et al., 2016). The gdpA promoter (PgdpA) and the fungal trpC terminator (TtrpC) were amplified from the pAN7-1 plasmid, the ORF of the Cdep1 gene was amplified from
M. Xie et al. / Journal of Invertebrate Pathology 138 (2016) 86–88
B. bassiana, and these three fragments were fused to form the PgdpA-Cdep-TtrpC (PCT), and then the expression vector of pBHt2PCT was produced as described previously (Zhang et al., 2016).
87
2.3. Protoplast preparation and transformation, and verification of transformants Protoplast preparation of L. attenuatum was performed as described for L. lecanii (Zhang et al., 2016) with some modifications; mycelia were used 14 h after inoculation of conidia and an enzymatic digestion time of 6 h was used. Transformation and verification of transformants were performed as described for L. lecanii (Zhang et al., 2016). 2.4. Biological characteristics, protease and nematicidal activities of transformants
Fig. 1. Molecular verification of transformants. PCR analysis (A). RT-PCR analysis (B). Western blotting analysis (C).
Colony growth, conidial yield and germination rates on PDA plates were tested as described for L. lecanii (Xie et al., 2015). Fungal conidia were incubated in protease-inducing (PI) liquid medium and protease activity was measured as described previously (Yang et al., 2007). Soybean cyst nematodes (SCN, H. glycines) were kept on the soybean cultivar of ‘Heihe-13’ on the Langfang
Fig. 2. Biological characteristics, protease and nematicidal activities of L. attenuatum. Colony growth (A). Conidia yield (B). Conidia germination (C). Protease activities in culture filtrates at various time points (D). Inhibition of hatching of SCN mature eggs by cell-free culture filtrates (E). Inhibition of SCN J2 motility by cell-free culture filtrates (F). The values were means ± standard deviation of five replicates. Different letters on error bars indicate significant difference (Tukey’s HSD, P < 0.05).
88
M. Xie et al. / Journal of Invertebrate Pathology 138 (2016) 86–88
Experimental Farm (in Hebei Province). SCN cysts were collected from the infested soils, and inhibition of hatching of mature eggs by cell-free fungal culture filtrates (i.e. without mycelia or conidia) and inhibition of the motility of second stage juveniles (J2) of SCN were determined as described previously (Shinya et al., 2008).
provide an example of increasing the nematicidal activity of L. attenuatum via expression of a protease implicated in insect virulence from B. bassiana (Zhang et al., 2008; Fan et al., 2010), paving the way for future studies improving the efficiency of using fungi for nematode control.
2.5. Data analysis
Acknowledgments
Data were subjected to the analyses of variance (ANOVA). The means were compared by the Tukey’s HSD test when the F-tests were statistically significant (P = 0.05). All analyses were run in the SPSS 19.0 software (SPSS Inc., Chicago, USA).
We thank Dr. Nan-Jun Wu (Vanderbilt University) for the critical revision of this manuscript. We also acknowledge supports from the Special Fund for Agri-scientific Research in the Public Interest of China (201503114), National Natural Science Foundation of China (31201571), Ministry of Science and Technology of China (2011AA10A203/204), National Key Technology Support Program (2012BAD15B03), International Cooperation Project of China (S2016G5103), National Special Program for Basic Work of Science and Technology (2006FY111000) and Agricultural Science and Technology Innovation Program (ASTIP).
3. Results and discussion The Cdep1 gene was successfully integrated into the L. attenuatum fungal genome (Fig. 1A), and the gene was transcribed (Fig. 1B) and expressed in the transformant (Fig. 1C). Colony growth, conidia yield and germination of the transformant on PDA plates were not significantly different from the wild-type strain (Fig. 2A–C). Serine proteases, secreted by nematophagous fungi, can degrade proteins in nematode cuticles facilitating fungal penetration into the nematode body (Yang and Zhang, 2014). Overexpression of the PII and Ver112 protease gene increased nematicidal activities of A. oligospora or P. lilacinus (Åhman et al., 2002; Yang et al., 2011). Here, we demonstrate that expression of the Cdep1 gene from the entomopathogenic fungus B. bassiana improved fungal virulence of the nematophagous fungus L. attenuatum to SCN. Overall protease activity was 2–3 fold higher in the transformant as compared to the wild type parent and reached the highest in PI medium on the fifth day of growth after incubation in the transformant. Onset of protease activity was earlier (at 3 d, i.e. 1 d earlier) in the transformant compared to the wild-type strain (Fig. 2D). Protease activity appeared to level-off and remained essentially constant over the time course of the experiment for both strains. A similar dynamics of protease activity was reported by Yang et al. (2011), in which the highest protease activity occurred on the sixth day of growth after incubation in a P. lilacinus strain expressing the Ver112 protease gene, while the wild-type strain showed the highest activity levels on the seventh day of growth after incubation. In order to examine whether cell-free culture supernatants could display biological activity against nematodes, culture supernatants derived from cells grown in PI medium for 6 d were prepared and assayed for their ability to inhibit nematode egg-hatching and to block the motility of nematode juveniles. Cell-free fungal culture filtrates derived from the Cdep1expressing L. attenuatum strain showed 17–76% increased inhibition of nematode egg-hatching activity 2–14 d after incubation as compared to the wild-type strain. In addition, inhibition of the motility of nematode J2 juveniles was increased by 43–152% 1–13 d after incubation (Fig. 2E and F). These data
References Åhman, J., Johanson, T., Olsson, M., Punt, P.J., van den Hondel, C.A.M.J.J., Tunlid, A.S., 2002. Improving the pathogenicity of a nematodetrapping fungus by genetic engineering of a subtilisin with nematotoxic activity. Appl. Environ. Microbiol. 689, 3408–3415. Chen, S.Y., Chen, F.J., 2003. Fungal parasitism of Heterodera glycines eggs as influenced by egg age and pre-colonization of cysts by other fungi. J. Nematol. 35, 271–277. Fan, Y.H., Borovsky, D., Hawkings, C., Ortiz-Urquiza, A., Keyhani, N.O., 2012. Exploiting host molecules to augment mycoinsecticide virulence. Nat. Biotechnol. 30, 35–37. Fan, Y.H., Pei, X.Q., Guo, S.J., Zhang, Y.J., Luo, Z.B., Liu, X.G., Pei, Y., 2010. Increased virulence using engineered protease-chitin binding domain hybrid expressed in the entomopathogenic fungus Beauveria bassiana. Microbial Pathogen. 49, 376– 380. Ortiz-Urquiza, A., Luo, Z.B., Keyhani, N.O., 2015. Improving mycoinsecticides for insect biological control. Appl. Microbiol. Biotechnol. 99, 1057–1068. Shinya, R., Aiuchi, D., Kushida, A., Tani, M., Kuramochi, K., Koike, M., 2008. Effects of fungal culture filtrates of Verticillium lecanii (Lecanicillium spp.) hybrid strains on Heterodera glycines eggs and juveniles. J. Invertebr. Pathol. 97, 291–297. Xie, M., Zhang, Y.J., Peng, D.L., Zhou, J., Zhang, X.L., Zhang, Z.R., Zhao, J.J., Wu, Y.H., 2015. Persistence and viability of Lecanicillium lecanii in Chinese agricultural soil. PLoS ONE 10, e0138337. Yang, J.K., Zhang, K.Q., 2014. Biological control of plant-parasitic nematodes by nematophagous fungi. In: Zhang, K.Q., Hyde, K.D. (Eds.), Nematode-Trapping Fungi. Springer, Dordrecht, the Netherlands, pp. 231–262. Yang, J.K., Tian, B.Y., Liang, L.M., Zhang, K.Q., 2007. Extracellular enzymes and the pathogenesis of nematophagous fungi. Appl. Microbiol. Biotechnol. 75, 21–31. Yang, J.K., Zhao, X.N., Liang, L.M., Xia, Z.Y., Lei, L.P., Niu, X.M., Zou, C.G., Zhang, K.Q., 2011. Overexpression of a cuticle-degrading protease Ver112 increases the nematicidal activity of Paecilomyces lilacinus. Appl. Microbiol. Biotechnol. 89, 1895–1903. Zhang, Y.J., Feng, M.G., Fan, Y.H., Luo, Z.B., Yang, X.Y., Wu, D., Pei, Y., 2008. A cuticledegrading protease (CDEP-1) of Beauveria bassiana enhances virulence. Biocontr. Sci. Technol. 18, 543–555. Zhang, Y.J., Xie, M., Zhao, X.L., Peng, D.L., Yu, W.B., Li, Q., Li, Q., Zhao, J.J., Zhang, Z.R., 2016. Establishment of polyethylene-glycol mediated protoplast transformation for Lecanicillium lecanii and development of virulenceenhanced strains against Aphis gossypii. Pest Manag. Sci. 2016. http://dx.doi. org/10.1002/ps.4236.