Extraction and Characterization of Cuticle Degrading Enzymes of Beauveria Bassiana for Enhanced Pathogenicity against Bactrocera Dorsalis

Sana Ullah, Hafsa Naeem, Aroma Murtaza, Umer Sharif, Sana Sarfaraz, Farhan Ali, Sami Ullah, Ghulam Ahmad Khan Sumbal, Ubaid Ullah, Mamoona Kanwal


Beauveria bassiana is a promising candidate for the biocontrol of many important insect pests. The cuticle is the site of attachment of fungal spores after invading the host body. Cuticle-degrading enzymes (CDE) are a set of enzymes possessed by entomopathogenic fungi that ensure successful penetration. The current study indicated the extraction of crude cuticle-degrading enzymes from B. bassiana, as a mixture of mycelium and CDE, offers enhanced pathogenicity to its host. Tris-HCL, calcium chloride, potassium hydrogen phosphate, sodium phosphate, magnesium sulfate, zinc chloride, and olive oil were mixed in a 50 ml Erlenmeyer flask to extract CDE from the lab strain of B. bassiana. After centrifugation of the mixture, the supernatant was separated as extracellular enzymes. The media was subjected to SDS-PAGE analysis for enzyme characterization. Stacking gel (4%) and resolving gel (12%) were used with a pH of 8.6 to determine the molecular masses of the samples (enzymes) in kDa. The results showed that after staining and de-staining the gel, different bands of various sizes appeared. When compared with a standard key, the bands were found at 19, 50, 25, 32, and 34.25 kDa, confirming the presence of proteases, lipases, and chitinase, respectively. The extracted CDE can be used with different combinations of mycelial medium of B. bassiana or other entomopathogenic fungi to enhance their pathogenicity against insect pests. The extracted crude enzymes were used against larvae, pupae, and adults at concentrations of 5, 10, 15, 20, and 25µl. The mortality rate in larvae and adults was recorded as 78.50±2.10% and 80±2.15% at 25µl/ml, respectively. At lower concentrations (5µl/ml), the mortality was 13.33±1.92%, followed by the control group. A low percentage of adult emergence (10±2.63%) from pupae was observed in treated insects, and a higher adult emergence (65.0±5.77%) was observed in the untreated group of insects. The results showed that mortality and adult emergence from pupae were concentration-dependent. Therefore, adding CDE to the mycelium of B. bassiana enhanced its pathogenicity against different life stages of Bactrocera dorsalis.


Beauveria bassiana; Pathogenicity; Bactrocera dorsalis; Enzyme characterization; Insect biocontrol

Full Text:



Ahmad, G., Khan, A., Khan, A.A., Ali, A., Mohammad, H.I., 2021. Biological control: a novel strategy for the control of the plant parasitic nematodes. Antonie van Leeuwenhoek 114(7), 885-912.

Ansari, M.S., Basri, R., Shekhawat, S.S., 2019. Insect pests infestation during field and storage of fruits and vegetables. Health and Safety Aspects of Food Processing Technologies 121-207.

Bara, G.T., Laing, M.D., 2020. Entomopathogens: potential to control thrips in avocado, with special reference to Beauveria bassiana. Horticultural Reviews 47, 325-368.

Beris, A.N., Horner, J.S., Jariwala, S., Armstrong, M.J., Wagner, N.J., 2021. Recent advances in blood rheology: a review. Soft Matter 17(47), 10591-10613.

Cheong, P.C., Glare, T.R., Rostás, M., Haines, S., Brookes, J.J., Ford, S., 2020. Lack of involvement of chitinase in direct toxicity of Beauveria bassiana cultures to the aphid Myzus persicae. Journal of Invertebrate Pathology 169, 107276.

Chergui, S., Boudjemaa, K., Benzehra, A., Karaca, I., 2020. Pathogenicity of indigenous Beauveria bassiana (Balsamo) against Ceratitis capitata Wiedemann (Diptera: Tephritidae) under laboratory conditions. Egyptian Journal of Biological Pest Control 30(1), 128.

Coutinho-Rodrigues, C.J.B., Perinotto, W.M.D.S., Beys da Silva, W.O., Santi, L., Berger, M., Marciano, A.F., Bittencourt, V.R.E.P., 2016. Virulence, proteolytic and lipolytic activities of Brazilian Beauveria bassiana sl isolates (Hypocreales: Clavicipitaceae) to Rhipicephalus microplus ticks (Acari: Ixodidae). Biocontrol Science and Technology 26(2), 239-249.

Demir, Y., Dikbaş, N., Beydemir, Ş., 2018. Purification and biochemical characterization of phytase enzyme from Lactobacillus coryniformis (MH121153). Molecular Biotechnology 60, 783-790.

Dhawan, M., Joshi, N., 2017. Enzymatic comparison and mortality of Beauveria bassiana against cabbage caterpillar Pieris brassicae LINN. Brazilian Journal of Microbiology 48, 522-529.

Dias, B.A., Neves, P.M.O.J., Furlaneto-Maia, L., Furlaneto, M.C., 2008. Cuticle-degrading proteases produced by the entomopathogenic fungus Beauveria bassiana in the presence ofcoffee berry borer cuticle. Brazilian Journal of Microbiology 39, 301-306.

Fernandes, E.G., Valério, H.M., Feltrin, T., Sand, S.T.V.D., 2012. Variability in the production of extracellular enzymes by entomopathogenic fungi grown on different substrates. Brazilian Journal of Microbiology 43, 827-833.

Grizanova, E.V., Coates, C.J., Dubovskiy, I.M., Butt, T.M., 2019. Metarhizium brunneum infection dynamics differ at the cuticle interface of susceptible and tolerant morphs of Galleria mellonella. Virulence 10(1), 999-1012.

Javed, K., Javed, H., Mukhtar, T., Qiu, D., 2019. Efficacy of Beauveria bassiana and Verticillium lecanii for the management of whitefly and aphid. Pakistan Journal of Agricultural Sciences 56(3), 669-674.

Kim, J.S., Roh, J.Y., Choi, J.Y., Je, Y.H., 2010. Influence of two FPLC fractions from Beauveria bassiana SFB-205 supernatant on the insecticidal activity against cotton aphid. Biocontrol Science and Technology 20(1), 77-81.

Lemoine, M.M., Engl, T., Kaltenpoth, M., 2020. Microbial symbionts expanding or constraining abiotic niche space in insects. Current Opinion in Insect Science 39, 14-20.

Malan, A.P., Von Diest, J.I., Moore, S.D., Addison, P., 2018. Control options for false codling moth, Thaumatotibia leucotreta (Lepidoptera: Tortricidae), in South Africa, with emphasis on the potential use of entomopathogenic nematodes and fungi. African Entomology 26(1), 14-29.

Motholo, L.F., 2019. Endophytic establishment of Beauveria bassiana in wheat (Triticum aestivum) and its impact on Diuraphis noxia. Doctoral dissertation, North-West University, South Africa, Potchefstroom Campus.

Mutamiswa, R., Nyamukondiwa, C., Chikowore, G., Chidawanyika, F., 2021. Overview of oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) in Africa: from invasion, bio-ecology to sustainable management. Crop Protection 141, 105492.

Ola-Oladimeji, F.A., Idowu, E.O., Adewumi, A.A., Fafowora, K.C., 2018. Heterogeneity studies of wild Clarias gariepinus (Osteichthyes, Clariidae) using SDS-Polyacrylamide Gel Electrophoresis. Vestnik Zoologii 52(6), 457-462.

Ortiz-Urquiza, A., Keyhani, N.O., Quesada-Moraga, E., 2013. Culture conditions affect virulence and production of insect toxic proteins in the entomopathogenic fungus Metarhizium anisopliae. Biocontrol Science and Technology 23(10), 1199—1212.

Petrisor, C., Stoian, G., 2017. The role of hydrolytic enzymes produced by entomopathogenic fungi in pathogenesis of insects mini review. Romanian Journal for Plant Protection 10, 66-72.

Rameshthangam, P., Solairaj, D., Arunachalam, G., Ramasamy, P., 2018. Chitin and Chitinases: biomedical and environmental applications of chitin and its derivatives. Journal of Enzymes 1(1), 20-43.

Shehzad, M., Tariq, M., Ali, Q., Aslam, A., Mukhtar, T., Akhtar, M.F., Gulzar, A., Faisal, M., 2022. Evaluation of insecticidal activity of Beauveria bassiana against different instar larvae of Plutella xylostella by using two different methods of application. International Journal of Tropical Insect Science 42, 1471-1476.

Shehzad, M., Tariq, M., Mukhtar, T., Gulzar, A., 2021. On the virulence of the entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae (Ascomycota: Hypocreales), against the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae). Egyptian Journal of Biological Pest Control 31(1), 86.

Silva, D.M., de Souza, V.H.M., Moral, R.D.A., Delalibera Júnior, I., Mascarin, G.M., 2022. Production of Purpureocillium lilacinum and Pochonia chlamydosporia by submerged liquid fermentation and Bioactivity against Tetranychus urticae and Heterodera glycines through seed inoculation. Journal of Fungi 8(5), 511.

Singh, M.K., Rajagopalan, A., Tanimu, H., Sukumaran, B.O., 2021. Purification, characterization and fibrino (geno) lytic activity of cysteine protease from Tabernaemontana divaricata latex. 3 Biotech 11(2), 106.

Stevenson, P.C., Belmain, S.R., Isman, M.B.(Eds.)., 2020. Pesticidal plants: From smallholder use to commercialisation. MDPI.

Svedese, V.M., Tiago, P.V., Bezerra, J.D.P., Paiva, L.M., Lima, E.Á.D.L.A., Porto, A.L.F., (2013). Pathogenicity of Beauveria bassiana and production of cuticle-degrading enzymes in the presence of Diatraea saccharalis cuticle. African Journal of Biotechnology 12(46), 6491-6497.

Wang, D., Liang, Q., Chen, M., Ye, H., Liao, Y., Yin, J., He, Y., 2021. Susceptibility of oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae) pupae to entomopathogenic fungi. Applied Entomology and Zoology 56, 269-275.

Zhang, S., Widemann, E., Bernard, G., Lesot, A., Pinot, F., Pedrini, N., Keyhani, N.O., 2012. CYP52X1, representing new cytochrome P450 subfamily, displays fatty acid hydroxylase activity and contributes to virulence and growth on insect cuticular substrates in entomopathogenic fungus Beauveria bassiana. Journal of Biological Chemistry 287(16), 13477-13486.

Zibaee, A., Bandani, A.R., 2009. Purification and characterization of the cuticle-degrading protease produced by the entomopathogenic fungus, Beauveria bassiana in the presence of Sunn pest, Eurygaster integriceps (Hemiptera: Scutelleridae) cuticle. Biocontrol Science and Technology 19(8), 797-808.

Zibaee, A., Ramzi, S., 2018. Cuticle-degrading proteases of entomopathogenic fungi: From biochemistry to biological performance. Archives of Phytopathology and Plant Protection 51(13-14), 779-794.

Zibaee, A., Sadeghi-Sefidmazgi, A., and Fazeli-Dinan, M. 2011. Properties of a lipase produced by Beauveria bassiana: Purification and biochemical studies. Biocontrol Science and Technology 21(3), 317-331.

DOI (PDF): https://doi.org/10.33804/pp.007.02.4691


  • There are currently no refbacks.