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  • 1 Qorveh Branch, Islamic Azad University, Qorveh, Iran
  • 2 University of Tabriz, P.O.B. 5166614766, Tabriz, Iran
  • 3 University of Trento, San Michele all’Adige, Italy
  • 4 Fondazione Edmund Mach (FEM), San Michele all’Adige, Italy
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Proteases constitute a significant part of cell wall-degrading enzymes (CWDEs) produced by fungal biocontrol agents and particularly crucial in mycoparasitism of fungal phytopathogens. Plate-based screening methods are routinely used for screening protease-producing microorganisms including fungi. Skim milk agar (SMA) is one of the most popular media for the detection of protease producing bacteria. However, SMA is not efficient to test fast growing fungi, because it does not give an estimation of the actual amount of secreted protease produced by fungal inocula. In the current study, the efficacy of two modified plate-screening methods, including split-SMA (SSMA) and minimal medium supplemented with skim milk (MSMW) was assessed for detection of protease production by three representative fungal strains including Trichoderma longibrachiatum strain N, Beauveria bassiana strain B and Purpureocillium lilacinum strain PL. Protease production was revealed on the three tested media by the three strains. However, the halo diameter of the fungal strains (a proxy for protease production) was the smallest on SMA. Furthermore, protease production could not be detected for T. longibrachiatum strain N on SMA due to its fast growth; while it showed the highest protease activity on both modified media compared with the other strains. According to the result of this study, the SSMA medium is an easy and more accurate method compared with the two other different methods as it displays the actual amount of protease produced by fungal strains and therefore this method is recommended for quantitative and qualitative detection of protease production by slow and fast growing fungi.

  • 1

    Agrawal, T. and Kotasthane, A. S. (2012): Chitinolytic assay of indigenous Trichoderma isolates collected from different geographical locations of Chhattisgarh in Central India. SpringerPlus 1, 73.

  • 2

    Benítez, T., Rincón, A. M., Limón, M. C. and Codón, A. C. (2004): Biocontrol mechanisms of Trichoderma strains. Int. Microbiol. 7, 249–260.

  • 3

    Chantawannakul, P., Oncharoen, A., Klanbut, K., Chukeatirote, E. and Lumyong, S. (2002): Characterization of proteases of Bacillus subtilis strain 38 isolated from traditionally fermented soybean in Northern Thailand. Sci. Asia 28, 241–245.

  • 4

    Goldman, G. H., Hayes, C. and Harman, G. E. (1994): Molecular and cellular biology of biocontrol by Tricho-derma spp. Tibtech. 12, 478–482.

  • 5

    Handelsman, J. and Stabb, E. V. (1996): Biocontrol of soilborn plant pathogens. The Plant Cell 8, 1855–1869. Harman, G. E., Howell, C. R., Viterbo, A., Chet, I. and Lorito, M. (2004): Trichoderma species–opportunistic, avirulent plant symbionts. Nat. Rev. Microbiol. 2, 43–56.

  • 6

    Karimi, K., Amini, J., Harighi, B. and Bahramnejad, B. (2012): Evaluation of biocontrol potential of Pseu-domonas and Bacillus spp. against fusarium wilt of chickpea. Aust. J. Crop. Sci. 6, 695–703.

  • 7

    Karimi, K., Ahari, A. B., Arzanlou, M., Amini, J. and Pertot, I. (2017): Comparison of indigenous Trichoderma spp. strains to a foreign commercial strain in terms of biocontrol efficacy against Colletotrichum nym-phaeae and related biological features. J. Plant. Dis. Prot. 124, 453–466.

  • 8

    Kasana, R. C., Salwan, R., Dhar, H., Dutt, S. and Gulati, A. (2008): A rapid and easy method for the detection of microbial cellulases on agar plates using gram’s iodine. Curr. Microbiol. 57, 503–507.

  • 9

    Kasana, R. C., Salwan, R. and Yadav, S. K. (2011): Microbial proteases: detection, production and genetic improvement. Crit. Rev. Microbiol. 37, 262–276.

  • 10

    Maheshwari, R., Bharadwaj, G. and Bhat, M. K. (2000): Thermophilic fungi: their physiology and enzymes. Microbiol. Mol. Biol. Rev. 64, 461–488.

  • 11

    Mehta, S. and Nautiyal, C. S. (2001): An efficient method for qualitative screening of phosphate solubilizing bacteria. Curr. Microbiol. 43, 51–56.

  • 12

    Rao, M. B., Tanksale, A. M., Ghatge, M. S. and Deshpande, V. V. (1998): Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62, 597–635.

  • 13

    Sanchez, V., Rebolledo, O., Picaso, R. M., Cardenas, E., Cordova, J., Gonzalez, O. and Samuels, G. J. (2007): In vitro antagonism of Thielaviopsis paradoxa by Trichoderma longibrachiatum. Mycopathologia 163, 49–58.

  • 14

    Savitha, S., Sadhasivam, S., Swaminathan, K. and Lin, F. H. (2011): Fungal protease: production, purification and compatibility with laundry detergents and their wash performance. J. Taiwan. Inst. Chem. Eng. 42, 298–304.

  • 15

    Silva, S. D., Carneiro, R. M., Faria, M., Souza, D. A., Monnerat, R. G. and Lopes, R. B. (2017): Evaluation of Pochonia chlamydosporia and Purpureocillium lilacinum for suppression of Meloidogyne enterolobii on tomato and banana. J. Nematol. 49, 77–85.

  • 16

    Souza, P. M. D., Bittencourt, M. L. D. A., Caprara, C. C., Freitas, M. D., Almeida, R. P. C. D., Silveira, D., Fonseca, Y. M., Ferreira Filho, E. X., Pessoa Junior, A. and Magalhães, P. O. (2015): A biotechnology perspective of fungal proteases. Braz. J. Microbiol. 46, 337–346.

  • 17

    Zhang, S., Gan, Y., Ji, W., Xu, B., Hou, B. and Liu, J. (2017): Mechanisms and characterization of Trichoderma longibrachiatum T6 in suppressing nematodes (Heterodera avenae) in wheat. Front. Plant. Sci. 8, 1491.

  • 18

    Zibaee, A. and 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. Sci. Techn. 19, 797–808.

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