Actinomycetes biocontrol agents against Botrytis cinerea, agent of tomato gray mold disease


  • Hind Lahmyed Ibn Zohr University
  • Rachid Bouharroud Research Unit of Integrated Crop Production, Regional Center of the Agronomic Research, Agadir (CRRA-AGADIR), National Institute of Agronomic Research (INRA), Morocco
  • Redouan Quessaoui Ibn Zohr University
  • Abdelhadi Ajerrar Ibn Zohr University
  • Abderrahim Amarraque Research Unit of Integrated Crop Production, Regional Center of the Agronomic Research, Agadir (CRRA-AGADIR), National Institute of Agronomic Research (INRA), Morocco
  • Moulay Abdelazize Aboulhassan Ibn Zohr University
  • Bouchra Chebli Ibn Zohr University



Botrytis cinerea, Actinomycetes, Tomato, Antagonist, grey mould, Siderophore, Chitinase, PGP


The present work aims to isolate actinomycete bacteria with antagonistic abilities towards Botrytis cinerea, the causal agent of grey mould, from a soil sample collected from the rhizosphere of healthy tomato grove. In vitro direct confrontation led to the isolation of 104 actinomycetes isolates, fifteen isolates have shown a most significant mortality rate of the mycelial growth of Botrytis cinerea (>50%). Based on the results of this screening, representative strains were selected to verify their in vivo antagonistic activity on tomato fruits, the reduction of B.cinerea has a percentage ranging from 52.38% to 96.19%. Following these tests, these bacterial strains were morphologically distinct on the basis of spore mass colour, reverse side colour, aerial and substrate mycelial formation and production of diffusible pigment. Furthermore, the actinomycetes isolates were evaluated for its plant growth promoting (PGP) properties and its ability to produce biocontrol related extracellular enzymes viz., amylase, protease, cellulase, chitinase, esterases and lecithinase. This study gives the possibility to develop new ways of protecting cultivated plants while reducing the risks of contamination and environmental pollution.


Lepoivre, P. (2003). Phytopathologie. De Boeck. 427p.

Vanachter, A., Van Wambeke, E., & Van Assche, C. (1982). Potential danger for infection and spread of root diseases of tomatoes in hydroponics. Nutrient Film Technique and Substrates, XXI IHC 133, 119-128.

Moënne-Loccoz, Y., Powell, J., Higgins, P., McCarthy, J., & O'Gara, F. (1998). An investigation of the impact of biocontrol Pseudomonas fluorescens F113 on the growth of sugarbeet and the performance of subsequent clover-Rhizobium symbiosis. Applied Soil Ecology, 7(3), 225-237.

Besnard, O., & Davet, P. (1993). Mise en évidence de souches de Trichoderma spp à la fois antagonistes de Pythium ultimum et stimulatrices de la croissance des plantes.

Adelere, I. A., & Lateef, A. (2016). Keratinases: emerging trends in production and applications as novel multifunctional biocatalysts. Kuwait Journal of Science, 43(3).

Whipps, J. M. (1997). Ecological considerations involved in commercial development of biological control agents for soil-borne diseases. Modern soil microbiology, 525-546.

Kalai, A. T., Kalai, E., Lehrer, E., & Samet, D. (2010). A commitment folk theorem. Games and Economic Behavior, 69(1), 127-137.

Hilali, L., Khattabi, A., Nssarlah, N., Malki, A., & Finance, C. (2002). Isolement des nouvelles souches d’Actinomycètes productrices de substances antifongiques a partir du milieu naturel marocain. Rev. biol. Biotechnol, 2, 49-53.

Ouhdouch, Y., Barakate, M., & Finance, C. (2001). Actinomycetes of Moroccan habitats: isolation and screening for antifungal activities. European Journal of Soil Biology, 37(2), 69-74.

Shirling, E. T., & Gottlieb, D. (1966). Methods for characterization of Streptomyces species. International journal of systematic bacteriology, 16(3), 313-340.

Hmouni, A., Hajlaoui, M. R., & Mlaiki, A. (1996). Résistance de Botrytis cinerea aux benzimidazoles et aux dicarboximides dans les cultures abritées de tomate en Tunisie. EPPO Bulletin, 26(3‐4), 697-705.

Berrada, I., Benkhemmar, O., Swings, J., Bendaou, N., & Amar, M. (2012). Selection of halophilic bacteria for biological control of tomato gray mould caused by Botrytis cinerea. Phytopathologia Mediterranea, 625-630.

Mehdi, R. B. A., Sioud, S., Fguira, L. F. B., Bejar, S., & Mellouli, L. (2006). Purification and structure determination of four bioactive molecules from a newly isolated Streptomyces sp. TN97 strain. Process biochemistry, 41(7), 1506-1513.

Nonomura, H. (1974). Key for classification and identification of 458 species of the streptomycetes included in ISP. J. Ferment. Technol., 52, 78-92.

Williams, S. T., & Cross, T. (1971). Chapter XI actinomycetes. In Methods in microbiology (Vol. 4, pp. 295-334). Academic Press.

Larkin, R. P., & Fravel, D. R. (1998). Efficacy of various fungal and bacterial biocontrol organisms for control of Fusarium wilt of tomato. Plant disease, 82(9), 1022-1028.

De Boer, W., Gunnewiek, P. J. K., Lafeber, P., Janse, J. D., Spit, B. E., & Woldendorp, J. W. (1998). Anti-fungal properties of chitinolytic dune soil bacteria. Soil Biology and Biochemistry, 30(2), 193-203.

Frändberg, E., Petersson, C., Lundgren, L. N., & Schnürer, J. (2000). Streptomyces halstedii K122 produces the antifungal compounds bafilomycin B1 and C1. Canadian journal of microbiology, 46(8), 753-758.

Müller, G., Matzanke, B. F., & Raymond, K. N. (1984). Iron transport in Streptomyces pilosus mediated by ferrichrome siderophores, rhodotorulic acid, and enantio-rhodotorulic acid. Journal of bacteriology, 160(1), 313-318.

Müller, G., & Raymond, K. N. (1984). Specificity and mechanism of ferrioxamine-mediated iron transport in Streptomyces pilosus. Journal of bacteriology, 160(1), 304-312.

Tokala, R. K., Strap, J. L., Jung, C. M., Crawford, D. L., Salove, M. H., Deobald, L. A., ... & Morra, M. J. (2002). Novel plant-microbe rhizosphere interaction involving Streptomyces lydicus WYEC108 and the pea plant (Pisum sativum). Appl. Environ. Microbiol., 68(5), 2161-2171.

Franceschi, V. R., Krokene, P., Christiansen, E., & Krekling, T. (2005). Anatomical and chemical defenses of conifer bark against bark beetles and other pests. New Phytologist, 167(2), 353-376.

Vassilev, N., Vassileva, M., & Nikolaeva, I. (2006). Simultaneous P-solubilizing and biocontrol activity of microorganisms: potentials and future trends. Applied microbiology and biotechnology, 71(2), 137-144.

Vassilev, S. V., & Vassileva, C. G. (2007). A new approach for the classification of coal fly ashes based on their origin, composition, properties, and behaviour. Fuel, 86(10-11), 1490-1512.

Prévost, K., Couture, G., Shipley, B., Brzezinski, R., & Beaulieu, C. (2006). Effect of chitosan and a biocontrol streptomycete on field and potato tuber bacterial communities. BioControl, 51(4), 533-546.

Lehr, N. A., Schrey, S. D., Hampp, R., & Tarkka, M. T. (2008). Root inoculation with a forest soil streptomycete leads to locally and systemically increased resistance against phytopathogens in Norway spruce. New Phytologist, 177(4), 965-976.

Challis, G. L., & Hopwood, D. A. (2003). Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proceedings of the National Academy of Sciences, 100(suppl 2), 14555-14561.

Jensen, S. E., & Paradkar, A. S. (1999). Biosynthesis and molecular genetics of clavulanic acid. Antonie Van Leeuwenhoek, 75(1-2), 125-133.

Liras, P. (1999). Biosynthesis and molecular genetics of cephamycins. Antonie Van Leeuwenhoek, 75(1-2), 109-124.

Cocito, C., Di Giambattista, M., Nyssen, E., & Vannuffel, P. (1997). Inhibition of protein synthesis by streptogramins and related antibiotics. The Journal of antimicrobial chemotherapy, 39(suppl_1), 7-13.

Burr, T. J., Schroth, M. N., & Suslow, T. (1978). Increased potato yields by treatment of seed pieces with specific strains of Pseudomonas fluorescens and P. putida. Phytopathology, 68(9), 1377-1383.

Elad, Y., & Baker, R. (1985). The role of competition for iron and carbon in suppression of chlamydospore germination of Fusarium spp. by Pseudomonas spp. Phytopathology, 75(9), 1053-1059.

James, D. W., & Gutterson, N. I. (1986). Multiple antibiotics produced by Pseudomonas fluorescens HV37a and their differential regulation by glucose. Appl. Environ. Microbiol., 52(5), 1183-1189.

Meyer, J. A., & Abdallah, M. A. (1978). The fluorescent pigment of Pseudomonas fluorescens: biosynthesis, purification and physicochemical properties. Microbiology, 107(2), 319-328.

Lynch, J. M. (1990). Introduction: some consequences of microbial rhizosphere competence for plant and soil. The rhizosphere., 1-10.

Sprusansky, O., Zhou, L., Jordan, S., White, J., & Westpheling, J. (2003). Identification of three new genes involved in morphogenesis and antibiotic production in Streptomyces coelicolor. Journal of bacteriology, 185(20), 6147-6157.

Kim, D. W., Chater, K., Lee, K. J., & Hesketh, A. (2005). Changes in the extracellular proteome caused by the absence of the bldA gene product, a developmentally significant tRNA, reveal a new target for the pleiotropic regulator AdpA in Streptomyces coelicolor. Journal of bacteriology, 187(9), 2957-2966.

Vijgenboom, E., & Keijser, B. (2002). Copper and the morphological development of Streptomyces. In Handbook of copper pharmacology and toxicology (pp. 503-525). Humana Press, Totowa, NJ.

Weiß, C. H. (2007). Statsoft, inc., tulsa, ok.: Statistica, version 8. AStA Advances in Statistical Analysis, 91(3), 339-341.

Li, Y., He, F., Lai, H., & Xue, Q. (2017). Mechanism of in vitro antagonism of phytopathogenic Scelrotium rolfsii by actinomycetes. European journal of plant pathology, 149(2), 299-311.

Dai, P., Zong, Z., Ma, Q., & Wang, Y. (2019). Isolation, evaluation and identification of rhizosphere actinomycetes with potential application for biocontrol of Valsa mali. European Journal of Plant Pathology, 153(1), 119-130.

Moline, H., Hubbard, J. E., Karns, J. S., Buyer, J. S., & Cohen, J. D. (1999). Selective isolation of bacterial antagonists of Botrytis cinerea. European journal of plant pathology, 105(1), 95-101.

Collins, C. H. (1967). Microbiological methods. Microbiological methods., (2nd Edition).

Hu, Q. P., & Xu, J. G. (2011). A simple double-layered chrome azurol S agar (SD-CASA) plate assay to optimize the production of siderophores by a potential biocontrol agent Bacillus. African Journal of Microbiology Research, 5(25), 4321-4327.

Pal, R. B., & Gokarn, K. (2010). Siderophores and pathogenecity of microorganisms. J. Biosci. Tech, 1(3), 127-134.

Morrissey, R. F., Dugan, E. P., & Koths, J. S. (1976). Chitinase production by an Arthrobacter sp. lysing cells of Fusarium roseum. Soil Biology and Biochemistry, 8(1), 23-28.

Lingappa, Y. (1962). Chitin media for selective isolation and culture of actinomycetes. Phytopathology, 52, 317-323.

Nelson, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. J. biol. Chem, 153(2), 375-380.

Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of biological chemistry, 193, 265-275.

Valois, D., Fayad, K., Barasubiye, T., Garon, M., Dery, C., Brzezinski, R., & Beaulieu, C. (1996). Glucanolytic actinomycetes antagonistic to Phytophthora fragariae var. rubi, the causal agent of raspberry root rot. Appl. Environ. Microbiol., 62(5), 1630-1635.

Passari, A. K., Mishra, V. K., Gupta, V. K., Yadav, M. K., Saikia, R., & Singh, B. P. (2015). In vitro and in vivo plant growth promoting activities and DNA fingerprinting of antagonistic endophytic actinomycetes associates with medicinal plants. PLoS one, 10(9), e0139468.

Prasad, P., Bedi, S., & Singh, T. (2012). In vitro cellulose rich organic material degradation by cellulolytic Streptomyces albospinus (MTCC 8768). Malaysian Journal of Microbiology, 8(3), 164-169.

Sierra, G. (1957). A simple method for the detection of lipolytic activity of micro-organisms and some observations on the influence of the contact between cells and fatty substrates. Antonie van Leeuwenhoek, 23(1), 15-22.

Raval, K. M., Vaswani, P. S., & Majumder, D. R. (2012). Biotransformation of a single amino-acid L-Tyrosine into a bioactive molecule L-DOPA. Int J Sci Res, 2, 2250-3153.

Roy, S., Das, I., Munjal, M., Karthik, L., Kumar, G., Kumar, S., & Rao, K. V. B. (2014). Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment. Frontiers in biology, 9(4), 306-316.

Garrity, G., Staley, J. T., Boone, D. R., De Vos, P., Goodfellow, M., Rainey, F. A., ... & Schleifer, K. H. (2006). Bergey's Manual® of systematic bacteriology: volume two: the proteobacteria. Springer Science & Business Media.

Saadoun, I., Rawashdeh, R., Dayeh, T., Ababneh, Q., & Mahasneh, A. (2007). Isolation, characterization and screening for fiber hydrolytic enzymes-producing streptomycetes of Jordanian forest soils. Biotechnology, 6(1), 120-128.

Tan, H., Deng, Z., & Cao, L. (2009). Isolation and characterization of actinomycetes from healthy goat faeces. Letters in applied microbiology, 49(2), 248-253.

Rania, A. B. A., Jabnoun-Khiareddine, H., Nefzi, A., Mokni-Tlili, S., & Daami-Remadi, M. (2016). Endophytic bacteria from Datura metel for plant growth promotion and bioprotection against Fusarium wilt in tomato. Biocontrol Science and Technology, 26(8), 1139-1165.

Passari, A. K., Mishra, V. K., Saikia, R., Gupta, V. K., & Singh, B. P. (2015). Isolation, abundance and phylogenetic affiliation of endophytic actinomycetes associated with medicinal plants and screening for their in vitro antimicrobial biosynthetic potential. Frontiers in microbiology, 6, 273.

HASTUTI, R. D., LESTARI, Y., SUWANTO, A., & SARASWATI, R. (2012). Endophytic Streptomyces spp. as biocontrol agents of rice bacterial leaf blight pathogen (Xanthomonas oryzae pv. oryzae). HAYATI Journal of Biosciences, 19(4), 155-162.

Goudjal, Y., Zamoum, M., Sabaou, N., Mathieu, F., & Zitouni, A. (2016). Potential of endophytic Streptomyces spp. for biocontrol of Fusarium root rot disease and growth promotion of tomato seedlings. Biocontrol science and technology, 26(12), 1691-1705.

Mendonsa, E. S., Vartak, P. H., Rao, J. U., & Deshpande, M. V. (1996). An enzyme from Myrothecium verrucaria that degrades insect cuticles for biocontrol of Aedes aegypti mosquito. Biotechnology letters, 18(4), 373-376.

Gonzalez-Franco, A. C., Deobald, L. A., Spivak, A., & Crawford, D. L. (2003). Actinobacterial chitinase-like enzymes: profiles of rhizosphere versus non-rhizosphere isolates. Canadian Journal of Microbiology, 49(11), 683-698.

Kumaran, S., Deivasigamani, B., & Uttara, V. (2012). Chitinase application—review.

Singh, P. P., Shin, Y. C., Park, C. S., & Chung, Y. R. (1999). Biological control of Fusarium wilt of cucumber by chitinolytic bacteria. Phytopathology, 89(1), 92-99.

Patke, D. S. (1996). Characterization enzyme and genetic studies of thermophilic streptomycetes.

Ravindra Chaphalkar, S. (1993). Studies of industrially important streptomyces with special reference to extracellular protease.

Vigal, T., Gil, J. A., Daza, A., García-González, M. D., & Martín, J. F. (1991). Cloning, characterization and expression of an $alpha$-amylase gene from Streptomyces griseus IMRU3570. Molecular and General Genetics MGG, 225(2), 278-288.

Stamford, T. L. M., Stamford, N. P., Coelho, L. C. B. B., & Araujo, J. M. (2001). Production and characterization of a thermostable $alpha$-amylase from Nocardiopsis sp. endophyte of yam bean. Bioresource Technology, 76(2), 137-141.

Chakraborty, S., Khopade, A., Kokare, C., Mahadik, K., & Chopade, B. (2009). Isolation and characterization of novel $alpha$-amylase from marine Streptomyces sp. D1. Journal of Molecular Catalysis B: Enzymatic, 58(1-4), 17-23.

Verma, J. N., & Khuller, G. K. (1983). Lipids of actinomycetes. Advances in lipid research.

Jang, H. D., & Chen, K. S. (2003). Production and characterization of thermostable cellulases from Streptomyces transformant T3-1. World journal of Microbiology and Biotechnology, 19(3), 263-268.

Loliam, B., Morinaga, T., & Chaiyanan, S. (2013). Biocontrol of Pythium aphanidermatum by the cellulolytic actinomycetes Streptomyces rubrolavendulae S4. Science Asia, 39(6), 584-590.

de Menezes, A. B., Lockhart, R. J., Cox, M. J., Allison, H. E., & McCarthy, A. J. (2008). Cellulose degradation by micromonosporas recovered from freshwater lakes and classification of these actinomycetes by DNA gyrase B gene sequencing. Appl. Environ. Microbiol., 74(22), 7080-7084.

Saratale, G. D., Saratale, R. G., Lo, Y. C., & Chang, J. S. (2010). Multicomponent cellulase production by Cellulomonas biazotea NCIM‐2550 and its applications for cellulosic biohydrogen production. Biotechnology Progress, 26(2), 406-416.

Stutzenberger, F. (1988). Production and activity of Thermomonospora curvata cellulases on protein-extracted lucerne fibers. Applied microbiology and biotechnology, 28(4-5), 387-393.

Shimizu, M. (2011). Endophytic actinomycetes: biocontrol agents and growth promoters. In Bacteria in agrobiology: Plant growth responses (pp. 201-220). Springer, Berlin, Heidelberg.