Abstract
Antifungal activity of chitosan on plant pathogenic fungi has been widely studied, but little is known about the effect of chitosan on fungal biocontrol agents. In this work, we characterize the increase of conidiation induced by chitosan in fungal pathogens of invertebrates (FPI). Chitosan increased conidiation of FPI, including Beauveria bassiana, widely used as mycoinsecticide, and did not affect conidia viability or pathogenicity. Increased conidiation induced by chitosan is shown to be concentration dependent and is not associated to growth inhibition as observed for the mycoparasitic fungus Trichoderma harzianum. Real-time reverse transcription polymerase chain reaction was used to study transcript levels of two genes involved in conidiation in B. bassiana, the regulatory G protein signaling gene Bbrgs1 and the hydrophobin gene hyd1, at different chitosan concentrations. Higher levels of Bbrgs1 and hyd1 transcripts were detected on chitosan-amended media. No correlation with chitosan concentration was observed for expression of Bbrgs1 unlike hyd1. Bbrgs1 deletion mutant ∆Bbrgs1 showed that chitosan-induced conidiation is independent of Bbrgs1, suggesting an alternative mechanism controlling conidiation in B. bassiana. Our data supports that sporulation increases by chitosan, with spores retaining their viability and pathogenicity, which makes chitosan a suitable compound to increase conidia production in fungi with applications in fungal biotechnology.
Similar content being viewed by others
References
Adams TH (1995) Asexual sporulation in higher fungi. In: Gow NAR, Gadd GM (eds) The growing fungus. Champan & Hall, London, pp 367–382
Adams TH, Wieser JK, Yu JH (1998) Asexual sporulation in Aspergillus nidulans. Microbiol Mol Biol Rev 62:35–54
Allan CR, Hadwiger LA (1979) The funcidal effect of chitosan on fungi of varying cell wall composition. Exp Mycol 3:285–287
Biever KD, Hostetter DL (1971) Activity of the nuclear-polyhedrosis virus of the cabbage looper evaluated at programmed temperature regimens. J Invertebr Pathol 18:81–84
Bölker M (1998) Sex and crime: heterotrimeric G proteins in fungal mating and pathogenesis. Fungal Genet Biol 25:143–156
Boucias DG, Pendland JC (1991) Attachment of Mycopathogens to cuticle. The initial event of mycoses in arthropod hosts. In: Cole GT, Hoch HC (eds) The fungal spore and disease initiation in plants and animals. Plenum Press, New York, pp 101–127
Butt TM, Jackson C, Magan N (2001) Fungal biological control agents: progress, problems and potential. In: Butt TM, Jackson C, Magan N (eds) Fungi as biocontrol agents. CABI, Oxfordshire, pp 1–8
Chittenden C, Singh T (2009) In vitro evaluation of combination of Trichoderma harzianum and chitosan for the control of sapstain fungi. Biol Control 50:262–266
Cho EM, Kirkland BH, Holder DJ, Keyhani NO (2007) Phage display cDNA cloning and expression analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. Microbiology 153:3438–3447
Dahlberg KR, Van Etten JL (1982) Physiology and biochemistry of fungal sporulation. Annu Rev Phytopathol 20:281–301
Fang W, Pei Y, Bidochka MJ (2007) A regulator of a G protein signalling (RGS) gene, cag8, from the insect-pathogenic fungus Metarhizium anisopliae is involved in conidiation, virulence and hydrophobin synthesis. Microbiology 153:1017–1025
Fang W, Scully LR, Zhang L, Pei Y, Bidochka MJ (2008) Implication of a regulator of G protein signalling (BbRGS1) in conidiation and conidial thermotolerance of the insect pathogenic fungus Beauveria bassiana. FEMS Microbiol Lett 279:146–156
Griesch J, Vilcinskas A (1998) Proteases released by entomopathogenic fungi impair phagocytic activity, attachment and spreading of plasmatocytes isolated from haemolymph of the greater wax moth Galleria mellonella. Bio Sci Tech 8:517–530
Hamm HE (1998) The many faces of G protein signaling. J Biol Chem 273:669–672
Hirano S, Nagao N (1989) Effects of chitosan, pectic acid, lysozyme, and chitinase on the growth of several phytopathogens. Agric Biol Chem 53:3065–3066
Laflamme P, Benhamou N, Bussieres G, Dessureault M (1999) Differential effect of chitosan on root rot fungal pathogens in forest nurseries. Can J Bot 77:1460–1468
Lee BN, Adams TH (1994) Overexpression of flbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brlA and premature initiation of development. Mol Microbiol 14:323–334
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the \( 2_T^{ - \Delta \Delta C} \) Method. Methods 25:402–408
Palma-Guerrero J, Lopez-Llorca LV, Jansson H-B, Salinas J, Güerri-Agulló B (2007) Use of chitosan to increase sporulation of fungi. WO2008102044
Palma-Guerrero J, Jansson HB, Salinas J, Lopez-Llorca LV (2008) Effect of chitosan on hyphal growth and spore germination of plant pathogenic and biocontrol fungi. J Appl Microbiol 104:541–553
Palma-Guerrero J, Huang IC, Jansson H-B, Salinas J, Lopez-Llorca LV, Read ND (2009) Chitosan permeabilizes the plasma membrane and kills cells of Neurospora crassa in an energy dependent manner. Fungal Genet Biol 46:585–594
Palma-Guerrero J, Lopez-Jimenez JA, Pérez-Berná AJ, Huang IC, Jansson HB, Salinas J, Villalaín J, Read ND, Lopez-Llorca LV (2010) Membrane fluidity determines sensitivity of filamentous fungi to chitosan. Mol Microbiol 75:1021–1032
Plascencia-Jatomea M, Viniegra G, Olayo R, Castillo-Ortega MM, Shirai K (2003) Effect of chitosan and temperature on spore germination of Aspergillus niger. Macromol Biosci 3:582–586
Ross EM, Wilkie TM (2000) GTPase-activating proteins for heterotrimeric G proteins: regulators of G-protein signaling (RGS) and RGS-like proteins. Annu Rev Biochem 69:795–827
Segers GC, Regier JC, Nuss DL (2004) Evidence for a role of the regulator of g-protein signaling protein CPRGS-1 in got subunit CPG-1-mediated regulation of fungal virulence, conidiation, and hydrophobin synthesis in the chestnut blight fungus Cryphonectria parasitica. Eukaryot Cell 3:1454–1463
Singh T, Vesentini D, Singh AP, Daniel G (2008) Effect of chitosan on physiological, morphological, and ultrastructural characteristics of wood-degrading fungi. Int Biodeterior Biodegrad 62:116–124
Stössel P, Leuba JL (1984) Effect of chitosan, chitin and some aminosugars on growth of various soilborne phytopathogenic fungi. J Phytopathol 111:82–90
Yu JH, Wieser J, Adams TH (1996) The Aspergillus FlbA RGS domain protein antagonizes G protein signaling to block proliferation and allow development. EMBO J 15:5184–5190
Acknowledgments
We thank Dr. M. J. Bidochka (Brock University, Canada) for providing the ∆Bbrgs1 mutant. We also wish to thank Dr. B. Kerry (Rothamsted Research, UK), Dr. K. Sivasithamparam (University of Western Australia, Perth, Australia), and Dr. J. Jimenez (Universidad Politecnica, Valencia, Spain) for providing fungal strains for our experiments. This study was supported by the Spanish Ministry of Science and Innovation (AGL2008-00716).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Palma-Guerrero, J., Larriba, E., Güerri-Agulló, B. et al. Chitosan increases conidiation in fungal pathogens of invertebrates. Appl Microbiol Biotechnol 87, 2237–2245 (2010). https://doi.org/10.1007/s00253-010-2693-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-010-2693-1