Effects of physical and nutritional stress conditions during mycelial growth on conidial germination speed, adhesion to host cuticle, and virulence of Metarhizium anisopliae, an entomopathogenic fungus
Introduction
Genomic studies on the pathogenicity of Metarhizium anisopliae, an insect-pathogenic fungus, were greatly advanced in 2005 with elegant studies by St Leger's group (Freimoser et al., 2005, Wang et al., 2005, Wang and St Leger, 2005) who demonstrated how pathogenicity-related and other genes are regulated differently when the fungus is grown on nutritionally rich haemolymph, or nutritionally poor culture media with or without insect cuticle supplementation. These studies answered questions that researchers had been asking for decades (Daoust and Roberts, 1982, Daoust and Roberts, 1983, Fargues and Robert, 1983, Ibrahim et al., 2002, Lane et al., 1991) on why several passages through a host or a particular culture medium may improve virulence of entomopathogenic fungi to insects. Their findings indicated that phenotypic plasticity in virulence, at least in some circumstances, can be traced to precise responses of the pathogen to specific environmental and/or nutritional conditions.
In addition, important information has been generated by Magan's group on the amounts of endogenous polyols and trehalose accumulated in conidia and the effects of these compounds on conidial performance. Conidia of the insect-pathogenic fungi Beauveria bassiana, M. anisopliae, and Isaria (syn. Paecilomyces) fumosoroseus produced on media with low water activity or with a high concentration of glycerol had increased accumulations of polyols and trehalose in conidia; and they were more virulent than conidia produced on a rich medium (Sabouraud–dextrose agar; SDA) without stress (Andersen et al., 2006, Hallsworth and Magan, 1994a, Magan, 2001). High concentrations of trehalose and polyols in conidia have also been related to increased stress tolerance (Thevelein 1984). Trehalose hydrolysis is a major event during early conidial germination and presumably provides glucose for energy (Cuber et al., 1997, Elbein et al., 2003, Lillie and Pringle, 1980, Thevelein, 1984). Both trehalose and polyols serve as easily mobilized energy reserves for rapid conidial germination; and this may explain, at least partially, why conidia produced under one culture condition have improved germination speed in relation to conidia from some other culture conditions when germination occurs in only one medium (Andersen et al., 2006, Rangel et al., 2004, Rangel et al., 2005, Shah et al., 2005). Higher germination speed, in some cases, has been associated with higher virulence (Al-Aidroos and Roberts, 1978, Altre et al., 1999, Andersen et al., 2006, Hassan et al., 1989, Samuels et al., 1989).
In this study, the in vitro germination rates, cuticular adhesion, and virulence to insects of conidia produced under nine different stress conditions were compared with conidia produced without stress on a rich medium (potato—dextrose agar (PDA) and agar supplemented with yeast extract).
Section snippets
Metarhizium anisopliae isolate
Metarhizium anisopliae var. anisopliae isolate ARSEF 2575 was obtained from the USDA-ARS Collection of Entomopathogenic Fungal Cultures (US Plant, Soil & Nutrition Laboratory, Ithaca, NY). ARSEF 2575 was isolated originally from Curculio caryae (Coleoptera: Curculionidae) in South Carolina. Isolate ARSEF 2575 also was deposited in the American Type Culture Collection (ATCC, Manassas, VA; ATCC #MYA-3093). Stock cultures were maintained at 4 °C in test-tube slants of PDA (Difco Laboratories,
Insect mortality (virulence)
Conidia produced on UV-A treated mycelium and the average of the two nutritive stress treatments (MML and MM) caused significantly greater insect mortality than conidia from the untreated control (PDAY; P = 0.03 and 0.002, respectively). There were no differences in mortality levels from conidia produced under other stress treatments (heat-shock, oxidative, and osmotic stress with exception of potassium chloride 0.8 m) and the untreated control PDAY (Fig 1A). There were no differences among the
Discussion
Conidia of Metarhizium anisopliae generated under nutritive stress (MML or MM) had increased virulence (Fig 1) and increased germination speed (Fig 2). In addition, conidial adhesion to host cuticle (Fig 3) and conidial fluorescence after calcofluor staining (Fig 4) were greater in conidia produced on MML than conidia produced on PDAY (optimal nutritive conditions). Ibrahim et al. (2002) and Shah et al. (2005) also found that M. anisopliae produced on nutrient-poor media had increased virulence
Acknowledgements
We are grateful to Susan Durham for the statistical analyses. We sincerely thank the National Council for Scientific and Technological Development (CNPq) of Brazil for a PhD fellowship for D.E.N.R. at Utah State University. We are grateful to Mycological Research Editor, Richard A. Humber and his two reviewers for their very insightful and helpful reviews of this manuscript. This research was supported in part by grants from the Utah State University Community/University Research Initiative and
References (40)
- et al.
Pathogenicity of Paecilomyces fumosoroseus isolates to diamondback moth, Plutella xylostella: correlation with spore size, germination speed, and attachment to cuticle
Journal of Invertebrate Pathology
(1999) - et al.
The role of the trehalose transporter during germination
Biochimica et Biophysica Acta
(1997) - et al.
Virulence of natural and insect-passaged strains of Metarhizium anisopliae to mosquito larvae
Journal of Invertebrate Pathology
(1982) - et al.
Studies on the prolonged storage of Metarhizium anisopliae conidia: effect of growth substrate on conidial survival and virulence against mosquitoes
Journal of Invertebrate Pathology
(1983) - et al.
Influence of accelerated germination of conidia on the pathogenicity of Metarhizium anisopliae for Manduca sexta
Journal of Invertebrate Pathology
(1989) - et al.
Effect of artificial culture media on germination, growth, virulence and surface properties of the entomopathogenic hyphomycete Metarhizium anisopliae
Mycological Research
(2002) - et al.
Influence of cultural conditions on the virulence of conidia and blastospores of Beauveria bassiana to the green leafhopper, Nephotettix virescens
Mycological Research
(1991) - et al.
Variations in UV-B tolerance and germination speed of Metarhizium anisopliae conidia produced on artificial and natural substrates
Journal of Invertebrate Pathology
(2004) - et al.
Influence of growth environment on tolerance to UV-B radiation, germination speed, and morphology of Metarhizium anisopliae var. acridum conidia
Journal of Invertebrate Pathology
(2005) - et al.
Growth of Metarhizium anisopliae on non-preferred carbon sources yields conidia with increased UV-B tolerance
Journal of Invertebrate Pathology
(2006)
The effect of heavy metal ions and their complexons upon the growth, sporulation and pathogenicity of the entomopathogenic fungus Verticillium lecanii
Journal of Invertebrate Pathology
Characteristics relating to the pathogenicity of Metarhizium anisopliae toward Nilaparvata lugens
Journal of Invertebrate Pathology
Nutrition influences growth and virulence of the insect-pathogenic fungus Metarhizium anisopliae
FEMS Microbiology Letters
Synthesis of proteins including a cuticle-degrading protease during differentiation of the entomopathogenic fungus Metarhizium anisopliae
Experimental Mycology
Differential gene expression by Metarhizium anisopliae growing in root exudate and host (Manduca sexta) cuticle or hemolymph reveals mechanisms of physiological adaptation
Fungal Genetics and Biology
Mutants of Metarhizium anisopliae with increased virulence toward mosquito larvae
Canadian Journal of Genetics and Cytology
Development of a population-based threshold model of conidial germination for analysing the effects of physiological manipulation on the stress tolerance and infectivity of insect pathogenic fungi
Environmental Microbiology
Stress tolerance and pathogenic potential of a mannitol mutant of Cryptococcus neoformans
Microbiology
Oxidative killing of Cryptococcus neoformans by human neutrophils. Evidence that fungal mannitol protects by scavenging reactive oxygen intermediates
Journal of Immunology
Increased mortality of Acanthoscelides obtectus by alkane-grown Beauveria bassiana
Biocontrol
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Current address: Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraíba, São José dos Campos, SP 12244-000 Brazil