Elsevier

Mycological Research

Volume 112, Issue 11, November 2008, Pages 1355-1361
Mycological Research

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

https://doi.org/10.1016/j.mycres.2008.04.011Get rights and content

Abstract

Growth under stress may influence pathogen virulence and other phenotypic traits. Conidia of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae (isolate ARSEF 2575) were produced under different stress conditions and then examined for influences on in vitro conidial germination speed, adhesion to the insect cuticle, and virulence to an insect host, Tenebrio molitor. Conidia were produced under non-stress conditions [on potato–dextrose agar plus 1 g l−1 yeast extract (PDAY; control)], or under the following stress conditions: osmotic (PDAY + sodium chloride or potassium chloride, 0.6 or 0.8 m); oxidative [(PDAY + hydrogen peroxide, 5 mm) or UV-A (irradiation of mycelium on PDAY)]; heat shock (heat treatment of mycelium on PDAY at 45 °C, 40 min); and nutritive [minimal medium (MM) with no carbon source, or on MM plus 3 g l−1 lactose (MML)]. Conidia were most virulent (based on mortality at 3 d) and had the fastest germination rates when produced on MML, followed by MM. In addition, conidial adhesion to host cuticle was greatest when the conidia were produced on MML. Media with high osmolarity (0.8 m) produced conidia with slightly elevated virulence and faster germination rates than conidia produced on the control medium (PDAY), but this trend did not hold for media with the lower osmolarity, (0.6 m). Conidia produced from mycelium irradiated with UV-A while growing on PDAY had somewhat elevated virulence levels similar to that of conidia produced on MM, but their germination rate was not increased. Hydrogen peroxide and heat shock treatments did not alter virulence. These results demonstrate that the germination, adhesion and virulence of M. anisopliae conidia can be strongly influenced by culture conditions (including stresses) during production of the conidia.

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

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    Current address: Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraíba, São José dos Campos, SP 12244-000 Brazil

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