Evaluating physical and nutritional stress during mycelial growth as inducers of tolerance to heat and UV-B radiation in Metarhizium anisopliae conidia

mycological research 112 (2008) 1362–1372

journal homepage: www.elsevier.com/locate/mycres

Evaluating physical and nutritional stress during mycelial growth as inducers of tolerance to heat and UV-B radiation in Metarhizium anisopliae conidia Drauzio E. N. RANGEL1, Anne J. ANDERSON, Donald W. ROBERTS* Department of Biology, Utah State University, Logan, UT 84322-5305, USA

article info

abstract

Article history:

Elevated tolerance to UV-B radiation and heat may be induced in conidia produced on fungi

Received 23 January 2007

exposed during mycelial growth to sublethal stresses other than heat or UV-B. This is due to

Received in revised form

a phenomenon referred to as ‘cross-protection’. Several mechanisms are associated with this

4 April 2008

increased conidial tolerance, one of which is the accumulation of trehalose and mannitol

Accepted 24 April 2008

within conidia. In the present study, conidia of the insect-pathogenic fungus Metarhizium ani-

Corresponding Editor:

sopliae var. anisopliae were produced on mycelium subjected to nutritive, heat-shock, osmotic,

Richard A. Humber

or oxidative stress. The tolerance levels to UV-B radiation and heat of the conidia from stressed mycelium were evaluated, and the amounts of trehalose and mannitol accumulated

Keywords:

in conidia were quantified. Conidia produced under nutritive stress (carbon and nitrogen star-

Biocontrol

vation) were two-times more heat and UV-B tolerant than conidia produced under rich (non-

Entomogenous fungi

stress) nutrient conditions [potato–dextrose agar with yeast extract (PDAY)], and they also ac-

Heat-shock stress

cumulated the highest concentrations of trehalose and mannitol. Conidia produced on heat-

Mannitol

shock stressed PDAY cultures had higher tolerance to UV-B radiation and heat than conidia

Nutritive stress

produced without heat shock; however, both the UV-B tolerance and trehalose/mannitol con-

Osmotic stress

centrations in conidia produced on heat-shocked mycelium were less than those of conidia

Oxidative stress

produced under nutritive stress. Conidia produced under osmotic stress (sodium or potas-

Trehalose

sium chloride added to PDAY) had elevated heat and UV-B tolerances similar to those of conidia produced under nutritive stress; however, they had the lowest levels of mannitol and trehalose, which indicates that accumulation of these compounds is not the only mechanism used by M. anisopliae for protection from heat and UV-B radiation. Oxidative stress from UV-A irradiation or hydrogen peroxide did not produce conidia with elevated UV-B or heat tolerances. Conidia produced under oxidative stress generated by menadione had increased or unchanged tolerances to heat or UV-B, respectively. The levels of mannitol or trehalose in conidia were similar to those in the unstressed controls. Conidial yield was reduced, in some cases severely, by nutritive and osmotic stress; whereas oxidative and heat-shock stress did not alter levels of spore production. ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: þ55 435 797 0049 E-mail address: [email protected] 1 Current address: Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraı´ba, Sa˜o Jose dos Campos, SP 12244-000, Brazil. 0953-7562/$ – see front matter ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2008.04.013

Stress tolerance of Metarhizium anisopliae conidia

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Introduction

Materials and methods

The insect-pathogenic fungus Metarhizium anisopliae is an important agent for biocontrol and of agricultural insect pests (Enserink 2004) and insect vectors of human diseases (Blanford et al. 2005; Lazzarini et al. 2006; Scholte et al. 2005). Solar ultraviolet radiation and heat severely reduce the viability of conidia in the field, and these stress factors are important obstacles to successful use of M. anisopliae in agriculture. Microorganisms that live in harsh environments usually have several mechanisms for protecting themselves from the stresses. One important mechanism is the endogenous accumulation of trehalose and mannitol. Trehalose protects cells against several stress factors by stabilizing proteins in their native state and by preserving the integrity of membranes (Herdeiro et al. 2006; Singer & Lindquist 1998). Mannitol protects cells by scavenging toxic oxygen intermediates from stresses (e.g. heat, osmotic, and oxidative stress) (Noventa-Jordao et al. 1999; Ruijter et al. 2003). Several heat-shock proteins are involved in the folding of newly synthesized proteins or in the repair of misfolded and/or aggregated proteins (Iwahashi et al. 1998; Parsell et al. 1994). Trehalose and heat-shock proteins (HSP) act complementarily in increasing thermotolerance: trehalose stabilizes native proteins during heat shock and reduces aggregation of denatured proteins, while HSP104 mediates resolubilization of those proteins that have aggregated (Singer & Lindquist 1998). Cells accumulate trehalose to remarkable levels in response to heat shock (Eleutherio et al. 1993; Hottiger et al. 1987; Singer & Lindquist 1998), cold shock (Gocheva et al. 2006; Kandror et al. 2004), freezing (Shima et al. 1999), dehydration (Crowe et al. 1984), osmotic stress (Hallsworth & Magan 1994), carbon starvation (Parrou et al. 1999; Rangel et al. 2006a), and stationary-phase growth (Thevelein & de Winde 1999). Accumulation of mannitol in cells also correlates with stress resistance (Ruijter et al. 2003). Cells exposed to mild stress develop tolerance not only to higher doses of the same stress, but also to stress caused by other agents. This phenomenon, known as ‘cross-protection’, suggests the existence of an integrating mechanism that senses and causes responses to different forms of stress (Hohmann & Mager 2003). The responses to several stresses that induce cross-protection to heat have been studied extensively in bacteria and fungi (Hohmann & Mager 2003; Neidhardt et al. 1996). However, with few exceptions (Hartke et al. 1995; Mitchel & Morrison 1983; Rangel et al. 2006a; Trautinger et al. 1996; Verma & Singh 2001), the effects of stresses on cross-protection to UV-B radiation in microorganisms have not been examined extensively. In the present study, M. anisopliae conidia produced under four different stress conditions (nutritive, heat-shock, osmotic, and oxidative stress) were tested for induction of increased tolerance (cross-protection) to heat and UV-B radiation. Also, the trehalose and mannitol levels in conidia from the treated mycelia were measured to identify possible correlations between levels of these compounds and the degree of induced heat and UV-B radiation tolerances.

Fungal 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 carryae (Coleoptera: Curculionidae) in South Carolina; its accession number in the American Type Culture Collection (Manassas, VA) is MYA-3093.

Culture of fungi Conidia were produced on 23 ml of the following media: (1) potato–dextrose agar supplemented with 1 g l1 yeast extract (Difco, Sparks, MD; PDAY); (2) minimal medium (MM ¼ Czapek medium without sucrose); (3) MM amended with 0.2, 1 or 3 % glucose (MMG); (4) MM amended with 0.2 % D(þ) trehalose (MMT); and (5) MM amended with 0.2 % myo-inositol (MMM), in 95-mm polystyrene Petri dishes. The pH of all media was adjusted before autoclaving to 6.9 with HCl or NaOH (both at 0.1 M). A conidial suspension (100 ml of 107 conidia ml1) was inoculated evenly onto the agar media with a turntable and glass rod spreader. The cultures were incubated in the dark at 28  1  C for 14 d. Each treatment was replicated three times, with a new batch of conidia produced for each replication.

Nutritive stress Metarhizium anisopliae conidia were produced on PDAY, MM, MMG, MMT, and MMM. The myo-inositol was added to the minimal medium (MMM) because it is abundant in soil and also because several microorganisms can grow on inositol as the sole carbon source (Yoshida et al. 2002). Trehalose is also abundant in soil, and it is utilized as a sole carbon source by several microorganisms (Jorge et al. 1997); it is an important carbon source for entomopathogenic fungi possibly because trehalose makes up more than 90 % of insect haemolymph soluble carbohydrates (Seyoum et al. 2002).

Heat-shock stress Cultures of Metarhizium anisopliae in the mycelial phase of growth on PDAY medium (grown as described above) were submitted to one high-temperature exposure on days 2, 3, or 4 after inoculation. In addition, one set of cultures was exposed three times (days 2, 3, and 4) to high temperature. Cultures were exposed for 40 min to sublethal heat stress in two different ways, either heated by convection in an incubator at 50  C or heated by visible light and infrared radiation under an Oriel Model: 6720 solar simulator (Oriel, Stamford, CT) for 25 min. The UV wavelengths of the solar simulator were removed by covering the plates with a 0.13 mm Llumar film (Norton Performance Plastics, Wayne, NJ) to block wavelengths shorter than 400 nm. In both heat treatments, the final temperature was approximately 45  C (see Supplementary Material Fig S1). After the heat-shock treatment, the cultures were held at 28  1  C in the dark.

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Osmotic stress

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The osmotic tolerance range of the Metarhizium anisopliae conidia was first determined by germination after 24 h incubation on PDAY with concentrations of potassium chloride or sodium chloride from 0.1 to 1.4 M with intervals of 0.1 M (Fig 1A). Based on these results, the fungus was grown on PDAY (control) or PDAY amended with potassium chloride or sodium chloride to 0.4, 0.6, 0.8, or 1.0 M (final concentration) in the media. Conidia were harvested from 14-d-old cultures grown on PDAY medium or PDAY medium with high osmolarity, and their tolerances to UV-B radiation and heat were measured as described below.

Survival curve – osmotic stress

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Oxidative stress Metarhizium anisopliae mycelium grown on PDAY was exposed to three types of oxidative stress: UV-A radiation, hydrogen peroxide, or menadione. Dosages of the oxidant were selected after construction of survival curves based on germination of conidia exposed to UV-A (Fig 1B) and hydrogen peroxide (Fig 1C). UV-A irradiation is known to cause indirect DNA damage through induction of reactive oxygen species (ROS) (Kozmin et al. 2005). On day 3 of growth, the mycelium was exposed to UV-A radiation for 1, 2, 4, or 6 h [Black-Ray Lamp Chamber Model 95-0042-07 (UVP Upland, CA) fitted with two blacklight lamps (Sankyo Denki 15 W, Japan)]. Mylar film (JSC Industries, La Miranda, CA) was used to filter UV wavelengths shorter than 315 nm. The UV-A irradiance at sample height inside this chamber was 169 mW m2, as measured with an Ocean Optics USB 2000 Spectroradiometer (Dunedin, FL) (Rangel et al. 2006b). The tolerance to oxidative stress from hydrogen peroxide was estimated by measuring germination at 24 and 48 h of conidia incubated on PDAY amended with this oxidant (concentrations 0 to 20 mM with intervals of 0.5 mM). The autoclaved PDAY medium was cooled to 46–48  C before addition of the hydrogen peroxide. Little germination occurred above 3 mM after either 24 (Fig 1C) or 48 h incubation (data not shown). After 7 d, a few colonies were observed on media with hydrogen peroxide concentrations above 10 mM. The hydrogen peroxide treatment was not lethal to M. anisopliae even at 20 mM. Hydrogen peroxide decomposes with time in the medium; so, apparently, it was fungistatic, not fungicidal. Therefore, hydrogen peroxide concentrations of 5, 10, 15, and 20 mM were selected for the oxidative-stress experiments. Exposures to hydrogen peroxide were by: (1) the fungus was grown on PDAY (control) or PDAY amended with 5, 10, 15, or 20 mM hydrogen peroxide, or (2) the fungus was grown on PDAY and on day 3 the mycelium flooded for 30 s with sterile distilled water (control) or the same hydrogen peroxide concentrations mentioned above. The third oxidative agent, menadione sodium bisulphate 2-methyl-1,4-naphtoquinone Sigma-Aldrich Corp., St. Louis, Missouri, USA was added to PDAY medium at a single concertration, 0.1 mM, as described elsewhere (Herdeiro et al. 2006).

Conidial production To measure conidial production under different stress conditions; plates were inoculated by spreading conidial

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Concentration (mM) Fig 1 – (A) Survival of Metarhizium anisopliae conidia exposed to various levels of osmotic stress. Each data point is the mean percent germination after 24 h incubation on PDAY medium plus concentrations of sodium chloride (NaCl) or potassium chloride (KCl) from 0 to 1.4 M. (B) Survival of M. anisopliae conidia exposed to various levels of oxidative stress generated by UV-A radiation. Each data point represents the mean percent germination at 24 h incubation following UV-A exposure. (C) Survival of M. anisopliae conidia exposed to oxidative stress generated by various concentrations of hydrogen peroxide. Each data point is the mean percent germination after 24 h incubation. Error bars are standard errors of three independent experiments.

suspensions over the agar with a turntable and bent glass rod (which afforded a lawn of mycelium followed by an almost even covering of the medium with conidia), three agar plugs (per plate) were removed with a cork borer (5 mm diam) at

Stress tolerance of Metarhizium anisopliae conidia

places on the medium surface with an even coverage of conidia, and the conidia were suspended in 1 ml sterile Tween 80 (0.01 %). Conidial concentrations were determined by haemocytometer counts. Each experiment was performed on three different dates and each experiment used a new batch of cultures.

UV-B radiation and heat exposure In each experiment, the same inocula prepared as described by Rangel et al. (2004, 2005) were used for both UV and heat exposure. Conidia were exposed to 978 mW m2 of weighted UVB for 2 h, providing a total dose of 7.04 kJ m2 (Rangel et al. 2006b). This UV-B dose kills approximately 50 % of conidia produced on PDAY medium (Rangel et al. 2006b). For heat exposure, 2 ml conidial suspension was placed in 16  125 mm screw-cap glass tubes (Pyrex, Corning, NY) and the tubes immediately placed in a 45  0.1  C waterbath. After heat exposure for 3 or 4 h, the conidial suspension (20 ml plate1) was inoculated onto PDAY medium, and germination was determined at 400 magnification after 48 h. The exposure times of 3 or 4 h were used because these heat exposures kill approximately 50 or 80 %, respectively, of conidia produced on PDAY medium. (Rangel et al. 2005, 2006b). The procedures used in these experiments are described elsewhere (Braga et al. 2001; Rangel et al. 2005, 2006b).

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trehalose. The eluant was 100 mmol l1 NaOH (Fisher) with a flow rate of 1 ml min1. Fifty microlitres of standard solutions of mannitol and trehalose (Sigma, St Louis, MO) or of conidial extracts were injected, and peak areas determined with the Dionex Peak Net Software. Each analysis was carried out with at least three replicates per treatment, and the mean polyol and trehalose content was calculated as milligrams per gram of dry conidia.

Statistical analyses Nutritive stress Differences in relative germination of conidia from the five media (PDAY, MM, MMG, MMT, and MMM) were assessed using an analysis of variance of a one-way factorial in a randomized block design. The five types of media were incorporated as fixed-effects factors. Blocks were defined by dates on which the experiments were repeated. Differences in conidial production among the five media were assessed using an analysis of variance of a one-way factorial in a randomized block design. Pair-wise comparisons among media means were controlled for experiment-wise type I error using the Tukey– Kramer method with a 0.1 significance level. Data were cube-root (i.e., y1/3) transformed prior to analysis to better meet assumptions of normality and homogeneity.

Heat-shock stress Conidial trehalose and mannitol Trehalose and mannitol levels in conidia produced under different stress conditions (heat-shock, nutritive, osmotic, and oxidative stress) were compared. In this series of experiments, the conidia were produced on minimal medium (MM) for nutritive stress; while heat-shock, osmotic, and oxidative stress conditions were administered as described above, but using MM þ 3 g l1 myo-inositol Sigma (MMMþ; in contrast to MMM, which has 0.2 g l1 myo-inositol). PDAY medium was not appropriate because it contains high concentrations of trehalose and mannitol that leach from this medium during harvesting of the conidia. Conidia were harvested by flooding a plate with 5 ml sterile MilliQ water and transferring this water from one plate to another successively. A sterile polyethylene cell lifter (Costar 3008, Corning International) was used to release the conidia from the mycelial mat. The washes were pooled, filtered through cheesecloth, and centrifuged (Refrigerated Sorvall Superspeed RC2-B centrifuge, Ivan Sorvall, Norwalk, CT) with an SA 600 rotor at 6500 rev min1 (6100 g) for 15 min at 5  C. The conidial pellets were washed with water, re-centrifuged, the pellets frozen (20  C), and then lyophilized. A modification of the method described by Hallsworth & Magan (1994) was used to extract polyols and trehalose from conidia. Briefly, 10 mg dry conidia were placed in a microtube with 1 ml MilliQ water, and the tubes were immersed in a boiling water-bath for 5.5 min. The tubes were centrifuged, and the supernatants (extracts) stored frozen (20  C) for later HPLC analysis. A Dionex HPLC series DX6000 fitted with a CarboPac PA1 (4  250 mm) resin-based column, a CarboPac PA1 guard column and a pulsed electrochemical detector (Dionex, Sunnyvale, CA) were used for the detection of mannitol and

The effects of the heat source (solar simulator or incubator) and time of application [control (no heat shock); single heat shock on days 2, 3 or 4; or three heat shocks (days 2, 3 and 4)] on tolerances to UV-B radiation and heat. The data were analysed using an analysis of variance of a two-way factorial in a complete block design. Blocks were defined as experiments repeated on three dates. Pair-wise comparisons among means were controlled for experiment-wise type I error using the Tukey–Kramer method with a 0.1 significance level. The data met assumptions of normality and homogeneity on their original scale, and so they were not transformed prior to analysis.

Osmotic stress The effects of salts (sodium chloride or potassium chloride at 0, 0.4, 0.6, 0.8, 1 M) on conidial response (relative germination) following UV-B or heat treatment. Also, the total production of conidia on each salt concentration was assessed using an analysis of variance of a two-way factorial in a randomized block design in which experimental trials defined blocks.

Oxidative stress Menadione or hydrogen-peroxide exposures or UV-A irradiation were used to generate oxidative stress. The effects of hydrogen peroxide concentration (0, 5, 10, 20 mM) and type of exposure (in medium or flooding) on the response to UV-B or heat of conidia produced under these conditions were measured and assessed using an analysis of variance of a two-way factorial in a randomized block design. For conidia from menadione and UV-A-treated mycelium (exposed to 0, 1, 2, 4, and 6 h) their relative germination after UV-B or heat exposure was assessed and compared using analysis of variance of a one-way factorial in a randomized block design.

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Trehalose and mannitol data were analysed separately. The means of the controls (MM þ 3 % myo-inositol ¼ MMMþ) were compared with each of the other treatments. Differences between treatments were assessed using a one-way analysis of variance in a randomized, complete-block design. Data were log-transformed prior to analysis to meet assumptions of normality and homogeneity of variance. Comparisons between each treatment mean and the control mean were controlled for experiment-wise type I error using Dunnett’s test. Computations were done using Proc MIXED in The SAS System for Windows Version 9 (SAS Institute 2002).

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Results and discussion 120

Heat-shock stress Heat-shock stress induces cross-protection in bacteria and fungi to oxidative stress, heat stress (Hottiger et al. 1989;

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In prokaryotes and eukaryotes, nutritive stress (carbon starvation) during growth is known to produce reproductive units with induced cross-protection to several other stresses, e.g. freeze–thaw tolerance (Park et al. 1997), heat, ethanol, acid, and osmotic and oxidative stresses (Hartke et al. 1994; Jenkins et al. 1990; Svensa¨ter et al. 2000). Conidia of the fungus Metarhizium anisopliae produced under nutritive stress (Czapek medium without sucrose ¼ MM ¼ carbon starvation) were two-fold more tolerant to UV-B radiation and heat than conidia produced on rich PDAY medium (F4,7 ¼ 17.74; P < 0.001, Fig 2A,B). Conidia produced on nutrient-limited media (MM þ 0.2 % glucose ¼ MMG, MM þ 0.2 % trehalose ¼ MMT, and MM þ 0.2 % myo-inositol ¼ MMM) were, in some cases, more tolerant to UV-B radiation and heat exposure than conidia from PDAY medium; but the differences were not always significant (F4,7 ¼ 4.89; P ¼ 0.034, Fig 2A,B). Therefore, even 0.2 % carbon caused catabolic repression of the stressprotective genes. Indeed, MM plus 1 or 3 % glucose produced conidia with tolerances to heat and UV-B radiation similar to that of conidia produced on PDAY (data not shown). Tolerance to both UV-B and heat were high in conidia produced on MM. However, there was a serious trade off, as the conidial production was much lower than on the nutrient rich PDAY medium (F4,6 ¼ 147.68; P < 0.001). Growth on MMG, MMT, and MMM also had conidial yields significantly lower than from PDAY, but not significantly different from each other (Fig 2C). High tolerance to UV-B radiation in M. anisopliae has been correlated with high accumulations of trehalose and mannitol (Rangel et al. 2006a). In the present study, these accumulations were greatest in the MM-grown conidia, and this again correlated with increased UV-B and thermotolerance (Figs 2 and 3). In addition to direct DNA damage, UV wavelengths (UV-B, UVA) also induce oxidative stress, protein denaturation, and lipid peroxidation (Gerhardt et al. 1999). Trehalose prevents protein denaturation and mannitol limits the effects of free radicals; these protective actions possibly contributed to increasing conidial tolerance.

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Medium Fig 2 – Effects of nutritive stress on Metarhizium anisopliae during mycelial growth on conidial UV-B and heat tolerance (expressed as mean relative percent germination) and on production of conidia. Conidia were produced on rich medium (PDAY), on carbon-deficient medium (MM), and on minimal medium supplemented with a single carbon source [0.2 % glucose (MMG), trehalose (MMT), or myo-inositol (MMM)]. Relative germination was calculated in relation to non-irradiated or unheated controls (see Braga et al. 2001). The germination counts were made at 48 h from the time conidia were inoculated on the media. Conidial yields were calculated from conidia on three 5-mm plugs of medium. Means with the same letter are not significantly different; error bars are standard errors of three independent experiments. Results after (A) exposure to 978 mW mL2 weighted UV-B irradiation for 2 h, providing total dose of 7.04 kJ mL2; or (B) 3 h heat exposure (45  C); and (C) conidial production at 14 d.

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Kapoor et al. 1990), freeze–thaw stress (Park et al. 1997), and UV radiation (Mitchel & Morrison 1983). We observed similar results in conidia produced on heat-shocked mycelium of Metarhizium anisopliae. Heat-shock treatment slightly improved conidial tolerance to UV-B radiation and heat, although the protective effects were not equal (UV-B ¼ F4,18 ¼ 20.46; P < 0.001, heat ¼ F4,18 ¼ 30.12; P < 0.0001, Fig 4). Both types of heat shock (incubator or solar simulator) generated similar tolerances to UV-B radiation and heat (UV-B ¼ F4,18 ¼ 5.03; P ¼ 0.007, heat ¼ F4,18 ¼ 2.40; P < 0.088, Fig 4). The culture phase at the time of exposure was important to the acquisition of tolerance to UV-B radiation and heat (Fig 4). When heat shock was applied on the 3rd day after inoculation, i.e. just before the completion of conidiogenesis, the conidial tolerances to UV-B radiation (Fig 4A) and heat (Fig 4B,C) were increased. Levels of trehalose rapidly increase when Neurospora crassa conidiospore germlings are submitted to a 45  C heat shock, and the levels decrease rapidly after return to a physiological temperature (Bonini et al. 1995; Neves et al. 1991). Trehalose production in fungi is known as a ‘futile cycle’ because the trehalose is rapidly used by the mycelium as a carbon source for growth, (Hottiger et al. 1987). Rapid degradation of trehalose after heat shock probably occurs because its continued presence would interfere with the refolding of denatured proteins by molecular chaperones (Singer & Lindquist 1998).

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Fig 3 – Accumulation of trehalose and polyols in conidia produced on nutritive or heat shock stressed mycelium. Cultures were grown on minimal medium plus 3 % myo-inositol (MMM) ([ control); MMM with mycelium heat shocked (HS) at different mycelial ages; minimal medium (MM); or MM plus 3 % glucose (MMG). Results: (A) endogenous mannitol; and (B) endogenous trehalose.

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Treatment Fig 4 – Effects of heat shock on Metarhizium anisopliae during mycelial growth on conidial UV-B and heat tolerance (expressed as mean relative percent germination). Conidia were produced on PDAY after the mycelia were treated by heat-shock exposure at 2, 3, or 4 d after inoculation. One set of cultures was exposed three times to high temperature, i.e. exposures on day 2, 3, and 4. Results after (A) exposure to UV-B for 2 h; (B) 3 h heat exposure (45  C); (C) 4 h heat exposure (45  C). Open bars, heat-shock treatment under solar simulator; closed bars, heat-shock treatment in incubator.

In Metarhizium, trehalose and mannitol accumulating in heat-shocked mycelium at day 2 were probably used as nutrients by the mycelium before conidial production (Fig 3), thus generating conidia with the same level of tolerance as control plates. Conversely, heat-shock treatment on day 3, when

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Osmotic stress induces cross-protection in prokaryotes and eukaryotes, e.g., induction of thermotolerance (Trollmo et al. 1988) and freeze–thaw tolerance (Park et al. 1997). With Metarhizium anisopliae osmotic stress induced cross-protection to heat and to UV-B radiation, and the tolerances were the same as found in response to nutritive stress. The tolerance to UV-B increased directly in proportion to the salt concentration (F4,27 ¼ 52.50; P < 0.001, Fig 5A). However, the heat tolerance increased greatly at the lower salt concentrations, but decreased when salt concentration increased (F4,27 ¼ 54.67; P < 0.001, Fig 5B). Different levels of the same stress have been reported to cause differences in response kinetics. For example, yeast cells exposed to a high dose of sodium chloride (0.8 M) had delayed gene expression in comparison with cells exposed to a lower salt concentration (0.4 M) (Posas et al. 2000). Conversely, in Beauveria bassiana, media with 0.4 M potassium chloride produced conidia that were less heat-tolerant than non-stressed controls conidia (Ying & Feng 2006). The conidial cell wall of M. anisopliae is normally thicker than that of B. bassiana, and this may be important to the differing heat-tolerances of B. bassiana and M. anisopliae.

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conidiogenesis was starting, resulted in conidia with higher heat and UV-B tolerances with slight (but not statistically significant) increases in trehalose and mannitol content (Figs 3A,B and 4). Initial experiments indicated that temperatures exceeding 50  C impaired the heat-shock response; i.e. conidia produced on mycelium treated at 50  C were more susceptible to UV-B and heat than conidia from the unheated control plates (data not shown). Similarly, heat-shock genes were not expressed by Beauveria bassiana above 50  C (Xavier et al. 1999). Furthermore, five strains of B. brongniartii showed maximal heatshock response (i.e. induction of new polypeptides and enhancement, as well as repression, of existing polypeptides) after 1 h at 45  C (Xavier & Khachatourians 1996). In Saccharomyces cerevisiae, the trehalose pool rapidly expanded at temperatures between 37 and 45  C, but at 50  C trehalose accumulation ceased and cells died. The maximal expansion of the trehalose pool was observed at 45  C (Hottiger et al. 1987). The levels of trehalose and mannitol accumulated in M. anisopliae conidia produced on heat-shocked mycelium were much lower than for conidia produced on MM without heat shock (Fig 3A,B). In comparison with the non-heated control, there was a detectable, but statistically nonsignificant, peak of trehalose and mannitol accumulation when the heat-shock treatment was undertaken at 60 h from start of incubation. With S. cerevisiae, trehalose was also not essential for thermotolerance acquired after sublethal heat-shock treatment (Van Dijck et al. 1995), and this suggests that other mechanisms of protection, such as heat-shock proteins, are involved (Nwaka et al. 1994). Heat-shock treated and untreated mycelium produced similar numbers of conidia (data not shown), even though the heat shock caused a transient growth arrest with conidial production resuming 10 d later. Growth arrest after heatshock treatment has also been reported with yeast (Shin et al. 1987).

D. E. N. Rangel et al.

0

0.0 M

0.4 M

0.6 M

0.8 M

1.0 M

Treatment Fig 5 – Effects of osmotic stress on Metarhizium anisopliae during mycelial growth on conidial UV-B and heat tolerance (expressed as mean relative percent germination) and on production of conidia. Conidia were produced on PDAY ([ control) or on PDAY supplemented with concentrations (from 0.4 to 1 M) of sodium chloride or potassium chloride. Results after (A) exposure to UV-B for 2 h; (B) 3 h heat exposure (45  C); (C) conidial production.

The UV-B tolerance was higher for conidia produced on sodium chloride than on potassium chloride-augmented PDAY (interaction effect; main effect of media; F1,27 ¼ 11.27; P ¼ 0.002, Fig 5A). However, thermotolerance was higher for conidia produced on PDAY with potassium chloride than

10

* P < 0.0001

20

* P = 0.0591

30

* P = 0.0201

* P = 0.0443

* P = 0.0569

40

0

* P = 0.0003

20 15 10 5

M M M

M M G

M M

aC l0 .8 +N

+N M M M

M M M M +K Cl 0. 6 M M M M +K Cl 0. 8 M

M

0 M M M

B

50

aC l0 .6

A

mg trehalose g-1 conidia

with sodium chloride, (interaction effect; main effect of media; F1,27 ¼ 55.12; P < 0.001, Fig 5B). Conidial production was highest on PDAY (F4,27 ¼ 143.69; P ¼ 0.001, Fig 5C), but conidial production decreased when salt concentrations increased (F1,27 ¼ 13.41; P < 0.001, Fig 5C). The highest salt concentration in which M. anisopliae was able to grow and to produce normal conidia was 1 M sodium chloride or potassium chloride, but conidial yields at this concentration were very low (Fig 5C). At 1.4–1.9 M of these salts the conidia were malformed, with a variety of shapes and sizes (data not shown). Similarly, Candida halophila cells grown at high salt concentration were spherical whereas those grown in the absence of salt were spheroid (Silva-Grac¸a 2004). Hyperosmotic stress causes water to diffuse out of the cell, thus resulting in cell shrinkage; this may lead to DNA and protein damage, cell-cycle arrest, and ultimately to cell death. Cells compensate for or adapt to osmotic stress by activating an osmotic-stress response, including synthesis of intracellular solutes, such as glycerol (Shen et al. 1999). In M. anisopliae, accumulation of glycerol was very low in conidia produced under osmotic stress (data not shown), but in Saccahromyces cerevisiae, when cells are washed with hypo-osmotic solutions there is a high glycerol loss, thus indicating that the membrane is highly glycerol-permeable (Shen et al. 1999). It is possible, therefore, that any glycerol present in M. anisopliae conidia was lost during the washing procedure preceding sugar alcohol extractions. Levels of trehalose and mannitol accumulated in M. anisopliae conidia produced under osmotic stress were low in comparison to conidia produced on MM (Fig 6A,B).The role of trehalose on osmotic stress is controversial. Trehalose may (Hounsa et al. 1998; Shen et al. 1999) or may not (Lewis et al. 1995; Rep et al. 2000) be accumulated by yeast cells in response to osmotic stress. Hallsworth & Magan (1995) reported high accumulations of trehalose in M. anisopliae conidia produced on trehalose medium with potassium chloride, but not in highnutrient Sabouraud–dextrose agar (SDA) medium with potassium chloride. It is not known why osmotic stress induced high tolerance to heat and UV-B in M. anisopliae, but as the levels of trehalose and mannitol were very low, other tolerance mechanisms apparently are involved. For example, the cytosolic catalase T (regulated by CTT1 gene) is induced under osmotic stress, is important for viability under severe osmotic stress in S. cerevisiae, and also plays a protective role during oxidative stress (Schu¨ller et al. 1994). In addition, the DNA damage-inducible gene DDR2 and the heat-shock protein gene HSP104 are induced by osmotic stress in S. cerevisiae (Schu¨ller et al. 1994). These genes, if present in M. anisopliae, may protect DNA and proteins in conidia from heat and UVB radiation. The decrease in conidial heat tolerance with increasing sodium chloride concentrations may relate to conidial cellwall and membrane disorganization, both of which increase water permeability. Fungi, as well other organisms, regulate membrane lipid composition in response to temperature and other stresses (Guerzoni et al. 2001). Higher concentrations of sodium chloride produce membranes with higher concentrations of unsaturated fatty acids (Guerzoni et al. 2001; Swan & Watson 1999) leading to enhanced water permeability (Guerzoni et al. 2001) and increased sensitivity to heat

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mg mannitol g-1 conidia

Stress tolerance of Metarhizium anisopliae conidia

Treatment Fig 6 – Accumulation of trehalose and polyols in conidia produced under osmotic or nutritive stress. Cultures were grown on MMM ([ control); MMM with different concentrations of sodium chloride (NaCl) or potassium chloride (KCl); MM; or MMG (see Fig 3 for abbreviations). Results: (A) endogenous mannitol; and (B) endogenous trehalose.

(Crisan 1973). Conidia of M. anisopliae produced on high osmolarity medium have thinner cell walls in comparison to cell walls of conidia produced on normal medium (Leland et al. 2005), and this may have been involved in the increased heat susceptibility noted in our experiments.

Oxidative stress Adaptation of microorganisms under oxidative stress generated by UV-A radiation, hydrogen peroxide, or other oxidants induces cross-protection to other stress conditions, such as acidity (Hartke et al. 1995; Svensa¨ter et al. 2000), UV radiation (Asad et al. 2000; Verma & Singh 2001), heat, hydrogen peroxide, ethanol (Hartke et al. 1995; Jamieson 1992; Kapoor et al. 1990). Nevertheless, hydrogen peroxide does not induce cross-protection against the lethal temperature for Saccharomyces. cerevisiae (Jamieson 1992). In addition, exposure of yeast cells to UV radiation does not confer crossprotection against heat or UV radiation (Dutta & Verma 1998). We found that M. anisopliae conidia generated under two conditions of oxidative stress (mycelium exposed to UV-A irradiation or to hydrogen peroxide) were not more tolerant to UV-B or heat than conidia produced under normal conditions (PDAY medium; see Supplementary Material Figs S2–S3). Conidial production under hydrogen peroxide or UV-A radiation oxidative stress was also similar to that on PDAY controls (data not shown). Metarhizium conidia produced on PDAY þ menadione (a strong superoxide-generating agent) had normal UV-B

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D. E. N. Rangel et al.

tolerance (i.e. similar to conidia produced on PDAY; Fig 7A); but their thermotolerance was increased to levels similar to that of conidia produced on MM (Fig 7B). Therefore, as with yeast (Herdeiro et al. 2006), not all oxidative stress agents will induce cross-protection against other stress conditions in M. anisopliae. Conidial production on PDAY þ menadione was similar to PDAY medium (Fig 7C). No significant accumulation of endogenous trehalose or mannitol was found in conidia produced under oxidative

A

UV-B - 2 h

120 100 80 60 40 20 0 PDAY

Menadione 0.1 mM

MM

Treatment

B

Heat 45 ºC - 3 h

120 100 80 60 40 20 0 PDAY

Menadione 0.1 mM

MM

Treatment

C

Conidial production

7000

Conidia ml-1 (× 104)

6000 5000 4000 3000 2000 1000 0 PDAY

Menadione 0.1 mM

stress (see Supplementary Material Fig S4). The role of trehalose as a protector against oxidative stress is still controversial. In S. cerevisiae, hydrogen peroxide caused only weak induction of trehalose genes (Parrou et al. 1997) and limited trehalose accumulation (Attfield 1987).

Conclusions Conidia produced under nutritive and osmotic stress conditions had enhanced tolerance to heat and UV-B radiation. High levels of trehalose and mannitol were found in conidia produced under nutritive stress, but not in conidia produced under osmotic stress. This indicates that the accumulation of trehalose and mannitol is not the only mechanism used by M. anisopliae to increase heat and UV-B tolerance. Further studies are needed to confirm which mechanisms are involved in protection of conidia produced under osmotic stress. Nutritive- and osmotic-stress treatments were continuous throughout the entire growth period, including during conidiogenesis. Heat-shock and oxidative stress (except menadione) treatments consisted of short-term exposures that caused temporary growth arrest in the first week of incubation, but growth and conidial production resumed soon thereafter. In general, only cultures that were grown under continuous stress conditions produced conidia with greatly improved thermo- and UV tolerance. Oxidative stress by exposure to menadione caused protection against heat, but not to UV-B radiation. Stress adaptation under menadione may use genes different from those used for protection against UV-B radiation. Heat-shock treatment improved thermotolerance more than UV-B tolerance in M. anisopliae. Interestingly, in S. cerevisiae, about 14 % of the genome had similar responses to several stress conditions, i.e. hydrogen peroxide, menadione, heat shock, hyperosmotic shock, starvation, and others (Gasch et al. 2000). The stress responses of both yeast and M. anisopliae appear to be generally stimulated by some types of stress, and this can lead to cross-protection. Heat-shock treatment, to be effective, should be synchronized with the beginning of conidiation. Accordingly, the time for heat-shock treatment will differ with fungal species and isolates. It has been suggested that stress tolerance in M. anisopliae conidia can be improved by manipulating the growth conditions (Hallsworth & Magan 1994, 1995, 1996). Our comparisons between stress tolerance and conidial production, particularly conidia produced under osmotic and nutritive stress, indicate that the benefits of producing very tolerant conidia have the enormous cost of low conidial production. Accordingly, other approaches to mass producing physiologically improved fungus for biological control of insects in agriculture should be sought.

MM

Treatment Fig 7 – Effects of oxidative stress (menadione) on Metarhizium anisopliae during mycelial growth on conidial UV-B and heat tolerances (expressed as mean relative percent germination). Conidia were produced on PDAY ([ control) or PDAY supplemented with 0.1 mM menadione. Results after (A) exposure to UV-B for 2 h; (B) 3 h heat exposure (45  C). (C) conidial production.

Acknowledgements We are grateful to Susan Durham, Statistician, USU Department of Biology, for the statistical analyses, to Philip Harrison and Jerry N. Chatterton (Forage and Range Research Laboratory, USDA, Logan, UT) for quantification of endogenous trehalose and mannitol, and to Anita D. Panek (Departamento de Bioquı´mica, Instituto de Quı´mica, UFRJ, Rio de Janeiro) for

Stress tolerance of Metarhizium anisopliae conidia

critical review of the manuscript. We are grateful to Mycological Research Editor, Richard A. Humber and his two reviewers for their very insightful and helpful reviews of this manuscript. We sincerely thank the National Council for Scientific and Technological Development (CNPq) of Brazil for a PhD fellowship for D.E.N.R. This research was supported in part by grants from the Utah State University Community/University Research Initiative and the Utah Department of Agriculture and Food, Division of Plant Industry. Paper #7848 of the Utah Agricultural Experiment Station.

Supplementary data Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.mycres.2008.04.013.

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