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

mycological research 112 (2008) 1355–1361

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

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 Drauzio E. N. RANGEL1, Diane G. ALSTON, Donald W. ROBERTS* Department of Biology, Utah State University, Logan, UT 84322-5305, USA

article info

abstract

Article history:

Growth under stress may influence pathogen virulence and other phenotypic traits. Coni-

Received 6 January 2007

dia of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae (isolate ARSEF

Received in revised form

2575) were produced under different stress conditions and then examined for influences

4 April 2008

on in vitro conidial germination speed, adhesion to the insect cuticle, and virulence to an

Accepted 24 April 2008

insect host, Tenebrio molitor. Conidia were produced under non-stress conditions [on po-

Corresponding Editor:

tato–dextrose agar plus 1 g l1 yeast extract (PDAY; control)], or under the following stress

Richard A. Humber

conditions: osmotic (PDAY þ sodium chloride or potassium chloride, 0.6 or 0.8 M); oxidative

Keywords:

(heat treatment of mycelium on PDAY at 45  C, 40 min); and nutritive [minimal medium

Biocontrol

(MM) with no carbon source, or on MM plus 3 g l1 lactose (MML)]. Conidia were most vir-

Heat-shock stress

ulent (based on mortality at 3 d) and had the fastest germination rates when produced on

Lactose

MML, followed by MM. In addition, conidial adhesion to host cuticle was greatest when the

Minimal medium

conidia were produced on MML. Media with high osmolarity (0.8 M) produced conidia with

Nutritive stress

slightly elevated virulence and faster germination rates than conidia produced on the con-

Osmotic stress

trol medium (PDAY), but this trend did not hold for media with the lower osmolarity,

[(PDAY þ hydrogen peroxide, 5 mM) or UV-A (irradiation of mycelium on PDAY)]; heat shock

Oxidative stress

(0.6 M). Conidia produced from mycelium irradiated with UV-A while growing on PDAY

UV-A radiation

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. ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

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 & 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

* Corresponding author. Tel.: þ435 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.2008.04.011

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questions that researchers had been asking for decades (Daoust & Roberts 1982, 1983; Fargues & 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 & 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 & 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, 2005; Shah et al. 2005). Higher germination speed, in some cases, has been associated with higher virulence (Al-Aidroos & 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 (potatoddextrose agar (PDA) and agar supplemented with yeast extract).

Materials and methods 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, Sparks, MD) supplemented with 1 g l1 yeast extract (Technical, Difco; PDAY) adjusted to pH 6.9.

Conidial production and stress treatments Conidia were produced as follows: (1) on PDAY medium as the control; (2) on PDAY medium and exposed on the third day

D. E. N. Rangel et al.

after inoculation to heat shock at 45  C for 40 min; (3) on PDAY medium and exposed on the third day to UV-A irradiation (196 mW m2 for 1 h); (4) on PDAY medium amended with 5 mM hydrogen peroxide; (5–6) on PDAY medium amended with 0.6 or 0.8 M sodium chloride; (7–8) on PDAY medium amended with 0.6 or 0.8 M potassium chloride; (9) on minimal medium (MM Czapek medium minus sucrose; 0.2 % (w/v) sodium nitrate, 0.1 % (w/v) dipotassium phosphate, 0.05 % (w/v) magnesium sulphate, 0.05 % (w/v) potassium chloride, 0.001 % (w/v) ferrous sulphide, 1.5 % (w/v) Bacto Agar (Becton Dickinson, Sparks, MD); or (10) on MM amended with 3 g l1 a-lactose (Sigma; MML). All media were pipetted (23 ml) into 95 mm polystyrene Petri dishes. The pH of each medium was adjusted to 6.9. Conidial suspensions (100 ml of 107 conidia ml1) were spread evenly on agar media with a bent glass rod, and the cultures were incubated in the dark at 28  1  C for 14 d. Five different batches of conidia were produced, one for each replication of the experiment. Some culture conditions/treatments afforded low production of conidia, in which case large numbers of plates were needed for those treatments to garner sufficient conidia for bioassay. For conidial production on MM, 120 plates were required for each replication. For conidial production on MML or PDAY supplemented with 0.8 M sodium chloride or potassium chloride, 25 plates were used, and 15 plates were used for PDAY supplemented with 0.6 M sodium chloride or potassium chloride. For conidial production on PDAY and the other three treatments (heat shock, UV-A, and hydrogen peroxide), only one plate was needed for each.

Insect mortality (virulence) Conidia were harvested by flooding the first plate with 5 ml sterile Tween 80 solution [0.1 % (v/v)] and transferring this liquid successively from one plate to another, for up to five plates. A sterile polyethylene cell lifter (Costar 3008, Corning International, Corning, NY) was used to liberate the conidia from the mycelial mat. The washes were pooled, centrifuged at 6500 rev min1 for 15 min and the pellet resuspended in sterile Tween 80 [0.1 % (v/v)]. The conidial suspensions were adjusted, based on haemocytometer counts, to 1  108 conidia ml1. Fifty Tenebrio molitor (3rd instar larvae; Rainbow Mealworms, Compton, CA), were dipped for 30 s into 10 ml of each conidial suspension in a 50 ml polystyrene conical tube (Falcon Becton Dickinson, New Jersey) or into sterile Tween 80 [0.1 % (v/v)] as a control. The insects were then removed from the suspensions by sterile-gauze filtration and excess moisture removed by allowing the larvae to crawl on dry sterile filter paper for 30 s. The insects were maintained in 60  15 mm Petri dishes, ten insects per dish, inside glass jars lined with wet filter paper and tightly sealed with Parafilm to maintain relative humidity at approximately 100 %. The jars were held at 28  0.5  C for 7 d. The insects were maintained without food. Insect mortality was recorded daily. Dead insects were removed daily, surface sterilized with 20 mM hydrogen peroxide for 5 min, placed in sterile Petri dishes lined with moist filter paper and kept in humid chambers (jars) as described above. Cadavers were monitored for 7 d for fungal growth. Five complete trials (repetitions) were conducted using a different culture batch and insect

Stress effects on Metarhizium anisopliae in culture

population for each trial. T. molitor is routinely used as a model host insect for studies with insect pathogenic fungi (Shah et al. 2005).

Conidial germination assays The germination speeds of conidia produced on the different media were assessed by inoculating 20 ml (105 conidia ml1) of the suspension on 4 ml PDAY medium in polystyrene Petri dishes (35  10 mm). The plates were incubated 3 or 6 h in the dark at 28  1  C, and germination was read at 400 magnification. A total of 300 conidia were scored for each treatment each time. Three independent repetitions of the experiment were performed.

Conidial adhesion assays To determine conidial adhesion to insect cuticles, three Tenebrio molitor larvae were exposed for 30 s to conidial suspensions of each fungal treatment and briefly blotted on filter paper as described above. Within 1 min of exposure to the conidia, the insects were immersed in 300 ml Tween 80 [0.1 % (v/v)] in 1.5 ml Eppendorf tubes, vortexed for 15 s, and the number of conidia washed from the insects’ cuticles were estimated immediately by haemocytometer counts. Trials were replicated at least six times, resulting in a total of 18 larvae tested for each fungal treatment.

Fluorescence microscopy assay Fluorescence microscopy with calcofluor white (Sigma), a vital stain that binds to b-glucans, was used to determine if culture media affected the carbohydrate composition at the surface of conidial cell walls. For this technique, we followed the method described by Ibrahim et al. (2002). Briefly, sporulating cultures of Metarhizium anisopliae grown on each of the culture media (stress conditions) were used to prepare separate conidial suspensions (107 conidia ml1). For each conidial suspension, 10 ml were centrifuged for 15 min at 2000 g, washed in 10 ml Tween 80 [0.03 % (v/v)] three times and re-suspended in 10 ml of 0.01 % aqueous fluorescent brightener 28 (Calcofluor White M2R, Sigma). The suspension was then incubated overnight at room temperature in the dark. Following incubation, the stained conidia were washed three times in sterile distilled water and resuspended in 10 ml of 0.03 % Tween 80 before examination under a fluorescence microscope. This assay was conducted two times.

Statistical analyses Insect mortality. Percentage mortality data were arcsinesquare root transformed before analysis to better meet assumptions of normality and homogeneity of variance. The effects of stress treatments and trial replication on percentage mortality of the host insect on day 3 after inoculation were assessed using a two-way factorial in a randomized block design [Proc Mixed, (SAS Institute 2002)]. Significance levels of pair-wise mean comparisons among treatments were controlled for experiment-wise type I error using the Tukey– Kramer method with overall a ¼ 0.10. Contrasts between stress groups and the untreated control (UV-A versus PDAY,

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heat versus PDAY, H2O2 versus PDAY, average of MM and MML versus PDAY, and average of potassium chloride and sodium chloride versus PDAY) were conducted with the ‘Estimates’ statement of SAS (SAS Institute 2002). Germination assay. The effect of treatment on percent germination was assessed with analysis of variance of a oneway factorial in a randomized block design. Significance levels of pair-wise mean comparisons among treatments were controlled for experiment-wise type I error using the Tukey–Kramer method with overall a ¼ 0.10. Data were log transformed before analysis to better meet assumptions of normality and homogeneity of variance. Separate analyses were computed for germination at 3 and 9 h. Conidial adhesion. The effect of treatment, based on conidia per insect, was assessed with an analysis of variance of a one-way factorial in a randomized block design. Significance levels of pair-wise mean comparisons among treatments were controlled for experiment-wise type I error using the Tukey–Kramer method with overall a ¼ 0.10. Data were log transformed before analysis to better meet assumptions of normality and homogeneity of variance.

Results 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 ¼ 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 five trials (P > 0.05). The total mortality in the negative control treatment immersed in Tween 80 solution was less than 20 % at the 5th day (Fig 1B), whereas, mortality of insects immersed in suspension with fungus reached almost 90 % on the 5th day (Fig 1B).

Germination assays After 3 h incubation on PDAY, germination was similar among treatments; except conidia produced on MM or 0.8 M sodium chloride had higher germination levels (Fig 2A). After 6 h, germination of conidia produced on MM or MML was significantly faster in comparison with the other treatments (P < 0.0001; Fig 2B) and conidia produced under all osmotic-stress treatments also germinated faster than conidia produced on PDAY medium, but germination was slower than that of conidia from MM and MML media.

Conidial adhesion assays and fluorescence microscopy assay Conidia produced on MML had considerably higher conidial adhesion to host cuticle (P ¼ 0.005; Fig 3) and greater fluorescence after calcofluor staining (Fig 4) than conidia from PDAY and the other treatments.

D. E. N. Rangel et al.

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Time (d) Fig 1 – Virulence of Metarhizium anisopliae conidia produced under different culture conditions. (A) Percentage mortality of Tenebrio molitor (instar 3) on day 3 after conidial application. Errors bars are standard errors of five independent experiments. Graph bars with the same letters are not significantly different (P < 0.001). (B) Mortality during the first 5 d after conidial application. Control [ Tween 80 [0.1 % (v/ v)]. Abbreviations for Figs 1–4: (1) MML, conidia produced on minimal medium amended with 3 g lL1 a-lactose (Sigma); (2) MM, conidia produced on minimal medium; (3) UV-A, conidia produced on PDAY medium with exposure on the third day of growth to UV-A irradiation (196 mW mL2 for 1 h); (4) potassium chloride, conidia produced on PDAY medium amended with 0.6 or 0.8 M potassium chloride; (5) sodium chloride, conidia produced on PDAY medium amended with 0.6 or 0.8 M sodium chloride; (6) HP, conidia produced on PDAY medium amended with hydrogen peroxide; (7) heat shock, conidia produced on PDAY medium, with the mycelium exposed on day 3 after inoculation to heat shock at 45  C for 40 min; (8) PDAY, conidia produced on potato–dextrose agar supplemented with 1 g lL1 yeast extract (non-stressed control).

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

Fig 2 – Germination speed on PDAY medium at 28  C of Metarhizium anisopliae conidia produced under different culture conditions. (A) Germination after 3 h incubation. (B) Germination after 6 h incubation. Errors bars are standard errors of three independent experiments. Graph bars with the same letters are not significantly different (P < 0.001).

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 to insects. Nutrient deprivation triggers increased Pr1 transcription and rapid secretion of this enzyme by M. anisopliae (Freimoser et al. 2005; St Leger et al. 1991b, 1994; Wang & St Leger 2005). The enzyme Pr1 is an extracellular chymoelastase that constitutes the major protein synthesized by this fungus during penetration through proteinaceous insect cuticles (Goettel et al. 1989; St Leger et al. 1987, 1989). The subtilisin Pr1 is upregulated on minimal medium (Freimoser et al. 2005) and on the insect cuticle (St Leger et al. 1991a). Conversely, protease synthesis is repressed in rich artificial media and in insect haemolymph (which also is nutrient rich) (St Leger et al. 1996). Three-day-old cultures exposed to short-term UV-A radiation also produced conidia more virulent to Tenebrio molitor larvae than conidia produced on the PDAY control medium. The virulence of conidia from UV-A treatment was similar to that of conidia produced on MM or MML (Fig 1), but both germination rates and conidial adhesion to cuticle of conidia from UV-A-treated mycelium were similar to that of conidia produced on PDAY medium (Figs 2 and 3). However, the heatshock treatment on the 3rd day of mycelial growth on PDAY produced conidia with similar virulence and germination

Stress effects on Metarhizium anisopliae in culture

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speed to that of conidia produced on PDAY without heat treatment (Fig 1A). Conidia produced under the higher osmotic stress (potassium chloride or sodium chloride at 0.8 M) also were more virulent than conidia produced on PDAY (Fig 1). However, conidial virulence decreased to the same level as the control (PDAY) when the salt concentration was lowered to 0.6 M. Increased virulence of conidia produced under osmotic stress has been reported [by addition of potassium chloride 0.75 M (Shah et al. 2005) or on glycerol media (Hallsworth & Magan 1994b)]. Also, conidia produced from mycelia submerged in a high osmolarity medium [with polyethylene glycol 200 (PEG)] had increased virulence to the grasshopper Schistocerca americana (Leland et al. 2005).

The medium MML produced conidia with greatest adhesion capacity and were the most hydrophobic when suspended in sterile MilliQ water. Conidial adhesion variability has been found in Isaria fumosoroseus isolates (Altre et al. 1999) and in M. anisopliae conidia or blastospores produced under different culture conditions (Ibrahim et al. 2002; Lane et al. 1991). In addition, conidia from SDAM medium (SDA media plus 0.75 M potassium chloride) had higher fluorescence with calcofluor treatment and adhered more readily to insect cuticle than conidia produced on MM (Ibrahim et al. 2002). In the present study, the surface properties of conidia produced on MML, as indicated by increased calcofluor-stain fluorescence and by greater attachment to insect cuticle, were much more reactive than PDAY-produced conidia (Fig 4); however, conidia produced with other stress treatments had similar brightness to conidia from PDAY (data not shown). This indicates that the MML culture medium influenced the surface properties of conidia produced, which, in turn, influenced binding of spores to insect cuticles by hydrophobic forces. Conidia produced on MM or MML germinated faster than conidia from the other media, especially in comparison with conidia produced on PDAY medium. Also, conidia produced on MM or MML have at least two-fold more trehalose and at least three-fold more mannitol than conidia produced on MM plus 3 % glucose medium (Rangel et al. 2006, 2008). Accordingly, it is probable that the increased germination rate of conidia produced on MM or MML is due to the high endogenous mannitol and trehalose in these conidia (Rangel et al. 2006). Indeed, intracellular trehalose and mannitol mobilization can contribute to some extent to the energy requirements of endogenous respiration and provide energy for spore germination (d’Enfert et al. 1999; Elbein et al. 2003; Thevelein 1984). Accumulations of endogenous mannitol also have been correlated with virulence in other microbes. A mutant of the fungus Cryptococcus neoformans that produces low amounts of

Fig 4 – Fluorescence of Metarhizium anisopliae conidia stained with calcofluor white (Sigma). (A) Conidia produced on MML. (B) Conidia produced on PDAY medium. Bar [ 10 mm.

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mannitol is less virulent in mice than the wild-type that produces higher amounts of endogenous mannitol (Chaturvedi et al. 1996a). Mannitol protects cells by scavenging reactive oxygen intermediates (Chaturvedi et al. 1996b). Because one of the defences of insects to pathogens is hydrogen peroxide production (Musser et al. 2005), the high levels of endogenous mannitol accumulated in M. anisopliae conidia produced on MML and MM (Rangel et al. 2006) may have aided in improving virulence by scavenging the protective peroxide. Conidia produced under osmotic stress also germinated faster than conidia from PDAY media, and this has been observed elsewhere (Leland et al. 2005; Shah et al. 2005). There is little information available on the influence of stress on the virulence of entomopathogenic fungi (Crespo et al. 2002; Ibrahim et al. 2002; Ropek & Para 2002). Recently it was demonstrated that osmotic stress made M. anisopliae conidia more virulent to insects (Leland et al. 2005; Shah et al. 2005). Our studies demonstrate that nutritive stress, UV-A radiation and osmotic stress during growth improved virulence of the resulting M. anisopliae conidia. Of interest was the finding that MML significantly improved virulence, germination speed and host adhesion. MML was previously reported to improve conidial UV-B tolerance (Rangel et al. 2006). Conidia from this medium were very difficult to suspend in pure water and, in fact, adhered to the walls of plastic tubes. This phenomenon warrants further investigation. In conclusion, we found high variations in virulence plasticity, as well as speed of germination of M. anisopliae conidia in response to different stress conditions during culture. However, the benefits of producing highly virulent conidia on MM or MML have the enormous cost of low conidial production (Rangel et al. 2006, 2008). Accordingly, searches for other approaches to mass production of physiologically improved fungi for biological control of insects in agriculture should be sought.

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 the Utah Department of Agriculture and Food, Division of Plant Industry. Paper # 7821 of the Utah Agricultural Experiment Station.

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