Correspondence: Donald W. Roberts, Department of Biology, Utah State University, Logan, UT 84322-5305, USA. Tel.: +1 435 797 0049; fax: +1 435 797 1575; e-mail: email@example.com
Light conditions during mycelial growth are known to influence fungi in many ways. The effect of visible-light exposure during mycelial growth was investigated on conidial tolerance to UVB irradiation and wet heat of Metarhizium robertsii, an insect-pathogenic fungus. Two nutrient media and two light regimens were compared. Conidia were produced on (A) potato dextrose agar plus yeast extract medium (PDAY) (A1) under dark conditions or (A2) under continuous visible light (provided by two fluorescent lamps with intensity 5.4 W m−2). For comparison, the fungus was also produced on (B) minimal medium (MM) under continuous-dark incubation, which is known to produce conidia with increased tolerance to heat and UVB radiation. The UVB tolerances of conidia produced on PDAY under continuous visible light were twofold higher than conidia produced on PDAY medium under dark conditions, and this elevated UVB tolerance was similar to that of conidia produced on MM in the dark. The heat tolerance of conidia produced under continuous light was, however, similar to that of conidia produced on MM or PDAY in the dark. Conidial yield on PDAY medium was equivalent when the fungus was grown either under continuous-dark or under continuous-light conditions.
Visible light during mycelial growth influences: (1) primary (Dunlap & Loros, 2006) and secondary metabolism (Bayram et al., 2008; Fischer, 2008); (2) induction of heat-shock proteins HSP100 in Phycomyces (Rodriguez-Romero & Corrochano, 2004, 2006), which are important in protecting the cells against several stress conditions by repairing misfolded and aggregated proteins; (3) trehalose accumulation in Neurospora crassa spores (Shinohara et al., 2002), which stabilizes proteins in their native state and preserves the integrity of membranes; and (4) pigment formation in several fungal species (Leach, 1971; Geis & Szaniszlo, 1984). All these light-affected mechanisms may be important to protect conidia against UVB radiation or to neutralize free radicals and oxidants.
The effect of visible light during mycelial growth on the stress tolerance of the resulting conidia is not known, but the influence of light on trehalose and heat-shock protein metabolism during mycelial growth suggests that conidia from light-exposed mycelium may exhibit enhanced tolerance to UVB and wet heat. This study explores this possibility with conidia of a well-known isolate (ARSEF 2575) of the insect-pathogenic fungus Metarhizium robertsii by testing conidia produced under light or dark conditions to detect differences in conidial tolerances to UVB radiation and heat. Metarhizium is an important biocontrol agent of agricultural insect pests (Li et al., 2010) and insect vectors of human diseases (Luz et al., 1998; Scholte et al., 2005).
Materials and methods
Metarhizium robertsii isolate ARSEF 2575 was obtained from the USDA–ARS Collection of Entomopathogenic Fungal Cultures (ARSEF) (RW Holley Center for Agriculture and Health, Ithaca, NY). ARSEF 2575 was isolated originally from Curculio caryae (Coleoptera: Curculionidae) in South Carolina. Stock cultures were maintained at 4 °C in test-tube slants of potato dextrose agar (Difco Laboratories, Sparks, MD) supplemented with 1 g L−1 yeast extract (Technical, Difco Laboratories) (PDAY) adjusted to pH 6.9.
Conidial production with and without visible-light treatment
Conidia were produced on 23 mL of PDAY medium in Petri dishes (polystyrene, 95 × 15 mm, Fisherbrand®, Pittsburgh, PA) with their lids in place, in a single layer (not stacked), under continuous dark or continuous light provided by two 15 W cool white Sylvania® fluorescent lamps suspended 25 cm from the samples. A sheet of 0.13-mm cellulose diacetate covered the plates to avoid medium dehydration. Spectrophotometric (Ocean Optics USB 2000 Spectroradiometer, Dunedin, FL) readings of the 290–750 nm output of the lamps that passed through the diacetate film plus the Petri dish lid were 5.4 W m−2 (Fig. 1), and the spectrum was almost identical to that of light passing through the diacetate, but without the Petri dish lid.
Conidia were also produced on a basal medium [minimal medium (MM)] 0.2% NaNO3, 0.1% K2HPO4, 0.05% MgSO4, 0.05% KCl, 0.001% FeSO4, and 1.5% Bacto agar (Becton, Dickinson and Co., Sparks, MD) adjusted to pH 6.9 under continuous dark, a condition that improves conidial tolerance of M. robertsii to UVB radiation and heat (Rangel et al., 2006a, b, 2008).
Conidial suspensions (100 μL of 107 conidia mL−1) were inoculated evenly with a glass spreader onto agar media. The cultures were incubated at 28 ± 1 °C for 14 days. Three different batches of conidia were produced, one for each replication of the experiment.
UV-radiation and wet-heat tolerances of conidia
The inoculum for each pair of treatments (UV and heat) was prepared for simultaneous exposures. Conidia were harvested after 14 days from colonies grown under continuous visible light or in the dark with a single pass of a microbiological loop through the spore layer of the fungal colonies without touching the substrate, and the conidia immediately suspended in 10 mL of sterile Tween 80 solution (0.01% v/v) in 15-mL polystyrene tubes (Corning®, Corning, NY). The suspensions (c. 105 conidia mL−1) were shaken vigorously using a vortex; filtered through a polycarbonate membrane (25 mm diameter, 8-μm pore size, Whatman® Nucleopore®, Acton, MA) to remove spore aggregates and mycelium; and the suspension was used immediately in the heat- and UVB-exposure experiments.
UVB irradiation experiments were conducted at 28 ± 1 °C in a Percival growth chamber (Boone, IA), with two UVB fluorescent lamps (TL 20W/12 RS, Philips, Eindhoven, Holland), with primarily UVB (peak at 313 nm) and minimal UVA radiation output. The Petri dish lids were removed during irradiation, but the plates were covered with cellulose diacetate filters (JCS Industries, Le Miranda, CA) to exclude UVC and short-wavelength UVB radiation. Spectral irradiance was measured as in Rangel et al. (2005a, b). The DNA damage (cyclobutane pyrimidine dimer formation) action spectrum developed by Quaite et al. (1992) and normalized to unity at 300 nm was used to calculate weighted UV irradiances in the chamber at sample height, which was 978 mW m−2. The total 2-h exposure afforded a dose of 7.04 kJ m−2.
For each of the three UVB-tolerance trials, conidial suspensions prepared from different growth treatments were inoculated (20 μL, without spreading) at the center of Petri dishes (polystyrene, 35 × 15 mm, Fisherbrand®) with 4 mL each of PDAY amended with 0.002% benomyl (25% active ingredient; Hi-Yield Chemical Company, Bonham, TX) (Milner et al., 1991). Controls for these experiments were conidia on plates that were not irradiated (placed in the chamber, but covered with an aluminum foil barrier). After exposure, the plates were incubated for 48 h in the dark at 28 °C, and then observed at × 400 magnification for germination. Conidia were considered germinated when a germ tube visibly projected from the conidium (Milner et al., 1991). At least 300 conidia per plate were evaluated, and the relative percent germination was calculated as described by Braga et al. (2001).
Two milliliters of the same filtered suspension used for UVB exposure was placed in pyrex screw-cap tubes (16 × 125 mm) and placed immediately in a 45 °C agitated (stirred) waterbath (Rangel et al., 2005a, b). After 3 h of wet-heat exposure, 20 μL of the conidial suspension was inoculated (dropped, but not spread) onto PDAY+benomyl medium and germination was determined as described above and elsewhere (Rangel et al., 2005a, b).
To measure conidial production after a 14-day incubation under the different culture conditions, three agar plugs were removed from each plate at random places in the medium with a cork borer (5 mm diameter) and all three (total surface area ∼60 mm2) were placed in 1 mL of sterile Tween 80 (0.01% v/v). The conidia were suspended by vigorous vortexing, and conidial concentrations were determined by hemacytometer counts. Each experiment was performed on three different dates, and each experiment used a new batch of cultures.
Assessment of the effects on conidia of continuous light or dark during their production, i.e., mycelial growth and conidiation, on PDAY medium was compared with the effects on conidia produced on MM in continuous dark as to differences in (1) relative conidial germination after heat or UVB treatment or (2) conidial production by one-way anova in a randomized block design in which trials were blocks. Relative germination data were arcsine-square root transformed and conidial production data were log transformed before analysis to better meet assumptions of normality and homogeneity of variance. Pairwise comparisons of means were controlled for experiment-wise type I error using the Tukey method at α=0.05. Computations were performed using the MIXED procedure in sas (SAS Institute Inc., 2002).
Results and discussion
UV-radiation and wet-heat tolerances of conidia
In many organisms, preadaptation to one stress may induce cross-protection to other stresses. This was found to be true for insect-pathogenic fungi M. robertsii (Rangel et al., 2006a, b, 2008) and Beauveria bassiana (Liu et al., 2009). When M. robertsii conidia were produced under nutritive stress (carbon starvation) or osmotic stress (NaCl or KCl), they were approximately twofold more tolerant to heat and UVB radiation than conidia produced under normal conditions on a rich (PDAY) medium (Rangel et al., 2006a, b, 2008). Also, other physical conditions of the environment during mycelial growth that may not necessarily be stress conditions might improve the stress tolerance of conidia. As reported here, this is true for M. robertsii mycelia grown under continuous visible-light exposure (5.4 W m−2), which induced significantly higher (almost twofold) conidial tolerance to UVB radiation (F2, 5=24.7, P<0.0025) (Fig. 2a). The UV-B tolerance of conidia produced on PDAY under constant visible light was similar to that of conidia produced on MM (nutritive stress), which is found elsewhere (Rangel et al., 2006a, b, 2008).
The survival rates of the light-grown dematiaceous fungus Wangiella dermatitidis revealed that the carotenoid-pigmented cells are considerably more resistant to UV radiation than nonpigmented ones grown in the dark (Geis & Szaniszlo, 1984). However, the pigment melanin, as well as the biosynthetic precursor of melanin (Rangel et al., 2006a, b; Fang et al., 2010), and carotenoids (Fang et al., 2010; Gonzales et al., 2010) have not been found in M. robertsii or Metarhizium anisopliae conidia. Therefore, these pigments are not involved in light-induced increases in the stress tolerance of M. robertsii conidia.
Conidia produced on PDAY under visible light had somewhat elevated tolerance to heat (45 °C for 3 h), but not significantly different from conidia produced on PDAY under continuous dark (F2, 4=7.8, P<0.0240) (Fig. 2b). It is well known that growth under nutritive stress induces cross-protection, providing the highest tolerance to heat and other stresses as found in this study and elsewhere (Steels et al., 1994; Park et al., 1997; Rangel et al., 2008; Rangel, 2010). Light during mycelial growth did not induce as much phenotypic plasticity in heat tolerance as it did for UVB radiation for the reason that microbial growth on different environmental conditions exhibits different levels of stress tolerance (Gasch & Werner-Washburne, 2002).
The growth of M. robertsii under osmotic or nutritive stress conditions decreased conidial production to approximately 20–40-fold, respectively, of that of conidia produced on PDAY medium (Rangel et al., 2008). When conidia were produced on PDAY under either continuous light or dark, the conidial yields were similar, but, as reported earlier (Rangel et al., 2006a, b, 2008), conidial yield on MM was extremely low (F2, 4=3566.5, P<0.0001) (Fig. 2c). Sporulation in many fungi is unaffected by light, as found here with M. robertsii (ARSEF 2575). In other species, however, light is very important for conidiogenesis (Griffin, 1996). A few reports indicate that continuous light influences conidial production in entomopathogenic fungi. For example, the maximum yield of Metarhizium acridum conidia was found when the fungus was grown under continuous light (Onofre et al., 2001) or with M. anisopliae s.l. under intermittent light (Alves et al., 1984). Continuous or intermittent light also resulted in prolific conidial production by the entomopathogenic fungi Isaria fumosorosea (=Paecilomyces fumosoroseus) (Sakamoto et al., 1985; Sanchez-Murillo et al., 2004) and B. bassiana (Zhang et al., 2009).
Conidia produced on a rich medium (PDAY) in the presence of continuous visible light were twofold more UVB tolerant and slightly more heat tolerant. The relative importance of the spectral elements and intensities of the visible light used in this study for producing conidia with increased stress tolerance is currently unknown; future studies will be directed to this question.
Growth under visible light on PDAY improved conidial stress tolerance, but unlike growth on MM, conidial production was not negatively influenced. Therefore, culture on rich media under light is proposed to be a promising approach for mass-producing conidia with improved UVB tolerance for the biological control of insect pests in agriculture. Because conidial mass production using Petri dishes or larger containers in a single layer during visible-light exposure would require excessive shelf space, new approaches for exposing production containers to effective levels of light are being sought.
Note added in proof
Recent experiments revealed that the average relative germination rate of conidia of M. robertsii produced under constant visible light was approximately 50% compared with approximately less than 1% germination of conidia produced under constant darkness. This is in contrast to responses following 3-h exposures to 45°C (see Fig. 2b), which did not afford a significant difference in germination levels between conidia produced under constant-light and constant-dark conditions. The higher germination of light-produced conidia in comparison to dark-produced ones after 4 h of heat treatment clearly indicates that light during mycelial growth can substantially improve heat tolerance of the resulting conidia.
We are grateful to Susan Durham (Utah State University, Logan, UT) for the statistical analyses. We sincerely thank the Brazilian National Council for Scientific and Technological Development (CNPq) for PhD fellowships #GDE 200382/02-0 for D.E.N.R. and #SWE 2006412005-0 for É.K.K.F. as well as the Utah Department of Agriculture and Food for research funds to D.W.R.