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Damage and recovery from UV-B exposure in conidia of the entomopathogens Verticillium lecanii and Aphanocladium album

     Department of Biology, Utah State University, Logan, Utah 84322-5305

Stephan D. Flint

     Department of Rangeland Resources and the Ecology Center, Utah State University, Logan, Utah 84322-5230

Charles D. Miller
Anne J. Anderson
Donald W. Roberts ¹

     Department of Biology, Utah State University, Logan, Utah 84322-5305

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

We evaluated the effects of exposure to doses supplied at an environmentally realistic intensity of UV-B radiation (800 mW m^-2 weighted irradiance) on the culturability and germination of selected strains of the entomopathogenic Hyphomycetes Verticillium lecanii and Aphanocladium album. Increased UV-B exposure decreased relative percent culturability for all strains. Four hours of exposure to UV-B were sufficient to reduce the culturability close to zero. The LT₅₀ (50% lethal time) ranged from 120 ± 5 min for the V. lecanii strain ARSEF 6430 to 86 ± 14 min for the A. album strain ARSEF 6433. A strong delay in the germination of surviving conidia was observed. To determine the occurrence of photoreactivation in these two genera, we evaluated the effect of exposure to visible light after exposure to UV-B radiation. There was no significant difference in relative culturability between conidia exposed to visible light after UV-B exposure compared to those incubated in the dark after UV-B exposure. This indicates that photoreactivation, if it occurs, must have limited importance in the repair of the damage induced by UV-B radiation in these two genera.

Key words: biological control, conidial germination, conidial photobiology, Hyphomycetes, photoreactivation, UV damage

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The deleterious effects of UV radiation have been demonstrated in several genera of entomopathogenic fungi such as Metarhizium, Beauveria, and Paecilomyces (Morley-Davis et al 1995, Fargues et al 1996, 1997, Braga et al 2001a, b, c, d). Although the Hyphomycete species V. lecanii and A. album are used for the control of various agricultural pests in different UV environments (Hall 1980, 1981, Lopez Lastra 1990, Lacey et al 1996, Steenberg and Humber 1999, Junqueira et al 1999), little is known about the effects of UV radiation on these genera. In general, a few hours of direct exposure to a radiation of an intensity frequently encountered in the environment are sufficient to fully inactivate the conidia of all the species studied. In addition to inducing inactivation, UV radiation has been demonstrated to delay the germination of surviving conidia (Zimmermann 1982, Moore et al 1993, Braga et al 2001a, b, c, d,). The reduction in inoculum due to conidial inactivation and the delay in germination are expected to reduce the efficiency of these organisms as bioinsecticides in situations with strong solar irradiation. Among these situations are those in which the strains are exposed during inoculation and/or germination to irradiances and doses above those occurring in their original habitats and to which they are not fully adapted. The reduction in the ozone layer and the consequent increase in UV-B irradiance, particularly at wavelengths between 290 and 315 nm, may aggravate the problem (Caldwell and Flint 1997, Caldwell et al 1998, Madronich et al 1998, McKenzie et al 1999). We have demonstrated previously that an increase in UV-B irradiance drastically reduces the culturability of conidia of various species of the genus Metarhizium (Braga et al 2001a, b, d) and that the effect of increased irradiance is greater during the growth phases in which the fungus is more susceptible to radiation, such as the end of germination (Braga et al 2001a). In general, attempts have been made to reduce the negative effects of UV radiation on entomopathogens by adding photoprotective agents to the formulations (Moore et al 1993, Alves et al 1998) and by selecting strains more tolerant to radiation. Strain selection has been facilitated by the wide intraspecific variability in tolerance observed in species such as Metarhizium anisopliae (Fargues et al 1996, Braga et al 2001b, c, d), M. flavoviride (Morley-Davies et al 1995, Fargues et al 1996), Beauveria bassiana (Morley-Davies et al 1995, Fargues et al 1996), and Paecilomyces fumosoroseus (Fargues et al 1996). Part of the variability in UV tolerance may be explained by the adaptation of the strains to specific habitats (Bidochka et al 2001) and to different environmental UV levels associated with the latitude of the sites of origin (Braga et al 2001d).

It is well established that UV-B radiation is the fraction of the spectrum that presents the highest biological effectiveness and the highest potential to damage entomopathogens within the wavelengths that reach the earth's surface (Moore et al 1993, Fargues et al 1997, Braga et al 2001c). In a recent series of field experiments, we demonstrated that solar UV-A also reduces the culturability and delays the germination of M. anisopliae conidia (Braga et al 2001c).

Both the conidial inactivation and the delay in the germination of the survivors are mainly caused by DNA damage induced by UV radiation. While UV-A usually causes only indirect damage to DNA through catalyzing the formation of chemical intermediates such as sensitizer radicals or reactive oxygen species, UV-B acts directly in DNA inducing the formation of several photoproducts (Friedberg et al 1995, Griffiths et al 1998). The most common photoproduct induced by UV-B is the cyclobutane pyrimidine dimer (Sancar 1994). This kind of lesion is removed by systems such as nucleotide excision repair (NER) and by photolyases, which are light-dependent enzymes that directly revert pyrimidine dimers into the original pyrimidines (Eker et al 1994, Sancar 1994, Deisenhofer 2000). The relative importance of these repair systems varies considerably among different biological systems (Sancar 1994, Kim and Sundin 2001). A simple way of detecting photoreactivation is to compare the survival of cells that remained in the dark to the survival of cells that were exposed to photoreactivating wavelengths (375–425 nm) after UV-B exposure. The greater the difference between the survival levels, the greater is the relative importance of photoreactivation (Tuveson and MacCoy 1982, Sametz-Baron et al 1997, Kim and Sundin 2001).

Our objective was to determine the effects of UV-B radiation on the conidia of selected strains of V. lecanii and A. album. Responses were measured as relative percent germination and relative percent culturability in relation to untreated spores. In order to determine the relative importance of photoreactivation on the repair of radiation-induced damage to the genetic material, we examined the effect on conidia of visible light exposure after UV-B radiation.

As far as we know, this is the first study showing the damage to, and recovery of, V. lecanii and A. album conidia after exposure to environmentally realistic UV doses and irradiance.

	MATERIALS AND METHODS

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V. lecanii and A. album strains – Strains were obtained from the USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF), US Plant, Soil & Nutrition Laboratory, Ithaca, New York, USA. The geographic origins and the insect host from which they were isolated are listed in Table I.

Irradiation chamber, lamps and filter – Irradiation experiments were conducted in a temperature-controlled Percival growth chamber (Boone, Iowa). The UV irradiance was provided by two TL 20W/12 RS fluorescent lamps (Philips, Eindhoven, Holland). To achieve a stable level of radiation during the different trials, lamps were aged prior to the start of the experiments. The temperature inside the chambers was maintained at 27 ± 1 C. The irradiated material was covered with a 0.13 mm-thick cellulose diacetate film (JCS Industries, Le Mirada, California), which blocked radiation below 290 nm. This permitted the passage of most UV-B and UV-A, but prevented sample exposure to UV-C (<280 nm) and short-wavelength UV-B. All UV radiation measurements were made with a double-grating spectroradiometer (Optronic 742, Orlando, Florida) as in Braga et al (2001a). The spectral distributions of the filtered lamps in the chamber is shown in Braga et al (2001a). The DNA damage action spectrum developed by Quaite et al (1992) and normalized to unity at 300 nm was used to calculate weighted UV irradiances (mW m^-2). We selected this spectral weighting function because Paul et al (1997) reviewed the spectral characteristics of nine fungal responses and concluded that this DNA damage spectrum closely approximated the fungal responses. The weighted irradiance was 800 mW m^-2. For our location, Logan, Utah, USA (41.5°N latitude, 1.5 km elevation), a model (Fiscus and Booker 1998) indicates that this irradiance value approximates noon sunlight in early spring (early April).

Effect of UV-B on conidial relative percent culturability – For each of three trials, the inoculum (40 µL, 2 x 10⁴ conidia mL^-1 for 1 and 2 h of exposure or 40 µL, 4 x 10⁴ conidia mL^-1 for 3 and 4 h of exposure) was spread over the surface of PDAY (8 mL) in Petri dishes (polystyrene, 60 x 15 mm, Fisherbrand®), using a sterile glass spreader. Conidia were immediately exposed to irradiances of 800 mW m^-2 for 1, 2, 3, or 4 h. Total doses supplied at the end of the periods of exposure were 2.9, 5.8, 8.8, and 11.8 kJ m^-2 [For comparison, we receive 25 kJ m^-2 d^-1 at our location (Logan, Utah) in mid-summer]. Four replicate dishes per exposure time were irradiated. The dishes were moved within the exposure area so that the replicates would receive the same amount of energy during the exposure period. Two control-dishes were prepared for each strain, for each exposure period, and protected from irradiation by aluminum foil inside the chambers. Two strains were exposed simultaneously in the chamber. After exposure, dishes were kept at 24 C in the dark for 4 d. Starting on the second day after exposure, colony-forming units (CFU) were counted. For each trial, conidial relative percent culturability after each exposure time was calculated by the following equation:

where M_t is the mean number of CFU of the 4 replicates at exposure time t and M_c is the mean number of CFU for all control dishes, regardless of exposure time.

Control-plate data were pooled over all exposure times because time in the chambers when plates were wrapped with aluminum foil did not influence level of culturability (data not shown).

The effects of exposure time (1, 2, 3, and 4 h), species (V. lecanii and A. album) and strains within species were assessed using a two-way factorial analysis of variance (exposure time and species) with strains nested within species. The design was a split-plot. The whole plot unit was a trial, and the whole plot factors were species and strains (nested within species). The subplot unit was repeated measurements within a trial, and the subplot factor was exposure time. Time of exposure to UV-B (in minutes) necessary to reduce culturability to 50% (LT₅₀) was estimated for all strains using culturability curves.

Effect of UV-B radiation on conidial germination – The effect of UV-B exposures on conidial germination was determined in the ARSEF 6430 strain of V. lecanii and in the ARSEF 6433 strain of A. album. Three trials were conducted on plates (polystyrene, 60 x 15 mm, Fisher Scientifics) containing 8 mL PDAY + 0.003% benomyl. The benomyl used (Hi-Yield Chemical Company, Bonham, Texas) was a formulated powder with 25% active ingredient. Low concentration of benomyl in the medium allowed germination to be monitored for longer periods of time because it inhibits the growth without adversely affecting germination as was observed in M. anisopliae previously by Milner et al (1991). The inoculum (two drops of 20 µL, 3 x 10⁵ conidia mL^-1) was placed on the medium and immediately exposed to the 800 mW m^-2 irradiance for 1, 2, 3, or 4 h. After exposure, the plates were incubated in the dark at 28 C. Germination was observed with 400 x magnification at 12, 24, and 36 h from the time when the conidia were placed on the medium. Conidia presenting a germ tube longer than the diameter of the conidium were considered to have germinated. A total of 300 conidia per treatment were evaluated. Conidia were stained with a drop of Methyl Blue solution [13g L^-1 in a 85% (w/w) lactic acid solution]. Relative percent germination after each period of incubation was calculated by the following equation:

Where W_t is the number of germlings at exposure time t and W_c is the number of germlings of the control plate.

Effect of visible light on conidial culturability recovery after UV-B exposure – The effect of visible light after UV-B exposures was determined in the ARSEF 6430 strain of V. lecanii and in the ARSEF 6433 strain of A. album. Conidia were exposed to visible light during their recovery period in order to check for the occurrence of photoreactivation. Sixteen plates per strain were inoculated for each UV-B exposure time (2 or 3 h). Eight plates were exposed to radiation and eight control plates were protected from radiation inside the chamber. After irradiation, 4 plates were exposed to two consecutive photoperiods (18:6, light:dark) and 4 plates were kept in the dark for a 48-h period of incubation. Eight control plates were similarly subdivided. During recovery at 28 ± 1 C, the plates remained closed and covered with a 0.1 mm-thick Llumar film (JSC Industries, Le Mirada, California) that blocks wavelengths shorter than 400 nm. At the end of the 48-h period, culturability of the conidia exposed to UV-B radiation was determined and compared to that of the respective controls. Three independent experiments were performed for each strain. Visible light during recovery was provided by two fluorescent lamps (Sylvania, cool white, 15 W and intensity of approximately 12 W m^-2). We also determined the effect of exposure to visible light at an intensity of 150 W m^-2 during the recovery period. In this case, light was provided by a 6000 W xenon lamp (Atlas Electric, Chicago, Illinois). Visible radiation (400–700 nm) was measured with an LI-1800 spectroradiometer (LiCor, Lincoln, Nebraska).

The effects of exposure time (2 and 3 h) and incubation (light and dark) on relative percent culturability were assessed using a two-way factorial analysis of variance in a randomized block design. Data for each of two strains were analyzed separately. Three trials for each strain formed three blocks. The response (relative percent culturability) was square root transformed prior to analysis to better meet assumptions of normality and homogeneity of variance. The results are shown in the original scale.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Effect of UV-B on conidial relative percent culturability – A strong negative effect of UV-B radiation on conidial culturability was observed with all V. lecanii and A. album strains (Fig. 1A–D). The LT₅₀ for all strains are shown in Fig. 1C. Four h of exposure were enough to reduce the culturability of all strains to near zero (Fig. 1D). Within a species, strain profiles vary in form (i.e., strain within species by exposure time interaction is significant; P = 0.005). However, the average profiles for the two species are not shown to be different (i.e., the species by exposure time interaction is not significant; P = 0.269); this result is consistent with the variability in profiles among strains within each species. Relative percent culturability decreases with increasing exposure time (P < 0.001) (Fig. 1A–C). Within a species, strains like V. lecanii ARSEF 6430 and A. album ARSEF 1329 have a consistently higher relative percent culturability than others (P = 0.050), but there is no apparent difference between species when averaged over strains (P = 0.979).

View larger version (28K): [in this window] [in a new window]    FIG. 1. Mean relative percent culturability of V. lecanii (black bars) and A. album (gray bars) strains after exposure for 1 (A), 2 (B), 3 (C) or 4 h (D) to the weighted irradiance of 800 mW m-2. Culturability was calculated in relation to non-irradiated controls. Error bars are standard deviations of three independent experiments. The LT50 ± SD (min) for all strains are shown in (C).

View larger version (17K): [in this window] [in a new window]    FIG. 2. Mean relative percent germination of V. lecanii strain ARSEF 6430 (A) and A. album strain ARSEF 6433 (B) after exposure for 1, 2, 3, or 4 h to the irradiance of 800 mW m-2. Relative percent germination was calculated in relation to non-irradiated controls after each incubation period (12, 24, or 36 h). Error bars are standard deviations of three independent experiments

View larger version (140K): [in this window] [in a new window]    FIG. 3. Effect of 1 and 2 h of UV-B exposure (800 mW m-2) on conidial swelling and on germ tube emergence in V. lecanii strain ARSEF 6430 and in A. album strain ARSEF 6433. 24-h germlings of V. lecanii from non-exposed conidia (A); from 1-h exposed conidia (B) and from 2-h exposed conidia (C). 24-h germlings of A. album from non-exposed conidia (D); from 1-h exposed conidia (E) and from 2-h exposed conidia (F). Bar = 15 µm.

View larger version (12K): [in this window] [in a new window]    FIG. 4. Effect of visible light (12 W m-2) on conidial recovery after UV-B exposure (800 mW m-2) for 2 or 3 h in V. lecanii strain ARSEF 6430 (A) and A. album strain ARSEF 6433 (B). Relative percent culturability was calculated in relation to non-irradiated controls. Error bars are standard deviations of three independent experiments.

View larger version (15K): [in this window] [in a new window]    FIG. 5. Effect of visible light (12 W m-2) on Trichoderma sp. of culturability of conidia recovery after UV-B exposure (800 mW m-2) for 2 or 3 h. Relative percent culturability was calculated in relation to non-irradiated controls. Error bars are standard deviations of three independent experiments. Experiments were carried out in Petri dishes (95 x 15 mm) containing 23 mL PDAY + 3 g L-1 of deoxycholic acid (Sigma). CFU counts were performed after 48 h of incubation at 24 C

View larger version (15K): [in this window] [in a new window]    FIG. 6. Effect of visible light (150 W m-2), on V. lecanii strain ARSEF 6430, on culturability of conidia after recovery from UV-B exposure (800 mW m-2) for 2 or 3 h. Relative percent culturability was calculated in relation to non-irradiated controls. Error bars are standard deviations of three independent experiments

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The failure to detect photoreactivation in V. lecanii and A. album does not mean that these repair mechanisms do not occur in other light environments, since it is virtually impossible to reproduce in the laboratory all the combinations between irradiances and spectral distribution that occur in natural envinronments (Gunasekera et al 1997). Since we have not observed significant levels of photoreactivation in strains of V. lecanii, A. album, and M. anisopliae using various combinations of lamps and filters, it may be that this mechanism, if present, is of limited importance in the repair of UV-B-induced damage in these genera.

The detrimental effect of exposure to the higher visible light intensity (150 W m^-2) after conidial exposure to UV-B suggests that there is an interaction between these two different spectral fractions. Interactions among different spectral fractions and between UV radiation and temperature have been observed in several genera of fungi and significantly change their survival in the environment (Rasanayagam et al 1995, Gunasekera et al 1997, Petin et al 1997, Braga et al 2001c). Visible radiation intensities used during our photoreactivation experiments (12 and 150 W m^-2) are not high in comparison to values observed in the environment. At our location, under clear-sky conditions, in mid-summer, the midday radiation between 400 and 700 nm typically reaches 500 W m^-2. Thus in addition to the deleterious effect of the UV-B fraction of the solar spectrum, the detrimental effect of visible radiation may lead to rapid inactivation of V. lecanni and A. album conidia upon direct exposure to solar radiation.

	ACKNOWLEDGMENTS

 
We are grateful to Susan Durham for the statistical analysis, Dr. Martyn M. Caldwell for supplying the spectroradiometer, and to the State of São Paulo Research Foundation (FAPESP), São Paulo, Brazil for financial support to G. U. L. B. This project was supported in part by a NRICGP/CSREES No. 99-35302-8052 grant, and a Utah State Mineral Lease funds grant.

	FOOTNOTES

 
¹ Corresponding author, dwroberts{at}biology.usu.edu

Accepted for publication April 9, 2002.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
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