Inactivation of Conidia ofPaecilomyces fumosoroseusby Near-Ultraviolet (UVB and UVA) and Visible Radiation

JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.

69, 70–78 (1997)

IN964637

Inactivation of Conidia of Paecilomyces fumosoroseus by Near-Ultraviolet (UVB and UVA) and Visible Radiation JACQUES FARGUES,* MARC ROUGIER,*,† ROBERT GOUJET,*,† NATHALIE SMITS,* CHRISTINE COUSTERE,* AND BERNARD ITIER† * Unite´ de Recherche en Lutte Biologique, INRA-Montpellier, Campus International de Baillarguet, 34982 Montferrier-sur-Lez Cedex, France; and †Unite´ de Recherche en Bioclimatologie, INRA-Grignon, 78850 Thiverval-Grignon, France Received March 5, 1996; accepted October 28, 1996

Moreover, it was established by means of selective screens that the decay of conidia resulted mainly from the lethal effect of the solar ultraviolet waveband. Solar radiation is, however, an extremely variable and unreliable source and its use is not recommended for experimental studies (Leach, 1971). Consequently, artificial sunlight is now commonly used (Ignoffo et al., 1977; Zimmermann, 1982). The objective of this study was to investigate the detrimental effects of solar radiation, especially nearUV, on quiescent conidia of Paecilomyces fumosoroseus, using simulated sunlight. This paper reports the first findings of a larger study devoted to system analysis and modeling of the short-term field persistence of hyphomycetous conidia in relation to the microclimate of the plant canopy.

The detrimental effects of solar radiation, especially the ultraviolet waveband, on quiescent conidia of Paecilomyces fumosoroseus were investigated. Conidia were irradiated by a high-intensity source, which emitted a continuous spectrum from 270 to 1100 nm and which was equipped with long-pass filters to block short wavelengths below 280, 295, 320, or 400 nm. After irradiation, conidia were tested for germinability, survival, and infectivity toward Spodoptera frugiperda larvae. It was demonstrated that the detrimental effects of light depended on irradiance in the shortest wavelengths. The UVB (280–320 and 295–320 nm) appeared to be the most detrimental part of natural radiation, although UVA (320–400 nm) was also harmful. Visible and near infrared radiations were less harmful than UV. Our results demonstrate that the irradiance of the UVB waveband should be considered as the pertinent factor for the detrimental effects of sunlight on the persistence of conidia of entomopathogenic fungi in insolated environments. r 1997 Academic

MATERIALS AND METHODS

Fungal Inocula

Press

KEY WORDS: Paecilomyces fumosoroseus; quiescent conidia; irradiation; simulated sunlight; UVB; UVA; photic effects; germinability; survival; infectivity.

The investigations were carried out with an isolate of P. fumosoroseus, No. 32, of the Culture Collection of INRA (Versailles, France), initially isolated from a dead larva of Scotia ipsilon and selected because of its infectivity toward the Fall armyworm, Spodoptera frugiperda (Maniania and Fargues, 1985; Fargues et al., 1994). Conidia were obtained from 3-week-old sporulating cultures grown at 25° 6 1°C on agar slants of a glucose–yeast extract medium (Fargues et al., 1994). Conidia were harvested directly by scraping and were suspended in sterile distilled water by shaking in 45-ml flasks containing five to six dozen glass beads (3-mmdiameter). No surfactant was added, since 5 min of agitation at 700 oscillations min21 (10-cm vertical travel) on a mechanical shaker was found to produce homogeneous suspensions of viable single conidia. Conidial densities were determined using a hemacytometer and adjusted to 107 conidia ml21. A volume of 10 ml of conidial suspension was deposited onto the 10-cm2

INTRODUCTION

A major problem in the use of pathogens as bioinsecticides is their relatively short persistence on leaf surfaces (Ignoffo and Hostetter, 1977; Roberts and Campbell, 1977; Ignoffo, 1992). Under field conditions, half-lives of 2 to 5 days or more have been reported for conidia of entomopathogenic Hyphomycetes such as Nomuraea rileyi (Ignoffo et al., 1977) or Beauveria bassiana (Gardner et al., 1977). Fargues et al. (1988) found that the inoculum half-life of N. rileyi conidia on the upper leaf surface of cabbage and bean plants decreased to 3.6 hr after conidia were exposed to sunlight, under a global irradiance of 800 W m22 at noon and a solar energy input of 2500 J cm22 per day. 0022-2011/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

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surface of 0.45-µm-pore-size mixed-ester membrane filters using a vacuum pump and were aseptically air dried for 30 min at room temperature prior to irradiation. The resulting deposit contained 107 conidia cm22 of membrane filter surface. Artificial Light Device Irradiation tests were carried out in 0.6-m3 controlledenvironment chambers, using an artificial sunlight device. Two Osram HQI-TS 400-W high-pressure metallic halogen lamps surrounded by a Radior reflector (Osram, Mu¨nchen, Germany) were installed over openings at the top of each chamber. These high-intensity sources emitted a continuous spectrum which included wavelengths of 270 to 1100 nm. The experimental chamber was ventilated to exhaust ozone, whose detrimental effect on fungal inocula has been demonstrated (Gotlib et al., 1985). A combination of instruments was employed for light measurement: a pyranometer calibrated for estimating the irradiance within a broad waveband including near-ultraviolet, visible, and near-infrared radiations (from 295 to 2550 nm) and a specific UV radiometer (UVCentra, Osram) equipped with two probes for estimating the irradiance within the UVB waveband (280–320 nm) and the UVA waveband (320–400 nm). The influence of light quality on conidial viability was investigated by using long-pass glass filters labeled WG280, WG295, WG320, and GG400 (250 3 200 3 3 mm) (Schott Glaswerke, Mainz, Germany) blocking short wavelengths at 280, 295, 320, and 400 nm, respectively, and transmitting long wavelengths over 2000 nm. The influence of the flux density (irradiance) was studied by varying the working distance from the source to the target. For example, the UVB irradiance was 4.8 W m22 at 0.5 m, 2.4 W m22 at 1 m, and 1.6 W m22 at 1.50 m. Using WG280 glass filters, at a 1-m distance the device provided a UVB density of ca. 2.0 W m22, a UVA density of ca. 21 W m22, and a total flux density of ca. 230 W m22. In comparison, sunlight irradiance at La Minie`re, France (48.8°N, 2.1°E) in June at 12:00 UTC (Universal Time Coordinate) was 1.6 W m22 of UVB, 40 W m22 of UVA, and 800 W m22 of the 300- to 3000-nm waveband. The spectral distributions of artificial light (transmitted through the filters) and natural sunlight were measured with an OL752 spectroradiometer (Optronic Laboratories, Orlando, FL) from 280 to 320 nm (Fig. 1). The stability of the beam power was ensured by using a highly stabilized power supply. Although no optical system was used (i.e., optical integrator and final collimating lens), the variations of the direct beam over the 0.144-m2 irradiated plate were relatively small: 65% at 1- and 1.5 m distance and 6 10% at 0.5 m. In fact, the position of each irradiated sample on the target surface was identified and its specific irradiance was used for estimating the dose (irradiation) received

FIG. 1. Distribution of spectral irradiance at the target level of sunlight and artificial light transmitted through the long-pass filters: (a) filters WG280 and WG295, UVB radiation; (b) filter WG320, UV radiation.

according to the duration of exposure. The influence of radiant heat was eliminated by exposing conidia on controlled-temperature platforms (Rougier et al., 1994). These platforms (620 3 620 3 35 mm) were constructed from a solid aluminium plate and temperature was regulated by circulated water. Because the temperature of the virtually still air around conidia would be close to that of the solid surface bearing them (Maddison and Manners, 1972), the surface temperature of the conidia-seeded membrane filters was measured by means of copper-constantan spot-soldered thermocouples connected to a datalogger (21X Micrologger, Campbell Scientific, Inc. Logan, Utah), which monitored each thermocouple every second and recorded averaged data 12 times per hour. The filters were attached to the bottom of opened plastic petri dishes (5-cm-diameter) with adhesive tape. A good thermal contact between the petri dishes and the thermally regulated plate surface was provided by a highly thermal-conductive silicone paste. Moreover, a thermocouple placed on the filter surface was used to monitor the thermal regulation of the platform (Rougier et al., 1994). The spatial variations of the temperature of the filters on the platform did not exceed 61°C. A box containing distilled liquid water was placed at the bottom of each chamber to prevent harsh dessication.

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Method of Exposure to Artificial Light

Statistical Analysis

Laboratory irradiation experiments consisted of exposing the dried fungal inocula on the membrane filters to broad wavebands greater than 280, 295, 320, or 400 nm, at various irradiance levels and increasing amounts of time, at 25° 6 1°C and 40 6 8% RH. Shaded conidia-seeded membrane filters were used as controls. After exposure, each membrane filter was mechanically shaken in 10-ml sterile distilled water in 45-ml flasks for 5 min to remove the conidia. Resuspended conidia were then checked for germinability, capability to form colonies, and infectivity toward S. frugiperda. For each dose of each wavelength tested, two membrane filters were exposed, and four samples of resuspended conidia were checked for each criterion.

A two-factor Analysis of Variance (ANOVA) (SAS Institute Inc., 1988) was conducted on either untransformed (% germination) or transformed (log10 CFU; arcsine % mortality) data. Moreover, for germinability tests, radiation inhibition was estimated via the exposure time or the effective dose to cause a 50% reduction in germination. Both inhibition time (IT50) and inhibition dose (ID50) were calculated by probit analysis (Finney, 1971). For survival, lethal times and lethal doses for killing 99% of conidia (LT99 and LD99) were calculated from a nonlinear decay curve (logistic model, STAT-ITCF, 1993) representing the logarithmic transformed average CFU counts versus time or as a function of the irradiation dose. For infectivity tests, mortality data were used for calculating the original activity remaining (OAR) (Ignoffo et al., 1977) by dividing the mortality obtained from the exposed samples by the mortality observed in unexposed samples. Untransformed OAR ratios were regressed against the exposure time for estimating the infectivity IT50s, i.e., the times of exposure to achieve a 50% reduction in infectivity. In Figs. 2–5, means are presented along with their standard deviations.

Germination Assay From each irradiated conidia-seeded filter, a 0.1-ml suspension, containing 3 3 105 suspended conidia, was spread onto each of four replicate plates containing the agar medium. Plates were incubated in the dark at 20° 6 1°C, inside plastic boxes under saturated humidity conditions. Plates were examined for the presence of germinated and ungerminated conidia at 24, 48, and 72 hr after plating. Conidia were considered to have germinated when the length of the germ tube exceeded the smallest diameter of the conidia. At least four batches of 100 conidia were examined on each plate. CFU Counts Suspensions of irradiated conidia were diluted to 105 conidia ml21 and 0.05 ml was plated on the agar medium using a spiral plating method (Gilchrist et al., 1973). Colonies were enumerated after 8 to 10 days of incubation in the dark at 15° 6 1°C. At least four plates were scanned for each treatment. Conidial survival was expressed as the number of colony-forming units (CFU) cm22 of exposed conidia-seeded filter surface. Bioassay The infectivity of irradiated conidia was determined using first-instar larvae of S. frugiperda (Maniania and Fargues, 1984, 1985; Fargues et al., 1994). Both surfaces of corn leaves were inoculated with 3 3 104 conidia cm22 of leaf surface in a spray tower (Burgerjon, 1956) using 9 ml of an aqueous suspension containing 107 conidia ml21. Four groups of 20 newly emerged larvae, 2–16 hr old, were exposed to these leaves for 24 hr. Larvae were then transferred to untreated leaves and incubated at 25° 6 1°C under a 16:8 L:D photoperiod. The larvae were checked daily and held until the completion of the fourth instar (ca. 10 days).

RESULTS

Persistence curves derived from each set of variables (germinability, survival, or infectivity) demonstrated that the detrimental effects of light depended on the irradiance in the shortest wavelengths (Figs. 2–5). The exposure required for complete inhibition of conidia at the lowest levels tested was in the order of 20 min at .280 nm; 50 min at .295 nm; 400 min at .320 nm; and 100 hr at .400 nm. During all experiments, the germination rate, viability, and infectivity of the shaded controls remained stable throughout irradiation, because of the short exposure durations. In the case of irradiation under l . 400 nm, germination of shaded controls declined slightly after more than 60 hr exposure. Total loss of germination occurred after only a 3-min exposure to UVB . 280 nm at 1.98 W m22 irradiance and after 20 min at 0.64 W m22 (Fig. 2a). There were highly significant differences between the times to inactivate germination at the two UVB irradiance levels tested (F 5 10721.67; P , 0.0001). The germination IT50s were 1.06 and 9.9 min (Table 1). As a function of the UVB dose (Fig. 2a8), the effect of irradiance was also highly significant (F 5 928.0; P , 0.0001). The germination ID50s were 124.8 and 381.7 J m22, respectively (Table 1). The loss of viability (CFU counts) occurred within 5 min of exposure at 1.98 W m22 and within 25 min at 0.64 W m22 (Fig. 2b). The LT99s were 3.6 min at 1.98 W m22 and 16.4 min at 0.64 W m22 (Table 1). Expressed as a function of UVB dose (Fig.

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FIG. 2. Effects of simulated sunlight on germinability (a), viability (b), and infectivity (c) of P. fumosoroseus conidia, expressed as a function of time (a, b, c) and as a function of UVB dose (a8, b8, c8); exposure to a continuous spectrum .280 nm, at two UVB irradiances, 0.64 W m22 (dashed line) and 1.98 W m22 (solid line) (error bars 5 SD of the mean of four replicates).

2b8), effects on survival did not differ significantly between both UVB irradiances tested (F 5 2.98; P . 0.05). The LD99s were 430.7 and 614.3 J m22 for the two irradiances tested (Table 1). When considering inhibition time, the effects on infectivity were significantly different between the two irradiances tested in the UVB . 280 nm (F 5 18.81; P , 0.0001) (Fig. 2c). The IT50s of the original pathogenic activity were 2.9 min at 1.98 W m22 and 7.4 min at 0.64 W m22 (Table 1). But when expressed as a function of the UVB dose, the reduction in infectivity did not differ significantly between both irradiances tested (F 5 0.25; P . 0.05) (Fig. 2c8).

When conidia were exposed to UVB . 295 nm (Fig. 3a), germination IT50s were 12.4 and 31.5 min at 1.56 and 0.71 W m22, respectively (Table 1). In contrast to the effects of UVB . 280 nm, there were few differences between the germination ID50s at both irradiances tested (Table 1). The reduction in viability of the inoculum, as a function of time (Fig. 3b), depended on the UVB irradiance (F 5 435.70; P , 0.0001); at 1.56 and 0.71 W m22, the CFU LT99s were 22.0 and 49.6 min, respectively (Table 1). In contrast, as a function of the UVB dose (Fig. 3b8), there was no significant effect of irradiance (F 5 0.08; P . 0.05); the CFU LD99s were

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FIG. 3. Effect of simulated sunlight on germinability (a), viability (b), and infectivity (c) of P. fumosoroseus conidia, expressed as a function of time (a, b, c) and as a function of UVB dose (a8, b8, c8); exposure to a continuous spectrum .295 nm, at two UVB irradiances, 0.71 W m22 (dashed line) and 1.56 W m22 (solid line) (error bars 5 SD of the mean of four replicates).

2096.4 and 2202.5 J m22, respectively (Table 1). The infectivity IT50s were 19.2 min at 1.56 W m22 and 47.7 min at 0.71 W m22 (Table 1), showing a significant effect of the UVB irradiance on infectivity (F 5 132.40; P , 0.0001) (Fig. 3c). However, as a function of the UVB dose, there was a weaker effect on infectivity between the two irradiances tested (F 5 17.43; P 5 0.001) (Fig. 3c8). The detrimental effect of radiation was delayed after exposure to UVA . 320 nm at 45.0 and 14.4 W m22 UVA irradiances (Fig. 4). At 14.4 W m22, the germination IT50 was 124.7 min and the ID50 was 107.5 kJ m22 of

UVA (Table 1). The survival of conidia, expressed as a function of time (Fig. 4b), differed significantly according to the UVA irradiance tested (F 5 40.56; P , 0.0001). However, when considering CFU counts as a function of UVA doses, there was no significant difference between the two irradiances tested (F 5 0.00017; P . 0.05) (Fig. 4b8). The CFU LT99s were 214.4 min at 14.4 W m22 and 95.3 min at 45.0 W m22, and the respective LD99s were 188.7 and 253.1 kJ m22 (Table 1). The reductions in infectivity of irradiated conidia at both irradiances tested were similar during the first 200 min of exposure (irradiance effect: F 5 2.23;

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FIG. 4. Effect of simulated sunlight on germinability (a), viability (b), and infectivity (c) of P. fumosoroseus conidia, expressed as a function of time (a, b, c) and as a function of UVA dose (a8, b8, c8); exposure to a continuous spectrum .320 nm, at two UVA irradiances, 14.4 W m22 (dashed line) and 45 W m22 (solid line) (error bars 5 SD of the mean of four replicates). Data for germination at 14.4 W m22 not available.

P 5 0.14) (Fig. 4c). The infectivity IT50s at 45.0 and 14.4 W m22 were 92.5 and 129.2 min, respectively. Visible light (.400 nm) was not as detrimental to conidia as those at lower wavelengths (Fig. 5). However, after 40 hr of exposure at 290 W m22 of global irradiance (.400 nm), the germinability declined markedly in comparison to that of nonirradiated conidia (F 5 847.32; P , 0.0001) (Fig. 5a). The IT50 of germination was 38.9 hr (Table 1), whereas the germinability of shaded conidia remained high for more than 72 hr (Fig. 5a). The viability of irradiated conidia, estimated by CFU counts, also decreased slowly but significantly

(F 5 473.11; P , 0.0001) (Fig. 5b). Infectivity tests showed a significant effect of light (.400 nm) (F 5 65.38; P , 0.0001) (Fig. 5c); the infectivity IT50 was 49.7 hr at 290 W m22 of global irradiance (Table 1). DISCUSSION

Experiments with shortwave cutoff filters clearly demonstrated that the susceptibility of quiescent conidia of P. fumosoroseus to polychromatic light depends on both spectral composition and intensity in the shortest wavelengths. However, irradiation at different

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FIG. 5. Effect of simulated sunlight on germinability (a), viability (b), and infectivity (c) of P. fumosoroseus conidia, expressed as a function of time (a, b, c); exposure to a continuous spectrum .400 nm, at an irradiance of 290 W m22 (solid line) and a control, nonirradiated treatment (dotted line) (error bars 5 SD of the mean of four replicates).

wavelengths (i.e., 310–500 nm) may be beneficial by promoting photoreactivation, a phenomenon whereby the damage in DNA produced by UV irradiation can be counteracted by the microorganism (Leach, 1971; Tuveson and McCoy, 1982; Tyrrell, 1982). To simulate sunlight effects under natural conditions as much as possible, including possible photoreactivation, we conducted our assays using polychromatic light at a temperature favorable to photoreactivation. Near-UV inactivation involved not only primary damage (i.e.,

photodimerization of pyrimidine bases) but also secondary effects (i.e., protein lesions, lipid peroxidation) (Tevini, 1993). Nevertheless, the results provided by irradiation tests with P. fumosoroseus conidia cannot be directly extrapolated to natural conditions. Under field conditions, the persistence of hyphomycetous conidia at the top of the canopy shows a slower decay than those obtained in our simulated studies (Daoust and Pereira, 1986; Fargues et al., 1988; Inglis et al., 1993; James et al., 1995). It is not surprising that photic effects can be overestimated through artificial irradiation, as the spectral irradiance of artificial and natural radiation differs in the lowest UVB wavelengths and the incidence angle of the sun’s rays on the foliage surface is very variable. For example, almost no radiation with a wavelength under 295 nm reaches the earth’s surface. Consequently, the artificial light we used provided higher irradiances in wavelengths below 300 nm than sunlight, as measured by a UV spectroradiometer (Fig. 1). Besides, the glass filters did not exactly suppress 100% of irradiance at the ‘‘cutoff’’ wavelength given by the manufacturer. Furthermore, the surfaces of leaves at the top of the canopy are rarely distributed on a horizontal plane, but show a great variability of positions (plane slope and azimuthal orientation) over space and time (Campbell and Norman, 1989), causing highly significant changes in both direct and diffuse natural UVB irradiances (Grant, 1993). Although different types of responses were obtained for the decay of irradiated conidia (germinability, survival, and infectivity), it is clear that the same trend occurred for all three parameters tested. However, there were apparent differences in the relationships between germination, viability, and infectivity which varied according to the spectral irradiance. For instance, with a UVB irradiance of 0.64 W m22 at wavelengths .280 nm, there was a closer relationship between germination (recorded after 48 hr incubation) and infectivity than with viability (Fig. 2). This was not apparent at 1.98 W m22. In addition, at wavelengths .400 nm (no UV), there was a closer relationship between viability and infectivity. Moreover, it was evident that the inhibition of germination at exposures of 40 hr or greater was temporary (Fig. 5). More detailed investigations on the effects of light on these relationships are warranted. The reason for the poor relationship between conidial germination, hyphal growth, and infectivity under the influence of climatic factors could be related to the difference between the complex biological processes involved at these different steps of the fungal life cycle (Kachatourians, 1991; Griffin, 1994). Selecting wavelengths using various long-pass filters permitted us to reveal that the UVB, UVA and, to a lesser extent, visible light components of the solar

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TABLE 1 Effect of Simulated Sunlight on Germinability, Viability, and Infectivity of Paecilomyces fumosoroseus Conidia, According to both UV Spectrum and UV Irradiance Irradition conditions Glass filters Shortest waveband (nm) UVB irradiance (W m22 ) UVA irradiance (W m22 ) Global irradiance (W m22 ) Germinability IT50 (min) a ID50 ( Jm22 ) b Survival LT99 (min) c LD99 ( Jm22 ) d Infectivity IT50 (min) e

WG 280 (280–320) 0.64 1.98 12.8 53.7 101 350

WG 295 (295–320) 0.71 1.56 26.9 58.0 210 400

WG 320 (320–400) 0.0 14.4 120

0.0 45.0 350

—

GG 400 (400–700) 0.0 0.0 290

9.9 (9.74–10.15) 381.7 (373.8–389.7)

1.1 (0.97–1.17) 124.8 (112.7–138.1)

31.5 (29.80–32.29) 923.7 (910.6–937.0)

12.4 (11.54–13.28) 1039.0 (1080–1243)

124.7 (121.4–127.3) 107.5 3 103 (104.8–110.0)

— —

38.9 (hr) (38.6–39.2) — —

16.4 614.3

3.6 430.7

49.6 2202.5

22.0 2096.4

214.4 188.7 3 103

95.3 253.1 3 103

86.4 (hr) —

2.9 (1.9–4.3)

47.7 (32.1–65.3)

19.2 (11.2–32.2)

129.2 (63.9–230.8)

92.5 (63.2–133.4)

49.7 (hr) (36.7–67.7)

7.4 (4.1–12.4)

a,b Exposure time (IT 50 with 95% CI) and effective dose (ID50 with 95% CI) of either UVB or UVA required to cause 50% decrease in germinability of irradiated conidia (probit analysis). c,d Lethal time (IT ) and lethal dose (ID ) of either UVB or UVA required for killing 99% of conidia (data calculated in using a logistic decay 99 99 model). e Inhibition time of 50% (IT with 95% CI) of the original pathogenic activity remaining (linear regression). 50

spectrum were all detrimental to germinability, viability, and infectivity of these fungal propagules. However, these irradiation tests showed that the detrimental effects of the polychromatic radiation, which simulated exposure to the solar spectrum (approximatively 290– 2200 nm), depended mainly on the quantity of UVB received by the inoculum. There is a great variability in the relationship between solar UVB irradiance and either visible or total radiation (Kromann et al., 1986; Webb, 1992; Webb and Steven, 1987). Solar UVB irradiance at the earth’s surface is influenced more by various atmospheric and environmental parameters than by total solar irradiance (Blumthaler et al., 1993). The results reported in the present paper clearly suggest that changes in the shorter wavelength part of the UVB waveband would not be masked by trends at the longer wavelengths. Because of these changes in natural UV irradiance, the dose of solar UVB radiation appears to be the most efficient variable to express the detrimental effects of sunlight on persistence of entomopathogenic conidia in exposed insolated field environments. In greenhouse environments, however, cover material drastically changes the radiation spectrum, particularly in blocking most UVB radiation (Tuller and Peterson, 1988; Nijskens et al., 1989). Under these conditions, according to our results, the dose of UVA radiation might be used as the pertinent variable for explaining the decay of exposed conidia. The susceptibility of P. fumosoroseus inocula to UV radiation suggests the use of greenhouse covers blocking UVA and B

radiation to improve the persistence of mycoinsecticides. But these wavebands, even at low irradiance, may be useful to integrated pest management by inhibiting the development of plant pathogenic fungi attacking greenhouse crops (Raviv, 1989). The possible conflict between these two scenarios remains to be investigated. ACKNOWLEDGMENTS The authors thank H. Vermeil de Conchard for his technical assistance. For assistance with the English translation of the manuscript, they are grateful to Mrs. Commeau (INRA Linguistic Services) and to Ms. K. Toohey. They also thank Dr. M. S. Goettel (Agriculture Canada) for reviewing the manuscript and for his useful suggestions and M. Rivault, Instrumat, Les Ulis, France, for his generous loan of the spectroradiometer. This research was supported by the French Ministry of the Environment (SRETIE/89337). REFERENCES Blumthaler, M., Huber, M., and Ambach, W. 1993. Measurements of direct and global UV spectra under varying turbidity. Atmos. Radiat. 2049, 194–198. Burgerjon, A. 1956. Pulve´risation et poudrage au laboratoire par des pre´parations pathoge`nes insecticides. Ann. Epiphyties I.N.R.A. 7, 675–684. Campbell, G. A., and Norman, J. M. 1989. The description and measurements of plant canopy structure. In ‘‘Plant Canopies, Their Growth, Form, and Function’’ (G. Russel, B. Marshall, and P. G. Jarvis, Eds.), pp. 1–19. Cambridge Univ. Press, Cambridge. Daoust, R. A., and Pereira, R. M. 1986. Stability of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae on beetleattracting tubers and cowpea foliage in Brazil. Environ. Entomol. 15, 1237–1243.

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