Inactivation of Paecilomyces fumosoroseus conidia by diffuse and total solar radiation

ELSEVIER

FEMS Microbiology

Ecology 21 (1996) 167-173

Inactivation of Paecilomycesjhmosoroseus and total solar radiation Nathalie Smits a3*, Marc Rougier a, Jacques Fargues Raymond Bonhomme b

conidia by diffuse a, Robert

Goujet b,

a Unit6 de Recherches en Lutte Biologique, I.N.R.A., Campus International de Baillarguet, 34982 Montferrier sur Let, France b Unite’de Bioclimatologie, I.N.R.A., 78850 Thiuerval-Grignon, France Received

12 April 1996; revised 8 July 1996; accepted 8 July 1996

Abstract The detrimental

photic effects of natural solar radiation on the conidial persistence of the entomopathogenic hyphomycete were investigated by exposing quiescent conidia either to total solar radiation or to its diffuse component. A given amount of UVB diffuse radiation was found to be as detrimental, and sometimes twice as detrimental, as the same amount of total solar radiation. The variability in quantity and spectral distribution of the diffuse component of UVB solar radiation reaching the earth’s surface, observed through spectral measurements, may be responsible for the difference in biological effects. Paecilomyces

fumosoroseus

Keywords: Paecilomyces fumosoroseus; Conidial persistence;

UVB solar radiation;

1. Introduction Numerous strains of entomopathogenic fungi have been studied as biocontrol agents of arthropod pests. When these agents are used as bioinsecticides, their survival in the field is a critical condition of success of biocontrol [l]. It has been established that the free-living stage of the microorganism is a determining step in quantifying disease development during epizootics [2,3]. However, all steps of fungal development are highly influenced by microclimatic factors. A better knowledge of inoculum persistence, and the environmental factors affecting it, is therefore needed to predict the success of biocontrol [1,4].

Diffuse radiation;

Biological

control

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Microbiology

Ecology 21 (19961 167-173

The entomopathogenic hyphomycete Paecilomyces fumosoroseus (Wize> Brown & Smith (strain INRA32). obtained from the entomopathogenic fungal collection of the Institut National de la Recherche Agronomique at La Mini&e. France, initially isolated from a larval cadaver of Scotia ipsilon Hbn (Lepidoptera: Noctuidae) and selected for its pathogenicity towards noctuids [12] was used. Conidia from three-week-old cultures grown on an agar yeast-glucose medium at 25 f 1°C were scraped from the surface of the culture and suspended in sterile distilled water. The conidial suspension, obtained by a 5-min agitation at 700 oscillations per min in 45ml flasks, was adjusted to lo7 spores ml-’ following a hemacytometer count. Conidia were deposited at a density of 10’ spores cm-’ onto a 0.45pm pore membrane filter using a vacuum pump and were then placed into open Petri dishes and aseptically air-dried for 0.5 h.

Exposures were carried out at La Mini&e INRA Research Center, near Paris, France (48”51’N, 2”06’E) on two different sunny summer days. The surface temperature of the inoculum was maintained at 25°C by means of cooled water circulating in the plate on which the filter-containing Petri dishes were placed. On July 10 1991, the sky was clear; the global radiation during the experiment varied between 800 and 850 Wm-’ ; the conidia were exposed between lo:45 a.m. and 12:30 p.m. UTC (Universal Time Coordinate). The UVB irradiance at the inoculum level was between 1.7 and 1.9 Wrn-* for the direct sunlight treatment, and quasi-constant at 0.8 Wrn-’ for the shaded treatment. For each treatment. one sample was kept in the dark and used as a control. Durations of exposure to total solar radiation varied from 20 to 80 min, providing UVB cumulative irradiations ranging from 2000 to 9000 Jrn-’ ; in the shaded treatment, conidia were exposed from 20 to 120 min and cumulative irradiations varied between 1000 and 6000 Jm-‘. On July 29 1992, the conidia were exposed between 1l:lO a.m. and 13:30 p.m. UTC under clear skies; the global radiation varied between 700 and 800 Wm-*. On the plate exposed to total sunlight, the UVB irradiance was between 1.4 and 1.6 Wm-‘, and it ranged between 0.7 and 0.9 Wm-’ on the shaded plate. Exposure times varied from 20 to 72 min for the plate exposed to total solar radiation, providing UVB cumulative irradiations ranging from 1800 to 6500 Jm -’ ; the inoculum was exposed for periods ranging from 25 to 115 min and received between 1300 and 6000 Jm-’ on the shaded plate.

2.2. Methods of exposure

2.3. Inoculum persistence

The spore-bearing filters were exposed to natural solar radiation at constant temperature, using a device described by Rougier et al. [ 131. Two thermoregulated plates were placed outdoors side by side; they were rotated and inclined so that their surface was kept perpendicular to the direct sunbeam. One of these plates was shaded from direct radiation by an opaque metal sheet so it received only the diffuse component of solar radiation, or skylight. The other one was kept unshielded and received total solar radiation.

Once exposed, irradiated conidia were resuspended in lo-ml sterile water, and assessed for germinating ability and colony forming ability. Petri dishes containing glucose-yeast agar medium were inoculated with 98 ~1 of suspension containing 3 x lo6 conidia ml-‘, and germination was monitored under 400 X magnification after 24, 48 and 72 h incubation in the dark at 20°C. For each exposure condition, four batches of 100 randomly chosen spores were checked for germination. A conidium was counted as germinated if the length of the germ

sity and spectral distribution. The parameters influencing these components act differently on diffuse than on direct radiation, and their action is highly wavelength-dependent [ 1 I]. In order to understand the effect of solar radiation on conidial persistence, the experiments presented in this paper were designed to study the survival of inocula exposed either to total solar radiation or to its diffuse component only.

2. Materials

and methods

2. I. Fungal inoculum

measurements

N. Smits et al. / FEMS Microbiology Ecology 21 (19961 167-l 73

tube was at least equal to the largest diameter of the spore. All of the percentages presented were recorded after 48 h incubation. Spore viability was measured by their ability to produce colonies after incubation in the dark for 5 to 10 days. For each exposure condition, four Petri dishes were inoculated with 49 ~1 of suspension containing 10’ conidia ml-’ using a spiral plate method [14]. Beginning on the fifth day after inoculation, the number of colonies was recorded daily until it stopped increasing. The surface density of colony forming units (cfu) was then detern-ined. 2.4. Radiation

measurements

Global radiation at the exposure level was monitored continually using a pyranometer CE 180 (Cimel, Paris, France) connected to a Campbell 21X datalogger (Campbell Scientific, Leicestershire, UK). UVA (320-400 nm) and UVB (280-320 nm) irradiantes received by the inoculum were measured using a radiometer Osram UV Centra (Osram, Molsheim, France). The spectra of global solar radiation and of its diffuse component were measured at Montferriersur-Lez (HCrault, France), located at 43”41’N and 3”53’E, and at an altitude of 85 m, on two different summer days (July 15 1994 and August 27 19941, using a commercially available spectroradiometer 0L752 (Optronics Laboratories, Orlando, FL, USA). This device is equipped with a 10.16 cm diameter integrating sphere, that can measure spectral irradiantes from 200 to 800 nm. It is suited to UV solar spectral radiation measurements because of its double monochromator, which achieve a low stray light level (10e8 at 285 nm). Its scan resolution is 0.1 nm and its wavelength accuracy within the UVB waveband is f0.2 nm. Our measurements were carried out scanning either the 280-320 or the 280-400 nm waveband, with wavelength increments of 1 nm. The weather on the measurement days was similar to that when conidia were exposed: clear summer days, with a little less direct sunlight on July 15 than on August 27.

169

greatest detrimental effect on fungal inocula [15]. Therefore, the persistence of conidia exposed to solar radiation was studied as a function of the UVB irradiation received by the inoculum (expressed in Jm-‘). This quantity was calculated as the integral of UVB irradiance (in Wm-’ > over time. Each set of data was analyzed using models describing germination ability and viability as functions of UVB irradiation. A logistic curve was therefore adjusted to each set of germination data, and an infinitely decreasing curve to viability data [9]. The logistic model is represented by Eq. (1): 1 +,-u*xo (3x1

= Go * 1 + en*(*-rI,)’

where ‘x0 ’ is the x-value of the inflection point of the response curve and corresponds to the UVB irradiation at which the rate of decrease in germination ability is at its maximum; ‘a’ is this maximum rate of decrease; and ‘Go’ is the probability that a non-irradiated spore is able to germinate. The viability model is represented by Eq. (2):

(2) where ‘Co’ is the y-value for non-irradiated conidia, i.e., the estimated logarithm of surface density of cfu in the initial inoculum deposit, ‘b’ is the general rate of decrease of y, and ‘a’ is the initial rate of decrease for x = 0. The parameter estimates were calculated using the statistical software Splus [16], and maximum likelihood and least squares estimators were used for germination ability and viability curves, respectively [ 171. These estimates were used to compare the response curves obtained under total and diffuse radiation. They were also used to calculate times (or doses) after which a given reduction of the initial inoculum was reached (e.g., half of the germination ability or one hundredth of the initial viability).

3. Results

2.5. Result analysis

3.1. Exposure

to solar radiation July 10 1991

The shortest wavelengths of the sun’s radiation reaching the earth, i.e. UVB, are known to have the

The loss of conidial persistence vs. UVB irradiation is presented in Fig. 1. The parameters of the

N. Smits et al. / FEMS Microbiology Ecology 21 (1996) 167-173

170

0 WE

IRRADlATlON

(J.m -‘)

the difference between the two treatments was smaller; germination ability was halved after exposure for 39 and 45 min to total and diffuse solar radiation, respectively. And a lOO-fold reduction of viability was achieved after exposure to total sunlight for 67 min and to diffuse radiation alone for 77 min. 3.2. Exposure to solar radiatim

G0 July 10 1991

Total radiation Diffuse radiation

July 29 1992

Total radiation Diffuse radiation Pooled data ^

1.00 (1.2 x 1.oo (5.3 x 1.oo (7.0 x 1.00 (6.3 x 1.00 (6.2 X

10-4) 10-h) lo-“) lO-5) 10~‘)

July 29 1992

Conidial persistence plotted against UVB irradiation is shown in Fig. 2. The curves representing adjusted models are defined by parameter estimates presented in Table 1. This experiment showed that a given quantity of UVB radiation received by the

Table 1 Parameter estimates (and S.D.) of germinability and viability models adjusted for each set of data collected fumosoroscus conidia to total and diffuse solar radiation during each outdoor experiment Germination

(Jm -‘)

Fig.

logistic curves adjusted on germination ability data and of the infinitely decreasing curves adjusted on viability data sets are presented in Table 1. It was found that a given reduction of spore germination ability or viability required a smaller UVB irradiation when exposed to diffuse radiation than when exposed to total solar radiation. For example, half of the conidia were unable to germinate after receiving 4171 Jm-’ total UVB radiation, whereas 2251 Jrn-” was enough when they received diffuse radiation only. Similarly, the surface density of viable conidia was reduced lOO-fold after receiving 7369 Jm-’ of total UVB radiation, and as little as 3850 Jm-’ UVB was necessary when the inoculum received only the diffuse component of solar radiation. However, when length of exposure is considered,

Mode of exposure

lRRADlATlON

2. Persistence of Paeciiomyces ~UMOSO~OSPUS conidia exposed to total (dots) or diffuse (circles) solar radiation on June 29 199 1. Solid lines represent adjusted models: (a) germination ability data and logistic curve; (b) viability data and infinitely decreasing curve.

Fig. 1. Persistence of Paecilomyces ~U~OSO~OS~US conidia exposed to total (dots) or diffuse (circles) solar radiation on July 10 1991. Solid lines represent adjusted models: (a) germination ability data and logistic curve; (b) viability data and infinitely decreasing curve.

Day of exposure

m4ooom UW

ability model

during

exposure

Viability model

x0 (J rn-‘)

a (J-’

rn’)

G,

a (J-’

m’)

b (J-’

4.17 (69) 2.25 (37) 2.85 (32) 2.95 (37) 2.90 (27)

1.76 (8.3 9.39 (3.5 1.78 (5.7 2.31 (9.4 1.97 (5.6

lo-’ IO-‘) 10-3 10-4) lO-3 10-51 lo-’ lo-a) 10-j

6.60 (0.11) 6.85 (0.087) 6.84 (0.02) 6.86 (0.03) 6.85 (0.04)

7.77 x (3.1 x 1.76 X (5.0 x 8.67 X (8.4 X 9.93 x (1.1 x 8.62 x (I.3 x

10-s 10-Y IO-’ 10-r) lo-’ IO- ‘) lo-5 1om5) lo-’ 10~~)

28.8 (8.1 4.87 (1.0 4.75 (2.7 4.91 (3.0 5.09 (4.1

x 107 x lo3 x IO7 x 10’ x lo3

of P.

x x x x x x x x x x

10-5)

* On July 29 1992, the two modes of exposure gave similar results, allowing a global estimation; because data collected under total and diffuse radiation were too different (see text).

m’) x lo-’

x 1o-s) x x x x x x x x

lO-4 lo-“) lo-” 10~‘) lomJ lo-S) lo10-S)

this was not possible on July 10 1991

N. Smifs et al. / FEMS Microbiology

WAVELENGTH

Fig. 3. Spectral distribution and of its diffuse component h UTC.

(nm)

of global solar radiation (solid line) (dashed line), on July 15 1994 at 12

inoculum always produced the same detrimental effect, whether the inoculum was exposed to total solar radiation or only to its diffuse component only. For example, half of the conidia exposed to total and diffuse solar radiation were unable to germinate after receiving 2853 and 2947 JmP2 UVB, respectively, and the density of cfu was reduced to one hundredth of its initial value after receiving 5222 and 4862 Jmm2 UVB in the same conditions. Since the UVB irradiance received by the conidia was lower when protected from direct sunlight, longer exposure to diffuse light was needed to obtain the same cumulative irradiation, and thus the same detrimental effect, was longer in this condition. For example, it took 31 min to suppress 50% germination ability of spores exposed to total solar radiation and 59 min for spores exposed to diffuse radiation only. Similarly, the density of cfu was reduced lOO-fold

Fig. 4. Spectral distribution of UVB global solar radiation (solid line) and of its diffuse component (dashed line), on August 27 1994 at 12 h UTC.

Ecology 21 (19961 167-173

WAVELENGTH

(nm)

Fig. 5. Percentage of diffuse radiation in global solar radiation within the UVB waveband, measured July 15 1994 at 12 h UTC (dashed line) and 13 h UTC (solid line), and August 27 1994 at 12 h UTC (dotted and dashed line) and 13 h UTC (dotted line).

after 58 min exposure to total solar radiation and after 98 min exposure to its diffuse component alone.

3.3. Spectral measurements

of solar radiation

The spectral distribution of UVB radiation, and of its diffuse component measured at 12 h UTC on July 15 and August 27 1994, are presented in Figs. 3 and 4, respectively. In both cases, irradiance increased with wavelength. A comparison between these two spectra shows that the spectral irradiance of total UVB did not change much between the two experiments; however the spectral irradiance of diffuse solar radiation was lower on August 27 than on July 15, within the whole UVB waveband. The proportion of the diffuse component in solar radiation is presented as a function of wavelength in Fig. 5. These profiles are almost horizontal, which corresponds to a proportion of diffuse radiation independent from the wavelength. There were slight variations, however, as on July 15 at 12 h UTC for example, this proportion ranged from 52.4% at 295 nm, to 60.5% at 310 nm. It was less variable on August 27 at 12 h UTC as the range of variation was between 43.7 and 48.2% diffuse radiation. Even though the weather was similar on the two measurement days, Fig. 5 shows that the proportion of diffuse radiation was different. For example, the proportion of diffuse UVB, calculated over the whole 295-320 nm range at 12 h UTC, was 60% on July 15, but only 47% on August 27.

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N. Stnirser al./ FEMS Microbiolog!:Ecology 21 (1996) 167-I 73

4. Discussion The two experiments presented in this paper are part of a series of trials conducted from 199 1 to 1993 to investigate the effects of solar radiation on conidial persistence of P. jiurnosoroseus. Survival data of various hyphomycetes collected under field conditions proved to be very variable. Half-lives of 2 to 5 days have been reported for conidia of entompathogenic fungi such as Nomuraea rileyi [4] or Beauueriu bassiana [18]. Fargues et al, found that viability of Nomuraea rileyi conidia was halved after 3.6 h of exposure on top of cabbage canopy at noon [5], and Moore et al. [ 191 found that germination of Metarhizium anisopliae spores exposed to natural sunlight in Benin was halved after less than 2 h of exposure. The influence on spore survival of their position within various canopies was studied [6,7], but very little is known so far about the influence of the different components of solar radiation and their spectral distribution. All our experiments were conducted in summer, under visually clear skies. These two were selected because they represent the large variation of results obtained when investigating the effect of diffuse solar radiation. On July 10 1991 a given amount of diffuse UVB radiation was twice as detrimental as the same amount of total radiation; conidial germination was halved after receiving 2251 Jmd2 diffuse UVB or 4171 Jm-’ total UVB. On July 29 1992, on the other hand, diffuse and total radiations were found to produce the same detrimental effect; germination ability was halved after conidia had received 2947 Jm-’ diffuse UVB or 2853 Jm-’ total UVB. During a series of spectral measurements of diffuse and total solar radiation, conducted in 1994 near Montpellier, France, we observed a large variability in the diffuse component of solar radiation reaching the earth’s surface. For example, the mean proportion of diffuse radiation, calculated over the UVB waveband (295-320 nm), was 60% on July 15 at 12 h UTC, and only 47% on August 27 at 12 h UTC. This showed that UVB irradiances recorded in similar conditions of atmosphere (visually clear), solar elevation and total solar radiation varied between our two experiments. It is probable that this observed variability was due to differences in total amount of atmospheric ozone, as well as vertical profiles of

ozone and aerosols [lo,21 -231. Since turbidity strongly influences UVB spectral distribution and its variations [ 11,20,24], we compared our measurements with data collected in Carpentras (Vaucluse, France), on the same days. At 12 h UTC, the Linke turbidity factor (T) was higher on July 15 (3.97), with a high proportion of diffuse UVB (60%), than on August 27 (2.55), with 47% diffuse UVB. This supports our observations, since turbidity has been found to be positively correlated with the part of diffuse radiation in global solar radiation [25]. Our spectral measurements also showed that the proportion of diffuse radiation at different wavelengths within the UVB waveband (e.g., 295 nm and 310 nm) changed between the two days of measurements. Taking these results into consideration, we can make the assumption that the variability of photobiological effects observed by exposing conidia may be linked to the spectral distribution of the radiation received by the spores, especially the distribution within the UVB waveband. Despite the great variability in the irradiance of total as well as diffuse solar radiation, we have demonstrated that diffuse radiation alone causes severe damage to fungal inocula. It should therefore not be overlooked when assessing spore survival in the field environment. In general, this study aids our understanding of the important role of diffuse solar radiation in microbial ecology and epidemiology.

Acknowledgements The authors are grateful to the Centre Radiomttrique of M&o-France in Carpentras (France) for providing turbidity data, and to M. Rivault, Instrumat. Les Ulis, France, for his generous loan of the spectroradiometer. They wish to thank Ms. K. Toohey for her contribution to the English translation, and for reviewing the manuscript, and Dr. M. Goettel, from Agriculture and Agri-Food Canada, for his critical review and editing of the manuscript. References [I] Ferron, P., Fargues, J. and Riba, G. (1991) Fungi as microbial insecticides against pests. In: Handbook of Applied Mycology (Arora, D.K., Ajello, L. and Mukerji, K.G., Eds.). Vol. 2. pp. 665-706. M. Dekker. New York. NY.

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