- Email: [email protected]
The effects of sunlight on a purified granulosis virus of Pieris brassicae applied to cabbage leaves
JOURNAL
OF INVERTEBRATE
PATHOLOGY
11,
496-501
(
1968)
The Effects of Sunlight on a Purified Granulosis Virus of Pieris bra&cue Applied to Cabbage Leaves W. A. L. DAVID, Agricultural
B. 0. C. GARDINER,
Research Council,
AND M. WOOLNER
34A Storey’s Way, Cambridge,
1
Engkznd
Received January 23, 1968 When a highly purified preparation of the known granulosis virus of Pieris bTassicae is exposed to direct sunlight, on the upper surface of cabbage leaves, carefully oriented so that no part is in shade, the virus is rapidly inactivated. For example, after an exposure of 3 hr, the suspension applied to the leaves gave very significantly less kill of larvae than a nonirradiated control suspension one-tenth its concentration. Total inactivation of the virus, under these circumstances, took between 12 and 19 hr. These results are discouraging from the point of view of using purified, unprotected, granulosis virus for biological control and emphasize the importance of finding some means of screening or protecting the virus from ultraviolet radiation.
MATERIALS
INTRODUCTION
AND METHODS
Certain preliminary observations on the effect of light on the activity of a granulosis virus of the European cabbageworm (or large white butterfly), Pieris bra&cue, led to the general conclusion that when the purified virus was exposed to sunlight it rapidly lost its capacity to cause lethal infections in the larvae (David, 1965; David, 1967). A crude virus preparation, on the other hand, retained some of its activity for 16 weeks on cabbage plants kept in the open during the winter 196263 (David and Gardiner, 1966a). Another crude preparation, in which there was solid matter from the dead larvae, showed very slight activity after being stored for 2 years as a dried film in a glass tube in a glasshouse (David and Gardiner, 1967). The tests reported in this paper are of an essentially practical nature. That is to say, the virus was exposed to direct sunlight, in the middle part of the day, on the upper surface of cabbage leaves, just as it might be after a field application.
The virus preparation exposed to the ultraviolet radiation (UVR) was highly purified to avoid any screening, protective, or chemical inactivating effect which might be associated with impurities. It was obtained by the usual process of grinding up larvae which had recently died of virus disease, suspending the material in distilled water, straining it through fine muslin, and filtering through Whatman No. 1 filter paper. Afterwards the crude virus was centrifuged in alternate cycles at low and high speeds and further purified by suspending it four or five times on sucrose gradients. The pooled virus samples were then washed free of sucrose and suspended in boiled distilled water which had been filtered through sintered glass. The concentrated stock was stored in the dark at about -20°C. All test suspensions were diluted from this and also stored under the same conditions. As applied to the leaves they contained 0.1% of a wetting agent (Teepol) .
1 M. Woolner was on study leave from Brunel University, London, W.3.
The grown 496
leaves of young cabbage plants in a screened glasshouse were used
EFFECTS
OF
SUNLIGHT
in the tests. They were left on the plants and treated on the upper, adaxial, surface. An area measuring 2 X 1% inches was marked off with a ballpoint pen and rubbed with absorbent tissue to remove some of the surface wax. The virus suspension was applied in droplets to the surface of each leaf, 0.25 ml being distributed over the prescribed area in 4060 drops. The applications were made when the leaves were already in the sun and the droplets evaporated completely within the next hour. During the exposure the leaves were arranged horizontally or oriented to face the sun. They were also kept as flat as possible by suitable supports and clips. Care was taken to see that even temporary shading caused by veins or irregularities was reduced to a minimum. Between exposures, when the leaves were not in sunlight, they were held in a shaded room, as were the control leaves which had been treated at the same time with the virus suspension. Light intensity readings were taken every hour by means of a commercial selenium cell light meter calibrated in foot-candles. Such an instrument is very unsatisfactory for measuring radiation in experiments of this type; all that can be concluded is that, in general, the higher the measured total intensity of the sunlight the greater the intensity of the active UVR. After the requisite exposure the leaves were brought in, trimmed to standard size and set up in shell vials of water as previously described (David and Gardiner, 1965, 1967). Twenty newly molted second-instar virus-free P. brmsicae larvae were placed on each leaf and the leaves were then put separately into sterilized jars (David and Cardiner, 1966b). At the same time, controls were also set up using the leaves treated with the virus which had not been exposed to sunlight. Except in two preliminary tests six replicates of the treated
ON
GRANULOSIS
VIRUS
497
and control leaves were used. The larvae were examined daily and the numbers dying of virus disease were recorded. Counting was continued for 5 days after the first virus death in any given jar (David and Gardiner, 1965). Control larvae (untreated with virus) were also kept in some tests but no virus deaths occurred among these larvae. The results are expressed as percentages in the tables and in any dubious cases the significance of each result has been assessed by the X” test on the original data. However, all the experiments reported show highly significant differences between the control and the irradiated leaves and the X’ figures are not given in order to simplify the tables. RESULTS
The experiments reported in this paper were carried out between the months of May and September in 1966 and 1967. In the preliminary tests, reported in Table 1, long exposures were given since it was not known what duration of exposure would be necessary to give a detectable reduction in activity of the virus. It can be seen that in one test some virus survived an exposure of 12 hr. This result seems a little anomalous (when compared with the results of tests in which a 6-hr exposure was given) and no active virus was detected after the longer exposures (19 hr and 43 hr), More detailed tests were carried out in which the leaves treated with virus were exposed for 6 hr and for 3 hr. The results of these tests (Table 2) show that the virus was substantially inactivated after an exposure of 3 hr to direct sunlight. Indeed the activity of the 0.05% virus suspension exposed was reduced to significantly less than that of the control tested at one-tenth of this concentration. (As stated previously X2 is not reported in the tables as the results are so evidently significant. )
TABLE
I
23-29.9.66 Control
10-11.5.67 Control 12-13.7.67 Control
19
12
conditions
8-16 Nil 9-16 Nil
lo-16 Nil
8-17 Nil Nil
Hours within which leaves were exposed GMT ‘r Mas.
3100-9300 Not exposed 4000-9500 Not exposed
Mostly sunny Not exposetl
Mostly sunny Not exposed Not exposed
Min.
Luminous intensity ( ft-c )
r
0.05 0.05 0.05 0.05
0.2 0.2
1.0 0.002 0.01
Virus concentration (%) b
6 6 6 6
10 2
2 2 2
No. of leaves treated
Biological
data
107 107 110 120
219 37
39 40 38
Total no. of test insects
TO THE UPPER SURFACE 12 HR OR MORE
(1 British Summer Time was corrected to Greenwich Mean Time, b Expressed as dilution of standard concentrates. Different standards wctre used in the 1966 and 1967 tests. ~The luminous intensity measurements given in Tables 1 and 2 are intended onIy as a guide to the light conditions under were made. Where they exceed the normally accepted maximum for sunlight ( 10,000 ft-c) this is probably due to reflection different spectral energy distribution of the light to the on e used for calibrating the cell. d X2 values are not reported in Tables 1 and 2, but the differences between the average percentage kills of the irradiated are highly significant in all cases.
l-7.6.66 Control ControI
43
Exposure
OF PUIUFIED PREPARATIONS OF A GRANULOSIS VLRLJS OF I’. brassicae, APPLIED FOLLOWING EXPOSURES TO DIRECT SUNLIGHT eon PERIODS TOTALLING
Dates on which exposures were given
LOSS OF ACTIWTY
Total duration of exposure ( hours )
THE
and
control
which the from clouds
LEAVES,
samples
exposures or to the
0 42 5 83
Average
OF CABBAGE
-. 8 s B
10-13
26.7.67
7.8.67 Nil Nil
10-13
TABLE
2
Max.
exposed
larvae
Time.
Not exposed 3109-3500 Not exposed 3100-3500 Not exposed 1 loo-12,000 Not exposed Not exposed 11,500-11,500 Not exposed Not exposed
-
to the
1,500
exposed exposed
3100-3500
Not Not
800&l
Not exposed 11,000-12,090 Not exposed Not exposed
3500-8000
Not
3500-8000
Min.
Luminous intensity ( ft-c )
other
two
b
run
0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.005 0.05 0.05 0.005
0.05 0.05 0.05 0.05 0.05 0.05 0.005 0.05 0.05 0.005
Virus concentration (%I
on 11.5.67.
6 6 6 6 6 6 6 6 6 6 6 G
6 6 6 6 6 6 6 6 6 6
No. of leaves treated
Biological
VIRUS OF P. brasicae, APPLIED TO THE UPPER SURFACE TO DIRECT SUNLIGHT FOR PERIODS OF 6 AND 3 HR
was corrected to Greenwich hlean of the same standard concentrate. out with a different batch of test
Control Control
Nil Nil
Nil 12-15 Nil 12-15 Nil
Control Control
12-15
11.5.67
Control 11.5.67 Control 11.5.67 Control
‘1 British Summer Time fi Expressed as dilution c This test was carried
3
lo-16
7.8.67 Nil Nil
Nil Nil
Control Control
Control Control
lo-16
26.7.67
Control
Nil
lo-16
10.5.67
Control
lo-16 Nil
10.5.67
6
Hours within which leaves were exposed GMT fl
conditions
GRANULOSIS EXPOSURE
Dates on which exposures were given
Exposure
OF A PURIFIED
Total duration of exposure ( hours )
THE LOSS OF ACTIVITY
data
129 118
107 112 113 117 107 120 118 117 124
117
111 118 117 128 119 118
113 107 118 113
Total no. of test insects
OF CABBAGE
LEAVES,
65
100
96 5c 42 57 98 78 16
100 65 20 82 32
82 12 96 3 98 78 2
10
Average kill (%I
FOLLOWING
Q
zvl
g $ 3 0CA E
8
8
[
8
9
4 2
500
DAVID,
GARDINER,
DISCUSSION
Action spectra show that, for several viruses, radiation of wavelength around 2600 A is most effective in producing inactivation and that the effectiveness of radiation beyond 3000 A is relatively low. However no radiation from the sun of wavelength less than 2915 A reaches the earth’s surface and there is, in fact, extremely little of this, since absorption by atmospheric ozone reaches a very high level for radiation of wavelength less than 3100 W. Smoke and clouds also further reduce the amount of UVR reaching the earth ( McCulloch, 1945; Hollaender, 1955). According to Kleczkowski (1957), viruses do not absorb visible light and so are not affected by it directly to any appreciable extent. If this is so we must conclude that the inactivating effect of sunlight on viruses depends on irradiation in the wavelength band 2915 A and 3800 A (violet). As so little radiation below 3100 A reaches the earth and reactivation phenomena begins at about 3600 A (Hollaender, 1955), the effective wavelengths in practice probably fall between these limits--that is to say, radiation between 3100 A with quanta of 4.00 ev and 3600 A with quanta of 3.43 ev. There is another possible explanation of the observed inactivation, namely that in bright sunlight toxic substances are produced on the leaf surface and these diffuse into the virus and affect its virulence. Such an effect, due to substances in the medium, has been observed with other test systems (Kleczkowski, 1957; Lauria, 1953). But
such
an explanation
seems
less likely
in the case of a granulosis virus which is protected by its relatively thick protein capsule and anyway, in this case, the virus is also rapidly destroyed when exposed to sunlight in a Spectrosil cell (David and Gardiner, unpublished). The results reported in this paper, with the granulosis virus of P. brassicae, obtained under conditions in England, are
AND
WOOLNER
very much in line with those recently reported by Cantwell (1967) for the granulosis virus of the salt-marsh caterpillar, Estigmene acrea, exposed to sunlight in Beltsville, Maryland. In the experiments described here highly purified virus preparations were used and since the virus was so rapidly destroyed by sunlight, it is obvious that, in this form, it would not be suitable for use in biological control. It is true that after a field application some of the virus may be lodged on the undersides of leaves and in other places where it will be protected from direct sunlight; nevertheless much of the virus is certain to be deposited on the upper surface of the leaves and, as this fraction will soon be inactivated, there will inevitably be a rapid loss of biological efficiency. Before the virus can be used effectively a way of protecting it from UVR must be found. In contrast with these results some earlier experiments suggest that when crude preparations of the virus are used the activity
is better
preserved
(David
and Gard-
iner, 1966a). The same is likely to be true of virus in the dried-up exudates of caterpillars which often occur on leaves during natural epizootics. This greater stability is to be expected since in the crude state the virus would be partly screened from the UVR by the dried up fluids and by tissue fragments present in the mixture. REFERENCES
G. E. 1967. Inactivation of biological insecticides by irradiation. J. Invertebrate Pathol., 9, 138-140. DAVID, W. A. L. 1965. The granulosis virus of Pi&s brassicae L. in relation to natural limitation and biological control. Ann. Appl. Biol., 56, 331-334. CANTWELL,
DAVID,
W. A. persistence
L. 1967. Factors influencing the of the granulosis virus of Pieris bmssicae in the environment. “Insect Pathol. and Microbial Control.” PTOC. Intern. Co&q. Insect Pathol. Microbial Control, Wageningen, Netherlands, 1966, pp. 174-178. (North-Holland Publ. Co., Amsterdam.)
EFFECTS
DAVID,
OF
SUNLIGHT
W. A. L., AND GARDINER, B. 0. C. 1965. The incidence of granulosis deaths in susceptible and resistant Pieris brassicae (Linnaeus) larvae following changes of population density, food, and temperature. J. Invertebrate Pathol., 7, 347-355. DAVID, W. A. L., AND CARDINER, B. 0. C. 1966a. Persistence of a granulosis virus of Pieris brassicae on cabbage leaves. J. Invertebrate Pathot., 8, 186-183. DAVID, W. A. L., AND GARDINER, B. 0. C. 1966b. Rearing Pieris bmssicae apparently free from granulosis virus. J. Invertebrate Pathol., 8, 325-333.
ON
GRANULOSIS
DAVID,
VIRUS
501
W. A. L., AND GARDINER, B. 0. C. 1967. The effect of heat, cold and prolonged storage on a granulosis virus of Pieris brassicae. J. Invertebrate Pathol., 9, 555-562. HOLLAENDER, A. (ed. ) . 1955. “Radiation Biology,” Vol. 2, 593 pp. McGraw-Hill, New York. KLECZKOWSKI, A. 1957. Effects of non-ionizing radiations on viruses. Advan. Virus Res., 4, 191-220. LAURIA, S. E. 1953. “General Virology,” 427 pp. Wiley, New York. MCCULLOCH, E. C. 1945. “Disinfection and Sterilization,” 2nd ed., 472 pp. Henry Kimpton, London.
OF INVERTEBRATE
PATHOLOGY
11,
496-501
(
1968)
The Effects of Sunlight on a Purified Granulosis Virus of Pieris bra&cue Applied to Cabbage Leaves W. A. L. DAVID, Agricultural
B. 0. C. GARDINER,
Research Council,
AND M. WOOLNER
34A Storey’s Way, Cambridge,
1
Engkznd
Received January 23, 1968 When a highly purified preparation of the known granulosis virus of Pieris bTassicae is exposed to direct sunlight, on the upper surface of cabbage leaves, carefully oriented so that no part is in shade, the virus is rapidly inactivated. For example, after an exposure of 3 hr, the suspension applied to the leaves gave very significantly less kill of larvae than a nonirradiated control suspension one-tenth its concentration. Total inactivation of the virus, under these circumstances, took between 12 and 19 hr. These results are discouraging from the point of view of using purified, unprotected, granulosis virus for biological control and emphasize the importance of finding some means of screening or protecting the virus from ultraviolet radiation.
MATERIALS
INTRODUCTION
AND METHODS
Certain preliminary observations on the effect of light on the activity of a granulosis virus of the European cabbageworm (or large white butterfly), Pieris bra&cue, led to the general conclusion that when the purified virus was exposed to sunlight it rapidly lost its capacity to cause lethal infections in the larvae (David, 1965; David, 1967). A crude virus preparation, on the other hand, retained some of its activity for 16 weeks on cabbage plants kept in the open during the winter 196263 (David and Gardiner, 1966a). Another crude preparation, in which there was solid matter from the dead larvae, showed very slight activity after being stored for 2 years as a dried film in a glass tube in a glasshouse (David and Gardiner, 1967). The tests reported in this paper are of an essentially practical nature. That is to say, the virus was exposed to direct sunlight, in the middle part of the day, on the upper surface of cabbage leaves, just as it might be after a field application.
The virus preparation exposed to the ultraviolet radiation (UVR) was highly purified to avoid any screening, protective, or chemical inactivating effect which might be associated with impurities. It was obtained by the usual process of grinding up larvae which had recently died of virus disease, suspending the material in distilled water, straining it through fine muslin, and filtering through Whatman No. 1 filter paper. Afterwards the crude virus was centrifuged in alternate cycles at low and high speeds and further purified by suspending it four or five times on sucrose gradients. The pooled virus samples were then washed free of sucrose and suspended in boiled distilled water which had been filtered through sintered glass. The concentrated stock was stored in the dark at about -20°C. All test suspensions were diluted from this and also stored under the same conditions. As applied to the leaves they contained 0.1% of a wetting agent (Teepol) .
1 M. Woolner was on study leave from Brunel University, London, W.3.
The grown 496
leaves of young cabbage plants in a screened glasshouse were used
EFFECTS
OF
SUNLIGHT
in the tests. They were left on the plants and treated on the upper, adaxial, surface. An area measuring 2 X 1% inches was marked off with a ballpoint pen and rubbed with absorbent tissue to remove some of the surface wax. The virus suspension was applied in droplets to the surface of each leaf, 0.25 ml being distributed over the prescribed area in 4060 drops. The applications were made when the leaves were already in the sun and the droplets evaporated completely within the next hour. During the exposure the leaves were arranged horizontally or oriented to face the sun. They were also kept as flat as possible by suitable supports and clips. Care was taken to see that even temporary shading caused by veins or irregularities was reduced to a minimum. Between exposures, when the leaves were not in sunlight, they were held in a shaded room, as were the control leaves which had been treated at the same time with the virus suspension. Light intensity readings were taken every hour by means of a commercial selenium cell light meter calibrated in foot-candles. Such an instrument is very unsatisfactory for measuring radiation in experiments of this type; all that can be concluded is that, in general, the higher the measured total intensity of the sunlight the greater the intensity of the active UVR. After the requisite exposure the leaves were brought in, trimmed to standard size and set up in shell vials of water as previously described (David and Gardiner, 1965, 1967). Twenty newly molted second-instar virus-free P. brmsicae larvae were placed on each leaf and the leaves were then put separately into sterilized jars (David and Cardiner, 1966b). At the same time, controls were also set up using the leaves treated with the virus which had not been exposed to sunlight. Except in two preliminary tests six replicates of the treated
ON
GRANULOSIS
VIRUS
497
and control leaves were used. The larvae were examined daily and the numbers dying of virus disease were recorded. Counting was continued for 5 days after the first virus death in any given jar (David and Gardiner, 1965). Control larvae (untreated with virus) were also kept in some tests but no virus deaths occurred among these larvae. The results are expressed as percentages in the tables and in any dubious cases the significance of each result has been assessed by the X” test on the original data. However, all the experiments reported show highly significant differences between the control and the irradiated leaves and the X’ figures are not given in order to simplify the tables. RESULTS
The experiments reported in this paper were carried out between the months of May and September in 1966 and 1967. In the preliminary tests, reported in Table 1, long exposures were given since it was not known what duration of exposure would be necessary to give a detectable reduction in activity of the virus. It can be seen that in one test some virus survived an exposure of 12 hr. This result seems a little anomalous (when compared with the results of tests in which a 6-hr exposure was given) and no active virus was detected after the longer exposures (19 hr and 43 hr), More detailed tests were carried out in which the leaves treated with virus were exposed for 6 hr and for 3 hr. The results of these tests (Table 2) show that the virus was substantially inactivated after an exposure of 3 hr to direct sunlight. Indeed the activity of the 0.05% virus suspension exposed was reduced to significantly less than that of the control tested at one-tenth of this concentration. (As stated previously X2 is not reported in the tables as the results are so evidently significant. )
TABLE
I
23-29.9.66 Control
10-11.5.67 Control 12-13.7.67 Control
19
12
conditions
8-16 Nil 9-16 Nil
lo-16 Nil
8-17 Nil Nil
Hours within which leaves were exposed GMT ‘r Mas.
3100-9300 Not exposed 4000-9500 Not exposed
Mostly sunny Not exposetl
Mostly sunny Not exposed Not exposed
Min.
Luminous intensity ( ft-c )
r
0.05 0.05 0.05 0.05
0.2 0.2
1.0 0.002 0.01
Virus concentration (%) b
6 6 6 6
10 2
2 2 2
No. of leaves treated
Biological
data
107 107 110 120
219 37
39 40 38
Total no. of test insects
TO THE UPPER SURFACE 12 HR OR MORE
(1 British Summer Time was corrected to Greenwich Mean Time, b Expressed as dilution of standard concentrates. Different standards wctre used in the 1966 and 1967 tests. ~The luminous intensity measurements given in Tables 1 and 2 are intended onIy as a guide to the light conditions under were made. Where they exceed the normally accepted maximum for sunlight ( 10,000 ft-c) this is probably due to reflection different spectral energy distribution of the light to the on e used for calibrating the cell. d X2 values are not reported in Tables 1 and 2, but the differences between the average percentage kills of the irradiated are highly significant in all cases.
l-7.6.66 Control ControI
43
Exposure
OF PUIUFIED PREPARATIONS OF A GRANULOSIS VLRLJS OF I’. brassicae, APPLIED FOLLOWING EXPOSURES TO DIRECT SUNLIGHT eon PERIODS TOTALLING
Dates on which exposures were given
LOSS OF ACTIWTY
Total duration of exposure ( hours )
THE
and
control
which the from clouds
LEAVES,
samples
exposures or to the
0 42 5 83
Average
OF CABBAGE
-. 8 s B
10-13
26.7.67
7.8.67 Nil Nil
10-13
TABLE
2
Max.
exposed
larvae
Time.
Not exposed 3109-3500 Not exposed 3100-3500 Not exposed 1 loo-12,000 Not exposed Not exposed 11,500-11,500 Not exposed Not exposed
-
to the
1,500
exposed exposed
3100-3500
Not Not
800&l
Not exposed 11,000-12,090 Not exposed Not exposed
3500-8000
Not
3500-8000
Min.
Luminous intensity ( ft-c )
other
two
b
run
0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.005 0.05 0.05 0.005
0.05 0.05 0.05 0.05 0.05 0.05 0.005 0.05 0.05 0.005
Virus concentration (%I
on 11.5.67.
6 6 6 6 6 6 6 6 6 6 6 G
6 6 6 6 6 6 6 6 6 6
No. of leaves treated
Biological
VIRUS OF P. brasicae, APPLIED TO THE UPPER SURFACE TO DIRECT SUNLIGHT FOR PERIODS OF 6 AND 3 HR
was corrected to Greenwich hlean of the same standard concentrate. out with a different batch of test
Control Control
Nil Nil
Nil 12-15 Nil 12-15 Nil
Control Control
12-15
11.5.67
Control 11.5.67 Control 11.5.67 Control
‘1 British Summer Time fi Expressed as dilution c This test was carried
3
lo-16
7.8.67 Nil Nil
Nil Nil
Control Control
Control Control
lo-16
26.7.67
Control
Nil
lo-16
10.5.67
Control
lo-16 Nil
10.5.67
6
Hours within which leaves were exposed GMT fl
conditions
GRANULOSIS EXPOSURE
Dates on which exposures were given
Exposure
OF A PURIFIED
Total duration of exposure ( hours )
THE LOSS OF ACTIVITY
data
129 118
107 112 113 117 107 120 118 117 124
117
111 118 117 128 119 118
113 107 118 113
Total no. of test insects
OF CABBAGE
LEAVES,
65
100
96 5c 42 57 98 78 16
100 65 20 82 32
82 12 96 3 98 78 2
10
Average kill (%I
FOLLOWING
Q
zvl
g $ 3 0CA E
8
8
[
8
9
4 2
500
DAVID,
GARDINER,
DISCUSSION
Action spectra show that, for several viruses, radiation of wavelength around 2600 A is most effective in producing inactivation and that the effectiveness of radiation beyond 3000 A is relatively low. However no radiation from the sun of wavelength less than 2915 A reaches the earth’s surface and there is, in fact, extremely little of this, since absorption by atmospheric ozone reaches a very high level for radiation of wavelength less than 3100 W. Smoke and clouds also further reduce the amount of UVR reaching the earth ( McCulloch, 1945; Hollaender, 1955). According to Kleczkowski (1957), viruses do not absorb visible light and so are not affected by it directly to any appreciable extent. If this is so we must conclude that the inactivating effect of sunlight on viruses depends on irradiation in the wavelength band 2915 A and 3800 A (violet). As so little radiation below 3100 A reaches the earth and reactivation phenomena begins at about 3600 A (Hollaender, 1955), the effective wavelengths in practice probably fall between these limits--that is to say, radiation between 3100 A with quanta of 4.00 ev and 3600 A with quanta of 3.43 ev. There is another possible explanation of the observed inactivation, namely that in bright sunlight toxic substances are produced on the leaf surface and these diffuse into the virus and affect its virulence. Such an effect, due to substances in the medium, has been observed with other test systems (Kleczkowski, 1957; Lauria, 1953). But
such
an explanation
seems
less likely
in the case of a granulosis virus which is protected by its relatively thick protein capsule and anyway, in this case, the virus is also rapidly destroyed when exposed to sunlight in a Spectrosil cell (David and Gardiner, unpublished). The results reported in this paper, with the granulosis virus of P. brassicae, obtained under conditions in England, are
AND
WOOLNER
very much in line with those recently reported by Cantwell (1967) for the granulosis virus of the salt-marsh caterpillar, Estigmene acrea, exposed to sunlight in Beltsville, Maryland. In the experiments described here highly purified virus preparations were used and since the virus was so rapidly destroyed by sunlight, it is obvious that, in this form, it would not be suitable for use in biological control. It is true that after a field application some of the virus may be lodged on the undersides of leaves and in other places where it will be protected from direct sunlight; nevertheless much of the virus is certain to be deposited on the upper surface of the leaves and, as this fraction will soon be inactivated, there will inevitably be a rapid loss of biological efficiency. Before the virus can be used effectively a way of protecting it from UVR must be found. In contrast with these results some earlier experiments suggest that when crude preparations of the virus are used the activity
is better
preserved
(David
and Gard-
iner, 1966a). The same is likely to be true of virus in the dried-up exudates of caterpillars which often occur on leaves during natural epizootics. This greater stability is to be expected since in the crude state the virus would be partly screened from the UVR by the dried up fluids and by tissue fragments present in the mixture. REFERENCES
G. E. 1967. Inactivation of biological insecticides by irradiation. J. Invertebrate Pathol., 9, 138-140. DAVID, W. A. L. 1965. The granulosis virus of Pi&s brassicae L. in relation to natural limitation and biological control. Ann. Appl. Biol., 56, 331-334. CANTWELL,
DAVID,
W. A. persistence
L. 1967. Factors influencing the of the granulosis virus of Pieris bmssicae in the environment. “Insect Pathol. and Microbial Control.” PTOC. Intern. Co&q. Insect Pathol. Microbial Control, Wageningen, Netherlands, 1966, pp. 174-178. (North-Holland Publ. Co., Amsterdam.)
EFFECTS
DAVID,
OF
SUNLIGHT
W. A. L., AND GARDINER, B. 0. C. 1965. The incidence of granulosis deaths in susceptible and resistant Pieris brassicae (Linnaeus) larvae following changes of population density, food, and temperature. J. Invertebrate Pathol., 7, 347-355. DAVID, W. A. L., AND CARDINER, B. 0. C. 1966a. Persistence of a granulosis virus of Pieris brassicae on cabbage leaves. J. Invertebrate Pathot., 8, 186-183. DAVID, W. A. L., AND GARDINER, B. 0. C. 1966b. Rearing Pieris bmssicae apparently free from granulosis virus. J. Invertebrate Pathol., 8, 325-333.
ON
GRANULOSIS
DAVID,
VIRUS
501
W. A. L., AND GARDINER, B. 0. C. 1967. The effect of heat, cold and prolonged storage on a granulosis virus of Pieris brassicae. J. Invertebrate Pathol., 9, 555-562. HOLLAENDER, A. (ed. ) . 1955. “Radiation Biology,” Vol. 2, 593 pp. McGraw-Hill, New York. KLECZKOWSKI, A. 1957. Effects of non-ionizing radiations on viruses. Advan. Virus Res., 4, 191-220. LAURIA, S. E. 1953. “General Virology,” 427 pp. Wiley, New York. MCCULLOCH, E. C. 1945. “Disinfection and Sterilization,” 2nd ed., 472 pp. Henry Kimpton, London.