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Inactivation of Baculovirus heliothis by ultraviolet irradiation, dew, and temperature
JOURNAL
OF INVERTEBRATE
Inactivation
PATHOLOGY
30,237-241
(1977)
of Baculovirus heliothis by Ultraviolet and Temperature1,2,3
Irradiation,
P. J. MCLEOD, W. C. YEARIAN, AND S. Y. YOUNG Virology
and Bio-Control
Laboratory, Department of Entomology, FayetteviNe, Arkansas 72701
University
Dew,
III of Arkansas,
Received November 23. 1976 The rate and extent of inactivation of Baculoviras heliothis by artificial ultraviolet (uv) irradiation, temperature, and dew collected from foliage of cotton and soybean plants were determined. Exposure to uv irradiation resulted in substantial inactivation of the virus. Increase in temperature from 15” to 45°C had little effect on viral activity. A significant loss in viral activity was detected as temperature was increased to 45°C with exposure to uv irradiation. Exposure topH 9.3 cotton dew resulted in substantial loss in activity during the initial dry-down of dew. Loss of activity appeared to result from exposure to highpH and high basic ion concentration. After the dew had dried; little additional activity loss occurred unless deposited ions were resuspended in water and allowed to redry. Exposure to cotton dews atpH 7.4 or 8.8 or soybean dew (pH 7.2) produced no significant viral inactivation.
cotton dew, and soybean dew at various temperatures.
INTRODUCTION
Studies on the persistence of &c&virus he&this on cotton foliage under field conditions indicated that substantial inactivation occurred during the first 24 hr of exposure (Bullock, 1967; Ignoffo and Batzer, 1971; Ignoffo et al., 1972; Yearian and Young, 1974; Young and Yearian, 1974). Inactivation of B. heliothis in the field has been attributed to solar irradiation, particularly in the ultraviolet spectrum (Bullock et al., 1970), high pH of dew on cotton foliage (Andrews and Sikorowski, 1973; Young et al., 1977), and high temperature (Gudauskas and Canerday, 1968). Although some data are available on the degree of viral inactivation caused by these factors, their interactions have not been investigated. This study reports on inactivation of B. heliothis exposed to artificial uv irradiation,
MATERIALS
AND METHODS
Heliothis zea larvae used in the study were from a stock culture maintained at the Virology-Bio-Control Laboratory, University of Arkansas, following procedures outlined by Phillips and Yearian ( 1966). The isolate of B. heliothis used was Biotrol VHZ Lot 17 (Nutrilite Products, Inc., Lakeview, California). The virus was propagated and purified as described by Young and Yearian (1974). The uv source was six GE F20T12/BL4 bulbs arranged parallel, 5.1 cm apart, 10.2 cm above shelves, in thermostatically controlled cabinets. Intensities of long- and short-wave ultraviolet irradiation 10.2 cm below the bulbs were 180 and 230 erg/set/ cmz, respectively, as determined by BlackRay ultraviolet intensity meters5. An area of 25.4 x 50.8 cm under each set of lights was uniformly exposed. Cotton dew was collected in late August
r Published with the approval of the Director, Arkansas Agricultural Experiment Station. * This research was supported in part by Cooperative State Research Service Grant 216-15-84. 3 Use of a trade name does not imply endorsement or guarantee of the product or the exclusion of other products of similar nature.
4 General Electric Co., Cleveland, Ohio. 5 Ultraviolet Products, Inc.. San Gabriel, California. 237
Copyright 0 1977 by Academic Press. Inc. All rights of reproductmn in any form reserved.
ISSN OW-201
I
238
McLEOD,
YEARIAN.
1973, between 5:00 and 8:00 AM from Delta Pine- 16 cotton planted at the Arkansas Agricultural Experiment Station Farm, Main Station, Fayetteville, following procedures outlined by Young et al. (1977). Soybean dew was collected in August 1973 from Hill soybeans also at Fayetteville. All dews were filtered through organdy and were centrifuged at 15OOg for 12 min. Samples were submitted to the Soil Testing Laboratory, University of Arkansas, for ion analysis (Young et al., 1976). The remainder was frozen for later use. One-milliliter aliquots of virus suspension containing 1 x 108 PIB were spread uniformly on the inside bottom surface of 150-mm glass Petri dishes and were allowed to air dry. Ater drying, a l-ml aliquot of dew was spread over the virus deposits in twothirds of the dishes and was air dried. A similar volume of distilled water was spread over the virus deposits in the remaining dishes. After drying, a l-ml aliquot of dew was again spread over the virus deposits in one-half of the dishes previously receiving dew. A similar volume of distilled water was again spread over the virus deposits in the remaining dishes. After drying, onehalf of the dishes were covered with glass lids, wrapped in aluminum foil, and placed in temperature cabinets set to maintain an air temperature of 15” + 2”, 30” t 2”. or 45” & 2°C. The remaining dishes, with lids off, were placed under the uv source in each of the three temperature cabinets. Thus, exposure regimes included exposure and nonexposure to uv irradiation and exposure to 0, 1, and 2 ml of cotton dew at 15”, 30”, and 45°C for 12, 24, and 48 hr. Each combination was replicated a minimum of four times. The virus was recovered from the dishes after appropriate exposure times by rinsing each dish with 5 ml of distilled water five times for a total of 25 ml of resuspended virus. A standard bioassay using neonate H. zea larvae was performed using four to nine concentrations per treatment (Ignoffo, 1965). Dosages were expressed in PIB/mm2
AND
YOUNG
of diet surface. All dosages were based on the assumption that 100% of the virus was recovered. The effect of soybean dew on inactivation of B. heliothis was assessed in a second series of tests. The procedure was similar to that used in tests on cotton dew, except that the soybean dew tests were replicated five times. Data from both tests were analyzed by a computer at the University of Arkansas Computing Center. The program was Probit Analysis by Method of Maximum Likelihood. Factorial analysis was performed on computed LD,, values. RESULTS
AND DISCUSSION
Exposure to the artificial uv source caused substantial virus inactivation. When all exposure regimes were considered, the mean LDsO for virus not exposed to uv irradiation was 10.6 PIB/mm2, while that for virus exposed to uv irradiation was 293.3 PIB/mm2 (Table 1). Inactivation of the virus by uv irradiation was directly related to the period of exposure. Losses in activity for virus exposed to uv between 0 and 12, 12 and 24, and 24 and 48 hr were 78.9, 86.9, and 84.1%, respectively. Virus with no uv exposure showed no statistically significant loss in activity. A significant interaction occurred between exposure to uv irradiation and temperature. Although some loss in activity occurred in virus not exposed to uv as temperature (15”, 30”, and 45°C) was increased, the loss was not significant (Table 2). There was a significant interaction in the effect of temperature and uv irradiation on virus activity in that inactivation of virus exposed to uv at 45°C was significantly more extensive than that of virus exposed at 30°C. Exposures to uv irradiation at 15” and 30°C did not cause significantly different inactivation. Only small increases in mean LD,,s were noted for the exposure time-temperature interaction, except for the exposure for 48 hr
INACTIVATION
OF ~A~ULO~I~US
at 45°C (Table 3). This loss in activity for virus exposed at 45°C for 48 hr was equivalent to activity losses of 92 and 82% over that for virus exposed for 48 hr at 1.5” and 3O”C, respectively. A substantial loss in activity was detected in virus bioassayed immediately afterpH 9.3 cotton dew had dried. The LDsO at 0 hr for virus not exposed to dew was 0.1 PIB/mm2, while the LDsO for virus exposed to 1 ml of cotton dew at 0 hr was 5.0 PIB/mm2 (Table 4). This was equivalent to a 98.0% activity loss over exposure to no dew. An additional activity loss of 55.8% resulted from addition of the second milliliter of dew. As length of exposure to dew was increased (12, 24, and 48 hr), a slight increase in activity loss was detected. Mean LD5,, values for exposure to 1 and 2 ml of dew at all temperatures was 11.4, 14.9, and 20.0 PIB/mm2 for 12, 24, and 48 hr, respectively. None of these activity losses were statistically significant. Although the addition of dew to virus exposed to uv irradiation increased inactivity, the dew-uv interaction was not significant as was the dew-temperature interaction. Chemical analysis of cotton dew showed an unusually highpH (9.3) and high concentrations of basic ions (Na, Mg, K, and NH,) in comparison to previous reports (Andrews and Sikorowski, 1973; Young et al., 1977). Most of the loss in activity occurred during drying of the dew, with little if any additional activity loss occurring unless deposited ions were resuspended by an addition of water. Young et al. (1977) reported that some loss in activity occurred when a suspension of virus and cotton dew, pH 8.8, was air dried and resuspended daily in deionized water. Two additional collections of cotton dew (collected September, 1974), withpH values of 7.4 and 8.8, were used to determine the effects of different dews on inactivation of B. ~~eli~t~~~.Procedures were as previously described, except treatments included only exposure to 0, 1, and 2 ml of dew at 0 hr. Each treatment was replicated a minimum of two times.
ffEL~~~~~S
239
TABLE
1
ACTIVITY OFBACULOV~RUSHELIOTHLY EXPOSED TO ARTIFICIAL ULTRAVIOLET IRRADIATION FOR 12, 24, AND 48 hr
Mean LD,,, (PIB/mmZ of diet surface)” Exposure (hr) 12 24 48 Mean
None”
UV
Mean
7.7 AC 10.0 AB 14.2 AB
20.6 B 136.5 c 722.9 D 293.3
14.2 73.2 368.6
10.6
a Each mean includes exposure to all combinations of temperature (W, 30”, and 45°C) and cotton dew (0, 1. and 2 ml). b None: Plates were covered. with aluminum foil. c Means not followed by the same letter are significantly different (P = 0.01). Duncan multiple-range test.
No significant loss in activity was detected in virus exposed to each dew. Exposure to 1 ml ofpH 7.4 dew resulted in a mean LDsD of 0.3 PIB/mm2, while the mean LD% for virus not exposed to dew was 0.3 PIBl mm2 (Table 4). Addition of the second milliliter ofpH 7.4 cotton dew caused no further activity loss. When virus was subjected to 1 ml ofpH 8.8 cotton dew, an activity loss TABLE
2
ACTIVITY ~~BACULOVIR~SHELIOT~IS EXPOSED TO ARTIFICIAL ULTRAVIOLET IRRADIATION AT t5”, 30”, AND 45°C
Mean LDS (PIB/mme of diet surface)” Temperature P-2 15 30 45 Mean
None0
“V
Mean
5.9 AC 11.6 A 14.4 AB 10.6
53.8 BC 142.2 C 684.0 D 293.3
29.8 76.9 349.2
n Each mean includes exposure to all combinations of cotton dew (0, 1, and 2 ml) and time lengths ( f 2.24, and 48 hr). b None: Plates were covered with aluminum foil. c Means not followed by the same letter are significantly different (P = 0.01). Duncan multiple-range test.
240
McLEOD. TABLE
YEARIAN,
3
ACTIVITY OF BACULOWRUSHELIOTHIS EXPOSED 15”, 30”, AND 45°C FOR 12, 24, AND 48 hr
AT
Mean LD5, (PIB/mmZ of diet surface)” Exposure temperature (“C) Exposure @r) 12 24 48
15
30
45
2.0 Ab 9.6 A 48.2 AB
4.8 A 24.7 ABC 124.4 BC
21.8 AB 112.1 BC 564.6 D
a Each mean includes exposure to all combinations of artificial uv irradiation (no uv and uv) and dew (0, 1, and 2 ml). b Means not followed by the same letter are significantly different (P = 0.01). Duncan multiple-range test.
of 39.2% was detected which, again, was nonsignificant. No significant loss in activity was detected upon exposure to the second milliliter ofpH 8.8 cotton dew. These results are in agreement with results presented by Ignoffo and Garcia (1966) in which B. heliothis was found to be stable after exposure to buffers at pH values as high as 9. In the other series of tests with pH 7.2 soybean dew being substituted for cotton dew, viral inactivation from exposure to uv irradiation at 15”, 30”, and 45°C for 12, TABLE
4
ACTIVITY OEBACULOVIRUSHELIOTHIS COTTON
DEWS
WITH
DIFFERENT
EXPOSED TO VALUES
pH
Mean LDso (PIB/mm2 of diet surface) Amount of dew (ml) Dew
0”
1*
2”
Cotton @H 9.3) Cotton (PH 8.8) Cotton @H 7.4)
0.1 0.3 0.3
5.0 0.4 0.3
11.3 0.8 0.3
a Exposure to 2 ml of water. b Exposure to 1 ml of dew + 1 ml of water. c Exposure to 2 ml of dew. d Virus bioassayed immediately after water or dew had dried with first-in&r Heliothis zea larvae.
AND YOUNG
24, and 48 hr was comparable with that in the first series of tests. In virus exposed to 1 ml of soybean dew, however, only a slight (nonsignificant) loss was detected. No additional loss in activity was detected upon exposure to the second milliliter of soybean dew during the initial drying. As length of exposure to soybean dew was increased (12, 24, and 48 hr), no additional loss in activity was detected for exposure to either the first or second milliliter of soybean dew. More activity was lost in virus nonexposed to dew than in that exposed. This could be attributed to the screening effect of particles in dew, preventing breakdown from uv exposure. Upon chemical analysis, soybean dew exhibited apH value of 7.2 which was substantially lower than the pH of 9.3 for cotton dew. Concentrations of basic ions (Na, Mg, K, and NH,) were also much greater in cotton dew than in soybean dew. Exposure topH 9.3 cotton dew produced a substantial loss in activity during the initial drying of the dew. This loss appeared to result from exposure to high concentrations of metallic ions at a high p H. After dew had dried, little if any additional activity loss occurred unless deposited ions were resuspended by addition of water. Exposure to cotton dews of pH 7.4 or 8.8 or soybean dew produced no significant viral breakdown. These results suggest that, under field conditions, the majority of cases of viral inactivation results from exposure to uv irradiation. It is doubtful that field temperatures ever become sufficiently high to cause viral inactivation, except in desert areas. Although exposure to dew of exceptionally high pH may play some role in virus inactivation, it is probably not substantial under field conditions. REFERENCES ANDREWS,
G. L., AND SIKOROWSKI,
P. P. 1973. Effects
of cotton leaf surfaces on the nuclear polyhedrosis virus of Heliothis zea and Heliothis virescens. J. Invertebr. Pathol., 22, 290. BULLOCK, H. R. 1%7. Persistence of Heliothis nuclear
INACTIVATION polyhedrosis Pathol., BULLOCK, STACK,
OF BACULOVIRUS
virus on cotton foliage. J. Inverkbr.
9,434-436. H. R., HOLLINGSWORTH, A. W., Jr. 1970. Virulence
J. R., AND HARTof Heliothis nuclear polyhedrosis virus exposed to monochromatic ultraviolet irradiation. J. Invertebr. Pathol., 16, 419-422. GUDAUSKAS, R. T., AND CANERDAY, D. 1%8. The effect of heat, buffer salt and H-ion concentration, and ultraviolet light on the infectivity of Heliothis and Trichoplusia nuclear-polyhedrosis viruses. J. Znvertebr. Pathol., 12, 405-411. IGNOFFO, C. M. 1%5. The nuclear-polyhedrosis virus of Heliothis zea (Boddie) and Heliothis virestens (Fabricius). Part IV. Bioassay of virus activity. J. Znvertebr. Puthol., 7, 315-319. IGNOFFO, C. M., AND BATZER, D. F. 1971. Microencapsulation and ultraviolet protectants to increase sunlight stability of an insect virus. J. Econ. Entomol., 64, 850-853. IGNOFFO, C. M., AND GARCIA,
C. 1%6. The relation of pH to the activity of inclusion bodies of a Helio-
HELIOTHIS
241
nuclear polyhedrosis. J. Invertebr. Pathol., 8, 426-427, IGNOFFO, C. M., BRADLEY, J. R., GILLILAND, F. R., HARRIS, F. A., FALCON, L. A., LARSON, L. V., MCGARR, R. L., SIKOROWSKI, P. P., WATSON, T. F., AND YEARIAN, W. C. 1972. Field studies on stability of the Heliothis nuclear polyhedrosis virus at various sites throughout the cotton belt. Environ. Entomol., 1,388-390. PHILLIPS, J. R., AND YEARIAN, W. C. 1966. Rearing insects for research purposes. Arkansas Farm Res., 15, 10. YEARIAN, W. C., AND YOUNG, S. Y. 1974. Persistence of Heliothis nuclear polyhedrosis virus on cotton plant parts. Environ. Entomol., 3, 1035- 1036. YOUNG, S. Y., AND YEARIAN, W. C. 1974. Persistence of Heliothis NPV on foliage of cotton soybean and tomato. Environ. Entomol., 3, 253-255. YOUNG, S. Y., YEARIAN, W. C., AND KIM, K. S. 1977. Effect of dew from cotton and soybean foliage on activity of Heliothis NPV. J. Invertebr. Puthol., 29, 105-111. this
OF INVERTEBRATE
Inactivation
PATHOLOGY
30,237-241
(1977)
of Baculovirus heliothis by Ultraviolet and Temperature1,2,3
Irradiation,
P. J. MCLEOD, W. C. YEARIAN, AND S. Y. YOUNG Virology
and Bio-Control
Laboratory, Department of Entomology, FayetteviNe, Arkansas 72701
University
Dew,
III of Arkansas,
Received November 23. 1976 The rate and extent of inactivation of Baculoviras heliothis by artificial ultraviolet (uv) irradiation, temperature, and dew collected from foliage of cotton and soybean plants were determined. Exposure to uv irradiation resulted in substantial inactivation of the virus. Increase in temperature from 15” to 45°C had little effect on viral activity. A significant loss in viral activity was detected as temperature was increased to 45°C with exposure to uv irradiation. Exposure topH 9.3 cotton dew resulted in substantial loss in activity during the initial dry-down of dew. Loss of activity appeared to result from exposure to highpH and high basic ion concentration. After the dew had dried; little additional activity loss occurred unless deposited ions were resuspended in water and allowed to redry. Exposure to cotton dews atpH 7.4 or 8.8 or soybean dew (pH 7.2) produced no significant viral inactivation.
cotton dew, and soybean dew at various temperatures.
INTRODUCTION
Studies on the persistence of &c&virus he&this on cotton foliage under field conditions indicated that substantial inactivation occurred during the first 24 hr of exposure (Bullock, 1967; Ignoffo and Batzer, 1971; Ignoffo et al., 1972; Yearian and Young, 1974; Young and Yearian, 1974). Inactivation of B. heliothis in the field has been attributed to solar irradiation, particularly in the ultraviolet spectrum (Bullock et al., 1970), high pH of dew on cotton foliage (Andrews and Sikorowski, 1973; Young et al., 1977), and high temperature (Gudauskas and Canerday, 1968). Although some data are available on the degree of viral inactivation caused by these factors, their interactions have not been investigated. This study reports on inactivation of B. heliothis exposed to artificial uv irradiation,
MATERIALS
AND METHODS
Heliothis zea larvae used in the study were from a stock culture maintained at the Virology-Bio-Control Laboratory, University of Arkansas, following procedures outlined by Phillips and Yearian ( 1966). The isolate of B. heliothis used was Biotrol VHZ Lot 17 (Nutrilite Products, Inc., Lakeview, California). The virus was propagated and purified as described by Young and Yearian (1974). The uv source was six GE F20T12/BL4 bulbs arranged parallel, 5.1 cm apart, 10.2 cm above shelves, in thermostatically controlled cabinets. Intensities of long- and short-wave ultraviolet irradiation 10.2 cm below the bulbs were 180 and 230 erg/set/ cmz, respectively, as determined by BlackRay ultraviolet intensity meters5. An area of 25.4 x 50.8 cm under each set of lights was uniformly exposed. Cotton dew was collected in late August
r Published with the approval of the Director, Arkansas Agricultural Experiment Station. * This research was supported in part by Cooperative State Research Service Grant 216-15-84. 3 Use of a trade name does not imply endorsement or guarantee of the product or the exclusion of other products of similar nature.
4 General Electric Co., Cleveland, Ohio. 5 Ultraviolet Products, Inc.. San Gabriel, California. 237
Copyright 0 1977 by Academic Press. Inc. All rights of reproductmn in any form reserved.
ISSN OW-201
I
238
McLEOD,
YEARIAN.
1973, between 5:00 and 8:00 AM from Delta Pine- 16 cotton planted at the Arkansas Agricultural Experiment Station Farm, Main Station, Fayetteville, following procedures outlined by Young et al. (1977). Soybean dew was collected in August 1973 from Hill soybeans also at Fayetteville. All dews were filtered through organdy and were centrifuged at 15OOg for 12 min. Samples were submitted to the Soil Testing Laboratory, University of Arkansas, for ion analysis (Young et al., 1976). The remainder was frozen for later use. One-milliliter aliquots of virus suspension containing 1 x 108 PIB were spread uniformly on the inside bottom surface of 150-mm glass Petri dishes and were allowed to air dry. Ater drying, a l-ml aliquot of dew was spread over the virus deposits in twothirds of the dishes and was air dried. A similar volume of distilled water was spread over the virus deposits in the remaining dishes. After drying, a l-ml aliquot of dew was again spread over the virus deposits in one-half of the dishes previously receiving dew. A similar volume of distilled water was again spread over the virus deposits in the remaining dishes. After drying, onehalf of the dishes were covered with glass lids, wrapped in aluminum foil, and placed in temperature cabinets set to maintain an air temperature of 15” + 2”, 30” t 2”. or 45” & 2°C. The remaining dishes, with lids off, were placed under the uv source in each of the three temperature cabinets. Thus, exposure regimes included exposure and nonexposure to uv irradiation and exposure to 0, 1, and 2 ml of cotton dew at 15”, 30”, and 45°C for 12, 24, and 48 hr. Each combination was replicated a minimum of four times. The virus was recovered from the dishes after appropriate exposure times by rinsing each dish with 5 ml of distilled water five times for a total of 25 ml of resuspended virus. A standard bioassay using neonate H. zea larvae was performed using four to nine concentrations per treatment (Ignoffo, 1965). Dosages were expressed in PIB/mm2
AND
YOUNG
of diet surface. All dosages were based on the assumption that 100% of the virus was recovered. The effect of soybean dew on inactivation of B. heliothis was assessed in a second series of tests. The procedure was similar to that used in tests on cotton dew, except that the soybean dew tests were replicated five times. Data from both tests were analyzed by a computer at the University of Arkansas Computing Center. The program was Probit Analysis by Method of Maximum Likelihood. Factorial analysis was performed on computed LD,, values. RESULTS
AND DISCUSSION
Exposure to the artificial uv source caused substantial virus inactivation. When all exposure regimes were considered, the mean LDsO for virus not exposed to uv irradiation was 10.6 PIB/mm2, while that for virus exposed to uv irradiation was 293.3 PIB/mm2 (Table 1). Inactivation of the virus by uv irradiation was directly related to the period of exposure. Losses in activity for virus exposed to uv between 0 and 12, 12 and 24, and 24 and 48 hr were 78.9, 86.9, and 84.1%, respectively. Virus with no uv exposure showed no statistically significant loss in activity. A significant interaction occurred between exposure to uv irradiation and temperature. Although some loss in activity occurred in virus not exposed to uv as temperature (15”, 30”, and 45°C) was increased, the loss was not significant (Table 2). There was a significant interaction in the effect of temperature and uv irradiation on virus activity in that inactivation of virus exposed to uv at 45°C was significantly more extensive than that of virus exposed at 30°C. Exposures to uv irradiation at 15” and 30°C did not cause significantly different inactivation. Only small increases in mean LD,,s were noted for the exposure time-temperature interaction, except for the exposure for 48 hr
INACTIVATION
OF ~A~ULO~I~US
at 45°C (Table 3). This loss in activity for virus exposed at 45°C for 48 hr was equivalent to activity losses of 92 and 82% over that for virus exposed for 48 hr at 1.5” and 3O”C, respectively. A substantial loss in activity was detected in virus bioassayed immediately afterpH 9.3 cotton dew had dried. The LDsO at 0 hr for virus not exposed to dew was 0.1 PIB/mm2, while the LDsO for virus exposed to 1 ml of cotton dew at 0 hr was 5.0 PIB/mm2 (Table 4). This was equivalent to a 98.0% activity loss over exposure to no dew. An additional activity loss of 55.8% resulted from addition of the second milliliter of dew. As length of exposure to dew was increased (12, 24, and 48 hr), a slight increase in activity loss was detected. Mean LD5,, values for exposure to 1 and 2 ml of dew at all temperatures was 11.4, 14.9, and 20.0 PIB/mm2 for 12, 24, and 48 hr, respectively. None of these activity losses were statistically significant. Although the addition of dew to virus exposed to uv irradiation increased inactivity, the dew-uv interaction was not significant as was the dew-temperature interaction. Chemical analysis of cotton dew showed an unusually highpH (9.3) and high concentrations of basic ions (Na, Mg, K, and NH,) in comparison to previous reports (Andrews and Sikorowski, 1973; Young et al., 1977). Most of the loss in activity occurred during drying of the dew, with little if any additional activity loss occurring unless deposited ions were resuspended by an addition of water. Young et al. (1977) reported that some loss in activity occurred when a suspension of virus and cotton dew, pH 8.8, was air dried and resuspended daily in deionized water. Two additional collections of cotton dew (collected September, 1974), withpH values of 7.4 and 8.8, were used to determine the effects of different dews on inactivation of B. ~~eli~t~~~.Procedures were as previously described, except treatments included only exposure to 0, 1, and 2 ml of dew at 0 hr. Each treatment was replicated a minimum of two times.
ffEL~~~~~S
239
TABLE
1
ACTIVITY OFBACULOV~RUSHELIOTHLY EXPOSED TO ARTIFICIAL ULTRAVIOLET IRRADIATION FOR 12, 24, AND 48 hr
Mean LD,,, (PIB/mmZ of diet surface)” Exposure (hr) 12 24 48 Mean
None”
UV
Mean
7.7 AC 10.0 AB 14.2 AB
20.6 B 136.5 c 722.9 D 293.3
14.2 73.2 368.6
10.6
a Each mean includes exposure to all combinations of temperature (W, 30”, and 45°C) and cotton dew (0, 1. and 2 ml). b None: Plates were covered. with aluminum foil. c Means not followed by the same letter are significantly different (P = 0.01). Duncan multiple-range test.
No significant loss in activity was detected in virus exposed to each dew. Exposure to 1 ml ofpH 7.4 dew resulted in a mean LDsD of 0.3 PIB/mm2, while the mean LD% for virus not exposed to dew was 0.3 PIBl mm2 (Table 4). Addition of the second milliliter ofpH 7.4 cotton dew caused no further activity loss. When virus was subjected to 1 ml ofpH 8.8 cotton dew, an activity loss TABLE
2
ACTIVITY ~~BACULOVIR~SHELIOT~IS EXPOSED TO ARTIFICIAL ULTRAVIOLET IRRADIATION AT t5”, 30”, AND 45°C
Mean LDS (PIB/mme of diet surface)” Temperature P-2 15 30 45 Mean
None0
“V
Mean
5.9 AC 11.6 A 14.4 AB 10.6
53.8 BC 142.2 C 684.0 D 293.3
29.8 76.9 349.2
n Each mean includes exposure to all combinations of cotton dew (0, 1, and 2 ml) and time lengths ( f 2.24, and 48 hr). b None: Plates were covered with aluminum foil. c Means not followed by the same letter are significantly different (P = 0.01). Duncan multiple-range test.
240
McLEOD. TABLE
YEARIAN,
3
ACTIVITY OF BACULOWRUSHELIOTHIS EXPOSED 15”, 30”, AND 45°C FOR 12, 24, AND 48 hr
AT
Mean LD5, (PIB/mmZ of diet surface)” Exposure temperature (“C) Exposure @r) 12 24 48
15
30
45
2.0 Ab 9.6 A 48.2 AB
4.8 A 24.7 ABC 124.4 BC
21.8 AB 112.1 BC 564.6 D
a Each mean includes exposure to all combinations of artificial uv irradiation (no uv and uv) and dew (0, 1, and 2 ml). b Means not followed by the same letter are significantly different (P = 0.01). Duncan multiple-range test.
of 39.2% was detected which, again, was nonsignificant. No significant loss in activity was detected upon exposure to the second milliliter ofpH 8.8 cotton dew. These results are in agreement with results presented by Ignoffo and Garcia (1966) in which B. heliothis was found to be stable after exposure to buffers at pH values as high as 9. In the other series of tests with pH 7.2 soybean dew being substituted for cotton dew, viral inactivation from exposure to uv irradiation at 15”, 30”, and 45°C for 12, TABLE
4
ACTIVITY OEBACULOVIRUSHELIOTHIS COTTON
DEWS
WITH
DIFFERENT
EXPOSED TO VALUES
pH
Mean LDso (PIB/mm2 of diet surface) Amount of dew (ml) Dew
0”
1*
2”
Cotton @H 9.3) Cotton (PH 8.8) Cotton @H 7.4)
0.1 0.3 0.3
5.0 0.4 0.3
11.3 0.8 0.3
a Exposure to 2 ml of water. b Exposure to 1 ml of dew + 1 ml of water. c Exposure to 2 ml of dew. d Virus bioassayed immediately after water or dew had dried with first-in&r Heliothis zea larvae.
AND YOUNG
24, and 48 hr was comparable with that in the first series of tests. In virus exposed to 1 ml of soybean dew, however, only a slight (nonsignificant) loss was detected. No additional loss in activity was detected upon exposure to the second milliliter of soybean dew during the initial drying. As length of exposure to soybean dew was increased (12, 24, and 48 hr), no additional loss in activity was detected for exposure to either the first or second milliliter of soybean dew. More activity was lost in virus nonexposed to dew than in that exposed. This could be attributed to the screening effect of particles in dew, preventing breakdown from uv exposure. Upon chemical analysis, soybean dew exhibited apH value of 7.2 which was substantially lower than the pH of 9.3 for cotton dew. Concentrations of basic ions (Na, Mg, K, and NH,) were also much greater in cotton dew than in soybean dew. Exposure topH 9.3 cotton dew produced a substantial loss in activity during the initial drying of the dew. This loss appeared to result from exposure to high concentrations of metallic ions at a high p H. After dew had dried, little if any additional activity loss occurred unless deposited ions were resuspended by addition of water. Exposure to cotton dews of pH 7.4 or 8.8 or soybean dew produced no significant viral breakdown. These results suggest that, under field conditions, the majority of cases of viral inactivation results from exposure to uv irradiation. It is doubtful that field temperatures ever become sufficiently high to cause viral inactivation, except in desert areas. Although exposure to dew of exceptionally high pH may play some role in virus inactivation, it is probably not substantial under field conditions. REFERENCES ANDREWS,
G. L., AND SIKOROWSKI,
P. P. 1973. Effects
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