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A leaf surface treatment bioassay for determining the activity of conidia of Beauveria bassiana against Leptinotarsa decemlineata
JOURNAL
OF INVERTEBRATE
PATHOLOGY
41, 385-386
(1983)
A Leaf Surface Treatment Bioassay for Determining Conidia of Beauveria bassiana against Leptinotarsa
the Activity of decemlineata
A leaf surface treatment bioassay was measuring 18 + 1 cm* were used for the successfully used to determine the activity bioassay. Each leaflet was treated with a of conidia of Beauveria bassiana. The suspension of conidia (0.1 ml/side) in sterile bioassay is a modification of that pre- distilled water (SDW) containing 0.05% viously used to determine the activity of co- Tween 80. The B. bassiana we used, named nidia of Nomurueu rileyi against several Beauverol, was a granular formulation of lepidopteran species (C. M. Ignoffo, B. dried, air-borne conidia prepared from a Puttler, D. L. Hostetter, and W. A. Dick- Czechoslovakian isolate (A. Samsinakova, erson, J. Invertebr. Pathol. 28, 259-262, S. Kalalova, and J. Kybol, J. Znvertebr. 1976). A similar bioassay also was used to Puthol., in press) provided by J. Weiser, A. determine the activity of conidia of a USSR Samsinakova, and S. Kalalova. This prepaformulation of B. bassiana (Boverin) ration contained 8.5 t 0.8 x lOlo total coagainst larvae of Trichoplusia ni and Pieris nidia/g and 8.7 x lOlo viable conidia/g. rupae (C. M. Ignoffo, C. Garcia, 0. A. Each series of tests consisted of seven deciAlyoshina, N. V. Lappa, J. Econ. Ento- mal dilutions of conidia suspensions rangmol. 72,562-565, 1979) and the susceptibiling from 0 to 100,000 conidia/mm* of leaflet. ity of Leptinotarsu decemlineata to Ba- Ten first instar larvae of L. decemlineLlta cillus thuringiensis (C. M. Ignoffo, C. (average 1.25 ? 0.06 mg/larva)* were conGarcia, and M. Kroha, J. Znvertebr. fined on a leaflet in a Petri dish (90 x Pathol. 39, 244-246, 1982). In other reports, 15 mm) containing an SDW-wetted filter preparations of B. bassiancr were bioas- paper. After 48 hr of incubation (25” + l”C), sayed against adult Musca domestica with the larvae from each leaflet were transferred a spraying technique (L. P. Sinitsyna, from the Petri dish to a cage fashioned from In “Proceedings of the 1st Joint USSR a plastic jar and a waxed-paper ice cream Conference on the Production Selection cup (C. M. Ignoffo, C. Garcia, and M. Kroha, J. Invertebr. Pathol. 39, 244-246, and Standardization of Entomopathogenic Fungi,” (C. M. Ignoffo, ed.), Nat. Sci. 1982. The area of leaflet consumed during 48 Publication, 1978) and against coleopterans hr of incubation was recorded. Fresh, un(Acanthoscelids obtectus, Scolytus scol- treated leaflets were placed in each cage at lytus) with either spraying or topical applito 2-day intervals until the bioassay was tercations of spores (G. Barson, J. Znvertebr. minated 5 days later. Larval mortality and Pllthol. 29, 361-366, 1977; P. Fen-on and body weight of surviving larvae were then P. H. Robert, J. Znvertebr. Pnthol. 25, recorded. Each rate was replicated three 379-388, 1975). times with 10 larvae/cage and two to three In this study, bioassays were conducted cages/replicate. Thus, a total of ca. 70-90 with first instar Colorado potato beetle lar- larvae/rate were used to calculate the revae (CPB), Leptinotarsa decemlineata, fed gression equation, LC-50, and ST-,, values. on greenhouse-grown potato leaflets surThe presence of B. bassiana on leaflets face treated with conidia of B. bussiana. did not inhibit feeding (except at IO5 coThe leaflet area was determined with a Cor nidia/mm*) by first instar larvae nor did it Model Li-3000 area-meter. l Only leaflets have any effect on the body weight of surviving larvae (Table 1). As far as mortality was concerned, less than 50% of the larvae 1 Mention of a proprietary product in this paper does survived doses of more than 100 conot constitute a recommendation for use by the U.S. Department
* + refers
of Agriculture.
to SE mean
in both
text
and table.
385 0022-2011183 $1.50 Copyright @ 1983 by Academic Al! ights
of reproduction
Press, Inc. in any form reserved.
NOTES
386
TABLE
SUSCEPTIBILITY OF LARVAEOF
Leptinotarsa
1
decemlineutu
TO INFECTION
BY CONIDIA
Mortality Rate” (conidia/mm2) 100,000 10,000 1,000 100 10 1 0
Area consumed”
(mm? 157 330 300 351 293 370 537
* 49 It_72 2 50 2 32 k 49 + 70 -t 146
Percentage 100.0 98.7 93.4 73.7 17.5 10.0 4.3
+ 2.1 2 7.8 2 3.1 + 5.9 ? 2.4 -+ 0.9
ST-50 (days)
OF Beaaveriu
hus~itrntr
Body weight surviving larvae’ (mg)
2.0 2.6 3.8 4.5 >lS.O
13.4 r+_0.0 51.0 r 4.0 60.8 k 2.5 56.1 + 5.1 50.1 * 4.1 61.6 2 8.3
a Total conidia/mm2. (I During 48 hr of exposure to 8. bassiunu. c 7 days postexposure.
nidia/mm2, and the ST-,,s for rates of lo* to lo5 conidia/mm2 ranged from 2.0 to 4.5 days, respectively (Table 1). The regression equation (Y = CI + bX) for rate mortality was Y = 3.555 + 1.074X (Y = probit - % mortality: X = log-rate conidia/mm2). The LC-5,, was 28.8 + 13.9 conidia/mm2. In a previous study the LC-,, of B. hssiana against T. ni and P. rapcre was ca. 200 and 165 conidia/mm2, respectively (Ignoffo et al., ibid, 1979). Although speculative, it is possible to calculate a possible field rate based upon the number of conidia/mm2 required to provide various levels of infection. To estimate these values, we calculated a total leaf surface area in a potato field of 25 billion mm2/hectare, and assumed a formulation of B. bassiana containing 1 x log conidia/g. Thus, to attain 99, 90, or 50% mortality of an incipient destructive field population of CPB larvae would require 2.0 x 10’” (ca. 200 kg), 1.6 X 1013 (ca. 16.25 kg), or 7.5 x 10” (0.75 kg) conidia/hectare, respectively. Only 2.5 x lOlo conidia/mm2 (ca. 25 g) would be needed to attain 10% mortality, a dose probably sufficient, if applied early in the season, to initiate an epizootic. Under field conditions in Czechoslovakia, efficient field suppression of CPB was obtained (accumulative control of greater than 90% of feeding larvae as well
as over-wintering larvae, pupae and adults) at rates of 40-50 g/hectare with preparations of Beauverol containing 10” conidia/g (J. Weiser, pers. commun.). Significant control of field populations of CPB with B. bassiuna was also recently reported in France at a dose of 1013 spores/hectare (J. Fargues, J. P. Cugier, and P. Van de Weghe, Actcl EcoiJEcol. Appl. 1, 49-61, 1980). KEY WORDS: Beaweria bassinna; Leptinotclrsa decemlineata; bioassay; Beuu\leria conidia. The authors acknowledge the contribution of Ben Puttler (USDA, SEA, AR, Biological Control of Insects Research Laboratory, Columbia, MO.) for providing eggs of Leptinotursa decemlineatu.
CARLO M. ICNOFFO CLEMENTE GARCIA MIKE KROHA U.S. Department of Agriculture Agriculturul Research Biological Control qf Insects Reseurch Columbia, Missouri 65205
Luborutory
SAMSINAKOVA SYLVIAKALALOVA
ANN
Academy of Science Institute of Entomology Laboratory of Insect Pathology Prugue, Czechoslovakia Received Januury 12, 1981; uccepted
April
21, 1982
OF INVERTEBRATE
PATHOLOGY
41, 385-386
(1983)
A Leaf Surface Treatment Bioassay for Determining Conidia of Beauveria bassiana against Leptinotarsa
the Activity of decemlineata
A leaf surface treatment bioassay was measuring 18 + 1 cm* were used for the successfully used to determine the activity bioassay. Each leaflet was treated with a of conidia of Beauveria bassiana. The suspension of conidia (0.1 ml/side) in sterile bioassay is a modification of that pre- distilled water (SDW) containing 0.05% viously used to determine the activity of co- Tween 80. The B. bassiana we used, named nidia of Nomurueu rileyi against several Beauverol, was a granular formulation of lepidopteran species (C. M. Ignoffo, B. dried, air-borne conidia prepared from a Puttler, D. L. Hostetter, and W. A. Dick- Czechoslovakian isolate (A. Samsinakova, erson, J. Invertebr. Pathol. 28, 259-262, S. Kalalova, and J. Kybol, J. Znvertebr. 1976). A similar bioassay also was used to Puthol., in press) provided by J. Weiser, A. determine the activity of conidia of a USSR Samsinakova, and S. Kalalova. This prepaformulation of B. bassiana (Boverin) ration contained 8.5 t 0.8 x lOlo total coagainst larvae of Trichoplusia ni and Pieris nidia/g and 8.7 x lOlo viable conidia/g. rupae (C. M. Ignoffo, C. Garcia, 0. A. Each series of tests consisted of seven deciAlyoshina, N. V. Lappa, J. Econ. Ento- mal dilutions of conidia suspensions rangmol. 72,562-565, 1979) and the susceptibiling from 0 to 100,000 conidia/mm* of leaflet. ity of Leptinotarsu decemlineata to Ba- Ten first instar larvae of L. decemlineLlta cillus thuringiensis (C. M. Ignoffo, C. (average 1.25 ? 0.06 mg/larva)* were conGarcia, and M. Kroha, J. Znvertebr. fined on a leaflet in a Petri dish (90 x Pathol. 39, 244-246, 1982). In other reports, 15 mm) containing an SDW-wetted filter preparations of B. bassiancr were bioas- paper. After 48 hr of incubation (25” + l”C), sayed against adult Musca domestica with the larvae from each leaflet were transferred a spraying technique (L. P. Sinitsyna, from the Petri dish to a cage fashioned from In “Proceedings of the 1st Joint USSR a plastic jar and a waxed-paper ice cream Conference on the Production Selection cup (C. M. Ignoffo, C. Garcia, and M. Kroha, J. Invertebr. Pathol. 39, 244-246, and Standardization of Entomopathogenic Fungi,” (C. M. Ignoffo, ed.), Nat. Sci. 1982. The area of leaflet consumed during 48 Publication, 1978) and against coleopterans hr of incubation was recorded. Fresh, un(Acanthoscelids obtectus, Scolytus scol- treated leaflets were placed in each cage at lytus) with either spraying or topical applito 2-day intervals until the bioassay was tercations of spores (G. Barson, J. Znvertebr. minated 5 days later. Larval mortality and Pllthol. 29, 361-366, 1977; P. Fen-on and body weight of surviving larvae were then P. H. Robert, J. Znvertebr. Pnthol. 25, recorded. Each rate was replicated three 379-388, 1975). times with 10 larvae/cage and two to three In this study, bioassays were conducted cages/replicate. Thus, a total of ca. 70-90 with first instar Colorado potato beetle lar- larvae/rate were used to calculate the revae (CPB), Leptinotarsa decemlineata, fed gression equation, LC-50, and ST-,, values. on greenhouse-grown potato leaflets surThe presence of B. bassiana on leaflets face treated with conidia of B. bussiana. did not inhibit feeding (except at IO5 coThe leaflet area was determined with a Cor nidia/mm*) by first instar larvae nor did it Model Li-3000 area-meter. l Only leaflets have any effect on the body weight of surviving larvae (Table 1). As far as mortality was concerned, less than 50% of the larvae 1 Mention of a proprietary product in this paper does survived doses of more than 100 conot constitute a recommendation for use by the U.S. Department
* + refers
of Agriculture.
to SE mean
in both
text
and table.
385 0022-2011183 $1.50 Copyright @ 1983 by Academic Al! ights
of reproduction
Press, Inc. in any form reserved.
NOTES
386
TABLE
SUSCEPTIBILITY OF LARVAEOF
Leptinotarsa
1
decemlineutu
TO INFECTION
BY CONIDIA
Mortality Rate” (conidia/mm2) 100,000 10,000 1,000 100 10 1 0
Area consumed”
(mm? 157 330 300 351 293 370 537
* 49 It_72 2 50 2 32 k 49 + 70 -t 146
Percentage 100.0 98.7 93.4 73.7 17.5 10.0 4.3
+ 2.1 2 7.8 2 3.1 + 5.9 ? 2.4 -+ 0.9
ST-50 (days)
OF Beaaveriu
hus~itrntr
Body weight surviving larvae’ (mg)
2.0 2.6 3.8 4.5 >lS.O
13.4 r+_0.0 51.0 r 4.0 60.8 k 2.5 56.1 + 5.1 50.1 * 4.1 61.6 2 8.3
a Total conidia/mm2. (I During 48 hr of exposure to 8. bassiunu. c 7 days postexposure.
nidia/mm2, and the ST-,,s for rates of lo* to lo5 conidia/mm2 ranged from 2.0 to 4.5 days, respectively (Table 1). The regression equation (Y = CI + bX) for rate mortality was Y = 3.555 + 1.074X (Y = probit - % mortality: X = log-rate conidia/mm2). The LC-5,, was 28.8 + 13.9 conidia/mm2. In a previous study the LC-,, of B. hssiana against T. ni and P. rapcre was ca. 200 and 165 conidia/mm2, respectively (Ignoffo et al., ibid, 1979). Although speculative, it is possible to calculate a possible field rate based upon the number of conidia/mm2 required to provide various levels of infection. To estimate these values, we calculated a total leaf surface area in a potato field of 25 billion mm2/hectare, and assumed a formulation of B. bassiana containing 1 x log conidia/g. Thus, to attain 99, 90, or 50% mortality of an incipient destructive field population of CPB larvae would require 2.0 x 10’” (ca. 200 kg), 1.6 X 1013 (ca. 16.25 kg), or 7.5 x 10” (0.75 kg) conidia/hectare, respectively. Only 2.5 x lOlo conidia/mm2 (ca. 25 g) would be needed to attain 10% mortality, a dose probably sufficient, if applied early in the season, to initiate an epizootic. Under field conditions in Czechoslovakia, efficient field suppression of CPB was obtained (accumulative control of greater than 90% of feeding larvae as well
as over-wintering larvae, pupae and adults) at rates of 40-50 g/hectare with preparations of Beauverol containing 10” conidia/g (J. Weiser, pers. commun.). Significant control of field populations of CPB with B. bassiuna was also recently reported in France at a dose of 1013 spores/hectare (J. Fargues, J. P. Cugier, and P. Van de Weghe, Actcl EcoiJEcol. Appl. 1, 49-61, 1980). KEY WORDS: Beaweria bassinna; Leptinotclrsa decemlineata; bioassay; Beuu\leria conidia. The authors acknowledge the contribution of Ben Puttler (USDA, SEA, AR, Biological Control of Insects Research Laboratory, Columbia, MO.) for providing eggs of Leptinotursa decemlineatu.
CARLO M. ICNOFFO CLEMENTE GARCIA MIKE KROHA U.S. Department of Agriculture Agriculturul Research Biological Control qf Insects Reseurch Columbia, Missouri 65205
Luborutory
SAMSINAKOVA SYLVIAKALALOVA
ANN
Academy of Science Institute of Entomology Laboratory of Insect Pathology Prugue, Czechoslovakia Received Januury 12, 1981; uccepted
April
21, 1982