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Laboratory and field cage tests with Bacillus thuringiensis against pink-bollworm larvae
JOURNAL
OF
INVERTEBRATE
Laboratory
PATHOLOGY
9,
and Field Against
390-394
(
1967)
Cage Tests with Pink-Bollworm
C. M. IGNOFFO~ AND H. M. Entomology U.S.
Research Department
Bacillus Larvae’
thuringiensis
GRAHAM
Division, Agricultural Research Service, of Agriculture, Brownsville, Texas
Accepted
September
15,
1966
Newly hatched and mature larvae of the pink bollwonn, Pectinophora gossypiella, were susceptible to spores of Bacillus thuringiensis in lactose dusts and in soil-spore mixtures, respectively. The pupation rate of survivors exposed to the spores as newly hatched larvae was inhibited, and pupal weights decreased with increases in concentration of spores. Application of spores to soil in field cages reduced overwintering populations of pink bollworms about 45%.
Control of the pink bollworm, Pectinophora gossypiella, with conventional insecticides is difficult because the larvae are exposed only as first instars before they enter fruiting forms or after they leave the fruiting form as last instars to pupate in the soil or surface debris. The pests overwinter as last-instar diapausing larvae in cottonseed or in cocoons in the soil and leave these hibernating sites in the spring to pupate near the surface of the soil. Such cultural control measures as shredding of stalks and plowing under of crop debris kill many overwintering larvae (Chapman et al, 1960; Martin and Lewis, 1962), but methods of increasing this larval mortality without use of chemical insecticides would be desirable. Previous laboratory tests demonstrated that both diapausing and nondiapausing pink-bollworm larvae were susceptible to a microbial insecticide containing Bacillus 1 In cooperation with the Texas Agricultural Experiment Station, Texas A&M University. ’ NOW with International Minerals and Chemical Corporation, Bioferm Division, Wasco, California.
thuringiensis ( see Ignoffo, 1962a), and that the usual temperatures and humidities in the field should not adversely affect spore stability and virulence (Ignoffo, 1962b; 1964). Additional laboratory and field tests were therefore conducted to determine whether first-instar larvae could be controlled with simulated foliar applications of spores and whether mature diapausing and nondiapausing larvae could be controlled by applications of the spores to the soil. LABORATORY
TESTS
Newly Hatched Larvae Exposed to Spores in Lactose Dusts Tests were conducted at Brownville, Texas, in 1961 to determine the effects of B. thuringiensis on first-instar pinkbollworm larvae. Thuricide (Bioferm preparation S9-517),3 a spore concentrate containing 1 X loll spores/g, was diluted with lactose to form dust mixtures containing 3 Mention of trade names imply endorsement by the Agriculture.
does not necessarily U.S. Department of
TESTS
OF
&Xi&is
AGAINST
1 X lOi, 5 X 107, 10 X 107, 50 X 107, and 100 X 10’ spores/g. One-gram samples of each of the five concentrations and a lactose dust without spores were placed in each of six 12-dram vials ( Table 1) with about 100 newly hatched first-instar larvae. The vials were stoppered, rolled gently for 30 seconds to ensure contact of the larvae with the dust, and then placed in an upright position. (Only larvae which migrated to the lips of the vials within 15 minutes of exposure were used in the tests.) Each treatment was replicated three times. At least 20 treated larvae from each of the six treatments of each replicate were then placed individually in 2-dram vials containing synthetic pinkbollworm diet (Adkisson et al.. 1960; Ouye, 1962) and reared at 29°C. Numbers of survivors, weight of pupae, pupation rate, and adult emergence were recorded. Also, more than 20 larvae were used at the higher concentrations of spores to ensure a sufficient sample of pupae for the studies of weight and adult emergence. Analysis of variance and Duncan’s multiple range test were used to compare the differences between means. The mortality of first-instar larvae exposed to spores of B. thuringiensis increased with increases in the concentration
PINK
BOLLWORM
391
of spores (Table 1). The LD,,, for larvae. was estimated at 25 X lo7 spores/g. Rates of pupation was influenced by exposun’ to spore dusts; at 17 days after exposurtb. pupation had hardly begun for larvae cxposed to the 100 X lo7 spores/g though 95% of the larvae in the check had ~IIpated by that time; also 50% of the untreated larvae pupated 8 days sooner thall 50% of the larvae receiving the highest concentration (Table 2). Pupal weight dccreased as the concentration of spores increased (Table 1). The weights of pupal-: treated with S, 10, 50, and 100 >(: 10; spores/g were significantly different from each other. Ifowever, pupal weights did not affect rclosion.
Tests were conducted in 1961 to detrrmine whether spores of B. thwingiensis could bc transmitted to diapausing fourthinstar pink-bollworm larvae exposed to spores mixed with soil. Eighteen grams of a spore-soil mixture obtained by seriallv diluting an original spore-soil sample WIItaining 18 g of air-dried top soil and 2 2 of spores (7 X lo!’ spores/g) were placczd in petri dishes. The original spore-soil sa111ple contained 7 >( lo!’ spores/g (126 ‘Y’ 10”
Pupae J,arvae surviving (averages)
(X lf)‘/d
Percent
0
Numbera
1
93 92
19 a 18 a
5
8s
10 50
68 32
18 a 14 b
100
I’ ralues
“LO
with
same letter
not significantly
_
.__--~
\l’eight (ma)”
6c
-21.3 21.0 19.8 18.1 17.ld
Id
lE.O
different
at lTp
level.
- -~_.
(averages)
Number of days to 50?$ pupation
a
14
a b
14
c e
16 17 20 “-2
392
IGNOFFO
TABLE
AND
2
PERCENT PUPATION OF PINK-BOLLWORM LARVAE AFTER EXPOSURE OF FIRST-INSTAR TO VARIOUS CONCENTR.4TIONS OF B. thuringiensis IN L.4CTOBE-SPORE DUSTS Days after exposure
Number of spores x 107/g of dust 0
1
5
10
50
100
12
4
5
2
13 14
29 61
27 48
5 17
15 16
75 87 95
82 94 95
42 65 72
15 38 51
5
82
64
24
8
88 90 92
84 89 93
38 52 57
20 48
95
67 71
50 52
86
58 65
91
75
95
78 80
17 18 19 20 91 22 23 24 25 26 27 28 29 31 32 33
96
4
85 88 92 95
spores/dish); the other dilutions were 7 x lo*, 7 x 107, 7 x 106, 7 x 105, and 7 X lo4 spores/g. Then 15-25 fourth-instar pink-bollworm larvae were placed in each petri dish (replicate) and held at 29” t 1°C. The six treatments were each replicated three times. Blood smears of dead larvae were made to verify B. thuringiensis septicemia on the day that the larvae died and were removed from the dishes. The characteristic increase in mortality with increases in concentration of spores was obtained (Table 3). However, no significant mortality occurred at concentrations of 7 X lo6 spores/g of soil or less. Mortality from septicemia was probably the result of spore entry through mechanical breaks in the body wall and subsequent spore germination and vegetative cell growth. In another test, mature larvae were
GRAHAM
pricked on the dorsal surface of the segment immediately behind the prothorax and dusted with a camel’s hair brush containing spores of B. thuringiensis. Of 15 larvae treated, 12 died within 72 hours with positive septicemia. Of 15 check larvae treated in the same manner but not dusted with spores, 13 pupated and 2 died from the injury. Nondiapausing, fourth-instar larvae which had recently emerged from firm, green field-infested bolls (collected between August 31 and September 16) were exposed to soil treated with spores in cages made of 1 lb, 5inch-diameter coffee cans. The following treatments were used: ( 1) untreated soil; (2) top soil dusted on the surface with 500 mg of 6 X lOlo spores/g; and (3) 500 mg of 6 X lOlo spores/g thoroughly mixed with 100 g of soil and then spread in a i/s-inch layer over 2 inches of untreated soil. In all three treatments, the soil contained 6-8 ml of water/100 g of soil. A hole was cut in the cover of the can and fitted with a bottle to trap the emerging adults. Fifty larvae/ treatment were used for the first replicate; 100 larvae/treatment were used for the other two replicates. The larvae were held at 28” -+ 1°C. The average number of living pink bollworms recovered (sum of adults, pupae, and larvae) from each treatTABLE
3
MORTALITY OF MATURE,DIAPAUSING PINK-ROLLWORM LARVAE EXPOSED TO SOILS TREATED WITH SPORES OF B. thuringiensis
Treatment (number of spores/g soil) 7 x 7 x 7 x
109 108 10’
7 x 7 x 7 x
10’ 105 104
Number of larvae treated 75 75 50 50 50 50
Percent mortality after 48 br 72 41 16 4 2 0
100 94 69 41 4 2 0
hr
TESTS
TABLE
OF
Bacillus
AGAINST
rntreated Spores drlsted on top soil Spores mixed in soil
Percent reduction
Average
Range
89.3
X3-96
-
“7 3
S-36
69
31.7
1ti-34
65
of 6 X lOto spores/g.
n 5(JO m g
ment 2-3 weeks after introduction of the larvae is shown in Table 4. Both soil treatments reduced the number of pinkbollworm larvae. FIELD
CAGE
TESTS
1961 Test Studies were conducted at Brownsville, Texas, from March 1 to June 20, 1961, in standard 3 X 3-ft pyramidal hibernation cages (Shiller, 1946) with Bakthane L-69,3 a commercial preparation of B. thuringiensis (Rohm and Haas, 75 X 10” spores/ a Mention of trade names imply endorsement 11y the Agriculture.
does U.S.
not necessarily Department of
TAB1.E STRJWAL
OF SPORES
Treatment
OF R. thuringiens;‘s
5 IN Son
Kumber of replicate treatments
IN FIELD Number
plate rounts
Check (no treatment) Treated cottona Treated soil6
1d 15 I.5
Check (no treatment) Treated cotton”
18 19 26
Treated
393
Ten cages/treatment were each stocked with 3?1, lb of infested seed cotton collected from plants grown at Torrcon, Coahuilla, Mexico. Treatments were as follows: ( 1) seed cotton buried under 2 inches of soil with no spore treatment: 112) seed cotton treated with I2 0% of ,5. tlmringiensis spores/cage (2.6 x 10’” spores/cage) and buried under 2 inches of !iOl ‘1; and (3) seed cotton buried undc~r .2 inches of soil that had been treated with 2 lb of the spore material/cage (68 X 10’” spores/cage). The potential adlIlt population, 431 larvae/cage, was bast~l on examination of IS;/,-lb samples. Plate counts were made to determine the number of viable spores/gram of soil (treatment 3 above) at the beginning (March 2) and end (June 21) of the test period as follows: About 50 g of surface soil was collected from each replicate, then 1 g from each collection was placed in sterile distilled water, pasteurized, and decimally diluted to obtain colonies in thca range of 3@-300/plate. All dilutions were plated in tryptose-phosphate-agar medium an d incubated for lS-24 hours at 28” -+ 1°C. Results are presented in Table 5. Moth emergence from cages containing treated seed cotton or treated soil \vas significantlv less (about 45% ) than from
Number of living pink hollworms
_----
BOLLWORM
E:).
4
EFFECTS OF SPORES OF B.thuringiensis ON STTHTIT.\L OF PINK BOLLWORMS
Treatment of soila
PINK
soil*
0 Cotton treated with 2.6 X 11F2 B. thuri?hgiPnvis 6 Soil treated with 68.0 X 1W spores/cage.
spores/cage.
CAGE
of
TESTS,
1!)61
Viable
~porcs of surface soil)
(x w/p
394
ICNOFFO
AND
cages containing untreated cotton and untreated soil (Table 6). Also, plate counts of viable spores in surface soil (Table 5) indicated that spores were not transferred from buried spore-treated cotton to the soil surface and that the spore count in treated soil was reduced only 1 log from 1.17 X lo* to 1.17 X lo7 spores/g of soil in 4 months despite a 7.28-inch rainfall total. Thus the initial level of control could be maintained by smaller, additional quantities of spores applied to the same area. For example, the initial application of 68.0 x 1013 spores/cage had raised the background spore count by about three logs, but only a l-log addition would be necessary to maintain this level. 1962 Test The procedures were the same used in 1961 tests. However, for the check, 5 lb of seed cotton estimated to contain 30 larvae/lb was buried under 2 inches of soil. Also 1, 2, 4, 8, or 16 oz of dust/cage (1 X 10” spores/g) was worked into the 2 inches of soil covering the seed cotton, TABLE
6
EMERGENCE OF PINK-BOLLWORM ADULTS FROM SOIL AND SEED COTTON TREATED WITH SPORES OF B. thuringiensis
Number of spores (X
Treatments 1961 Check Treated Treated
(no treatment) seed cotton soil
(no treatment) Treated soil [spores (oz)/cagel 1 e 4 8 16
Test 0
105.8 59.2 58.2
0
176.1
“2,600 68,000 1962
(‘heck
109)
/cage
Mean number of moths emerging per cage
Test
28 57 113 %?a7 454
“00.0 “06.0 184.6 119.8
107.0
GRAHAM
Thus spore levels ranged from 0 to 450 billion spores/cage. Five replicates were made of each treatment. The results are shown in Table 6. When the no-spore treatment was included in the analysis, the unexplained low survival in two untreated cages resulted in no statistically significant differences among treatments. However, when the no-spore treatment was excluded, applications of 8 and 16 oz of spores/cage caused significantly lower moth emergence than the other treatments at the 1% probability level, a reduction of more than 40%. The rates of application necessary to achieve 40% control (2420 and 4840 lb/ acre) would probably be uneconomical unless only one subsequent log dose (2.4 + 4.8 lb/acre) would be needed.
REFERENCES ADKISSON, P. L., VANDEHZANT, E. S., BULL, D. L., AND ALLISON, W. E. 1960. A wheat germ medium for rearing the pink bollworm. J. Econ. Entomol., 53, 759-762. CHAPMAN, A. J., NOBLE, L. W., ROBERTSON, 0. T., AND FIFE, L. C. 1960. Survival of the pink bollworm under various cultural and climatic conditions. U.S. Dept. Agr., Agr. Res. Sero., Prod. Res. Rept. No. 34, 21 pp. IGNOFFO, C. M. 1962a. The susceptibility of Pectinophora gossypiella (Saunders) to intrahemocoelic injections of Bacillus thuringiensis Berliner. J. Insect Pathol., 4, 34-40. IGNOFFO, C. M. 1962b. The effects of temperature and humidity on mortality of Pectinophoru gossypiellu (Saunders) with Bacillus thuringiensis Berliner. J. insect Pathol., 4, 63-71. IGNOFFO, C. M. 1964. Effects of temperature and water on viability and virulence of Bacihs thuringiensis var. thuringiensis Berliner. Entomophaga, Mem. No. 2, 293-298. MARTIN, D. F., AND LEWIS, R. ,D., eds. 1962. A summary of recent research basic to the cultural control of pink bollworm. Texas Agr. Expt. Sta. Misc. Publ. 579, 28 pp. OUYE, M. T. 1962. Effects of antimicrobial agents on micro-organisms and pink bollworm development. J. Econ. Entomol., 55, 854-857. SHILLER, I. 1946. A hibernation cage for the pink bollworm. U.S. BUT. Entomol. Plunt f&art. ET-226, 6 pp.
OF
INVERTEBRATE
Laboratory
PATHOLOGY
9,
and Field Against
390-394
(
1967)
Cage Tests with Pink-Bollworm
C. M. IGNOFFO~ AND H. M. Entomology U.S.
Research Department
Bacillus Larvae’
thuringiensis
GRAHAM
Division, Agricultural Research Service, of Agriculture, Brownsville, Texas
Accepted
September
15,
1966
Newly hatched and mature larvae of the pink bollwonn, Pectinophora gossypiella, were susceptible to spores of Bacillus thuringiensis in lactose dusts and in soil-spore mixtures, respectively. The pupation rate of survivors exposed to the spores as newly hatched larvae was inhibited, and pupal weights decreased with increases in concentration of spores. Application of spores to soil in field cages reduced overwintering populations of pink bollworms about 45%.
Control of the pink bollworm, Pectinophora gossypiella, with conventional insecticides is difficult because the larvae are exposed only as first instars before they enter fruiting forms or after they leave the fruiting form as last instars to pupate in the soil or surface debris. The pests overwinter as last-instar diapausing larvae in cottonseed or in cocoons in the soil and leave these hibernating sites in the spring to pupate near the surface of the soil. Such cultural control measures as shredding of stalks and plowing under of crop debris kill many overwintering larvae (Chapman et al, 1960; Martin and Lewis, 1962), but methods of increasing this larval mortality without use of chemical insecticides would be desirable. Previous laboratory tests demonstrated that both diapausing and nondiapausing pink-bollworm larvae were susceptible to a microbial insecticide containing Bacillus 1 In cooperation with the Texas Agricultural Experiment Station, Texas A&M University. ’ NOW with International Minerals and Chemical Corporation, Bioferm Division, Wasco, California.
thuringiensis ( see Ignoffo, 1962a), and that the usual temperatures and humidities in the field should not adversely affect spore stability and virulence (Ignoffo, 1962b; 1964). Additional laboratory and field tests were therefore conducted to determine whether first-instar larvae could be controlled with simulated foliar applications of spores and whether mature diapausing and nondiapausing larvae could be controlled by applications of the spores to the soil. LABORATORY
TESTS
Newly Hatched Larvae Exposed to Spores in Lactose Dusts Tests were conducted at Brownville, Texas, in 1961 to determine the effects of B. thuringiensis on first-instar pinkbollworm larvae. Thuricide (Bioferm preparation S9-517),3 a spore concentrate containing 1 X loll spores/g, was diluted with lactose to form dust mixtures containing 3 Mention of trade names imply endorsement by the Agriculture.
does not necessarily U.S. Department of
TESTS
OF
&Xi&is
AGAINST
1 X lOi, 5 X 107, 10 X 107, 50 X 107, and 100 X 10’ spores/g. One-gram samples of each of the five concentrations and a lactose dust without spores were placed in each of six 12-dram vials ( Table 1) with about 100 newly hatched first-instar larvae. The vials were stoppered, rolled gently for 30 seconds to ensure contact of the larvae with the dust, and then placed in an upright position. (Only larvae which migrated to the lips of the vials within 15 minutes of exposure were used in the tests.) Each treatment was replicated three times. At least 20 treated larvae from each of the six treatments of each replicate were then placed individually in 2-dram vials containing synthetic pinkbollworm diet (Adkisson et al.. 1960; Ouye, 1962) and reared at 29°C. Numbers of survivors, weight of pupae, pupation rate, and adult emergence were recorded. Also, more than 20 larvae were used at the higher concentrations of spores to ensure a sufficient sample of pupae for the studies of weight and adult emergence. Analysis of variance and Duncan’s multiple range test were used to compare the differences between means. The mortality of first-instar larvae exposed to spores of B. thuringiensis increased with increases in the concentration
PINK
BOLLWORM
391
of spores (Table 1). The LD,,, for larvae. was estimated at 25 X lo7 spores/g. Rates of pupation was influenced by exposun’ to spore dusts; at 17 days after exposurtb. pupation had hardly begun for larvae cxposed to the 100 X lo7 spores/g though 95% of the larvae in the check had ~IIpated by that time; also 50% of the untreated larvae pupated 8 days sooner thall 50% of the larvae receiving the highest concentration (Table 2). Pupal weight dccreased as the concentration of spores increased (Table 1). The weights of pupal-: treated with S, 10, 50, and 100 >(: 10; spores/g were significantly different from each other. Ifowever, pupal weights did not affect rclosion.
Tests were conducted in 1961 to detrrmine whether spores of B. thwingiensis could bc transmitted to diapausing fourthinstar pink-bollworm larvae exposed to spores mixed with soil. Eighteen grams of a spore-soil mixture obtained by seriallv diluting an original spore-soil sample WIItaining 18 g of air-dried top soil and 2 2 of spores (7 X lo!’ spores/g) were placczd in petri dishes. The original spore-soil sa111ple contained 7 >( lo!’ spores/g (126 ‘Y’ 10”
Pupae J,arvae surviving (averages)
(X lf)‘/d
Percent
0
Numbera
1
93 92
19 a 18 a
5
8s
10 50
68 32
18 a 14 b
100
I’ ralues
“LO
with
same letter
not significantly
_
.__--~
\l’eight (ma)”
6c
-21.3 21.0 19.8 18.1 17.ld
Id
lE.O
different
at lTp
level.
- -~_.
(averages)
Number of days to 50?$ pupation
a
14
a b
14
c e
16 17 20 “-2
392
IGNOFFO
TABLE
AND
2
PERCENT PUPATION OF PINK-BOLLWORM LARVAE AFTER EXPOSURE OF FIRST-INSTAR TO VARIOUS CONCENTR.4TIONS OF B. thuringiensis IN L.4CTOBE-SPORE DUSTS Days after exposure
Number of spores x 107/g of dust 0
1
5
10
50
100
12
4
5
2
13 14
29 61
27 48
5 17
15 16
75 87 95
82 94 95
42 65 72
15 38 51
5
82
64
24
8
88 90 92
84 89 93
38 52 57
20 48
95
67 71
50 52
86
58 65
91
75
95
78 80
17 18 19 20 91 22 23 24 25 26 27 28 29 31 32 33
96
4
85 88 92 95
spores/dish); the other dilutions were 7 x lo*, 7 x 107, 7 x 106, 7 x 105, and 7 X lo4 spores/g. Then 15-25 fourth-instar pink-bollworm larvae were placed in each petri dish (replicate) and held at 29” t 1°C. The six treatments were each replicated three times. Blood smears of dead larvae were made to verify B. thuringiensis septicemia on the day that the larvae died and were removed from the dishes. The characteristic increase in mortality with increases in concentration of spores was obtained (Table 3). However, no significant mortality occurred at concentrations of 7 X lo6 spores/g of soil or less. Mortality from septicemia was probably the result of spore entry through mechanical breaks in the body wall and subsequent spore germination and vegetative cell growth. In another test, mature larvae were
GRAHAM
pricked on the dorsal surface of the segment immediately behind the prothorax and dusted with a camel’s hair brush containing spores of B. thuringiensis. Of 15 larvae treated, 12 died within 72 hours with positive septicemia. Of 15 check larvae treated in the same manner but not dusted with spores, 13 pupated and 2 died from the injury. Nondiapausing, fourth-instar larvae which had recently emerged from firm, green field-infested bolls (collected between August 31 and September 16) were exposed to soil treated with spores in cages made of 1 lb, 5inch-diameter coffee cans. The following treatments were used: ( 1) untreated soil; (2) top soil dusted on the surface with 500 mg of 6 X lOlo spores/g; and (3) 500 mg of 6 X lOlo spores/g thoroughly mixed with 100 g of soil and then spread in a i/s-inch layer over 2 inches of untreated soil. In all three treatments, the soil contained 6-8 ml of water/100 g of soil. A hole was cut in the cover of the can and fitted with a bottle to trap the emerging adults. Fifty larvae/ treatment were used for the first replicate; 100 larvae/treatment were used for the other two replicates. The larvae were held at 28” -+ 1°C. The average number of living pink bollworms recovered (sum of adults, pupae, and larvae) from each treatTABLE
3
MORTALITY OF MATURE,DIAPAUSING PINK-ROLLWORM LARVAE EXPOSED TO SOILS TREATED WITH SPORES OF B. thuringiensis
Treatment (number of spores/g soil) 7 x 7 x 7 x
109 108 10’
7 x 7 x 7 x
10’ 105 104
Number of larvae treated 75 75 50 50 50 50
Percent mortality after 48 br 72 41 16 4 2 0
100 94 69 41 4 2 0
hr
TESTS
TABLE
OF
Bacillus
AGAINST
rntreated Spores drlsted on top soil Spores mixed in soil
Percent reduction
Average
Range
89.3
X3-96
-
“7 3
S-36
69
31.7
1ti-34
65
of 6 X lOto spores/g.
n 5(JO m g
ment 2-3 weeks after introduction of the larvae is shown in Table 4. Both soil treatments reduced the number of pinkbollworm larvae. FIELD
CAGE
TESTS
1961 Test Studies were conducted at Brownsville, Texas, from March 1 to June 20, 1961, in standard 3 X 3-ft pyramidal hibernation cages (Shiller, 1946) with Bakthane L-69,3 a commercial preparation of B. thuringiensis (Rohm and Haas, 75 X 10” spores/ a Mention of trade names imply endorsement 11y the Agriculture.
does U.S.
not necessarily Department of
TAB1.E STRJWAL
OF SPORES
Treatment
OF R. thuringiens;‘s
5 IN Son
Kumber of replicate treatments
IN FIELD Number
plate rounts
Check (no treatment) Treated cottona Treated soil6
1d 15 I.5
Check (no treatment) Treated cotton”
18 19 26
Treated
393
Ten cages/treatment were each stocked with 3?1, lb of infested seed cotton collected from plants grown at Torrcon, Coahuilla, Mexico. Treatments were as follows: ( 1) seed cotton buried under 2 inches of soil with no spore treatment: 112) seed cotton treated with I2 0% of ,5. tlmringiensis spores/cage (2.6 x 10’” spores/cage) and buried under 2 inches of !iOl ‘1; and (3) seed cotton buried undc~r .2 inches of soil that had been treated with 2 lb of the spore material/cage (68 X 10’” spores/cage). The potential adlIlt population, 431 larvae/cage, was bast~l on examination of IS;/,-lb samples. Plate counts were made to determine the number of viable spores/gram of soil (treatment 3 above) at the beginning (March 2) and end (June 21) of the test period as follows: About 50 g of surface soil was collected from each replicate, then 1 g from each collection was placed in sterile distilled water, pasteurized, and decimally diluted to obtain colonies in thca range of 3@-300/plate. All dilutions were plated in tryptose-phosphate-agar medium an d incubated for lS-24 hours at 28” -+ 1°C. Results are presented in Table 5. Moth emergence from cages containing treated seed cotton or treated soil \vas significantlv less (about 45% ) than from
Number of living pink hollworms
_----
BOLLWORM
E:).
4
EFFECTS OF SPORES OF B.thuringiensis ON STTHTIT.\L OF PINK BOLLWORMS
Treatment of soila
PINK
soil*
0 Cotton treated with 2.6 X 11F2 B. thuri?hgiPnvis 6 Soil treated with 68.0 X 1W spores/cage.
spores/cage.
CAGE
of
TESTS,
1!)61
Viable
~porcs of surface soil)
(x w/p
394
ICNOFFO
AND
cages containing untreated cotton and untreated soil (Table 6). Also, plate counts of viable spores in surface soil (Table 5) indicated that spores were not transferred from buried spore-treated cotton to the soil surface and that the spore count in treated soil was reduced only 1 log from 1.17 X lo* to 1.17 X lo7 spores/g of soil in 4 months despite a 7.28-inch rainfall total. Thus the initial level of control could be maintained by smaller, additional quantities of spores applied to the same area. For example, the initial application of 68.0 x 1013 spores/cage had raised the background spore count by about three logs, but only a l-log addition would be necessary to maintain this level. 1962 Test The procedures were the same used in 1961 tests. However, for the check, 5 lb of seed cotton estimated to contain 30 larvae/lb was buried under 2 inches of soil. Also 1, 2, 4, 8, or 16 oz of dust/cage (1 X 10” spores/g) was worked into the 2 inches of soil covering the seed cotton, TABLE
6
EMERGENCE OF PINK-BOLLWORM ADULTS FROM SOIL AND SEED COTTON TREATED WITH SPORES OF B. thuringiensis
Number of spores (X
Treatments 1961 Check Treated Treated
(no treatment) seed cotton soil
(no treatment) Treated soil [spores (oz)/cagel 1 e 4 8 16
Test 0
105.8 59.2 58.2
0
176.1
“2,600 68,000 1962
(‘heck
109)
/cage
Mean number of moths emerging per cage
Test
28 57 113 %?a7 454
“00.0 “06.0 184.6 119.8
107.0
GRAHAM
Thus spore levels ranged from 0 to 450 billion spores/cage. Five replicates were made of each treatment. The results are shown in Table 6. When the no-spore treatment was included in the analysis, the unexplained low survival in two untreated cages resulted in no statistically significant differences among treatments. However, when the no-spore treatment was excluded, applications of 8 and 16 oz of spores/cage caused significantly lower moth emergence than the other treatments at the 1% probability level, a reduction of more than 40%. The rates of application necessary to achieve 40% control (2420 and 4840 lb/ acre) would probably be uneconomical unless only one subsequent log dose (2.4 + 4.8 lb/acre) would be needed.
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