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Comparative susceptibility in the laboratory of larval instars of Trichoplusia ni and Pieris rapae to Bacillus thuringiensis var. thuringiensis
JOURNAL
OF
INVERTEBRATE:
Comparative Trichoplusia
PATHOLOGY
12, M-181
( 1968)
Susceptibility in tbe Laboratory ni and Pieris rapae to Bacillus thuringiemis 1
of Larval Instars of thuringiensis var.
JAMES V. BELL AND C. S. CREIGHTON Entomology Research Divtiion, Agricultural Research Service, U. S. Department of Agriculture, Charleston, Sou,th Carolina 29407 Received February 6, 1968 The comparative pathogenicity between four commercial preparations of Bacillus thudngiensis var. thuringiensis and their laboratory-cultured counterparts was determined by feeding the materials on foliage to all instars of the cabbage looper, Trichoplwia ni, and the imported cabbageworm, Pieris rapae. Mortality counts were recorded daily up to 72 hr. Two of the commercial products were as effective against the imported cabbageworm as their laboratory cultures, but the other two were consistently less effective. Against the cabbage looper, the differences between the commercial products and their laboratory cultures were inconsistent up to 48 hr, but after 72 hr, the laboratory cultures were consistently more effective. When each of the five larval instars of both species was allowed a choice between treated and untreated foliage for 72 hr, the daily mortality of first-instar larvae of both species was similar. The daily mortality of second-instar larvae of each species was inconsistent, but that of the third- to fifth-instar cabbageworm larvae was higher, All instars of cabbageworms that were allowed a feeding choice had similar mortalities after 24 hr, but after they fed for 48 to 72 hr. the mortality was highest among the fourth and fifth instars; in contrast, mortality of the first and second instars of cabbage-looper larvae after 24 hr was similar, but that of all instars after 48 and 72 hr was erratic (no trend toward increased mortality among older instars as in cabbageworms). When larvae were fed only treated foliage, the mortality between all instars of cabbageworms was similar after each daily observation period, but mortality between instars of the cabbage looper was similar only after 72 hr. Furthermore, when each instar of both species was compared, the percentage mortality was the same after the 72-hr feeding period.
The southeastern United States produces a considerable quantity of cole crops, and in some sections two crops a year can be harvested. However, plants such as cabbage have leaves that expand quickly and mature at a rapid rate, therefore, treatments with chemicals or pathogens by conventional means often do not cover plant surfaces thoroughly and sometimes remain 1 In cooperation with the South Carolina cultural Experiment Station.
Agri180
effective for only short periods. Also, the heavy rainfall that may occur in this area during the growing season sometimes continues long enough to delay scheduled treatment applications. Consequently, when high temperatures occur, large populations of insects can build up rapidly and cause severe damage if they are not quickly controlled, especially lepidopterous pests such as the cabbage looper, Trichoplusiu ni, that are most resistant to chemicals in the late larval stages (Kerr and Rrazzel, 19sO).
B.
thUTingienS&
AGAINST CABBAGE INSECTS
The cabbage looper, which is a major pest through wide geographical areas, has a broad host range, is often unpredictable, and is sometimes not efficiently controlled by registered chemical insecticides or pathogens (Chalfant and Brett, 1965; Grigarick and Tanada, 1959; Hall et al., 1961; Schuster, 1966; Semel, 1961). In field testing of two noncruciferous crops in California, Shorey and Hall ( 1962, 1963), controlled the cabbage looper with dusts of Bacillus thuringiensis var. thuringiensis. The imported cabbageworm, Pieris rapae, which is often found in similar environments and on the same crops as the cabbage looper, is effectively controlled by 9 variety of materials including B. t1~urir~giensi.s(Creighton et al., 1964; McEwen and Hervey, 1959; McEwen et al., 1969; Tanada, 1956). Standardized laboratory experiments were therefore conducted at Charleston, South Carolina during 1966-1967 to explore possible reasons for the difficulties that often occur in controlling the cabbage looper, and an explanation for the relative ease with which populations of the imported cabbageworm are reduced with B. thuringiensis. Although the results reported here are not always consistent with field observations, we hope they will provide data that might aid in controlling the cabbage looper in the field. MATERIALS
AND METHODS
Bacterial Formulations. Ten digerent preparations of Bacillus thuringiensis were used. Six fresh laboratory cultures were grown from five wettable powders and one liquid suspension supplied by commercial companies; however, only three commercial wettable powders and the liquid suspension were in ample supply for comparative testing. The three wettable powders were each suspended in water equivalent to a field application of 8 lb. in 50 gal. of water/acre; and the com-
lS1
mercial liquid suspension was made up equivalent to 4 quarts/acre. Then l-inchdiameter disks of sterilized cabbage or collard foliage cut with a No, 17 cork-borer tool were immersed in the suspensions. Laboratory cultures were grown by placing small amounts of six commercial preparations onto tryptose phosphate agar culture plates. The resultant bacterial colonies were given 7-14 days to grow away from the carrier materials, and the peripheral portion, free of the inert materials, was then painted by brush on the sterilized foliage disks. Larual Instars. Insectary-reared cabbagelooper and imported cabbageworm larvae were used. All originated from parental stock collected in South Carolina fields and fed sterilized field-grown cabbage or collards during captivity. The newly hatched larvae were separated daily and maintained in the rearing room at 75” -t 3°F and a relative humidity of 50% t 3% until the desired instar appeared. Separate tests of the susceptibility of the instars were made between the two species (interspecific) and within each species (intraspecific). Treatment. The larvae were tested in standard lOO-mm petri plates which were lined in the bottom with a circle of Whatman No. 3 filter paper (g-cm diam) that had been numbered from 1 to 4 at equal distances around the periphery. Each filter paper was moistened by adding 2.5 ml of distilled water just before the test began; thus the foliage was kept moist and palatable during the full 72 hr of the test. Each of the 10 bacterial preparations were assayed against 20 larvae placed in two replicated petri plates of 10 larvae each, and the untreated controls included 20 1arvae fed unsterilized foliage and 20 fed sterilized foliage disks, All instars of both insect species were compared by using 240 larvae for each instar tested, and the treat-
182
BELL
AND
ments included conditions of choice and no choice feeding. In choice testing, half the number of foliage disks fed to larvae were coated on both sides with the bacterial material, and these were placed on the filter paper numbered positions 2 and 4; the remaining untreated foliage disks were then placed on positions 1 and 3. Thus, when larvae were introduced into the center of the petri plate, they could migrate to the foliage of their choice. In no-choice testing, all foliage disks were treated and the larvae could not select their diet. For the first and second instars, four single foliage disks were quantitatively sufficient to feed the 10 larvae in each replicate; larger larvae were provided with disks stacked two or three high. Mortality was determined after 24,48, and 72 hr of feeding. The laboratory was maintained at a constant temperature of 75” -I- 5°F and a relative humidity of 50% + 5% during the tests.
Postmortem
Examination.
Cadavers were examined with an oil immersion microscope after acetocarmine-stained smears were made to clear the mounts and facilitate observations. Usually satisfactory microscopic slides could be made by pulling the larvae apart with two forceps and examining the strungout gut portion. The occurrence of numerous characteristic rods was considered adequate evidence of the bacterial infection. RESULTS
Comparison
AND
DEKXJSSION
of Bacterial Preparations
Results of 24 comparisons comprised of four commercial preparations of B. thuringie&.s (A, B, C, and D), and their laboratory-grown counterparts which were compared daily for 72 hr are shown in Table 1. When significant differences existed, the data indicated that the highest mortality occurred among larvae that fed on laboratory-grown cultures. Laboratory cultures were responsible for a higher
CRJZIGHToN
mortality of cabbage loopers in nine comparisons and for imported cabbageworms in six comparisons; the remaining nine comparisons disclosed no significant difference in mortality between the commercial and laboratory materials for either species. Against the imported cabbageworm, commercial products A and D were as effective as their laboratory counterparts; commercial materials B and C were less effective. Against the cabbage looper, mortality was erratic during 48 hr of feeding, but after 72 hr, the four commercial preparations were less effective than their laboratory-grown counterparts. The greater effectiveness of the laboratory-grown cultures may be explained by one or more of the following factors: (a) Laboratory cultures may have herent qualities which are diminished commercial formulation methods.
inby
(b) Laboratory cultures contain no inert materials that may reduce maximum ingestion of infective units. (c) Laboratory cultures lack added ingredients that might interfere, repel, or otherwise alter the normal feeding habits of larvae. (d) Laboratory cultures of bacteria are mucilaginous and therefore stick readily and spread uniformly.
Comparative
Mortality
of Instars
Table 2 shows the comparative mortality of each instar when both species were given a choice between treated and untreated foliage or no choice of treated foliage only. After 24 hr of choice feeding by cabbage loopers, the first and second instars died in greater numbers than later instars, but the mortality between instars was erratic after 48 and 72 hr. With imposed no-choice feeding, the first- through fifth-instar cabbage-looper mortality was erratic during the 24- and 48-hr feeding periods, but after 72 hr all instars had the
1
0.0 3.0
41.0 * 18.0 * 52.5 *
12.9
0.0 2.5
11.5 57.0
T. ni P. rapae
T. ni P. rapaci
58.4 72.5
a Percentages based on 100 larvae (20 larvae for each of five instars). + Significant at the 5% level of confidence. **Significant at the 1% level of confidence, based on Student’s “t” test.
25.2 * 57.5
33.1 Mated
14.0
0.4
T. ni P. rapae
Material
Material 2.9
Material
Commercial pathogen
D
C
B
A
48 hr
mortality a at indicated
51.8 66.5
Laboratorygrown pathogen
larval
28.9 37.9
14.3 39.5
T. ni P. mpae
Insect
Commercial pathogen
24 hr
TABLE PREPARATIONS
P. rapae
69.1 79.0
48.0 ** 79.5 **
54.0 ** 74.0 **
82.3 ** 69.7
Laboratorygrown pathogen
hour of feeding-
OF COM~RCUU. AND LABORATORY-GROWN thuringiensk AGAINST LARVAE OF T. ni AND
Percentage
B.
PATHOGENICITY
66.8 80.5
1.0 16.5
15.4 54.5
62.6 77.5
Commercial pathogen
OF
72 hr
83.5 * 84.0
66.5 ** 83.5 **
71.4 ** 80.0 **
91.6** 80.6
-.Laboratorygrown pathogen
-- -.-
?
E z 2
I84
BELL
AND
CREIGHTON
TABLE MORTALITIES
Insect and instar
OF CABBAGE-LOOPER AND FOLMGE TREATED Choice feeding % Mortality a after 48
24
2
IMPORTED CABBAGEWORM WITH B. thuringierwis
LARVAE
FED
ON
No-choice feeding % Mortality Q after (hr)-
( hr)72
24
72
48
T. ni 1 2 3 4 5
14.1 14.0 5.2 10.9 0.5
a a ab a b
33.9 42.5 20.0 35.3 22.5
ab a b ab b
43.6 55.5 30.1 52.7 34.5
ab a b a b
32.0 14.0 51.5 5.5 5.5
b bc a c c
72.0 46.5 77.5 57.5 74.0
ab c a bc a
76.0 78.5 80.0 74.0 82.0
a a a a a
26.0 20.0 25.5 27.0 25.0
a a a a a
41.5 31.0 36.5 57.5 67.0
b b b a a
43.0 33.5 58.5 71.0 78.0
c c b a a
52.4 64.0 53.3 53.5 49.5
a a a a a
80.3 80.0 74.6 80.0 81.5
a a a a a
91.5 82.0 89.1 89.5 86.0
a a a a A
P. rapae 1 2 3 4 5 a Mean significantly
percentage different
mortality based at the 5% level
on 20 larvae/instar; values followed by different of confidence according to Duncan’s multiple range
same percentage mortality. In choice feeding, all imported cabbageworm instars died in equal numbers during the first 24-hr period; however, after 48 and 72 hr, the highest mortality was among fourth and fifth instars. Cabbageworms responded to no-choice feeding with a similar mortality for all five instars within each time period. In Table 3, choice and no-choice cabbage-looper mortalities were inconsistent for 24 and 48 hr of feeding, but after 72 hr, greatest mortalities occurred from nochoice feeding among all five instars; however imported cabbageworm comparisons disclosed that the first four instars died in greatest numbers at all time periods, but that fifth instars showed no differences in mortality. Table 4 shows that when cabbage loopers and cabbageworms were allowed choice feeding, equal numbers of iirst instars died in both species after feeding for 24, 48, or 72 hr, the mortality of second-instar larvae of both species was erratic, and third-, fourth-, and fifth-instar cabbage-
letters test.
are
worms always exceeded the mortality of cabbage loopers. After 72 hr of feeding on treated foliage only, the mortality of each instar of both species was the same. Thus, for reliable laboratory results with cabbage-looper larvae, more than 48 hr of feeding is necessary; reliable data for the imported cabbageworm can be obtained after 24 hr of feeding. The cabbage looper may therefore be more tolerant to or more repelled by the bacterial preparation. If a hereditary resistance is involved or if insect sensory stimuli are present that cause repulsion to the bacterium, then more thorough coverage or augmented applications may be necessary for satisfactory plant protection as the leaf surfaces expand. However, observations in the field indicate that the cabbageworm is often found on the upper, more exposed portions of the plant where pathogenic materials are most likely to be applied; the cabbage looper is usually found beneath the leaf surfaces. Then, since the extent of coverage, particularly by microbials which must be ingested,
rapae
FEEDING
22.5 67.0
5th
35.3 57.5
4th
20.0 36.5
3rd
42.5 31.0
2nd
33.9 41.5
Instar
Znstar
Instar
lnstar
1st Instar
Choice
u Mean percentage based on 20 larvae. * Significant at the 5% level of confidence. **Significant at the 1% level of confidence based on Strident’s “t” test.
P. rapae
T. ni
P.
5.5 49.5
10.9
T. ni
0.5 25.0
51.5 ** 53.3 **
5.2 25.5
P. mpae
27.0
14.0 64.0 **
14.0 20.0
T. ni P. mpae
5.5 53.5 **
32.0 52.4 ** **
14.1 26.0
T. ni P. rapae
ni
No-choice
T.
3
LOOPERS
TABLE CABBAGE
AND NO-CHOICE
OF ON
AND
IASPORTED
74.0 ** 81.5
57.5 ** 80.0 **
77.5 ** 74.6 **
46.5 80.0 +*
72.0 80.3 ** **
No-choice
hour-
B. thuringiensis
Percentage larval mortality a at indicated 48 hr
CHOICE
Choice
24 hr
AFTER
MORTALITY
insect
Instar and
INTRASPECIFIC
CABBAGEWORMS
34.5 78.0
52.7 71.0
30.1 58.5
55.5 33.5
43.6 43.0
Choice
72 hr
86.0
82.0*4:
74.0 e*: 89.5 **
go.0 ** 89.1 **
78.5 ** 82.0 **
76.0 *1; *;I: 91.5
No-choice
2 h
E !?
w’
g
3.
F: 3.
P B
4
20.0 64.0 **
25.5 ** 53.3
27.0 * 53.5 **
25.0 ** 49.5 **
14.0 14.0
5.2 51.5
10.9 5.5
0.5 5.5
Choice No choice
Choice No choice
Choice No choice
Choice No choice
22.5 74.0
5th Znstar
35.3 57.5
4th Instar
20.0 77.5
3rd Znstar
42.5 46.5
2nd Instar
33.9 72.0
1st Instar
T. ni
48 hr
hour-
67.0 ** 81.5
57.5 * 80.0 *
36.5 * 74.6
31.0 80.0 **
41.5 80.3
P. mpae
larval mortality a at indicated
a Mean percentage based on 20 larvae. *Significant at the 5% level of confidence. **Sign&ant at the 1% level of confidence based on Student’s “t” test.
26.0 52.4 *
14.1 32.0
P. rapaf3
Choice No choice
24 hr
T. ni
Instar and treatment
Percentage
34.5 82.0
52.7 74.0
30.1 80.0
55.5 * 78.5
43.6 76.0
T. ni
INTERSPECIFIC SUSCEPTIBILITY OF CABBAGE-LOOPER AND IMPORTED CABBAGEWORM LARVAE TO FEEDING ON B. thuringknsk
TABLE
72 hr
78.0 ** 86.0
71.0 * 89.5
58.5 ** 89.1
33.5 82.0
43.0 91.5
P. rapae
B. thuringimsis AGAINST CABBAGE INSECTIS is of great importance in suppressing insect populations, we may perhaps need new techniques of application or a new design of crop planting, so the spray or dust more adequateIy covers the under surfaces of leaves of such plants as cabbage and other leafy vegetables. Also, the effectiveness of pathogens that are applied to the undersides of leaves is enhanced by the high relative humidity in that locality, and by the partial protection that is afforded from sun, wind, and rain. ACKNOWLEDGMENTS
The authors wish to thank Dr. Frank E. Giles, Robert J. Hamalle, T. L. McFadden, Mrs. E. L. Rice and Mrs. E. D. Welch, all of the U. S. Department of Agriculture, Entomology Research Division, Charleston, South Carolina, who assisted in this study. REFERENCES R. B., AND BRETT, C. H. 1965. Cabbage loopers and imported cabbageworms; feeding damage and control on cabbage in Western North Carolina. J. Econ. Entomol., 58, 28-33. CREIGHTON, C. S., CUTHBERT, JR., F. P., AND REID, JR., W. J. 1964. Evaluation of Bacillus thudngiensb var. thuringiensis Berliner in control of caterpillars on cabbage. J. Insect Pathol., 6, 102-110. CRIGARICK, A. A., AND TANADA, Y. 1959. A field test for the control of Trichoplusiu ni (Hubn. ) on celery with several insecticides and CHALFANT,
187
Bacillus thuringiends Berliner. J. Econ. Entomol., 52, 1013-1014. HALL, I. M., HALE, R. L., SHOREY, H. H., AND ARAKAWA, K. Y. 1961. Evaluation of chemical and microbial materials for control of the cabbage Iooper. .J. Econ. Entomol., 54, 141146. KERR, W. P., AND BRAZZEL, J. R. 1960. Laboratory tests of insecticides against eggs and larvae of the cabbage looper. I. Econ. Entomol., 53, 991-992. MCEWEN, F. L., AND HERVEY, G. E. R. 1959. Microbial control of two cabbage insects. I. Insect Pathol., 1, 86-94. MCEWEN, F. L., GLASS, E. H., DAVIS, H. C., AND SPLITTSTOESSER, C. M. 1960. Field tests with Bacillus thuringiensis Berliner for control of four lepidopterous pests. 1. Insect Pathol., 2, 152-164. SCHUSTER, M. F. 1966. Insecticide and microbial dusts and application intervals for control of lettuce insects. J. Econ. Entomol., 59, 469471. SEMEL, M. 1961. The efficiency of a polyhedrosis virus and Bacillus thuringiensis for control of the cabbage looper on cauliflower. I. Econ. Entomol., 54, 698-701. SHOREY, H. H., AND HALL, I. M. 1962. Effect of chemical and microbial insecticides on several insect pests of lettuce in southern California. I. Econ. Entomol., 55, 169-174. SHOREY, H. H., AND HALL, I. M. 1963. Toxici@ of chemical and microbial insecticides to pest and beneficial insects on poled tomatoes. J. Econ. Entomol., 56, 8X3-817. TANADA, Y. 1956. Microbial control of some lepidopterous pests of crucifers. I. Econ. Entomol., 49, 320-329.
OF
INVERTEBRATE:
Comparative Trichoplusia
PATHOLOGY
12, M-181
( 1968)
Susceptibility in tbe Laboratory ni and Pieris rapae to Bacillus thuringiemis 1
of Larval Instars of thuringiensis var.
JAMES V. BELL AND C. S. CREIGHTON Entomology Research Divtiion, Agricultural Research Service, U. S. Department of Agriculture, Charleston, Sou,th Carolina 29407 Received February 6, 1968 The comparative pathogenicity between four commercial preparations of Bacillus thudngiensis var. thuringiensis and their laboratory-cultured counterparts was determined by feeding the materials on foliage to all instars of the cabbage looper, Trichoplwia ni, and the imported cabbageworm, Pieris rapae. Mortality counts were recorded daily up to 72 hr. Two of the commercial products were as effective against the imported cabbageworm as their laboratory cultures, but the other two were consistently less effective. Against the cabbage looper, the differences between the commercial products and their laboratory cultures were inconsistent up to 48 hr, but after 72 hr, the laboratory cultures were consistently more effective. When each of the five larval instars of both species was allowed a choice between treated and untreated foliage for 72 hr, the daily mortality of first-instar larvae of both species was similar. The daily mortality of second-instar larvae of each species was inconsistent, but that of the third- to fifth-instar cabbageworm larvae was higher, All instars of cabbageworms that were allowed a feeding choice had similar mortalities after 24 hr, but after they fed for 48 to 72 hr. the mortality was highest among the fourth and fifth instars; in contrast, mortality of the first and second instars of cabbage-looper larvae after 24 hr was similar, but that of all instars after 48 and 72 hr was erratic (no trend toward increased mortality among older instars as in cabbageworms). When larvae were fed only treated foliage, the mortality between all instars of cabbageworms was similar after each daily observation period, but mortality between instars of the cabbage looper was similar only after 72 hr. Furthermore, when each instar of both species was compared, the percentage mortality was the same after the 72-hr feeding period.
The southeastern United States produces a considerable quantity of cole crops, and in some sections two crops a year can be harvested. However, plants such as cabbage have leaves that expand quickly and mature at a rapid rate, therefore, treatments with chemicals or pathogens by conventional means often do not cover plant surfaces thoroughly and sometimes remain 1 In cooperation with the South Carolina cultural Experiment Station.
Agri180
effective for only short periods. Also, the heavy rainfall that may occur in this area during the growing season sometimes continues long enough to delay scheduled treatment applications. Consequently, when high temperatures occur, large populations of insects can build up rapidly and cause severe damage if they are not quickly controlled, especially lepidopterous pests such as the cabbage looper, Trichoplusiu ni, that are most resistant to chemicals in the late larval stages (Kerr and Rrazzel, 19sO).
B.
thUTingienS&
AGAINST CABBAGE INSECTS
The cabbage looper, which is a major pest through wide geographical areas, has a broad host range, is often unpredictable, and is sometimes not efficiently controlled by registered chemical insecticides or pathogens (Chalfant and Brett, 1965; Grigarick and Tanada, 1959; Hall et al., 1961; Schuster, 1966; Semel, 1961). In field testing of two noncruciferous crops in California, Shorey and Hall ( 1962, 1963), controlled the cabbage looper with dusts of Bacillus thuringiensis var. thuringiensis. The imported cabbageworm, Pieris rapae, which is often found in similar environments and on the same crops as the cabbage looper, is effectively controlled by 9 variety of materials including B. t1~urir~giensi.s(Creighton et al., 1964; McEwen and Hervey, 1959; McEwen et al., 1969; Tanada, 1956). Standardized laboratory experiments were therefore conducted at Charleston, South Carolina during 1966-1967 to explore possible reasons for the difficulties that often occur in controlling the cabbage looper, and an explanation for the relative ease with which populations of the imported cabbageworm are reduced with B. thuringiensis. Although the results reported here are not always consistent with field observations, we hope they will provide data that might aid in controlling the cabbage looper in the field. MATERIALS
AND METHODS
Bacterial Formulations. Ten digerent preparations of Bacillus thuringiensis were used. Six fresh laboratory cultures were grown from five wettable powders and one liquid suspension supplied by commercial companies; however, only three commercial wettable powders and the liquid suspension were in ample supply for comparative testing. The three wettable powders were each suspended in water equivalent to a field application of 8 lb. in 50 gal. of water/acre; and the com-
lS1
mercial liquid suspension was made up equivalent to 4 quarts/acre. Then l-inchdiameter disks of sterilized cabbage or collard foliage cut with a No, 17 cork-borer tool were immersed in the suspensions. Laboratory cultures were grown by placing small amounts of six commercial preparations onto tryptose phosphate agar culture plates. The resultant bacterial colonies were given 7-14 days to grow away from the carrier materials, and the peripheral portion, free of the inert materials, was then painted by brush on the sterilized foliage disks. Larual Instars. Insectary-reared cabbagelooper and imported cabbageworm larvae were used. All originated from parental stock collected in South Carolina fields and fed sterilized field-grown cabbage or collards during captivity. The newly hatched larvae were separated daily and maintained in the rearing room at 75” -t 3°F and a relative humidity of 50% t 3% until the desired instar appeared. Separate tests of the susceptibility of the instars were made between the two species (interspecific) and within each species (intraspecific). Treatment. The larvae were tested in standard lOO-mm petri plates which were lined in the bottom with a circle of Whatman No. 3 filter paper (g-cm diam) that had been numbered from 1 to 4 at equal distances around the periphery. Each filter paper was moistened by adding 2.5 ml of distilled water just before the test began; thus the foliage was kept moist and palatable during the full 72 hr of the test. Each of the 10 bacterial preparations were assayed against 20 larvae placed in two replicated petri plates of 10 larvae each, and the untreated controls included 20 1arvae fed unsterilized foliage and 20 fed sterilized foliage disks, All instars of both insect species were compared by using 240 larvae for each instar tested, and the treat-
182
BELL
AND
ments included conditions of choice and no choice feeding. In choice testing, half the number of foliage disks fed to larvae were coated on both sides with the bacterial material, and these were placed on the filter paper numbered positions 2 and 4; the remaining untreated foliage disks were then placed on positions 1 and 3. Thus, when larvae were introduced into the center of the petri plate, they could migrate to the foliage of their choice. In no-choice testing, all foliage disks were treated and the larvae could not select their diet. For the first and second instars, four single foliage disks were quantitatively sufficient to feed the 10 larvae in each replicate; larger larvae were provided with disks stacked two or three high. Mortality was determined after 24,48, and 72 hr of feeding. The laboratory was maintained at a constant temperature of 75” -I- 5°F and a relative humidity of 50% + 5% during the tests.
Postmortem
Examination.
Cadavers were examined with an oil immersion microscope after acetocarmine-stained smears were made to clear the mounts and facilitate observations. Usually satisfactory microscopic slides could be made by pulling the larvae apart with two forceps and examining the strungout gut portion. The occurrence of numerous characteristic rods was considered adequate evidence of the bacterial infection. RESULTS
Comparison
AND
DEKXJSSION
of Bacterial Preparations
Results of 24 comparisons comprised of four commercial preparations of B. thuringie&.s (A, B, C, and D), and their laboratory-grown counterparts which were compared daily for 72 hr are shown in Table 1. When significant differences existed, the data indicated that the highest mortality occurred among larvae that fed on laboratory-grown cultures. Laboratory cultures were responsible for a higher
CRJZIGHToN
mortality of cabbage loopers in nine comparisons and for imported cabbageworms in six comparisons; the remaining nine comparisons disclosed no significant difference in mortality between the commercial and laboratory materials for either species. Against the imported cabbageworm, commercial products A and D were as effective as their laboratory counterparts; commercial materials B and C were less effective. Against the cabbage looper, mortality was erratic during 48 hr of feeding, but after 72 hr, the four commercial preparations were less effective than their laboratory-grown counterparts. The greater effectiveness of the laboratory-grown cultures may be explained by one or more of the following factors: (a) Laboratory cultures may have herent qualities which are diminished commercial formulation methods.
inby
(b) Laboratory cultures contain no inert materials that may reduce maximum ingestion of infective units. (c) Laboratory cultures lack added ingredients that might interfere, repel, or otherwise alter the normal feeding habits of larvae. (d) Laboratory cultures of bacteria are mucilaginous and therefore stick readily and spread uniformly.
Comparative
Mortality
of Instars
Table 2 shows the comparative mortality of each instar when both species were given a choice between treated and untreated foliage or no choice of treated foliage only. After 24 hr of choice feeding by cabbage loopers, the first and second instars died in greater numbers than later instars, but the mortality between instars was erratic after 48 and 72 hr. With imposed no-choice feeding, the first- through fifth-instar cabbage-looper mortality was erratic during the 24- and 48-hr feeding periods, but after 72 hr all instars had the
1
0.0 3.0
41.0 * 18.0 * 52.5 *
12.9
0.0 2.5
11.5 57.0
T. ni P. rapae
T. ni P. rapaci
58.4 72.5
a Percentages based on 100 larvae (20 larvae for each of five instars). + Significant at the 5% level of confidence. **Significant at the 1% level of confidence, based on Student’s “t” test.
25.2 * 57.5
33.1 Mated
14.0
0.4
T. ni P. rapae
Material
Material 2.9
Material
Commercial pathogen
D
C
B
A
48 hr
mortality a at indicated
51.8 66.5
Laboratorygrown pathogen
larval
28.9 37.9
14.3 39.5
T. ni P. mpae
Insect
Commercial pathogen
24 hr
TABLE PREPARATIONS
P. rapae
69.1 79.0
48.0 ** 79.5 **
54.0 ** 74.0 **
82.3 ** 69.7
Laboratorygrown pathogen
hour of feeding-
OF COM~RCUU. AND LABORATORY-GROWN thuringiensk AGAINST LARVAE OF T. ni AND
Percentage
B.
PATHOGENICITY
66.8 80.5
1.0 16.5
15.4 54.5
62.6 77.5
Commercial pathogen
OF
72 hr
83.5 * 84.0
66.5 ** 83.5 **
71.4 ** 80.0 **
91.6** 80.6
-.Laboratorygrown pathogen
-- -.-
?
E z 2
I84
BELL
AND
CREIGHTON
TABLE MORTALITIES
Insect and instar
OF CABBAGE-LOOPER AND FOLMGE TREATED Choice feeding % Mortality a after 48
24
2
IMPORTED CABBAGEWORM WITH B. thuringierwis
LARVAE
FED
ON
No-choice feeding % Mortality Q after (hr)-
( hr)72
24
72
48
T. ni 1 2 3 4 5
14.1 14.0 5.2 10.9 0.5
a a ab a b
33.9 42.5 20.0 35.3 22.5
ab a b ab b
43.6 55.5 30.1 52.7 34.5
ab a b a b
32.0 14.0 51.5 5.5 5.5
b bc a c c
72.0 46.5 77.5 57.5 74.0
ab c a bc a
76.0 78.5 80.0 74.0 82.0
a a a a a
26.0 20.0 25.5 27.0 25.0
a a a a a
41.5 31.0 36.5 57.5 67.0
b b b a a
43.0 33.5 58.5 71.0 78.0
c c b a a
52.4 64.0 53.3 53.5 49.5
a a a a a
80.3 80.0 74.6 80.0 81.5
a a a a a
91.5 82.0 89.1 89.5 86.0
a a a a A
P. rapae 1 2 3 4 5 a Mean significantly
percentage different
mortality based at the 5% level
on 20 larvae/instar; values followed by different of confidence according to Duncan’s multiple range
same percentage mortality. In choice feeding, all imported cabbageworm instars died in equal numbers during the first 24-hr period; however, after 48 and 72 hr, the highest mortality was among fourth and fifth instars. Cabbageworms responded to no-choice feeding with a similar mortality for all five instars within each time period. In Table 3, choice and no-choice cabbage-looper mortalities were inconsistent for 24 and 48 hr of feeding, but after 72 hr, greatest mortalities occurred from nochoice feeding among all five instars; however imported cabbageworm comparisons disclosed that the first four instars died in greatest numbers at all time periods, but that fifth instars showed no differences in mortality. Table 4 shows that when cabbage loopers and cabbageworms were allowed choice feeding, equal numbers of iirst instars died in both species after feeding for 24, 48, or 72 hr, the mortality of second-instar larvae of both species was erratic, and third-, fourth-, and fifth-instar cabbage-
letters test.
are
worms always exceeded the mortality of cabbage loopers. After 72 hr of feeding on treated foliage only, the mortality of each instar of both species was the same. Thus, for reliable laboratory results with cabbage-looper larvae, more than 48 hr of feeding is necessary; reliable data for the imported cabbageworm can be obtained after 24 hr of feeding. The cabbage looper may therefore be more tolerant to or more repelled by the bacterial preparation. If a hereditary resistance is involved or if insect sensory stimuli are present that cause repulsion to the bacterium, then more thorough coverage or augmented applications may be necessary for satisfactory plant protection as the leaf surfaces expand. However, observations in the field indicate that the cabbageworm is often found on the upper, more exposed portions of the plant where pathogenic materials are most likely to be applied; the cabbage looper is usually found beneath the leaf surfaces. Then, since the extent of coverage, particularly by microbials which must be ingested,
rapae
FEEDING
22.5 67.0
5th
35.3 57.5
4th
20.0 36.5
3rd
42.5 31.0
2nd
33.9 41.5
Instar
Znstar
Instar
lnstar
1st Instar
Choice
u Mean percentage based on 20 larvae. * Significant at the 5% level of confidence. **Significant at the 1% level of confidence based on Strident’s “t” test.
P. rapae
T. ni
P.
5.5 49.5
10.9
T. ni
0.5 25.0
51.5 ** 53.3 **
5.2 25.5
P. mpae
27.0
14.0 64.0 **
14.0 20.0
T. ni P. mpae
5.5 53.5 **
32.0 52.4 ** **
14.1 26.0
T. ni P. rapae
ni
No-choice
T.
3
LOOPERS
TABLE CABBAGE
AND NO-CHOICE
OF ON
AND
IASPORTED
74.0 ** 81.5
57.5 ** 80.0 **
77.5 ** 74.6 **
46.5 80.0 +*
72.0 80.3 ** **
No-choice
hour-
B. thuringiensis
Percentage larval mortality a at indicated 48 hr
CHOICE
Choice
24 hr
AFTER
MORTALITY
insect
Instar and
INTRASPECIFIC
CABBAGEWORMS
34.5 78.0
52.7 71.0
30.1 58.5
55.5 33.5
43.6 43.0
Choice
72 hr
86.0
82.0*4:
74.0 e*: 89.5 **
go.0 ** 89.1 **
78.5 ** 82.0 **
76.0 *1; *;I: 91.5
No-choice
2 h
E !?
w’
g
3.
F: 3.
P B
4
20.0 64.0 **
25.5 ** 53.3
27.0 * 53.5 **
25.0 ** 49.5 **
14.0 14.0
5.2 51.5
10.9 5.5
0.5 5.5
Choice No choice
Choice No choice
Choice No choice
Choice No choice
22.5 74.0
5th Znstar
35.3 57.5
4th Instar
20.0 77.5
3rd Znstar
42.5 46.5
2nd Instar
33.9 72.0
1st Instar
T. ni
48 hr
hour-
67.0 ** 81.5
57.5 * 80.0 *
36.5 * 74.6
31.0 80.0 **
41.5 80.3
P. mpae
larval mortality a at indicated
a Mean percentage based on 20 larvae. *Significant at the 5% level of confidence. **Sign&ant at the 1% level of confidence based on Student’s “t” test.
26.0 52.4 *
14.1 32.0
P. rapaf3
Choice No choice
24 hr
T. ni
Instar and treatment
Percentage
34.5 82.0
52.7 74.0
30.1 80.0
55.5 * 78.5
43.6 76.0
T. ni
INTERSPECIFIC SUSCEPTIBILITY OF CABBAGE-LOOPER AND IMPORTED CABBAGEWORM LARVAE TO FEEDING ON B. thuringknsk
TABLE
72 hr
78.0 ** 86.0
71.0 * 89.5
58.5 ** 89.1
33.5 82.0
43.0 91.5
P. rapae
B. thuringimsis AGAINST CABBAGE INSECTIS is of great importance in suppressing insect populations, we may perhaps need new techniques of application or a new design of crop planting, so the spray or dust more adequateIy covers the under surfaces of leaves of such plants as cabbage and other leafy vegetables. Also, the effectiveness of pathogens that are applied to the undersides of leaves is enhanced by the high relative humidity in that locality, and by the partial protection that is afforded from sun, wind, and rain. ACKNOWLEDGMENTS
The authors wish to thank Dr. Frank E. Giles, Robert J. Hamalle, T. L. McFadden, Mrs. E. L. Rice and Mrs. E. D. Welch, all of the U. S. Department of Agriculture, Entomology Research Division, Charleston, South Carolina, who assisted in this study. REFERENCES R. B., AND BRETT, C. H. 1965. Cabbage loopers and imported cabbageworms; feeding damage and control on cabbage in Western North Carolina. J. Econ. Entomol., 58, 28-33. CREIGHTON, C. S., CUTHBERT, JR., F. P., AND REID, JR., W. J. 1964. Evaluation of Bacillus thudngiensb var. thuringiensis Berliner in control of caterpillars on cabbage. J. Insect Pathol., 6, 102-110. CRIGARICK, A. A., AND TANADA, Y. 1959. A field test for the control of Trichoplusiu ni (Hubn. ) on celery with several insecticides and CHALFANT,
187
Bacillus thuringiends Berliner. J. Econ. Entomol., 52, 1013-1014. HALL, I. M., HALE, R. L., SHOREY, H. H., AND ARAKAWA, K. Y. 1961. Evaluation of chemical and microbial materials for control of the cabbage Iooper. .J. Econ. Entomol., 54, 141146. KERR, W. P., AND BRAZZEL, J. R. 1960. Laboratory tests of insecticides against eggs and larvae of the cabbage looper. I. Econ. Entomol., 53, 991-992. MCEWEN, F. L., AND HERVEY, G. E. R. 1959. Microbial control of two cabbage insects. I. Insect Pathol., 1, 86-94. MCEWEN, F. L., GLASS, E. H., DAVIS, H. C., AND SPLITTSTOESSER, C. M. 1960. Field tests with Bacillus thuringiensis Berliner for control of four lepidopterous pests. 1. Insect Pathol., 2, 152-164. SCHUSTER, M. F. 1966. Insecticide and microbial dusts and application intervals for control of lettuce insects. J. Econ. Entomol., 59, 469471. SEMEL, M. 1961. The efficiency of a polyhedrosis virus and Bacillus thuringiensis for control of the cabbage looper on cauliflower. I. Econ. Entomol., 54, 698-701. SHOREY, H. H., AND HALL, I. M. 1962. Effect of chemical and microbial insecticides on several insect pests of lettuce in southern California. I. Econ. Entomol., 55, 169-174. SHOREY, H. H., AND HALL, I. M. 1963. Toxici@ of chemical and microbial insecticides to pest and beneficial insects on poled tomatoes. J. Econ. Entomol., 56, 8X3-817. TANADA, Y. 1956. Microbial control of some lepidopterous pests of crucifers. I. Econ. Entomol., 49, 320-329.