Comparative tests of various species of Bacillus of the “cereus Group” on larvae of Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae)

266

NOTES

examination of diseased fat-body tissues has revealed the presence of hexagonal particles, possibly icosahedra, approximately 60 rnp in diameter in the cytoplasm of the cells. The particles may be a noninclusion body type of insect virus and appear to be similar to those reported by Krieg and Huger (J. Znsect Pathol., 2, 274-288, 1960), by S’teinhausand Leutenegger (J. Insect Pathol., 5, 266-270, 1963), and by Leutenegger (Virology, 24, 200-204, 1964). No such particles were found in the healthy tissues. Huger (personal communication) has suggested that these particles may be parallels to the virus Moratorvirus lamellicornium Krieg and Huger in the fat cells of Melolontha in which there appears to be a breakdown of

Comparative of

Tests

the

JEAN

ADAMS

A.

WILCOX

Entomology Research Division Agricultural Research Service U. S. Department of Agriculture Beltsville, Maryland Accepted

Group” fumiferana

(Lepidoptera

R.

THEODORE

of Various

“cereus

Choristoneura

albuminoid sphereswith a transformation into virogenic stroma which disappear during the course of the disease (Figs. 2 and 3). The hexagonal shape of the pathogen is shown in Fig. 4. As diseased specimens become available, transmission and isolation and purification studies will be conducted.

April

12, 1965

Species

of

on Larvae

Bacillus of

(Clemens)

: Tortricidae)

’

Among the six species of entomopathogenous Bacillus of the so-called “cereus Group” (Heimpel and Angus, Bacterial. Rev., 24, 266-288, 1960), only two have been tested on the spruce budworm, Choristoneura jumijerana (Clemens): Bacillus thuringiensis var. thuringiensis Berliner (Angus et al., Can.

spraying of fir twigs with bacterial suspensions; (b) buccal administration of droplets of the Bacillus suspensionsto larvae; (c) immersion of larvae for 10 seconds in bacterial suspensions;(d) oral administration of Bacillus suspensionsto larvae reared on artificial diet (Berger, U. S. Dept. Agr. Agr. Res. Dept. Forestry, Forest Entomol. Pathol. Serv., Puhl. X3-84, 4 pp. (October 1963). The following speciesof spore- and crystalBranch, Bimonthly Progr. Rept., 17 (3) forming Bacillus were tested: B. thuringiensis 1961; Smirnoff, Can. Entomologist, 95, 127Berliner; B. thuringiensis 133, 1963) and Bacillus ceyeus Franklin and var. thuringiensis Franklin (Smirnoff, Can. Entomologist, 95, var. sotto Aoki and Chigasaki: B. cereus var. 127-133, 1963). Biological control studies of dendrolimus Talalaev; B. t huringiensis var. the spruce budworm warranted further tests alesti Toumanoff and Vago; B. entomocidus with species of Bacillus to establish their var. entomoridus Heimp and Angus; while comparative efficacy. This note reports on the B. cereus Franklin and Franklin (strain 44)” results obtained. 2 B. WY~US No. 44 was isolated by the author Four methods of infection were used: (a) from larvae of Trichiocamp~ts viminalis (Fallen) 1 Contribution No. 1204, Forest Entomology and Pathology Branch, Department of Forestry, Ottawa, Canada.

(Hymenoptera: Tenthredinidae), 1959. All varieties of Racillus were supplied by Dr. Angus, Insect Pathology Research Institute, Ste. Marie, Ontario, Canada.

other T. A. Sault

267

NOTES

PERCENTAGE

OF LARVAL

Varieties and

of Bacillus tested treatment used

B. thuringiensis Sprayed

MORTALITY

var.

foliage

Sprayed

var.

Buccal administration on foliage

Sprayed

var.

foliage

B. thuringiensis

var.

var.

50

96

4

SO

70

100

5

20

50

100

30.7

53.5

96.0

4 6.5

5

15

85

11

30

50

80

8

20

30

40

9

20

50

100

5

18.8

36.2

14

0

8;

10 5

76.2

9

50

100

70

80 100

5 -

20

SO

28.5

42.5

91.3

20

30

70

12

30

60

100

3

50

0

80

6

30

90

100

3

32.5

45.0

87.5

35

45

75

1.1

30

60

80

8

SO

80

90

6

20

70

33.7

63.7

100 -_ 86.2

5 8

6.7

6

to larvae

larvae

Oral administration on artificial diet

20 60

entomocidus:

foliage

Buccal administration on foliage

Average

20

to larvae

.i\verage

Immersed

13

44

to larvae

larvae

Oral administration on artificial diet

Sprayed

88

33

alesti:

foliage

Buccal administration on foliage

B. entomocidus

120

to larvae

Average

Immersed

48

Sumber of days for total mortality

to larvae

larvae

Oral administration on artificial diet

Sprayed

mortality hours

.-~DMINISTERED

dendrolimzrs:

Buccal administration on foliage Immersed

24

VARIOUSLY

to larvae

Average B. ceretu

Percent after

Bacillus

to larvae

larvae

Oral administration on artificial diet

OF

sotto:

foliage

Immersed

SPECIES

to larvae

Average B. thuriagiensis

1

to larvae

larvae

Oral administration on artificial diet

TABLE BY DIFFERENT

thuringiensis:

Buccal administration on foliage Immersed

CAUSED

to larvae

268

NOTES TABLE

Varieties of Bacillus tested and treatment used

1 (Continued) Percent after 24

mortality hours

Number of days for total mortality

48

120

26

0

53

14

60

70 70

70

7

30

80

9

60

70

80

7 -

44.0

52.5

70.7

9.2

33

49

82

9

30

60

80

5

50

70

100

5

30

80

100

35.7

64.7

B. cereus: Sprayed

foliage

Buccal administration on foliage Immersed

to larvae

larvae

Oral administration on artificial diet

to larvae

Average Mechanical Sprayed

mixture: foliage

Buccal administration on foliage Immersed

larvae

Oral administration on artificial diet Average

to larvae

to larvae

was the only acrystalliferous sporeforming BacilZus tested. All cultures were grown on Difco nutrient agar and incubated at 28’C for 10 days, i.e. until lysis was completed. The bacilli were collected and kept in peptonized water (0.25 g peptone/lOO ml of distilled water) at a concentration of one part of Bacillus to 10 parts of peptonized water. The concentrations of bacterial suspensions were standardized by transparency and the number of spores was estimated (2 X log) by counting with the aid of a microscope. In addition to the six pure cultures, two mixtures were also tested: (a) a mechanical mixture of suspensions from cultures of the six species grown separately; (b) a mixture of the six species grown together. In the feeding tests, 1700 fourth-instar larvae of C. jumijerana were used. These larvae were obtained from the Insect Pathology Research Institute at Sault Ste. Marie, Ontario, and were free of the native microsporidia Per&a fumiferanae Thomson. The larvae were reared individually in wellaerated glass vials, at 21-22’C and 7070 relative humidity. Dead larvae were collected

90.5

7 .s

daily and microscopically analyzed; only those containing bacterial cells in the hemolymph were considered as having died from Bacillus infection. The results of the tests with the six pure cultures and the mechanical mixture are presented in Table 1. The data obtained with the six cultures grown together were not significant and have been deleted from the table. From the tests reported in this paper I have drawn the following conclusions: 1. The larvae of C. jumijerana are susceptible to the five spore- and crystal-forming Bacillus and also to the sporeforming B. cereus. The microorganisms were pathogenic to C. jumijerana and caused mortality only when bacterial cells were present in the hemolymph. 2. The period of mortality due to B. thuvingiensis and B. cereus was twice as long (13-14 days) in this case as in previous tests with larvae infected by the native microsporidia (Smirnoff, Can. Entomologist, 95, 127-133, 1963). This suggests that the microsporidia act as a stressor for the Bacillus. 3. The following order of efficacy of the

269

NOTES

TABLE AVERAGE

PERCENT

MORTALITY

CAUSED

BY EACH

2 OF THE

FOUR

Percent after Methods

of

treatment

Sprayed

foliage

Buccal

administration

on

on

to

larvae

administration artificial

to diet

hours

of total

davs for mortality

120

20.7

22.8

70.3

13.6

27.5

53.7

80.7

6.5

40.0

52.5

77.5

7.5

26.2

62.5

91.2

5.1

of

of a grant-in-aid Council

to of

A. J. Musgrave Canada

method, for each of the six pure cultures, and the two mixtures of cultures depended not only on the species tested, but also on the method of treatment (Table 2). The difference in percentage and in rate of mortality between buccal administration and spraying of foliage is as yet unknown but could possibly be due to bactericidal and bacteriostatic effect of the fir foliage on the Bacillus.

W. A.

SMIRNOFF

Forest Research Laboratory, Sillery, Quebec, Canada. Accepted

Nuclear

March

29, 1965

Equivalents

in

Microorganisms in Sitophilus granarius (Linnaeus) ’

The mycetomal microorganisms of the granary weevil, Sitophilus granarius (Linnaeus), supposedly play a useful but not always essentialpart in that insect’s nutrition (Musgrave, Can. Entomologist, 96, 377-389, 1964; Schneider, Z. Morphol. Oekol. Tiere, 44, 555-625, 1956). Kolb (2. Morphol. Oekol. Tiere, 48, 1-71, 1959) using Giemsaand other stains was able

acknowledged.

.

48

Evidence

Mycetomal

Research

.4verage number

mortality

larvae

Histochemical

1 Receipt

USED

24

Bacillus was determined. The percentage given is the average mortality obtained after 120 hours for the four methods of treatment described: (a) B. thuringiensis, 96.0%; (b) B. dendrolimus, 91.3% ; (c) B. alesti, 87.5%; (d) B. entomocidus, 86.2%; (e) B. sotto, 76.2r0; (f) B. cereus, 70.770. The mechanical mixture of the six species gave 90.5% mortality, while the six speciesgrown together gave 41.270 mortality. In the control rearings the larval mortality never passed 5-77oafter 120 hours. The reasons for the low percentage obtained with the mixed insemination are the object of a separate study. 4. The average percent mortality for each

National

OF TREATMENT

larvae

foliage

Immersed Oral

used

METHODS

is

gratefully

by

to identify DNA in the symbiotes of the ant, Camponotus, and the fruit fly, Ceratitis, but was unable to identify it in those of Sitophi1US.

Recently, after a number of preliminary trials, successin detecting DNA in the microorganisms of the mycetome in Sitophilus granarius has been achieved in this laboratory by the following technique. Mycetomes of larvae and mesentera of adults were prepared as smears on glass slides. Following fixation for 3 minutes in the