JOURNALOFINVERTEBRATE

PATHOLOGY

s&33-39(1991)

Two New Isolates of Bacillus thuringiensis Spodoptera litura V. H. WHITLOCK,’ Agricultural

Biotechnology

Division,

M. C. Lo, M. H. Kuo, Development Taipei, Taiwan,

Center Republic

Pathogenic

to

AND T. S. SOONG~

for Biotechnology, of China

81 Chang

Hsing

Street,

Received May 9, 1990; accepted September 25, 1990 Both the standard Bacillus thuringiensis karstaki (HD-1) and the formulated commercial product resulted from this strain have shown limited pathogenicity against the tobacco cutworm (Spodoptera litura). However, two new isolates of Bacillus thuringiensis (K-2074 and K-2178) isolated from Taiwan have been identified through an active screening program to be highly pathogenic against the tobacco cutworm. In this paper, we present results of characterization and the pathogenicity of these two new isolates. o 1~1 Academic PRSS. IK. KEY WORDS: pathogenic bacteria; Bacillus thuringiensis; Spodoptera litura; biological control; endotoxin.

with S. littoralis, but again, no mention was made of S. litura; in fact, no reference could be found to any isolate having proved effective against this species in either laboratory or field assays. Based on the assumption that the possibility exists of discovering new strains with new pathogenic spectra or host ranges, many samples were collected from different areas in Taiwan, and the crystal-producing isolates were assayed for insecticidal activity against several insect species. We report here on the pathogenicity of two of these isolates to S. litura.

INTRODUCTION Spodopteru lituru (tobacco cutworm) is a serious pest of agricultural crops in many parts of the world, and at present only chemical pesticides are effective in its control. The development of biological pesticides is being encouraged in many countries, and various isolates of Bacillus thuringiensis are in commercial use against a number of lepidopterous pests. S. litura is, however, not susceptible to any of these, although a related species, S. littoralis, has been found to be susceptible to serovars aizawi, kenyae, and entomocidus (Kalfon and de Barjac, 1985). Salma (1984) also found S. littoralis to be susceptible to a single culture of the serovar entomocidis and demonstrated the pathogenicity of this culture to another related species, viz., S. exigua. He further investigated the effect of a number of isolates on various lepidoptera species, but no mention was made of S. fituru. Broza et al. (1984) had similar results

MATERIALS

AND METHODS

Bacterial isolates. Sampling was done by collecting 100 g of soil l-5 cm below the surface and transported to the laboratory in sterile plastic bags where 5 g of the soil was suspended in 50 ml sterile-distilled water and heated to 60°C for 30 mm. The suspension was cooled to room temperature, plated out on nutrient agar, and incubated at 28°C for 3 days. All the crystal-producing isolates were kept in the collection at the Development Center for Biotechnology, Taiwan, ROC, and assayed against various insect species.

r Current address: Department of Microbiology, University of the Witwatersrand, Johannesburg, P.O. Wits, 2050, South Africa. ’ To whom correspondence should be addressed.

33

0022-2011/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

34

WHITLOCK

ET AL.

The two isolates used in these bioassays were designated K-2178, collected from a groundnut field on Pen-hu island, and K2074, collected from a guava orchard near Taitung. The “K” preceding the identification number is derived from an internal indexing system and does not identify the isolates as having been typed as Kurstaki strains. Purification of the crystals. A 72-hr broth culture (90% lysis) was centrifuged at 10,OOOgfor 15 min at 4”C, and the pellet was washed with distilled water twice before being resuspended in distilled water. This sample will be referred to as a cell suspension. To separate the crystals from the other components of the cell suspension, the latter was centrifuged on a 45-80% (w/ v) sucrose gradient at 25000 rpm for 4 hr at 4°C. The crystal suspension was mixed with an equal volume of dissolving buffer (0.1 M Na*CO, + 0.1 M P-mercaptoethanol, pH 10) and incubated at 35°C for 2 hr centrifuged at 1,000 rpm for 5 min, and assayed using the BioRad protein assay procedure. Infection of insects. The insects used were third instar larvae from a laboratory culture maintained on a kidney bean/wheat

LARVAL

WEIGHT (5 DAYS pi). PUPAL INSTAR Spodoptera litura Concentralion

Isolate K-2178 (crystal)

(mgflarva)

WEIGHT, LARVAE

germ diet. The larvae were placed in translucent plastic containers with clip-on lids containing a small plug (7 mm diameter, 10 mg) of medium infected with 20 ~1 of the inoculum. The inocula were various dilutions of cell suspensions and purified crystals of both isolates (Table 1). Five hundred larvae were infected with each dilution of the four samples, and larvae that had not consumed the entire plug within 72 hr were discarded and the rest were transferred to similar containers with enough medium to complete the larval stage. When more than 10% had to be discarded. the experiment was repeated. This procedure made it possible to infect each larva with an exact dose of the inoculum and was expressed as milligrams per larva. Controls were treated with distilled water. Mortality counts were made daily until pupation, and pupal weights of the survivors were recorded. An assay with B. thuringiensis subsp. kurstaki (HD-1), which is the isolate used in a commercial product, was done in the same way to compare the efficacy of the two new isolates with a commercially used one.

TABLE 1 AND LARVAL MORTALITY AFTER EXPOSURE TO Two STRAINS OF Bacillus thuringiensis

Mean larval wt (mg)

OF THIRD

Mean pupal

wt (rng) -

% Mortality

0.1 0.06 0.03

16.2 15.6 44.9

421.1 425.1

85 74

0.07 0.03 0.02 -

90.2 260.5 235.1

419.3 435.7 428.1

94 62 54

316.2

443.2

10

K-2178 cell suspension

0.5 0.1 0.05

21.9 18.9 190.9

441.1 420.4

99 93 41

K-2074 cell suspension

0.6 0.1 0.03

144.2 147. I 146.6

434.3 422.5 423.2

70 26 52

K-2074 (crystal)

Control

100

PATHOGENIC

RESULTS

BACTERIA

TO

AND DISCUSSION

TOBACCO

35

CUTWORM

K-2178 had a marked effect on the growth of the larvae (Table l), and although pupae of the surviving larvae did not weigh significantly less than the untreated controls, it took longer to reach pupation (Figs. 1, 2). The same phenomenon was observed for K-2074, but it was not as pronounced as for K-2178 (Figs. 3, 4). All the pupae devel-

S. lituru is susceptible to both isolates, either as a purified crystal or as cell suspensions, with K-2178 being the more pathogenie of the two (Table 1, Figs. l-4). Both isolates were far superior to HD-1 (Fig. 5).

A

zs. s

60

1 1

4

+ -+ -

A

0 1 mg/larva 0 5 mgilarva 0 05mgilarva ConkI

Q

1

00 0

10

20

30

Days 100

I----

B

l 0 01 mgilarva + 0005mgilarva -A- Control

80

0

10

12

14

18

16

20

22

Days FIG.

iensis

1. Cumulative mortality and pupation of Spodoptera strain K-2178 cell suspension.

litura

infected with a Bacillus

thuring-

36

WHITLOCK

0

lb

ET

AL.

30

2b

Days 100 B

60

* *

.-iii

0 06mgilarva 0.03mgilarva 0 01 mgilarva 0 005mgilarva Control

P

90

40

0

10

15

20

25

Days FIG.

iensis

2. Cumulative mortality and pupation of Spodoptera strain K-2178 toxin.

oped into normal adults, and oviposition and fecundity were not affected. Both isolates are very effective against S. litura in laboratory assays. Since neither the crystal nor the spore content of the cell suspensions was determined, it was not possible to make a comparison of the relative efficacy of the cell suspensions of these

litura

infected with a Bacillus

thuring

two isolates. While these results may seem to suggest that K-2178 produces more toxin or a more potent toxin than K2074 per unit of dry weight (Table 1)) it still remains to be proven. The retarding effect of especially K-2178 on the development of the larvae is also a phenomenon not recorded for other strains

PATHOGENIC

BACTERIA

TO

TOBACCO

37

CUTWORM

100

/

FIG. iensis

-m-

0 07 mgi,arvan

0-a-

0n 03 nnAmn/lnr”n 02 mg/larva

3. Cumulative mortality and pupation of Spodopreru litura infected with a strain K-2074 toxin.

of B. thuringiensis. Mathavan et al. (1989) showed that B. thuringiensis var. israelensis crystals act on the midgut epithelium of the silkworm, causing it to swell and eventually disintegrate, resulting in death. They do not mention the effect of a sublethal dose, but Percy and Fast (1983) state that it is necessary for direct contact between the

Bacillus

thuring-

epithelial cells and the crystals to produce pathological changes. They also found that unaffected and seriously affected cells can be observed in the same region, and only in dead larvae is the epithelium completely affected. In our work, a partially affected epithelium could account for the slowing of the

38

WHITLOCK

ET AL.

80 A -A * + -

I 60

I 0

I

0 OGmgilarva 0 1 mgilarva 0 03mgilarva Control

I 20

10

, 30

Days B -A + + +

80

i

I

10

12

!

I

I

r

14

16

FIG. 4. Cumulative mortality and pupation of iensis strain K-2074 cell suspension.

Spodoptera

0

I

I

18

0 OGmgilarva 0 1 mgilarva O.OBmg/larva Control

I,

I

20

I

I

22

Days

growth rate, but for the larvae to provide normal pupae, albeit on average 5 days later than the controls (Figs. 14), regeneration of the epithelium after a simple sublethal dose would be necessary. If this is the case, a repeated exposure to sublethal doses would also have a suppressing effect on any field population because it was fur-

litura

infected with a

Bacillus

thuring-

ther noted in this study that infected insects were very lethargic and often exhibited a negative geotropic reaction-both phenomena which can contribute to the insect’s vulnerability to environmental factors and to natural enemies. Additional research with different pest species will be necessary to determine the

PATHOGENIC

BACTERIA

TO TOBACCO

CUTWORM

39

BI K2178 E&K2074 Bfk

o!

/ 0

20

40

Toxin FIG.

and

5. Mortality of Spodoptera

B. thuringiensis

kurstaki

litura

level ( x 0.01 mg/larva)

larvae induced by Bacillus

K-2178 and K-2074

thuringiensis

toxins.

host ranges of these two isolates, but we believe that their effectiveness against S. Zitura alone is of significance (Fig. 5). REFERENCES B.. YAWETZ, A., ORON, U., AND 1984. Commercial application of Bacillus thurirzgiensis var. entomocicus to cotton fields for the control of Spodoptera littoralis. Boisduval (Lepidoptera:Noctuidae). J. Econ. Entomol., 77(8), 1530-1533. KALFON, A. R., AND DE BARJAC, H. 1985. Screening of the insecticidal activity of Bacillus thuringiensis BROZA, M., SNEH, HONIGMAN, A.

60

strains against the Egyptian Spodoptera

littoralis.

cotton leaf worm.

Entomophaga,

30(2).

177-

186. S.. SUDHA. P. M., AND PECHIMUTHA. M. 1989. Effect of Bacillus thuringiensis israelenon the midgut cells of Bombyx mori larvae: A histopathological and histochemical study. J. fnver-

MATHAVAN, S. sis

tebr.

Pathol..

53,

217-227.

PERCY, J., AND FAST, F. G. 1983.

crystal toxin: Ultrastructural worm midgut cells. J. Invertebr.

Bacillus

thuringien-

studies on silkPathol.. 41, 86-89. SALMA, H. S. 1984. Bacillus thuringiensis Berliner and its role as a biological control agent in Egypt. Z. sis

Anger.

Entomol.

98,

206220.