Biocontrol Science and Technology (1999) 9, 29-33 Laboratory Evaluation of the Pathogenicity of Three Isolates of the Entomopathogenic Fungus Beauveria bassiana (Bals.) Vuillemin on the American Cockroach (Periplaneta americana) CH. MURALI MOHAN, K. ARUNA LAKSHMI AND K. UMA DEVI1 Department of Botany, Andhra University, Visakhapatnam-530 003, AP, India (Received/or publication 21 May 1998; revised manuscript accepted 25 October 1998)

Mycopesticides can be ideal for the biocontrol of cockroaches because the habitat of these insects promotes initial fungal infection and its subsequent spread. The pathogenicity of three isolates of Beauveria bassiana to the American cockroach was tested. The insects were treated in three different ways, by direct contact with spore mass, a spore-wheat flour mixture and a spray of an aqueous spore suspension. A mortality of 100% in the first treatment, 67-100% in the second treatment and 17-75% in the third treatment was observed. These results suggest that B. bassiana spore formulations in food baits can be developed for cockroaches. Keywords: biological control, entomopathogenic fungi, Beauveria bassiana, American cockroach, food bait, Periplaneta Americana. INTRODUCTION Periplaneta americana, the American cockroach, is considered to be a nuisance pest in households. Cockroaches leave an odour when they crawl on food and leave stains on the furniture. The use of chemical insecticides against cockroaches poses a health risk because these agents have to be applied in living spaces. Entomopathogenic fungi are receiving renewed interest as biocontrol agents in instances where chemical pesticides present a risk to human health (McCoy, 1990). Fungi enter the host through the cuticle and a contact with the insect can cause infection. Profuse mycosis develops on the cadavers of the insects that have died due to fungal infection. These mycotic cadavers perpetuate the lethal fungal infection in the insect population. Hyphomycetous fungi have been reported to infect cockroaches (Hywel-Jones, 1995; Kaakeh et al., 1996; Zukowski & Bajan, 1996). It should be easy to induce an entomopathogenic fungal infection artificially in cockroaches because the development of profuse mycosis on the insects is favoured under the humid conditions prevailing in their ecological niches. A rapid spread of the infection in the insect population is therefore highly likely. A commercial formulation of Metarhiziurn anisopliae has been developed for cockroach control in the US by the Ecoscience Corporation (Andes, 1994; Kaakeh et al., 1996). Beauveria bassiana, an entomogenous fungus with a wide host range, occurs more commonly than Metarhiziurn spp. in the tropics (Vanninen, 1995). It has been reported to infect American and German cockroaches (Steinhaus, 1949; Zukowski & Bajan, 1996). Strains of B. bassiana differ in their host range (McCoy, 1990). Therefore, the pathogenicity of three local isolates of B. bassiana was tested on the American cockroach. The infectivity of the fungal spores when mixed with food baits was also examined. Correspondence to: K. U. Devi. Tel: +91891-27554871 extn 342; Fax: +91891-27555547; E-mail: [email protected] 1

0958-3157/99/010029-05 $9.00 ©1999 Taylor & Francis Ltd

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CH. MURALI MOHAN ET AL. MATERIALS AND METHODS Three isolates of B. bassiana, Bbl, Bb2, and Bb3, were used. These had been isolated from fieldcollected Spodoptera litura, Helicoverpa armigera and soil respectively, and were supplied by the Regional Agricultural University/Research Station. The fungal spores were stored in 20% glycerol at - 20°c. Freshly renovated cultures of glycerol stocks were subcultured on Sabouraud dextrose agar yeast (SDAY) slants in test tubes and used in the experiment. The spores from 14-day-old cultures were used. The viability of the spores was tested by a germination bioassay in SD medium before they were used for insect treatment, and more than 95% of the spores were found to be viable. The cockroaches were collected from households in Visakhapatnam (a coastal city in south India). Small insects about 3 months old (with an average body weight of 540 mg) and fully-grown adults (with body weights of about 800 mg) were included in equal numbers for each treatment. Twenty treated insects and 20 control insects were used in each treatment. All experiments were carried out in triplicate. The insects were kept in batches of five each in perforated plastic boxes (12 × 18 cm) with lids. The boxes containing the insects were placed in an environmental chamber at 251: 1°C, with a 16: 8 h (light:dark) regime and > 90% humidity. The cockroaches were treated as follows. (1) The insects were made to crawl singly on the spore mat on SDAY slants in test tubes for 30 s. (2) A 4.5 × 106 spores/ml concentration of an aqueous spore suspension (with 0.01% Tween 20) was applied with a Gilson pipette (Gilson, France) (500 III for small cockroaches and 1 ml for large ones) on the ventral side of each insect. In two other treatments, the cockroaches were left in batches of five each for 12 h on: (3) a Bbl-killed cockroach cadaver with profuse mycosis; (4) wheat flour mixed with the spores of Bbl. Spore mass harvested (by scraping with a spatula) from a 14-day-old culture on a SDAY slant in a test-tube (25 × 150 mm) was mixed with 65 g of wheat flour by vortexing. Approximately 15 g of this mixture was placed in each box with five insects. The control insects in treatment 2 were sprayed with 0.01% Tween 20 and in treatment 3 they were left on plain flour. The cockroach oothecae were treated with B. bassiana. The spores were dusted on the oothecae with a 3 mm paintbrush. Ten oothecae were treated with spores of each isolate and 10 untreated oothecae served as the controls. The number of spores that were attached to each insect in treatment 1 was calculated as follows. The treated insect was dipped in 10 ml of water with 0.01 % Tween 20 and vortexed for 5 min. The insect was then removed. The number of spores in this solution (spores/ insect) was estimated from the spore count made of an aliquot of this solution with a haemocytometer. The spore count for five large and five small insects/treatment was thus made, and the mean of the five values is given in Table 1. An aqueous (0.01% Tween 20) suspension of the wheat flour-spore mixture was made for the spore count. The viability of the spores incubated in wheat flour for 12 h was found to be 90%.

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After treatment, the insects were fed on raw potatoes and observed for 10 days. Dead insects were washed with water and wiped dry on blotting paper. They were placed in a Petri dish lined with moist blotting paper (to facilitate mycosis) and the lid was sealed with tape. RESULTS AND DISCUSSION The three isolates were found to be pathogenic to cockroaches. From treatment 2, the Bb3 isolate was identified as being the most virulent (Table I). Differences in virulence to a host among isolates is reported in all the entomopathogenic fungi assayed (Glare & Milner, 1991). The exposure of cockroaches to spores in wheat flour was found to be effective. No significant difference in the death rates was found between the large and small insects. In the control cockroach population, 3-15% mortality was observed by the 10th day and 80-90% of the untreated cockroaches remained alive for up to 6 months, after which time the experiment was terminated. Among the insects that died within 3 days after treatment, only 3-13% developed mycosis, while 30-74% of the insects that died on later days showed profuse mycosis. In the insects that died within 3 days, death could have been caused by a toxaemic reaction. Such insects do not support external sporulation (Glare & Milner, 1991). Where the insects showed mycosis, the fungus was first evident on the antennae and legs of the cadavers. By the third day after death, profuse mycosis was evident on both the dorsal and ventral sides (Figure I). Some of the treated insects showed mycosis when they were still alive and mobile. Pre-death sporulation has not been reported for B. bassiana infections, but is known for infections by Verticillum lecanii and a few other entomopathogenic fungi. This can improve the spread of the infective spores by utilizing host movement (Glare & Milner, 1991). None of the dead untreated insects showed mycosis. The B. bassiana treated oothecae showed fungal growth on their outer covering by the third or fourth day after treatment and failed to hatch. The control (untreated) oothecae hatched in 15-20 days and 12-16 nymphs emerged from each of them.

FIGURE I. Mycotic cadavers (life size) of P. americana that died of B. bassiana infection. (a) Dorsal view; (b) Ventral view.

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TABLE 1. Laboratory bioassays with spores of entomopathogenic fungus B. bassiana on the American cockroach (P. americana) TreatIsolate menta DC

Bb1

Spore Concentration

Bb3

A

3

4

5

6

7

L

7 ± 1.8

32 ± 0.3

100

-

-

-

S

7 ± 1.2

32 ± 0.1

100

2 ± 2.3

53 ± 5.8 99 ± 0.4

100

2 ± 1.6

55 ± 6.9

99± 0.6

100

8

1.7 × 10 /insect

L

9.2 × 105/insect

S

7

-

L

-

-

41 +9.6

100

-

5

S

-

-

43 ±10.8

100

-

L

-

3 ± 3.0

S

-

L

-

Bb1 --

Bb1 4.5 × 106/ml

S 6

Bb2 4.5 × 10 /ml

Bb3 4.5 × 106/ml

WF

Bb1 3.9 × 107/g 6

Bb2 6.5 × 10 /g 5

Bb3 5.3 × 10 /g

a

8

9

10 -

-

9.8 × 10 /insect 8.2 × 10 linsect

SI

2

5

4.2 × 10 /insect Bb2

1

7

6.2 × 10 /insect

Cumulative mortality on day (%)C

Insectb

-

Dead insects with sporulation (%)

69± 5.3 -

-

71 ± 5.4 50 ± 3.1

-

-

-

-

56 ± 0.3

-

63 ± 7.8

-

-

-

68 ± 5.3

42 ± 0.1 90 ± 2.6

100

-

-

-

-

-

49 ± 6.3

-

48 ± 0.2

92 ±1.7

100

-

-

-

-

-

57 ± 4.7

-

-

-

-

-

-

-

2 ± 1.2

14± 0.1

76 ± 2.8

-

-

-

-

-

-

-

2 ± 0.7

18 ± 0.8

78 ± 3.1

L

-

-

-

-

-

-

-

1 ± 0.3

11± 0.2

45 ± 0.2

85 ± 4.2

S

-

-

-

-

-

-

-

1 ± 0.1

13 ± 0.3 49 ± 0.4

89 ± 1.8

L

-

-

3 ± 1.7

37 ± 0.1 47 ± 0.3 56 ± 0.2 75 ± 0.0 75 ± 0.0 75 ± 0.0 75 ± 0.0

64 ± 0.0

S

-

-

5 ± 1.8

39 ± 0.1 51 ± 0.6 58 ± 0.7 75 ± 0.0 75 ± 0.0 75 ± 0.0 75 ± 0.0

70 ± 0.0

L

64 ± 0.2 74 ± 0.0 98 ± 0.7

100

-

-

-

-

S

70 ± 0.3 80 ± 0.0

100

100

-

-

-

-

36 ± 0.0 -

44 ± 0.0

L

-

-

-

-

-

6 ± 0.8 17 ± 2.9 29 ± 0.8 30 ± 0.4 68 ± 0.1

68 ± 3.3

S

-

-

-

-

-

6 ± 1.4 19± 3.7 29 ± 1.3 36 ± 0.4 76 ± 0.1

78 ± 2.9

L

-

-

-

-

6 ± 1.0 11± 0.0 21± 0.3 20 ± 0.2 30 ± 0.6 66 ± 0.1

71 ± 5.0

S

-

-

-

-

6 ± 0.8 13 ± 0.0 23 ± 0.8 22 ± 0.2 36 ± 0.8 68 ± 0.1

81 ± 4.9

DC, direct contact of the insect with spore mat on SDAY slant (the spore concentration values are for large/small insects); SI, secondary infection from a mycotic cadaver infected by Bbl; A, aqueous spore suspension 1 ml/500 µl per large/small insect; WF, spores in wheat flour. b L, large insect, S, small insect. c Values are corrected for control mortality (Abbott, 1925) and angular transformed (arcsine √percentage) before analysis, back-transformed and rounded.

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PATHOGENICITY OF B. BASSIANA TO P. AMERICANA From the results of this study, B. bassiana shows promise for the management of cockroaches. A formulation of B. bassiana spores in food baits with a reasonable shelf-life needs to be developed. ACKNOWLEDGEMENTS Ch. Murali Mohan and K. Aruna Lakshmi are grateful to University Grants Commission (UGC) and Council for Scientific and Industrial Research (CSIR) respectively for providing financial assistance. REFERENCES ABBOTT, WS. (1925) A method of computing the effectiveness of an insecticide. Journal of Economic Entomology 18, 265_267. ANDES, M. (1994) The biopath cockroach control chamber uses nature to control nature's pests. Pest Control 62, 44-48. GLARE, T.R. & MILNER, R.J. (1991) Ecology of entomopathogenic fungi, in Handbook of Applied MycologyHumans, Animals, and Insects, Vol. 2 (ARORA, D.K., AJELLO, L & MUKERJI, G, Eds) Marcel Dekker, New York, pp. 547-612. HYWEL-JONES, N.L (1995) Hymenostilbe ventricosa Sp.nov., a pathogen of cockroaches in Thailand. Mycological Research 99, 1201-1204. KAAKEH, W., REID, B.L & BENNETT, GW (1996) Horizontal transmission of the entomopathogenic Metarhizium anisopliae (Imperfect fungi: Hyphomycetes) and hydromethylnon among German cockroaches (Dictyophora: Blatellidae). Journal of Entomological Science 31, 378-390. McCoy, C.W (1990) Entomogenous fungi as microbial pesticides, in New Directions in Biological Control: Alternatives for Suppressing Agricultural Pests and Diseases (BAKER, R.R. & DUNN, PE., Eds) Alan R. Liss, New York, pp. 139-159. STEINHAUS, E.A. (1949) Principles of Insect Pathology. McGraw-Hill Inc., New York. VANNINEN, I. (1995) Distribution and occurrence of four entomopathogenic fungi in Finland: effect of geographical location, habitat type and soil type. Mycological Research 100, 93101. ZUKOWSKI, K. & BAJAN, C. (1996) Studies of the usefulness of Beauveria bassiana for eradication of cockroaches (Blattella germanica L). Roczniki Panstwowy Zakled Higieny 47, 343-349.