APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1991,
Vol. 57, No. 8
p. 2277-2282
0099-2240/91/082277-06$02.00/0 Copyright C 1991, American Society for Microbiology
Toxicity of Bacillus thuringiensis to Laboratory Populations of the Olive Fruit Fly (Dacus oleae) KARAMANLIDOU,l A.
LAMBROPOULOS,l S.
I. KOLIAIS,1* T. MANOUSIS,2 D. ELLAR,2 C. KASTRITSIS' Department of Biology, Aristotelian University of Thessaloniki, 54006 Salonica, Greece,' and Department of
G.
F.
AND
Biochemistry, University of Cambridge, Cambridge CB2 IQW, United Kingdom2 Received 10 December 1990/Accepted 11 May 1991
A survey of Bacillus thuringiensis recovered from the environments of olive groves in Greece was carried out. Of 80 soil samples, 24 were found to contain B. thuringiensis with parasporal crystal inclusions; these were tested for toxicity against the olive fruit fly (Dacus okae). Mortality levels of larvae caused by the different isolates varied from 7 to 87%. Higher levels of mortality were observed if a mixture of relatively pure crystals and spores was used compared with the mortality resulting from either fraction alone. We were able to show that the toxicity of the most active isolate is likely to be specific for D. oleae.
isolated from these areas were tested against D. oleae by using laboratory populations of the olive pest.
Because of the importance of the olive in the economies of several Mediterranean countries, the control of the olive fruit fly, Dacus oleae (Insecta, Diptera), which is a common pest of the olive, is considered vital. The losses of the olive crop due to the damage inflicted mainly by D. oleae range from 10 to 70% (7, 14). The control of this pest up to now has been based on spraying wide areas with various chemical pesticides which pollute the environment and may affect other useful species. Other methods such as pheromone
MATERIALS AND METHODS Isolation of bacilli containing crystalline structures. The isolation of the spore-forming aerobic bacteria was performed according to the method described by Ohba and Aizawa (12). In brief, 1 g of the sample (soil or mud from the proximity of olive trees or olive-oil-producing factories or stalk material from the olive trees) was suspended in 10 ml of sterile distilled water and was heated at 70°C for 30 min. This treatment should exclude most of the vegetative forms of bacteria. The suspensions were plated on nutrient agar (pH 7.4; GIBCO), and the bacterial colonies were formed after incubation at 30°C for 3 days. The isolates were searched for the presence of endospores and parasporal inclusions by phase-contrast microscopy. Selective growth of B. thuringiensis. Essentially, the method described by Travers et al. (16) was followed. In brief, bacteria from colonies isolated as described above were shaken in L broth buffered with 0.25 M sodium acetate for 4 h at 200 rpm at 30°C; a small sample of this was heated at 80°C for 3 min and then streaked onto nutrient agar plates. The new colonies formed after overnight incubation at 30°C were observed for the presence of crystal inclusion. Separation of crystals and endospores. Bacteria were grown in petri dishes or in suspension cultures. The spores were collected from nutrient agar washed three times in ice-cold distilled water. Pellets (spores and crystals) were resuspended in small volumes of distilled water. The bacterial suspension cultures were prepared as follows. Loopfuls from bacterial colonies with spores and crystals were transferred to 1 ml of distilled water. Heat-shocked (70°C for 30 min) suspensions were transferred to 250 ml of PWYE medium (5% peptone, 0.1% yeast extract, 0.5% NaCl, pH 7.5) and incubated at 30°C for 8 to 15 h with shaking at 180 rpm. Two milliliters of the PWYE culture was used to inoculate 1 liter of CCY minimal sporulation medium and was incubated at 30°C for 3 to 4 days with shaking at 180 rpm; at least 90% of bacterial cells were lysed releasing spores and crystals after this incubation. Spores and crystals were collected by centrifugation (10,000 x g for 10 min). Pellets were washed three times with ice-cold distilled water,
traps have been used with various degrees of efficiency. The application of biological control is important and requires
investigation. Bacillus thuringiensis is an aerobic sporeformer which during the sporulation process produces some proteinaceous crystalline structures (delta endotoxins) that are toxic to a variety of insects (4, 10, 12, 15). After being ingested, the protein of the crystals dissolves in the alkaline gut of the insect; the epithelial gut wall loses integrity and gut contents mix with the blood, which becomes alkaline. These phenomena lead to paralysis of the insect. Since spores and crystals of B. thuringiensis are not known to be toxic to higher-order animals or plants, preparations of sporulating cells of B. thuringiensis have had extensive applications as biological insecticides in agriculture. As a result of these uses as insect controls, since 1960 several patents for the application of B. thuringiensis have been issued (9). A detailed study on the worldwide abundance and distribution of B. thuringiensis has revealed that B. thuringiensis is a common soil microorganism, since it has been isolated in 70% of soil samples from all over the world. Approximately half of the B. thuringiensis isolates do not match known varieties of B. thuringiensis (11). Because of the serious disease caused by B. thuringiensis in silkworms (1), its isolation from silkworm breeding environments in sericultural farms is very common and its distribution in such environments has been studied (13). As a result of our interest in the isolation of B. thuringiensis strains that could be toxic for the olive tree pest D. oleae, we have studied the distribution of B. thuringiensis in the environments of olive trees and the olive-oil-producing mills. The various strains of B. thuringiensis (and their toxins) *
Corresponding author. 2277
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KARAMANLIDOU ET AL.
Limenaria
Almiros C
A
01
cV P
BRodos
e
d'
op
go
6
oG FIG. 1. Map of Greece indicating the positions where the samples for the isolation of B. thuringiensis were collected.
and final pellets were resuspended in 20 ml of water and stored at -20°C. To purify crystals from spores and cellular debris, samples were sonicated on a Vibra cell sonicator (Sonics and Materials Inc., Danbury, Conn.) for 60 s at 70 W and were centrifuged on discontinuous sucrose density gradients (67 to 72 to 79% [wt/vol] sucrose) at 15,000 x g for 2 h. Crystal bands and spore pellets were purified by three centrifugations and washed with distilled water. Final pellets were resuspended in small volumes of distilled water and stored at -20°C. D. oklae populations. D. oleae pupae were supplied by J. A. Tsitsipis of Democritos Nuclear Research Center in Athens, Greece. Emerging D. oleae flies were kept at 25°C and fed a diet containing sugar, yeast hydrolysate, fresh egg yolk, and streptomycin. Eggs were collected from paraffin cones and transferred to petri dishes that contained filter paper soaked with 0.3% propionic acid. After hatching, eggs were transferred to food containing yeast, soya hydrolysate, sugar, olive oil, Tween, cellulose, 2 N HCl, sorbic potassium, and Nipagine (17). Toxicity tests. Toxicity tests were performed on 5-day-old larvae and 1- to 7-day-old flies. Twenty flies were fed a diet containing 0.7 ml of B. thuringiensis (crystals, crystals and spores, or spores) plus 0.3 ml of liquid fly food. The concentration of the B. thuringiensis component was approximately 109 crystals and/or spores per milliliter. Control flies were fed 0.7 ml of H20 plus 0.3 ml of liquid fly food. Similarly, 20 larvae were kept in petri dishes containing wet filter paper and 0.5 g of larva food which contained 0.7 ml of spores and/or crystals or H20 for controls. Fly and larva mortality was recorded daily.
RESULTS Distribution of bacteria with parasporal bodies. Of 80 soil samples and samples from the environments of olive groves from various regions of Greece shown in Fig. 1 (islands; central and northern Greece), 24 were found to contain parasporal crystal inclusions with a variety of shapes and sizes (Fig. 2). At least one sample from each site contained bacteria with crystals. In Table 1, it is shown that all 24 isolates exhibit, to some extent, toxicity against the olive pest D. oleae. These isolates were tested by the sodium acetate test that is known to eliminate most other sporeforming bacteria, allowing only B. thuringiensis and Bacillus sphaericus to be selected. None of our 24 isolates were inhibited by sodium acetate and after subsequent growth showed formation of parasporal crystals. Two of the toxic isolates, one with high and one with intermediate levels of toxicity, were sent to M. Ohba (Institute of Biological Control, Faculty of Agriculture, Kyushu University, Fukuoka, Japan) for serotyping. Strain Redina a was identified as serotype 4a:4c, and strain Ormilia (subspecies fukuokaensis) was found to correspond to a new serotype, H3a:3d:3e, first identified in Japan (13). Toxicity of B. thuringiensis against D. oleae. The toxicity of the 24 isolates of B. thuringiensis from Greece against D. oleae (adults or larvae) was examined. If the pellet of a sporulated culture was included for 48 h in the diet of D. oleae larvae, the mortality varied from 7 to 87% (Table 1). The toxicity of the same preparations of the microorganisms was considerably lower if the spores and crystals were included in the diet of adult D. oleae; 5-day-old larvae and up to 7-day-old adults were used for these experiments.
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FIG. 2. Phase-contrast microscopy of sporulated culture of B. thuringiensis. Isolate names, derived from regions from which specimens were collected, and shapes of inclusions are as follows: (a) Thessaloniki, irregular; (b) Redina a, cuboidal; (c) Limenaria 3, bipyramidal; (d) Rodos 4(7)b, spherical; (e) Eleonas 1, irregular. The arrows show characteristic crystals of the particular types.
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TABLE 1. lToxicity of B. thuringiensis isolated from the environment of olive groves in Greece against D. oleae
olathuringienss
Cystal shape
Ormiliab Rodos 4(7b) Rodos 4(5) Rodos 4(10) Amphissa 1 Amphissa 5 Amphissa 4(13) Kavala b Kavala d Redina a Redina b Almiros
Spherical Spherical Spherical Cuboidal Irregular Bipyramidal
B.
isolate
Prinos 4 Prinos 2 Limenaria 4b Limenaria 3 Eleonas 5 Eleonas 1 Thessaloniki Epanomi Limenaria 4a Kokkinohoma Amphissa 4 Limenaria 1
Spherical Bipyramidal Spherical Cuboidal Spherical Irregular (double crystal) Bipyramidal Irregular Spherical Bipyramidal Cuboidal Irregular Cuboidal Bipyramidal Spherical Spherical Spherical Bipyramidal
TABLE 3. Mortality caused by toxins from B. thuringiensis against D. melanogaster
% Mortality' of % Mortalitya of D. oleae D. oleae in 48 larvae h in 48 adults h
87 ± 5.50c 37 27 12 37 20 52 ± 8.29c 37 70 ± 21.20c 35 18 20
15 0 15 0 0 0 13 Q 15 15 0 0
70 ± 7.07c 50 ± 0C 60 ± 10.60c 20 32 20 7 19 25 13 20 19
10 5 0 10 7 0 5 0 5 5 15 5
B. thuringiensis isolate and period of incubation (h)
% D. oleae
Ormilia 24 48 Kavala 24 48
Mortality' of larvae D. melanogaster
60 80
10 10
30 50
0 0
a Mortality was estimated from the death rate of 10 larvae that were used for each experiment.
phenomenon was monitored by testing the toxicity of the cultures after storing them at 4°C for different numbers of days. Thus, mortality levels of D. oleae caused by B. thuringiensis are eleVated if the culture of the sporulated microorganism is kept at 4°C for at least 11 days before testing (Fig. 3).
a Mortality was estimated from the death rate of 20 larvae or flies for each isolate. b New serotype H3a:3d:3e (subspecies fukuokaensis) first identified in Japan by M. Ohba (13). c For those experiments showing the highest toxicities, the runs were repeated four times and the standard deviations are given along with the mean values.
Both purified crystals and endospores from B. thuringiensis Ormilia (serotype H3a:3d:3e), exhibited high levels of toxicity against the larvae of D. oleae after 48 h (Table 2). However, when purified crystals and endospores were combined, they always induced, for the 24-h treatment, higher levels of mortality than the same quantity of either crystals or spores (Table 2). Two isolates with high levels of toxicity against D. oleae were tested also for toxicity against another dipteran, a laboratory strain of Drosophila melanogaster (third instar larvae). The results presented in Table 3 show that the toxins from the two isolates of B. thuringiensis are inactive against D. melanogaster. An interesting finding is that cultures that have been left at 4°C for several days had elevated levels of toxicity. This
DISCUSSION The proportion of the Greek soil samples from the environments of the olive trees or olive-oil-producing mills that contain bacteria with parasporal inclusions is higher than what is reported in the literature (12). This, of course, is related to the method followed for the sampling of the various regions and the selection of the particular soils. Because of our interest in the possible effects of B. thuringiensis on the D. oleae populations, the samples examined for B. thuringiensis were from the ground under olive trees, from the stalk material of the trees, and from local olive-oil-
100
-
?75 -
50-
25
-
TABLE 2. Toxicity of crystals and endospores isolated from B. thuringiensis Ormilia B. thuringiensis Ormilia componentsa
Crystals Spores Crystals and spores
% Mortality' of D. oleae larvae after time (h)
24
48
22.0 12.5 60.0
72 100 95
Equal numbers of each component (measured by microscopy) were used. Mortality was estimated from the death rate of 20 larvae that were used for each experiment. a
b
8
16
24
32
Time (days) FIG. 3. Toxicity against D. oleae of B. thuringiensis Ormilia as a function of the time for which the bacterium was stored at 40C. Mortality is calculated as the percentage of the larvae that died after having been fed toxin-containing food for 24 h. The experiment was done with 20 control and 20 treated larvae.
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producing mills. It is possible that these particular grounds are biased towards the existence of these bacilli. The morphological data from the isolates show that there is a considerable variety of B. thuringiensis strains since most known sizes and shapes of crystals were observed in the various isolates. It should be pointed out that in most samples, one type of spore-forming bacillus is observed; very rarely have we isolated from the same sample more than one strain of bacillus. It has been shown that the toxins of these isolates possess various levels of activity when tested for toxicity against laboratory populations of D. oleae. It would be of interest to test our isolates of B. thuringiensis against natural populations of D. oleae, but such populations are difficult to establish in the laboratory. The activity of some of the isolates is significant and comparable to the toxicity rates against other insects (10, 12) and against nematodes reported in the literature (4). Ohba and Aizawa (12) have suggested a possible relationship between the architecture of the crystal inclusions and the selective toxicity to mosquitoes since they found that most of their toxic strains contained spherical crystals. Our data show that the isolate most toxic against D. oleae also contains a spherical crystal inclusion (Table 1), and this could extend the suggestion of these investigators to other diptera, although several other spherical isolates do not exhibit this property and some crystals of other architecture also prove to be toxic. The mortality levels of D. oleae caused by the toxins of the different isolates of B. thuringiensis are always higher if the experiments are carried out with larvae rather than the adults. This might be a consequence of the different modes of feeding larvae and adults; it is possible that with the filter-feeding mode, the concentration of delta endotoxin delivered to larvae is increased. The data presented in Table 2 show that fractionated endospores possess high levels of activity that cannot be interpreted as being caused by contamination of the spore fraction with small amounts of crystals. Similar observations on the mortality caused to lepidoptera by B. thuringiensis were reported by others (10, 18). The fact that the toxicity of unfractionated material is consistently higher at 24 h than the activity of the crystals or the endospores alone supports the view that the endospores are, on their own, carriers of activity that is different from that of the crystalline inclusions. This view is supported by the findings of Bone and his collaborators, who distinguish two toxic activities of B. thuringiensis, each being specific for larvae or adults of the nematode Trichostrongylus colubriformis (2, 3). Alternatively, the increased toxicity of the crystalline inclusions in the presence of the spores might be due to the possibility that the spore itself provides the necessary factors that are required for the processing of the protoxins into active toxins (15). The variable toxicity of the delta endotoxins of B. thuringiensis against different taxonomic groups of insects has been interpreted by the different mechanisms that lead to the activation of the delta endotoxin. The activation of the protoxins requires the action of proteolytic enzymes (6) after their solubilization in alkali (5, 8, 15). Differences in the conditions in insect gut environments could be the cause of the differences that are observed with the action of the toxins on various insects. It has also been suggested that insects which are not good targets for the toxins could lack the necessary receptor which can be exploited by soluble delta endotoxin (15). These different gut conditions or the lack of
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receptors could very well explain the differences observed with the Ormilia toxin against D. oleae and D. melanogaster. The toxin from the Ormilia isolate that shows little or no activity against D. melanogaster is an interesting specific toxin and therefore merits further investigation. Another aspect that needs more experimentation is the finding that the mortality caused by the toxins of the different strains of B. thuringiensis against D. oleae is elevated if the microbial culture is allowed to stand at 4°C for several days. In similar experiments against the eggs of the nematode T. colubriformis, no such activation was observed (3). ACKNOWLEDGMENTS This work was supported by grant STJ-0137-1-GR under the Stimulation programme of the E.E.C. We thank J. A. Tsitsipis of Democritos Nuclear Research Center, Athens, Greece, for supplying us with the initial cultures of D. oleae and M. Ohba for serotyping the two isolates of B. thuringiensis. REFERENCES 1. Aizawa, K., T. Takasu, and K. Kurata. 1961. Isolation of Bacillus thuringiensis from the dust of silkworm rearing houses of farmers. J. Sericult. Sci. Jpn. 30:451-455. (In Japanese with
English summary.) 2. Bone, L. W. 1989. Activity of commercial Bacillus thuringiensis preparations against Trichostrongylus colubriformis and Nippostrongylus brasiliensis. J. Invertebr. Pathol. 53:276-277. 3. Bone, L. W., and K. P. Bottjer. 1988. Factors affecting the larvicidal activity of Bacillus thuringiensis israelensis toxin for Trichostrongylus colubriformis (Nematoda). J. Invertebr. Pathol. 52:102-107. 4. Bone, L. W., K. P. Bottjer, and S. S. Gill. 1986. Trichostrongylus colubriformis: isolation and characterization of ovicidal activity from Bacillus thuringensis israelensis. Exp. Parasitol. 62:247-253. 5. Bulla, L. A., Jr., K. J. Kramer, D. J. Cox, B. L. Jones, L. J. Davidson, and G. L. Lookhart. 1981. Purification and characterization of the entomocidal protoxin of Bacillus thuringiensis. J. Biol. Chem. 256:3000-3004. 6. Fast, P. G. 1981. The crystal toxin of Bacillus thurigiensis, p. 223-248. In H. D. Burges (ed.), Microbial control of pest and plant diseases 1970-1980. Academic Press, Inc. (London), Ltd., London. 7. Fiori, G. 1982. Proceedings of the Second Meeting on Dacus oleae (Gmel.), Perugia, 5-6 March 1982. Frustula Entomol., vol. 4. 8. Huber, H. E., P. Luthy, H. R. Ebersold, and J. L. Cordier. 1981. The subunits of the parasporal crystal of Bacillus thuringiensis: size, linkage and toxicity. Arch. Microbiol. 129:14-18. 9. Ignoffo, C. M., and R. F. Anderson. 1979. Bioinsecticides, p. 1-28. In H. J. Peppler and D. Perlmann (ed.), Microbial technology. Academic Press, Inc., New York. 10. Jarrett, P., R. S. Li, and H. D. Burges. 1987. Importance of spores, crystals, and 5-endotoxins in the pathogenicity of different varieties of Bacillus thuringiensis in Galleria mellonella and Pieris brassicae. J. Invertebr. Pathol. 50:277-284. 11. Martin, P. A. W., and R. S. Travers. 1989. Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appl. Environ. Microbiol. 55:2437-2442. 12. Ohba, M., and K. Aizawa. 1986. Insect toxicity of Bacillus thuringiensis isolated from soils of Japan. J. Invertebr. Pathol. 47:12-20. 13. Ohba, M., and K. Aizawa. 1989. New flagellar (H) antigenic subfactors in Bacillus thuringiensis H serotype 3 with description of two new subspecies, Bacillus thuringiensis subsp. sumiyoshiensis (H serotype 3a:3d) and Bacillus thuringiensis subsp. fukuokaensis (H serotype 3a:3d:3e). J. Invertebr. Pathol. 54: 208-212. 14. Pelerents, C. 1980. The contribution of the European community in the domain of integrated control, p. 383-386. In K. Russ and H. Berger (ed.), Proceedings of the National International Symposium of the IOBC/WPRS on Integrated Control in Agri-
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culture and Forestry, Vienna, 8-12 October 1979. 15. Thomas, W. E., and D. J. Ellar. 1983. Bacillus thuringiennsis var. israelensis crystal 8-endotoxin: effects of insect and mammalian cells in vitro and in vivo. J. Cell Sci. 60:181-197. 16. Travers, R. S., P. A. W. Martin, and C. F. Reichelderfer. 1987. Selective process for efficient isolation of soil Bacillus spp. Appl. Environ. Microbiol. 53:1263-1266.
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17. Tsitsipis, J. A. 1977. An improved method for the mass rearing of the olive fruit fly (Dacus oleae, Gmel.) (Diptera, Tephritidae). Z. Angew. Entomol. 83:419-426. 18. Tyrell, D. J., L. A. Bulla, Jr., and L. I. Davidson. 1981. Characterization of spore coat proteins of Bacillus thuringiensis and Bacillus cereus. Comp. Biochem. Physiol. B Comp. Biochem. 70:535-539.
