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Survival and conjugation of Bacillus thuringiensis in a soil microcosm
FEMS Microbiology Ecology 31 (2000) 255^259
www.fems-microbiology.org
Survival and conjugation of Bacillus thuringiensis in a soil microcosm Laurival A. Vilas-Boªas a;c , Gislayne F.L.T. Vilas-Boªas a;c , Halha O. Saridakis b , Manoel Victor F. Lemos c , Didier Lereclus d;e , Olivia M.N. Arantes a; * a
Bio/CCB, Universidade Estadual de Londrina 86051-970, Londrina, Brazil Micro/CCB, Universidade Estadual de Londrina 86051-970, Londrina, Brazil c FCAV/UNESP, Rod. Carlos Tonanni Km5, 14870-000, Jaboticabal, Brazil Unite¨ de Biochimie Microbienne, lnstitut Pasteur, 25, rue du Dr. Roux, 75724 Paris, Cedex 15, France e Station de Recherches de Lutte Biologique, INRA, La Minie©re, 78285 Guyancourt, Cedex, France b
d
Received 2 August 1999; received in revised form 21 December 1999; accepted 22 December 1999
Abstract The survival and conjugation ability of sporogenic and asporogenic Bacillus thuringiensis strains were investigated in broth, in nonamended sterile clay soil monoculture and in mixed soil culture. The 75 kb pHT73 plasmid carrying an erythromycin resistance determinant and a cry1Ac gene was transferred in mating broth and soil microcosm. Survival of strains was assessed in soil monoculture and in mixed soil culture for up to 20 days. Sporogenic strains rapidly formed viable spores which were maintained until the end of the experiment. The asporogenic strains were no longer recovered after 8 days of incubation. This study shows that the environmental impact of asporogenic B. thuringiensis strains is lower than that of sporogenic B. thuringiensis strains. Thus, the use of asporogenic strains may significantly reduce any potential risk (gene transfer, soil and plant contamination) due to the dissemination of B. thuringiensis-based biopesticides in the environment. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Sporulation mutant; Plasmid; Conjugation; Soil microcosm; Bacillus thuringiensis
1. Introduction The Gram-positive ubiquitous bacterium Bacillus thuringiensis produces a proteinaceous crystal during the stationary phase. The crystal proteins, designated Cry proteins, are toxic against the larval forms of several insects of agronomic and medical importance [1]. Strains of B. thuringiensis have been isolated around the world from various sources including soil, stored product dust, dead and living insects [2]. The use of commercial preparations of B. thuringiensis as agricultural insecticides has increased in recent years, and they now account for more than 90% of the biopesticides used. B. thuringiensis formulations present many advantages such as no toxicity for animals, high speci¢city, low development of insect resistance and they control insect pests resistant to other insecticides. Two factors, however, have limited the use of B. thuringiensis
* Corresponding author. Tel. : +55 (43) 3714527; Fax: +55 (43) 3284440; E-mail: [email protected]
as a biopesticide : the poor persistence of its toxins and the spread of spores in the environment. B. thuringiensis spores could germinate and multiplicate in particular conditions in the insect larvae, which allows the spreading in surrounding areas. Occasionally, plasmid transfer could occur among B. thuringiensis strains (12) and correlated bacteria during growth within an insect. In addition, it has been established that Bacillus cereus and B. thuringiensis are opportunistic pathogens and some strains are responsible for various infections [3,4]. It was recently shown that B. thuringiensis produces a variety of potential virulence factors including phospholipases, hemolysins and enterotoxin [4]. The use of asporogenic strains that do not produce viable spores has been suggested [5,6]. This would protect the insecticidal crystal, which remains encapsulated in the vegetative cells, and would prevent the dissemination of viable spores in the environment. However, the survival and fate of asporogenic strains in environmental conditions has not been investigated. The ecological role of B. thuringiensis in the soil ecosystem is poorly understood. Generally, B. thuringiensis spores persist in the soil for several years, although there
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is a rapid decline in spore viability during the ¢rst few weeks after application [7^9]. Spore germination has never been demonstrated in non-amended soil. Gene transfer in soil systems has been reported for several bacterial species [10]. However, little is known about the transfer of genetic material in B. thuringiensis cells growing in environmental conditions. Haack et al. [11] observed a conjugation-like event between Bacillus subtilis and B. thuringiensis in non-amended soil, using Tn916mediated genetic exchange. Plasmid conjugation between two B. thuringiensis strains has been observed in nonamended sterile soils with neutral pH and various organic matter contents [12]. In this study, we report the vegetative cell dynamics of asporogenic and sporogenic B. thuringiensis strains in terms of their survival, sporulation rate and plasmid conjugation in non-amended sterile soil. 2. Materials and methods 2.1. Bacterial strains The B. thuringiensis strains used in this study are listed in Table 1. B. thuringiensis var. kurstaki KT0(pHT73-EmR ) harbors the 75 kb pHT73-EmR plasmid, which bears an erythromycin resistance marker and the cry1Ac gene encoding active toxin. 2.2. Soil For all experiments, samples of a clay soil were collected from a site (Londrina, Parana¨ State, Brazil) with no plant cover. Soil samples were collected to a maximum depth of 10 cm. The samples were sieved to remove debris and stored at room temperature. The same batch of soil was used for all experiments. The soil had an initial pH of 5.1 and its mineral content was: P (ppm): 0.2, K: 0.008, Ca: 1.7, Mg: 0.77, H+Al: 3.79, Al: 0.00 (all in milli equivalents per 100 g of soil). It contained 0.12% organic matter. The collected soil was dried, and 35 g was placed in Petri dishes and sterilized thrice at 121³C for 1 h each to prevent the indigenous soil microbial population from affecting the characteristics of the soil.
2.3. Conjugal transfer in broth An initial experiment was conducted to determine the conjugation ability of the asporogenic strain. The procedure was as described by Andrup et al. [13]. Equal volumes (250 Wl) of donor and recipient strain cultures grown until an OD600 of 1.0 (107 CFU ml31 ) were added to 7 ml of fresh prewarmed Luria-Bertani broth (LB) without antibiotics and the mixture was incubated at 30³C with moderate shaking (40 rpm) for 2 h. The pHT73-EmR plasmid was transferred using strain KT0(pHT73-EmR ) as the donor and strains 407-1 and 407-0A as recipients. Exconjugants were selected on LB agar plates containing either erythromycin (10 Wl ml31 ) and streptomycin (200 Wl ml31 ) or erythromycin (10 Wl ml31 ) and kanamycin (200 Wl ml31 ). Dilutions of the bacteria were plated onto LB agar containing the appropriate antibiotic, for counting of the recipient and donor cells. Conjugation frequencies were calculated by dividing the number of exconjugants by the number of donors. The recipient and donor controls, cultured separately, were tested in parallel. 2.4. Mating and survival in mixed soil culture The method used was similar to that described by VilasBoªas et al. [12]. In all conjugation experiments, sterilized soil samples were used without pH correction (5.1) and with no addition of nutrients. The donor and recipient cells were recovered in the exponential phase of growth, suspended in 0.85% NaCl and appropriate dilutions were incorporated into 35 g of soil in Petri dishes, to obtain 108 ^109 cells g31 of soil. The moisture content was corrected to 60% by adding sterile water. The KT0(pHT73EmR ) strain was used as the donor and the 407-1 and 4070A strains as recipient strains. The microcosm experiments were performed at 30³C for 20 days. Bacteria were extracted by mixing 3.2 g of soil with 18.8 ml of saline and shaking for 10 min in a pendular shaker. Appropriate dilutions were then plated on selective medium containing either erythromycin and streptomycin or erythromycin and kanamycin, and were incubated at 30³C for 18 h. Surviving recipients and donors (vegetative cells and spores) were counted using the same method as for conjugal transfer in broth. The transfer frequency was
Table 1 Strains of B. thuringiensis Subspecies/strain kurstaki KT0(pHT73-EmR ) thuringiensis 407-1 407-0A
Major characteristics
Resistance markers
Source
Cry
EmR
[12]
Cry3 , Pig Cry3 , Spo3
StrR KmR
[12] [5]
Cry : produces an insecticidal crystal; Cry3 : does not produce an insecticidal crystal; Pig : produces a brown pigment; Spo3 : asporogenic ; EmR : erythromycin resistant (10 Wg ml31 ); StrR : streptomycin resistant (200 Wg ml31 ); KmR : kanamycin resistant (200 Wg ml31 ).
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estimated from the number of exconjugants divided by the number of donors. The sporulation rate of the sporogenic strains was estimated by heating the original soil suspensions at 70³C for 20 min, diluting and plating on selective media. The ¢rst evaluation was made immediately after inoculation of the soil (t0 ) to assess the survival of the inoculated strains and to check that there were no cells resistant to both antibiotics in the bacterial population. Soil control samples containing only donor or recipient bacteria were also prepared under similar conditions using the appropriate antibiotics. It was previously demonstrated that genetic transfer occurs via conjugation rather than transformation or transduction. This was done using pHT301, a non-conjugative plasmid carrying an erythromycin resistance cassette. No transfer of pHT301 was observed in conditions similar to those described here [12]. 2.5. Survival in soil monoculture To study vegetative cell fate in soil monoculture, exponentially growing cells suspended in saline were used to inoculate separate soil samples, giving a ¢nal concentration of 108 ^109 cells g31 of soil. Each experiment was carried out in a Petri dish containing 35 g of dried soil incubated at 30³C. Cells were harvested at various times, up to the 20th day. Bacteria were counted and the sporulation rate determined as described above. We checked for the presence of pHT73-EmR in vegetative cells and spores by plating appropriate dilutions of the original soil suspensions onto LB agar plates without erythromycin. The obtained colonies were picked o¡ onto LB agar plates containing erythromycin to determine the proportion of plasmid-containing bacteria. The number of CFU for each plate was determined after incubation at 30³C for 18 h. 2.6. Statistical treatment Analysis of variance was used to assess the e¡ects of incubation in soil on various strains of B. thuringiensis and Student's t-test was used to test the signi¢cant di¡erences in plasmid transfer. The data were analyzed using SAS/STAT version 6.11 (Statistical Analysis Systems. SAS Institute, Cary, NC, USA). 3. Results and discussion 3.1. Conjugal transfer in broth In all experiments in which the KT0(pHT73-EmR ) strain was the donor, exconjugant clones were selected by double antibiotic resistance, by monitoring crystal
257
Fig. 1. Survival and sporulation of B. thuringiensis in soil monoculture. Strain KT0(pHT73-EmR ), b ; strain 407-0A, S; spores of KT0(pHT73EmR ), O. Vertical bars correspond to the MSD values. Strain 407-0A, S after the 8th day of incubation was below the detection limit (about 30 CFU g31 of soil).
and spore production by microscopy and assessment of brown pigment production. Conjugation occurred in all mating pairs tested. The plasmid transfer frequency was 1.2 þ 0.1U1034 and 7.5 þ 4.2U1034 exconjugants per donor, if sporogenic and asporogenic strains were used as the recipient, respectively (three replicates for each experiment). The di¡erence between these two frequencies is not signi¢cant. The transfer of genetic material between B. thuringiensis sporogenic strains in broth has often been described [14^17]. However, conjugation using a genetically characterized asporogenic strain has never been described. This study shows that a spo0A mutation that completely abolishes the initiation of sporulation did not a¡ect the ability to receive plasmids. 3.2. Survival in soil B. thuringiensis survival was studied in sterilized soil monoculture samples. In four identical and independent experiments the number of viable KT0(pHT73-EmR ) cells decreased from 4.0U108 cells g31 to 8.3U107 g31 of soil after 24 h of incubation and stabilized over the following days (Fig. 1). An initial decrease in the cell population has often been observed in soil microcosms [9,18,19], and in ¢eld experiments [9,20,21]. We found that the stabilization in the number of viable cells corresponded to spore formation. Indeed, after 24 h of incubation, about 30% of the viable cells were recovered as heat-resistant spores and after 4 days of incubation, 100% of the cells were heatresistant spores (Fig. 1). This suggests that the vegetative cells did not multiply and that the spores did not germinate in the soil microcosm. The asporogenic strain showed a decrease from 1.5U109 to 1.5U108 g31 of soil in the number of cells recovered after 24 h of incubation (Fig. 1). The number of viable cells did not change signi¢cantly thereafter until the 5th
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Fig. 2. Conjugation of B. thuringiensis in mixed soil culture. Frequency of exconjugants originating from the mating pair KT0(pHT73-EmR )/ 407-1, b. Frequency of exconjugants originating from the mating pair KT0(pHT73-EmR )/407-0A, E. Vertical bars correspond to the MSD values.
day of incubation when it decreased substantially, and after the eighth day of incubation, the number of asporogenic cells was below the detection threshold (about 30 CFU g31 of soil). The rate of survival of the asporogenic strain was signi¢cantly di¡erent from that of the sporogenic strain. We tested whether pHT73-EmR was maintained in KT0(pHT73-EmR ) and asporogenic exconjugant strains in soil monocultures. The plasmid was present and stable in both strains throughout the entire incubation period, because 99 to 100% of the cells recovered were resistant to erythromycin. For strains incubated in mixed soil culture, the dynamics of sporogenic and asporogenic cells were similar to the dynamics in soil monoculture. 3.3. Mating in soil Fig. 2 shows the frequency of exconjugants obtained at various times of incubation of B. thuringiensis cells in soil. No plasmid transfer was detected at time zero of the experiment. The frequency of exconjugant cells recovered from soil was 0.5 þ 0.2U1036 per donor cell for the mating pair KT0(pHT73-EmR )/407-1 and 1.0 þ 0.2U1036 per donor cell for the mating pair KT0(pHT73-EmR )/407-0A, detected after 4 h of incubation. In four identical and independent experiments, the frequency of exconjugants showed a 10-fold increase for both mating pairs between t4 and t24 and then, stabilized. No signi¢cant di¡erence in the frequency of conjugation was observed between the mating pairs. After the 5th day of incubation, the number of exconjugants, resulting from the mating pair KT0(pHT73EmR )/407-0A, decreased. This was due to the poor persistence of asporogenic cells in soil.
The stabilization in the rate of exconjugants for sporogenic mating pairs coincided with the appearance of heatresistant spores and was associated with the onset of the stationary phase. The conjugation e¤ciency of pHT73-EmR in unamended soil microcosms was similar to that in soil at pH 7 [12]. This suggests that pH di¡erences (5.1 to 7) did not a¡ect the conjugation process. This is not consistent with other studies reporting that pH is a limiting factor for conjugation in Pseudomonas and Bacillus species [22,23]. However, it is di¤cult to make comparisons because the di¡erence in results may be due to the speci¢c characteristics of the soils used in the experiments. The commercial use of B. thuringiensis as a biopesticide is increasing and results in a massive release of spores into the environment (1015 spores per hectare, 3000 tons per year) [24]. This study shows that the environmental impact of asporogenic B. thuringiensis strains is lower than that of sporogenic B. thuringiensis strains because asporogenic strains do not persist in the environment. In addition, the use of asporogenic strains could minimize the risk of gene transfer in soil, because conjugation does not seem to occur at a detectable level in such strains during the stationary phase in which the bacteria are released for agricultural applications. Acknowledgements We thank Joa¬o de Godoy Bueno for technical assistance, Michel Gohar for statistical advice, Weda A. Westin for secretarial assistance and Julie Knight for revising the English manuscript. This work was supported by research funds from the Fundac°a¬o Banco do Brasil, Universidade Estadual de Londrina, Institut National de la Recherche Agronomique and Institut Pasteur. L.A.V.-B. and G.F.L.T.V.-B. were supported by fellowships from CAPES.
References [1] Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E., van Rie, J., Lereclus, D., Baum, J. and Dean, D.H. (1998) Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 807^813. [2] Schnepf, E., Crickmore, N., van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R. and Dean, D.H. (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62, 775^806. [3] Drobniewski, F.A. (1993) Bacillus cereus and related species. Clin. Microbiol. Rev. 6, 324^338. [4] Agaisse, H., Gominet, M., Okstad, O.A., Kolsto, A.B. and Lereclus, D. (1999) PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Mol. Microbiol. 32, 1043^1053. [5] Lereclus, D., Agaisse, H., Gominet, M. and Chaufaux, J. (1995)
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[6]
[7]
[8] [9]
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[13]
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[15]
Overproduction of encapsulated insecticidal crystal proteins in a Bacillus thuringiensis spo0A mutant. Biotechnology 13, 67^71. Sanchis, V., Agaisse, H., Chaufaux, J. and Lereclus, D. (1996) Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site speci¢c recombinant vector. J. Biotechnol. 48, 81^96. Pruett, C.J.H., Burges, H.D. and Wyborn, C.H. (1980) E¡ect of exposure to soil on potency and spore viability of Bacillus thuringiensis. J. Invert. Pathol. 35, 168^174. Petras, S.F. and Casida Jr., L.E. (1985) Survival of Bacillus thuringiensis spores in soil. Appl. Environ. Microbiol. 50, 1496^1501. Pedersen, J.C., Damgaard, P.H., Eilenberg, J. and Hansen, B.M. (1995) Dispersal of Bacillus thuringiensis var. kurstaki in an experimental cabbage ¢eld. Can. J. Microbiol. 41, 118^125. Hill, K.E. and Top, E.M. (1998) Gene transfer in soil using microcosms. FEMS Microbiol. Ecol. 25, 319^329. Haack, B.J., Andrews, R.E. and Loynachan, T.E. (1996) Tn916mediated genetic exchange in soil. Soil Biol. Biochem. 28, 765^771. Vilas-Boªas, G.F.L.T., Vilas-Boªas, L.A., Lereclus, D. and Arantes, O.M.N. (1998) Bacillus thuringiensis conjugation in environmental conditions. FEMS Microbiol. Ecol. 25, 369^374. Andrup, L., Jorgensen, O., Wilcks, A., Smidt, L. and Jensen, J.B. (1996) Mobilization of `nonmobilizable' plasmids by the aggregationmediated conjugation system of Bacillus thuringiensis. Plasmid 36, 1^ 11. Gonza¨lez Jr., J.M., Brown, B.J. and Carlton, B.C. (1982) Transfer of Bacillus thuringiensis plasmids coding for N-endotoxin among strains of Bacillus thuringiensis and Bacillus cereus. Proc. Natl. Acad. Sci. USA 79, 6951^6955. Chapman, J.S. and Carlton, B.C. (1985) Conjugal plasmid transfer in Bacillus thuringiensis. In: Plasmids in Bacteria (Helinski, D.R., Cohen, S.N., Clewell, D.B., Jackson, D.A. and Hollaender, A., Eds.), pp. 453^467. Plenum Publishing Corporation, New York.
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[16] Andrup, L., Damgaard, J. and Wassermann, K. (1993) Mobilization of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by speci¢c aggregation. J. Bacteriol. 175, 6530^6536. [17] Jensen, G.B., Wilcks, A., Petersen, S.S., Damgaard, J., Baum, J.A. and Andrup, L. (1995) The genetic basis of the aggregation system in Bacillus thuringiensis subsp. israelensis is located on the large conjugative plasmid pXO16. J. Bacteriol. 177, 2914^2917. [18] Saleh, S.M., Harris, R.F. and Allen, O.N. (1970) Fate of Bacillus thuringiensis in soil: e¡ect of soil pH and organic amendment. Can. J. Microbiol. 16, 677^680. [19] Akiba, Y. (1986) Microbial ecology of Bacillus thuringiensis VI. Germination of Bacillus thuringiensis spores in the soil. Appl. Entomol. Zool. 21, 76^80. [20] Salamitou, S., Marchal, M. and Lereclus, D. (1996) Bacillus thuringiensis: un pathoge©ne facultatif. Ann. Inst. Pasteur 7 (4), 285^296. [21] Eskils, K. and Lo«vgren, A. (1997) Release of Bacillus thuringiensis subsp. israelensis in Swedish soil. FEMS Microbiol. Ecol. 43, 1142^ 1150. [22] Van Elsas, J.D., Govaert, J.M. and van Veen, J.A. (1987) Transfer of plasmid pFT30 between bacilli in soil as in£uenced by bacterial population dynamics and soil conditions. Soil Biol. Biochem. 19, 639^ 647. [23] Ramos-Gonzalez, M.I., Duque, E. and Ramos, J.L. (1991) Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids. Appl. Environ. Microbiol. 57, 3020^3027. [24] Bernhard, K. and Utz, R. (1993) Production of Bacillus thuringiensis insecticides for experimental and commercial uses. In: Bacillus thuringiensis, an Environmental Biopesticide : Theory and Practice (Entwistle, P., Cory, J., Bailey, M. and Higgs, S., Eds.), pp. 255^267. John Wiley and Sons, Chichester.
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Survival and conjugation of Bacillus thuringiensis in a soil microcosm Laurival A. Vilas-Boªas a;c , Gislayne F.L.T. Vilas-Boªas a;c , Halha O. Saridakis b , Manoel Victor F. Lemos c , Didier Lereclus d;e , Olivia M.N. Arantes a; * a
Bio/CCB, Universidade Estadual de Londrina 86051-970, Londrina, Brazil Micro/CCB, Universidade Estadual de Londrina 86051-970, Londrina, Brazil c FCAV/UNESP, Rod. Carlos Tonanni Km5, 14870-000, Jaboticabal, Brazil Unite¨ de Biochimie Microbienne, lnstitut Pasteur, 25, rue du Dr. Roux, 75724 Paris, Cedex 15, France e Station de Recherches de Lutte Biologique, INRA, La Minie©re, 78285 Guyancourt, Cedex, France b
d
Received 2 August 1999; received in revised form 21 December 1999; accepted 22 December 1999
Abstract The survival and conjugation ability of sporogenic and asporogenic Bacillus thuringiensis strains were investigated in broth, in nonamended sterile clay soil monoculture and in mixed soil culture. The 75 kb pHT73 plasmid carrying an erythromycin resistance determinant and a cry1Ac gene was transferred in mating broth and soil microcosm. Survival of strains was assessed in soil monoculture and in mixed soil culture for up to 20 days. Sporogenic strains rapidly formed viable spores which were maintained until the end of the experiment. The asporogenic strains were no longer recovered after 8 days of incubation. This study shows that the environmental impact of asporogenic B. thuringiensis strains is lower than that of sporogenic B. thuringiensis strains. Thus, the use of asporogenic strains may significantly reduce any potential risk (gene transfer, soil and plant contamination) due to the dissemination of B. thuringiensis-based biopesticides in the environment. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Sporulation mutant; Plasmid; Conjugation; Soil microcosm; Bacillus thuringiensis
1. Introduction The Gram-positive ubiquitous bacterium Bacillus thuringiensis produces a proteinaceous crystal during the stationary phase. The crystal proteins, designated Cry proteins, are toxic against the larval forms of several insects of agronomic and medical importance [1]. Strains of B. thuringiensis have been isolated around the world from various sources including soil, stored product dust, dead and living insects [2]. The use of commercial preparations of B. thuringiensis as agricultural insecticides has increased in recent years, and they now account for more than 90% of the biopesticides used. B. thuringiensis formulations present many advantages such as no toxicity for animals, high speci¢city, low development of insect resistance and they control insect pests resistant to other insecticides. Two factors, however, have limited the use of B. thuringiensis
* Corresponding author. Tel. : +55 (43) 3714527; Fax: +55 (43) 3284440; E-mail: [email protected]
as a biopesticide : the poor persistence of its toxins and the spread of spores in the environment. B. thuringiensis spores could germinate and multiplicate in particular conditions in the insect larvae, which allows the spreading in surrounding areas. Occasionally, plasmid transfer could occur among B. thuringiensis strains (12) and correlated bacteria during growth within an insect. In addition, it has been established that Bacillus cereus and B. thuringiensis are opportunistic pathogens and some strains are responsible for various infections [3,4]. It was recently shown that B. thuringiensis produces a variety of potential virulence factors including phospholipases, hemolysins and enterotoxin [4]. The use of asporogenic strains that do not produce viable spores has been suggested [5,6]. This would protect the insecticidal crystal, which remains encapsulated in the vegetative cells, and would prevent the dissemination of viable spores in the environment. However, the survival and fate of asporogenic strains in environmental conditions has not been investigated. The ecological role of B. thuringiensis in the soil ecosystem is poorly understood. Generally, B. thuringiensis spores persist in the soil for several years, although there
0168-6496 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 0 0 2 - 7
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is a rapid decline in spore viability during the ¢rst few weeks after application [7^9]. Spore germination has never been demonstrated in non-amended soil. Gene transfer in soil systems has been reported for several bacterial species [10]. However, little is known about the transfer of genetic material in B. thuringiensis cells growing in environmental conditions. Haack et al. [11] observed a conjugation-like event between Bacillus subtilis and B. thuringiensis in non-amended soil, using Tn916mediated genetic exchange. Plasmid conjugation between two B. thuringiensis strains has been observed in nonamended sterile soils with neutral pH and various organic matter contents [12]. In this study, we report the vegetative cell dynamics of asporogenic and sporogenic B. thuringiensis strains in terms of their survival, sporulation rate and plasmid conjugation in non-amended sterile soil. 2. Materials and methods 2.1. Bacterial strains The B. thuringiensis strains used in this study are listed in Table 1. B. thuringiensis var. kurstaki KT0(pHT73-EmR ) harbors the 75 kb pHT73-EmR plasmid, which bears an erythromycin resistance marker and the cry1Ac gene encoding active toxin. 2.2. Soil For all experiments, samples of a clay soil were collected from a site (Londrina, Parana¨ State, Brazil) with no plant cover. Soil samples were collected to a maximum depth of 10 cm. The samples were sieved to remove debris and stored at room temperature. The same batch of soil was used for all experiments. The soil had an initial pH of 5.1 and its mineral content was: P (ppm): 0.2, K: 0.008, Ca: 1.7, Mg: 0.77, H+Al: 3.79, Al: 0.00 (all in milli equivalents per 100 g of soil). It contained 0.12% organic matter. The collected soil was dried, and 35 g was placed in Petri dishes and sterilized thrice at 121³C for 1 h each to prevent the indigenous soil microbial population from affecting the characteristics of the soil.
2.3. Conjugal transfer in broth An initial experiment was conducted to determine the conjugation ability of the asporogenic strain. The procedure was as described by Andrup et al. [13]. Equal volumes (250 Wl) of donor and recipient strain cultures grown until an OD600 of 1.0 (107 CFU ml31 ) were added to 7 ml of fresh prewarmed Luria-Bertani broth (LB) without antibiotics and the mixture was incubated at 30³C with moderate shaking (40 rpm) for 2 h. The pHT73-EmR plasmid was transferred using strain KT0(pHT73-EmR ) as the donor and strains 407-1 and 407-0A as recipients. Exconjugants were selected on LB agar plates containing either erythromycin (10 Wl ml31 ) and streptomycin (200 Wl ml31 ) or erythromycin (10 Wl ml31 ) and kanamycin (200 Wl ml31 ). Dilutions of the bacteria were plated onto LB agar containing the appropriate antibiotic, for counting of the recipient and donor cells. Conjugation frequencies were calculated by dividing the number of exconjugants by the number of donors. The recipient and donor controls, cultured separately, were tested in parallel. 2.4. Mating and survival in mixed soil culture The method used was similar to that described by VilasBoªas et al. [12]. In all conjugation experiments, sterilized soil samples were used without pH correction (5.1) and with no addition of nutrients. The donor and recipient cells were recovered in the exponential phase of growth, suspended in 0.85% NaCl and appropriate dilutions were incorporated into 35 g of soil in Petri dishes, to obtain 108 ^109 cells g31 of soil. The moisture content was corrected to 60% by adding sterile water. The KT0(pHT73EmR ) strain was used as the donor and the 407-1 and 4070A strains as recipient strains. The microcosm experiments were performed at 30³C for 20 days. Bacteria were extracted by mixing 3.2 g of soil with 18.8 ml of saline and shaking for 10 min in a pendular shaker. Appropriate dilutions were then plated on selective medium containing either erythromycin and streptomycin or erythromycin and kanamycin, and were incubated at 30³C for 18 h. Surviving recipients and donors (vegetative cells and spores) were counted using the same method as for conjugal transfer in broth. The transfer frequency was
Table 1 Strains of B. thuringiensis Subspecies/strain kurstaki KT0(pHT73-EmR ) thuringiensis 407-1 407-0A
Major characteristics
Resistance markers
Source
Cry
EmR
[12]
Cry3 , Pig Cry3 , Spo3
StrR KmR
[12] [5]
Cry : produces an insecticidal crystal; Cry3 : does not produce an insecticidal crystal; Pig : produces a brown pigment; Spo3 : asporogenic ; EmR : erythromycin resistant (10 Wg ml31 ); StrR : streptomycin resistant (200 Wg ml31 ); KmR : kanamycin resistant (200 Wg ml31 ).
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estimated from the number of exconjugants divided by the number of donors. The sporulation rate of the sporogenic strains was estimated by heating the original soil suspensions at 70³C for 20 min, diluting and plating on selective media. The ¢rst evaluation was made immediately after inoculation of the soil (t0 ) to assess the survival of the inoculated strains and to check that there were no cells resistant to both antibiotics in the bacterial population. Soil control samples containing only donor or recipient bacteria were also prepared under similar conditions using the appropriate antibiotics. It was previously demonstrated that genetic transfer occurs via conjugation rather than transformation or transduction. This was done using pHT301, a non-conjugative plasmid carrying an erythromycin resistance cassette. No transfer of pHT301 was observed in conditions similar to those described here [12]. 2.5. Survival in soil monoculture To study vegetative cell fate in soil monoculture, exponentially growing cells suspended in saline were used to inoculate separate soil samples, giving a ¢nal concentration of 108 ^109 cells g31 of soil. Each experiment was carried out in a Petri dish containing 35 g of dried soil incubated at 30³C. Cells were harvested at various times, up to the 20th day. Bacteria were counted and the sporulation rate determined as described above. We checked for the presence of pHT73-EmR in vegetative cells and spores by plating appropriate dilutions of the original soil suspensions onto LB agar plates without erythromycin. The obtained colonies were picked o¡ onto LB agar plates containing erythromycin to determine the proportion of plasmid-containing bacteria. The number of CFU for each plate was determined after incubation at 30³C for 18 h. 2.6. Statistical treatment Analysis of variance was used to assess the e¡ects of incubation in soil on various strains of B. thuringiensis and Student's t-test was used to test the signi¢cant di¡erences in plasmid transfer. The data were analyzed using SAS/STAT version 6.11 (Statistical Analysis Systems. SAS Institute, Cary, NC, USA). 3. Results and discussion 3.1. Conjugal transfer in broth In all experiments in which the KT0(pHT73-EmR ) strain was the donor, exconjugant clones were selected by double antibiotic resistance, by monitoring crystal
257
Fig. 1. Survival and sporulation of B. thuringiensis in soil monoculture. Strain KT0(pHT73-EmR ), b ; strain 407-0A, S; spores of KT0(pHT73EmR ), O. Vertical bars correspond to the MSD values. Strain 407-0A, S after the 8th day of incubation was below the detection limit (about 30 CFU g31 of soil).
and spore production by microscopy and assessment of brown pigment production. Conjugation occurred in all mating pairs tested. The plasmid transfer frequency was 1.2 þ 0.1U1034 and 7.5 þ 4.2U1034 exconjugants per donor, if sporogenic and asporogenic strains were used as the recipient, respectively (three replicates for each experiment). The di¡erence between these two frequencies is not signi¢cant. The transfer of genetic material between B. thuringiensis sporogenic strains in broth has often been described [14^17]. However, conjugation using a genetically characterized asporogenic strain has never been described. This study shows that a spo0A mutation that completely abolishes the initiation of sporulation did not a¡ect the ability to receive plasmids. 3.2. Survival in soil B. thuringiensis survival was studied in sterilized soil monoculture samples. In four identical and independent experiments the number of viable KT0(pHT73-EmR ) cells decreased from 4.0U108 cells g31 to 8.3U107 g31 of soil after 24 h of incubation and stabilized over the following days (Fig. 1). An initial decrease in the cell population has often been observed in soil microcosms [9,18,19], and in ¢eld experiments [9,20,21]. We found that the stabilization in the number of viable cells corresponded to spore formation. Indeed, after 24 h of incubation, about 30% of the viable cells were recovered as heat-resistant spores and after 4 days of incubation, 100% of the cells were heatresistant spores (Fig. 1). This suggests that the vegetative cells did not multiply and that the spores did not germinate in the soil microcosm. The asporogenic strain showed a decrease from 1.5U109 to 1.5U108 g31 of soil in the number of cells recovered after 24 h of incubation (Fig. 1). The number of viable cells did not change signi¢cantly thereafter until the 5th
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Fig. 2. Conjugation of B. thuringiensis in mixed soil culture. Frequency of exconjugants originating from the mating pair KT0(pHT73-EmR )/ 407-1, b. Frequency of exconjugants originating from the mating pair KT0(pHT73-EmR )/407-0A, E. Vertical bars correspond to the MSD values.
day of incubation when it decreased substantially, and after the eighth day of incubation, the number of asporogenic cells was below the detection threshold (about 30 CFU g31 of soil). The rate of survival of the asporogenic strain was signi¢cantly di¡erent from that of the sporogenic strain. We tested whether pHT73-EmR was maintained in KT0(pHT73-EmR ) and asporogenic exconjugant strains in soil monocultures. The plasmid was present and stable in both strains throughout the entire incubation period, because 99 to 100% of the cells recovered were resistant to erythromycin. For strains incubated in mixed soil culture, the dynamics of sporogenic and asporogenic cells were similar to the dynamics in soil monoculture. 3.3. Mating in soil Fig. 2 shows the frequency of exconjugants obtained at various times of incubation of B. thuringiensis cells in soil. No plasmid transfer was detected at time zero of the experiment. The frequency of exconjugant cells recovered from soil was 0.5 þ 0.2U1036 per donor cell for the mating pair KT0(pHT73-EmR )/407-1 and 1.0 þ 0.2U1036 per donor cell for the mating pair KT0(pHT73-EmR )/407-0A, detected after 4 h of incubation. In four identical and independent experiments, the frequency of exconjugants showed a 10-fold increase for both mating pairs between t4 and t24 and then, stabilized. No signi¢cant di¡erence in the frequency of conjugation was observed between the mating pairs. After the 5th day of incubation, the number of exconjugants, resulting from the mating pair KT0(pHT73EmR )/407-0A, decreased. This was due to the poor persistence of asporogenic cells in soil.
The stabilization in the rate of exconjugants for sporogenic mating pairs coincided with the appearance of heatresistant spores and was associated with the onset of the stationary phase. The conjugation e¤ciency of pHT73-EmR in unamended soil microcosms was similar to that in soil at pH 7 [12]. This suggests that pH di¡erences (5.1 to 7) did not a¡ect the conjugation process. This is not consistent with other studies reporting that pH is a limiting factor for conjugation in Pseudomonas and Bacillus species [22,23]. However, it is di¤cult to make comparisons because the di¡erence in results may be due to the speci¢c characteristics of the soils used in the experiments. The commercial use of B. thuringiensis as a biopesticide is increasing and results in a massive release of spores into the environment (1015 spores per hectare, 3000 tons per year) [24]. This study shows that the environmental impact of asporogenic B. thuringiensis strains is lower than that of sporogenic B. thuringiensis strains because asporogenic strains do not persist in the environment. In addition, the use of asporogenic strains could minimize the risk of gene transfer in soil, because conjugation does not seem to occur at a detectable level in such strains during the stationary phase in which the bacteria are released for agricultural applications. Acknowledgements We thank Joa¬o de Godoy Bueno for technical assistance, Michel Gohar for statistical advice, Weda A. Westin for secretarial assistance and Julie Knight for revising the English manuscript. This work was supported by research funds from the Fundac°a¬o Banco do Brasil, Universidade Estadual de Londrina, Institut National de la Recherche Agronomique and Institut Pasteur. L.A.V.-B. and G.F.L.T.V.-B. were supported by fellowships from CAPES.
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