Release of Bacillus thuringiensis subsp. israelensis in Swedish Soil

Abstract

A streptomycin-resistant strain of Bacillus thuringiensis subsp. israelensis (BtN14) was released in a field experiment and the effect on the indigenous bacterial flora was analyzed. A minor, transient increase of the concentration of B. thuringiensis/cereus-like bacteria was detected. The released strain was able to establish itself at a low number, but the total concentration of B. thuringiensis subsp. israelensis was not affected. BtN14 was detected outside the release site, but decreased to a concentration below the detection limit (20 cfu g⁻¹ soil) at the end of the test period. Laboratory experiments showed that the released strain can grow in non-sterilized as well as sterilized soil, but that growth was very slow and transient in non-sterilized soil. Seven weeks after release, no remaining effects on the total bacterial population could be detected.

Bacillus thuringiensis subsp. israelensis, Biopesticide, Release, Soil bacteria

1 Introduction

Bacillus thuringiensis is a Gram-positive spore-forming bacterium, pathogenic to a variety of insect species. Strains of B. thuringiensis have been isolated from sources all over the world, most commonly from soil [1], but also from dead insect larvae [2], and the phylloplane [3]. A number of subspecies and strains are identified based on serotyping, biochemical assays, toxicity or other features [4], and new strains and toxin variants are frequently reported [5]. B. thuringiensis is closely related to B. cereus, and is distinguished from this species only by the presence of a protein crystal produced during sporulation. Different subspecies are commercially used against insect pests [6,7].B. thuringiensis subsp. kurstaki has been used as an insecticide for more than 30 years, mainly against Lepidopteran larvae [7]. B. thuringiensis subsp. israelensis is also available in commercial preparations for use against Dipteran larvae [6–8]. B. thuringiensis insecticides consist of a mixture of crystals and spores, the latter being either viable or UV-inactivated.

Although effects of releasing B. thuringiensis biopesticides have been studied previously [9–12], most studies consider the fate of B. thuringiensis toxin or the released strain over cultivated areas. No study deals with possible effects on indigenous B. thuringiensis/cereus-like and B. thuringiensis subsp. israelensis populations at a site with no previous agricultural activity, where a viable B. thuringiensis preparation is released. Before releasing such a viable biopesticide, safety aspects must be taken into consideration. B. thuringiensis is considered harmless to man [6], but the long-term effect on indigenous soil and sediment flora and fauna is so far poorly investigated. Commonly desired traits of an introduced organism are non-virulence against non-pest organisms and low probability of gene transfer, for instance low probability of transfer of antibiotic resistance plasmids and/or genes. Other important factors to consider are the abilities of the strain to survive, grow, or eventually become the dominant species, as well as the ability to spread to surrounding areas.

In this study, we applied a streptomycin-resistant derivative of B. thuringiensis subsp. israelensis over a small area (10 m²) and followed the effects of some indigenous microflora, especially the B. thuringiensis/cereus-like and B. thuringiensis subsp. israelensis populations. We also screened for the released strain outside the application site at a swampland in Tärnsjö in central Sweden. The area is known for its abundance of mosquitoes, against which the use of B. thuringiensis insecticides is currently being discussed.

2 Materials and methods

2.1 Strains and media

A commercially used B. thuringiensis subsp. israelensis strain from Novo Nordisk A/S, Denmark (Skeetal), was plated on LA (1% NaCl, 1% tryptone, 0.5% yeast extract, 1% agar) containing streptomycin (100 μg ml⁻¹) and incubated overnight at 30°C. Of several selected colonies, one had a growth rate in LB (LA without agar) similar to the parental strain. The streptomycin-resistant strain, BtN14, was fermented in streptomycin-supplemented (100 μg ml⁻¹) fermentation broth (4% maltodextrin (Maltrin® M500 obtained from Bröste AB, Mölndal, Sweden) (w/v), 1.5% corn steep liquor (Sigma) (v/v), 3% peptone (w/v) and 0.3% K₂HPO₄ with a final pH of 7.0) at 30°C for 80 h. During fermentation, the pH was held at 7.0 using 2 M H₃PO₄ and 5 M NaOH. Spore and crystal production was monitored by phase-contrast microscopy. Viable counts on unheated and heated (80°C, 10 min) fermented BtN14 on LA plates gave total and spore content. After incubation overnight at 30°C, the total and spore-forming concentrations were calculated.

2.2 Release of B. thuringiensis subsp. israelensis

For the field release of BtN14, a 2×5 m plot was selected close to the water in a swampland area at Tärnsjö, Sweden. The fermented strain was diluted with sterile water to the appropriate concentration and was thoroughly spread over the test area with a hand dispenser, to a final concentration of 1.3×10⁷ cfu cm⁻², similar to concentrations recommended for commercial use. Samples were taken from the plot before and 30 minutes after release. Subsequent samples were collected at intervals during the following 100 days (Fig. 3).

3

A: Concentrations of (a) culturable bacteria, (b) B. thuringiensis/cereus-like bacteria, (c) total B. thuringiensis subsp. israelensis, and (d) released BtN14. Concentrations are indicated as cfu g⁻¹ soil as dry weight in the application plot during the 100-day test period. Three samples were collected at each time point and each sample was analyzed in duplicate. B: Concentrations presented as relative amounts of (a) B. thuringiensis/cereus-like bacteria to total culturable bacteria, (b) B. thuringiensis subsp. israelensis to B. thuringiensis/cereus-like bacteria, (c) B. thuringiensis subsp. israelensis to total culturable bacteria, (d) BtN14 to B. thuringiensis subsp. israelensis, and (e) BtN14 to total culturable bacteria.

2.3 Soil sampling and analysis

Two 50 ml plastic tubes were aseptically filled with soil at each sampling site and kept at −20°C until analyzed. The soil samples from the test plot were taken as above, but each time three samples were taken, one from each short end and one from the middle of the plot, to minimize sample variations by calculating a mean from the three samples. Soil samples were from the top 10 cm of soil and were rich in more or less decayed organic matter. 1 g duplicates of soil from each tube were mixed in 9 ml 0.9% NaCl, diluted to appropriate concentrations and spread on LA, the B. thuringiensis or B. cereus selective T3 plates [13], and LA-Str (LA with streptomycin; 100 μg ml⁻¹). The plates were incubated overnight at 30°C. To calculate spore content, 3 ml of the original suspension was heated to 80°C for 10 min, diluted, and plated as above. Total bacterial concentration was calculated, as was the concentration of B. thuringiensis/cereus-like bacteria (selection based on colony morphology; white, flat, large, non-mucoid colonies were designated B. thuringiensis/cereus-like). For estimation of B. thuringiensis subsp. israelensis content, plates with 10–1000 colonies were selected for colony blotting and immunodetection. The released strain BtN14 was detected using LA-Str and the immunodetection method.

2.4 Immunodetection of B. thuringiensis subsp. israelensis

Antiserum was raised in rabbits against purified B. thuringiensis subsp. israelensis flagella, as described in Lövgren et al. [14]. The serum was filter sterilized after pre-adsorption with whole cell cultures of Escherichia coli, B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. gelechiae.

Colonies were transferred to nitrocellulose membrane filters by placing the filters on the plates until wetted. To kill the bacteria, membranes were soaked in 0.5 M sodium hydroxide and then washed in buffer 1 (1 M Tris-HCl, pH 7.5), blocked in 5% BSA in PBS (w/v), washed in washing buffer 2 (0.05% Tween 20 in PBS (v/v)) and incubated overnight at room temperature with diluted antiserum against purified B. thuringiensis subsp. israelensis flagella. After washing three times in washing buffer 2, the filters were incubated with alkaline phosphatase conjugated secondary antibodies (Sigma A-7778), washed, and developed according to the manufacturer's instructions. A control membrane with B. thuringiensis subsp. gelechiae, B. cereus, BtN14 and B. thuringiensis subsp. israelensis was always included.

2.5 Statistical analysis of data from the release plot

For the different samples at the release area, the relative amounts of B. thuringiensis/cereus-like colonies were analyzed with respect to average where y=sum of concentrations through sample number and standard deviation where s=square root of the sum of ((concentration−y)² through (sample number−1)). Based on the total high number of samples and the central limit theorem [15], the statistical analysis could be performed as for a normally distributed population. Student's t-test was performed as described by Box et al. [15] to identify discrepancies between the samples at the different time points.

2.6 Growth of BtN14 in sterile soil

50 g of humus-rich forest soil was heat-sterilized three times at 121°C for 20 min at a pressure of 1.1 bar, with overnight incubation between sterilizations to allow spore germination. The water loss was compensated for by the addition of different concentrations of BtN14 diluted in sterile 0.9% NaCl. The BtN14-amended soil was incubated at 30°C for 15 days. Samples were taken at specific intervals in 1 g duplicates from each flask, suspended in sterile 0.9% NaCl as above, and plated on LA. The plates were incubated overnight at 30°C after which the resulting colonies were counted.

2.7 Growth of BtN14 in non-sterile soil

BtN14 was added at different concentrations to non-sterilized soil lacking indigenous streptomycin-resistant B. thuringiensis subsp. israelensis. The microcosms were incubated at room temperature. At specific time intervals, samples were taken as above and plated on streptomycin-amended (100 μg ml⁻¹) LA plates. After incubation, colonies with the typical B. thuringiensis subsp. israelensis morphology were counted.

3 Results

3.1 Description of release area

The area chosen for investigation is situated close to the water line of a shallow lake and swampland that are connected to the river system of Dalälven, approximately 30 km north-east of Sala, Sweden. Flooding of the swampland and areas surrounding the water system is common in the spring; this provides excellent breeding sites for mosquitoes. Because of the abundance of mosquitoes and birds, the latter including some rare species, Bacillus thuringiensis subsp. israelensis has been proposed as a biological pesticide. Only at the peaks of mosquito numbers would control be proposed; long-term effects are undesired. The consequences of a B. thuringiensis subsp. israelensis release were, however, not easily predictable, since virtually nothing was known about the indigenous occurrence of B. thuringiensis subsp. israelensis and the ability of a released strain to persist and grow in the local area.

The vegetation at the test site consisted mainly of grass and young leaf trees such as oak (Quercus robur), birch (Betula sp.), hazel (Corylus avellana) and Salix sp., and the soil is humus-rich, sandy clay moraine.

The map in Fig. 1 shows the sampling sites 9 months before release. Sites 1, 2, and 3 are located at the edge of the swampland. Site 4 is in a nearby forest and site 5 lies at the edge of a field. Our release area of 10 m² is located at sample site 1. The water line was up to 1 m from the plot at the release time, but was back to its normal small stream after 4 weeks. Because of the changes in water level during the test period, no samples to assay for spreading were taken on the stream side of the test area.

1

A map of the study area at Tärnsjö, Sweden. The numbers indicate soil sampling sites 9 months before release of a streptomycin-resistant B. thuringiensis subsp. israelensis strain, BtN14. Sites 1, 2, and 3 lie close to the wetter area at the water system. Site 4 is a control in the drier forest, and site 5 another control at the edge of a field. At site 1, BtN14 was released in a plot of 2×5 m at a concentration of 10⁷ cfu cm⁻². The broken lines indicate paths across the area. Open areas are shaded and wet are striped. (Published with permission from Lantmäteriverket 1997-03-24, Dnr 601-97-1242.)

3.2 Bacterial community structure before release

We started our investigation by analyzing the indigenous microbial population prior to the release of BtN14. Nine months before release, soil samples at three sites close to and in the swampland were collected, as were samples from the forest and field (Fig. 1). The soil samples were analyzed on the different media, and B. thuringiensis subsp. israelensis was detected by the immunoassay method. The concentrations of total culturable bacteria, B. thuringiensis/cereus-like bacteria and indigenous B. thuringiensis subsp. israelensis were determined (Fig. 2). The three sites close to water were shown to have markedly higher ratios of B. thuringiensis/cereus-like and B. thuringiensis subsp. israelensis colonies, while control sites 4 and 5 had lower concentrations of B. thuringiensis subsp. israelensis. The total fraction of B. thuringiensis/cereus-like bacteria appears similar.

2

Bacterial concentrations at five different sites at Tärnsjö, Sweden, 9 months before release. Open bars indicate total culturable bacteria, striped bars B. thuringiensis/cereus-like bacteria and dotted bars total B. thuringiensis subsp. israelensis. Concentrations are indicated as cfu g⁻¹ soil (wet weight). Sites 1, 2, and 3 are close to water and mosquito breeding holes.

3.3 Bacterial community structure after release

Three samples taken at different time points after release from the 2×5 m release plot at sampling site 1 (Fig. 1) were analyzed as described earlier (Fig. 3). We released BtN14 in late June and sampled at intervals for 100 days. During the experiment, climate conditions varied notably from start to end. The overall drop in bacterial numbers correlates well with a period of high mean temperature and no or little rain (Table 1). In Fig. 3B, the B. thuringiensis/cereus-like, total B. thuringiensis subsp. israelensis and released BtN14 are given as relative amounts in varied combinations. The total culturable bacteria were calculated only for use as a factor to minimize the effects of the varied climate conditions and other unknown factors. Soil samples are corrected for dry weight in Fig. 3A for the same reason. All points in the figure are the result of at least three independent double samples with correction for soil dry weights (data not shown). The selective T3 plates showed no difference in total culturable bacterial concentrations, nor in the B. thuringiensis/cereus-like population, compared to LA plates. We believe this is due to the unusually high number of B. thuringiensis/cereus-like bacteria and that our growth method favors B. thuringiensis/cereus. Therefore, both media were used for statistical analysis, giving a greater sample population. Concentrations were analyzed with respect to mean value and standard deviation. Student's t-test was performed for the different concentrations and the only significant difference in relative amounts is shown immediately after release of BtN14, when the fraction of B. thuringiensis/cereus-like colonies rose from 81% to 98% of the total culturable population (P=0.05). After 7 weeks, the ratio decreased to a level indistinguishable from that before release (no significant difference shown). B. thuringiensis subsp. israelensis was already present at a relatively high concentration (approx. 10⁶ cfu g⁻¹ soil) at the site before release (Figs. 2 and 3). The proportion of total B. thuringiensis subsp. israelensis (including BtN14) in total bacteria returned to initial levels after 7 weeks. BtN14, the released strain, never comprised more than 5% of total B. thuringiensis subsp. israelensis concentrations. Compared to total colony counts, released B. thuringiensis subsp. israelensis stabilized at a level of approximately 1% (Fig. 3).

1

Climate and soil bacteria at Tärnsjö in the period from time of release to the dry period

Week	Temp (°C)	Rain (mm)	Soil water (%)	Soil bacteria (cfu g−1)
0	16.0±3.6	17.1	62.5	1.8×106±5.6×105
1	15.5±2.9	0.9	59	1.8×106±7.0×105
2	14.4±2.1	8.2	60	1.5×106±4.1×105
3	17.7±1.6	18.5	NDa	ND
4	16.5±1.5	10.7	43	1.2×106±3.6×105
5	18.6±2.9	0.0	ND	ND
6	19.1±2.1	10.3	ND	ND
7	16.7±2.7	1.9	31	7.4×105±3.4×105
8	18.8±2.2	6.2	ND	ND
9	15.8±3.5	21.1	ND	ND

Temperature and rain data were obtained from SMHI (Swedish Meteorology and Hydrology Institute).

a

^aND=no data available.

3.4 Spreading of BtN14 outside the application site

Screening for streptomycin-resistant B. thuringiensis subsp. israelensis outside the release plot was done to test for spreading of the released strain. After 1 month, samples were taken in three directions at distances of 10, 25, and 50 m. The samples were spread on LA-Str and presumptive B. thuringiensis subsp. israelensis colonies were immunoassayed. As seen in Figure 4, the highest concentrations of streptomycin-resistant B. thuringiensis subsp. israelensis were detected south of the release site, closer to the water line. One month after release, the concentration was about 500 cfu g⁻¹ soil 10 m from the original site. At 25 m, the concentration was somewhat lower, about 100 cfu g⁻¹, but at 50 m, the concentration was highest, around 1000 cfu g⁻¹ soil. Three weeks later, the concentration at 10 m had dropped; and at 25 m no released BtN14 were detected. At 50 m, the strain was still detectable (about 300 cfu g⁻¹). No streptomycin-resistant B. thuringiensis subsp. israelensis were detected outside the release plot at the end of the examination period (7 weeks post release). To the north and east, the concentrations of streptomycin-resistant B. thuringiensis subsp. israelensis decreased to below the detection limit (20 cfu g⁻¹ soil wet weight) 3 weeks post release. In all directions outside the plot, BtN14 was present mainly as vegetative cells, with spores detected only occasionally (i.e. only one colony on a LA-Str at a 20 times dilution after 10 min heat treatment).

4

Re-isolation of the released strain BtN14 outside the release plot in three directions: (A) north, (B) east, and (C) south. Included in graphs are concentrations of total B. thuringiensis subsp. israelensis (Bti) within the plot (cfu g⁻¹ soil dry weight), the released strain BtN14 within the plot (cfu g⁻¹ soil dry weight), as well as at 50 m, 25 m, and 10 m from the plot in each direction (cfu g⁻¹ soil wet weight). The broken line indicates a BtN14 detection level of 20 cfu g⁻¹ soil (wet weight). All samples outside the plot were analyzed in duplicate.

3.5 Growth of BtN14 in soil

A possible explanation for the results shown in Fig. 4 is growth of the released strain BtN14 in the soil. To test this hypothesis, we added different concentrations of BtN14 to non-sterilized as well as heat-sterilized forest soil and followed the growth.

The extent to which BtN14 grows depends on the size of the original inoculum. Fig. 5A shows that B. thuringiensis subsp. israelensis grows well in sterilized soil. Under the laboratory conditions used, a maximum level of 10⁸ cfu g⁻¹ soil was observed, and the doubling time was about 1 day. In non-sterile soil, BtN14 added to give 10⁵ cfu g⁻¹ shows a slight increase in cell number, not strongly statistically significant (using the t-test, the higher concentrations after 3 days had a significance level between 0.025 and 0.05), and the concentration declines after 1 week (Fig. 5B).

5

Growth of BtN14 in (A) heat-sterilized and (B) non-sterilized forest soil. Inoculations were done at four different concentrations: 300 cfu g⁻¹, 10³ cfu g⁻¹, 10⁵ cfu g⁻¹, and 10⁷ cfu g⁻¹. Duplicate samples were analyzed at different time points and the mean values are indicated in the graph. All samples were calculated as wet weight.

4 Discussion

In spite of the extensive use of Bacillus thuringiensis insecticides, the ecological consequences of their release over non-cultivated soil have been poorly investigated. The increasing use of biopesticides has, however, highlighted the need for such investigations. Of major concern is the risk that the spreading of large amounts of viable microorganisms will permanently alter the resident microbial population at the release site. Previous papers have reported the fate of B. thuringiensis and its crystal in agriculture [9,11], mulberry plantations [10], water [16] or under laboratory control [17,18]. A review of the effects and persistence in soil of B. thuringiensis[12] presents laboratory experiments in microcosms amended with B. thuringiensis strains, but so far, no study has involved B. thuringiensis subsp. israelensis in a field experiment at concentrations used when spread as a biopesticide.

In this study, we examined the effect on a population of culturable soil bacteria when BtN14, a streptomycin-resistant strain of B. thuringiensis subsp. israelensis, was released at concentrations normally used for such a biopesticide. The concentrations of B. thuringiensis/cereus-like bacteria, B. thuringiensis subsp. israelensis, and released BtN14 were followed during the test period. For the study, a swampland at Tärnsjö in northern Uppland was chosen (Fig. 1).

Based on analyses of samples collected before and after the release, we conclude that there was no dramatic change in the studied populations (Fig. 3). In fact, only a minor increase in the B. thuringiensis/cereus-like fraction was seen immediately after the release. After slowly decreasing, the relative amounts of B. thuringiensis/cereus-like bacteria stabilized at the initial levels after 7 weeks. For the total B. thuringiensis subsp. israelensis population the change was not statistically significant, while the released strain BtN14 remained around 1% of the total culturable population during the whole test period. Thus, the release of BtN14 is not likely to alter the general ecological situation in soil, even if the strain seems to at least temporarily replace 5% of the total B. thuringiensis subsp. israelensis population (Fig. 3).

To study spreading of the released organism, we screened for streptomycin-resistant B. thuringiensis subsp. israelensis in three directions (Fig. 4) and found it outside the test plot in all tested directions. In one direction (south), the highest concentrations were about 10³ cfu g⁻¹ soil. The BtN14 concentration decreased below the detection limit (20 cfu g⁻¹ soil) at the screening sites towards the end of the test period.

During favorable conditions such as major disturbances in the upper soil microflora, BtN14 has the ability to grow and establish itself. But as long as the soil is relatively undisturbed, our released strain has little chance of changing the overall population structure more than temporarily, even if BtN14 shows the ability to persist in soil as a minor part of the natural microflora when added at high concentrations (Fig. 3). Fig. 5 shows the situation in microcosms amended with BtN14. Heat sterilization of soil constitutes a major disturbance. Nutrients are freely available for any microorganism [19], and without competing microorganisms and predators the added bacteria grow well. A minor disturbance caused by flooding and subsequent drying could have caused the relatively high concentrations of BtN14 south of the release plot by growth of animal- or water-transported BtN14.

BtN14 was not capable of permanent establishment outside the release site, probably because the concentration was too low and the strain was not fit enough to compete with the remaining indigenous microbial flora. Our data in Fig. 5 indicate a small increase (about 2 times) when 10⁵ cfu g⁻¹ is added to the non-sterilized soil but not with a smaller inoculum, which supports the hypothesis that the colonization ability is connected with the size of the inoculum.

Theoretically, BtN14 may have spread from the release site due to flagellar movement [20], [21], soil animal transport [9] and/or physical deposition by slow water streams and diffusion. The southern sampling sites lie between the water and the release site. Physical transport and diffusion of BtN14 is possible in that direction, although this cannot fully explain the relatively high (10³ cfu g⁻¹) concentrations found. Pedersen et al. [9] reported B. thuringiensis subsp. kurstaki transport by soil beetles for as far as 135 m. A combination of soil animal transport, physical movement and growth is a probable explanation for the relatively high concentrations south of the release plot. Further supporting this hypothesis is the dominance of vegetative BtN14 cells in the samples, and the fact that BtN14 disappears at the end of the test period. Another, less likely possibility is genetic exchange from conjugation [22] or transduction [23], but this requires germination of the spores and subsequent growth.

Our three sampling sites located at the edge of the swampy area at Tärnsjö (Fig. 1) were found to contain high natural concentrations of B. thuringiensis subsp. israelensis compared to the sites in a nearby forest and at a more distant field (Fig. 2). Bacterial relatives already present at high concentrations may influence the ability of a released strain to establish itself at a favorable site. The local abundance of both mosquitoes and B. thuringiensis subsp. israelensis is an interesting possible correlation. The ecological aspects of interactions between insect pathogens and their hosts largely remain to be investigated.

Acknowledgements

This work was supported by grants from the Swedish Environmental Protection Agency (SNV) and Philip Sørensens Stiftelse to A.L. The authors would like to thank Alexandra Berg and Jonas Åsberg for technical assistance, and Steve Muir for critical reading of the manuscript.