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. 2010 May 20;6(5):e1000905.
doi: 10.1371/journal.ppat.1000905.

Environmental Factors Determining the Epidemiology and Population Genetic Structure of the Bacillus Cereus Group in the Field

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Free PMC article

Environmental Factors Determining the Epidemiology and Population Genetic Structure of the Bacillus Cereus Group in the Field

Ben Raymond et al. PLoS Pathog. .
Free PMC article

Abstract

Bacillus thuringiensis (Bt) and its insecticidal toxins are widely exploited in microbial biopesticides and genetically modified crops. Its population biology is, however, poorly understood. Important issues for the safe, sustainable exploitation of Bt include understanding how selection maintains expression of insecticidal toxins in nature, whether entomopathogenic Bt is ecologically distinct from related human pathogens in the Bacillus cereus group, and how the use of microbial pesticides alters natural bacterial populations. We addressed these questions with a MLST scheme applied to a field experiment in which we excluded/added insect hosts and microbial pesticides in a factorial design. The presence of insects increased the density of Bt/B. cereus in the soil and the proportion of strains expressing insecticidal toxins. We found a near-epidemic population structure dominated by a single entomopathogenic genotype (ST8) in sprayed and unsprayed enclosures. Biopesticidal ST8 proliferated in hosts after spraying but was also found naturally associated with leaves more than any other genotype. In an independent experiment several ST8 isolates proved better than a range of non-pathogenic STs at endophytic and epiphytic colonization of seedlings from soil. This is the first experimental demonstration of Bt behaving as a specialized insect pathogen in the field. These data provide a basis for understanding both Bt ecology and the influence of anthropogenic factors on Bt populations. This natural population of Bt showed habitat associations and a population structure that differed markedly from previous MLST studies of less ecologically coherent B. cereus sample collections. The host-specific adaptations of ST8, its close association with its toxin plasmid and its high prevalence within its clade are analogous to the biology of Bacillus anthracis. This prevalence also suggests that selection for resistance to the insecticidal toxins of ST8 will have been stronger than for other toxin classes.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The effect of the presence of insect hosts and biopesticide application on bacterial abundance of the Bacillus cereus group in experimental enclosures.
Populations of host insects (P. xylostella) were established in host addition cages six weeks before the first sampling time point (time point 0). Soil samples were taken immediately before the application of a B. t. kurstaki biopesticidal spray (DiPel DF) (time point T0) and then at 10 and 28 days after spray application. Leaf samples were taken at time points T10 and T28 only. Treatment labels insect/no insect indicate the experimental exclusion of Lepidopteran hosts or the experimental addition of diamondback moth populations; the labels spray/no spray indicate the application or absence of biopesticide. The data are colony-forming units per gram of sampled material from leaf and soil samples (two samples per experimental cage, twenty four cages in all). Data are means ± SEs.
Figure 2
Figure 2. A CLONALFRAME genealogy of the Bacillus cereus group indicating the niche/habitat associations of STs recovered in this study.
Sequence types (STs) isolated in this study have been mapped onto the genealogical tree with circular symbols, the remaining unmarked STs were recovered from the pubMLST isolates database. Filled black circles indicate STs were recovered from soil in this field study; open circles were recovered from leaf material only. Partially filled circles represent STs that were recovered from both soil and leaf material; the extent of the black fill inside indicates the frequency of isolates recovered from soil. The size of the circular symbols also indicates the abundance of that ST within the field population. Diamond symbols indicate which STs expressed Cry toxin parasporal inclusions. The extent of the blue fill within each diamond indicates the frequency of Cry-producing isolates within that ST in the field. The dominant sequence type (ST8) is also the genotype of the Bt strain (Dipel – Btk HD-1) that was used to spray experimental enclosures.
Figure 3
Figure 3. Clade level ecological differentiation of all Bacillus cereus group sequence types (STs) in the pubMLST isolates database.
These charts show the frequency of sequence types within each clade that have a particular host associated niche or have been recovered from a particular habitat. Data for 298 STs and 773 isolates were recovered from this study, the pubMLST database and the MLST literature , , , with 59, 94 and 106 STs from clades 1–3 respectively and 39 STs from both clades 4 and 5. Ecological niche was initially defined by the possession of ecologically significant plasmids: pX01 pX02 define anthracis; Cry toxin expression defines a strain as having an insect host and STs carrying the cereulide emetic toxin plasmid are also denoted as such. If this information was not available STs were defined according to clinical symptoms with which they were associated or the habitat from which they were isolated. STs were designated as of faecal origin rather than associated with food poisoning when there was no clear documentation of disease symptoms in hosts. If multiple isolates with a single ST had more than one ecological association this ST divided its contribution to the ecological categories in proportion to the frequency of isolates associated with each habitat/host, the sum of all these contributions being 1.
Figure 4
Figure 4. The allelic ancestry (entomopathogenic or non-pathogenic) of samples from experimentally defined isolate groups.
Alleles from STs isolated from leaf samples were defined as being of pathogenic origin – these were predominantly from clade 2. All other alleles, those associated with STs that were only recovered from soil (predominantly clade 3) were defined as non-pathogenic. Ancestry was inferred using a no-admixture model in STRUCTURE, and is given for alleles associated with putatively pathogenic (dark grey) and non-pathogenic (light grey) genotypes. A CLONALFRAME genealogy was used to define background population structure (POPFLAG).
Figure 5
Figure 5. Habitat (sample origin) and experimental treatment affect the proportion of sampled Bacillus cereus isolates expressing Cry toxin parasporal inclusions.
All the isolates collected during the 2006 field experiment were scored microscopically for the presence of proteinaceous parasporal inclusions (306 isolates in total). The data have been plotted according to sample origin (soil or leaf) and according to the treatments imposed upon experimental enclosures. Data are mean proportions for the six enclosures per treatment ±SEs.
Figure 6
Figure 6. The association between habitat and genotype in unsprayed experimental enclosures naturally colonised by Bacillus cereus group bacteria.
The dark bars represent the number of isolates identified as ST8, the light bars represent all other STs. Leaf and soil labels indicate sample origin; “insect” and “none” indicate the experimental addition or exclusion of host insects in experimental enclosures. Data are genotype frequencies ±SEs.
Figure 7
Figure 7. Variation in the ability of B. cereus clade 3 bacteria and B. thuringiensis (ST8) to colonize growing plants from experimentally inoculated soil.
Cabbage seedlings (from surface sterilized seeds) were grown in autoclaved compost inoculated with either a clade 3 B. cereus strain; an ST8 isolate or in controls (without inoculation). Bacteria were recovered on B. cereus group selective media from leaf washes (epiphytic bacteria- solid bars) or homogenized surface sterilized leaf tissue (endophytic bacteria- cross hatched bars) after seedlings were at the 6-leaf stage. Data are the mean colony forming units (cfu) of B. cereus group bacteria per sample from four independent experimental replicates +SEs; no B. cereus group bacteria were recovered from leaf washes of control plants.

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