The ecology of Bacillus thuringiensis on the phylloplane
Two selective media which specifically allow the cultivation of Bacillus thuringiensis while it is in the vegetative as opposed to the spore form were developed. Using these media it was conclusively proved for the first time that B. thuringiensis can reside on the phylloplane in a metabolically active form. This was corroborated by evidence, also for the first time, that conjugation can take place on the phylloplane between such endemic strains. A new bacteriophage, infecting one of the endemic stains, which was activated by the process of genetic recombination, was isolated and characterized. The appearance of naturally occurring strains of B. thuringiensis in vegetative and spore form was followed over a growing season on clover (Trifolium hybridum) in the field. Simultaneous and sudden rises and declines of both spore and vegetative cell densities were observed. These could not be correlated with weather conditions. A genetically stable population of strains seemed to be maintained throughout the growing season. The most common other spore-former on these leaves was Bacillus cereus but the fluctuations in appearance of these two very closely related species were not co-incident. Using specific PCR primers, a considerably diversity of toxin types was found with the majority of isolates possessing multiple d-endotoxin genes. Bioassays against a lepidopteran insect (Pieris brassicae) of purified d-endotoxins showed that they were not more potent than those from a laboratory-adapted strain. A high percentage, however, of the endemic isolates (82%) possessed the ‘Vegetative insecticidal protein’ (Vip) gene, vip3. This might indicate an involvement of Vips in the establishment of these strains on the phylloplane. A mechanism for colonization of annual plants by B. thuringiensis was demonstrated for the first time. It was shown that spores added to seeds, even in non-sterile soil, can germinate and replicate on the resulting seedlings. The level of colonization achieved did not have a consistent influence on the feeding behaviour of third instar larvae of P. brassicae which were present on the plants for three days. Nevertheless, the fact that the number of CFU of B. thuringiensis recovered per gram of insect increased with time is evidence of proliferation of the bacterium inside the insects. Four isolates of B. thuringiensis that had been recovered in the vegetative phase from the phylloplane of T. hybridum were grown for 500 generations in a rich medium. Changes were observed in all of the strains but one isolate changed remarkably in all of the characteristics assessed which included: structure; plasmid profile; fatty acid composition; and d-endotoxin production. Moreover, the sequence of the Vip3 protein harboured by the evolved strain showed changes when compared with that of the parental strain.