An efficient procedure for the isolation of PCR-competent DNA from Bacillus endospores germinated in soil

DNA extraction techniques for endospore-forming bacteria in soil are often labour-intensive and unreliable. Our objective in this work was to investigate whether good quality DNA could be obtained from spores germinated in soil. To this end, endospores from Bacillus subtilis, B. megaterium and B. thuringiensis were inoculated into soil microcosms and germination was induced by addition of LB medium supplemented with l-alanine, glucose, fructose and KCl. Heat resistance count was reduced to 80% for B. subtilis and more than 90% for B. thuringiensis and B. megaterium after a few minutes. Isolation of DNA from soil with a procedure which did not work on spores was shown to be as efficient for in situ-germinated spores as for inoculated vegetative cells. Furthermore, we developed a simple procedure that allowed us to use the recovered DNA in PCR amplifications. The present methodology is simple and efficient; it avoids the use of special equipment and harsh spore rupturing methods and can be carried out with multiple samples.     

Tagging of Bacillus subtilis soil isolates through illegitimate recombination for PCR detection

Although there are numerous bacteria of the genus Bacillus of great importance for biological control, little is known about their ecology in the soil. We wanted to test illegitimate recombination as a tagging system that would allow us to study selected or genetically engineered Bacillus soil isolates. Strains carrying the plasmid integrated into the chromosome were obtained by growing at a non-permissive temperature after transformation with a plasmid carrying a thermo-sensitive replication origin with selection for erythromycin. A laboratory strain, a commercial strain (Kodiak), and four other soil isolates were generated through this procedure and analysed. In all of these strains the integrated plasmid was maintained in multicopy. The erythromycin resistance gene (ermB) placed on the plasmid was used as a target for polymerase chain reaction (PCR). The tagged strains could be then detected when inoculated into microcosms prepared with non-sterile soil.     

Antagonism of Bacillus spp. Isolated from Marine Biofilms Against Terrestrial Phytopathogenic Fungi

We aimed at determining the antagonistic behavior of bacteria derived from marine biofilms against terrestrial phytopathogenic fungi. Some bacteria closely related to Bacillus mojavensis (three isolates) and Bacillus firmus (one isolate) displayed antagonistic activity against Colletotrichum gloeosporioides ATCC 42374, selected as first screen organism. The four isolates were further quantitatively tested against C. gloeosporioides, Colletotrichum fragariae, and Fusarium oxysporum on two culture media, potato dextrose agar (PDA) and a marine medium-based agar [yeast extract agar (YEA)] at different times of growth of the antagonists (early, co-inoculation with the pathogen and late). Overall antagonistic assays showed differential susceptibility among the pathogens as a function of the type of culture media and time of colonization (P < 0.05). In general, higher suppressive activities were recorded for assays performed on YEA than on PDA; and also when the antagonists were allowed to grow 24 h earlier than the pathogen. F. oxysporum was the most resistant fungus while the most sensitive was C. gloeosporioides ATCC 42374. Significant differences in antagonistic activity (P < 0.05) were found between the different isolates. In general, Bacillus sp. MC3B-22 displayed a greater antagonistic effect than the commercial biocontrol strain Bacillus subtilis G03 (Kodiak). Further incubation studies and scanning electronic microscopy revealed that Bacillus sp. MC3B-22 was able to colonize, multiply, and inhibit C. gloeosporioides ATCC 42374 when tested in a mango leaf assay, showing its potential for fungal biocontrol. Additional studies are required to definitively identify the active isolates and to determine their mode of antifungal action, safety, and biocompatibility.