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Bacillus subtilis CL27 and B. pumilus CL45 showed similar activity against Botrytis cinerea in in vitro plate assays. In a seedling bioassay, however, B. subtilis CL27 had activity similar to a commercial fungicide while B. pumilus CL45 failed completely to prevent seedling damping-off caused by Bot. cinerea. Antibiotic production by the two Bacillus strains was found to depend on the growth substrate and highest antibiotic production was found on media based on homogenized cabbage tissue. Antibiotic activity was found to depend on the pH and nutrient concentration in the assay medium. Antifungal antibiotics produced by B. subtilis CL27 and B. pumilus CL45 in different fermentation media were separated by thin layer chromatography. As suspected from the activity spectrum, three antibiotics (one with activity against Alternaria brassicicola, one with activity against Botrytis cinerea and one with activity against both fungi) could be detected in the fermentation broth of CL27, but only one in the fermentation broth of CL45. The two antibiotics produced by strain CL27 with activity against A. brassicicola were identified as peptides since their bands on the TLC plates developed a green to blue/green colour after treatment with 4,4'-tetramethyldiamino-diphenylmethane (TDM) reagent. The third antibiotics produced by strain CL27 and antibiotic produced by CL45 had a similar Rf-value and appeared not to be peptides based on the reaction with TDM. However, they showed a slightly different activity spectrum when tested against a range of different fungi. Antibiotic production was clearly indicated as the mode of action of in vivo biocontrol by strain CL27 against damping off caused by Bot. cinerea of Astilbe micro-plants, because a u.v.-induced antibiotic negative mutant strain CL27b showed no activity in seedling bioassays in vivo. Also the mutant strain CL27a which produced the two peptide antibiotics but had lost the ability to produce the non-peptide antibiotic, showed greatly reduced in vivo activity.

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We describe here the identification and characterization of two Listeria monocytogenes (Tn917-LTV3) relA and hpt transposon insertion mutants that were impaired in growth after attachment to a model surface. Both mutants were unable to accumulate (p)ppGpp in response to amino acid starvation, whereas the wild-type strain accumulated (p)ppGpp within 30 min of stress induction. The induction of transcription of the relA gene after adhesion was demonstrated, suggesting that the ability to mount a stringent response and undergo physiological adaptation to nutrient deprivation is essential for the subsequent growth of the adhered bacteria. The absence of (p)ppGpp in the hpt mutant, which is blocked in the purine salvage pathway, is curious and suggests that a functional purine salvage pathway is required for the biosynthesis of (p)ppGpp. Both mutants were avirulent in a murine model of listeriosis, indicating an essential role for the stringent response in the survival and growth of L. monocytogenes in the host. Taken as a whole, this study provides new information on the role of the stringent response and the physiological adaptation of L. monocytogenes for biofilm growth and pathogenesis.The ubiquitous gram-positive pathogen L. monocytogenes is responsible for the clinical syndromes of listeriosis in humans and animals (27, 28). The principle mode of transmission of the organism to humans is believed to be the consumption of contaminated food (44). Normally, the consumption of foodborne L. monocytogenes does not result in overt gastrointestinal disease. Rather, in serious cases, listeriosis presents as bacteremia, meningitis, or miscarriage (30).The ability of L. monocytogenes to adhere to and colonize surfaces during food preparation and storage is important in the contamination of food products prior to consumption. While much is known about the virulence of L. monocytogenes (reviewed in references 7 and 24), detailed knowledge about the mechanisms of attachment and proliferation of L. monocytogenes on surfaces is scant. Previously, the adhesion of L. monocytogenes to plant surfaces has been shown to be a consequence of hydrophobic bonds between the plant surface and outer surface components of the bacterium rather than a specific ligand-receptor binding process (3).It is known that biofilm formation is a complex process, and a number of cell surface structures have been implicated in the initial stages of biofilm formation. In Pseudomonas aeruginosa these include flagella (motility) (35) and the expression of type IV pili for microcolony formation (36). In streptococci a number of cell surface adhesins, together with autolysins, have been identified as being important in biofilm development (12,17,19,21,26). The subsequent development and differentiation of an ordered three-dimensional biofilm requires the expression of extracellular polysaccharides (9) and may involve cell-to-cell signaling molecules (11,26). During the initial establishment of a sessile community, it is likely that bacteria have to adapt their...

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