In this study the efficacy of two different methods for extracting lipopeptides produced by five Bacillus strains - ethyl acetate extraction, and acid precipitation followed by methanol extraction - was investigated using mass spectrometry. High performance thin layer chromatography (HPTLC) was also used for the simultaneous separation of complex mixtures of lipopeptide extracts and for the determination of antimicrobial activity of their components. The mass spectra clearly showed well-resolved groups of peaks corresponding to different lipopeptide families (kurstakins, iturins, surfactins and fengycins). The ethyl acetate extracts produced the most favorable results. The extracts of SS-12.6, SS-13.1 and SS-38.4 showed the highest inhibition zones. An iturin analogue is responsible for the inhibition of Xanthomonas arboricola and Pseudomonas syringae phytopathogenic strains. HPTLC bioautography effectively identified the active compounds from a mixture of lipopeptide extracts, proving in situ its potential for use in direct detection and determination of antimicrobials. In the test of potential synergism among individual extracts used in different mixtures, stronger antimicrobial effects were not observed. Biochemical and phylogenetic analysis clustered isolates SS-12.6, SS-13.1, SS-27.2 and SS-38.4 together with Bacillus amyloliquefaciens, while SS-10.7 was more closely related to Bacillus pumilus.

Pseudomonas syringae is the most frequently emerging group of plant pathogenic bacteria. Because this bacterium is ubiquitous as an epiphyte and on various substrates in non-agricultural settings, there are many questions about how to assess the risk for plant disease posed by strains in the environment. Although P. syringae is considered to have discrete host ranges in defined pathovars, there have been few reports of comprehensive comparisons of host range potential. Here we present results of host range tests for 134 strains, representing eight phylogroups, from epidemics and environmental reservoirs on 15 to 22 plant species per test conducted in four separate tests to determine the patterns and extent of host range. We sought to identify trends that are indicative of distinct pathotypes and to assess if strains in the P. syringae complex are indeed restricted in their host range. We show that for each test, strains display a diversity of host ranges from very restricted to very broad regardless of the gamut of phylogroups used in the test. Overall, strains form an overlapping continuum of host range potential with equal representation of narrow, moderate and broad host ranges. Groups of distinct pathotypes, including strains with currently the same pathovar name, could not be identified. The absence of groupings was validated with statistical tests for pattern recognition. The extent of host range was positively correlated with the capacity of strains to swarm on semi-solid agar medium and with the abundance of genes in biosynthetic clusters and was inversely correlated with the abundance of genes for proteins with transmembrane domains in their genomes. Our results are consistent with the current paradigm that disease symptoms are the result of multiple molecular interactions between P. syringae and its plant host that are modulated by abiotic and biotic conditions. This leads us to propose that pathovar denominations do not correspond to the underlying biology of P. syringae. A new concept of pathogenicity that accounts for the continuum of pathogenic potential in P. syringae would open new perspectives to understand the evolution of pathogenicity in this bacterium and new insights to anticipate disease and to manage plant health.

New information has come to light about the biological activity of propolis and the quality of natural products which requires a rapid and reliable assessment method such as High Performance Thin-Layer Chromatography (HPTLC) fingerprinting. This study investigates chromatographic and chemometric approaches for determining the antimicrobial activity of propolis of Serbian origin against various bacterial species. A linear multivariate calibration technique, using Partial Least Squares, was used to extract the relevant information from the chromatographic fingerprints, i.e. to indicate peaks which represent phenolic compounds that are potentially responsible for the antimicrobial capacity of the samples. In addition, direct bioautography was performed to localize the antibacterial activity on chromatograms. The biological activity of the propolis samples against various bacterial species was determined by a minimum inhibitory concentration assay, confirming their affiliation with the European poplar type of propolis and revealing the existence of two types (blue and orange) according to botanical origin. The strongest antibacterial activity was exhibited by sample 26 against Staphylococcus aureus, with a MIC value of 0.5 mg/mL, and Listeria monocytogenes, with a MIC as low as 0.1 mg/mL, which was also the lowest effective concentration observed in our study. Generally, the orange type of propolis shows higher antimicrobial activity compared to the blue type. PLS modelling was performed on the HPTLC data set and the resulting models might qualitatively indicate compounds that play an important role in the activity exhibited by the propolis samples. The most relevant peaks influencing the antimicrobial activity of propolis against all bacterial strains were phenolic compounds at R_F values of 0.37, 0.40, 0.45, 0.51, 0.60 and 0.70. The knowledge gained through this study could be important for attributing the antimicrobial activity of propolis to specific chemical compounds, as well as the verification of HPTLC fingerprinting as a reliable method for the identification of compounds that are potentially responsible for antimicrobial activity. This is the first report on the activity of Serbian propolis as determined by several combined methods, including the modelling of antimicrobial activity by HPTLC fingerprinting.