In vitro antifungal activity of Burkholderia gladioli pv. agaricicola against some phytopathogenic fungi.

The trend to search novel microbial natural biocides has recently been increasing in order to avoid the environmental pollution from use of synthetic pesticides. Among these novel natural biocides are the bioactive secondary metabolites of Burkholderia gladioli pv. agaricicola (Bga). The aim of this study is to determine antifungal activity of Bga strains against some phytopathogenic fungi. The fungicidal tests were carried out using cultures and cell-free culture filtrates against Botrytis cinerea, Aspergillus flavus, Aspergillus niger, Penicillium digitatum, Penicillium expansum, Sclerotinia sclerotiorum and Phytophthora cactorum. Results demonstrated that all tested strains exert antifungal activity against all studied fungi by producing diffusible metabolites which are correlated with their ability to produce extracellular hydrolytic enzymes. All strains significantly reduced the growth of studied fungi and the bacterial cells were more bioactive than bacterial filtrates. All tested Bulkholderia strains produced volatile organic compounds (VOCs), which inhibited the fungal growth and reduced the growth rate of Fusarium oxysporum and Rhizoctonia solani. GC/MS analysis of VOCs emitted by strain Bga 11096 indicated the presence of a compound that was identified as 1-methyl-4-(1-methylethenyl)-cyclohexene, a liquid hydrocarbon classified as cyclic terpene. This compound could be responsible for the antifungal activity, which is also in agreement with the work of other authors.

The structure of the O-specific polysaccharide of the lipopolysaccharide from Burkholderia gladioli pv. agaricicola.

A neutral O-specific polysaccharide containing d-mannose, d-rhamnose and d-galactose was obtained by mild acid hydrolysis of the lipopolysaccharide of the plant pathogenic bacterium Burkholderia gladioli pv. agaricicola. By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as a linear trisaccharide of the structure shown below, in which the mannose residue was quantitatively acetylated at C-2. [carbohydrate structure: see text]

The structure of a putative exopolysaccharide of Burkholderia gladioli pv. agaricicola.

A putative capsular polysaccharide containing D-rhamnose was isolated from the phytopathogenic bacterium Burkholderia gladioli pv. agaricicola by phenol/water extraction followed by ultracentrifugation of the separated water phase and gel-permeation chromatography of the thus obtained supernatant. By means of chemical analyses and NMR spectroscopy, the repeating unit of the polymer was shown to be a linear tetrasaccharide with the structure. [carbohydrates: see text].

A new syringopeptin produced by bean strains of Pseudomonas syringae pv. syringae.

Two strains (B728a and Y37) of the phytopathogenic bacterium Pseudomonas syringae pv. syringae isolated from bean (Phaseolus vulgaris) plants were shown to produce in culture both syringomycin, a lipodepsinonapeptide secreted by the majority of the strains of the bacterium, and a new form of syringopeptin, SP(22)Phv. The structure of the latter metabolite was elucidated by the combined use of mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy and chemical procedures. Comparative phytotoxic and antimicrobial assays showed that SP(22)Phv did not differ substantially from the previously characterized syringopeptin 22 (SP(22)) as far as toxicity to plants was concerned, but was less active in inhibiting the growth of the test fungi Rhodotorula pilimanae and Geotrichum candidum and of the Gram-positive bacterium Bacillus megaterium.

Antibacterial activity of Cuminum cyminum L. and Carum carvi L. essential oils.

Essential oils extracted by hydrodistillation from fruits of Cuminum cyminum L. and Carum carvi L. were analyzed by gas chromatography (GC) and GC-mass spectrometry (MS). The main components of C. cyminum oil were p-mentha-1,4-dien-7-al, cumin aldehyde, gamma-terpinene, and beta-pinene, while those of the C. carvi oil were carvone, limonene, germacrene D, and trans-dihydrocarvone. Antibacterial activity, determined with the agar diffusion method, was observed against Gram-positive and Gram-negative bacterial species in this study. The activity was particularly high against the genera Clavibacter, Curtobacterium, Rhodococcus, Erwinia, Xanthomonas, Ralstonia, and Agrobacterium, which are responsible for plant or cultivated mushroom diseases worldwide. In general, a lower activity was observed against bacteria belonging to the genus Pseudomonas. These results suggest the potential use of the above essential oils for the control of bacterial diseases.

Bioactivity of volatile organic compounds produced by Pseudomonas tolaasii.

Pseudomonas tolaasii is the main bacterial pathogen of several mushroom species. In this paper we report that strains of P. tolaasii produce volatile substances inducing in vitro mycelia growth inhibition of Pleurotus ostreatus and P. eryngii, and Agaricus bisporus and P. ostreatus basidiome tissue blocks brown discoloration. P. tolaasii strains produced the volatile ammonia but not hydrogen cyanide. Among the volatiles detected by GC-MS, methanethiol, dimethyl disulfide (DMDS), and 1-undecene were identified. The latter, when assayed individually as pure compounds, led to similar effects noticed when P. tolaasii volatiles natural blend was used on mushrooms mycelia and basidiome tissue blocks. Furthermore, the natural volatile mixture resulted toxic toward lettuce and broccoli seedling growth. In contrast, pure volatiles showed different activity according to their nature and/or doses applied. Indeed, methanethiol resulted toxic at all the doses used, while DMDS toxicity was assessed till a quantity of 1.25 µg, below which it caused, together with 1-undecene (≥10 µg), broccoli growth increase.

Biocide effects of volatile organic compounds produced by potential biocontrol rhizobacteria on Sclerotinia sclerotiorum.

Six rhizobacteria isolated from common bean and able to protect bean plants from the common bacterial blight (CBB) causal agent, were in vitro evaluated for their potential antifungal effects toward different plant pathogenic fungi, mostly soil-borne. By dual culture assays, the above bacteria resulted producing diffusible and volatile metabolites which inhibited the growth of the majority of the pathogens under study. In particular, the latter substances highly affected the mycelium growth of Sclerotinia sclerotiorum strains, one of which was selected for further studies either on mycelium or sclerotia. Gas chromatographic analysis of the bacterial volatiles led to the identification of an array of volatile organic compounds (VOCs). Time course studies showed the modification of the VOCs profile along a period of 5 days. In order to evaluate the single detected VOC effects on fungal growth, some of the pure compounds were tested on S. sclerotiorum mycelium and their minimal inhibitory quantities were determined. Similarly, the minimal inhibitory quantities on sclerotia germination were also defined. Moreover, observations by light and transmission electron microscopes highlighted hyphae cytoplasm granulation and ultrastructural alterations at cell organelles, mostly membranes, mitochondria, and endoplasmic reticulum. The membranes appeared one of the primary targets of bacterial volatiles, as confirmed by hemolytic activity observed for the majority of pure VOCs. However, of interest is the alteration observed on mitochondria as well.

A left-handed alpha-helix containing both L- and D-amino acids: the solution structure of the antimicrobial lipodepsipeptide tolaasin.

The 18-amino acid cytolytic lipodepsipeptide tolaasin, produced in culture by virulent strains of Pseudomonas tolaasii, is the causal agent of the brown blotch disease of the cultivated mushroom. Tolaasin has a sequence of D-amino acids in its N-terminal region, then alternates L- and D-amino acids, and bears a C-terminal lactone macrocycle composed of 5-residues. The solution structure of tolaasin in sodium dodecyl sulfate was studied by 2D-NMR spectroscopy and molecular dynamics simulated annealing calculations. Tolaasin forms an amphipathic left-handed alpha-helix in the regionDPro2-DalloThr14 comprising the sequence of seven D-amino acids and the adjacent L-D-L-D-D-region. To the best of our knowledge, this is the first recognized example of a left-handed alpha-helix including both D- and L-amino acids. The lactone macrocycle adopts a "boat-like" conformation and is shifted from the helical axis as to form a "golf-club" overall conformation. These structural features will be of importance in understanding, and preventing, tolaasin's role in the bacterial colonization of the host plant, and its toxic action on cells. Furthermore, the observed antimicrobial activity together with the potential resistance to enzymatic degradation and the increased antigenicity (both due to the presence of L- and D-amino acids) strongly suggests for tolaasin a potential role as a template model for the design of new therapeutic antibacterial molecules.

Antibacterial activity of Coriandrum sativum L. and Foeniculum vulgare Miller Var. vulgare (Miller) essential oils.

Essential oils were extracted from the fruits of Coriandrum sativum L. and Foeniculum vulgare Miller var. vulgare (Miller) and assayed in vitro for antibacterial activity to Escherichia coli and Bacillus megaterium, bacteria routinely used for comparison in the antimicrobial assays, and 27 phytopathogenic bacterial species and two mycopathogenic ones responsible for cultivated mushroom diseases. A significant antibacterial activity, as determined with the agar diffusion method, was shown by C. sativum essential oil whereas a much reduced effect was observed for F. vulgare var. vulgare oil. C. sativum and F. vulgare var. vulgare essential oils may be useful natural bactericides for the control of bacterial diseases of plants and for seed treatment, in particular, in organic agriculture. The significant antibacterial activity of essential oils to the bacterial pathogens of mushrooms appears promising.

Acylhomoserine lactone production by bacteria associated with cultivated mushrooms.

The main bacterial pathogens of cultivated mushroom as well as mushroom-associated bacteria, which were isolated from Agaricus bisporus, Pleurotus ostreatus and Pleurotus eryngii mushroom niches, were evaluated for the production of N-acyl-L-homoserine lactones (AHLs) by using four bioreporters. Furthermore, identification of AHLs by LC-ESI-FTICR MS was performed on culture filtrates of selected pathogens and mushroom-associated bacteria strains, which resulted in inducing at least one of the four bioreporters. Strains of Burkolderia gladioli pv. agariciola, Pseudomonas agarici and Pseudomonas gingeri, but not those of Pseudomonas tolaasii and Pseudomonas reactans, produced an array of AHLs depending on the strain. This is the first report of AHL production by mushroom bacterial pathogens. Forty-four of 236 bacterial isolates obtained from different niches of cultivated mushrooms, in part identified by the Biolog identification system, were demonstrated to produce AHLs. Among them, seven mushroom-associated bacterial species were for the first time demonstrated to produce the above signal molecules. In the culture filtrates of a certain number of isolates/strains the AHL-hydrolyzed forms were also present. The minimal signal inducing concentration (MSIC) of selected pure AHLs was also determined for the four bioreporters used in this study.

Tolaasins A--E, five new lipodepsipeptides produced by Pseudomonas tolaasii.

Pseudomonas tolaasii, the causal organism of brown blotch disease of Agaricus bisporus and of the yellowing of Pleurotus ostreatus, was shown to produce in culture tolaasin I (1), tolaasin II (2), and five other minor metabolites, tolaasins A, B, C, D, and E (3-7). These compounds were demonstrated to be important in the development of the disease symptoms. This paper reports on the structural elucidation, based essentially on NMR studies and MS spectra, and biological activity of the above lipodepsipeptides (3-7). All the above analogues showed differences in the peptide moiety, as observed in other lipodepsipeptides of bacterial origin, and maintained the beta-hydroxyoctanoyl phi chain at the N-terminus, except tolaasin A, in which the acyl moiety was a gamma-carboxybutanoyl phi moiety. Among the target microorganisms used (fungi, yeast, and bacteria) the Gram-positive bacteria were the most sensitive, although the antimicrobial activity appeared to be correlated to the structural modification in the different analogues. The structure-activity relationships of these toxins are discussed.