4 International Symposium on Biological Control of Bacterial Plant Diseases Species interations within themicrobiomemediate potential for disease suppression

During the winter of 2004, symptoms were observed in commercial cauliflower (Brassica oleracea L. convar. botrytis (L.) Alef. var. italica) fields of "romanesco" type (cv. Navona) in Apulia, southern Italy. These symptoms were noted on inflorescences that were almost ready for harvest, and a bacterial etiology was suspected. In particular, the corymbs showed water-soaked and brown discolored areas which then rotted. The above alterations involved the whole inflorescences, or in some cases, only a few florets. Longitudinal sections of the symptomatic inflorescences or the single floret showed brown discoloration and rotting of the internal tissues. The disease caused severe crop losses (approximately 100% either in the field or after harvest). Bacteria were isolated from water-soaked and soft-rotted cauliflower heads on King's medium B (KB). The strains were purified on nutrient agar and assayed for pathogenicity on subcorymbs from freshly harvested cauliflower heads. Bacterial suspensions containing approximately 108 CFU/ml were then sprayed on the surface of subcorymbs (3 subcorymbs per strain). Furthermore, in other pathogenicity assays, the florets were dipped in 108 CFU/ml bacterial suspensions or small aliquots of inoculum were injected into the peduncle of subcorymbs with a sterile syringe. Cauliflower heads treated with sterile distilled water were used as controls. After inoculation, the subcorymbs were maintained at 25°C and approximately 100% relative humidity for 48 h. All bacterial strains either applied to cauliflower subcorymbs by spray inoculation or dipping reproduced the disease symptoms. Intensity of symptoms varied with the inoculation method. Injection of bacteria caused water soaking and soft rot of cauliflower internal tissues. No symptoms were observed in negative control subcorymbs inoculated with sterile water. All bacterial strains were gram negative and fluorescent on KB. Isolates (17 of 18) showed the LOPAT characters of group Vb (++-+-) fluorescent pseudomonads, and only strain USB1237 showed characters of group IVb (-+++-) (3). The pectolytic activity of the latter strain was confirmed by the pectinase plate assay (4). The identity of representative strains was confirmed by the nutritional profile obtained with the Biolog Identification System (MicroLogTM System Release 4.2; Biolog, Inc., Hayward, CA). Strains USB1224, USB1226, USB1228, USB1231, USB1235, USB1236, USB1238, and USB1239 were identified as Pseudomonas fluorescens with similarity indices of 0.86, 0.52, 0.73, 0.81, 0.73, 0.74, 0.69, and 0.85, respectively. The pectolytic strain USB1237 was identified as a Pseudomonas spp. that is closely related to P. putida (similarity index = 0.45). In conclusion, the above results indicate that P. fluorescens is responsible for head rot of cauliflower. A similar disease has been previously reported on broccoli in different areas (1,2), but to our knowledge, this is the first report of head rot of cauliflower caused by P. fluorescens. References: (1) C. H. Canaday et al. Phytopathology 77:1712, 1987. (2) P. D. Hildebrand. Can. J. Plant Pathol. 8:350, 1986. (3) R. A. Lelliott and D. E. Stead. Methods for the diagnosis of bacterial diseases of plants. In: Methods in Plant Pathology. Vol. 2, T. F. Preece, ed. Blackwell Scientific Publications, Oxford, UK, 1987. (4) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St. Paul, MN, 2001.

The activity of the White Line Inducing Principle (WLIP) and tolaasin 1, produced by virulent strains of Pseudomonas reactans and Pseudomonas tolaasii, respectively, was comparatively evaluated on lipid membranes. Both lipodepsipeptides were able to induce the release of calcein from large unilamellar vesicles. Their activity was dependent on the toxin concentration and liposome composition and in particular it increased with the sphingomyelin content of the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (> 27 mu M). This effect was not detected for tolaasin I at the concentrations tested (< 28 mu M). Differences were also observed in lipodepsipeptides secondary structure. In particular, the conformation of the smaller WLIP changed slightly when it passed from the buffer solution to the lipid environment. On the contrary, we observed a valuable increment in the helical content of tolaasin I which was inserted in the membrane core and oriented parallel to the lipid acyl chains. (c) 2006 Elsevier B.V. All rights reserved.

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.

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.

From superficial brown discolorations on the caps and stalks of deformed sporophores of Agaricus bisporus, bacteria were consistently isolated. On the basis of biochemical, nutritional and pathogenic characteristics, both on tissue blocks and on whole sporophores of A. bisporus, the above bacteria were identified as strains of Pseudomonas agarici.

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.

In this paper, we report the structure of the O-specific polysaccharide of the LPS fraction of the strain type NCPPB416 of X. campestris pv. pruni. It is built up of three different monosaccharides - glucose, rhamnose and xylose - in an intricate block-wise polymer. Herein, the primary structure is elucidated by means of chemical degradation and 2D NMR spectroscopy. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003).

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 α‐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 α‐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. Proteins 2003;52:534–543. © 2003 Wiley‐Liss, Inc.

Shaken culture in KB of strain NCPPB2192 of Pseudomonas tolaasii showed an activity of 3,200 and 800 units ml−1 toward Bacillus megaterium and Rhodotorula pilimanae, respectively. On the contrary, an activity of 400 units ml−1 toward B. megaterium was observed with the cultures of strain NCPPB1311 of P. “reactans”. The purification of lipodepsipeptides from the cultures of P. tolaasii and P. “reactans” yielded about 13 mg l−1 of tolaasin and 169 mg l−1 of White Line Inducing Principle (WLIP), respectively. Antimicrobial assays showed that HPLC grade tolaasin I inhibited the growth of filamentous fungi and yeasts and Gram positive bacteria. Only a limited activity was observed toward some Gram negative bacteria. Of particular interest is the activity of tolaasin I toward phytopathogenic fungi, Pleurotus and Agaricus spp., as well as yeasts and filamentous fungi responsible for mycoses of mammals. WLIP inhibited the growth of fungi and Gram positive bacteria, though at higher concentration when compared to tolaasin I, but not that of Gram negative bacteria. Assays of the two toxins on blocks of A. bisporus confirmed the antifungal activity of the two lipodepsipeptides and suggested their possible role in the symptoms caused by the mushroom bacterial pathogens.

The activity of tolaasin I and White .Line !nducing £rinciple (WLIP), produced in vitro by virulent strains of Pseudomonas tolaasii and P. reactans, respectively, was comparatively evaluated on red blood cells and large unilamellar vesicles. Tolaasin I and WLIP caused red blood cell haemolysis through a colloid-osmotic shock apparently caused by transmembrane pores formation. Both lipodepsipeptides were able to form channels in lipid vesicles. Their activity was dependent on the toxin concentration and liposomes composition, and independent on the pH. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (higher than 27 /lM). This effect was not detected for tolaasin I at the concentrations tested (up to 28.2 1lM).

Recent investigations have shown that brown blotch of Agaricus bisporus and the yellowing of Pleurotus ostreatus, caused by Pseudomonas tolaasii, are actually complex diseases. In fact, besides P. tolaasii, which may be considered the main causal agent of the above diseases, also P. "reactans" and not yet characterised fluorescent pseudomonads participated to the expression of the diseases symptoms. Furthermore, P. "reactans" has been demonstrated to be the causal agent of P. eryngii yellowing. The demonstration that P. "reactans", a bacterial entity considered a saprotroph associated to cultivated mushrooms and useful for the specific identification of P. tolaasii in the "white line" assay, is a pathogen of A. bisporus and Pleurotus spp. cultivated mushrooms is a novelty. However, a strong variability in the pathogenicity, antagonistic activity and response in "white line" assay was observed among the strains of P. "reactans". This behaviour is apparently due to the formation in the cultures of morphological variants which were avirulent, did not produce the White Line Inducing Principle (WLIP) and were negative in the "white line" assay. The presence of an high ratio of the above variants in the cultures may explain the unravelled pathogenicity of this bacterium.

Essential oils extracted from fruits of cumin, caraway, fennel and coriander, were assayed in vitro for antibacterial activity toward strains of ten Pseudomonas syringae pathovars. Significant antibacterial activity was shown by essential oils of cumin, caraway and coriander. In general a lower activity was shown by essential oil from fennel. These preliminary results indicated the potential use of the above essential oils in the control of diseases caused by P. syringae pathovars on important crops.