Plant-pathogenic bacteria as biological weapons - real threats?

At present, much attention is being given to the potential of plant pathogens, including plant-pathogenic bacteria, as biological weapons/bioterror weapons. These two terms are sometimes used interchangeably and there is need for care in their application. It has been claimed that clandestine introduction of certain plant-pathogenic bacteria could cause such crop losses as to impact so significantly on a national economy and thus constitute a threat to national security. As a separate outcome, it is suggested that they could cause serious public alarm, perhaps constituting a source of terror. Legislation is now in place to regulate selected plant-pathogenic bacteria as potential weapons. However, we consider it highly doubtful that any plant-pathogenic bacterium has the requisite capabilities to justify such a classification. Even if they were so capable, the differentiation of pathogens into a special category with regulations that are even more restrictive than those currently applied in quarantine legislation of most jurisdictions offers no obvious benefit. Moreover, we believe that such regulations are disadvantageous insofar as they limit research on precisely those pathogens most in need of study. Whereas some human and animal pathogens may have potential as biological or bioterror weapons, we conclude that it is unlikely that any plant-pathogenic bacterium realistically falls into this category.

First Report of Head Rot of Brassica oleracea convar. botrytis var. italica Caused by Pseudomonas fluorescens in Southern Italy.

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.

WLIP and tolaasin I, lipodepsipeptides from Pseudomonas reactans and Pseudomonas tolaasii, permeabilise model membranes.

The activity of the White Line Inducing Principle (WLIP) and tolaasin I, 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 microM). This effect was not detected for tolaasin I at the concentrations tested (< 28 microM). 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.

Structure elucidation of the O-chain from the major lipopolysaccharide of the Xanthomonas campestris strain 642.

A novel O-polysaccharide consisting of D-Xylp and L-Rhap in the molar ratio of 1:2.5 was identified as the major component in the lipopolysaccharide fraction of Xanthomonas campestris strain 642, which is responsible for a new bacterial disease of the strawberry plant. Its structure was mainly determined using chemical analysis, Smith degradation and 1D and 2D NMR spectroscopy experiments as: carbohydrate sequence [see text].

Structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae.

The covalent structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae isolates, pathogenic on various species of citrus trees, has been deduced from 1D and 2D 1H- and 13C-NMR spectra combined with extensive FAB-MS data and results of some chemical reactions. Similarly to syringomicins and syringostatins, produced by other plant pathogenic strains of P. syringae pv. syringae, syringotoxin is a lipodepsinonapeptide. Its peptide moiety corresponds to Ser-Dab-Gly-Hse-Orn-aThr-Dhb-(3-OH)Asp-(4-Cl)Thr with the terminal carboxy group closing a macrocyclic ring on the OH group of the N-terminal Ser, which in turn is N-acetylated by 3-hydroxytetradecanoic acid.

The structure of syringomycins A1, E and G.

By a combination of 1D and 2D 1H- and 13C-NMR, FAB-MS, and chemical and enzymatic reactions carried out at the milligram level, it has been demonstrated that syringomycin E, the major phytotoxic antibiotic produced by Pseudomonas syringae pv. syringae, is a new lipodepsipeptide. Its amino acid sequence is Ser-Ser-Dab-Dab-Arg-Phe-Dhb-4(Cl)Thr-3(OH)Asp with the beta-carboxy group of the C-terminal residue closing a macrocyclic ring on the OH group of the N-terminal Ser, which in turn is N-acylated by 3-hydroxydodecanoic acid. Syringomycins A1 and G, two other metabolites of the same bacterium, differ from syringomycin E only in their fatty acid moieties corresponding, respectively, to 3-hydroxydecanoic and 3-hydroxytetradecanoic acid.

Long-term storage of plant-pathogenic bacteria in sterile distilled water.

This study was made to determine the effectiveness of the preservation of plant-pathogenic bacteria in sterile distilled water. After 20 or 24 years of storage in distilled water, a very high percentage (90 to 92%) of the isolates of Agrobacterium tumefaciens and Pseudomonas spp. were still alive. Moreover, 12 of 13 viable (after 24 years) isolates of P. syringae subsp. syringae maintained their ability to produce syringomycin and were pathogenic to bean seedlings. The water-stored cells of two isolates of P. syringae subsp. syringae, when observed by electron microscopy, were smaller than cells of 24-h-old subcultures of bacterial cells grown in nutrient broth; the water-stored cells appeared plasmolysed with an electron-dense cytoplasm and thickened cell wall.