Carlo Bazzi

The inhibition of susceptible and hypersensitive reactions by protein-lipopolysaccharide complexes from phytopathogenic pseudomonads: relationship to polysaccharide antigenic determinants

Abstract Protein-lipopolysaccharide (pr-LPS) complexes were purified from a virulent strain of Pseudomonas tabaci (NCPPB 1427) and two incompatible strains ( P. aptata NCPPB 2664 and P. lachrymans NCPPB 1436). They all contained two major polypeptides with mol. wts of 20 000 and 40 000. Slide agglutination tests with O- or pr-LPS-antisera produced against antigens from P. aptata NCPPB 2664 showed that the surface antigens of P. aptata had common determinants with P. tabaci NCPPB 1427, but not with P. lachrymans NCPPB 1436. Precipitin ring tests of heat-stable antigens (121 °C) in the presence of pr-LPS antiserum indicated that most determinants were carried by lipopolysaccharides. Pr-LPS complexes from the three strains, pre-injected into tobacco leaves, prevented hypersensitive confluent necrosis evoked by seven incompatible pseudomonads, and delayed or inhibited the development of the susceptible reaction induced by five compatible strains. These effects were unrelated to the degree of virulence of compatible strains and to the type of surface antigenic determinants of the whole bacterium, as recognized by the rabbit immune system. This suggests a common mechanism of interaction in the protected tissue, causing plant tolerance to various degrees towards different strains. Pr-LPS complexes of P. tabaci NCPPB 1427 were most effective in inducing protection, consistent with the possibility that compatible strains may produce surface substances having higher tolerance-inducing activity.

Induction of antimicrobial 3-deoxyflavonoids in pome fruit trees controls fire blight.

Abstract Fire blight, a devastating bacterial disease in pome fruits, causes severe economic losses worldwide. Hitherto, an effective control could only be achieved by using antibiotics, but this implies potential risks for human health, livestock and environment. A new approach allows transient inhibition of a step in the flavonoid pathway, thereby inducing the formation of a novel antimicrobial 3-deoxyflavonoid controlling fire blight in apple and pear leaves. This compound is closely related to natural phytoalexins in sorghum. The approach does not only provide a safe method to control fire blight: Resistance against different pathogens is also induced in other crop plants.

Biological control of grape crown gall by strain f2/5 is not associated with agrocin production or competition for attachment sites on grape cells.

ABSTRACT Agrocin-minus mutants of nontumorigenic Agrobacterium vitis strain F2/5 controlled grape crown gall as well as the wild-type strain, indicating that agrocin is not a major factor in the mechanism of biological control. Relative levels of attachment to grape cells by tumorigenic and biocontrol strains were also measured. Attachment of tumorigenic strains (CG49 and K306) and biological control strains (F2/5 and agrocin-minus mutant 1077) was often reduced when mixtures of the strains were applied. However, high populations (10(3) to 10(5) CFU/ml) of all strains attached following mixed inoculations, suggesting that competition for attachment sites is also not a factor in the mechanism of biological control. Transfer of T-DNA to grape by CG49 was prevented or greatly inhibited in the presence of F2/5 or 1077 as measured by expression of the GUS reporter gene. The Ti plasmid virulence genes, however, were induced by exudates from grape shoots that had been inoculated with F2/5. Sonicated and autoclaved preparations of F2/5 and 1077 did not control crown gall or inhibit T-DNA transfer. Control by F2/5 is specific to grape, since gall formation on tomato, sunflower, and Kalanchoe daigremontiana were not inhibited.

Survival and tumorigenicity of agrobacterium vitis in living and decaying grape roots and canes in soil

Agrobacterium vitis was recovered from living and decaying grape roots and canes 23 months after grapes were artificially inoculated with a mixture of six strains of the bacterium. Each strain contained a unique plasmid profile. Following inoculation, some plants were treated with the herbicide Roundup to speed up plant death and tissue decay. Roots and canes were assayed over time, and by comparing plasmid profiles of recovered strains it was determined that certain A. vitis strains used in the inoculum mixture were recovered more frequently than others. Profiles identical to those identified for each strain used in the inoculum mixture were observed at least twice in strains recovered during the experiment. Of 133 plasmid profiles that were observed, only 18 did not resemble any of the strains used in the inoculum mixture. Of 333 strains recovered from roots and canes, 321 were tumorigenic, indicating that this trait was stable throughout the experiment. A group of six strains having plasmid profiles identical to strain CG49 that were recovered over an 16-month period were further characterized using restriction fragment length polymorphic analysis of plasmid DNA, random amplified polymorphic DNA analysis of total genomic DNA, and ribofingerprinting of a chromosomal region including 1,479 bp (99.5%) of the 16S rDNA, the intergenic spacer between 16S and 23S rDNA genes, and 132 bp of the 23S rDNA gene. All six strains were shown to be identical to CG49.

Identification and in planta detection of Pseudomonas syringae pv. tomato using PCR amplification of hrpZPst.

A rapid detection method based on PCR amplification of Pseudomonas syringae pv. tomato chromosomal sequences was developed. Primer design was based on the P. syringae DC3000 hrpZPst gene, which maps on a pathogenicity-associated operon of the hrp/hrc pathogenicity island.A 532 bp product corresponding to an internal fragment of hrpZPst was amplified from 50 isolates of P. syringae pv. tomato belonging to a geographically representative collection. The amplification product was also obtained from three coronatine-deficient strains of P. syringae pv. tomato.On the other hand, PCR did not produce any such products from 100 pathogenic and symbiotic bacterial strains of the genera Pseudomonas, Xanthomonas, Erwinia, and Rhizobium and 75 unidentified bacterial saprophytes isolated from tomato plants. The method was tested using leaf and fruit spots from naturally-infected tomato plants and asymptomatic nursery plants and artificially contaminated tomato seeds. The results confirmed the high specificity observed using pure cultures.

Encapsulation of Pseudomonas syringae pv. tabaci in relation to its growth in tobacco leaves both pretreated and not pretreated with protein-lipopolysaccharide complexes

Abstract Protein-lipopolysaccharide (pr-LPS) complexes from Pseudomonas aptata NCPPB 2664 were purified and used to pretreat tobacco leaves, thus inducing protection against P. syringae pv. tabaci NCPPB 1427. Protection consisted in the delaying of the susceptible reaction. The growth of P. syringae pv. tabaci was approximately the same in the control as in the pretreated tissue during the first hours of the experiment, but the number of bacteria became significantly greater in the control tissue after 24 h. This peak was followed by a population decline. In contrast, the maximum level of growth was reached after 48 h in the pretreated tissue, and was followed by a gradual decline. P. syringae pv. tabaci appeared to be encapsulated, both in the control and in the protected tissue, during a period which fell between the lag phase and the maximum of the growth curves. When the population reached their maximum, the bacteria appeared to be entrapped within a network of fibrillar material in the intercellular spaces. The encapsulation of P. syringae pv. tabaci leaves is interpreted as a critical phase which enables the bacteria to survive until achieve is established.

Detection of Agrobacterium vitis by PCR using novel virD2 gene-specific primers that discriminate two subgroups

Tumour tissue samples were collected from vines grown in various regions of Italy and other parts of Europe and extracted for detection of Agrobacterium vitis. Fifty strains were isolated on agar plates and screened by PCR with consensus primers from the virD2 gene. They were confirmed as A. vitis with a species-specific monoclonal antibody. The isolates were further analyzed by PCR for their opine synthase genes and ordered into octopine, nopaline and vitopine strains. Primers designed on the octopine synthase gene did not detect octopine strains of Agrobacterium tumefaciens. For quantitative PCR, virD2 fragments were sequenced: two classes of virD2 genes were found and two primer sets designed, which detected octopine and nopaline strains or only vitopine strains. For simultaneous identification of all opine-type strains, multiplex real-time PCR with either primer pair and SYBR Green was performed: the combined sets of primers gave signals with DNA from any A. vitis strain. Specificity of the new primers for real-time PCR was evaluated using several unidentified bacterial isolates from grapevines and other plant species. An elevated level of non-specific background was observed when the combined primer sets were used in multiplex PCR assays. The real-time PCR protocol was also used to detect A. vitis cells directly from grapevine tumours; avoiding direct isolation procedures a sensitivity in the range of one to ten cells per assay was found. Inhibition of the PCR reaction by plant material was overcome by treating tumour extracts with a DNA purification kit as a step for the isolation of nucleic acids.

Electronic nose as an innovative tool for the diagnosis of grapevine crown gall

For the first time, a portable electronic nose was used to discriminate between healthy and galled grapevines, experimentally inoculated with two tumourigenic strains of Agrobacterium vitis. The volatile profile of target cutting samples was analysed by headspace solid phase microextraction coupled with gas chromatography-mass spectrometry. Spectra from tumoured samples revealed the presence of styrene which is compatible with decarboxylation of cinnamic acid involved in secondary metabolism of plants. Principal Component Analysis confirmed the difference in volatile profiles of infected vines and their healthy controls. Linear Discriminant Analysis allowed the correct discrimination between healthy and galled grapevines (83.3%, cross-validation). Although a larger number of samples should be analysed to create a more robust model, our results give novel interesting clues to go further with research on the diagnostic potential of this innovative system associated with multi-dimensional chemometric techniques.

Characterization of Erwinia amylovora strains from Croatia

Erwinia amylovora is the causative agent of fire blight, a destructive disease of rosaceous plants. The European population can be divided into several subtypes according to differences in restriction fragment length polymorphism of the XbaI genomic DNA digest analysed with pulsed-field gel electrophoresis. This technique was also used to determine the genetic relatedness of six Croatian isolates to the E. amylovora types found in the countries surrounding Croatia. The isolates belong to the Pt2 pattern type that is characteristic of the East Mediterranean basin. All tested isolates gave essentially the same total cell protein pattern in SDS-polyacrylamide gel electrophoresis. The number of short-sequence DNA repeats in plasmid pEA29 of six isolates was determined by PCR assays and ranged from four to seven. The isolates examined showed high pathogenicity in immature pear fruits. Differences were also revealed in microbiological assays such as amylovoran synthesis, levan formation, siderophore production and colour on coliform medium.

Molecular Differentiation of Erwinia amylovora Strains from Europe and the Mediterranean Region

Fire blight is a necrotic disease, which affects fruit trees like apple and pear and some ornamentals (e.g. Cotoneaster and Crataegus spp.) (6). Most European countries and the Eastern Mediterranean area are affected (7). The spreading of the causative bacterium E. amylovora can occur over short distances for example via insects but also over longer distances via birds, wind and asymptomatic plant propagation materials. Recent outbreaks of the disease in Italy (9) and Spain (4) renewed the question about the origin of the pathogen. To describe the epidemic spread of fire blight we have applied two molecular methods: Pulsed Field Gel Electrophoresis (PFGE) (8,9) and determination of short sequence DNA repeats (SSR analysis) (2,3,5).