Enzyme-linked immunosorbent assay for the detection and identification of plant pathogenic bacteria (in particular for Erwinia amylovora and Clavibacter michiganensis subsp. sepedonicus).

Enzyme-linked immunosorbent assay (ELISA) is the most commonly used serological diagnostic technique. A number of different ELISA formats can be used for the detection of bacterial plant pathogens and in particular Erwinia amylovora and Clavibacter michiganensis subsp. sepedonicus.

Indirect immunofluorescence microscopy for the detection and identification of plant pathogenic bacteria (in particular for Ralstonla solanacearum).

Immunofluorescence microscopy is a very sensitive serological test which harnesses both the power of antibodies to bind to targets along with the use of the fluorescence microscope to visualise the structures to which they bind. Antibody binding is visualised by the fluorescent emission from a marker molecule bound to the antibody. The technique is of particular use in bacteriology and pathology where the location and morphology of the bacterial cells can be viewed along with the location of the fluorescently labelled antibodies.

Pseudomonas syringae pv. coryli, the Causal Agent of Bacterial Twig Dieback of Corylus avellana.

ABSTRACT Thirty-eight bacterial strains isolated from hazelnut (Corylus avellana) cv. Tonda Gentile delle Langhe showing a twig dieback in Piedmont and Sardinia, Italy, were studied by a polyphasic approach. All strains were assessed by fatty acids analysis and repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using BOX and ERIC primer sets. Representative strains also were assessed by sequencing the 16S rDNA and hrpL genes, determining the presence of the syrB gene, testing their biochemical and nutritional characteristics, and determining their pathogenicity to hazelnut and other plants species or plant organs. Moreover, they were compared with reference strains of other phytopathogenic pseudomonads. The strains from hazelnut belong to Pseudomonas syringae (sensu latu), LOPAT group Ia. Both fatty acids and repetitive-sequence-based PCR clearly discriminate such strains from other Pseudomonas spp., including P. avellanae and other P. syringae pathovars as well as P. syringae pv. syringae strains from hazelnut. Also, the sequencing of 16S rDNA and hrpL genes differentiated them from P. avellanae and from P. syringae pv. syringae. They did not possess the syrB gene. Some nutritional tests also differentiated them from related P. syringae pathovars. Upon artificial inoculation, these strains incited severe twig diebacks only on hazelnut. Our results justify the creation of a new pathovar because the strains from hazelnut constitute a homogeneous group and a discrete phenon. The name of P. syringae pv. coryli is proposed and criteria for routine identification are presented.

Collimonas fungivorans gen. nov., sp. nov., a chitinolytic soil bacterium with the ability to grow on living fungal hyphae.

A polyphasic approach was used to describe the phylogenetic position of 22 chitinolytic bacterial isolates that were able to grow at the expense of intact, living hyphae of several soil fungi. These isolates, which were found in slightly acidic dune soils in the Netherlands, were strictly aerobic, Gram-negative rods. Cells grown in liquid cultures were flagellated and possessed pili. A wide range of sugars, alcohols, organic acids and amino acids could be metabolized, whereas several di- and trisaccharides could not be used as substrates. The major cellular fatty acids were C(16 : 0), C(16 : 1)omega7c and C(18 : 1)omega7c. DNA G+C contents were 57-62 mol%. Analysis of nearly full-length 16S rDNA sequences showed that the isolates were related closely to each other (>98.6 % sequence similarity) and could be assigned to the beta-Proteobacteria, family 'Oxalobacteraceae', order 'Burkholderiales'. The most closely related species belonged to the genera Herbaspirillum and Janthinobacterium, exhibiting 95.9-96.7 % (Herbaspirillum species) and 94.3-95.6 % (Janthinobacterium species) 16S rDNA sequence similarity to the isolates. Several physiological and biochemical properties indicated that the isolates could be distinguished clearly from both of these genera. Therefore, it is proposed that the isolates described in this study are representatives of a novel genus, Collimonas gen. nov. Genomic fingerprinting (BOX-PCR), detailed analysis of 16S rDNA patterns and physiological characterization (Biolog) of the isolates revealed the existence of four subclusters. The name Collimonas fungivorans gen. nov., sp. nov. has been given to one subcluster (four isolates) that appears to be in the centre of the novel genus; isolates in the other subclusters have been tentatively named Collimonas sp. The type strain of Collimonas fungivorans gen. nov., sp. nov. is Ter6(T) (=NCCB 100033(T)=LMG 21973(T)).