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Leaves of olive and oleander were sprayed with suspensions of their homologous strains (PVBa230 and ITM519, respectively) of Pseudomonas syringae subsp. savastanoi and examined by SEM. It was found that both strains multiplied on the lower surface of the leaves of both species. Preferred sites for survival and multiplication were the shields of peltate hairs on olive and the stomatal pits on oleander. The findings suggest that, at least in olive leaves, some bacteria entered the leaf tissue through the stomata but that this was not important since cells which entered the host did not cause any disease symptoms. Cells of both strains were agglomerated and cells of ITM519 were further attached to the surface of the leaf hairs on oleander by fibrillar material. Inoculated leaves did not show any disease symptoms except at leaf abscission scars on olive plants where leaves had been excised prior to inoculation. It is suggested that on both olive and oleander preexisting wounds are necessary for symptoms to develop. [ABSTRACT FROM AUTHOR]

Pinus nigra(Austrian pine) is a tree widely planted in central and southern Europe in the reforestation of areas with poor soils. One of the major constrains ofP. nigrais a fungal disease caused bySphaeropsis sapineawhich causes shoot tip dieback and tree mortality. This paper describes a real-time quantitative PCR assay based on the partial sequence of the small subunit ribosomal RNA ofS. sapineato detect and quantify DNA of the fungal pathogen inS. sapineainoculated shoots. The study found that real-time quantitative PCR was a reliable technique to diagnose this disease and suggests that it can also be used to study fungal behaviour in host tissue and particularly to quantify fungal growth in the latent or endophytic phase. [ABSTRACT FROM AUTHOR]

Aims: To develop a PCR-based protocol for the rapid, sensitive and specific detection of Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) in bean seeds. Methods and Results: A pair of PCR primers (CffFOR2-CffREV4), targeting the sequence of a cloned DNA fragment of 550 bp amplified in Repetitive-sequence-based-PCR (Rep-PCR) experiments, were designed and shown to specifically amplify a 306-bp DNA fragment using CffDNA as template. Moreover, this PCR protocol was demonstrated to successfully detect Cff in naturally infected bean seeds in 36 h. Conclusions: A specific, highly sensitive and rapid PCR assay for the detection of Cff was achieved. Significance and Impact of the Study: Cff is a seed-borne bacterium on the EPPO A2 quarantine list; this procedure may be useful for routine diagnosis of Cff, overcoming the problems of conventional techniques. [ABSTRACT FROM AUTHOR]

Summary To verify the role of phytoplasma infections in a decline of Ulmus chenmoui in Central Italy, two plots of seedlings established in two locations were studied. Roots and twigs with leaves were sampled in June and in September 1996 from 11 U. chenmoui plants located at San Rossore (Pisa), and in July 1996 and September 1997 from nine U. chenmoui plants at Presiola (Ravenna). The plants at San Rossore showed a generalized decline consisting of small, pale green leaves and death of small twigs, whereas those at Presiola had an healthy appearance in 1996 and started to decline in 1997. Molecular analyses resulted in the identification of elm yellows-related phytoplasmas in three out of the twenty plants examined (two from San Rossore and 1 from Presiola). The same phytoplasma was identified in both locations in yellows-diseased elm hybrids employed as positive controls. Phytoplasmas belonging to other groups were also detected in one U. chenmoui plant and in weeds and insects tested from the Presiola plot. Restriction fragment length polymorphism analyses on 16Sr DNA, spacer region and ribosomal protein fragment coding for the 3′ end of rpl22 and the entire rps3 genes, did not show a genetic variability in elm yellows phytoplasmas detected in U. chenmoui . No differences among these isolates, phytoplasmas infecting other elm species in these plots and the reference elm yellows isolates were found. [ABSTRACT FROM AUTHOR]

The present study compares the pathogenicity on olive and oleander plants of three wild-type strains of Pseudomonas syringae subsp. savastanoi (ITM317 and PBa230 from olive and ITM519 from oleander) and three phytohormone-deficient mutants of ITM519: ITM519-41 (Iaa+/cytokinins+), ITM519-7 (Iaa+/cytokinins+), ITM519-6 (Iaa+/cytokinins+). Mutants not producing IAA (ITM519-7 and ITM519-6) only induced necrosis of the inoculated tissues (ITM519-6) or swellings on the stems attributed to cytokinin production accompanied by necrosis (ITM519-7). By contrast, the Iaa+/cytokinins+ mutant (ITM519-41) induced attenuated symptoms on stems and knots similar to those obtained with the parent strain on oleander leaves. Olive strains induced necrosis of oleander leaves and were virulent and avirulent, respectively, on olive and oleander stems. Strain ITM519 and its three mutants were all able to multiply in oleander leaves at similar rates, reaching the same final populations. By contrast, the two olive strains multiply poorly, reaching populations c.10²-fold lower. These results confirm that expression of IAA genes alone is sufficient to initiate the development of knots on oleander while cytokinins are necessary for the full expression of the disease symptoms (determining knot size). The findings also indicate that the plant tissues (stems and leaves) react differently to the various strains of the bacterium and, furthermore, suggest that, besides phytohormones, other pathogenetic factors could be involved in this host-pathogen interaction. The necrotic reaction of oleander leaves heavily inoculated with olive strains was interpreted as a possible form of hypersensitivity reaction. [ABSTRACT FROM AUTHOR]

Over 85 % of total cellular fatty acids of 30 strains of P. syringae pv. savastanoi, grown for one day at 28 °C on King's medium B (KB) agar, were 12 : 0 (5.0 %), 16 : 0 (27.5 %), 16 : 1 (36.7 %) and 18 : 1 (16.8 %). Three hydroxy-substituted fatty acids comprised 7.2 % of the total and 22 other minor components, each occurring at concentrations of less than 1 %, comprised an additional 4 %. Three percent were unidentified components. Cells grown for 3 and 6 days on KB agar contained lower concentrations of the unsaturated 16 : 1 (30.4 and 21.1 %, respectively), and higher concentrations of branched-chain and cyclopropane fatty acids than one-day old cells. No consistent differences in fatty acid composition could be detected between virulent and avirulent strains, nor between pv. savastanoi and other pathovars of P. syringae. However, when cells were grown on a chemically- defined medium for 6 days, concentrations of 16 : 0 and a tentatively-identified 17-carbon hydroxy fatty acid were higher, and those of 12 : 0 and 16 : 1 were lower in strains from Fraxinus than from Olea. P. fluorescens (7 strains) and P. viridiflava (6 strains) could be differentiated from each other but not from P. syringae. [ABSTRACT FROM AUTHOR]

The cytokinin and auxin production by Pseudomonas amygdali was studied in relation to bacteria virulence on almond plants. All isolates of Pseudomonas amygdali used in this study secreted indoles and cytokinins in culture medium although their relative amount varied among the strains-1-zeatin (t-Z) and indole-3-acetic acid (IAA), which proved to be the main components of cytokinin and indole mixtures, ranged respectively, as determined by HPLC, from 0.013 to 4.621 mg I"1 and from 0.1 to 15.14 mg I"1. The production of IAA and t-Z appeared to be correlated to virulence of P. amygdali. The highly virulent strains, having the shortest incubation period and inducing the longest cankers produced high levels of IAA and t-Z in vitro. On the other hand, the weakly virulent strains, having longer incubation periods and inducing shorter cankers, secreted low levels of IAA and t-Z. Strain A142, producing high level of IAA and low level of t-Z had the same incubation period as the highly virulent strains but a canker length similar to that of the other weakly virulent strains. These results suggest that in the disease process by P. amygdali on almond the amount of IAA and cytokinins are correlated respectively to the length of the incubation period and to the final length of the cankers. [ABSTRACT FROM AUTHOR]

The ability to produce indole‐3‐acetic acid (IAA) through the indole‐3‐acetamide (IAM) pathway as well as cytokinins is a common trait of Pseudomonas savastanoi populations causing disease on oleander and olive. These phytohormones are required for the induction and development of an outgrowth of plant cell tissue termed a knot. However, in myrtle orchards of Sardinia (Italy), strains of P. savastanoi unable to produce cytokinins were found coexisting with cytokinin‐producing strains. Data presented here show that the ability to produce IAA through the IAM pathway is also a variable trait within this population, raising questions on the exact role of these plant growth substances in the disease process on myrtle. Three P. savastanoi strains were selected based on their differential ability to produce phytohormones in vitro, and their interaction with the host was investigated over a period of 8 months using histological methods. All strains successfully invaded the infected twigs, moving systemically (unhalted by host defences) upward and downward from the inoculation point, both by completely degrading the cell walls and by taking advantage of the xylem vessels and intercellular spaces. Moreover, all strains induced the development of cankers, which slowly evolved into typical knots only on the twigs inoculated with the phytohormone‐producing strains. This study further demonstrates that cytokinins and IAA are essential for knot development; moreover, it ascertains that bacterial production of cytokinins is not necessary for host colonization and for the expression of pathogenicity (i.e. the ability to cause disease) of P. savastanoi on myrtle. [ABSTRACT FROM AUTHOR]

A flow cytometry technique that unequivocally identifies some of the toxic metabolites produced by Phaco,noniei/a chiamydospora, one of the main fungal pathogens causing esca disease of grapevine (Vitis vinifera), was developed. Antibodies raised against exopolysaccharides (EPS)metabolites produced by Pa. chlarnydospora that have been reported to be phytotoxic-were used as antigen to immunize rats. The specificity of these antibodies was assayed by flow cytometry against Pa. chiamydospora polysaccharides and against EPS with a different structure isolated from other phytopathogenic fungi, including Phaeoacremonium aleophilum and the Botryosphaeriaceae species Neofusicoccum Iuteuin and N. parvuin. Using this method, Pa. clilainydospora polysaccharides were detected in the symptomatic leaves of esca-affected grapevines, while healthy and asymptomatic leaves from both healthy and diseased vines did not produce a binding reaction. This method potentially could be used to develop a simple kit to study the mechanisms underlying the development of esca foliar symptoms and to indirectly assess the presence of Pa. chlainydospora in grapevine material. [ABSTRACT FROM AUTHOR]

The manner in which the bacterium Pseudomonas savastanoi pv. savastanoi ( Pss), the causal agent of knot disease, infects olive plants is erratic and has not been fully documented. To investigate the process of Pss invasion, olive explants were inoculated in vitro and examined visually and by light microscopy at 2-weekly intervals for 10 weeks. In all host genotypes tested, interaction with the pathogen resulted in: (i) a progressive collapse of the stem, originating at the inoculation site at the apex of the explant, and proceeding downwards; and (ii), localized outgrowths on the stem located at various distances from the inoculation site. Histological analysis revealed that the anatomy of the outgrowths closely resembled that of knots formed in vivo; they showed that Ps. savastanoi also diffused within the olive explants through the xylem vessels, and that the olive host reacted to pathogen invasion, possibly by producing substances of polysaccharidic and/or phenolic nature. [ABSTRACT FROM AUTHOR]

Results of a survey of olive knot disease in central Italy in 2002 and 2003 showed that Pantoea agglomerans was found associated with the pathogen Pseudomonas savastanoi pv. savastanoi ( Ps. savastanoi) in 70% of the olive knots examined. Pathogenicity tests in which these two bacteria were co-inoculated on the stems of 1-year-old olive plants at ratios of 1:1, 1:100 and 100:1 showed that the growth of P. agglomerans was apparently aided by the presence of an actively growing population of Ps. savastanoi. At the same time, however, a dominant population of P. agglomerans at the inoculation site tended to depress the growth of Ps. savastanoi, probably because of competition for space and nutrients between these bacteria and by means of antibiotic production by P. agglomerans. In some cases the association of P. agglomerans, which in culture was found to produce indole-3-acetic acid but not cytokinins, with Ps. savastanoi resulted in an increase in the size of knots. This boosting effect of P. agglomerans on proliferation was probably due to the release of IAA by this bacterium at the inoculation sites. [ABSTRACT FROM AUTHOR]

A selected group of strains of Pseudomonas syringae subsp. savastanoi from olive, oleander and ash were compared with pathogenicity tests and with DNA restriction fingerprinting using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The strains from each host were distinguishable by their pathogenicity to the same host and to the other two plant species. A division into the same groups was obtained with unweighted pair-group method with averages (UPGMA) clustering of the data from genomic fingerprinting, even though high overall similarity between the strains also indicated that they formed a single, well characterized taxon. It seems clear that the subspecies savastanoi of P. syringae comprises at least 3 groups of strains that differ in their precise host range, in the nature of the symptoms induced on the individual hosts, and in their genomic profile. [ABSTRACT FROM AUTHOR]