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In this study, the efficacy of the strain BB20 of P. fluorescens (Pf) bv.1 (Patent No. PCT/IB2012/050694; European Application No./Patent No. 12706932.6 – 1401) was in vitro and in vivo evaluated against several bacterial pathogens, agents of disease of different plant species. Moreover, preliminary solid and liquid formulations of the antagonist were firstly assayed for their time stability when stored at 4 °C and assayed against bacterial pathogens in in vivo experiments. In in vitro experiments BB20 inhibited the growth of several bacterial pathogen species by producing a peptidic antibacterial compound. Moreover, the antagonist suspension was able to prevent infections of Erwinia amylovora (Ea), Xanthomonas vesicatoria (Xv) and X. arboricola pv. pruni (Xap) by providing a relative protection of 62%, 64% and 58% when applied on pear, tomato and peach plants, respectively, under greenhouse and outdoor conditions. The solid and liquid formulations resulted stable and maintained the initial concentration for 6monthsat4°C. The in vivo efficacy of both solid and liquid formulations was comparable to that of fresh bacterial suspensions and resulted able to significantly reduce the fire blight incidence on pear plantlets and to statistically decrease the bacterial leaf spot severity on tomato plants and peach one-year-scions. The in vitro efficacy against a broad spectrum of bacterial pathogen species and the ability to prevent bacterial plant infections under controlled and field conditions pose the strain BB20 as a versatile tool in the frame of integrated control measures.

A molecular diagnostic technique (polymerase chain reaction enzyme-linked immunosorbent assay [PCR-ELISA]) for detection of Erwinia amylovora was developed. The protocol is based on the immunoenzymatic determination of PCR products. For in vitro amplification, we used previously published primers able to detect the cryptic plasmid pEA29, which is ubiquitous in E. amylovora. Amplicons were labeled with 11-digoxigenin (DIG)-dUTP during the amplification reaction, captured by hybridization to a biotinylated oligonucleotide in streptavidin-coated ELISA microplates, and then detected with anti-DIG-Fab′-peroxidase conjugated antibodies. The specificity of the assay was verified using E. amylovora strains from different host plants and geographical origins in addition to other plant-associated bacteria (either phytopathogenic or saprophytic) belonging to the genera Erwinia, Pseudomonas, and Agrobacterium. In detection threshold experiments with pure cultures, as few as 30 and 3 CFU/reaction tube were detected when the ABTS (colorimetric) and ECL (chemiluminescent) detection assays, respectively, were used. PCR-ELISA coupled with chemiluminescent detection was able to detect as few as 4 × 102 CFU/g of artificially infested pear twigs. The assay was further shown to be suitable for detection of E. amylovora in naturally infected plant organs, and the results were compared to those obtained using standard PCR assays with electrophoretic separation of amplicons.

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.

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.

The efficacy of the dioxygenase inhibitor prohexadione-Ca (ProCa) as an inducer of resistance responses against apple scab (Venturia inaequalis/Spilocaea pomi) was evaluated in two sets of experiments under glasshouse conditions. ProCa (200 ppm) was applied on seedlings of open-pollinated Malus domestica L. cv. Golden Delicious, and 10 days after application, a group of seedlings was inoculated with a conidial suspension of S. pomi. Total RNA was extracted from leaf samples collected at different times from both inoculated and noninoculated seedlings, and expression of the pathogenesis-related proteins PR-1, PR-2, PR-5, PR-8, PR-10 and chitinase was studied by reverse transcription-polymerase chain reaction and Northern blot hybridisation. Disease severity was significantly lower on seedlings treated with ProCa than on the water-treated control seedlings. ProCa triggered a general increase in gene expression of almost all the PR genes; only the level of chitinase and PR-10 did not show a detectable increase. A strong upregulation of all the assayed genes was observed in leaves of apple seedlings first treated with ProCa and then inoculated with the pathogen, whereas no expression and/or a constitutive signal was found in samples from the control seedlings.

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.

Crown gall is considered one of the most important and widespread bacterial diseases of grapevine (Vitis vinifera L.) throughout the world. It is known in Europe for more than 150 years and can be still of great phytopathologic significance in the vineyards and nurseries, especially in cold-climate regions. The disease is predominantly caused by tumorigenic strains of Agrobacterium vitis, more rarely by tumorigenic A. tumefaciens and A. rhizogenes. Unlike A. tumefaciens and A. rhizogenes, that are broad-host-range pathogens, A. vitis is specific to grapevine. Crown gall reduces vigor and yield of grapevines and severe disease may cause partial or complete death of infected plants. High losses occur in nurseries where different graft combinations with visible symptoms are unmarketable and must be discarded. Typical symptoms of crown gall are tissue proliferation (tumors) formed mostly on the lower areas of the trunk and on aerial canes. Tumorigenic and nontumorigenic strains of A. vitis are also able to cause specific root decay and it has been hypothesized that both types may be factors involved in the “replant disease” syndrome. Wounds mainly caused by freezing temperatures or grafting serve as a crucial entry points for the pathogen and its complex infection process. During the infection process DNA fragment from the bacterial tumor inducing (Ti) plasmid is transferred and integrated into the plant genome (interkingdom gene transfer). This leads to the overproduction of the phytohormones auxin and cytokinin, resulting in an uncontrolled proliferation of plant cells and tumor formation. A. vitis is unevenly distributed within systemically infected grapevines and able to survive in vineyard soil, particularly in the vicinity of infected plants and in their debris. Another important aspect is the ability of the pathogen to be latently present within the grapevine, providing an important means of spread over short and long distances by asymptomatic propagation material. Management of grapevine crown gall is not easy considering that no effective chemical control measures are available. However, production of A. vitis-free grapevines is an essential prerequisite for an effective prevention of the disease, and great efforts should be done in this direction. For this reason, shoot tip propagation of grapevine and thermotherapy are available as control measures. Planting of crown gall and cold-resistant cultivars and rootstocks would be a good practice when establishing new vineyards. Biological control of crown gall is another promising approach in the control of the disease and several antagonistic bacterial strains have shown a certain level of efficiency in preventing tumor formation. Indexing of grapevines for the endophytic presence of A. vitis is a very important preventive measure. Differentiation and identification of tumorigenic strains can be rapidly assessed by PCR using primer combinations specific for bacterial Ti plasmid and chromosomal genes. However, a high level of genetic diversity among Agrobacterium strains may limit the efficiency of PCR. In our studies virC, virD, virF, pehA and 23S rRNA gene-specific primers (Bini et al., Vitis 47:181, 2008; Pulawska et al., Syst. Appl. Microbiol. 29:470, 2006; Suzaki et al., J. Gen. Plant Pathol. 70:342, 2004; Szegedi and Bottka, Vitis 41:37, 2002) were reliable in routine detection and identification of a broad range of Agrobacterium strains occurring in grapevine. However, there is necessity for development and standardization of indexing procedures including protocols of analysis and sampling methods. In the EU and many other European countries, A. vitis is not listed as a quarantine pathogen and is considered as a “quality organism” which significantly reduces the value of propagation material. Therefore, the importance of proper phytosanitary measures in grapevine nurseries and on commercial lots should be emphasized.

The main objective of this EU project is the study of natural defense mechanisms of pome fruit against fire blight, a disease of relevant importance in several rosaceous species all over the world and quite recently in Italy. The recent outbreaks of the disease in Italy as well as the situation in other European countries justify the attention given by several research institutions to face this disease. A multidisciplinary approach was chosen to achieve the objective of the project including horticultural, bacteriological, biochemical, and molecular biology methods. The goals partially achieved by the project partners concern the availability of dioxygenase inhibitors, of plant material susceptible and tolerant to Erwinia amylovora to test, of preliminary results of practical applications on pome fruit species and varieties and the involvement of some flavonoids involved in defense reactions versus Erwinia amylovora. The most significant results obtained up to now are presented here.

The experiments were conducted following the "Guideline for the Efficacy Evaluation of Bactericides - E. amylovora" of the European and Mediterranean Plant Protecton Organization. Bacillus subtilis BS-F3 strain gave the best relative protection (62%) against blossom infections, when applied prior to and after inoculation with the pathogen. The highest shoot protection level (95%) was obtained when the bacterial antagonist was applied as a suspension from lyophilized cells. A spontaneous rifampicin resistant mutant (Rifr) of BS-F3 strain was used to study bacterial population dynamics on flowers and leaves in a pear orchard under field conditions throughout the growing season. On flowers, viable bacteria were found till petal fall, reaching a population size of about 103 cfu/sample after 14 days. On leaves, the antagonist was still present at a high population level 10 days after initial application; thereafter, the population size decreased gradually, and at 150 days no live bacteria were detected on the recovery medium. BS-F3 strain was found to produce peptide antibiotic(s) with a wide-spectrum of action against E. amylovora strains from different geographic origin and different hosts. The timing of synthesis and release of antimicrobial compounds coincided with spore formation. None of the amino acids tested (asparagine, glutamic acid, aspartic acid, serine and alanine) were able to inactivate antimicrobial compounds produced by BS-F3.

The purpose of the studies carried out in the spring of 2000 and 2001 under greenhouse conditions, was to evaluate the role of honeybees in the dispersal of Erwinia amylovora in pear blossoms and successive fire blight development. It was demonstrated that honeybees, while visiting for 24 h pear flowers experimentally inoculated with E. amylovora (10⁶ cfu/ml), became contaminated with the pathogen and were able to disperse the bacteria to healthy pear trees in full bloom; 48 h after initial contact of honeybees with inoculated flowers no viable bacteria were detected on honeybee bodies and in honeybee intestines. In other trials, it was demonstrated that honeybees were able to disseminate E. amylovora from experimentally contaminated beehives (10⁸ cfu/ml) to healthy pear flowers for less than 48 h after initial contamination of the beehives. No viable bacteria were re-isolated from analysed honeybee body and honeybee intestine samples 48 h after initial contamination of beehives. E. amylovora detection was based on quantitative re-isolations on CCT medium. The identity of the representative colonies was determined by Bio-PCR (Schaad et al., 1995) using the primers of Bereswill et al. (1992).

Patient de 62 ans, insuffisant renal, dialyse a partir de 2007 sur un abord d’hemodialyse brachial droit. Cet abord a ete utilise jusqu’a 2011, annee de la transplantation renale, puis conserve en raison d’un mediocre capital veineux. De nombreuses angioplasties du reseau veineux de drainage de cet abord vasculaire ont ete effectuees de 2007 a 2015. En 2015, l’abord vasculaire etait bien fonctionnel, debit a 1,6 L/min. En decembre 2015, une angiographie par resonance magnetique (ARM) realisee dans le cadre de cephalees chroniques, mettait en evidence une opacification precoce et asymetrique du sinus veineux lateral droit et de la jugulaire interne droite, faisant evoquer une fistule durale. Une arteriographie debut 2016 confirmait un retour veineux precoce vers la jugulaire interne droite, sans dilatation arterielle, conduisant au diagnostic de fistule durale de type 1, sans risque hemorragique et donc abstention therapeutique. Cette association fistule durale et abord d’hemodialyse brachiale homolaterale a debit important nous a conduits a nous interroger sur le caractere fortuit ou non de cette association. L’interrogatoire retrouvait des cephalees temporo-occipitales droites frequentes et des acouphenes droits pulsatiles moderes depuis 4 ans, croissant depuis 1 an. L’echo-Doppler d’aout 2016 retrouvait un abord d’hemodialyse brachial droit bien fonctionnel a debit eleve a 1,7 L/min, un tronc innomine normalement permeable, une hyperemie veineuse retrograde de la jugulaire interne droite. Lors de la compression de l’abord d’hemodialyse, le flux de la jugulaire droite redevenait normal anterograde et les acouphenes disparaissaient. L’hyperemie de la jugulaire droite et du sinus transverse droit semblait donc liee au drainage d’une partie de la fistule de dialyse par la jugulaire. La relecture de la sequence d’ARM dynamique initiale permettait de confirmer le caractere retrograde de l’hyperemie jugulaire. Une fermeture de l’abord d’hemodialyse etait alors organisee, en accord avec le nephrologue. A l’echo-Doppler postoperatoire, le flux de la jugulaire interne droite etait redevenu de sens physiologique anterograde, avec diminution conjointe des acouphenes. Une ARM de controle realisee 1 semaine plus tard, confirmait la normalisation de la circulation veineuse avec flux anterograde dans la jugulaire droite, symetrique par rapport a la jugulaire gauche et disparition de l’hyperemie du sinus lateral, eliminant ainsi formellement une fistule durale.

La paraplegie est responsable de dysfonctionnements majeurs sensitivomoteurs et neurovegetatifs sous-lesionnels. A l'exercice, les troubles de la vasomotricite s'associent a la perte de la «pompe musculaire» des membres inferieurs pour constituer une stase sanguine sous-lesionnelle, avec diminution du retour veineux, de la precharge, du remplissage cardiaque et de la composante volume d'ejection systolique du debit cardiaque. Nous avons etudie l'effet d'une contention veineuse des membres inferieurs et de l'abdomen sur l'adaptation cardiaque a l'effort et sur la performance de 16 blesses medullaires et de dix temoins valides, au cours d'exercices maximaux sur ergocycle a bras, effectues sans et avec contention de l'abdomen et des membres inferieurs par pantalon antigravite. Les frequences cardiaques de repos et d'effort ainsi que les VO 2 max des temoins n'ont pas ete modifiees par la contention. Les VO 2 max des blesses medullaires ont ete significativement (p < 0,005) ameliorees par la contention, de 12 % en moyenne (21,4 ± 6,2 mL.min -1 kg -1 sans contention, 23,9 ± 5,9 mL.min -1 kg -1 avec contention). Les frequences cardiaques des blesses medullaires ont ete tres significativement abaissees aux differents paliers d'exercice lors de la contention (p < 0,001). Cette etude a mis en evidence l'amelioration des performances et de l'adaptation cardiaque a l'effort des paraplegiques, lors de la contention sous-lesionnelle, probablement par augmentation de la reserve de debit cardiaque, avec cependant variabilite individuelle des reponses (role du niveau et de l'anciennete de la lesion, de la spasticite, de l'entrainement).

Resume Les paraplegiques ont une aptitude physique reduite en raison de leur moindre masse musculaire fonctionnelle et des moins bonnes qualites aerobies des muscles des membres superieurs. Le niveau de l'atteinte medullaire conditionne la masse musculaire fonctionnelle ainsi que l'etendue des perturbations neurovegetatives associees. L'interruption des efferences sympathiques est responsable de troubles vasomoteurs, qui, associes a la perte de la fonction pompe des muscles des membres inferieurs est responsable d'une stase sanguine sous-lesionnelle. La diminution du retour veineux est responsable d' un moindre volume de precharge et volume d'ejection systolique, le debit cardiaque etant maintenu par cardio-acceleration. La contention veineuse permet de reduire le volume de stase et done d'ameliorer le remplissage cardiaque. Nous avons etudiel' effet de la contention veineuse sous-lesionnelle sur l' adaptation cardiaque a l'effort et sur la performance physique des blesses medullaires. Quatorze sujets paraplegiques et dix sujets temoins ont ete evalues au cours d'un exercice maximal des bras, effectue sans contention et avec contention des membres inferieurs et de l'abdomen par pantalon antigravite. La consolation maximale d'oxygene (VO2max) des sujets valides de 24 ± 5.8mL·min−1·kg−1 sans contention, n'etait pas modifiee par la contention, leur profil de frequence cardiaque a l'effort etait superposable dans les deux situations. La VO2max des blesses medullaires de 21.5 ± 6.5mL·min−1 kg−1 sans contention, etait significativement amelioree par la contention, atteignant 23,8 ± 6,3 mL·min−1·kg−1 (p

Monospecific antibodies were successfully obtained by using unique protein bands of the quarantine bacterium Xylella fastidiosa , the causal agent of Pierce's disease of grapevine, and by applying gel electrophoresis and electroblotting. The monospecific antibodies directed against the two best suited protein bands (20.7 kD, 19.8 kD) did not show any cross-reaction on a Western blot, a solid phase ELISA (Enzyme-Linked Immunosorbent Assay) or an IFAS (Indirect Fluorescent Antibody Staining). A monospecific antiserum was generated in rabbits directed against the 20.7 kD and 19.8 kD protein bands, and was applied in an ELISA. Formerly cross-reacting bacteria isolated from grapevine cuttings did not show any antibody reaction with this antiserum. When grapevine cuttings were pre-infiltrated with a suspension of 4.15 × 10 7 bacteria the detection limit was found to be 10 6 −10 5 cells in the sap extract using ELISA. Titer used in all assays of the monospecific antiserum was 1:100. Using pure cultures of Xylella fastidiosa the detection limit of the monospecific antiserum in an indirect ELISA was 8.30 × 10 5 cells/ml.

Cell-associated oligosaccharides were extracted with trichloroacetic acid treatment from Pseudomonas syringae pv. aptata, purified by column chromatography, analysed by GC-MS and fast atom bombardment-mass spectrometry, and bioassayed on tobacco leaves. The material was shown to be a mixture of neutral oligosaccharides, with a mol. wt of 800–1800, degree of polymerization 5–11, and linear glucose and mannose chains. Local infiltrations of the mixture, in concentrations of 1 mg, 100, 10 and 1 μg ml−1, did not cause tobacco tissue necrosis within 1 week. When the mixture was infiltrated in the interveinal leaf panels, in concentrations of 1 μg, 100, 10 and 1 ng ml−1, 48 h before challenge inoculation, it delayed or prevented normosensitive necrosis by P. syringae pv. tabaci but not hypersensitive necrosis by P. syringae pv. aptata. Five days after inoculation with P. syringae pv. tabaci, inhibition of normosensitive necrosis, as compared with the control tissue, was 80·55, 77·21, 56·36 and 45·46 % for the four concentrations. In the bioassay with P. syringae pv. tabaci the efficacy threshold was assessed at approx. 1 ng ml−1. Pre-treatment with oligosaccharides (1 μg ml"−1) significantly inhibited the growth of P. syringae pv. tabaci between 3 and 5 days after inoculation and, between day 6 and 8, it delayed the death of bacteria when the tissue was not subject to necrosis. An interval of 6 h was necessary between the infiltration of oligosaccharides (1 μg ml−1) and inoculation of P, syringae pv. tabaci for a clear inhibition of normosensitive necrosis. These oligosaccharides acted as signal molecules by modifying, at nanomolar concentrations, the P. syringae pv. tabaci—tobacco leaf interaction, but they were not elicitors of the hypersensitive reaction.

There is a real risk of introducing Xylella fastidiosa into the EPPO region where this quarantine pest has not yet been found and constitutes a serious threat to the Mediterranean grapevine industry. Direct isolation of endophytic bacteria from imported propagation material, combined with one-dimensional SDS-PAGE of the total soluble proteins of the bacterial cell envelope, can be considered as a test to be used in grapevine certification schemes. The gel electrophoretic pattern of Xylella fastidiosa is characterized by a constant distribution of 14 major diagnostic bands. Even without gel scanning, this relatively simple technique allows discrimination between grapevine strains of X. fastidiosa and other xylem-inhabiting, slow-growing bacteria.

Studies were carried out in 2002 and 2003 to evaluate the suitability of the highly efficient pollinator of pear Osmia cornuta (Hymenoptera Megachilidae) as a carrier of a rifampicin resistant mutant of the Bacillus subtilis strain BS-F4 (BS-F4rif) on pear flowers cultiver 'Abbé Fétel' for the biological control of fire blight, and its capacity was compared with that of Apis mellifera. A dispenser for the spread of bacteria by O. cornuta was successfully developed. Females exited and entered the dispenser through the proper pathways 81.4% and 97.7% of the attempts, respectively, and the number of BS-F4rif reisolated from the body of O. cornuta exiting the dispenser ranged from 104 to 107 CFU/insect. Individuals of A. mellifera carried an average population of 104 CFU/insect. In net screened tunnel conditions, the efficiency of O. cornuta to carry and deposit the BS-F4rif on pear flowers during a single visit was higher than that of A. mellifera. Both in 2002 and 2003, the dispersal ability of bees was assessed in a 7 ha pear orchard. Trees were labelled at 2, 10, 50, 110 m from the nesting shelter, in the direction of the four cardinal points. Flowers on branches covered with fine netting acted as controls. Mason bees distributed 30 g of the biocontrol agent (BCA). Fifty flowers from both exposed and netted branches were sampled at 3, 7 and 10 days (the latter only in 2003) after the beginning of distribution. BS-F4rif was not recovered from any of the flowers on net-covered branches, whereas the bacterium was consistently recovered on the flowers of non-screened plants. Variation in distribution and population density of the BCA among flowers were observed, with greater colonisation in 2002 than 2003. We conclude that O. cornuta may serve as a carrier for the dispersal of powdered BCA formulations to pear flowers.

Experimental and clinical studies show that proteinuria is an independent risk factor for the progression of chronic glomerular diseases and is associated with the extent of tubulo-interstitial damage. The accumulation of proteins in tubular cells induces the increased expression of a variety of inflammatory and fibrogenic cytokines, with the consequent development of interstitial inflammation, a proliferation of fibroblasts, the increased production of extracellular matrix, and the formation of interstitial fibrosis. Laboratory methods, such as immunonephelometry, can easily evaluate the glomerular component of proteinuria, due to the alteration of the structural integrity of the glomerular capillary wall. This alteration allows the tubular lumen to pass proteins of high and middle molecular weight (HMW and MMW proteins: IgM, alpha2-macroglobulin, IgG, transferrin, albumin). Using the same method and SDS-PAGE, it is possible to evaluate those tubular components of proteinuria that are composed of low molecular weight (LMW) proteins, such as alpha1-microglobulin (alpha1m) and beta2-microglobulin (beta2m), whose reabsorption by tubular cells-almost complete in physiological conditions-is impaired in pathological conditions. Recent studies clarified some aspects of the relationships between the components of proteinuria, histological lesions, prediction of outcome, and response to therapy. The extent of tubulo-interstitial damage is correlated with selectivity of proteinuria and IgG excretion, suggesting a possible tubulo-toxic role for IgG or for some other protein of similar molecular weight. The tubulo-interstitial lesions are also correlated with the excretion of LMW proteins, due to their impaired reabsorption. The remission of nephrotic syndrome, not predicted by the amount of proteinuria, is highly predicted by the selectivity index or IgG excretion in membranous glomerulonephritis (MGN) and focal segmental glomerulosclerosis (FSGS). Progression to chronic renal failure is better predicted both by the glomerular component (selectivity index, IgG excretion) and by the tubular component of proteinuria (alpha1m, beta1m, LMW proteins), than by 24-hour proteinuria. The response to therapy in MGN and FSGS is dependent on the excretion of IgG and alpha1m. In conclusion the composition of proteinuria can easily be assessed using automated methods, and it is useful to evaluate the relationship of proteinuria with histological lesions to predict the functional outcome and response to therapy in primary glomerulonephritis.

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.

Pseudomonas syringae pv. tomato is the causal agent of bacterial speck of tomato. This bacterium may cause spots on leaves, with or without a chlorotic halo, and on fruits, with consequent economic damage. The identification of the P. s. pv. tomato is mainly based on isolation of bacteria on semiselective media (e.g. VBTar) coupled to DNA hybridisation using coronatine synthesis genes and on serological techniques such as indirect fluorescent antibody staining (IFAS) and enzyme-linked immunosorbent assay (ELISA). A method based on PCR amplification of coronatine gene sequences was reported as well. Unluckily not only are cor genes conserved among several P. syringae pathovars, but also Cot P. s. pv. tomato strains have been described, leading to potential false negative or positive misdiagnosis. In this study we present a molecular detection method based on PCR amplification of stable chromosomal sequences that are located in the HrpPST pathogenicity island. Pathovar-specific oligonucleotide primers were designed on nonconserved regions of the hrpZ gene. A specific amplification product of 532 bp was obtained from all of 35 isolates of P. s. pv. tomato but not from 98 pathogenic and symbiotic bacterial isolates belonging to the genera Pseudomonas, Xanthomonas, Erwinia, and Rhizobium. Moreover, no amplicons were obtained from a plethora of unidentified bacterial saprophytes isolated from tomato plants in the field. On experimentally and naturally infected tomato plant materials, the expected amplicon was always obtained using crud extracts from leaves and fruit spots.

Summary Fire blight has been detected recently in several areas of northern Spain and north-eastern Italy. To follow spread of the disease within Europe, more than 120 Erwinia amylovora strains isolated from 1957-2000 in England, France, Germany, The Netherlands, Belgium, Poland, Italy and Spain were assayed using pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA after XbaI digestion. Pattern types Pt1 and Pt4 were found for strains from England. Pt1 was also found in central Europe and eastern France, Pt4 in western France. Pt2 appeared first in Egypt, from where strains with this pattern disseminated north- wards as far as into the Balkans. Pt3 was typical for northern France and Belgium. Strains from Spain dis- played the pattern types Pt3 and Pt4. In Italy, Pt2 was found in the south-eastern areas, Pt3 in the north- eastern areas, and Pt1 was found very recently in orchards adjacent to the Austrian border, together with Pt3. Despite barely controlled trade with fire blight host plants and associated plant products within Europe, the PFGE patterns of the E. amylovora isolates were ordered indicating sequential spread. On the other hand, the appearance of Pt3 in northern Italy and central Spain can be explained by the import of contaminated plants by nurseries.

Agrobacterium radiobacter (Beijerinc and van Delden) Conn strain K-84 failed to control raspberry (Rubus idaeus L.) crown gall caused by A. tumefaciens (E.F. Smith and Townsend) Conn. Agrobacterium tumefaciens strains isolated from galls on plants that had been treated with K-84 were not sensitive to agrocin 84 in vitro. These strains were isolated from 'Titan' and 'Hilton' raspberry in New York state and from 'Himbo Queen' and 'Schonemann' raspberry in Italy. Almost all strains were identified as A. tumefaciens biovar 2. Raspberry crown gall was not controlled by K-84 in three field experiments in New York state. In two of the experiments, plants were produced by micropropagation and were known to be pathogen-free. The other plant source was shown to be contaminated with the pathogen before treatment with K-84. Crown gall was not controlled either on raspberry in a greenhouse experiment or on Kalanchoe diagremintiana (Hamet. and Perrier) plants that were coinoculated with K-84 and strains of A. tumefaciens isolated from galls on raspberry. Agrobacterium radiobacter strain K-84 is a highly effective biological control of crown gall on certain plant species (Kerr, 1980). K- 84 produces a bacteriocin, agrocin 84, that is an important factor contributing to its biologi- cal control activity (Kerr and Htay, 1974). The sensitivity of A. tumefaciens strains to agrocin

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).

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 (103to 105CFU/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.

Fatiy acid profiles were prepared from 122 bacterial strains isolated from cereals grown in different Italian areas. Almost all of them were typical fluorescent pseudomonads with the three key hydroxy fatty acids 10:0 3OH, 12:0 2OH and 12:0 3OH, and belonged to two major groups. In group 1 most of the isolates were iisted as Pseudomonas syringae pathovars including pv. atrofaciens. These results fit with those obtained using SDS-PAGE. Strains in group 2 were identified as members of the P. fluorescens complex, and some were pathogenic to durum wheat seedlings. Experimentai evidence suggests that, apart from P. atrofaciens, a second pathogen may be associated with basal glume rot/leaf blight of cereais and play a roie in the disease epidemiology.

Paraplegics have low aerobic capacity because of the spinal cord injury. Their functional muscle mass is reduced and usually untrained. They have to use upperbody muscles for displacements and daily activities. Sympathic nervous system injury is responsible of vasomotricity disturbances in leg vessels and possible abdominal vessels, proportionally to level injury. If cord injury level is higher than T5, then sympathic cardiac efferences may be damaged. Underbody muscles atrophy and vasomotricity disturbances contribute to phlebostasis. This stasis may decrease venous return, preload and stroke volume (Starling). To maintain appropriate cardiac output, tachycardia is necessary, especially during exercise. Low stroke volume, all the more since it is associated with cardio-acceleration disturbances, may reduce cardiac output reserve, and so constitutes a limiting factor for adaptation to exercise. The aim of this study was to verify if use of an underlesional pressure suit may increase cardiac output reserve because of lower venous stasis, and increase performance. We studied 10 able-bodied and 14 traumatic paraplegic subjects. Able-bodied subjects were 37 +/- 6 years old, wellbeing, not especially trained with upperbody muscles: there were 2 women and 8 men. Paraplegics were 27 +/- 7 years old, wellbeing except paraplegia, five of them practiced sport regularly (athletism or basket for disabled), and the others just daily propelled their wheelchair; there were 5 women and 9 men. For 8 of them, cord injury levels were located below T7, between T1 and T6 for the others. The age disability varied from 6 months to 2 years for 9 of them, it was approximately five years for 4 of them, and 20 years for one. We used a maximal triangular arm crank exercise with an electro-magnetic ergocycle Gauthier frame. After five minutes warm up, it was proceeded in one minute successive stages until maximal oxygen consumption is raised. VO2, VCO2, RER were measured by direct method with an Ergostar analyser every 30 seconds. Heart rate was registered continuously using a cardio-frequence-meter Baumann, and ECG was observed on a Cardiovit electro-cardiograph. Each subject reached maximal exercises on different days: one without any contention, and the other one with abdomen and legs contention using an antigravity suit, inflated to 45-50 mm Hg for legs and 30-40 mm Hg for abdomen. The able-bodied subjects VO2 peak was 24 +/- 5.8 mL min-1 kg-1, without any change on peak VO2 and on cardiac frequency when pressure suit was used. Results were different for paraplegics: peak VO2 was significantly higher (21.5 +/- 6.5 mL min-1 kg-1 without contention and 23.8 +/- 6.3 mL min-1 kg-1 with contention), heart rate was significantly lower at all stages of exercise with antigravity suit and comfort was better during exercise and rest. In our study, contention contributed to increase paraplegics's performances, but responses depend also on spinal cord level, injury age, spasticity. Therefore, testing paraplegics using an antigravity suit may be useful to determine if neurovegetative disturbances significantly modify their cardiac adaptation and capability. If gravity suit is efficient, contention tights might be prescribed, with respect to subject's legs measurements. But, because these tights are very difficult to put on, their efficiency has to be proved before, the motivation of the subject is essential too.

Hearing loss has been described in patients with chronic renal failure on regular dialysis treatment (RDT) with very different frequency, ranging from 20 to 75%; RDT does not seem to worsen hearing function for at least the first 5 years of treatment; no studies are available on patients on RDT for more than 10 years.We performed an audiometric evaluation in 91 patients on RDT for various periods: group I (34 patients),5 years; group II (32 patients), 5-10 years; group III (25 patients),10 years; patients with histories of chronic otitis, ototoxic drug treatment, and chronic auditory trauma were excluded; the possible correlations with some biochemical parameters (urea, creatinine, PTH) were also looked for.Hearing loss was present in 77% of patients and 69.2% of ears; the percentage of patients with hypoacusia was higher in group III (84%) than in group I (76.3%) and II (71.7%), but the differences were not statistically significant. Hypoacusia was cochlear neurosensory in 61.5%, conductive in 6.5%, and mixed in 9.0% of patients; the percentage of patients with cochlear neurosensory hypoacousia was similar in the three groups (I, 61.7%; II, 59.3%; III, 64%). Hearing loss was of slight to moderate degree and not different in the three groups (I, 22.7 +/- 15 dB; II, 26.9 +/- 6.0 dB; III, 29.1 +/- 8.9 dB). There were no correlations between hearing loss and plasma creatinine and PTH values; patients with plasma urea200 mg/dl had higher percentage of hypoacousia (86%) than patients with plasma urea200 mg/dl (69%) (P = 0.06).Hearing loss, mainly cochlear neurosensory in type, is present in a high percentage of patients on RDT even at the beginning of treatment, but no negative effects on hearing can be correlated with the duration of dialysis.

Careful investigation of the clinical conditions of patients on maintenance hemodialysis for about 20 years in a single dialysis unit was of great interest for evaluation of the pathological consequences in long-term survivors of insufficient correction of uremia and of the dialysis treatment "per se". We analyzed the outcomes for a cohort of 116 patients who started RDT before 1976 and the clinical conditions of the 24 patients still on RDT in our unit at the end of 1991 (average duration of treatment = 222 +/- 23 months). Actuarial survival was 72% at 10 years and 43% at 20 years. Rehabilitation of the 24 survivors was rather good: 13 were able to work, 8 were retired or unable to work, but able to care for most personal needs. Actual body weight, anthropometric parameters and biochemical parameters revealed a well-preserved nutritional status. Anemia improved from 23 +/- 7 at the start of RDT to 31 +/- 8 in the 21 patients never treated with erythropoietin. Blood pressure was normal without therapy in 18 patients and elevated in 6. Mild-to-moderate left ventricular hypertrophy was present in all the 6 patients with arterial hypertension and in only 6 of the 18 normotensive patients. The ratio of early diastolic filling to filling during atrial contraction (E/A ratio) was1 in 16 patients: it was 1.05 +/- 0.43 in 9 patients with stable intradialysis blood pressure and significantly lower (0.73 +/- 0.15) in 12 patients with recurrent intradialysis hypotension. Supraventricular arrhythmias were detected by Holter monitoring in 41% and ventricular arrhythmias in 35% of patients. Extensive vascular calcifications were present (in 100% of patients in the abdominal aorta), but only 4 patients showed clinical signs of peripheral vascular disease. Subperiosteal resorption was detected radiologically in the hands of 59% of patients. Bone histology, interpretable for only 20 patients, revealed no bone lesions in 1 case (5%), mild mixed osteodystrophy in 3 cases (15%), advanced mixed osteodystrophy in 5 cases (25%), osteodystrophy with predominant hyperparathyroidism in 2 cases (10%), osteodystrophy with predominant osteomalacia in 6 cases (30%), and aplastic bone disease in 3 cases (15%). Moderate aluminum staining was found in only 4 patients and was more marked in earlier biopsies taken before withdrawal of the aluminium-containing phosphate-binding drugs.(ABSTRACT TRUNCATED AT 400 WORDS)

Proteinuria was characterized by SDS-PAGE and by immunoblotting with anti-human albumin sera for the detection of urinary polymers of albumin (PA) in 40 patients with biopsy proven lupus glomerulonephritis (LN) (6 pts class III WHO, 24 pts class IV, 10 pts class V) with various clinical presentations (nephrotic syndrome with normal or impaired renal function, 14 pts; urinary abnormalities with normal or impaired renal function, 21 pts; clinical remission, 5 pts); in 25 pts, for whom the characterization of proteinuria and the renal biopsy were performed at the same time, the activity and chronicity index scores were calculated. The mixed SDS-PAGE patterns, characterized by the presence of low molecular weight proteins, were the more frequently found; the mixed patterns were significantly associated with interstitial leukocyte infiltration (p = 0.05) and glomerular sclerosis (p = 0.046) and nonsignificantly associated with higher values of serum creatinine; no SDS-PAGE pattern had predictive value on functional outcome at 36 months. Albumin polymers were present in 67% of pts; in active disease they were present in 33% of class III, in 100% of class IV and in 45% of class V WHO (p = 0.026); PA were not present in 5 pts with clinical remission (4 class IV and 1 class V WHO). The presence of PA was significantly associated with high values (10) of activity index (p = 0.009) and with extracapillary proliferation (p = 0.041). Serum creatinine was lower in patients without PA (Scr 1.0 +/- 0.4 mg/dl) than in those with PA (Scr 1.5 +/- 1.0 mg/dl), but the difference was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

During recent years, recurrent attacks of basal glume rot/leaf blight attributed to Pseudomonas syringae pv. atrofaciens have been observed on cultivars of durum wheat grown in the Po valley (IT). The main aim of this study was to test for this pathogen and Xanthomonas campestris pathovars in commercial seed lots of cereals produced in different regions of Italy, as well as in symptomatic plants collected in the field. None of the analyses led to the detection of xanthomonads. In testing for P.s. atrofaciens, representative bacterial isolates were selected and characterized; this was done by combining conventional identification tests and computerized densitometric analysis of electrophoretic patterns of cell proteins obtained using the SDS-PAGE technique. P.s. atrofaciens was detected in seed samples (barley, wheat and durum wheat) and symptomatic plants (durum wheat) grown in northern and central Italy. The possible pathogenic role of other unrelated fluorescent pseudomonads in this interaction was also examined. Difficulties in discriminating pvs syringae and atrofaciens of P. syringae are discussed and the uncertain taxonomic relationship between these two pathovars is emphasized.

To evaluate the presence and the severity of uraemic encephalopathy (UE) in regular dialysis treatment patients in relation to dialytic age, pattern reversal visual evoked potentials (PRVEPs) and brainstem auditory evoked potentials (BAEPs) were respectively performed in 86 and 98 patients on haemodialysis for 1-194 months, divided into three subgroups according to dialytic age (group 1,5 years of regular dialysis; group 2, 5-10 years; group 3,10 years). VEPs in the whole group of 86 patients and in each subgroup with different dialytic age differed significantly from controls for both eyes, 41.7% of whom had pathological P100; no differences were observed between the three subgroups. BAEPs were pathological in 9.7% of the ears and 18.4% of patients. On the right ear the three subgroups were significantly different from controls in the latencies of peaks III and V; subgroup 2 and 3 differed from controls in the I-V interpeak, while the interpeak I-III was different from controls only in subgroup 3. On the left ear the three subgroups differed significantly from controls in the latencies of peak V; subgroup 2 and 3 were significantly different from controls in the latency of peak I; subgroup 3 was different in the peak III latency; subgroup 1 and 3 were different from controls in the interpeak I-V; no differences were observed in BAEPs between the three subgroups with increasing dialytic age. No significant correlations were found between the neurophysiological parameters and some biochemical parameters (urea, creatinine, PTH).(ABSTRACT TRUNCATED AT 250 WORDS)

In an uncontrolled trial, patients with IgA nephropathy (IgAN) were treated with drugs that can alter the intestinal mucosal permeability to food antigens. These drugs are known to ameliorate urinary abnormalities and histological lesions of IgAN associated with ulcerative colitis or Crohn's disease [5-aminosalicylic acid (5-ASA)] or to prevent, in mice, the induction of IgAN-like disease by oral immunization [disodium cromoglycate (SCG)]. Nine patients [serum creatinine (s-Cr) less than 2 mg/dl; 24-hour proteinuria higher than 1.5 g, but not nephrotic) were treated with 5-ASA (2.4 g/day for 6 months); 9 similar patients were treated with SCG (400 mg/day for 6 months); the follow-up extended to 6 months after stopping therapy. The 5-ASA group showed a slight but not significant decrease in s-Cr, 24-hour/proteinuria, IgA circulating immune complexes (IgA-CIC) and IgA rheumatoid factor (IgA-RF); serum beta 2-microglobulin and serum IgA were unchanged; 2 of 9 treated patients showed, after 6 months of therapy, a reduction in proteinuria of more than 50% that lasted for the subsequent 18 months. The SCG-treated group showed a slight but not significant increase in 24-hour proteinuria and a significant decrease in serum IgA; unchanged were s-Cr, IgA-CIC, IgA-RF, serum beta 2-microglobulin; no patient treated with SCG showed a reduction in proteinuria of more than 50%. At the dosages and for the periods used, 5-ASA and SCG did not show a significant influence on clinical and laboratory parameters of disease in IgAN; other trials with increased dosages are warranted to definitely ascertain the possible therapeutic role of these drugs in IgAN.

Twenty neurophysiological parameters were employed to evaluate the presence and the degree of peripheral neuropathy (PNP) in a cohort of 135 patients (pts) on regular dialysis treatment (RDT) for 2 to 184 months. The 135 pts were divided into 3 groups according to the duration of RDT (group I: 52 pts with less than 5 yrs; group II: 46 pts 5 to 10 yrs; group III: 37 pts 10 to 15 yrs). Each group was then divided into two subgroups according to age (less or more than 47 yrs) to evaluate the influence of age on PNP. Correlations of electrophysiological parameters with some biochemical parameters (urea, creatinine, PTH) were looked for. The presence of clinical PNP was evaluated according to the Bolton classification: in group I, 50% of pts have mild PNP; in group II, 45.7% of pts have mild PNP; in group III, 81.1% have mild, 10.8% have moderate and 2.7% of pts have severe PNP. In as many as 84.4% of the 135 pts at least one of the 20 parameters studied had abnormal values and in 63% two or more parameters were abnormal. Of 20 parameters evaluated separately in the 3 groups only three showed abnormal mean values: sural nerve latency in all 3 groups; sural nerve Sensory Conduction Velocity (SCV) and peroneal nerve Max. Motor Conduction Velocity (MCV) in group III. Five parameters referring to ulnar nerves and two referring to the sural nerve were significantly more impaired in the group of pts with the longest duration of RDT and in this group the impairment was more severe in older patients.(ABSTRACT TRUNCATED AT 250 WORDS)

As in other countries, pear production in Italy requires an efficient control of excessive shoot growth. Chlormequat chloride, the only available plant growth regulator, does not necessarily represent the ideal solution because it requires high dosages, thus posing the risk of residues in fruit. Fire blight, which has invaded Italy in at the beginning of 1990, is of great concern because `Abate Fetel', one of the predominant cultivars, is highly susceptible and no preparations for control are currently available. To evaluate solutions for both aspects, prohexadione-Ca (BAS 125 11 W) has been tested on pears under growth chamber and orchard conditions. In each case, reductions of shoot growth and, if present, lowered incidences of fire blight could be achieved. Six-year-old trees of `Abate Fetel', located in an area with high fire-blight infestation pressure, were sprayed with prohexadione-Ca four times each at either 50 or 100 ppm of active ingredient at a new shoot length of 6, 12, 33, and 45 cm. The resulting final shoot length was 96% and 75%, respectively, of the control, whereas an average of 15 fire blight incidences could be found in the controls. This number was reduced to 8 and 4, respectively, in the treated trees. Fruit yield and quality was not affected.