First Report of Knot Disease Caused by Pseudomonas savastanoi on Sweet Olive in Central Italy.

In April 2012 the presence of hyperplastic outgrowths on trunks, branches, and twigs of sweet olive plants, Osmanthus fragrans Lour (Fam. Oleaceae), was recorded in two ornamental hedges made up of five and four plants, respectively, in the city center of Montecatini (Pistoia-Italy). All sweet olive plants were seriously affected by the disease with outgrowths appearing either singly or close together, often forming a single mass that could extend up to 20 cm along the stems, occasionally surrounding the entire circumference. The symptoms observed on O. fragrans closely resembled those induced by the bacterium Pseudomonas savastanoi on Olea europea (common olive) and other plant species. Suspecting a bacterial origin of the disorder, young knots were collected from four diseased plants and used for bacterial isolation with standard techniques on nutrient sucrose agar medium (1). After 3 days of incubation at 26°C, non-levan forming colonies about 3 mm in diameter that were circular, convex, smooth, and cream colored with entire margins appeared on the surface of the agar medium. Purified isolates were gram negative, levan negative, oxidase negative, potato rot negative, arginine dihydrolase negative, showed a tobacco hypersensitive reaction, and tested positive to PCR screening for the presence of the iaaM (tryptophan-2-monooxygenase), iaaH (indoleacetamide hydrolase), ptz (isopentenyl transferase) (1) and iaaL (IAA-lysine synthethase) (3) genes. Three isolates were selected arbitrarily and further characterized by sequencing a fragment of the housekeeping genes rpoD (sigma factor 70) and pgi (phosphoglucose isomerase) (2). All sequenced gene fragments, of 620 bp and 552 bp for the rpoD and pgi genes, respectively, were identical to those of P. savastanoi pv. savastanoi strain NCPPB3335. The pathogenicity of the three isolates was verified on three O. fragrans plants and three Olea europea (cv. Frantoio) plants. Per each isolate, three 1-cm wounds were made on the branches of each plant using a sterile scalpel dipped in a bacterial suspension (1 × 108 CFU/ml). P. savastanoi pv. savastanoi PVFi-t2b isolated from olive was also inoculated as reference strain. After 30 days, all isolates including the reference strain induced typical knots on both plant species while no symptoms were observed on wounds inoculated with sterile water. Bacteria were reisolated from induced knots and Koch's postulates were confirmed. On the basis of biochemical tests, PCR screening, pathogenicity testing, and sequence analyses, the causal agent of knot disease on O. fragrans was identified as P. savastanoi. The potential susceptibility of O. aquifolium Sieb. to the causal agent of olive knot disease has been demonstrated in the past by means of artificial inoculations but interestingly, in the same trials, O. fragrans had tested negative (4). To the best of our knowledge, this is the world's first report of O. fragrans as natural host of P. savastanoi, which extends the growing list of cultivated and ornamental plant species affected by this phytopathogenic bacterium. References: (1) G. Marchi et al. Eur J. Plant Pathol. 112:101, 2005. (2) N. Parkinson et al. Plant Pathol. 60:338, 2011. (3) R. Penyalver et al. Appl. Environ. Microbiol. 66:2673, 2000. (4) C. O. Smith. Phytopathology 12:271, 1922.

Bacterial Leaf Spot Caused by the Quarantine Pathogen Xanthomonas arboricola pv. pruni on Cherry Laurel in Central Italy.

In June 2010, 1-year-old potted plants of cherry laurel (Prunus laurocerasus L.) cv. Novita showing leaf spot symptoms were collected in a commercial nursery in the district of Pistoia (Tuscany, central Italy). Red-purple necrotic lesions (measuring a few millimeters up to 1 cm) surrounded by a brilliant light green halo were observed on the abaxial surface of symptomatic leaves. With age, the necrotic areas drop out, leaving a "shot-hole" appearance. Microscopic observation revealed the absence of fungal structures, whereas bacteria were isolated from symptomatic tissue on nutrient sucrose agar medium. Purified single colonies appeared mucoid, convex, and yellow on yeast extract-dextrose-CaCO3 agar (YDCA) medium, were positive to the KOH test, and induced hypersensitive responses on tobacco (cv. Virginia Bright). Three isolates were selected arbitrarily for further analysis. A fragment of approximately 500 bp of the 16S rRNA gene was amplified via PCR with the universal primer pair 27f/519r and sequenced. Subsequent database searches in the INSD (GenBank, EMBL, and DDBJ) indicated that the resulting sequences had 100% identity over 490 bp with the corresponding gene of a Xanthomonas sp. The isolates were further identified as Xanthomonas arboricola pv. pruni on the basis of quinate metabolism and starch hydrolysis tests and by sequencing the PCR products obtained with the gyrB (4) and X. arboricola pv. pruni-specific (3) primer sets. Pathogenicity tests were conducted on cvs. Novita and Caucasica following the detached leaf bioassay procedure (1) and by injecting with a hypodermic needle a bacterial suspension (1 × 107 CFU/ml) in the leaf mesophyll of 1-year-old potted plants (three plants per cultivar and three leaves per isolate on each plant). Incubation was carried out at 25°C under fluorescent lights with a 16-h photoperiod. After seven (detached leaves) and four (potted plants) days, all leaves inoculated with X. arboricola pv. pruni isolates showed brown necrotic spots delimited by a chlorotic margin. Reisolated bacteria on YDCA showed the same colony morphology as described above and tested positive to the X. arboricola pv. pruni-specific primer set, confirming the causal agent of the disease. Leaf tissue inoculated with sterile distilled water remained symptomless. Bacterial leaf spot on cherry laurel was reported in Lombardy (northern Italy) by the local plant protection service in 2005 but without a confirmatory diagnosis of the causal agent (2). To our knowledge, this is the first confirmed report on the occurrence of X. arboricola pv. pruni on cherry laurel in Italy. The pathogen could have a significant impact on the commercial cherry laurel production in the district of Pistoia, which is the most important area for ornamental plants nurseries (4,536 ha of cultivated surface in 2005) in Italy. X. arboricola pv. pruni is included in the EPPO A2 list of pests recommended for regulation to the member countries. References: (1) Anonymous. EPPO Bull. 36:129, 2006. (2) EPPO Reporting Service. Online publication. Retrieved from archives.eppo.org/EPPOReporting/2006/Rse-0606.pdf , 2006. (3) M. C. Pagani. Ph.D. diss. North Carolina State University. Online publication. http://repository.lib.ncsu.edu/ir/bitstream/1840.16/4540/1/etd.pdf , 2004. (4) N. Parkinson et al. Int. J. Syst. Evol. Microbiol. 59:264, 2009.

A New Flow Cytometry Technique to Identify Phaeomoniella chlamydospora Exopolysaccharides and Study Mechanisms of Esca Grapevine Foliar Symptoms.

A flow cytometry technique that unequivocally identifies some of the toxic metabolites produced by Phaeomoniella chlamydospora, one of the main fungal pathogens causing esca disease of grapevine (Vitis vinifera), was developed. Antibodies raised against exopolysaccharides (EPS)-metabolites produced by Pa. chlamydospora 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. chlamydospora polysaccharides and against EPS with a different structure isolated from other phytopathogenic fungi, including Phaeoacremonium aleophilum and the Botryosphaeriaceae species Neofusicoccum luteum and N. parvum. Using this method, Pa. chlamydospora 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. chlamydospora in grapevine material.

Molecular and phenotypic characterisation of novel Phaeoacremonium species isolated from esca diseased grapevines.

Petri disease and esca are very destructive grapevine decline diseases that occur in most countries where grapevine (Vitis vinifera) is cultivated. Phaeoacremonium species are among the principal hyphomycetes associated with symptoms of the two diseases, producing a range of enzymes and phytotoxic metabolites. The present study compared the phylogeny of a global collection of 118 Phaeoacremonium isolates from grapevines, in order to gain a better understanding of their involvement in Petri disease and esca. Phylogenetic analyses of combined DNA sequence datasets of actin and beta-tubulin genes revealed the presence of 13 species of Phaeoacremonium isolated from esca diseased grapevines. Phaeoacremonium aleophilum was the most frequently isolated species with an incidence up to 80 % of all isolates investigated. Species previously described mainly as human pathogenic species, namely Pm. alvesii, Pm. griseorubrum and Pm. rubrigenum are newly reported on grapevine from Turkey, Italy and Croatia, respectively. Phaeoacremonium viticola and Pm. scotyli represent new records for Italy, as well as Pm. mortoniae for Hungary and Croatia. In addition, four new species of Phaeoacremonium, namely Pm. croatiense, Pm. hungaricum, Pm. sicilianum and Pm. tuscanum are newly described from grapevine based on morphology, cultural characteristics, as well as molecular phylogeny.

Fluctuation of endophytic bacteria and phytoplasmosis in elm trees.

A total of 658 heterotrophic bacterial colonies isolated from phloem tissues of roots and branches in four months (April, June, September and December) from two elm plants, one of which affected by phytoplasmosis, were typed by means of ARDRA. This analysis revealed the existence of a high degree of variability within the community and was able to detect 84 different ARDRA groups. The Analysis of Molecular Variance was applied to ARDRA patterns to analyze the differentiation between communities isolated from the various samplings. Data obtained were compared with those from a previous work (Mocali et al. 2003). Results indicated that plants with symptoms of phytoplasmosis showed marked alterations in the extent of the fluctuations of the community along the seasons in the different plant organs.

PCR-based assay for the detection of Curtobacterium flaccumfaciens pv. flaccumfaciens in bean seeds.

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 Cff DNA 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.

Parenteral iron supplementation for the treatment of iron deficiency anemia in children.

Iron-deficiency anemia impairs growth and intellectual development in children, which can be reversed only by early diagnosis and iron supplementation. Oral supplementation can efficiently replace stores, but in many cases parenteral iron is needed. Unfortunately some adverse reactions have limited its use in children. We compared the efficacy and safety of intramuscular and intravenous administration in 33 evaluable children with severe iron deficiency and/or iron-deficiency anemia who failed to respond to oral iron supplementation. Nineteen children received intravenous infusion and 14 intramuscular injections. All children showed recovery from iron-deficiency anemia, with statistically similar improvement in hemoglobin levels. The duration of treatment was longer in those receiving intramuscular injection. Parenteral iron therapy for the treatment of iron-deficiency anemia is a rapid, easy, and definitive solution to a long-troubling situation. We suggest the use of parenteral iron, in particular intravenous iron, in children who do not recover from severe iron-deficiency anemia after oral therapy. We should consider the physical and neuropsychological sequelae of long-lasting iron deficiency in children and the fact that oral supplementation is less likely to replace iron stores.

Impairment of nasal mucociliary clearance after radiotherapy for childhood head cancer.

BACKGROUND: Radiotherapy of the head region in children is known to cause long-term sequelae, such as facial, dental, and ocular abnormalities. We investigated whether a decreased nasal mucociliary function occurs after radiotherapy of the head in children. METHODS: A saccharin/charcoal test was performed in 20 children treated with radiotherapy of the head and in 20 controls, age-matched and gender-matched. RESULTS: We found a decreased nasal mucociliary clearance (lower percentage of responses (p = 0083) and longer mucociliary transport times (p =.0001) in the patients compared with the controls. The radiotherapy dosage influenced the response to the test (p =.0046). CONCLUSIONS: Irradiation of the head in children may cause impairment of mucociliary function, even permanently, which may predispose children to upper respiratory infections. We would suggest careful monitoring of such patients to detect as early as possible the clinical effects of the functional changes and to prevent the evolution to chronic diseases.

Angiogenesis extent and expression of matrix metalloproteinase-2 and -9 correlate with progression in human neuroblastoma.

In human tumors changes in angiogenesis and expression of extracellular matrix-degrading enzymes occur simultaneously during invasion and metastasis. Tissues from 20 biopsies of human neuroblastoma (NB) were investigated immunohistochemically by using an antibody against factor VIII to determine their microvessel number, and by in situ hybridisation to determine the expression of mRNA of the matrix metalloproteinase-2 (MMP-2) and MMP-9. The extent of angiogenesis and the expression of the MMP-2 and MMP-9 mRNA were upregulated in advancing stages. These in situ data suggest that angiogenesis and degradation of extracellular matrix occur simultaneously with NB tumor progression.

Concurrent Langerhans cell histiocytosis and myelodysplasia in children.

BACKGROUND: Langerhans cell histiocytosis (LCH) is characterized by the proliferation of abnormal histiocytes (Langerhans cells), whose origin as a reactive process or a neoplastic disorder is still poorly understood. Although LCH has been recorded as being associated with malignant neoplasms, concurrence of LCH and myelodysplastic syndrome has not been reported so far. PROCEDURE: We report on four children aged 23, 25, 26, and 53 months with multisystem LCH with organ dysfunction (bone marrow and liver) whose bone marrow pictures, taken at diagnosis, revealed the presence of myelodysplastic abnormalities (RA, RAEB, RAEB-t). RESULTS: We suggest that the commonly used expression of "organ dysfunction," which refers to clinical and functional alterations, could be explained by a myelodysplastic-like disorder. CONCLUSIONS: The contemporary presence of both events may provide a better understanding of the pathogenesis of LCH, especially in young children with multisystem disease and organ dysfunction, who are known to have a very poor outcome.

Ear involvement in childhood Langerhans' cell histiocytosis.

BACKGROUND: Ear involvement (EI) in Langerhans' cell histiocytosis (LCH) occurs quite often. We reviewed the Italian pediatric population of 251 children with LCH diagnosed between 1982 and 1995, focusing on EI, to highlight the prevalence, the clinical presentation, the outcome during follow-up, and the prognostic impact of otologic LCH. METHODS: EI was defined by chronic otorrhea and/or mastoid infiltration, external auditory meatus lesions, and middle/internal EI. The age at diagnosis, sex, system involved, organ dysfunction, treatment, disease control, and outcome were recorded. RESULTS: EI was noted at presentation in 34 children (13. 5%). They had a younger age at diagnosis (p=.0013) and near totality of multisystem disease (93.8% of patients with EI). Among patients with multisystem disease, children with EI seemed to have a higher risk of poor response and a higher percentage of second line treatment (p=.003). CONCLUSIONS: EI seems to identify patients with a particular disease behavior, which requires a more accurate evaluation at diagnosis, staging and treatment, and a strict follow-up, considering the possibility of an unfavorable outcome.

Polyclonal hypergammaglobulinemia at the onset of acute myeloid leukemia in children.

Polyclonal hypergammaglobulinemia (PHG) associated with hematological malignancies is a rare occurrence. We reviewed our series of 47 children with AML in order to define the prevalence of PHG and its prognostic value in achieving complete remission (CR) after induction treatment. Patients were stratified by immunoglobulin levels into two groups: with PHG and without PHG. CR reached after induction chemotherapy was considered a positive response. Conditional exact tests were used for the statistical analysis; conditional maximum likelihood estimates of the odds ratio (OR) were obtained. Significance levels (p) were determined from two-tailed tests. Twenty-two of 38 (57.9%) evaluable children showed PHG. Children with PHG and AML were more likely to be in CR after first induction treatment (OR=6.25, p=0.021), independent of sex, age at diagnosis, white blood cell count, percentage of blasts in the bone marrow, FAB phenotype, and treatment protocol. Infections seemed to positively influence early treatment response (p=0.038). PHG and infections were not statistically associated (p=0.16). PHG may result from the uncontrolled stimulation of B lymphocytes by cytokines. Infections or transfusions may act as triggers for the immune system, leading to the antileukemic effect seen in patients with AML and PHG going into spontaneous remission. It could be that this activation caused the larger number of CRs observed in our series. Clarification of why PHG exerts a positive influence on children with AML could help us to understand the ways by which the organism is able to control a malignant disease.

Bloodstream infections in children with cancer: a multicentre surveillance study of the Italian Association of Paediatric Haematology and Oncology. Supportive Therapy Group-Infectious Diseases Section.

A one-year prospective, multicentre surveillance study on aetiology, main clinical features and outcome of bloodstream infections in children with cancer was conducted in 18 paediatric haematology centres belonging to the Italian Association for Paediatric Haematology and Oncology. A total of 191 bloodstream infections were reported during the study period. Of them, 123 (64%) occurred in neutropenic and 68 (36%) in non-neutropenic patients. Gram-positive cocci caused 45% (85/191) of the episodes, gram-negative rods 41% (78/191), and fungi 9% (18/191). The remaining 5% (10/191) of the episodes were poly-microbial infections. A total of 204 pathogens were isolated (46% gram-positive cocci; 44% gram-negative rods; and 10% fungi). The aetiologic distribution was similar among neutropenic and non-neutropenic patients. A correlation between the infection and the presence of an indwelling central venous catheter was found in 20% (23/114) of the episodes among neutropenic patients and in 55% (23/62) among non-neutropenic patients. Gram-negative micro-organisms were isolated in an unusually high proportion of catheter-related infections (48%). The overall mortality rate from any cause within 30 days from the first positive blood culture was 11%, and was higher among patients who were neutropenic at the onset of the infection than among those who were not neutropenic (15 versus 4%, P = 0.03). In addition, the mortality was significantly higher in recipients of bone marrow transplantation than in patients with acute leukaemia or solid tumour (21, 11 and 6%, respectively) and was also higher in fungaemias and poly-microbial infections (22 and 30%) than in single gram-positive and gram-negative bacteraemias (11 and 6%).

Isolation and characterisation of a new antagonistic Burkholderia strain from the rhizosphere of healthy tomato plants.

A new Burkholderia strain (PVFi5A) which exhibits antagonism towards many bacterial and fungal plant pathogens has been partially characterised. This strain was isolated from the rhizosphere of tomato plants and was referred to the Burkholderia cepacia complex on the basis of cultural, morphological and biochemical characteristics, including determination of the 16S ribosomal DNA sequence and fatty acid profile. Strain PVFi5A is a Gram-negative, aerobic bacterium, oxidase- and catalase-positive, motile with a polar tuft of flagella, able to grow on a variety of media without producing diffusible pigments; it is avirulent to onion, able to grow at 41 degrees C and resistant to several antibiotic substances. Its fatty acid profile contains the hydroxy acids 18:1 20H, 14:0 3OH and 16:0 3OH, but not the hydroxy acids 16:0 2OH. The antagonistic activity of strain PVFi5A is due to its production of various, as yet unidentified, antimicrobial compounds, one or more of which may differ from those reported previously for certain 'B. cepacia' strains. The ability of PVFi5A to suppress the growth of important bacterial and fungal phytopathogens makes this strain a potential biocontrol agent.

Chemical structure of two phytotoxic exopolysaccharides produced by Phomopsis foeniculi.

The two main exocellular polysaccharides produced in vitro by Phomopsis foeniculi, a fungal pathogen of fennel, were isolated and characterized by chemical and spectroscopic methods as a galactan with the known structure [-->6)-beta-D-Galf-(1-->5)-beta-D- Galf-(1-->5)-beta-D-Galf-(1-->]n and a mannan. The latter consists of a backbone of alpha-(1-->6)-linked mannopyranose units. Almost all of these are branched at the 2 position with arms containing 2- and 3-linked mannopyranose units. The crude polysaccharide fraction and its components, galactan and mannan, showed phytotoxic effects, i.e. chlorosis, necrosis and/or wilting, on fennel and on two non-host plants, tobacco and tomato.

Structural determination of the O-deacetylated O-chain of lipopolysaccharide from Burkholderia (Pseudomonas) cepacia strain PVFi-5A.

On the basis of chemical degradation methods and one-and two-dimensional 1H and 13C NMR experiments the novel following structure was established for the O-deacetylated repeating unit of the O-chain of the main Burkholderia (Pseudomonas) cepacia (strain PVFi-5A) lipopolysaccharide: -->4)-beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->6)-alpha-D-GlcpNAc-(1-->.

Lipopolysaccharides from three phytopathogenic pseudomonads.

Analysis of the polysaccharide and lipid moieties of the lipopolysaccharides (LPSs) of the phytopathogenic bacteria, Pseudomonas amygdali and P. syringae pv. ciccaronei has demonstrated that for both bacteria, the O-chain consists of a tetrasaccharide repeating unit of three alpha-L-Rhap and one terminal nonreducing alpha-D-Fucp3NAc. Two of the rhamnosyl residues are 3-linked, the third one 2,3-linked. This structure had been previously found for the O-chains of three phytopathogenic strains of P. syringae subsp. savastanoi, but this is the first report on its occurrence in P. amygdali and P. syringae pv. ciccaronei. The results of the LPS lipid residue analysis made it possible to make some chemotaxonomic considerations and therefore classify P. amygdali as a chemotype, which is different from that of the other two bacteria examined.

[Association of infantile nephroblastomatosis and Wilms' tumor: discussion of a clinical case and therapeutic problems, analysis of the literature]. / Associazione della nefroblastomatosi infantile e tumore di Wilms: discussione di un caso clinico e delle problematiche terapeutiche, analisi della letteratura.

This is a case report of bilateral nephroblastomatosis in a 19 month child, who underwent a unilateral nephrectomy and chemotherapy. Further review of the nephrectomy specimen and biopsies of the contralateral kidney revealed mature features of nephrogenic rests progressing to Wilms' tumor. We have reviewed the literature and discuss the presentation and different therapeutic approaches.

Langerhans cell histiocytosis in childhood: results from the Italian Cooperative AIEOP-CNR-H.X '83 study.

Ninety patients with biopsy-proven Langerhans cell histiocytosis (LCH) were enrolled from June, 1983, to December, 1988, in the multicenter AIEOP-CNR-H.X. '83 study. They were divided into two groups: poor prognosis (PP), comprising 11 children with organ dysfunction (OD), and good prognosis (GP), made up of 79 patients without OD. Eighty-four patients were evaluable for treatment results. Among GP patients, 16 with a single lesion received only local treatment, while 59 entered a clinical trial of immunotherapy and/or monochemotherapy with vinblastine (VBL). Nonresponders, sequentially received doxorubicin (ADM) and then etoposide (VP16). PP patients were treated with 4 week cycles of vincristine, ADM, cyclophosphamide, and prednisone for nine courses. The overall survival was 92.8% (100% for GP patients and 45.5% for PP patients) at 48 months. The complete response (CR) rates for immunotherapy, VBL, ADM, and VP16 were 10%, 62.9%, 42.8%, and 88.2%, respectively. Two of the 11 PP patients had a CR (18.2%), while six died and three are still alive with recurrent disease. The overall incidence of disease-related disabilities was 47.7%, while that of diabetes insipidus was 20%. Monochemotherapy is probably adequate in GP patients, while more effective treatments are needed for PP patients.