Conjunctival fungal flora in healthy donkeys

OBJECTIVE: To identify and quantify ocular fungi from healthy donkeys living in the center of Italy. ANIMALS STUDIED: One hundred and two Amiata donkeys were examined. PROCEDURES: Conjunctival swabs from both eyes were seeded onto Sabouraud dextrose agar (SDA) and malt extract agar (MEA), and incubated at 25 degrees C over a 10-day period. Filamentous fungi identification was achieved to the genus level; yeast colonies were identified for macro-micromorphologic and physiological characteristics. RESULTS: Eighty-one donkeys out of 102 (79.4%) were positive for fungi; 47/102 (46.1%) had positive cultures from both eyes. Most frequently recovered fungal genera were Aspergillus spp., Penicillium spp., Cladosporium spp., Acremonium spp. Different fungal genera and/or species were recovered from the same donkey in 43 cases (42.1%). Yeasts were isolated from five subjects; the yeasts were never associated with molds. The number of colony forming units (CFU) ranged from 1 to 100. CONCLUSIONS: Aspergillus was the most commonly isolated fungal genus (33%). This result agrees with the findings of similar surveys carried out in horses. There was a remarkable presence of fungi and perfect forms. These observations may be explained by the optimal conditions for presence and development of fungi in the conjunctival fornix microenvironment in Amiata donkeys.     

Hydrogen peroxide localization and antioxidant status in the recovery of apricot plants from European Stone Fruit Yellows

Hydrogen peroxide (H₂O₂) localization and roles of peroxidases, malondialdehyde and reduced glutathione were compared in leaves of apricot (Prunus armeniaca) plants asymptomatic, European Stone Fruits Yellows (ESFY)-symptomatic and recovered. Nested PCR analysis revealed that Candidatus Phytoplasma prunorum, is present in asymptomatic, symptomatic and recovered apricot trees, confirming previous observations on this species, in which recovery does not seem to be related to the disappearance of phytoplasma from the plant.H₂O₂was detected cytochemically by its reaction with cerium chloride, which produces electron-dense deposits of cerium perhydroxides. H₂O₂was present in the plasmalemma of the phloem cells of recovered apricot plant leaves, but not in the asymptomatic or symptomatic material. Furthermore, by labelling apricot leaf tissues with diaminobenzidine DAB, no differences were found in the localization of peroxidases.Protein content in asymptomatic, symptomatic and recovered leaves was not significantly different from one another. In contrast, guaiacol peroxidase activity had the following trend: symptomatic > recovered > asymptomatic, whereas reduced glutathione content followed the opposite trend: asymptomatic > recovered > symptomatic. Moreover, no differences were observed in malondialdehyde concentrations between asymptomatic, symptomatic and recovered leaves. The overall results suggest that H₂O₂ and related metabolites and enzymes appear to be involved in lessening both pathogen virulence and disease symptom expression in ESFY-infected apricot plants.     

Hymenoptera wasps associated with the Asian gall wasp of chestnut (Dryocosmus kuriphilus) in Calabria,Italy

The parasitoid complex of the Asian chestnut gall wasp Drycosmus kuriphilus Yasumatsu was studied in Calabria (Italy). A total of 14 different species of parasitoids were collected, of which three are recorded on the Asian gall wasp for the first time. The composition of the parasitoid complex collected in Calabria was compared with that reported from Italy and from Europe. The altitude of the sites of collection seemed to have an effect on the distribution and abundance of the single species of parasitoids.     

Temporal and spatial propagule deposition patterns of the emerging fungal pathogen of chestnut Gnomoniopsis castaneae in orchards of north-western Italy

Two chestnut (Castanea sativa) orchards of north-western Italy were sampled with passive spore traps 35 times over 24 months. Samples were analysed through a newly developed quantitative PCR assay to quantify propagule loads of the emerging fungal pathogen Gnomoniopsis castaneae. Average propagule deposition patterns were assessed along with temporal and climatic variables, including sampling month and season, temperatures, relative humidity, precipitations, and wind. Machine learning algorithms combining information theory, fractal analysis, unbiased recursive partitioning, ordinary least squares and logistic regressions, were used to model propagule deposition patterns. The trained models were validated on independent data gathered from 24 samplings conducted in a third chestnut orchard during the same timeframe. Results showed that propagule deposition rate (DR) was variable within and among sites, with a site average ranging from 173 to 765 spores ⋅m⁻² ⋅h⁻¹. Propagule deposition was observed across all seasons, although the DR dropped substantially during wintertime (p < 0.05). Mean, maximum, and minimum temperatures, the growing degree days at 0 and 5℃ thresholds, and wind gust were all positively correlated (p < 0.05) with DR of G. castaneae. The trained models were all significant (p < 0.05), as well as their validation (p < 0.05). Fluctuations of propagule deposition throughout the year were consistent among sites and proved to be driven by temperatures. Wind gust was associated with the overall amount of propagules deposited at site level. In future, the increase in temperatures and strong winds as a result of climate change may boost the spread of G. castaneae.     

Identifying resistance in wild and ornamental cherry towards bacterial canker caused by Pseudomonas syringae

Bacterial canker is a major disease of stone fruits and is a critical limiting factor to sweet cherry (Prunus avium) production worldwide. One important strategy for disease control is the development of resistant varieties. Partial varietal resistance in sweet cherry is discernible using shoot or whole tree inoculations; however, these quantitative differences in resistance are not evident in detached leaf assays. To identify novel sources of resistance to canker, we used a rapid leaf pathogenicity test to screen a range of wild cherry, ornamental Prunus species and sweet cherry × ornamental cherry hybrids with the canker pathogens, Pseudomonas syringae pvs syringae, morsprunorum races 1 and 2, and avii. Several Prunus accessions exhibited limited symptom development following inoculation with each of the pathogens, and this resistance extended to 16 P. syringae strains pathogenic on sweet cherry and plum. Resistance was associated with reduced bacterial multiplication after inoculation, a phenotype similar to that of commercial sweet cherry towards nonhost strains of P. syringae. Progeny resulting from a cross of a resistant ornamental species Prunus incisa with susceptible sweet cherry (P. avium) exhibited resistance indicating it is an inherited trait. Identification of accessions with resistance to the major bacterial canker pathogens is the first step towards characterizing the underlying genetic mechanisms of resistance and introducing these traits into commercial germplasm.     

Molecular characterization of <Emphasis Type="Italic">Xiphinema brevicollum</Emphasis> (Nematoda: Longidoridae) from the Czech Republic

Populations of Xiphinema brevicollum occurring in the Czech Republic were described morphologically and molecularly. Published species-specific primer set BL18 and BV3 was used to amplify three populations of X. brevicollum from the Czech Republic. These primers were tested against 9 species of Xiphinema and 11 species of Longidorus. Amplification was also observed for X. inaequale, X. italiae and X. lambertii. Three additional markers, mitochondrial cytochrome c oxidase subunit 1, ribosomal D2/D3 expansion segment of the 28S gene and 18S gene, were amplified and sequenced for X. brevicollum, X. inaequale and X. lambertii belonging to X. americanum-group. Comparison of cox1 sequences of X. brevicollum from the Czech Republic with X. taylori from the Slovak Republic (accession number AM086702) suggested that these populations represent the same species.     

Oleuropein-Specific-13-Glucosidase Activity Marks the Early Response of Olive Fruits （Olea europaea） to Mimed Insect Attack

Olive fruits are seriously deteriorated by pre and postharvest damage due to the attack of insects, such as Bactrocera olaea, which strongly alters the quality of olives. Defence response in olive fruits injured both by pathogens and by mechanical damages has been associated with the enzyme β-glucosidase, which specifically hydrolyses oleuropein, producing highly reactive aldehyde molecules. In situ detection of ~-glucosidase activity in olive fruit tissues following injury, which simulates Bactrocera oleae punctures, is reported. The assay was performed in two cultivars showing different degrees of susceptibilities to fly infestation. In both cultivars, the histochemical assay for β-glucosidase showed that within 20 min after the injury, a strong ~-glucosidase activity could be observed in the damaged tissues. Thereafter a progressive enzyme inactivation occurred starting from tissues around the boundary of the injury with decrease of the enzyme activity and stopped after 3 h. Whereas the mass of active cells reached a distance of （300±50） μm from the edge of the injury. Biochemical analyses showed that in extracts of the injured fruit, β-glucosidase activity rapidly increased within 20 min from injury, thereafter decreasing and reaching values comparable with those in intact fruits. Following puncture, the oleuropein contents did not change significantly in the high susceptibility cultivar, whereas it rapidly decreased in the cultivar showing low susceptibility. The results strongly suggest that olive fruits susceptible towards fly infestation could be related to the ability of the oleuropein-degrading-β-glucosidase to produce the highly reactive molecules in the damaged tissues. As a consequence of puncture, high level of peroxidase activity was detected. This feature also suggested that this enzyme could play a key role in the defence response against insect injuries.     

Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning

Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID.