Lídia Ruz

An indigenous virulent strain of Erwinia amylovora lacking the ubiquitous plasmid pEA29.

ABSTRACT An atypical strain of Erwinia amylovora was isolated near an outbreak of fire blight at a nursery in Spain in 1996. It was obtained from a Crataegus plant showing typical symptoms and was identified as E. amy-lovora by biochemical tests and enrichment-enzyme-linked immuno-sorbent assay, but not by polymerase chain reaction using primers based on the pEA29 sequence. Nevertheless, with primers from chromosomal regions, the isolate gave the expected amplification band. This strain carries one plasmid of approximately 70 kb, with no homology with the 29-kb plasmid common to all pathogenic strains, or with a large plasmid present in some E. amylovora strains. Growth of the strain in minimal medium without thiamine was slower compared with cultures in the same medium with thiamine, a characteristic typical of strains cured of the 29-kb plasmid. Nevertheless, aggressiveness assays on pear, apple, and Pyracantha plants and in immature pear fruit showed that this strain exhibited a virulence level similar to other strains containing pEA29. To the best of our knowledge, this is the first report of the isolation from naturally infected plant material of a pathogenic strain of E. amylovora without pEA29, but with a plasmid of approximately 70 kb not previously described.

An update on control of brown spot of pear

Brown spot of pear is a fungal disease producing high economical losses in several pear-growing areas in Europe. Fungicide applications during the growing period either at fixed schedule or delivered according to the BSPcast forecasting system are not enough to control the disease under favorable conditions. New strategies have been introduced to control the inoculum production using sanitation methods. These methods are based on combinations of leaf litter removal during winter and biological control agent applications during late winter, spring and summer. These practices reduce both the inoculum pressure and disease levels. Therefore, the resulting optimized disease management consists of a combination of sanitation methods applied during the whole year with chemical fungicides scheduled according to the BSPcast forecasting model during the vegetative period. It is expected that the control of brown spot could be further refined upon availability of rapid methods for inoculum potential analysis. However, this analysis is difficult due to the variability in pathogenicity within the pathogen population.

Postinfection Activity of Synthetic Antimicrobial Peptides Against Stemphylium vesicarium in Pear

Brown spot of pear is a fungal disease of economic importance caused by Stemphylium vesicarium that affects the pear crops in Europe. Due to the characteristics of this disease and the moderate efficacy of available fungicides, the effectiveness of control measures is very limited; however, synthetic antimicrobial peptides (AMPs) may be a complement to these fungicides. In the present study, 12 AMPs of the CECMEL11 library were screened for fungicidal activity against S. vesicarium. In vitro experiments showed that eight AMPs significantly reduced the germination of conidia. The most effective peptides, BP15, BP22, and BP25, reduced fungal growth and sporulation at concentrations below 50 μM. Leaf assays showed that preventive application of BP15 and BP22 did not reduce infection; however, when the peptides were applied curatively, infection was significantly reduced. The use of a BP15 fluorescein 5-isothiocyanate conjugate revealed that the peptide binds to hyphae and germ tubes and produces malformations that irreversibly stop their development.

Interaction of antifungal peptide BP15 with Stemphylium vesicarium, the causal agent of brown spot of pear.

Peptide BP15 has shown antifungal activity against several plant pathogenic fungi, including Stemphylium vesicarium, the causal agent of brown spot of pear. BP15 inhibits the germination, growth and sporulation of S. vesicarium and displays post-infection activity by stopping fungal infection in pear leaves. In this work, live-cell imaging was undertaken to understand the antifungal mechanism of BP15. A double-staining method based on the combination of calcofluor white and SYTOX green coupled with epifluorescence microscopy was used to investigate fungal cell permeabilization and alterations in fungal growth induced by BP15. GFP-transformants of S. vesicarium were obtained and exposed to rhodamine-labelled BP15. Confocal laser microscopy provided evidence of peptide internalization by hyphae, resulting in fungal cell disorganization and death. S. vesicarium membrane permeabilization by BP15 was found to be peptide-concentration dependent. BP15 at MIC and sub-MIC concentrations (10 and 5 μM, respectively) inhibited S. vesicarium growth and produced morphological alterations to germ tubes, with slow and discontinuous compromise of fungal cell membranes. Fungal cell membrane disruption was immediately induced by BP15 at 100 μM, and this was accompanied by rapid peptide internalization by S. vesicarium hyphae. Peptide BP15 interacted with germ tubes and hyphae of S. vesicarium but not with conidial cells.

Combined morphological and molecular approach for identification of Stemphylium vesicarium inoculum in pear orchards.

Stemphylium vesicarium is the causal agent of brown spot of pear, an important disease reported in pear-growing areas of Europe. The pathogen is able to colonize pear leaf debris and dead tissues of herbaceous plants and produce abundant ascospores and conidia that are capable of infecting pear trees. Inoculum monitoring in pear orchards is mainly achieved through spore traps and species identification is based on conidial morphology, but the similarities on conidial traits among species of Stemphylium make correct identification difficult. In this work a total of thirty-seven Stemphylium isolates from pear orchards were characterized at the morphological, pathogenic, and molecular level. Correspondence among ITS and gpd sequences and morphological traits were evaluated. Species identification based exclusively on morphological data was not feasible. Combined morphological and molecular data were necessary for unambiguous identification of isolates in the S. vesicarium species group. Only isolates identified as S. vesicarium were pathogenic on pear. The study revealed that several species of Stemphylium coexist in pear orchards with S. vesicarium, the causal agent of BSP, and that combined morphological and molecular data are needed to differentiate them. Consequently, direct measurements of the airborne inoculum using volumetric spore traps may overestimate the actual pathogen population.

Controlling Brown Spot of Pear by a Synthetic Antimicrobial Peptide Under Field Conditions

Brown spot of pear, caused by Stemphylium vesicarium, is a fungal disease of increasing importance in several pear-growing areas of Europe. Disease control measures include the application of fungicides and sanitation methods. Antimicrobial peptides may be a complement or alternative to conventional fungicides used to manage brown spot disease. In a previous study, the synthetic peptide BP15 showed postinfection fungicidal activity against S. vesicarium in in vitro and detached-leaf assays. In the present study, the efficacy of BP15 (KKLFKKILKVL-NH2) in controlling brown spot of pear was evaluated under field conditions using potted plants and pear trees in orchards. In field trials, the treatments with BP15 or with the fungicide thiram were scheduled according to the infection risk predicted by the BSPcast model. Potted pear plants treated with BP15 showed a disease reduction of about 42 to 60% in five of seven trials. In three of four tree trials, the disease severity on shoots treated with BP15 was significantly lower than in the nontreated controls, with a mean efficacy of 38.2%. It was concluded that BP15 is a good candidate to be further developed as a fungicide for controlling brown spot of pear.

Epidemiological Features and Trends of Brown Spot of Pear Disease Based on the Diversity of Pathogen Populations and Climate Change Effects

Brown spot of pear, caused by the fungus Stemphylium vesicarium, is an emerging disease of economic importance in several pear-growing areas in Europe. In recent years, new control strategies combining sanitation practices and fungicide applications according to developed forecasting models have been introduced to manage the disease. However, the pathogenic and saprophytic behavior of this pathogen makes it difficult to manage the disease. In addition, climate change can also result in variations in the severity and geographical distribution of the disease. In this study, ecological and epidemiological aspects of brown spot of pear disease related to inoculum characterization and climate change impact were elucidated. The pathogenic variation in S. vesicarium populations from pear orchards and its relationship to inoculum sources (air samples, leaf debris, and infected host and nonhost tissues) was determined using multivariate analysis. In total, six variables related to infection and disease development on cultivar Conference pear detached leaves of 110 S. vesicarium isolates were analyzed. A high proportion of isolates (42%) were nonpathogenic to pear; 85% of these nonpathogenic isolates were recovered from air samples. Most isolates recovered from lesions (93%) and pseudothecia (83%) were pathogenic to pear. A group of pathogenic isolates rapidly infected cultivar Conference pear leaves resulted in disease increase that followed a monomolecular model, whereas some S. vesicarium isolates required a period of time after inoculation to initiate infection and resulted in disease increase that followed a logistic model. The latter group was mainly composed of isolates recovered from pseudothecia on leaf debris, whereas the former group was mainly composed of isolates recovered from lesions on pear fruit and leaves. The relationship between the source of inoculum and pathogenic/aggressiveness profile was confirmed by principal component analysis. The effect of climate change on disease risk was analyzed in two pear-growing areas of Spain under two scenarios (A2 and B1) and for three periods (2005 to 2009, 2041 to 2060, and 2081 to 2100). Simulations showed that the level of risk predicted by BSPcast model increased to high or very high under the two scenarios and was differentially distributed in the two regions. This study is an example of how epidemiological models can be used to predict not only the onset of infections but also how climate change could affect brown spot of pear. [Formula: see text] Copyright