Evaluation of four whole-plant inoculation methods to analyze the pathogenicity of Erwinia amylovora under quarantine conditions

Four methods were tested to assess the fire-blight disease response on grafted pear plants. The leaves of the plants were inoculated with Erwinia amylovora suspensions by pricking with clamps, cutting with scissors, local infiltration, and painting a bacterial suspension onto the leaves with a paintbrush. The effects of the inoculation methods were studied in dose-time-response experiments carried out in climate chambers under quarantine conditions. A modified Gompertz model was used to analyze the disease-time relatiobbnships and provided information on the rate of infection progression (rg) and time delay to the start of symptoms (t0). The disease-pathogen-dose relationships were analyzed according to a hyperbolic saturation model in which the median effective dose (ED50) of the pathogen and maximum disease level (ymax) were determined. Localized infiltration into the leaf mesophile resulted in the early (short t0) but slow (low rg) development of infection whereas in leaves pricked with clamps disease symptoms developed late (long t0) but rapidly (high rg). Paintbrush inoculation of the plants resulted in an incubation period of medium length, a moderate rate of infection progression, and low ymax values. In leaves inoculated with scissors, fire-blight symptoms developed early (short t0) and rapidly (high rg), and with the lowest ED50 and the highest ymax

Improvement of strategies for the management of fire blight (Erwinia amylovora). Evaluation and optimization of physical and chemical control methods, and use of decision support systems

El foc bacterià és una malaltia que afecta a plantes de la família de la rosàcies, causada pel bacteri Erwinia amylovora. El seu rang d'hostes inclou arbres fruiters, com la perera, la pomera o el codonyer, i plantes ornamentals de gran interès comercial i econòmic. Actualment, la malaltia s'ha dispersat i es troba àmpliament distribuïda en totes les zones de clima temperat del món. A Espanya, on la malaltia no és endèmica, el foc bacterià es va detectar per primer cop al 1995 al nord del país (Euskadi) i posteriorment, han aparegut varis focus en altres localitzacions, que han estat convenientment eradicats. El control del foc bacterià, és molt poc efectiu en plantes afectades per la malaltia, de manera que es basa en mesures encaminades a evitar la dispersió del patogen, i la introducció de la malaltia en regions no endèmiques. En aquest treball, la termoteràpia ha estat avaluada com a mètode d'eradicació d'E. amylovora de material vegetal de propagació asimptomàtic. S'ha demostrat que la termoteràpia és un mètode viable d'eradicar E. amylovora de material de propagació. Gairebé totes les espècies i varietats de rosàcies mantingudes en condicions d'humitat sobrevivien 7 hores a 45 ºC i més de 3 hores a 50 ºC, mentre que més d'1 hora d'exposició a 50 ºC amb calor seca produïa danys en el material vegetal i reduïa la brotació. Tractaments de 60 min a 45 ºC o 30 min a 50 ºC van ser suficients per reduir la població epífita d'E. amylovora a nivells no detectables (5 x 102 ufc g-1 p.f.) en branques de perera. Els derivats dels fosfonats i el benzotiadiazol són efectius en el control del foc bacterià en perera i pomera, tant en condicions de laboratori, com d'hivernacle i camp. Els inductors de defensa de les plantes redueixen els nivells de malaltia fins al 40-60%. Els intervals de temps mínims per aconseguir el millor control de la malaltia van ser 5 dies pel fosetil-Al, i 7 dies per l'etefon i el benzotiadiazol, i les dosis òptimes pel fosetil-Al i el benzotiadiazol van ser 3.72 g HPO32- L-1 i 150 mg i.a. L-1, respectivament. Es millora l'eficàcia del fosetil-Al i del benzotiadiazol en el control del foc bacterià, quan es combinen amb els antibiòtics a la meitat de la dosi d'aquests últims. Tot i que l'estratègia de barrejar productes és més pràctica i fàcil de dur a terme a camp, que l'estratègia de combinar productes, el millor nivell de control de la malaltia s'aconsegueix amb l'estratègia de combinar productes. Es va analitzar a nivell histològic i ultrastructural l'efecte del benzotiadiazol i dels fosfonats en la interacció Erwinia amylovora-perera. Ni el benzotiadiazol, ni el fosetil-Al, ni l'etefon van induir canvis estructurals en els teixits de perera 7 dies després de la seva aplicació. No obstant, després de la inoculació d'E. amylovora es va observar en plantes tractades amb fosetil-Al i etefon una desorganització estructural cel·lular, mentre que en les plantes tractades amb benzotiadiazol aquestes alteracions tissulars van ser retardades. S'han avaluat dos models (Maryblyt, Cougarblight) en un camp a Espanya afectat per la malaltia, per determinar la precisió de les prediccions. Es van utilitzar dos models per elaborar el mapa de risc, el BRS-Powell combinat i el BIS95 modificat. Els resultats van mostrar dos zones amb elevat i baix risc de la malaltia. Maryblyt i Cougarblight són dos models de fàcil ús, tot i que la seva implementació en programes de maneig de la malaltia requereix que siguin avaluats i validats per un període de temps més llarg i en àrees on la malaltia hi estigui present.Fire blight, caused by the bacterium Erwinia amylovora, is a serious disease of rosaceous plants that affects fruit trees such as pear, apple or quince, and ornamental plants with great commercial and economic interest. The disease is spread and well distributed in all temperate regions of the world. In Spain, where the disease is non endemic, fire blight was first detected in 1995 in the North of the country (Euskadi) and later, several new outbreaks have appeared in other locations that have been properly eradicated. Control of fire blight is very slightly effective in affected plants and is based on measures to avoid the spread of pathogen, and the introduction of disease in non-endemic regions. In this work, thermotherapy has been evaluated as a method for eradication of E. amylovora from symptomless propagating plant material. It has been demonstrated that heat is a viable method for eradicating E. amylovora from the propagation material of the pear. Almost all rosaceous species and cultivars maintained under moist conditions survived 7 hours at 45 ºC and up to 3 hours at 50 ºC, while more than 1 hour of exposure at 50 ºC under dry heat injured plants and reduced shooting. However, 60 min at 45 ºC or 30 min at 50 ºC were enough to reduce epiphytic E. amylovora population on pear budwoods to non-detectable level (5 x 102 cfu g-1 f.w.). Phosphonate derivatives and benzothiadiazole were effective in fire blight control in pear and apple, under laboratory, greenhouse and field conditions. Plant defense inducers reduced disease levels to 40-60%. The minimal time intervals to achieve the best control of disease were 5 days for fosetyl-Al, and 7 days for ethephon and benzothiadiazole, and the optimal doses of fosetyl-Al and benzothiadiazole were 3.72 g HPO32- L-1 and 150 mg a.i. L-1, respectively. The efficacy of fosetyl-Al and benzothiadiazole in fire blight control was improved when consecutively sprayed (combined strategy) with a half-reduced dose of antibiotics. Although the mixed strategy is more practical and easier to apply in the orchard than the combined one, the best level of fire blight control was achieved with the combined strategy. The effect of benzothiadiazole and phosphonates in Erwinia amylovora-pear interaction was analyzed at histological and ultrastructural level. Neither benzothiadiazole, nor fosetyl-Al, nor ethephon induced structural changes in pear leaf tissues 7 days after their application. However, after E. amylovora inoculation structural cell disorganization was observed in fosetyl-Al and ethephon-sprayed plants, while in benzothiadiazole-sprayed plants these tissue alterations were delayed. Two predictive models (Maryblyt and Cougarblight) were evaluated in an orchard naturally affected by fire blight in Spain, to determine the accuracy of the predictions. The combined BRS-Powell model and the modified BIS95 model were also evaluated. Results showed two clearly differentiated geographical areas with high and low fire blight risk. Maryblyt and Cougarblight are easy models to use, but their implementation in disease management programs must be evaluated and validated for more seasons and in areas where the disease is present

Evaluation of four whole-plant inoculation methods to analyze the pathogenicity of Erwinia amylovora under quarantine conditions

Four methods were tested to assess the fire-blight disease response on grafted pear plants. The leaves of the plants were inoculated with Erwinia amylovora suspensions by pricking with clamps, cutting with scissors, local infiltration, and painting a bacterial suspension onto the leaves with a paintbrush. The effects of the inoculation methods were studied in dose-time-response experiments carried out in climate chambers under quarantine conditions. A modified Gompertz model was used to analyze the disease-time relatiobbnships and provided information on the rate of infection progression (rg) and time delay to the start of symptoms (t0). The disease-pathogen-dose relationships were analyzed according to a hyperbolic saturation model in which the median effective dose (ED50) of the pathogen and maximum disease level (ymax) were determined. Localized infiltration into the leaf mesophile resulted in the early (short t0) but slow (low rg) development of infection whereas in leaves pricked with clamps disease symptoms developed late (long t0) but rapidly (high rg). Paintbrush inoculation of the plants resulted in an incubation period of medium length, a moderate rate of infection progression, and low ymax values. In leaves inoculated with scissors, fire-blight symptoms developed early (short t0) and rapidly (high rg), and with the lowest ED50 and the highest yma

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 pearThis research was supported in part by grants from Ministerio de Educación y Ciencia (AGL2009-09829/AGR) of Spain; from the University of Girona (BR10/17 and MPCUdG2016/085); and from the CIRIT of the Catalonian Government (2009SGR812

Criteria for efficient prevention of dissemination and successful eradication of Erwinia amylovora (the cause of fire blight) in Aragón, Spain

Erwinia amylovora was detected on pome fruits in the Aragón region (North-Eastern Spain), in a ca. 5 km radius area located in the mid Jalón river (mid Ebro Valley) in the province of Zaragoza, during 2000‒2003. Eight years have now passed since this pathogen was last detected, without new infections being reported in the same area. The bases for surveys and rapid eradication performed have been analyzed in detail to understand the reasons for the success in removing fireblight. The results demonstrate that intensive surveillance, risk assessment, plant analyses using accurate identification methods, and, especially, rapid total or selective eradication of infected trees in the plots have been very effective in preventing the generalized spread of fireblight and in delaying economic losses associated with this disease. Eradication and compensation to growers, estimated to cost approx. € 467,000, were clearly counterbalanced by the economic value of apple and pear production in the 2000‒2003 period (approx. € 368 million). Fire blight risk-assessment, using the MARYBLYT system, showed that climatic conditions in the studied area were favourable to infections during the analyzed period (1997‒2006). Molecular characterization of E. amylovora strains had revealed their homogeneity, suggesting that these fire blight episodes could have been caused by just one inoculum source, supporting the hypothesis that there was a unique introduction of E. amylovora in the studied area. Spatial spread of E. amylovora to trees was analyzed within six orchards, indicating an aggregated distribution model. This Spanish experience demonstrates the success of scientifically-based prevention methods that lead to the deployment of a fast and strict containment strategy, useful for other Mediterranean area