Project

molecular characterization of current races of Plasmopara halstedii in Argentina, the causal agent of sunflower downy mildew

Goal: To know the genetics of the current races of the pathogen in Argentina. Do a survey of the curent pathotypes and a molecular characterization using tools like sequencing.

Methods: Sequencing, 2D Agarose Gel Electrophoresis, MEGA, BioEdit, DNA Extraction

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Ana Laura Martinez
added 2 research items
Desde 2013 se observa un incremento de mildiu de girasol por Plasmopara halstedii en SE de Bs As y N de Santa Fé. Esto se atribuye a cambios genéticos del patógeno que confirieron tolerancia al curasemilla metalaxyl y/o superaron algunos genes Pl de resistencia de los híbridos cultivados. Ante este problema, se ha conformado una red compuesta por el INTA, universidades (UNS y UNMdP), laboratorios y semilleros que llevan a cabo: i) colección de muestras, ii) inoculación de líneas diferenciales de razas, iii) caracterización genética del patógeno por marcadores de ADN y secuencia, iv) evaluación de fungicidas. El set original de líneas diferenciales fue ampliado para mantener la capacidad de discriminación de razas presentes en Argentina. Se determinó presencia de razas 770, 730 y 710, a las que se suman la raza 713 que quebró la resistencia de Pl13 y Pl14 y una variante de la raza 710 que afecta híbridos con Pl15. Ensayos de control químico sobre variantes tolerantes al metalaxyl demostraron que azoxistrobina+metalaxyl controla hasta el 100% en estadios tempranos de infección. Se integrará la información de próximas campañas para comprender el proceso de expansión de la enfermedad, estudiar la generación/dinámica de variantes patogénicas y diseñar las estrategias de protección.
The objective of this work was to find practical procedures to overcome methodological drawbacks encountered during studies on sunflower downy mildew. Techniques for recovering living isolates of Plasmopara halstedii from the field and for the preservation of infected leaf samples for further molecular analysis were developed. A Polymerase Chain Reaction (PCR)-based test for the detection of P. halstedii in sunflower leaves and a method to remove azoxystrobin from fungicide-treated seeds are proposed. In situ -inoculations of pre-germinated seeds allowed the recovery of living isolates from the field. Three sample-preservation methods were evaluated (silica, heating and lyophilization) resulting in high yield and quality of the DNA extract. It was detected the presence of the pathogen in symptomless leaves through PCR using molecular markers based on expressed sequence tags. A treatment using sodium hypochlorite is recommended for the removal of azoxystrobin from fungicide treated seeds.
Ana Laura Martinez
added 2 research items
Downy mildew caused by Plasmopara halstedii is an economically challenging disease of sunflower (Helianthus annuus) worldwide. Our purpose was to evaluate naturalized wild sunflower populations as potential sources of resistance. Three populations were selected from contrasting environments in Argentina (Buenos Aires (BRW), Córdoba (RCU) and Entre Rios (DIA)). The response to the pathogen of 80 individuals of each population was evaluated in comparison with that of two susceptible genotypes. Analysis comprised inoculation (race 710, 45.000 zoosporangia ml-1) followed by phenotyping through macroscopic and microscopic observations and molecular SSRs-SNPs screening of genes associated with vertical resistance (Pl8, Plarg, Pl17, Pl19). Controls consisting in non-inoculated seeds were included. Phenotyping was done by either categorizing all treated seedlings into six categories (0:no disease − 5:dead), from which an index was calculated, or by assessing the proportion of affected seedlings. In both cases, there was a larger proportion of presumably resistant plants in BRW and DIA populations. Differences in responses among seedlings could be partially explained by the extent of pathogen infection in hypocotyls. Around 20% of the seedlings categorized as 0 showed sporulation on roots after an induction treatment. On average, 13% of the individuals from the studied populations showed no signs of the pathogen whatsoever thus expressing the highest level of resistance. Molecular screening demonstrated that genomic regions associated to Pl genes are highly polymorphic although correlation with previously described resistance alleles could not be established. These results indicate the presence of resistance determinants in the wild sunflower populations of Argentina.
Ana Laura Martinez
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Ana Laura Martinez
added an update
Our major objective is the study of the genetic constitution of the current breeds of P.h and knowing their geographic distribution in Argentina. We must know the genetic variability that causes the breaking of resistance in sunflower commercial hybrids. Besides, we expect to apply a specific method for detecting this pathogen in sunflower seeds.
 
Ana Laura Martinez
added a project goal
To know the genetics of the current races of the pathogen in Argentina. Do a survey of the curent pathotypes and a molecular characterization using tools like sequencing.