Screening of sensitivity to mandipropamid of Plasmopara viticola populations from Italian vineyards by molecular and biological methods.

UNLABELLED: The objective of this work was to study the sensitivity to mandipropamid of 33 Plasmopara viticola populations utilizing both molecular and biological techniques. The PCR-RFLP technique was developed in order to detect the single point mutation, G1105S, occurring on the PvCesA3 gene. The sensitivity was also studied using the leaf-disc bioassay. Thirty-three downy mildew-infected leaf samples, collected from 2010 to 2013 from Italian vineyards, were used in the study. PCR-RFLP revealed the presence of 7 resistant, 12 sensitive, 14 mixed (sensitive and resistant) mutation profiles. Effective concentration for 50% inhibition rate (EC50 ) calculated from the bioassays showed an EC50  < 1 mg l(-1) for samples that showed sensitive profiles, while for those samples that had a mixed profile, EC50 ranged from <1 to >300 mg l(-1) , and values for resistant profiles ranged from 200·28 to >300 mg l(-1) . The results suggest that P. viticola populations infecting Italian vineyards are under a selection pressure due to CAA-based fungicide applications. SIGNIFICANCE AND IMPACT OF THE STUDY: We characterized Plasmopara viticola populations utilizing PCR-RFLP technique to detect a point mutation known to cause resistance to carboxylic acid amides (CAA) fungicides. Sensitivity of these samples to the mandipropamid fungicide was assayed by a leaf-disc method. In this work, we provide the first evidence about the presence of mandipropamid-resistant populations of P. viticola from commercial vineyards in Italy. Improving the knowledge about development of resistant populations could enhance the current grapevine downy mildew management strategies and minimize the risk of the spread of mandipropamid and other CAA-resistant populations.

Development and evaluation of different complex media for phytoplasma isolation and growth.

The focus of this research was the development and evaluation of different complex liquid and solid media for the isolation and growth of phytoplasma strains infecting grapevine plants. Previously reported media supporting phytoplasma isolation are commercial and not easy to modify in order to improve performance and selectivity towards obtaining pure cultures of 'Candidatus Phytoplasma' species. Three media (Piv®, CB and MB) were therefore evaluated for phytoplasma isolation and colony formation under microaerophilic growing conditions, using grapevine canes from plants showing yellows symptoms, and infected by "flavescence dorée", "bois noir" and aster yellows phytoplasmas as sources. The newly developed methodology was applied for two years at three sample collection times. Broad applicability and a good repeatability in supporting phytoplasma colony formation were obtained in Pivs® and CBs media. While the MB medium did not support phytoplasma isolation and growth, the CB media support a phytoplasma growth comparable to the one obtained in the previously reported media. This medium has the advantage of a formulation that allow its modification to implement specificity towards selective phytoplasma growth. Moreover preliminary trials on serial dilutions and tetracycline addition confirmed some phytoplasma growth behaviours.