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.

First Report of Resistance to Cyflufenamid in Podosphaera xanthii, Causal Agent of Powdery Mildew, from Melon and Zucchini Fields in Italy.

The fungicide cyflufenamid (phenyl-acetamide, Fungicide Resistance Action Committee [FRAC] code U6) was approved for use in Italy in 2011 as Takumi (Certis Europe, Utrecht, The Netherlands) to control Podosphaera xanthii (Castagne) U. Braun. & N. Shishkoff, the main causal agent of cucurbit powdery mildew. Considering that strains of this pathogen have developed resistance to strobilurin (5) and demethylation inhibitor (DMI) (4) fungicides, cyflufenamid represented a viable alternative to control this disease. However, this fungicide is also prone to resistance development as illustrated by resistance of P. xanthii in Japan (3). In the 2012 and 2013 growing seasons, significant declines in cyflufenamid efficacy were observed in two experimental fields in the Apulia (AP) and Emilia-Romagna (ER) regions of Italy on Cucumis melo and Cucurbita pepo, respectively. Takumi had been applied four times at the recommended field rate of 0.15 liter/ha (15 µg/ml of active ingredient [a.i.]) each growing season since 2010 in each field. Powdery mildew-infected leaf samples were collected in 2012 from both fields (25 isolates from AP and 19 from ER), and from five gardens (one isolate per garden); while in 2013, samples were collected only from the ER field (two polyconidial isolates). Isolates were maintained on detached zucchini cotyledons (1). Sensitivity of the isolates to cyflufenamid was determined by leaf disk bioassays (4) using Takumi at 0.01, 0.1, 1, 10, 20, and 50 µg a.i./ml. The 50% effective concentration (EC50) and the minimum inhibitory concentration (MIC) values were calculated (2). Isolates collected in ER and the gardens in 2012 all had an EC50< 0.01 µg/ml, and the MIC ranged from <0.01 to <1 µg/ml. Isolates from AP in 2012 had elevated EC50 values, from 0.230 to >50 µg/ml, and MIC values from <10 to >50 µg/ml; by 2013, the EC50 values of ER isolates ranged from 3.35 to >50 µg/ml. Based on the mean EC50 value of 0.0019 µg/ml for sensitive isolates of P. xanthii in Japan (2), isolates from both the ER field and gardens in 2012 were considered sensitive to cyflufenamid. Additionally, EC50 values of AP isolates from 2012 and ER isolates from 2013 were greater than those of sensitive isolates, indicating a shift in sensitivity toward resistance to cyflufenamid (resistance factor >100 [2]). Consequently, poor control of powdery mildew with cyflufenamid applications in the AP and ER trials was most likely a result of fungicide resistance. Isolates from these fields were exposed to selection pressure for fungicide resistance because cyflufenamid was applied more times than permitted in the label instructions. However, control of powdery mildew in 2013 was not as effective as in previous years in commercial fields in AP (C. Dongiovanni, personal communication). This observation, combined with proof of reduced sensitivity of some P. xanthii strains in Italy to cyflufenamid, highlights the need for implementing resistance management strategies to minimize the risk of fungicide resistant strains developing in cucurbit fields. References: (1) B. Álvarez and J. A. Torés. Bol. San. Veg. Plagas 23:283, 1997. (2) M. Haramoto et al. J. Pest. Sci. 31:397, 2006. (3) H. Hosokawa et al. Jpn. J. Phytopathol. 72:260, 2006. (4) M. T. McGrath et al. Plant Dis. 80:697, 1996. (5) M. T. McGrath and N. Shishkoff. Plant. Dis. 87:1007, 2003.