First report of quinone outside inhibitor stigmatellin binding type (QoSI) resistance in Plasmopara viticola in India

Grape (Vitis vinifera) is an important fruit crop in India with about 3.5 million tonnes produced annually from an area of 111,000 ha. The Indian state of Karnataka is the major producer of grapes contributing about 25% of the national production. Downy mildew disease caused by Plasmopara viticola (Berk. & Curt.) is a threat to grape production throughout the world. Several fungicides have been used to manage downy mildew in grapevine, the most important being the quinone outside inhibitor stigmatellin binding type (QoSI) fungicide, ametoctradin, which has been used successfully in India for about a decade. However, in recent years’ farmers have noticed the failure of ametoctradin in managing downy mildew, and evolved resistance has been observed in France (Fontaine et al., 2019). In the present study, 41 downy mildew samples were collected from the major grapevine-growing districts of Karnataka state during 2022–23 and analysed for the presence of QoSI resistance. Sensitivity to QoSI fungicide was determined using a modified 24-well leaf-disc bioassay (Fungicide Resistance Action Committee, 2003). Healthy leaves were taken from the 6th node from the apex of a growing shoot of a downy mildew-susceptible grapevine cultivar (cv. Thomson Seedless) and 15 mm discs were cut from the leaves. The leaf discs were placed upside down in wells containing 1 mL of 0.5% water agar amended with 0, 100, 500, 1500, 2500 or 3000 µg mL−1 of either ametoctradin (300 g/l active ingredient) or dimethomorph (225 g/l active ingredient) (Zampro, BASF) as described by Sawant et al. (2016). Each treatment was repeated four times. Leaf disks were then inoculated with 10 µL of a suspension of P. viticola sporangia (50,000 sporangia mL−1) collected from a single lesion. Plates were incubated at 22°C with alternating periods of 12 hours light and dark. After six days, lesion area was measured and EC50 value was calculated by regression analysis of the per cent area of infection versus log10 fungicide concentration and resistance factor was also determined as described by Massi et al. (2021). The EC50 value of a sensitive isolate to the ametoctradin and dimethomorph mix was 2.36 µg/mL. The EC50 value of moderately resistant isolates ranged from 389 to 874 g/mL with RF (164-370), for resistant isolates the value ranged from 569 to 1126 µg/mL with RF (241-477) and for highly resistant isolates it ranged from 956 to 1423 µg/mL with RF (405-603). The BN-2 isolate from Babanagar, Vijayapur district had the largest EC50 value (1423 µg/mL). The resistance to QoSI fungicides in P. viticola developed due to a mutation in the cytochrome b (Pv-Cyt b) gene at the S34L site. This was detected using allele-specific primers 5′-ATTATTTTTATGGATTCGGTTT-3′ (forward) and 5′-ACCAACCGTTATTTACATCAC-3′ (reverse) as described by Fontaine et al. (2019). Total DNA was isolated from the isolates using a NucleoSpin Plant II kit as per the manufacturer's protocol (Macherey-Nagel, Germany) and the quality was assessed using a Qubit® 3.0 fluorometer (Thermo Fisher Scientific, USA). Of the 41 isolates tested, the 37 resistant isolates produced amplicons of the expected size (152 bp) and no amplicon was produced from the four sensitive isolates. The detection of this amplicon indicates an S34L amino acid mutation in the Pv-Cyt b gene associated with reduced sensitivity to quinone inside outside inhibitors (QoSI), viz. ametoctradin, and commercial products in which it is a constituent (Fontaine et al., 2019). To our knowledge this is the first occurrence of QoSI (ametoctradin) resistance in P. viticola in India. It is interesting to note that the related oomycete, Phytophthora litchii, has also developed resistance to ametoctradin which has been attributed to changes in PlCyt b (S33L and D228N) (Gao et al., 2022); S33L in P. litchi is orthologous to S34L in Plasmopara (Oliver et al., 2023). Further work is required to monitor resistance to QoSI fungicides in the P. viticola population and to alert the grower community to use alternative fungicides to effectively manage the disease.