First report of the <i>Phytophthora infestans</i> EU_43_A1 clonal lineage and associated PiCesA3 mutation G1105S in Ireland

Abstract

Potato late blight (PLB), caused by Phytophthora infestans continues to be the most economically destructive disease of potato crops in Ireland. The Irish P. infestans population is typically dominated by a small number of genotypes or lineages, identified using a nomenclature devised by the Euroblight network, e.g. EU_x_A1, to reflect geographical region in which it was first identified, number of clonal lineages assigned in that region and mating type. The combination of cool and moist weather with a predominance of PLB susceptibility amongst the potato cultivars grown mean that in Ireland growers use between 12–15 fungicide applications annually to control PLB, often on a weekly basis from crop emergence. In seasons where weather conditions are highly conducive to the development and spread of PLB increased fungicide applications can be expected. Even though a range of fungicides belonging to modes of action are available for PLB control (see for example https://agro.au.dk/forskning/internationale-platforme/euroblight/control-strategies/late-blight-fungicide-table), their application and effectiveness can depend on the growth stage of the crop. Given the requirement for season-long control and potential specificity of the different fungicides for crop growth, the emergence of fungicide resistance in P. infestans to one or more fungicide modes of action can have significant consequences for entire control programmes. Unfortunately in 2019 instances of reduced efficacy following treatment with the carboxylic acid amide (CAA) mandipropamid were reported in Denmark, with similar reports in 2020 and 2021, and with subsequent analysis confirming resistance to the CAA fungicides in the P. infestans lineage EU_43_A1 (Abuley et al., 2023). Although resistance to CAA has previously been identified in other oomycete species, this represented the first detection of CAA resistance in P. infestans (Fungicide Resistance Action Committee, 2024). As part of annual fungicide efficacy and cultivar resistance trials conducted by Teagasc at Oak Park, Carlow, Ireland (Stellingwerf et al., 2018) 140 PLB lesions were sampled at random from naturally infected potato plants throughout the 2023 season. Upon collection single lesions were separately pressed onto FTA cards (Qiagen, UK), air-dried and stored at ambient room temperature. Genotyping was conducted using 12 simple sequence repeat (SSR) markers in accordance with Li et al. (2013). Almost half (47.9%) of the samples had SSR profiles typical of EU_37_A2, 39.6% EU_36_A2, 7.9 % EU_6_A1 and 1.4% EU_12_A1. For both EU_37_A2 and EU_36_A2 a number of variants were detected, signifying both lineages are now established in the Irish P. infestans population (Figure 1). Single lesions (0.7%) belonging to EU_13_A2 and EU_43_A1 were also detected. To screen for potential mutation(s) associated with CAA resistance a nested PCR approach was used to amplify a fragment (500 bp) of the PiCesA3 gene encompassing the nucleotide triplet for amino acid 1105 previously reported to confer resistance (Blum et al., 2010). Samples from 96 P. infestans FTA card lesions from the 2019 and 2023 seasons and representative of the lineages identified above, plus EU_8_A1 which was previously dominant in Ireland were amplified. Briefly, FTA card samples were prepared as per the SSR markers and subjected to 35 cycles of PCR amplification using primer pair PiCesA3F1 (5′-AGGACTTTGAAGGAGACGCA-3′) and PiCesA3R1 (5′-GCCTTCCTCGTCTTCCTCTT-3′) at Tm = 58°C. The PCR product (1 μL) was used as template for a second round PCR using primer pair PiCesA3F2 (5′-CCTGTACTCTTTCTTGCCCG-3′) and PiCesA3R2 (5′-GTTGCCCTCGTAGTACACCT-3′) at Tm = 62°C. The PCR products were sequenced using the PiCesA3F2 and PiCesA3R2 primers by LGC Biosciences (Germany), confirming the presence of the nucleotide adenine at position 3416 in a homozygous state in only the sample confirmed as EU_43_A1, and leading to the amino acid substitution serine for glycine at amino acid position 1105 (G1105S). As CAA resistance is recessive (Blum et al., 2010) the detection of G1105S in a homozygous state confirms the first detection of CAA resistance in the Irish P. infestans population. In eight of the ten samples confirmed as EU_6_A1 and the EU_12_A1 sample, a synonymous nucleotide mutation, cytosine to thymine, was also identified at nucleotide position 3163, however it was always found in a heterozygous state. The EU_43_A1 lesion carrying the PiCesA3 substitution G1105S was one of 140 lesions sampled at the end of the season (between 05.09.2023 and 15.09.2023), possibly suggesting that its detection is a secondary infection, with a primary infection possibly elsewhere in Ireland. Although it was only detected at <1% of the entire collection sampled and tested, evidence from Denmark suggests that the lineage is fit and will be selected following CAA use if anti-resistance measures are not implemented (Abuley et al., 2023). To minimise selection, all measures to minimise initial PLB infections should be taken, with careful consideration given to the fungicide programme employed, including ensuring each application contains fungicides with different modes of action and alternating different modes of action at each application. This work was supported by Teagasc (project no. 0837) and the Teagasc Research Leaders 2025 fellowship funded by European Union's Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement no. 754380. David Cooke acknowledges the technical support from Louise Sullivan and funding from the Scottish Government Rural and Environment Science and Analytical Services Division.

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