ToxA and ToxB have minimal effect on tan spot epidemics of spring wheat in the Nordics

ToxA and ToxB have minimal effect on tan spot epidemics of spring wheat in the Nordics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ToxA and ToxB have minimal effect on tan spot epidemics of spring wheat in the Nordics Annika Johansson, Petteri Karisto, Satu Latvala, Pao Theen See, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6547012/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Dec, 2025 Read the published version in European Journal of Plant Pathology → Version 1 posted 6 You are reading this latest preprint version Abstract Tan spot, caused by the fungus Pyrenophora tritici-repentis ( Ptr ), is one of the major diseases of spring wheat ( Triticum aestivum L.) in the Nordic countries. However, little is known on the role of Ptr necrotrophic effectors (NEs), ToxA, ToxB and ToxC, behind tan spot epidemics in the region. A total of 217 Nordic Ptr isolates were screened for ToxA and ToxB genes using PCR. ToxA was found to be prevalent (64.5%), whereas ToxB was absent. A subset of 25 Ptr isolates was further tested on a set of wheat differentials. Majority ( n = 21) of these isolates induced susceptible reaction on ToxC sensitive genotype ‘6B365’, indicating the presence of ToxC effector. The symptoms observed on ToxA and ToxB differentials were not completely in line with the PCR results, suggesting the presence of ‘atypical’ Ptr isolates. In a collection of 197 spring wheat genotypes, ToxA and ToxB sensitivities were determined and found in 46.2% and 23.9% of the lines, respectively. Additionally, 179 of the wheat accessions were tested in the field over three years at two locations in Finland, and NE sensitivities were found to explain very little of the variation in tan spot susceptibility. Interestingly, ToxB sensitivity was found to have a counter-intuitive effect in European spring wheat germplasm, as ToxB-sensitive genotypes were less susceptible to tan spot disease compared to insensitive ones. The results presented here provide the first comprehensive overview of the importance of Ptr NEs in tan spot epidemics in the Nordic countries. Pyrenophora tritici-repentis Triticum aestivum necrotrophic effector field resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction In the Nordic countries, at the northernmost limit of wheat cultivation, spring wheat ( Triticum aestivum L.) has maintained its high importance as a food and feed crop alongside winter wheat. On average, 80% of the wheat cultivated in Finland and 65% in Norway consists of spring wheat (Luke, 2024 ; Statistics Norway, 2024 ). Spring wheat has its role in crop rotation also in Sweden and Denmark, although its share of total wheat hectares in these countries is substantially lower, 10% and 4%, respectively (Statistics Sweden, 2024 ; Statistics Denmark, 2024 ). The special status of spring wheat with requirements set by the unique northern environment demands local breeding efforts for improved spring wheat cultivars. Disease resistance stands high in breeders’ priorities when striving towards more resilient spring wheat varieties. One of the major diseases of spring wheat is tan spot caused by the ascomycete Pyrenophora tritici-repentis (Died.) Drechsler ( Ptr ). Typical symptoms elicited by this necrotrophic fungus are oval, necrotic lesions on leaves, often surrounded by a chlorotic halo. Ptr has greatly benefitted from the widespread adoption of reduced tillage practices over the last decades, facilitating the pathogen's survival in the field into the subsequent growing season (Jalli et al., 2020 ). Under high tan spot disease pressure, the average yield loss in Finland is 535 kg ha − 1 (Kauppi et al., 2021 ), corresponding to approximately 16% of Finnish spring wheat yield average (Luke, 2024 ). Yield losses by tan spot can however exceed 30% under favourable conditions (Bhathal et al., 2003 ). Successful resistance breeding requires knowledge on the underlying interactions between the plant host and the pathogen. Genetic resistance against a disease is usually not a binary trait but a complex quality built on numerous quantitatively inherited genes. Although the latter is true also for tan spot resistance in wheat, some major factors contributing to tan spot susceptibility have been identified (Faris et al., 2013 ). Studies on tan spot have heavily focused on the role of necrotrophic effectors (NEs) in Ptr virulence. NEs are small, extracellularly secreted molecules that are targeted to interfere with plant defenses. NE–host interactions work in an inverse manner compared to the classical gene-for-gene model (Flor, 1956 ). Recognition of NE by the host leads to susceptibility instead of resistance (Friesen & Faris, 2010 ). To date, three different Ptr NEs have been described in the literature: ToxA and ToxB, which are proteinaceous and encoded by the ToxA and ToxB genes, respectively; and ToxC, which is a small, polar molecule (Ballance et al., 1989 , Effertz et al., 2002 ; Martinez et al., 2001 ; Orolaza et al., 1995 ; Strelkov et al., 1999 ; Tomas et al., 1990 ; Tuori et al., 1995 ). ToxA triggers strong necrotic symptoms in wheat genotypes carrying a dominant sensitivity allele, Tsn1 (Faris et al., 1996 , 2010 ), and ToxB induces a chlorotic response in plants carrying the dominant Tsc2 allele (Friesen & Faris, 2004 ; Orolaza et al., 1995 ). Similar to ToxB, ToxC induces chlorosis, but the underlying molecular interactions behind ToxC-triggered sensitivity are still under investigation (Running et al., 2022 ; Shi et al., 2022 ). Ptr isolates can be divided into eight races based on which of the three known effectors they carry (Lamari & Bernier, 1989 ; Lamari et al., 1995 ; Lamari et al., 2003 ; Strelkov et al., 2002 ). Race classification is a commonly adopted measure for describing virulence patterns and diversity in many pathogens. With Ptr , race classification is traditionally made by using a differential set containing wheat genotypes with known reactions to each three NEs (Lamari et al., 2003 ). Ptr isolates that do not fit under the current eight races have also been described in the literature, demonstrating the diversity of this pathogen (Ali et al., 2010 ; Andrie et al., 2007 ; Guo et al., 2018 ; Laribi et al., 2022 ). Multiple PCR primers are available for screening ToxA and ToxB (e.g. Andrie et al., 2007 ; Antoni et al., 2010 ; Martinez et al., 2001 , 2004 ). PCR primers have also been developed for the ToxC1 gene that seems to have an important role in the ToxC producing pathway but is not sufficient alone for ToxC production (Shi et al., 2022 ). Isolates harbouring ToxC1 but lacking the ability to induce chlorosis in ToxC sensitive wheat differential have been detected (See et al., 2023 ). At present, visual assessment of symptoms on ToxC differential wheat currently stands as the only method to confirm whether ToxC is produced by fungal strains. Despite the diligent research on Ptr over the last decades, there is only little knowledge about the factors behind tan spot epidemics in the Nordic countries. To fill the knowledge gaps and to support the Nordic spring wheat breeding efforts (1) a total of 217 Ptr isolates were screened for the effector genes ToxA and ToxB with PCR to resolve the frequency of these genes in the Nordic Ptr population; (2) a subset of 25 Ptr isolates were further selected and tested for their virulence using a wheat differential set to gain more information on the virulence patterns and the presence of ToxC; (3) a collection of 197 spring wheat genotypes was tested for ToxA and ToxB sensitivities to better understand the distribution of NE sensitivity alleles in the European spring wheat population and (4) from these wheat genotypes, 179 were further tested for their tan spot susceptibility in the field over three years at two locations in Finland to study how NE sensitivities contribute to tan spot susceptibility under field conditions. Materials and Methods ToxA and ToxB presence in the Nordic Ptr population A total of 217 Ptr isolates (Supplementary Table 1) were collected from various locations in the Nordic and Baltic countries between 2004 and 2022 (Fig. 1 ) and screened for the presence of ToxA and ToxB genes using PCR. As positive controls for ToxA , Danish isolates ‘Efecta 4–4’ and ‘DTR 10–89’ (kindly provided by Lise Nistrup Jørgensen from Aarhus University, Denmark) were used, and as a positive control for ToxB , ‘DW-2’ was used (kindly provided by Dr. Jana Palicova from Crop Research Institute, Czech Republic and Prof. Shaukat Ali from South Dakota State University, USA). For genomic DNA (gDNA) extraction, isolates were recovered from mycelial plugs stored in liquid nitrogen (–196°C) and grown on V8 agar plates in 12 h near ultraviolet light (NUV)/12 h dark, at 18 ℃ for two weeks. gDNA was extracted by using DNeasy® Plant Mini Kit (Qiagen, Hilden, Germany). The mycelium on each agar plate was scraped into a 2 mL centrifuge tube containing one 6.3 mm ceramic sphere (MP Biomedicals, Eschwege, Germany). Samples were kept at − 81 ℃ for ½–1 h before addition of 400 µL AP1 buffer and 4 µL RNase A (100 µg µL − 1 ) from DNeasy® Plant Mini kit (Qiagen). Samples were heavily vortexed for 30 s, 2–3 times, and after this the gDNA extraction protocol was continued according to the manufacturer’s instructions. The gDNA samples were eluted in 100 µL of sterile distilled water, and the concentration was measured by NanoDrop 2000C spectrophotometer (Thermo-Scientific, Waltham, MA, USA). Species identity was confirmed by using Ptr species-specific primers PtrUniqueF2 and PtrUniqueR2 (Antoni et al., 2010 ). Total reaction volume was 25 µL with approximately 20 ng template DNA, 10X Taq buffer, 0.2 mM dNTP, 1.5 mM Mg 2+ , 1 U Taq DNA Polymerase (Thermo Fisher Scientific, Massachusetts, USA) and 0.2 µM of each primer. PCR amplification was carried out using a Biorad S1000 Thermal Cycler (Biorad, Hercules, CA, USA) with the following amplification conditions: 95°C/1 min, (95°C/30 s, 64°C/30 s, 72°C/30 s) × 35, 72°C/5 min. ToxA presence in the isolates collected in 2021 was analysed with singleplex PCR using primer pair ToxAscreeningF and ToxAscreeningR1 (Antoni et al., 2010 ; Moolhuijzen et al., 2018 ). Reagent concentrations were the same as with PtrUniqueF2 and PtrUniqueR2, with the following amplification conditions: 95°C/2 min, (95°C/30 s, 53°C/30 s, 72°C/1 min) × 35, 72°C/10 min. ToxA presence in the isolates collected in 2004–2019 and 2022, and ToxB presence in all isolates were screened with multiplex PCR that included a primer pair for chitin synthase 1 ( CHS-1 ) to confirm the presence of fungal DNA, a primer pair for detecting ToxA , and/or a primer pair for detecting ToxB (Table 1). The total reaction volume was 50 µL with approximately 20 ng template DNA, 10X Taq buffer, 0.2 mM dNTP, 1.5 mM Mg 2+ , 1 U Taq polymerase and 0.2 µM of each primer, except 0.4 µM of ToxA primers. Amplification conditions for multiplex PCR were adopted from Kamel et al. ( 2019 ) with slight modifications: 95°C/1 min, (95°C/30 s, 57°C/30 s, 72°C/30 s) × 38, 72°C/10 min. The size and quality of all the PCR products were analysed by electrophoresis on an 1.5% agarose gel in TBE buffer. The gels were stained with ethidium bromide and the DNA was visualized under UV-transilluminator (SCIE-PLAS, Cambridge, UK). Table 1. Primers used in the singleplex and multiplex PCR analyses for ToxA and ToxB genes. Gene Primer Sequence Product size Reference Singleplex Ptr-specific PtrUniqueF2 5´-GGACTTTGGCTTTCTATTGTGC-3´ 490 bp Antoni et al. ( 2010 ) PtrUniqueR2 5´-CTTGGTGAATGGTGAAGATGG-3´ ToxA ToxAscreeningF 5´-CCTCGTACTTCTTTTCAGCG-3´ 832 bp Antoni et al. ( 2010 ), Moolhuijzen et al. ( 2018 ) ToxAscreeningR1 5´-TGTAGAAGACAAGATTTTGA-3´ Multiplex ToxA TA51F 5′-GCGTTCTATCCTCGTACTTC-3′ 573 bp Andrie et al. ( 2007 ) TA52R 5′-GCATTCTCCAATTTTCACG-3 ToxB TB71F 5′-GCTACTTGCTGTGGCTATC-3′ 232 bp Andrie et al. ( 2007 ), Martinez et al. ( 2004 ) TB60R 5′-ACTAACAACGTCCTCCACTTTG-3′ CHS-1 CHS-79F 5′-TGGGGCAAGGATGCTTGGAAGAAG-3′ 275 bp Carbone & Kohn ( 1999 ), Andrie et al. ( 2007 ) CHS-354R 5′-TGGAAGAACCATCTGTGAGAGTTG-3′ Virulence of Nordic Ptr isolates A subset of 25 Nordic Ptr isolates collected in 2021 was selected for virulence testing with a wheat differential set. Isolates were selected 1) to represent all countries where isolates were available from the 2021 season and 2) based on the PCR results so that both ToxA positive and negative fungal individuals from each country would be present in the subset. The isolates used in the virulence testing are marked in Supplementary Table 1. The wheat differential set used in this study consisted of nine wheat genotypes (Table 2). Four genotypes, (‘Salamouni’, ‘Glenlea’, ‘6B662’ and ‘6B365’, kindly provided by Lise Nistrup Jørgensen from Aarhus University, Denmark) have known sensitivities to ToxA, ToxB and ToxC (Lamari et al., 2003 ). The remaining five genotypes consisted of Nordic spring wheat cultivars or breeding lines demonstrating variable susceptibility to tan spot from susceptible to resistant (‘Wappu’, ‘Alarm’, ‘Demonstrant’, ‘BOR_17’ and ‘BOR_19’). For these genotypes, data on the NE sensitivities and tan spot resistance were obtained in this study. Plants were sown in 3.5 L plastic pots filled with W HS R80301 seedling peat mix (Kekkilä Professional, Vantaa, Finland). One seed of each of the nine wheat differentials was sown in each pot in a fixed order. Plants were grown in the greenhouse at 18 ℃/15 ℃ day/night, 16 h photoperiod, watered as needed, and fertilized once a week with Yara Ferticare (Yara International, Oslo, Norway). Approximately two weeks after sowing, when the second leaf was fully expanded, plants were inoculated with Ptr spore and mycelium suspension. For inoculum, fungal isolates were grown on V8 agar in 12 h NUV/12 h dark at 18 ℃ for 3 weeks, 20 agar plates per isolate. Two millilitres of sterilized water was added on each agar plate and fungal colonies were scraped with a sterile glass spreader into falcon tubes. Inoculum was stirred gently and filtered through a sterile cheesecloth. As the sporulation rate was low in all isolates, the amount of mycelium in each sample was visually estimated to be approximately even among isolates via microscope. Just before inoculation, one drop of Tween 20 was added into each tube. Two hours before inoculation, misting was turned on in the greenhouse room and temperature raised from 18 to 20 ℃. Inoculation was started when the relative humidity reached approximately 100%. Pots were protected with plastic cones, and 3.6 mL inoculum per pot (0.4 mL per plant) was sprayed with 3 bar pressure using a modified paint sprayer. Plastic cones were removed after the inoculum was settled on plant leaves. Greenhouse ventilation and misting were switched off for the duration of inoculation and turned back on after. Lights were switched off during inoculation and switched back on after 24 h, to prevent leaf surfaces from drying. Misting was applied twice per day until observations were made: 2 h in the morning and 2 h in the evening, to maintain relative humidity of > 80%. Table 2. Differential wheat genotypes used in the virulence testing trial. R = resistant, MR = moderately resistant, S = susceptible, - = insensitive, + = sensitive, NA = data not available or not known. Wheat genotype ToxA sensitivity ToxB sensitivity ToxC sensitivity Known resistance level Salamouni - - - R Glenlea + - - NA 6B662 - + - NA 6B365 - - + NA Wappu + NA NA S Alarm - - NA S Demonstrant + - NA MR BOR_17 - + NA R BOR_19 - - NA R The pots were arranged in four blocks of 26 pots in a randomized complete block design (RCBD), so that each isolate was present once in each of the four blocks. One negative control pot inoculated with sterile water and a drop of Tween 20 was included in each block. Symptoms were evaluated 10 days after inoculation by using a scale from 1 to 5 described by Lamari and Bernier ( 1989 ). ToxA and ToxB sensitivities in spring wheat A collection of 179 spring wheat genotypes (Supplementary Table 2), generated by the CResWheat project (Pre-breeding for Nordic climate resilient spring wheat), and 18 Nordic spring wheat landraces, received from NordGen, were tested for sensitivity to purified ToxA and ToxB effector proteins in a controlled greenhouse environment. The majority of the 179 CResWheat genotypes were registered cultivars and breeding lines of European plant breeding companies, but he collection also included 28 non-European genotypes, mostly bred by CIMMYT. Landraces were tested separately from other accessions for seed availability reasons. ToxA and ToxB effector sensitivities were tested in separate trials. Seeds were sown in 1 L plastic pots filled with W HS R80301 seedling peat mix (Kekkilä Professional), two seeds per pot (a pot comprising one replicate). In each pot, one plant was treated while the second one was left untreated. Plants were grown at 18 ℃/15 ℃ day/night, 16 h photoperiod and watered as needed. Plants were fertilized once, two weeks after sowing, with Yara Ferticare (Yara International). Wheat accessions ‘Salamouni’ (ToxA and ToxB insensitive), ‘BG261’ (ToxA sensitive), ‘6B662’ (ToxB sensitive) and ‘BR34’ (ToxA insensitive) were used as controls in the experiments. In the trial setting for the 179 CResWheat genotypes, in addition to the four replicate pots of each genotype, 27 water control pots were included with a maximum of seven different genotypes sown in one pot (one seed per genotype), and the plants were treated with sterilized water. All pots were randomized according to the row-column experiment design using the CycDesigN -software (VSN International, Hemel Hempstead, UK) onto four tables in two different greenhouse rooms. For the 18 landraces, the trial setting was RCBD with five blocks, of which the fifth block included the water controls treated with sterilized water. Four replicates were treated with a necrotrophic effector. Purified ToxA and ToxB effector proteins produced in E. coli (See et al., 2019 ) were kindly provided by Curtin University, Australia. Freeze-dried effector proteins were dissolved in sterilized water to obtain the concentration of 10 ng/µL for ToxA and 200 ng/µL for ToxB, and infiltrated with needleless 1 mL syringe into the fully opened second leaf of two-week-old seedlings. The infiltration site was marked using a non-toxic, waterproof pen. Symptoms were observed 5 to 7 days after infiltration and scored on a scale from 0 to 5 adopted from See et al. ( 2019 ), where 0 = no visible symptoms, 1 = mild chlorosis, 2 = chlorosis, 3 = very strong chlorosis, 4 = strong chlorosis with necrosis, 5 = complete necrosis with tissue collapse. Field trials In addition to ToxA and ToxB effector sensitivity, the 179 CResWheat spring wheat genotypes (Supplementary Table 2) were tested for their tan spot susceptibility under field conditions in 2022, 2023 and 2024 at two locations in Finland: Inkoo (60°04'02.1"N 23°53'21.2"E) and Jokioinen (60°48'57.9"N 23°28'30.2"E). The 18 Nordic landrace accessions were not included in the field trials for seed availability reasons. The Inkoo field had a natural infection through all the three years, whereas in Jokioinen, artificial inoculation and sprinkler irrigation were used to promote uniform tan spot infection across the trial. In Inkoo, wheat genotypes were sown in 2 m 2 plots arranged in RCBD with two replicates. Sowing dates were May 25th in 2022, May 22nd in 2023, and May 17th in 2024. Plots were fertilized in 2022 with YaraMila Y3 HIVEN, 560 kg ha − 1 , in 2023 with YaraMila Y3, 450 kg ha − 1 and in 2024 with Yara Mila Y2, 400 kg ha − 1 (Yara International). Herbicides were sprayed as needed in all three years. In 2022 and 2024, plots were also treated once with insecticides. Observations were made twice in 2022 at BBCH growth stages GS65–GS71 and GS77–GS85 (Meier, 1997 ), and once in 2023 and 2024 at GS65–GS71. In Jokioinen, wheat genotypes were sown in hill plots arranged in RCBD with two replicates, 20 seeds per plot and distance between each hill plot being 30 cm. Sowing dates in Jokioinen were June 7th in 2022, June 5th in 2023, and May 29th in 2024. Plots were fertilized both in 2022 and 2023 with BeFert NPK, 370 kg ha − 1 and in 2024 with Yara Mila Y2, 370 kg ha − 1 (Yara International). Herbicides were sprayed as needed at GS21–GS30. Insecticides were sprayed each year, mainly against flea beetle ( Phyllotreta sp). Observations were made three three times every year at GS47–GS61, GS65–71 and GS77–GS83. Inoculum for the Jokioinen field trial was produced during preceding winters in a greenhouse by spraying a susceptible spring wheat cultivar ‘Wanamo’ at GS12–GS13 with a mixture of five Ptr isolates representing the natural variation in Finland (marked in Supplementary Table 1). After two weeks the infected leaves were cut into pieces and dried at room temperature. In the field, artificial inoculation was then performed by distributing approximately 1 g of infected wheat leaf material at the base of each hill plot at GS12. Sprinkler irrigation was used to maintain continuous near 100% humidity in the canopy for 24 h after inoculation. After this, sprinkler irrigation was applied in the evenings to promote disease development, depending on weather conditions and precipitation. Disease severity was assessed from the four uppermost leaves using a rating scale from 1 to 9, commonly used by Nordic breeders, where 1 = no symptoms, 2 = one lesion per 10 wheat individuals, 3 = one lesion per each wheat individual, 4 = two lesions per each wheat individual, 5 = lesions start to merge and cover the leaves, 6 = nethermost leaves are covered with lesions, 6 = approximately half of the leaves are covered by lesions, 7 = leaves are more diseased than healthy, 8 = very little green tissue left, 9 = leaves are completely necrotic. Statistical analyses The randomization for each RCBD was conducted using Microsoft Excel (v16.89; Microsoft, 2024). A random number was generated for each genotype, within each block separately, using the `RAND` function. The genotypes were subsequently sorted based on these values to achieve randomization. All statistical analyses were performed and figures drawn using R Statistical Software (v4.3.1 and v4.4.0; R Core Team, 2023 ). In NE sensitivity testing data, arithmetic means across the four replicates were taken as descriptors of sensitivity or insensitivity of each wheat genotype. The mean value of 2.5 was used as a threshold for ToxA sensitivity, and the mean value of 1 for ToxB sensitivity (Corsi et al., 2020 ). From virulence testing data, medians of the four replicates were calculated for each isolate + wheat genotype combination, and used as the estimates for virulence. The effect of ToxA on the overall virulence of the Ptr isolates was estimated with Kruskal-Wallis rank sum test, using the data from the five Nordic wheat genotypes in the differential set. In the field data, each timepoint was tested separately for spatial row-column effects by using Cumulative Link Mixed Model (CLMM) provided by R package ‘ordinal’ (v2023.12-4; Christensen, 2023 ). Adjustment for row and/or column effect was included in the model if the effect was significant. If there were multiple observations in a location in a year, the area under disease progress stairs (AUDPS) was calculated with the following equation (Simko & Piepho, 2012 ): Where Y is the disease assessment score, t is the observation timepoint and n is the total number of observations. To assess the impact of ToxA and ToxB sensitivities on tan spot susceptibility in the field, European and non-European wheat germplasm were analysed separately. This approach was necessary as the differences in genetic backgrounds between these two groups led to masking effects when treated as one dataset. The influence of ToxA and ToxB sensitivity in individual years was tested with Wilcoxon signed-rank test. To examine the effect of NE sensitivities across years, a linear mixed model was applied to the Jokioinen dataset using the R package ‘lme4’ (Bates et al., 2015 ), with year included as a random factor. Effect sizes for ToxA and ToxB sensitivities were estimated by extracting marginal R² (R 2 m ) values from the model using the R package ‘MuMIn’ (v1.48.4; Bartoń, 2024 ). Since the Inkoo dataset was not normally distributed, Nagelkerke’s pseudo-R² (R 2 N ) values were calculated using the R package ‘performance’ (Lüdecke et al., 2021 ). Results ToxA and ToxB presence in the Nordic Ptr population The Ptr isolate collection in years 2004–2022 consisted of 217 isolates of which 178 (82%) originated from Finland. The most extensively sampled years were 2004 ( n = 83), 2009 ( n = 23) and 2021 ( n = 53). Based on the PCR screening, 64.5% of the isolates carried the ToxA gene ( n = 140). The frequency of ToxA in Ptr isolates in each year and country is presented in Table 3. ToxB was not detected in any of the isolates. Table 3. Percentages (%) of ToxA positive Pyrenophora tritici-repentis isolates based on PCR screening, and the total number (N) of isolates per year and country of origin. Norway Denmark Sweden Finland Estonia Latvia Year % ( N ) % ( N ) % ( N ) % ( N ) % ( N ) % ( N ) 2004 . . . 77.1 (83) . . 2005 . . . 80.0 (10) . . 2006 . . . . . 0.0 (2) 2007 . . 25.0 (4) . . . 2008 . . 25.0 (4) . 37.5 (8) . 2009 . . . 73.9 (23) . . 2010 50.0 (2) . 0.0 (1) . . 100.0 (2) 2019 . . . 61.5 (13) . . 2021 50.0 (2) . 33.3 (6) 60.0 (45) . . 2022 0.0 (7) 100.0 (1) . . . . Unknown . . . 100.0 (4) . . Total 18.2 (11) 100.0 (1) 26.7 (15) 71.9 (178) 37.5 (8) 50.0 (4) Virulence of Nordic Ptr isolates Virulence scores of the 25 Ptr isolates tested with the wheat differential set indicated the presence of ToxC effector among the Nordic strains. The majority of the isolates ( n = 21) induced a susceptible reaction including strong chlorosis and necrosis in the ToxC differential ‘6B365’ (Fig. 2 c and Fig. 3 ). Virulence scorings were not fully consistent with the results from PCR screening of ToxA and ToxB genes (Fig. 3 ). Three isolates lacking ToxA were virulent on ToxA sensitive differential ‘Glenlea’ by inducing varying levels of necrosis, while four isolates carrying ToxA did not elicit notable necrosis on ‘Glenlea’ (Fig. 2 a). On the ToxB differential line ‘6B662’, four isolates that do not carry ToxB induced strong necrotic symptoms upon inoculation (Fig. 2 b). Overall disease response on the five Nordic wheat genotypes in the differential set showed that ToxA- positive isolates were more virulent compared to ToxA negative ones ( p = 0.0052). Intriguingly, the most susceptible wheat genotype was ‘Alarm’, despite being insensitive to both ToxA and ToxB based on NE infiltrations. Definitive conclusions on race classification were not drawn as the scorings were based more on the lesion type than the production of spreading necrosis or chlorosis. None of the isolates induced susceptible symptoms on ‘Salamouni’ which is insensitive to all of the three Ptr NEs. ToxA and ToxB sensitivities in spring wheat Greenhouse infiltrations with purified necrotrophic effectors revealed that 46.2% ( n = 91) of the 197 spring wheat genotypes were sensitive to ToxA (mean score ≥ 2.5), while 53.8% ( n = 106) were insensitive (mean score < 2.5). Only reactions in classes 0 (no reaction) and 5 (full necrosis) were observed. Thirteen accessions showed mixed sensitivity to ToxA, as replicates of the same genotype displayed both full insensitivity and complete sensitivity. In response to ToxB, 23.9% ( n = 47) of the accessions were sensitive (mean score ≥ 1), while 76.1% ( n = 150) were insensitive (mean score < 1). Observed reactions ranged from 0 to 3, with none of the accessions exhibiting necrotic symptoms. Ten accessions displayed mixed sensitivity to ToxB. The frequencies of ToxA and ToxB sensitivity only, and in combinations across wheat genotypes are illustrated in Fig. 4 . Field trials Tan spot disease incidence was the highest in 2022 and the lowest in 2023 in both Inkoo (natural infection, rainfed) and Jokioinen (artificial inoculation, sprinkler irrigation). Majority of the wheat genotypes were highly susceptible to tan spot. Spearman correlation between disease severity in Inkoo and Jokioinen was 0.56 in 2022, 0.34 in 2023 and 0.54 in 2024 (all p -values < 0.001). In Inkoo, ToxA sensitivity had no effect on the severity of tan spot infection either among European or non-European subset (Fig. 5 a and 5 b). In contrast, ToxB-sensitive genotypes in the European subset consistently exhibited lower tan spot severity compared to ToxB-insensitive ones (R 2 N = 0.142, 0.068 and 0.054 in 2022, 2023 and 2024, respectively) (Fig. 6 a). In the non-European subset, the difference between ToxB-sensitive and insensitive genotypes drew close to statistical significance only in 2022 (Fig. 6 b). In Jokioinen, wheat genotypes carrying ToxA-sensitivity had higher AUDPS over the three years compared to ToxA-insensitive genotypes in European subset ( p = 0.0274, R 2 m = 0.0150). When the three years were analysed individually, the difference was statistically significant only in 2024 in the European subset (Fig. 5 c and 5 d). ToxB sensitive wheat genotypes had lower AUDPS over the years in the European subset ( p = 0.0042, R 2 m = 0.0252), and higher in the non-European subset ( p = 0.0006, R 2 m = 0.1934), but the statistical significance varied between years (Fig. 6 c and Fig. 6d). Discussion The results presented here provide the first comprehensive overview of the importance of known Ptr necrotrophic effectors ToxA, ToxB and ToxC in tan spot epidemics in the Nordic countries. Despite the high prevalence of ToxA in the tested Ptr isolates and the presence of both ToxA and ToxB sensitivities in the tested spring wheat material, the effect of NE sensitivity was negligible on the observed tan spot epidemic under field conditions. ToxC presence in the Nordic Ptr isolates was studied for the first time and the results suggest ToxC to be frequently produced by local isolates. Among the Ptr isolates screened for the presence of the effector genes ToxA and ToxB , the Finnish samples yielded the most extensive dataset. Despite the samples from other countries were not as numerous, it can be concluded that ToxA is present at varying frequencies in all sampled Ptr populations. From all the tested isolates, 65% carried the ToxA gene. The results align with earlier observations of ToxA presence in the Baltics and Denmark, although different estimates on the gene frequency have been obtained depending on the fungal collections studied (Abdullah et al., 2017a ; Justesen et al., 2021 ; Kaneps et al., 2022). The ToxA gene was found present in isolates from all sampled years, although a slight decreasing trend was observed in Finnish samples, from approximately 80% in 2004–2005 to 60% in 2021. The result indicates, however, a likely benefit of ToxA for Ptr as the gene’s frequency in the fungal population has stayed relatively high over the last 20 years. The absence of ToxB in all screened isolates aligns well with previously reported results from Europe (Abdullah et al., 2017a ; Justesen et al., 2021 ; Kaneps et al., 2022). ToxB has been found prevalent predominantly in Ptr isolates originating from Northern Africa, near the wheat center of origin (Benslimane, 2018 ; Kamel et al., 2019 ; Laribi et al., 2022 ). Contrasting with the PCR results, three isolates lacking the ToxA gene induced necrosis on ToxA differential wheat ‘Glenlea’ in the virulence test of 25 Ptr isolates. Furthermore, four isolates that carried ToxA did not elicit notable necrosis on ‘Glenlea’. Previously, Abdullah et al. ( 2017a ) found that Ptr isolates lacking ToxA but inducing necrosis on ‘Glenlea’ were quite common in Lithuania. Similar observation was also reported in the Tunisian Ptr population although these isolates were collected from durum wheat (Laribi et al., 2022 ). Despite the total absence of the ToxB gene in the PCR analysis, four of the 25 isolates elicited susceptible reaction on the ToxB differential wheat ‘6B662’. However, as the observed symptoms were more necrotic than chlorotic, the disease phenotype warrants further investigation. By using knockout strains, Guo et al. ( 2018 ) previously demonstrated that Ptr isolates lacking both ToxA and ToxB genes were still virulent on various bread and durum wheat genotypes. Altogether, these results indicate the possibility of additional NE–host sensitivity locus interactions as well as the presence of other factors than NEs affecting the overall aggressiveness and virulence of these ‘atypical’ isolates (Abdullah et al., 2017a ; Ali et al., 2010 ; Andrie et al., 2007 ; Guo et al., 2018 ; Laribi et al., 2022 ). ToxC differential ‘6B365’ reacted to the majority of the isolates (21/25) with strong chlorosis rapidly progressing to necrosis, indicating high prevalence of ToxC producing isolates in the Nordic Ptr population. This result agrees with the previous studies showing ToxA and ToxC producing ‘race 1’ to be highly common and the dominant one in most of the wheat growing areas (Abdullah et al., 2017b ; Aboukhaddour et al., 2013 ; Gamba et al., 2012 ; Lepoint et al., 2010 ; MacLean et al., 2017 ; See et al., 2023 ). The high frequency of ToxC-producing isolates in the Nordic Ptr population and the significant role of ToxC sensitivity locus Tsc1 in tan sport resistance in different genetic backgrounds (Liu et al., 2020 ; Peters Haugrud et al., 2023 ) highlights the importance for Nordic breeders to eliminate Tsc1 -carrying wheat material from spring wheat breeding programs. On the host side, 47% of the 197 tested spring wheat accessions exhibited ToxA sensitivity, indicating ToxA sensitivity gene Tsn1 to be common. A similar level of ToxA sensitivity was observed by Ruud et al. ( 2018 ) in spring wheat accessions predominantly of Nordic origin. Another screening from Europe, focusing mostly on winter wheat, found ToxA sensitivity to be rare (Downie et al., 2018 ). However, a quarter of the ToxA-sensitive accessions in the study of Downie et al. ( 2018 ) was spring wheat despite representing only 6% of the genotypes. Tsn1 might thus be more common among spring wheat compared to winter wheat. It is also possible that spring wheat accessions adapted to Nordic climate share a high level of genetic similarity and common ancestors, resulting in the enrichment of Tsn1 in the germplasm. Despite the prevalence of ToxA sensitivity among spring wheat genotypes, as well as the high frequency of ToxA gene in the fungal population, ToxA sensitivity seems to play a minor role in the epidemic outcome in the field among the spring wheat genotypes of European origin. In the European spring wheat germplasm, ToxA sensitivity explained only about 1.5% of the tan spot susceptibility in the Jokioinen experiment, and in Inkoo, the effect of ToxA was not detected. In contrast, ToxA-sensitive genotypes of non-European origin had higher tan spot severity under natural infection, suggesting that ToxA was present in the local Ptr population in Inkoo. ToxA- Tsn1 interaction has been found to have a varying effect on tan spot susceptibility depending on the genetic background of the host, and also of the pathogen (Faris et al., 2012 ; Friesen et al., 2003 ; Liu et al., 2017 ; Peters Haugrud et al., 2023 ; See et al., 2018 ; Virdi et al., 2016 ; Wei et al., 2021 ). These studies have suggested ToxA- Tsn1 interaction to include additional regulatory components, from either on the host or the pathogen side, affecting the disease outcome. The effect of ToxA has also been found to be more pronounced at the seedling stage (Dinglasan et al., 2018 ; Taylor et al., 2023 ), which could also explain the lack of strong association here, as the plants were assessed for tan spot at the adult stage. Surprisingly, 24% of the screened spring wheat accessions here were found to be sensitive to ToxB. Previously, ToxB sensitivity has been reported to be rare in European wheat material (Corsi et al., 2020 ). ToxB sensitivity is highly heritable (Corsi et al., 2020 ) as it is governed by a single gene, Tsc2 (Abeysekara et al., 2010 ). Wheat genotypes screened here might share a common relative or relatives carrying Tsc2 , affecting the relatively high frequency of ToxB sensitivity. Interestingly, ToxB sensitivity had a counter-intuitive effect on tan spot severity among the European spring wheat genotypes. ToxB-sensitive accessions of European origin were less diseased compared to ToxB-insensitive ones, while the non-European genotypes with ToxB sensitivity displayed higher tan spot scorings in Jokioinen, even though ToxB was absent in the Ptr isolates used in the inoculation of the field. The results might suggest that the ToxB-sensitive wheat accessions in the European subset carry an unknown source of tan spot resistance, possibly linked to the Tsc2 locus. Detailed genetic studies would be necessary to confirm this assumption. Therefore, before removing ToxB-sensitive genotypes from their breeding material, Nordic breeders should take this possibility into account. Factors other than ToxA and ToxB seem to have a major role in tan spot epidemics in the Nordics. Tan spot resistance has a quantitative genetic makeup and many resistance QTLs in wheat have been found that are not connected to the known NE–host sensitivity locus interactions (Faris et al., 2013 ). Most probably there are also numerous effectors on the fungal side, awaiting to be detected. Recently, Rawlinson et al. ( 2024 ) described two novel secondary metabolites of Ptr , ToxE1 and ToxE2, that induce chlorosis on wheat in a genotype-specific manner. In conclusion, our comprehensive analysis and testing of both the pathogen and the host revealed that Ptr NEs have a rather small role in the tan spot epidemics in the Nordic region. Breeding against effector sensitivity is potentially simple, due to straight-forward testing, and there is no reason to avoid that. However, our results indicate the role of other factors than the known NE–sensitivity gene interactions in disease resistance of tan spot in the Nordics. Hence, resistance testing with relevant isolates and the use of naturally infected field nurseries in epidemic areas is still essential for breeding progress in this region. Statements and Declarations The authors have no competing interests to declare that are relevant to the content of this article. Acknowledgements : In addition to the ones mentioned in the text, we thank the CResWheat project partners, especially the Nordic breeders at Boreal Plant Breeding Ltd, Graminor AS, Lantmännen, Nordic Seed A/S and Sejet Plant Breeding, for providing spring wheat germplasm for the study. Special thanks to Tarja Niemelä, Boreal Plant Breeding Ltd, for helping with the field observations in Inkoo. Warm thanks to Senja Tuominen and Auli Kedonperä, Luke, and Elyce Iagallo, Curtin University, for their highly skilled technical assistance. Thanks to Daniel Fischer and Lauri Jauhiainen, Luke, for their helpful insights with the trial design and statistical analyses. This work was supported by the Nordic Council of Ministers (NMR), as in-kind from private partners in the CResWheat project, and by The Finnish Association of Academic Agronomists/Henrik and Ellen Tornberg trust fund (grant numbers 20220050 and 20230045). Author contributions : Annika Johansson, Satu Latvala and Marja Jalli designed the experiments. Annika Johansson collected the data. Annika Johansson and Petteri Karisto performed data analyses. Pao Theen See provided essential material, and instructions for the experiments and interpretation of the data. Morten Lillemo provided advice on the data analysis and results interpretation. Pernille Bjarup Hansen supplied breeding material. Annika Johansson wrote the first draft of the manuscript. All authors contributed in reviewing and editing the manuscript. Marja Jalli and Petteri Karisto supervised the work. All authors read and approved the final manuscript. References Abdullah, S., Sehgal, S. K., Ali, S., Liatukas, Z., Ittu, M., & Kaur, N. (2017a). Characterization of Pyrenophora tritici-repentis (tan spot of wheat) races in Baltic States and Romania. Plant Pathology Journal, 33 (2), 133–139. https://doi.org/10.5423/PPJ.OA.10.2016.0214 Abdullah, S., Sehgal, S. K., & Ali, S. (2017b). Race Diversity of Pyrenophora tritici-repentis in South Dakota and Response of Predominant Wheat Cultivars to Tan Spot. Journal of Plant Pathology & Microbiology, 8 (5), 409. https://doi.org/10.4172/2157-7471.1000409 Abeysekara, N. S., Friesen, T. L., Liu, Z., McClean, P. E., & Faris, J. D. (2010). Marker Development and Saturation Mapping of the Tan Spot Ptr Toxb Sensitivity Locus Tsc2 in Hexaploid Wheat. Plant Genome, 3 (3), 179–189. https://doi.org/10.3835/plantgenome2010.07.0017 Aboukhaddour, R., Turkington, T. K., & Strelkov, S. E. (2013). Race structure of Pyrenophora tritici-repentis (tan spot of wheat) in Alberta, Canada. Canadian Journal of Plant Pathology, 35 (2), 256–268. https://doi.org/10.1080/07060661.2013.782470 Ali, S., Gurung, S., & Adhikari, T. B. (2010). Identification and characterization of novel isolates of Pyrenophora tritici-repentis from Arkansas. Plant Disease, 94 (2), 229–235. https://doi.org/10.1094/PDIS-94-2-0229 Andrie, R., Pandelova, I., & Ciuffetti, L. M. (2007). Combination of phenotypic and genotypic characterization strengthens Pyrenophora tritici-repentis race identification. Phytopathology, 97 (6), 694–701. https://doi.org/10.1094/PHYTO-97-6-0694 Antoni, E. A., Rybak, K., Tucker, M. P., Hane, J. K., Solomon, P. S., Drenth, A., Shankar, M., & Oliver, R. P. (2010). Ubiquity of ToxA and absence of ToxB in Australian populations of Pyrenophora tritici-repentis . Australasian Plant Pathology, 39 (1), 63–68. https://doi.org/10.1071/AP09056 Ballance, G. M., Lamari, L., & Bernier, C. C. (1989). Purification and characterization of a host-selective necrosis toxin from Pyrenophora tritici-repentis . Physiological and Molecular Plant Pathology, 35 (3), 203–213. https://doi.org/10.1016/0885-5765(89)90051-9 Bartoń, K. (2024). MuMIn: Multi-Model Inference. R package version 1.48.4. Retrieved from https://CRAN.R-project.org/package=MuMIn Bates, D., Mächler, M., Bolker, B., & Walker, S. (2015). Fitting linear mixed-effects models using lme4. Journal of Statistical Software, 67 (1), 1–48. https://doi.org/10.18637/jss.v067.i01 Benslimane, H. (2018). Virulence phenotyping and molecular characterization of a new virulence type of Pyrenophora tritici-repentis , the causal agent of tan spot. Plant Pathology Journal, 34 (2), 139–142. https://doi.org/10.5423/PPJ.NT.07.2017.0150 Bhathal, J. S., Loughman, R., & Speijers, J. (2003). Yield reduction in wheat in relation to leaf disease from yellow (tan) spot and septoria nodorum blotch. European Journal of Plant Pathology, 109 , 435–443. https://doi.org/10.1023/A:1024277420773 Carbone, I., & Kohn, L. M. (1999). A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia, 91 (3), 553–556. https://doi.org/10.2307/3761358 Christensen, R. (2023). ordinal—Regression Models for Ordinal Data . R package version 2023.12-4.1, https://CRAN.R-project.org/package=ordinal Corsi, B., Percival-Alwyn, L., Downie, R. C., Venturini, L., Iagallo, E. M., Campos Mantello, C., McCormick-Barnes, C., See, P. T., Oliver, R. P., Moffat, C. S., & Cockram, J. (2020). Genetic analysis of wheat sensitivity to the ToxB fungal effector from Pyrenophora tritici-repentis , the causal agent of tan spot. Theoretical and Applied Genetics 133 (3), 935–950. https://doi.org/10.1007/s00122-019-03517-8 Dinglasan, E. G., Godwin, I. D., Phan, H. T. T., Tan, K.-C., Platz, G. J., & Hickey, L. T. (2018). Vavilov wheat accessions provide useful sources of resistance to tan spot (syn. yellow spot) of wheat. Plant Pathology, 67 , 1076–1087. https://doi.org/10.1111/ppa.12822 Downie, R. C., Bouvet, L., Furuki, E., Gosman, N., Gardner, K. A., Mackay, I. J., Campos Mantello, C., Mellers, G., Phan, H. T. T., Rose, G. A., Tan, K.-C., Oliver, R. P., & Cockram, J. (2018). Assessing European Wheat Sensitivities to Parastagonospora nodorum Necrotrophic Effectors and Fine-Mapping the Snn3-B1 Locus Conferring Sensitivity to the Effector SnTox3. Frontiers in Plant Science, 9 , 881. https://doi.org/10.3389/fpls.2018.00881 Effertz, R. J., Meinhardt, S. W., Anderson, J. A., Jordahl, J. G., & Francl, L. J. (2002). Identification of a chlorosis-inducing toxin from Pyrenophora tritici-repentis and the chromosomal location of an insensitivity locus in wheat. Phytopathology, 92 (5), 527–533. https://doi.org/10.1094/PHYTO.2002.92.5.527 Faris, J. D., Abeysekara, N. S., McClean, P. E., Xu, S. S., & Friesen, T. L. (2012). Tan spot susceptibility governed by the Tsn1 locus and race-nonspecific resistance QTL in a population derived from the wheat lines Salamouni and Katepwa. Molecular Breeding, 30 , 1669–1678. https://doi.org/10.1007/s11032-012-9750-7 Faris, J. D., Anderson, J. A., Francl, L. J., & Jordahl, J. G. (1996). Chromosomal location of a gene conditioning insensitivity in wheat to a necrosis-inducing culture filtrate from Pyrenophora tritici-repentis . Phytopathology, 8 (5), 459–463. https://doi.org/10.1094/Phyto-86-459 Faris, J. D., Liu, Z., & Xu, S. S. (2013). Genetics of tan spot resistance in wheat. Theoretical and Applied Genetics, 126 , 2197–2217. https://doi.org/10.1007/s00122-013-2157-y Faris, J. D., Zhang, Z., Lu, H., Lu, S., Reddy, L., Cloutier, S., Fellers, J. P., Meinhardt, S. W., Rasmussen, J. B., Xu, S. S., Oliver, R. P., Simons, K. J., & Friesen, T. L. (2010). A unique wheat disease resistance-like gene governs effector-triggered susceptibility to necrotrophic pathogens. Proceedings of the National Academy of Sciences USA, 107 (30), 13544–13549. https://doi.org/10.1073/pnas.1004090107 Flor, H. H. (1956). The complementary systems in flax and flax rust. Advances in Genetics, 8 , 29–54. https://doi.org/10.1016/S0065-2660(08)60498-8 Friesen, T. L., & Faris, J. D. (2004). Molecular mapping of resistance to Pyrenophora tritici-repentis race 5 and sensitivity to Ptr ToxB in wheat. Theoretical and Applied Genetics, 109 (3), 464–471. https://doi.org/10.1007/s00122-004-1678-9 Friesen, T. L., & Faris, J. D. (2010). Characterization of the wheat- Stagonospora nodorum disease system: What is the molecular basis of this quantitative necrotrophic disease interaction? Canadian Journal of Plant Pathology, 32 (1), 20–28. https://doi.org/10.1080/07060661003620896 Friesen, T. L., Ali, S., Kianian, S., Francl, L. J., & Rasmussen, J. B. (2003). Role of host sensitivity to Ptr ToxA in the development of tan spot of wheat. Phytopathology, 93 , 397–401. https://doi.org/10.1094/PHYTO.2003.93.4.397 Gamba, F. M., Strelkov, S. E., & Lamari, L. (2012). Virulence of Pyrenophora tritici-repentis in the southern cone region of South America. Canadian Journal of Plant Pathology , 34 (4), 545–550. https://doi.org/10.1080/07060661.2012.695750 Guo, J., Shi, G., & Liu, Z. (2018). Characterizing virulence of the Pyrenophora tritici-repentis isolates lacking both ToxA and ToxB genes. Pathogens, 7 (3), 74. https://doi.org/10.3390/pathogens7030074 Jalli, M., Kaseva, J., Andersson, B., Ficke, A., Nistrup-Jørgensen, L., Ronis, A., Kaukoranta, T., Ørum, J.-E., & Djurle, A. (2020). Yield increases due to fungicide control of leaf blotch diseases in wheat and barley as a basis for IPM decision-making in the Nordic-Baltic region. European Journal of Plant Pathology, 158 , 315–333. https://doi.org/10.1007/s10658-020-02075-w Justesen, A. F., Corsi, B., Ficke, A., et al. (2021). Hidden in plain sight: A molecular field survey of three wheat leaf blotch fungal diseases in North-Western Europe shows co-infection is widespread. European Journal of Plant Pathology, 160 , 949–962. https://doi.org/10.1007/s10658-021-02298-5 Kamel, S., Cherif, M., Hafez, M., Despins, T., & Aboukhaddour, R. (2019). Pyrenophora tritici–repentis in Tunisia: Race Structure and Effector Genes. Frontiers in Plant Science, 10 , 1562. https://doi.org/10.3389/fpls.2019.01562 Kaņeps, J., Moročko-Bičevska, I., Bankina, B., et al. (2022). Diversity in morphotypes and necrotrophic effectors of Pyrenophora tritici-repentis strains in Latvia and Belarus. Cereal Research Communications, 50 , 1037–1043. https://doi.org/10.1007/s42976-022-00255-4 Kauppi, K., Rajala, A., Huusela, E., Kaseva, J., Ruuttunen, P., Jalli, H., Alakukku, L., & Jalli, M. (2021). Impact of pests on cereal grain and nutrient yield in boreal growing conditions. Agronomy, 11 (3), 592. https://doi.org/10.3390/agronomy11030592 Lamari, L., & Bernier, C. C. (1989). Virulence of isolates of Pyrenophora tritici-repentis on 11 wheat cultivars and cytology of the differential host reactions. Canadian Journal of Plant Pathology , 11 , 284–290. https://doi.org/10.1080/07060668909501114 Lamari, L., Sayoud, R., Boulif, M., & Bernier, C. C. (1995). Identification of a new race in Pyrenophora tritici-repentis : Implications for the current pathotype classification system. Canadian Journal of Plant Pathology , 17 (4), 312–318. https://doi.org/10.1080/07060669509500668 Lamari, L., Strelkov, S. E., Yahyaoui, A., Orabi, J., & Smith, R. B. (2003). The identification of two new races of Pyrenophora tritici-repentis from the host center of diversity confirms a one-to-one relationship in tan spot of wheat. Phytopathology, 93 (4), 391–396. https://doi.org/10.1094/PHYTO.2003.93.4.391 Laribi, M., Akhavan, A., Ben M’Barek, S., Yahyaoui, A. H., Strelkov, S. E., & Sassi, K. (2022). Characterization of Pyrenophora tritici-repentis in Tunisia and comparison with a global pathogen population. Plant Disease, 106 (2), 464–474. https://doi.org/10.1094/PDIS-04-21-0763-RE Lepoint, P., Renard, M. E., Legrève, A., Duveiller, E., & Maraite, H. (2010). Genetic diversity of the mating type and toxin production genes in Pyrenophora tritici-repentis . Phytopathology, 100 (5), 474–483. https://doi.org/10.1094/PHYTO-100-5-0474 Liu, Y., Salsman, E., Wang, R., et al. (2020). Meta-QTL analysis of tan spot resistance in wheat. Theoretical and Applied Genetics, 133 , 2363–2375. https://doi.org/10.1007/s00122-020-03604-1 Liu, Z., Zurn, J. D., Kariyawasam, G., Faris, J. D., Shi, G., Hansen, J., Rasmussen, J. B., & Acevedo, M. (2017). Inverse gene-for-gene interactions contribute additively to tan spot susceptibility in wheat. Theoretical and Applied Genetics , 130 (6), 1267–1276. https://doi.org/10.1007/s00122-017-2886-4 Lüdecke, D., Ben-Shachar, M. S., Patil, I., Waggoner, P., & Makowski, D. (2021). performance: An R package for assessment, comparison, and testing of statistical models. Journal of Open Source Software, 6 (60), 3139. https://doi.org/10.21105/joss.03139 LUKE (2024). Retrieved from https://statdb.luke.fi/PxWeb/pxweb/fi/LUKE/LUKE__02%20Maatalous__04%20Tuotanto__14% 20Satotilasto/01a_Viljelykasvien_sato_mk.px/table/tableViewLayout2/. MacLean, D. E., Aboukhaddour, R., Tran, V. A., Askarian, H., Strelkov, S. E., Turkington, T. K., & Kutcher, H. R. (2017). Race characterization of Pyrenophora tritici-repentis and sensitivity to propiconazole and pyraclostrobin fungicides. Canadian Journal of Plant Pathology , 39 (4), 433–443. https://doi.org/10.1080/07060661.2017.1387178 Martinez, J. P., Oesch, N. W., & Ciuffetti, L. M. (2004). Characterization of the multiple-copy host-selective toxin gene, ToxB , in pathogenic and nonpathogenic isolates of Pyrenophora tritici-repentis . Molecular Plant-Microbe Interactions, 17 , 467–474. https://doi.org/10.1094/MPMI.2004.17.5.467 Martinez, J. P., Ottum, S. A., Ali, S., Francl, L. J., & Ciuffetti, L. M. (2001). Characterization of the ToxB gene from Pyrenophora tritici-repentis . Molecular Plant-Microbe Interactions , 14 (5), 675–677. https://doi.org/10.1094/MPMI.2001.14.5.675 Meier, U. (1997). Growth stages of mono- and dicotyledonous plants: BBCH-Monograph . Blackwell Wissenschafts-Verlag. Microsoft Corporation. (2024). Microsoft Excel. https://office.microsoft.com/excel Moolhuijzen, P. M., See, P. T., Oliver, R. P., & Moffat, C. S. (2018). Genomic distribution of a novel Pyrenophora tritici-repentis ToxA insertion element. PLoS ONE 13 (10), e0206586. https://doi.org/10.1371/journal.pone.0206586 Orolaza, N. P., Lamari, L., & Ballance, G. M. (1995). Evidence of a host-specific chlorosis toxin from Pyrenophora tritici-repentis , the causal agent of tan spot of wheat. Phytopathology, 85 (10), 1282–1287. https://doi.org/10.1094/Phyto-85-1282 Peters Haugrud, A. R. P., Shi, G., Seneviratne, S., Running, K. L., Zhang, Z., Singh, G., et al. (2023). Genome-wide association mapping of resistance to the foliar diseases septoria nodorum blotch and tan spot in a global winter wheat collection. Molecular Breeding, 43 (7), 54. https://doi.org/10.1007/s11032-023-01400-5 Rawlinson, C., Nealon, G., Chooi, Y. H., Oliver, R. P., Moffat, C. S., & See, P.T. (2024). Discovery of two novel phthalide phytotoxins from the wheat tan spot fungal pathogen Pyrenophora tritici-repentis . Journal of Agricultural and Food Chemistry, 72 (36), 19594–19603. https://doi.org/10.1021/acs.jafc.4c02533 R Core Team (2023). R: A Language and Environment for Statistical Computing . R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/ Running K. L. D., Momotaz A., Kariyawasam G. K., Zurn J. D., Acevedo M., Carter A. H., Liu Z. and Faris J. D. (2022). Genomic Analysis and Delineation of the Tan Spot Susceptibility Locus Tsc1 in Wheat. Frontiers in Plant Science, 13 , 793925. https://doi.org/10.3389/fpls.2022.793925 Ruud, A. K., Dieseth, J. A., & Lillemo, M. (2018). Effects of three Parastagonospora nodorum necrotrophic effectors on spring wheat under Norwegian field conditions. Crop Science, 58 , 159–168. https://doi.org/10.2135/cropsci2017.05.0281 See, P. T., Marathamuthu, K., Cupitt, C., Iagallo, E., & Moffat, C. S. (2023). A race profile of tan spot in Australia reveals race 2 isolates harbouring ToxC1 . Phytopathology, 113 (7), 1202–1209. https://doi.org/10.1094/PHYTO-11-22-0422-R See P.T., Iagallo E.M., Oliver R.P. & Moffat C.S. (2019) Heterologous Expression of the Pyrenophora tritici-repentis Effector Proteins ToxA and ToxB, and the Prevalence of Effector Sensitivity in Australian Cereal Crops. Frontiers in Microbiology 10 (182). https://doi.org/10.3389/fmicb.2019.00182 See, P. T., Marathamuthu, K. A., Iagallo, E. M., Oliver, R. P., & Moffat, C. S. (2018). Evaluating the importance of the tan spot ToxA- Tsn1 interaction in Australian wheat varieties. Plant Pathology, 67 , 1066–1075. https://doi.org/10.1111/ppa.12835 Shi, G., Kariyawasam, G., Liu, S., Leng, Y., Zhong, S., Ali, S., Moolhuijzen, P., Moffat, C. S., Rasmussen, J. B., Friesen, T. L., Faris, J. D., & Liu, Z. (2022). A conserved hypothetical gene is required but not sufficient for Ptr ToxC production in Pyrenophora tritici-repentis . Molecular Plant-Microbe Interactions, 35 (4), 336–348. https://doi.org/10.1094/MPMI-12-21-0299-R Simko, I., & Piepho, H.-P. (2012). The area under the disease progress stairs: Calculation, advantage, and application. Phytopathology, 102 , 381–389. https://doi.org/10.1094/PHYTO-07-11-0216 Statistics Denmark (2024). Retrieved from https://www.statbank.dk/20471 Statistics Norway (2024). Retrieved from https://www.ssb.no/en/jord-skog-jakt-og-fiskeri/jordbruk/statistikk/korn-og-oljevekster-areal-og-avlinger Statistics Sweden (2024). Retrieved from https://www.scb.se/en/finding-statistics/statistics-by-subject-area/agriculture-forestry-and-fishery/ Strelkov, S. E., Lamari, L., & Ballance, G. M. (1999). Characterization of a host-specific protein toxin (Ptr ToxB) from Pyrenophora tritici-repentis . Molecular Plant-Microbe Interactions, 12 (8), 728–732. https://doi.org/10.1094/MPMI.1999.12.8.728 Strelkov, S. E., Lamari, L., Sayoud, R., & Smith, R. B. (2002). Comparative virulence of chlorosis-inducing races of Pyrenophora tritici-repentis . Canadian Journal of Plant Pathology , 24 (1), 29–35. https://doi.org/10.1080/07060660109506967 Taylor, J., Jorgensen, D., Moffat, C. S. et al. (2023). An international wheat diversity panel reveals novel sources of genetic resistance to tan spot in Australia. Theoretical and Applied Genetics, 136 , 61. https://doi.org/10.1007/s00122-023-04332-y Tomas, A., Feng, G. H., Reeck, G. R., Bockus, W. W., & Leach, J. E. (1990). Purification of a cultivar-specific toxin from Pyrenophora tritici-repentis , causal agent of tan spot of wheat. Molecular Plant-Microbe Interactions , 3 , 221–224. https://doi.org/10.1094/MPMI-3-221 Tuori, R. P., Wolpert, T. J., & Ciuffetti, L. M. (1995). Purification and immunological characterization of toxic components from cultures of Pyrenophora tritici-repentis . Molecular Plant-Microbe Interactions , 8 , 41–48. https://doi.org/10.1094/mpmi-8-0041 Virdi, S. K., Liu, Z., Overlander, M. E., Zhang, Z., Xu, S. S., Friesen, T. L., & Faris, J. D. (2016). New insights into the roles of host gene-necrotrophic effector interactions in governing susceptibility of durum wheat to tan spot and septoria nodorum blotch. G3 Genes/Genomes/Genetics (Bethesda), 6 (12), 4139–4150. https://doi.org/10.1534/g3.116.036525 Wei, B., Despins, T., Fernandez, M. R., Strelkov, S. E., Ruan, Y., Graf, R., & Aboukhaddour, R. (2021). Race distribution of Pyrenophora tritici-repentis in relation to ploidy level and susceptibility of durum and winter bread wheat. Canadian Journal of Plant Pathology , 43 (4), 582–598. https://doi.org/10.1080/07060661.2020.1870002 Supplementary Files SupplementaryTable1isolates.xlsx SupplementaryTable2wheatgenotypes.xlsx Cite Share Download PDF Status: Published Journal Publication published 08 Dec, 2025 Read the published version in European Journal of Plant Pathology → Version 1 posted Editorial decision: Revision 03 Jul, 2025 Reviewers agreed at journal 14 May, 2025 Reviewers invited by journal 08 May, 2025 Editor invited by journal 01 May, 2025 Editor assigned by journal 30 Apr, 2025 First submitted to journal 28 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6547012","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":453665562,"identity":"ac7d5b46-c637-4502-b088-8d192a1558a4","order_by":0,"name":"Annika Johansson","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-1041-7507","institution":"University of Helsinki: Helsingin Yliopisto","correspondingAuthor":true,"prefix":"","firstName":"Annika","middleName":"","lastName":"Johansson","suffix":""},{"id":453665563,"identity":"618ebf7a-de6a-4805-96f8-76f108e43bcc","order_by":1,"name":"Petteri Karisto","email":"","orcid":"","institution":"Natural Resources Institute Finland Jokioinen: Luonnonvarakeskus Jokioinen","correspondingAuthor":false,"prefix":"","firstName":"Petteri","middleName":"","lastName":"Karisto","suffix":""},{"id":453665564,"identity":"18ef973e-5f99-4cfb-9973-b20f85126f2d","order_by":2,"name":"Satu Latvala","email":"","orcid":"","institution":"Natural Resources Institute Finland Jokioinen: Luonnonvarakeskus Jokioinen","correspondingAuthor":false,"prefix":"","firstName":"Satu","middleName":"","lastName":"Latvala","suffix":""},{"id":453665565,"identity":"2293e484-45fc-4823-a1d5-1c52913ce976","order_by":3,"name":"Pao Theen See","email":"","orcid":"","institution":"Centre of Crop and Disease Management, Curtin University","correspondingAuthor":false,"prefix":"","firstName":"Pao","middleName":"Theen","lastName":"See","suffix":""},{"id":453665566,"identity":"efec9707-37e0-4f5f-8856-a0a1f28472bb","order_by":4,"name":"Morten Lillemo","email":"","orcid":"","institution":"Norwegian University of Life Sciences, Faculty of Biosciences, Department of Plant Sciences","correspondingAuthor":false,"prefix":"","firstName":"Morten","middleName":"","lastName":"Lillemo","suffix":""},{"id":453665567,"identity":"582f6389-2912-4bfd-95a5-c8b66b50a5cf","order_by":5,"name":"Pernille Bjarup Hansen","email":"","orcid":"","institution":"Nordic Seed A/S","correspondingAuthor":false,"prefix":"","firstName":"Pernille","middleName":"Bjarup","lastName":"Hansen","suffix":""},{"id":453665568,"identity":"29bc5bc3-b975-472b-b448-66e001c32e4c","order_by":6,"name":"Marja Jalli","email":"","orcid":"","institution":"Natural Resources Institute Finland Jokioinen: Luonnonvarakeskus Jokioinen","correspondingAuthor":false,"prefix":"","firstName":"Marja","middleName":"","lastName":"Jalli","suffix":""}],"badges":[],"createdAt":"2025-04-28 10:46:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6547012/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6547012/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10658-025-03173-3","type":"published","date":"2025-12-08T15:56:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82590910,"identity":"e9dd353d-152e-4bfc-99ce-9a0002878b4e","added_by":"auto","created_at":"2025-05-13 08:02:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":163412,"visible":true,"origin":"","legend":"\u003cp\u003eLocations where Pyrenophora tritici-repentis isolates were collected in 2004–2022.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6547012/v1/51f6a1da037137eaaf498e2f.png"},{"id":82592100,"identity":"ca820930-54c4-4d17-990c-d60d78af81ab","added_by":"auto","created_at":"2025-05-13 08:10:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":345996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Atypical symptoms elicited on ToxA sensitive differential ‘Glenlea’. Isolates D191, D246 and D247 induced necrosis on ‘Glenlea’ despite not carrying the ToxA gene. In contrast, isolates D193, D228, D243 and D250 are ToxA-positive but failed to induce necrosis on ‘Glenlea’. \u003cstrong\u003eB.\u003c/strong\u003e Four isolates, D200, D247, D256 and D265 induced necrosis on ToxB differential ‘6B662’’ despite lacking the ToxB gene. The symptoms elicited on ‘6B662’ by ToxB-carrying isolates usually exhibit strong chlorosis. \u003cstrong\u003eC.\u003c/strong\u003e The range of symptoms induced on ToxC sensitive differential ‘6B365’. Most of the 21 isolates virulent on ‘6B365’ induced strong chlorosis that quickly evolved into necrosis and full tissue collapse on the leaves. All pictures are taken 10 days post inoculation.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6547012/v1/478e426a53491038fda4a7c2.png"},{"id":82593905,"identity":"fb940019-8a95-4de6-8615-8d5faed6f9f9","added_by":"auto","created_at":"2025-05-13 08:26:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":405219,"visible":true,"origin":"","legend":"\u003cp\u003eThe presence of necrotrophic effector genes in the fungal strains, and the medians of virulence scoring (from 0 to 5 (Lamari \u0026amp; Bernier, 1989)) of the 25 selected Pyrenophora tritici-repentis isolates at 10 days post inoculation on the wheat differential set. Isolates are sorted in descending order according to overall virulence. Necrotrophic effector sensitivities of the wheat genotypes used in race profiling, as well as tan spot resistance levels of the Nordic spring wheat differentials, are marked on top of the figure (I = insensitive, R = resistant, MR = medium resistant, S = susceptible).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6547012/v1/2f10e381179d3ece258d608f.png"},{"id":82594427,"identity":"c7c56f72-e050-46db-8163-a12231cbdc31","added_by":"auto","created_at":"2025-05-13 08:34:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74238,"visible":true,"origin":"","legend":"\u003cp\u003eThe amounts of different combinations of necrotrophic effector (NE) sensitivities among the tested 197 European spring wheat genotypes.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6547012/v1/074b12a5161cdff9a76b2fcb.png"},{"id":82592540,"identity":"0529c2d6-fda5-4b8b-9f09-247bb7994b62","added_by":"auto","created_at":"2025-05-13 08:18:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90521,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of ToxA sensitivity on tan spot severity in 2022–2024 field trials. Disease severity is presented as the Area Under Disease Progress Stairs (AUDPS), except for Inkoo 2023 and 2024 data with only one observation timepoint. Wilcoxon rank-sum test p-values are shown in the plots. \u003cstrong\u003eA.\u003c/strong\u003e Inkoo, European subset \u003cstrong\u003eB. \u003c/strong\u003eInkoo, non-European subset \u003cstrong\u003eC.\u003c/strong\u003e Jokioinen, European subset \u003cstrong\u003eD.\u003c/strong\u003e Jokioinen, non-European subset. Green represents ToxA sensitive genotypes and blue insensitive genotypes.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6547012/v1/db6003de5dbbcbdfd15b6777.png"},{"id":82590916,"identity":"68fa835f-56bf-403a-8f43-6806ff361cdb","added_by":"auto","created_at":"2025-05-13 08:02:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":83889,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of ToxB sensitivity on tan spot severity in 2022–2024 field trials. Disease severity is presented as the Area Under Disease Progress Stairs (AUDPS), except for Inkoo 2023 and 2024 data with only one observation timepoint. Wilcoxon rank-sum test p-values are shown in the plots. \u003cstrong\u003eA.\u003c/strong\u003e Inkoo, European subset \u003cstrong\u003eB. \u003c/strong\u003eInkoo, non-European subset \u003cstrong\u003eC.\u003c/strong\u003e Jokioinen, European subset \u003cstrong\u003eD.\u003c/strong\u003e Jokioinen, non-European subset. Green represents ToxB sensitive genotypes and blue insensitive genotypes.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6547012/v1/5b2572ecbc97eedef30f4cc2.png"},{"id":98243506,"identity":"a1d24b3b-fe1b-455e-b3a9-921c16598e95","added_by":"auto","created_at":"2025-12-15 16:07:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2008915,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6547012/v1/183af738-963d-4600-a435-91a0cde30b01.pdf"},{"id":82590919,"identity":"1f2d1ff1-0d08-4c4e-bdd8-5d69b9f62515","added_by":"auto","created_at":"2025-05-13 08:02:47","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":20569,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1isolates.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6547012/v1/53f09ad3b32169e155ed109a.xlsx"},{"id":82592537,"identity":"1a801b4a-d948-4dcf-96df-e4c4470ba81d","added_by":"auto","created_at":"2025-05-13 08:18:47","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":21344,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2wheatgenotypes.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6547012/v1/5d5ace64690210f07a19d526.xlsx"}],"financialInterests":"","formattedTitle":"ToxA and ToxB have minimal effect on tan spot epidemics of spring wheat in the Nordics","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the Nordic countries, at the northernmost limit of wheat cultivation, spring wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) has maintained its high importance as a food and feed crop alongside winter wheat. On average, 80% of the wheat cultivated in Finland and 65% in Norway consists of spring wheat (Luke, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Statistics Norway, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Spring wheat has its role in crop rotation also in Sweden and Denmark, although its share of total wheat hectares in these countries is substantially lower, 10% and 4%, respectively (Statistics Sweden, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Statistics Denmark, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The special status of spring wheat with requirements set by the unique northern environment demands local breeding efforts for improved spring wheat cultivars.\u003c/p\u003e \u003cp\u003eDisease resistance stands high in breeders\u0026rsquo; priorities when striving towards more resilient spring wheat varieties. One of the major diseases of spring wheat is tan spot caused by the ascomycete \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e (Died.) Drechsler (\u003cem\u003ePtr\u003c/em\u003e). Typical symptoms elicited by this necrotrophic fungus are oval, necrotic lesions on leaves, often surrounded by a chlorotic halo. \u003cem\u003ePtr\u003c/em\u003e has greatly benefitted from the widespread adoption of reduced tillage practices over the last decades, facilitating the pathogen's survival in the field into the subsequent growing season (Jalli et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Under high tan spot disease pressure, the average yield loss in Finland is 535 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Kauppi et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), corresponding to approximately 16% of Finnish spring wheat yield average (Luke, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Yield losses by tan spot can however exceed 30% under favourable conditions (Bhathal et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSuccessful resistance breeding requires knowledge on the underlying interactions between the plant host and the pathogen. Genetic resistance against a disease is usually not a binary trait but a complex quality built on numerous quantitatively inherited genes. Although the latter is true also for tan spot resistance in wheat, some major factors contributing to tan spot susceptibility have been identified (Faris et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Studies on tan spot have heavily focused on the role of necrotrophic effectors (NEs) in \u003cem\u003ePtr\u003c/em\u003e virulence. NEs are small, extracellularly secreted molecules that are targeted to interfere with plant defenses. NE\u0026ndash;host interactions work in an inverse manner compared to the classical gene-for-gene model (Flor, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1956\u003c/span\u003e). Recognition of NE by the host leads to susceptibility instead of resistance (Friesen \u0026amp; Faris, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo date, three different \u003cem\u003ePtr\u003c/em\u003e NEs have been described in the literature: ToxA and ToxB, which are proteinaceous and encoded by the \u003cem\u003eToxA\u003c/em\u003e and \u003cem\u003eToxB\u003c/em\u003e genes, respectively; and ToxC, which is a small, polar molecule (Ballance et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1989\u003c/span\u003e, Effertz et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Martinez et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Orolaza et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Strelkov et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Tomas et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Tuori et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). ToxA triggers strong necrotic symptoms in wheat genotypes carrying a dominant sensitivity allele, \u003cem\u003eTsn1\u003c/em\u003e (Faris et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1996\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and ToxB induces a chlorotic response in plants carrying the dominant \u003cem\u003eTsc2\u003c/em\u003e allele (Friesen \u0026amp; Faris, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Orolaza et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Similar to ToxB, ToxC induces chlorosis, but the underlying molecular interactions behind ToxC-triggered sensitivity are still under investigation (Running et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shi et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePtr\u003c/em\u003e isolates can be divided into eight races based on which of the three known effectors they carry (Lamari \u0026amp; Bernier, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Lamari et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Lamari et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Strelkov et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Race classification is a commonly adopted measure for describing virulence patterns and diversity in many pathogens. With \u003cem\u003ePtr\u003c/em\u003e, race classification is traditionally made by using a differential set containing wheat genotypes with known reactions to each three NEs (Lamari et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). \u003cem\u003ePtr\u003c/em\u003e isolates that do not fit under the current eight races have also been described in the literature, demonstrating the diversity of this pathogen (Ali et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Andrie et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Laribi et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMultiple PCR primers are available for screening \u003cem\u003eToxA\u003c/em\u003e and \u003cem\u003eToxB\u003c/em\u003e (e.g. Andrie et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Antoni et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Martinez et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). PCR primers have also been developed for the \u003cem\u003eToxC1\u003c/em\u003e gene that seems to have an important role in the ToxC producing pathway but is not sufficient alone for ToxC production (Shi et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Isolates harbouring \u003cem\u003eToxC1\u003c/em\u003e but lacking the ability to induce chlorosis in ToxC sensitive wheat differential have been detected (See et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). At present, visual assessment of symptoms on ToxC differential wheat currently stands as the only method to confirm whether ToxC is produced by fungal strains.\u003c/p\u003e \u003cp\u003eDespite the diligent research on \u003cem\u003ePtr\u003c/em\u003e over the last decades, there is only little knowledge about the factors behind tan spot epidemics in the Nordic countries. To fill the knowledge gaps and to support the Nordic spring wheat breeding efforts (1) a total of 217 \u003cem\u003ePtr\u003c/em\u003e isolates were screened for the effector genes \u003cem\u003eToxA\u003c/em\u003e and \u003cem\u003eToxB\u003c/em\u003e with PCR to resolve the frequency of these genes in the Nordic \u003cem\u003ePtr\u003c/em\u003e population; (2) a subset of 25 \u003cem\u003ePtr\u003c/em\u003e isolates were further selected and tested for their virulence using a wheat differential set to gain more information on the virulence patterns and the presence of ToxC; (3) a collection of 197 spring wheat genotypes was tested for ToxA and ToxB sensitivities to better understand the distribution of NE sensitivity alleles in the European spring wheat population and (4) from these wheat genotypes, 179 were further tested for their tan spot susceptibility in the field over three years at two locations in Finland to study how NE sensitivities contribute to tan spot susceptibility under field conditions.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eToxA\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eToxB\u003c/strong\u003e \u003cstrong\u003epresence in the Nordic\u003c/strong\u003e \u003cstrong\u003ePtr\u003c/strong\u003e \u003cstrong\u003epopulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 217 \u003cem\u003ePtr\u003c/em\u003e isolates (Supplementary Table 1) were collected from various locations in the Nordic and Baltic countries between 2004 and 2022 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and screened for the presence of \u003cem\u003eToxA\u003c/em\u003e and \u003cem\u003eToxB\u003c/em\u003e genes using PCR. As positive controls for \u003cem\u003eToxA\u003c/em\u003e, Danish isolates \u0026lsquo;Efecta 4\u0026ndash;4\u0026rsquo; and \u0026lsquo;DTR 10\u0026ndash;89\u0026rsquo; (kindly provided by Lise Nistrup J\u0026oslash;rgensen from Aarhus University, Denmark) were used, and as a positive control for \u003cem\u003eToxB\u003c/em\u003e, \u0026lsquo;DW-2\u0026rsquo; was used (kindly provided by Dr. Jana Palicova from Crop Research Institute, Czech Republic and Prof. Shaukat Ali from South Dakota State University, USA).\u003c/p\u003e\n\u003cp\u003eFor genomic DNA (gDNA) extraction, isolates were recovered from mycelial plugs stored in liquid nitrogen (\u0026ndash;196\u0026deg;C) and grown on V8 agar plates in 12 h near ultraviolet light (NUV)/12 h dark, at 18 ℃ for two weeks. gDNA was extracted by using DNeasy\u0026reg; Plant Mini Kit (Qiagen, Hilden, Germany). The mycelium on each agar plate was scraped into a 2 mL centrifuge tube containing one 6.3 mm ceramic sphere (MP Biomedicals, Eschwege, Germany). Samples were kept at \u0026minus;\u0026thinsp;81 ℃ for \u0026frac12;\u0026ndash;1 h before addition of 400 \u0026micro;L AP1 buffer and 4 \u0026micro;L RNase A (100 \u0026micro;g \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) from DNeasy\u0026reg; Plant Mini kit (Qiagen). Samples were heavily vortexed for 30 s, 2\u0026ndash;3 times, and after this the gDNA extraction protocol was continued according to the manufacturer\u0026rsquo;s instructions. The gDNA samples were eluted in 100 \u0026micro;L of sterile distilled water, and the concentration was measured by NanoDrop 2000C spectrophotometer (Thermo-Scientific, Waltham, MA, USA).\u003c/p\u003e\n\u003cp\u003eSpecies identity was confirmed by using \u003cem\u003ePtr\u003c/em\u003e species-specific primers PtrUniqueF2 and PtrUniqueR2 (Antoni et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Total reaction volume was 25 \u0026micro;L with approximately 20 ng template DNA, 10X Taq buffer, 0.2 mM dNTP, 1.5 mM Mg\u003csup\u003e2+\u003c/sup\u003e, 1 U Taq DNA Polymerase (Thermo Fisher Scientific, Massachusetts, USA) and 0.2 \u0026micro;M of each primer. PCR amplification was carried out using a Biorad S1000 Thermal Cycler (Biorad, Hercules, CA, USA) with the following amplification conditions: 95\u0026deg;C/1 min, (95\u0026deg;C/30 s, 64\u0026deg;C/30 s, 72\u0026deg;C/30 s) \u0026times; 35, 72\u0026deg;C/5 min.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eToxA\u003c/em\u003e presence in the isolates collected in 2021 was analysed with singleplex PCR using primer pair ToxAscreeningF and ToxAscreeningR1 (Antoni et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e; Moolhuijzen et al., \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Reagent concentrations were the same as with PtrUniqueF2 and PtrUniqueR2, with the following amplification conditions: 95\u0026deg;C/2 min, (95\u0026deg;C/30 s, 53\u0026deg;C/30 s, 72\u0026deg;C/1 min) \u0026times; 35, 72\u0026deg;C/10 min.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eToxA\u003c/em\u003e presence in the isolates collected in 2004\u0026ndash;2019 and 2022, and \u003cem\u003eToxB\u003c/em\u003e presence in all isolates were screened with multiplex PCR that included a primer pair for chitin synthase 1 (\u003cem\u003eCHS-1\u003c/em\u003e) to confirm the presence of fungal DNA, a primer pair for detecting \u003cem\u003eToxA\u003c/em\u003e, and/or a primer pair for detecting \u003cem\u003eToxB\u003c/em\u003e (Table 1). The total reaction volume was 50 \u0026micro;L with approximately 20 ng template DNA, 10X Taq buffer, 0.2 mM dNTP, 1.5 mM Mg\u003csup\u003e2+\u003c/sup\u003e, 1 U Taq polymerase and 0.2 \u0026micro;M of each primer, except 0.4 \u0026micro;M of \u003cem\u003eToxA\u003c/em\u003e primers. Amplification conditions for multiplex PCR were adopted from Kamel et al. (\u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) with slight modifications: 95\u0026deg;C/1 min, (95\u0026deg;C/30 s, 57\u0026deg;C/30 s, 72\u0026deg;C/30 s) \u0026times; 38, 72\u0026deg;C/10 min. The size and quality of all the PCR products were analysed by electrophoresis on an 1.5% agarose gel in TBE buffer. The gels were stained with ethidium bromide and the DNA was visualized under UV-transilluminator (SCIE-PLAS, Cambridge, UK).\u003c/p\u003e\n\u003cp\u003eTable 1. Primers used in the singleplex and multiplex PCR analyses for ToxA and ToxB genes.\u003c/p\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePrimer\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSequence\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eProduct size\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eSingleplex\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePtr-specific\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePtrUniqueF2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026acute;-GGACTTTGGCTTTCTATTGTGC-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e490 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAntoni et al. (\u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePtrUniqueR2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026acute;-CTTGGTGAATGGTGAAGATGG-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eToxA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eToxAscreeningF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026acute;-CCTCGTACTTCTTTTCAGCG-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e832 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAntoni et al. (\u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e),\u003c/p\u003e\n \u003cp\u003eMoolhuijzen et al. (\u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eToxAscreeningR1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026acute;-TGTAGAAGACAAGATTTTGA-3\u0026acute;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eMultiplex\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eToxA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTA51F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-GCGTTCTATCCTCGTACTTC-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e573 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAndrie et al. (\u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTA52R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-GCATTCTCCAATTTTCACG-3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eToxB\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTB71F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-GCTACTTGCTGTGGCTATC-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e232 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAndrie et al. (\u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e), Martinez et al. (\u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTB60R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-ACTAACAACGTCCTCCACTTTG-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eCHS-1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCHS-79F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-TGGGGCAAGGATGCTTGGAAGAAG-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e275 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCarbone \u0026amp; Kohn (\u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e), Andrie et al. (\u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCHS-354R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026prime;-TGGAAGAACCATCTGTGAGAGTTG-3\u0026prime;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVirulence of Nordic\u003c/strong\u003e \u003cstrong\u003ePtr\u003c/strong\u003e \u003cstrong\u003eisolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA subset of 25 Nordic \u003cem\u003ePtr\u003c/em\u003e isolates collected in 2021 was selected for virulence testing with a wheat differential set. Isolates were selected 1) to represent all countries where isolates were available from the 2021 season and 2) based on the PCR results so that both \u003cem\u003eToxA\u003c/em\u003e positive and negative fungal individuals from each country would be present in the subset. The isolates used in the virulence testing are marked in Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003eThe wheat differential set used in this study consisted of nine wheat genotypes (Table 2). Four genotypes, (\u0026lsquo;Salamouni\u0026rsquo;, \u0026lsquo;Glenlea\u0026rsquo;, \u0026lsquo;6B662\u0026rsquo; and \u0026lsquo;6B365\u0026rsquo;, kindly provided by Lise Nistrup J\u0026oslash;rgensen from Aarhus University, Denmark) have known sensitivities to ToxA, ToxB and ToxC (Lamari et al., \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). The remaining five genotypes consisted of Nordic spring wheat cultivars or breeding lines demonstrating variable susceptibility to tan spot from susceptible to resistant (\u0026lsquo;Wappu\u0026rsquo;, \u0026lsquo;Alarm\u0026rsquo;, \u0026lsquo;Demonstrant\u0026rsquo;, \u0026lsquo;BOR_17\u0026rsquo; and \u0026lsquo;BOR_19\u0026rsquo;). For these genotypes, data on the NE sensitivities and tan spot resistance were obtained in this study.\u003c/p\u003e\n\u003cp\u003ePlants were sown in 3.5 L plastic pots filled with W HS R80301 seedling peat mix (Kekkil\u0026auml; Professional, Vantaa, Finland). One seed of each of the nine wheat differentials was sown in each pot in a fixed order. Plants were grown in the greenhouse at 18 ℃/15 ℃ day/night, 16 h photoperiod, watered as needed, and fertilized once a week with Yara Ferticare (Yara International, Oslo, Norway).\u003c/p\u003e\n\u003cp\u003eApproximately two weeks after sowing, when the second leaf was fully expanded, plants were inoculated with \u003cem\u003ePtr\u003c/em\u003e spore and mycelium suspension. For inoculum, fungal isolates were grown on V8 agar in 12 h NUV/12 h dark at 18 ℃ for 3 weeks, 20 agar plates per isolate. Two millilitres of sterilized water was added on each agar plate and fungal colonies were scraped with a sterile glass spreader into falcon tubes. Inoculum was stirred gently and filtered through a sterile cheesecloth. As the sporulation rate was low in all isolates, the amount of mycelium in each sample was visually estimated to be approximately even among isolates via microscope. Just before inoculation, one drop of Tween 20 was added into each tube.\u003c/p\u003e\n\u003cp\u003eTwo hours before inoculation, misting was turned on in the greenhouse room and temperature raised from 18 to 20 ℃. Inoculation was started when the relative humidity reached approximately 100%. Pots were protected with plastic cones, and 3.6 mL inoculum per pot (0.4 mL per plant) was sprayed with 3 bar pressure using a modified paint sprayer. Plastic cones were removed after the inoculum was settled on plant leaves. Greenhouse ventilation and misting were switched off for the duration of inoculation and turned back on after. Lights were switched off during inoculation and switched back on after 24 h, to prevent leaf surfaces from drying. Misting was applied twice per day until observations were made: 2 h in the morning and 2 h in the evening, to maintain relative humidity of \u0026gt;\u0026thinsp;80%.\u003c/p\u003e\n\u003cp\u003eTable 2. Differential wheat genotypes used in the virulence testing trial. R = resistant, MR = moderately resistant, S = susceptible, - = insensitive, + = sensitive, NA = data not available or not known.\u003c/p\u003e\n\u003ctable id=\"Tabb\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eWheat genotype\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eToxA sensitivity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eToxB sensitivity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eToxC sensitivity\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eKnown resistance level\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSalamouni\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlenlea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6B662\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6B365\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWappu\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlarm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDemonstrant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBOR_17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBOR_19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eNA\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003eThe pots were arranged in four blocks of 26 pots in a randomized complete block design (RCBD), so that each isolate was present once in each of the four blocks. One negative control pot inoculated with sterile water and a drop of Tween 20 was included in each block.\u003c/p\u003e\n\u003cp\u003eSymptoms were evaluated 10 days after inoculation by using a scale from 1 to 5 described by Lamari and Bernier (\u003cspan class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eToxA and ToxB sensitivities in spring wheat\u003c/h2\u003e\n \u003cp\u003eA collection of 179 spring wheat genotypes (Supplementary Table\u0026nbsp;2), generated by the CResWheat project (Pre-breeding for Nordic climate resilient spring wheat), and 18 Nordic spring wheat landraces, received from NordGen, were tested for sensitivity to purified ToxA and ToxB effector proteins in a controlled greenhouse environment. The majority of the 179 CResWheat genotypes were registered cultivars and breeding lines of European plant breeding companies, but he collection also included 28 non-European genotypes, mostly bred by CIMMYT. Landraces were tested separately from other accessions for seed availability reasons. ToxA and ToxB effector sensitivities were tested in separate trials.\u003c/p\u003e\n \u003cp\u003eSeeds were sown in 1 L plastic pots filled with W HS R80301 seedling peat mix (Kekkil\u0026auml; Professional), two seeds per pot (a pot comprising one replicate). In each pot, one plant was treated while the second one was left untreated. Plants were grown at 18 ℃/15 ℃ day/night, 16 h photoperiod and watered as needed. Plants were fertilized once, two weeks after sowing, with Yara Ferticare (Yara International). Wheat accessions \u0026lsquo;Salamouni\u0026rsquo; (ToxA and ToxB insensitive), \u0026lsquo;BG261\u0026rsquo; (ToxA sensitive), \u0026lsquo;6B662\u0026rsquo; (ToxB sensitive) and \u0026lsquo;BR34\u0026rsquo; (ToxA insensitive) were used as controls in the experiments.\u003c/p\u003e\n \u003cp\u003eIn the trial setting for the 179 CResWheat genotypes, in addition to the four replicate pots of each genotype, 27 water control pots were included with a maximum of seven different genotypes sown in one pot (one seed per genotype), and the plants were treated with sterilized water. All pots were randomized according to the row-column experiment design using the CycDesigN -software (VSN International, Hemel Hempstead, UK) onto four tables in two different greenhouse rooms.\u003c/p\u003e\n \u003cp\u003eFor the 18 landraces, the trial setting was RCBD with five blocks, of which the fifth block included the water controls treated with sterilized water. Four replicates were treated with a necrotrophic effector.\u003c/p\u003e\n \u003cp\u003ePurified ToxA and ToxB effector proteins produced in \u003cem\u003eE. coli\u003c/em\u003e (See et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e) were kindly provided by Curtin University, Australia. Freeze-dried effector proteins were dissolved in sterilized water to obtain the concentration of 10 ng/\u0026micro;L for ToxA and 200 ng/\u0026micro;L for ToxB, and infiltrated with needleless 1 mL syringe into the fully opened second leaf of two-week-old seedlings. The infiltration site was marked using a non-toxic, waterproof pen. Symptoms were observed 5 to 7 days after infiltration and scored on a scale from 0 to 5 adopted from See et al. (\u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e), where 0\u0026thinsp;=\u0026thinsp;no visible symptoms, 1\u0026thinsp;=\u0026thinsp;mild chlorosis, 2\u0026thinsp;=\u0026thinsp;chlorosis, 3\u0026thinsp;=\u0026thinsp;very strong chlorosis, 4\u0026thinsp;=\u0026thinsp;strong chlorosis with necrosis, 5\u0026thinsp;=\u0026thinsp;complete necrosis with tissue collapse.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eField trials\u003c/h3\u003e\n\u003cp\u003eIn addition to ToxA and ToxB effector sensitivity, the 179 CResWheat spring wheat genotypes (Supplementary Table\u0026nbsp;2) were tested for their tan spot susceptibility under field conditions in 2022, 2023 and 2024 at two locations in Finland: Inkoo (60\u0026deg;04\u0026apos;02.1\u0026quot;N 23\u0026deg;53\u0026apos;21.2\u0026quot;E) and Jokioinen (60\u0026deg;48\u0026apos;57.9\u0026quot;N 23\u0026deg;28\u0026apos;30.2\u0026quot;E). The 18 Nordic landrace accessions were not included in the field trials for seed availability reasons. The Inkoo field had a natural infection through all the three years, whereas in Jokioinen, artificial inoculation and sprinkler irrigation were used to promote uniform tan spot infection across the trial.\u003c/p\u003e\n\u003cp\u003eIn Inkoo, wheat genotypes were sown in 2 m\u003csup\u003e2\u003c/sup\u003e plots arranged in RCBD with two replicates. Sowing dates were May 25th in 2022, May 22nd in 2023, and May 17th in 2024. Plots were fertilized in 2022 with YaraMila Y3 HIVEN, 560 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, in 2023 with YaraMila Y3, 450 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and in 2024 with Yara Mila Y2, 400 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Yara International). Herbicides were sprayed as needed in all three years. In 2022 and 2024, plots were also treated once with insecticides. Observations were made twice in 2022 at BBCH growth stages GS65\u0026ndash;GS71 and GS77\u0026ndash;GS85 (Meier, \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e), and once in 2023 and 2024 at GS65\u0026ndash;GS71.\u003c/p\u003e\n\u003cp\u003eIn Jokioinen, wheat genotypes were sown in hill plots arranged in RCBD with two replicates, 20 seeds per plot and distance between each hill plot being 30 cm. Sowing dates in Jokioinen were June 7th in 2022, June 5th in 2023, and May 29th in 2024. Plots were fertilized both in 2022 and 2023 with BeFert NPK, 370 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and in 2024 with Yara Mila Y2, 370 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Yara International). Herbicides were sprayed as needed at GS21\u0026ndash;GS30. Insecticides were sprayed each year, mainly against flea beetle (\u003cem\u003ePhyllotreta\u003c/em\u003e sp). Observations were made three three times every year at GS47\u0026ndash;GS61, GS65\u0026ndash;71 and GS77\u0026ndash;GS83.\u003c/p\u003e\n\u003cp\u003eInoculum for the Jokioinen field trial was produced during preceding winters in a greenhouse by spraying a susceptible spring wheat cultivar \u0026lsquo;Wanamo\u0026rsquo; at GS12\u0026ndash;GS13 with a mixture of five \u003cem\u003ePtr\u003c/em\u003e isolates representing the natural variation in Finland (marked in Supplementary Table 1). After two weeks the infected leaves were cut into pieces and dried at room temperature. In the field, artificial inoculation was then performed by distributing approximately 1 g of infected wheat leaf material at the base of each hill plot at GS12. Sprinkler irrigation was used to maintain continuous near 100% humidity in the canopy for 24 h after inoculation. After this, sprinkler irrigation was applied in the evenings to promote disease development, depending on weather conditions and precipitation.\u003c/p\u003e\n\u003cp\u003eDisease severity was assessed from the four uppermost leaves using a rating scale from 1 to 9, commonly used by Nordic breeders, where 1\u0026thinsp;=\u0026thinsp;no symptoms, 2\u0026thinsp;=\u0026thinsp;one lesion per 10 wheat individuals, 3\u0026thinsp;=\u0026thinsp;one lesion per each wheat individual, 4\u0026thinsp;=\u0026thinsp;two lesions per each wheat individual, 5\u0026thinsp;=\u0026thinsp;lesions start to merge and cover the leaves, 6\u0026thinsp;=\u0026thinsp;nethermost leaves are covered with lesions, 6\u0026thinsp;=\u0026thinsp;approximately half of the leaves are covered by lesions, 7\u0026thinsp;=\u0026thinsp;leaves are more diseased than healthy, 8\u0026thinsp;=\u0026thinsp;very little green tissue left, 9\u0026thinsp;=\u0026thinsp;leaves are completely necrotic.\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eThe randomization for each RCBD was conducted using Microsoft Excel (v16.89; Microsoft, 2024). A random number was generated for each genotype, within each block separately, using the `RAND` function. The genotypes were subsequently sorted based on these values to achieve randomization.\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed and figures drawn using R Statistical Software (v4.3.1 and v4.4.0; R Core Team, \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). In NE sensitivity testing data, arithmetic means across the four replicates were taken as descriptors of sensitivity or insensitivity of each wheat genotype. The mean value of 2.5 was used as a threshold for ToxA sensitivity, and the mean value of 1 for ToxB sensitivity (Corsi et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). From virulence testing data, medians of the four replicates were calculated for each isolate\u0026thinsp;+\u0026thinsp;wheat genotype combination, and used as the estimates for virulence. The effect of \u003cem\u003eToxA\u003c/em\u003e on the overall virulence of the \u003cem\u003ePtr\u003c/em\u003e isolates was estimated with Kruskal-Wallis rank sum test, using the data from the five Nordic wheat genotypes in the differential set.\u003c/p\u003e\n\u003cp\u003eIn the field data, each timepoint was tested separately for spatial row-column effects by using Cumulative Link Mixed Model (CLMM) provided by R package \u0026lsquo;ordinal\u0026rsquo; (v2023.12-4; Christensen, \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Adjustment for row and/or column effect was included in the model if the effect was significant. If there were multiple observations in a location in a year, the area under disease progress stairs (AUDPS) was calculated with the following equation (Simko \u0026amp; Piepho, \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e):\u003c/p\u003e\n\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/div\u003e\n\u003c/div\u003e\n\u003cp\u003eWhere \u003cem\u003eY\u003c/em\u003e is the disease assessment score, \u003cem\u003et\u003c/em\u003e is the observation timepoint and \u003cem\u003en\u003c/em\u003e is the total number of observations.\u003c/p\u003e\n\u003cp\u003eTo assess the impact of ToxA and ToxB sensitivities on tan spot susceptibility in the field, European and non-European wheat germplasm were analysed separately. This approach was necessary as the differences in genetic backgrounds between these two groups led to masking effects when treated as one dataset. The influence of ToxA and ToxB sensitivity in individual years was tested with Wilcoxon signed-rank test. To examine the effect of NE sensitivities across years, a linear mixed model was applied to the Jokioinen dataset using the R package \u0026lsquo;lme4\u0026rsquo; (Bates et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e), with year included as a random factor. Effect sizes for ToxA and ToxB sensitivities were estimated by extracting marginal R\u0026sup2; (R\u003csup\u003e2\u003c/sup\u003e\u003csub\u003em\u003c/sub\u003e) values from the model using the R package \u0026lsquo;MuMIn\u0026rsquo; (v1.48.4; Bartoń, \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e). Since the Inkoo dataset was not normally distributed, Nagelkerke\u0026rsquo;s pseudo-R\u0026sup2; (R\u003csup\u003e2\u003c/sup\u003e\u003csub\u003eN\u003c/sub\u003e) values were calculated using the R package \u0026lsquo;performance\u0026rsquo; (L\u0026uuml;decke et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eToxA\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eToxB\u003c/strong\u003e \u003cstrong\u003epresence in the Nordic\u003c/strong\u003e \u003cstrong\u003ePtr\u003c/strong\u003e \u003cstrong\u003epopulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ePtr\u003c/em\u003e isolate collection in years 2004\u0026ndash;2022 consisted of 217 isolates of which 178 (82%) originated from Finland. The most extensively sampled years were 2004 (\u003cem\u003en\u003c/em\u003e = 83), 2009 (\u003cem\u003en\u0026nbsp;\u003c/em\u003e=\u0026nbsp;23) and 2021 (\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 53). Based on the PCR screening, 64.5% of the isolates carried the \u003cem\u003eToxA\u0026nbsp;\u003c/em\u003egene (\u003cem\u003en\u003c/em\u003e = 140). The frequency of \u003cem\u003eToxA\u0026nbsp;\u003c/em\u003ein \u003cem\u003ePtr\u003c/em\u003e isolates in each year and country is presented in Table 3. \u003cem\u003eToxB\u0026nbsp;\u003c/em\u003ewas not detected in any of the isolates.\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003eTable 3. Percentages (%) of ToxA positive Pyrenophora tritici-repentis isolates based on PCR screening, and the total number (N) of isolates per year and country of origin.\u003c/div\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tabc\" border=\"1\"\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNorway\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDenmark\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSweden\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFinland\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEstonia\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLatvia\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYear\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% (\u003cem\u003eN\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% (\u003cem\u003eN\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% (\u003cem\u003eN\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% (\u003cem\u003eN\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% (\u003cem\u003eN\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e% (\u003cem\u003eN\u003c/em\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77.1 (83)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80.0 (10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.0 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.0 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.5 (8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2009\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73.9 (23)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.0 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.0 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61.5 (13)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.0 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.3 (6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.0 (45)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.0 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnknown\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.0 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.2 (11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100.0 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e26.7 (15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e71.9 (178)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e37.5 (8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.0 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eVirulence of Nordic\u003c/strong\u003e \u003cstrong\u003ePtr\u003c/strong\u003e \u003cstrong\u003eisolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVirulence scores of the 25 \u003cem\u003ePtr\u003c/em\u003e isolates tested with the wheat differential set indicated the presence of ToxC effector among the Nordic strains. The majority of the isolates (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21) induced a susceptible reaction including strong chlorosis and necrosis in the ToxC differential \u0026lsquo;6B365\u0026rsquo; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eVirulence scorings were not fully consistent with the results from PCR screening of \u003cem\u003eToxA\u003c/em\u003e and \u003cem\u003eToxB\u003c/em\u003e genes (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Three isolates lacking \u003cem\u003eToxA\u003c/em\u003e were virulent on ToxA sensitive differential \u0026lsquo;Glenlea\u0026rsquo; by inducing varying levels of necrosis, while four isolates carrying \u003cem\u003eToxA\u003c/em\u003e did not elicit notable necrosis on \u0026lsquo;Glenlea\u0026rsquo; (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). On the ToxB differential line \u0026lsquo;6B662\u0026rsquo;, four isolates that do not carry \u003cem\u003eToxB\u003c/em\u003e induced strong necrotic symptoms upon inoculation (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). Overall disease response on the five Nordic wheat genotypes in the differential set showed that \u003cem\u003eToxA-\u003c/em\u003epositive isolates were more virulent compared to \u003cem\u003eToxA\u003c/em\u003e negative ones (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0052). Intriguingly, the most susceptible wheat genotype was \u0026lsquo;Alarm\u0026rsquo;, despite being insensitive to both ToxA and ToxB based on NE infiltrations.\u003c/p\u003e\n\u003cp\u003eDefinitive conclusions on race classification were not drawn as the scorings were based more on the lesion type than the production of spreading necrosis or chlorosis. None of the isolates induced susceptible symptoms on \u0026lsquo;Salamouni\u0026rsquo; which is insensitive to all of the three \u003cem\u003ePtr\u003c/em\u003e NEs.\u003c/p\u003e\n\u003ch3\u003eToxA and ToxB sensitivities in spring wheat\u003c/h3\u003e\n\u003cp\u003eGreenhouse infiltrations with purified necrotrophic effectors revealed that 46.2% (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;91) of the 197 spring wheat genotypes were sensitive to ToxA (mean score\u0026thinsp;\u0026ge;\u0026thinsp;2.5), while 53.8% (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;106) were insensitive (mean score\u0026thinsp;\u0026lt;\u0026thinsp;2.5). Only reactions in classes 0 (no reaction) and 5 (full necrosis) were observed. Thirteen accessions showed mixed sensitivity to ToxA, as replicates of the same genotype displayed both full insensitivity and complete sensitivity.\u003c/p\u003e\n\u003cp\u003eIn response to ToxB, 23.9% (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;47) of the accessions were sensitive (mean score\u0026thinsp;\u0026ge;\u0026thinsp;1), while 76.1% (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;150) were insensitive (mean score\u0026thinsp;\u0026lt;\u0026thinsp;1). Observed reactions ranged from 0 to 3, with none of the accessions exhibiting necrotic symptoms. Ten accessions displayed mixed sensitivity to ToxB. The frequencies of ToxA and ToxB sensitivity only, and in combinations across wheat genotypes are illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eField trials\u003c/h2\u003e\n \u003cp\u003eTan spot disease incidence was the highest in 2022 and the lowest in 2023 in both Inkoo (natural infection, rainfed) and Jokioinen (artificial inoculation, sprinkler irrigation). Majority of the wheat genotypes were highly susceptible to tan spot. Spearman correlation between disease severity in Inkoo and Jokioinen was 0.56 in 2022, 0.34 in 2023 and 0.54 in 2024 (all \u003cem\u003ep\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\n \u003cp\u003eIn Inkoo, ToxA sensitivity had no effect on the severity of tan spot infection either among European or non-European subset (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). In contrast, ToxB-sensitive genotypes in the European subset consistently exhibited lower tan spot severity compared to ToxB-insensitive ones (R\u003csup\u003e2\u003c/sup\u003e\u003csub\u003eN\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.142, 0.068 and 0.054 in 2022, 2023 and 2024, respectively) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). In the non-European subset, the difference between ToxB-sensitive and insensitive genotypes drew close to statistical significance only in 2022 (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e\n \u003cp\u003eIn Jokioinen, wheat genotypes carrying ToxA-sensitivity had higher AUDPS over the three years compared to ToxA-insensitive genotypes in European subset (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0274, R\u003csup\u003e2\u003c/sup\u003e\u003csub\u003em\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.0150). When the three years were analysed individually, the difference was statistically significant only in 2024 in the European subset (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed). ToxB sensitive wheat genotypes had lower AUDPS over the years in the European subset (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0042, R\u003csup\u003e2\u003c/sup\u003e\u003csub\u003em\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.0252), and higher in the non-European subset (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0006, R\u003csup\u003e2\u003c/sup\u003e\u003csub\u003em\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1934), but the statistical significance varied between years (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec and Fig. 6d).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results presented here provide the first comprehensive overview of the importance of known \u003cem\u003ePtr\u003c/em\u003e necrotrophic effectors ToxA, ToxB and ToxC in tan spot epidemics in the Nordic countries. Despite the high prevalence of \u003cem\u003eToxA\u003c/em\u003e in the tested \u003cem\u003ePtr\u003c/em\u003e isolates and the presence of both ToxA and ToxB sensitivities in the tested spring wheat material, the effect of NE sensitivity was negligible on the observed tan spot epidemic under field conditions. ToxC presence in the Nordic \u003cem\u003ePtr\u003c/em\u003e isolates was studied for the first time and the results suggest ToxC to be frequently produced by local isolates.\u003c/p\u003e \u003cp\u003eAmong the \u003cem\u003ePtr\u003c/em\u003e isolates screened for the presence of the effector genes \u003cem\u003eToxA\u003c/em\u003e and \u003cem\u003eToxB\u003c/em\u003e, the Finnish samples yielded the most extensive dataset. Despite the samples from other countries were not as numerous, it can be concluded that \u003cem\u003eToxA\u003c/em\u003e is present at varying frequencies in all sampled \u003cem\u003ePtr\u003c/em\u003e populations. From all the tested isolates, 65% carried the \u003cem\u003eToxA\u003c/em\u003e gene. The results align with earlier observations of \u003cem\u003eToxA\u003c/em\u003e presence in the Baltics and Denmark, although different estimates on the gene frequency have been obtained depending on the fungal collections studied (Abdullah et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e; Justesen et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kaneps et al., 2022). The \u003cem\u003eToxA\u003c/em\u003e gene was found present in isolates from all sampled years, although a slight decreasing trend was observed in Finnish samples, from approximately 80% in 2004\u0026ndash;2005 to 60% in 2021. The result indicates, however, a likely benefit of \u003cem\u003eToxA\u003c/em\u003e for \u003cem\u003ePtr\u003c/em\u003e as the gene\u0026rsquo;s frequency in the fungal population has stayed relatively high over the last 20 years. The absence of \u003cem\u003eToxB\u003c/em\u003e in all screened isolates aligns well with previously reported results from Europe (Abdullah et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e; Justesen et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kaneps et al., 2022). \u003cem\u003eToxB\u003c/em\u003e has been found prevalent predominantly in \u003cem\u003ePtr\u003c/em\u003e isolates originating from Northern Africa, near the wheat center of origin (Benslimane, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kamel et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Laribi et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eContrasting with the PCR results, three isolates lacking the \u003cem\u003eToxA\u003c/em\u003e gene induced necrosis on ToxA differential wheat \u0026lsquo;Glenlea\u0026rsquo; in the virulence test of 25 \u003cem\u003ePtr\u003c/em\u003e isolates. Furthermore, four isolates that carried \u003cem\u003eToxA\u003c/em\u003e did not elicit notable necrosis on \u0026lsquo;Glenlea\u0026rsquo;. Previously, Abdullah et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e) found that \u003cem\u003ePtr\u003c/em\u003e isolates lacking \u003cem\u003eToxA\u003c/em\u003e but inducing necrosis on \u0026lsquo;Glenlea\u0026rsquo; were quite common in Lithuania. Similar observation was also reported in the Tunisian \u003cem\u003ePtr\u003c/em\u003e population although these isolates were collected from durum wheat (Laribi et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the total absence of the \u003cem\u003eToxB\u003c/em\u003e gene in the PCR analysis, four of the 25 isolates elicited susceptible reaction on the ToxB differential wheat \u0026lsquo;6B662\u0026rsquo;. However, as the observed symptoms were more necrotic than chlorotic, the disease phenotype warrants further investigation. By using knockout strains, Guo et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) previously demonstrated that \u003cem\u003ePtr\u003c/em\u003e isolates lacking both \u003cem\u003eToxA\u003c/em\u003e and \u003cem\u003eToxB\u003c/em\u003e genes were still virulent on various bread and durum wheat genotypes. Altogether, these results indicate the possibility of additional NE\u0026ndash;host sensitivity locus interactions as well as the presence of other factors than NEs affecting the overall aggressiveness and virulence of these \u0026lsquo;atypical\u0026rsquo; isolates (Abdullah et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e; Ali et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Andrie et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Laribi et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eToxC differential \u0026lsquo;6B365\u0026rsquo; reacted to the majority of the isolates (21/25) with strong chlorosis rapidly progressing to necrosis, indicating high prevalence of ToxC producing isolates in the Nordic \u003cem\u003ePtr\u003c/em\u003e population. This result agrees with the previous studies showing ToxA and ToxC producing \u0026lsquo;race 1\u0026rsquo; to be highly common and the dominant one in most of the wheat growing areas (Abdullah et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Aboukhaddour et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Gamba et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lepoint et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; MacLean et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; See et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The high frequency of ToxC-producing isolates in the Nordic \u003cem\u003ePtr\u003c/em\u003e population and the significant role of ToxC sensitivity locus \u003cem\u003eTsc1\u003c/em\u003e in tan sport resistance in different genetic backgrounds (Liu et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Peters Haugrud et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) highlights the importance for Nordic breeders to eliminate \u003cem\u003eTsc1\u003c/em\u003e-carrying wheat material from spring wheat breeding programs.\u003c/p\u003e \u003cp\u003eOn the host side, 47% of the 197 tested spring wheat accessions exhibited ToxA sensitivity, indicating ToxA sensitivity gene \u003cem\u003eTsn1\u003c/em\u003e to be common. A similar level of ToxA sensitivity was observed by Ruud et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) in spring wheat accessions predominantly of Nordic origin. Another screening from Europe, focusing mostly on winter wheat, found ToxA sensitivity to be rare (Downie et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, a quarter of the ToxA-sensitive accessions in the study of Downie et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) was spring wheat despite representing only 6% of the genotypes. \u003cem\u003eTsn1\u003c/em\u003e might thus be more common among spring wheat compared to winter wheat. It is also possible that spring wheat accessions adapted to Nordic climate share a high level of genetic similarity and common ancestors, resulting in the enrichment of \u003cem\u003eTsn1\u003c/em\u003e in the germplasm.\u003c/p\u003e \u003cp\u003eDespite the prevalence of ToxA sensitivity among spring wheat genotypes, as well as the high frequency of \u003cem\u003eToxA\u003c/em\u003e gene in the fungal population, ToxA sensitivity seems to play a minor role in the epidemic outcome in the field among the spring wheat genotypes of European origin. In the European spring wheat germplasm, ToxA sensitivity explained only about 1.5% of the tan spot susceptibility in the Jokioinen experiment, and in Inkoo, the effect of ToxA was not detected. In contrast, ToxA-sensitive genotypes of non-European origin had higher tan spot severity under natural infection, suggesting that \u003cem\u003eToxA\u003c/em\u003e was present in the local \u003cem\u003ePtr\u003c/em\u003e population in Inkoo. ToxA-\u003cem\u003eTsn1\u003c/em\u003e interaction has been found to have a varying effect on tan spot susceptibility depending on the genetic background of the host, and also of the pathogen (Faris et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Friesen et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Peters Haugrud et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; See et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Virdi et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wei et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These studies have suggested ToxA-\u003cem\u003eTsn1\u003c/em\u003e interaction to include additional regulatory components, from either on the host or the pathogen side, affecting the disease outcome. The effect of ToxA has also been found to be more pronounced at the seedling stage (Dinglasan et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Taylor et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), which could also explain the lack of strong association here, as the plants were assessed for tan spot at the adult stage.\u003c/p\u003e \u003cp\u003eSurprisingly, 24% of the screened spring wheat accessions here were found to be sensitive to ToxB. Previously, ToxB sensitivity has been reported to be rare in European wheat material (Corsi et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). ToxB sensitivity is highly heritable (Corsi et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) as it is governed by a single gene, \u003cem\u003eTsc2\u003c/em\u003e (Abeysekara et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Wheat genotypes screened here might share a common relative or relatives carrying \u003cem\u003eTsc2\u003c/em\u003e, affecting the relatively high frequency of ToxB sensitivity.\u003c/p\u003e \u003cp\u003eInterestingly, ToxB sensitivity had a counter-intuitive effect on tan spot severity among the European spring wheat genotypes. ToxB-sensitive accessions of European origin were less diseased compared to ToxB-insensitive ones, while the non-European genotypes with ToxB sensitivity displayed higher tan spot scorings in Jokioinen, even though \u003cem\u003eToxB\u003c/em\u003e was absent in the \u003cem\u003ePtr\u003c/em\u003e isolates used in the inoculation of the field. The results might suggest that the ToxB-sensitive wheat accessions in the European subset carry an unknown source of tan spot resistance, possibly linked to the \u003cem\u003eTsc2\u003c/em\u003e locus. Detailed genetic studies would be necessary to confirm this assumption. Therefore, before removing ToxB-sensitive genotypes from their breeding material, Nordic breeders should take this possibility into account.\u003c/p\u003e \u003cp\u003eFactors other than ToxA and ToxB seem to have a major role in tan spot epidemics in the Nordics. Tan spot resistance has a quantitative genetic makeup and many resistance QTLs in wheat have been found that are not connected to the known NE\u0026ndash;host sensitivity locus interactions (Faris et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Most probably there are also numerous effectors on the fungal side, awaiting to be detected. Recently, Rawlinson et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) described two novel secondary metabolites of \u003cem\u003ePtr\u003c/em\u003e, ToxE1 and ToxE2, that induce chlorosis on wheat in a genotype-specific manner.\u003c/p\u003e \u003cp\u003eIn conclusion, our comprehensive analysis and testing of both the pathogen and the host revealed that \u003cem\u003ePtr\u003c/em\u003e NEs have a rather small role in the tan spot epidemics in the Nordic region. Breeding against effector sensitivity is potentially simple, due to straight-forward testing, and there is no reason to avoid that. However, our results indicate the role of other factors than the known NE\u0026ndash;sensitivity gene interactions in disease resistance of tan spot in the Nordics. Hence, resistance testing with relevant isolates and the use of naturally infected field nurseries in epidemic areas is still essential for breeding progress in this region.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to the ones mentioned in the text, we thank the CResWheat project partners, especially the Nordic breeders at Boreal Plant Breeding Ltd, Graminor AS, Lantmännen, Nordic Seed A/S and Sejet Plant Breeding, for providing spring wheat germplasm for the study. Special thanks to Tarja Niemelä, Boreal Plant Breeding Ltd, for helping with the field observations in Inkoo. Warm thanks to Senja Tuominen and Auli Kedonperä, Luke, and Elyce Iagallo, Curtin University, for their highly skilled technical assistance. Thanks to Daniel Fischer and Lauri Jauhiainen, Luke, for their helpful insights with the trial design and statistical analyses.\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Nordic Council of Ministers (NMR), as in-kind from private partners in the CResWheat project, and by The Finnish Association of Academic Agronomists/Henrik and Ellen Tornberg trust fund (grant numbers 20220050 and 20230045).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthor contributions\u003c/em\u003e:\u003c/p\u003e\n\u003cp\u003eAnnika Johansson, Satu Latvala and Marja Jalli designed the experiments. Annika Johansson collected the data. Annika Johansson and Petteri Karisto performed data analyses. Pao Theen See provided essential material, and instructions for the experiments and interpretation of the data. Morten Lillemo provided advice on the data analysis and results interpretation. Pernille Bjarup Hansen supplied breeding material. Annika Johansson wrote the first draft of the manuscript. All authors contributed in reviewing and editing the manuscript. Marja Jalli and Petteri Karisto supervised the work. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbdullah, S., Sehgal, S. K., Ali, S., Liatukas, Z., Ittu, M., \u0026amp; Kaur, N. (2017a). Characterization of Pyrenophora tritici-repentis (tan spot of wheat) races in Baltic States and Romania. \u003cem\u003ePlant Pathology Journal, 33\u003c/em\u003e(2), 133\u0026ndash;139. https://doi.org/10.5423/PPJ.OA.10.2016.0214\u003c/li\u003e\n \u003cli\u003eAbdullah, S., Sehgal, S. K., \u0026amp; Ali, S. (2017b). Race Diversity of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e in South Dakota and Response of Predominant Wheat Cultivars to Tan Spot. \u003cem\u003eJournal of Plant Pathology \u0026amp; Microbiology, 8\u003c/em\u003e(5), 409. https://doi.org/10.4172/2157-7471.1000409\u003c/li\u003e\n \u003cli\u003eAbeysekara, N. S., Friesen, T. L., Liu, Z., McClean, P. E., \u0026amp; Faris, J. D. (2010). Marker Development and Saturation Mapping of the Tan Spot Ptr Toxb Sensitivity Locus \u003cem\u003eTsc2\u003c/em\u003e in Hexaploid Wheat. \u003cem\u003ePlant Genome, 3\u003c/em\u003e(3), 179\u0026ndash;189. https://doi.org/10.3835/plantgenome2010.07.0017\u003c/li\u003e\n \u003cli\u003eAboukhaddour, R., Turkington, T. K., \u0026amp; Strelkov, S. E. (2013). Race structure of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e (tan spot of wheat) in Alberta, Canada. \u003cem\u003eCanadian Journal of Plant Pathology, 35\u003c/em\u003e(2), 256\u0026ndash;268. https://doi.org/10.1080/07060661.2013.782470\u003c/li\u003e\n \u003cli\u003eAli, S., Gurung, S., \u0026amp; Adhikari, T. B. (2010). Identification and characterization of novel isolates of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e from Arkansas. \u003cem\u003ePlant Disease, 94\u003c/em\u003e(2), 229\u0026ndash;235. https://doi.org/10.1094/PDIS-94-2-0229\u003c/li\u003e\n \u003cli\u003eAndrie, R., Pandelova, I., \u0026amp; Ciuffetti, L. M. (2007). Combination of phenotypic and genotypic characterization strengthens \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e race identification. \u003cem\u003ePhytopathology, 97\u003c/em\u003e(6), 694\u0026ndash;701. https://doi.org/10.1094/PHYTO-97-6-0694\u003c/li\u003e\n \u003cli\u003eAntoni, E. A., Rybak, K., Tucker, M. P., Hane, J. K., Solomon, P. S., Drenth, A., Shankar, M., \u0026amp; Oliver, R. P. (2010). Ubiquity of ToxA and absence of ToxB in Australian populations of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e. \u003cem\u003eAustralasian Plant Pathology, 39\u003c/em\u003e(1), 63\u0026ndash;68. https://doi.org/10.1071/AP09056\u003c/li\u003e\n \u003cli\u003eBallance, G. M., Lamari, L., \u0026amp; Bernier, C. C. (1989). Purification and characterization of a host-selective necrosis toxin from \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e. \u003cem\u003ePhysiological and Molecular Plant Pathology, 35\u003c/em\u003e(3), 203\u0026ndash;213. https://doi.org/10.1016/0885-5765(89)90051-9\u003c/li\u003e\n \u003cli\u003eBartoń, K. (2024). MuMIn: Multi-Model Inference. R package version 1.48.4. Retrieved from https://CRAN.R-project.org/package=MuMIn\u003c/li\u003e\n \u003cli\u003eBates, D., M\u0026auml;chler, M., Bolker, B., \u0026amp; Walker, S. (2015). Fitting linear mixed-effects models using lme4. \u003cem\u003eJournal of Statistical Software, 67\u003c/em\u003e(1), 1\u0026ndash;48. https://doi.org/10.18637/jss.v067.i01\u003c/li\u003e\n \u003cli\u003eBenslimane, H. (2018). Virulence phenotyping and molecular characterization of a new virulence type of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e, the causal agent of tan spot. \u003cem\u003ePlant Pathology Journal, 34\u003c/em\u003e(2), 139\u0026ndash;142. https://doi.org/10.5423/PPJ.NT.07.2017.0150\u003c/li\u003e\n \u003cli\u003eBhathal, J. S., Loughman, R., \u0026amp; Speijers, J. (2003). Yield reduction in wheat in relation to leaf disease from yellow (tan) spot and septoria nodorum blotch. \u003cem\u003eEuropean Journal of Plant Pathology, 109\u003c/em\u003e, 435\u0026ndash;443. https://doi.org/10.1023/A:1024277420773\u003c/li\u003e\n \u003cli\u003eCarbone, I., \u0026amp; Kohn, L. M. (1999). A method for designing primer sets for speciation studies in filamentous ascomycetes. \u003cem\u003eMycologia, 91\u003c/em\u003e(3), 553\u0026ndash;556. https://doi.org/10.2307/3761358\u003c/li\u003e\n \u003cli\u003eChristensen, R. (2023). \u003cem\u003eordinal\u0026mdash;Regression Models for Ordinal Data\u003c/em\u003e. R package version 2023.12-4.1, https://CRAN.R-project.org/package=ordinal\u003c/li\u003e\n \u003cli\u003eCorsi, B., Percival-Alwyn, L., Downie, R. C., Venturini, L., Iagallo, E. M., Campos Mantello, C., McCormick-Barnes, C., See, P. T., Oliver, R. P., Moffat, C. S., \u0026amp; Cockram, J. (2020). Genetic analysis of wheat sensitivity to the ToxB fungal effector from \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e, the causal agent of tan spot. \u003cem\u003eTheoretical and Applied Genetics 133\u003c/em\u003e(3), 935\u0026ndash;950. https://doi.org/10.1007/s00122-019-03517-8\u003c/li\u003e\n \u003cli\u003eDinglasan, E. G., Godwin, I. D., Phan, H. T. T., Tan, K.-C., Platz, G. J., \u0026amp; Hickey, L. T. (2018). Vavilov wheat accessions provide useful sources of resistance to tan spot (syn. yellow spot) of wheat. \u003cem\u003ePlant Pathology, 67\u003c/em\u003e, 1076\u0026ndash;1087. https://doi.org/10.1111/ppa.12822\u003c/li\u003e\n \u003cli\u003eDownie, R. C., Bouvet, L., Furuki, E., Gosman, N., Gardner, K. A., Mackay, I. J., Campos Mantello, C., Mellers, G., Phan, H. T. T., Rose, G. A., Tan, K.-C., Oliver, R. P., \u0026amp; Cockram, J. (2018). Assessing European Wheat Sensitivities to \u003cem\u003eParastagonospora nodorum\u003c/em\u003e Necrotrophic Effectors and Fine-Mapping the Snn3-B1 Locus Conferring Sensitivity to the Effector SnTox3. \u003cem\u003eFrontiers in Plant Science, 9\u003c/em\u003e, 881. https://doi.org/10.3389/fpls.2018.00881\u003c/li\u003e\n \u003cli\u003eEffertz, R. J., Meinhardt, S. W., Anderson, J. A., Jordahl, J. G., \u0026amp; Francl, L. J. (2002). Identification of a chlorosis-inducing toxin from \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e and the chromosomal location of an insensitivity locus in wheat. \u003cem\u003ePhytopathology, 92\u003c/em\u003e(5), 527\u0026ndash;533. https://doi.org/10.1094/PHYTO.2002.92.5.527\u003c/li\u003e\n \u003cli\u003eFaris, J. D., Abeysekara, N. S., McClean, P. E., Xu, S. S., \u0026amp; Friesen, T. L. (2012). Tan spot susceptibility governed by the \u003cem\u003eTsn1\u003c/em\u003e locus and race-nonspecific resistance QTL in a population derived from the wheat lines Salamouni and Katepwa. \u003cem\u003eMolecular Breeding, 30\u003c/em\u003e, 1669\u0026ndash;1678. https://doi.org/10.1007/s11032-012-9750-7\u003c/li\u003e\n \u003cli\u003eFaris, J. D., Anderson, J. A., Francl, L. J., \u0026amp; Jordahl, J. G. (1996). Chromosomal location of a gene conditioning insensitivity in wheat to a necrosis-inducing culture filtrate from \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e. \u003cem\u003ePhytopathology, 8\u003c/em\u003e(5), 459\u0026ndash;463. https://doi.org/10.1094/Phyto-86-459\u003c/li\u003e\n \u003cli\u003eFaris, J. D., Liu, Z., \u0026amp; Xu, S. S. (2013). Genetics of tan spot resistance in wheat. \u003cem\u003eTheoretical and Applied Genetics, 126\u003c/em\u003e, 2197\u0026ndash;2217. https://doi.org/10.1007/s00122-013-2157-y\u003c/li\u003e\n \u003cli\u003eFaris, J. D., Zhang, Z., Lu, H., Lu, S., Reddy, L., Cloutier, S., Fellers, J. P., Meinhardt, S. W., Rasmussen, J. B., Xu, S. S., Oliver, R. P., Simons, K. J., \u0026amp; Friesen, T. L. (2010). A unique wheat disease resistance-like gene governs effector-triggered susceptibility to necrotrophic pathogens. \u003cem\u003eProceedings of the National Academy of Sciences USA, 107\u003c/em\u003e(30), 13544\u0026ndash;13549. https://doi.org/10.1073/pnas.1004090107\u003c/li\u003e\n \u003cli\u003eFlor, H. H. (1956). The complementary systems in flax and flax rust.\u0026nbsp;\u003cem\u003eAdvances in Genetics, 8\u003c/em\u003e, 29\u0026ndash;54.\u003cbr\u003ehttps://doi.org/10.1016/S0065-2660(08)60498-8\u003c/li\u003e\n \u003cli\u003eFriesen, T. L., \u0026amp; Faris, J. D. (2004). Molecular mapping of resistance to \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e race 5 and sensitivity to Ptr ToxB in wheat. \u003cem\u003eTheoretical and Applied Genetics, 109\u003c/em\u003e(3), 464\u0026ndash;471. https://doi.org/10.1007/s00122-004-1678-9\u003c/li\u003e\n \u003cli\u003eFriesen, T. L., \u0026amp; Faris, J. D. (2010). Characterization of the wheat-\u003cem\u003eStagonospora nodorum\u003c/em\u003e disease system: What is the molecular basis of this quantitative necrotrophic disease interaction? \u003cem\u003eCanadian Journal of Plant Pathology, 32\u003c/em\u003e(1), 20\u0026ndash;28. https://doi.org/10.1080/07060661003620896\u003c/li\u003e\n \u003cli\u003eFriesen, T. L., Ali, S., Kianian, S., Francl, L. J., \u0026amp; Rasmussen, J. B. (2003). Role of host sensitivity to Ptr ToxA in the development of tan spot of wheat. \u003cem\u003ePhytopathology, 93\u003c/em\u003e, 397\u0026ndash;401. https://doi.org/10.1094/PHYTO.2003.93.4.397\u003c/li\u003e\n \u003cli\u003eGamba, F. M., Strelkov, S. E., \u0026amp; Lamari, L. (2012). Virulence of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e in the southern cone region of South America. \u003cem\u003eCanadian Journal of Plant Pathology\u003c/em\u003e, \u003cem\u003e34\u003c/em\u003e(4), 545\u0026ndash;550. https://doi.org/10.1080/07060661.2012.695750\u003c/li\u003e\n \u003cli\u003eGuo, J., Shi, G., \u0026amp; Liu, Z. (2018). Characterizing virulence of the \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e isolates lacking both \u003cem\u003eToxA\u003c/em\u003e and \u003cem\u003eToxB\u003c/em\u003e genes. \u003cem\u003ePathogens, 7\u003c/em\u003e(3), 74. https://doi.org/10.3390/pathogens7030074\u003c/li\u003e\n \u003cli\u003eJalli, M., Kaseva, J., Andersson, B., Ficke, A., Nistrup-J\u0026oslash;rgensen, L., Ronis, A., Kaukoranta, T., \u0026Oslash;rum, J.-E., \u0026amp; Djurle, A. (2020). Yield increases due to fungicide control of leaf blotch diseases in wheat and barley as a basis for IPM decision-making in the Nordic-Baltic region. \u003cem\u003eEuropean Journal of Plant Pathology, 158\u003c/em\u003e, 315\u0026ndash;333. https://doi.org/10.1007/s10658-020-02075-w\u003c/li\u003e\n \u003cli\u003eJustesen, A. F., Corsi, B., Ficke, A., et al. (2021). Hidden in plain sight: A molecular field survey of three wheat leaf blotch fungal diseases in North-Western Europe shows co-infection is widespread. \u003cem\u003eEuropean Journal of Plant Pathology, 160\u003c/em\u003e, 949\u0026ndash;962. https://doi.org/10.1007/s10658-021-02298-5\u003c/li\u003e\n \u003cli\u003eKamel, S., Cherif, M., Hafez, M., Despins, T., \u0026amp; Aboukhaddour, R. (2019). \u003cem\u003ePyrenophora tritici\u0026ndash;repentis\u003c/em\u003e in Tunisia: Race Structure and Effector Genes. \u003cem\u003eFrontiers in Plant Science, 10\u003c/em\u003e, 1562. https://doi.org/10.3389/fpls.2019.01562\u003c/li\u003e\n \u003cli\u003eKaņeps, J., Moročko-Bičevska, I., Bankina, B., et al. (2022). Diversity in morphotypes and necrotrophic effectors of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e strains in Latvia and Belarus. \u003cem\u003eCereal Research Communications, 50\u003c/em\u003e, 1037\u0026ndash;1043. https://doi.org/10.1007/s42976-022-00255-4\u003c/li\u003e\n \u003cli\u003eKauppi, K., Rajala, A., Huusela, E., Kaseva, J., Ruuttunen, P., Jalli, H., Alakukku, L., \u0026amp; Jalli, M. (2021). Impact of pests on cereal grain and nutrient yield in boreal growing conditions. \u003cem\u003eAgronomy, 11\u003c/em\u003e(3), 592. https://doi.org/10.3390/agronomy11030592\u003c/li\u003e\n \u003cli\u003eLamari, L., \u0026amp; Bernier, C. C. (1989). Virulence of isolates of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e on 11 wheat cultivars and cytology of the differential host reactions. \u003cem\u003eCanadian Journal of Plant Pathology\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e, 284\u0026ndash;290. https://doi.org/10.1080/07060668909501114\u003c/li\u003e\n \u003cli\u003eLamari, L., Sayoud, R., Boulif, M., \u0026amp; Bernier, C. C. (1995). Identification of a new race in \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e: Implications for the current pathotype classification system. \u003cem\u003eCanadian Journal of Plant Pathology\u003c/em\u003e, \u003cem\u003e17\u003c/em\u003e(4), 312\u0026ndash;318. https://doi.org/10.1080/07060669509500668\u003c/li\u003e\n \u003cli\u003eLamari, L., Strelkov, S. E., Yahyaoui, A., Orabi, J., \u0026amp; Smith, R. B. (2003). The identification of two new races of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e from the host center of diversity confirms a one-to-one relationship in tan spot of wheat. \u003cem\u003ePhytopathology, 93\u003c/em\u003e(4), 391\u0026ndash;396. https://doi.org/10.1094/PHYTO.2003.93.4.391\u003c/li\u003e\n \u003cli\u003eLaribi, M., Akhavan, A., Ben M\u0026rsquo;Barek, S., Yahyaoui, A. H., Strelkov, S. E., \u0026amp; Sassi, K. (2022). Characterization of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e in Tunisia and comparison with a global pathogen population. \u003cem\u003ePlant Disease, 106\u003c/em\u003e(2), 464\u0026ndash;474. https://doi.org/10.1094/PDIS-04-21-0763-RE\u003c/li\u003e\n \u003cli\u003eLepoint, P., Renard, M. E., Legr\u0026egrave;ve, A., Duveiller, E., \u0026amp; Maraite, H. (2010). Genetic diversity of the mating type and toxin production genes in \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e. \u003cem\u003ePhytopathology, 100\u003c/em\u003e(5), 474\u0026ndash;483. https://doi.org/10.1094/PHYTO-100-5-0474\u003c/li\u003e\n \u003cli\u003eLiu, Y., Salsman, E., Wang, R., et al. (2020). Meta-QTL analysis of tan spot resistance in wheat. \u003cem\u003eTheoretical and Applied Genetics, 133\u003c/em\u003e, 2363\u0026ndash;2375. https://doi.org/10.1007/s00122-020-03604-1\u003c/li\u003e\n \u003cli\u003eLiu, Z., Zurn, J. D., Kariyawasam, G., Faris, J. D., Shi, G., Hansen, J., Rasmussen, J. B., \u0026amp; Acevedo, M. (2017). Inverse gene-for-gene interactions contribute additively to tan spot susceptibility in wheat. \u003cem\u003eTheoretical and Applied Genetics\u003c/em\u003e, \u003cem\u003e130\u003c/em\u003e(6), 1267\u0026ndash;1276. https://doi.org/10.1007/s00122-017-2886-4\u003c/li\u003e\n \u003cli\u003eL\u0026uuml;decke, D., Ben-Shachar, M. S., Patil, I., Waggoner, P., \u0026amp; Makowski, D. (2021). performance: An R package for assessment, comparison, and testing of statistical models. \u003cem\u003eJournal of Open Source Software, 6\u003c/em\u003e(60), 3139. https://doi.org/10.21105/joss.03139\u003c/li\u003e\n \u003cli\u003eLUKE (2024). Retrieved from https://statdb.luke.fi/PxWeb/pxweb/fi/LUKE/LUKE__02%20Maatalous__04%20Tuotanto__14%\u003cbr\u003e20Satotilasto/01a_Viljelykasvien_sato_mk.px/table/tableViewLayout2/.\u003c/li\u003e\n \u003cli\u003eMacLean, D. E., Aboukhaddour, R., Tran, V. A., Askarian, H., Strelkov, S. E., Turkington, T. K., \u0026amp; Kutcher, H. R. (2017). Race characterization of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e and sensitivity to propiconazole and pyraclostrobin fungicides. \u003cem\u003eCanadian Journal of Plant Pathology\u003c/em\u003e, \u003cem\u003e39\u003c/em\u003e(4), 433\u0026ndash;443. https://doi.org/10.1080/07060661.2017.1387178\u003c/li\u003e\n \u003cli\u003eMartinez, J. P., Oesch, N. W., \u0026amp; Ciuffetti, L. M. (2004). Characterization of the multiple-copy host-selective toxin gene, \u003cem\u003eToxB\u003c/em\u003e, in pathogenic and nonpathogenic isolates of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e. \u003cem\u003eMolecular Plant-Microbe Interactions, 17\u003c/em\u003e, 467\u0026ndash;474. https://doi.org/10.1094/MPMI.2004.17.5.467\u003c/li\u003e\n \u003cli\u003eMartinez, J. P., Ottum, S. A., Ali, S., Francl, L. J., \u0026amp; Ciuffetti, L. M. (2001). Characterization of the \u003cem\u003eToxB\u003c/em\u003e gene from \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e. \u003cem\u003eMolecular Plant-Microbe Interactions\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(5), 675\u0026ndash;677. https://doi.org/10.1094/MPMI.2001.14.5.675\u003c/li\u003e\n \u003cli\u003eMeier, U. (1997). \u003cem\u003eGrowth stages of mono- and dicotyledonous plants: BBCH-Monograph\u003c/em\u003e. Blackwell Wissenschafts-Verlag.\u003c/li\u003e\n \u003cli\u003eMicrosoft Corporation. (2024). Microsoft Excel. https://office.microsoft.com/excel\u003c/li\u003e\n \u003cli\u003eMoolhuijzen, P. M., See, P. T., Oliver, R. P., \u0026amp; Moffat, C. S. (2018). Genomic distribution of a novel \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e ToxA insertion element.\u0026nbsp;\u003cem\u003ePLoS ONE 13\u003c/em\u003e(10), e0206586.\u003cbr\u003ehttps://doi.org/10.1371/journal.pone.0206586\u003c/li\u003e\n \u003cli\u003eOrolaza, N. P., Lamari, L., \u0026amp; Ballance, G. M. (1995). Evidence of a host-specific chlorosis toxin from \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e, the causal agent of tan spot of wheat.\u0026nbsp;\u003cem\u003ePhytopathology, 85\u003c/em\u003e(10), 1282\u0026ndash;1287.\u003cbr\u003ehttps://doi.org/10.1094/Phyto-85-1282\u003c/li\u003e\n \u003cli\u003ePeters Haugrud, A. R. P., Shi, G., Seneviratne, S., Running, K. L., Zhang, Z., Singh, G., et al. (2023). Genome-wide association mapping of resistance to the foliar diseases septoria nodorum blotch and tan spot in a global winter wheat collection. \u003cem\u003eMolecular Breeding, 43\u003c/em\u003e(7), 54. https://doi.org/10.1007/s11032-023-01400-5\u003c/li\u003e\n \u003cli\u003eRawlinson, C., Nealon, G., Chooi, Y. H., Oliver, R. P., Moffat, C. S., \u0026amp; See, P.T. (2024). Discovery of two novel phthalide phytotoxins from the wheat tan spot fungal pathogen \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e. \u003cem\u003eJournal of Agricultural and Food Chemistry, 72\u003c/em\u003e(36), 19594\u0026ndash;19603. https://doi.org/10.1021/acs.jafc.4c02533\u003c/li\u003e\n \u003cli\u003eR Core Team (2023). \u003cem\u003eR: A Language and Environment for Statistical Computing\u003c/em\u003e. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/\u003c/li\u003e\n \u003cli\u003eRunning K. L. D., Momotaz A., Kariyawasam G. K., Zurn J. D., Acevedo M., Carter A. H., Liu Z. and Faris J. D. (2022). Genomic Analysis and Delineation of the Tan Spot Susceptibility Locus \u003cem\u003eTsc1\u003c/em\u003e in Wheat. \u003cem\u003eFrontiers in Plant Science, 13\u003c/em\u003e, 793925. https://doi.org/10.3389/fpls.2022.793925\u003c/li\u003e\n \u003cli\u003eRuud, A. K., Dieseth, J. A., \u0026amp; Lillemo, M. (2018). Effects of three \u003cem\u003eParastagonospora nodorum\u003c/em\u003e necrotrophic effectors on spring wheat under Norwegian field conditions. \u003cem\u003eCrop Science, 58\u003c/em\u003e, 159\u0026ndash;168. https://doi.org/10.2135/cropsci2017.05.0281\u003c/li\u003e\n \u003cli\u003eSee, P. T., Marathamuthu, K., Cupitt, C., Iagallo, E., \u0026amp; Moffat, C. S. (2023). A race profile of tan spot in Australia reveals race 2 isolates harbouring \u003cem\u003eToxC1\u003c/em\u003e. \u003cem\u003ePhytopathology, 113\u003c/em\u003e(7), 1202\u0026ndash;1209. https://doi.org/10.1094/PHYTO-11-22-0422-R\u003c/li\u003e\n \u003cli\u003eSee P.T., Iagallo E.M., Oliver R.P. \u0026amp; Moffat C.S. (2019) Heterologous Expression of the \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e Effector Proteins ToxA and ToxB, and the Prevalence of Effector Sensitivity in Australian Cereal Crops. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e \u003cem\u003e10\u003c/em\u003e(182). https://doi.org/10.3389/fmicb.2019.00182\u003c/li\u003e\n \u003cli\u003eSee, P. T., Marathamuthu, K. A., Iagallo, E. M., Oliver, R. P., \u0026amp; Moffat, C. S. (2018). Evaluating the importance of the tan spot ToxA-\u003cem\u003eTsn1\u003c/em\u003e interaction in Australian wheat varieties. \u003cem\u003ePlant Pathology, 67\u003c/em\u003e, 1066\u0026ndash;1075. https://doi.org/10.1111/ppa.12835\u003c/li\u003e\n \u003cli\u003eShi, G., Kariyawasam, G., Liu, S., Leng, Y., Zhong, S., Ali, S., Moolhuijzen, P., Moffat, C. S., Rasmussen, J. B., Friesen, T. L., Faris, J. D., \u0026amp; Liu, Z. (2022). A conserved hypothetical gene is required but not sufficient for Ptr ToxC production in \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e. \u003cem\u003eMolecular Plant-Microbe Interactions, 35\u003c/em\u003e(4), 336\u0026ndash;348. https://doi.org/10.1094/MPMI-12-21-0299-R\u003c/li\u003e\n \u003cli\u003eSimko, I., \u0026amp; Piepho, H.-P. (2012). The area under the disease progress stairs: Calculation, advantage, and application. \u003cem\u003ePhytopathology, 102\u003c/em\u003e, 381\u0026ndash;389. https://doi.org/10.1094/PHYTO-07-11-0216\u003c/li\u003e\n \u003cli\u003eStatistics Denmark (2024). Retrieved from https://www.statbank.dk/20471\u003c/li\u003e\n \u003cli\u003eStatistics Norway (2024). Retrieved from https://www.ssb.no/en/jord-skog-jakt-og-fiskeri/jordbruk/statistikk/korn-og-oljevekster-areal-og-avlinger\u003c/li\u003e\n \u003cli\u003eStatistics Sweden (2024). Retrieved from https://www.scb.se/en/finding-statistics/statistics-by-subject-area/agriculture-forestry-and-fishery/\u003c/li\u003e\n \u003cli\u003eStrelkov, S. E., Lamari, L., \u0026amp; Ballance, G. M. (1999). Characterization of a host-specific protein toxin (Ptr ToxB) from \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e. \u003cem\u003eMolecular Plant-Microbe Interactions, 12\u003c/em\u003e(8), 728\u0026ndash;732. https://doi.org/10.1094/MPMI.1999.12.8.728\u003c/li\u003e\n \u003cli\u003eStrelkov, S. E., Lamari, L., Sayoud, R., \u0026amp; Smith, R. B. (2002). Comparative virulence of chlorosis-inducing races of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e. \u003cem\u003eCanadian Journal of Plant Pathology\u003c/em\u003e, \u003cem\u003e24\u003c/em\u003e(1), 29\u0026ndash;35. https://doi.org/10.1080/07060660109506967\u003c/li\u003e\n \u003cli\u003eTaylor, J., Jorgensen, D., Moffat, C. S. et al. (2023). An international wheat diversity panel reveals novel sources of genetic resistance to tan spot in Australia. \u003cem\u003eTheoretical and Applied Genetics, 136\u003c/em\u003e, 61. https://doi.org/10.1007/s00122-023-04332-y\u003c/li\u003e\n \u003cli\u003eTomas, A., Feng, G. H., Reeck, G. R., Bockus, W. W., \u0026amp; Leach, J. E. (1990). Purification of a cultivar-specific toxin from \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e, causal agent of tan spot of wheat. \u003cem\u003eMolecular Plant-Microbe Interactions\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e, 221\u0026ndash;224. https://doi.org/10.1094/MPMI-3-221\u003c/li\u003e\n \u003cli\u003eTuori, R. P., Wolpert, T. J., \u0026amp; Ciuffetti, L. M. (1995). Purification and immunological characterization of toxic components from cultures of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e. \u003cem\u003eMolecular Plant-Microbe Interactions\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e, 41\u0026ndash;48. https://doi.org/10.1094/mpmi-8-0041\u003c/li\u003e\n \u003cli\u003eVirdi, S. K., Liu, Z., Overlander, M. E., Zhang, Z., Xu, S. S., Friesen, T. L., \u0026amp; Faris, J. D. (2016). New insights into the roles of host gene-necrotrophic effector interactions in governing susceptibility of durum wheat to tan spot and septoria nodorum blotch. \u003cem\u003eG3 Genes/Genomes/Genetics (Bethesda), 6\u003c/em\u003e(12), 4139\u0026ndash;4150. https://doi.org/10.1534/g3.116.036525\u003c/li\u003e\n \u003cli\u003eWei, B., Despins, T., Fernandez, M. R., Strelkov, S. E., Ruan, Y., Graf, R., \u0026amp; Aboukhaddour, R. (2021). Race distribution of \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e in relation to ploidy level and susceptibility of durum and winter bread wheat. \u003cem\u003eCanadian Journal of Plant Pathology\u003c/em\u003e, \u003cem\u003e43\u003c/em\u003e(4), 582\u0026ndash;598. https://doi.org/10.1080/07060661.2020.1870002\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pyrenophora tritici-repentis, Triticum aestivum, necrotrophic effector, field resistance","lastPublishedDoi":"10.21203/rs.3.rs-6547012/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6547012/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTan spot, caused by the fungus \u003cem\u003ePyrenophora tritici-repentis\u003c/em\u003e (\u003cem\u003ePtr\u003c/em\u003e), is one of the major diseases of spring wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) in the Nordic countries. However, little is known on the role of \u003cem\u003ePtr \u003c/em\u003enecrotrophic effectors (NEs), ToxA, ToxB and ToxC, behind tan spot epidemics in the region. A total of 217 Nordic \u003cem\u003ePtr \u003c/em\u003eisolates were screened for \u003cem\u003eToxA \u003c/em\u003eand \u003cem\u003eToxB \u003c/em\u003egenes using PCR. \u003cem\u003eToxA\u003c/em\u003e was found to be prevalent (64.5%), whereas \u003cem\u003eToxB \u003c/em\u003ewas absent. A subset of 25 \u003cem\u003ePtr \u003c/em\u003eisolates was further tested on a set of wheat differentials. Majority (\u003cem\u003en \u003c/em\u003e= 21) of these isolates induced susceptible reaction on ToxC sensitive genotype ‘6B365’, indicating the presence of ToxC effector. The symptoms observed on ToxA and ToxB differentials were not completely in line with the PCR results, suggesting the presence of ‘atypical’ \u003cem\u003ePtr \u003c/em\u003eisolates.\u003c/p\u003e\n\u003cp\u003eIn a collection of 197 spring wheat genotypes, ToxA and ToxB sensitivities were determined and found in 46.2% and 23.9% of the lines, respectively. Additionally, 179 of the wheat accessions were tested in the field over three years at two locations in Finland, and NE sensitivities were found to explain very little of the variation in tan spot susceptibility. Interestingly, ToxB sensitivity was found to have a counter-intuitive effect in European spring wheat germplasm, as ToxB-sensitive genotypes were less susceptible to tan spot disease compared to insensitive ones. The results presented here provide the first comprehensive overview of the importance of \u003cem\u003ePtr \u003c/em\u003eNEs in tan spot epidemics in the Nordic countries.\u003c/p\u003e","manuscriptTitle":"ToxA and ToxB have minimal effect on tan spot epidemics of spring wheat in the Nordics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 08:02:42","doi":"10.21203/rs.3.rs-6547012/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision","date":"2025-07-03T06:41:23+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-05-14T05:19:31+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-08T08:26:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"European Journal of Plant Pathology","date":"2025-05-02T01:45:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-30T05:18:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Plant Pathology","date":"2025-04-28T06:45:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"35d7a9f8-8753-42bd-862f-f8d2ce612ea0","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T16:00:49+00:00","versionOfRecord":{"articleIdentity":"rs-6547012","link":"https://doi.org/10.1007/s10658-025-03173-3","journal":{"identity":"european-journal-of-plant-pathology","isVorOnly":false,"title":"European Journal of Plant Pathology"},"publishedOn":"2025-12-08 15:56:54","publishedOnDateReadable":"December 8th, 2025"},"versionCreatedAt":"2025-05-13 08:02:42","video":"","vorDoi":"10.1007/s10658-025-03173-3","vorDoiUrl":"https://doi.org/10.1007/s10658-025-03173-3","workflowStages":[]},"version":"v1","identity":"rs-6547012","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6547012","identity":"rs-6547012","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}