1. Year, location and crop where the seeds were collected The resistant Galium aparine biotype (hereinafter referred to as GALAP-R) was collected from a field near St. Valentin (Austria) grown with an ALS-tolerant sugar beet cultivar (Conviso Smart). In the field, a splitting application of the maximum registered dose rate of Conviso® One (2 x 0.5 L ha-1 Conviso® One; 50 g L-1 foramsulfuron + 30 g L-1 thiencarbazone; Bayer CropScience) at an interval of 19 days had provided inadequate control of G. aparine in spring 2024. Herbicide resistance was suspected since ALS herbicides have been frequently applied in the crop rotation in the three previous years. Seeds from surviving G. aparine plants were collected from the field before harvest of the sugar beets in August 2024. A known susceptible G. aparine population (hereinafter referred to as GALAP-S) was continuously propagated in the “weed garden” at the Julius Kühn-Institute in Braunschweig (Germany) without any herbicide treatment. GALAP-S served as the herbicide-susceptible reference biotype.   
   
   
2. Materials and Methods
   Dose-response bioassay and ALS gene analysis Seeds of the GALAP-R and GALAP-S biotype, respectively, were stratified in a cold room at 4°C for 12 days. Afterwards the seeds were germinated in germination trays on potting soil in a climate cabinet at 20°C/10°C with a 16 h light period for 10 days until the seedlings were ready for transplanting. Germinated seedlings of similar growth stage were transplanted into pots (8x8 cm) containing a standardized soil (50% sand, 38% silty loam, 12.2% clay) with four seedlings per pot and four replicates (pots) per herbicide dose rate. When the first leaf whorl was developed, the plants were sprayed with nicosulfuron (40 g L^-1, trade name: Stretch, Agraria SA, Bulgaria) at dose rates adjusted to the nicosulfuron sensitivity of each biotype (Table 1). The herbicide was applied with the use of a movingnozzle cabinet sprayer equipped with a flat-fan nozzle tip (TeeJet 8002EVS, TeeJet Technologies GmbH, Ludwigsburg, Germany) calibrated to deliver 300 L ha^-1 of spray solution at 210 kPa in a single pass over the foliage. For the dose-response analysis, nicosulfuron was chosen instead of foramsulfuron + thiencarbazone (Conviso One) as a herbicide since it has been applied to GALAP-R before in the field. In addition, it contains only one active which eases interpretation of the dose-response results. After herbicide treatment, plants were maintained in a growth cabinet 16 h light with 206-208 μmolm⁻² s⁻¹ and 8 h dark periods at a temperature of 20 °C/10 °C. Plant survival and shoot fresh weight per pot were recorded 18 days after herbicide treatment. Plants with new green leaf tissue were considered as survivors. The mean fresh weight was expressed as a percentage of the untreated control. The GR₅₀ (herbicide dose to reduce biomass by 50%) and relative standard errors for the mean percentage fresh weight were calculated using non-linear regression analysis and the R software (drc add-on package), the fourparameter log-logistic model was fitted to the data and GR₅₀ values were estimated for both biotypes. A resistance index (RI) was calculated as ratio between the GR₅₀ of the GALAP-R population with the corresponding GR₅₀ of the susceptible GALAP-S biotype.  Table 1. Dose rates of nicosulfuron (40 g L^-1, trade name: Stretch, Agraria SA, Bulgaria) applied to the two GALAP biotypes. The registered dose rate of nicosulfuron in Germany is 40 g ha^-1. Biotype   nicosulfuron dose (g ha^-1) GALAP-R 0, 10, 20, 40, 80, 160, 320, 640 GALAP-S 0, 0.652, 1.25, 2.5, 5, 10, 20, 40   For ALS gene sequence analysis, DNA was extracted from four plants of GALAP-R surviving the full registered dose treatment of 40 g ha^-1 nicosulfuron and 2 plants of GALAP-S for reference using the InviSorb® Spin Plant Mini Kit (Invitek Diagnostics, Germany) according to manufacturer’s instructions. Partial ALS gene was amplified using self-designed primers covering most of the ALS gene sequence and Q5 High Fidelity DNA polymerase (New England Biolabs, USA). PCR was performed as follows: 5 min at 95°C, 37 cycles of 30 s at 95°C, 30 s at 62°C, 30 s at 72°C and 2 min at 72°C. PCR product size was confirmed by gel electrophoresis. Subsequent attempts of commercial Sanger sequencing failed, potentially due to the polyploid nature of G. aparine. Therefore, PCR products were sent to commercial amplicon sequencing using Oxford Nanopore technology (ONT, Microsynth Seqlab, Switzerland). A bioinformatic pipeline was run to map and cluster the sequencing results according to the reference sequence GU377313.1 provided by NCBI.  Sequence comparisons between GALAP-R and the reference sequence were performed on CLC main workbench (Qiagen, Denmark).  
   
   3. Results
   The dose-response study confirmed resistance to nicosulfuron in the GALAP-R biotype. The GALAP-R biotype had a GR₅₀ of 150.6 g ha^-1 nicosulfuron, while the GR₅₀ of the GALAP-S biotype was 16.6 g ha^-1, making GALAP-R nine-fold more resistant than the susceptible GALAP-S biotype (Figure 1, Table 2).     Figure 1. Dose-response curves (biomass relative to the untreated control, %) for GALAP-S (solid) and GALAP-R (dashed) biotypes of Galium aparine treated with nicosulfuron.    Table 2. Estimated GR₅₀ (herbicide dose to reduce biomass by 50%) and resistance index (RI) values for Galium aparine biotypes treated with nicosulfuron (40 g L^-1, trade name: Stretch, Agraria SA, Bulgaria). Values in parentheses are standard errors.  Biotype GR50 (L ha-1) RI GALAP-R 150.6 (11.17) 9.072 GALAP-S 16.6 (8.24)     Amplicon sequencing using ONT sequencing technology revealed a G to T substitution in the second nucleotide of the codon coding for Trp574 leading to a Trp574Leu substitution in the ALS protein (Figure 2). This substitution was found in two out of four identified ALS genes in GALAP-R Figure 2. Comparison of partial ALS gene sequences of GALAP-R to the reference sequence GU377313.1 provided in the NCBI database.    

Contributors
Dagmar Rissel Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants Institute for Plant Protection in Field Crops and Grassland Messeweg 11-12 38104 Braunschweig Germany E-Mail: dagmar.rissel@julius-kuehn.de  

Lena Ulber Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants Institute for Plant Protection in Field Crops and Grassland Messeweg 11-12 38104 Braunschweig Germany E-Mail: lena.ulber@julius-kuehn.de  

Wibke Imgenberg Agrana Research & Innovation Center GmbH Josef-Reither-Strasse 21-23 3430 Tulln Austria E-Mail: wibke.imgenberg@agrana.com