38 SEED TESTING INTERNATIONAL www.seedtest.org RULES DEVELOPMENT • and other organisms or samples which do not contain the target. Sixty-three isolates (44 target and 19 non- target isolates) were selected and plated on two non-selective media (PDA and MA). When necessary, isolates were subcultured on SNA and CLA to enhance sporulation. Based on colony morphology, analytical specificity was validated and reached 100% performance for both inclusivity (morphology found as expected for the target pathogens) and exclusivity (morphology different to the prescribed morphology for the target pathogens) on both media. The analytical sensitivity is the lowest quantity or concentration of a pest that can be reliably detected with a given analytical method. The performance of the analytical sensitivity was based on the ability to detect one infected seed in a sample of 399 seeds (0.25% of contamination). Thirty-eight seed samples of wheat, barley or oat containing 399 seeds free from F. culmorum and F. tricinctum were spiked with one seed contaminated by one of these Fusarium (culmorum or tricinctum). Examination resulted in 100% detection of each contaminated seed in all the samples. Analytical sensitivity was therefore validated. Robustness is the ability of a method to produce results that do not vary, even if there are small parameter variations in the method. To evaluate the robustness, three parameters were studied (light conditions, duration of incubation and plating design). For the light conditions, incubation under darkness and NUV were compared on both media (PDA and MA) and resulted in having better sporulation most of the time under NUV. The results on the duration of incubation (6, 7 and 9 d), led us to propose that the examination is best to be executed after 7 d incubation. Plating design was validated with no differences for both conditions: five and ten seeds per plate. Comparative Test To demonstrate the repeatability, reproducibility and diagnostic performance of the method a CT was performed. In a CT it is extremely important that participants execute the protocol on provided samples, adhering as strictly as possible to the protocol. Selection and Quality Monitoring of Test Sample Sets The CT was conducted in seven globally dispersed laboratories between March and April 2023. Each laboratory received eight coded Fusarium species isolates for identification and ten samples consisting of three highly contaminated oat seed samples positive for F. poae (A); four medium-contaminated wheat seed samples positive for F. poae and F. avenaceum (B); and three wheat seed samples negative for F. poae and F. avenaceum (D). Prior to shipment of the samples, the level of seed lot contamination and the homogeneity were evaluated. All selected seed lots were proven to be homogeneous. A stability test was conducted after the last participant had indicated having begun the experiment, and was validated with stable contamination percentages over time. CT Results Isolate identification The media, the incubation conditions and the morphological criteria allowed the seven laboratories to identify all the different species of Fusarium. Detection on seed samples For each of the seven participating laboratories, all negative samples were detected as negative (0+/3) as expected, and all the expected Fusarium species were found to be positive in the samples (F. poae: 7+/7; F. avenaceum: 4+/4). Therefore, accordance (repeatability) and concordance (reproducibility) of results for the negative and positive seed lots were calculated according to Langton et al. (2002) and the result was 100% for both performance criteria. The results of the seven participating laboratories were analysed with Hampel’s method and boxplot (Fig. 3) for medium and high levels. Conclusion All the performance criteria were validated by this study. The CT organised with seven participants allowed the reproducibility of the method to be evaluated. Nevertheless, the CT results on naturally infected seeds showed that the identification of the species by morphological criteria is difficult. The difficulty is related to the presence of several different species and not due to the lack of laboratories’ expertise. All laboratories identified the species by morphological criteria for the target isolates. This conclusion led us to propose that the expression of results will: • Specify the identified Fusarium species and/or • Be given in complex following Crous et al. (2021), O’Donnel et al. (2022) and/or • Be given as Fusarium sp. when the identification is not possible • Or be a mix of these expressions depending on the Fusarium species present To help with the identification of Fusarium species, this method will be enriched in the coming years through an ongoing project, by an optional identification using barcoding. References 1. Crous, P.W., Lombard, L., Sandoval-Denis, M., Seifert, K.A., Schroers, H.-J., Chaverri, P. et al. (2021). Fusarium: more than a node or a foot cell. Studies in Mycology, 98, 1–184. 2. Dean, R., Van Kan, J.A.L., Pretorius, Z.A., Hammond-Kosack, K.E., Di Pietro, A., Spanu, P.D. et al. (2012). The top ten fungal pathogens in molecular plant pathology. Molecular Plant Pathology, 13, 414–430. 3. Desjardins, A.E. (2006). Fusarium Mycotoxins: Chemistry, Genetics, and Biology. American Phytopathological Society (APS Press), St. Paul, MN, USA. 260 pp. 4. ISTA (2020). 7-022: Detection of Microdochium nivale and Microdochium majus in Triticum spp. (wheat) seed. International Rules for Seed Testing, 2020: Validated Seed Health Testing Methods. International Seed Testing Association, Wallisellen, Switzerland. 5. Langton, S.D., Chevennement, R., Nagelkerke, N. and Lombard, B. (2002). Analysing collaborative trials for qualitative microbiological methods: accordance and concordance. International Journal of Food Microbiology, 79(3), 175–181. 6. Nelson, P.E., Toussoun, T.A. and Marasas, W.F.O. (1983). Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State University Press, USA. 183 pp. 7. O’Donnell, K., Whitaker, B.K., Laraba, I., Proctor, R.H., Brown, D.W., Broders, K. et al. (2022). DNA sequence-based identification of Fusarium: a work in progress. Plant Disease, 106(6), 1597–1609. Figure 3. Boxplot comparison of homogeneity, participating laboratories and stability test at high level (Fusarium poae)
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