Unveiling Race Diversity and Fungicide Sensitivity Profiles of <em>Pyrenophora Tritici Repentis</em> Populations in South Dakota Collected From 2021-2023

Tan spot, a foliar disease of wheat caused by the ascomycetes fungus Pyrenophora tritici repentis (Ptr), poses a significant threat to wheat crops globally. The disease gained economic importance in the late 1970s due to shifts in farming practices, such as minimal tillage and planting on crop residues, providing favorable conditions for the pathogen to overwinter and cause infections. Ptr is classified into eight races based on the production of necrotrophic effectors—Ptr ToxA (necrosis-inducing), Ptr ToxB, and Ptr ToxC (chlorosis-inducing on respective susceptible and sensitive cultivars)—resulting in distinct symptoms on susceptible wheat differentials. The inverse gene-for-gene hypothesis of Ptr suggests an interaction between toxin-encoding genes and host genes, rendering the host susceptible to the disease. Widely grown wheat varieties in South Dakota exhibit susceptibility to prevalent Ptr races, hence the disease continues to emerge annually for which farmers commonly resort to fungicide applications, however frequent and intensive use of the same fungicides raises concerns about development of resistance or reduced sensitivity among pathogen population. Necessitating the regular monitoring for race structure and fungicide sensitivity in the pathogen population. Additionally, an unexplained correlation between Ptr geographical origin and its ability to produce various effector combinations suggests a divergent evolution affecting wheat crops. Therefore, the study was planned with following objectives to 1) race characterization and assessing fungicide sensitivity among the population of Ptr collected from 2021-2023 in South Dakota: 2) assessing the diversity of necrotrophic effectors in a globally collected population of Pyrenophora tritici repentis, causal agent of tan spot of wheat. For the first objective, we genotyped 251 Ptr isolates collected during 2021-2023 growing season using Ptr ToxA, Ptr ToxB, and Ptr ToxC gene-specific markers with further race confirmation by phenotyping on wheat differentials. The same set of collected isolates were also tested to determine their sensitivity to pyraclostrobin and propiconazole based on conidial germination, mycelial growth inhibition and in molecular analysis. Of the 251 isolates, 186 ( have Ptr ToxA and ToxC), 26 (have Ptr ToxA), 21 ( have only Ptr ToxC), 1 ( have only Ptr ToxB), and 4 (containing Ptr ToxA and Ptr ToxB) were designated as race 1, race 2, race 3, race 5, and race 7, respectively. 13 of 251 genotyped isolates lacked all three effectors genes and were designated as race 4. To best of our knowledge, race 7 was identified for the first time in the USA. A subset of forty isolates carrying Ptr ToxA and Ptr ToxC were phenotyped on tan spot wheat differentials, confirming established host-pathogen interactions. 122 of 252 isolates were found to carry mutation at G143A site. Conidial germination assays revealed complete inhibition at the maximum concentration (1 μg ml-1) for pyraclostrobin, while 100% spores germinated for the highest concentrations of propiconazole, indicating the effectiveness of pyraclostrobin to populations tested as compared to propiconazole. This study suggests a diverse Ptr population in South Dakota with reduced to insensitive sensitivity to pyraclostrobin and propiconazole. The second objective involve sequencing isolates and determining the variations present among the toxins genes, also, effector protein which can be potential candidates were predicted in this study. In this, 25 Ptr isolates representing diverse geographical regions (Canada, Romania, Lithuania, Latvia, and USA) and hosts (wheat, rye, and triticale) were studied to determine the diversity in Ptr necrotrophic effectors, race structure, phenotypic and genotypic components of the Ptr host range. High molecular weight genomic DNA of these isolates was extracted and a subset of 25 isolates were sequenced using Illumina MiSeq platform. Sequencing data analysis reveals the highly conserved nature of the coding sequenced of toxins. We also found single copies of Ptr ToxB, indicating the isolate sequenced being less virulent. An inactive Ptr ToxB homolog (toxb) was also found present in a non-pathogenic, race 4 as well as a pathogenic strain belonging to race 3. Most of the isolates collected represented race1 (Ptr ToxA + Ptr ToxC), but races 2 (Ptr ToxA), 3 (Ptr ToxC), 4 (None) and 5 (Ptr ToxB) were also seen. The use of different references for annotating isolates sequenced with Nanopore MinION highlighted variability among isolates. Effector protein predictions from the sequenced isolates indicated a prevalence of cytoplasmic effectors over apoplastic ones. Additionally, only a small subset of predicted effectors were found to contain signal peptides. This study provides a comprehensive overview of toxin variations and underscores the conservation of these effector proteins over evolutionary processes. Overall, this study shows the presence of diverse population of Pyrenophora tritici repentis present in South Dakota. Presence of mutations responsible for reduced fungicide sensitivity, suggests that Pyrenophora tritici repentis isolates might be developing resistance towards fungicide labelled to manage foliar disease of wheat including pyraclostrobin (strobilurins) and propiconazole (triazoles). In addition, investigation into the genetic aspects of necrotrophic effectors reveals the conserved nature of these pathogenicity regulating toxins among isolates irrespective of geographical locations, host, time year when they were collected.

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