Genotypic discrimination of chytrid fungus lineages in the amphibian trade

The international amphibian trade raises concerns regarding its impact on native populations due to the potential introduction of different lineages of the chytrid fungus ( Batrachochytrium dendrobatidis or Bd) into new environments. Current diagnostic methods, particularly qPCR, lack the capability to differentiate between these lineages, necessitating more accurate genotyping approaches. Here we propose the application of TaqMan SNP genotyping assays to discriminate Bd genotypes (Bd-GPL, Bd-ASIA2/Bd-BRAZIL [hereafter Bd-BRAZIL], and Hybrid) in samples from Brazilian bullfrog farms. We collected samples, including skin swabs, tadpole mouthparts, and pure Bd cultures, from bullfrog farms across Brazil. Employing two assays utilizing both qPCR and dPCR, we identified genotype presence and analyzed the relationship between Bd load and genotype determination. The genotyping assay achieved a 56.6 % success rate, notably improving with higher Bd loads, reaching 81.8 % for loads over 1,000 genomic equivalents. Culture samples achieved a 100 % success rate. We identified all Bd genotypes in the bullfrog farms, highlighting the issue of coinfections and hybrids in densely populated farms. We hereby present an efficient method for discriminating Bd genotypes, applicable to both pure cultures and field samples with low Bd loads. We emphasize the need for advanced discriminatory methods and comprehensive genetic studies, particularly regarding national regulations governing breeding sites and the global amphibian trade. Our research underscores the feasibility and significance of the proposed method and advocates for further investigations into infection dynamics by different Bd lineages to inform amphibian conservation efforts and trade regulatory policies.