Morphological characterization and multi-locus DNA sequence analysis of fungal isolates obtained from 32 clinical cases of equine fungal keratitis (FK) was performed to identify species and determine associations with antifungal susceptibility, response to therapy and clinical outcome. Two species of Aspergillus ( A . flavus and A . fumigatus ) and three species of Fusarium ( F . falciforme , F . keratoplasticum , and F . proliferatum ) were the most common fungi isolated and identified from FK horses. Most (91%) equine FK Fusarium nested within the Fusarium solani species complex (FSSC) with nine genetically diverse strains/lineages, while 83% of equine FK Aspergillus nested within the A . flavus clade with three genetically diverse lineages. Fungal species and evolutionary lineage were not associated with clinical outcome. However, species of equine FK Fusarium were more likely ( p = 0.045) to be associated with stromal keratitis. Species of Aspergillus were more susceptible to voriconazole and terbinafine than species of Fusarium , while species of Fusarium were more susceptible to thiabendazole than species of Aspergillus . At the species level, A . fumigatus and A . flavus were more susceptible to voriconazole and terbinafine than F . falciforme . Natamycin susceptibility was higher for F . falciforme and A . fumigatus compared to A . flavus . Furthermore, F . falciforme was more susceptible to thiabendazole than A . flavus and A . fumigatus . These observed associations of antifungal sensitivity to natamycin, terbinafine, and thiabendazole demonstrate the importance of fungal identification to the species rather than genus level. The results of this study suggest that treatment of equine FK with antifungal agents requires accurate fungal species identification.

Aspergillus flavus is an agriculturally important fungus that causes ear rot of maize and produces aflatoxins, of which B1 is the most carcinogenic naturally-produced compound. In the US, the management of aflatoxins includes the deployment of biological control agents that comprise two nonaflatoxigenic A. flavus strains, either Afla-Guard (member of lineage IB) or AF36 (lineage IC). We used genotyping-by-sequencing to examine the influence of both biocontrol agents on native populations of A. flavus in cornfields in Texas, North Carolina, Arkansas, and Indiana. This study examined up to 27,529 single-nucleotide polymorphisms (SNPs) in a total of 815 A. flavus isolates, and 353 genome-wide haplotypes sampled before biocontrol application, three months after biocontrol application, and up to three years after initial application. Here, we report that the two distinct A. flavus evolutionary lineages IB and IC differ significantly in their frequency distributions across states. We provide evidence of increased unidirectional gene flow from lineage IB into IC, inferred to be due to the applied Afla-Guard biocontrol strain. Genetic exchange and recombination of biocontrol strains with native strains was detected in as little as three months after biocontrol application and up to one and three years later. There was limited inter-lineage migration in the untreated fields. These findings suggest that biocontrol products that include strains from lineage IB offer the greatest potential for sustained reductions in aflatoxin levels over several years. This knowledge has important implications for developing new biocontrol strategies.

Aspergillus flavus is an agriculturally important fungus that causes ear rot of maize and produces aflatoxins (AFs), of which B1 is the most potent carcinogen known. In the US, the management of AFs includes the deployment of biological control agents that comprise two nonaflatoxigenic A. flavus strains, either Afla-Guard (member of lineage IB) or AF36 (lineage IC). We used genotyping-by-sequencing to examine the influence of both biocontrol agents on native populations of A. flavus in cornfields in Texas, North Carolina, Arkansas, and Indiana. This study examined up to 27,529 single-nucleotide polymorphisms (SNPs) in a total of 815 A. flavus isolates, and 353 genome-wide haplotypes sampled before biocontrol application, three months after biocontrol application, and up to three years after initial application. Here, we report that the two distinct A. flavus evolutionary lineages IB and IC differ significantly in their frequency distributions across states. We provide evidence of increased unidirectional gene flow from lineage IB into IC, inferred to be due to the applied Afla-Guard biocontrol strain. Genetic exchange and recombination of biocontrol strains with native strains was detected in as little as three months after biocontrol application and up to one and three years later. There was limited inter-lineage migration in the untreated fields. These findings suggest that biocontrol products that include strains from lineage IB offer the greatest potential for sustained reductions in aflatoxin levels over several years. This knowledge has important implications for developing new biocontrol strategies.

Host resistance is an important tool in the management of black shank disease of tobacco. While race development leads to rapid loss of single-gene resistance, the adaptation by Phytophthora nicotianae to sources of partial resistance from Beinhart 1000, Florida 301, and the Wz gene region introgressed from Nicotiana rustica is poorly characterized. In greenhouse environments, host genotypes with QTLs conferring resistance from multiple sources were initially inoculated with an aggressive isolate of race 0 or race 1 of P. nicotianae. The most aggressive isolate was selected after each of six host generations to inoculate the next generation of plants. The race 0 isolate demonstrated a continuous gradual increase in disease severity and percent root rot on all sources of resistance except the genotype K 326 Wz/--, where a large increase in both was observed between generations two and three. Adaptation by the race 0 isolate on Beinhart 1000 represents the first report of adaptation to this genotype by P. nicotianae. The race 1 isolate did not exhibit significant increases in aggressiveness over generations, but also exhibited a large increase in aggressiveness on K 326 Wz/-- between generations 3 and 4. Molecular characterization of isolates recovered during selection was completed using ddRADseq, but no polymorphisms were associated with the observed changes in aggressiveness. The rapid adaptation to Wz resistance and the gradual adaptation to other QTLs highlights the need to study the nature of Wz resistance and for conducting field studies on efficacy of resistance-gene rotation for disease management.