Sensitivity of Aspergillus flavus isolates from peanut seeds in Georgia to Azoxystrobin, a Quinone outside Inhibitor (QoI) fungicide

Authors: ALI, MD EMRAN - University Of Georgia; GUNN, MACKENZIE - University Of Georgia; STACKHOUSE, TAMMY - University Of Georgia; WALIULLAH, SUMYYA - University Of Georgia; Guo, Baozhu; CULBREATH, ALBERT - University Of Georgia; BRENNEMAN, TIMOTHY - University Of Georgia

Submitted to: Toxins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/7/2021
Publication Date: 4/9/2021
Citation: Ali, M., Gunn, M., Stackhouse, T., Waliullah, S., Guo, B., Culbreath, A., Brenneman, T. 2021. Sensitivity of Aspergillus flavus isolates from peanut seeds in Georgia to Azoxystrobin, a Quinone outside Inhibitor (QoI) fungicide. Toxins. 7, 284:1-11. https://doi.org/10.3390/jof7040284.
DOI: https://doi.org/10.3390/jof7040284

Interpretive Summary: Aspergillus flavus infects peanuts and produces aflatoxin, a potent human carcinogen. In infected peanuts, it can also affect peanut seed quality by causing seed rot and reducing seed viability, resulting in low germination. In 2020, peanut seeds in Georgia had lower than expected germination rate and high frequency of A. flavus infection. Dynasty PD (azoxystrobin, fludioxonil, and mefenoxam) has been the standard peanut commercial seed treatment in the U.S. for prevention of A. flavus infection. One of the active ingredients in Dynasty PD is azoxystrobin, the class of quinone outside inhibitors (QoI) fungicides. The objective of this study was to investigate the cause of the low seed quality in 2020. We isolated 76 A. flavus isolates from seven seed lots and conducted in vitro study to test isolate-specific reaction to QoI fungicide (azoxystrobin). Identification of these isolates was confirmed as A. flavus by morphological characteristics and a PCR-based method. In vitro study, isolate-specific reaction to QoI tests resulted in average EC50 values ranged from 0.12 µg/m to 297.22 µg/m, suggesting that some isolates were resistant to this seed treatment fungicide. By sequencing these isolates, we identified a new single nucleotide mutation in these isolates, and this mutation exists in 36.8% of these fungal isolates, which was linked to total resistance to the QoI fungicide. We detected a new major single mutation in A. flavus natural population associated with 2020 bad peanut seed quality. Therefore, new seed treatment fungicides are needed.

Technical Abstract: Aspergillus flavus infects peanuts and produces a mycotoxin called aflatoxin, a potent human carcinogen. In infected peanuts, it can also affect peanut seed quality by causing seed rot and reducing seed viability, resulting in low germination. In 2020, peanut seeds in Georgia had lower than expected germination and a high frequency of A. flavus contamination. A total of 76 Aspergillus isolates were collected from seven seed lots and their identity and in vitro reaction QoI fungicide (azoxystrobin) were studied. The isolates were confirmed as A. flavus by morphological characteristics and a PCR-based method using species-specific primers. In vitro, these isolates were tested for sensitivity to azoxystrobin. The mean EC50 values were ranged from 0.12 to 297.22 µg/m, suggesting that some isolates were resistant or tolerate to this fungicide. The sequences of cytochrome b gene from these isolates were compared and a single nucleotide mutation (36.8% isolates) was find as Cyt B G143A, which was associated with the total resistance to the QoI's. Another single mutation (15.8% isolates) was also observed as Cyt B F129L, which had been documented for QoI resistance. Therefore, a new major single mutation was detected in A. flavus natural population in this study, and it might explain the cause of the bad seed quality in 2020. The high frequency of this new single nucleotide mutation exists in the natural population of A. flavus and results in the ineffectiveness of using azoxystrobin seed treatment. New seed treatment fungicides are needed.