First Report of Resistance to QoI Fungicides in Alternaria alternata Isolates from Potato in South Africa.

Abstract

Since the first report of brown spot of potatoes (Solanum tuberosum) caused by Alternaria alternata (Fr.) Keissl in South Africa (3), disease intensity has steadily increased. No fungicides are registered for control of brown spot of potatoes in South Africa but growers attempt to control the disease with products registered for early blight, which include various QoI fungicides. Failure to control brown spot with QoI fungicides led to an investigation on putative development of resistance among A. alternata populations. In the summer of 2012, diseased leaves were collected from five potato growing regions. Isolations were made from the margin of brown spot lesions by plating surface disinfested tissue on V8 agar medium (200 ml V8 juice, 3 g CaCO3, 20 g agar). Plates were incubated at 25°C in darkness for 7 days, purified, and single-spore cultures transferred to fresh potato dextrose agar (PDA) plates. Identity of isolates was confirmed using conidial morphology and PCR amplification using species-specific primers AAF2 and AAR3 (1). Eight A. alternata isolates (PPRI 13607, 13608, 13609, 13610, 13611, 13612, 13613, and 13614) were obtained and screened for sensitivity to azoxystrobin in vitro by evaluating relative conidial germination on media amended with 0, 1.0, 2.5, 5.0, 10, 25, 50, 75, 85, and 100 μg of azoxystrobin per ml of media. The dose effect of the fungicide on germination and the EC50 of each isolate were computed using the probit analysis. Isolates were subjected to DNA extraction and the partial cytochrome b (cyt b) was amplified using primer pair CBF1 and CBR2 (2). PCR products were transformed into DH5α competent cells using a pGEM-T Easy vector. Both strands of the cloned fragments were sequenced using primers T7 and SP6 (4). Isolates PPRI 13611 and 13614 had low EC50 values of 0.11 and 0.23 μg/ml, respectively, and a mean EC50 of 0.17 μg/ml, showing their relative sensitivity to azoxystrobin. The other six isolates had EC50 values ranging from 51.88 to 114.92 μg/ml, and a mean EC50 of 71.60 μg/ml, showing their resistance to azoxystrobin. Sequence analysis of the partial cyt b gene showed strong association of resistance in isolates PPRI 13607, 13608, 13609, 13610, 13612, and 13613 to a base substitution resulting in an amino acid substitution at position 143 (G143A). Isolates PPRI 13611 and 13614 did not exhibit this mutation. Although resistance has been reported on other crops where QoI fungicides, including azoxystrobin, have been used to control different pathogens, this is the first report of resistance to a QoI fungicide in field isolates of A. alternata from potatoes in South Africa. Identification of resistance will help to explain failure to control this disease using QoI fungicides. Further monitoring of resistance to azoxystrobin and other QoI fungicides is warranted.