Resistance in Strawberry Isolates of Colletotrichum acutatum from Florida to Quinone-Outside Inhibitor Fungicides

Abstract

Anthracnose fruit rot of strawberry, caused by Colletotrichum acutatum, is a major disease in Florida and frequent quinone-outside inhibitor (QoI) fungicide applications are needed for disease control. From 1994 to 2014, 181 C. acutatum isolates were collected from multiple strawberry fields in Florida with or without QoI spray history. Sensitivity to azoxystrobin and pyraclostrobin was tested based upon mycelial growth and germ tube elongation inhibition. Mean effective concentration where growth was reduced by 50% (EC50) values for isolates collected prior to 2013 based upon mycelial growth were 0.22 and 0.013 μg/ml and upon germ tube elongation were 0.57 and 0.03 μg/ml for azoxystrobin and pyraclostrobin, respectively. Mycelial growth and germ tube elongation of 48 isolates collected in 2013 and 2014 were not inhibited with azoxystrobin at 3 μg/ml and pyraclostrobin at 0.110 μg/ml. A fungicide discriminatory dose assay indicated that 43 of the 48 isolates had EC50 values higher than 100 and 10 μg/ml for azoxystrobin and pyraclostrobin, respectively. Azoxystrobin and pyraclostrobin sprayed preventively on strawberry fruit inoculated with C. acutatum failed to control resistant isolates. Sequencing of the cytochrome b gene of sensitive and resistant isolates showed that QoI-resistant isolates contained either G143A or F129L amino acid substitutions.

Fungicide sensitivity and spatial and temporal dynamics of <i>Botrytis cinerea</i> and <i>Colletotrichum</i> spp. in conventional and organic strawberry fields

“…However, chemical control can lead to the emergence of fungicide resistance of Colletotrichum spp. in strawberries and other crops, such as previously reported (FORCELINI et al, 2016;MONDAL et al, 2005;YOUNG et al, 2010a). …”

“…The disease can be controlled by applying fungicides beginning before or at bloom and continuing until harvest (MERTELY; MACKENZIE; XIAO et al, 2001). Single-and multi-site fungicides are widely sprayed and their alternate usage as well as the use of fungicides belonging to groups with different modes of actions must be adopted by growers, since the emergence of B. cinerea resistance to systemic fungicides have been widely reported by several authors (AMIRI; HEATH; FERNÁNDEZ-ORTUÑO et al, 2015;FORCELINI et al, 2016;GRABKE et al, 2014).…”

“…HEATH; FERNÁNDEZ-ORTUÑO et al, 2016;FORCELINI et al, 2016;. For instance, some mechanisms of fungicide resistance can be identified in field strains of pathogens, such as reduced sensitivity of the fungicide target site; reduced penetration of toxicants; increased fungicide efflux; increased detoxification; decreased conversion to toxic metabolites; increased production of target enzyme; and target site detour through alternative pathway (SISLER, 1994).…”

“…acutatum isolates, especially related to point mutations at codons 129, 137 and 143, the partial cytb gene was amplified using the primers gramiF1 (5'-GAAGAGGTATGTACTACGGTTCATATAG-3') and gramiR1 (5'-TAGCAGCTGGAGTTTGCATAG-3') (FORCELINI et al, 2016). PCR was performed in a final volume of 25 µL containing 1.9 µL of 10X Buffer (Thermo Scientific), 1.6 µL of 25 mM MgCl 2 , 0.5 µL of 5 M betaine (Sigma-Aldrich®), 0.5 µL of 10 mM dNTPs (Promega Corp.), 0.25 µL of each 10 µM primer, 0.3 µL of Taq polymerase (Thermo Scientific) and 2 µL of DNA at 75 ng/ml.…”

“…PCR was performed in a final volume of 25 µL containing 1.9 µL of 10X Buffer (Thermo Scientific), 1.6 µL of 25 mM MgCl 2 , 0.5 µL of 5 M betaine (Sigma-Aldrich®), 0.5 µL of 10 mM dNTPs (Promega Corp.), 0.25 µL of each 10 µM primer, 0.3 µL of Taq polymerase (Thermo Scientific) and 2 µL of DNA at 75 ng/ml. PCR was performed using the following parameters: an initial denaturation at 95 °C for 3 min; 34 cycles of 95 °C for 40 s, 60 °C for 50 s, and 72 °C for 1 min; and a final extension at 72 °C for 5 min (FORCELINI et al, 2016). PCR products were separated in ethidium-bromide-stained 1 % agarose gel as previously described.…”