Sensitivity of Alternaria alternata from Citrus to Boscalid and Polymorphism in Iron-Sulfur and in Anchored Membrane Subunits of Succinate Dehydrogenase.

Boscalid, a succinate dehydrogenase inhibitor (SDHI), was registered in 2011 to control Alternaria brown spot (ABS) of citrus, caused by Alternaria alternata. In this study, the effect of boscalid on mycelial growth, conidial germination, and resazurin reduction was established in a subset of 16 sensitive isolates using three different media. Conidial germination and mycelial growth inhibition were not suppressed even at higher concentrations of boscalid, although effective concentration to inhibit 50% growth (EC50) values were established with each method. Resazurin reduction produced the lowest EC50 values and was selected for further sensitivity tests. In total, 419 isolates, never exposed to boscalid and collected from Florida tangerine orchards between 1996 to 2012, were tested for boscalid sensitivity. The sensitivity distribution was a unimodal curve with a mean EC50 value of 0.60 µg/ml and a range of 0.07 to 5.84 µg/ml. The molecular characterization of the succinate dehydrogenase (SDH) genes were also determined in a subset of 15 isolates, exhibiting great variability in boscalid sensitivity, by cloning and sequencing the sdhB, sdhC, and sdhD genes. Sequence comparisons of the SDH complex revealed the presence of mutations in 14 of 15 isolates. In total, 21 mutations were identified. Double and multiple mutations were observed in SDHC and SDHD, respectively. In SDHB, 4 mutations were observed while, in SDHC and SDHD, 5 and 12 mutations were detected, respectively. No mutations were found in the highly conserved histidine residues at positions 277 in SDHB, 134 in SDHC, and 133 in SDHD, typically observed in SDHI-resistant isolates. Our findings suggest that A. alternata populations from Florida are sensitive to boscalid and it could be used in ABS spray programs. Boscalid resistance is currently not a problem, although further monitoring for resistance is advisable.

QoI-Resistance Stability in Relation to Pathogenic and Saprophytic Fitness Components of Alternaria alternata from Citrus.

The phenotypic stability, fitness components, and ability to cause disease of isolates of the Alternaria alternata tangerine pathotype resistant to quinone-outside inhibitors (QoIs) were studied. Stability of resistance to azoxystrobin (AZ) and pyraclostrobin (PYR) was determined after consecutive transfers on potato dextrose agar (PDA). The sensitivity to QoIs did not change significantly after 10 transfers on PDA compared with the initial sensitivity of all isolates tested. Fitness components evaluated in vitro were mycelial growth, conidial germination, and conidial production. Incubation period, number of lesions per leaf area, and virulence were determined with detached leaf assays using four cultivars: Dancy, Minneola, Murcott, and Sunburst. Variability in fitness components was observed among isolates within the same sensitivity group. As a group, no significant differences in the mean values of these fitness components were observed between resistant and sensitive phenotypes, except for virulence. Resistant isolates were significantly (P < 0.05) more virulent than the sensitive isolates on Dancy, Minneola, and Sunburst but not on Murcott (P = 0.3506). There was no significant correlation between individual fitness components and the level of sensitivity to AZ and PYR. Preventive applications of Abound (commercial formulation of AZ) at full field rates failed to control disease caused by QoI-resistant isolates under greenhouse conditions. Our results suggest that QoI resistance in A. alternata tangerine pathotype is stable in the absence of QoI selection pressure and that resistance development did not affect the fitness of resistant isolates.

Distribution of QoI Resistance in Populations of Tangerine-Infecting Alternaria alternata in Florida.

Chemical control, based on copper and quinone outside inhibitor (QoI) fungicides, has been essential for the management of brown spot of citrus, caused by Alternaria alternata. However, QoI control failures were detected recently in Florida. From 2008 to 2012, 817 monoconidial isolates of A. alternata from 46 citrus orchards were examined for sensitivity to azoxystrobin (AZ) and pyraclostrobin (PYR). Of the isolates, 57.6% were resistant to both fungicides, with effective concentration to inhibit 50% growth (EC50) values greater than 5 µg/ml for AZ and 1 µg/ml for PYR. The mean EC50 values for sensitive isolates were 0.139 and 0.020 µg/ml for AZ and PYR, respectively. The EC50 values of both fungicides were highly correlated (P < 0.0001), indicating cross resistance. The proportion of resistant isolates differed significantly (P < 0.0001) among cultivars and with QoI application frequency (P < 0.0001). However, resistance was not significantly related (P = 0.364) to disease severity in the field (low, moderate, and high) or isolate virulence (P = 0.397). The molecular basis for QoI resistance was determined for a subset of 235 isolates using polymerase chain reaction restriction fragment length polymorphism of the cytochrome b gene. All resistant isolates showed the point mutation G143A. Based on the presence of one or two introns, isolates were classified as profile I and profile II, respectively. The resistance frequency was significantly higher (P < 0.0001) in isolate profile II, suggesting a higher selection pressure for resistant population profile II.

A Rapid Resazurin-Based Microtiter Assay to Evaluate QoI Sensitivity for Alternaria alternata Isolates and Their Molecular Characterization.

Chemical management of Alternaria brown spot of citrus is based upon the timely application of site-specific fungicides, many of which are vulnerable to the development of fungicide resistance. A rapid microtiter bioassay based on the colorimetric changes of resazurin (RZ) dye was developed to evaluate the sensitivity of Alternaria alternata to quinone outside inhibitor (QoI) fungicides. Four liquid media (complete medium, minimal medium, potato dextrose broth, and yeast peptone dextrose broth), five conidia concentrations (from 101 to 105 conidia/ ml), and five RZ concentrations (10, 20, 30, 40, and 50 µM) were evaluated. Complete medium at 105 conidia/ml and 40 µM RZ were identified as optimal for measuring RZ reduction. The effective concentration of two QoI fungicides (azoxystrobin and pyraclostrobin) needed to reduce RZ by 50% (EC50) was calculated and compared with those obtained from conidia germination tests on fungicide-amended media. Concordant EC50 values were observed (R2 = 0.923; P < 0.0001) from both methods. Resistant phenotypes were further characterized by the partial sequencing of the cytochrome b gene. Genetic variability associated with the presence or absence of two introns was observed among isolates. The identified resistant isolates had the amino acid substitution G143A, typical of QoI resistance in other fungi.