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.

Vega, B., and Dewdney, M. M. 2014. Distribution of Qol resistance in populations of tangerine-infecting Alternada alternata in Florida. Plant Dis. 98:67-76.Chemical control, based on copper and quinone outside inhibitor (Qol) fungicides, has been essential for the management of brown spot of citrus, caused by Alternarla alternata. However, Qol 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 (EC,,,) values greater than 5 |ag/ml for AZ and 1 |ig/ml for PYR. The mean EC5,, values for sensitive isolates were 0.139 and 0.020 pg/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 sig-nificantly (P < 0.0001) among cultivars and with Qol applicadon 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 Qol resistance was determined for a subset of 235 isolates using polymerase chain reaction restricdon fragment length polymorphism of the cytochrome b gene. All resistant isolates showed the point mutadon 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, suggesdng a higher selecdon pressure for resistant population profile II.Alternaria brown spot (ABS), caused by Alternarla alternata (Fr.;Fr.) Keissl., is a serious disease of tangerine cultivars {Citrus reticulata Blanco) and their hybrids in commercial citrus orchards in humid subtropical and semiarid growing regions (58). This fungus produces the host-selective ACT toxin (32,33), which plays an important role in host specificity and isolate virulence (23,34). Florida has 7,000 ha of tangerine and tangerine hybrids for freshfruit production (21). More than two-thirds of the tangerine cultivars planted in Florida are susceptible to ABS, including 'Minneola', 'Nova', and 'Orlando' tángelo; 'Dancy' and 'Sunburst' tangerine; 'Murcott' tangor, and some Dancy hybrids such as 'Lee' (56). The fungus can affect leaves, twigs, and young fruit, cause considerable yield loss, and produce blemishes on fruit that reduce marketability (56,58). Management of ABS includes the use of cultural methods such as disease-free nursery stock, wide tree spacing, reduction of excessive vegetadve growth, the elimination of overhead irrigation, and fungicide applications (16,57). Although cultural practices reduce disease severity, ABS control is largely dependent on fungicide applications (8,16,56). Copper-based fungicides (Fungicide Resistance Action Committee [FRAC] code Ml), ferbam (FRAC code M3), and ...

Vega, B., and Dewdney, M. M. 2015. Sensitivity of Alternaria alternata from citrus to boscalid and polymorphism in iron-sulfur and in anchored membrane subunits of succinate dehydrogenase. Plant Dis. 99:231-239.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 (EC 50 ) values were established with each method. Resazurin reduction produced the lowest EC 50 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 EC 50 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.Alternaria brown spot (ABS), caused by Alternaria alternata (Fr.) Keissl., is the most important foliar fungal disease of tangerine plants (Citrus reticulata Blanco) and their hybrids (34,35). The disease affects the leaves, twigs, and young fruit. The first symptoms observed on young leaves is the presence of small brown to black spots surrounded by a yellow halo. Lesions expand rapidly, due to the production of the host-specific ACT toxin (1,46,47), and form irregular necrotic areas. On mature fruit, blemishes that reduce marketability can vary from small necrotic spots to large and depressed dark brown areas (12,51). Although cultural practices such as disease-free nursery stock, wide tree spacing, avoidance of excessive vegetative growth, and the elimination of overhead irrigation (15,47) can help to reduce disease severity, multiple fungicide applications are essential to achieve optimal disease control.Over the last decade, qui...