Resistance of Botrytis cinerea from strawberry (Fragaria x ananassa Duch.) to fungicides in Michoacan Mexico

Plant Protection Resistance of Botrytis cinerea from strawberry (Fragaria x ananassa Duch.) to fungicides in Michoacan Mexico Adán Álvarez-Medina1  Hilda V. Silva-Rojas2  Santos G. Leyva-Mir1  Nahum Marbán-Mendoza1  Ángel Rebollar-Alviter3  *  1Protección Vegetal. Universidad Autónoma Chapingo. 56230. Chapingo, Estado de Mexico. 2Laboratorio de Biotecnología de Semillas. Campus Montecillo. Colegio de Postgraduados. 56230. Montecillo, Estado de México. 3Centro Regional Morelia-Universidad Autónoma Chapingo. Periférico Paseo de la República No.1000. 58170. Morelia, Michoacán, México. (). Abstract Gray mold caused by Botrytis cinerea Pers. is one of the most important diseases of strawberry (Fragaria x ananassa Duch.), and fungicide resistance is a concern. The objective of this research was to evaluate the sensitivity of B. cinerea isolates to the fungicides iprodione and thiophanate-methyl in two strawberry-producing areas in Mexico. Sixty-two B. cinerea isolates collected from strawberry fruits from Maravatio Valley (MV) and Zamora-Jacona Valley (ZJ) in Michoacan, Mexico, were used to determine the sensitivity distribution based on mycelium growth (MG) and conidial germination (CG). Each monosporic isolate was grown in 4 plates containing media amended with 0, 0.1, 0.5, 1, 10, 50 and 100 µg mL-1 of iprodione and 0, 0.1, 10, 100, 1000 and 2000 µg mL-1 thiophanate-methyl, and the experiment was conducted twice. The effective concentration (EC50) that inhibits 50 % of mycelial growth and conidial germination was determined for each isolate-fungicide combination by fitting the log-logistic model to the data. Fungicide sensitivity distributions were illustrated by box plots and histograms, and compared by the Kolmogorov-Smirnov non-parametric test. For iprodione, there were significant differences between the sensitivity distributions (p≤0.0001) of both of the sampling areas for mycelia. In MV, the median EC50 was 0.35 µg mL-1, while that of the ZJ isolates was 1.5 µg mL-1. There were no significant differences in the sensitivity distributions of CG (p=0.47) between producing areas for conidia (median EC50 = 3.73 µg mL-1). For thiophanate-methyl, the sensitivity distribution for mycelia was scattered and bimodal, with significant differences between the sampling areas (p≤0.002). The median EC50 of MG was 3.11 µg mL-1 in MV, and 2000 µg mL-1 in ZJ. There were no significant differences between the sensitivity distributions from MV and ZJ in the conidial germination assay (p=0.13, Ks=1.17). The EC50 varied from 0.01 to >2000 µg mL-1 (median=2.01 µg mL-1). Resistance in B. cinerea to thiophanate-methyl and iprodione is widespread in strawberry-producing areas in Michoacan. Key words: fungicide resistance; benzimidazole; dicarboximides Introduction he commercial cultivation of strawberry (Fragaria x ananassa) in Mexico began in the late 1940s in the state of Guanajuato and in the 1950s in the crop extended to the state of Michoacan in the Zamora Valley, and later to other regions such as Panindicuaro and Maravatio. Production of this crop has expanded significantly in the north, especially in the state of Baja California (Medina y Aguirre, 2004; Sánchez, 2008). Mexico grows more than 9000 ha of strawberry and more than 50 % of this crop is grown in the state of Michoacan, mainly in the valleys of Zamora-Jacona and Maravatío (SIAP, 2014). Among the most important diseases is gray mold caused by the fungus Botrytis cinerea, which can cause severe losses when conditions favor disease development, especially if they coincide with the flowering period (Sutton, 1998). Given the national and international increase in demand for this berry, the cultivated area has increased in recent years. The advancement in technology for strawberry production has also favored an increase in the intensity of fungicide applications to manage the gray mold, because the geographic area for strawberry production has favorable conditions for the disease development. Cultural management of the disease is based on the establishment of healthy plants and crop debris removal. However, chemical fungicides, especially those with a specific mode of action, provide a better disease management. Among these fungicides, iprodione (dicarboximide), thiophanate-methyl (benzimidazole), pyraclostrobin and azoxystrobin (QoI), fenhexamide (hydroxianilide), cyprodinil + fludioxonil (phenylpyrrole + anilinopyrimidine) and pyrimethanil (anilinopyrimidine) are classified as medium-high risk fungicides for the selection of resistance in B. cinerea (FRAC, 2008). Resistance of B. cinerea to single-site fungicides with different modes of action is documented, even shortly after the development and use of these fungicides in the late 1960s (Leroux, 2007). There are reports about development of resistance of B. cinerea to benzimidazole and dicarboximide fungicides (Yourman and Jeffers, 1999; Lennox and Spotts, 2003; Li et al., 2007; Leroux, 2007; Banno et al., 2008), as well as to QoI (Quinone Outside Inhibitors), anilinopyrimidines, hydroxianilides and phenylpyrroles (Myresiotis et al., 2007; Grabke et al., 2014). Applying different discriminatory doses against strawberry isolates from California (USA), Mercier et al. (2010) found that 92 % of the strawberry isolates were resistant to thiophanate-methyl, 25 % to fenhexamide, 28 % to cyprodinil + fludioxonil and 66 % to pyraclostrobin + boscalid. Out of the active ingredients tested, 85 % of the isolates were resistant to at least two of them. Similarly, in a study with 353 blueberry, raspberry and currant isolates of B. cinerea in Germany, 40.5 % of the isolates were highly resistant to thiophanate-methyl, 64 % to iprodione, 45 % to fenhexamide, 76.8 % to trifloxystrobin, 21 % to boscalid and 14 % to cyprodinil. In addition, 18 % of the isolates were resistant to the five tested active ingredients, and between 6 % and 23 % of the isolates were highly or moderately resistant to one of the five tested active ingredients (Weber, 2011). In Florida (USA), resistance to multiple fungicides was reported in B. cinerea, frequently associated with subpopulations that are resistant to three fungicides (boscalid-QoI-anilinopyrimidines or boscalid-QoI-hydroxianilides), revealing the wide distribution of B. cinerea resistance to multiple fungicides that contain the most common point mutations associated with each of these chemical groups (Amiri et al., 2014). Similar results were obtained in isolates from the Carolinas (USA), where 66.7 % of the collected B. cinerea isolates were resistant to pyraclostrobin and boscalid-pyraclostrobin, in most cases related to the G143A mutation conferring high levels of resistance to strobilurin and H272R and H272Y to boscalid (Fernández-Ortuño and Schnabel, 2012). Benzimidazole fungicides are systemic, broad-spectrum fungicides used commercially for controlling plant diseases since the ea (...truncated)