Fitness of Cercospora Beticola Field Isolates – Resistant And – Sensitive to Demethylation Inhibitor Fungicides

Isolates of Cercospora beticola resistant to fungicides that inhibit sterol demethylation (DMIs) were collected from sugar beet fields in Northern Greece. Fitness of these isolates was compared to that of DMI-sensitive isolates. The parameters measured were competitive ability both under growth chamber and field conditions, mycelial growth, spore germination, germ tube length, incubation period, virulence and spore production. The competitive ability under growth chamber conditions was measured for 4 pairs of one resistant and one sensitive isolate. Results showed that after 4 disease cycles, in 2 out of 4 pairs tested, the resistant isolates competed well with the sensitive isolates, but in the remaining two pairs the frequency of the resistant isolate decreased significantly. The competition experiment in the field was carried out by inoculating field plots with a conidial suspension consisting of a spore mixture from all the resistant and all the sensitive isolates used in this study. Results showed that at the end of the growing period the frequency of the resistant isolates had slightly decreased (P < 0.05). The measurements of fitness components of individual isolates showed that the resistant isolates had significantly lower (P < 0.05) virulence and spore production than the sensitive isolates, while no significant differences (P > 0.05) to the remaining 4 fitness components, were detected. With correlation analysis it was determined whether there is a relationship between values of each fitness component and the level of sensitivity to flutriafol of individual isolates. The correlation coefficients for virulence (r = 0.45) and spore production (r = 0.41) were significantly different from 0 (P > 0.05), indicating that resistance to DMIs affected, to some degree, the fitness of the resistant isolates.     

Detection of resistance to sterol demethylation-inhibiting (DMI) fungicides in<Emphasis Type="Italic">Cercospora beticola</Emphasis> and efficacy of control of resistant and sensitive strains with flutriafol

Single-lesion isolates ofCercospora beticola (n=150) were collected in 1998 from sugar beet fields in the area of Serres, N. Greece. In this area, sterol demethylation-inhibiting (DMI) fungicides have been used for almost 20 years to control sugar beet leaf spot. The sensitivity of these isolates to the DMI fungicides flutriafol and difenoconazole (EC₅₀ values) was determined on the basis of inhibition of mycelial growth at several fungicide concentrations. The relative growth (RG) of isolates was correlated at all tested concentrations with the respective EC₅₀ values, indicating that RG provides a reliable estimate for the sensitivity of the isolates. The highest correlation coefficients were obtained for concentrations of 1 μg ml⁻¹ flutriafol and of 0.05 μg ml⁻¹ difenoconazole, respectively. Consequently, they are proposed for monitoring of DMI sensitivity inC. beticola populations, as single discriminatory concentrations in a simplified test method. Based on the RG values at the discriminatory concentration of 1 μg ml⁻¹ flutriafol,C. beticola isolates were classified as either resistant or sensitive. The efficacy of flutriafol, applied at the commercially recommended dose, in controlling Cercospora leaf spot was examined in field experiments conducted during 1999 and 2000. Disease incidence in plots artificially inoculated with resistant isolates and treated with flutriafol was significantly higher than in similar plots inoculated with sensitive strains. These results suggest that poor disease control after application of flutriafol may be based on the presence of resistant strains within the pathogen population in northern Greece. This emphasizes the risk of the development of practical resistance if there is increased frequency of such strains within the population. http://www.phytoparasitica.org posting July 13, 2003.     

Reduced sensitivity of Cercospora beticola isolates to sterol-demethylation-inhibiting fungicides

In a survey conducted during October 1995, single-lesion isolates of the sugar beet leaf-spot fungus, Cercospora beticola , were tested for sensitivity to the sterol demethylation inhibiting fungicides (DMIs) flutriafol and bitertanol. The isolates were collected from fields in three different areas of northern Greece. Fields at Serres and Imathia had been sprayed with DMIs for about 15 years to control sugar beet leaf-spot. At the third site, Amyndeon, DMI fungicides had not been used. From each area 150 isolates were tested. ED₅₀ values were calculated for individual isolates by regressing the relative inhibition of colony growth against the natural logarithm of the fungicide concentration. The mean ED₅₀ values for flutriafol for the Serres, Imathia and Amyndeon populations were 1·07, 0·73 and 0·5 µg mL⁻¹, respectively (significantly different at P  = 0·05). For bitertanol the mean ED₅₀ values for the Serres and Imathia populations were 0·72 and 0·81 µg mL⁻¹, respectively, which were not significantly different at P  = 0·05. The mean ED₅₀ value of the Amyndeon population was 0·48 µg mL⁻¹, which was significantly lower than those of the other two populations ( P  < 0·05). A cross-resistance relationship was found to exist between the two triazole fungicides tested when log transformed ED₅₀ values of 60 isolates were subjected to a linear regression analysis ( r  = 0·81).     

Lack of influence of host plant disease resistance on the evolution of resistance to sterol demethylation-inhibiting (DMI) fungicides in<Emphasis Type="Italic">Cercospora beticola</Emphasis>

Field experiments were conducted during 1997 and 1998 to determine the effects of sugar beet cultivar susceptibility to Cercospora leaf-spot on the sensitivity ofCercospora beticola isolates to the triazole fungicide flutriafol. Four cultivars with different levels of disease resistance were treated in experimental plots with six spray applications of flutriafol. Disease assessments were carried out at 15-day intervals. Sensitivity to flutriafol was measured on isolates collected from the plots ∼15 days after the last flutriafol application. Measurements of disease severity and calculations of AUDPC (area under disease progress curve) values showed a distinct differentiation among cultivars, reflecting their level of disease resistance. Disease severity was significantly lower in cvs. ‘Bianca’ and ‘Areth’ than in ‘Univers’ and ‘Rizor’ both in the untreated and in the flutriafol-treated plots. Fungal isolates from flutriafol-treated plots were less sensitive to the fungicide than were isolates from untreated plots. However, no differences in isolate sensitivity were observed among the cultivars, as regards their level of disease resistance. Despite the fact that the use of resistant cultivars cannot eliminate selectively the resistant strains, it can eliminate both resistant and sensitive isolates. Reducing the number of treatments with DMIs, by applying them only when environmental conditions are favorable for disease development, is a prerequisite for successful resistance management; therefore, the use of disease-resistant varieties could aid toward management of DMIs resistance inC. beticola. http://www.phytoparasitica.org posting May 6, 2003.     

Identification of the G143A mutation associated with QoI resistance in Cercospora beticola field isolates from Michigan,United States

BACKGROUND: Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, is the most serious foliar disease of sugar beet (Beta vulgaris L.) worldwide. Disease control is mainly achieved by timely fungicide applications. In 2011, CLS control failures were reported in spite of application of quinone outside inhibitor (QoI) fungicide in several counties in Michigan, United States. The purpose of this study was to confirm the resistant phenotype and identify the molecular basis for QoI resistance of Michigan C. beticola isolates. RESULTS: Isolates collected in Michigan in 1998 and 1999 that had no previous exposure to the QoI fungicides trifloxystrobin or pyraclostrobin exhibited QoI EC₅₀ values of ?0.006 µg mL^?1. In contrast, all isolates obtained in 2011 exhibited EC₅₀ values of > 0.92 µg mL^?1 to both fungicides and harbored a mutation in cytochrome b (cytb) that led to an amino acid exchange from glycine to alanine at position 143 (G143A) compared with baseline QoI‐sensitive isolates. Microsatellite analysis of the isolates suggested that QoI resistance emerged independently in multiple genotypic backgrounds at multiple locations. A real‐time PCR assay utilizing dual‐labeled fluorogenic probes was developed to detect and differentiate QoI‐resistant isolates harboring the G143A mutation from sensitive isolates. CONCLUSION: The G143A mutation in cytb is associated with QoI resistance in C. beticola. Accurate monitoring of this mutation will be essential for fungicide resistance management in this pathosystem. Copyright © 2012 Society of Chemical Industry