Beticola Isolates Research Articles

Characterization of cytochrome b from European field isolates of Cercospora beticola with quinone outside inhibitor resistance

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most important foliar disease of sugar beet worldwide. Control strategies for CLS rely heavily on quinone outside inhibitor (QOI) fungicides. Despite the dependence on QOIs for disease control for more than a decade, a comprehensive survey of QOI sensitivity has not occurred in the sugar beet growing regions of France or Italy. In 2010, we collected 866 C. beticola isolates from sugar beet growing regions in France and Italy and assessed their sensitivity to the QOI fungicide pyraclostrobin using a spore germination assay. In total, 213 isolates were identified with EC50 values greater than 1.0 μg ml−1 to pyraclostrobin, all of which originated from Italy. To gain an understanding of the molecular basis of QOI resistance, we cloned the full-length coding region of Cbcytb, which encodes the mitochondrial QOI-target enzyme cytochrome b in C. beticola. Cbcytb is a 1,162-bp intron-free gene with obvious homology to other fungal cytb genes. Sequence analysis of Cbcytb was carried out in 32 QOI-sensitive (<0.080 μg ml−1) and 27 QOI-resistant (>1.0 μg ml−1) isolates. All tested QOI-resistant isolates harboured a point mutation in Cbcytb at nucleotide position 428 that conferred an exchange from glycine to alanine at amino acid position 143 (G143A). A PCR assay developed to discriminate QOI-sensitive and QOI-resistant isolates based on the G143A mutation could detect and differentiate isolates down to approximately 25 pg of template DNA. Microsatellite analyses suggested that QOI resistance emerged independently in multiple genotypic backgrounds at multiple locations. Our results indicate that QOI resistance has developed in some C. beticola populations in Italy and monitoring the G143A mutation is essential for fungicide resistance management in this pathosystem.

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

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. Isolates collected in Michigan in 1998 and 1999 that had no previous exposure to the QoI fungicides trifloxystrobin or pyraclostrobin exhibited QoI EC(50) values of ≤ 0.006 µg mL(-1) . In contrast, all isolates obtained in 2011 exhibited EC(50) 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. 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.

Uneven distribution of mating type alleles in Iranian populations of Cercospora beticola , the causal agent of Cercospora leaf spot disease of sugar beet

Normal 0 14 false false false IT ZH-TW X-NONE MicrosoftInternetExplorer4 Cercospora beticola , the causal agent of Cercospora leaf spot disease on sugar beet, is thought to be exclusively asexual because no teleomorph has yet been found. The possibility of a clandestine sexual cycle in the Iranian population of Cercospora beticola was evaluated by analyzing the distribution and frequency of the mating type alleles on a microspatial and a macrogeographical scale. A total of 89 single-conidial Cercospora beticola isolates were obtained from sugar beet fields in the Moghan, the Talesh and the Khoy regions. The isolates were identifed using a Cercospora beticola -specifc primer set in a PCR assay. A multiplex PCR method using previously designed mating type primers was used to study the distribution and the frequency of the mating type alleles. All isolates showed either the 805-bp fragment or the 442-bp fragment of the MAT1-1 and MAT1-2 genes, but no isolate had both fragments. The distribution of the mating type genes in the sampled areas was uneven. From three sugar beet fields sampled in the Moghan region, two fields had only MAT1-1 isolates; while in the third field all isolates had only the MAT1-2 allele. In the Talesh region only MAT1-1 isolates occurred, and in the Khoy region the mating type alleles were uniformly distributed amongst the isolates. The skewed distribution of mating type alleles in Northwestern Iran was in line with the lack of a sexual cycle for this species and may also indicate that sugar fields in the Moghan region were infected by C. beticola populations of different origins. Normal 0 false false false EN-US X-NONE FA

Molecular diagnostic for detecting the cytochrome b G143S – QoI resistance mutation in Cercospora beticola

The mutation G143S has been associated with high-level strobilurin resistance in laboratory mutant strains of Cercospora beticola, one of the most destructive pathogens in sugar beet plants. By using allele specific primers (PASA-PCR) and agarose gel visualization, a molecular diagnostic was developed for the detection of the G143S resistance mutation. This assay is simple and applicable in low tech laboratory settings, with high reliability when a relatively large proportion of mutated mitochondrial alleles are present in the resistant strains. To achieve detection of resistant alleles at low frequencies, a more sensitive Real Time PCR based assay capable of discriminating resistant (S143) genotypes in frequencies as low as 1:10,000 resistant:sensitive alleles was developed. Both diagnostics were successfully validated in laboratory strains. Subsequently, a large number of C. beticola isolates from QoI-treated sugar beet experimental fields in Greece were screened for resistance to Qo fungicides using these diagnostics and classic bioassays. No proportion of the 143S resistant allele was detected in all field isolates tested, which was in agreement with the phenotypes revealed by the biotests confirming that the efficacy of QoIs against C. beticola has been sustained in Greece 7 years after their introduction.

Detection of Pathotypes and Genetic Diversity of Cercospora beticola

The pathotypes of Cercospora beticola, causal agent of sugar beet leaf spot disease, were identified by application of pathogenicity test using 100 isolates obtained from the provinces with intensive sugar beet cultivation. For the identification of pathotypes, five sugar beet cultivars were used each with different resistance factors. Cultivar reactions were determined by inoculation of cultivars with the isolates under controlled conditions and measuring disease severity on the <TEX>$15^{th}$</TEX> day according to the 1-9 KWS Scale. Based on the reactions of the five cultivars, a total of 15 pathotypes were detected. All employed sugar beet cultivars were resistant to Pathotype no:1 comprising most of the isolates. Genetic diversity of the causal agent was characterized by AFLP reaction. The products acquired at the end of AFLP reaction were detected by means of Beckman CEQ 8800 DNA Capillary Series Analysis and the results obtained were evaluated according to the similarity index UPGMA. For the genetic analysis of C. beticola isolates, 9874 polymorphic fragments of sizes between 100 and 500 bp were analysed which were generated by nine primers. The dendrogram derived from AFLP analysis depicted the existence of five different subgroups. The polymorphism rate among isolates was 91.13% and the dendrogram distribution of the pathotypes obtained by pathogenicity indicated that pathotypes were not discriminated and did not compose any groups.

Indirect evidence for sexual reproduction in Cercospora beticola populations from sugar beet

Cercospora beticola is the main causal agent of cercospora leaf spot on sugar beet and has a large negative impact on the yield and quality of sugar beet production worldwide. Previous studies have shown that both mating type idiomorphs of C. beticola are present in natural populations, suggesting that C. beticola is heterothallic and may be reproducing sexually. Cercospora beticola isolates are diverse in the morphology of their conidia, onset of disease symptoms and fungicide resistance. To find the source of this diversity and to determine if sexual reproduction occurs in this fungus, C. beticola populations were collected from Western Europe, Iran and New Zealand. The mating types of these isolates were determined and AFLP analyses were used to study the genetic diversity in these populations. The mating type ratios did not deviate significantly from a 1:1 ratio in most of the populations and AFLP analyses showed high levels of genetic variation within and between the populations, with 86·4% of the isolates having unique genotypes. All populations were in significant linkage disequilibrium but levels of disequilibrium were low, and loci from only one primer pair were in significant gametic equilibrium in populations from the Netherlands and Italy. From these results there is the possibility that C. beticola reproduces sexually. High levels of gene flow among the samples from Europe demonstrated a single panmictic European population. This study confirms C. beticola to be a genetically highly diverse species, supporting the assumption that some populations are reproducing sexually.

Control of Benzimidazole- and DMI-Resistant Strains of Cercospora beticola with Strobilurin Fungicides.

The control efficacy of two new strobilurin fungicides, trifloxystrobin and pyraclostrobin, against Cercospora beticola isolates resistant and sensitive to sterol demethylation-inhibiting (DMI) fungicides and benzimidazole fungicides and the effects on evolution of resistance were tested in the current study. Control efficacy of strobilurin fungicides was measured using three C. beticola isolates, one DMI-resistant (DMIR), one benzimidazole-resistant (BENR), and one of wild-type sensitivity (WCB). Both pyraclostrobin and trifloxystrobin provided satisfactory control of all the three isolates used in the study, when applied at 5 μg ml-1 and very high levels of control when applied at 10 μg ml-1. Control was independent of the isolate sensitivity to benomyl and difenoconazole. In contrast, benomyl applied at 10 μg ml-1 failed to control sufficiently the benzimidazole-resistant isolate, whereas difenoconazole applied at either 5 or 10 μg ml-1 failed to provide satisfactory control of the DMI-resistant isolate of the pathogen. The effects of strobilurin fungicide applications on the evolution of resistance to benzimidazole and DMI fungicides were tested under field conditions in a 2-year experiment (2003 to 2004). Applications of either trifloxystrobin or pyraclostrobin provided high levels of disease control during both years of the study, whereas applications of either benomyl or difenoconazole provided a moderate control efficacy. Measurements of resistance frequencies to benomyl and to difenoconazole showed that successive applications of benomyl tended to select for high frequencies of benzimidazole-resistant phenotypes, whereas successive applications of difenoconazole tended to select for high frequencies of DMI-resistant phenotypes. In contrast, applications of either trifloxystrobin or pyraclostrobin prevented an increase of benzimidazole- or DMI-resistant phenotypes compared with the plots treated with benomyl or difenoconazole, respectively, and decreased frequency of resistance compared with untreated control plots.

Safflower: A New Host of Cercospora beticola.

Safflower is an oilseed crop adapted to the small-grain production areas of the western Great Plains, including the Northern Plains Area (NPA). In the NPA, safflower production is being evaluated for potential rotation with sugar beet. Safflower is susceptible to Cercospora carthami, whereas sugar beet is susceptible to C. beticola C. carthami has not been observed on safflower in the NPA but C. beticola is ubiquitous on sugar beet. Observation of unusual leaf spots on irrigated safflower cv. Centennial at Sidney, MT prompted this investigation of safflower as a potential alternate host of C. beticola. Safflower plants were inoculated with four isolates of C. beticola (C1, C2, Sid1, and Sid2) and incubated in growth chambers; leaf spot symptoms appeared between 3 and 4 weeks later. Polymerase chain reaction (PCR) amplification of extracts from lesion leaf tissue with C. beticola-specific primers produced fragments comparable with amplified fragments from purified cultures of control C. beticola. PCR assay of cultures of single spores from diseased safflower leaf lesions also produced fragments comparable with fragments from C. beticola cultures. Antibody that was raised from isolate C2 also bound to antigens from the single-spore cultures of the four C. beticola isolates. Inoculum from single-spore cultures from infected safflower also infected sugar beet and produced typical Cercospora leaf spot symptoms. Assay of these leaf lesions by PCR resulted in amplification of target fragments with the C. beticola-specific primers. Our results demonstrate that safflower is a new host of C. beticola.

Lack of influence of host plant disease resistance on the evolution of resistance to sterol demethylation-inhibiting (DMI) fungicides inCercospora beticola

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.

Sensitivity of Cercospora beticola populations to fentin-acetate, benomyl and flutriafol in Greece

Cercospora beticola isolates were collected, over 2 years (2000–2001), from sugar beet fields of 4 areas of Greece (Imathia, Larissa, Serres and Orestiada) with different histories of fungicide use. The isolates were used to determine their sensitivity to benomyl, fentin-acetate and flutriafol. In Orestiada and Larissa, where benomyl has been used in one spray application per year since 1996 or 2000, the resistance frequency was more than 50%. In contrast to Imathia and Serres, where benomyl has not been used since the detection of resistance in the early 1970s. Resistance to fentin-acetate, was significantly decreased since previous surveys in 1978. The resistance frequency ranged from 2–3% in the Serres’ population to 8–11% in the Orestiada’ population. In Larissa and Orestiada no population shift towards decreased sensitivity to flutriafol was detected but in Serres and Imathia, which were more heavily treated with sterol demethylation-inhibiting fungicides (DMIs), populations clearly showed decreased sensitivity, although to a smaller frequency than in earlier surveys when more DMI sprays were being applied.

Fungicide Sensitivity Characteristics of Cercospora beticola Isolates Recovered from the High Plains of Colorado, Montana, Nebraska, and Wyoming. 2. Mancozeb, Propiconazole. and Azoxystrobin

Sugarbeet (Beta vulgaris L.) growing areas in northeastern Colorado, southeastern Montana and southwestern Nebraska were surveyed in 1998 through 2001 to determine if insensitive Cercospora beticola isolates were present in fields. One field in southeast Wyoming was included in the 1999 and 2000 surveys. A total of 328 isolates recovered from 110 fields in 1998, 305 isolates recovered from 101 fields in 1999, 255 isolates recovered from 72 fields in 2000, and 256 isolates recovered from 85 fields in 2001 was tested in this study. The radial growth of each isolate on potato dextrose agar (PDA) was compared to its radial growth on PDA amended with 5 μg mL -1 mancozeb, 1 and 10 μg mL -1 propiconazole and 1 and 10 μg mL -1 azoxystrobin. The overall percentages of isolates that grew in the presence of 5 μg mL -1 mancozeb were 92% in 1999, 88% in 2000 and 90% in 2001. Overall percentages of isolates that grew in the presence of 1 μg mL -1 propiconazole were 2% in 1999, 0.4% in 2000 and 0% in 2001. The overall percentages of isolates that grew in the presence of 1 μg -1 mL azoxystrobin were 99.7% in 1998 and 98% in both 2000 and 2001. Data reported here establishes baseline characteristics of the current C. beticola population infecting sugarbeet in the High Plains regions of Colorado, Montana, Nebraska and Wyoming.

Laboratory-induced fungicide resistance to benzimidazole and azole fungicides in Cercospora beticola

Strains of Cercospora beticola resistant to sterol 14α-demethylation inhibitors (DMI) were found 30 times more frequently than strains resistant to carbendazim (MBC) following mutagenesis. In contrast, pathogenicity and resistance tests under greenhouse conditions revealed only 16% of the highly resistant DMI resistant strains to be pathogenic as compared to 67% of the highly resistant MBC resistant strains which were pathogenic. Strains selected on one DMI fungicide were cross-resistant to all other DMI fungicides tested but were not highly resistant to fenpropimorph which inhibits the 14-reductase and the 8-7 isomerase step in sterol biosynthesis. The DMI sensitive parent strain contains ergosta-5,7-dienol, not ergosterol, as its primary sterol. In 24 of 25 different C. beticola isolates which were resistant to flusilazole, the major sterol present was ergosterol. All strains accumulated 14α-methyl sterols as a result of DMI fungicide treatment, suggesting that the 14α-demethylase enzyme was still sensitive to the fungicide. The results of this laboratory study indicate that resistance to DMI fungicides is incremental and associated with reduced fitness, and may suggest that there is a greater risk of resistance development to benzimidazoles than to DMI fungicides.