Beticola Isolates Research Articles

Characterization of Stemphylium species associated with Stemphylium leaf spot of spinach (Spinacia oleracea).

Stemphylium leaf spot can result in significant losses to spinach seed, processing, and fresh market crops. Stemphylium isolates (n = 1,775) collected from 2000 to 2022 from spinach seed, leaves, and seed crop stem residues were used to assess the diversity of species associated with spinach. Eleven Stemphylium species were identified based on cmdA sequences: S. vesicarium (63.6% of isolates), S. beticola (48.9%), S. amaranthi (5.1%), S. eturmiunum (4.5%), S. astragali (4.0%), S. simmonsii (3.4%), and S. lucomagnoense, S. drummondii, S. gracilariae, S. lycopersici, and S. chrysanthemicola (each 0.6 to 1.7%). Only isolates of S. beticola, S. drummondii, and S. vesicarium were pathogenic to spinach. The incidence of spinach seed on which Stemphylium was observed ranged from 2.5 to 73.5% per seed lot, with S. vesicarium and S. beticola predominant. However, only 60.7 and 62.3% of isolates tested for these two species were pathogenic to spinach, respectively. Therefore, the incidence of Stemphylium species on spinach seed may not reflect accurately the risk of a seed lot carrying pathogenic isolates. Fused MAT1-1 and MAT1-2 genes were detected in isolates of S. vesicarium, but only MAT1-1 was detected in S. beticola isolates, which corroborates previous studies that have proposed the two species to be self-fertile. The duration of ascospore dispersal of S. beticola and S. vesicarium from spinach seed crop stem residues in western Washington, the primary region of spinach seed production in the USA, occurred from mid-winter to late spring or early fall, potentially serving as inoculum for the next season's spinach seed crops. Growers should incorporate residues into the soil after harvest to reduce inoculum production of these pathogens on spinach seed crop residues.

A new method for single spore isolation and fungicide resistance monitoring of Cercospora beticola, and the first report of QoI‐resistant isolates with G143A or F129L mutations of the CbCyt b gene in China

AbstractCercospora leaf spot (CLS) caused by Cercospora beticola is the most destructive foliar disease of sugar beet worldwide and is mainly controlled by timely fungicide applications. Recently, CLS control by pyraclostrobin exhibited significantly reduced efficiency in some fields of the Chifeng area in Inner Mongolia Autonomous Region of China. Purification of fungi by single spore isolation is essential for the study of fungal and fungicide resistance. However, the conventional single spore isolation and fungicide sensitivity assay for C. beticola are time‐consuming with a risk of contamination. We developed a new method for single spore isolation of C. beticola, which facilitated monitoring fungicide sensitivity of infected field samples. First, the composite spore suspension was prepared from typical lesions by vortex in the sterilized centrifuge tube. Then, the diluted spore suspension was spread on the streptomycin‐amended potato dextrose agar (PDA) plate or the PDA plate contained a threshold dosage of specific fungicide. The C. beticola colony derived from a single spore could be observed with naked eye and easily isolated by a sterilized scalpel knife after 36 h of incubation at 25°C. Meanwhile, the percentage of fungicide‐resistant or ‐tolerant isolates could be analysed based on the growth of different PDA media within 3 days. Based on this method, we identified that 22.88% of the C. beticola isolates were resistant to pyraclostrobin in the Chifeng area, indicating a high risk of emergence of C. beticola‐resistant strains to QoI fungicides in this area. Furthermore, the G143A or F129L mutation of the cytochrome b (Cyt‐b) gene was found in these resistant isolates. To our knowledge, this is the first report of C. beticola QoI‐resistant isolates with G143A or F129L mutation of CbCyt‐b gene in China. Our approach provides considerable potential for studying the biology, fitness and fungicide resistance of C. beticola.

Diversity and fungicidal resistance of Cercospora beticola

The fungus Cercospora beticola Sacc. belongs to the most important pathogens on sugar beet. The Cercospora leaf spot disease is important problem for growers in all growing areas of sugar beet. This study was focused on the morphological diversity of C. beticola isolates and occurrence of their fungicidal resistance in Slovakia. Isolates involved in this work were collected from sugar beet leaf during 2016-2018. Average growth rate of the tested C. beticola isolates on five different media showed the major role of cultivation temperature. The fastest growth was measured by 30°C on TE medium, followed by PDA, SBLEA, AWSBL, and V8. The colour of aerial mycelium varied from olive-green to grey with white powdery appearance, per grey colour with wrinkled texture, to black colour. There is no significant correlation among isolates origin, colony morphology parameters and growth rate. The highest sporulation rate was recorded at cultivation temperature 25°C by using of TE growth medium, the lowest one by V8 medium. The inhibition effect of azoxystrobin + cyproconazole was significantly different among the tested isolates from different localities and years. The results showed reduced sensitivity of C. beticola population in Slovakia strictly depending of the locality. The most reduced sensitivity was measured on localities Hronovce and Nové Zámky, followed by localities Mojmírovce, Senec, and Dolné Saliby with similar values. There is no positive correlation between inhibition effect and mycelial growth rate. The results showed increasing fungicidal resistance of C. beticola to azoxystrobin + cyproconazole in some localities of Slovakia.

The first detection of multiple resistant (MBC and QoI) strains of Cercospora beticola Sacc. in Poland

Long-term and intensive use of fungicides may lead to fungal resistance to active ingredients, even with different modes of action. In Poland, Cercospora leaf spot (CLS), the most remarkable disease of sugar beet, is mainly controlled with benzimidazoles (MBC), triazoles (DMI), and strobilurins (QoI). A constant monitoring of the emergence and the development of resistant fungal strains is required by the limited number of active ingredients. On the basis of RFLP analysis application, the aim of the study was to determine and monitor simultaneous appearing of C. beticola strains with E198A and G143A mutations. Mutation E198A in the β-tubulin gene is the main reason for MBC-resistance. Whereas mutation G143A in the cytochrome b is responsible for QoI-resistance. Most of the 414 tested C. beticola isolates (90%) were able to grow (growth inhibition percentage<50%) on the medium amended with 1 μg/ml thiophanate-methyl, and 34% of the isolates on the medium amended with 1 μg/ml azoxystrobin. Based on the PCR-RFLP analysis both mutations were detected in 44% of the tested isolates. The current study is the pioneer detection of multiple resistances to MBC and QoI fungicides in C. beticola in Poland and the European Union. The presence of multiple resistant isolates in the population of pathogens is adverse and this phenomenon requires thorough monitoring and registration of new active ingredients, allowing effective control of CLS.

Characterization of the Mycovirome from the Plant-Pathogenic Fungus Cercospora beticola.

Cercospora leaf spot (CLS) caused by Cercospora beticola is a devastating foliar disease of sugar beet (Beta vulgaris), resulting in high yield losses worldwide. Mycoviruses are widespread fungi viruses and can be used as a potential biocontrol agent for fugal disease management. To determine the presence of mycoviruses in C. beticola, high-throughput sequencing analysis was used to determine the diversity of mycoviruses in 139 C. beticola isolates collected from major sugar beet production areas in China. The high-throughput sequencing reads were assembled and searched against the NCBI database using BLASTn and BLASTx. The results showed that the obtained 93 contigs were derived from eight novel mycoviruses, which were grouped into 3 distinct lineages, belonging to the families Hypoviridae, Narnaviridae and Botourmiaviridae, as well as some unclassified (−)ssRNA viruses in the order Bunyavirales and Mononegavirales. To the best of our knowledge, this is the first identification of highly diverse mycoviruses in C. beticola. The novel mycoviruses explored in this study will provide new viral materials to biocontrol Cercospora diseases. Future studies of these mycoviruses will aim to assess the roles of each mycovirus in biological function of C. beticola in the future.

Genome-Wide Association and Selective Sweep Studies Reveal the Complex Genetic Architecture of DMI Fungicide Resistance in Cercospora beticola.

The rapid and widespread evolution of fungicide resistance remains a challenge for crop disease management. The demethylation inhibitor (DMI) class of fungicides is a widely used chemistry for managing disease, but there has been a gradual decline in efficacy in many crop pathosystems. Reliance on DMI fungicides has increased resistance in populations of the plant pathogenic fungus Cercospora beticola worldwide. To better understand the genetic and evolutionary basis for DMI resistance in C. beticola, a genome-wide association study (GWAS) and selective sweep analysis were conducted for the first time in this species. We performed whole-genome resequencing of 190 C. beticola isolates infecting sugar beet (Beta vulgaris ssp. vulgaris). All isolates were phenotyped for sensitivity to the DMI tetraconazole. Intragenic markers on chromosomes 1, 4, and 9 were significantly associated with DMI fungicide resistance, including a polyketide synthase gene and the gene encoding the DMI target CbCYP51. Haplotype analysis of CbCYP51 identified a synonymous mutation (E170) and nonsynonymous mutations (L144F, I387M, and Y464S) associated with DMI resistance. Genome-wide scans of selection showed that several of the GWAS mutations for fungicide resistance resided in regions that have recently undergone a selective sweep. Using radial plate growth on selected media as a fitness proxy, we did not find a trade-off associated with DMI fungicide resistance. Taken together, we show that population genomic data from a crop pathogen can allow the identification of mutations conferring fungicide resistance and inform about their origins in the pathogen population.

Evidence for the association of target-site resistance in cyp51 with reduced DMI sensitivity in European Cercospora beticola field isolates.

Cercospora leaf spot caused by Cercospora beticola is the most relevant foliar disease in sugar beet cultivation. In the last decade a decreasing sensitivity of C. beticola towards demethylation inhibitors (DMIs) occurred. Different mechanisms mediating a reduced sensitivity towards DMIs have been identified in different plant pathogens to date, such as target site mutations, over-expression or active excretion of the fungicide. A sequencing of the cytochrome P450-dependent sterol 14α-demethylase gene sequence (cyp51) of diverse C. beticola isolates collected in different European countries with reduced DMI sensitivity was performed in order to find a possible correlation of mutations with higher EC50 values. The amino acid alterations Y464S, L144F and I309T combined with L144F were found to be associated with a reduced sensitivity. Furthermore, mutations I387M, M145W and M145W with E460Q were found uniquely. Additionally, constitutive and fungicide triggered expression of cyp51 was assayed by means of RT-qPCR. A very strong induction of cyp51 mRNA expression in sensitive isolates suggests that the fungal cells upregulate expression to maintain ergosterol biosynthesis in DMI presence. The less intensive cyp51 induction in isolates with higher EC50 values underlines the possible correlation of the found target-site mutations with reduced sensitivity. This study provides new results about possible alterations in the target gene mediating reduced sensitivity of C. beticola towards DMIs and hypothesized a fungicide induced over-expression of the target enzyme CYP51 as natural reaction of the fungus to fungicide application. © 2020 Society of Chemical Industry.

Microtiter plate test using liquid medium is an alternative method for monitoring metyltetraprole sensitivity in Cercospora beticola.

BACKGROUNDMetyltetraprole is a new quinone outside inhibitor (QoI) fungicide showing potent activity against QoI‐resistant fungi that possess the G143A cytochrome b mutation, which confers resistance to existing QoIs such as trifloxystrobin. For its sustainable use, monitoring of metyltetraprole sensitivity is necessary and the establishment of appropriate methodology is important in each pathogen species.RESULTSIn Cercospora beticola, the causal agent of sugar beet leaf spot, some isolates were less sensitive to metyltetraprole (EC50 > 1 mg L–1, higher than the saturated concentration) using the common agar plate method, even with 100 mg L−1 salicylhydroxamic acid, an alternative oxidase inhibitor. However, microtiter tests (EC50 < 0.01 mg L−1), conidial germination tests (EC50 < 0.01 mg L−1) and in planta tests (>80% control at 75 mg L−1 run‐off spraying) confirmed that all tested isolates were highly sensitive to metyltetraprole. For trifloxystrobin, G143A mutants were clearly resistant upon microtiter plate tests (median EC50 > 2 mg L−1) and distinct from wild‐type isolates (median EC50 < 0.01 mg L−1). Notably, mycelium fragments were usable for the microtiter plate tests and the test was applicable for isolates that do not form sufficient conidia. Our monitoring study by microtiter plate tests did not indicate the presence of metyltetraprole‐resistant C. beticola isolates in populations in Hokkaido, Japan.CONCLUSIONThe microtiter tests were revealed to be useful for monitoring the sensitivity of C. beticola to metyltetraprole and trifloxystrobin. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

First Report of Cercospora Leaf Spot Caused by Cercospora beticola on Spinach in Pakistan

Spinach (Spinacia oleracea L.) is one of the most important leafy vegetables, cultivated throughout Pakistan. During September to November 2016, five spinach (cultivar ‘Kandiari’) fields (31°36′17″N, 74°34′23″E) were inspected near Lahore city, Pakistan. Leaf spot symptoms on plants were observed in 10 to 30% of inspected fields. Symptoms began as small circular to subcircular light-brown spots with a distinct dark brown margin. In mature more or less sunken lesions, the center was whitish gray to black, owing to sporulation. Microscopic study of the whitish-gray lesions revealed the presence of small and black stromata within the leaf substomatal cavities. The stromata produced loose fascicles of conidiophores, up to 15 conidiophores in a fascicle. Conidiophores were simple, one to four septate, golden brown but subhyaline at the apex, straight, 4.5 to 5 × 20 to 100 µm (n = 50), geniculate, having distinctive spore scars, and unbranched. Conidia were solitary, hyaline, filiform, straight to slightly curved, with obtuse to subacute at the apex and subtruncate bases, multiseptate (one to 18), nonconstricted at the septa, 28 to 230 × 2.5 to 4 µm (n = 50). On the basis of morphological characteristics, the causal organism was identified as Cercospora beticola Sacc. (Chupp 1954; Crous and Braun 2003). To obtain single-spore isolates from samples, conidial suspension from one lesion per sample was inoculated on separate 2% water agar plates and incubated at 24°C. After 24 h, individual germinating conidia were planted on separate potato dextrose agar (PDA) plates and incubated at 24°C for 10 days. Pure cultures of six C. beticola isolates were used for DNA extraction. The partial internal transcribed spacer (ITS) regions of the fungal rDNA and calmodulin (CAL) gene were amplified using the primers ITS1/ITS4 and CAL228F/CAL737R and sequenced. Obtained DNA sequences of the six isolates were identical; therefore, ITS (MH318663) and CAL (MK333232) sequences of a representative isolate (IM084) were submitted to GenBank. The resulting ITS and CAL sequences of IM084 showed 99 to 100% similarity with C. beticola accessions (MH42448, MF681169, JX142819, AY752227, and FJ473438). A pathogenicity test was accomplished by placing PDA discs (0.5 mm²) from a 10-day-old representative fungal culture onto leaves of six 3-week-old potted spinach (cv. Kandiari) and sugar beet (cv. ‘9597’) (three plants of each). For noninoculated controls, three plants of each type were inoculated with noncolonized PDA plugs. After inoculation, plants were kept 48 h in a dew chamber at 25°C and then transferred to the greenhouse under 12/12-h light/dark conditions at 25°C. Lesions developed on fungal inoculated leaves of spinach and sugar beet plants with similar characteristic symptoms as those observed on naturally infected spinach leaves within 10 days after inoculation. Control spinach and sugar beet plants remained asymptomatic. Sporulation of C. beticola was observed in lesions similar to initial symptoms observed in field samples. The pathogen was reisolated from infected leaf lesions and confirmed as C. beticola. Previously, C. beticola causing Cercospora leaf spot has been reported on sugar beet (Khan et al. 2007); however, to our knowledge this is a new record of C. beticola on spinach in Pakistan. Spinach is one of the high demand leafy vegetables, and incidence of this new pathogen can be a significant threat for spinach production.

Genetic Diversity and Structure in Regional Cercospora beticola Populations from Beta vulgaris subsp. vulgaris Suggest Two Clusters of Separate Origin.

Cercospora leaf spot, caused by Cercospora beticola, is a highly destructive disease of Beta vulgaris subsp. vulgaris worldwide. C. beticola populations are usually characterized by high genetic diversity, but little is known of the relationships among populations from different production regions around the world. This information would be informative of population origin and potential pathways for pathogen movement. For the current study, the genetic diversity, differentiation, and relationships among 948 C. beticola isolates in 28 populations across eight geographic regions were investigated using 12 microsatellite markers. Genotypic diversity, as measured by Simpson's complement index, ranged from 0.18 to 1.00, while pairwise index of differentiation values ranged from 0.02 to 0.42, with the greatest differentiation detected between two New York populations. In these populations, evidence for recent expansion was detected. Assessment of population structure identified two major clusters: the first associated with New York, and the second with Canada, Chile, Eurasia, Hawaii, Michigan, North Dakota, and one population from New York. Inferences of gene flow among these regions suggested that the source for one cluster likely is Eurasia, whereas the source for the other cluster is not known. These results suggest a shared origin of C. beticola populations across regions, except for part of New York, where population divergence has occurred. These findings support the hypothesis that dispersal of C. beticola occurs over long distances.

Temporal Genetic Differentiation of Cercospora beticola Populations in New York Table Beet Fields.

Annual epidemics of Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, can result in substantial defoliation in table beet fields in New York. High allelic and genotypic diversity have been described within C. beticola populations; however, information on the temporal stability of populations is lacking. C. beticola isolates were obtained from symptomatic leaves in three table beet fields in successive years. Two of the fields were organic mixed-cropping farms and the third was managed conventionally in a broad-acre cropping system. C. beticola isolates (n = 304) were genotyped using 12 microsatellite markers. Genotypic diversity (Simpson's complement index = 0.178 to 0.990), allele frequencies, and indices of differentiation between years varied. Pairwise index of differentiation values ranged from 0.02 to 0.25 for clone-corrected data, and indicated significant genetic differentiation at Farm 2. No multilocus genotype was shared between years. The shift in multilocus genotypes between years questions the role of clonally reproducing primary inoculum. Collectively, these results suggest that a dominant inoculum source for initiating annual CLS epidemics is external to the field of interest. These findings have implications for CLS disease management in conventional and organic table beet production.

Horticultural Characteristics and Susceptibility of Table Beet Cultivars to Cercospora Leaf Spot in New York

Table beet (Beta vulgaris ssp. vulgaris) production in New York is increasing for direct sale, use in value-added products, or processing. One of the most important diseases affecting table beet is cercospora leaf spot (CLS) caused by the fungus Cercospora beticola. CLS causes lesions on leaves that coalesce and leads to premature defoliation. The presence of CLS may cause buyer rejection at fresh markets. Defoliation from CLS may also result in crop loss because of the inability to harvest with top-pulling machinery. The susceptibility of popular table beet cultivars (Boldor, Detroit, Falcon, Merlin, Rhonda, Ruby Queen, and Touchstone Gold) to CLS was tested using C. beticola isolates representative of the New York population. Two trials were conducted by inoculating 6-week-old plants in the misting chamber. A small-plot replicated field trial was also conducted to examine horticultural characteristics of the cultivars. In the misting chamber trials, disease progress measured by the area under the disease progress stairs (AUDPS) was not significantly different between the red cultivars, Detroit and Ruby Queen, and was significantly higher in ‘Boldor’ than the other yellow cultivar Touchstone Gold. In the field trial, the number of CLS lesions per leaf at the final disease assessment and AUDPS were significantly lower in cultivar Ruby Queen than others and not significantly different between the yellow cultivars. The dry weight of roots was not significantly different among cultivars at first harvest (77 days after planting). At 112 days after planting, the dry weight of roots was significantly higher in cultivar Detroit than Rhonda and Boldor. Leaf blade length and the length:width ratio were cultivar-dependent, which may facilitate selection for specific fresh markets. Significant associations between canopy reflectance in the near infrared (IR) (830 nm), dry weight of foliage, and number of CLS lesions per leaf suggested that this technique may have utility for remote assessment of these variables in table beet research. Implications of these findings for the management of CLS in table beet are discussed.

Management of Cercospora Leaf Spot in Conventional and Organic Table Beet Production.

Cercospora leaf spot (CLS; Cercospora beticola) is the most important foliar disease affecting table beet. Epidemics occur annually and fungicides extend the survival of foliage to enable mechanized harvest. However, a high frequency of strobilurin-resistant C. beticola isolates necessitates the identification of fungicides with different modes of action for tactical rotation. There is also substantial demand for organically produced table beet, for which synthetic fungicides are prohibited. Five small-plot, replicated field trials were conducted over two years to evaluate conventional and Organic Materials Review Institute (OMRI)-listed products for CLS control in table beet cv. Ruby Queen at Geneva and Ithaca, New York. Benzovindiflupyr + difenoconazole significantly reduced temporal disease progress (measured by the area under the disease progress stairs; AUDPS) by 86.7 to 97.3% compared with nontreated plots, and mean survival time of leaves was significantly extended. The demethylation inhibitor, propiconazole, also provided significant disease control in two trials in 2016. Disease severity in plots treated with succinate dehydrogenase inhibitors (boscalid, fluxapyroxad + pyraclostrobin, and penthiopyrad) was significantly decreased compared with nontreated plots but less than other fungicides. Efficacious fungicides significantly increased the dry weight of foliage but did not significantly affect the dry weight of roots, and root shoulder diameter. The enhanced longevity of leaves and increased dry weight of foliage may extend opportunities for mechanized harvesting without deleteriously affecting root yield parameters which are strictly regulated for the processing markets. In two trials, copper octanoate + Bacillus amyloliquefaciens strain D747 (as Cueva + Double Nickel LC) resulted in significantly improved disease control in comparison with application of either product alone and provided comparable and reproducible disease control equivalent to conventional fungicides at both locations. The implications of these findings for CLS control in conventional and organic table beet production systems are discussed.

Molecular and experimental evidence of multi-resistance of Cercospora beticola field populations to MBC, DMI and QoI fungicides

Cercospora leaf spot (CLS) caused by Cercospora beticola occurs annually in Serbia causing severe yield losses of sugar beet, which requires intensive use of fungicides. In recent years we have observed unsatisfactory control of CLS originating from northwestern Serbia. Frequency of C. beticola populations resistant to Quinone outside inhibitors (QoI) was 81% (51/63 isolates), 98% (62/63) to sterol-demethylation inbibitors (DMI) and 54% (34/63) to methyl-2-benzimidazole carbamate fungicides (MBC). The genetic basis underlying the resistance was tested by characterizing the cob, CYP51 and s-tubulin genes, associated with resistance to QoI, DMI and MBC fungicides, respectively. Isolates that were resistant to QoI fungicides had the G143A mutation in the cob gene. Characterization of the CYP51 gene revealed seven diverse haplotypes; however, no correlation with sensitivity or resistance to DMI fungicides could be identified. Resistance to MBC fungicides was associated with the presence of the E198A mutation in the s-tubulin gene of all resistant isolates. From a total of 63 isolates originating from sugar beet fields of northwestern Serbia, 62 isolates showed resistance to multiple modes of action. Three multi-resistant phenotypes were identified: MR1 (N = 29) - resistant to QoI and DMI fungicides (QoI-R and DMI-R) but sensitive to MBC fungicides (MBC-S); MR2 (N = 11, QoI-S, DMI-R and MBC-R); and MR3 (N = 22), resistant to all three groups of fungicides (QoI-R, DMI-R and MBC-R). This is the first report of C. beticola resistance to QoI fungicides in Serbia. This study revealed development of multi-resistance of C. beticola isolates to MBC, DMI and QoI fungicides, which represents the first record of this phenomenon in C. beticola populations.

Molecular analysis of Cercospora beticola isolates for strobilurin resistance from the Central High Plains, USA

Cercospora leaf spot (CLS), caused by Cercospora beticola, is the most destructive foliar disease and is a problem in sugar beet production areas, such as Central High Plains (states of Colorado, Montana, Nebraska and Wyoming) in the United States. The disease can be controlled by strobilurin fungicides, referred to as quinone outside inhibitors (QoIs), with a single target site on C. beticola. Strobilurin resistance has been reported in beet production areas from the United States, including the Central High Plains. Although strobilurin resistance is quantitatively inherited, it is considered that it has low to medium heritability in the population. Effective diagnostic tools are required for the rapid detection of C. beticola strobilurin resistance. The study obtained a partial nucleotide sequence of the C. beticola cytochrome b gene and determined to a putative protein with ~386 amino acid residues. Eighty C. beticola isolates (2004–2011) from the Central High Plains were analyzed for mutations. We found a single nucleotide polymorphic (SNP) site which led to G143A mutation and was present in 2 C. beticola QoI-resistant isolates. Partial sequences obtained from 82 C. beticola QoI-sensitive isolates showed identical cytochrome b gene. We developed a PCR-RFLP assay that involved an in vitro digestion using Fnu4HI restriction enzyme for the rapid molecular detection of G143A mutation in the C. beticola population. Results indicated the PCR-RFLP assay was reliable, sensitive, and can be used for the rapid detection of C. beticola strobilurin resistance.

Genotypic Diversity and Resistance to Azoxystrobin of Cercospora beticola on Processing Table Beet in New York.

Cercospora leaf spot (CLS), caused by Cercospora beticola, is one of the major diseases affecting productivity and profitability of beet production worldwide. Fungicides are critical for the control of this disease and one of the most commonly used products is the quinone outside inhibitor (QOI) azoxystrobin. In total, 150 C. beticola isolates were collected from two commercial processing table beet fields in Batavia, NY in 2014. The mating types of the entire population were determined, and genetic diversity of a subset of samples (n = 48) was assessed using five microsatellite loci. Sensitivity to azoxystrobin was tested using a spore germination assay. The cytochrome b gene was sequenced to check for the presence of point mutations known to confer QOI resistance in fungi. High allelic diversity (He = 0.50) and genotypic diversity (D* = 0.96), gametic equilibrium of the microsatellite loci, and equal ratios of mating types were suggestive of a mixed mode of reproduction for C. beticola. Resistance to azoxystrobin was prevalent because 41% of the isolates had values for effective concentrations reducing spore germination by 50% (EC50) > 0.2 μg/ml. The G143A mutation, known to cause QOI resistance in C. beticola, was found in isolates with EC50 values between 0.207 and 19.397 μg/ml. A single isolate with an EC50 of 0.272 μg/ml carried the F129L mutation, known to be associated with low levels of QOI resistance in fungi. This is the first report of the F129L mutation in C. beticola. The implications of these findings for the epidemiology and control of CLS in table beet fields in New York are discussed.

Occurrence of Cercospora beticola populations resistant to benzimidazoles and demethylation-inhibiting fungicides in Serbia and their impact on disease management

The emergence of Cercospora beticola populations that are resistant to benzimidazoles (MBC) and demethylation-inhibiting fungicides (DMI) has been recently reported in Serbia and has resulted in a reduced efficacy of fungicides in controlling Cercospora leaf spot (CLS). Between 2008 and 2011, using a discriminatory concentration method in sugar beet fields in two separate regions of Serbia, we determined that 93.3%–98.6% of collected C. beticola isolates were resistant to MBCs, whereas 6.2%–42.4% were resistant to DMI fungicides. At the same localities, field trials were conducted to investigate the impact of resistant C. beticola populations on disease management. From the MBC group of fungicides, both thiophanate methyl and carbendazim failed to suppress the spread of CLS at both of the tested localities. Between 2008 and 2010, DMI fungicides expressed moderate efficacy at a South Banat locality (79.8%–84.6%) whether they were applied individually (flutriafol, epoxiconazole) or in combination with MBCs (epoxiconazole/carbendazim, thiophanate-methyl/epoxiconazole). The frequency of resistant isolates in these test trials ranged from 6.2% to 10.9%. In 2011, at the same locality, conditions were observed to change in favor of the occurrence of resistant populations, which comprised up to 18.7% of the population, at the expense of DMI efficacy in CLS management. At a Srem locality, the frequency of C. beticola isolates that were resistant to DMIs was high during all four years of field testing (30.5%–42.4%), and the efficacy of these fungicides ranged from 48.4% to 68.0%. A combination of DMI and a protective chlorothalonil had a stable, moderate impact on disease management regardless of the frequency of DMI resistance, whereas a combination of the cyproconazole DMI with trifloxystrobin from the strobilurin group of fungicides expressed the highest efficacy. High correlation coefficient values (r = 0.87) indicated how strongly the frequencies of resistant populations affected disease severity in the trial plots that were treated with carbendazim and thiophanate methyl, as well as in the plots that were treated with flutriafol and epoxiconazole (r = 0.98). In shift sensitivity trials, MBC-resistant C. beticola isolates were found at equally high frequencies both before and after the treatments, indicating a complete loss of efficacy in CLS control. This test revealed the significant impact of multiple DMI applications in terms of increasing the frequency of resistant C. beticola populations following treatments with flutriafol, epoxiconazole, epoxiconazole/carbendazim, thiophanate-methyl/epoxiconazole and flutriafol/chlorothalonil, except when used in combination with trifloxystrobin, in which case CLS suppression was substantially high.

Przyczyny odporności izolatów Cercospora beticola (chwościk buraka) na strobiluryny w Wielkopolsce

Leaf spot caused by Cercospora beticola is one of the most serious diseases of sugar beet. Various fungicide groups are used to control this disease and among them also strobilurin based products. The aim of the study was to analyze the causes of strobilurin resistance of C. beticola isolates collected in Wielkopolska region. The strobilurin inhibits mitochondrial respiratory processes and mutations occurring in the cytochrome b gene (cytb) are the most important reason of C. beticola strobilurin resistance. An increasing activity of an alternative oxidase (AOX) also plays an important role in resistance occurring in C. beticola isolates. The sequence of cytochrome b gene of C. beticola isolates were analyzed. Mutations G143 and F129L – the main determinants of strobilurin resistance were no found. However, an increased strobilurin resistance level of the isolates is caused by higher activity of alternative oxidase, which replaces the function of cytochrome b in the respiratory pathway. Chwościk wywoływany przez Cercospora beticola należy do najpoważniejszych chorób buraka cukrowego. Do ochrony upraw buraka wykorzystywane są fungicydy z różnych grup chemicznych, także strobiluryny, które hamują procesy oddechowe zachodzące w mitochondriach. Mutacje pojawiające się w genie cytochromu b (cytb) są najważniejszą przyczyną nabywania odporności na strobiluryny przez izolaty C. beticola. Wśród pozostałych przyczyn odporności wymienia się wzrost aktywności alternatywnej oksydazy. Celem badań była analiza przyczyn podwyższonej odporności na piraklostrobinę izolatów C. beticola zbieranych na terenie Wielkopolski. W badanym fragmencie genu cytochromu b izolatów C. beticola nie stwierdzono mutacji G143A oraz F129L, które wymieniane są wśród podstawowych czynników warunkujących odporność na strobiluryny. Zwiększona odporność badanych izolatów spowodowana jest wzmożoną aktywnością alternatywnej oksydazy, zastępującej funkcję cytochromu b w szlaku oddechowym.

Odporność na fungicydy izolatów Cercospora beticola pochodzących z terenu Wielkopolski

Leaf spot caused by Cercospora beticola is one of the most serious disease of sugar beet. For the disease control benzimidazole, triazole, strobilurine, morpholine and pyrimidine fungicides have been used. The aim of the study was to evaluate the resistance level of C. beticola isolates collected in Wielkopolska region to selected fungicides. Most of the tested isolates were resistant to carbendazim and thiophanate-methyl, and only a few of the isolates were sensitive to benzimidazoles. Most of the tested isolates were sensitive to the other tested fungicides. However, several isolates resistant to triazoles and piraclostrobin were identified. Chwościk wywoływany przez Cercospora beticola należy do najważniejszych chorób buraka cukrowego. Do ochrony upraw buraka wykorzystywane są fungicydy zawierające związki z grup benzimidazoli, triazoli, strobiluryn, morfolin i pirymidyn. Celem badań była ocena odporności izolatów C. beticola zbieranych na terenie Wielkopolski na wybrane fungicydy. Większość badanych izolatów C. beticola była odporna na karbendazym i tiofanat metylu, jedynie kilkanaście spośród badanych izolatów było wrażliwych na benzimidazole. Większość badanych izolatów była wrażliwa na pozostałe z badanych fungicydów. Wśród badanych izolatów zidentyfikowane zostały także szczepy odporne na triazole i piraklostrobinę.

CbCTB2, an O-methyltransferase is essential for biosynthesis of the phytotoxin cercosporin and infection of sugar beet by Cercospora beticola

BackgroundCercospora leaf spot disease, caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugar beets (Beta vulgaris) worldwide. Cercosporin, a light-inducible toxin, is essential for necrosis of the leaf tissue and development of the typical leaf spots on sugar beet leaves.ResultsIn this study we show that the O-methyltransferase gene CTB2 is essential for cercosporin production and pathogenicity in two C. beticola isolates. We established a transformation system for C. beticola protoplasts, disrupted CTB2, and transformed the Δctb2 strains as well as a wild type strain with the DsRed reporter gene. The Δctb2 strains had lost their pigmentation and toxin measurements demonstrated that the Δctb2 strains were defective in cercosporin production. Infection of sugar beets with the wild type and Δctb2 DsRed strains showed that the deletion strain was severely impaired in plant infection. Histological analysis revealed that the CTB2-deficient isolate cannot enter the leaf tissue through stomata like the wild type.ConclusionsTaken together, these observations indicate that cercosporin has a dual function in sugar beet infection: in addition to the well-known role in tissue necrosis, the toxin is required for the early phase of sugar beet infection.