Cercospora Beticola Research Articles

Fungicide Sensitivity Monitoring of Alternaria spp. Causing Leaf Spot of Sugarbeet (Beta vulgaris) in the Upper Great Lakes.

Alternaria leaf spot (ALS), caused by Alternaria spp., can occur wherever sugarbeet is grown. Infection by Alternaria spp. and disease management has historically been considered a minor issue in sugarbeet production in the United States. An increase of both incidence and severity in 2016 of ALS high enough to cause yield loss has been observed in Michigan. With a renewed need to consider potential management of this disease, the sensitivity was determined for populations of Alternaria spp. to three classes of fungicides currently labeled for management of leaf spot on sugarbeet, including demethylase inhibitor (DMI), quinone outside inhibitor (QoI), and organo-tin fungicides. Leaves with symptoms of ALS were sampled from sugarbeet fields in east-central Michigan and southwestern Ontario, Canada. Monoconidial isolates were obtained to determine sensitivity to each fungicide class above. A spiral gradient dilution method was used to estimate the fungicide effective concentration (in milligrams per liter) that caused a 50% inhibition of fungal growth in vitro for all isolates. Significant temporal shifts were detected in the frequencies of sensitivity phenotypes to DMI and QoI but not organo-tin fungicides from 2016 through 2017. Individual isolates of Alternaria spp. were recovered with cross-resistance to DMI and multiple resistance to DMI, QoI, and triphenyltin hydroxide fungicides. To our knowledge, this is the first report of a fungus other than Cercospora beticola with resistance to organo-tin fungicides. Fungicide sensitivity monitoring indicates that an effective integrated disease management approach combining fungicide efficacy trials and monitoring pathogen biology is essential for developing effective resistance management recommendations.

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.

An improved PCR assay for species-specific detection and quantification of Cercospora beticola

Cercospora beticola causes cercospora leaf spot (CLS) on sugar beet and table beet. Accurate identification of this pathogen is critical to disease diagnosis and effective research outcomes for improved management. Several PCR assays have been described for identifying C. beticola; however, the specificity has either not been adequately tested or cross-reactions with related Cercospora species have occurred. Comparison of the three published assays for specificity to C. beticola indicated that each also amplified DNA from closely related Cercospora species. This study describes the development and subsequent assessment of conventional and quantitative PCR assays specific for detection of C. beticola. Assay specificity was confirmed across a broad range of Cercospora and other common fungal species using public DNA sequence databases and PCR. A conventional PCR assay was designed with fast PCR conditions and completed in under 40 min. The quantitative PCR assay detected 0.001–10 ng of C. beticola DNA. The effectiveness of the quantitative PCR assay to detect C. beticola DNA in diseased leaf tissue and diseased leaf tissue mixed with soil was also demonstrated. These assays provide an improved method for specific identification and quantification of C. beticola, and a valuable tool for enhancing studies into the biology of C. beticola and epidemiology of CLS.

Alternative Hosts of Cercospora beticola in Field Surveys and Inoculation Trials.

Cercospora beticola, the cause of Cercospora leaf spot (CLS) of sugar beet and table beet, has a broad range of potential alternative hosts. The role of these hosts as inoculum sources in the field is unclear and has had limited investigation since the advent of DNA-based pathogen identification. The presence of C. beticola on alternative hosts associated with table beet fields of New York was assessed in field surveys during 2016. Lesions were collected, and 71 cercosporoid conidia were isolated for phylogenetic comparison. C. beticola was identified from Solanum ptycanthum (n = 4), Chenopodium album (n = 2), and Spinacia oleracea (n = 1), whereas C. chenopodii was identified on Chenopodium album (n = 51). Artificial inoculation of 21 plants species demonstrated that C. beticola was pathogenic to Brassica kaber, Chenopodium album, Carthamus tinctorius, Rumex obtusifolius, and Spinacia oleracea. These results indicate that although C. beticola may be pathogenic to a range of plant species, the role of symptomatic tissue for inoculum production on alternative hosts in the field appears limited. Observations of C. beticola on necrotic and naturally senescent tissue suggest saprophytic survival on plant debris of a range of species, which has implications for CLS epidemics and disease management.

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.

A LysM effector protein from the basidiomycete Rhizoctonia solani contributes to virulence through suppression of chitin-triggered immunity

Rhizoctonia solani is a fungal species that belongs to the fungal division Basidiomycota. It is a soil-borne pathogen that attacks a broad range of plant species and crops. Disease symptoms are commonly seen as damping off of seedlings and root rot, although it can infect plants at any developmental stage. Despite the severity of this disease, many aspects in R. solani infection biology remain unclear. Here we investigated the role of a LysM effector, previously predicted from the genome of a R. solani AG2-2IIIB strain that has sugar beet as a host. Gene expression analysis showed that RsLysM was highly induced upon sugar beet infection. When RsLysM was heterologously expressed in Cercospora beticola, necrotic lesion size and fungal colonization ability were increased, indicating a role in virulence. RsLysM displayed chitin-binding affinity and suppression of chitin-triggered immunity but could not protect hyphae from hydrolysis. Overall, this study is the first characterization of a LysM effector from Basidiomycota, suggesting that this necrotrophic fungal species relies on perturbation of chitin-triggered immunity to establish a successful infection.

Mass spectrometry-based metabolomic discrimination of Cercospora leaf spot resistant and susceptible sugar beet germplasms

A better understanding of the plant metabolites produced in response to disease infection may be useful for the development of disease-resistant crop varieties. In the present study, ultra high-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (QTOF-MS) was used to identify differentially accumulated metabolites in a subset of sugar beet genotypes harbouring different levels of resistance to Cercospora leaf spot (CLS), a disease caused by the fungal pathogen Cercospora beticola. Leaves of three susceptible (S1, S2 and S3) and two resistant (R1 and R2) genotypes were subjected to QTOF-MS for metabolite profiling. A wide range of metabolites was identified in sugar beet genotypes using metabolomics. Results of Partial Least Squares-Discriminant Analysis indicated that 15 metabolites could better discriminate resistant and susceptible genotypes. A Volcano Plot analysis indicated that the flavonoid quercetin 3-O-(6″-O-p-coumaroyl)-glucoside and gibberellin A51 with the highest absolute fold change (FC = 16), were repressed in resistant samples. Among the 3 metabolites (isovitexin-7-O-xyloside, 3-demethylubiquinol-8 and apigenin 7-O-d-glucoside) showing significant up accumulation in CLS-resistant samples, the flavonoid isovitexin-7-O-xyloside (FC = 4825.634) is associated with resistance to infection with fungal species causing the disease in other crops. Although further studies are still necessary to better elucidate the mechanism of resistance, our results suggest that breeders might exclude susceptible plants based on discriminating metabolites without the need for field inoculation tests. The results also create a solid basis for metabolite-associated reverse genetics and single nucleotide polymorphism discovery based on significantly differentially accumulated metabolites, whose identification is a next strategic priority. The results obtained also underline the role of metabolic signature in CLS resistance mechanisms and provide a platform for the metabolic engineering of sugar beet with higher resistance against C. beticola pathogen.

A Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid and Specific Detection of Airborne Inoculum of Uromyces betae (Sugar Beet Rust).

Sugar beet rust disease (causal agent Uromyces betae) represents a serious threat to worldwide sugar beet (Beta vulgaris) crops, causing yield losses of up to 10% in the United Kingdom. Currently, the disease is managed mainly by application of fungicides after rust disease symptoms appear. Development of a future forecasting system, incorporating data on environmental factors and U. betae inoculum levels, would enable better disease control by more targeted application of fungicides. In this study, we developed a first molecular diagnostic, targeted to cytochrome b DNA sequences and based on loop-mediated isothermal amplification (LAMP) technology, for rapid (<30 min) and specific detection of U. betae. The new assay only detected U. betae strains (collected from across eastern England, the main sugar beet growing region in the United Kingdom) and Denmark; it did not detect other closely related pathogens (e.g., Puccinia sp., U. fabae) or others that are commonly found on sugar beet (Cercospora beticola, Erysiphe betae, Ramularia beticola). The assay could consistently detect down to small amounts of U. betae DNA (10 pg). Application of the new LAMP diagnostic to air spore tape samples collected between mid-June and mid-September from a single U.K. sugar beet field site revealed differences in temporal patterns of pathogen inoculum between the 2015 and 2016 seasons. The described LAMP assay could now be used as a component of a future automated inoculum-based forecasting system, enabling more targeted control of sugar beet rust disease.

Optimizing management of cercospora leaf spot (Cercospora beticola) of sugarbeet in the wake of fungicide resistance

Cercospora leaf spot (CLS), caused by Cercospora beticola, is the most detrimental disease of sugar beet in temperate climates. In the Great Lakes region, CLS was well managed by pyraclostrobin-based programmes using the decision support tool BEETcast™. Due to C. beticola resistance to pyraclostrobin in the region, other groups of fungicides were evaluated. Field experiments were conducted at six sites from 2013 to 2015 in Pain Court (PC) and Ridgetown (RT), Ontario. BEETcast™ application schedules for prothioconazole and mancozeb as well as carrier volume (115 and 235 L ha−1) were compared with label-based (calendar) applications. When disease intensity was high, the conservative calendar application schedule reduced the standardized area under the disease progress curve (sAUDPC) by an average of 84% compared with all BEETcast™ schedules, and the BEETcast™ 50/35 schedule (235 L ha−1) reduced sAUDPC by 48% compared with the 55/50 schedules. However, there were fewer differences among application schedules at sites with low or moderate disease intensity. BEETcast™ application schedules reduced the number of fungicide applications by 34–55%. All application schedules increased sucrose (%) and recoverable white sugar (RWS) to an equivalent level, but none of the schedules increased profit margin compared with the non-treated control. Using a carrier volume of 235 L ha−1 reduced sAUDPC by 28% compared with 115 L ha−1 at one site when disease severity exceeded 95% in non-treated control plots. Thus, management recommendations should consider that CLS severity is reduced by application schedules using a shortened interval and appropriate carrier volume, but this does not necessarily result in higher beet or sucrose yield or increases in profit margin.

Ocena zasadności stosowania tiofanatu metylowego w zwalczaniu chwościka buraka (Cercospora beticola Sacc.) na podstawie analizy RFLP

Ocena zasadności stosowania tiofanatu metylowego w zwalczaniu chwościka buraka (Cercospora beticola Sacc.) na podstawie analizy RFLP

Chwościk buraka cukrowego (Cercospora beticola Sacc.) Część II. Zapobieganie występowaniu, ochrona przed chwościkiem, odporność na fungicydy

Chwościk buraka cukrowego (Cercospora beticola Sacc.) Część II. Zapobieganie występowaniu, ochrona przed chwościkiem, odporność na fungicydy

Chwościk buraka cukrowego (Cercospora beticola Sacc.) Część I. Biologia i występowanie

Chwościk buraka cukrowego (Cercospora beticola Sacc.) Część I. Biologia i występowanie

Induction of Induced Systemic Resistance in Fodder Beet (Beta vulgaris L.) to Cercospora Leaf Spot Caused by (Cercospora beticola Sacc.)

Induction of Induced Systemic Resistance in Fodder Beet (Beta vulgaris L.) to Cercospora Leaf Spot Caused by (Cercospora beticola Sacc.)

Gene cluster conservation identifies melanin and perylenequinone biosynthesis pathways in multiple plant pathogenic fungi.

SummaryPerylenequinones are a family of structurally related polyketide fungal toxins with nearly universal toxicity. These photosensitizing compounds absorb light energy which enables them to generate reactive oxygen species that damage host cells. This potent mechanism serves as an effective weapon for plant pathogens in disease or niche establishment. The sugar beet pathogen Cercospora beticola secretes the perylenequinone cercosporin during infection. We have shown recently that the cercosporin toxin biosynthesis (CTB) gene cluster is present in several other phytopathogenic fungi, prompting the search for biosynthetic gene clusters (BGCs) of structurally similar perylenequinones in other fungi. Here, we report the identification of the elsinochrome and phleichrome BGCs of Elsinoë fawcettii and Cladosporium phlei, respectively, based on gene cluster conservation with the CTB and hypocrellin BGCs. Furthermore, we show that previously reported BGCs for elsinochrome and phleichrome are involved in melanin production. Phylogenetic analysis of the corresponding melanin polyketide synthases (PKSs) and alignment of melanin BGCs revealed high conservation between the established and newly identified C. beticola, E. fawcettii and C. phlei melanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs in C. beticola and E. fawcettii coupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.

Determination of Sugar Beet Leaf Spot Disease Level (Cercospora Beticola Sacc.) with Image Processing Technique by Using Drone

Determination of Sugar Beet Leaf Spot Disease Level (Cercospora Beticola Sacc.) with Image Processing Technique by Using Drone

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.

Challenges and Prospects for Building Resilient Disease Management Strategies and Tactics for the New York Table Beet Industry

The New York table beet industry is expanding and has unique challenges to minimize crop loss in both conventional and organic production. Diseases may reduce plant population density and increase heterogeneity in a stand, reduce the duration of time foliage is healthy, and decrease the yield of marketable roots. Rhizoctonia solani Kuhn and Pythiumultimum Trow are dominant in the pathogen complex affecting crop stand and root health. Cercospora leaf spot (CLS) caused by the fungus, Cercospora beticola Sacc., is a highly destructive disease affecting foliar health. In conventional table beet production, fungicides are applied in-furrow and at emergence for early season and root disease control, and applied to foliage periodically thereafter for foliar disease control. Resistance within C. beticola populations to single-site mode-of-action fungicides poses the most significant threat to the resilience of conventional disease management. An integrated approach to reduce pesticide application when not economically warranted (i.e., a false positive) is urgently required. For foliar disease, improved scheduling of fungicides may reduce usage without loss of disease control. For soilborne diseases, pre-plant quantification of soilborne inoculum may support the selection of fields with lower inoculum densities to minimize risk of early season and root disease. For organic production, some approved products have moderate efficacy for foliar disease control, but strategies to reduce inoculum and select fields at lowest risk of disease will be paramount. Crop rotation has shown promise for disease management, but broad host range of several of the major soilborne pathogens limits the utility of this method in the production region. Enhanced knowledge of cultivar susceptibility to local populations of fungal pathogens responsible for foliar and root diseases is paramount, and adoption of commercially acceptable cultivars with improved resistance to CLS and Rhizoctonia crown and root rot has potential to transform disease management strategies for the New York table beet industry.

First report of benzimidazole, DMI and QoI‐insensitive Cercospora beticola in sugar beet in Morocco

Sugar beet (Beta vulgaris) is one of most important industrial crops in Morocco. In 2016, the cultivated area exceeded 61,000 ha and it's anticipated that this area will increase to 77,500 ha by 2020. However, sugarbeet yield is often…

Evaluation of certain Penicillium frequentans isolates against Cercospora leaf spot disease of sugar beet

The impact of six local isolates of Penicillium frequentans recovered from healthy sugar beet (Beta vulgaris L.) leaves was evaluated against Cercospora beticola, the causal pathogen of Cercospora sugar beet leaf spot under laboratory and field conditions. In in vitro studies, all the six isolates were able to inhibit the mycelial growth of C. beticola with variation in their antagonistic capability. P. frequentans isolates produce pectinase and cellulase at different degrees. There was a correlation between enzyme activity and the antagonistic ability for each isolate. The high antagonistic ability isolates had the most enzyme activity. In field studies, some adhesives such as agar, starch flour, white glue, gum, and commercial adhesive (Triton Mok) were added to conidia spore suspensions of P. frequentans at 1% to improve conidial adhesion to sugar beet plant surface. Data also showed that all adhesives increased (P = 0.05) the efficiency of the spore suspension of P. frequentans to control the disease. The starch flour at 1% gave a significant reduction in disease severity from 43.23 to 10.42% pre-infection and from 43.23 to 10.52% post-infection. The application of P. frequentans led to improved root yield and the sugar percent of sugar beet crop in two tested seasons.

Draft genome sequence of Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina, and Morchella septimelata

Draft genomes of the species Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina and Morchella septimelata are presented. Both mating types (MAT1-1 and MAT1-2) of Cercospora beticola are included. Two strains of Coleophoma cylindrospora that produce sulfated homotyrosine echinocandin variants, FR209602, FR220897 and FR220899 are presented. The sequencing of Aspergillus mulundensis, Coleophoma cylindrospora and Phialophora cf. hyalina has enabled mapping of the gene clusters encoding the chemical diversity from the echinocandin pathways, providing data that reveals the complexity of secondary metabolism in these different species. Overall these genomes provide a valuable resource for understanding the molecular processes underlying pathogenicity (in some cases), biology and toxin production of these economically important fungi.