Search

Cercospora beticola is a hemibiotrophic fungus that causes cercospora leaf spot disease of sugar beet (Beta vulgaris). After an initial symptomless biotrophic phase of colonization, necrotic lesions appear on host leaves as the fungus switches to a necrotrophic lifestyle. The phytotoxic secondary metabolite cercosporin has been shown to facilitate fungal virulence for several Cercospora spp. However, because cercosporin production and subsequent cercosporin‐initiated formation of reactive oxygen species is light‐dependent, cell death evocation by this toxin is only fully ensured during a period of light. Here, we report the discovery of the effector protein CbNip1 secreted by C. beticola that causes enhanced necrosis in the absence of light and, therefore, may complement light‐dependent necrosis formation by cercosporin. Infiltration of CbNip1 protein into sugar beet leaves revealed that darkness is essential for full CbNip1‐triggered necrosis, as light exposure delayed CbNip1‐triggered host cell death. Gene expression analysis during host infection shows that CbNip1 expression is correlated with symptom development in planta. Targeted gene replacement of CbNip1 leads to a significant reduction in virulence, indicating the importance of CbNip1 during colonization. Analysis of 89 C. beticola genomes revealed that CbNip1 resides in a region that recently underwent a selective sweep, suggesting selection pressure exists to maintain a beneficial variant of the gene. Taken together, CbNip1 is a crucial effector during the C. beticola–sugar beet disease process., The fungus Cercospora beticola secretes necrosis‐inducing molecules during infection of sugar beet that are optimized to be fully functional during the day or night.

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most destructive disease of sugar beet worldwide. Although growing CLS-tolerant varieties is helpful, disease management currently requires timely application of fungicides. However, overreliance on fungicides has led to the emergence of fungicide resistance in many C. beticola populations, resulting in multiple epidemics in recent years. Therefore, this study focused on developing a fungicide resistance detection “toolbox” for early detection of C. beticola in sugar beet leaves and mutations associated with different fungicides in the pathogen population. A loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of C. beticola in infected sugar beet leaves. The LAMP primers specific to C. beticola (Cb-LAMP) assay was able to detect C. beticola in inoculated sugar beet leaves as early as 1 day postinoculation. A quinone outside inhibitor (QoI)-LAMP assay was also developed to detect the G143A mutation in cytochrome b associated with QoI resistance in C. beticola. The assay detected the mutation in C. beticola both in vitro and in planta with 100% accuracy. We also developed a probe-based quantitative PCR (qPCR) assay for detecting an E198A mutation in β-tubulin associated with benzimidazole resistance and a probe-based qPCR assay for detection of mutations in cytochrome P450-dependent sterol 14α-demethylase (Cyp51) associated with resistance to sterol demethylation inhibitor fungicides. The primers and probes used in the assay were highly efficient and precise in differentiating the corresponding fungicide-resistant mutants from sensitive wild-type isolates.

Plants counter disease with an array of responses to styme pathogen ingress. In contrast to this cacophony, plant pathogens orchestrate a finely tuned repertoire of virulence mechanisms in their attempt to cause disease. One such example is the production of secondary metabolite effectors (SMEs). Despite many attempts to functionally categorize SMEs, their many roles in plant disease have proven they march to the beat of their producer's drum. Some lesser studied features of SMEs in plant disease include self-resistance (SR) and manipulation of the microbiome to enhance pathogen virulence. SR can be accomplished in three general compositions, with the first being the transport of the SME to a benign location; the second being modification of the SME so it cannot harm the producer; and the third being metabolic regulation of the SME or the producer homolog of the SME target. SMEs may also play an interlude prior to disease by shaping the plant microbial community, allowing producers to better establish themselves. Taken together, SMEs are integral players in the phytopathology canon.

Cercospora leaf spot (CLS) is a globally important disease of sugar beet (

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.

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.

Cercospora leaf spot is the most important disease of sugar beet worldwide. The disease is caused by the fungus Cercospora beticola and is managed principally by timely application of fungicides including those of the sterol demethylation inhibitor (DMI) class. However, reliance on DMIs has caused an increase in resistance to this class of fungicides in multiple C. beticola populations. To better understand the genetic and evolutionary basis for resistance in C. beticola, a genome-wide association study (GWAS) and selective sweep analysis were conducted for the first time in this fungal plant pathogen. We performed whole genome resequencing of 190 C. beticola isolates predominantly from North Dakota and Minnesota that were phenotyped for sensitivity to tetraconazole, the most widely used DMI fungicide in this region. GWAS identified mutations in genes associated with DMI fungicide resistance including a Regulator of G-protein Signaling (RGS) protein, an ATP-binding cassette (ABC) pleiotropic drug resistance transporter, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK), and a gene annotated as a hypothetical protein. A SNP upstream of CbCYP51, the gene encoding the target of DMI fungicides, was also identified via GWAS. Haplotype analysis of CbCYP51 identified a synonymous mutation (E170) in high linkage disequilibrium with the upstream SNP, and multiple non-synonymous mutations (L144F, I387M and Y464S) associated with DMI resistance. Additionally, a putative codon bias effect for the L144F substitution was identified that generated different resistance potentials. We also identified a CbCYP51 paralog in C. beticola, CbCYP51-like, with high protein homology to CYP51C found uniquely in Fusarium species but CbCYP51-like does not appear to influence DMI sensitivity. 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 suggesting that resistance mutations can persist in C. beticola populations. 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.

Cercospora leaf spot (CLS), caused by the fungal pathogen

Cercospora leaf spot, caused by the fungal pathogen Cercospora beticola, is the most destructive foliar disease of sugar beet worldwide. This review discusses C. beticola genetics, genomics, and biology and summarizes our current understanding of the molecular interactions that occur between C. beticola and its sugar beet host. We highlight the known virulence arsenal of C. beticola as well as its ability to overcome currently used disease management strategies. Finally, we discuss future prospects for the study and management of C. beticola infections in the context of newly employed molecular tools to uncover additional information regarding the biology of this pathogen. Taxonomy Cercospora beticola Sacc.; Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes, Order Capnodiales, Family Mycosphaerellaceae, Genus Cercospora. Host range Well‐known pathogen of sugar beet (Beta vulgaris subsp. vulgaris) and most species of the Beta genus. Reported as pathogenic on other members of the Chenopodiaceae (e.g., lamb's quarters, spinach) as well as members of the Acanthaceae (e.g., bear's breeches), Apiaceae (e.g., Apium), Asteraceae (e.g., chrysanthemum, lettuce, safflower), Brassicaceae (e.g., wild mustard), Malvaceae (e.g., Malva), Plumbaginaceae (e.g., Limonium), and Polygonaceae (e.g., broad‐leaved dock) families. Disease symptoms Leaves infected with C. beticola exhibit circular lesions that are coloured tan to grey in the centre and are often delimited by tan‐brown to reddish‐purple rings. As disease progresses, spots can coalesce to form larger necrotic areas, causing severely infected leaves to wither and die. At the centre of these spots are black spore‐bearing structures (pseudostromata). Older leaves often show symptoms first and younger leaves become infected as the disease progresses. Management Application of a mixture of fungicides with different modes of action is currently performed although elevated resistance has been documented in most employed fungicide classes. Breeding for high‐yielding cultivars with improved host resistance is an ongoing effort and prudent cultural practices, such as crop rotation, weed host management, and cultivation to reduce infested residue levels, are widely used to manage disease. Useful website https://www.ncbi.nlm.nih.gov/genome/11237?genome_assembly_id=352037, The hemibiotrophic fungus Cercospora beticola applies various virulence strategies to infect sugar beet and is currently only managed in‐field through integrated practices.

Jasmonic acid is a natural plant hormone that induces native defense responses in plants. Sugarbeet (Beta vulgaris L.) root unigenes that were differentially expressed 2 and 60 days after a postharvest jasmonic acid treatment are presented. Data include changes in unigene expression relative to water-treated controls, unigene annotations against nonredundant (Nr), Swiss-Prot, Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases, and unigene annotations with Gene Ontology (GO) terms. Putative defense unigenes are compiled and annotated against the sugarbeet genome. Differential gene expression data were generated by RNA sequencing. Interpretation of the data is available in the research article, “Jasmonic acid causes short- and long-term alterations to the transcriptome and the expression of defense genes in sugarbeet roots” (K.K. Fugate, L.S. Oliveira, J.P. Ferrareze, M.D. Bolton, E.L. Deckard, F.L. Finger, 2017) [1]. Public dissemination of this dataset will allow further analyses of the data.

Cercospora leaf spot caused by Cercospora beticola is a significant threat to the production of sugar and table beet worldwide. A de novo genome assembly of C. beticola was used to develop eight polymorphic and reproducible microsatellite markers for population genetic analyses. These markers were used, along with five previously described microsatellite loci to genotype two C. beticola populations from table beet fields in New York, USA. High allelic and genotypic diversity and low population differentiation were found between fields. Linkage disequilibrium of loci after clone-correction of datasets was attributed to the presence of two distinct clonal lineages within the populations. Linkage equilibrium of loci in one of the clusters supported the presence of sexual reproduction. The draft de novo genome assembly will help elucidate the reproductive system of C. beticola through investigating evidence of recombination in the C. beticola genome.

Jasmonic acid (JA) induces native defense responses in plants and increases the resistance of postharvest sugarbeet roots to three common storage-rot causing organisms. To gain insight into the defense responses induced by JA in harvested sugarbeet roots, RNA was isolated from roots treated with water or 10 μM JA and incubated for 2 or 60 d post-treatment. RNA was sequenced, and sequence data was analyzed for short-term (2 d) and long-term (60 d) effects of JA on the sugarbeet root transcriptome. A total of 283 and 326 differentially expressed unigenes were identified in JA-treated roots at 2 and 60 d after treatment, respectively. Of these, > 96% were uniquely altered in expression at 2 or 60 d, indicating that long-term JA-induced changes to the sugarbeet root transcriptome were substantially different from short-term JA-induced changes. Categorization of differentially expressed unigenes by gene ontology (GO) or Kyoto Encyclopedia of Genes and Genomes (KEGG) identifiers indicated that JA altered expression of a large number of unigenes involved in metabolism, including many unigenes encoding enzymes and unigenes that participate in secondary metabolism. In addition, 88 unigenes, or 15% of all unigenes whose expression were altered by JA, were putatively involved in plant defense. Included were unigenes for pathogenesis-related proteins, regulatory proteins, plant disease resistance proteins, secondary metabolite biosynthetic enzymes, oxidases, and plant cell wall-modifying proteins. Detailed, time-course expression analysis of 19 JA-altered defense unigenes revealed similarities in expression for three plant disease resistant (R) genes and three laccase genes, indicating possible co-regulation of these genes. No relationships, however, were observed between enzyme activities and transcript levels for any of four major families of upregulated defense genes for which enzyme activities were determined, consistent with JA functioning as a primer, rather than an inducer, of plant defenses. Overall, this research supports JA's role as a signaling molecule for plant defense and provides evidence that a variety of defense mechanisms, including the production of antifungal and antimicrobial compounds, stimulation of antioxidant defenses, and stiffening and strengthening of cell walls, may contribute to JA-induced storage rot resistance in sugarbeet roots.

Beet necrotic yellow vein virus (BNYVV) is the causal agent of rhizomania, a disease of global importance to the sugar beet industry. The most widely implemented resistance gene to rhizomania to date is Rz1, but resistance has been circumvented by resistance-breaking (RB) isolates worldwide. In an effort to gain greater understanding of the distribution of BNYVV and the nature of RB isolates in Minnesota and eastern North Dakota, sugar beet plants were grown in 594 soil samples obtained from production fields and subsequently were analyzed for the presence of BNYVV as well as coding variability in the viral P25 gene, the gene previously implicated in the RB pathotype. Baiting of virus from the soil with sugar beet varieties possessing no known resistance to rhizomania resulted in a disease incidence level of 10.6% in the region examined. Parallel baiting analysis of sugar beet genotypes possessing Rz1, the more recently introgressed Rz2, and with the combination of Rz1 + Rz2 resulted in a disease incidence level of 4.2, 1.0, and 0.8%, respectively. Virus sequences recovered from sugar beet bait plants possessing resistance genes Rz1 and/or Rz2 exhibited reduced genetic diversity in the P25 gene relative to those recovered from the susceptible genotype while confirming the hypervariable nature of the coding for amino acids (AAs) at position 67 and 68 in the P25 protein. In contrast to previous reports, we did not find an association between any one specific AA signature at these positions and the ability to circumvent Rz1-mediated resistance. The data document ongoing virulence development in BNYVV populations to previously resistant varieties and provide a baseline for the analysis of genetic change in the virus population that may accompany the implementation of new resistance genes to manage rhizomania.

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 establishment. The sugar beet pathogenCercospora beticolasecretes 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 ofElsinoё fawcettiiandCladosporium phlei,respectively, based on gene cluster conservation with theCTBand 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 identifiedC. beticola, E. fawcettii,andC. phleimelanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs inC. beticolaandE. fawcettiicoupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.Originality and significance statementGenes involved in secondary metabolite (SM) production are often clustered together to form biosynthetic pathways. These pathways frequently have highly conserved keystone enzymes which can complicate allocation of a biosynthetic gene cluster (BGC) to the cognate SM. In our study, we utilized a combination of comparative genomics, phylogenetic analyses and biochemical approaches to reliably identify BGCs for perylenequinone toxins and DHN-melanin in multiple plant pathogenic fungi. Furthermore, we show that earlier studies that aimed to identify these perylenequinone pathways were misdirected and actually reported DHN-melanin biosynthetic pathways. Our study outlines a reliable approach to successfully identify fungal SM pathways.

Cercospora leaf spot, caused by

Sugar beet (Beta vulgaris L.) and canola (Brassica napus L.) are major crops in North Dakota, with sugar beet production primarily in the eastern part of the state in the Red River Valley and canola production across the northern half of the state. Both crops are hosts of sugar beet cyst nematode (SBCN), Heterodera schachtii Schmidt. In April 2011, soil samples were collected from four sugar beet fields belonging to three growers who believed the fields were infested with SBCN. The fields were located in a 65-km2 area in the Yellowstone Valley of western North Dakota. Cysts were extracted by sieving and Heterodera-like cysts with eggs were observed in all four soil samples. Population densities in the four fields ranged from 100 to 1,750 eggs/100 cm3 soil. Sugar beet seedlings (cv. M832224) were grown in a potting mix for 6 weeks in the greenhouse and then transferred to conetainers (type D40; volume 656 ml) containing autoclaved river sand. Conetainers were placed in sand in plastic pots immersed in a water bath at 27°C. Three plants were each infested with 800 eggs from field No. 2. After 55 days of incubation, the average number of females was 115 per plant. A similar experiment was conducted with canola cvs. Hyclass 940, Caliber 30, and Westar, which were inoculated with 500 eggs each from field No. 2. After 53 days of incubation, there was an average of 39, 20, and 30 females for each respective cultivar. Flask-shaped cysts (n = 26) from canola roots were light to dark brown; the vulval cone was ambifinestrate with dark brown, molar-shaped bullae positioned underneath the vulval bridge. Body length (excluding neck) ranged from 600 to 850 μm (mean 701.2 μm); body width, 350 to 580 μm (mean 469.2 μm); and length/width ratio, 1.2 to 1.8 (mean 1.5). Second-stage juvenile (J2) (n = 21) body length ranged from 400 to 485 μm (mean 437.1 μm); stylet length was 25 μm (no variation) with forwardly directed knobs; conical tail with rounded tip ranged from 37.5 to 55.0 μm long (mean 46.6 μm) with hyaline region from 20.0 to 32.5 μm (mean 27.3 μm); and lateral field presented four incisures. These morphometrics were used to identify H. schachtii according to Subbotin et al. (4). Confirmation of identification was by amplification and sequencing of a 28S rDNA gene fragment (1) from individual females (GenBank Accession No. JQ040526), which was 100% identical to H. schachtii 28S rDNA sequence (GenBank Accession No. GU475088). To our knowledge, this is the first confirmed report of H. schachtii in North Dakota. A 1958 report of SBCN in North Dakota (2) was not subsequently confirmed (3). Because there is extensive canola production across the northern part of the state bordering western and eastern sugar beet- production areas, canola may serve as a bridge for movement of SBCN from west to east. SBCN is a potential threat to these two important crops. References: (1) A. Amiri et al. Eur. J. Plant Pathol. 108:497, 2002. (2) F. Caveness. J. Sugar Beet Res. 10:544, 1958. (3) P. Donald and R. Hosford. Plant Dis. 64:45, 1980. (4) S. A. Subbotin et al. Systematics of Cyst Nematodes (Nematoda: Heteroderinae). Nematology Monographs and Perspectives. Vol. 8B. Brill, The Netherlands. 2010.

Cercospora leaf spot (CLS) of sugar beet is caused by the fungus Cercospora beticola. CLS management practices include the application of the sterol demethylation inhibitor (DMI) fungicides tetraconazole, difenoconazole, and prothioconazole. Evaluating resistance to DMIs is a major focus for CLS fungicide resistance management. Isolates were collected in 1997 and 1998 (baseline sensitivity to tetraconazole, prothioconazole, or difenoconazole) and 2007 through 2010 from the major sugar-beet-growing regions of Minnesota and North Dakota and assessed for in vitro sensitivity to two or three DMI fungicides. Most (47%) isolates collected in 1997–98 exhibited 50% effective concentration (EC50) values for tetraconazole of –1, whereas no isolates could be found in this EC50 range in 2010. Since 2007, annual median and mean tetraconazole EC50 values have generally been increasing, and the frequency of isolates with EC50 values >0.11 μg ml–1 increased from 2008 to 2010. In contrast, the frequency of isolates with EC50 values for prothioconazole of >1.0 μg ml–1 has been decreasing since 2007. Annual median difenoconazole EC50 values appears to be stable, although annual mean EC50 values generally have been increasing for this fungicide. Although EC50 values are important for gauging fungicide sensitivity trends, a rigorous comparison of the relationship between in vitro EC50 values and loss of fungicide efficacy in planta has not been conducted for C. beticola. To explore this, 12 isolates exhibiting a wide range of tetraconazole EC50 values were inoculated to sugar beet but no tetraconazole was applied. No relationship was found between isolate EC50 value and disease severity. To assess whether EC50 values are related to fungicide efficacy in planta, sugar beet plants were sprayed with various dilutions of Eminent, the commercial formulation of tetraconazole, and subsequently inoculated with isolates that exhibited very low, medium, or high tetraconazole EC50 values. The high EC50 isolate caused significantly more disease than isolates with medium or very low EC50 values at the field application rate and most reduced rates. Because in vitro sensitivity testing is typically carried out with the active ingredient of the commercial fungicide, we investigated whether loss of disease control was the same for tetraconazole as for the commercial product Eminent. The high EC50 isolate caused more disease on plants treated with tetraconazole than Eminent but disease severity was not different between plants inoculated with the very low EC50 isolate.

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.

&ldquo, Rhizomania&rdquo, of sugar beet is a soilborne disease complex comprised of beet necrotic yellow vein virus (BNYVV) and its plasmodiophorid vector, Polymyxa betae. Although BNYVV is considered the causal agent of rhizomania, additional viruses frequently accompany BNYVV in diseased roots. In an effort to better understand the virus cohort present in sugar beet roots exhibiting rhizomania disease symptoms, five independent RNA samples prepared from diseased beet seedlings reared in a greenhouse or from field-grown adult sugar beet plants and enriched for virus particles were subjected to RNAseq. In all but a healthy control sample, the technique was successful at identifying BNYVV and provided sequence reads of sufficient quantity and overlap to assemble &gt, 98% of the published genome of the virus. Utilizing the derived consensus sequence of BNYVV, infectious RNA was produced from cDNA clones of RNAs 1 and 2. The approach also enabled the detection of beet soilborne mosaic virus (BSBMV), beet soilborne virus (BSBV), beet black scorch virus (BBSV), and beet virus Q (BVQ), with near-complete genome assembly afforded to BSBMV and BBSV. In one field sample, a novel virus sequence of 3682 nt was assembled with significant sequence similarity and open reading frame (ORF) organization to members within the subgenus Alphanecrovirus (genus Necrovirus, family Tombusviridae). Construction of a DNA clone based on this sequence led to the production of the novel RNA genome in vitro that was capable of inducing local lesion formation on leaves of Chenopodium quinoa. Additionally, two previously unreported satellite viruses were revealed in the study, one possessing weak similarity to satellite maize white line mosaic virus and a second possessing moderate similarity to satellite tobacco necrosis virus C. Taken together, the approach provides an efficient pipeline to characterize variation in the BNYVV genome and to document the presence of other viruses potentially associated with disease severity or the ability to overcome resistance genes used for sugar beet rhizomania disease management.

Species in the genus

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae. Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.

Fungi represent an ecologically diverse group of microorganisms that includes plant pathogenic species able to cause considerable yield loses in crop production systems worldwide. In order to establish compatible interactions with their hosts, pathogenic fungi rely on the secretion of molecules of diverse nature during host colonization to modulate host physiology, manipulate other environmental factors or provide self-defence. These molecules, collectively known as effectors, are typically small secreted cysteine-rich proteins, but may also comprise secondary metabolites and sRNAs. Here, we discuss the most common strategies that fungal plant pathogens employ to subvert their host plants in order to successfully complete their life cycle and secure the release of abundant viable progeny.

Objective The ubiquitous soil pathogen Rhizoctonia solani causes serious diseases in different plant species. Despite the importance of this disease, little is known regarding the molecular basis of susceptibility. SuperSAGE technology and next-generation sequencing were used to generate transcript libraries during the compatible Nicotiana tabacum–R. solani interaction. Also, we used the post-transcriptional silencing to evaluate the function of a group of important genes. Results A total of 8960 and 8221 unique Tag sequences identified as differentially up- and down-regulated were obtained. Based on gene ontology classification, several annotated UniTags corresponded to defense response, metabolism and signal transduction. Analysis of the N. tabacum transcriptome during infection identified regulatory genes implicated in a number of hormone pathways. Silencing of an mRNA induced by salicylic acid reduced the susceptibility of N. tabacum to R. solani. We provide evidence that the salicylic acid pathway was involved in disease development. This is important for further development of disease management strategies caused by this pathogen. Electronic supplementary material The online version of this article (10.1186/s13104-017-2934-9) contains supplementary material, which is available to authorized users.

The taxonomy and evolutionary species boundaries in a global collection of Cercospora isolates from Beta vulgaris was investigated based on sequences of six loci. Species boundaries were assessed using concatenated multi-locus phylogenies, Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Processes (PTP), and Bayes factor delimitation (BFD) framework. Cercospora beticola was confirmed as the primary cause of Cercospora leaf spot (CLS) on B. vulgaris. Cercospora apii, C. cf. flagellaris, Cercospora sp. G, and C. zebrina were also identified in association with CLS on B. vulgaris. Cercospora apii and C. cf. flagellaris were pathogenic to table beet but Cercospora sp. G and C. zebrina did not cause disease. Genealogical concordance phylogenetic species recognition, GMYC and PTP methods failed to differentiate C. apii and C. beticola as separate species. On the other hand, multi-species coalescent analysis based on BFD supported separation of C. apii and C. beticola into distinct species; and provided evidence of evolutionary independent lineages within C. beticola. Extensive intra- and intergenic recombination, incomplete lineage sorting and dominance of clonal reproduction complicate evolutionary species recognition in the genus Cercospora. The results warrant morphological and phylogenetic studies to disentangle cryptic speciation within C. beticola.

This study characterized a novel sugar beet (Beta vulgaris L.) pathogen from the Red River Valley in north central USA, which was formally named Fusarium secorum. Molecular phylogenetic analyses of three loci (translation elongation factor1α, calmodulin, mitochondrial small subunit) and phenotypic data strongly supported the inclusion of F. secorum in the Fusarium fujikuroi species complex (FFSC). Phylogenetic analyses identified F. secorum as a sister taxon of F. acutatum and a member of the African subclade of the FFSC. Fusarium secorum produced circinate hyphae sometimes bearing microconidia and abundant corkscrew-shaped hyphae in culture. To assess mycotoxin production potential, 45 typical secondary metabolites were tested in F. secorum rice cultures, but only beauvericin was produced in detectable amounts by each isolate. Results of pathogenicity experiments revealed that F. secorum isolates are able to induce half- and full-leaf yellowing foliar symptoms and vascular necrosis in roots and petioles of sugar beet. Inoculation with F. acutatum did not result in any disease symptoms. The sugar beet disease caused by F. secorum is named Fusarium yellowing decline. Since Fusarium yellowing decline incidence has been increasing in the Red River Valley, disease management options are discussed.

Jasmonic acid is a natural plant hormone that induces native defense responses in plants. Sugarbeet (Beta vulgaris L.) root unigenes that were differentially expressed 2 and 60 days after a postharvest jasmonic acid treatment are presented. Data include changes in unigene expression relative to water-treated controls, unigene annotations against nonredundant (Nr), Swiss-Prot, Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases, and unigene annotations with Gene Ontology (GO) terms. Putative defense unigenes are compiled and annotated against the sugarbeet genome. Differential gene expression data were generated by RNA sequencing. Interpretation of the data is available in the research article, “Jasmonic acid causes short- and long-term alterations to the transcriptome and the expression of defense genes in sugarbeet roots” (K.K. Fugate, L.S. Oliveira, J.P. Ferrareze, M.D. Bolton, E.L. Deckard, F.L. Finger, 2017) [1]. Public dissemination of this dataset will allow further analyses of the data.

Fusarium spp. are responsible for significant yield losses in sugar beet (Beta vulgaris) with Fusarium oxysporum f. sp. betae most often reported as the primary causal agent. Recently, a new species, F. secorum, was reported to cause disease in sugar beet but little is known on the range of virulence within F. secorum or how this compares to the virulence and phylogenetic relationships previously reported for Fusarium pathogens of sugar beet. To initiate this study, partial translation elongation factor 1‐α (TEF1) sequences from seven isolates of F. secorum were obtained and the data were added to a previously published phylogenetic tree that includes F. oxysporum f. sp. betae. Unexpectedly, the F. secorum strains nested into a distinct group that included isolates previously reported as F. oxysporum f. sp. betae. These results prompted an expanded phylogenetic analysis of TEF1 sequences from genomes of publicly available Fusarium spp., resulting in the additional discovery that some isolates previously reported as F. oxysporum f. sp. betae are F. commune, a species that is not known to be a sugar beet pathogen. Inoculation of sugar beet with differing genetic backgrounds demonstrated that all Fusarium strains have a significant range in virulence depending on cultivar. Taken together, the data suggest that F. secorum is more widespread than previously thought. Consequently, future screening for disease resistance should rely on isolates representing the full diversity of the Fusarium population that impacts sugar beet.

Exogenous application of salicylic acid (SA) reduces storage rots in a number of postharvest crops. SA's ability to protect sugarbeet ( Beta vulgaris L.) taproots from common storage rot pathogens, however, is unknown. To determine the potential of SA to reduce storage losses caused by three common causal organisms of sugarbeet storage rot, freshly harvested roots were treated with 0.01, 0.1, 1.0 or 10 mM SA, inoculated with Botrytis cinerea , Penicillium claviforme , or Phoma betae , and evaluated for the severity of rot symptoms after incubation at 20 °C and 90% relative humidity. Roots were obtained from plants that received sufficient water or were water-stressed prior to harvest. Roots from water-stressed plants were included since water-stress increases sugarbeet root susceptibility to storage rot and SA mitigates drought effects in other plant species. SA at concentrations of 0.01–10 mM had no effect on the severity of storage rot caused by B. cinerea , P. claviforme , or P. betae in roots from plants that received sufficient water prior to harvest. However, SA at these same concentrations reduced the severity of rot symptoms for all three pathogens in roots from plants that were water stressed before harvest. For water-stressed roots, all concentrations of SA produced statistically equivalent reductions in the weight of rotted tissue for each pathogen, and on average, SA reduced rot severity due to B. cinerea , P. claviforme , and P. betae by 54, 45, and 58%, respectively. SA reduced rot from all three pathogens by reducing lesion size, but did not affect the incidence of infection. The ability of SA to reduce rot severity in water-stressed roots, but not in roots that received sufficient water before harvest suggests that SA alleviated the negative impact of water stress but did not directly protect sugarbeet roots against storage rots.

Jasmonic acid (JA) treatment significantly reduces rot due to several sugarbeet (Beta vulgaris L.) storage pathogens. However, the mechanisms by which JA protects postharvest sugarbeet roots from disease are unknown. In other plant species and organs, alterations in antioxidant defense mechanisms and elevations in common pathogenesis-related defense enzymes have been implicated in jasmonate-induced disease resistance. To investigate whether these mechanisms are involved in JA-induced disease resistance in stored sugarbeet roots, the activities of several reactive oxygen species (ROS)-scavenging and pathogenesis-related defense enzymes and the total concentration of antioxidant compounds were determined in harvested sugarbeet roots in the 60 d following treatment with JA. ROS-scavenging and pathogenesis-related defense enzymes and the concentration of antioxidant compounds were largely unaffected by JA as JA-treated roots exhibited small declines in superoxide dismutase (SOD) and chitinase activities, and were generally unaltered in ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), β-1,3-glucanase (β-Gluc), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) activities or antioxidant compounds concentration. The lack of increase in enzyme activities or metabolites related to defense against oxidative stress or pathogens suggests that JA-induced disease resistance in postharvest sugarbeet roots does not arise from a direct increase in any of the ROS-scavenging and defense-related enzymes examined, or the concentration of total antioxidant compounds. However, ROS-scavenging enzymes and pathogenesis-related defense enzymes were affected by storage duration with POD, SOD, β-Gluc, chitinase, and PPO activities elevated and APX and CAT activities reduced in roots stored for 10 d or more. Storage-related changes in activities of ROS-scavenging enzymes and defense-related enzymes provide further evidence that these enzymes are uninvolved in sugarbeet root disease resistance during storage since many of these enzymes increased in activity after prolonged storage when disease resistance generally declines.

Sexual recombination drives genetic diversity in eukaryotic genomes and fosters adaptation to novel environmental challenges. Although strictly asexual microorganisms are often considered as evolutionary dead ends, they comprise many devastating plant pathogens. Presently, it remains unknown how such asexual pathogens generate the genetic variation that is required for quick adaptation and evolution in the arms race with their hosts. Here, we show that extensive chromosomal rearrangements in the strictly asexual plant pathogenic fungus Verticillium dahliae establish highly dynamic lineage-specific (LS) genomic regions that act as a source for genetic variation to mediate aggressiveness. We show that such LS regions are greatly enriched for in planta-expressed effector genes encoding secreted proteins that enable host colonization. The LS regions occur at the flanks of chromosomal breakpoints and are enriched for retrotransposons and other repetitive sequence elements. Our results suggest that asexual pathogens may evolve by prompting chromosomal rearrangements, enabling rapid development of novel effector genes. Likely, chromosomal reshuffling can act as a general mechanism for adaptation in asexually propagating organisms.

Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including those that study non-model organisms. Thus, hundreds of fungal genomes have been sequenced and are publically available today, although these initiatives have typically yielded considerably fragmented genome assemblies that often lack large contiguous genomic regions. Many important genomic features are contained in intergenic DNA that is often missing in current genome assemblies, and recent studies underscore the significance of non-coding regions and repetitive elements for the life style, adaptability and evolution of many organisms. The study of particular types of genetic elements, such as telomeres, centromeres, repetitive elements, effectors, and clusters of co-regulated genes, but also of phenomena such as structural rearrangements, genome compartmentalization and epigenetics, greatly benefits from having a contiguous and high-quality, preferably even complete and gapless, genome assembly. Here we discuss a number of important reasons to produce gapless, finished, genome assemblies to help answer important biological questions.

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 ( 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.

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

Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1–5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5–14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening “tyrosine-and lysine-rich protein,” suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more influenced by harvest than genotype. Importantly, StTLRP was the only gene examined that may be involved in phellogen cell wall thickening after cessation of phellogen cell division.