Beet Soil-Borne Virus Is a Helper Virus for the Novel Beta vulgaris Satellite Virus 1A.

Sugar beet (Beta vulgaris) is grown in temperate regions around the world as a source of sucrose used for natural sweetening. Sugar beet is susceptible to a number of viral diseases, but identification of the causal agent(s) under field conditions is often difficult due to mixtures of viruses that may be responsible for disease symptoms. In this study, the application of RNAseq to RNA extracted from diseased sugar beet roots obtained from the field and from greenhouse-reared plants grown in soil infested with the virus disease rhizomania (causal agent beet necrotic yellow vein virus; BNYVV) yielded genome-length sequences from BNYVV, as well as beet soil-borne virus (BSBV). The nucleotide identities of the derived consensus sequence of BSBV RNAs ranged from 99.4 to 96.7% (RNA1), 99.3 to 95.3% (RNA2), and 98.3 to 95.9% (RNA3) compared with published BSBV sequences. Based on the BSBV genome consensus sequence, clones of the genomic RNAs 1, 2, and 3 were obtained to produce RNA copies of the genome through in vitro transcription. Capped RNA produced from the clones was infectious when inoculated into leaves of Chenopodium quinoa and B. vulgaris, and extracts from transcript-infected C. quinoa leaves could infect sugar beet seedling roots through a vortex inoculation method. Subsequent exposure of these infected sugar beet seedling roots to aviruliferous Polymyxa betae, the protist vector of both BNYVV and BSBV, confirmed that BSBV derived from the infectious clones could be transmitted by the vector. Co-inoculation of BSBV synthetic transcripts with transcripts of a cloned putative satellite virus designated Beta vulgaris satellite virus 1A (BvSat1A) resulted in the production of lesions on leaves of C. quinoa similar to those produced by inoculation with BSBV alone. Nevertheless, accumulation of genomic RNA and the encoded protein of the satellite virus in co-inoculated leaves was readily detected on Northern and Western blots, respectively, whereas no accumulation of satellite virus products occurred when satellite virus RNA was inoculated alone. The predicted sequence of the detected protein encoded by BvSat1A bears hallmarks of coat proteins of other satellite viruses, and virions of a size consistent with a satellite virus were observed in samples testing positive for the virus. The results demonstrate that BSBV is a helper virus for the novel satellite virus BvSat1A.

Fungal social influencers: secondary metabolites as a platform for shaping the plant-associated community.

Fungal secondary metabolites (FSMs) are capable of manipulating plant community dynamics by inhibiting or facilitating the establishment of co-habitating organisms. Although production of FSMs is not crucial for survival of the producer, their absence can indirectly impair growth and/or niche competition of these fungi on the plant. The presence of FSMs with no obvious consequence on the fitness of the producer leaves questions regarding ecological impact. This review investigates how fungi employ FSMs as a platform to mediate fungal-fungal, fungal-bacterial and fungal-animal interactions associated with the plant community. We discuss how the biological function of FSMs may indirectly benefit the producer by altering the dynamics of surrounding organisms. We introduce several instances where FSMs influence antagonistic- or alliance-driven interactions. Part of our aim is to decipher the meaning of the FSM 'language' as it is widely noted to impact the surrounding community. Here, we highlight the contribution of FSMs to plant-associated interaction networks that affect the host either broadly or in ways that may have previously been unclear.

Seedborne Cercospora beticola Can Initiate Cercospora Leaf Spot from Sugar Beet (Beta vulgaris) Fruit Tissue.

Cercospora leaf spot (CLS) is a globally important disease of sugar beet (Beta vulgaris) caused by the fungus Cercospora beticola. Long-distance movement of C. beticola has been indirectly evidenced in recent population genetic studies, suggesting potential dispersal via seed. Commercial sugar beet "seed" consists of the reproductive fruit (true seed surrounded by maternal pericarp tissue) coated in artificial pellet material. In this study, we confirmed the presence of viable C. beticola in sugar beet fruit for 10 of 37 tested seed lots. All isolates harbored the G143A mutation associated with quinone outside inhibitor resistance, and 32 of 38 isolates had reduced demethylation inhibitor sensitivity (EC50 > 1 µg/ml). Planting of commercial sugar beet seed demonstrated the ability of seedborne inoculum to initiate CLS in sugar beet. C. beticola DNA was detected in DNA isolated from xylem sap, suggesting the vascular system is used to systemically colonize the host. We established nuclear ribosomal internal transcribed spacer region amplicon sequencing using the MinION platform to detect fungi in sugar beet fruit. Fungal sequences from 19 different genera were identified from 11 different sugar beet seed lots, but Fusarium, Alternaria, and Cercospora were consistently the three most dominant taxa, comprising an average of 93% relative read abundance over 11 seed lots. We also present evidence that C. beticola resides in the pericarp of sugar beet fruit rather than the true seed. The presence of seedborne inoculum should be considered when implementing integrated disease management strategies for CLS of sugar beet in the future.

Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum.

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.

Rapid Detection of Cercospora beticola in Sugar Beet and Mutations Associated with Fungicide Resistance Using LAMP or Probe-Based qPCR.

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 ß-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.

Tools of the crook- infection strategies of fungal plant pathogens.

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.

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

Perylenequinones 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.

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.

Prevalence and Distribution of Beet Necrotic Yellow Vein Virus Strains in North Dakota and Minnesota.

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.

Microbial small molecules - weapons of plant subversion.

Covering: up to 2018 Plants live in close association with a myriad of microbes that are generally harmless. However, the minority of microbes that are pathogens can severely impact crop quality and yield, thereby endangering food security. By contrast, beneficial microbes provide plants with important services, such as enhanced nutrient uptake and protection against pests and diseases. Like pathogens, beneficial microbes can modulate host immunity to efficiently colonize the nutrient-rich niches within and around the roots and aerial tissues of a plant, a phenomenon mirroring the establishment of commensal microbes in the human gut. Numerous ingenious mechanisms have been described by which pathogenic and beneficial microbes in the plant microbiome communicate with their host, including the delivery of immune-suppressive effector proteins and the production of phytohormones, toxins and other bioactive molecules. Plants signal to their associated microbes via exudation of photosynthetically fixed carbon sources, quorum-sensing mimicry molecules and selective secondary metabolites such as strigolactones and flavonoids. Molecular communication thus forms an integral part of the establishment of both beneficial and pathogenic plant-microbe relations. Here, we review the current knowledge on microbe-derived small molecules that can act as signalling compounds to stimulate plant growth and health by beneficial microbes on the one hand, but also as weapons for plant invasion by pathogens on the other. As an exemplary case, we used comparative genomics to assess the small molecule biosynthetic capabilities of the Pseudomonas genus; a genus rich in both plant pathogenic and beneficial microbes. We highlight the biosynthetic potential of individual microbial genomes and the population at large, providing evidence for the hypothesis that the distinction between detrimental and beneficial microbes is increasingly fading. Knowledge on the biosynthesis and molecular activity of microbial small molecules will aid in the development of successful biological agents boosting crop resiliency in a sustainable manner and could also provide scientific routes to pathogen inhibition or eradication.

Extensive chromosomal reshuffling drives evolution of virulence in an asexual pathogen.

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.

Mind the gap; seven reasons to close fragmented genome assemblies.

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.

RNA-sequencing of Cercospora beticola DMI-sensitive and -resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction.

Cercospora beticola causes Cercospora leaf spot of sugar beet. Cercospora leaf spot management measures often include application of the sterol demethylation inhibitor (DMI) class of fungicides. The reliance on DMIs and the consequent selection pressures imposed by their widespread use has led to the emergence of resistance in C. beticola populations. Insight into the molecular basis of tetraconazole resistance may lead to molecular tools to identify DMI-resistant strains for fungicide resistance management programs. Previous work has shown that expression of the gene encoding the DMI target enzyme (CYP51) is generally higher and inducible in DMI-resistant C. beticola field strains. In this study, we extended the molecular basis of DMI resistance in this pathosystem by profiling the transcriptional response of two C. beticola strains contrasting for resistance to tetraconazole. A majority of the genes in the ergosterol biosynthesis pathway were induced to similar levels in both strains with the exception of CbCyp51, which was induced several-fold higher in the DMI-resistant strain. In contrast, a secondary metabolite gene cluster was induced in the resistance strain, but repressed in the sensitive strain. Genes encoding proteins with various cell membrane fortification processes were induced in the resistance strain. Site-directed and ectopic mutants of candidate DMI-resistance genes all resulted in significantly higher EC50 values than the wild-type strain, suggesting that the cell wall and/or membrane modified as a result of the transformation process increased resistance to tetraconazole. Taken together, this study identifies important cell membrane components and provides insight into the molecular events underlying DMI resistance in C. beticola.

The Genome of the Saprophytic Fungus Verticillium tricorpus Reveals a Complex Effector Repertoire Resembling That of Its Pathogenic Relatives.

Vascular wilts caused by Verticillium spp. are destructive plant diseases affecting hundreds of hosts. Only a few Verticillium spp. are causal agents of vascular wilt diseases, of which V. dahliae is the most notorious pathogen, and several V. dahliae genomes are available. In contrast, V. tricorpus is mainly known as a saprophyte and causal agent of opportunistic infections. Based on a hybrid approach that combines second and third generation sequencing, a near-gapless V. tricorpus genome assembly was obtained. With comparative genomics, we sought to identify genomic features in V. dahliae that confer the ability to cause vascular wilt disease. Unexpectedly, both species encode similar effector repertoires and share a genomic structure with genes encoding secreted proteins clustered in genomic islands. Intriguingly, V. tricorpus contains significantly fewer repetitive elements and an extended spectrum of secreted carbohydrate- active enzymes when compared with V. dahliae. In conclusion, we highlight the technical advances of a hybrid sequencing and assembly approach and show that the saprophyte V. tricorpus shares many hallmark features with the pathogen V. dahliae.

Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing.

Fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium albo-atrum, but the corresponding Verticillium effector remained unknown thus far. By high-throughput population genome sequencing, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains, and subsequent transcriptome sequencing of Verticillium-infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for Avirulence on Ve1 tomato). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis. Interestingly, Ave1 is homologous to a widespread family of plant natriuretic peptides. Besides plants, homologous proteins were only found in the bacterial plant pathogen Xanthomonas axonopodis and the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola, and Fusarium oxysporum f. sp. lycopersici. The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens.

The heterothallic sugarbeet pathogen Cercospora beticola contains exon fragments of both MAT genes that are homogenized by concerted evolution.

Dothideomycetes is one of the most ecologically diverse and economically important classes of fungi. Sexual reproduction in this group is governed by mating type (MAT) genes at the MAT1 locus. Self-sterile (heterothallic) species contain one of two genes at MAT1 (MAT1-1-1 or MAT1-2-1) and only isolates of opposite mating type are sexually compatible. In contrast, self-fertile (homothallic) species contain both MAT genes at MAT1. Knowledge of the reproductive capacities of plant pathogens are of particular interest because recombining populations tend to be more difficult to manage in agricultural settings. In this study, we sequenced MAT1 in the heterothallic Dothideomycete fungus Cercospora beticola to gain insight into the reproductive capabilities of this important plant pathogen. In addition to the expected MAT gene at MAT1, each isolate contained fragments of both MAT1-1-1 and MAT1-2-1 at ostensibly random loci across the genome. When MAT fragments from each locus were manually assembled, they reconstituted MAT1-1-1 and MAT1-2-1 exons with high identity, suggesting a retroposition event occurred in a homothallic ancestor in which both MAT genes were fused. The genome sequences of related taxa revealed that MAT gene fragment pattern of Cercospora zeae-maydis was analogous to C. beticola. In contrast, the genome of more distantly related Mycosphaerella graminicola did not contain MAT fragments. Although fragments occurred in syntenic regions of the C. beticola and C. zeae-maydis genomes, each MAT fragment was more closely related to the intact MAT gene of the same species. Taken together, these data suggest MAT genes fragmented after divergence of M. graminicola from the remaining taxa, and concerted evolution functioned to homogenize MAT fragments and MAT genes in each species.

Characterization of CbCyp51 from field isolates of Cercospora beticola.

The hemibiotrophic fungus Cercospora beticola causes leaf spot of sugar beet. Leaf spot control measures include the application of sterol demethylation inhibitor (DMI) fungicides. However, reduced sensitivity to DMIs has been reported recently in the Red River Valley sugar beet-growing region of North Dakota and Minnesota. Here, we report the cloning and molecular characterization of CbCyp51, which encodes the DMI target enzyme sterol P450 14α-demethylase in C. beticola. CbCyp51 is a 1,632-bp intron-free gene with obvious homology to other fungal Cyp51 genes and is present as a single copy in the C. beticola genome. Five nucleotide haplotypes were identified which encoded three amino acid sequences. Protein variant 1 composed 79% of the sequenced isolates, followed by protein variant 2 that composed 18% of the sequences and a single isolate representative of protein variant 3. Because resistance to DMIs can be related to polymorphism in promoter or coding sequences, sequence diversity was assessed by sequencing >2,440 nucleotides encompassing CbCyp51 coding and flanking regions from isolates with varying EC(50) values (effective concentration to reduce growth by 50%) to DMI fungicides. However, no mutations or haplotypes were associated with DMI resistance or sensitivity. No evidence for alternative splicing or differential methylation of CbCyp51 was found that might explain reduced sensitivity to DMIs. However, CbCyp51 was overexpressed in isolates with high EC(50) values compared with isolates with low EC(50) values. After exposure to tetraconazole, isolates with high EC(50) values responded with further induction of CbCyp51, with a positive correlation of CbCyp51 expression and tetraconazole concentration up to 2.5 µg ml(-1).

Efficacy of Variable Tetraconazole Rates Against Cercospora beticola Isolates with Differing In Vitro Sensitivities to DMI Fungicides.

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 <0.01 µg ml-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.

The unsung roles of microbial secondary metabolite effectors in the plant disease cacophony.

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.

Mechanism of Sugarbeet Seed Germination Enhanced by Hydrogen Peroxide.

Seed germination is a critical first stage of plant development but can be arrested by factors including dormancy and environmental conditions. Strategies to enhance germination are of interest to plant breeders to ensure the ability to utilize the genetic potential residing inside a dormant seed. In this study, seed germination in two sugarbeet (Beta vulgaris ssp. vulgaris L.) lines F1004 and F1015 through incubating seeds in hydrogen peroxide (H2O2) solution was improved over 70% relative to germinating seeds through water incubation. It was further found that low germination from water incubation was caused by physical dormancy in F1015 seeds with initial seed imbibition blocked by the seed pericarp, and physiological dormancy in F1004 seeds with germination compromised due to the physiological condition of the embryo. To identify genes that are differentially expressed in response to cellular activities promoted by H2O2 during overcoming different type of dormancies, an RNA-Seq study was carried out and found H2O2 treatment during germination accelerated the degradation of seed stored mRNAs that were synthesized before or during seed storage to provide protections and maintain the dormant state. Comparison of transcripts in H2O2-treated seeds between the two sugarbeet lines identified differentially expressed genes (DEGs) that were higher in F1004 for alleviating physiological dormancy were known to relative to gene expression regulation. The research established that H2O2 overcomes both physical and physiological dormancies by hastening the transition of seeds from dormancy into germination. More DEGs related to gene expression regulation were involved in relieving physiological dormancy which provides new knowledge about the role of exogenous H2O2 as a signaling molecule for regulating gene activities during germination. Moreover, the protocol using H2O2 to promote germination will be useful for rescuing plant germplasms with poor germination.