Viviana Rivera-Varas

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).

Mycoparasitism of Helminthosporium solani by Acremonium strictum

ABSTRACT Isolates of Helminthosporium solani, the causal agent of silver scurf of potato, collected from multiple locations consistently show white sectoring and rings, differential coloration, and reduced sporulation in culture. It has been accepted that this growth pattern is normal for H. solani cultures. Scanning electron microscopy confirmed the presence of a contaminating fungus in close association with cultures of H. solani. Repeated hyphal tip isolation techniques were used to separate H. solani from the fungal contaminant. Resultant pure cultures of H. solani were uniformly black in color, without white sectors or rings. The contaminating fungus was identified as Acremonium strictum. The purpose of this study was to elucidate the relationship between A. strictum and H. solani, and evaluate the impact of the fungicolous A. strictum on the growth and biology of H. solani. In vitro studies demonstrated that A. strictum significantly reduced sporulation of H. solani isolates from 65 to 35%, spore germination from 53 to 43%, and mycelial growth from 40 to 32% compared with noncontaminated cultures of H. solani. These data indicate that A. strictum is antagonistic to H. solani, and can be considered a mycoparasite. A. strictum reduced H. solani conidia production on minitubers, thereby reducing inoculum for infection. However, treatment with A. strictum does not reduce silver scurf of previously infected tubers. Further studies are warranted to determine the full potential of A. strictum as a biological control agent of H. solanii-incited silver scurf of stored potato tubers and the most effective manner of use.

Trichothecene Mycotoxins Associated with Potato Dry Rot Caused by Fusarium graminearum

Fusarium graminearum, a known producer of trichothecene mycotoxins in cereal hosts, has been recently documented as a cause of dry rot of potato tubers in the United States. Due to the uncertainty of trichothecene production in these tubers, a study was conducted to determine the accumulation and diffusion of trichothecenes in potato tubers affected with dry rot caused by F. graminearum. Potato tubers of cv. Russet Burbank were inoculated with 14 F. graminearum isolates from potato, sugar beet, and wheat and incubated at 10 to 12 degrees C for 5 weeks to determine accumulation of trichothecenes in potato tubers during storage. Twelve of the isolates were classified as deoxynivalenol (DON) genotype and two isolates were as nivalenol (NIV) genotype. Trichothecenes were detected only in rotted tissue. DON was detected in all F. graminearum DON genotype isolates up to 39.68 microg/ml in rotted potato tissue. Similarly, both NIV genotype isolates accumulated NIV in rotted potato tissue up to 18.28 microg/ml. Interestingly, isolates classified as genotype DON accumulated both DON and NIV in the dry rot lesion. Potato tubers were then inoculated with two isolates of F. graminearum chemotype DON and incubated up to 7 weeks at 10 to 12 degrees C and assayed for DON diffusion. F. graminearum was recovered from >53% of the isolations from inoculated tubers at 3 cm distal to the rotted tissue after 7 weeks of incubation but DON was not detected in the surrounding tissue. Based in this data, the accumulation of trichothecenes in the asymptomatic tissue surrounding dry rot lesions caused by F. graminearum is minimal in cv. Russet Burbank potato tubers stored for 7 weeks at customary processing storage temperatures.

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.

Characterization of Fusarium secorum, a new species causing fusarium yellowing decline of sugar beet in north central USA.

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.

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.