Niloofar Vaghefi

One stop shop: backbones trees for important phytopathogenic genera: I (2014)

Many fungi are pathogenic on plants and cause significant damage in agriculture and forestry. They are also part of the natural ecosystem and may play a role in regulating plant numbers/density. Morphological identification and analysis of plant pathogenic fungi, while important, is often hampered by the scarcity of discriminatory taxonomic characters and the endophytic or inconspicuous nature of these fungi. Molecular (DNA sequence) data for plant pathogenic fungi have emerged as key information for diagnostic and classification studies, although hampered in part by non-standard laboratory practices and analytical methods. To facilitate current and future research, this study provides phylogenetic synopses for 25 groups of plant pathogenic fungi in the Ascomycota, Basidiomycota, Mucormycotina (Fungi), and Oomycota, using recent molecular data, up-to-date names, and the latest taxonomic insights. Lineage-specific laboratory protocols together with advice on their application, as well as general observations, are also provided. We hope to maintain updated backbone trees of these fungal lineages over time and to publish them jointly as new data emerge. Researchers of plant pathogenic fungi not covered by the present study are invited to join this future effort. Bipolaris, Botryosphaeriaceae, Botryosphaeria, Botrytis, Choanephora, Colletotrichum, Curvularia, Diaporthe, Diplodia, Dothiorella, Fusarium, Gilbertella, Lasiodiplodia, Mucor, Neofusicoccum, Pestalotiopsis, Phyllosticta, Phytophthora, Puccinia, Pyrenophora, Pythium, Rhizopus, Stagonosporopsis, Ustilago and Verticillium are dealt with in this paper.

Stagonosporopsis spp. associated with ray blight disease of Asteraceae

Ray blight disease of pyrethrum (Tanacetum cinerariifolium) is shown to be caused by more than one species of Stagonosporopsis. The Australian pathogen, previously identified as Phoma ligulicola var. inoxydabilis, represents a new species described as Stagonosporopsis tanaceti based on morphological characters and a five-gene phylogeny employing partial sequences of the actin, translation elongation factor 1-alpha, internal transcribed spacers and 5.8S of the nrDNA, 28S large subunit and beta-tubulin 2 gene sequences. Furthermore, the two varieties of Stagonosporopsis ligulicola are elevated to species level as S. chrysanthemi and S. inoxydabilis based on their DNA phylogeny and morphology.

IMA Genome-F 4: Draft genome sequences of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygamai, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata.

The genomes of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygami, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata are presented in this genome announcement. These seven genomes are from endophytes, plant pathogens and economically important fungal species. The genome sizes range from 26.6 Mb in the case of Leptographium lundbergii to 44 Mb for Chrysoporthe austroafricana. The availability of these genome data will provide opportunities to resolve longstanding questions regarding the taxonomy of species in these genera, and may contribute to our understanding of the lifestyles through comparative studies with closely related organisms.

Identification of the MAT1 locus in Stagonosporopsis tanaceti, and exploring its potential for sexual reproduction in Australian pyrethrum fields

Stagonosporopsis chrysanthemi, S. inoxydabilis, and S. tanaceti are closely related Ascomycetes associated with ray blight of the Asteraceae. To date, only S. tanaceti has been identified in Australia, incurring substantial losses to the pyrethrum industry. In contrast to the homothallic S. chrysanthemi and S. inoxydabilis, a sexual state has not been observed for S. tanaceti. The MAT1 locus in S. tanaceti was identified through de novo assembly of shotgun reads, and was further used to develop primers for amplification of the full-length MAT1/2 locus in S. chrysanthemi and S. inoxydabilis. As expected, S. chrysanthemi and S. inoxydabilis possessed a MAT1/2 locus typical of homothallic Dothideomycetes with two adjacent MAT1-1 and MAT1-2 idiomorphs. However, only MAT1-1 could be detected in the assembled genome of S. tanaceti. Although a sexual mode of reproduction cannot be ruled out for S. tanaceti, evidence so far suggests this is absent or occurring at very low frequency in Australian pyrethrum fields.

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

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

Rapid Changes in the Genetic Composition of Stagonosporopsis tanaceti Population in Australian Pyrethrum Fields.

A novel set of microsatellite markers were developed and employed for geographical and temporal population analyses of Stagonosporopsis tanaceti, the cause of ray blight of pyrethrum in Australia. Genotyping of 407 isolates, using 13 markers, suggested an asexual mode of reproduction with significant linkage disequilibrium and high levels of clonality. Low geographical differentiation and widespread distribution of a few multilocus genotypes (MLGs), in the absence of airborne ascospores, suggested the role of human-mediated movement of seed as a major means of long-distance pathogen dispersal. The genetic composition of S. tanaceti was stable for a decade then changed rapidly in only 2 years. Bayesian clustering analyses and minimum spanning networks determined only two major clonal lineages in and prior to 2010. However, in 2012, a previously unobserved cluster of MLGs was detected, which significantly increased in frequency and displaced the historically dominant MLGs by 2013. This rapid change in the genetic composition of S. tanaceti could indicate a second introduction then a selective sweep, or strong selection pressures from recently introduced fungicides or pyrethrum varieties. These results may have serious implications for durability of management strategies for this disease.

Genetic structure of Cercospora beticola populations on Beta vulgaris in New York and Hawaii

Cercospora leaf spot (CLS), caused by Cercospora beticola, is a major disease of Beta vulgaris worldwide. No sexual stage is known for C. beticola but in its asexual form it overwinters on infected plant debris as pseudostromata, and travels short distances by rain splash-dispersed conidiospores. Cercospora beticola infects a broad range of host species and may be seedborne. The relative contribution of these inoculum sources to CLS epidemics on table beet is not well understood. Pathogen isolates collected from table beet, Swiss chard and common lambsquarters in mixed-cropping farms and monoculture fields in New York and Hawaii, USA, were genotyped (n = 600) using 12 microsatellite markers. All isolates from CLS symptoms on lambsquarters were identified as C. chenopodii. Sympatric populations of C. beticola derived from Swiss chard and table beet were not genetically differentiated. Results suggested that local (within field) inoculum sources may be responsible for the initiation of CLS epidemics in mixed-cropping farms, whereas external sources of inoculum may be contributing to CLS epidemics in the monoculture fields in New York. New multiplex PCR assays were developed for mating-type determination for C. beticola. Implications of these findings for disease management are discussed.

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

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

Global genotype flow in Cercospora beticola populations confirmed through genotyping-by-sequencing

Genotyping-by-sequencing (GBS) was conducted on 333 Cercospora isolates collected from Beta vulgaris (sugar beet, table beet and swiss chard) in the USA and Europe. Cercospora beticola was confirmed as the species predominantly isolated from leaves with Cercospora leaf spot (CLS) symptoms. However, C. cf. flagellaris also was detected at a frequency of 3% in two table beet fields in New York. Resolution of the spatial structure and identification of clonal lineages in C. beticola populations using genome-wide single nucleotide polymorphisms (SNPs) obtained from GBS was compared to genotyping using microsatellites. Varying distance thresholds (bitwise distance = 0, 1.854599 × 10−4, and 1.298 × 10−3) were used for delineation of clonal lineages in C. beticola populations. Results supported previous reports of long distance dispersal of C. beticola through genotype flow. The GBS-SNP data set provided higher resolution in discriminating clonal lineages; however, genotype identification was impacted by filtering parameters and the distance threshold at which the multi-locus genotypes (MLGs) were contracted to multi-locus lineages. The type of marker or different filtering strategies did not impact estimates of population differentiation and structure. Results emphasize the importance of robust filtering strategies and designation of distance thresholds for delineating clonal lineages in population genomics analyses that depend on individual assignment and identification of clonal lineages. Detection of recurrent clonal lineages shared between the USA and Europe, even in the relaxed-filtered SNP data set and with a conservative distance threshold for contraction of MLGs, provided strong evidence for global genotype flow in C. beticola populations. The implications of intercontinental migration in C. beticola populations for CLS management are discussed.

Horticultural characteristics and susceptibility of table beet cultivars to Cercospora Leaf Spot in New York

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