Nighttime Applications of Germicidal Ultraviolet Light (UV-C) to Suppress Cercospora Leaf Spot in Table Beet.

Cercospora leaf spot (CLS), caused by the hemibiotrophic fungus Cercospora beticola, is a destructive disease affecting table beet. Multiple applications of fungicides are needed to reduce epidemic progress to maintain foliar health and enable mechanized harvest. The sustainability of CLS control is threatened by the rapid development of fungicide resistance, the need to grow commercially acceptable yet CLS-susceptible cultivars, and the inability to manipulate agronomic conditions to mitigate disease risk. Nighttime applications of germicidal ultraviolet light (UV-C) have recently been used to suppress several plant diseases, notably those caused by ectoparasitic biotrophs such as powdery mildews. We evaluated the efficacy of nighttime applications of UV-C for suppression of CLS in table beet. In vitro lethality of UV-C to germinating conidia increased with increasing dose, with complete suppression at 1,000 J/m2. Greenhouse-grown table beet tolerated relatively high doses of UV-C without lethal effects despite some bronzing on the leaf blade. A UV-C dose >1,500 J/m2 resulted in phytotoxicity severities greater than 50%. UV-C exposure to ≤750 J/m2 resulted in negligible phytotoxicity. Older (6-week-old) greenhouse-grown plants were more susceptible to UV-C damage than younger (2- and 4-week-old) plants. Suppression of CLS by UV-C was greater when applied within 6 days of C. beticola inoculation than if delayed until 13 days after infection in greenhouse-grown plants. In field trials, there were significant linear relationships between UV-C dose and CLS control and phytotoxicity severity, and a significant negative linear relationship between phytotoxicity and CLS severity at the final assessment. Significant differences between UV-C doses on the severity of CLS and phytotoxicity indicated an efficacious dose near 800 J/m2. Collectively, these findings illustrate significant and substantial suppression by nighttime applications of UV-C for CLS control on table beet, with potential for incorporation in both conventional and organic table beet broadacre production systems.

Enabling Population Biology Studies of Stemphylium vesicarium from Onion with Microsatellites.

Stemphylium leaf blight (SLB), caused by the fungus Stemphylium vesicarium, is dominant within the foliar disease complex affecting onion production in New York (NY). The disease causes premature defoliation and significant reductions in bulb weight and quality. Foliar diseases of onion are usually managed by an intensive fungicide program, but SLB management is complicated by resistance to multiple single-site modes of action. The design of integrated disease management strategies is limited by incomplete knowledge surrounding the dominant sources of S. vesicarium inoculum. To facilitate genomic-based studies of S. vesicarium populations, nine microsatellite markers were developed. The markers were multiplexed into two PCR assays containing four and five fluorescently labeled microsatellite markers. Initial testing of the S. vesicarium isolates found the markers were highly polymorphic and reproducible with an average of 8.2 alleles per locus. The markers were used to characterize 54 S. vesicarium isolates from major NY onion production regions in 2016 (n = 27) and 2018 (n = 27). Fifty-two multilocus genotypes (MLGs) were identified between these populations. Genotypic and allelic diversities were high in both the 2016 and 2018 populations. A greater degree of genetic variation was observed within populations than between years. No distinct pattern of MLGs according to population was identified and some MLGs were closely related between 2016 and 2018. The lack of evidence for linkage among loci also was strongly suggestive of clonal populations with only minor differences between the two populations. These microsatellite markers will be a foundational resource for the testing of hypotheses surrounding the population biology of S. vesicarium and therefore informing disease management.

Confirmation of Itersonilia perplexans infecting pyrethrum (Tanacetum cinerariifolium) in Australia.

Pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.) is grown to extract pyrethrins which are active ingredients for insecticides (Greenhill 2007). The Australian pyrethrum industry supplies over 50% of the world market. Surveys of Tasmanian crops in spring 2013, detected the presence of a fungus putatively identified as Itersonilia perplexans Derx. on foliage in 54 of 86 surveyed fields (Hay et al. 2015). This fungus was associated with necrotic leaf tips often spreading to encompass whole leaves. However, pathogenicity to pyrethrum was not confirmed. To isolate, tissue was excised from foliar lesions, surface sterilised using 0.4% NaClO, placed onto 2% water agar and incubated at 20°C for 5 days. Colonies were pure-cultured by hyphal-tip transfer onto potato-dextrose agar. Eleven isolates were cultured onto yeast mold agar (YMA) for 14 days at 15°C in the dark (Horita and Yasuoka 2002). Colonies were slow growing (1.9 to 2.3 mm/day) white to buff on both surfaces, with a darker center visible on lower surfaces. Mycelia were straight and hyaline with clamp connections at the septa. Squares transferred from the edge of YMA colonies onto microscope slides produced ballistoconidia that were aseptate, granular and lunate, kidney or lemon-shaped after 24 h. Ballistoconidia lengths and widths (n = 50/isolate) ranged from 14.6 to 20.4 µm and 10.0 to 13.6 µm. Chlamydospores were not observed. These observations were consistent with descriptions of I. perplexans (Koike and Tjosvold 2001; Liu et al. 2015). All 11 isolates were sequenced across the internal transcribed spacer (ITS) region of rDNA (ITS; primers V9G/ITS4; de Hoog and van den Ende 1998; White et al. 1990), and large (LSU; primers LROR/LR7; Rehner and Samuels 1995), and small (SSU; NS1/NS4; White et al. 1990) subunits of rDNA (Genbank accession nos. KU563626 to KU563658). The ITS (673 bp), SSU (1,047 bp) and LSU (1,318 bp) differed by 3, 1 and 0 bp, respectively, across isolates. Maximum parsimony and maximum likelihood analyses of a concatenated 3 loci alignment with Cystofilobasidiales representatives (Liu et al. 2015) placed all isolates and the I. perplexans ex-neotype strain CBS 363.85 within a single monophyletic clade with 100% bootstrap support. Two representative isolates are stored at the Plant Pathology Herbarium (accession nos. BRIP 57986 and 57987). Leaves of 46-day-old pyrethrum plants (n = 45), generated from surface sterilised seed, were inoculated with a 1.5 × 105 ballistoconidia/ml suspension (equal mix of eight isolates) and maintained between 10 and 22°C under a 12-h photoperiod for 14 days. Brown necrotic leaf tips, consistent with reported field symptoms were observed on 71% of plants and I. perplexans was recovered from 69% of symptomatic plants. For flower inoculations, pyrethrum plants were removed from fields as vegetative plants in spring and maintained in a greenhouse set at 20:14°C and 14:10 h day:night. Open flowers (10 per plant) were dipped into a 1.2 × 104 ballistoconidia/ml suspension mix of three isolates. Brown withered ray florets were observed on 10/12 plants 18 days post-inoculation, matching those described in petal blight of chrysanthemum (McRitchie et al. 1973). I. perplexans was re-isolated from 11/12 inoculated plants and 1 control plant (of 12) which exhibited the same symptoms. In both experiments, I. perplexans was identified based on its distinctive morphology. This confirms the pathogenicity of I. perplexans to both pyrethrum leaves and flowers.

First Report of Halo Blight on Hop Caused by Diaporthe humulicola in New York.

In late July and August 2015, foliar disease was observed in three hop (Humulus lupulus; unknown cultivars) yards in Ontario, Otsego, and Putnam counties, New York (NY). Disease incidence ranged between 70 and 90% of plants, and up to 25% of the leaves per plant were affected. Leaf symptoms were large, necrotic patches with a chlorotic halo (2 to 10 cm diam.). Leaves and dry, easily shattered cones were placed at high humidity for 10 days. Pycnidia were abundant in leaf lesions which extruded conidia. Pycnidia were also observed on cone bracts and bracteoles. Fifteen isolations were made from each yard by placing a pycnidium onto 2% water agar + 0.02% (w/v) ampicillin. Colonies were hyphal tipped and transferred to potato dextrose agar (PDA) before incubation at 20°C with a 12-h photoperiod. Colonies on PDA had flat mycelia and were white to cream in color. The isolation frequency was 100%. To induce sporulation, five isolates were grown on PDA with autoclaved alfalfa stems for 7 to 10 days. Alpha conidia were hyaline, and oval with obtuse ends. Mean alpha conidial dimensions were (n = 20): 9.1 m × 3.4 µm (BE1; Ontario Co.); 11.8 × 3.8 µm (BE34; Ontario Co.); 9.6 × 4.1 µm (BE10; Ontario Co.); 10.2 × 3.7 µm (BE52; Otsego Co.); and 10.3 × 3.6 µm (BE69; Putnam Co.). Beta conidia were not observed. DNA was extracted and PCR performed to amplify the internal transcribed spacer (ITS) region (primers ITS1/ITS4; White et al. 1990), translation elongation factor 1-α (TEF; EF1-728F/EF1-986R; Carbone and Kohn 1999), a partial region of ß-tubulin (TUB; Bt2a/Bt2b; Glass and Donaldson 1995), a partial region of histone 3 (H3) (H3; CYLH3F/H3-1b Crous et al. 2004), and calmodulin (CAL; CAL-228F/CAL2Rd; Groenewald et al. 2013) genes. For all NY isolates, sequence similarity was >99% to D. humulicola CT2018-3 for the ITS region, and TEF, HIS, and CAL genes. Sequence similarity to CT2018-3 for the TUB region ranged from 86.96% (BE-1) to 96.15% (BE-10). . Analyses with the ITS, TEF, CAL, and HIS sequences supported our identification of the NY isolates as D. humulicola. Sequences were deposited in GenBank (OM370960 to OM370984). For pathogenicity testing, BE-34 and BE-69 were grown on PDA + autoclaved alfalfa stems at room temperature and a 12-h photoperiod for 10 days. Conidia were harvested by flooding the plate with sterile water. Conidial concentration was quantified, and the inoculum suspension diluted to ~5  105 (+ 0.01% polysorbate-20)/ml. Five cv. Cascade plants were sprayed with inoculum until run-off and covered with a plastic bag for 72 h. Non-inoculated control plants were sprayed with 0.01% polysorbate-20 and bagged. Plants were placed in a misting chamber and exposed to alternating 25°C light/18°C dark with a 16 h photoperiod. Mist was applied for 1 h daily. Necrotic lesions like the field specimens were observed on all inoculated plants after 28 days with no symptoms on control plants. Diseased leaves were detached and placed in a humid chamber for 2 days, and pycnidia observed in lesions. The reisolation frequency of D. humulicola was 100%. Conidia from the isolates had similar morphology to the original isolates. This is the first report of halo blight caused by D. humulicola on hop in NY. Halo blight has been reported on hop and associated with significant yield loss through cone shattering in MI (Higgins et al. 2021), CT (Allan-Perkins et al. 2020), and Quebec, Canada (Hatlen et al. 2021). Research is needed to determine if management is warranted.

Control of Phoma Leaf Spot and Root Decay of Table Beet in New York.

Disease caused by Neocamarosporium betae (syn. Phoma betae, Pleospora betae) results in reductions in plant populations, foliar disease (Phoma leaf spot [PLS]), and root disease and decay in table beet. Disease caused by N. betae has reemerged as prevalent in organic table beet production in New York. The disease can also cause substantial issues in conventional table beet production. To evaluate in-field control options for conventional and organic table beet production, small-plot, replicated trials were conducted in each of two years (2019 and 2021). The fungicides, propiconazole and difenoconazole, and premixtures, pydiflumetofen + fludioxonil or pydiflumetofen + difenoconazole, provided excellent PLS and root decay control. Azoxystrobin provided excellent (69.9%) control of PLS in 2019 and lesser (40%) control in 2021. Field trial results complemented in vitro sensitivity testing of 30 New York N. betae isolates that were all highly sensitive to azoxystrobin (mean effective concentration to reduce mycelial growth by 50%, EC50 = 0.0205 µg/ml) and propiconazole (mean EC50 = 0.0638 µg/ml). Copper octanoate and microbial biopesticides containing either Bacillus amyloliquefaciens D747 or B. mycoides strain J provided moderate (68.5 to 74.6%) PLS control as reflected in epidemic progress. The Gompertz model provided the best fit to PLS epidemics reflecting a polycyclic epidemic. Reductions in PLS severity were associated with significant decreases in Phoma root decay and increases in canopy health and the time-to-death of leaves compared with nontreated control plots. Prolonging leaf survival is critical for mechanical harvest of roots. These findings underpin the design of programs for foliar disease control in conventional and organic table beet production. Assessment of PLS severity in the field will better inform postharvest management decisions.

Spatiotemporal Dynamics of Stemphylium Leaf Blight and Potential Inoculum Sources in New York Onion Fields.

Stemphylium leaf blight (SLB) caused by Stemphylium vesicarium is the dominant foliar disease affecting large-scale onion production in New York. The disease is managed by fungicides, but control failures are prevalent and are attributed to fungicide resistance. Little is known of the relative role of inoculum sources in initiation and spread of SLB epidemics. Plate testing of 28 commercially available organic onion seedlots from 2016 and 2017 did not detect S. vesicarium. This finding suggests that although S. vesicarium has been reported as seed-transmitted, this is unlikely to be a significant inoculum source in commercially available organic seed lots and even less so in fungicide-treated seed used to establish conventional fields. The spatial and spatiotemporal dynamics of SLB epidemics in six onion fields were evaluated along linear transects in 2017 and 2018. Average SLB incidence increased from 0 to 100% throughout the cropping seasons with an average final lesion length of 28.3 cm. Disease progress was typical of a polycyclic epidemic and the logistic model provided the best fit to 83.3% of the datasets. Spatial patterns were better described by the beta-binomial than binomial distribution in half of the datasets (50%) and random patterns were more frequently observed by the index of dispersion (59%). Geostatistical analyses also found a low frequency of datasets with aggregation (60%). Spatiotemporal analysis of epidemics detected that the aggregation was influenced by disease incidence. However, diseased units were not frequently associated with the previous time period according to the spatiotemporal association function of spatial analyses by distance indices. Variable spatial patterns suggested mixed inoculum sources dependent upon location, and likely an external inoculum source at the sampling scale used in this study. A small-plot replicated trial was also conducted in each of 2 years to quantify the effect of S. vesicarium-infested onion residue on SLB epidemics in a field isolated from other onion fields. SLB incidence was significantly reduced in plots without residue compared with those in which residue remained on the soil surface. Burial of infested residue also significantly reduced epidemic progress in 1 year. The effect of infested onion residue on SLB epidemics in the subsequent onion crop suggests rotation or residue management may have a substantial effect on epidemics. However, the presence of an inoculum source external to fields in onion production regions, as indicated by a lack of spatial aggregation, may reduce the efficacy of in-field management techniques.

Stemphylium Leaf Blight: A Re-Emerging Threat to Onion Production in Eastern North America.

Stemphylium leaf blight (SLB), caused by Stemphylium vesicarium, is a foliar disease of onion worldwide, and has recently become an important disease in the northeastern United States and Ontario, Canada. The symptoms begin as small, tan to brown lesions on the leaves that can progress to defoliate plants. Crop loss occurs through reduced photosynthetic area, resulting in smaller, lower-quality bulbs. Leaf necrosis caused by SLB also can compromise bulb storage, as green leaves are required for the uptake of sprout inhibitors applied prior to harvest. The pathogen can overwinter on infested onion residue and infected volunteer plants. Asymptomatic weedy hosts near onion fields may also be a source of inoculum. Production of ascospores of the teleomorph (Pleospora allii) peaks in early spring in northeastern North America, often before the crop is planted, and declines rapidly as daily mean air temperatures rise. Conidia are usually present throughout the growing season. Application of fungicides is a standard practice for management of the complex of fungi that can cause foliar diseases of onion in this region. Recent assessments have shown that populations of S. vesicarium in New York and Ontario are resistant to at least three single-site mode-of-action fungicides. Three disease prediction systems have been developed and evaluated that may enable growers to reduce the frequency and/or number of fungicide applications, but the loss of efficacious fungicides due to resistance development within S. vesicarium populations threatens sustainability. The lack of commercially acceptable onion cultivars with sufficient resistance to reduce the number of fungicides for SLB also limits the ability to manage SLB effectively. Integrated disease management strategies for SLB are essential to maintain profitable, sustainable onion production across eastern North America.

Genome Resource for Two Stemphylium vesicarium Isolates Causing Stemphylium Leaf Blight of Onion in New York.

Stemphylium leaf blight caused by Stemphylium vesicarium was recently identified as an emerging disease and dominant in the foliar disease complex affecting onion in New York. Here, we report the genomes of two isolates of S. vesicarium, On16-63 and On16-391. The availability of the genomes will accelerate genomic studies of S. vesicarium, including population biology, sexual reproduction, and fungicide resistance. Additionally, comparative genomics with the other published genome of S. vesicarium causing brown spot of pear will help understand pathogen biology and underpin the development of management strategies for this disease.

Emergence of Stemphylium Leaf Blight of Onion in New York Associated With Fungicide Resistance.

A complex of foliar diseases affects onion production in New York, including Botrytis leaf blight (Botrytis squamosa), purple blotch (Alternaria porri), Stemphylium leaf blight (SLB; Stemphylium vesicarium), and downy mildew (Peronospora destructor). Surveys were conducted in 2015 and 2016 to evaluate the cause of severe premature foliar dieback in New York onion fields. SLB was the most prevalent disease among fields with the greatest incidence, surpassing downy mildew, purple blotch, and Botrytis leaf blight. Sequencing of the internal transcribed spacer region of ribosomal DNA and the glyceraldedyhe-3-phosphate dehydrogenase and calmodulin genes identified S. vesicarium as the species most commonly associated with SLB. S. vesicarium was typically associated with a broad range of necrotic symptoms but, most commonly, dieback of leaf tips and asymmetric lesions that often extended over the entire leaf. Because of the intensive use of fungicides for foliar disease control in onion crops in New York, the sensitivity of S. vesicarium populations to various fungicides with site-specific modes of action was evaluated. Sensitivity of S. vesicarium isolates collected in 2016 to the quinone outside inhibitor (QoI) fungicide, azoxystrobin, was tested using a conidial germination assay. Isolates representing a broad range of QoI sensitivities were selected for sequencing of the cytochrome b gene to evaluate the presence of point mutations associated with insensitivity to azoxystrobin. The G143A mutation was detected in all 74 S. vesicarium isolates with an azoxystrobin-insensitive phenotype (effective concentrations reducing conidial germination by 50%, EC50 = 0.2 to 46.7 µg of active ingredient [a.i.]/ml) and was not detected in all 31 isolates with an azoxystrobin-sensitive phenotype (EC50 = 0.01 to 0.16 µg a.i./ml). The G143A mutation was also associated with insensitivity to another QoI fungicide, pyraclostrobin. Sensitivity to other selected fungicides commonly used in onion production in New York was evaluated using a mycelial growth assay and identified isolates with insensitivity to boscalid, cyprodinil, and pyrimethanil, but not difenoconazole. The frequency of isolates sensitive to iprodione, fluxapyroxad, and fluopyram was high (93.5 to 93.6%). This article discusses the emergence of SLB as dominant in the foliar disease complex affecting onion in New York and the complexities of management posed by resistance to fungicides with different modes of action.

Development of a Species-Specific PCR for Detection and Quantification of Meloidogyne hapla in Soil Using the 16D10 Root-Knot Nematode Effector Gene.

The Northern root-knot nematode (Meloidogyne hapla) is an important soilborne pathogen of numerous agricultural crops in temperate regions. Accurate detection and quantification is vital to supporting informed pest management decisions. However, traditional methods of manual nematode extraction and morphology-based identification are time-consuming and require highly specialized training. Molecular methods may expand the diagnostician's toolkit beyond those methods that rely on this disappearing specialized skillset. However, molecular assays targeting the internal transcribed spacer region may lead to inaccurate results because of intraspecific variability. The Meloidogyne spp. effector gene 16D10 was assessed as a target for a SYBR Green I quantitative PCR (qPCR) assay for detection and quantification of M. hapla. M. hapla-specific qPCR primers were developed and evaluated for specificity against five M. hapla isolates and 14 other plant-parasitic nematodes. A standard curve was generated by relating the quantification cycle (Cq) to the log of M. hapla population densities artificially introduced into soil. The influence of soil inhibitors on quantitative amplification was assessed by generating a dilution series from DNA extracted from pure nematode cultures and inoculated soil. Extracts from soil produced significantly higher Cq values than those produced from pure culture extracts. The utility of the qPCR was evaluated using soil samples collected from three naturally infested potato fields, resulting in a significant positive relationship between populations estimated using qPCR and populations derived from manual counting. The qPCR developed in this study provides a useful method for detecting and quantifying M. hapla in soil and demonstrates the utility of effector genes in plant-parasitic nematode diagnostics. The ability to use effector genes as targets for qPCR and other molecular detection and quantification methods may open additional avenues of novel research and support development of improved species-level diagnostics.

Evidence for Sexual Recombination in Didymella tanaceti Populations, and Their Evolution Over Spring Production in Australian Pyrethrum Fields.

Tan spot, caused by Didymella tanaceti, is one of the most important foliar diseases affecting pyrethrum in Tasmania, Australia. Population dynamics, including mating-type ratios and genetic diversity of D. tanaceti, was characterized within four geographically separated fields in both late winter and spring 2012. A set of 10 microsatellite markers was developed and used to genotype 774 D. tanaceti isolates. Isolates were genotypically diverse, with 123 multilocus genotypes (MLG) identified across the four fields. Fifty-eight MLG contained single isolates and Psex analysis estimated that, within many of the recurrent MLG, there were multiple clonal lineages derived from recombination. Isolates of both mating types were at a 1:1 ratio following clone correction in each field at each sampling period, which was suggestive of sexual recombination. No evidence of genetic divergence of isolates of each mating type was identified, indicating admixture within the population. Linkage equilibrium in two of the four field populations sampled in late winter could not be discounted following clone correction. Evaluation of temporal changes in gene and genotypic diversity identified that they were both similar for the two sampling periods despite an increased D. tanaceti isolation frequency in spring. Genetic differentiation was similar in populations sampled between the two sampling periods within fields or between fields. These results indicated that sexual reproduction may have contributed to tan spot epidemics within Australian pyrethrum fields and has contributed to a genetically diverse D. tanaceti population.

Cryptic diversity, pathogenicity, and evolutionary species boundaries in Cercospora populations associated with Cercospora leaf spot of Beta vulgaris.

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.

Spatiotemporal Attributes and Crop Loss Associated with Tan Spot Epidemics in Baby Lima Bean in New York.

Tan spot, caused by the pycnidial fungi Didymella americana and Boeremia exigua var. exigua, is a foliar disease affecting processing baby lima bean production in New York. Tan spot epidemics are prevalent, occur annually, and may result in substantial defoliation. The disease is controlled by the prophylactic application of fungicides to maximize green leaf area. Information on yield losses due to tan spot on baby lima bean yield and the benefits of fungicide applications is needed to justify investments in disease management. Four small-plot, replicated trials were conducted over 2 years in commercial baby lima bean fields to evaluate the efficacy of fungicides for tan spot control at Piffard and Leicester, NY. Applications of pyraclostrobin or boscalid significantly reduced tan spot incidence and severity compared with nontreated plots, and increased the number of leaves per stem. In 2016, the increase in green leaf area associated with fungicide application was also documented in canopy reflectance values at 830 nm. Despite the decrease in tan spot incidence and corresponding increase in crop health obtained from fungicides, this effect did not translate into significant increases in pod yield. This finding suggested that the relationship between green leaf area and yield is highly variable in baby lima bean. The spatial and spatiotemporal patterns of naturally occurring tan spot epidemics were also characterized in baby lima bean fields across western New York using disease incidence data collected in transects and grids. The spatial pattern of data collected in transects was analyzed using median runs analysis. Disease incidence data collected in two-dimensional grids were analyzed to quantify spatial pattern using spatial analysis by distance indices (SADIE). The association function of SADIE was used to quantify the spatiotemporal patterns of tan spot epidemics after crop emergence and at harvest. These findings suggested that tan spot is likely to initiate at relatively frequent, randomly positioned foci, and that subsequent, limited spread results in significant local aggregation. Hypotheses for inoculum sources and recommendations for tan spot control in baby lima bean fields in New York are discussed.

Mycoflora Associated With Pyrethrum Seed and the Integration of Seed Steam Treatment Into Foliar Disease Management Strategies.

A complex of foliar diseases can affect pyrethrum in Australia, but those of greatest importance are ray blight, caused by Stagonosporopsis tanaceti, and tan spot, caused primarily by Didymella tanaceti. Isolation of fungi from pyrethrum seed lots produced over 15 years resulted in recovery of six known pathogens: S. tanaceti, D. tanaceti, Alternaria tenuissima, Colletotrichum tanaceti, Stemphylium botryosum, and Botrytis cinerea. The incidence of S. tanaceti and D. tanaceti isolated from seed varied between 0.9 and 19.5% (mean = 7.7%) and 0 and 24.1% (mean = 5.3%) among years, respectively. Commercial heat treatment of pyrethrum seed via steaming reduced the incidence of D. tanaceti from 10.9 to 0.06% and the incidence of S. tanaceti from 24.6% to nondetectable levels (<0.18%). In a second experiment, both species were reduced to nondetectable levels (<0.20%) from their initial incidences of 22.4 and 2.4%, respectively. In a field study in 2013, colonization of pyrethrum foliage by S. tanaceti was reduced from 21.1 to 14.3% in early winter when heat-treated seed was planted. However, isolation frequency of D. tanaceti was not affected significantly by seed treatment in this year. In a related experiment in 2015, the isolation frequency of D. tanaceti in plots planted from heat-treated seed depended on both prior application of an industry-standard fungicide program and proximity to another pyrethrum field in autumn. The fungus was recovered at a similar frequency in fungicide-treated and nontreated plots located near other pyrethrum fields (13.8 versus 16.3%, respectively), whereas recovery of the pathogen was reduced by fungicide applications in geographically remote pyrethrum fields (6.7 versus 1.4%, respectively). However, these differences in isolation frequency of D. tanaceti in autumn did not obviate the need for later fungicide applications to suppress foliar disease intensity in spring or flower yield in summer, independent of the proximity to other pyrethrum fields. This study suggests that steam treatment of seed can delay development of the foliar disease complex on pyrethrum, although an extremely low level of remaining infected seed or exogenous sources of inoculum necessitates the use of foliar fungicide applications in spring.

Mating-Type Gene Structure and Spatial Distribution of Didymella tanaceti in Pyrethrum Fields.

Tan spot of pyrethrum (Tanacetum cinerariifolium) is caused by the ascomycete Didymella tanaceti. To assess the evolutionary role of ascospores in the assumed asexual species, the structure and arrangement of mating-type (MAT) genes were examined. A single MAT1-1 or MAT1-2 idiomorph was identified in all isolates examined, indicating that the species is heterothallic. The idiomorphs were flanked upstream and downstream by regions encoding pyridoxamine phosphate oxidase-like and DNA lyase-like proteins, respectively. A multiplex MAT-specific polymerase chain reaction assay was developed and used to genotype 325 isolates collected within two transects in each of four fields in Tasmania, Australia. The ratio of isolates of each mating-type in each transect was consistent with a 1:1 ratio. The spatial distribution of the isolates of the two mating-types within each transect was random for all except one transect for MAT1-1 isolates, indicating that clonal patterns of each mating-type were absent. However, evidence of a reduced selection pressure on MAT1-1 isolates was observed, with a second haplotype of the MAT1-1-1 gene identified in 4.4% of MAT1-1 isolates. In vitro crosses between isolates with opposite mating-types failed to produce ascospores. Although the sexual morph could not be induced, the occurrence of both mating-types in equal frequencies suggested that a cryptic sexual mode of reproduction may occur within field populations.

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.

Prediction of Potato Tuber Damage by Root-Knot Nematodes using Quantitative DNA Assay of Soil.

Root-knot nematodes (Meloidogyne fallax and M. hapla) cause significant reductions in potato yield by reducing tuber quality. Concentrations of M. fallax and M. hapla DNA in soil were determined by quantitative polymerase chain reaction following sampling at planting and harvest within 78 fields across 3 years in Australia. Meloidogyne spp. were also detected using a tomato bioassay. M. fallax was more prevalent than M. hapla and DNA concentrations of M. fallax in soil were significantly higher in samples collected at harvest compared with those at planting. In contrast, M. hapla DNA in soil did not significantly change from planting to harvest. Using receiver operating characteristic curve analysis, M. fallax DNA in soil at planting and harvest was a highly accurate predictor of tuber damage at harvest and galling on tomato. Prediction accuracy for tuber damage was highest for M. fallax DNA compared with M. hapla or M. fallax + M. hapla. Both Meloidogyne spp. were detected in the peel of asymptomatic certified seed. For M. fallax, the addition of seedborne inoculum did not improve tuber damage predictions. This suggested that soilborne M. fallax populations contributed most substantially to tuber damage. These findings highlight the utility of this approach for predicting risk of crop damage from nematodes. The use of this technique as a practical management tool is discussed.

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.

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.

Changes in Distribution and Frequency of Fungi Associated With a Foliar Disease Complex of Pyrethrum in Australia.

In Australia, pyrethrum (Tanacetum cinerariifolium) is affected by a foliar disease complex that can substantially reduce green leaf area and yield. Historically, the most important foliar disease of pyrethrum in Australia has been ray blight, caused by Stagonosporopsis tanaceti, and other fungi generally of minor importance. Temporal fluctuations in the frequency of fungi associated with foliar disease were quantified in each of 83 fields in northern Tasmania, Australia, during 2012 and 2013. Sampling was conducted throughout winter (April to July), spring (August to September), and summer (November) representing different phenological stages. Microsphaeropsis tanaceti, the cause of tan spot, was the pathogen most prevalent and isolated at the highest frequency, irrespective of sampling period. The next most common species was S. tanaceti, whose isolation frequency was low in winter and increased in spring and summer. Known pathogens of pyrethrum, Alternaria tenuissima, Colletotrichum tanaceti, and Stemphylium botryosum were recovered sporadically and at low frequency. Two species of potential importance, Paraphoma chrysanthemicola and Itersonilia perplexans, were also found at low frequency. This finding suggests a substantial shift in the dominant pathogen associated with foliar disease, from S. tanaceti to M. tanaceti, and coincides with an increase in defoliation severity in winter, and control failures of the spring fungicide program. Factors associated with this finding were also investigated. Sensitivity of M. tanaceti and S. tanaceti populations to the fungicides boscalid and cyprodinil collected prior to and following disease control failures in the field were tested under in vitro conditions. A high proportion (60%) of the M. tanaceti isolates obtained from fields in which no response to the spring fungicide program was found were insensitive to 50 µg a.i./ml boscalid. This represented a 4.2-fold increase in the frequency of this phenotype within the M. tanaceti population over 2 years. No shifts in sensitivities to cyprodinil of M. tanaceti and S. tanaceti, or S. tanaceti to boscalid, were observed. Considering the increase in defoliation severity over winter, the benefits of applying fungicides in autumn, in addition to the commercial standard (spring only), were quantified in 14 individual field trials conducted in 2011 and 2012. Mixed-model analysis suggested fungicide application in autumn may improve pyrethrum growth during late winter and early spring, although effects on defoliation and yield were minimal. The increasing prevalence and isolation frequency of M. tanaceti and boscalid resistance within the population is of concern and highlights the urgent need for adoption of nonchemical methods for disease management in Australian pyrethrum fields.