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.

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.

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.

Characterization of mating type genes supports the hypothesis that Stagonosporopsis chrysanthemi is homothallic and provides evidence that Stagonosporopsis tanaceti is heterothallic.

To understand the organization of the mating type locus of Stagonosporopsis tanaceti and Stagonosporopsis chrysanthemi, and its potential role in the epidemiology of ray blight of pyrethrum and chrysanthemum, respectively, the mating type (MAT) locus of these species was cloned and characterized using PCR-based techniques. The complete MAT locus of each species was cloned and annotated including complete and/or partial hypothetical genes flanking the idiomorphs. Analysis of the MAT locus organization indicated that S. chrysanthemi is likely homothallic with both MAT1-2-1 and MAT1-1-1 co-located within the idiomorph, and this was supported by production of the teleomorph in cultures of single-conidial-derived isolates. Sequencing of the MAT locus and flanking genes of S. tanaceti demonstrated that only a single MAT gene, MAT1-1-1, was located within this idiomorph and suggesting that S. tanaceti is heterothallic. MAT-specific PCR primers were developed and used to determine mating type of isolates sampled from diseased pyrethrum fields in Australia. These results indicated that only one mating type of S. tanaceti was present in Tasmania, Australia. The absence of a second mating type suggests that this species does not reproduce sexually in Tasmania, Australia and that ascospores are unlikely to be a source of inoculum for ray blight of pyrethrum. The MAT-specific PCR assay will be a valuable tool to distinguish mating types present among isolates of S. tanaceti, to monitor populations of S. tanaceti for the introduction of a second mating type and to differentiate S. tanaceti from S. chrysanthemi.

Crop Damage from Sclerotinia Crown Rot and Risk Factors in Pyrethrum.

Sclerotinia crown rot, caused by Sclerotinia sclerotiorum and S. minor, is a prevalent disease in pyrethrum fields in Australia. Management involves fungicide applications during the rosette stage of plant development from autumn to early spring in fields approaching first harvest. However, estimates of crop damage and the efficacy of these tactics are poorly understood; therefore, plots were established in 86 pyrethrum fields in Tasmania, Australia during 2010 to 2012 to quantify these and to identify risk factors for disease outbreaks. On average, commercial management for Sclerotinia crown rot reduced disease incidence 43 to 67% compared with nontreated plots. There was a weak but significant relationship between relative increase in flower yield when fungicides were applied and the incidence of crown rot (R2 = 0.09, P = 0.006), although the mean number of flowers produced was similar regardless of fungicide applications. Flower yield was positively associated with canopy density in spring (S = 0.39, P = 0.001). Moreover, canopy density in spring was linked by both direct and indirect effects to canopy density during autumn and winter which, in turn, were associated with planting date and previous rain events. Modeling canopy density and disease incidence in autumn correctly categorized disease incidence in spring relative to a threshold of 2% in 72% of fields. In a subset of 22 fields monitored over 2 years, canopy density in the autumn following the first harvest had a negative relationship with Sclerotinia crown rot incidence the preceding year (R2 = 0.23, P = 0.006). On average, however, current commercial management efforts provided only small increases in flower yield in the current season and appear best targeted to fields with well-developed plant canopies and Sclerotinia crown rot present during early autumn.

Spatiotemporal Characterization of Sclerotinia Crown Rot Epidemics in Pyrethrum.

Sclerotinia crown rot, caused by Sclerotinia minor and S. sclerotiorum, is a disease of pyrethrum in Australia that may cause substantial decline in plant density. The spatiotemporal characteristics of the disease were quantified in 14 fields during three growing seasons. Fitting the binary power law to disease incidence provided slope (b = 1.063) and intercept (ln(Ap) = 0.669) estimates significantly (P ≤ 0.0001) greater than 1 and 0, respectively, indicating spatial aggregation at the sampling unit scale that was dependent upon disease incidence. Covariate analyses indicated that application of fungicides did not significantly influence these estimates. Spatial autocorrelation and spatial analysis by distance indices indicated that spatial aggregation above the sampling unit scale was limited to 20 and 17% of transects analyzed, respectively. The range of significant aggregation was limited primarily to neighboring sampling units only. Simple temporal disease models failed to adequately describe disease progress, due to a decline in disease incidence in spring. The relationships between disease incidence at the scales of individual plants within quadrats and quadrats within a field was modeled using four predictors of sample size. The choice of the specific incidence-incidence relationship influenced the classification of disease incidence as greater than or less than 2% of plants, a provisional commercial threshold for fungicide application. Together, these studies indicated that epidemics of Sclerotinia crown rot were dominated by small-scale aggregation of disease. Larger scale patterns of diseased plants, when present, were associated with severe disease outbreaks. The spatial and temporal analyses were suggestive of disease epidemics being associated with localized primary inoculum and other factors that favor disease development at a small scale.

Minimizing Crop Damage Through Understanding Relationships Between Pyrethrum Phenology and Ray Blight Disease Severity.

The most damaging foliar disease of pyrethrum in Australia is ray blight caused by Stagonosporopsis tanaceti. The probability of growers incurring economic losses caused by this disease has been substantially reduced by the implementation of a prophylactically applied spring fungicide program. This has been traditionally initiated when 50% of the stems have reached between 5 and 10 cm in height. Data collected on the emergence of stems from semidormant plants over late winter from 27 fields across northern Tasmania from 2009 to 2011 were used to develop a degree-day model to assist with initiation of the fungicide program. Temporal changes in cumulative proportion of plants with elongated stems were well described by a logistic growth model (R2 ≥ 0.97 across all fields). These models were used to calculate the number of days until 50% of the sampling units had at least one elongated stem for the calculation of simple degree-days, assuming a nominal biofix date of the austral winter solstice. The median date for 50% stem elongation was estimated as 30 August in these data sets. Mean error and root mean square error of degree-day models were minimized when a base of 0°C was selected. Mixed-model analysis found prediction errors to be significantly affected by geographic region, requiring the use of scalar correction factors for specific production regions. In the Western region, 50% stem emergence was predicted at 590.3 degree-days (mean prediction error = 0.7 days), compared with 644.6 (mean prediction error = 7.7 days) in the Coastal region and 684.7 (mean prediction error = 0.7 days) degree-days in the Inland region. The importance of fungicide timing for initiation of the spring disease management program in minimizing losses (expressed as percent disease control in October) was also quantified. This relationship was best explained by a split-line regression with a significant break-point of 513.8 degree-days, which corresponded to 10.7% of sampling units with elongated stems. Overall, this research indicated that disease management may be improved by applying the first fungicide of the program substantially earlier in phenological development of the stems than currently recommended.

Strain Characterization of Potato virus S Isolates from Tasmania, Australia.

Potato virus S (PVS) is prevalent within potato (Solanum tuberosum) production worldwide. Traditionally, PVS has been split into two strains, Ordinary (PVSO) and Andean (PVSA), based on reaction in herbaceous indicator species such as Chenopodium quinoa. However, recent research has identified further strain designations, such as PVSO-CS (Ordinary and Chenopodium systemic). Forty-four isolates of PVS were collected from potato seed lines in different geographical regions within Tasmania, Australia. Isolates were initially characterized by reactions in C. quinoa. Nineteen isolates were characterized as PVSO, based on the development of local lesions and serological detection in inoculated leaves only. Three isolates were identified as PVSA-like, based on local lesion development in inoculated leaves, mild mottling or chlorotic spots on noninoculated leaves, and serological detection in both inoculated and noninoculated leaves. Thirteen isolates produced no symptoms, and were detected serologically in inoculated leaves only (PVSO-like). Four isolates produced no symptoms but were detected serologically in both inoculated and noninoculated leaves (PVSA-like). Five isolates produced symptoms in inoculated leaves only but were detected serologically in both inoculated and noninoculated leaves (also PVSA-like). The ability of isolates to infect tomato has also been used as a criterion to assist in PVS strain differentiation. A subsample of isolates (n = 16) was unable to infect tomato 'Grosse Lisse'. Seventeen isolates representative of these groupings based on reactions in C. quinoa were also characterized by coat-protein sequencing. Phylogenetic comparisons suggested that all isolates were PVSO rather than PVSA. Therefore, whereas some of these PVS isolates were systemic in C. quinoa, findings from this study suggest that they were not PVSA, and that only PVSO and PVSO-CS isolates are present in Tasmania. The implications of this finding for disease management are discussed.

Lack of Evidence for Recombination or Spatial Structure in Phoma ligulicola var. inoxydabilis Populations from Australian Pyrethrum Fields.

Ray blight, caused by Phoma ligulicola var. inoxydabilis, causes substantial annual losses in Australian pyrethrum fields. Fifty-nine P. ligulicola var. inoxydabilis isolates were randomly selected from fields in three distinct geographical regions in Tasmania, Australia. Genetic diversity was characterized using random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Based on genetic similarities of less than 99%, 56 distinct genotypes (putative clones) were observed. Mean haploid gene diversity of clone-corrected populations ranged between 0.05 and 0.31, and 0.11 and 0.32, for the RAPD and AFLP data sets, respectively. Cluster analysis indicated two distinct groups of isolates supported by all bootstrap replicates. The first cluster contained all but four isolates with representatives from all three populations. The second cluster contained two isolates from the Western and Central populations, respectively, while the remaining isolates were not able to be grouped with any distinct cluster. Analysis of the population structure suggested no evidence for spatial autocorrelation at the smallest distance classes. The presence of linkage disequilibrium was indicated regardless of population scale. Collectively, these findings provided further evidence for the absence or minor role of the teleomorph in the epidemiology of ray blight in Australian pyrethrum fields.

Epidemics of ray blight on pyrethrum are linked to seed contamination and overwintering inoculum of Phoma ligulicola var. inoxydabilis.

Ray blight, caused by Phoma ligulicola var. inoxydabilis, is the most damaging disease of pyrethrum (Tanacetum cinerariifolium) in Australia. Data collected from 72 plots in commercial pyrethrum fields since 2001 to 2009 revealed substantial annual variations in isolation frequency of the pathogen during semidormancy of the crop in autumn and winter. Isolation frequency of the pathogen during this time and subsequent outbreaks of ray blight in spring were similar across the eight production regions where sampling was conducted, and isolation frequency of the pathogen was linearly correlated (r = 0.88; P < 0.0001) with subsequent defoliation severity when plants commenced growth in spring. Isolation frequency and defoliation severity also were correlated with the incidence of seed infested with P. ligulicola var. inoxydabilis (r = 0.71 and 0.44, respectively; P < 0.0001 in both correlations). Highly accurate risk algorithms for the occurrence of severe epidemics of ray blight were constructed using logistic regression. A model based solely on isolation frequency of the pathogen over autumn and winter correctly predicted epidemic development in 92% of fields. Another model utilizing the incidence of infested seed and rain-temperature interactions in early autumn (austral March and April) and late winter (austral June and July) had similar predictive ability (92% accuracy). Path analysis modeling was used to disentangle interrelationships among the explanatory variables used in the second logistic regression model. The analysis indicated that seedborne inoculum of P. ligulicola var. inoxydabilis contributes indirectly to ray blight defoliation severity through directly increasing overwintering frequency of the pathogen. Autumn and fall weather variables were modeled to have indirect effects on defoliation severity through increasing overwintering success of the pathogen but also direct effects on defoliation severity. Collectively, the analyses point to several critical stages in the disease cycle that can be targeted to minimize the probability of regional epidemics of ray blight in this perennial pathosystem.