Evolution of the Genetic Structure of the Didymella tanaceti Population During Development of Succinate Dehydrogenase Inhibitor Resistance.

Emergence of pathogens with decreased sensitivity to succinate dehydrogenase inhibitor fungicides is a global agronomical issue. Analysis of Didymella tanaceti isolates (n = 173), which cause tan spot of pyrethrum (Tanacetum cinerariifolium), collected prior to (2004 to 2005) and after (2009, 2010, 2012, and 2014) the commercial implementation of boscalid in Tasmanian pyrethrum fields identified that insensitivity developed over time and has become widespread. To evaluate temporal change, isolates were characterized for frequency of mutations in the succinate dehydrogenase (Sdh) B, C, and D subunits associated with boscalid resistance, mating type, and SSR genotype. All isolates from 2004 and 2005 exhibited wild-type (WT) Sdh alleles. Seven known Sdh substitutions were identified in isolates collected from 2009 to 2014. In 2009, 60.7% had Sdh substitutions associated with boscalid resistance in D. tanaceti. The frequency of WT isolates decreased over time, with no WT isolates identified in 2014. The frequency of the SdhB-H277Y genotype increased from 10.7 to 77.8% between 2009 and 2014. Genotypic evidence suggested that a shift in the population structure occurred between 2005 and 2009, with decreases in gene diversity (uh; 0.51 to 0.34), genotypic evenness (E5; 0.96 to 0.67), genotypic diversity (G; 9.3 to 6.8), and allele frequencies. No evidence was obtained to support the rapid spread of Sdh genotypes by clonal expansion of the population. Thus, insensitivity to boscalid has developed and become widespread within a diverse population within 4 years of usage. These results suggest that D. tanaceti can disperse insensitivity through repeated frequent mutation, sexual recombination, or a combination of both.

Pathogen Incursions - Integrating Technical Expertise in a Socio-Political Context.

The incursion of a plant pathogen into a new geographic area initiates a series of decisions about appropriate control or eradication efforts. Incomplete, erroneous, and/or selective information may be used by diverse stakeholders to support individual goals and positions on how an incursion should be managed. We discuss the complex social, political, and technical factors that shape a biosecurity response prior to reviewing information needs and common stakeholder misunderstandings. Selected examples focus on the rust fungi (order Pucciniales). We then explore how plant pathologists, as technical experts, can interact with biosecurity stakeholders to build empathy and understanding that in turn allows a shift from being a distant subject matter expert to an active participant helping to structure problems and shape knowledge flows for better outcomes.

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.

Population Structure of Colletotrichum tanaceti in Australian Pyrethrum Reveals High Evolutionary Potential.

Colletotrichum tanaceti, the causal agent of anthracnose, is an emerging pathogen of commercially grown pyrethrum (Tanacetum cinerariifolium) in Australia. A microsatellite marker library was developed to understand the spatio-genetic structure over three sampled years and across two regions where pyrethrum is cultivated in Australia. Results indicated that C. tanaceti was highly diverse with a mixed reproductive mode; comprising both sexual and clonal reproduction. Sexual reproduction of C. tanaceti was more prevalent in Tasmania than in Victoria. Little differentiation was observed among field populations likely due to isolation by colonization but most of the genetic variation was occurring within populations. C. tanaceti was likely to have had a long-distance gene and genotype flow among distant populations within a state and between states. Anthropogenic transmission of propagules and wind dispersal of ascospores are the most probable mechanisms of long-distance dispersal of C. tanaceti. Evaluation of putative population histories suggested that C. tanaceti most likely originated in Tasmania and expanded from an unidentified host onto pyrethrum. Victoria was later invaded by the Tasmanian population. With the mixed mode of reproduction and possible long-distance gene flow, C. tanaceti is likely to have a high evolutionary potential and thereby has ability to adapt to management practices in the future.

Detection of Two Peronospora spp., Responsible for Downy Mildew, in Opium Poppy Seed.

Downy mildew is a serious threat to opium poppy production globally. In recent years, two pathogen species, Peronospora somniferi and Peronospora meconopsidis, which induce distinct symptoms, have been confirmed in Australia. In order to manage the spread of these pathogens, identifying the sources of inoculum is essential. In this study, we assessed pathogen presence associated with poppy seed. We developed PCR and qPCR assays targeting the coxI and coxII gene regions, for the detection, differentiation, and quantification of P. somniferi and P. meconopsidis in poppy seed. These results were complemented and compared with direct seed histological examination and a seed washing combined with viability staining for oospore detection. The majority of seed lots from all harvest years contained detectable P. meconopsidis, the earliest (1987) predating the first official record of the disease in Tasmania (1996). In contrast, only seed lots harvested in 2012 or later contained P. somniferi, evidence of its more recent introduction. P. meconopsidis contamination was estimated to be as high as 33.04 pg DNA/g of seed and P. somniferi as high as 35.17 pg DNA/g of seed. Incidence of pathogen contamination of seeds, estimated via a group testing protocol, ranged from 0 to 9% (P. meconopsidis) or 0 to 11% (P. somniferi). Mycelia were predominately found external to the seed coat. Seed washing and viability staining demonstrated that putatively viable oospores were present in the majority of seed lots. Transmission testing confirmed both pathogens can be successfully transmitted from infested seed to infected seedling. PCR and qPCR pathogen assays were found to be reliable and offer a routine test for determining pathogen inoculum in poppy seeds.

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.

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.

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.

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.

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.

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.

Comparative genome analysis indicates high evolutionary potential of pathogenicity genes in Colletotrichum tanaceti.

Colletotrichum tanaceti is an emerging foliar fungal pathogen of commercially grown pyrethrum (Tanacetum cinerariifolium). Despite being reported consistently from field surveys in Australia, the molecular basis of pathogenicity of C. tanaceti on pyrethrum is unknown. Herein, the genome of C. tanaceti (isolate BRIP57314) was assembled de novo and annotated using transcriptomic evidence. The inferred putative pathogenicity gene suite of C. tanaceti comprised a large array of genes encoding secreted effectors, proteases, CAZymes and secondary metabolites. Comparative analysis of its putative pathogenicity gene profiles with those of closely related species suggested that C. tanaceti likely has additional hosts to pyrethrum. The genome of C. tanaceti had a high repeat content and repetitive elements were located significantly closer to genes inferred to influence pathogenicity than other genes. These repeats are likely to have accelerated mutational and transposition rates in the genome, resulting in a rapid evolution of certain CAZyme families in this species. The C. tanaceti genome showed strong signals of Repeat Induced Point (RIP) mutation which likely caused its bipartite nature consisting of distinct gene-sparse, repeat and A-T rich regions. Pathogenicity genes within these RIP affected regions were likely to have a higher evolutionary rate than the rest of the genome. This "two-speed" genome phenomenon in certain Colletotrichum spp. was hypothesized to have caused the clustering of species based on the pathogenicity genes, to deviate from taxonomic relationships. The large repertoire of pathogenicity factors that potentially evolve rapidly due to the plasticity of the genome, indicated that C. tanaceti has a high evolutionary potential. Therefore, C. tanaceti poses a high-risk to the pyrethrum industry. Knowledge of the evolution and diversity of the putative pathogenicity genes will facilitate future research in disease management of C. tanaceti and other Colletotrichum spp.

Multilocus sequence analysis of Fusarium pseudograminearum reveals a single phylogenetic species.

Fusarium pseudograminearum causes crown rot of wheat in Australia and most other wheat growing regions, but its evolutionary history is largely unknown. We demonstrate for the first time that F. pseudograminearum is a single phylogenetic species without consistent lineage development across genes. Isolates of F. pseudograminearum, F. graminearum sensu lato, and F. cerealis, were collected from four countries and four single copy, nuclear genes were partially sequenced, aligned with previously published sequences of these and related species, and analysed by maximum parsimony and Bayesian inference. Evolutionary divergence varied between genes, with high phylogenetic incongruence occurring between the gene genealogies. The absence of geographic differentiation between isolates indicates that the introduction of new fungal strains to a region has the potential to introduce new pathogenic and toxigenic genes into the native population through sexual recombination.

Phylogenetic analysis of the downy mildew pathogen of oilseed poppy in Tasmania, and its detection by PCR.

Downy mildew of oilseed poppy (Papaver somniferum) has become a serious disease issue for the Tasmanian poppy industry since its first record in 1996. Previous reports have reported the pathogen as Peronospora arborescens, which is differentiated from the related species P. cristata, also known to infect Papaver spp., by conidium dimensions alone. This study investigated the taxonomic status of the downy mildew pathogen, using both morphological characters and molecular analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA). The inherent variability of conidium dimensions made differentiation of species difficult. Sequence homology and phylogenetic analyses of the ITS region showed the pathogen to be more closely related to P. cristata than P. arborescens. It is therefore proposed that downy mildew of oilseed poppy in Tasmania be reattributed to the pathogen P. cristata. In addition to this work, PCR primers have been developed for the specific detection of the downy mildew pathogen in Tasmania.