Pyrethrum In Australia Research Articles

Globisporangium and Pythium Species Associated with Yield Decline of Pyrethrum (Tanacetum cinerariifolium) in Australia

Pyrethrum (Tanacetum cinerariifolium) cultivation in Australia, which accounts for the majority of global production of natural insecticidal pyrethrins, is affected by a persistent yield decline which in part is caused by a complex of pathogens. Globisporangium and Pythium species were isolated from crown and roots of pyrethrum plants showing stunting and brown discoloration of crown tissue, and from soil adjacent to diseased plants from yield-decline-affected sites in Tasmania and Victoria, Australia. Ten known Globisporangium species (Globisporangium attrantheridium, G. erinaceum, G. intermedium, G. irregulare, G. macrosporum, G. recalcitrans, G. rostratifingens, G. sylvaticum, G. terrestris and G. ultimum var. ultimum), two new Globisporangium species (Globisporangium capense sp. nov. and Globisporangium commune sp. nov.) and three Pythium species (Pythium diclinum/lutarium, P. tracheiphilum and P. vanterpoolii) were identified through morphological studies and multigene phylogenetic analyses using ITS and Cox1 sequences. Globisporangium ultimum var. ultimum, G. sylvaticum, G. commune sp. nov. and G. irregulare were most abundant. Globisporangium attrantheridium, G. macrosporum and G. terrestris were reported for the first time in Australia. Seven Globisporangium species were pathogenic on both pyrethrum seeds (in vitro assays) and seedlings (glasshouse bioassays), while two Globisporangium species and three Pythium species only caused significant symptoms on pyrethrum seeds. Globisporangium irregulare and G. ultimum var. ultimum were the most aggressive species, causing pyrethrum seed rot, seedling damping-off and significant plant biomass reduction. This is the first report of Globisporangium and Pythium species causing disease in pyrethrum globally and suggests that oomycete species in the family Pythiaceae may have an important role in the yield decline of pyrethrum in Australia.

Ray blight of pyrethrum in Australia: a review of the current status and future opportunities

Ray blight caused by Stagonosporopsis tanaceti is one of the most important diseases of pyrethrum (Tanacetum cinerariifolium), a perennial herbaceous plant cultivated for the extraction of insecticidal pyrethrins in Australia. The disease is responsible for complete yield loss in severe outbreaks. Infected seed is considered as the principal source of S. tanaceti. Infection hyphae remain only in the seed coat and not in the embryo, resulting in pre‐ and post‐emergence death of seedlings and latent infection. Therefore, quantification of the level of infection by S. tanaceti within seed using a qPCR assay is important for efficient management of the disease. Stagonosporopsis tanaceti completes its life cycle within 12 days after leaf infection through production of pycnidia and can infect every tissue of the pyrethrum plant except the vascular and root tissues. Ray blight epidemics occur in pyrethrum fields through splash dispersal of pycnidiospores between adjacent plants. Besides steam sterilization, thiabendazole/thiram and fludioxonil are effective seed‐treating chemicals in controlling S. tanaceti before planting begins. Ray blight is currently managed in the field through the foliar application of strobilurin fungicides in the first 1–2 years of crop establishment. Later on, difenoconazole and multisite specific fungicides in the next 2–3 years during early spring successfully reduce ray blight infestation. Avoiding development of resistance to fungicides will require more sustainable management of ray blight including the development and deployment of resistant cultivars.

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.

Fusarium oxysporum and Fusarium avenaceum associated with yield-decline of pyrethrum in Australia

Fusarium species were isolated from roots, crowns, basal petioles but rarely from the leaves of infected pyrethrum (Tanacetum cinerariifolium) plants showing poor growth and stunting in yield-decline sites of northern Tasmania and the Ballarat region of Victoria, Australia. Multigene phylogenetic analyses using the internal transcribed spacer (ITS), the polymerase II second largest subunit (RPB2) and the partial translation elongation factor 1-α (EF1) sequences, identified F. oxysporum and F. avenaceum as the most frequently isolated species associated with root and crown diseases of pyrethrum, and F. equiseti and F. venenatum at a low incidence. Pathogenicity trials confirmed that F. oxysporum and F. avenaceum significantly affected growth of pyrethrum plants causing Fusarium crown rot; however, F. avenaceum was less pathogenic than F. oxysporum. Fusarium oxysporum and F. avenaceum may be part of a complex of pathogens that are involved in pyrethrum yield-decline.

Alternaria infectoria and Stemphylium herbarum, two new pathogens of pyrethrum (Tanacetum cinerariifolium) in Australia

Perithecia at the base of dead flower stems of pyrethrum (Tanacetum cinerariifolium) plants in yield-decline affected fields of northern Tasmania were identified as Alternaria infectoria and Stemphylium herbarum. Identification was based on morphological description of cultures established from single ascospores; and conidiospores; ascospore shape and septation; and multigene phylogenetic analyses using the internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1), polymerase II second largest subunit (RPB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes. Stemphylium herbarum produced necrotic leaf lesions on both sides of the spray inoculated pyrethrum leaves which coalesced to encompass the entire leaf in in vivo and in vitro experiments. Alternaria infectoria produced small necrotic leaf lesions on both sides of the leaves two weeks after inoculation which did not expand and hence, was considered as a minor pathogen of pyrethrum. This is the first report of A. infectoria and S. herbarum as pathogens of pyrethrum in Australia. The role of A. infectoria and S. herbarum in pyrethrum yield-decline in northern Tasmania needs to be evaluated.

Development of a multiplex PCR diagnostic assay for the detection of Stagonosporopsis species associated with ray blight of Asteraceae

Ray blight, a destructive disease of Asteraceae worldwide, is caused by three morphologically similar but phylogenetically distinct species; Stagonosporopsis chrysanthemi, S. inoxydabilis and S. tanaceti. Stagonosporopsis chrysanthemi has been reported as a specific pathogen of chrysanthemum while S. inoxydabilis has been found associated with various Asteraceae. Stagonosporopsis tanaceti has only been reported in Australia, causing substantial crop loss on pyrethrum. All three species were shown to infect and cause disease on in vitro grown pyrethrum plants, hence, S. chrysanthemi and S. inoxydabilis may pose a significant biosecurity threat to the Australian pyrethrum industry. All these Stagonosporopsis species are also Level 2 quarantine pathogens in Europe. Rapid and accurate detection and differentiation of these species is a priority for ray blight management in Australia and in Europe. Accordingly, three species-specific PCR-based assays, targeted to the intergenic spacer of the nuclear ribosomal DNA, were developed. The specificity of each assay was confirmed against 21 Stagonosporopsis spp. as well as 14 pathogenic and saprophytic fungal species commonly found in association with pyrethrum in Australia. The primers were highly sensitive and specific to the target species, detecting down to 4 fg of genomic DNA. These primers were further used in a multiplex PCR to differentiate the presence of the three Stagonosporopsis spp. based on variable sized amplicons in a single reaction.

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.

Carpogenic germination of sclerotia of Sclerotinia minor and ascosporic infection of pyrethrum flowers

Carpogenic germination of sclerotia and infection of flowers by ascospores of Sclerotinia minor is rare and seldom documented in most hosts. During 2007–2009, S. minor isolates were obtained from surface-sterilized pyrethrum flowers collected from fields in Australia. The isolation frequency of S. minor from flowers in 2007, 2008 and 2009 was 15.8%, 5% and 1.4%, respectively. During these years, the prevalence of S. minor in flowers amongst pyrethrum fields varied between 10.3% and 60%. Sclerotia with apothecia, consistent in size with S. minor, were collected in one field. Colonies from individual ascospores from this isolate were identified as S. minor. A subsample of 10 S. minor isolates was selected for further studies. Phylogenetic analysis based on the internal transcribed spacer region grouped these isolates with S. minor, and distinct from published sequences of other Sclerotinia spp. Species-specific primers developed previously to differentiate the four major Sclerotinia spp. (S. sclerotiorum, S. minor, S. homoeocarpa and S. trifoliorum) were used to confirm identity. Of the 10 S. minor isolates, eight were able to carpogenically germinate in vitro. Pathogenicity of S. minor to flowers was confirmed in the greenhouse using ascospores. This study is one of the few instances documenting the ability of S. minor to infect floral tissues and the first documentation of S. minor causing flower disease of pyrethrum. These findings serve as a scaffold for further investigations into the mechanisms of flower infection by S. minor and have implications for the management of the Sclerotinia disease complex affecting pyrethrum in Australia.

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.

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.

Quantifying the effects of uniconazole on growth and yield of pyrethrum in Australia

The efficacy of the plant growth regulator uniconazole (Sumagic®) for manipulating pyrethrum canopy architecture and enhancing yield was evaluated in each of 3 years across northern Tasmania, Australia. Trials examined the efficacy of, and factors influencing, reliability and magnitude of responses, including product rate, application timing, the benefits of adding a non-ionic organosilicone surfactant (Activator®) and combining application with urea. Results suggested that a rate of 100 g L−1 uniconazole (2 L ha−1 Sumagic®) was the most efficacious and that the addition of Activator® was unnecessary. However, at the highest rate, the cost of using the product became prohibitive and therefore trials in the latter 2 years of the study were conducted using the lower rate. Timing of application was not critical for efficacy and yield enhancements were observed when uniconazole was applied when stem length ranged from 16 to 36 cm, coinciding with a 30-day period in spring. Physiological effects of uniconazole were significant reductions in the height of stems (approximately 6 cm at flowering), increases in green leaf area and the number of flowers produced per unit area; the effects were independent of field age. This information has formed the basis for a cost-benefit analysis for the adoption of uniconazole into pyrethrum production in Australia.

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.

Baseline Sensitivity of Australian Phoma ligulicola Isolates from Pyrethrum to Azoxystrobin and Difenoconazole

AbstractRay blight caused by Phoma ligulicola is an important disease of pyrethrum in Australia, and successful management relies upon the fungicides, azoxystrobin and difenoconazole. Azoxystrobin and difenoconazole were introduced into pyrethrum production in 2001. The sensitivity of P. ligulicola isolates collected in 2003 to azoxystrobin (n = 56) and difenoconazole (n = 61) was tested. Testing for sensitivity to azoxystrobin and difenoconazole used a conidial germination and mycelial growth assay respectively. For each fungicide, the effective dose required to reduce mycelial growth or conidial germination by 50% (EC50) was determined by probit analysis. The EC50 values ranged from 0.007 to 0.193 μg/ml for azoxystrobin and 0.04 to 13.8 μg/ml for difenoconazole. No evidence was found for cross‐resistance between azoxystrobin and difenoconazole in this baseline population. This information serves as important baseline data for tracking future changes in sensitivities of P. ligulicola to these fungicides.

Confirmation of ray blight disease of pyrethrum in Australia

Isolations from affected plant parts of pyrethrum exhibiting flower bud abortion and necrotic symptoms consistently yielded a Phoma sp., identified as the anamorph of Didpella ligulicola, the causal agent of ray blight of chrysanthemum. Pathogenicity of both mycelial and conidial inoculum of the isolated fungus to pyrethrum was proven. Infection of stem pieces occurred by direct penetration of the fungal hyphae with invasion being both inter- and intracellular. Infection was associated with red pigmentation and cytoplasmic crystalline inclusions of host tissues. This is the first confirmed record of this disease of pyrethrum in Australia.