Jb Scott

Pathogen population structure and epidemiology are keys to wheat crown rot and Fusarium head blight management

This paper summarises the key findings from recent research on the population genetics and epidemiology of Fusarium pathogens causing head blight and crown rot of wheat in Australia and how this information has enabled the screening and selection of wheat germplasm with improved resistance to Fusarium. By relating new findings to the current state of knowledge, the paper serves as a timely and critical review of the international literature. In Australia, both Fusarium pseudograminearum and F. graminearum can cause both crown rot and Fusarium head blight under artificial inoculation. However, the former species is more widespread and is predominantly associated with crown rot whereas F. graminearum is mainly associated with Fusarium head blight, with limited geographical distribution in and around the Liverpool Plains in northern New South Wales. Studies of population structure and genetics have revealed that both species are genotypically diverse with similar levels of genetic recombination despite Gibberella zeae, the teleomorph of F. graminearum, being homothallic and G. coronicola, the teleomorph of F. pseudograminearum, being heterothallic. A high-throughput and reliable crown rot bioassay has been developed and used to screen over 1500 wheat germplasms to select 17 lines with putative crown rot resistance. Key differences in pathogen biology and epidemiology between Australia and the USA have emerged from other recent collaborative studies, which show that macroconidia constitute the bulk of aerial Fusarium head blight inoculum in Australia, whereas ascospores are the dominant primary inoculum for Fusarium head blight worldwide. The limited spread of splash-dispersed macroconidia of F. graminearum probably explains the restricted geographical distribution of this species in Australia. Other research collaboration has compared the aggressiveness, mycotoxin production and genotypic polymorphisms of the pathogen population from Australia and the USA. These and other differences in pathogen adaptation emphasise that research outcomes from elsewhere must be tested for relevance before applying them to Australian farming systems.

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.

Genetic relationships among isolates of Phoma ligulicola from pyrethrum and chrysanthemum based on ITS sequences and its detection by PCR

Variation within the internal transcribed spacer (ITS 1, 5.8S gene and ITS2) region of the rDNA (ITS) was used to characterise the phylogenetic relationships among Phoma ligulicola isolates infecting pyrethrum crops in Tasmania, P. ligulicola isolates from the USA, Germany and mainland Australia, and other closely related fungal species. This study reports the first characterisation of the ITS region of P. ligulicola. Sequence homology within P. ligulicola isolates varied between 99.3 and 100%. For 9 of the 11 isolates from Tasmania, Australia, the nucleotide sequences in this region were identical, whereas the sequences for the remaining two isolates differed only by two nucleotides in the ITS 1 region. Isolates from Australia and the USA failed to metabolise NaOH on malt-extract agar and were characterised as P. ligulicola var. inoxydablis. The two isolates from ray blight disease of chrysanthemum in Germany (DSMZ 63133 and DSMZ 62547) were classified as P. ligulicola var. ligulicola. Phylogenetic analyses suggested that the ITS sequences of P. ligulicola isolates were more similar to other Phoma species than selected representatives of the Mycosphaerella genus. Didymella bryoniae had the greatest interspecific homology with P. ligulicola of the fungi used in this study. This information was used to design specific primers within the ITS regions for the detection of P. ligulicola.

Spatiotemporal Analysis of Epiphytotics of Downy Mildew of Oilseed Poppy in Tasmania, Australia

Downy mildew, caused by Peronospora arborescens, has become the major disease affecting oilseed poppy (Papaver somniferum) since its first record in Tasmania in 1996. Two field trials conducted in 2000 and 2001 studied the progression and spatial distribution of downy mildew epiphytotics. The logistic and exponential models best described the progression of disease incidence and severity, respectively. Incidence and severity increased rapidly following canopy closure. In 2001, incidence increased from 0.16%, prior to canopy closure, to 100% at late flowering (40 days). Spatial analyses of epiphytotics were conducted by fitting the beta-binomial and binomial distributions, median runs analysis, and the spatial analysis by distance indices. All analyses demonstrated that the distribution of incidence and severity was strongly spatially aggregated from canopy closure until at least late flowering. These results suggest that secondary spread from a few primary infections is the major factor in epiphytotics.

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.

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.

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.

Identification of 11 polymorphic simple sequence repeat loci in the phytopathogenic fungus Fusarium pseudograminearum as a tool for genetic studies

Simple sequence repeat (SSR) markers for Fusarium pseudograminearum with 2 to 3 bp repeat motifs were identified by screening the genome database of the related species Fusarium graminearum. Twelve SSRs amplified single loci in both F. graminearum and F. pseudograminearum. Forty F. pseudograminearum and six F. graminearum individual isolates were screened to determine levels of polymorphism, with all SSRs displaying three to 14 alleles across all isolates. Eleven SSRs were polymorphic across F. pseudograminearum isolates tested proving the usefulness of genome databases of closely related species in identifying genetic markers.

Paraphoma chlamydocopiosa sp. nov. and Paraphoma pye sp. nov., two new species associated with leaf and crown infection of pyrethrum

Two new pathogens of pyrethrum, described as Paraphoma chlamydocopiosa and Paraphoma pye, isolated from necrotic leaf lesions on pyrethrum plants in northern Tasmania, Australia, were identified using morphological characters, phylogenetic analysis of the internal transcribed spacer (ITS), elongation factor 1-α (EF1-α) and β-tubulin (TUB) genes, and pathogenicity bioassays. Bootstrap support in the combined and individual gene region phylogenetic trees supported the two species that were significantly different from the closely related P. chrysanthemicola and P. vinacea. Morphological characteristics also supported the two new species, with conidia of P. chlamydocopiosa being considerably longer and wider than either P. chrysanthemicola or P. vinacea, and P. pye being distinct in forming bilocular pycnidia. Glasshouse pathogenicity tests based on root dip inoculation resulted in P. chlamydocopiosa and P. pye infecting the crown and upper root tissues of pyrethrum plants, and significant reduction in biomass 2 months after inoculation. Both of these Paraphoma species caused leaf lesions during in vitro and in vivo bioassays 2 weeks after foliar spray inoculation. Although P. chlamydocopiosa and P. pye were shown to be crown rot pathogens, they were also commonly isolated from leaves of diseased plants in pyrethrum fields of northern Tasmania.