Global Spread, Genetic Differentiation, and Selection of Barley Spot Form Net Blotch Isolates.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a significant necrotrophic disease of barley that spread worldwide in the twentieth century. Genetic relationships were analyzed to determine the diversity, survival, and dispersal of a diverse collection of 346 isolates from Australia, Southern Africa, North America, Asia Minor, and Europe. The results, based on genome-wide DArTseq data, indicated that isolates from Turkey were the most differentiated with regional sub-structuring, together with individuals closely related to geographically distant genotypes. Elsewhere, population subdivision related to country of origin was evident, although low levels of admixturing was found that may represent rare genotypes or migration from unsampled populations. Canadian isolates were the next most diverged, and Australian and South African the most closely related. With the exception of Turkish isolates, multiple independent Cyp51A mutation events (which confer insensitivity to demethylation inhibitor fungicides) between countries and within regions was evident, with strong selection for a transposable element insertion at the 3' end of the promoter and counterselection elsewhere. Individuals from Western Australia shared genomic regions and Cyp51A haplotypes with South African isolates, suggesting a recent common origin. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Exploiting long read sequencing to detect azole fungicide resistance mutations in Pyrenophora teres using unique molecular identifiers.

Resistance to fungicides is a global challenge as target proteins under selection can evolve rapidly, reducing fungicide efficacy. To manage resistance, detection technologies must be fast and flexible enough to cope with a rapidly increasing number of mutations. The most important agricultural fungicides are azoles that target the ergosterol biosynthetic enzyme sterol 14α-demethylase (CYP51). Mutations associated with azole resistance in the Cyp51 promoter and coding sequence can co-occur in the same allele at different positions and codons, increasing the complexity of resistance detection. Resistance mutations arise rapidly and cannot be detected using traditional amplification-based methods if they are not known. To capture the complexity of azole resistance in two net blotch pathogens of barley we used the Oxford Nanopore MinION to sequence the promoter and coding sequence of Cyp51A. This approach detected all currently known mutations from biologically complex samples increasing the simplicity of resistance detection as multiple alleles can be profiled in a single assay. With the mobility and decreasing cost of long read sequencing, we demonstrate this approach is broadly applicable for characterizing resistance within known agrochemical target sites.

Workflows for detecting fungicide resistance in net form and spot form net blotch pathogens.

BACKGROUND: Fungicide resistance in Pyrenophora teres f. maculata and P. teres f. teres has become an important disease management issue. Control of the associated barley foliar diseases, spot form and net form net blotch, respectively, relies on three major groups of fungicides, demethylation inhibitors (DMIs), succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs). However, resistance has been reported for the DMI and SDHI fungicides in Australia. To enhance detection of different resistance levels, phenotyping and genotyping workflows were designed. RESULTS: The phenotyping workflow generated cultures directly from lesions and compared growth on discriminatory doses of tebuconazole (DMI) and fluxapyroxad (SDHI). Genotyping real-time polymerase chain reaction (PCR) assays were based on alleles associated with sensitivity or resistance to the DMI and SDHI fungicides. These workflows were applied to spot form and net form net blotch collections from 2019 consisting predominantly of P. teres f. teres from South Australia and P. teres f. maculata from Western Australia. For South Australia the Cyp51A L489-3 and SdhC-R134 alleles, associated with resistance to tebuconazole and fluxapyroxad, respectively, were the most prevalent. These alleles were frequently found in single isolates with dual resistance. This study also reports the first detection of a 134 base pair insertion located at position-66 (PtTi-6) in the Cyp51A promoter of P. teres f. maculata from South Australia. For Western Australia, the PtTi-1 insertion was the most common allele associated with resistance to tebuconazole. CONCLUSION: The workflow and PCR assays designed in this study have been demonstrated to efficiently screen P. teres collections for both phenotypic and genetic resistance to DMI and SDHI fungicides. The distribution of reduced sensitivity and resistance to DMI and SDHI fungicides varied between regions in south-western Australia, suggesting the emergence of resistance was impacted by both local pathogen populations and disease management programmes. The knowledge of fungicide resistance in regional P. teres collections will be important for informing appropriate management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Detection of Ramularia collo-cygni from barley in Australia using triplex quantitative and droplet digital PCR.

BACKGROUND: Ramularia leaf spot (RLS), caused by Ramularia collo-cygni, is an emerging threat to barley (Hordeum vulgare L.) production. RLS has been reported in Australia, however only minimal information is available regarding its detection and distribution. Due to initial asymptomatic growth in planta, slow growth in vitro and symptomatic similarities to net blotch and physiological leaf spots, detection of this pathogen can be challenging. Quantitative polymerase chain reaction (PCR)-based methods for R. collo-cygni-specific identification and detection have been described, however these assays have been demonstrated to lack specificity. False-positive detections may have serious implications, thus we aimed to design a robust R. collo-cygni-specific PCR method. RESULTS: Using the phylogenetically informative RNA polymerase II second largest subunit (rpb2) and translation elongation factor 1-alpha (tef1-α) genes, along with the tef1-α gene of H. vulgare, a triplex assay was developed for both quantitative and droplet digital PCR. The triplex assay detected R. collo-cygni DNA in barley leaves from New South Wales, South Australia, Tasmania, Victoria and Western Australia. No R. collo-cygni DNA was detected in barley seed grown in Western Australia. CONCLUSION: The presence of R. collo-cygni DNA has been confirmed in Australian barley crops, suggesting a distribution ranging across the southern barley growing regions of Australia. The R. collo-cygni-specific assay will be a valuable tool to assist with monitoring the distribution and impact of R. collo-cygni in Australia and other regions. © 2021 Society of Chemical Industry.

Population Structure of Pyrenophora teres f. teres Barley Pathogens from Different Continents.

Net form net blotch disease, caused by Pyrenophora teres f. teres, results in significant yield losses to barley industries. Up-to-date knowledge of the genetic diversity and structure of pathogen populations is critical for elucidating the disease epidemiology and unraveling pathogen survival and dispersal mechanisms. Thus, this study investigated long-distance dispersal and adaptation by analyzing the genetic structure of 250 P. teres f. teres isolates collected from Australia, Canada, Hungary, and Republic of South Africa (RSA), and historical isolates from Canada, Denmark, Japan, and Sweden. The population genetic structure detected by discriminant analysis of principal components, with the use of 5,890 Diversity Arrays Technology markers, revealed the presence of four clusters. Two of these contained isolates from all regions, and all isolates from RSA were grouped in these two. Australia and Hungary showed three clusters each. One of the Australian clusters contained only Australian isolates. One of the Hungarian clusters contained only Hungarian isolates and one Danish isolate. STRUCTURE analysis indicated that some isolates from Australia and Hungary shared recent ancestry with RSA, Canada, and historical isolates and were thus admixed. Subdivisions of the neighbor joining network indicated that isolates from distinct countries were closely related, suggesting that multiple introduction events conferred genetic heterogeneity in these countries. Through a neighbor joining analysis and amplification with form-specific DNA markers, we detected two hybrid isolates, CBS 281.31 from Japan and H-919 from Hungary, collected in 1931 and 2018, respectively. These results provide a foundation for exploring improved management of disease incursions and pathogen control through strategic deployment of resistance.

Detection of Cercospora beticola and Phoma betae on Table Beet Seed using Quantitative PCR.

Cercospora beticola and Phoma betae are important pathogens of table beet, sugar beet, and Swiss chard (Beta vulgaris subsp. vulgaris), causing Cercospora leaf spot (CLS) and Phoma leaf spot, root rot, and damping-off, respectively. Both pathogens may be seedborne; however, limited evidence is available for seed infestation by C. beticola. Due to the limitations of culture-based seed assessment methods, detection of these pathogens was investigated using PCR. A P. betae-specific quantitative PCR assay was developed and used in conjunction with a C. beticola-specific assay to assess the presence of pathogen DNA in 12 table beet seed lots. DNA of C. beticola and P. betae was detected in four and eight seed lots, respectively. Plate tests and BIO-PCR confirmed the viability of each pathogen; however, competitive growth of other microbes and low incidence limited the frequency and sensitivity of detection in some seed lots. The results for P. betae support previously described infestation of seed. Further investigation of C. beticola-infested seed lots indicated the ability of seedborne C. beticola to cause CLS on plants grown from infested seed. Detection of viable C. beticola on table beet seed demonstrates the potential for pathogen dispersal and disease initiation via infested seed, and provides valuable insight into the epidemiology of CLS. Surveys of commercial table beet seed are required to determine the frequency and source of C. beticola seed infestation and its role as primary inoculum for epidemics, and to evaluate the effectiveness of seed treatments.

Alternative Hosts of Cercospora beticola in Field Surveys and Inoculation Trials.

Cercospora beticola, the cause of Cercospora leaf spot (CLS) of sugar beet and table beet, has a broad range of potential alternative hosts. The role of these hosts as inoculum sources in the field is unclear and has had limited investigation since the advent of DNA-based pathogen identification. The presence of C. beticola on alternative hosts associated with table beet fields of New York was assessed in field surveys during 2016. Lesions were collected, and 71 cercosporoid conidia were isolated for phylogenetic comparison. C. beticola was identified from Solanum ptycanthum (n = 4), Chenopodium album (n = 2), and Spinacia oleracea (n = 1), whereas C. chenopodii was identified on Chenopodium album (n = 51). Artificial inoculation of 21 plants species demonstrated that C. beticola was pathogenic to Brassica kaber, Chenopodium album, Carthamus tinctorius, Rumex obtusifolius, and Spinacia oleracea. These results indicate that although C. beticola may be pathogenic to a range of plant species, the role of symptomatic tissue for inoculum production on alternative hosts in the field appears limited. Observations of C. beticola on necrotic and naturally senescent tissue suggest saprophytic survival on plant debris of a range of species, which has implications for CLS epidemics and disease management.

Genetic Diversity and Differentiation in Phoma betae Populations on Table Beet in New York and Washington States.

Phoma betae is an important seedborne pathogen of table beet worldwide that is capable of causing foliar, root, and damping-off diseases. Ten microsatellite and mating type markers were developed to investigate the genetics of P. betae populations in table beet root crops in New York and in table beet seed crops in Washington, from where table beet seed is predominantly sourced. The markers were used to characterize 175 isolates comprising five P. betae populations (two from New York and three from Washington), and they were highly polymorphic with an allelic range of 4 to 33 and an average of 11.7 alleles per locus. All populations had high genotypic diversity (Simpson's complement index = 0.857 to 0.924) and moderate allelic diversity (Nei's unbiased gene diversity = 0.582 to 0.653). Greater differentiation observed between populations from the two states compared with populations within the same state suggested that an external inoculum source, such as windblown ascospores, may be homogenizing the populations. However, most genetic diversity (87%) was among individual isolates within populations (pairwise index of population differentiation = 0.127; P = 0.001), suggesting that local within-field inoculum source(s), such as infested field debris or infected weeds, may also be important in initiating disease outbreaks. Standardized index of association, proportion of compatible pairs of loci, and mating type ratio calculations showed evidence for a mixed reproduction mode in all populations. These findings could be useful in designing more effective management strategies for diseases caused by P. betae in table beet production.

Genetic Diversity and Structure in Regional Cercospora beticola Populations from Beta vulgaris subsp. vulgaris Suggest Two Clusters of Separate Origin.

Cercospora leaf spot, caused by Cercospora beticola, is a highly destructive disease of Beta vulgaris subsp. vulgaris worldwide. C. beticola populations are usually characterized by high genetic diversity, but little is known of the relationships among populations from different production regions around the world. This information would be informative of population origin and potential pathways for pathogen movement. For the current study, the genetic diversity, differentiation, and relationships among 948 C. beticola isolates in 28 populations across eight geographic regions were investigated using 12 microsatellite markers. Genotypic diversity, as measured by Simpson's complement index, ranged from 0.18 to 1.00, while pairwise index of differentiation values ranged from 0.02 to 0.42, with the greatest differentiation detected between two New York populations. In these populations, evidence for recent expansion was detected. Assessment of population structure identified two major clusters: the first associated with New York, and the second with Canada, Chile, Eurasia, Hawaii, Michigan, North Dakota, and one population from New York. Inferences of gene flow among these regions suggested that the source for one cluster likely is Eurasia, whereas the source for the other cluster is not known. These results suggest a shared origin of C. beticola populations across regions, except for part of New York, where population divergence has occurred. These findings support the hypothesis that dispersal of C. beticola occurs over long distances.

Temporal Genetic Differentiation of Cercospora beticola Populations in New York Table Beet Fields.

Annual epidemics of Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, can result in substantial defoliation in table beet fields in New York. High allelic and genotypic diversity have been described within C. beticola populations; however, information on the temporal stability of populations is lacking. C. beticola isolates were obtained from symptomatic leaves in three table beet fields in successive years. Two of the fields were organic mixed-cropping farms and the third was managed conventionally in a broad-acre cropping system. C. beticola isolates (n = 304) were genotyped using 12 microsatellite markers. Genotypic diversity (Simpson's complement index = 0.178 to 0.990), allele frequencies, and indices of differentiation between years varied. Pairwise index of differentiation values ranged from 0.02 to 0.25 for clone-corrected data, and indicated significant genetic differentiation at Farm 2. No multilocus genotype was shared between years. The shift in multilocus genotypes between years questions the role of clonally reproducing primary inoculum. Collectively, these results suggest that a dominant inoculum source for initiating annual CLS epidemics is external to the field of interest. These findings have implications for CLS disease management in conventional and organic table beet production.

Colonization of Durum Wheat (Triticum turgidum L. var. durum) Culms Exhibiting Premature Senescence (Dead Heads) Associated with Fusarium pseudograminearum Crown Rot.

Fusarium crown rot is a significant disease of durum wheat (Triticum turgidum L. var. durum), which exhibits high levels of disease susceptibility. The most extreme symptom of crown rot is a prematurely senescing culm that typically fails to set grain. Individual crown rot-affected durum wheat plants displaying both nonsenescent and prematurely senescent culms were harvested to compare visual discoloration, Fusarium pseudograminearum biomass, and vascular colonization in culm sections sampled at three different heights above the crown. Field samples of EGA Bellaroi were collected at Wellcamp, QLD, in 2011, 2012, 2013, and 2014, and of Hyperno at Narrabri, NSW, in 2014. Prematurely senescent culms exhibited greater visual discoloration, F. pseudograminearum biomass, and vascular colonization than nonsenescent culms in each year they were examined. The extent of these differences varied between environments and timing of collection in each year. Vascular colonization initially occurred in xylem vessels and spread into phloem tissues as disease severity increased. The increased presence of hyphae in vascular bundles of prematurely senescing culms provides strong evidence for the hypothesis that restriction of water and nutrient movement in a diseased culm is a key factor in crown rot severity.

Assessment of Fusarium pseudograminearum and F. culmorum Biomass in Seedlings of Potential Host Cereal Species.

Fusarium crown rot is a major disease of wheat and barley worldwide, with the most frequently isolated causal agents being Fusarium pseudograminearum and F. culmorum. This study has successfully designed a quantitative polymerase chain reaction assay that is specific for F. culmorum, which has been used in conjunction with a previously established F. pseudograminearum-specific assay to compare the location and extent of infection by each fungus across a range of potential hosts, including six winter and three summer cereal species. All common winter cereals, excluding oat, demonstrated a similar range of visual and fungal biomass results when inoculated with either F. pseudograminearum or F. culmorum. Oat exhibited the lowest visual disease ratings and fungal biomass values of the winter cereals, while the sorghum, maize, and rice cultivars returned the lowest values overall. The ranking of host species according to visual discoloration was strongly correlated for both pathogens. Visual reactions to F. pseudograminearum were greater than those caused by F. culmorum in all potential hosts trialed; however, fungal biomass results only indicated this trend for barley. These results demonstrate significant variation in the ability of these pathogens to colonize the range of cereal species examined and also suggest differences between the pathogens in their patterns of host colonization.

Histopathological Assessment of Fusarium pseudograminearum Colonization of Cereal Culms During Crown Rot Infections.

Histopathological assessment of the crown rot pathogen Fusarium pseudograminearum was performed using fluorescence microscopy of culm tissues of six cereal genotypes grown in inoculated field conditions. Tissue samples were collected at 10, 16, and 22 weeks after planting (WAP). Colonization of culm tissues was initiated through epidermal penetration, most distinctly through stomatal apertures, and progressed into the parenchymatous hypoderm, which exhibited the discoloration used as the basis for visual assessment of disease. Hyphae spread from the culm base vertically through the tissues, initially via the hypoderm and pith cavity. Colonization of sclerified cells occurred later in the disease process. Both xylem and phloem tissues became colonized by 16 WAP in all host genotypes, with colonization being less extensive in the more resistant genotypes. Culms displaying dead head symptoms revealed dense colonization in at least the first three internodes, with frequent xylem vessel and phloem cell occlusions. Paired living culms from the same plants exhibited less extensive colonization. These observations have revealed the ability of F. pseudograminearum to colonize all cell types of nodal and internodal sections, including vascular tissues, across all host genotypes. This study is the first detailed examination of the pattern of F. pseudograminearum colonization in adult hosts and indicates a potential vascular mechanism by which the effects of crown rot are produced.

Assessment of Infection by Fusarium pseudograminearum in Wheat Seedling Tissues Using Quantitative PCR and a Visual Discoloration Scale.

Assessment among cereal genotypes of relative seedling resistance to the crown rot pathogen Fusarium pseudograminearum has been primarily based on visual discoloration of the leaf sheaths. This study is the first to investigate the relationship between the widely used visual rating of seedling leaf sheath discoloration and the degree of colonization of these tissues by the pathogen, based on quantitative polymerase chain reaction (qPCR) of fungal DNA using primers specific for the translation elongation factor α sequence. Fourteen-day-old seedlings of four hard white spring wheat genotypes which differ in their degree of resistance to the pathogen, based on the expression of visible symptoms, were inoculated using a droplet method and assessed weekly from 7 to 35 days after inoculation (dai) for both discoloration and fungal DNA content per unit of tissue weight. Both visual assessment of disease symptoms and qPCR of fungal biomass indicated significant differences between the partially resistant and susceptible wheat genotypes from 14 dai. Visual discoloration of leaf sheath tissues was strongly correlated with fungal biomass estimated by qPCR in all four genotypes; however, this correlation became weaker with increasing time after inoculation. Significant correlations between these parameters were indicated at 14, 21, and 28 dai whereas, by 35 dai, the correlation was not significant. Evaluation of plants at 14 dai provided a rapid test which gave clear discrimination between lines for both parameters and was the time point of closest correlation between fungal colonization and disease symptoms. Symptom expression at all times following inoculation was accompanied by tissue infection, and at no time was symptomless infection observed under this screening environment. These qPCR results confirm that visual assessments of disease symptoms reflect the extent of tissue colonization by the pathogen in recently colonized tissues and confirm the validity of visual assessments for disease rating in high-throughput screening of breeding materials.