Phytopathology®

Australia is the largest producer of Pyrethrum (Tanacetum cinerariifolium) globally. Amongst the constraints on production are the fungal pathogens Didymella tanaceti and Stagonosporopsis tanaceti, which pose a significant threat to the industry, causing substantial yield losses. While the infection biology of S. tanaceti is well characterised, knowledge of D. tanaceti and its potential interaction with S. tanaceti on plants remains limited, hindering disease management. We developed fluorescently labelled strains of both pathogens via Agrobacterium tumefaciens-mediated transformation (ATMT). Binary vectors carrying the mNeonGreen or tdTomato fluorescent protein genes were introduced into D. tanaceti and S. tanaceti, respectively, and expression of the fluorescent proteins was confirmed by microscopy. Genome sequencing revealed single-copy T-DNA insertions in all transformants, with minor genomic rearrangements at insertion sites. Detached leaf assays demonstrated that transformed strains retained pathogenicity, producing disease symptoms indistinguishable from those of the wild type. These fluorescently labelled variants enabled detailed visualisation of D. tanaceti infection biology and its interactions with S. tanaceti, including co-infection dynamics. Co-infection assays using fluorescent strains further facilitated simultaneous visualisation and differentiation of both pathogens within host tissues. Importantly, these tools also allowed the first description of the early stages of infection by D. tanaceti in pyrethrum leaves. This study represents the first successful transformation of D. tanaceti and S. tanaceti, providing valuable resources to investigate their infection processes.

Beyond the significant impact of Cassava witches broom disease (CWBD), caused by the fungus Rhizoctonia (syn. Ceratobasidium) theobromae on cassava cultivation in French Guiana and Brazil, this disease also poses a potential threat to cacao trees in the region, since the fungus is responsible for Vascular Streak Dieback (VSD) of cacao in South East Asia. Cross-pathogenicity trials were conducted in several cassava fields in French Guiana by planting young cacao plants adjacent to diseased cassava plants. Vascular necrosis was observed in some cacao plants, and the presence of R. theobromae in the cacao tissues was confirmed through PCR diagnostics using primers specific to the fungus. Sequence analysis indicated 100% similarity between samples from both hosts and 97.53 to 99.74% identity with R. theobromae isolates previously reported from cassava in the Americas and Southeast Asia. Additionally, symptomatic cacao in a mixed cacao-cassava farm yielded R. theobromae-positive PCR results, suggesting a natural infection. Ongoing work includes artificial inoculations and controlled cross-pathogenicity trials under screenhouse conditions to attempt reproduction of the symptoms. While current data do not yet establish definitive causality, the findings indicate potential host jump and warrant rapid communication to researchers, policy makers, and farmers to safeguard cacao production and Theobroma biodiversity in the Amazon region.

Cucurbit yellow vine disease (CYVD), caused by the bacterium Serratia ureilytica, is a phloem-associated disease of cucurbits. This study characterized the spatial and temporal distribution of S. ureilytica in Cucurbita pepo cultivar Delicata plants under greenhouse conditions using a GFP-tagged isolate (P01). Seedlings were sampled weekly for four weeks. Transverse sections from the stem, petiole, leaf, shoot apex, and root were imaged by laser scanning confocal and fluorescent dissecting microscopy. In parallel, bacterial abundance in each plant tissue was assessed by quantifying colony-forming units (CFU) via droplet plating over a 4-week time course. Across plant tissues and time points, S. ureilytica fluorescent signal was primarily concentrated in the inner and outer periphery of the bicollateral vascular bundles, with higher magnification images revealing mainly symplastic localization within phloem-associated cells. Consistent with the imaging results, bacterial quantification data showed a high abundance of CFUs in the main stem across weeks, with an irregular pattern of presence in the distal tissues at later time points. These results suggest that S. ureilytica is predominantly localized within phloem-associated cells and spreads both acropetally and basipetally during infection.

The fastidious basidiomycete Rhizoctonia (Ceratobasidium) theobromae is a biotrophic pathogen that causes Vascular-Streak Dieback (VSD) of Theobroma cacao (cocoa). The fungus has also been identified as the cause of an emergent disease known as Cassava Witches Broom Disease (CWBD) raising concerns that the pathogen is spreading to alternative hosts and to new regions. Interestingly, while VSD of cocoa and CWBD are reported as co-present in several countries, there is currently no evidence for cross-infection between species. The fungus is difficult to culture in vitro due its slow growth and Kochs postulates have not been definitive on either host. The complete fungus life cycle therefore remains enigmatic, though studies have progressed knowledge on pathology within the both the cocoa and cassava hosts. We have conducted limited field trials and sequenced mating (MAT) and ITS loci of isolates from various infected hosts and regions. We hypothesize that (i) genetic variation at MAT loci correlates with region or host (ii) long amplicon ITS sequences between isolates are more definitive for polymorphisms (iii) life-cycle traits of R. theobromae may be inferred from MAT loci (iv) cassava grown under VSD infected cocoa will be infected and develop symptoms of CWBD. We did not find any cross-infection in field trials, and we show that the pathogen is highly homozygous, despite undergoing meiosis, indicating a predominantly homothallic life cycle. Our data indicate that the pathogen is likely host specific and regionally divergent and suggests that host specificity on cocoa and cassava evolved by selection from a common ancestor rather than a host jump.

Phytophthora species are globally significant soilborne oomycetes responsible for widespread ecosystem decline. Standard soil sampling protocols, originally developed for qualitative baiting assays, typically require collecting substantial soil volumes in order to capture viable propagules. While effective for culture-based detection, these protocols are labour-intensive and can damage the shallow root systems of sensitive host species such as New Zealand kauri (Agathis australis). Phytophthora agathidicida (PA), the pathogen associated with kauri dieback disease, is routinely surveyed using these methods. However, quantitative data describing the vertical distribution of PA in natural forest soils are lacking. Consequently, it remains unclear whether extensive depth sampling is necessary to ensure consistent molecular detection. In this study, we applied a quantitative oospore DNA (oDNA) qPCR assay to characterise the fine-scale vertical distribution of PA across four soil depth increments (0-5, 5-10, 10-15, 15-20 cm) from 12 kauri trees representing a range of disease stages. Results revealed distinct vertical stratification, with PA DNA concentrations peaking within the upper 0-10 cm of soil in non-symptomatic and possibly symptomatic trees. In symptomatic trees, the absolute peak occasionally reached 10-15 cm, while pathogen signals remained consistently detectable within the top 10 cm. Field validation from an additional eight trees confirmed that targeted 0-10 cm "shallow" sampling yielded higher PA concentrations than deeper sampling protocols. These findings provide a data-driven basis for refining soil sampling strategies, enabling more sensitive molecular detection while minimising disturbance and logistical effort in fragile ecosystems. IMPORTANCEPhytophthora species are among the most destructive soilborne pathogens globally, requiring robust diagnostic protocols for both agricultural and conservation settings. Traditional sampling frameworks were established to meet the biological requirements of baiting assays, which often necessitate collecting large soil volumes from broad depth profiles to ensure the capture of viable, infectious propagules. However, these extensive requirements are labour-intensive and can cause significant soil disturbance in sensitive forest ecosystems. Using P. agathidicida as a model, this study provides a high-resolution quantitative assessment of how pathogen DNA is distributed vertically across different disease stages. We demonstrate that while absolute peak abundance can shift within the 0-15 cm range as infection progresses, the pathogen signal remains consistently detectable within the top 10 cm. This evidence-based approach suggests that targeted, shallow sampling enhances sensitivity by reducing signal dilution, offering a lower-impact path for monitoring soilborne oomycetes worldwide.

Vertical transmission of plant pathogenic viruses is an important component of viral persistence, survival, and spread in agricultural production systems. This type of transmission is of considerable economic significance as it can cause major crop losses by serving as the initial focus of infection for future epidemics. Vertical transmission occurs when a virus is passed on to offspring either by direct invasion of the developing seed embryo from infected mother plants or through infected pollen grains after fertilization. We have recently demonstrated by high throughput sequencing that mature seeds of the agriculturally important forage crop alfalfa (Medicago sativa L.) are associated with a broad range of viruses some of which could potentially spread over long distances via seed. Aside from alfalfa mosaic virus, little is currently known about viral transmission via alfalfa pollen and its role in the epidemiology in this crop. This research was conducted to screen the pollen obtained from unique alfalfa genotypes for the presence of pathogenic viruses and their potential for dissemination. The plants from which the pollen was collected were alfalfa genotypes selected for fungal plant disease resistance and agronomic performance in a USDA ARS pre-breeding program in Prosser, WA.

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola and other Brassica crops. Polymerase chain reaction (PCR), particularly probe-based quantitative PCR (qPCR), is widely used for the detection of P. brassicae in soil samples. To improve consistency in clubroot detection while maintaining efficiency, diagnostic laboratories would benefit from adopting a single, highly efficient qPCR system for routine testing. In this study, we analyzed the primer and probe sequences of all published PCR and qPCR systems for P. brassicae detection. Based on these analyses, three independently developed probe-based qPCR systems were selected and their performance was evaluated using synthesized target DNA (gBlock). One probe-based qPCR system exhibiting superior sensitivity on gBlock was subsequently evaluated on P. brassicae genomic DNA. This system consistently detected DNA equivalent to four resting spores per reaction, corresponding to a soil sample containing 1,000 spores per g soil when the DNA extraction protocol was considered as a component of the qPCR system. The sensitivity of the system was further validated using DNA extracted from soil samples collected from multiple locations across Alberta, where P. brassicae was detected at levels below those associated with visible clubroot symptoms. Based on these results, we recommend this qPCR system for routine clubroot diagnostics in laboratories across Canada.

RNA interference (RNAi) shows great potential to protect crops against fungal diseases, yet reported protection efficiencies vary greatly, and our understanding of the factors responsible for this variance remains limited. In this meta-analysis, we evaluated 89 studies that compare the efficiency of host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS) in controlling fungal diseases, focusing on biotrophic, hemibiotrophic, and necrotrophic fungi, the use of formulations, and the dsRNA design as explanatory factors for differences between reported efficiency values. Our results indicate that SIGS is slightly more effective, particularly in biotrophs. Surprisingly, SIGS studies using formulations did not outperform those applying naked dsRNA. We also assessed parameters of RNA design. Differences in dsRNA length and the number of constructs, and number of targets showed no consistent significant effect on resistance in either HIGS or SIGS. Interestingly, however, HIGS studies reported significantly higher efficiency when targeting genes closer to the 3 end and SIGS when targeting genes closer to the 5 end. We discuss potential reasons for the reported patterns, such as variability in dsRNA uptake mechanisms, intercellular trafficking and Dicer processing, and conclude that more research is needed to understand the biological mechanisms determining RNAi efficiency for fungal control.

Summer squash (Cucurbita pepo) is a popular vegetable in Mississippi. These are harvested during the tender and immature stages. This vegetable is known to be a good source of vitamins A and C, as well as potassium, and iron. Small farms typically sell summer squash directly to consumers through local farmers markets. In this study, we isolated and identified a soft rot causing bacteria, Pectobacterium brasiliense strain 25ASUB12 (GenBank: PX884501), from symptomatic fruit of field-grown summer squash in Mississippi. We deployed both phenotypic and molecular techniques to identify this important pathogen which has a wide host range, including cucumbers, potatoes, and tomatoes.

Developing grapevine cultivars with genetic resistance to pathogens is a key strategy to reduce fungicide use and enhance sustainability. The French INRAE-ResDur program aims to pyramid several resistance loci against Plasmopara viticola (Rpv), the causal agent of downy mildew, while integrating factors against Erysiphe necator (Ren/Run) which causes powdery mildew. We evaluated in field the performance of grapevine genotypes carrying single or pyramided Rpv loci during the exceptionally severe downy mildew epidemic of 2024. Disease severity was quantified as the proportion of leaf foliage exhibiting symptoms. Susceptible controls averaged 66.6 % symptomatic leaves, Rpv1/3.1 combination remained below 16.1 %. whereas the Rpv1/Rpv3.1/Rpv10 pyramid showed only 4.9 % symptomatic leaves. The single loci provided partial protection, but the effect varied with genetic background. Pyramiding improved resistance effectiveness and stability, indicating synergistic interactions among loci. These findings demonstrate that pyramiding Rpv loci is an effective strategy for durable downy mildew resistance and should be the preferred strategy in grapevine breeding programs and genetic resistance deployment strategies.

Gosss wilt and leaf blight of maize is caused by Clavibacter nebraskensis and has reemerged as an important disease in North America. Despite its epidemiological relevance, this species remains poorly characterized in terms of population structure, functional diversity, and ecological differentiation, particularly among strains reported from Mexico. In this study, long-read whole-genome sequencing and phenotypic assays were used to characterize genomic diversity, virulence, and fitness-associated traits in C. nebraskensis. We generated 24 long-read genomes, including 20 contemporary Mexican isolates and four historical United States strains collected between 1969 and 1996, and compared them with publicly available genomes from North America and South Africa. Phylogenomic analyses confirmed that all strains cluster within the C. nebraskensis clade, and gene accumulation curves supported a closed pangenome with accessory gene variation linked to geographic origin and isolation period. Functional assays showed strain-level variation in virulence, enzymatic activity, bacteriocin antagonism, polysaccharide production, biofilm formation, and pigmentation. Cellulolytic activity was associated with disease severity, whereas pigment-related traits were linked to thiamine metabolism. Overall, these results indicate that C. nebraskensis comprises ecologically heterogeneous populations, structured around alternative survival and competition strategies. Integrating genome-wide comparisons with functional characterization of fitness-related traits provides a framework for understanding the biological factors underlying Gosss wilt. dynamics

Leaf diseases pose a serious threat to faba bean production. Leaf blotch of faba bean, caused by Alternaria spp., has become increasingly widespread and destructive in several countries. Leaf diseases pose a serious threat to faba bean production. The infection of plant by pathogens can be influenced by various factors associated with the host plant, environmental conditions and presence of other microorganisms. The phyllosphere and endosphere play a critical role in plant health and disease development. This study aimed to evaluate the factors shaping the structure and diversity of fungal communities associated with faba beans. Plant samples were collected in 2004 from two intensively managed faba bean production fields in the central region of Latvia. Fungal assemblages were characterized using an ITS region metabarcoding approach based on Illumina MiSeq sequencing. Among the assigned amplicon sequence variant (AVS), 65% belonged to the phylum Ascomycota, while approximately 4% were classified as Basidiomycota. Alternaria and Cladosporium were the dominant genera across samples. The alfa and beta diversities of fungal communities was higher during flowering of faba beans to compare with ripening. The higher abundance of Basidiomycota yeasts were observed during flowering, in contrast, Cladosporium genus was significantly more abundant during ripening. Alternaria DNA was found on leaves that showed no symptoms of the disease. The diversity and composition of fungal communities were significantly influenced by sampling time and presence of leaf blotch, caused by Alternaria spp.

Wild potatoes are long-evolving relatives of the cultivated potatoes we have today. These wild Solanum species harbor traits that can be exploited to develop more nutritive and resilient potato varieties, providing the genetic basis for resistance to abiotic and biotic stresses such as drought, low temperatures, diseases and pests. Wild potato species are widely used as valuable genetic resources in breeding programs, including efforts aimed at improving resistance to bacterial wilt caused by Ralstonia solanacearum. Among the wild species native to Uruguay, Solanum malmeanum has emerged as a particularly valuable source of resistance. The aim of this work was to investigate weather differences in bacterial wilt resistance among S. malmeanum accessions are associated with structural and compositional changes of rhizosphere bacterial communities. Two S. malmeanum accessions were compared, one susceptible (RN9P2) and the other resistant (A11P1) to bacterial wilt. The impact of plant resistance and pathogen colonization on the structure of rhizosphere bacterial communities were evaluated using high throughput 16S rRNA gene amplicon-sequencing. Significant differences were observed between accessions and pronounced shifts in rhizosphere bacterial communities were detected in response to pathogen inoculation. Cryseobacterium, Sphingobacterium, Komagataeibacter, Gluconobacter, Lactobacillus and Dyella were differential genera and enriched in the rhizosphere of the resistant accession. Several of these genera have been previously associated with disease suppression. Overall, these results suggest that the rhizosphere bacterial community associated with resistant S. malmeanum accessions may contribute to protection against R. solanacearum infection.

Potato common scab (Streptomyces sp.) is an economically important disease that reduces the quality and market value of tubers. A key aspect in developing management strategies involves accurately quantifying the disease. Due to the three-dimensional nature of the tuber and the heterogeneous distribution of lesions across its surface, visual estimates of severity can be challenging. Therefore, the objectives of this study were to develop and validate a standard area diagram (SAD) for estimating common scab severity on potato tubers and to compare validation outcomes obtained using real tubers and digital images. A SAD comprising six severity levels (from 1.3 to 66.8%) was developed based on image analysis of naturally infected tubers. Validation was conducted using two complementary approaches in which inexperienced raters evaluated either real potato tubers or digital images of the same tubers under unaided and aided conditions. Accuracy, bias components, and inter-rater reliability were quantified using absolute error metrics, Lins concordance correlation coefficient, intraclass correlation coefficients, and overall concordance correlation coefficients. Use of the SAD significantly improved accuracy, reduced systematic bias, and increased inter-rater reliability across both validation approaches. No significant differences were detected between assessments conducted on real tubers and images, although image-based evaluations showed a slight, non-significant tendency toward reduced scale and location bias under aided conditions. These results demonstrate that a dimension-aware SAD integrating information across the full tuber surface enhances the reliability and reproducibility of visual severity assessments and supports the use of image-based evaluations for training, large-scale surveys, and remote or collaborative applications involving three-dimensional plant organs.

Blackleg disease in potato, caused by soft rot Pectobacteriaceae, is a substantial cause of loss in seed potato production. Recent research has attempted to identify bacteria with antagonistic activity against several diseases, among which blackleg. However, most biocontrol agents have been tested only in-vitro or in the greenhouse. In this study, we tested the effect of bacterial biocontrol agents in a four-year field experiment against blackleg caused by Pectobacterium brasiliense and Dickeya solani. Effects of the treatments on disease incidence was highly variable between years and also differed between cultivars, soil type and even replicates. Disease incidence was on average higher in sandy soil compared to clay soil and higher in the cultivar Kondor than Mozart. For a subset of the bacterial isolates genome mining could detect the presence of genes involved in the production of antibiotics and siderophores, but this was not correlated with disease incidence in the field. Moreover, most isolates were able to survive in storage on tubers from inoculation until planting. Thus, we conclude that while the used isolates showed the potential for antagonistic activity and were present on tubers when planting, no antagonist treatment could consistently decrease disease incidence. Inoculation of the isolates on the tuber surface might have been insufficient for plant colonization.

BackgroundPotato is an important crop worldwide, yet its production is severely threatened by Phytophthora infestans, the causal agent of late blight. Alternatives to the current control strategies are needed, as these rely heavily on environmentally harmful treatments. The recruitment of beneficial microbes by plants upon stress ("cry-for-help" mechanism) may represent an opportunity to find new biocontrol agents but this has not yet been reported for potato. The aim of this study was to analyse whether foliar late blight infection induces shifts in the phyllosphere, rhizosphere and soil bacterial communities associated with two potato cultivars of differing sensitivity to late blight. Moreover, we aimed at isolating members of the plant microbiota to test whether bacteria putatively recruited upon infection would be particularly active in protecting the plant against late blight. ResultsControlled foliar infection triggered substantial, cultivar-specific shifts in the rhizosphere communities across two successive generations. Despite the number of differentially abundant ASVs detected being ten times higher in the second generation than in the first one, the same taxonomic groups were concerned by the shifts: Burkholderiales, Flavobacteriales, and Bacillales. Furthermore, the communities linked to the susceptible cultivar consistently shifted more strongly than the communities linked to the resistant cultivar. The obtained ASV sequences were used to identify 163 corresponding isolates. The inhibition potential of these strains against P. infestans spores was assessed through biological assays, which revealed the biocontrol potential of strains otherwise not yet known to inhibit phytopathogenic organisms, such as Advenella, Nocardioides and Phyllobacterium strains. Although we found no correlation between the relative abundance shift of the ASVs upon infection and the activity of the corresponding strains, we observed that the overall activity of strains isolated from the resistant cultivar was higher than that of the strains isolated from the susceptible one. ConclusionTaken together, the higher activity of the strains isolated from the resistant cultivar, along with its comparatively modest microbiome shifts upon infection suggest that the investigated resistant cultivar might harbour specific microbiota enriched in strains with efficient protective abilities against their host plants pathogens, which possibly contribute to its higher resistance against P. infestans.

Host resistance is a critical component of oat crown rust disease management. Pc94 is a qualitative resistance locus derived from diploid Avena strigosa with several independent introgressions into A. sativa that have been used in cultivar deployment. Quantitative trait locus (QTL) analysis combining previously published data for a historic A. strigosa population segregating for Pc94 revealed a large effect QTL on the distal end of A. strigosa chromosome 7A. Genome assembly of the parents identified a cluster of five nucleotide binding site leucine-rich repeat receptor (NLR) candidate genes within the QTL region. A single candidate NLR with an integrated zinc finger BED domain, AstNLR94, was determined as necessary for Pc94 resistance based on map-based cloning and forward mutagenesis. A presence/absence allele specific PCR marker was designed in AstNLR94 and verified for accuracy and specificity in a diverse panel of A. strigosa and A. sativa. Pc94 introgressions in A. sativa ranged in size from 1.7-71 Mbp and two different introgression locations appear to have occurred. In A. sativa Leggett, a 6.3 Mbp Pc94 introgression is located at the end of chromosome 2A, and the same sized introgression was discovered in the OT3098 v2 genome. Finally, a QTL analysis identified an additional minor resistance locus on A. strigosa chromosome 4A, which has complicated previous efforts to characterize the Pc94 locus. This is the first report of an NLR gene underlying disease resistance in Avena spp. and delivers a Pc94 marker for marker assisted selection to produce disease resistant cultivars. Key messageWe mapped a zfBED-NLR encoding gene necessary for Pc94 resistance, developed a diagnostic marker, and revealed diverse introgression sizes, clarifying Pc94s history and utility for durable oat crown rust resistance.

Colletotrichum sublineola (Cs) is a hemibiotrophic fungal pathogen that causes anthracnose in Sorghum bicolor, leading to significant yield losses. To enable infection, Cs secretes effectors - proteins, small RNAs, and metabolites - that damage the plant cell wall or enter the plant cell to suppress immune responses and manipulate host metabolism. Effectors can detoxify host antimicrobials, alter nutrient processing, and evade host immunity. Paradoxically, some effectors can also trigger pattern-triggered immunity (PTI), especially in biotrophic and necrotrophic fungi. More than half of fungal protein effectors lack conserved domains and functional network annotations. In this study, we identified prospective Cs effectors, separating those with non-conserved domains and classifying those with conserved domains by protein families. Comparative genomics is employed to predict effector functions and analyze their roles. Using their predicted locations and domains, we mapped the effectors into functional subsystems related to PTI. These include interactions in the apoplast, oxidative stress response, protein modification and degradation systems, and Cysteine-rich Fungus-specific Epidermal Growth Factor-like Module (CFEM) domain proteins involved in immune regulation. Our functional network analysis advances the understanding of Cs pathogenicity and offers insights into effector infection mechanisms.

Glutathione has important roles in diverse infections, yet its involvement in the interaction between the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd) and its amphibian hosts is still unclear. Using in vitro assays and a cell infection model, we examined how glutathione influences Bd virulence traits and cellular host disease resistance. For Bd, inhibition of glutathione reductase rapidly killed zoospores, indicating that glutathione is essential for this pathogen. In addition, exposure to exogenous glutathione promoted the potential for virulence through accelerated and increased zoospore release. In host amphibian cells, Bd infection decreased intracellular glutathione content and increased reactive oxygen species, suggesting that chytridiomycosis pathogenesis may involve oxidative stress. Depletion of host glutathione before exposure to Bd increased infection severity and Bd growth, whereas amphibian cells with slightly elevated glutathione levels were partially protected against Bd. However, manipulation of host glutathione levels after the establishment of Bd infection did not impact its intracellular growth, implying that the host glutathione-mediated resistance only occurs during the initial Bd invasion process. Importantly, this effect of glutathione on host resistance is not a general response to pathogens, as it was not observed in cells exposed to viral pathogen FV3. As glutathione increased both infectious zoospore production and host resistance to zoospore infection, our study suggests that this antioxidant may play an important role in the host/pathogen interaction during chytridiomycosis. Thus, environmental conditions and therapeutic approaches that affect glutathione systems in the host and/or pathogen have the potential to alter chytridiomycosis dynamics and should be further explored.

Peanut smut, caused by the fungus Thecaphora frezzii, is a significant disease of peanuts in Argentina. Infected plants have seeds replaced by a mass of dark teliospores, reducing yield and seed quality. To prevent the spread of the pathogen, several countries have limited import of raw peanuts from Argentina, a major grower and exporter. Following successful in vitro culture of the fungus in its haploid stage, we produced a chromosome-level genome assembly of the species for the first time. We compare this genome with those of 49 other species of true smut fungi, or Ustilaginomycetes, including species of medical, agricultural, and industrial importance, some of which are known as pathogens and others only as saprotrophic yeasts. At almost 39 Mb, T. frezzii has the largest genome of the smut fungi sequenced to date and the highest repetitive content. While it shares some core effectors with species of the distantly related and better studied Ustilago and related fungi, predicted effectors only found in T. frezzii or in Thecaphora suggest unique infection strategies. Comparisons among the 50 smut genomes also show that the 14 smut fungi observed only as yeasts share genomic traits such as low repeat content and generally smaller genomes. This supports the hypothesis that some smut fungi are adapted to saprotrophic growth as yeasts. The high-quality, annotated genome for T. frezzii will be a valuable resource for investigating the population dynamics and evolution of an economically important pathogen, as well as illuminating an understudied clade of smut fungi. Article summaryPeanut smut is a destructive and costly disease of peanuts in Argentina. For the first time, a high quality, annotated genome is presented for the causal agent, Thecaphora frezzii. This fungus has the largest and most repetitive genome of the true smut fungi, thus prompting comparison with 49 other species of smut fungi with available genomes, including non-pathogenic ones. While it shares some likely pathogenicity factors with well-studied smuts, it has many unique genes, a trait reflective of its evolutionary distance and likely novel mechanism for infection. This important genomic resource will benefit research regarding the evolution and adaption of T. frezzii, the development of diagnostic tools that enable rapid detection of it, and the study of smut fungi broadly.