Saprophytic Stage Research Articles

Sclerotinia sclerotiorum (Lib.) de Bary: Insights into the Pathogenomic Features of a Global Pathogen.

Sclerotinia sclerotiorum (Lib.) de Bary is a broad host-range fungus that infects an inclusive array of plant species and afflicts significant yield losses globally. Despite being a notorious pathogen, it has an uncomplicated life cycle consisting of either basal infection from myceliogenically germinated sclerotia or aerial infection from ascospores of carpogenically germinated sclerotia. This fungus is unique among necrotrophic pathogens in that it inevitably colonizes aging tissues to initiate an infection, where a saprophytic stage follows the pathogenic phase. The release of cell wall-degrading enzymes, oxalic acid, and effector proteins are considered critical virulence factors necessary for the effective pathogenesis of S. sclerotiorum. Nevertheless, the molecular basis of S. sclerotiorum pathogenesis is still imprecise and remains a topic of continuing research. Previous comprehensive sequencing of the S. sclerotiorum genome has revealed new insights into its genome organization and provided a deeper comprehension of the sophisticated processes involved in its growth, development, and virulence. This review focuses on the genetic and genomic aspects of fungal biology and molecular pathogenicity to summarize current knowledge of the processes utilized by S. sclerotiorum to parasitize its hosts. Understanding the molecular mechanisms regulating the infection process of S. sclerotiorum will contribute to devising strategies for preventing infections caused by this destructive pathogen.

Lifecycle of Pyrenopeziza protrusa (Helotiales, Dermateaceae sensu lato) in Magnolia obovata revealed by field observation and molecular quantification.

Fungi exhibit saprophytic, parasitic, and symbiotic lifestyles, and flexibly switching between them by the environmental changes and host conditions. However, only a few studies have elucidated the detailed changes in fungal DNA or morphology, including the formation of reproductive structures along with lifestyle switching. We hypothesized that Pyrenopeziza protrusa, which occurs abundantly and specifically on Magnolia obovata as a saprophyte, is also associated with living hosts and switches its lifestyles as part of its lifecycle. To elucidate this hypothesis, we periodically sampled the fresh/fallen leaves of M. obovata to observe the seasonal occurrence of reproductive structures for the isolation and detection/quantification of P. protrusa DNA with newly developed species-specific primers. The isolation frequency and amount of P. protrusa DNA drastically increased in the fresh leaves just before defoliation in autumn, but remained high in fallen leaves from autumn to spring. Abundant production of conidiomata and apothecia was also observed in the fallen leaves with increasing DNA content. These results clarified a large part of the lifecycle of P. protrusa, suggesting that the lifestyle is switched from symbiotic to saprophytic stage by significantly increasing the amount of DNA in response to host conditions according to the seasonal variations.

Tec1, a member of the TEA transcription factors family, is involved in virulence and basidiocarp development in Ustilago maydis.

The life cycle of Ustilago maydis involves alternation of a haploid saprophytic yeast-like stage and a dikaryotic hyphal virulent form. Under in vitro conditions, basidiocarps are formed. Analysis of the transcriptional network of basidiocarp formation revealed the possible involvement of a Tec transcription factor (Tec1, UMAG_02835) in the process. In some Ascomycota, Tec factors are involved in mycelial formation, pathogenesis, and interaction with other regulatory elements, but their role in Basidiomycota species is almost unknown. Accordingly, we proceeded to determine the role of this gene in U. maydis by its mutation. Tec1 was found to be a crucial factor for normal mating, basidiocarp development, and virulence, all of the functions related to the dikaryotic stage dependent of the b genes, whereas dimorphism and resistance to different stress conditions occurring in the haploid stage were not affected in tec1 mutants. The observation that mutants showed a low residual wild-type phenotype suggests the presence of a secondary mechanism that partially compensates the loss of Tec1.

A Unique Combination of Two Different Quorum Sensing Systems in the β-Rhizobium Cupriavidus taiwanensis.

Cupriavidus taiwanensis LMG19424, a β-rhizobial symbiont of Mimosa pudica, harbors phc and tqs quorum sensing (QS), which are the homologous cell-cell communication systems previously identified from the plant pathogen Ralstonia solanacearum and the human pathogen Vibrio cholerae, respectively. However, there has been no experimental evidence reported that these QS systems function in C. taiwanensis LMG19424. We identified (R)-methyl 3-hydroxymyristate (3-OH MAME) and (S)-3-hydroxypentadecan-4-one (C15-AHK) as phc and tqs QS signals, respectively, and characterized these QS systems. The expression of the signal synthase gene phcB and tqsA in E. coli BL21(DE3) resulted in the high production of 3-OH MAME and C15-AHK, respectively. Their structures were elucidated by comparison of EI-MS data and GC/chiral LC retention times with synthetic standards. The deletion of phcB reduced cell motility and increased biofilm formation, and the double deletion of phcB/tqsA caused the accumulation of the metal chelator coproporphyrin III in its mutant culture. Although the deletion of phcB and tqsA slightly reduced its ability to nodulate on aseptically grown seedlings of M. pudica, there was no significant difference in nodule formation between LMG19424 and its QS mutants when commercial soils were used. Taken together, this is the first example of the simultaneous production of 3-OH MAME/C15-AHK as QS signals in a bacterial species, and the importance of the phc/tqs QS systems in the saprophytic stage of C. taiwanensis LMG19424 is suggested.

Aerobiology, epidemiology and management strategies in apple scab: science and its applications

Apple Scab, caused by Venturia inaequalis (Cke.) Wint. (anamorph Spilocea pomi Fr.), is considered to be one of the most important fungal diseases of apple, which occurs almost every year in severe epidemic form under favourable environmental conditions wherever apple is cultivated. The disease has two distinct phases in its life cycle i.e. the primary saprophytic stage on the fallen dead leaves and the secondary parasitic phase on the trees. The intensity of the disease varied with the environmental conditions, cultivar and orchard management practices. Yield losses during epidemic years in 1996, 2008 and 2013 in the Gangotri fruit valley of Dist. Uttarkashi went up to 84%. Among the environmental parameters, temperature, moisture and relative humidity have the highest influence on the epidemic development. The formations of pseudothecia and ascospore discharge were associated mainly with late autumn (October) infection. Urea @ 5% concentration had a significant effect on breaking of the life cycle of V. inaequalis in overwintered leaves. The pseudothecia formation takes 32–48 days more after sexual reproduction at temperature around 8–10 °C and this process completes between February and March every year. The maximum ascospores productivity was recorded at fruit development stage (pea size to walnut size) of apple with ascospore productivity of 1, 23,000/ml and thereafter declined gradually. The ascospore emission period was 64–78 days and mean numbers of cumulative degree days for 50 and 95% spore release were 456 and 960, respectively. Data accumulated over 17 years were analyzed for validation of Mills criteria to establish its relevance in rescheduling fungicide applications under monitored spray programme. On examination of the primary infection period from Gangotri valley, some differences were observed between our results and Mills table for ascospores infection. Potential ascospore dose is a useful tool for predicting the total amount of inoculums in an orchard and has been shown to effectively improve apple scab management. PAD values were 50 times higher in the poorly managed orchards than in the integrated orchards. However, integrated orchards were considered well-managed in the investigated years and showed that the epidemic risk was low and medium, while it was very high in the poorly managed orchards on the basis of PAD. Based on the long term studies carried out at different locations from 1993 to 2018, a computer based warning system was developed to forewarn the farmers for adoption of suitable management practices. The information generated are passed on to the growers by blowing a characteristic signaling, local news paper, SMS, telephonic communication, Govt. organization and through personal contacts or messages flashed 4–5 times through radio stations. In addition, growing season kisan mela, press releases and newsletters are issued via the extension workers of the University and Govt. department.

Development of two strains of Fusarium culmorum with a different aggressiveness in the soil and on the roots of barley of two genotypes

Fusarium culmorum can affect plants including cereals or develop saprophytically in the soil. It is unknown whether its saprophytic ability is different in strains with a different aggressiveness. This knowledge could be used for effective breeding of resistant cultivars. Here, we aimed to study the development of two F. culmorum strains with a different aggressiveness in the soil under barley, to compare their saprophytic ability and to reveal the influence, if any, of the host plant on the development of strains in the soil and on the roots. Saprophytic development of the strains was studied on membranes inoculated with macroconidia and placed into non-sterile soil under barley of two genotypes. The fungus was identified on the membranes and on barley roots by indirect immunofluorescent method. Both strains could develop saprophytically. The more aggressive strain (Fc538) showed a lesser saprophytic fitness than the less aggressive strain (Fc885): its mycelial density was lower and the number of chlamydospores was greater. Barley genotypes influenced the development of the fungal strains. Interestingly, conditions for saprophytic development of both strains were more favourable in the soil under barley of the resistant genotype. The more aggressive strain colonized barley of the resistant genotype more actively. Rot symptoms appeared earlier in the barley of the resistant genotype, but the number of diseased plants was greater in the barley of the susceptible genotype. The presence of a saprophytic stage in life cycle should slow down the accumulation of aggressive races in field populations of F. culmorum. Possible interactions between F. culmorum strains and barley plants are discussed.

Purification and characterization of an extracellular β-glucosidase from Sporothrix schenckii.

An extracellular β‐glucosidase (E.C. 3.2.1.21), induced by cellulose in the mycelial form of human pathogen fungus Sporothrix schenckii, was purified to homogeneity using hydroxyapatite (HAp) adsorption chromatography in batch and Sephacryl S200‐HR size exclusion chromatography. The molecular mass of the purified enzyme was estimated to be 197 kDa by size exclusion chromatography with a subunit of 96.8 kDa determined by SDS/PAGE. The β‐glucosidase exhibited optimum catalytic activity at pH 5.5/45 °C and was relatively stable for up to 24 h at 45 °C. Isoelectric focusing displayed an enzyme with a pI value of 4.0. Its activity was inhibited by Fe2+ but not by any other ions or chelating agents. K m and V max values of the purified enzyme were 0.012 mm and 2.56 nmol·min−1·mg−1, respectively, using 4‐methylumbelliferyl β‐D‐glucopyranoside (4‐MUG) as the substrate and 44.14 mm and 22.49 nmol·min−1·mg−1 when p‐nitrophenyl β‐D‐glucopyranoside (p‐NPG) was used. The purified β‐glucosidase was active against cellobioside, laminarin, 4‐MUG, and p‐NPG and slightly active against 4‐methylumbelliferyl β‐D‐cellobioside and p‐nitrophenyl β‐D‐cellobioside but did not hydrolyze 4‐methylumbelliferyl β‐D‐xyloside, 4‐methylumbelliferyl β‐D‐galactopyranoside nor 4‐methylumbelliferyl α‐D‐glucopyranoside. In addition, the enzyme showed transglycosylation activity when it was incubated along with different oligosaccharides. Whether the transglycosylation and cellulase activities function in vivo as a mechanism involved in the degradation of cellulolytic biomass in the saprophytic stage of S. schenckii remains to be determined.

Phytohormone sensing in the biotrophic fungus Ustilago maydis - the dual role of the transcription factor Rss1.

SummaryThe phenolic compound salicylic acid (SA) is a key signalling molecule regulating local and systemic plant defense responses, mainly against biotrophs. Many microbial organisms, including pathogens, share the ability to degrade SA. However, the mechanism by which they perceive SA is unknown. Here we show that Ustilago maydis, the causal agent of corn smut disease, employs a so far uncharacterized SA sensing mechanism. We identified and characterized the novel SA sensing regulator, Rss1, a binuclear zinc cluster protein with dual functions as putative SA receptor and transcriptional activator regulating genes important for SA and tryptophan degradation. Rss1 represents a major component in the identified SA sensing pathway during the fungus’ saprophytic stage. However, Rss1 does not have a detectable impact on virulence. The data presented in this work indicate that alternative or redundant sensing cascades exist that regulate the expression of SA‐responsive genes in U. maydis during its pathogenic development.

Saprophytic growth of the alder rust fungus Melampsoridium hiratsukanum on artificial media

The first axenic culture of a free living saprophytic stage of the exotic rust fungus Melampsoridium hiratsukanum is reported. Colonies were obtained from one-celled, dikaryotic urediniospores on eight nutrient media out of twelve. Modified Harvey and Grasham (HG) and Schenk and Hildebrandt (HS) media HG1 and SH1 and their bovine serum albumin (BSA)-enriched derivatives gave abundant mycelial growth, but modified Murashige and Skoog (MS) QMS media and their BSA-enriched modifications performed poorly, colony growth being low on QMS-1 and QMS1+BSA, and nil on QMS-5 and QMS-6, with or without BSA. Colonies initially grew poorly when subcultured for one month in purity, but much better after re-transfer to fresh media later: presumably because only the most exploitative genotypes survived, best able to cope with an uncongenial medium. Stabilised cultures survived, and remained vegetative, but only few reproductive colonies produced spore-like bodies. Though the agarised medium remains an inhospitable environment for this biotrophic parasite, it is shown that non-living media can nevertheless sustain the growth and sporulation of this fungus outside its natural hosts and habitat. Axenic culture promises important advances in basic and applied research on this rust, leading to a better understanding of its nutrition, metabolism, diversity and pathogenicity.

Effect of sanitation treatment and cultivar on saprophytic development of Blumeriella jaapii in integrated and organic sour cherry orchards

A three-year study was conducted to determine the effect of urea and lime sulphur sanitation treatments on three saprophytic development stages of Blumeriella jaapii on two sour cherry cultivars in integrated and organic orchards. The sanitation treatments in the integrated orchard included sprays of urea applied in the autumn, sprays of urea followed by mulch cover, and non-sanitized control. Urea was replaced with lime sulphur in the treatments in the organic orchard. The percent survival of three saprophytic development stages of B. jaapii (i.e., saprophytic hyphal growth, mycelial aggregates, and presence of apothecia and/or winter acervuli) ranged between 11 and 70 % and between 21 and 67 % in the urea and lime sulphur treatments, in the integrated and organic orchards, respectively. In all years, all urea and lime sulphur treatments significantly (P < 0.05) reduced the percent survival of the three development stages of B. jaapii compared to non-sanitized plots in both orchards and on both cultivars. Treatments that included a mulch cover resulted in a generally lower percent survival of the three development stages of B. jaapii than treatments that did not include the mulch cover, but their significance varied among the B. jaapii development stages, for years, cultivars and orchards. An interpretation of the importance of these sanitation treatments for orchard management practices is discussed.

Compost suppressiveness against Fusarium oxysporum was not reduced after one-year storage under various moisture and temperature conditions

The effect of storage conditions on compost suppressiveness against fusarium wilt of melon, caused by Fusarium oxysporum f. sp. melonis (FOM) was studied in relation to the dynamics of compost microbial activity and biodegradability. For this purpose, mature suppressive compost, prepared from tomato plants and separated cow manure, was divided into four portions and stored for one year under cool/warm (12 or 28 °C) or dry/wet (15–35 or 55–65% moisture content) conditions, in four different combinations: cool-dry, warm-dry, cool-wet and warm-wet. All composts retained and even enhanced their suppressive capacity during storage, with no significant differences among them by the end of the storage period. However, significant differences were found in the dynamics of some of the measured chemical and microbial properties. The microbial activity of composts stored under wet conditions was higher than that of those stored under dry condition, which resulted in a substantial decrease in dissolved organic matter content (expressed as dissolved organic carbon; DOC) and increase in its recalcitrance to biological degradation, decrease in basal heat emission, slower response to added glucose or citric acid, and higher NO 3 concentration, indicating increased nitrification under wet conditions. The DOC significantly correlated with several microbial properties as well as with compost suppressiveness of fusarium wilt of melon seedlings, and may be regarded as a most suitable general index for compost maturity. A best-subset multiple linear regression analysis revealed that the three best predictors, namely dissolved organic carbon (DOC), basal heat, and mesophilic bacterial counts, could explain as much as 83% of the total variance in compost suppressiveness. The generally agreed association between compost maturity and suppressiveness was verified in this case. It appears that compost microbial populations might compete and interfere with the saprophytic stage of FOM conidia, between germination and host invasion. In conclusion, it was demonstrated that compost suppressiveness against fusarium wilt of melon can be maintained for at least one year under a wide range of storage conditions, without any loss of suppressive capacity. This fact has positive logistical implications for the use of suppressive composts against FOM.

Listeria monocytogenes — from saprophyte to intracellular pathogen

Listeria monocytogenes is a bacterium that lives in the soil as a saprophyte but is capable of making the transition into a pathogen following its ingestion by susceptible humans or animals. Recent studies suggest that L. monocytogenes mediates its saprophyte-to-cytosolic-parasite transition through the careful modulation of the activity of a virulence regulatory protein known as PrfA, using a range of environmental cues that include available carbon sources. In this Progress article we describe the regulation of PrfA and its role in the L. monocytogenes transition from the saprophytic stage to the virulent intracellular stage.

Seasonal Dynamics of Taphrina deformans Inoculum in Peach Orchards

ABSTRACT The dynamics of the inoculum of Taphrina deformans were studied during a 4-year period by (i) inspecting curled leaves for the presence of asci, (ii) placing deposition spore samplers within the tree canopies, and (iii) exposing potted peach plants (trap plants). These three approaches produced consistent results. Four main periods characterized the dynamics of the inoculum: the first period coincides with the parasitic stage of the pathogen's life cycle and the other periods with the saprophytic stage. Mid- to late spring (first period) was characterized by the presence of asci on infected leaves which produced and ejected large quantities of ascospores in 96% of the samplings. Rainfall was not necessary for ascospore dispersal, which was favored by air temperature <20 degrees C and relative humidity >/=80% or wetness duration >8 h. In summer and autumn (second period), blastospores were trapped in 54 and 24% of samplings, respectively, with low spore numbers. In the winter (third period), blasto-spores were trapped in the lowest numbers and in only 6% of samplings. In late winter to early spring (fourth period), blastospores were found in 56% of samples, with increasing numbers. Rainfall significantly influenced blastospore dispersal and temperature was correlated with the seasonality found during the saprophytic stage.

Malassezia yeasts and pityriasis versicolor

To analyze the relationships among different Malassezia species and pityriasis versicolor, the only human disease in which the etiologic role of these fungi is fully accepted (although the species implicated remains a matter of discussion). Since 1996, after the taxonomic revision of the genus, a limited number of papers analyzing the role of the different Malassezia species in pityriasis versicolor have been published or were the subject of presentations in congresses; there were only four in the past year. This paper discusses the results of these works, comparing them with results of the authors' most recent study in this field, conducted over the past 16 months. Most of the studies published thus far now show that Malassezia globosa is the predominant species found in the lesions of pityriasis versicolor, at least in temperate climates. The authors' recent findings confirm these results. The etiologic role of M. globosa in pityriasis versicolor is based, even more than on its isolation in a high percentage of cultures, on its identification by direct microscopy as typical globose yeast cells producing pseudohyphae in almost 100% of cases. The confirmation of the pathogenic role of this species in pityriasis versicolor could help in understanding these conditions, which are still unclear, which promote its transformation from the saprophytic stage present in healthy skin to the parasitic one, and could also help in selecting the best therapeutic measures.

Phenotypic and genotypic identification and phylogenetic characterisation of Taphrina fungi on alder

All Taphrina species are dimorphic with a mycelium stage biotrophic on vascular plants and a saprophytic yeast stage. European species of Taphrina on Alnus species (Betulaceae) were identified using morphological, physiological and molecular characteristics, the latter including determination of PCR fingerprints and of nucleotide sequences from selected nuclear ribosomal DNA regions. PCR fingerprinting gives a good overview of species identification, as do nucleotide sequences, which in addition, help to clarify phylogenetic relationships. Taphrina alni is a homogeneous species that exhibited more than 50% similarity in PCR fingerprinting with three different primers. Morphologically, it produces tongue-like outgrowths from female catkins of Alnus incana. Taphrina robinsoniana from A. rugosa and A. serrulata in North America is phylogenetically closely related to T. alni, but the two species could be separated by their PCR fingerprints, partial sequences of 26S rDNA (D1/D2) and ITS1/ITS2 sequences. T. epiphylla and T. sadebeckii are two phylogenetically closely related species. T. epiphylla causes witches brooms in crowns of A. incana. In addition, T. epiphylla forms slightly yellow white-grey leaf spots in midsummer on A. incana. Yellow white-grey leaf spots up to 10 mm on A. glutinosa are characteristic for T. sadebeckii. Both species can be separated well by PCR fingerprinting. Different from T. epiphylla, T. sadebeckii is genotypically more heterogeneous. Only two out of three different primers showed similarity values above 50% in different European strains of T. sadebeckii. Although genetic variability was not detected in complete sequences of the 18S ribosomal DNA of T. sadebeckii, ITS1/ITS2 sequences appeared to be more heterogeneous, too. Taphrina tosquinetii is a genotypically homogeneous species causing leaf curl on Alnus glutinosa. It was not possible to distinguish the yeast phases from different Taphrina species on Alnus using morphological and physiological characteristics only.

Adiaspores of Emmonsia parva var. crescens in Lungs of Small Rodents in a Rural Area

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Arthroconidia in coccidioidoma: Case report and literature review

Coccidioides immitis is a dimorphic fungus capable of causing a diverse spectrum of disease in humans. Although the diagnostic pathologic finding in tissue is a mature endosporulating spherule, hyphal structures can also be found in over 50% of pathologic specimens. This report presents a case of coccidioidomycosis in which there were no intact spherules, but characteristic barrel shaped arthrospores present in tissue and cultures positively identified the organism as C. immitis. This case was further complicated by presentation in a nonendemic area for the fungus. Coccidioides immitis is a soil fungus with two distinct phases: the saprophytic and the parasitic. This biphasic life cycle was first described by Ophöls,1 and more fully characterized by Baker and colleagues. The saprophytic stage occurs in the environment with the organism existing in a mycelial state. As the mold matures, barrel-shaped arthroconidia form and alternate with empty cells. The arthroconidia subsequently fracture from hyphae and are dispersed as an aerosol. The saprophytic cycle repeats itself upon infection of a new soil site. However, if the airborne arthroconidia are inhaled by a susceptible host, the parasitic phase is initiated. In the parasitic cycle, the arthrospore swells and becomes a rounded structure, known as a spherule. The protoplasm of the spherule divides to form large numbers of endospores. If the spherule ruptures, the endospores are released. Each endospore can develop into a new spherule, or, upon expulsion via contaminated secretions from the host, return to the environment where it transforms into its mycelial form. Although the characteristic histologic finding in coccidioidomycosis is the spherule with endospores, hyphal forms also can be found. The mycelial form of C. immitis cannot be definitively identified morphologically and requires confirmation by culture techniques or genetic probes. This report presents a pulmonary coccidioidoma in which arthroconidia were the predominant forms found in tissue.

Diversity of cutinases from plant pathogenic fungi: differential and sequential expression of cutinolytic esterases byAlternaria brassicicola

Plant cuticles provide a protective layer that has to be penetrated by fungal pathogens. Evidence is provided for a differential and sequential induction of two classes of cutinolytic esterases by Alternaria brassicicola. Serine esterases with cutinolytic activities were expressed by conidia germinating on host surfaces. The enzymes were not induced by surface wax or cutin monomers. They were only expressed during initial (24 h) contact of conidia with cutin on host surfaces freed from wax, and with cutin in aqueous suspensions. In contrast, contact with cutin had no immediate effect on the expression of CUTAB1, a gene encoding two cutinase isozymes with crucial functions in the saprophytic utilization of cutin. Presence of a cutin monomer or prolonged exposure to cutin was required for the induction of CUTAB1 expression. The differential induction of cutinolytic esterases indicates a sequential recognition of cutin as a barrier to be penetrated and to be utilized as a carbon source in saprophytic stages.

Diversity of cutinases from plant pathogenic fungi: differential and sequential expression of cutinolytic esterases by Alternaria brassicicola

Plant cuticles provide a protective layer that has to be penetrated by fungal pathogens. Evidence is provided for a differential and sequential induction of two classes of cutinolytic esterases by Alternaria brassicicola. Serine esterases with cutinolytic activities were expressed by conidia germinating on host surfaces. The enzymes were not induced by surface wax or cutin monomers. They were only expressed during initial (24 h) contact of conidia with cutin on host surfaces freed from wax, and with cutin in aqueous suspensions. In contrast, contact with cutin had no immediate effect on the expression of CUTAB1, a gene encoding two cutinase isozymes with crucial functions in the saprophytic utilization of cutin. Presence of a cutin monomer or prolonged exposure to cutin was required for the induction of CUTAB1 expression. The differential induction of cutinolytic esterases indicates a sequential recognition of cutin as a barrier to be penetrated and to be utilized as a carbon source in saprophytic stages.

New CJD strain

In contrast to most biotrophic plant pathogens, smut fungi belonging to the family of Ustilaginaceae are facultative parasites that have a yeastlike, saprophytic phase contrasting the filamentous, parasitic growth during plant infection. This dimorphic lifestyle allows easy culturing, reverse genetic modifications and cell biological analyses in the saprophytic stage; impact on virulence or pathogenicity can then been monitored easily in infection assays. Such excellent preconditions made the maize smut fungus Ustilago maydis the most advanced model organism for fungal biotrophs. A recent key step in Ustilago research was the high-quality sequencing and manual annotation of its genome, which identified a wealth of novel virulence genes that are arranged in physical gene clusters. In next steps, the genomes of the maize head smut Sporisorium reilianum f. sp. zeae and the barley covered smut Ustilago hordei have been sequenced. These closely related pathogens exhibit significant differences regarding genome organization and gene equipment, likely reflecting different infection styles compared to U. maydis, whose unique feature is to cause tumours locally to sites of infection in vegetative tissue. Comparative analysis of these genomes as well as transcriptome profiling approaches allowed the identification of regulatory elements controlling pathogenic development as well as of secreted effector genes with important functions in plant interaction. In this chapter, we summarize the most important findings gained by genome analyses of smut fungi and define important questions that need to be addressed in the future.