Teliospore Production Research Articles

Gene expression in the smut fungus Ustilago esculenta governs swollen gall metamorphosis in Zizania latifolia

Ustilago esculenta, a smut fungus, can induce the formation of culm galls in Zizania latifolia, a vegetable consumed in many Asian countries. Specifically, the mycelia-teliospore (M-T) strain of U. esculenta induces the Jiaobai (JB) type of gall, while the teliospore (T) strain induces the Huijiao (HJ) type. The underlying molecular mechanism responsible for the formation of the two distinct types of gall remains unclear. Our results showed that most differentially expressed genes relevant to effector proteins were up-regulated in the T strain compared to those in the M-T strain during gall formation, and the expression of teliospore formation-related genes was higher in the T strain than the M-T strain. Melanin biosynthesis was also clearly induced in the T strain. The T strain exhibited stronger pathogenicity and greater teliospore production than the M-T strain. We evaluated the implications of the gene regulatory networks in the development of these two type of culm gall in Z. latifolia infected with U. esculenta and suggested potential targets for genetic manipulation to modify the gall type for this crop.

Sexual recombination within the “Kranich” race of the yellow rust fungus Puccinia striiformis f.sp. tritici on Berberis vulgaris

An isolate of the “Kranich” race of the basidiomycete Puccinia striiformis f.sp. tritici (Pst), which causes yellow (stripe) rust on wheat, was selfed on Berberis vulgaris, the alternate (sexual) host for several rust fungi infecting cereals and grasses. Since 2011, the “Kranich” race has been detected in several European countries and it has contributed to the replacement of the pre-existing European Pst population. A sexual origin of the “Kranich” race has been suggested due to the high capacity of teliospore production and the genetic similarity to the populations in the centre of diversity of Pst in the near-Himalayan region. For the completion of the sexual life cycle, B. vulgaris was inoculated with basidiospores derived from germinating teliospores. Pycnia appeared on the adaxial side of the leaves 8 days after inoculation (dai) and subsequent fertilization resulted in the development of aecia on the abaxial side of the leaves from 15 dai. Inoculation of wheat seedlings with aeciospores from bulked aecia resulted in 124 progeny isolates. Twenty-three of these were randomly selected and genotyped using 19 simple sequence repeat (SSR) markers, which confirmed the parental origin of the progeny isolates. Eight heterozygous markers in the parental isolate revealed segregation in the progenies resulting in 18 novel multilocus genotypes, confirming recombination following sexual reproduction. This study demonstrated a high sexual capacity of the “Kranich” race, which highlights the risks associated with sexual reproduction under suitable conditions for rust development.

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection.

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Phytohormone profiling reveals fungal signatures and strong manipulation of infection cycle in theGymnosporangium juniper-virginianaedual-host plant system

The aim of this work was to examine the function of phytohormones in the pathogenesis of cedar-apple rust, a fungal disease caused by Gymnosporangium juniper-virginianae Schwein. on Eastern red cedar (Juniperus virginiana L.) and crabapple trees (Malus spp. Mill.). Control cedar branchlets, gall tissues, fungal telial horns, as well as healthy and infected apple leaves were collected throughout fungal and plant development and used for profiling endogenous cytokinins (CK) and abscisic acid (ABA) by high performance liquid chromatography – electrospray ionization – tandem mass spectrometry. Phytohormone composition implicates cytokinin involvement in the development of rust infection. Moreover, increased levels of total CKs, as well as the unique profiles of sporulating galls on cedar trees, telial horns, and infected apple leaves, dominated by cis-Zeatin type CK, suggest that the fungus can synthesize hormones to facilitate the infection process. Distribution of ABA in the fungal and plant tissues indicates an important function of this stress hormone in regulating rapid changes in osmotic pressure during teliospore production by the galls. This study of the cedar-apple rust disease cycle is the first elucidation of phytohormones profiling between a pathogenic fungus and the attacked plant in a dual-host infection system.

Three regulators of G protein signaling differentially affect mating, morphology and virulence in the smut fungus Ustilago maydis.

Regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling negatively. To broaden an understanding of the roles of RGS proteins in fungal pathogens, we functionally characterized the three RGS protein-encoding genes (rgs1, rgs2 and rgs3) in the phytopathogenic fungus Ustilago maydis. It was found that RGS proteins played distinct roles in the regulation of development and virulence. rgs1 had a minor role in virulence when deleted in a solopathogenic strain. In crosses, rgs1 was dispensable for mating and filamentation, but was required for teliospore production. Haploid rgs2 mutants were affected in cell morphology, growth, mating and were unable to cause disease symptoms in crosses. However, virulence was unaffected when rgs2 was deleted in a solopathogenic strain, suggesting an exclusive involvement in pre-fusion events. These rgs2 phenotypes are likely connected to elevated intracellular cAMP levels. rgs3 mutants were severely attenuated in mating, in their response to pheromone, virulence and formation of mature teliospores. The mating defect could be traced back to reduced expression of the transcription factor rop1. It was speculated that the distinct roles of the three U. maydis RGS proteins were achieved by direct modulation of the Gα subunit-activated signaling pathways as well as through Gα-independent functions.

Natural antisense transcripts are linked to the modulation of mitochondrial function and teliospore dormancy in Ustilago maydis.

The basidiomycete smut fungus Ustilago maydis causes common smut of corn. This disease is spread through the production of teliospores, which are thick-walled dormant structures characterized by low rates of respiration and metabolism. Teliospores are formed when the fungus grows within the plant, and the morphological steps involved in their formation have been described, but the molecular events leading to dormancy are not known. In U. maydis, natural antisense transcripts (NATs) can function to alter gene expression and many NATs have increased levels in the teliospore. One such NAT is as-ssm1 which is complementary to the gene for the mitochondrial seryl-tRNA synthetase (ssm1), an enzyme important to mitochondrial function. The disruption of ssm1 leads to cell lysis, indicating it is also essential for cellular viability. To assess the function of as-ssm1, it was ectopically expressed in haploid cells, where it is not normally present. This expression led to reductions in growth rate, virulence, mitochondrial membrane potential and oxygen consumption. It also resulted in the formation of as-ssm1/ssm1 double-stranded RNA and increased ssm1 transcript levels, but no change in Ssm1 protein levels was detected. Together, these findings suggest a role for as-ssm1 in facilitating teliospore dormancy through dsRNA formation and reduction of mitochondrial function.

Some dynamics of spread and infection by aeciospores of Puccinia punctiformis, a biological control pathogen of Cirsium arvense

Systemic disease of Cirsium arvense caused by Puccinia punctiformis depends on teliospores, from telia that are formed from uredinia, on C. arvense leaves. Uredinia result from infection of the leaves by aeciospores which are one main source of dispersal of the fungus. However, factors governing aeciospore spread, germination, infection, and conversion to uredinia and telia have not been extensively investigated. In this study, effective spread of aeciospores from a source area in a field was fitted to an exponential decline model with a predicted maximum distance of spread of 30m from the source area to observed uredinia on one leaf of one C. arvense shoot. However, the greatest number of shoots bearing leaves with uredinia/telia was observed within 12m of the source area, and there were no such shoots observed beyond 17m from the source area. Aeciospore germination under laboratory conditions was low, with a maximum of about 10%. Temperatures between 18°C and 25°C were most favorable for germination with maximum germination at 22°C. Temperature and dew point data collected from the Frederick, MD airport indicated that optimum temperatures for aeciospore germination occurred in late spring from about May 18 to June 20. Dew conditions during this period were favorable for aeciospore germination. A total of 122 lower leaves, 2 per shoot, on 61 C. arvense shoots were individually inoculated in a dew tent in a greenhouse by painting suspensions of aeciospores onto the leaves. Of these inoculated leaves, 47 produced uredinia within an average of 21.2±6.9days after inoculation. Uredinia were also produced, in the absence of dew, on 17 non-inoculated leaves of 12 shoots. These leaves were up to 4 leaves above leaves on the same shoots that had been individually and separately inoculated. Results of PCR tests for the presence of the fungus in non-inoculated leaves that were not bearing uredinia, showed that 44 leaves above inoculated leaves on 27 shoots were positive for the presence of the fungus. These leaves were up to 5 leaves above inoculated leaves on the same shoot. Uredinia production and positive PCR results on leaves above inoculated leaves on the same shoot indicated that aeciospore infection was weakly systemic. In other tests in which all leaves of plants were spray-inoculated with aeciospores, uredinia were produced by 10days after inoculation and converted to telia and sole production of teliospores in about 63days after inoculation. Successful systemic aeciospore infections in late spring would be expected to result in uredinia production in excess of a 1:1 ratio of aeciospore infections to uredinia and ultimately telia production in late summer. In this manner, systemic aeciospore infections would promote increased density of telia that lead to systemic infections of roots in the fall.

The autophagy genes atg8 and atg1 affect morphogenesis and pathogenicity in Ustilago maydis

Autophagy is a complex degradative process in which cytosolic material, including organelles, is randomly sequestered within double-membrane vesicles termed autophagosomes. In Saccharomyces cerevisiae, the autophagy genes ATG1 and ATG8 are crucial for autophagy induction and autophagosome assembly, respectively, and their deletion has an impact on the autophagic potential of the corresponding mutant strains. We were interested in the role of autophagy in the development and virulence of U. maydis. Using a reverse genetic approach, we showed that the U. maydis ATG8 orthologue, atg8, is associated with autophagy-dependent processes. Deletion of atg8 abolished autophagosome accumulation in the vacuoles of carbon-starved cells and drastically reduced the survival of U. maydisDeltaatg8 mutant strains during these conditions. In addition, atg8 deletion had an impact on the budding process during saprobic haploid growth. The infection of maize with compatible Deltaatg8 strains resulted in fewer galled plants, and fungal gall colonization was strongly reduced, as reflected by the very low hyphal density in these tissues. Deltaatg8 infections resulted in the formation of very few teliospores. To corroborate the role of autophagy in U. maydis development, we also deleted the ATG1 orthologue, atg1. Deletion of atg1 yielded phenotypes similar to the Deltaatg8 strains during saprobic growth, but of lower magnitude. The Deltaatg1 strains were only slightly less pathogenic than the wild-type and teliospore production was not affected. Surprisingly, atg1 deletion in the Deltaatg8 background exacerbated those phenotypes already observed in the Deltaatg8 and Deltaatg1 single-mutant strains, strongly suggesting an additive phenotype. In particular, the double mutant was completely suppressed for plant gall induction.

Puccinia jaceae var. solstitialis Teliospore Priming on Yellow Starthistle

Following the introduction of Puccinia jaceae var. solstitialis to California for biological control of yellow starthistle (Centaurea solstitialis, Asteraceae), teliospores, pycnia, and multiple urediniospore generations have been observed in the field. Because urediniospores have a relatively short life span in the field, functioning teliospores are expected to be necessary for the permanent establishment of P. jaceae var. solstitialis in California. To determine if conditions in California were conducive to this, teliospore emergence and priming were evaluated in the field. A factorial experiment in the laboratory with five incubation times and three incubation temperatures was used to determine teliospore priming requirements. Teliospore production coincided with plant senescence in August and September at two sites in 2 years; fewer teliospores were produced in 2006, suggesting inconsistent teliospore production may limit population growth and contribute to local extinctions in some areas. When teliospores were primed in the field, germination was low through the fall and abruptly peaked in January during both years. In the laboratory, teliospore germination increased as incubation time increased from 2 to 6 weeks and temperatures decreased from 12 to 4 degrees C. A degree-hour model derived from laboratory data accurately predicts when teliospores are primed for germination in the field. Based on the results obtained in this study, it is apparent that teliospore germination can occur over a range of priming conditions. However, lower temperatures and longer incubation periods are superior in breaking teliospore dormancy.

First record of the rust Puccinia lagenophorae on Emilia spp. in the UK

Emilia coccinea (tasselflower) and Emilia sonchifolia (Cupid's shaving brush) are widely available ornamentals that are grown in the UK for their attractive display of small red flowers during the summer. In July 2004, plants showing symptoms of rust were found at the Royal Horticultural Society Garden, Wisley, Surrey. Aeciospores were orange, round to obovate, 16–18 × 16–20 µm in diameter. Aecia appeared on both sides of the leaves and stems. Production of teliospores was noted during August. Telia appeared as black, raised blisters on the stems, sometimes several centimetres long. Teliospores were stalked, two-celled, rarely three-celled, constricted between the cells, and their colour ranged from light to dark-brown, 38–56 µm long and 16–21 µm across. The terminal cell was rounded sometimes with a pointed apex. One-celled mesospores were not observed. The diameter of the stalk measured at the point of attachment of the spore body was 7·4 µm (average made from 30 spores). Based on morphological characters described above, this rust pathogen was identified as Puccinia lagenophorae (Weber et al., 1998). The 5′ end of the large ribosomal subunit (LSU) and the ITS region were amplified using the primers NL1/NL4 and ITS4/ITS5, respectively, and the amplification procedures described by Maier et al. (2003) and Weber et al. (2003). The ITS and LSU amplicons were sequenced and their sequences deposited in GenBank (accession numbers AY808060 and AY808061, respectively). The ITS sequence was identical to that of P. lagenophorae (AF468041). As this was the first LSU sequence submitted for this fungus, no comparison was possible. Puccinia lagenophorae was first discovered in Britain in 1961 on Senecio spp. The rust originated from Australia, and is now causing epidemics on Senecio spp. in Europe. Other hosts affected in the UK are Calendula officinalis, Pericallis hybrida and Bellis perennis (Henderson, 2000), although natural infection of this fungus on the latter host has been questioned (Weber et al., 1998). This rust has never been recorded on Emilia spp. in the UK or in other parts of the world. The only rust recorded so far on Emilia spp. in the UK is Coleosporium tussilaginis (Henderson, 2000). We would like to thank Andrew Halstead for drawing our attention to this new disease in the garden.

Life cycle and nuclear behavior in three rust fungi (Uredinales)

Kuehneola japonica has a microcyclic life cycle with a regular alternation of generations. Single basidiospore inoculations onto Rosa wichuraiana resulted in teliospore production, indicating its homothallic nature. Dikaryotization in a vegetative mycelium in the host seemed to occur through nuclear division that was not followed by septum formation. Karyogamy and meiosis took place through teliospore and metabasidium development; this fungus was considered to reproduce genetically homogeneous progenies. Puccinia lantanae and P. patriniae were also microcyclic in their life cycle; however, these fungi differed from K. japonica in the mode of nuclear behavior. In the former two fungi, both vegetative and reproductive cells were uninucleate. No karyogamy was observed, and nuclear division in the metabasidium development was thought to be mitotic. In P. lantanae, a basidiospore was formed on a sterigma, whereas a whiplike hypha emerged from each metabasidium cell in P. patriniae. Inoculations of Justicia procumbens with a single basidiospore of P. lantanae resulted in teliospore production. The fungus seemed to remain uninucleate, either haploid or diploid, throughout the life cycle. Thus, reproduction was considered to be apomictic.

A substance inducing teliospore production in wheat leaf rust, Puccinia recondita f. sp. tritici

A substance inducing teliospore production inPuccinia racondita f. sp.tritici was found in water and methanol extracts of wheat leaves with telia of the wheat leaf rust just before harvest time. Methanol (MeOH) and water extracts from uninfected wheat leaves also showed telia-inducing activity. However, the MeOH and water extracts from wheat leaves covered with telia showed much stronger activity than those from uninfected wheat leaves. We obtained a fraction (0.2 mg) showing activity at 2 ng/ml by purification of the water extract.

Induction of Lytic Enzymes by the Interaction of Ustilago maydis with Zea mays Tissues

Cano-Canchola, C., Acevedo, L., Ponce-Noyola, P., Flores-Martı́nez, A., Flores-Carreón, A., and Leal-Morales, C. A. 2000. Induction of lytic enzymes by the interaction of Ustilago maydis with Zea mays tissues. The nonpathogenic (FB-2) and pathogenic (FB-D12) strains of Ustilago maydis were grown in medium supplemented with different carbon sources including monosaccharides, polysaccharides, and plant tissues. Both strains were able to grow on all substrates, with doubling times varying from 2 to 25 h depending on the carbon source. Plant tissues supplied as carbon source induced lytic enzymes differentially; pectate lyase and cellulase activities were induced preferentially by apical stem meristem in strain FB-D12, whereas leaves preferentially induced xylanase and cellulase activities in strain FB2. Stems induced polygalacturonase activity in both strains. All enzyme activities, except cellulase in the FB-D12 strain, were detected at a low level when U. maydis was grown on glucose. In planta, chlorosis and production of teliospores were paralleled by an increase in pectate lyase activity. Anthocyanin production and formation of galls and teliospores correlated with polygalacturonase expression whereas cellulase activity increased only during the stage of anthocyanin production and gall formation. Expression of xylanase activity coincided with the last stage of teliospore formation.

Mendelian inheritance of teliospore production in Uromyces appendiculatus

The change from urediniospore to teliospore production is a crucial developmental stage in the life cycle of many rust fungi. Uromyces appendiculatus has five different spore stages during its life cycle and alternates between asexual and sexual reproduction. Teliospore production is a prerequisite to the formation of the spore stages associated with the sexual cycle (basidiospores, pycniospores, and aeciospores) in this rust. Many naturally occurring isolates apparently lack the ability to produce teliospores and are restricted to asexual reproduction by production of urediniospores. A cross was previously made by using urediniospores from an asexual isolate to fertilize pycnia of a sexual isolate. The parents and progeny from this cross were analyzed for the ability to produce teliospores. When inoculated onto Phaseolus vulgaris L. (cv. Top Crop), teliospores first appeared in uredinial pustules produced by the teliospore-forming parent 14–16 days after inoculation, compared with 20–22 days for the F1 progeny of this cross. Teliospore production relative to urediniospore production was quantified for the parents, the F1, and 67 F2 progeny. At 38 days after inoculation, pustules produced by the teliospore forming parent contained 68% teliospores and 32% urediniospores, whereas pustules of the F1 contained 33% teliospores and 67% urediniospores. The number of F2 progeny that did not produce teliospores versus those that produced teliospores, fit a single gene 1:3 ratio. Thirty-five backcross progeny produced from backcrossing the F1 to the teliospore-producing parent were also analyzed for the ability to produce teliospores. The backcross progeny segregated into two groups which fit a 1:1 ratio of intermediate versus high teliospore production. This supports the hypothesis that a single gene segregated for control of teliospore production in this cross.

Mendelian Inheritance of Teliospore Production in Uromyces appendiculatus

The change from urediniospore to teliospore production is a crucial developmental stage in the life cycle of many rust fungi. Uromyces appendiculatus has five different spore stages during its life cycle and alternates between asexual and sexual reproduction. Teliospore production is a prerequisite to the formation of the spore stages associated with the sexual cycle (basidiospores, pycniospores, and aeciospores) in this rust. Many naturally occurring isolates apparently lack the ability to produce teliospores and are restricted to asexual reproduction by production of urediniospores. A cross was previously made by using urediniospores from an asexual isolate to fertilize pycnia of a sexual isolate. The parents and progeny from this cross were analyzed for the ability to produce teliospores. When inoculated onto Phaseolus vulgaris L. (cv. Top Crop), teliospores first appeared in uredinial pustules produced by the teliospore-forming parent 14–16 days after inoculation, compared with 20–22 days for the F1 progeny of this cross. Teliospore production relative to urediniospore production was quantified for the parents, the F1, and 67 F2 progeny. At 38 days after inoculation, pustules produced by the teliospore forming parent contained 68% teliospores and 32% urediniospores, whereas pustules of the F1 contained 33% teliospores and 67% urediniospores. The number of F2 progeny that did not produce teliospores versus those that produced teliospores, fit a single gene 1:3 ratio. Thirty-five backcross progeny produced from backcrossing the F1 to the teliospore-producing parent were also analyzed for the ability to produce teliospores. The backcross progeny segregated into two groups which fit a 1:1 ratio of intermediate versus high teliospore production. This supports the hypothesis that a single gene segregated for control of teliospore production in this cross.

Changes in Carbohydrate, Protein and Chlorophyll Content, and Enzyme Activity During the Switch from Uredinio‐ to Teliospore Sporulation in the Bean‐rust Fungus Uromyces appendiculatus (Pers.) Link

AbstractChanges in carbohydrate metabolism and senescence during the development of the bean‐rust fungus Uromyces appendiculatus (Pers.) Link were investigated. At different stages of uredinio‐ and teliospore production, samples were taken from infected and non‐infected bean leaves, beginnig with the flecking stage. The unsoluble‐carbohydrate, sucrose, glucose and fructose content, as well as fluctuations in soluble acidinvertase activity and parameters of leaf senescence were determined.While fructose remained constant without significant differences between infected leaf sections and the control, differences in the other carbohydrates and in soluble‐invertase activity could be detected. Parameters indicating leaf senescence were retarded and decreased with age, except for peroxidase activity, which was enhanced and increasd with age in infected tissue to a higher degree than in the control. The results are discussed in relation to their significance for fungal development.

Experimental Induction of Teliospores of Puccinia Paullula F. Sp. Monsterae on Monstera Deliciosa.

High constant temperatures and 12 h112 h lowhigh temperature regimes in controlled environment cabinets induced the formation of teliospores of Puccinia paullula f. sp. Monsterae on Monstera deliciosa. In most experiments, 28–28.5°C was the optimum temperature for teliospore production; below this they were produced more slowly, if at all, and very few teliospores (or urediniospores) were produced at 30.5° and 31.5°C. Transfer of teliospore-producing material from a high to a low temperature led to a decrease and then cessation of teliospore production and the resumption of urediniospore production. Production of teliospores was slower in small lesions than in larger ones, suggesting that the physiological state of the thallus also affects rate of teliospore production and that the time of sampling is also important. Subjecting the host/pathogen system to 28.5°C constant while the fungus was still incubating later produced resistant-type symptoms with very few uredinio- or teliospores. Some abnormal spores (which are described and illustrated) occurred mainly around the time of spore form change.

Anther-Smut Infection in Silene dioica: Variation in Floral Morphology and Patterns of Spore Deposition

The anther-smut Microbotryum violaceum, causes a systemic perennial infection of its host Silene dioica. Infection results in sterility and the production of teliospores in flowers. These spores are transmitted to healthy plants by flower visiting insects. The behavioural responses of flower visiting insects to a variation in floral characters are therefore likely to affect rates of pollen export/import, the rate of spore deposition and probability of disease. Since infected plants are sterilised, they are effectively removed from the gene pool. It is therefore often assumed that in this host-pathogen system there is a considerable potential for a pathogen-pollinator mediated selection on floral characters

Rhodosporidium lusitaniae sp. nov., a Novel Homothallic Basidiomycetous Yeast Species from Portugal that Degrades Phenolic Compounds

Summary Three strains of an undescribed basidiomycetous yeast, for which the name Rhodosporidium lusitaniae is proposed, were isolated from different substrates using either protocatechuic acid or vanillic acid as the sole source of carbon and energy in the isolation medium. They showed identical morphological, physiological and biochemical properties and shared the following characteristics: absence of ballistospores, production of carotenoid pigments, homothallic production of teliospores, formation of phragmo-metabasidia and failure to grow with inositol as sole source of carbon. This led us to classify them in the genus Rhodosporidium Banno. The new strains have some phenotypic similarity with the species Rhodosporidium dacryoidum . The molar G+C content of nDNA of the new strains is identical to the value determined for the type strain of Ro. dacryoidum but their nDNAs showed no hybridization indicating lack of conspecificity.

Growth and sporulation of Ustilago scitaminea, in vivo and in vitro

Two hyphal forms of Ustilago scitaminea were observed in vivo: dikaryotic vegetative hyphae in the sugarcane stems and monokaryotic sporogenous hyphae in the peripheral layers of the whip, a sorus-bearing structure on infected stalks. The fungus was easily isolated from sugarcane tissue and cultured on six different media. A brown cerebriform colony was usually the first form to grow from tissue. A white mycelial colony commonly arose from the brown colony after 1–2 wk and grew rapidly over its surface. On a medium containing extracts of sugarcane, the fungus developed into black colonies in which teliospores were formed. Teliospore production was enhanced and development of black colonies prolonged if covered by a thin film of liquid medium. Without a moist covering, black colonies were commonly overgrown by white mycelium.