Interaction between crown rust (Puccinia coronata) and two viruses of ryegrass

In Lemtal Italian and S.24 perennial ryegrass plants, two isolates of ryegrass mosaic virus (RMV) suppressed the amount of crown rust emerging on leaves inoculated with Puccinia coronata uredospores by up to 75% compared with the amount on virus-free plants. Severity of rust infection on barley yellow dwarf virus (BYDV) infected plants generally did not differ significantly from that on virus-free plants. When both RMV and BYDV were present, rust was restricted in Lemtal plants to a level intermediate between those occurring on plants infected by either virus alone, and in S.24 plants to a level below that obtained with either virus alone. The mean water soluble carbohydrate (WSC) content of Lemtal plants was reduced more than 20% by RMV, but was not significantly altered by BYDV. In S.24 plants the WSC content was increased by 10% by RMV and by 60% by BYDV. Rust reduced the WSC content of healthy and virus-infected plants, the reduction being positively correlated with the level of rust on the sampled leaves. In plants of Lemtal, but not of S.24, the degree of rust infection was positively correlated with the WSC content of leaves from rust-free control plants.     

Microtubules and organelle movements in the rust fungus Uromyces phaseoli var. vignae

Direct visual observation and time lapse films of in vitro differentiating infection structures of the cowpea rust fungus Uromyces phaseoli var. vignae revealed three categories of movement: a) general movement of cytoplasm, plus organelles, into the developing portions of the fungus during which the nuclei, in particular, maintained their characteristic position with remarkable constancy, b) relatively slow movements of various organelles such that they became displaced relative to one another and to the growing fungal tip, and c) erratic, rapid, saltations of small organelles over short distances. Serial section ultrastructural analysis showed that microtubules were typically orientated parallel to the direction of cytoplasm migration. Simple statistical analyses showed that the microtubules were non-randomly associated with mitochondria but only rarely associated with lipid droplets or microbodies. All microtubules were typically short (less than 2 micrometer) and, in various parts of the cell, were often intimately associated with 3 to 6 nm diameter filaments of unidentified material. Interphase nuclei characteristically lacked microtubules emanating from their variously laterally or posteriorly located NAOs (nucleus associated organelle) but were associated with groups of laterally placed microtubules. The correlations between the observed types of movement and the ultrastructure of the cells discussed in terms of various models for organelle motility.     

Morphology of the rust fungus Puccinia boroniae revisited

Puccinia boroniae Henns. is a rust fungus endemic to Australia, infecting various Boronia spp. This study describes and illustrates, using light and scanning electron microscopy, the telial stage, teliospore germination and basidiospore production of specimens collected from commercial Boronia plantations in Western Australia. Unusual formation of a single basidiospore per germinating teliospore, and the pycnial stage, observed on Boronia megastigma leaves, are reported for the first time for P. boroniae.     

Polyphyly of mtDNA lineages in the Russian sturgeon, Acipenser gueldenstaedtii: forensic and evolutionary implications

Molecular species identification methods are an important component of CITES monitoring programs for trade in sturgeon and caviar. To date, obtaining molecular evidence for distinguishing caviar from four closely related Eurasian sturgeon species Acipenser baerii (Siberian sturgeon), A. gueldenstaedtii (osetra), A. persicus (Persian sturgeon), A. naccarii (Italian sturgeon) remains problematic. Using approximately 2.3 kb of mtDNA sequence data (cytochrome b, NADH5, control region), we find this to be attributable to the polyphyletic nature of these mitochondrial DNA markers in the Russian sturgeon, A. gueldenstaedtii. Two mitochondrial lineages are present within this species: one is phylogenetically affiliated with A. persicus and A. naccarii, while the other clusters with A. baerii. These findings have a direct impact on molecular testing of commercial caviar and demonstrate the necessity of using large sample sizes when constructing forensic databases. Furthermore, the results affect current taxonomic designations for these species as well as hypotheses concerning their evolutionary origins.     

Histological Observations on Uromyces phaseoli and Puccinia recondita Infection in Allopurinol-treated Susceptible Plants

The development of rust after administering allopurinol, a specific inhibitor of xanthine oxidoreductase, via roots was studied at the histological level in leaves of susceptible‘Pinto 111’bean plants inoculated with Uromyces phaseoli and‘Thatcher',‘Mentana’and‘Leopardo’wheat plants challenged with Puccinia recondita. A marked reduction and delay in fungal growth was observed in allopurinol-treated plants starting between 24 h and 48 h post-inoculation, i.e. after differentiation of the first haustoria (onset of the biotrophic plant-parasite relationship). Infection hyphae often grew twisted and convoluted in treated hosts, sometimes producing small, irregularly shaped colonies. Differentiation of subepidermal stromata in fungal colonies was delayed and restricted by the treatment and uredospore yield severely reduced. Allopurinol administration also tended to increase the proportion of haustoria which became embedded in thick translucent sheaths during the late stages of infection. These results support the view that plant xanthine oxidoreductase activity is necessary for biotrophic development of rust fungi and suggest that the inhibition of this enzyme, which impairs the pathogen metabolism, may favour some natural host responses to attack such as haustorial sheath formation.     

Ultrastructure of intercellular hypha and haustorium of the rust fungus,Uromyces euphorbiae

The ultrastructure of intercellular hyphae and dikaryotic haustoria of Uromyces euphorbiae, and the host response to haustorial invasion was investigated. The intercellular hyphae share common characteristics with those of other uredinial stages of rust fungi. Three types of septa were recognized inside the intercellular hypha. This study showed that the extrahaustorial membrane was possibly formed before the development of the haustorium. The periodic acid-thiocharbohydrazide-silver proteinate technique showed that the haustorial mother cell wall at the penetration site, and the haustorial wall contained more carbohydrates than other fungal structures. In addition, the neckband, present around the haustorial neck, contains different material from those of the rest of the haustorial neck wall. The close associations of host organelles, such as the nucleus, chloroplasts, mitochondria, endoplasmic reticulum and microtubules, with the haustorium, is described.     

Characterization of an infection structure-specific gene from the rust fungus Uromyces appendiculatus

Uredospores of the plant pathogen, Uromyces appendiculatus, infect leaves of the bean plant, Phaseolus vulgaris, through stomata. Physical stimuli provided by the stomate induce differentiation of the germ tube to form a series of infection structures involved in host colonization. Contact between the uredospores and the oil-collodion membranes induces formation of infection structures in the absence of the host. This report describes the characterization of a Uromyces gene, INF24, that is induced by the physical stimulus of an oil-collodion membrane. INF24 contains a 450-bp open reading frame which encodes a 16.4-kDa polypeptide. The N terminus of the INF24-encoded protein, and the C terminus of human single-stranded DNA-binding protein are both glycine-rich and share homology.     

Ultrastructure of mitosis in the cowpea rust fungus Uromyces phaseoli var. Vignae

Aspects of the ultrastructure of mitotic nuclei of the fungus Uromyces phaseoli var. vignae are described from both intercellular hyphae in the cowpea host and infection structures induced to differentiate in vitro. The interphase nucleus-associated organelle (NAO) consists of two trilamellar acircular disks connceted by an osmiophilic bar. The intranuclear spindle develops between these disks when they separate. The spindle contains pole to pole, interdigitating, chromosomal, and fragmentary microtubules arranged to form a central bundle along the surface of which lie the metaphase chromosomes. No metaphase plate is found. There are up to three microtubules per kinetochore and approximately 14 chromosomes on the haploid spindle. Telophase elongation appears to involve extension of pole to pole microtubules with no evidence for the remaining presence of interdigitating microtubules. Concomitantly, numerous cytoplasmic microtubules develop from each NAO disk where few or none are present in other phases. Reformation of the interphase NAO involves the formation of a sausage- shaped intermediate at late telophase. The nuclear envelope remains intact and the nucleolus persists throughtout division. Various aspects of the spindle and NAOs appear to be evolutionary intermediates between Ascomycetes and higher Basidiomycetes, thus supporting the theory of Basidiomycete evolution from the former group and demonstrating an encouraging correlation between mitotic characteristics and other phylogenetic markers.     

Uromyces vankyorum sp. nov. and Uromyces atriplicis, putative vicariant rust species of Patagonian and Australian semi-desert biomes

Uromyces vankyorum (Basidiomycota, Uredinales) growing on Atriplex lampa in the cool Patagonian dwarf shrub semi-desert is described as a new species and a new member of the Argentinean rust mycota. The fungus is morphologically similar to Uromyces atriplicis on Atriplex vesicaria from halophytic shrublands of South Australia. U. atriplicis seems to be derived from U. vankyorum as indicated by its shortened life cycle lacking the urediniospore stage. The species are interpreted as trans-pacific vicariants and represent one of the rare examples for the austral South American-Australasian element among rust fungi. The disjunct distribution of rust fungi between austral South America, Australia and New Zealand is reviewed and discussed. Available data suggest migration of rust fungi along Gondwanan land bridges rather than long-distance dispersal of spores by air currents. Part 198 of the series “Studies in Heterobasidiomycetous Fungi” of the Botanical Institute, University of Tübingen     

Ribosomal DNA-ITS sequence polymorphism in the sugarcane rust,Puccinia kuehnii

The two highly divergent ITS types previously observed in isolates ofPuccinia kuehnii were amplified from the same isolates through the use of ITS type-specific primers for PCR and are therefore considered as polymorphisms of ITS regions within the species. Although homology of sequences of one of the ITS types with otherPuccinia species was of expected levels, significantly high homology of sequences of the other type with those ofCronartium members indicates that abnormal genetic events led to the occurrence of these polymorphic regions. These results indicate that ITS gene tree phylogeny may not reflect true species phylogeny in this group of rust fungi. Rather, D1/D2 region tree phylogeny, which was concordant with the differences in morphology with and among related rusts, more correctly reflects phylogenetic relationships of sugarcane and related grass rusts. contribution No. 159, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Shotgun identification of proteins from uredospores of the bean rust Uromyces appendiculatus

We are interested in learning more about the proteome of Uromyces appendiculatus, the fungus that causes common bean rust. Knowledge of the proteins that differentiate life-cycle stages and distinguish infectious bodies such as uredospores, germlings, appressoria, and haustoria may be used to define host-pathogen interactions or serve as targets for chemical inhibition of the fungus. We have used 2-D nanoflowLC-MS/MS to identify more than 400 proteins from asexual uredospores. A majority of the proteins appear to have roles in protein folding or protein catabolism. We present a model by which an abundance of heat shock proteins and translation elongation factors may enhance a spore's ability to survive environmental stresses and rapidly initiate protein production upon germination.     

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum

Summary Changes in the spindle pole body (SPB) and meiotic nuclei from interphase I through interphase II in the hollyhock rustPuccinia malvacearum are analyzed ultrastructurally by three-dimensional reconstructions from serial sections. Interphase I nuclei undergo a coordinated migration and rotation during which the SPBs approach the convex face of the lateral promycelial wall. During the transition from interphase I to prometaphase II, the collateral disc (co-disc) apparently enlarges and fuses with the main disc of the SPB. The resulting single SPB nucleates two confluent half spindles and about 225 astral microtubules (MTs). Co-discs and middle pieces (MPs) are absent during division II. SPBs separate and form metaphase II intranuclear spindles oriented in a predictable manner. Tubular cisternae are present within the spindle at early metaphase II. The architecture of the spindle at division II is essentially identical to that reported for division I except that the spindle is about half as long. Anaphase-telophase II nuclear envelope constriction, separation of the sibling nuclei, and externalization of the SPBs is identical to that reported for division I. Genesis of the duplicated interphase II SPB apparently occurs rapidly and involves formation of the MP followed by the three-layered SPB discs. General aspects of the division II spindle are discussed. A model for the meiotic SPB cycle in a rust is presented and its phylogenetic and functional significance in relation to other basidiomycetes and ascomycetes is discussed.     

Polymorphism and evolution of vulval precursor cell lineages within two nematode genera, Caenorhabditis and Oscheius

BACKGROUND: The cell lineage of nematodes is mostly invariant for a given species, but varies between species. One can thus wonder how a cell lineage varies during evolution. We have started a microevolutionary approach within two genera by observing lineage variations of vulval precursor cells in different natural nematode populations of the same and closely related species. RESULTS: In Caenorhabditis elegans, the P3.p cell lineage is variable within a genetically homogeneous population and polymorphic between wild strains. Irrespective of its division pattern, P3.p is competent to form vulval tissue in different C. elegans strains, whereas it is not competent in C. briggsae. In Oscheius sp. 1, P4.p and P8.p lineages are strongly polymorphic. Within each genus, these intraspecies polymorphisms in cell lineages are amplified between closely related species. In Oscheius sp. 1, the large polymorphisms in P4.p and P8.p lineages allowed us to undertake a genetic analysis of the variation between two pairs of strains. Multiple loci are involved in cell lineage differences, and variation at one locus appears to have a relatively strong effect. In addition to these large lineage variations in cells that do not normally contribute to the vulva, we find minor variations (errors) in vulval lineages, which represent the precision level of the vulval-patterning process and point to a selection pressure for maintenance of a large vulval equivalence group. CONCLUSIONS: Polymorphisms in vulval cell lineage are found within a given nematode species, and could be instrumental in explaining evolutionary variations between closely related species.     

Genome complexity of the maize rust fungus,Puccinia sorghi

The base composition and complexity of genomic DNA fromPuccinia sorghi have been estimated by thermal denaturation, analytical ultracentrifugation, and reassociation kinetics. The buoyant density of genomic DNA in CsCl was found to be 1.7021 g/ml, which corresponds to a GC content of 43%. From thermal denaturation curves the GC content was estimated to be 41%. The haploid genome size ofP. sorghi was estimated to be 4.7 × 10⁷ bp, half of which represented a moderately repetitive fraction. The size of theP. sorghi genome is similar to that of other basidiomycete fungi; however, the amount of repetitive DNA is greater than that reported for most other fungi.     

Uromyces umiamensis, a new rust species on Momordica cochinchinensis in India

Uromyces umiamensis is described as a new rust fungus (Basidiomycota, Uredinales) of Momordica cochinchinensis (Cucurbitaceae) from Meghalaya, in northeastern India. The species differs from the known Uromyces species on members of Cucurbitaceae in terms of the character of the teliospores and aeciospores and its apparently demicyclic life cycle. Besides U. yakushimensis and the present species, all Uromyces species known from cucurbits occur in the New World. A key to known Uromyces species on Cucurbitaceae is provided.     

Transient transformation of the obligate biotrophic rust fungus Uromyces fabae using biolistics

Obligate biotrophic pathogens like the rust fungi are important plant pathogens causing enormous losses on food, forage and biomass crops. The analysis of the molecular details underlying obligate biotrophic host-parasite interactions is mainly hampered by the fact that no system for transformation is available for most obligate biotrophic organisms. Here we report the transient transformation of Uromyces fabae, an obligate biotrophic rust fungus using a biolistic approach. Biolistic bombardment of U. fabae urediospores was used to deliver different color markers (β-glucuronidase (GUS), intron green fluorescent protein (iGFP) and red fluorescent protein (DsRed) and/or a selection marker. Endogenous regulatory elements from U. fabae plasma membrane ATPase (Uf-PMA1) were used to drive expression of the transgenes. In addition to the delivery of color markers, an in planta selection procedure using the fungicide Carboxin was established allowing the propagation of transformants. In addition to mere cytoplasmic expression of the color markers, a nuclear localization signal was fused to DsRed (pRV115-NLS) targeting the fluorescent marker protein to the nuclei. A?procedure for the genetic modification of U. fabae was established. The method can be easily adapted for use with other obligate biotrophic fungi. This provides the basis for a more in depth analysis of the molecular principles governing the obligate biotrophic lifestyle.     

Catabolic activity of two phosphoric diester hydrolases in wheat leaves inoculated with brown rust,Puccinia recondita

Changes in total phospholipids and activities of two phosphoric diester hydrolases,i.e. phospholipase C and phospholipase D have been studied in inoculated wheat leaves of 25-day old plants, interacting differentially with three races of brown rust,Puccinia recondita, during early stages of pathogenesis. With progressive rust iafection the lipids and phospholipids were found to decrease in all the three interactions except at later stages (48 h and 72 h) in race 104-interaction. However, total phospholipids at various stages were found to be higher in resistant interaction (race 63) as compared to the other two interactions. An initial increase in activities of phospholipase C and D in all the three interactions was followed by a decline at the intermediate stages which continued in case of phospholipase C activity while a reverse trend was observed in phospholipase D activity towards later stages of infection. Further the enzymatic activities at various stages were found to be higher in the susceptible and intermediate interactions (with races 77 and 104 respectively) as compared to the resistant interaction.     

Chitin deacetylase activity of the rust Uromyces viciae-fabae is controlled by fungal morphogenesis

Abstract The broad bean rust fungus Uromyces viciae-fabae exhibits chitin only on surfaces of those infection structures which in nature are formed on the plant cuticle, but not on those differentiated in the intercellular space of the host leaf. Chitin deacetylase, an enzyme which converts chitin to chitosan, has been studied during in vitro differentiation of rust infection structures. Radiometrie and gel electrophoretic analyses of crude extracts and extracellular washing fluids have shown that chitin deacetylase activity massively increases when the fungus starts to penetrate through the stomata, and that formation of the enzyme is strictly differentiation-specifically controlled. The extracellular portion of chitin deacetylase activity was about 53% in 24-h-old differentiated infection structures. Five isoforms with apparent molecular masses of 48.1, 30.7, 25.2, 15.2 and 12.7 kDa were detectable after substrate SDS-PAGE. The enzyme is temperature-sensitive and has a pH optimum of 5.5-6.0.