Identification of thigmoresponsive loci for cell differentiation inUromyces germlings

Summary The sites alongUromyces appendiculatus germ tubes that are responsive to topographical induction for appressorium formation were determined using glass micropipettes. The germ tubes were perturbed with the micropipettes at different sites and durations. The most responsive region of the germ tubes for appressorium formation was within 0–10 m from the cell apex where >90% of the perturbed germ tubes developed appressoria. Furthermore, only the cell surface in contact with the substratum was responsive. Appressoria could not be induced to form, under any conditions, by perturbing cell-substratum regions of the germ tubes more than 40 m from the apex. Maximum appressorium formation occurred when the perturbing micropipette was left in place for >20 min.     

Inhibition of thigmostimulated cell differentiation with RGD-peptides inUromyces germlings

Summary Germlings of the plant pathogenic fungusUromyces appendiculatus sense and respond to topographical signals of various substrata by undergoing a cell differentiation process that culminates in a structure termed an appressorium. In some cell systems, recognition and mediation of extracellular signals is via transmembrane glycoproteins known as integrins that often exhibit specific affinities to the tripeptide sequence Arg-Gly-Asp (RGD) found in several extracellular matrix components. Germlings grown on substrata inductive for appressorium formation in the presence of buffered synthetic peptides containing the amino acid sequence RGD, e.g., RGD, RGDS, GRGD, and GRGDGSPK (0.5–2.0 mM), were inhibited from developing appressoria. Two non-RGD peptides (GGGG and RGES) as well as two RGD peptides (GRGDS and RGDSPASSKP) did not inhibit appressorium formation. Germling growth was not significantly affected by any of the peptides. Furthermore, 0.5 m diameter micropipettes that are normally inductive for appressorium formation when positioned between the germling apex and the substratum did not induce appressorium formation when coated with the RGD peptide. Silanized micropipettes left uncoated or coated with RGES were inductive for appressorium formation. Those observations lead to the hypothesis that an integrin-like protein may be involved in the process of signaling for initiation of appressorium formation inUromyces. An RGDSPC-affinity column was used to isolate proteins fromUromyces germlings with affinity to the RGD sequence. Elution with RGD or EDTA, but not with RGES, yielded at least 12 proteins of which one protein (95 kDa) expressed affinity on immunoblots to two different antibodies of ₁-integrin; one to the carboxyl-terminus of a synthetic peptide of integrin from chicken, and the other from the amino terminus of integrin from human placenta.Abbreviations ECM extracellular matrix - EDTA ethylenediamine-tetraacetic acid - MAb monoclonal antibody - PAb polyclonal antibody - PVDF polyvinylidene difluoride     

The hyphae ofUromyces appendiculatus within the leaf tissue after high pressure freezing and freeze substitution

Summary The fine structure of the intercellular dikaryotic hyphae of the biotrophic fungusUromyces appendiculatus was studied. High pressure freezing and freeze substitution were used to achieve a closer approximation of the native state than with conventional fixation and dehydration techniques. In addition to organelles previously described in rust fungi, heavily decorated multivesicular bodies (star bodies) were found close to the nuclei. Two types of tubular-vesicular complexes were distributed randomly within the cytoplasm of the hyphae. Furthermore, a more or less pronounced brush-like fibrillar layer on the hyphal walls was detected. The possibility that the latter two structures are correlated with the biotrophic phase of this fungus is discussed.Abbreviations TVC tubular-vesicular complex - MVB multivesicular body - M mitochondrion - N nucleus - NP nuclear pore - S septum - MT microtubule     

Further evidence for geographic differentiation in R. appendiculatus (Acari: Ixodidae) from Eastern and Southern provinces of Zambia

Studies in the biology, ecology and behaviour of R. appendiculatus in Zambia have shown considerable variation within and between populations often associated with their geographical origin. We studied variation in the mitochondrial COI (mtCOI) gene of adult R. appendiculatus ticks originating from the Eastern and Southern provinces of Zambia. Rhipicephalus appendiculatus ticks from the two provinces were placed into two groups on the mtCOI sequence data tree. One group comprised all haplotypes of specimens from the Eastern province plateau districts of Chipata and Petauke. The second group consisted of a single haplotype of specimens from the Southern province districts and Nyimba, an Eastern province district on the fringes of the valley. This variation provides additional evidence to the earlier observations in the 12S rDNA and ITS2 data for the geographic subdivision of R. appendiculatus from Southern province and Eastern province plateau. The geographic subdivision further corresponds with differences in body size and diapause between R. appendiculatus from these geographic areas. The possible implications of these findings on the epidemiology of East Coast fever (ECF) the disease for which R. appendiculatus is one of the vectors are discussed. Nucleotide sequence data reported in this paper are available in the GenBank^TM under the Accession Numbers DQ859259–DQ859266     

Repellent and acaricidal properties of Ocimum suave against Rhipicephalus appendiculatus ticks

An oil extracted from the leaves of a tropical shrub Ocimum suave was found to repel as well as kill all stages of the tick Rhipicephalus appendiculatus. In an in vitro bioassay for the larvae, the LC₅₀ of the oil in liquid paraffin was 0.024%. A 10% solution was found to kill all immatures and more than 70% of adults feeding on rabbits. Rabbits were protected for 5 days against attaching larvae using a 10% solution. Preliminary experiments undertaken with cattle kept in the field suggest that the oil may have potential in tick control, and a role in integrated tick management.     

Diapause induction in adults of three Rhipicephalus appendiculatus stocks

Newly-moulted adults of three Rhipicephalus appendiculatus stocks, originating from Kenya, Zambia and Zimbabwe, were kept in different photoperiods (12h : 12h, 13h : 11h and 14h : 10h light : dark). The stock originating from Kenya showed almost no behavioural diapause in any of these day lengths, whereas virtually all individuals of the Zimbabwean stock entered a behavioural diapause irrespective of day length. Adults from the Eastern Province of Zambia, a transition zone between the multivoltine phenology in equatorial Africa and the univoltine in southern Africa, exhibited a photoperiod-dependent diapause response. The possible mechanisms of diapause regulation and their origin are discussed.     

Inheritance of weight in Rhipicephalus appendiculatus ticks (Acari: Ixodidae) in the laboratory

A selection of the 10% lightest and 10% heaviest males and females of a population of individually weighed Rhipicephalus appendiculatus Neumann adults was made in two experiments. The offspring of homologous pairs were followed until the next adult stage (light x light, control x control and heavy x heavy). The engorged nymphal weights, unfed adult weights, engorged female weights of the parents, egg mass weights, egg weights, larval scutal lengths, engorged larval weights, unfed nymphal weights, engorged nymphal weights and adult weights of the progeny were determined. No significant differences could be demonstrated between the two lines for egg weight, larval scutal length, engorged larval weight and unfed nymphal weight. Significant differences were found between the egg masses, engorged nymphal weights and adult weights of the two lines. The heritability coefficients of body weight determined from adult to adult were 0.14 and 0.10, respectively, during the first and second experiments. Considering females and males separately, the coefficients were 0.10 and 0.18 during the frist experiment and 0.12 and 0.09 during the repeat experiment respectively.     

Meiosis,basidiospore development and post-meiotic mitosis in the basidiomycete,Phallus impudicus

The meiotic spindles of Phallus impudicus had a similar ultrastructure to those seen in other basidiomycetes. After meiosis the basidal apex differentiated to form a number of broadly based protuberances which subsequently developed into basidiospores (up to 9 per basidium). The nuclei then underwent a post-meiotic mitosis within the cytoplasm of each basidiospore. After this mitosis one nucleus remained in the basidiospore and a complete septum developed at the base. The significance of this arrangement of meiosis, spore production and post-meiotic mitosis is discussed with respect to the taxonomy and life cycle of this unusual basidiomycete.     

Survival of Theileria parva-infected adult Rhipicephalus appendiculatus under laboratory and quasi-natural conditions

Adult Rhipicephalus appendiculatus Muguga, having high or low intensities of Theileria parva Muguga infection in their salivary glands, were exposed to 20 °C and 85% relative humidity in the laboratory or quasi-natural conditions. Survival of the ticks and T. parva infections in their salivary glands was then monitored over a two year period. Ticks, having an average infection level of 2 infected acini per female, survived for up to 70 or 106 weeks after moulting under the laboratory or quasi-natural conditions respectively. Those having an infection level of 26 infected acini per female, survived for a similar duration except that those under quasi-natural conditions survived for a slightly shorter duration (102 weeks). Similarly, T. parva parasites survived for much longer periods under quasi-natural conditions than under the laboratory conditions. They survived for up to 38 or 78 weeks post salivary gland infection under the laboratory or quasi-natural conditions respectively in both categories of infection levels. There was apparently a density dependent relationship in T. parva survival, with a dramatic fall in infection occurring in ticks with high levels of infection between weeks 10 and 18 or weeks 38 and 46 post salivary gland infection in those exposed to laboratory or quasi-natural conditions before levelling off. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rhipicephalus appendiculatus: characterization of a testis-associated protein

A novel gene coding for Rhipicephalus appendiculatus Male-specific Protein (RAMP) was identified in a cDNA library constructed from the testis/vas deferens of R. appendiculatus ticks. This gene encodes a secreted protein exclusively expressed in the testis/vas deferens. The putative RAMP amino acid sequence contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMP mRNA was up-regulated in male ticks during blood feeding. RAMP was detected not only in the testis/vas deferens of males but also in postcoitum female ticks based on Western blotting, indicating that this protein is transferred to the female tick during copulation. Virgin female ticks, microinjected with recombinant RAMP, had significantly prolonged attachment duration during feeding, but there was no effect on fed weight. These results suggest that RAMP is a male-specific molecule in the spermatophore, and is related to female attachment behavior in R. appendiculatus ticks.     

Three serine proteinases from midguts of the hard tick Rhipicephalus appendiculatus; cDNA cloning and preliminary characterization

Rhipicephalus appendiculatus is one of the most economically important ticks distributed in south central and eastern Africa where little or no progress has been made on attempts to develop a vaccine. We have used a combination of RT-PCR, the 3 and 5rapid amplification of cDNA ends (RACE) to clone and sequence three cDNAs encoding full-length R. appendiculatus midgut serine proteinases (RAMSP). RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His⁵⁷ and Ser¹⁹⁵ conserved among most known serine proteinase-like genes . Northern blotting analysis of total RNA extracted from unfed and partially fed adult ticks revealed that mRNAs for RAMSP-1 and -2 were expressed only in partially fed ticks, while RAMSP-3 mRNA was not only expressed in both unfed and partially fed ticks, it was also up-regulated as tick feeding progressed. Expression analysis by RT-PCR revealed that RAMSP-3 was predominantly expressed in midguts when compared to salivary glands. For RAMSP-1 and -2, they were expressed at equivalent levels in both midguts and salivary glands. Based on key amino acid sequence features as well as similarity comparisons from the database, we speculated that polypeptides encoded by RAMPSP-1 to -3 are structurally more closely related to chymotrypsin- than trypsin-like serine proteinases. We have based our comments on the potential of serine proteinases as candidates for tick vaccines.     

Life cycle of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> var. <Emphasis Type="Italic">azukicola</Emphasis> on <Emphasis Type="Italic">Vigna angularis</Emphasis>

Field observations and inoculation experiments revealed that Uromyces appendiculatus var. azukicola has an autoecious and macrocyclic life cycle and produces spermogonia, aecia, uredinia, and telia on Vigna angularis var. angularis and V. angularis var. nipponensis. From inoculation experiments, it was suggested that this rust fungus has different host relationships from other varieties. Morphological examinations revealed that the characteristics of urediniospores and teliospores are different among varieties, although aeciospores are morphologically similar to each other.Contribution no. 182, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Low-temperature stratification strategies and growth regulators for rapid induction of Paris polyphylla var. yunnanensis seed germination

The seeds of Paris polyphylla var. yunnanensis are deeply dormant, and they remain dormant for 18 months or longer in their natural environment. Periodic exposure of the seeds to a low-temperature of 4 °C broke the dormancy in about 16 weeks (112 days). The most effective temperature stratification scheme was an interval of 14 days at 4 °C and 14 days at 22 °C. Both GA₃ and ethephon significantly enhanced the germination rate during the stratification treatment. The seed coat, particularly the mesophyll outer layer of the seed coat, strongly inhibited the germination. With removal of the seed coat and exposure of the uncoated seeds to 600 mg/l GA₃ for 48 h before the temperature stratification of 14 days at 4 °C and 14 days at 22 °C for 112 days, a germination percentage as high as 95.3% of the seeds was attained in about 160 days.     

Comparative studies on the teliospore morphology and ontogeny ofSpumula serispora,spec. nova,andRavenelia texensis (Uredinales,Raveneliaceae)

A new rust fungus,Spumula serispora, is described. The morphology of the teliospores and the telia was studied. In the teliospores ofS. serispora andS. quadrifida, the type species of the genus, sterile cells could be found, which are comparable to apical cells of otherRaveneliaceae. Similar sterile cells were also present in the telial heads ofRavenelia texensis. The ontogeny of the teliospores ofS. serispora and the teliospore heads ofRavenelia texensis was studied and compared. In both species the ontogeny of the spores heads was similar. InR. texensis hygroscopic cysts sustaining the spore heads were produced by division and not by conversion of basal sterile cells of the teliospore heads.Part 114 of the series Studies inHeterobasidiomycetes.     

Comparison of Preservation Methods of Rhipicephalus appendiculatus (Acari: Ixodidae) for Reliable DNA Amplification by PCR

Five differently preserved groups of adult Rhipicephalus appendiculatus specimens were compared for quality of DNA extracted. Three methods were used to extract DNA from specimens i.e. two simple mosquito validated DNA extraction methods and a tick validated method. Extraction of DNA from tick legs was attempted. The quality of DNA extracted was evaluated by the success of PCR amplification of the ITS2 gene and the mitochondrial COI gene fragment. Fresh specimens (i.e. killed just before extraction) had the highest success of DNA amplification followed by specimens killed in ethanol and subsequently stored in the refrigerator (4 °C). There was no significant difference in amplification success between cryopreserved and 70% ethanol preserved specimens. It was possible to amplify DNA from legs of ticks. Sequenced ITS2 amplicon of template obtained from legs of ticks was as legible as those from whole tick extract. The two mosquito validated DNA extraction methods showed a significantly lower amplification success than the tick validated protocol.     

Flavonoids induce germination of basidiospores of the ectomycorrhizal fungus <Emphasis Type="Italic">Suillus bovinus</Emphasis>

Under laboratory conditions, spores of ectomycorrhizal fungi usually germinate very poorly or not at all. In a previous study, we showed that spores of the ectomycorrhizal fungus Suillus bovinus germinated through the combination of activated charcoal treatment of media and co-culture with seedlings of Pinus densiflora, which suggested that some substances contained in root exudates induced the germination. Among the compounds reported from root exudates, flavonoids have been elucidated to play various and substantial roles in plant–microbe interactions; we therefore investigated the effects of flavonoids on basidiospore germination of S. bovinus by the diffusion gradient assay on water agar plates pretreated with charcoal powder. Seven out of the 11 flavonoids tested, hesperidin, morin, rutin, quercitrin, naringenin, genistein, and chrysin, had greater effects than controls, whereas flavone, biochanin A, luteolin, and quercetin showed no positive effects. The effective concentration presumably corresponded to several micromolar levels, which was equivalent to those effective for pollen development, nod gene induction, and spore germination of F. solani f. sp. pisi and AM fungi. The results suggest that flavonoids play a role as signaling molecules in symbiotic relationships between woody plants and ectomycorrhizal fungi.     

Teaching techniques for mycology: 24. Patterns of basidiospore germination in Auricularia (Heterobasidiomycetes)

‘Basidiocarps of Auricularia can be stored dry, discharging ballistospores upon rehydration. This article describes methods to demonstrate basidiospore germination by repetition or formation of a primary mycelium and/or microconidia’     

Control of the bean rust fungus <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> by means of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: leaf disc assays on the antibiotic effect of spore suspensions and culture filtrates

Several species of the fungal genus Trichoderma act as antagonists of other fungi. A number of strains from the Trichoderma species T. harzianum Rifai are used as biological control agents for the control of soilborne as well as foliar plant pathogens. Six T. harzianum strains, five of them isolated from commercial preparations, were evaluated for their capability to control the bean rust fungus Uromyces appendiculatus (Pers. ex Pers.) Unger. Different kinds of leaf disc assays were performed with conidial spore suspensions and sterile culture filtrates of the T. harzianum strains. Great differences were observed concerning the efficacy of the Trichoderma strains to reduce the number of the uredial pustules developing after rust inoculation which followed the application of the particular Trichoderma strains. Efficacy values ranged from 1 to over 50%. Increasing spore or culture filtrate concentrations of the two most effective isolates T12 and T_U led to decreases in the number of developing uredial pustules. Culture filtrate applications had a protective but no curative effect. T12 spore suspensions maintained their disease reducing activity even when autoclaved. This and some other evidence for an antibiotic interaction between T. harzianum and U. appendiculatus are discussed. Handling Editor: Reijo Karjalainen.     

Salivary fluid secretion in the ixodid tick Rhipicephalus appendiculatus is inhibited by Thogoto virus infection

Adult Rhipicephalus appendiculatus ticks, infectedwith Thogoto (THO) virus or control, were fed on guinea pigs and removed atintervals throughout the feeding cycle. Salivary fluid secretion was measuredbyan in vitro technique. The salivary glandsof infected, partially-fed ticks secreted fluid in vitro at about 75% the rateof controls, but the difference between infected and controls among engorgedticks was not statistically significant. Basal and DA-stimulated levels ofcyclic AMP (cAMP) were determined in isolated glands and were significantlyaffected by THO virus infection. The differences in secretory rate amongcontroland infected ticks could not be explained in terms of altered cAMP levels.Haemolymph volume was measured by a tracer-dilution technique using³H-inulin. The mean haemolymph volume for both THO-infected andcontrol groups was between 23–24% body weight throughout the feedingcycle, indicating that infection by this arbovirus did not influence salivaryfluid secretion via altered haemolymph volume. The mechanism by which THO virusaffects secretory activity of its tick vector remains unknown.     

Coupling- and repulsion-phase RAPDs for marker-assisted selection of PI 181996 rust resistance in common bean

The Guatemalan black bean (Phaseolus vulgaris L.) plant introduction (PI) 181996 is resistant to all known US races of the bean rust fungus Uromyces appendiculatus (Pers. ex Pers.) Unger var. appendiculatus [syn. U. phaseoli (Reben) Wint.]. We report on two random amplified polymorphic DNA (RAPD) markers OAC20₄₉₀ tightly linked (no recombinants) in coupling phase and OAE19₈₉₀ linked in repulsion phase (at 6.2±2.8 cM) to PI 181996 rust resistance. These RAPDs, generated by single decamer primers in the polymerase chain reaction, were identified in near-isogenic bulks of non-segregating resistant and susceptible BC₄F₂ (NX-040*4/PI 181996) lines. Linkage of the RAPD markers was confirmed by screening 19 BC₄F₂ and 57 BC₄F₃ individuals segregating for PI 181996 resistance. Utility of the RAPDs OAC20₄₉₀ and OAE19₈₉₀ was investigated in a diverse group of common bean cultivars and lines. All cultivars into which the PI 181996 resistance was introgressed had the RAPD OAC20₄₉₀. A RAPD similar in size to OAC20₄₉₀, observed in some susceptible common bean lines, was confirmed by Southern blotting to be homologous to the RAPD OAC20₄₉₀. Use of the RAPDs OAC20₄₉₀ and OAE19₈₉₀ in marker-assisted selection (MAS) is proposed. The coupling-phase RAPD is most useful for MAS of resistant BC_nF₁individuals during traditional backcross breeding. The repulsion-phase RAPD has greatest utility in MAS of homozygous-resistant individuals in F₂ or later-segregating generations.Mention of a trademark or a proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable.