Impact of carbon sources on growth and oxalate synthesis by the phytopathogenic fungus <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

The impact of various supplemental carbon sources (oxalate, glyoxylate, glycolate, pyruvate, formate, malate, acetate, and succinate) on growth and oxalate formation (i.e., oxalogenesis) by Sclerotinia sclerotiorum was studied. With isolates D-E7, 105, W-B10, and Arg-L of S. sclerotiorum, growth in an undefined broth medium (0.1% soytone; pH 5) with 25 mM glucose and 25 mM supplemental carbon source was increased by the addition of malate and succinate. Oxalate accumulation occurred in the presence of glucose and a supplemental carbon source, with malate, acetate, and succinate supporting the most oxalate synthesis. With S. sclerotiorum Arg-L, oxalate-to-biomass ratios, an indicator of oxalogenic potential, were dissimilar when the organism was grown in the presence of different carbon sources. The highest oxalate-to-biomass ratios were observed with pyruvate, formate, malate, acetate, and succinate. Time-course studies with acetate-supplemented cultures revealed that acetate and glucose consumption by S. sclerotiorum D-E7 coincided with oxalogenesis and culture acidification. By day 5 of incubation, oxalogenesis was halted when cultures reached a pH of 3 and were devoid of acetate. In succinate-supplemented cultures, oxalogenesis essentially paralleled glucose and succinate utilization over the 9-day incubation period; during this time period, culture pH declined but never fell below 4. Overall, these results indicate that carbon sources can regulate the accumulation of oxalate, a key pathogenicity determinant for S. sclerotiorum.     

Mycelial growth of strains of the genera <Emphasis Type="Italic">Suillus</Emphasis> and <Emphasis Type="Italic">Boletinus</Emphasis> in media with a wide range of concentrations of carbon and nitrogen sources

Suillus and Boletinus were studied using Ohta medium. In media with glucose or trehalose, all tested strains grew well. With mannose and cellobiose, strains generally grew well, except for one strain of Suillus. Utilization of dextrin and soluble starch differed with each strain, and that of sucrose and glycerol was low for all strains. Utilization of four amino acids, arginine, glutamic acid, aspartic acid, and alanine, was similar to that of ammonium tartrate for Suillus strains, but mycelial growth with amino acids was clearly lower than with ammonium tartrate for the Boletinus strain. The effect of glucose and ammonium tartrate concentrations for nine strains of the genera Suillus and Boletinus was studied with ranges for glucose of 1–100 and 200g/l, respectively, and for ammonium tartrate of 0.2–5 and 20g/l, respectively. Six strains showed maximal growth at a glucose concentration greater than 25g/l, and one strain showed maximal growth at 70g/l. The results indicate that these fungi are adapted to relatively high concentrations of carbon sources. In general, glucose concentration at mycelial growth maximum decreased as ammonium tartrate concentration increased, and at higher concentrations of glucose, mycelial growth decreased more rapidly in higher concentrations of ammonium tartrate.     

Application of a formulation of the nematophagous fungus <Emphasis Type="Italic">Duddingtonia</Emphasis><Emphasis Type="Italic">flagrans</Emphasis> in the control of cattle gastrointestinal nematodiosis

The viability of a formulation of Duddingtonia flagrans was assessed in the control of parasite gastrointestinal nematodes of cattle. Two groups (A and B) of eight crossbred Holstein × Zebu cattle, approximately one year old, were placed in Brachiaria decumbens pasture. Each animal in group B (treated) received orally 20 g sodium alginate pellets containing mycelial mass of the D. flagrans fungus, while the animals in the group A (control) received pellets without fungus for seven months, starting in March 2005. The egg per gram of feces counting the gastrointestinal nematodes showed a difference (P < 0.05) in the treated group in June, July and August, with reductions of 58% (June), 47% (July) and 51% (August) compared to the control group. The infective larvae recovered in the pastures collected up to 20 cm from distance of the fecal dung in group B differed (P < 0.01) from the larvae recovered in group A. At the end of the experimental period, the animals in group B presented a greater weight gain (P < 0.01) compared to the untreated group (A). The treatment of cattle with pellets containing the D. flagrans nematophagous fungus, at the dose and duration used was effective in controlling the infective larvae of gastrointestinal nematodes of cattle.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Extensive host range of an endophytic fungus, <Emphasis Type="Italic">Guignardia endophyllicola</Emphasis> (anamorph: <Emphasis Type="Italic">Phyllosticta capitalensis</Emphasis>)

Isolation of endophytic species of Guignardia (anamorph: Phyllosticta) from healthy leaves of 94 plants (91 species and 3 varieties) in 69 genera, 42 families, was carried out in a test site (Kyoto Herbal Garden) to investigate the host range of Guignardia endophyllicola (anamorph: Phyllosticta capitalensis). Species of Guignardia and Phyllosticta were isolated from the leaves of 67 plants (66 species and 1 variety) belonging to 54 genera, 38 families. Among them, 53 isolates from different plants belonging to 43 genera in 36 families were similar in morphology, and sequence analysis of internal transcribed spacer (ITS) regions of ribosome DNA revealed these isolates to be conspecific with G. endophyllicola. In addition, this fungus was isolated from leaves of various plants collected in different places in Japan and Thailand. Thus, this endophytic fungus has been revealed to live within various vascular plants, angiosperms, gymnosperms, and pteridophytes.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

Improvement of nisin production in pH feed-back controlled,fed-batch culture by <Emphasis Type="Italic">Lactococcus lactis</Emphasis>subsp<Emphasis Type="Italic">. lactis</Emphasis>

Nisin production by Lactococcus lactis subsp. lactisin fed-batch culture was doubled by using a pH feed-back controlled method. Sucrose concentration was controlled at 10 g l^–1 giving 5010 IU nisin ml^–1 compared to 2660 IU nisin ml^–1 in batch culture.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-<Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Helminthosporium turcicum</Emphasis>, the maize leaf-blight fungus

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.     

Incidence of <Emphasis Type="Italic">Wolbachia</Emphasis> and <Emphasis Type="Italic">Cardinium</Emphasis> endosymbionts in the <Emphasis Type="Italic">Osmia</Emphasis> community in Korea

Sex ratio distorting endosymbionts induce reproductive anomalies in their arthropod hosts. They have recently been paid much attention as firstly texts of evolution of host-symbiont relationships and secondly potential biological control agents to control arthropod pests. Among such organisms, Wolbachia and Cardinium bacteria are well characterized. This study aims at probing such bacteria in the Osmia community to evaluate their potential utilization to control arthropod pests. Among 17 PCR tested species, Osmia cornifrons and a parasitic fly are infected with Wolbachia and a mite species is infected with Cardinium. Phylogenetic tree analyses suggest that horizontal transfer of the bacteria occurred between phylogenetically distant hosts.     

Leaf spot disease on <Emphasis Type="Italic">Tilia cordata</Emphasis> caused by the fungus <Emphasis Type="Italic">Cercospora microsora</Emphasis>

Increased incidence of leaf spots on many tree species, up to the presence of peripheral importance only, including linden trees was noticed recently. First massive and continuous occurence of the fungus Cercospora microsora Sacc. [teleomorph Mycosphaerella millegrana (Cook.) Schröet., Mycosphaerella microsora Syd.], causal agent of anthracnose on linden trees (Tilia cordata Mill.) grown in urban plantings in Slovakia was reported. Along with this, certain of the important growth characteristics of this fungus were studied under laboratory conditions. To specify Cercospora biology mycelial growth of C. microsora in pure hyphal cultures was observed in relation to medium and locality. One-way ANOVA has confirmed a statistically significant influence of both factors, culture medium and locality on growth rate values of C. microsora. The effect of these factors has not proved unambiguously in all cases. In the case of one locality (Nitra), the significant influence of used media has not been proved (P > 0.05). PDAg showed generally as the most suitable medium, inducing the most intensive growth in three localities (41.06 mm/week on average). Comparing three localities, the effect of this factor is not so unambiguous. Growth rate values from the localities Bratislava and Pribeta indicate unsuitability of medium A for the fast radial growth. A Tukey test separately conducted for the factors medium and the locality revealed the significant combinations of means (P ≤ 0.05).     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

<Emphasis Type="Italic">Funastrum rupicola</Emphasis> (<Emphasis Type="Italic">Apocynaceae:</Emphasis><Emphasis Type="Italic">Asclepiadoideae</Emphasis>), a new species from Bolivia

Summary   Funastrum rupicola Goyder, a new species of Apocynaceae: Asclepiadoideae from Bolivia, is described and illustrated. The conservation status of this species is assessed.     

<Emphasis Type="Italic">Phuphanochloa,</Emphasis> a new bamboo genus (<Emphasis Type="Italic">Poaceae</Emphasis>: <Emphasis Type="Italic">Bambusoideae</Emphasis>) from Thailand

Summary  A new monotypic bamboo genus Phuphanochloa (Poaceae: Bambusoideae) from north-eastern Thailand is described, together with a new species, P. speciosa.