<Emphasis Type="Italic">In vitro</Emphasis> rooting of two <Emphasis Type="Italic">Eucalyptus urophylla X Eucalyptus grandis</Emphasis> mature clones

Abstract   The rooting capacity of microshoots derived from two mature Eucalyptus urophylla X Eucalyptus grandis half-sib clones kept for 3 y under intensive micropropagation was assessed in different in vitro conditions. A first set of experiments established that clone 147 microshoots rooted earlier and in greater proportions, while producing more adventitious roots overall than their homologs from clone 149. Modifying the composition of the basal 1/2-MS-derived rooting medium by 1/4-MS or Knop macronutrients, or reducing sucrose concentration to 10 g l⁻¹ did not enhance the rooting rates. However, together with the growth regulators added, they had a significant effect on the number of adventitious roots formed. With rooting rates reaching 81%, the higher rootability of clone 147 over clone 149 was further confirmed by the second set of experiments with significant effects of the various auxins tested and strong clone × auxin interactions on the proportions of rooted microshoots and on the number of adventitious roots. The best rooting scores were given by 5 μM indole-3-butyric acid (IBA) and 12.5 μM 1-naphthaleneacetic acid (NAA), whereas the microshoots exposed to 5 or 12.5 μM indole-3-acetic acid (IAA) were less responsive. Lower light intensities did not improve significantly root capacities, although differences might exist according to the genotype. Overall, root and shoot elongation was stimulated by light. At the end of the experiment, the rooted microshoots were markedly taller than the non-rooted ones, with significant influences of auxins and light intensity, and to a lesser extent, of the genotypes.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Genetic mapping of <Emphasis Type="Italic">Eef1</Emphasis>, a major effect QTL for early flowering in <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>

An early flowering mutant plant of Eucalyptus grandis with normal vegetative growth was found in a nursery in northern Brazil. This mutant plant flowers at approximately 90 days from germination. A cross between a wild-type (normal flowering) tree and the mutant was carried out, generating a progeny of 88 individuals where early flowering segregated in an approximate 1:1 ratio. A genome scan with 100 microsatellite markers distributed across the genome was carried out using bulk segregant analysis (BSA) on two contrasting bulks of 15 plants each. Linkages (LOD>3.0) with a major effect early flowering quantitative trait locus (QTL) were detected and confirmed by a full scale cosegregation analysis for markers EMBRA27, EMBRA60, EMBRA164, EMBRA158, EMBRA91, and EMBRA65. A localized linkage map involving the six loci and the early flowering QTL named Eucalyptus early flowering 1 (Eef1) was constructed belonging to linkage group #2 in the existing microsatellite reference map. The Eef1 locus was mapped between markers EMBRA27 and EMBRA164, with distances of 21.8 and 6.4 cM, respectively. In introgression experiments, these two markers could be successfully used with an expected precision of 98% to select plants carrying the Eef1 mutant allele, assuming no recombination interference in the genomic segment. Early flowering could be a very useful trait both in breeding as well as experimental genetics of Eucalyptus.     

A new <Emphasis Type="Italic">Dactylella</Emphasis> species from <Emphasis Type="Italic">Orbilia alba</Emphasis>

A new Dactylella species, Dactylella alba was isolated from the ascospores of Orbilia alba collected in Wenshan County, Yunnan Province, China. Conidiophores were either not branched or occasionally branched, bearing divergent sterigmata on the tip with single conidium on each. Conidia were elongated ellipsoids, 1–2 septate, mostly 1 septate. By combining the ITS sequence with morphological characteristics, a new anamorphic species is described and illustrated together with its teleomorph.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

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.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

A <Emphasis Type="Italic">gyrB</Emphasis>-targeted PCR for Rapid Identification of <Emphasis Type="Italic">Salmonella</Emphasis>

Salmonella causes the majority of infections in humans and homeothermic animals. This article describes a specific polymerase chain reaction (PCR) method developed for a rapid identification of Salmonella. A gyrB-targeted species-specific primer pair, S-P-for (5′-GGT GGT TTC CGT AAA AGT A-3′) and S-P-rev (5′-GAA TCG CCT GGT TCT TGC-3′), was successfully designed. PCR with all the Salmonella strains produced a 366- bp DNA fragment that was absent from all the non-Salmonella strains tested. The detection limit of the PCR was 0.01 ng with genomic DNA or 3.2 cells per assay. Good specificity was also demonstrated by fecal samples, from which only the gyrB gene of Salmonella was amplified. Using the culture-PCR method, 27 isolates on Salmonella-Shigella (SS) medium were rapidly identified as Salmonella, which was confirmed by the sequencing of the gyrB gene.     

New species in <Emphasis Type="Italic">Barleria</Emphasis> sect. <Emphasis Type="Italic">Stellatohirta</Emphasis> (<Emphasis Type="Italic">Acanthaceae</Emphasis>) from Africa

Summary  Three new species are described in Barleria L. sect. Stellatohirta M. Balkwill from tropical Africa: B. aristata from south-central Tanzania, B. aenea from south-western Tanzania and northeast Zambia, and B. purpureotincta from south-western Zambia. Their affinities and conservation status are discussed.     

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.     

A new species of <Emphasis Type="Italic">Gymnopilus</Emphasis> Sect. <Emphasis Type="Italic">Microspori</Emphasis> from Japan

Gymnopilus ombrophilus sp. nov., growing on rotten wood of conifers and hardwoods from Niigata, Japan is described and illustrated. It is characterized by its medium-sized, brownish-orange basidiocarps with a finely squamulose pileus, stipe lacking an annulus, and mild taste, and microscopically by dextrinoid, small, broadly ellipsoid basidiospores. The new species belongs to the section Microspori. The differences between the taxon and similar species are briefly discussed.     

A new methanol assimilating yeast, <Emphasis Type="Italic">Ogataea</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>, the ascosporic state of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>

Ogataea parapolymorpha sp. n. (NRRL YB-1982, CBS 12304, type strain), the ascosporic state of Candida parapolymorpha, is described. The species appears homothallic, assimilates methanol as is typical of most Ogataea species and forms hat-shaped ascospores in asci that become deliquescent. O. parapolymorpha is closely related to Ogataea angusta and Ogataea polymorpha. The three species can be resolved from gene sequence analyses but are unresolved from fermentation and growth reactions that are typically used for yeast identification. On the basis of multiple isolates, O. angusta is known only from California, USA, in association with Drosophila and Aulacigaster flies, O. parapolymorpha is predominantly associated with insect frass from trees in the eastern USA but O. polymorpha has been isolated from various substrates in the USA, Brazil, Spain and Costa Rica.     

<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.     

Ectomycorrhizae of <Emphasis Type="Italic">Tuber huidongense</Emphasis> and <Emphasis Type="Italic">T. liyuanum</Emphasis> with <Emphasis Type="Italic">Castanea mollissima</Emphasis> and <Emphasis Type="Italic">Pinus armandii</Emphasis>

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees—Castanea mollissima and Pinus armandii—under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.     

A new species of <Emphasis Type="Italic">Elaphoglossum</Emphasis> sect. <Emphasis Type="Italic">Lepidoglossa</Emphasis> subsect. <Emphasis Type="Italic">Muscosa</Emphasis> (Dryopteridacae) with cristate spores

A new species from the Bolivian highlands is described as Elaphoglossum cristatum. It is very similar to E. engelii but is characterized by a (for subsect. Muscosa unique) cristate perispore structure with irregular deposits (versus papillate spores), more densely ciliate petiole scales (50–80 versus 10–30 cilia per scale), somewhat thicker blade texture, denser scale cover, and paler, more reddish rhizome scales.     

PCR Detection of <Emphasis Type="Italic">Serratia</Emphasis> spp<Emphasis Type="Italic">.</Emphasis> Using Primers Targeting <Emphasis Type="Italic">pfs</Emphasis> and <Emphasis Type="Italic">luxS</Emphasis> Genes Involved in AI-2-Dependent Quorum Sensing

Quorum sensing is the ability of bacteria to communicate and coordinate behavior emitting signaling molecules. A series of primers for PCR detection of Serratia spp. has been designed using as targets the pfs and luxS genes involved in AI-2-dependent quorum sensing. The identities of the PCR products (193 and 102 bp) were confirmed by commercial sequencing. Twenty-seven Serratia strains (representing 10 different species) tested positive for the presence of the pfs and luxS genes, while a total of 7 different species of non-Serratia (25 strains) were tested and gave negative results. The sensitivity and specificity of the pfs- and luxS-based PCR assay were also checked in artificially contaminated bacterial samples. In this study we established a novel method to detect Serratia using quorum-sensing genes as diagnostic markers.     

Identification of novel miRNAs and their target genes in <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>

Despite Eucalyptus grandis being the most widely planted hardwood tree globally, along with the availability of a sequenced genome and easily accessible functional genetic tools, the quantities and roles of miRNA in its developmental processes remains largely unknown. In this study, we constructed small RNA libraries by high-throughput sequencing from Eucalyptus grandis samples, and 386 novel miRNAs were identified by miRDeep2. We found 179 novel miRNAs, 41 miRNA families, and 456 target genes in leaf samples, and 257 novel miRNAs, 61 miRNA families, and 483 target genes in stem samples. The function of the MIR396 family of miRNAs in Eucalyptus grandis was found to be mainly associated with the process of cell growth. By annotation analysis of miRNA targets, we found that some target genes, such as GRF, expansin-A15, and RPS2, had a close correlation in stem. Finally, the three randomly selected members of the MIR396 family were confirmed to express in Eucalyptus grandis by qRT-PCR, indicating that our reported miRNAs were existed. The identification of miRNAs and their target genes will lead to a greater understanding of the role of miRNAs in the physiology, growth, and development of Eucalyptus grandis trees.