Identification and characterization of <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> mutants defective in the solubilization of phosphorus and zinc

Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium, which is able to colonize sugarcane and other plant species of economic importance. The potentially beneficial effects promoted by this bacterium on plants are nitrogen-fixation, production of phythormones, action against pathogens and mineral nutrient solubilization. In this study, the molecular mechanisms associated with phosphorus and zinc solubilization were analyzed. A transposon mutant library was constructed and screened to select for mutants defective for phosphorous [Ca₅(PO₄)₃OH] and zinc (ZnO) solubilization. A total of five mutants were identified in each screen. Both screenings, performed independently, allowed to select the same mutants. The interrupted gene in each mutant was identified by sequencing and the results demonstrate that the production of gluconic acid is a required pathway for solubilization of such nutrients in G. diazotrophicus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. C. Intorne and M. V. V. de Oliveira contributed equally to this work.     

Ecological Occurrence of <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> and Nitrogen-fixing <Emphasis Type="Italic">Acetobacteraceae</Emphasis> Members: Their Possible Role in Plant Growth Promotion

Gluconacetobacter diazotrophicus has a long-standing history of bacterial-plant interrelationship as a symbiotic endophyte capable of fixing atmospheric nitrogen. In low nitrogen fertilized sugarcane fields it plays a significant role and its occurrence was realised in most of the sugarcane growing countries. In this mini review, the association of G. diazotrophicus with sugarcane, other crop plants and with various hosts is discussed. The factors affecting survival in the rhizosphere and the putative soil mode of transmission are emphasized. In addition, other N₂-fixing Acetobacteraceae members, including Gluconacetobacter azotocaptans, Gluconacetobacter johannae and Swaminathania salitolerans, occurring in coffee, corn and rice plants are also covered. Lastly, the plant-growth-promoting traits identified in this group of bacteria, including N₂ fixation, phytohormone synthesis, P and Zn solubilization and biocontrol, are analysed.     

Colonization of sorghum and wheat by seed inoculation with <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis>

Colonization of sorghum and wheat after seed inoculation with Gluconacetobacter diazotrophicus strains PAL 5 and UAP 5541/pRGS561 (containing the marker gene gusA) was studied by colony counting and microscopic observation of plant tissues. Inoculum levels as low as 10² CFU per seed were enough for root colonization and further spreading in aerial tissues. Rhizoplane colonization was around 7 log CFU g^?1 (fresh weight). G. diazotrophicus was found inside sorghum and wheat roots with populations higher than 5 log CFU g^?1 (fresh weight). Stem colonization remained stable for 30 days post inoculation with endophyte concentrations from 4 to 5 log CFU g^?1 (fresh weight) (in both plants). Population in leaves decreased continuously being undetectable after 17 days post inoculation.     

<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">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.     

<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.     

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.     

The synthesis of <Emphasis Type="Italic">N</Emphasis><Superscript>2</Superscript>,<Emphasis Type="Italic">N</Emphasis><Superscript>6</Superscript>-substituted diaminopurine ribosides

A series of new 2,6-substituted diaminopurine riboside derivatives were synthesized by activation of protected xantosine with sulfonyl chlorides followed by treatment with various amines. The relationship between the reactivity of intermediates and the nature of the activating agents was studied.     

Studies on mineral phosphate solubilization by cyanobacteria <Emphasis Type="Italic">Westiellopsis</Emphasis> and <Emphasis Type="Italic">Anabaena</Emphasis>

Two diazotrophic cyanobacteria, Westiellopsis prolifica and Anabaena variabilis were evaluated for elucidating the possible mechanism of mineral phosphate solubilization. Phosphate starved cyanobacteria evaluated for the presence of organic acids, extracellular compounds or enzymes that might have been produced and promoted the mineral phosphate solubilization with Mussorie Rock Phosphate and Tricalcium Phosphate as substrates. Both the cultures did not reveal production of organic acids throughout the incubation period when checked for decrease in media pH of the media and thin layer chromatography. Thin layer chromatography of culture filtrates showed the presence of hydrocarbon like compound. Further analysis of the culture filtrates with gas liquid chromatography, a single peak near to the retention time of 7.6 was observed in all extracts of culture filtrates irrespective of phosphate source. UV-visible spectra of culture filtrates revealed the absorption maxima of 276 nm. Gas chromatographic-mass spectrometric analysis of culture filtrates showed most intense peak in the electron impact (EI) ionization was at m/z 149 and molecular ion peaks at m/z 207 and 167, inferring the presence of phthalic acid. Among the mechanisms in mineral phosphate solubilization, it was evident that these cyanobacteria used phthalic acid as possible mode of P solubilization.     

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.     

Phosphate solubilization by <Emphasis Type="Italic">Rhizobium</Emphasis> strains

Forty-six Rhizobium isolates from legume root and stem nodules were examined for their phosphate-solubilizing ability on Pikovskaya’s agar medium. Rhizobium isolates from root nodules of Cassia absus, Vigna trilobata and three strains from Sesbania sesban showed zone of tricalcium phosphate (TCP) solubilization. The isolate from C. absus showed maximum solubilization (620 μg/ml) after 12 d of incubation, while the Rhizobium sp. strain 26 (from S. sesban) showed the least amount (150 μg/ml) of phosphate solubilization. Among the carbon sources tested for their ability to solubilize TCP, maximum solubilization (620 μg/ml) was observed in glucose by Rhizobium isolate from C. absus. Phosphate solubilization increased with increase in glucose concentration steeply up to 2% and slowly above this concentration in four isolates. Among the nitrogen sources tested, maximum solubilization (620 μg/ml) was observed in ammonium sulphate by Rhizobium isolate from C. absus.     

<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.     

Population Dynamics of <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> in Sugarcane Cultivars and Its Effect on Plant Growth

Different experiments have estimated that the contribution of biological nitrogen fixation (BNF) is largely variable among sugarcane cultivars. Which bacteria are the most important in sugarcane-associated BNF is unknown. However, Gluconacetobacter diazotrophicus has been suggested as a strong candidate responsible for the BNF observed. In the present study, bacteria-free micropropagated plantlets of five sugarcane cultivars were inoculated with three G. diazotrophicus strains belonging to different genotypes. Bacterial colonization was monitored under different nitrogen fertilization levels and at different stages of plant growth. Analysis of the population dynamics of G. diazotrophicus strains in the different sugarcane varieties showed that the bacterial populations decreased drastically in relation to plant age, regardless of the nitrogen fertilization level, bacterial genotype or sugarcane cultivars. However, the persistence of the three strains was significantly longer in some cultivars (e.g., MEX 57-473) than in others (e.g., MY 55-14). In addition, some strains (e.g., PAl 5^T) persisted for longer periods in higher numbers than other strains (e.g., PAl 3) inside plants of all the cultivars tested. Indeed, the study showed that the inoculation of G. diazotrophicus may be beneficial for sugarcane plant growth, but this response is dependent both on the G. diazotrophicus genotype and the sugarcane variety. The most positive response to inoculation was observed with the combination of strain PAl 5^T and the variety MEX 57-473. Although the positive effect on sugarcane growth apparently occurred by mechanisms other than nitrogen fixation, the results show the importance of the sugarcane variety for the persistence of the plant–bacteria interaction, and it could explain the different rates of BNF estimated among sugarcane cultivars.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

Mosaic analysis of small intestinal development using the<Emphasis Type="Italic"> spf</Emphasis><Superscript><Emphasis Type="Italic">ash</Emphasis></Superscript>-heterozygous female mouse

Mosaic analysis using the spf(ash)-heterozygous female mouse was performed to clarify the cell lineage and cell behavior during small intestinal development with special attention given to the villus and crypt formation. The spf(ash) mutation, located on the X-chromosome, causes ornithine transcarbamylase (OTC) deficiency, which leads to mosaic expression of this enzyme in the small intestine of the heterozygous female mouse. In the small intestine in heterozygous fetuses, very small patches, which were aggregates of OTC-positive cells or negative cells, with no definite orientation to the villus structures were observed. In the neonatal small intestine, the intervillus region (the presumptive crypts) was polyclonal, and the majority of crypts were comprised exclusively cells of either genotype in 2-week-old small intestine. These results suggest that extensive migration and cell mixing of small intestinal epithelial cells, which have no definite correlation with the villus formation, occur in fetal stages of development, and that the crypt morphogenesis commences after birth independently of the monoclonality of the epithelial cells. Our data with the mosaic mice also reconfirmed the monoclonality of the adult small intestinal crypts demonstrated in mouse aggregation chimeras.