Investigations on influencing plant-associated bacteria in tissue cultures of black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.)

During tissue culture of black locust (Robinia pseudoacacia L.), serious problems with plant-associated bacteria led to a reduction of propagation potential in several clones. Four dominant strains of plant-associated bacteria could be isolated and were assigned to the genera Acidovorax, Dyella, Microbacterium and Sphingomonas. Out of five essential oils tested, thyme and lemongrass oil at a concentration of 0.03% each and 0.015% of both oils in combination clearly inhibited the growth of these bacteria strains on bacteriologic medium. There were no significant differences in total bacterial population density when penicillin, thyme and lemongrass oil or thyme plus lemongrass oil were added to the plant propagation media. The use of lemongrass oil changed the proportion of dominant bacterial strains.     

Methyl jasmonate regulated diploid and tetraploid black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.) tolerance to salt stress

Methyl jasmonate (MeJA) is an essential and promising plant growth regulation factor that can improve plant development and growth. Here, we explored the mechanism by which MeJA regulates the tolerance of black locust (Robinia pseudoacacia L.) to salt stress. In this study, diploid and tetraploid R. pseudoacacia were subjected to three treatments: 500 mM NaCl; 100 μM MeJA; and 500 mM NaCl and 100 μM MeJA, and the changes in plant growth, endogenous MeJA levels and the anti-oxidative metabolism of leaves were investigated. The results showed that salt stress significantly inhibited plant growth and induced the accumulation of Na⁺ and Cl^? ions, malondialdehyde (MDA) content and reactive oxygen species. However, these adverse effects could be alleviated by applying MeJA, which was followed by a marked increase in the activities of antioxidant enzymes. In addition, some genes encoding several antioxidant enzymes were also up-regulated. Simultaneously, the endogenous MeJA content in MeJA-treated plants was lower than in salt-treated plants. It is noteworthy that tetraploids always possessed higher salt tolerance and obtained greater positive effects from MeJA than diploids. These results suggested that MeJA might play a protective role in defense responses, enabling diploid and tetraploid black locust, especially tetraploid, to better tolerate the adverse effects of salt stress.     

Water entry for the black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.) seeds observed by dedicated micro-magnetic resonance imaging

Water entry at germination for black locust (Robinia pseudoacacia L.) seeds which are known as hard seeds with impermeable seed coat to water, was examined using micro-magnetic resonance imaging (MRI). The MRI apparatus equipped with a low-field (1 T; Tesla) permanent magnet was used, which is open access, easy maintenance, operable and transportable. The excellent point of the apparatus is that T ₁-enhancement of water signals absorbed in dry seeds against steeping free water is stronger than the apparatuses with high-field superconducting magnets, which enabled clear detection of water entry. Water hardly penetrated into the seeds for more than 8 h but approximately 60 % of seeds germinated by incubating on wet filter papers for several days. Hot water treatments above 75 °C for 3 min effectively induced water gap; scarification was 70 % at 100 °C and 75 °C, declined to 15 % at 50 °C and decreased further at room temperature. Water entered into the scarified seeds exclusively through the lens, spread along the dorsal side of the seeds and reached the hypocotyl, whereas water migrated slowly through hilum side to radicle within 3 h.     

Photosynthetic characteristics and nitrogen allocation in the black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.) grown in a FACE system

Key message

The black locust is adapted to elevated [CO ₂ ] through changes in nitrogen allocation characteristics in leaves.

Abstract

The black locust (Robinia pseudoacacia L.) is an invasive woody legume within Japan. This prolific species has a high photosynthetic rate and growth rate, and undergoes symbiosis with N₂-fixing micro-organisms. To determine the effect of elevated CO₂ concentration [CO₂] on its photosynthetic characteristics, we studied the chlorophyll (Chl) and leaf nitrogen (N) content, and the leaf structure and N allocation patterns in the leaves and acetylene reduction activity after four growing seasons, in R. pseudoacacia. Our specimens were grown at ambient [CO₂] (370 μmol mol^?1) and at elevated [CO₂] (500 μmol mol^?1), using a free air CO₂ enrichment (FACE) system. Net photosynthetic rate at growth [CO₂] (A _growth) and acetylene reduction activity were significantly higher, but maximum carboxylation rate of RuBisCo (V _cmax), maximum rate of electron transport driving RUBP regeneration (J _max), net photosynthetic rate under enhanced CO₂ concentration and light saturation (A _max), the N concentration in leaf, and in leaf mass per unit area (LMA) and ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCo) content were significantly lower grown at elevated [CO₂] than at ambient [CO₂]. We also found that RuBisCo/N were less at elevated [CO₂], whereas Chl/N increased significantly. Allocation characteristics from N in leaves to photosynthetic proteins, N_L (Light-harvesting complex: LHC, photosystem I and II: PSI and PSII) and other proteins also changed. When R. pseudoacacia was grown at elevated [CO₂], the N allocation to RuBisCo (N_R) decreased to a greater extent but N_L and N remaining increased relative to specimens grown at ambient [CO₂]. We suggest that N remobilization from RuBisCo is more efficient than from proteins of electron transport (N_E), and from N_L. These physiological responses of the black locust are significant as being an adaptation strategy to global environmental changes.

     

Analysis of interactions between the invasive tree-of-heaven (<Emphasis Type="Italic">Ailanthus altissima</Emphasis>) and the native black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>)

Invasive exotic plants can persist and successfully spread within ecosystems and negatively affect the recruitment of native species. The exotic invasive Ailanthus altissima and the native Robinia pseudoacacia are frequently found in disturbed sites and exhibit similar growth and reproductive characteristics, yet each has distinct functional roles such as allelopathy and nitrogen fixation, respectively. A four-month full additive series in the greenhouse was used to analyze the intraspecific and interspecific interference between these two species. In the greenhouse experiment, the inverse of the mean total biomass (g) response per plant for each species was regressed on the density of each species and revealed that the performance of the plants was significantly reduced by interspecific interference and not by intraspecific interference (p < 0.05). Other biomass traits such as root dry weight, shoot dry weight, stem dry weight, and leaf dry weight were also negatively affected by interspecific interference. Competition indices such as Relative Yield Total and Relative Crowding Coefficient suggested that A. altissima was the better competitor in mixed plantings. Ailanthus altissima consistently produced a larger above ground and below ground relative yield while R. pseudoacacia generated a larger aboveground relative yield in high density mixed species pots.     

Characterization of Phages Virulent for <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> Rhizobia

Three lytic phages (ΦRP1, ΦRP2, and ΦRP3) specific for Robinia pseudoacacia rhizobia were isolated from the soil under black locust. They were characterized by their morphology, host range, and some other properties including DNA molecular weights. Studied phages have been found to belong to Siphoviridae family that comprises viruses with long, and noncontractile tails. They had broad host ranges and effectively lysed not only Robinia pseudoacacia microsymbionts but also different Mesorhizobium species. The phages were homogenous in latent periods (300 min) but heterogeneous in burst sizes (100–200 phage particles per one infected cell) and rise periods (90–120 min). They showed a distinct adsorption rate to Robinia pseudoacacia rhizobia (70.4–93.94%). The molecular weights of phage DNAs estimated from restriction enzyme digests were in the range from ca. 82 kb to ca. 105 kb.     

Evaluation of protein <Emphasis Type="BoldItalic">in vitro</Emphasis> digestibility of <Emphasis Type="BoldItalic">Palmaria palmata</Emphasis> and <Emphasis Type="BoldItalic">Gracilaria verrucosa</Emphasis>

Palmaria palmata and Gracilaria verrucosa are edible red seaweeds and potential protein sources for human or animal nutrition, so studies were conducted on their in vitro protein digestibility. After 30 min predigestion by pepsin followed by 6 h digestion into a cell dialysis containing porcine pancreatin, the in vitro protein digestibility of P. palmata and G. verrucosa, expressed in regard to casein digestibility, was 4.9% and 42.1%, respectively. The level of protein digestibility seems to be related to the amount of soluble fibre, which was 45.3% and 30.5%, respectively.     

<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> in Poland and Japan is nodulated by <Emphasis Type="Italic">Mesorhizobium amorphae</Emphasis> strains

Robinia pseudoacacia microsymbionts from plants growing in Poland and Japan were evaluated for phylogeny and taxonomic position by genomic approach. Based on the comparative analyses of atpD (368 bp) and dnaK (573 bp) gene sequences as well as 16S rDNA restriction analysis (RFLP-16S rDNA), R. pseudoacacia microsymbionts were identified as Mesorhizobium strains. In dnaK and atpD gene phylograms R. pseudoacacia nodulators formed robust, monophyletic clusters with Mesorhizobium species with the nucleotide sequence similarity of 91–98% and 90–98%, respectively. The classification of R. pseudoacacia rhizobia to the genus Mesorhizobium was also supported by amplified 16S rDNA restriction analysis. The studied bacteria formed common clusters with Mesorhizobium species, and their DNA patterns were identical or nearly identical to Mesorhizobium genus strains. When DNA-DNA hybridization was performed, the total DNA of the representative R. pseudoacacia rhizobia exhibited 51–75% relatedness to DNA of Mesorhizobium amorphae ICMP15022 strain and below 41% to DNA of other Mesorhizobium species. These results showed that R. pseudoacacia and M. amorphae belong to the same genomospecies. The G+C content of DNA of R. pseudoacacia two microsymbionts was 59.7 and 60.6 mol% compared to 61–64 mol% across M. amorphae strains.     

Leaf movement and photosynthetic plasticity of black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) alleviate stress under different light and water conditions

Leaf morphological, physiological and biochemical characteristics of Robinia pseudoacacia L. seedlings were studied under different stress conditions. The plants were subjected to drought and shade stress for one month. Leaf inclination, chlorophyll fluorescence and chlorophyll content were measured at the first day (short-term stress) and at the end of the stress period (long-term stress) and in the recovery period. Leaf inclination was affected mainly by light; a low level of irradiance caused leaves to be arranged horizontally. Diurnal rhythmicity was lost after the long-term stress, but resumed, in part, in the recovery period. Drought stress caused leaves to tilt more obviously and decreased damage to the photosystem. Sun avoiding movement in a single leaf and sun tracking movement in the whole plant coexisted. Significant physiological changes occurred under different conditions of light. Increased energy dissipation and light capture were the main responses to high and low level of irradiance, respectively, and these were reflected by changes of chlorophyll fluorescence and chlorophyll content. Phenotypic plasticity in the leaflet enhanced the protective response to stress. These adaptive mechanisms may explain better survival of R. pseudoacacia seedlings in the understory, especially during the drought periods, and made it to be the preponderant reforestation species in Shandong Province of China.     

Isolation and characterization of the major histocompatibility complex <Emphasis Type="BoldItalic">DQA1</Emphasis> and <Emphasis Type="BoldItalic">DQA2</Emphasis> genes in gayal (<Emphasis Type="BoldItalic">Bos frontalis</Emphasis>)

The species origin of Yunnan gayal has been controversial since many years. However, few recent genetic studies have suggested that it has perhaps originated from the hybridization between male Bos frontalis and female B. taurus or B. indicus. Being an important semi-wild bovid species, this has also been listed under the red list of International Union of Conservation of Nature and Natural Resources. However, there is limited information available about the immunogenicity of this precarious species of Bos. Major histocompatibility complex (MHC) plays a pivotal role in immune response to infectious diseases in vertebrates. In the present study, we have investigated the structural and functional characteristics and possible duplication of the MHC-DQA genes in gayal (B. frontalis). Two full-length cDNA clones of the MHC-DQA genes were amplified and designated as Bofr-DQA1 (DQA*0101) and Bofr-DQA2 (DQA*2001) with GenBank accession numbers KT318732 and KT318733, respectively. A comparison between Bofr-DQA1, Bofr-DQA2 and to other MHC-DQA molecules from different animal species showed that nucleotide and encoded amino acid sequences of these two identified MHC-DQA genes have more similarity to alleles of specific DQA1 and DQA2 molecules from other Ruminantia species than to each other. The phylogenic investigation also demonstrated a large genetic distance between these two genes than to homologous from the other species. The large genetic distance between Bofr-DQA1 and Bofr-DQA2, and the presence of different bovine DQA putative motifs clarify that these sequences are nonallelic type. These results could suggest that duplication of the DQA genes has also occurred in gayal. The findings of the present study have strengthened our understanding to MHC diversity in rare ruminants and mutation of immunological functions, selective and evolutionary forces that affect MHC variation within and between species.     

Preparation and regeneration of spheroplasts from <Emphasis Type="BoldItalic">Arthrospira</Emphasis><Emphasis Type="BoldItalic">platensis</Emphasis> (Spirulina)

Ultrasonic pretreatment, lysozyme, inorganic osmotics and bovine albumin were used to prepare the spheroplasts of Arthrospira platensis (Spirulina platensis). The average cell number of the fragments from the filaments of strain A9 was about 2.2 cells after 80-s ultrasonic pretreatment. These fragments could regenerate and were suitable material for isolating spheroplasts, so the optimum conditions for doing this were investigated. The best enzymolysis parameters were designed. During the isolation process, gentle shaking of the enzymolysis sample for several times greatly enhanced the proportion of spheroplasts. However, no spheroplasts were obtained when organic compounds were used as osmotics. The spheroplasts could form typical colonies on plate of inorganic medium, with a regeneration rate of about 3%. These spheroplasts might be used as competence cells to carry on the research of genetic transformation.     

4-Coumarate:coenzyme A ligase in black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>) catalyses the conversion of sinapate to sinapoyl-CoA

4-Coumarate:coenzyme A (CoA) ligase (4CL, EC 6.2.1.12) in crude enzyme preparation from the developing xylem of black locust (Robinia pseudoacacia) converted sinapate to sinapoyl CoA. The sinapate-converting activity was not inhibited by other cinnamate derivatives, such as p-coumarate, caffeate or ferulate, in the mixed-substrate assay. The crude extract prepared from the developing xylem was separated by anion-exchange chromatography into three different 4CL isoforms. The isoform 4CL1 had a strong substrate preference for p-coumarate, but lacked the activity for ferulate and sinapate. On the other hand, 4CL2 and 4CL3 displayed activity toward sinapate and also possessed high activity toward caffeate as well as p-coumarate. The crude extract from the shoots exhibited a very similar substrate preference to that of the developing xylem; therefore, 4CL2 may be a major isoform in both crude enzyme preparations. These results support the hypothesis that sinapate-converting 4CL isoform is constitutively expressed in lignin-forming cells.     

Cells of <Emphasis Type="BoldItalic">Candida utilis</Emphasis> for <Emphasis Type="BoldItalic">in vitro</Emphasis> (<Emphasis Type="BoldItalic">R</Emphasis>)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 °C, a screen of several 1-alcohols (C4–C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l⁻¹ (PDC activity 2.5 U ml⁻¹), PAC levels of 103 g l⁻¹ in the octanol phase and 12.8 g l⁻¹ in the aqueous phase were produced in 15 h at 21 °C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 °C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U⁻¹ h⁻¹) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l⁻¹ h⁻¹) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g⁻¹).*Dedicated with gratitude to Prof. Dr. Franz Lingens – “Theo”.     

High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA^® (4 mg l^?1) containing MS medium followed by BASTA^® foliar spray on 15-day-old black gram plants (35 mg l^?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.     

Ammonium chloride: a novel effective and inexpensive salt solution for phycocyanin extraction from <Emphasis Type="BoldItalic">Arthrospira</Emphasis> (<Emphasis Type="BoldItalic">Spirulina</Emphasis>) <Emphasis Type="BoldItalic">platensis</Emphasis>

Phycocyanin, a blue pigment, is a type of phycobiliproteins. Because of its various potential properties, phycocyanin is applied to various fields, such as nutraceutical, pharmaceutical, medicine, cosmetics, and biotechnological research. The cost and application of phycocyanin are highly dependent on its purity index. In this study, ammonium chloride is presented as a novel, effective, and inexpensive salt for phycocyanin extraction. Compared with sodium phosphate, which is commonly used during phycocyanin extraction process, ammonium chloride solution efficiently extracted phycocyanin with high purity from Arthrospira platensis FACHB-314. In addition, ammonium phosphate solution is also presented as an alternative precipitation agent in phycocyanin purification that may replace the widely used ammonium sulfate. Statistical analysis shows that there is no significant difference in phycocyanin concentration between crude extracts (overall mean of 0.208 and 0.215 for extraction using sodium phosphate and ammonium chloride, respectively). However, the difference in phycocyanin purity ratio (A₆₂₀/A₂₈₀) between these two extractions is significant (overall mean of 0.742 and 1.428 for extraction using sodium phosphate and ammonium chloride, respectively). With ammonium chloride, the purity indexes of phycocyanin are 1.5 and 2.81 after the optimum extraction step, and precipitation used as the primary purification step, respectively. The present study describes a novel purification method to achieve phycocyanin with analytical grade without multiple purification steps.     

Compatibility of <Emphasis Type="Italic">Piriformospora indica</Emphasis> and <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> as dual inoculants in black pepper (<Emphasis Type="Italic">Piper nigrum</Emphasis> L.)

The compatibility of two biological inoculants, Trichoderma harzianum, a mycoparasitic biological control fungus and Piriformospora indica, a root colonizing plant-growth promoting endophytic fungus was evaluated using tissue cultured black pepper plantlets. We report, for the first time, the ability of P. indica to colonize black pepper, a perennial climber. T. harzianum inhibited the growth of P. indica in an in vitro dual culture plate assay. Simultaneous inoculation with both biological inoculants of tissue cultured black pepper plantlets negatively influenced root colonization by P. indica. However, when P. indica was applied initially followed 30 days later by T. harzianum, there was increased root colonization by the root endophyte P. indica and beneficial effects were found on the growth of the black pepper plants. The present study also showed that the efficacy of inoculation of the two fungal biological agents can be increased by sequential application of P. indica at the hardening stage followed by T. harzianum during transplanting into a soil-sand mixture.     

Environmental assessment of cod (<Emphasis Type="BoldItalic">Gadus morhua</Emphasis>) from autoline fisheries

Purpose  

The main purpose of this study has been to document the environmental performance of products based on autoline-caught cod and the distribution of environmental impacts in the value chain from fishing to retail. Another aim has been to document the performed environmental improvement analyses.     

Comparing EST-based genetic maps between <Emphasis Type="BoldItalic">Pinus sylvestris</Emphasis> and <Emphasis Type="BoldItalic">Pinus taeda</Emphasis>

A genetic map of Pinus sylvestris was constructed using ESTP (expressed sequence tag polymorphism) markers and other gene-based markers, AFLP markers and microsatellites. Part of the ESTP markers (40) were developed and mapped earlier in Pinus taeda, and additional markers were generated based on P. sylvestris sequences or sequences from other pine species. The mapping in P. sylvestris was based on 94 F₁ progeny from a cross between plus-tree parents E635C and E1101. AFLP framework maps for the parent trees were first constructed. The ESTP and other gene sequence-based markers were added to the framework maps, as well as five published microsatellite loci. The separate maps were then integrated with the aid of AFLPs segregating in both trees (dominant segregation ratios 3:1) as well as gene markers and microsatellites segregating in both parent trees (segregation ratios 1:1:1:1 or 1:2:1). The integrated map consisted of 12 groups corresponding to the P. taeda linkage groups, and additionally three and six smaller groups for E1101 and E635C, respectively. The number of framework AFLP markers in the integrated map is altogether 194 and the number of gene markers 61. The total length of the integrated map was 1,314 cM. The set of markers developed for P. sylvestris was also added to existing maps of two P. taeda pedigrees. Starting with a mapped marker from one pedigree in the source species resulted in a mapped marker in a pedigree of the other species in more than 40% of the cases, with about equal success in both directions. The maps of the two species are largely colinear, even if the species have diverged more than 70 MYA. Most cases of different locations were probably due to problems in identifying the orthologous members of gene families. These data provide a first ESTP-containing map of P. sylvestris, which can also be used for comparing this species to additional species mapped with the same markers.Communicated by C. Möllers