<Emphasis Type="Italic">Nibribacter flagellatus</Emphasis> sp. nov., isolated from rhizosphere of <Emphasis Type="Italic">Hibiscus syriacus</Emphasis> and emended description of the genus <Emphasis Type="Italic">Nibribacter</Emphasis>

A Gram-stain negative, aerobic, motile by flagella, rod-shaped strain (THG-T16^T) was isolated from rhizosphere of Hibiscus syriacus. Growth occurred at 10–40 °C (optimum 28–30 °C), at pH 6.0–8.0 (optimum 7.0) and at 0–1.0% NaCl (optimum 0%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-T16^T were identified as Nibribacter koreensis KACC 16450^T (98.6%), Rufibacter roseus KCTC 42217^T (94.7%), Rufibacter immobilis CCTCC AB 2013351^T (94.5%) and Rufibacter tibetensis CCTCC AB 208084^T (94.4%). The DNA G+C content of strain THG-T16^T was determined to be 46.7 mol%. DNA–DNA hybridization values between strain THG-T16^T and N. koreensis KACC 16450^T, R. roseus KCTC 42217^T, R. immobilis CCTCC AB 2013351^T, R.tibetensis CCTCC AB 208084^T were 33.5?±?0.5% (31.7?±?0.7% reciprocal analysis), 28.1?±?0.2% (25.2?±?0.2%), 17.1?±?0.9% (10.2?±?0.6%) and 8.1?±?0.3% (5.2?±?0.1%). The polar lipids were identified as phosphatidylethanolamine, two unidentified aminophospholipids, an unidentified aminolipid and three unidentified lipids. The quinone was identified as MK-7 and the polyamine as sym-homospermidine. The major fatty acids were identified as C_16:1 ω5c, C_17:1 ω6c, iso-C_15:0, summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c) and summed feature 4 (iso-C_17:1 I and/or anteiso-C_17:1 B). On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics, and DNA–DNA hybridization data, strain THG-T16^T represents a novel species of the genus Nibribacter, for which the name Nibribacter flagellatus sp. nov. is proposed. The type strain is THG-T16^T(=?KACC 19188^T?=?CCTCC AB 2016246^T).     

The new bacterial strain <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. IB-1: A producer of exopolysaccharide and biologically active substances with phytohormonal and antifungal activities

The bacterial strain IB-1, which exhibits antagonism towards phytopathogens and stimulates the growth of agricultural plants, was isolated from the soil. Analysis of the cultural, morphological, physiological, and biochemical features and nucleotide sequences of the genes 16S rRNA and gyrB, as well as the fatty acid composition, made it possible to attribute the strain IB-1 to the genus Paenibacillus; however, the results did not provide an unambiguous conclusion on its species. The strain Paenibacillus sp. IB-1 possesses nitrogenase activity, the ability to synthesize indoleacetic acid and cytokinin-like compounds, and antagonistic activity towards phytopathogenic fungi, which indicates prospects for its use as a biological product for agricultural purposes. A high-viscous exopolysaccharide was isolated from the cultural fluid of Paenibacillus sp. IB-1. Based on the data from IR and NMR spectroscopy, it was shown to be a heteropolymer comprised of one to four linked α-L-guluronic acid and β-D-mannuronic acid residues. The exopolysaccharide was successfully tested as an adhesive for presowing treatment of barley and wheat seeds with biofungicides.     

Down-regulation of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">DND1</Emphasis> orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death 1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.     

A New record of four <Emphasis Type="Italic">Penicillium</Emphasis> species isolated from <Emphasis Type="Italic">Agarum clathratum</Emphasis> in Korea

Agarum clathratum, brown algae, play important ecological roles in marine ecosystem, but can cause secondary environment pollution when they pile up on the beach. In order to resolve the environment problem by A. clathratum, we focus to isolate and identify Penicillium because many species are well known to produce extracellular enzymes. A total of 32 Penicillium strains were isolated from A. clathratum samples that collected from 13 sites along the mid-east coast of Korea in summer. They were identified based on morphological characters and phylogenetic analysis using β-tubulin DNA sequences as well as a combined dataset of β-tubulin and calmodulin. A total of 32 strains were isolated and they were identified to 13 Penicillium species. The commonly isolated species were Penicillium citrinum, P. roseomaculatum, and Penicillium sp. Among 13 Penicillium species, four species – P. bilaiae, P. cremeogriseum, P. madriti, and P. roseomaculatum – have not been previously recorded in Korea. For these four new species records to Korea, we provide morphological characteristics of each strain.     

Altered somatic mutation level and DNA repair gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> exposed to ultraviolet C,salt, and cadmium stresses

It is known that somatic mutations arising during animal growth and ageing contribute to the development of neurodegenerative and other animal diseases. For plants, several studies showed that small-scale somatic DNA mutations accumulated during Arabidopsis life cycle. However, there is a lack of data on the influence of environmental stresses on somatic DNA mutagenesis in plants. In this study, we analyzed the effects of ultraviolet C (UV-C) irradiation, high soil salinity, and cadmium (CdI₃) stresses on the level of small-scale somatic DNA mutations in Arabidopsis thaliana. The number of DNA mutations was examined in the Actin2 3′UTR (Actin-U1), ITS1-5.8rRNA-ITS2 (ITS), and ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) DNA regions. We found that somatic mutation levels considerably increased in CdI₃-treated Arabidopsis plants, while the mutation levels declined in the UV-C- and NaCl-treated A. thaliana. Cadmium is a mutagen that is known to inhibit DNA repair processes. The detected stress-induced alterations in somatic DNA mutation levels were accompanied by markedly increased expression of base excision repair genes (AtARP, AtDME, AtDML2, AtDML3, AtMBD4, AtROS, AtUNG, and AtZDP), nucleotide excision repair genes (AtDDB1a, AtRad4, and AtRad23a), mismatch repair genes (AtMSH2, AtMSH3, and AtMSH7), and photoreactivation genes (AtUVR2, AtUVR3). Thus, the results demonstrated that UV-C, high soil salinity, and cadmium stresses influence both the level of DNA mutations and expression of DNA repair genes. Salt- and UV-induced activation of DNA repair genes could contribute to the stress-induced decrease in somatic mutation level.     

Natural variation of potato <Emphasis Type="Italic">allene oxide synthase 2</Emphasis> causes differential levels of jasmonates and pathogen resistance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Natural variation of plant pathogen resistance is often quantitative. This type of resistance can be genetically dissected in quantitative resistance loci (QRL). To unravel the molecular basis of QRL in potato (Solanum tuberosum), we employed the model plant Arabidopsis thaliana for functional analysis of natural variants of potato allene oxide synthase 2 (StAOS2). StAOS2 is a candidate gene for QRL on potato chromosome XI against the oömycete Phytophthora infestans causing late blight, and the bacterium Erwinia carotovora ssp. atroseptica causing stem black leg and tuber soft rot, both devastating diseases in potato cultivation. StAOS2 encodes a cytochrome P450 enzyme that is essential for biosynthesis of the defense signaling molecule jasmonic acid. Allele non-specific dsRNAi-mediated silencing of StAOS2 in potato drastically reduced jasmonic acid production and compromised quantitative late blight resistance. Five natural StAOS2 alleles were expressed in the null Arabidopsis aos mutant under control of the Arabidopsis AOS promoter and tested for differential complementation phenotypes. The aos mutant phenotypes evaluated were lack of jasmonates, male sterility and susceptibility to Erwinia carotovora ssp. carotovora. StAOS2 alleles that were associated with increased disease resistance in potato complemented all aos mutant phenotypes better than StAOS2 alleles associated with increased susceptibility. First structure models of ‘quantitative resistant’ versus ‘quantitative susceptible’ StAOS2 alleles suggested potential mechanisms for their differential activity. Our results demonstrate how a candidate gene approach in combination with using the homologous Arabidopsis mutant as functional reporter can help to dissect the molecular basis of complex traits in non model crop plants.     

Revision of <Emphasis Type="Italic">Protohydnum</Emphasis> (Auriculariales,Basidiomycota)

Three species currently addressed to Protohydnum (Auriculariales) are studied with morphological and DNA methods. The genus Protohydnum is retained for the type species only, P. cartilagineum, recently re-collected in Brazil. The European species, P. piceicola, is not congeneric with P. cartilagineum and, therefore, placed in its own genus, Hyalodon, gen. nov. Another Hyalodon species, H. antui, is described from East Asia. The third member of Protohydnum sensu lato, P. sclerodontium from South-East Asia, is transferred to Elmerina.     

Characterization of phosphate solubilizing rhizobacteria associated with pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) isolated from two agricultural soils

Phosphorous (P) availability is a major concern in European agriculture where reserves are limited. In the case of pea (Pisum sativum L.), one of the most important legumes in the human diet, P has specific effects on nodulation and N₂ fixation. Therefore, when biofertilization schemes are considered for pea cropping, it is very important to include symbiotic dinitrogen-fixing bacteria as well as phosphate-solubilizing bacteria (PSB). In this study sixteen PSB were isolated from the rhizosphere of two pea cultivars in two French soils with different characteristics. They were phenotypically and genotypically diverse displaying 9 different Two Primers-Random Amplified Polymorphic DNA (TP-RAPD) patterns. The 16S rRNA gene analysis of representative strains showed that they belong to four highly divergent phylogenetic groups. Most of the PSB strains belonged to the genus Pseudomonas and were closely related to Pseudomonas baetica, P. lutea, P. azotoformans, P. jessenii and P. frederiksbergensis. Other strains from the genus Burkholderia were closely related to B. caledonica and those from the genus Rhizobium to R. grahamii. The single strain of genus Bacillus was close to Bacillus toyonensis. Some phylogenetic groups to which our PSB strains belong are widely distributed in plant rhizospheres in different countries and continents. This is particularly interesting in the case of strains from the phylogenetic group of P. fluorescens which includes PSB strains with high ability to solubilize phosphate indicating that they may be used as biofertilizers in many soils.     

Evaluation of antifungal,phosphate solubilisation,and siderophore and chitinase release activities of endophytic fungi from <Emphasis Type="Italic">Pistacia vera</Emphasis>

Fungal endophytes use different strategies to protect host plants from abiotic and biotic stress. In this study, we isolated endophytic fungi from Pistacia vera and characterised their antifungal activity against Aspergillus flavus, Rhizoctonia solani and Sclerotinia sclerotiorum, and their release of some factors that can alter plant growth capability. Trichoderma harzianum TH 5-1-2, T. harzianum TH 10-2-2 and T. atroviride TA 2-2-1 exhibited the highest growth inhibition percentages in dual culture assays against A. flavus, R. solani and S. sclerotiorum, respectively. Among the fungal endophyte cultures, ethyl acetate extracts of T. harzianum TH 10-2-2, T. harzianum TH 5-1-2 and T. atroviride TA 2-2-1 exhibited the highest growth inhibition of S. sclerotiorum, R. solani and A. flavus, respectively. Phosphate solubilisation was induced by Byssochlamys nivea BN 1-1-1 in culture. Large amounts of siderophore production were observed with Quambalaria cyanescens QC 11-3-2 and Epicoccum nigrum EN1, but Trichoderma spp. also produced siderophore in lower amounts. Trichoderma harzianum TH 5-1-2 produced the highest chitinase activity (2.92 U/mL). In general, among the endophytes isolated, Trichoderma spp. appear to have the most promise for promoting healthy growth of P. vera.     

Differential expression of two <Emphasis Type="Italic">CONSTANS-LIKE1</Emphasis> genes in potato

Although the CONSTANS gene and its CONSTANS-LIKE1 (COL1) orthologs are known to control the photoperiod-dependent floral transition in many plant species, the role of these genes in Solanum development has not been sufficiently elucidated. Previously we characterized two forms of CONSTANS-LIKE1 genes, sCOL1 and lCOL1, in potato (Solanum tuberosum ssp. tuberosum). To prove that these genes were functional, we followed their expression in potato cv. Early Rose with the real-time PCR technique. Both sCOL1 and lCOL1 displayed characteristic day-night patterns of expression under long-day and short-day conditions. The profiles and amplitudes of expression dramatically differed in two genes, with the maximum sCOL1 expression exceeding that of lCOL1 by an order of magnitude.     

Defense Responses and Changes in Symbiotic Gut Microflora in the Colorado Potato Beetle <Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis> under the Effect of Endophytic Bacteria from the Genus Bacillus

Phytophagous insects and host plants have a complex of microsymbionts and make up a united co-evolving system with them. Microsymbiotic complexes are actively involved in stress responses of macrosymbionts. We established that a treatment of potato plants with endophytic bacterial strains Bacillus thuringiensis var. thuringiensis-5689, B. th. var. kurstaki-5351, and Bacillus subtilis 26D decreased the survival rate of the plant feeder, Colorado potato beetle Leptinotarsa decemlineata Say. The B. th. strains suppressed phenoloxidase and acetylcholinesterase activities in the beetle hemolymph. An antagonistic relationship was found between endophytic bacteria B. subtilis 26D and beetle symbiotic bacteria from the genera Acinetobacter and Enterobacter, with the former being able to suppress the growth of endophytic colonies. The recombinant B. subtilis strain 26D Cry, containing the B. th. var. kurstaki δ-endotoxin cry1Ia gene, combined the ability of the original B. subtilis 26D strain to suppress the development of beetle symbionts and immune responses with a production of the Cry toxin, thus leading to a high mortality of the phytophage.     

Bioprospecting of <Emphasis Type="Italic">Diaporthe terebinthifolii</Emphasis> LGMF907 for antimicrobial compounds

Antibiotic-resistant bacteria have been observed with increasing frequency over the past decades, driving the search for new drugs and stimulating the interest in natural products sources. Endophytic fungi from medicinal plants represent a great source of novel bioactive compounds useful to pharmaceutical and agronomical purposes. Diaporthe terebinthifolii is an endophytic species isolated from Schinus terebinthifolius, a plant used in popular medicine for several health problems. The strain D. terebinthifolii LGMF907 was previously reported by our group to produce secondary metabolites with biological activity against phytopathogens. Based on these data, strain LGMF907 was chosen for bioprospecting against microorganisms of clinical importance and for characterization of major secondary metabolites. In this study, different culture conditions were evaluated and the biological activity of this strain was expanded. The crude extracts demonstrated high antibacterial activity against Escherichia coli, Micrococcus luteus, Saccharomyces cerevisiae, methicillin-sensitive Staphylococcus aureus, and methicillin-resistant S. aureus. The compounds diaporthin and orthosporin were characterized and also showed activity against the clinical microorganisms evaluated. This study discloses the first isolation of diaporthin and orthosporin from D. terebinthifolii, and revealed the potential of this endophytic fungus to produce secondary metabolites with antimicrobial activity.     

<Emphasis Type="Italic">Phenylobacterium terrae</Emphasis> sp. nov., isolated from a soil sample of Khyber-Pakhtun-Khwa,Pakistan

A Gram-stain negative, aerobic, motile, non-spore-forming and rod-shaped bacterial strain, designated YIM 730227^T, was isolated from a soil sample, collected from Karak district, Khyber-Pakhtun-Khwa, Pakistan. The bacterium was characterized using a polyphasic taxonomic approach. Pairwise comparison of the 16S rRNA gene sequences showed that strain YIM 730227^T is closely related to Phenylobacterium lituiforme FaiI3^T (97.5% sequence similarity), Phenylobacterium muchangponense A8^T (97.4%), Phenylobacterium panacis DCY109^T (97.1%), Phenylobacterium immobile E^T (97.1%) and Phenylobacterium composti 4T-6^T (97.0%), while also sharing 98.0% sequence similarity with Phenylobacterium hankyongense HKS-05^T after NCBI blast, showing it represents a member of the family Caulobacteraceae. The major respiratory quinone was Q-10 and the major fatty acids were C_16:0, summed feature 8 (comprising C_18:1ω7c and/or C_18:1ω6c), C_18:1ω7c 11-methyl and C_17:0. The polar lipids were phosphatidylglycerol, unidentified glycolipids, phospholipid and unidentified lipid. The G?+?C content of the genomic DNA was 68.2 mol%. The DNA–DNA relatedness values of strain YIM 730227^T with P. hankyongense HKS-05^T, P. lituiforme FaiI3^T, P. muchangponense A8^T, P. panacis DCY109^T, P. immobile E^T and P. composti 4T-6^T were 31.3?±?0.6, 26.1?±?0.2, 24.3?±?0.1, 21.8?±?0.9, 19.8?±?0.6 and 18.2?±?1.1%, respectively, values lower than 70%. Besides the morphological and chemotaxonomic characteristics, phylogenetic analyses of 16S rRNA gene sequences and the biochemical characteristics indicated that the strain YIM 730227^T represents a novel member of the genus Phenylobacterium, for which the name Phenylobacterium terrae sp. nov. (type strain YIM 730227^T =?KCTC62324^T?=?CGMCC 1.16326^T) is proposed.     

Biosynthesis of poly(2-hydroxybutyrate-co-lactate) in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polyhydroxyalkanoates containing 2-hydroxyacid monomers such as lactate and 2-hydroxybutyrate in recombinant Escherichia coli strains by the expression of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Here, we report the biosynthesis of poly(2-hydroxybutyrate-co-lactate)[P(2HB-co-LA)] by direct fermentation of metabolically engineered E. coli strain. Among E. coli strains WL3110, XL1-Blue, and BL21(DE3), recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 produced P(76.4mol%2HB-co-23.6mol%LA) to the highest content of 88 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 2-hydroxybutyrate. When recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 was cultured in a chemically defined medium containing 20 g/L of glucose and varying concentration of sodium 2-hydroxybutyrate, 2HB monomer fraction in P(2HB-co-LA) increased proportional to the concentration of sodium 2-hydroxybutyrate added to the culture medium. P(2HB-co-LA)] could also be produced from glucose as a sole carbon source without sodium 2-hydroxybutyrate into the culture medium. Recombinant E. coli XL1-Blue strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene, successfully produced P(23.5mol%2HB-co-76.5mol%LA)] to the polymer content of 19.4 wt% when it cultured in a chemically defined medium containing 20 g/L of glucose. The metabolic engineering strategy reported here should be useful for the production of novel copolymer P(2HB-co-LA)].     

Analysis of endophytic actinobacteria species diversity in the stem of <Emphasis Type="Italic">Gynura cusimbua</Emphasis> by 16S rRNA gene clone library

Endophytic actinobacteria that lived in any associations with plant tissues represented a rather unexplored area of actinobacteria compared with soils. Gynura cusimbua was a kind of medicinal plant which had prevention effects for high blood pressure, coronary heart disease, Alzheimer’s disease, atherosclerosis, etc. Endophytic actinobacteria of G. cusimbua might produce some secondary metabolites which had the same function as their host. Stem samples of G. cusimbua collected from Hainan Province were used to study their endophytic actinobacteria to find some new compounds. In order to avoid vast proportions of the host plant DNA in the metagenomic library, the strategies of enrichment of the microorganism cells after tissue digestion and exclusion of 16S rRNA gene derived from the plastid by digested with PvuII were used. Two sets of actinobacteria specific primers were used for targeting endophytic actinobacteria from metagenomic library. 63 positive clones of actinobacteria were selected for sequencing and constructing the phylogenetic tree of 16S rRNA gene, and the 16S rRNA gene sequence of 59 strains among them had higher similar to the closest type strain and belonged to Microbacterium, Arthrobacter, Micrococcus, Curtobacterium, Okibacterium, Quadrisphaera and Kineococcus, respectively. Others were in low similarity and belonged to unclassified Micrococcineae, unclassified Intrasporangiaceae and unclassified Microbacteriaceae.     

Usefulness of the Non-conventional <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Model to Assess <Emphasis Type="Italic">Candida</Emphasis> Virulence

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.