Hephaestia mangrovi sp. nov., a novel endophytic bacterium isolated from Aegiceras corniculatum.

A Gram-stain-negative, aerobic, motile, rod-shaped bacterium, designated CMS5P-6T, was isolated from a surface-sterilized bark of Aegiceras corniculatum collected from Guangxi Zhuang Autonomous Region, PR China, and investigated by a polyphasic approach to determine its taxonomic position. Strain CMS5P-6T was found to grow optimally with 0-1 % (w/v) NaCl, at 30 °C and pH 6.0-7.0. Substrate mycelia and aerial mycelia were not formed, and no diffusible pigments were observed on the media tested. Phylogenetic analysis showed that strain CMS5P-6T showed high 16S rRNA gene sequence similarity of 96.7 % to Hephaestia caeni DSM 25527T and Sphingomonas colocasiea CC-MHH0539T. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values between strain CMS5P-6T and H. caeni DSM 25527T were 78.0, 21.7 and 70.8 %, respectively. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values between strain CMS5P-6T and S. colocasiea JCM 31229T were 74.0, 19.9 and 61.4 %, respectively. Phylogenomic analyses based on genome sequences showed that strain CMS5P-6T and H. caeni DSM 25527T formed a distinct cluster within the family Sphingomonadaceae and far away from S. colocasiea JCM 31229T. The DNA G+C content of strain CMS5P-6T was determined to be 65.6 mol%. The cell-wall peptidoglycan was found to contain meso-diaminopimelic acid as the diagnostic diamino acid and ubiquinone Q-10 was identified as the respiratory lipoquinone. The polar lipids were found to comprise diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, sphingoglycolipid and two unidentified aminolipids, and the major fatty acids were identified as C18 : 1 ω7c, C19 : 0 cycloω8c and C16 : 0. On the basis of phylogenetic, genomic, chemotaxonomic and phenotypic data, strain CMS5P-6T can be concluded to represent a novel species of the genus Hephaestia, for which the name Hephaestia mangrovi sp. nov. is proposed. The type strain is CMS5P-6T (=JCM 33125T=CGMCC 1.13868T).

Pseudooceanicola endophyticus sp. nov., a novel endophytic bacterium isolated from bark Aegiceras corniculatum.

A Gram-stain-negative, aerobic, short-rod-shaped bacterium, designated strain CBS1P-1T, was isolated from a surface-sterilized bark of Aegiceras corniculatum. Growth of strain CBS1P-1T was observed with between 0 and 12.0 % (w/v) NaCl (optimally with 5.0 %) and at between pH 6.0-9.0. It grew at temperatures between 25-37 °C (optimum, 30 °C). Chemotaxonomic analysis showed that ubiquinone-10 was the respiratory quinone. The lipids comprised diphosphatidylglycerol, phosphatidylglycerol, an unidentified glycolipid, an unidentified phospholipid and an unidentified aminolipid. The major fatty acids of strain CBS1P-1T were C18 : 1 ω7c, C16 : 0 and C19 : 0 cyclo ω8c. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain CBS1P-1T was most related to Pseudooceanicola antarcticus CGMCC 1.12662T with a sequence similarity of 96.5 %. The average nucleotide identity and digital DNA-DNA hybridization values between strain CBS1P-1T and P. antarcticus 1.12662T were 77.5 and 21.1 %, respectively. The G+C content of the genomic DNA was 67.3 mol%. Based on phylogenetic, chemotaxonomic and phenotypic data, strain CBS1P-1T is considered to represent a novel species of the genus Pseudooceanicola, for which the name Pseudooceanicola endophyticus is proposed. The type strain is CBS1P-1T (=KCTC 62836T=CGMCC 1.13743T).

Comparative Analysis of Bacterial Diversity and Community Structure in the Rhizosphere and Root Endosphere of Two Halophytes, Salicornia europaea and Glaux maritima, Collected from Two Brackish Lakes in Japan.

Microbial community structures associated with halophytes and their compositions among different habitats, particularly natural saline sites, have not yet been investigated in detail. In the present study, we examined the diversity and composition of the rhizosphere and root endosphere bacteria of two halophytes, Salicornia europaea L. and Glaux maritima L., collected from two adjacent brackish lakes, Lake Notoro and Lake Tofutsu, in Japan. The bacterial species richness and diversity indices of the two halophytes collected from both lakes showed no significant differences in the rhizosphere or root endosphere. In contrast, beta diversity and taxonomic analyses revealed significant differences in the bacterial communities from each halophyte between the two lakes even though the two locations were natural saline sites, indicating that the bacterial communities for S. europaea and G. maritima both fluctuated in a manner that depended on the geographical location. Common and abundant genera associated with each halophyte across the two lakes were then identified to verify the bacterial genera specifically inhabiting each plant species. The results obtained showed that the composition of abundant genera inhabiting each halophyte across two lakes was distinct from that reported previously in other saline soil areas. These results suggest that each halophyte in different geographical sites had an individual complex bacterial community.

Mangrovicella endophytica gen. nov., sp. nov., a new member of the family Aurantimonadaceae isolated from Aegiceras corniculatum.

A Gram-negative, aerobic, motile and short-rod-shaped bacterium, designated strain 5T4P-12-1T, was isolated from a piece of surface-sterilized bark of Aegiceras corniculatum collected from Cotai Ecological Zones in Macao, China and tested by a polyphasic approach to clarify its taxonomic position. Strain 5T4P-12-1T grew optimally with 0-1 % (w/v) NaCl at 30 °C and at pH 7.0-8.0. The 16S rRNA gene sequence of strain 5T4P-12-1T had the highest similarity (96.7 %) to Aureimonas altamirensis DSM 21988T. Phylogenic analysis based on 16S rRNA gene sequences and coding sequences of 98 protein clusters showed that the strain represented a novel genus of the family Aurantimonadaceae. The predominant quinone system of strain 5T4P-12-1T was ubiquinone 10. The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylmethylethanolamine, an unidentified aminophospholipid, three unidentified aminolipids, three unidentified phospholipids and three unidentified lipids. The major fatty acids (>10 % of total fatty acids) were C18 : 1ω7c (55.4 %) and C18 : 1 2-OH (15.6 %). The DNA G+C content of strain 5T4P-12-1T was 66.5 mol%. Based on the phylogenic, phenotypic and chemotaxonomic features, strain 5T4P-12-1T is considered to represent a novel species of a new genus in the family Aurantimonadaceae, for which the name Mangrovicella endophytica gen. nov., sp. nov. is proposed. The type strain is 5T4P-12-1T (=KCTC 62053T=CGMCC 1.16279 T).

Amnibacterium endophyticum sp. nov., an endophytic actinobacterium isolated from Aegiceras corniculatum.

A Gram-stain-positive, aerobic, non-motile, non-spore-forming and short-rod-shaped actinobacterium, designated strain 1T4Z-3T, was isolated from a piece of surface-sterilized branch of Aegiceras corniculatum collected from the Cotai Ecological Zones in Macao, China. Comparative 16S rRNA gene sequence analysis showed that strain 1T4Z-3T was clearly affiliated to the genus Amnibacterium and exhibited 97.9 % gene sequence similarity to Amnibacterium kyonggiense JCM 16463T, 97.3 % gene sequence similarity to Amnibacterium soli JCM 19015T and less than 96.4 % gene sequence similarities to other genera of the family Microbacteriaceae. Strain 1T4Z-3T had L-2,4-diaminobutyric acid as the diagnostic cell-wall diamino acid. The major fatty acids (>10 % of total fatty acids) were iso-C16 : 0 (46.6 %) and anteiso-C15 : 0 (27.3 %). The predominant menaquinones of strain 1T4Z-3T were MK-11 (81.4 %) and MK-12 (14.1 %). The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, six unidentified glycolipids, four unidentified phospholipids and two unidentified lipids. The DNA G+C content of strain 1T4Z-3T was 71.4 mol%. Based on the phylogenetic, phenotypic and chemotaxonomic features, strain 1T4Z-3T is considered to represent a novel species of the genus Amnibacterium, for which the name Amnibacterium endophyticum sp. nov. is proposed. The type strain of Amnibacterium endophyticum is 1T4Z-3T (=KCTC 39983T=CGMCC 1.16066T).

Rathayibacter oskolensis sp. nov., a novel actinobacterium from Androsace koso-poljanskii Ovcz. (Primulaceae) endemic to the Central Russian Upland.

A rod-shaped, non-endospore-forming and non-motile bacterium, strain DL-329T, was isolated from the above-ground part of a plant, Androsace koso-poljanskii Ovcz. (Primulaceae), at the the State Natural Reserve 'Belogorie', Russia. On the basis of 16S rRNA gene sequence comparisons, the strain clustered with members of the genus Rathayibacter, showing the highest sequence similarity to Rathayibacter tritici (98.89 %), Rathayibacter rathayi (98.82 %) and Rathayibacter festucae (98.82 %). The DNA hybridization experiments demonstrated that strain DL-329T represents a separate genomic species. The results of comparative studies of physiological and chemotaxonomic characteristics, including cell-wall sugar patterns, polar lipid profiles, and the matrix-assisted laser desorption/ionization time-of-flight mass spectra of bacterial cells, allowed clear differentiation of VKM Ac-2121T from the recognized Rathayibacter species at the phenotypic level. Based on the data obtained, a new species, Rathayibacter oskolensis sp. nov., is proposed, with DL-329T (=VKM Ac-2121T=LMG 22542T) as the type strain.

Leaf nodule symbiosis: function and transmission of obligate bacterial endophytes.

Various plant species establish intimate symbioses with bacteria within their aerial organs. The bacteria are contained within nodules or glands often present in distinctive patterns on the leaves, and have been used as taxonomic marker since the early 20th century. These structures are present in very diverse taxa, including dicots (Rubiaceae and Primulaceae) and monocots (Dioscorea). The symbionts colonize the plants throughout their life cycles and contribute bioactive secondary metabolites to the association. In this review, we present recent progress in the understanding of these plant-bacteria symbioses, including the modes of transmission, distribution and roles of the symbionts.

Aureimonas endophytica sp. nov., a novel endophytic bacterium isolated from Aegiceras corniculatum.

A Gram-negative, motile, aerobic and coccoid rod-shaped bacterium, designated strain 2T4P-2-4T, was isolated from a piece of surface-sterilized bark of Aegiceras corniculatum collected from Cotai Ecological Zones in Macao, China, and tested by a polyphasic approach to clarify its taxonomic position. Strain 2T4P-2-4T grew optimally without NaCl at 28-30 °C, pH 7.0-8.0. The 16S rRNA gene sequence of strain 2T4P-2-4T had the highest similarity (96.2 %) to Aureimonas rubiginis CC-CFT034T. Phylogenetic analysis showed that the strain grouped with species of the genus Aureimonas. The predominant quinone system of strain 2T4P-2-4T was ubiquinone 10 (Q-10). The polar lipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, sulfoquinovosyldiacylglycerol, phosphatidylmethylethanolamine, two unidentified amino lipids, an unidentified aminophospholipid and five unidentified lipids. The predominant cellular fatty acid was C18 : 1ω7c (61.2 %). The DNA G+C content of strain 2T4P-2-4T was 69.8 mol%. Based on the phylogenetic, phenotypic and chemotaxonomic features, strain 2T4P-2-4T is a representative of a novel species of the genus Aureimonas, for which the name Aureimonas endophytica sp. nov. is proposed. The type strain of Aureimonas endophytica sp. nov. is 2T4P-2-4T (=KCTC 52217T=CGMCC 1.15367T).

Anti-proliferative ambuic acid derivatives from Hawaiian endophytic fungus Pestalotiopsis sp. FT172.

Five previously undescribed ambuic acid derivatives, pestallic acids A-E and three known analogs were isolated from the cultured broth of Pestalotiopsis sp. FT172. The structures of the pestallic acids A-E were determined through the analysis of HRMS and NMR spectroscopic data. The absolute configurations (ACs) of pestallic acids B-E were assigned by comparison of the experimental electric circular dichroism (ECD) spectra or the optical rotations with those in the literature. All compounds were tested against A2780 and cisplatin resistant A2780 (A2780CisR) cell lines. Pestallic acid E and (+)-ambuic acid showed potent activities with IC50 values from 3.3 to 17.0 µM.

Wounds on Rapanea melanophloeos provide habitat for a large diversity of Ophiostomatales including four new species.

Rapanea melanophloeos, an important canopy tree in Afromontane forests, is commonly utilised for medicinal bark harvesting. Wounds created from these activities provide entrance for many fungi, including arthropod-associated members of the Ophiostomatales and Microascales (ophiostomatoid fungi). In this study we assessed the diversity of wound-associated Ophiostomatales on storm-damaged R. melanophloeos trees in the Afromontane forests of South Africa. Five species were identified based on micro-morphological and molecular phylogenetic analyses. These included Ophiostoma stenoceras and four newly described taxa Sporothrix itsvo sp. nov., S. rapaneae sp. nov., S. uta sp. nov. and O. noisomeae sp. nov. Four of these are members of the S. schenckii-O. stenoceras complex (O. stenoceras, S. itsvo sp. nov., S. rapaneae sp. nov., S. uta sp. nov.) while O. noisomeae groups basal in the Ophiostomatales alongside the S. lignivora complex and Graphilbum. In addition to other taxa known from this host, the present study shows that there is a rich, yet still poorly explored, diversity of Ophiostomatales associated with R. melanophloeos in Afromontane forests. More taxa are likely to be discovered with increased research effort. These must be assessed in terms of pathogenicity towards this ecologically and economically important tree.

Pestalols A-E, new alkenyl phenol and benzaldehyde derivatives from endophytic fungus Pestalotiopsis sp. AcBC2 isolated from the Chinese mangrove plant Aegiceras corniculatum.

Five alkenyl phenol and benzaldehyde derivatives, pestalols A-E (1-5), as well as seven known compounds (6-12), were isolated from endophytic fungus Pestalotiopsis sp. AcBC2 derived from the Chinese mangrove plant Aegiceras corniculatum. Their structures were determined by spectroscopic analyses. Compounds 2 and 3 showed cytotoxicity against a panel of 10 tumor cell lines. Compounds 1-5, 8, 9, 11, and 12 showed inhibitory activities against Influenza A virus subtype (H3N2) and Swine Flu (H1N1) viruses. Compound 2 also showed inhibitory activity against tuberculosis.

Neighboring Deschampsia flexuosa and Trientalis europaea harbor contrasting root fungal endophytic communities.

Fungal endophytic communities and potential host preference of root-inhabiting fungi of boreal forest understory plants are poorly known. The objective of this study was to find out whether two neighboring plant species, Deschampsia flexuosa (Poaceae) and Trientalis europaea (Primulaceae), share similar root fungal endophytic communities and whether the communities differ between two sites. The study was carried out by analysis of pure culture isolates and root fungal colonization percentages. A total of 84 isolates from D. flexuosa and 27 isolates from T. europaea were obtained. The roots of D. flexuosa harbored 16 different isolate types based on macromorphological characteristics, whereas only 4 isolate types were found in T. europaea. The root colonization by dark septate and hyaline septate hyphae correlated with isolate numbers being higher in D. flexuosa compared to T. europaea. The different isolate types were further identified on the basis of internal transcribed spacer sequence and phylogenetic analysis. An isolate type identified as dark septate endophyte Phialocephala fortinii colonized 50 % of the T. europaea and 21 % of the D. flexuosa specimens. In addition, Meliniomyces variabilis, Phialocephala sphaeroides, and Umbelopsis isabellina were found colonizing the grass, D. flexuosa, for the first time and Mycena sp. was confirmed as an endophyte of D. flexuosa. Site-specific differences were observed in the abundance and diversity of endophytic fungi in the roots of both study plants, but the differences were not as predominant as those between plant species. It is concluded that D. flexuosa harbors both higher amount and more diverse community of endophytic fungi in its roots compared to T. europaea.

Biosynthesis of archetypal plant self-defensive oxylipins by an endophytic fungus residing in mangrove embryos.

A tree's travel companion: a fungal endophyte (Fusarium incarnatum) isolated from a viviparous propagule (embryo) of a mangrove tree produces typical plant defense oxylipins. Stable-isotope labeling experiments revealed that the endophyte biosynthesizes coriolic acid, didehydrocoriolic acid, and an epoxy fatty acid derived from linoleic acid by a process involving Δ(15)-desaturation and 13-lipoxygenation.

Identification of Hortaea werneckii Isolated from mangrove plant Aegiceras comiculatum based on morphology and rDNA sequences.

Hortaea werneckii is a black yeast-like ascomycetous fungi associated with the human superficial infection tinea nigra, which commonly occurs in tropical and subtropical countries. Now, this fungus has been found in the halophilic environment all over the world and recognized as a new model organism in exploring the mechanisms of salt tolerance in eukaryotes. During a survey of endophytic fungi of mangrove forest at South China Sea, two isolates of H. werneckii were recovered from medicinal plant of Aegiceras comiculatum. The isolates were identified by morphological characters and phylogenetic analyses (e.g., ITS rDNA, LSU rDNA and translation elongation factor EF1α). Some physiological tests such as thermotolerance, acid tolerance (pH) and NaCl tolerance as well as pathogenicity test in vitro for the strains of Hortaea were performed. It is the first report that H. werneckii was isolated from medicinal plant of A. comiculatum in south sea of China as the endophytic fungi.

Agroinfiltration of intact leaves as a method for the transient and stable transformation of saponin producing Maesa lanceolata.

UNLABELLED: A method has been developed to genetically transform the medicinal plant Maesa lanceolata. Initially, we tested conditions for transient expression of GFP-bearing constructs in agroinfiltrated leaves. Leaf tissues of M. lanceolata were infiltrated with Agrobacterium tumefaciens carrying a nuclear-targeted GFP construct to allow the quantification of the transformation efficiency. The number of transfected cells was depended on the bacterial density, bacterial strains, the co-cultivation time, and presence of acetosyringone. The transient transformation assay generated the highest ratio of transfected cells over non-transfected cells upon 5 days post-infiltration using A. tumefaciens strain LBA4404 at an OD600 = 1.0 in the presence of 100 µM acetosyringone and in the absence of a viral suppressor construct. In a second series of experiments we set up a stable transformation protocol that resulted in the regeneration of kanamycin-resistant plants expressing nuclear GFP. This transformation protocol will be used to introduce overexpression and RNAi constructs into M. lanceolata plants that may interfere with triterpenoid saponin biosynthesis. KEY MESSAGE: We have developed a transformation protocol for saponin producing Maesa lanceolata. Using the protocol reported here, now we are able to generate the tools for the modification of saponin production.

Cytotoxic alkaloids from Fusarium incarnatum associated with the mangrove tree Aegiceras corniculatum.

Several unusual alkaloids, N-2-methylpropyl-2-methylbutenamide (1), 2-acetyl-1,2,3,4-tetrahydro-ß-carboline (2), fusarine (3), fusamine (4), and 3-(1-aminoethylidene)-6-methyl-2H-pyran-2,4(3H)-dione (5), were isolated from the culture broth of Fusarium incarnatum (HKI0504), an endophytic fungus of the mangrove plant Aegiceras corniculatum. Compounds 2, 4, and 5 exhibit weak antiproliferative and cytotoxic activities against HUVEC, K-562, and HeLa human cell lines, respectively.

Antiviral isoindolone derivatives from an endophytic fungus Emericella sp. associated with Aegiceras corniculatum.

Chemical investigation of the endophytic fungus Emericella sp. (HK-ZJ) isolated from the mangrove plant Aegiceras corniculatum led to isolation of six isoindolones derivatives termed as emerimidine A and B and emeriphenolicins A and D, and six previously reported compounds named aspernidine A and B, austin, austinol, dehydroaustin, and acetoxydehydroaustin, respectively. Their structures were elucidated on the basis of NMR spectroscopic evidence while the anti-influenza A viral (H1N1) activities of eight compounds were also evaluated using the cytopathic effect (CPE) inhibition assay.

Bacterial leaf symbiosis in Ardisia (Myrsinoideae, Primulaceae): molecular evidence for host specificity.

The association between bacteria and leaves in Ardisia has been described as a cyclic and obligate symbiosis in which bacteria are maintained throughout all stages of the plant's life cycle to guarantee normal growth and survival of the host. This intimate interaction suggests that both partners have co-diversified together. To test this co-speciation hypothesis, we constructed an endosymbiont (16S rDNA and gyrB) and host (rps16, trnL, matK and ITS) phylogeny. Phylogenetic analyses of the endosymbionts revealed a pattern of strict host specificity and recovered a single clade in the genus Burkholderia (ß-proteobacteria), which was closely related to the endosymbionts of leaf-nodulated Rubiaceae. Comparison of symbiont and host phylogenies suggests a single origin of bacterial leaf symbiosis in the nodulated ancestor of Ardisia and does not reject the co-speciation hypothesis.

p-Aminoacetophenonic acids produced by a mangrove endophyte Streptomyces sp. (strain HK10552).

Four new p-aminoacetophenonic acids, named (2E)-11-(4'-aminophenyl)-5,9-dihydroxy-4,6,8-trimethyl-11-oxo-undec-2-enoic acid (1), 9-(4'-aminophenyl)-3,7-dihydroxy-2,4,6-trimethyl-9-oxo-nonoic acid(2), (2E)-11-(4'-aminophenyl)-5,9-O-cyclo-4,6,8-trimethyl-11-oxo-undec-2-enoic acid (3) and 9-(4'-aminophenyl)-3,7-O-cyclo-2,4,6-trimethyl-9-oxo-nonoic acid(4), were isolated from an endophyte Streptomyces sp. (strain HK10552) of the mangrove plant Aegiceras corniculatum. The structures of 1-4 were elucidated by using spectroscopic analyses. The relative stereoconfigurations of compounds 3 and 4 were determined by NOESY experiments. In the bioassay test, 1-4 showed no cytotoxicity against the Hela cell lines. Compound 4 also showed no inhibitory bioactivity on HCV protease and SecA ATPase and wasn't active against VSVG/HIV-luc pseudotyping virus.

Pyrrole and indole alkaloids from an endophytic Fusarium incarnatum (HKI00504) isolated from the mangrove plant Aegiceras corniculatum.

Two new pyrrole alkaloids, N-[4-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-butyl]-acetamide (1) and N-[5-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-pentyl]-acetamide (2), and a new indole derivative (3aR,8aR)-3a-acetoxyl-1,2,3,3a,8,8a-hexahydropyrrolo-[2,3-b]indol (3) were isolated, together with ( - )-3a-hydroxyfuroindoline, (3aR,8aS)-1-acetyl-1,3,3a,8,8a-hexahydropyrrolo-[2,3-b]indol-3a-ol, and N-acetyltryptamine A, from an endophytic ascomycetous fungus, Fusarium incarnatum (HKI00504), which was isolated from the mangrove plant Aegiceras corniculatum. The structures of compounds 1-3 were determined on the basis of extensive spectroscopic data analyses.