Production of Alginate Oligosaccharides (AOSs) Using Enhanced Physicochemical Properties of Immobilized Alginate Lyase for Industrial Application.

Alginate lyase (AL) is a polysaccharide-degrading enzyme that can degrade alginate by hydrolyzing glycosidic bonds and produces unsaturated alginate oligosaccharides (AOSs). These AOSs have wide therapeutic and nutraceutical applications. However, to produce alginate oligosaccharides in a cost-effective manner is challenging due to the low availability and high cost of this degrading enzyme. Immobilization of the enzyme facilitates industrial applications owing to its stability, reusability, and cost-effectiveness. This study was focused on the enhancement of the properties of alginate lyase and improvement of the production of AOS. Alginate lyase was immobilized on magnetic nanoparticles (NPs) using glutaraldehyde as the crosslinker. The study showed that the maximum binding achieved between NPs and protein in the enzyme was 71% at a ratio of 1:150 NP:protein. As a result of immobilization, the optimum activity of free enzyme which was obtained at 37 °C and pH 7.4 changed to 45 °C and pH 9. Furthermore, the enzyme was thermostable at 45 °C for 3 h with up to 50% reusability for six consecutive cycles. Storage stability after 15 days showed ~67% relative hydrolysis of alginate. The free alginate lyase (25 IU) showed 76% raw biomass (seaweed) hydrolysis which is higher compared to 63% provided by the immobilized enzyme. As a result of efficient hydrolysis, AOSs with molecular weight profile of 370-1040 kDa were produced and detected using HPLC.

Oleaginous Microbial Lipids' Potential in the Prevention and Treatment of Neurological Disorders.

The products of oleaginous microbes, primarily lipids, have gained tremendous attention for their health benefits in food-based applications as supplements. However, this emerging biotechnology also offers a neuroprotective treatment/management potential for various diseases that are seldom discussed. Essential fatty acids, such as DHA, are known to make up the majority of brain phospholipid membranes and are integral to cognitive function, which forms an important defense against Alzheimer's disease. Omega-3 polyunsaturated fatty acids have also been shown to reduce recurrent epilepsy seizures and have been used in brain cancer therapies. The ratio of omega-3 to omega-6 PUFAs is essential in maintaining physiological function. Furthermore, lipids have also been employed as an effective vehicle to deliver drugs for the treatment of diseases. Lipid nanoparticle technology, used in pharmaceuticals and cosmeceuticals, has recently emerged as a biocompatible, biodegradable, low-toxicity, and high-stability means for drug delivery to address the drawbacks associated with traditional medicine delivery methods. This review aims to highlight the dual benefit that lipids offer in maintaining good health for disease prevention and in the treatment of neurological diseases.

Factors affecting the isolation and diversity of marine sponge-associated bacteria.

Marine sponges are an ideal source for isolating as yet undiscovered microorganisms with some sponges having about 50% of their biomass composed of microbial symbionts. This study used a variety of approaches to investigate the culturable diversity of the sponge-associated bacterial community from samples collected from the South Australian marine environment. Twelve sponge samples were selected from two sites and their bacterial population cultivated using seven different agar media at two temperatures and three oxygen levels over 3 months. These isolates were identified using microscopic, macroscopic, and 16S rRNA gene analysis. A total of 1234 bacterial colonies were isolated which consisted of four phyla: Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes, containing 21 genera. The diversity of the bacterial population was demonstrated to be influenced by the type of isolation medium, length of the incubation period and temperature, sponge type, and oxygen level. The findings of this study showed that marine sponges of South Australia can yield considerable bacterial culturable diversity if a comprehensive isolation strategy is implemented. Two sponges, with the highest and the lowest diversity of culturable isolates, were examined using next-generation sequencing to better profile the bacterial population. A marked difference in terms of phyla and genera was observed using culture-based and culture-independent approaches. This observed variation displays the importance of utilizing both methods to reflect a more complete picture of the microbial population of marine sponges. KEY POINTS: Improved bacterial diversity due to long incubations, 2 temperatures, and 3 oxygen levels. Isolates identified by morphology, restriction digests, and 16S rRNA gene sequencing. At least 70% of culturable genera were not revealed by NGS methods.

Evaluation of ACC-deaminase-producing rhizobacteria to alleviate water-stress impacts in wheat (Triticum aestivum L.) plants.

Application of plant-growth-promoting rhizobacteria (PGPR) is an environmentally sustainable option to reduce the effects of abiotic and biotic stresses on plant growth and productivity. Bacteria isolated from rain-fed agriculture field soils in the Central Himalaya Kumaun region, India, were evaluated for the production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. Those producing ACC deaminase in high amounts were evaluated for their potential to improve wheat (Triticum aestivum L.) plant growth under irrigated and water-stress conditions in two glasshouse experiments. Some of the isolates also showed other plant-growth-promoting (PGP) traits, e.g., N2 fixation, siderophore production, and phosphate solubilization; however, strains with higher ACC deaminase activity showed the greatest effects. These were Variovorax paradoxus RAA3; Pseudomonas spp. DPC12, DPB13, DPB15, DPB16; Achromobacter spp. PSA7, PSB8; and Ochrobactrum anthropi DPC9. In both simulated irrigated and water-stress conditions, a single inoculation of RAA3 and a consortium of DPC9 + DPB13 + DPB15 + DPB16 significantly improved wheat plant growth and foliar nutrient concentrations and caused significant positive changes in antioxidant properties compared with noninoculated plants especially under water stress. These findings imply that PGPB having ACC deaminase activity together with other PGP traits could potentially be effective inoculants to improve the growth of wheat plants in water-stressed rain-fed environments.

The antifungal action mode of the rice endophyte Streptomyces hygroscopicus OsiSh-2 as a potential biocontrol agent against the rice blast pathogen.

Microbial antagonists and their bioactive metabolites provide one of the best alternatives to chemical pesticides to control crop disease for sustainable agriculture and global food security. The rice endophyte Streptomyces hygroscopicus OsiSh-2, with remarkable antagonistic activity towards the rice blast fungus Magnaporthe oryzae, was reported in our previous study. The present study deciphered the possible direct interaction mode of OsiSh-2 against M. oryzae. An in vitro antibiotic assay for OsiSh-2 culture filtrate revealed strong suppression of mycelial growth, conidial germination and appressorial formation of M. oryzae. Meanwhile, severe morphological and internal abnormalities in M. oryzae hyphae were observed under a scanning electron microscope and transmission electron microscope. Foliar treatment of rice seedlings by OsiSh-2 culture filtrate in the greenhouse and in the field showed 23.5% and 28.3% disease reduction, respectively. Correspondingly, OsiSh-2 culture filtrate could induce disorganized chitin deposition in the cell wall and lowered ergosterol content in the cell membrane of M. oryzae. Additionally, cell wall integrity pathway activation, large cell electrolytes release, reactive oxygen species accumulation and tricarboxylic acid cycle-related enzyme activity changes were found in M. oryzae. All these results suggested that the direct antagonistic activity of OsiSh-2 against M. oryzae may be attributed to damaging the integrity of the cell wall and membrane and disrupting mitochondrial function in the pathogen.

Promicromonospora callitridis sp. nov., an endophytic actinobacterium isolated from the surface-sterilized root of an Australian native pine tree.

A new strain of the genus Promicromonospora, CAP94T, was isolated from the surface sterilized root of Callitrispreissii (Australian native pine tree). This strain was a Gram-stain-positive, aerobic actinobacterium with hyphae breaking up into fragments which were non-motile, rod-like, coccoid elements. Phylogenetic evaluation based on 16S rRNA gene sequence analysis placed this isolate as a member of the family Promicromonospora ceae, and most closely to Promicromonospora sukumoe NBRC 14650T (99.4 %), Promicromonospora kroppenstedtii DSM 19349T (99.2 %) and Promicromonosporaaerolata V54AT (99.1 %). Chemotaxonomic data including cell-wall components, major menaquinone and major fatty acids confirmed the affiliation of strain CAP94T to the genus Promicromonospora. The results of the phylogenetic analysis, including physiological and biochemical studies in combination with DNA-DNA hybridization, allowed the genotypic and phenotypic differentiation of strain CAP94T and the closest species with validly published names. The name proposed for the new species is Promicromonospora callitridis sp. nov. The type strain is CAP94T (=DSM 103339T=TBRC 6025T).

The role of sponge-bacteria interactions: the sponge Aplysilla rosea challenged by its associated bacterium Streptomyces ACT-52A in a controlled aquarium system.

Sponge-associated bacteria play a critical role in sponge biology, metabolism and ecology, but how they interact with their host sponges and the role of these interactions are poorly understood. This study investigated the role of the interaction between the sponge Aplysilla rosea and its associated actinobacterium, Streptomyces ACT-52A, in modifying sponge microbial diversity, metabolite profile and bioactivity. A recently developed experimental approach that exposes sponges to bacteria of interest in a controlled aquarium system was improved by including the capture and analysis of secreted metabolites by the addition of an absorbent resin in the seawater. In a series of controlled aquaria, A. rosea was exposed to Streptomyces ACT-52A at 106 cfu/ml and monitored for up to 360 h. Shifts in microbial communities associated with the sponges occurred within 24 to 48 h after bacterial exposure and continued until 360 h, as revealed by TRFLP. The metabolite profiles of sponge tissues also changed substantially as the microbial community shifted. Control sponges (without added bacteria) and Streptomyces ACT-52A-exposed sponges released different metabolites into the seawater that was captured by the resin. The antibacterial activity of compounds collected from the seawater increased at 96 and 360 h of exposure for the treated sponges compared to the control group due to new compounds being produced and released. Increased antibacterial activity of metabolites from treated sponge tissue was observed only at 360 h, whereas that of control sponge tissue remained unchanged. The results demonstrate that the interaction between sponges and their associated bacteria plays an important role in regulating secondary metabolite production.

Acetylated Triterpene Glycosides and Their Biological Activity from Holothuroidea Reported in the Past Six Decades.

Sea cucumbers have been valued for many centuries as a tonic and functional food, dietary delicacies and important ingredients of traditional medicine in many Asian countries. An assortment of bioactive compounds has been described in sea cucumbers. The most important and abundant secondary metabolites from sea cucumbers are triterpene glycosides (saponins). Due to the wide range of their potential biological activities, these natural compounds have gained attention and this has led to their emergence as high value compounds with extended application in nutraceutical, cosmeceutical, medicinal and pharmaceutical products. They are characterized by bearing a wide spectrum of structures, such as sulfated, non-sulfated and acetylated glycosides. Over 700 triterpene glycosides have been reported from the Holothuroidea in which more than 145 are decorated with an acetoxy group having 38 different aglycones. The majority of sea cucumber triterpene glycosides are of the holostane type containing a C18 (20) lactone group and either Δ(7(8)) or Δ(9(11)) double bond in their genins. The acetoxy group is mainly connected to the C-16, C-22, C-23 and/or C-25 of their aglycone. Apparently, the presence of an acetoxy group, particularly at C-16 of the aglycone, plays a significant role in the bioactivity; including induction of caspase, apoptosis, cytotoxicity, anticancer, antifungal and antibacterial activities of these compounds. This manuscript highlights the structure of acetylated saponins, their biological activity, and their structure-activity relationships.

Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity.

Structure elucidation of new acetylated saponins, Lessoniosides A, B, C, D, and E, and non-acetylated saponins, Lessoniosides F and G, from the viscera of the sea cucumber Holothuria lessoni.

Sea cucumbers produce numerous compounds with a wide range of chemical structural diversity. Among these, saponins are the most diverse and include sulfated, non-sulfated, acetylated and methylated congeners with different aglycone and sugar moieties. In this study, MALDI and ESI tandem mass spectrometry, in the positive ion mode, were used to elucidate the structure of new saponins extracted from the viscera of H. lessoni. Fragmentation of the aglycone provided structural information on the presence of the acetyl group. The presence of the O-acetyl group was confirmed by observing the mass transition of 60 u corresponding to the loss of a molecule of acetic acid. Ion fingerprints from the glycosidic cleavage provided information on the mass of the aglycone (core), and the sequence and type of monosaccharides that constitute the sugar moiety. The tandem mass spectra of the saponin precursor ions [M + Na]+ provided a wealth of detailed structural information on the glycosidic bond cleavages. As a result, and in conjunction with existing literature, we characterized the structure of five new acetylated saponins, Lessoniosides A-E, along with two non-acetylated saponins Lessoniosides F and G at m/z 1477.7, which are promising candidates for future drug development. The presented strategy allows a rapid, reliable and complete analysis of native saponins.

Kribbella endophytica sp. nov., an endophytic actinobacterium isolated from the surface-sterilized leaf of a native apricot tree.

A new strain of the genus Kribbella, PIP 118(T), was isolated from the leaf of an Australian native apricot tree (Pittosporum angustifolium), or Gumbi Gumbi in the indigenous language. This strain is an aerobic actinobacterium consisting of hyphae that fragment into short to elongated rod-like elements. Phylogenetic evaluation based on 16S rRNA gene sequence analysis placed this isolate as a member of the family Nocardioidaceae and most closely related to Kribbella antibiotica YIM 31530(T) (98.6 %) and Kribbella koreensis LM 161(T) (98.4 %). Chemotaxonomic data including cell wall components, major menaquinone and major fatty acids confirmed the affiliation of strain PIP 118(T) to the genus Kribbella. The results of the phylogenetic analysis, including physiological and biochemical studies in combination with DNA-DNA hybridization, allowed the genotypic and phenotypic differentiation of strain PIP 118(T) and members of the most closely related species with validly published names. The name proposed for the new species is Kribbella endophytica sp. nov. The type strain is PIP 118(T) ( = DSM 23718(T) = NRRL B-24812(T)).

Streptomyces kebangsaanensis sp. nov., an endophytic actinomycete isolated from an ethnomedicinal plant, which produces phenazine-1-carboxylic acid.

A spore-forming streptomycete designated strain SUK12(T) was isolated from a Malaysian ethnomedicinal plant. Its taxonomic position, established using a polyphasic approach, indicates that it is a novel species of the genus Streptomyces. Morphological and chemical characteristics of the strain were consistent with those of members of the genus Streptomyces. Analysis of the almost complete 16S rRNA gene sequence placed strain SUK12(T) in the genus Streptomyces where it formed a distinct phyletic line with recognized species of this genus. The strain exhibited highest sequence similarity to Streptomyces corchorusii DSM 40340(T) (98.2 %) followed by Streptomyces chrestomyceticus NRRL B-3310(T) (98.1 %). The G+C content of the genomic DNA was 74 mol%. Chemotaxonomic data [MK-9(H8) as the major menaquinone; LL-diaminopimelic acid as a component of cell-wall peptidoglycan; C12 : 0, C14 : 0, C15 : 0 and C17 : 1 as the major fatty acids; phospholipid type II] supported the affiliation of strain SUK12(T) to the genus Streptomyces. The results of the phylogenetic analysis and phenotypic data derived from this and previous studies allowed the genotypic and phenotypic differentiation of strain SUK12(T) from the related species of the genus Streptomyces. The DNA-DNA relatedness value between strain SUK12(T) and S. corchorusii DSM 40340(T) is 18.85±4.55 %. Strain SUK12(T) produces phenazine-1-carboxylic acid, known as tubermycin B, an antibacterial agent. It is proposed, therefore, that strain SUK12(T) ( = DSM 42048(T) = NRRL B-24860(T)) be classified in the genus Streptomyces as the type strain of Streptomyces kebangsaanensis sp. nov.

Rational approaches to improving the isolation of endophytic actinobacteria from Australian native trees.

In recent years, new actinobacterial species have been isolated as endophytes of plants and shrubs and are sought after both for their role as potential producers of new drug candidates for the pharmaceutical industry and as biocontrol inoculants for sustainable agriculture. Molecular-based approaches to the study of microbial ecology generally reveal a broader microbial diversity than can be obtained by cultivation methods. This study aimed to improve the success of isolating individual members of the actinobacterial population as pure cultures as well as improving the ability to characterise the large numbers obtained in pure culture. To achieve this objective, our study successfully employed rational and holistic approaches including the use of isolation media with low concentrations of nutrients normally available to the microorganism in the plant, plating larger quantities of plant sample, incubating isolation plates for up to 16 weeks, excising colonies when they are visible and choosing Australian endemic trees as the source of the actinobacteria. A hierarchy of polyphasic methods based on culture morphology, amplified 16S rRNA gene restriction analysis and limited sequencing was used to classify all 576 actinobacterial isolates from leaf, stem and root samples of two eucalypts: a Grey Box and Red Gum, a native apricot tree and a native pine tree. The classification revealed that, in addition to 413 Streptomyces spp., isolates belonged to 16 other actinobacterial genera: Actinomadura (two strains), Actinomycetospora (six), Actinopolymorpha (two), Amycolatopsis (six), Gordonia (one), Kribbella (25), Micromonospora (six), Nocardia (ten), Nocardioides (11), Nocardiopsis (one), Nonomuraea (one), Polymorphospora (two), Promicromonospora (51), Pseudonocardia (36), Williamsia (two) and a novel genus Flindersiella (one). In order to prove novelty, 12 strains were characterised fully to the species level based on polyphasic taxonomy. One strain represented a novel genus in the family Nocardioides, and the other 11 strains were accepted as novel species. In summary, the holistic isolation strategies were successful in obtaining significant culturable actinobacterial diversity within Australian native trees that includes rare and novel species.

Development of sustainable downstream processing for nutritional oil production.

Nutritional oils (mainly omega-3 fatty acids) are receiving increased attention as critical supplementary compounds for the improvement and maintenance of human health and wellbeing. However, the predominant sources of these oils have historically shown numerous limitations relating to desirability and sustainability; hence the crucial focus is now on developing smarter, greener, and more environmentally favourable alternatives. This study was undertaken to consider and assess the numerous prevailing and emerging techniques implicated across the stages of fatty acid downstream processing. A structured and critical comparison of the major classes of disruption methodology (physical, chemical, thermal, and biological) is presented, with discussion and consideration of the viability of new extraction techniques. Owing to a greater desire for sustainable industrial practices, and a desperate need to make nutritional oils more available; great emphasis has been placed on the discovery and adoption of highly sought-after 'green' alternatives, which demonstrate improved efficiency and reduced toxicity compared to conventional practices. Based on these findings, this review also advocates new forays into application of novel nanomaterials in fatty acid separation to improve the sustainability of nutritional oil downstream processing. In summary, this review provides a detailed overview of the current and developing landscape of nutritional oil; and concludes that adoption and refinement of these sustainable alternatives could promptly allow for development of a more complete 'green' process for nutritional oil extraction; allowing us to better meet worldwide needs without costing the environment.

Promicromonospora endophytica sp. nov., an endophytic actinobacterium isolated from the root of an Australian native Grey Box tree.

A novel aerobic actinobacterium, strain EUM 273(T), was isolated from the root of a Grey Box tree (Eucalyptus microcarpa Maiden). Cells were Gram-staining-positive with well-developed substrate mycelia which were non-motile and rod-like, with coccoid elements. Phylogenetic evaluation based on 16S rRNA gene sequence analysis placed the isolate as a member of the family Promicromonosporaceae that was most closely related to Promicromonospora xylanilytica YIM 61515(T) (98.2%) and Promicromonospora vindobonensis V45(T) (98%). Chemotaxonomic data including cell wall components, major menaquinone and major fatty acids confirmed the affiliation of strain EUM 273(T) to the genus Promicromonospora. The results of the phylogenetic analysis, including physiological and biochemical studies in combination with DNA-DNA hybridization, allowed the genotypic and phenotypic differentiation of strain EUM 273(T) from the closest related species with validly published names. The name proposed for the novel species is Promicromonospora endophytica sp. nov. The type strain is EUM 273(T) (=DSM 23716(T)=NRRL B-24816(T)).

Cytobacts: Abundant and Diverse Vertically Seed-Transmitted Cultivation-Recalcitrant Intracellular Bacteria Ubiquitous to Vascular Plants.

We have recently described 'Cytobacts' as abundant intracellular endophytic bacteria inhabiting live plant cells based on the observations with callus and cell suspension cultures of grapevine and other plant species with the origin ascribable to field explants. In this study, we investigated the prevalence of such cytoplasmic bacterial associations in field plants across different taxa, their cultivability, and the extent of taxonomic diversity and explored the possibility of their embryo-mediated vertical transmission. Over 100 genera of field plants were surveyed for 'Cytobacts' through bright-field live-cell imaging as per our previous experience using fresh tissue sections from surface-sterilized shoot-tissues with parallel cultivation-based assessments. This revealed widespread cellular bacterial associations visualized as copious motile micro-particles in the cytoplasm with no or sparse colony forming units (CFU) from the tissue-homogenates indicating their general non-cultivability. Based on the ease of detection and the abundance of 'Cytobacts' in fresh tissue sections, the surveyed plants were empirically classified into three groups: (i) motile bacteria detected instantly in most cells; (ii) motility not so widely observed, but seen in some cells; and (iii) only occasional motile units observed, but abundant non-motile bacterial cells present. Microscopy versus 16S-rRNA V3-V4 amplicon profiling on shoot-tip tissues of four representative plants-tomato, watermelon, periwinkle, and maize-showed high bacterial abundance and taxonomic diversity (11-15 phyla) with the dominance of Proteobacteria followed by Firmicutes/Actinobacteria, and several other phyla in minor shares. The low CFU/absence of bacterial CFU from the tissue homogenates on standard bacteriological media endorsed their cultivation-recalcitrance. Intracellular bacterial colonization implied that the associated organisms are able to transmit vertically to the next generation through the seed-embryos. Microscopy and 16S-rRNA V3-V4 amplicon/metagenome profiling of mature embryos excised from fresh watermelon seeds revealed heavy embryo colonization by diverse bacteria with sparse or no CFU. Observations with grapevine fresh fruit-derived seeds and seed-embryos endorsed the vertical transmission by diverse cultivation-recalcitrant endophytic bacteria (CREB). By and large, Proteobacteria formed the major phylum in fresh seed-embryos with varying shares of diverse phyla. Thus, we document 'Cytobacts' comprising diverse and vertically transmissible CREBs as a ubiquitous phenomenon in vascular plants.

Pseudonocardia eucalypti sp. nov., an endophytic actinobacterium with a unique knobby spore surface, isolated from roots of a native Australian eucalyptus tree.

A novel strain, designated EUM 374(T), was isolated from the root of a native Australian eucalyptus tree, Eucalyptus microcarpa, and subjected to a range of morphological, phylogenetic and chemotaxonomic analyses. The strain was Gram-reaction-positive with well-developed aerial mycelia, which fragmented into rod-shaped spores that had unique knobby protrusions on the spore surface. Substrate mycelia were not present in the media used. Strain EUM 374(T) grew as a film on the surface of static liquid culture medium but did not grow under shaking conditions. Phylogenetic evaluation based on 16S rRNA gene sequences identified the new isolate as belonging to the family Pseudonocardiaceae with sequence similarities of 96.1 and 96.3 % to Pseudonocardia acaciae GMKU095(T) and Pseudonocardia spinosispora LM 141(T), respectively, and 93-96 % sequence similarity to other members of the genus Pseudonocardia. The results of comprehensive phylogenetic analyses, including physiological and biochemical tests, differentiated strain EUM 374(T) from related members of the genus Pseudonocardia. Based on the phenotypic, phylogenetic and chemotaxonomic evidence, strain EUM 374(T) represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia eucalypti sp. nov. is proposed. The type strain is EUM 374(T) ( = DSM 45351(T)  = ACM 5285(T)).

Flindersiella endophytica gen. nov., sp. nov., an endophytic actinobacterium isolated from the root of Grey Box, an endemic eucalyptus tree.

A novel endophytic actinobacterium, designated strain EUM 378(T), was isolated from the surface-sterilized root tissue of Eucalyptus microcarpa, a eucalyptus tree known as Grey Box. Phylogenetic evaluation based on 16S rRNA gene sequence analysis, including alignment with taxon-specific 16S rRNA gene signature nucleotides, placed this isolate as a member of the family Nocardioidaceae. Strain EUM 378(T) showed >5.5 % 16S rRNA gene sequence divergence from other members of this family and was related most closely to Actinopolymorpha alba YIM 48868(T) (94.2 %) and Actinopolymorpha singaporensis IM 7744(T) (94.4 %). This Gram-positive, aerobic actinobacterium has well-developed substrate mycelia that fragment into small rods. Chemotaxonomic data revealed that the cell wall contains LL-diaminopimelic acid, ribose, glucose and rhamnose. MK-10(H6) is the predominant menaquinone. Chemotaxonomic and phylogenetic evidence confirmed that strain EUM 378(T) represents a novel species of a new genus, for which the name Flindersiella endophytica gen. nov., sp. nov. is proposed. The type strain is EUM 378(T) ( = DSM 45355(T) = ACM 5289(T)).

Actinopolymorpha pittospori sp. nov., an endophyte isolated from surface-sterilized leaves of an apricot tree (Pittosporum phylliraeoides).

A member of the genus Actinopolymorpha, designated PIP 143(T), was isolated from the leaves of an Australian native apricot tree (Pittosporum phylliraeoides). The isolate was a Gram-reaction-positive, aerobic actinobacterium, with a well-developed substrate mycelium that fragmented into small rods. Phylogenetic evaluation based on 16S rRNA gene sequences placed the isolate in the family Nocardioidaceae. Strain PIP 143(T) was most closely related to Actinopolymorpha cephalotaxi I06-2230(T) (98.7 %) and Actinopolymorpha rutila YIM 45725(T) (98.1 %). Chemotaxonomic data, including cell-wall components, menaquinones and fatty acids, confirmed the affiliation of strain PIP 143(T) to the genus Actinopolymorpha. Phylogenetic analysis and physiological and biochemical studies, in combination with DNA-DNA hybridization studies, allowed the differentiation of strain PIP 143(T) from its closest phylogenetic neighbours with validly published names. Therefore, a novel species is proposed, with the name Actinopolymorpha pittospori sp. nov. The type strain is PIP 143(T) ( = DSM 45354(T) = ACM 5288(T) = NRRL B-24810(T)).

Intracellular Bacteria in Plants: Elucidation of Abundant and Diverse Cytoplasmic Bacteria in Healthy Plant Cells Using In Vitro Cell and Callus Cultures.

This study was initiated to assess whether the supposedly axenic plant cell cultures harbored any cultivation-recalcitrant endophytic bacteria (CREB). Adopting live-cell imaging with bright-field, fluorescent and confocal microscopy and bacterial 16S-rRNA gene taxonomic profiling, we report the cytoplasmic association of abundant and diverse CREBs in long-term actively maintained callus and cell suspension cultures of different plant species. Preliminary bright-field live-cell imaging on grape cell cultures showed abundant intracellular motile micro-particles resembling bacteria, which proved uncultivable on enriched media. Bacterial probing employing DNA stains, transmission electron microscopy, and Eubacterial FISH indicated abundant and diverse cytoplasmic bacteria. Observations on long-term maintained/freshly established callus stocks of different plant species-grapevine, barley, tobacco, Arabidopsis, and medicinal species-indicated intracellular bacteria as a common phenomenon apparently originating from field shoot tissues.Cultivation-independent 16S rRNA gene V3/V3-V4 amplicon profiling on 40-year-old grape cell/callus tissues revealed a high bacterial diversity (>250 genera), predominantly Proteobacteria, succeeded by Firmicutes, Actinobacteria, Bacteriodetes, Planctomycetes, and 20 other phyla, including several candidate phyla. PICRUSt analysis revealed diverse functional roles for the bacterial microbiome, majorly metabolic pathways. Thus, we unearth the widespread association of cultivation-recalcitrant intracellular bacteria "Cytobacts" inhabiting healthy plant cells, sharing a dynamic mutualistic association with cell hosts.