Auxin production and plant growth promotion by Microbacterium albopurpureum sp. nov. from the rhizoplane of leafless Chiloschista parishii Seidenf. orchid.

The strains of the genus Microbacterium, with more than 150 species, inhabit diverse environments; plant-associated bacteria reveal their plant growth-promoting activities due to a number of beneficial characteristics. Through the performance of diverse techniques and methods, including isolation of a novel Microbacterium strain from the aerial roots of leafless epiphytic orchid, Chiloschista parishii Seidenf., its morphological and biochemical characterization, chemotaxonomy, phylogenetic and genome analysis, as well as bioassays and estimation of its auxin production capacity, a novel strain of ET2T is described. Despite that it shared 16S rRNA gene sequence similarity of 99.79% with Microbacterium kunmingense JXJ CY 27-2T, so they formed a monophyletic group on phylogenetic trees, the two strains showed clear divergence of their genome sequences. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values of ET2T differed greatly from phylogenetically close JXJ CY 27-2T. Based on the differences being below the threshold for species similarity, together with the unique chemotaxonomic characteristics, strain ET2T represents a novel species of the genus Microbacterium. Several genes, putatively involved in auxin biosynthesis were predicted. This strain revealed obvious plant growth-promoting activities, including diazotrophy and biosynthesis of tryptophan-dependent auxins (indole-3-acetic and indole-3-pyruvic acids). Microbial auxins directly stimulated the rhizogenesis, so that the ET2T-inoculated seeds of wheat, cucumber and garden cress showed evident promotion in their growth and development, both under optimal and under cold stress conditions. Based on phenotypic, chemotypic and genotypic evidences, the strain ET2T belongs to the genus Microbacterium, order Micrococcales, class Actinomycetes, and it represents a novel species, for which the name Microbacterium albopurpureum sp. nov. is proposed, with strain ET2T (VKPM Ac-2212, VKM Ас-2998) as the type strain.

Complete genome sequence of new Microbacterium sp. strain ET2, isolated from roots of leafless orchid.

Whole-genome sequence of ET2 strain, isolated from the roots of leafless orchid, constitutes a single circular chromosome of 3,604,840 bp (69.44% G + C content). BLAST+-based average nucleotide identity (ANIb) and digital DNA-DNA hybridization values indicate that ET2 may be a novel Microbacterium species. Genes putatively involved in plant-microbial interactions were predicted.

Cyanobacterial Root Associations of Leafless Epiphytic Orchids.

The leafless orchids are rare epiphytic plants with extremely reduced leaves, and their aerial roots adopted for photosynthesis. The beneficial plant-microbial interactions contribute significantly to host nutrition, fitness, and growth. However, there are no data available on the bacterial associations, inhabiting leafless orchids. Here, we describe the diversity of cyanobacteria, which colonize the roots of greenhouse Microcoelia moreauae and Chiloschista parishii. The biodiversity and structure of the cyanobacterial community were analyzed using a complex approach, comprising traditional cultivable techniques, denaturing gradient gel electrophoresis (DGGE), and phylogenetic analysis, as well as the light and scanning electron microscopy (SEM). A wide diversity of associated bacteria colonize the root surface, forming massive biofilms on the aerial roots. The dominant populations of filamentous nitrogen-fixing cyanobacteria belonged to the orders Oscillatoriales, Synechococcales, and Nostocales. The composition of the cyanobacterial community varied, depending on the nitrogen supply. Two major groups prevailed under nitrogen-limiting conditions, belonging to Leptolyngbya sp. and Komarekiella sp. The latter was characterized by DGGE profiling and sequencing, as well as by its distinctive features of morphological plasticity. The leading role of these phototrophophic and diazotrophic cyanobacteria is discussed in terms of the epiphytic lifestyle of the leafless orchids.

Megasporogenesis, megagametogenesis, and embryogenesis in Dendrobium nobile (Orchidaceae).

The orchid reproductive strategy, including the formation of numerous tiny seeds, is achieved by the elimination of some stages in the early plant embryogenesis. In this study, we documented in detail the formation of the maternal tissues (the nucellus and integuments), the structures of female gametophyte (megaspores, chalazal nuclei, synergids, polar nuclei), and embryonic structures in Dendrobium nobile. The ovary is unilocular, and the ovule primordia are formed in the placenta before the pollination. The ovule is medionucellate: the two-cell postament and two rows of nucellar cells persist until the death of the inner integument. A monosporic eight-nucleated embryo sac is developed. After the fertilization, the most common central cell nucleus consisted of two joined but not fused polar nuclei. The embryogenesis of D. nobile is similar to the Caryophyllad-type, and it is characterized by the formation of all embryo cells from the apical cell (ca) of a two-celled proembryo. The only exception is that there is no formation of the radicle and/or cotyledons. The basal cell (cb) does not divide during the embryogenesis, gradually transforming into the uninuclear suspensor. Then the suspensor goes through three main stages: it starts with an unbranched cell within the embryo sac, followed by a branched stage growing into the integuments, and it ends with the cell death. The stage-specific development of the female gametophyte and embryo of D. nobile is discussed.

Comparative "Omics" of the Fusarium fujikuroi Species Complex Highlights Differences in Genetic Potential and Metabolite Synthesis.

Species of the Fusarium fujikuroi species complex (FFC) cause a wide spectrum of often devastating diseases on diverse agricultural crops, including coffee, fig, mango, maize, rice, and sugarcane. Although species within the FFC are difficult to distinguish by morphology, and their genes often share 90% sequence similarity, they can differ in host plant specificity and life style. FFC species can also produce structurally diverse secondary metabolites (SMs), including the mycotoxins fumonisins, fusarins, fusaric acid, and beauvericin, and the phytohormones gibberellins, auxins, and cytokinins. The spectrum of SMs produced can differ among closely related species, suggesting that SMs might be determinants of host specificity. To date, genomes of only a limited number of FFC species have been sequenced. Here, we provide draft genome sequences of three more members of the FFC: a single isolate of F. mangiferae, the cause of mango malformation, and two isolates of F. proliferatum, one a pathogen of maize and the other an orchid endophyte. We compared these genomes to publicly available genome sequences of three other FFC species. The comparisons revealed species-specific and isolate-specific differences in the composition and expression (in vitro and in planta) of genes involved in SM production including those for phytohormome biosynthesis. Such differences have the potential to impact host specificity and, as in the case of F. proliferatum, the pathogenic versus endophytic life style.

The biotransformation of brewer's spent grain into biogas by anaerobic microbial communities.

The present study reports on the biotransformation of the brewer's spent grain (BSG) in co-digestion with Jerusalem artichoke (JA, Helianthus tuberosus L.) phytomass by thermophilic (+55 °C) and mesophilic (+30 °C) anaerobic methanogenic communities. BSG is a by-product of the beer-brewing process generated in large amounts, in which utilization provokes a negative effect on the environment. In this study, we will show an effective conversion of BSG into biogas by selected microbial communities, obtained from different sources (animal manure and previously isolated microbial consortia). The stimulation of methanogenesis was reached by the co-digestion of JA's phytomass (stem and leaves). The optimized conditions for microbial stable cultivation included the use of nutrient medium, containing yeast extract and trace element solution. The optimal BSG concentration in biogas production was 50 and 100 g L(-1). Under thermophilic conditions, the maximum total methane production reached 64%, and it comprised around 6-8 and 9-11 of L CH4 per 100 g of fermented BSG without and with co-digested JA, respectively, when the fresh inoculum was added. Although, after a year of re-cultivation, the values reduced to around 6-7, and 6-10 L CH4/100 g BSG, correspondingly, the selected microbial communities showed effective biotransformation of BSG. The supplementation of soil with the residual fermented BSG (10%, w/w) resulted in the promotion of lettuce (Lepidium sativum L.) growth. The results obtained demonstrate a potential for complete BSG utilization via biogas production and application as a soil additive.

Asymbiotic in vitro seed propagation of Dendrobium.

The ability to germinate orchids from seeds in vitro presents a useful and viable method for the propagation of valuable germplasm, maintaining the genetic heterogeneity inherent in seeds. Given the ornamental and medicinal importance of many species within the genus Dendrobium, this review explores in vitro techniques for their asymbiotic seed germination. The influence of abiotic factors (such as temperature and light), methods of sterilization, composition of basal media, and supplementation with organic additives and plant growth regulators are discussed in context to achieve successful seed germination, protocorm formation, and further seedling growth and development. This review provides both a basis for the selection of optimal conditions, and a platform for the discovery of better ones, that would allow the development of new protocols and the exploration of new hypotheses for germination and conservation of Dendrobium seeds and seedlings.

Symbiotic in vitro seed propagation of Dendrobium: fungal and bacterial partners and their influence on plant growth and development.

The genus Dendrobium is one of the largest genera of the Orchidaceae Juss. family, although some of its members are the most threatened today. The reason why many species face a vulnerable or endangered status is primarily because of anthropogenic interference in natural habitats and commercial overexploitation. The development and application of modern techniques and strategies directed towards in vitro propagation of orchids not only increases their number but also provides a viable means to conserve plants in an artificial environment, both in vitro and ex vitro, thus providing material for reintroduction. Dendrobium seed germination and propagation are challenging processes in vivo and in vitro, especially when the extreme specialization of these plants is considered: (1) their biotic relationships with pollinators and mycorrhizae; (2) adaptation to epiphytic or lithophytic life-styles; (3) fine-scale requirements for an optimal combination of nutrients, light, temperature, and pH. This review also aims to summarize the available data on symbiotic in vitro Dendrobium seed germination. The influence of abiotic factors as well as composition and amounts of different exogenous nutrient substances is examined. With a view to better understanding how to optimize and control in vitro symbiotic associations, a part of the review describes the strong biotic relations of Dendrobium with different associative microorganisms that form microbial communities with adult plants, and also influence symbiotic seed germination. The beneficial role of plant growth-promoting bacteria is also discussed.

Identification and functional characterization of indole-3-acetamide-mediated IAA biosynthesis in plant-associated Fusarium species.

The plant hormone indole-3-acetic acid (IAA) can be synthesized from tryptophan via the intermediate indole-3-acetamide (IAM). The two genes, IaaM (encoding tryptophan monooxygenase) and IaaH (encoding indole-3-acetamide hydrolase) that constitute the IAM pathway have been described in plant-associated bacteria. We have identified putative homologs of the bacterial IaaM and IaaH genes in four Fusarium species -Fusarium proliferatum, Fusarium verticillioides, Fusarium fujikuroi, and Fusarium oxysporum. In all four species the two genes are organized next to each other in a head to head orientation and are separated by a short non-coding region. However, the pathway is fully functional only in the orchid endophytic strain F. proliferatum ET1, which produces significant amounts of IAM and IAA. Minor amounts of IAM are produced by the corn pathogen F. verticillioides strain 149, while in the two other species, the rice pathogen F. fujikuroi strain m567 and the tomato pathogen F. oxysporum f. sp. lycopersici strain 42-87 the IAM pathway is inactive. Deletion of the entire gene locus in F. proliferatum ET1 resulted in drastic reduction of IAA production. Conversely, transgenic strains of F. fujikuroi over-expressing the F. proliferatum IAM genes produced elevated levels of both IAM and IAA. Analysis of the intergenic promoter region in F. proliferatum showed that transcriptional activation in direction of the IaaH gene is about 3-fold stronger than in direction of the IaaM gene. The regulation of the IAM genes and the limiting factors of IAA production via the IAM pathway are discussed.

Production of gibberellic acids by an orchid-associated Fusarium proliferatum strain.

The rice pathogen Fusarium fujikuroi is well known for its ability to produce the plant hormones gibberellins (GAs). However, the majority of closely related Fusarium species is unable to produce GAs although the GA gene cluster is present in their genomes. In this study, we analyzed five orchid-associated Fusarium isolates for their capacity to produce GAs. Four of them did not produce any GAs and were shown not to contain any GA biosynthetic genes. However, the fifth isolate, which has been identified as F. proliferatum based on five molecular markers, produced significant amounts of GAs in contrast to previously characterized F. proliferatum strains. We focused on the molecular characterization of two GA-specific genes, ggs2 and cps/ks, both inactive in F. proliferatum strain D-02945. Complementation of a F. fujikuroi Deltaggs2 mutant with the ET1 ggs2 gene fully restored GA biosynthesis, confirming that the orchid-associated isolate contains an active gene copy. A possible correlation between GA production and their role in plant-fungal interactions is discussed.

Orchid-associated bacteria produce indole-3-acetic acid, promote seed germination, and increase their microbial yield in response to exogenous auxin.

Germination of orchid seeds is a complex process. In this paper we focus on interactions between the host-plant and its bacterial partners via indole-3-acetic acid (IAA). Originally isolated from the roots of the epiphytic orchid Dendrobium moschatum, the strains of Rhizobium, Microbacterium, Sphingomonas, and Mycobacterium genera were among the most active IAA producers. Addition of exogenous tryptophan significantly enhanced auxin formation both in mineral and complex media. The presence of IAA and indole-3-acetaldehyde was confirmed by HPLC. Indole-3-pyruvic and indole-3-lactic acids were also detected in supernatants of culture filtrates of Sphingomonas sp., Rhizobium sp., and Microbacterium sp., while indole-3-acetamide was identified only in Mycobacterium sp. Some concentration- and strain-dependent effects of exogenous IAA on bacterial development were also established. Treatment of the cultures with 10 and 100 microg/ml of auxin resulted in an increase in microbial yield. None of the investigated strains was able to utilize IAA as a source of carbon and energy. Furthermore, inoculation of D. moschatum seeds with Sphingomonas sp. and Mycobacterium sp. resulted in considerable enhancement of orchid seeds germination. This growth-promoting activity was observed in the absence of any plant growth stimulators or mycorrhizal fungi, usually required for orchid germination.

Bacteria associated with orchid roots and microbial production of auxin.

Associative bacteria of terrestrial (Paphiopedilum appletonianum) and epiphytic (Pholidota articulata) tropical orchids were investigated. Microbial community of epiphytic plant differed from that of the terrestrial one. Streptomyces, Bacillus, Pseudomonas, Burkholderia, Erwinia and Nocardia strains populated Paphiopedilum roots, whereas Pseudomonas, Flavobacterium, Stenotrophomonas, Pantoea, Chryseobacterium, Bacillus, Agrobacterium, Erwinia, Burkholderia and Paracoccus strains colonized Pholidota roots. Endophytic bacteria populations were represented with less diversity: Streptomyces, Bacillus, Erwinia and Pseudomonas genera were isolated from P. appletonianum, and Pseudomonas, Bacillus, and Flavobacterium genera were isolated from Ph. articulata. Microorganisms produced indole-3-acetic acid (IAA). Variations in its biosynthesis among the strains of the same genus were also observed. The highest auxin level was detected during the stationary growth phase. Biological activity of microbial IAA was proved by treatment of kidney bean cuttings with bacterial supernatants, revealing considerable stimulation of root formation and growth.