A new genus, Planticonsortium (Mucoromycotina), and new combination (P. tenue), for the fine root endophyte, Glomus tenue (basionym Rhizophagus tenuis).

In 1977, the fine root endophyte, originally named Rhizophagus tenuis, was transferred into the genus Glomus as G. tenue, thus positioning the species with all other known arbuscular mycorrhizal fungi (Glomeromycota, Glomeromycotina). Recent molecular evidence, however, places it in a different subphylum, Mucoromycotina in the Mucoromycota. No suitable genus exists in the Mucoromycotina to accommodate G. tenue, so it is moved to Planticonsortium gen. nov. as P. tenue comb. nov.

Defining the pathways of symbiotic Epichloë colonization in grass embryos with confocal microscopy.

Asexual cool-season grass endophytes of the genus Epichloë (Ascomycota: Clavicipitaceae) are strictly vertically disseminated. The hosts of these mutualistic fungi express no symptoms during the fungal lifecycle that takes place entirely within the plant, while their hosts receive beneficial outcomes. These fungi are distributed in two major locations within the mature seeds of their hosts; namely, within the embryo (including the scutellum, coleoptile, plumule, radicle, and coleorhiza tissues) and between the aleurone and pericarp layers, with the latter hyphae playing no role in transmission of the fungus to the next plant generation. Conflicting evidence remains in the literature on the timing of embryo colonization. In a detailed investigation, utilizing confocal microscopy to observe the distribution of Epichloë coenophiala strain AR601 in tall fescue (Lolium arundinaceum), we tracked endophyte hyphal colonization in the ovary (pre-fertilization) through to the fully mature seed stage. Confocal microscopy images revealed that at the early and mature developmental stages of the embryo sac, before host grass fertilization, there were large quantities of endophyte mycelium present, especially around the antipodal cells, indicating that this endophyte enters the embryo sac before the fertilization stage. After host fertilization, fungal hyphae could be seen in the true embryo and early nonstarchy endosperm. Understanding the mechanisms of transmission to the seed is important for commercial seed producers and end users.

Isolation, characterization, and evaluation of bacterial root and nodule endophytes from chickpea cultivated in Northern India.

Endophytic bacteria from roots (12 isolates) and nodules (76 isolates) of chickpea legume grown under CCS Haryana Agricultural University farm were isolated. Among the endophytic bacteria, 50% from roots and 93.4% from nodules were Gram positive spore formers. Large number of endophytes from roots and nodules solubilized phosphate and produced ammonia. Isolate CRE3, and CNE215, were most efficient P solubilizers and. CRE12 and CNE76 being most efficient ammonia producer. Interestingly, few endophytic bacteria produced organic acid. Further selected 62 isolates were used to determine molecular diversity by RFLP of PCR amplified 16S rDNA. Endophytes from roots formed five separate clusters and nodule endophytes formed 13 clusters. Isolate CNE215 from nodules and CRE1 from roots possessed multiple beneficial traits and belonged two different clusters. These two isolates were identified after amplification and sequencing of 16S rRNA gene. Isolate CNE215 showed more than 98% similarity with partial sequence of 16S rRNA gene of Bacillus subtilis, whereas CRE1 showed more than 98% similarity with Bacillus licheniformis. Efficacy of these two strains was evaluated under field conditions and an increase up to 22.5% in grain yield over uninoculated control was observed with B. subtilis strain CNE215, whereas all the recommended biofertilizers were able to record an increase upto 14.4%.

Holoparasitic Rafflesiaceae possess the most reduced endophytes and yet give rise to the world's largest flowers.

BACKGROUND AND AIMS: Species in the holoparasitic plant family Rafflesiaceae exhibit one of the most highly modified vegetative bodies in flowering plants. Apart from the flower shoot and associated bracts, the parasite is a mycelium-like endophyte living inside their grapevine hosts. This study provides a comprehensive treatment of the endophytic vegetative body for all three genera of Rafflesiaceae (Rafflesia, Rhizanthes and Sapria), and reports on the cytology and development of the endophyte, including its structural connection to the host, shedding light on the poorly understood nature of this symbiosis. METHODS: Serial sectioning and staining with non-specific dyes, periodic-Schiff's reagent and aniline blue were employed in order to characterize the structure of the endophyte across a phylogenetically diverse sampling. KEY RESULTS: A previously identified difference in the nuclear size between Rafflesiaceae endophytes and their hosts was used to investigate the morphology and development of the endophytic body. The endophytes generally comprise uniseriate filaments oriented radially within the host root. The emergence of the parasite from the host during floral development is arrested in some cases by an apparent host response, but otherwise vegetative growth does not appear to elicit suppression by the host. CONCLUSIONS: Rafflesiaceae produce greatly reduced and modified vegetative bodies even when compared with the other holoparasitic angiosperms once grouped with Rafflesiaceae, which possess some vegetative differentiation. Based on previous studies of seeds together with these findings, it is concluded that the endophyte probably develops directly from a proembryo, and not from an embryo proper. Similarly, the flowering shoot arises directly from the undifferentiated endophyte. These filaments produce a protocorm in which a shoot apex originates endogenously by formation of a secondary morphological surface. This degree of modification to the vegetative body is exceptional within angiosperms and warrants additional investigation. Furthermore, the study highlights a mechanical isolation mechanism by which the host may defend itself from the parasite.

New species and records of Trichoderma isolated as mycoparasites and endophytes from cultivated and wild coffee in Africa.

A survey for species of the genus Trichoderma occurring as endophytes of Coffea, and as mycoparasites of coffee rusts (Hemileia), was undertaken in Africa; concentrating on Cameroon and Ethiopia. Ninety-four isolates of Trichoderma were obtained during this study: 76 as endophytes of healthy leaves, stems and berries and, 18 directly from colonized rust pustules. A phylogenetic analysis of all isolates used a combination of three genes: translation elongation factor-1α (tef1), rpb2 and cal for selected isolates. GCPSR criteria were used for the recognition of species; supported by morphological and cultural characters. The results reveal a previously unrecorded diversity of Trichoderma species endophytic in both wild and cultivated Coffea, and mycoparasitic on Hemileia rusts. Sixteen species were delimited, including four novel taxa which are described herein: T. botryosum, T. caeruloviride, T. lentissimum and T. pseudopyramidale. Two of these new species, T. botryosum and T. pseudopyramidale, constituted over 60% of the total isolations, predominantly from wild C. arabica in Ethiopian cloud forest. In sharp contrast, not a single isolate of Trichoderma was obtained using the same isolation protocol during a survey of coffee in four Brazilian states, suggesting the existence of a 'Trichoderma void' in the endophyte mycobiota of coffee outside of Africa. The potential use of these African Trichoderma isolates in classical biological control, either as endophytic bodyguards-to protect coffee plants from Hemileia vastatrix, the fungus causing coffee leaf rust (CLR)-or to reduce its impact through mycoparasitism, is discussed, with reference to the on-going CLR crisis in Central America.

An endophyte of Macrochloa tenacissima (esparto or needle grass) from Tunisia is a novel species in the Fusarium redolens species complex.

Here, we report on the morphological, molecular, and chemical characterization of a novel Fusarium species recovered from the roots and rhizosphere of Macrochloa tenacissima (halfa, esparto, or needle grass) in central Tunisia. Formally described here as F. spartum, this species is a member of the Fusarium redolens species complex but differs from the other two species within the complex, F. redolens and F. hostae, by its endophytic association with M. tenacissima and its genealogical exclusivity based on multilocus phylogenetic analyses. To assess their sexual reproductive mode, a polymerase chain reaction (PCR) assay was designed and used to screen the three strains of F. spartum, 51 of F. redolens, and 14 of F. hostae for mating type (MAT) idiomorph. Genetic architecture of the MAT locus in the former two species suggests that if they reproduce sexually, it is via obligate outcrossing. By comparison, results of the PCR assay indicated that 13/14 of the F. hostae strains possessed MAT1-1 and MAT1-2 idiomorphs and thus might be self-fertile or homothallic. However, when the F. hostae strains were selfed, 11 failed to produce perithecia and one only produced several small abortive perithecia. Cirrhi with ascospores, however, were only produced by 8/28 and 4/84 of the variable size perithecia, respectively, of F. hostae NRRL 29888 and 29890. The potential for the three F. redolens clade species to produce mycotoxins, pigments, and phytohormones was assessed by screening whole genome sequence data and by analyzing extracts on cracked maize kernel cultures via liquid chromatography-mass spectrometry.

Evaluating antibacterial and anticancer activity of crude extracts of bacterial endophytes from Crinum macowanii Baker bulbs.

The results from this study revealed that crude extracts isolated from bacterial endophytes obtained from Crinum macowanii bulbs showed activity against both Gram-positive and Gram-negative pathogenic bacteria, while Acinetobacter guillouiae crude extracts displayed anticancer activity. This study aimed to isolate and characterize bacterial endophytes and their crude extracts from C. macowanii bulbs. Endophytes were isolated using validated surface sterilization techniques, followed by phenotypic and genotypic profiles of the isolates. Crude extracts were extracted from the endophytes using ethyl acetate, while methanol:dichloromethane (1:1) was used to obtain crude extracts from the bulbs. Antibacterial activity of crude extract from each endophyte was investigated against selected pathogenic strains using the broth microdilution method, and anticancer activity against U87MG glioblastoma and A549 lung carcinoma cells was determined by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Acinetobacter guillouiae, Pseudomonas moraviensis, Pseudomonas sp., Rahnella aquatilis, Bacillus cereus, Novosphingobium sp., Raoultella ornithinolytica, and Burkholderia tropica were successfully isolated. The crude extracts from the majority of endophytes showed antibacterial activity, ranging from 0.125 to >16.00 mg/ml against Gram-negative and Gram-positive pathogenic bacteria. Acinetobacter guillouiae extracts showed a high bioactive potential against U87MG glioblastoma cell lines by reducing their growth by 50% at concentrations of 12.5, 6.25, and 3.13 µg/ml. Crude extracts isolated from C. macowanii bulbs showed potential for possible drug lead against common pathogenic bacteria.

A comparative analysis of Lophodermium fissuratum, sp. nov., found in haploxylon pine needles in the Pacific Northwest, and other Lophodermium endophytes.

Lophodermium is a large fungal genus consisting of over 100 named species, with ca. 38 of these commonly found as endophytes of pine needles. In this study, we use both morphological and sequencing data to describe a new Lophodermium species associated with haploxylon pines from the Pacific Northwest. This new species resembled the morphology of L. nitens, another commonly occurring species from the same geographic regions and host species. They both present dark subcuticular ascocarps without lips. However, the upper walls of their ascocarps are different, as the new species forms an inward V-shaped folding, not present in L. nitens. Phylogenies using nuc rDNA internal transcribed spacer barcodes (ITS1-5.8S-ITS2 = ITS), partial D1-D2 domains of nuc rDNA 28S, and partial sequences of the nuc actin gene confirmed that this species represents a unique lineage not closely related to L. nitens. We discuss the current state of the phylogeny in light of all currently available sequences from pine-associated Lophodermium species.

Fungal root symbionts of high-altitude vascular plants in the Himalayas.

Arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) form symbiotic relationships with plants influencing their productivity, diversity and ecosystem functions. Only a few studies on these fungi, however, have been conducted in extreme elevations and none over 5500 m a.s.l., although vascular plants occur up to 6150 m a.s.l. in the Himalayas. We quantified AMF and DSE in roots of 62 plant species from contrasting habitats along an elevational gradient (3400-6150 m) in the Himalayas using a combination of optical microscopy and next generation sequencing. We linked AMF and DSE communities with host plant evolutionary history, ecological preferences (elevation and habitat type) and functional traits. We detected AMF in elevations up to 5800 m, indicating it is more constrained by extreme conditions than the host plants, which ascend up to 6150 m. In contrast, DSE were found across the entire gradient up to 6150 m. AMF diversity was unimodally related to elevation and positively related to the intensity of AMF colonization. Mid-elevation steppe and alpine plants hosted more diverse AMF communities than plants from deserts and the subnival zone. Our results bring novel insights to the abiotic and biotic filters structuring AMF and DSE communities in the Himalayas.

Epichloë festucae endophytic growth in florets, seeds, and seedlings of perennial ryegrass (Lolium perenne).

Many symbiotic Epichloë species are seed-transmitted in their grass hosts. For a detailed investigation of Epichloë festucae colonization throughout the life cycle of its host, the authors transformed strain Fl1 with a fungal-active gene for enhanced cyan-fluorescent protein (eCFP), introduced it into perennial ryegrass (Lolium perenne), and used confocal microscopy to track its growth in the shoot apex, floral primordium, floral organs, seeds, and seedlings. Hyphae intercellularly colonized leaf sheaths, blades, true stems, and leaf primordia, and among floral primordia the endophyte exhibited different levels of colonization. In preanthesis florets, E. festucae colonized the pistil and stamen, but not pollen grains, and ramified throughout the ovule nucellus, but not the integument or embryo sac. Generally, only a single hypha was observed extended from the ovary placenta into the ovule. Within 4 d after anthesis, fungal hyphae had ramified throughout the developing seed and embryo. As the embryo matured, fungal hyphae became abundant between the testa and aleurone layer, and around the shoot apex and radical of the embryonic axis. During germination, hyphae accumulated in the mesocotyl and invaded the newly formed shoot apex near the meristem. In this host-fungus symbiosis, transmission to seedlings averaged 41% in 2010 and 76% in 2011. Each year, the frequency of ovary infection was similar to the frequency of infecting embryos and seedlings, indicating that colonization of the ovary and embryo was required for seed transmission.

Root endophytic fungal communities associated with pitch pine, switchgrass, and rosette grass in the pine barrens ecosystem.

Almost all plants in nature harbour fungi in their roots but the knowledge on distribution and the underlying principles of assemblage is still poorly developed for the root-associated fungi. In this study we analysed the root endophytic fungal communities associated with switchgrass, rosette grass, and pitch pine in the acidic, oligotrophic pine barrens ecosystem. A total of 434 fungal isolates were obtained from 600 root segments of 60 plant samples. DNA barcoding and morphological analyses identified 92 fungal species, which belong to 39 genera in six classes. Compared to other ecosystems, the pine barrens has a higher proportion of Leotiomycetes. The fungal community associated with pitch pine was significantly different from those associated with the grasses, while less difference was found between those associated with the two grasses. Our results suggest that edaphic factors and host specificity play a role in shaping root endophytic fungal community. This study also corroborates our previous finding that plant roots in the pine barrens are a rich reservoir of novel fungi.

Brevicollum, a new genus in Neohendersoniaceae, Pleosporales.

A new genus, Brevicollum, is established for two new species, B. hyalosporum on Syzygium samarangense and B. versicolor on Volkameria inermis, in Japan. This genus is characterized by the presence of immersed ascomata with an excentric to central, short cylindrical ostiolar neck, thin ascomatal wall, clavate asci with a shallow ocular chamber, and broadly fusiform, hyaline to pale brown ascospores with a sheath. Molecular phylogenetic analyses based on a combined data set of sequences of 18S and 28S nuc rDNA genes and the second largest subunit of RNA polymerase II (RPB2) revealed that Brevicollum is a member of Neohendersoniaceae (Pleosporales, Dothideomycetes). The genera Brevicollum, Crassiparies, Medicopsis, and Neohendersonia are accepted in Neohendersoniaceae. Revised descriptions of Crassiparies and its type species, C. quadrisporus, are provided based on the holotype and a newly obtained specimen and isolate. An asexual morph of C. quadrisporus is reported for the first time. The nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) phylogenetic tree of the newly collected species (B. hyalosporum, B. versicolor, and C. quadrisporus), and the unnamed or provisionally identified endophytic species, suggests a broader distribution of Brevicollum and Crassiparies species.

A consortium of non-rhizobial endophytic microbes from Typha angustifolia functions as probiotic in rice and improves nitrogen metabolism.

Endophytic microbes isolated from plants growing in nutrient-deficient environments often possess properties that improve nutrition of agriculturally important plants. A consortium of non-rhizobial endophytic microbes isolated from a macrophyte Typha angustifolia growing in the marginal wetlands associated with a Uranium mine was characterized for their beneficial effect on rice and the mechanisms of growth promotion were investigated. The microbes were identified and characterized for their potential plant growth promoting (PGP) properties. Effect of these microbes on nitrogen (N)-metabolism of rice was tested as Typha endophytes were predominantly (N)-fixing. Relative N-use efficiency and expression of genes involved in N-uptake and assimilation were investigated in treated plants. Evidence of horizontal gene transfer (HGT) of dinitrogen reductase gene was observed within the consortium from a Pseudomonas stutzeri strain. The consortium behaved as plant probiotic and showed substantial growth benefits to Typha, their natural host as well as to rice. Typha endophytes colonized rice endosphere significantly increasing biomass, shoot length and chlorophyll content in rice plants both under N-sufficient and N-deficient conditions. N-uptake and assimilation genes were upregulated in plants treated with the endophytes even after three weeks post infection. Our results suggested, HGT of nitrogen-fixation trait to be highly prevalent among endophytes isolated from nutrient-poor habitats of the uranium mine. A long-term nitrogen deficiency response in the treated plants was elicited by the consortium improving N-uptake, assimilation and relative N-use efficiency of rice plants. This appeared to be at least one of the main strategies of plant growth promotion.

Fungal endophytic communities on twigs of fast and slow growing Scots pine (Pinus sylvestris L.) in northern Spain.

Most plant species harbour a diverse community of endophytic, but their role is still unknown in most cases, including ecologically and economically important tree species. This study describes the culturable fungal endophytic community of Pinus sylvestris L. twigs in northern Spain and its relationship with diametric growth of the host. In all, 360 twig samples were collected from 30 Scots pines in fifteen stands. Isolates were obtained from all twig samples and 43 fungal taxa were identified by morphogrouping and subsequent ITS rDNA sequencing. All isolates were Ascomycetes, being Dothideomycetes and Sordariomycetes the most abundant classes. Half of the species were host generalists while the others were conifer or pine specialists. We found three new endophytic species for the Pinaceae: Biscogniauxia mediterranea, Phaeomoniella effusa and Plectania milleri, and additional six new species for P. sylvestris: Daldinia fissa, Hypocrea viridescens, Nigrospora oryzae, Ophiostoma nigrocarpum, Penicillium melinii and Penicillium polonicum. The endophytic community of fast and slow growing trees showed differences in species composition, abundance and evenness, but not in diversity. Phoma herbarum was associated to fast growing trees and Hypocrea lixii to those growing slow. Our results support the hypothesis that some endophytic species may affect growth of P. sylvestris.

Azoarcus sp. CIB, an anaerobic biodegrader of aromatic compounds shows an endophytic lifestyle.

BACKGROUND: Endophytic bacteria that have plant growth promoting traits are of great interest in green biotechnology. The previous thought that the Azoarcus genus comprises bacteria that fit into one of two major eco-physiological groups, either free-living anaerobic biodegraders of aromatic compounds or obligate endophytes unable to degrade aromatics under anaerobic conditions, is revisited here. METHODOLOGY/PRINCIPAL FINDINGS: Light, confocal and electron microscopy reveal that Azoarcus sp. CIB, a facultative anaerobe ß-proteobacterium able to degrade aromatic hydrocarbons under anoxic conditions, is also able to colonize the intercellular spaces of the rice roots. In addition, the strain CIB displays plant growth promoting traits such nitrogen fixation, uptake of insoluble phosphorus and production of indoleacetic acid. Therefore, this work demonstrates by the first time that a free-living bacterium able to degrade aromatic compounds under aerobic and anoxic conditions can share also an endophytic lifestyle. The phylogenetic analyses based on the 16S rDNA and nifH genes confirmed that obligate endophytes of the Azoarcus genus and facultative endophytes, such as Azoarcus sp. CIB, locate into different evolutionary branches. CONCLUSIONS/SIGNIFICANCE: This is the first report of a bacterium, Azoarcus sp. CIB, able to degrade anaerobically a significant number of aromatic compounds, some of them of great environmental concern, and to colonize the rice as a facultative endophyte. Thus, Azoarcus sp. CIB becomes a suitable candidate for a more sustainable agricultural practice and phytoremediation technology.

Novel and highly diverse fungal endophytes in soybean revealed by the consortium of two different techniques.

Fungal endophytes were isolated from the leaves of soybean cultivars in Brazil using two different isolation techniques - fragment plating and the innovative dilution-to-extinction culturing - to increase the species richness, frequency of isolates and diversity. A total of 241 morphospecies were obtained corresponding to 62 taxa that were identified by analysis of the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA). The Phylum Ascomycota predominated, representing 99% and 95.2% of isolates in the Monsoy and Conquista cultivars, respectively, whereas the Phylum Basidiomycota represented 1% and 4.8% of isolates, respectively. The genera Ampelomyces, Annulohypoxylon, Guignardia, Leptospora, Magnaporthe, Ophiognomonia, Paraconiothyrium, Phaeosphaeriopsis, Rhodotorula, Sporobolomyces, and Xylaria for the first time were isolated from soybean; this suggests that soybean harbours novel and highly diverse fungi. The yeasts genera Rhodotorula and Sporobolomyces (subphylum Pucciniomycotina) represent the Phylum Basidiomycota. The species richness was greater when both isolation techniques were used. The diversity of fungal endophytes was similar in both cultivars when the same isolation technique was used except for Hill's index, N1. The use of ITS region sequences allowed the isolates to be grouped according to Order, Class and Phylum. Ampelomyces, Chaetomium, and Phoma glomerata are endophytic species that may play potential roles in the biological control of soybean pathogens. This study is one of the first to apply extinction-culturing to isolate fungal endophytes in plant leaves, thus contributing to the development and improvement of this technique for future studies.

An endophytic Nodulisporium sp. from Central America producing volatile organic compounds with both biological and fuel potential.

A Nodulisporium sp. (Hypoxylon sp.) has been isolated as an endophyte of Thelypteris angustifolia (Broadleaf Leaf Maiden Fern) in a rainforest region of Central America. It has been identified both on the basis of its morphological characteristics and by scanning electron microscopy as well as ITS sequence analysis. The endophyte produces volatile organic compounds (VOCs) that have both fuel (mycodiesel) and use for biological control of plant disease. When grown on potato dextrose agar, the organism uniquely produces a series of ketones, including acetone; 2-pentanone; 3-hexanone, 4-methyl; 3-hexanone, 2,4- dimethyl; 2-hexanone, 4-methyl, and 5-hepten, 2-one and these account for about 25% of the total VOCs. The most abundant identified VOC was 1,8 cineole, which is commonly detected in this group of organisms. Other prominent VOCs produced by this endophyte include 1-butanol, 2- methyl, and phenylethanol alcohol. Moreover, of interest was the presence of cyclohexane, propyl, which is a common ingredient of diesel fuel. Furthermore, the VOCs of this isolate of Nodulisporium sp. were selectively active against a number of plant pathogens, and upon a 24 h exposure caused death to Phytophthora palmivora, Rhizoctonia solani, and Sclerotinia sclerotiorum and 100% inhibition to Phytophthora cinnamomi with only slight to no inhibition of the other pathogens that were tested. From this work, it is becoming increasingly apparent that each isolate of this endophytic Nodulisporium spp., including the Daldina sp. and Hypoxylon spp. teleomorphs, seems to produce its own unique set of VOCs.

A polyphasic approach for the characterization of endophytic Alternaria strains isolated from grapevines.

A polyphasic approach was set up and applied to characterize 20 fungal endophytes belonging to the genus Alternaria, recovered from grapevine in different Italian regions. Morphological, microscopical, molecular and chemical investigations were performed and the obtained results were combined in a pooled cluster analysis. Following morphological analyses, all strains were grouped according to their three-dimensional sporulation pattern on PCA and to the colony characteristics on different substrates. After DNA extraction, all strains were analyzed by RAPD-PCR and the resulting profiles were subjected to cluster analysis. The metabolites extracted from the 20 Alternaria endophytes were analyzed by a HPLC and the resulting metabolite profiles were subjected to multivariate statistic analyses. In comparison with reference 'small-spored' Alternaria species, the 20 strains were segregated into two morphological groups: one belonging to the A. arborescens species-group and a second to the A. tenuissima species-group. RAPD analysis also showed that grapevine endophytes belonged to either the A. arborescens or the A. tenuissima species-group and that they were molecularly distinct from strains belonging to A. alternata. Chemotaxonomy gave the same grouping: the grapevine endophytic strains belong to A. arborescens or A. tenuissima species-groups producing known metabolites typical of these species-groups. Interestingly, the 20 grapevine endophytes were able to produce also a number of unknown metabolites, whose characterization could be useful for a more precise segregation of the two species-groups. The results show how complementary morphological, molecular and chemical data can clarify relationships among endophyte species-groups of low morphological divergence.

An endophytic Pseudonocardia species induces the production of artemisinin in Artemisia annua.

Endophytic actinobacteria colonize internal tissues of their host plants and are considered as a rich and reliable source of diverse species and functional microorganisms. In this study, endophytic actinobacterial strain YIM 63111 was isolated from surface-sterilized tissue of the medicinal plant Artemisia annua. We identified strain YIM 63111 as a member of the genus Pseudonocardia. A. annua seedlings grown under both sterile and greenhouse conditions were inoculated with strain YIM 63111. The growth of A. annua seedlings was strongly reduced when YIM 63111 was inoculated at higher concentrations under sterile conditions. However, no growth inhibition was observed when A. annua was grown under greenhouse conditions. Using an enhanced green fluorescent protein (EGFP) expressing YIM 63111 strain, we also observed the endophytic colonization of A. annua seedling using confocal laser-scanning microscopy. The transcription levels of the key genes involved in artemisinin biosynthesis were investigated using real time RT-PCR, revealing that cytochrome P450 monooxygenase (CYP71AV1) and cytochrome P450 oxidoreductase (CPR) expression were up-regulated in A. annua upon inoculation with strain YIM 63111 under certain conditions. The up-regulation of these genes was associated with the increased accumulation of artemisinin. These results suggest that endophytic actinobacteria effectively stimulate certain plant defense responses. Our data also demonstrate the use of Pseudonocardia sp. strain YIM 63111 as a promising means to enhance artemisinin production in plants.

Fungi associated with the southern Eurasian orchid Spiranthes spiralis (L.) Chevall.

The hitherto unknown relationships between the European orchid Spiranthes spiralis (L.) Chevall and its internally associated fungi were explored by a combined approach involving microscopy-based investigations at a morpho-histological level as well as by molecular analyses of the identity of the eukaryotic endophytes present in the root tissue of the plant. We found that this orchid which is currently reported to have a vulnerable status in northern Italy, can host and interact with at least nine types of fungi. Some of these fungi show similarity to mycorrhizal genera found in orchids such as the Ceratobasidium-Rhizoctonia group. Other fungi found are from the genera Davidiella (Ascomycota), Leptosphaeria (Ascomycota), Alternaria (Ascomycota), and Malassezia (Basidiomycota), some of which until have not previously been reported to have an endophytic relationship with plants. The repeated occurrence of often pathogenic fungi such as Fusarium oxysporum, Bionectria ochroleuca, and Alternaria sp., within healthy specimens of this orchid suggests a tempered interaction with species that are sometimes deleterious to non-orchid plants. The fact is reminiscent of the symbiotic compromise established by orchids with fungi of the rhizoctonia group.