Endophyte roles in nutrient acquisition, root system architecture development and oxidative stress tolerance.

Plants associate with communities of microbes (bacteria and fungi) that play critical roles in plant development, nutrient acquisition and oxidative stress tolerance. The major share of plant microbiota is endophytes which inhabit plant tissues and help them in various capacities. In this article, we have reviewed what is presently known with regard to how endophytic microbes interact with plants to modulate root development, branching, root hair formation and their implications in overall plant development. Endophytic microbes link the interactions of plants, rhizospheric microbes and soil to promote nutrient solubilization and further vectoring these nutrients to the plant roots making the soil-plant-microbe continuum. Further, plant roots internalize microbes and oxidatively extract nutrients from microbes in the rhizophagy cycle. The oxidative interactions between endophytes and plants result in the acquisition of nutrients by plants and are also instrumental in oxidative stress tolerance of plants. It is evident that plants actively cultivate microbes internally, on surfaces and in soils to acquire nutrients, modulate development and improve health. Understanding this continuum could be of greater significance in connecting endophytes with the hidden half of the plant that can also be harnessed in applied terms to enhance nutrient acquisition through the development of favourable root system architecture for sustainable production under stress conditions.

Bacillus amyloliquefaciens alters gene expression, ROS production and lignin synthesis in cotton seedling roots.

AIMS: Previous research demonstrated that applying Bacillus amyloliquefaciens to cotton seeds promotes growth, alters root architecture and alleviates salt stress of cotton seedlings. This research was undertaken to further study the genetic responses elicited in cotton seedlings by this growth promoting bacterium. METHODS AND RESULTS: GeneChip microarrays and RT-qPCR were used to detect changes in gene expression in seedling roots inoculated with B. amyloliquefaciens. Roots were stained with 3'3-diaminobenzidine and phloroglucinol-HCl to determine whether treated seedlings had a greater accumulation of reactive oxygen species and lignin. Two hundred and fifty-two transcripts were differentially expressed in inoculated cotton seedling roots; 139 transcripts were up-regulated and 113 were down-regulated. Some up-regulated transcripts were related to nitrate assimilation, cell growth, hormones, transport, transcription factors and antioxidants. Five genes identified to be up-regulated using microarrays were determined to be up-regulated using RT-qPCR. Inoculated cotton seedling roots had a greater accumulation of reactive oxygen species and lignin. CONCLUSIONS: The differential expression of genes associated with diverse functions supports that B. amyloliquefaciens elicits a complex genetic response in seedling roots. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that beneficial bacteria can alter gene expression of cotton that leads to growth promotion.

Induction of abiotic stress tolerance in plants by endophytic microbes.

Endophytes are micro-organisms including bacteria and fungi that survive within healthy plant tissues and promote plant growth under stress. This review focuses on the potential of endophytic microbes that induce abiotic stress tolerance in plants. How endophytes promote plant growth under stressful conditions, like drought and heat, high salinity and poor nutrient availability will be discussed. The molecular mechanisms for increasing stress tolerance in plants by endophytes include induction of plant stress genes as well as biomolecules like reactive oxygen species scavengers. This review may help in the development of biotechnological applications of endophytic microbes in plant growth promotion and crop improvement under abiotic stress conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing human populations demand more crop yield for food security while crop production is adversely affected by abiotic stresses like drought, salinity and high temperature. Development of stress tolerance in plants is a strategy to cope with the negative effects of adverse environmental conditions. Endophytes are well recognized for plant growth promotion and production of natural compounds. The property of endophytes to induce stress tolerance in plants can be applied to increase crop yields. With this review, we intend to promote application of endophytes in biotechnology and genetic engineering for the development of stress-tolerant plants.

Indigenous endophytic seed bacteria promote seedling development and defend against fungal disease in browntop millet (Urochloa ramosa L.).

AIMS: This study was conducted to investigate indigenous seed endophyte effects on browntop millet seedling development. We report that seed-inhabiting bacterial endophytes are responsible for promoting seedling development, including stimulation of root hair formation, increasing root and shoot length growth and increasing photosynthetic pigment content of seedlings. Bacterial endophytes also improved resistance of seedlings to disease. METHODS AND RESULTS: A total of four endophytic bacteria were isolated from surface-sterilized seeds and identified by 16S rDNA sequencing as Curtobacterium sp. (M1), Microbacterium sp. (M2), Methylobacterium sp. (M3) and Bacillus amyloliquefaciens (M4). Removal of bacteria with streptomycin treatment from the seeds compromised seedling growth and development. When endophytes were reinoculated onto seeds, seedlings recovered normal development. Strains M3 and M4 were found to be most potent in promoting growth of seedlings. Bacteria were found to produce auxin, solubilize phosphate and inhibit fungal pathogens. Significant protection of seedlings from Fusarium infection was found using strain M4 in microcosm assays. The antifungal lipopeptide genes for surfactin and iturin were detected in M4; culture extracts of M4 showed a positive drop collapse result for surfactins. CONCLUSIONS: This study demonstrates that browntop millet seeds vector indigenous endophytes that are responsible for modulation of seedling development and protection of seedlings from fungal disease. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is significant and original in that it is the first report of seed-inhabiting endophytes of browntop millet that influence seedling development and function in defence against soilborne pathogens. This study suggests that conservation and management of seed-vectored endophytes may be important in development of more sustainable agricultural practices.

Seed-vectored endophytic bacteria modulate development of rice seedlings.

AIM: The aim of the present study was to evaluate the effects of the removal of indigenous bacteria from rice seeds on seedling growth and development. Here we report the presence of three indigenous endophytic bacteria in rice seeds that play important roles in modulating seedling development (shoot and root lengths, and formation of root hairs and secondary roots) and defence against pathogens. METHODS AND RESULTS: Seed-associated bacteria were removed using surface sterilization with NaOCl (bleach) followed by antibiotic treatment. When bacteria were absent, growth of seedlings in terms of root hair development and overall seedling size was less than that of seedlings that contained bacteria. Reactive oxygen staining of seedlings showed that endophytic bacteria became intracellular in root parenchyma cells and root hairs. Roots containing endophytic bacteria were seen to stain densely for reactive oxygen, while roots free of bacteria stained lightly for reactive oxygen. Bacteria were isolated and identified as Enterobacter asburiae (VWB1), Pantoea dispersa (VWB2) and Pseudomonas putida (VWB3) by 16S rDNA sequencing. Bacteria were found to produce indole acetic acid (auxins), inhibited the pathogen Fusarium oxysporum and solubilized phosphate. Reinoculation of bacteria onto seedlings derived from surface-disinfected rice and Bermuda grass seeds significantly restored seedling growth and development. CONCLUSION: Rice seeds harbour indigenous bacterial endophytes that greatly influence seedling growth and development, including root and shoot lengths, root hair formation and disease susceptibility of rice seedlings. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that seeds of rice naturally harbour bacterial endophytes that play key roles in modulation of seedling development.

Application of bacteria from non-cultivated plants to promote growth, alter root architecture and alleviate salt stress of cotton.

AIMS: Cotton seeds are frequently treated with acid to remove fibres and reduce seed-transmitted diseases. This process also eliminates beneficial bacteria on the seed surface. The goal of this research was to seek and apply beneficial bacteria to acid delinted cotton seeds to evaluate their growth-promoting and salt stress alleviating effects in seedlings. METHODS AND RESULTS: Bacteria were isolated from non-cultivated plants in the Malvaceae. Seeds were collected from Portia tree (Thespesia populnea) and wild cotton (Gossypium hirsutum) from coastal and arid areas of Puerto Rico. Bacillus amyloliquefaciens, Curtobacterium oceanosedimentum and Pseudomonas oryzihabitans were inoculated onto acid delinted cotton seeds. Bacteria increased cotton seed germination and length of emerging seedling radicles. Cotton seeds were inoculated with B. amyloliquefaciens to evaluate growth and root architecture of non-stressed and salt stressed seedlings. Inoculating cotton seeds with B. amyloliquefaciens led to a greater percentage of seedlings with expanded cotyledons after 8 days, enhanced primary and lateral root growth, and altered root architecture. Similar results were obtained when okra seeds were inoculated with B. amyloliquefaciens. CONCLUSION: The data supported the hypothesis that non-cultivated plants in the Malvaceae growing in stressful environments possess bacteria that promote growth, alter root architecture and alleviate salt stress of cotton and okra seedlings. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the effects of applying beneficial bacteria on acid delinted cotton seeds. Inoculating seeds with salt stress alleviating bacteria could improve the growth of crop seedlings that are vulnerable to soil salinization.

Functional Role of Bacteria from Invasive Phragmites australis in Promotion of Host Growth.

We hypothesize that bacterial endophytes may enhance the competitiveness and invasiveness of Phragmites australis. To evaluate this hypothesis, endophytic bacteria were isolated from P. australis. The majority of the shoot meristem isolates represent species from phyla Firmicutes, Proteobacteria, and Actinobacteria. We chose one species from each phylum to characterize further and to conduct growth promotion experiments in Phragmites. Bacteria tested include Bacillus amyloliquefaciens A9a, Achromobacter spanius B1, and Microbacterium oxydans B2. Isolates were characterized for known growth promotional traits, including indole acetic acid (IAA) production, secretion of hydrolytic enzymes, phosphate solubilization, and antibiosis activity. Potentially defensive antimicrobial lipopeptides were assayed for through application of co-culturing experiments and mass spectrometer analysis. B. amyloliquefaciens A9a and M. oxydans B2 produced IAA. B. amyloliquefaciens A9a secreted antifungal lipopeptides. Capability to promote growth of P. australis under low nitrogen conditions was evaluated in greenhouse experiments. All three isolates were found to increase the growth of P. australis under low soil nitrogen conditions and showed increased absorption of isotopic nitrogen into plants. This suggests that the Phragmites microbes we evaluated most likely promote growth of Phragmites by enhanced scavenging of nitrogenous compounds from the rhizosphere and transfer to host roots. Collectively, our results support the hypothesis that endophytic bacteria play a role in enhancing growth of P. australis in natural populations. Gaining a better understanding of the precise contributions and mechanisms of endophytes in enabling P. australis to develop high densities rapidly could lead to new symbiosis-based strategies for management and control of the host.

Induction of salt tolerance and up-regulation of aquaporin genes in tropical corn by rhizobacterium Pantoea agglomerans.

UNLABELLED: Bacteria were isolated from surface disinfected seeds of eight modern corn types and an ancestor of corn, 'teosinte' and identified using 16S rDNA sequences. From each of the modern corn types we obtained Bacillus spp. (including, Bacillus amyloliquefaciens and Bacillus subtilis); while from teosinte we obtained only Pantoea agglomerans and Agrobacterium species. Of these bacteria, only P. agglomerans could actively grow under hypersaline conditions and increase salt tolerance of tropical corn seedlings. In laboratory and greenhouse experiments where plants were watered with a 0.2 mol l(-1) NaCl solution, P. agglomerans was found to enhance the capacity of tropical corn to grow compared to uninoculated controls. The total dry biomass was significantly higher in P. agglomerans-treated plants compared to controls under saline water. Gene expression analysis showed the up-regulation of the aquaporin gene family especially plasma membrane integral protein (ZmPIP) genes in P. agglomerans-treated plants. The plasma membrane integral protein type 2 (PIP2-1) gene in tropical corn seedlings was highly up-regulated by P. agglomerans treatment under salt stress conditions. Microscopic examination of P. agglomerans inoculated seedlings revealed that the bacterium colonized root meristems densely, and as roots developed, the bacterium became sparsely located in cell junctions. SIGNIFICANCE AND IMPACT OF THE STUDY: The enhancement of salt tolerance capacity in tropical corn, an important food crop, has the capacity to increase its cultivation area and yield in saline soils. The application of rhizobacteria to improve salt tolerance of tropical corn is ecofriendly and cost effective. We show that P. agglomerans isolated from teosinte (an ancestor of corn) induces salt tolerance in tropical corn and up-regulation of aquaporin genes. This study shows that microbes that increase salt tolerance may be used to enhance crop growth in saline soils.

Temporal changes in the biomechanical properties of endometrial mesenchymal stem cell seeded scaffolds in a rat model.

Use of synthetic clinical meshes in pelvic organ prolapse (POP) repair can lead to poor mechanical compliance in vivo, as a result of a foreign body reaction leading to excessive scar tissue formation. Seeding mesh with mesenchymal stem cells (MSCs) prior to implantation may reduce the foreign body reaction and lead to improved biomechanical properties of the mesh-tissue complex. This study investigates the influence of seeding human endometrial mesenchymal stem cells (eMSCs) on novel gelatin-coated polyamide scaffolds, to identify differences in scaffold/tissue biomechanical properties and new tissue growth following up to 90 days' implantation, in a subcutaneous rat model of wound repair. Scaffolds were subcutaneously implanted, either with or without eMSCs, in immunocompromised rats and following 7, 30, 60 and 90 days were removed and assessed for their biomechanical properties using uniaxial tensile testing. Following 7, 30 and 90 days' implantation scaffolds were assessed for tissue ingrowth and organization using histological staining and scanning electron microscopy. The eMSCs were associated with altered collagen growth and organization around the mesh filaments of the scaffold, affecting the physiologically relevant tensile properties of the scaffold-tissue complex, in the toe region of the load-elongation curve. Scaffolds seeded with eMSCs were significantly less stiff on initial stretching than scaffolds implanted without eMSCs. Collagen growth and organization were enhanced in the long-term in eMSC-seeded scaffolds, with improved fascicle formation and crimp configuration. Results suggest that neo-tissue formation and remodelling may be enhanced through seeding scaffolds with eMSCs.

Impact of heparan sulfate chains and sulfur-mediated bonds on the mechanical properties of bovine lens capsule.

We assessed the importance of glycosaminoglycans and sulfur-mediated bonds for the mechanical properties of lens capsules by comparing the stress-strain responses from control and treated pairs of bovine source. No significant change in mechanical properties was observed upon reduction of disulfide bonds. However, removal of glycosaminoglycan chains resulted in a significantly stiffer lens capsule, whereas high concentrations of reducing agent, which is expected to reduce the recently reported sulfilimine bond of collagen IV, resulted in a significantly less stiff lens capsule. A comparison of the diffraction patterns of the control and strongly reduced lens capsules indicated structural rearrangements on a nanometer scale.

A systematic account of the genus Plagiostoma (Gnomoniaceae, Diaporthales) based on morphology, host-associations, and a four-gene phylogeny.

Members of the genus Plagiostoma inhabit leaves, stems, twigs, and branches of woody and herbaceous plants predominantly in the temperate Northern Hemisphere. An account of all known species of Plagiostoma including Cryptodiaporthe is presented based on analyses of morphological, cultural, and DNA sequence data. Multigene phylogenetic analyses of DNA sequences from four genes (ß-tubulin, ITS, rpb2, and tef1-α) revealed eight previously undescribed phylogenetic species and an association between a clade composed of 11 species of Plagiostoma and the host family Salicaceae. In this paper these eight new species of Plagiostoma are described, four species are redescribed, and four new combinations are proposed. A key to the 25 accepted species of Plagiostoma based on host, shape, and size of perithecia, perithecial arrangement in the host, and microscopic characteristics of the asci and ascospores is provided. Disposition of additional names in Cryptodiaporthe and Plagiostoma is also discussed.

Biodegradable and injectable cure-on-demand polyurethane scaffolds for regeneration of articular cartilage.

This paper describes the synthesis and characterization of an injectable methacrylate functionalized urethane-based photopolymerizable prepolymer to form biodegradable hydrogels. The tetramethacrylate prepolymer was based on the reaction between two synthesized compounds, diisocyanato poly(ethylene glycol) and monohydroxy dimethacrylate poly(epsilon-caprolactone) triol. The final prepolymer was hydrated with phosphate-buffered saline (pH 7.4) to yield a biocompatible hydrogel containing up to 86% water. The methacrylate functionalized prepolymer was polymerized using blue light (450 nm) with an initiator, camphorquinone and a photosensitizer, N,N-dimethylaminoethyl methacrylate. The polymer was stable in vitro in culture media over the 28 days tested (1.9% mass loss); in the presence of lipase, around 56% mass loss occurred over the 28 days in vitro. Very little degradation occurred in vivo in rats over the same time period. The polymer was well tolerated with very little capsule formation and a moderate host tissue response. Human chondrocytes, seeded onto Cultispher-S beads, were viable in the tetramethacrylate prepolymer and remained viable during and after polymerization. Chondrocyte-bead-polymer constructs were maintained in static and spinner culture for 8 weeks. During this time, cells remained viable, proliferated and migrated from the beads through the polymer towards the edge of the polymer. New extracellular matrix (ECM) was visualized with Masson's trichrome (collagen) and Alcian blue (glycosaminoglycan) staining. Further, the composition of the ECM was typical for articular cartilage with prominent collagen type II and type VI and moderate keratin sulphate, particularly for tissue constructs cultured under dynamic conditions.

Evaluation of photo-crosslinked fibrinogen as a rapid and strong tissue adhesive.

Tissue adhesives and sealants are commonly used in surgery either as an adjunct to, or replacement for, sutures. Previously, we have shown that fibrinogen can be crosslinked rapidly to give a high-strength bond in the presence of a ruthenium(II) complex, a persulfate and irradiation with visible light, and that the crosslinked fibrinogen is nontoxic to cells in vitro. This approach addresses limitations to current fibrin sealants that typically have relatively slow curing times and low bond strengths. In the present study, we have evaluated the efficacy and safety of this new biological scaffold sealant in various animal models. When placed as solid implants into rats, the crosslinked fibrinogen persisted for at least 8 weeks but was fully resorbed by 18 weeks with minimal inflammatory responses. When used as a tissue adhesive for repair of skin incisions in rats or as an arterial haemostat in pig, the photo-crosslinked fibrinogen sealed tissue or arrested bleeding within 20 s of application. For the skin incisions, the fibrinogen sealant promoted rapid tissue vascularization and cellular infiltration with no adverse foreign body cell generation. New collagen deposition occurred and with time the matrix had remodelled to acquire large mature collagen fiber bundles which were accompanied by maximum regenerated tensile strength. This biomaterial system may find useful applications in surgical procedures where rapid curing and/or high strength tissue sealing is required.

Fungal endophytes: diversity and functional roles.

All plants in natural ecosystems appear to be symbiotic with fungal endophytes. This highly diverse group of fungi can have profound impacts on plant communities through increasing fitness by conferring abiotic and biotic stress tolerance, increasing biomass and decreasing water consumption, or decreasing fitness by altering resource allocation. Despite more than 100 yr of research resulting in thousands of journal articles, the ecological significance of these fungi remains poorly characterized. Historically, two endophytic groups (clavicipitaceous (C) and nonclavicipitaceous (NC)) have been discriminated based on phylogeny and life history traits. Here, we show that NC-endophytes represent three distinct functional groups based on host colonization and transmission, in planta biodiversity and fitness benefits conferred to hosts. Using this framework, we contrast the life histories, interactions with hosts and potential roles in plant ecophysiology of C- and NC-endophytes, and highlight several key questions for future work in endophyte biology.

Leaf-inhabiting genera of the Gnomoniaceae, Diaporthales.

The Gnomoniaceae are characterised by ascomata that are generally immersed, solitary, without a stroma, or aggregated with a rudimentary stroma, in herbaceous plant material especially in leaves, twigs or stems, but also in bark or wood. The ascomata are black, soft-textured, thin-walled, and pseudoparenchymatous with one or more central or eccentric necks. The asci usually have a distinct apical ring. The Gnomoniaceae includes species having ascospores that are small, mostly less than 25 mum long, although some are longer, and range in septation from non-septate to one-septate, rarely multi-septate. Molecular studies of the Gnomoniaceae suggest that the traditional classification of genera based on characteristics of the ascomata such as position of the neck and ascospores such as septation have resulted in genera that are not monophyletic. In this paper the concepts of the leaf-inhabiting genera in the Gnomoniaceae are reevaluated using multiple genes, specifically nrLSU, translation elongation factor 1-alpha (tef1-alpha), and RNA polymerase II second largest subunit (rpb2) for 64 isolates. ITS sequences were generated for 322 isolates. Six genera of leaf-inhabiting Gnomoniaceae are defined based on placement of their type species within the multigene phylogeny. The new monotypic genus Ambarignomonia is established for an unusual species, A. petiolorum. A key to 59 species of leaf-inhabiting Gnomoniaceae is presented and 22 species of Gnomoniaceae are described and illustrated.

First Report of Aspergillus flavus Colonizing Naturally Dispersed Seeds of Oxandra acuminata, Pseudomalmea diclina, and Unonopsis matthewsii in Peru.

Tree species Oxandra acuminata, Pseudomalmea diclina, and Unonopsis matthewsii (Annonaceae) are sources of wood for people of the Amazonian Region where the trees are harvested from natural populations. With increased human population and agriculture in the Amazonian Region, forest diversity is affected. To manage the forest communities, it is necessary to understand the dynamics of regeneration of forest plants. Diseases that affect seed and seedling survival are critical in determining the ultimate species composition. During the dry season in June of 2006, rotten seeds of U. matthewsii (approximately 90% of 380 seeds) were observed in seven natural locations over an area of 150 ha in lowland tropical forest in Manu National Park, Peru. Colonized seeds were open and covered with yellow, dry, powdery, easily liberated conidia. One month later, seeds of O. acuminata in the same locations showed the same symptoms. In August of 2007, P. diclina seeds were naturally dispersed (by animals) in these plots and showed similar symptoms. The disease affecting O. acuminata was found in two other sampling sites along the river at the Los Amigos Research Station located 80 km southeast of Manu National Park. In all cases, the pathogen was identified as Aspergillus flavus based on morphological characteristics (1,2). Isolation of the pathogen was made on potato dextrose agar (PDA) amended with chloramphenicol (100 mg/l) and incubated at 28°C for 5 to 7 days. Single-spore isolations were made from each plant species and maintained as stock cultures. Colonies grown on PDA were granular, flat, and yellow at first, but quickly became bright to dark yellow-green. The radiate conidial heads measured approximately 400 µm in diameter. Older globose vesicles measured from 28 to 45 µm in diameter. Conidia were globose or subglobose with roughened walls and measured 3 to 5.5 µm in diameter. For pathogenicity tests, healthy seeds from the three species obtained from several trees, were previously surface sterilized by dipping in a 0.1% chlorine solution and allowed to dry. To inoculate seeds, a small scalpel was used to make a superficial cut on the seeds, after which a conidial suspension (3 × 105 conidia/ml of distilled water) was pipetted over each wound. For each plant species, 20 seeds were inoculated and 20 were used as control. This procedure was repeated twice. Each seed was maintained in a petri plate at ambient temperature in a field station lab and evaluated daily for 10 days. Inoculated seeds of all three species showed symptoms identical to those seen in field populations. Colonized seeds died after 2 to 7 days, and dry, yellow conidia were produced inside the open seed after approximately 1 to 2 days of decay. A. flavus was reisolated from colonized seeds. Control seeds remained healthy. A. flavus is ubiquitous and has been reported on numerous host plants worldwide (1,2). However, to our knowledge, this is the first report of A. flavus causing high seed mortality of species of Annonaceae in Peru. Our observations suggest that A. flavus is an important fungus affecting survival of seeds of O. acuminata, P. diclina, and U. matthewsii in the natural plant communities where we conducted this study. References: (1) B. W. Horn. Mycologia 97:202, 2005. (2) K. B. Raper and D. I. Fennell. The Genus Aspergillus. Williams and Wilkins, Baltimore, MD, 1965.

Phylogenetic evidence for an animal pathogen origin of ergot and the grass endophytes.

Grass-associated fungi (grass symbionts) in the family Clavicipitaceae (Ascomycota, Hypocreales) are species whose host range is restricted to the plant family Poaceae and rarely Cyperaceae. The best-characterized species include Claviceps purpurea (ergot of rye) and Neotyphodium coenophialum (endophyte of tall fescue). They have been the focus of considerable research due to their importance in agricultural and grassland ecosystems and the diversity of their bioactive secondary metabolites. Here we show through multigene phylogenetic analyses and ancestral character state reconstruction that the grass symbionts in Clavicipitaceae are a derived group that originated from an animal pathogen through a dynamic process of interkingdom host jumping. The closest relatives of the grass symbionts include the genera Hypocrella, a pathogen of scale insects and white flies, and Metarhizium, a generalist arthropod pathogen. These data do not support the monophyly of Clavicipitaceae, but place it as part of a larger clade that includes Hypocreaceae, a family that contains mainly parasites of other fungi. A minimum of 5-8 independent and unidirectional interkingdom host jumps has occurred among clavicipitaceous fungi, including 3-5 to fungi, 1-2 to animals, and 1 to plants. These findings provide a new evolutionary context for studying the biology of the grass symbionts, their role in plant ecology, and the evolution of host affiliation in fungal symbioses.

Black Choke Disease Caused by an Ephelis sp. on Purple Fountain Grass in Maryland.

Purple fountain grass (Pennisetum alopecuroides) is indigenous to Asia, prized for its foxtail-like purple flowers, and widely used as an ornamental. During October 1999, black choke disease was found on P. alopecuroides cv. Hameln (L.) Spreng at a plant nursery in Maryland. Disease symptoms include mummification of inflorescences by black conidial stromata, distorted leaf tissue, and a dense layer of white epiphytic mycelium on the adaxial leaves and culms. Stromata were initially white but became black with age. Microscopic analysis of the isolated fungus indicated that the causal organism was an Ephelis sp., American Type Culture Collection No. MYA-3317. The ephelidial conidia developed in sporodochia on stromata and were hyaline, filiform to acicular, and 18 to 21 × 1 µm. Cultures on potato dextrose agar were off-white and 50 mm in diameter after 14 days at 23°C. Analysis of herbarium specimens of several Balansia spp. revealed that the Ephelis sp. isolate bears morphological resemblance to Asian and not American Balansieae. In fact, the infection observed on Pennisetum sp. forms similarly to Ephelis sp. infection on Oryza sativa L. (Asian) that also results in development of stromata on panicles and a mycelial network enclosing the panicles, preventing maturation and expansion. On both plants, the infected inflorescence becomes black with age and appears mummified as pseudosclerotia form. Furthermore, flag leaves and tillers of both plants appear slightly distorted and silver due to the epibiotic mycelia. The causal agent of black choke disease on rice is Ephelis oryzae Syd. (teleomorph = Balansia oryzae-sativae Hashioka). The mature stroma of E. oryzae forms on the inflorescence and is embedded with a layer of ovate perithecia. Immature stromata bear conidiomata that are cupulate to cushion shaped and black, producing hyaline, branched conidiophores that terminate in phialides. Conidia are ephelidial, filiform to acicular, hyaline, and 18 to 22 × 1.5 µm (2). To determine the phylogenetic relationship between other balansioid fungi and the Ephelis sp. isolate, the nuclear ribosomal internal transcribed spacer (ITS1) region was amplified with primers ITS4 and ITS5 (3). Maximum parsimony analysis of the ITS1 sequences showed that the Ephelis sp. infecting P. alopecuroides cv. Hameln grouped (100% bootstrap support) in a clade with Ephelis oryzae, Balansia sclerotica, Balansia andropogonis, and Balansia sp.; all endemic to Asia and tightly groups with the Asian rice pathogen Ephelis oryzae (100% bootstrap support). Further phylogenetic analysis using topological constraints indicated that Ephelis sp. is not appropriately grouped with American balansioid species. Since P. alopecuroides is often imported to North America from Asia (1), it is likely that Ephelis sp. on P. alopecuroides is endemic to Asia and perhaps was transported along with its host to North America. The disease ontogeny, morphology, and sequence similarities between the Ephelis sp. isolated from Pennisetum sp. and E. oryzae suggests that these fungi are evolutionarily close, sibling species, or conspecific. To our knowledge, this is the first report of choke disease on P. alopecuroides in the United States. References: (1) A. S. Hitchcock. Manual of the Grasses of the United States. A. Chase, ed. U.S. Government Print Office, Washington DC, 1951 (2) F. N. Lee and P. S. Gunnell. Udbatta. Page 29 in: Compendium of Rice Diseases. R. K. Webster and P. S. Gunnell, eds. The American Phytopathological Society, St. Paul. MN, 1992. (3) J. F. White Jr. et al. Mycologia 89:408, 1997.

Ultrasound-assisted hydrogenation of cinnamaldehyde.

The hydrogenation, employing hydrogen gas, of cinnamaldehyde was performed using Pd-black and Raney Ni catalysts at 298+/-3 K in a water-cooled (jacketed) reaction vessel. Sampling at pre-determined time intervals and GC/MS analysis yielded time-dependent product state distribution information. A kinetic modeling of the data revealed that cinnamaldehyde was both hydrogenated directly to the final product benzenepropanol, as well as a fraction being converted to the intermediate benzenepropanal, where the latter was subsequently hydrogenated to benzenepropanol. Comparing the ultrasound-assisted and blank (stirred) experiments revealed that a higher maximum relative concentration of the intermediate benzenepropanal was formed in the ultrasound experiments compared to the stirred experiment. The activity of the ultrasound experiments compared to blank were 9-fold and 20-fold greater for the Pd-black and Raney Ni catalysts, respectively. Finally, an application of the Bell-Evans-Polanyi principle to yield an estimate of the ratio of rate coefficients for benzenepropanal and benzenepropanol formation was performed by considering chemical group energy differences and surface adsorption energy differences in the first mechanistic step of hydrogenation.

Phylogenetic placement of the anamorphic tribe Ustilaginoideae (Hypocreales, Ascomycota).

Tribe Ustilaginoideae (Hypocreales, Ascomycetes) is made up of three anamorph genera, Munkia, Neomunkia and Ustilaginoidea. Species of Munkia and Neomunkia develop on the culms of bamboo (Chusquea spp.) and have a neotropical distribution while species of Ustilaginoidea infect the florets of various grasses and are pantropical in distribution. In this study we evaluated the phylogeny of the tribe and assessed hypotheses regarding its affinity to clavicipitalean teleomorphic groups. To support phylogenetic analyses, morphology of representatives of several key species of Ustilaginoideae was examined also. Phylogenetic analyses using sequences of the large subunit of the ribosomal RNA gene suggest that members of Ustilaginoideae are distinct from teleomorphic genera of Clavicipitaceae and that Ustilaginoideae should be recognized as a monophyletic group within Hypocreales. However, phylogenetic analyses did not resolve the placement of Ustilaginoideae in Clavicipitaceae or Hypocreaceae, suggesting that it might be a distinct lineage within Hypocreales. This evaluation supported the monophyly of tribes Balansieae and Clavicipeae in the family Clavicipitaceae.