The Experts below are selected from a list of 1206 Experts worldwide ranked by ideXlab platform
Christopher L Schardl - One of the best experts on this subject based on the ideXlab platform.
-
MOLECULAR SYSTEMATICS OF CLAVICIPITACEAE SUPPORTING MONOPHYLY OF GENUS EPICHLOE AND FORM GENUS EPHELIS
Mycologia, 2018Co-Authors: Gretchen Anna Kuldau, Malcolm R. Siegel, James F White, Christopher L SchardlAbstract:The family Clavicipitaceae (Ascomycota) is comprised of fungi with perithecia borne on stro- mata, unitunicate asci, and filamentous, multiseptate ascospores. All are biotrophic symbionts, either mu- tualistic with plant hosts or pathogenic to plants, in- vertebrate animals or other fungi. Genera of plant- associated Clavicipitaceae (tribes Balansieae and Clavicipieae) are distinguished, in part, by stromal and ascus morphology, ascospore germination pat- terns, whether sclerotia are formed, and host inter- actions. Their anamorphs include enteroblastic mi- croconidial states, classified in anamorphic genera Neotyphodium and Sphacelia (for teleomorphs Atkin- sonella, Claviceps, Echinodothis, and Epichloe), and ho- loblastic macroconidia, classified in the anamorphic genus Ephelis (teleomorphs Atkinsonella, Balansia, and Myriogenospora). Epichloe' species often are mu- tualistic with grass hosts, and are ancestral to asexual, seed transmitted endophytes symbiotic with many cool-season grasses. Partial 28S nuclear rDNA se- quences were determined from isolates of five species and two undescribed mating populations of Epichloe, one asexual Epichloe hybrid (Neotyphodium coeno- phialum), and representatives of six other genera in the family. Results from phylogenetic analysis of the sequences supported monophyly of plant-associated Clavicipitaceae, with insect-pathogenic Cordyceps spe- cies more deeply rooted. Four clades were distin-
-
nomenclatural realignment of Neotyphodium species with genus epichloe
Mycologia, 2014Co-Authors: Adrian Leuchtmann, Christopher L Schardl, Charles W. Bacon, James F White, Mariusz TadychAbstract:Nomenclatural rule changes in the International Code of Nomenclature for algae, fungi and plants, adopted at the 18th International Botanical Congress in Melbourne, Australia, in 2011, provide for a single name to be used for each fungal species. The anamorphs of Epichloe species have been classified in genus Neotyphodium, the form genus that also includes most asexual Epichloe descendants. A nomenclatural realignment of this monophyletic group into one genus would enhance a broader understanding of the relationships and common features of these grass endophytes. Based on the principle of priority of publication we propose to classify all members of this clade in the genus Epichloe. We have reexamined classification of several described Epichloe and Neotyphodium species and varieties and propose new combinations and states. In this treatment we have accepted 43 unique taxa in Epichloe, including distinct species, subspecies, and varieties. We exclude from Epichloe the two taxa Neotyphodium starrii, as nom...
-
beneficial effects of Neotyphodium tembladerae and Neotyphodium pampeanum on a wild forage grass
Grass and Forage Science, 2012Co-Authors: Leopoldo Javier Iannone, Christopher L Schardl, Padmaja Nagabhyru, Albertina Daniela Pinget, J P De BattistaAbstract:Abstract Asexual, vertically transmitted fungal endophytes of thegenus Neotyphodium are considered to enhance growth,stress resistance and competitiveness of agronomicgrasses, but have been suggested to have neutral ordeleterious effects on wild grasses. We studied whetherthe associations between Bromus auleticus and theirendophytes,NeotyphodiumpampeanumandNeotyphodiumtembladerae,arebeneficialforthiswildforagegrassnativeto South America. In a greenhouse experiment,3-month-old endophyte-infected plants (E+) showedenhanced growth relative to their endophyte-free coun-terparts(E)).Inaone-year-longexperimentinthefield,E+plantsshowedhighersurvivalandregrowthrate,andproduced more biomass and seeds than E) plants. OnlywithrespecttotheseedoutputdidN. tembladeraeseemtobedetrimental,whereasN. pampeanumdidnotaffectthisparameter. No differences were observed with respect tothe germination of seeds produced by E+ or E) plants.LolinealkaloidsweredetectedinN. pampeanum-infectedplants. Our results show that, similar to results withagronomic grasses, in wild grasses, the symbiosis withNeotyphodium species could be mutualistic.Keywords: endophytes, wild grasses, Bromus, growth,reproduction
-
Elimination of marker genes from transformed filamentous fungi by unselected transient transfection with a Cre-expressing plasmid.
Fungal Genetics and Biology, 2009Co-Authors: Simona Florea, Kalina Andreeva, Caroline Machado, Peter M Mirabito, Christopher L SchardlAbstract:Abstract A convenient method to remove selectable markers from fungal transformants permits the markers to be used for sequential transformations, and should also reduce public concerns and regulatory impediments to applications involving environmental release of genetically modified fungi. We report a method for marker removal that requires no genetic selection. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum and Epichloe festucae transformants containing a hygromycin B phosphotransferase gene ( hph ) flanked by loxP sites in direct orientation were transiently transfected with a Cre-recombinase expression plasmid, and then cultured without selection. The marker was eliminated in 0.5–2% of the colonies, leaving a single loxP sequence and no other exogenous DNA in the genome. This approach was also applied to the yA gene of Aspergillus nidulans as a laboratory exercise to demonstrate multiple principles of transformation and genome manipulation. Thus, the Cre-expression plasmid and transient transfection approach was rapid, flexible and useful for diverse filamentous fungi.
-
phylogenetic divergence morphological and physiological differences distinguish a new Neotyphodium endophyte species in the grass bromus auleticus from south america
Mycologia, 2009Co-Authors: Leopoldo Javier Iannone, Christopher L Schardl, Daniel Cabral, Maria Susana RossiAbstract:The fungi of genus Neotyphodium are systemic, constitutive, symbionts of grasses of subfamily Pooideae. In the southern hemisphere most of these asexual endophytes are the result of the hybridization between two sexual species, Epichlo" festucae and E. typhina, from the northern hemisphere. However the ancestral sexual species have not been detected in this region. Several grasses from Argentina are infected by Neotyphodium species. These endophytes are in general very similar macro- and micromorphologically and phylogenetically conform to species N. tembladerae. However the Neotyphodium spp. endophytes of some hosts, Bromus auleticus and Poa spicifomis var. spiciformis, have not been included in this species. In this work we studied the incidence and characterized the diversity of Neotyphodium species in populations of the native grass Bromus auleticus from Argentina. The incidence of endophytes was 100% in all populations investigated. Two groups of endophytes were differentiated by their morphologies, growth rates, conidial ontogenies and by relative resistance to the fungicide benomyl. Phylogenetic trees inferred from tefA and tubB intron sequences indicated that both N. tembladerae and the novel morphotype were hybrids of E. festucae and E. typhina, but the ancestral E. typhina genotype distinguished them. Isolates from plants that inhabit coastal dunes, xerophytic forests, savannahs and hills were similar morphologically and phylogenetically to N. tembladerae, whereas the endophytes from the humid pampa plains conformed to the novel group. We propose the endophyte of Bromus auleticus from humid pampas as a new species, Neotyphodium pampeanum.
Stanley H. Faeth - One of the best experts on this subject based on the ideXlab platform.
-
Appendix E. Significant rarefaction curves for differences in species richness of total arthropods, parasites, natural enemies (predators and parasites), omnivores, and detrivores on plants infected or uninfected with the Neotyphodium endophyte over the four sampling periods of the study.
2016Co-Authors: Stanley H. Faeth, Eyal ShochatAbstract:Significant rarefaction curves for differences in species richness of total arthropods, parasites, natural enemies (predators and parasites), omnivores, and detrivores on plants infected or uninfected with the Neotyphodium endophyte over the four sampling periods of the study
-
hybridization of Neotyphodium endophytes enhances competitive ability of the host grass
New Phytologist, 2012Co-Authors: Susanna Saari, Stanley H. FaethAbstract:Associations with microbial symbionts may lead to niche differentiation of their host. Vertically transmitted Neotyphodiumendophytes of grasses often hybridize in nature. Infection by these hybrid symbionts may result in different host–plant phenotypes from those caused as a result of infection by nonhybrid symbionts. Observations of wild Arizona fescue (Festuca arizonica) populations show that hybrid Neotyphodium-infected (H+) grasses dominate in resource-poor environments, whereas nonhybrid endophyte-infected (NH+) grasses dominate in environments with more resources. We studied the hypothesis that hybridization of endophytes increases stress tolerance of the host.To test whether hybridization of Neotyphodium affects performance and competitive abilities of the host depending on resources, we conducted a glasshouse experiment where competition, nutrients and watering were manipulated.H+ plants had greater wet biomass than NH+ and endophyte-free plants, when grown in competition, but only in low-water and low-nutrient treatments. By contrast, NH+ plants did not perform better than H+ or endophyte-free plants regardless of the treatment combination.Our results suggest that hybridization of symbiotic Neotyphodium endophytes may increase competitive potential of the host in stressful environments and that this hybridization may be underlying niche expansion of Arizona fescue in the environments with low resources.
-
Local adaptation in Festuca arizonica infected by hybrid and nonhybrid Neotyphodium endophytes.
Microbial ecology, 2007Co-Authors: T J Sullivan, Stanley H. FaethAbstract:Cool-season grasses often harbor obligate fungal symbionts from the genus Neotyphodium, and these symbiota can function as a single ecological unit. Previous studies have shown that gene flow in Neotyphodium in Festuca arizonica is low enough such that populations could diverge and form local adaptations. A reciprocal transplant experiment was performed between two F. arizonica/Neotyphodium populations in Arizona, Clint’s Well and Flagstaff, using symbiota with the most common Neotyphodium genotypes in each population, to test for local adaptations. The genetic difference between populations is potentially large as Neotyphodium from Clint’s Well are of hybrid origin. Local environmental variation was the most important source of variation for F. arizonica/Neotyphodium symbiota growth, with individuals at Flagstaff growing larger and individuals at Clint’s Well not reproducing. Local environment and the source population of the symbiota interacted to affect vegetative growth. Symbiota from Clint’s Well, which harbor hybrid Neotyphodium, had higher volume/wet mass and volume/dry mass ratios but only in the marginal Clint’s Well habitat. The local environment also affected F. arizonica/Neotyphodium reproduction because only symbiota transplanted to Flagstaff reproduced. Symbiota from Clint’s Well produced more panicles, whereas symbiota from Flagstaff with nonhybrid Neotyphodium produced greater seed mass per panicle. Overall seed mass production was not different, suggesting that the two strategies are functionally equivalent. We find that F. arizonica/Neotyphodium symbiota vary geographically, but potential local adaptations are only apparent in marginal habitats and may be related to the evolutionary history of the Neotyphodium part of the symbiota.
-
Neotyphodium interactions with a wild grass are driven mainly by endophyte haplotype
Functional Ecology, 2007Co-Authors: L J Morse, Stanley H. Faeth, Thomas A DayAbstract:Summary 1Strong mutualistic associations are expected to arise between host and microbial symbionts when symbionts lose sexuality, and rely strictly on their host for reproduction via vertical transmission. The Neotyphodium endophyte is a vertically transmitted, asexual symbiont of pooid grasses. In agronomic grasses, Neotyphodium typically interacts mutualistically with its host by increasing drought resistance and other properties. However, the interaction likely depends on host and endophyte genotypic variation, yet little is known how this variation influences host physiological and morphological responses, especially in wild grasses. 2We used four different Neotyphodium-infected maternal lines of a wild grass, Arizona fescue (Festuca arizonica). Two lines harboured one haplotype and two lines harboured a different haplotype. We experimentally removed the endophyte from some ramets of the four lines. We grew infected (E+) and uninfected (E−) plants in a greenhouse under varying water availability. 3We examined the effect of endophyte infection, endophyte and host plant maternal genotype, and water availability on traditional growth parameters. We also measured leaf net photosynthesis and dark respiration, leaf conductance to water vapour, leaf water potential, leaf rolling and stomatal density to provide explanations for differences in biomass production and relative growth rates (RGR). 4Our general findings show that Neotyphodium infection, Neotyphodium haplotype and its interaction with host maternal genotype, and varying water availability influence Arizona fescue physiology, growth and biomass production. Based only on infection status, the direction of interaction between endophyte and host is not mutualistic in terms of host growth. Overall, endophyte haplotype and its interaction with host maternal genotype is the most critical, and consistent factor in influencing host growth and physiological outcomes. Variation due to endophyte haplotype in terms of growth and physiological parameters is often greater than that between infected and uninfected hosts. 5Endophyte–host interactions are likely to be enormously complex because of the genetic and environmental variation that exists in natural populations. The outcome of these interactions in natural grass–endophyte systems is exceedingly difficult to predict based simply on the presence or absence of the endophyte.
-
Temporal and Spatial Variation in Alkaloid Levels in Achnatherum robustum, a Native Grass Infected with the Endophyte Neotyphodium
Journal of Chemical Ecology, 2006Co-Authors: Stanley H. Faeth, Dale R. Gardner, Cinnamon J. Hayes, Andrea Jani, Sally K. Wittlinger, Thomas A. JonesAbstract:The native North American perennial grass Achnatherum robustum (Vasey) Barkworth [= Stipa robusta (Vasey) Scribn.] or sleepygrass is toxic and narcotic to livestock. The causative agents are alkaloidal mycotoxins produced from infections by a systemic and asexual Neotyphodium endophyte. Recent studies suggest that toxicity is limited across the range of sleepygrass in the Southwest USA. We sampled 17 populations of sleepygrass with varying distance from one focal population known for its high toxicity levels near Cloudcroft, NM, USA. For some, we sampled individual plants twice within the same growing season and over successive years (2001–2004). We also determined infection levels in each population. In general, all populations were highly infected, but infection levels were more variable near the focal population. Only infected plants within populations near the Cloudcroft area produced alkaloids. The ergot alkaloid, ergonovine, comprised the bulk of the alkaloids, with lesser amounts of lysergic and isolysergic acid amides and ergonovinine alkaloids. Levels of all alkaloids were positively correlated among individual plants within and between growing seasons. Infected plants that produced no alkaloids in 1 yr did not produce any alkaloids within the same growing season or in other years. Levels of alkaloids in sleepygrass populations declined with distance from the Cloudcroft population, although infection levels increased. Infected plants in populations in northern New Mexico and southern Colorado produced no alkaloids at all despite 100% infectivity. Our results suggest that only specific Neotyphodium haplotypes or specific Neotyphodium –grass combinations produce ergot alkaloids in sleepygrass. The Neotyphodium haplotype or host–endophyte combination that produces toxic levels of alkaloids appears restricted to one locality across the range of sleepygrass. Because of the wide variation in alkaloid levels among populations, interactions between the endophyte and host, and consequences for herbivores, competitors, and pathogens and other components of the community, are likely to vary widely across the geographic range of this native grass.
Barry Scott - One of the best experts on this subject based on the ideXlab platform.
-
indole diterpene biosynthetic capability of epichloe endophytes as predicted by ltm gene analysis
Applied and Environmental Microbiology, 2009Co-Authors: Carolyn A Young, Christina D Moon, Christopher L Schardl, B A Tapper, Barry ScottAbstract:Bioprotective alkaloids produced by Epichloe and closely related asexual Neotyphodium fungal endophytes protect their grass hosts from insect and mammalian herbivory. One class of these compounds, known for antimammalian toxicity, is the indole-diterpenes. The LTM locus of Neotyphodium lolii (Lp19) and Epichloe festuce (Fl1), required for the biosynthesis of the indole-diterpene lolitrem, consists of 10 ltm genes. We have used PCR and Southern analysis to screen a broad taxonomic range of 44 endophyte isolates to determine why indole-diterpenes are present in so few endophyte-grass associations in comparison to that of the other bioprotective alkaloids, which are more widespread among the endophtyes. All 10 ltm genes were present in only three epichloe endophytes. A predominance of the asexual Neotyphodium spp. examined contained 8 of the 10 ltm genes, with only one N. lolii containing the entire LTM locus and the ability to produce lolitrems. Liquid chromatography-tandem mass spectrometry profiles of indole-diterpenes from a subset of endophyte-infected perennial ryegrass showed that endophytes that contained functional genes present in ltm clusters 1 and 2 were capable of producing simple indole-diterpenes such as paspaline, 13-desoxypaxilline, and terpendoles, compounds predicted to be precursors of lolitrem B. Analysis of toxin biosynthesis genes by PCR now enables a diagnostic method to screen endophytes for both beneficial and detrimental alkaloids and can be used as a resource for screening isolates required for forage improvement.
-
a complex ergovaline gene cluster in epichloe endophytes of grasses
Applied and Environmental Microbiology, 2007Co-Authors: Barry Scott, Damien J Fleetwood, Geoffrey A Lane, Aiko Tanaka, Richard D JohnsonAbstract:Clavicipitaceous fungal endophytes of the genera Epichloe and Neotyphodium form symbioses with grasses of the subfamily Pooideae, in which they can synthesize an array of bioprotective alkaloids. Some strains produce the ergopeptine alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyte-infected grasses. Cloning and analysis of a nonribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii revealed a putative gene cluster for ergovaline biosynthesis containing a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Despite conservation of gene sequence, gene order is substantially different between the N. lolii, C. purpurea, and A. fumigatus ergot alkaloid gene clusters. Southern analysis indicated that the N. lolii cluster was linked with previously identified ergovaline biosynthetic genes dmaW and lpsA. The ergovaline genes are closely associated with transposon relics, including retrotransposons and autonomous and nonautonomous DNA transposons. All genes in the cluster were highly expressed in planta, but expression was very low or undetectable in mycelia from axenic culture. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.
-
a complex gene cluster for indole diterpene biosynthesis in the grass endophyte Neotyphodium lolii
Fungal Genetics and Biology, 2006Co-Authors: Carolyn A Young, Silvina Andrea Felitti, Katherine Shields, German Spangenberg, Richard D Johnson, Gregory T Bryan, Sanjay Saikia, Barry ScottAbstract:Abstract Lolitrems are a structurally diverse group of indole-diterpene mycotoxins synthesized by Epichloe/Neotyphodium endophytes in association with Pooid grasses. Using suppression subtractive hybridization combined with chromosome walking, two clusters of genes for lolitrem biosynthesis were isolated from Neotyphodium lolii, a mutualistic endophyte of perennial ryegrass. The first cluster contains five genes, ltmP, ltmQ, ltmF, ltmC, and ltmB, four of which appear to be orthologues of functionally characterized genes from Penicillium paxilli. The second cluster contains two genes, ltmE and ltmJ, that appear to be unique to lolitrem biosynthesis. The two clusters are separated by a 16 kb AT-rich sequence that includes two imperfect direct repeats. A previously isolated ltm cluster composed of ltmG, ltmM, and ltmK, is linked to these two new clusters by 35 kb of AT-rich retrotransposon relic sequence. All 10 genes at this complex LTM locus were highly expressed in planta but expression was very low or undetectable in mycelia. ltmM and ltmC were shown to be functional orthologues of P. paxilli paxM and paxC, respectively. This work provides a genetic foundation for elucidating the metabolic grid responsible for the diversity of indole-diterpenes synthesized by N. lolii.
-
Elimination of ergovaline from a grass-Neotyphodium endophyte symbiosis by genetic modification of the endophyte.
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Daniel G Panaccione, Barry Scott, Carolyn A Young, Richard D Johnson, Jinghong Wang, Prapassorn Damrongkool, Christopher L SchardlAbstract:The fungal endophytes Neotyphodium lolii and Neotyphodium sp. Lp1 from perennial ryegrass (Lolium perenne), and related endophytes in other grasses, produce the ergopeptine toxin ergovaline, among other alkaloids, while also increasing plant fitness and resistance to biotic and abiotic stress. In the related fungus, Claviceps purpurea, the biosynthesis of ergopeptines requires the activities of two peptide synthetases, LPS1 and LPS2. A peptide synthetase gene hypothesized to be important for ergopeptine biosynthesis was identified in C. purpurea by its clustering with another ergot alkaloid biosynthetic gene, dmaW. Sequence analysis conducted independently of the research presented here indicates that this gene encodes LPS1 [Tudzynski, P., Holter, K., Correia, T., Arntz, C., Grammel, N. & Keller, U. (1999) Mol. Gen. Genet. 261, 133–141]. We have cloned a similar peptide synthetase gene from Neotyphodium lolii and inactivated it by gene knockout in Neotyphodium sp. Lp1. The resulting strain retained full compatibility with its perennial ryegrass host plant as assessed by immunoblotting of tillers and quantitative PCR. However, grass–endophyte associations containing the knockout strain did not produce detectable quantities of ergovaline as analyzed by HPLC with fluorescence detection. Disruption of this gene provides a means to manipulate the accumulation of ergovaline in endophyte-infected grasses for the purpose of determining the roles of ergovaline in endophyte-associated traits and, potentially, for ameliorating toxicoses in livestock.
-
the evolutionary origins of epichloe endophytes from annual ryegrasses
Mycologia, 2000Co-Authors: Christina D Moon, Christopher L Schardl, Barry Scott, Michael J. ChristensenAbstract:The annual Lolium (ryegrass) species were surveyed for seedborne, asexual Epichloe endo- phytes (i.e., Neotyphodium spp.). Endophytes that were extremely slow growing in culture were identi- fied from all seven annual ryegrass spp. Sequences of ,3-tubulin gene (tub2) introns and the internal tran- scribed spacers of the nuclear ribosomal RNA (rDNA- ITS) were determined, as were profiles based on five microsatellite loci. Two taxonomic groups of annual ryegrass endophytes were distinguishable by mor- phology, culturability and genotypes. One group, for which the name Neotyphodium occultans is proposed, comprised only isolates that could not be subcultured on potato dextrose agar medium. These isolates con- tained multiple tub2 genes and multiple alleles at mi- crosatellite loci, suggestive of an interspecific hybrid origin. The results of phylogenetic analysis implied that the ancestors of N. occultans included Epichloe baconii and E. bromicola, or close relatives. The sec- ond taxonomic group of annual ryegrass endophytes, exemplified by isolate Lc4, was found only in L. can- ariense. This taxon had a single tub2 sequence and microsatellite genotype that was nearly identical to those of extant Epichloi typhina isolates that infect Poa nemoralis. Therefore, it is proposed that Lc4 is an asexual derivative of an E. typhina genotype close- ly related to extant strains from P nemoralis. We pro- pose the name Neotyphodium typhinum var. canariense for this taxon. These two new endophyte taxa from the annual ryegrasses are further examples of asexual Neotyphodium species that have arisen from sexual Ep-
Michael J. Christensen - One of the best experts on this subject based on the ideXlab platform.
-
epichloe endophytes grow by intercalary hyphal extension in elongating grass leaves
Fungal Genetics and Biology, 2008Co-Authors: Michael J. Christensen, Wayne R. Simpson, Raymond J Bennett, Richard D Johnson, Gregory T Bryan, H A Ansari, Hironori Koga, John Koolaard, Elizabeth M Nickless, Christine R VoiseyAbstract:A fundamental hallmark of fungal growth is that vegetative hyphae grow exclusively by extension at the hyphal tip. However, this model of apical growth is incompatible with endophyte colonization of grasses by the symbiotic Neotyphodium and Epichloe species. These fungi are transmitted through host seed, and colonize aerial tissues that develop from infected shoot apical meristems of the seedling and tillers. We present evidence that vegetative hyphae of Neotyphodium and Epichloe species infect grass leaves via a novel mechanism of growth, intercalary division and extension. Hyphae are attached to enlarging host cells, and cumulative growth along the length of the filament enables the fungus to extend at the same rate as the host. This is the first evidence of intercalary growth in fungi and directly challenges the centuries-old model that fungi grow exclusively at hyphal tips. A new model describing the colonization of grasses by clavicipitaceous endophytes is described.
-
growth of epichloe Neotyphodium and p endophytes in leaves of lolium and festuca grasses
Fungal Biology, 2002Co-Authors: Michael J. Christensen, Raymond J Bennett, Jan SchmidAbstract:Epichloe spp. (Clavicipitaceae) and their close asexual relatives, Neotyphodium spp., form systemic endophytic associations with Pooideae grasses. Interactions between Lolium and Festuca host grasses and fungal endophytes were examined in studies focusing on leaves of natural associations and also of plants into which the endophyte was introduced by seedling inoculation. Light microscopy as well as fungal isolation was used to locate the position of hyphae, while transmission electron microscopy was used to examine host/fungus interactions at the cellular level. These studies provide support for synchronised plant and endophyte growth. This characteristic pattern of growth was maintained when both Epichloe and Neotyphodium spp. were introduced by seedling inoculation into new host species. Hyphae, with few exceptions, grew rapidly as leaves grew and ceased when leaf growth ceased. This pattern of growth offers an explanation for the characteristic appearance of hyphae in leaf sheaths of host grasses; seldom branched and for all species other than N. occultans, aligned parallel to the leaf axis. Hyphal growth of a second group of endophytic fungi, referred to as p-endophytes, was not regulated in the same way, with growth continuing as leaves aged. This pattern of growth gives rise to high concentrations of branched ramifying hyphae in old leaf sheaths. Although host genotype did not effect the basic pattern of hyphal growth of Epichloe/Neotyphodium endophytes, it strongly influenced the concentration and distribution of hyphae throughout leaves. Examination by TEM revealed no evidence that penetration of hyphae into dense tissue was aided by the secretion of pectic enzymes that loosen the middle lamella connecting host cells. Instead it appeared that penetration occurred by hyphae physically pushing between cells. The absence of enzymatic loosening of the middle lamella during penetration could explain why hyphae of these endophytes apparently do not elicit host defence reactions in natural associations. However, subtle interactions between the hyphae of Epichloe/Neotyphodium and host cells in natural associations were observed.
-
the evolutionary origins of epichloe endophytes from annual ryegrasses
Mycologia, 2000Co-Authors: Christina D Moon, Christopher L Schardl, Barry Scott, Michael J. ChristensenAbstract:The annual Lolium (ryegrass) species were surveyed for seedborne, asexual Epichloe endo- phytes (i.e., Neotyphodium spp.). Endophytes that were extremely slow growing in culture were identi- fied from all seven annual ryegrass spp. Sequences of ,3-tubulin gene (tub2) introns and the internal tran- scribed spacers of the nuclear ribosomal RNA (rDNA- ITS) were determined, as were profiles based on five microsatellite loci. Two taxonomic groups of annual ryegrass endophytes were distinguishable by mor- phology, culturability and genotypes. One group, for which the name Neotyphodium occultans is proposed, comprised only isolates that could not be subcultured on potato dextrose agar medium. These isolates con- tained multiple tub2 genes and multiple alleles at mi- crosatellite loci, suggestive of an interspecific hybrid origin. The results of phylogenetic analysis implied that the ancestors of N. occultans included Epichloe baconii and E. bromicola, or close relatives. The sec- ond taxonomic group of annual ryegrass endophytes, exemplified by isolate Lc4, was found only in L. can- ariense. This taxon had a single tub2 sequence and microsatellite genotype that was nearly identical to those of extant Epichloi typhina isolates that infect Poa nemoralis. Therefore, it is proposed that Lc4 is an asexual derivative of an E. typhina genotype close- ly related to extant strains from P nemoralis. We pro- pose the name Neotyphodium typhinum var. canariense for this taxon. These two new endophyte taxa from the annual ryegrasses are further examples of asexual Neotyphodium species that have arisen from sexual Ep-
-
Infection of tall fescue and perennial ryegrass plants by combinations of different Neotyphodium endophytes.
Fungal Biology, 2000Co-Authors: Michael J. Christensen, Wayne R. Simpson, T. Al SamarraiAbstract:Individual tall fescue (Festuca arundinacea) plants infected with both Neotyphodium coenophialum and N. lolii endophytes, and individual perennial ryegrass (Lolium perenne) plants infected with both N. lolii and Neotyphodium LpTG-2, were obtained following inoculation of naturally infected seedlings with the second endophyte. Differences in the ability of the endophytes to produce conidia, together with colony characteristics, enabled the endophytes in plants to be identified following incubation of excised leaf tissue on potato dextrose agar. Most tillers of dually infected plants were infected with just a single endophyte, but tillers infected with two endophytes were identified in three tall fescue plants. In these tillers one endophyte was always present at a much higher concentration than the other. Over time the incidence of dually infected tillers decreased and no tillers with both N. coenophialum and N. lolii were present 5 months after the associations were established. No evidence was obtained of exchange of nuclei between different endophytes present in dually infected tillers giving rise to heterokaryons or interspecific hybrids.
Carolyn A Young - One of the best experts on this subject based on the ideXlab platform.
-
prevalence of an intraspecific Neotyphodium hybrid in natural populations of stout wood reed cinna arundinacea l from eastern north america
Mycologia, 2011Co-Authors: Sita R Ghimire, Jennifer A. Rudgers, Carolyn A Young, Nikki D Charlton, Kelly D CravenAbstract:Members of genus Neotyphodium are asex- ual derivatives of sexual Epichloespecies and maintain endophytic relationships with many cool-season grass- es. Most Neotyphodium species analyzed so far are interspecific hybrids with combined or partial ge- nomes of two or three ancestral species. In this study we characterized Neotyphodium isolates from Cinna arundinacea, a perennial cool-season grass from eastern North America. A total of 23 isolates grouping into two distinct morphotypes were obtained from five local populations of C. arundinacea .P CR amplification and cloning of translation-elongation factor 1-a (tefA) and b-tubulin (tubB) genes of 10 isolates comprising both morphotypes (two isolates per location) revealed that all 10 contain two copies of tefA and tubB genes. Surprisingly phylogenetic analysis of mainly non-coding sequence from these genes revealed that both copies in each isolate were inherited from Epichloetyphina ancestors, indicating that the C. arundinacea endophytes arose through intraspecific hybridization between two E. typhina progenitors with extant relatives infecting hosts Poa nemoralis and Poa pratensis. Furthermore the tefA sequences were identical between isolates, as were tubB sequences, despite obvious morphological dif- ferences. Profiling of alkaloid biosynthetic genes from these isolates indicated the presence of the peramine biosynthetic gene (perA) and the absence of genes required for biosynthesis of lolines, indole-diterpenes and ergot alkaloids. Thus this endophyte is potentially capable of producing peramine in planta and providing protection to its host from insect pests. The absence of genes for indole-diterpenes and ergot alkaloid biosynthesis makes this endophyte a candi- date for agricultural applications. Based on our phylogenetic analysis, alkaloid profiling and descrip- tion of morphological characteristics, we propose the name Neotyphodium schardlii for these isolates from C. arundinacea, a new member of genus Neotypho- dium and the first described to have arisen through
-
indole diterpene biosynthetic capability of epichloe endophytes as predicted by ltm gene analysis
Applied and Environmental Microbiology, 2009Co-Authors: Carolyn A Young, Christina D Moon, Christopher L Schardl, B A Tapper, Barry ScottAbstract:Bioprotective alkaloids produced by Epichloe and closely related asexual Neotyphodium fungal endophytes protect their grass hosts from insect and mammalian herbivory. One class of these compounds, known for antimammalian toxicity, is the indole-diterpenes. The LTM locus of Neotyphodium lolii (Lp19) and Epichloe festuce (Fl1), required for the biosynthesis of the indole-diterpene lolitrem, consists of 10 ltm genes. We have used PCR and Southern analysis to screen a broad taxonomic range of 44 endophyte isolates to determine why indole-diterpenes are present in so few endophyte-grass associations in comparison to that of the other bioprotective alkaloids, which are more widespread among the endophtyes. All 10 ltm genes were present in only three epichloe endophytes. A predominance of the asexual Neotyphodium spp. examined contained 8 of the 10 ltm genes, with only one N. lolii containing the entire LTM locus and the ability to produce lolitrems. Liquid chromatography-tandem mass spectrometry profiles of indole-diterpenes from a subset of endophyte-infected perennial ryegrass showed that endophytes that contained functional genes present in ltm clusters 1 and 2 were capable of producing simple indole-diterpenes such as paspaline, 13-desoxypaxilline, and terpendoles, compounds predicted to be precursors of lolitrem B. Analysis of toxin biosynthesis genes by PCR now enables a diagnostic method to screen endophytes for both beneficial and detrimental alkaloids and can be used as a resource for screening isolates required for forage improvement.
-
a complex gene cluster for indole diterpene biosynthesis in the grass endophyte Neotyphodium lolii
Fungal Genetics and Biology, 2006Co-Authors: Carolyn A Young, Silvina Andrea Felitti, Katherine Shields, German Spangenberg, Richard D Johnson, Gregory T Bryan, Sanjay Saikia, Barry ScottAbstract:Abstract Lolitrems are a structurally diverse group of indole-diterpene mycotoxins synthesized by Epichloe/Neotyphodium endophytes in association with Pooid grasses. Using suppression subtractive hybridization combined with chromosome walking, two clusters of genes for lolitrem biosynthesis were isolated from Neotyphodium lolii, a mutualistic endophyte of perennial ryegrass. The first cluster contains five genes, ltmP, ltmQ, ltmF, ltmC, and ltmB, four of which appear to be orthologues of functionally characterized genes from Penicillium paxilli. The second cluster contains two genes, ltmE and ltmJ, that appear to be unique to lolitrem biosynthesis. The two clusters are separated by a 16 kb AT-rich sequence that includes two imperfect direct repeats. A previously isolated ltm cluster composed of ltmG, ltmM, and ltmK, is linked to these two new clusters by 35 kb of AT-rich retrotransposon relic sequence. All 10 genes at this complex LTM locus were highly expressed in planta but expression was very low or undetectable in mycelia. ltmM and ltmC were shown to be functional orthologues of P. paxilli paxM and paxC, respectively. This work provides a genetic foundation for elucidating the metabolic grid responsible for the diversity of indole-diterpenes synthesized by N. lolii.
-
Elimination of ergovaline from a grass-Neotyphodium endophyte symbiosis by genetic modification of the endophyte.
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Daniel G Panaccione, Barry Scott, Carolyn A Young, Richard D Johnson, Jinghong Wang, Prapassorn Damrongkool, Christopher L SchardlAbstract:The fungal endophytes Neotyphodium lolii and Neotyphodium sp. Lp1 from perennial ryegrass (Lolium perenne), and related endophytes in other grasses, produce the ergopeptine toxin ergovaline, among other alkaloids, while also increasing plant fitness and resistance to biotic and abiotic stress. In the related fungus, Claviceps purpurea, the biosynthesis of ergopeptines requires the activities of two peptide synthetases, LPS1 and LPS2. A peptide synthetase gene hypothesized to be important for ergopeptine biosynthesis was identified in C. purpurea by its clustering with another ergot alkaloid biosynthetic gene, dmaW. Sequence analysis conducted independently of the research presented here indicates that this gene encodes LPS1 [Tudzynski, P., Holter, K., Correia, T., Arntz, C., Grammel, N. & Keller, U. (1999) Mol. Gen. Genet. 261, 133–141]. We have cloned a similar peptide synthetase gene from Neotyphodium lolii and inactivated it by gene knockout in Neotyphodium sp. Lp1. The resulting strain retained full compatibility with its perennial ryegrass host plant as assessed by immunoblotting of tillers and quantitative PCR. However, grass–endophyte associations containing the knockout strain did not produce detectable quantities of ergovaline as analyzed by HPLC with fluorescence detection. Disruption of this gene provides a means to manipulate the accumulation of ergovaline in endophyte-infected grasses for the purpose of determining the roles of ergovaline in endophyte-associated traits and, potentially, for ameliorating toxicoses in livestock.
