Heather H. Wilkinson

Bioprotective Alkaloids of Grass-Fungal Endophyte Symbioses

Symbiotic interactions of C3 grasses with fungal endophytes, Epichloe species and their asexual relatives Neotypkodium, often provide the grass hosts with major fitness enhancements (for review, see Siegel and Bush [1994]). The endophytes protect host plants from both biotic and abiotic environmental stresses. Documented enhancements to host fitness include greater resistance to mammalian and insect herbivores, pathogens, and nematodes, as well as increased drought tolerance and competitiveness. Our understanding of the mechanisms responsible for a11 of these effects on host fitness is not well established. However, the antiherbivore fitness enhancements are largely attributable to the accumulation of four groups of alkaloids: lolines, peramine, ergot alkaloids, and lolitrems. The objective of this Update is to present the current status of knowledge on these alkaloids, focusing on their biological activities and the potential for genetically manipulating their expression in grasslendophyte symbiota.

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

BackgroundMycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.ResultsHere we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.ConclusionsThe data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Contribution of fungal loline alkaloids to protection from aphids in a grass-endophyte mutualism.

Fungal endophytes provide grasses with enhanced protection from herbivory, drought, and pathogens. The loline alkaloids (saturated 1-aminopyrrolizidines with an oxygen bridge) are fungal metabolites often present in grasses with fungal endophytes of the genera Epichloë or Neotyphodium. We conducted a Mendelian genetic analysis to test for activity of lolines produced in plants against aphids feeding on those plants. Though most loline-producing endophytes are asexual, we found that a recently described sexual endophyte, Epichloë festucae, had heritable variation for loline alkaloid expression (Lol+) or nonexpression (Lol-). By analyzing segregation of these phenotypes and of linked DNA polymorphisms in crosses, we identified a single genetic locus controlling loline alkaloid expression in those E. festucae parents. We then tested segregating Lol+ and Lol- full-sibling fungal progeny for their ability to protect host plants from two aphid species, and observed that alkaloid expression cosegregated with activity against these insects. The in planta loline alkaloid levels correlated with levels of anti-aphid activity. These results suggested a key role of the loline alkaloids in protection of host plants from certain aphids, and represent, to our knowledge, the first Mendelian analysis demonstrating how a fungal factor contributes protection to plant-fungus mutualism.

Shifts in microbial community structure along an ecological gradient of hypersaline soils and sediments

Studies of hypersaline ecosystems often yield novel organisms and contribute to our understanding of extreme environments. Soils and sediments from La Sal del Rey, a previously uncharacterized, hypersaline lake located in southern Texas, USA, were surveyed to characterize the structure and diversity of their microbial communities. Samples were collected along a transect that spanned vegetated uplands, exposed lakebed sediments, and water-logged locations, capturing a wide range of environments and physical and chemical gradients. Community quantitative PCR (qPCR) was used in combination with tag-encoded pyrosequencing, 16S rRNA gene cloning, and Sanger sequencing to characterize the lakes soil and sediment microbial communities. Further, we used multivariate statistics to identify the relationships shared between sequence diversity and heterogeneity in the soil environment. The overall microbial communities were surprisingly diverse, harboring a wide variety of taxa, and sharing significant correlations with site water content, phosphorus and total organic carbon concentrations, and pH. Some individual populations, especially of Archaea, also correlated with sodium concentration and electrical conductivity salinity. Across the transect, Bacteria were numerically dominant relative to Archaea, and among them, three phyla—the Proteobacteria, Bacteroidetes, and Firmicutes—accounted for the majority of taxa detected. Although these taxa were detected with similar abundances to those described in other hypersaline ecosystems, the greater depth of sequencing achieved here resulted in the detection of taxa not described previously in hypersaline sediments. The results of this study provide new information regarding a previously uncharacterized ecosystem and show the value of high-throughput sequencing in the study of complex ecosystems.

Production of loline alkaloids by the grass endophyte, Neotyphodium uncinatum, in defined media.

Lolines (saturated 1-aminopyrrolizidines with an oxygen bridge) are insecticidal alkaloids produced in symbioses of certain Epichloë (anamorph-Neotyphodium) species (fungal endophytes) with grasses, particularly of the genera Lolium and Festuca. Prior to the present study, it was unknown whether lolines were of plant or fungal origin. Neotyphodium uncinatum, the common endophyte of meadow fescue (Lolium pratense=Festuca pratensis) produced loline, N-acetylnorloline, and N-formylloline when grown in the defined minimal media at pH 5.0-7.5, with both organic and inorganic nitrogen sources and sugars as carbon sources. In contrast, lolines were not detected in complex medium cultures. GC-MS and 13C NMR spectroscopic analyses confirmed the identity of the alkaloids isolated from the defined medium cultures. Lolines accumulated to ca. 700 mg/l (4 mM) in cultures with 16.7 mM sucrose and 15-30 mM asparagine, ornithine or urea. Kinetics of loline production and fungal growth were assessed in defined medium with 16.7 mM sucrose and 30 mM ornithine. The alkaloid production rate peaked after the onset of stationary phase, as is common for secondary metabolism in other microbes.

DIVERGENCE IN SYMBIOTIC COMPATIBILITY IN A LEGUME-BRADYRHIZOBIUM MUTUALISM

Geographic variation in the mutualism between the legume Amphicarpaea bracteata and its nitrogen‐fixing root nodule bacteria (Bradyrhizobium sp.) was analyzed by sampling genotypes from 11 sites separated by distances ranging from 0.6 km to more than 1000 km. Cross inoculation experiments revealed that plants were genetically differentiated in traits determining compatibility with mutualist partners from different sites. Combinations of plant and bacterial genotypes native to the same local habitat yielded 26% higher plant growth relative to non‐native combinations (range across 4 experiments; 9% to 48%). Among non‐native symbioses, plant growth was unrelated to the geographic distance between sites of plant and bacterial origin. However, compatibility varied significantly with the genetic distance among host populations (inferred by multilocus enzyme electrophoresis): genetically similar plants from separate sites showed superior growth with each others mutualist partners. Nevertheless, the tree structure of population genetic similarity was not congruent in plants versus bacteria. This implies that adaptive variation in symbiotic compatibility has evolved without strictly parallel divergence in the two species.

Increased conidiation associated with progression along the sterigmatocystin biosynthetic pathway

The Aspergillus nidulans sterigmatocystin (ST) gene cluster contains both regulatory (aflR) and biosynthetic genes (stc genes) required for ST production. A total of 26 genes are in the cluster, 13 of which have been assigned a known function in the biosynthetic pathway. This complex secondary pathway represents a physiological cost to the fungus. We tested the amount of asexual spore production using a series of isogenic lines of A. nidulans, differing only in a mutation in aflR (resulting in a strain containing no ST intermediates) or a mutation in three stc genes that produced either no ST intermediates (ΔstcJ), an early ST intermediate, norsoloroinic acid (ΔstcE) or a late ST intermediate, versicolorin A (ΔstcU). In two independently replicated experiments we compared the numbers of conidia produced by each of these mutant strains and a wild type ST producer in a neutral (growth media) and a host (corn seed) environment. A stepwise increase in asexual spore production was observed with each progressive step in the ST pathway. Thus, the data suggest that recruitment or loss of these secondary metabolite pathway genes has a selective advantage apart from the physiological activity of the metabolite.

FSR1 Is Essential for Virulence and Female Fertility in Fusarium verticillioides and F. graminearum

Fusarium verticillioides (teleomorph Gibberella moniliformis) and F. graminearum (teleomorph G. zeae) are well known to cause devastating diseases on cereal crops. Despite their importance, our understanding of the molecular mechanisms involved in these host-pathogen interactions is limited. The FSR1 locus in F. verticillioides was identified by screening REMI mutants for loss of virulence in maize stalk rot inoculation studies. FSR1 encodes an 823-codon open reading frame interrupted by two introns. The Fsr1 protein shares 60% sequence identity with the Sordaria macrospora Pro11, a multimodular protein with four putative protein-protein binding domains (caveolin-binding domain, coiled-coil structure, calmodulin-binding motif, and seven-WD40 repeats), which plays a regulatory role in cell differentiation and ascocarp development. Our data demonstrate that FSR1 is essential for female fertility and virulence in F. verticillioides. Significantly, targeted disruption of the FSR1 ortholog in F. graminearum (FgFSR1) reduced virulence on barley and deterred perithecia formation. Cross-complementation experiments demonstrated that the gene function is conserved in the two Fusarium species. FSR1 is expressed constitutively, and we hypothesize that Fsr1 regulates virulence by acting as a scaffold for a signal transduction pathway. A survey of available genome databases indicates Fsr1 homologs are present in a number of filamentous fungi and animal systems but not in budding yeast or plants. A maximum likelihood analysis of this gene family reveals well-supported monophyletic clades associated with fungi and animals.

Identification and Quantification of Methanogenic Archaea in Adult Chicken Ceca

ABSTRACT By using molecular methods for the identification and quantification of methanogenic archaea in adult chicken ceca, 16S rRNA genes of 11 different phylotypes, 10 of which were 99% similar to Methanobrevibacter woesei, were found. Methanogen populations, as assessed by cultivation, and the 16S rRNA copy number were between 6.38 and 8.23 cells/g (wet weight) and 5.50 and 7.19 log10/g (wet weight), respectively.

NONRANDOM GENOTYPIC ASSOCIATIONS IN A LEGUME- BRADYRHIZOBIUM MUTUALISM

Genetically divergent lineages often coexist within populations of the annual legume Amphicarpaea bracteata. At one site dominated by two such lineages (termed biotypes “C” and “S”), isolates of root‐nodule bacteria (Bradyrhizobium sp.) were sampled from both hosts and analyzed by enzyme electrophoresis. Symbiont populations on the two plant biotypes were highly distinct. Out of 15 bacterial multilocus genotypes detected (among 51 isolates analyzed), only one was shared in common by the two plant biotypes. Cluster analysis revealed three bacterial lineages (designated I, II, and III), with lineage I found exclusively on biotype C plants, and the two other lineages almost completely restricted to biotype S hosts. Laboratory inoculation tests indicated that lineage I bacteria were strictly specialized on biotype C hosts, forming few or no nodules on plants of the other host biotype. Bacterial lineages II and III were capable of forming nodules on both kinds of plants, but nodule numbers were often significantly higher on biotype S hosts. The nonrandom association between plant and bacterial lineages at this site implies that genetic diversity of hosts is an important factor in the maintenance of polymorphism within the symbiont population.