Wade J. Mace

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

Semi-quantitative and structural metabolic phenotyping by direct infusion ion trap mass spectrometry and its application in genetical metabolomics

The identification of quantitative trait loci (QTL) for plant metabolites requires the quantitation of these metabolites across a large range of progeny. We developed a rapid metabolic profiling method using both untargeted and targeted direct infusion tandem mass spectrometry (DIMSMS) with a linear ion trap mass spectrometer yielding sufficient precision and accuracy for the quantification of a large number of metabolites in a high-throughput environment. The untargeted DIMSMS method uses top-down data-dependent fragmentation yielding MS2 and MS3 spectra. We have developed software tools to assess the structural homogeneity of the MS2 and MS3 spectra hence their utility for phenotyping and genetical metabolomics. In addition we used a targeted DIMS(MS) method for rapid quantitation of specific compounds. This method was compared with targeted LC/MS/MS methods for these compounds. The DIMSMS methods showed sufficient precision and accuracy for QTL discovery. We phenotyped 200 individual Lolium perenne genotypes from a mapping population harvested in two consecutive years. Computational and statistical analyses identified 246 nominal m/z bins with sufficient precision and homogeneity for QTL discovery. Comparison of the data for specific metabolites obtained by DIMSMS with the results from targeted LC/MS/MS analysis showed that quantitation by this metabolic profiling method is reasonably accurate. Of the top 100 MS1 bins, 22 ions gave one or more reproducible QTL across the 2 years. Copyright

The use of genomics and metabolomics methods to quantify fungal endosymbionts and alkaloids in grasses.

The association of plants with endosymbiotic micro-organisms poses a particular challenge to metabolomics studies. The presence of endosymbionts can alter metabolic profiles of plant tissues by introducing non-plant metabolites such as fungal specific alkaloids, and by metabolic interactions between the two organisms. An accurate quantification of the endosymbiont and its metabolites is therefore critical for studies of interactions between the two symbionts and the environment.Here, we describe methods that allow the quantification of the ryegrass Neotyphodium lolii fungal endosymbiont and major alkaloids in its host plant Lolium perenne. Fungal concentrations were quantified in total genomic DNA (gDNA) isolated from infected plant tissues by quantitative PCR (qPCR) using primers specific for chitinase A from N. lolii. To quantify the fungal alkaloids, we describe LC-MS based methods which provide coverage of a wide range of alkaloids of the indolediterpene and ergot alkaloid classes, together with peramine.

Variation in Alkaloid Production from Genetically Diverse Lolium Accessions Infected with Epichloë Species.

Widespread infection of Epichloë occultans in annual ryegrass in Australia suggests that infection provides its weedy host, Lolium rigidum, some ecological advantage. Initial studies determined the distribution and profiles of known Epichloë alkaloids (epoxy-janthitrems, ergovaline, lolines, lolitrem B, and peramine) in plant extracts using a combination of GC-FID and HPLC techniques utilizing a single accession of Australian L. rigidum. However, the lolines N-acetylnorloline (NANL) and N-formylloline (NFL) were the only alkaloids detected and were highly concentrated in the immature inflorescences of mature plants. Additional glasshouse studies subjected a wide range of Australian L. rigidum haplotypes and international annual Lolium accessions to a suite of analyses to determine alkaloid levels and profiles. Again, NFL and NANL were the key lolines produced, with NFL consistently predominating. Considerable variation in alkaloid production was found both within and between biotypes and accessions evaluated under identical conditions, at the same maturation stage and on the same tissue type. The pyrrolopyrazine alkaloid peramine was also present in 8 out of 17 Australian biotypes of L. rigidum and 7 out of 33 international accessions infected with Epichloë spp.; the highest peramine concentrations were observed in seed extracts from L. rigidum collected from Australia. This study represents the first report of alkaloids from a geographically diverse collection of annual ryegrass germplasm infected with Epichloë spp. when grown under identical controlled conditions.

Endophyte-Mediated Resistance to Black Cutworm as a Function of Plant Cultivar and Endophyte Strain in Tall Fescue

ABSTRACT To improve Neotyphodium endophyte-mediated resistance to black cutworm Agrotis ipsilon (Hufnagel) (BCW), a series of experiments was conducted by using several different cultivars of tall fescue, Schedonorus arundinaceus (Schreb.) Dumort. in combination with several different haplotypes of the endophyte Neotyphodium coenophialum (Morgan-Jones & Gams) (plant cultivar × endophyte haplotype = plant line), each producing unique alkaloid profiles. BCW settling response, survival at 5 and 10 d, and larval biomass varied significantly among plant lines. In general, greater variation BCW performance was observed within a single plant cultivar infected with different endophyte haplotypes than among different plant cultivars infected with the same endophyte haplotype, but comparisons among the former were far more numerous. Although five endophytemediated alkaloids representing three alkaloid classes were quantified in the plants, the pyrrolizidine alkaloid N-acetyl norloline was consistently the single best predictor of BCW performance. BCW settling response, 5-d survival, and 10-d survival decreased as levels of the alkaloid N-acetyl norloline increased. The same three response variables also decreased with increasing levels of peramine, but increased with increasing levels of ergovaline. Minor variation in endophyte infection levels occurring among infected plant lines had no significant influence on BCW performance. Results indicate a potentially important role for N-acetyl norloline and peramine in providing resistance to black cutworm whereas ergovaline appears to be much less important. Therefore, endophyte haplotypes expressing high levels of N-acetyl norloline and peramine may be of particular importance for developing ‘friendly’ endophyte-enhanced turf and pasture grasses that resist challenging lepidopteran pests, although remaining safe for wildlife and grazing mammals.

Variation in the expression of ergot alkaloids between individual tillers of perennial ryegrass

Epichloë fungal endophytes of cool season grasses are well-known to produce a range of alkaloids of benefit to the host. Some of these compounds are advantageous to agriculture due to qualities that promote pasture persistence (e.g., the loline class of alkaloids confer insect protection) while others are detrimental to the well-being of grazing livestock. The ergot alkaloids (e.g., ergovaline), produced in ryegrass and tall fescue associations, causes poor animal health in farming regions in many countries around the world and further study is required to improve our knowledge on this class of compounds. Here we present the application of a quantitative LC-MS/MS (liquid chromatography coupled to mass spectrometry) method measuring eight ergot alkaloids (chanoclavine, agroclavine, elymoclavine, lysergol, lysergic acid, ergine, lysergyl-alanine, ergovaline) produced by endophyte infected grasses, to monitor levels in individual tillers from multiple plants of a single cultivar of perennial ryegrass (Lolium perenne cv. “Grasslands Samson”) infected with a common toxic endophyte strain (Epichloë festucae var. lolii). Monitoring the expression in individual tillers allows an estimation of the variability within a plant (between tillers) as well as between plants. The study showed that there is significant variation in the concentration of the ergot alkaloids between tillers of a single plant, at or exceeding the level of variation observed between individual plants of a population. This result emphasizes the fundamental importance of robust experimental design and sampling procedures when alkaloid expression assessment is required and these need to be rigorously tailored to the hypothesis being tested.

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

ABSTRACT 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.

The plant hormone salicylic acid interacts with the mechanism of anti-herbivory conferred by fungal endophytes in grasses: Effects of salicylic acid on fungal endophytes

The plant hormone salicylic acid (SA) is recognized as an effective defence against biotrophic pathogens, but its role as regulator of beneficial plant symbionts has received little attention. We studied the relationship between the SA hormone and leaf fungal endophytes on herbivore defences in symbiotic grasses. We hypothesize that the SA exposure suppresses the endophyte reducing the fungal-produced alkaloids. Because of the role that alkaloids play in anti-herbivore defences, any reduction in their production should make host plants more susceptible to herbivores. Lolium multiflorum plants symbiotic and nonsymbiotic with the endophyte Epichloë occultans were exposed to SA followed by a challenge with the aphid Rhopalosiphum padi. We measured the level of plant resistance to aphids, and the defences conferred by endophytes and host plants. Symbiotic plants had lower concentrations of SA than did the nonsymbiotic counterparts. Consistent with our prediction, the hormonal treatment reduced the concentration of loline alkaloids (i.e., N-formyllolines and N-acetylnorlolines) and consequently decreased the endophyte-conferred resistance against aphids. Our study highlights the importance of the interaction between the plant immune system and endophytes for the stability of the defensive mutualism. Our results indicate that the SA plays a critical role in regulating the endophyte-conferred resistance against herbivores.

The occurrence of ryegrass staggers and heat stress in sheep grazing ryegrass-endophyte associations with diverse alkaloid profiles

Abstract AIMS: To compare ryegrass pastures infected with endophytes producing diverse alkaloids for their ability to cause ryegrass staggers in grazing lambs; to compare respiration rates and rectal temperatures of these lambs after exposure to heat stress, and to compare liveweight gains during the study period. METHODS: Ryegrass pastures of cultivar Trojan infected with NEA endophytes, branded NEA2 (T-NEA2), endophyte-free Trojan (T-NIL), Samson infected with standard endophyte (S-STD), Samson infected with AR37 endophyte (S-AR37) and endophyte-free Samson (S-NIL), were grazed by lambs (n=30 per cultivar) for up to 48 days in February and March of 2012 and 2013. Pasture samples were analysed for alkaloid concentrations and lambs were scored for ryegrass staggers at intervals during the study period. Liveweight was recorded at the start (Day 0) and end of the study, and rectal temperatures and respiratory rates were measured in lambs exposed to heat stress on Days 23 and 26, in 2012 and 2013, respectively. RESULTS: Concentrations of alkaloids were lower in 2012 than 2013, associated with warmer and drier conditions in 2013, and the prevalence of ryegrass staggers was low in 2012. In 2013, concentrations of ergovaline were similar in T-NEA2 and S-STD, but concentrations of lolitrem B were lower in T-NEA2 than S-STD. S-AR37 produced epoxy-janthitrems but no lolitrem B or ergovaline. In 2013, by Day 20, 9/30 (30%) sheep grazing S-STD had severe staggers (score ≥4), and by Day 47 all sheep had been removed from this cultivar due to severe staggers. By Day 47, 18/30 (60%), 4/30 (13%) and 0/30 (0%) sheep grazing S-AR37, T-NEA2 and T-NIL pastures, respectively, had severe staggers. There were no differences in mean daily weight gain of lambs between cultivars in either year. In both years, mean rectal temperature and respiration rate following exposure to heat stress were highest in sheep grazing S-STD and T-NEA2, and lowest in sheep grazing T-NIL. CONCLUSIONS: In lambs grazing different ryegrass pastures infected with endophytes, ryegrass staggers was most severe on S-STD, less severe on S-AR37 and least on T-NEA2. When under heat stress, lambs grazing ergovaline-producing S-STD and T-NEA2 pastures had increased respiration rates and rectal temperatures compared with lambs grazing T-NIL. CLINICAL RELEVANCE: If ambient temperatures are suitable, NEA2-branded endophytes have the potential to express concentrations of ergovaline sufficient to induce heat stress in grazing sheep.

Endophyte species influence the biomass production of the native grass Achnatherum sibiricum (L.) Keng under high nitrogen availability

Abstract Research on the interaction of endophytes and native grasses normally takes infection status into account, but less often considers the species of endophyte involved in the interaction. Here, we examined the effect of endophyte infection, endophyte species, nitrogen availability, and plant maternal genotype on the performance of a wild grass, Achnatherum sibiricum. Six different Epichloë‐infected maternal lines of A. sibiricum were used in the study; three lines harbored Epichloë gansuensis (Eg), while three lines harbored Epichloë sibirica (Es). These endophytes are vertically transmitted, while Eg also occasionally produces stromata on host tillers. We experimentally removed the endophyte from some ramets of the six lines, with the infected (E+) and uninfected (E−) plants grown under varying levels of nitrogen availability. Eg hosts produced more aboveground biomass than Es hosts only under high nitrogen supply. Endophyte species did not show any influence on the maximum net photosynthetic rate (P max), photosynthetic nitrogen use efficiency, or total phenolics of A. sibiricum under all nitrogen conditions. However, the plant maternal genotype did influence the P max and shoot biomass of A. sibiricum. Our results show that endophyte species influenced the shoot biomass of A. sibiricum, and this effect was dependent on nitrogen supply. As with most coevolutionary interactions, A. sibiricum that harbored Eg and Es may show pronounced geographic variation in natural habitats with increased nitrogen deposition. In addition, stroma‐bearing endophyte (Eg) provides positive effects (e.g., higher biomass production) to A. sibiricum plants during the vegetative growth stage.