Variation in Alkaloid Production from Genetically Diverse Lolium

Article pubs.acs.org/JAFC

Variation in Alkaloid Production from Genetically Diverse Lolium Accessions Infected with Epichloë Species Joseph R. Moore,*,† James E. Pratley,† Wade J. Mace,‡ and Leslie A. Weston† †

Graham Centre for Agricultural Innovation (An alliance between NSW Department of Primary Industries and Charles Sturt University), School of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga, New South Wales 2650, Australia ‡ Grasslands Research Centre, AgResearch Limited, Palmerston North 4442, New Zealand ABSTRACT: 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 Nformylloline (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. KEYWORDS: alkaloids, vertical transmission, secondary metabolites, plant defense, defensive mutualism

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between individual plants,22and also with plant tissue type2320,21,24 and positively correlates with endophyte mycelium levels.21,25,26 Levels of alkaloids found in the seed coat and embryo of infected plants are often higher in strictly vertically transmitted endophytes than in horizontally transmitted endophytes.24,27,28 Alkaloid levels tend to be concentrated in the leaf sheath and developing inflorescence, although mature seeds often have high alkaloid levels relative to plant tissue.24,29 Biotic and abiotic factors influence the level of alkaloid production. Endophyte infected plants that have been clipped are reported to contain higher levels of loline than unclipped infected plants.8,30 Abiotic factors such as nitrogen application and moisture status are also known to affect alkaloid production. Increasing nitrogen has produced contrasting results; some studies show that tall fescue contained increased alkaloids at elevated N availability20,31 whereas Faeth et al. (2002)32 found no increase for Arizona fescue. Similarly, L. perenne showed no pattern for alkaloid accumulation based on nitrogen in one study33 while another34 reported decreased alkaloid concentrations as N increased. Altering soil moisture levels has produced contrasting results and is highly dependent on the grass−fungal association studied.32,35−37 The success of the endophyte relies heavily on the adaptability of its plant hosts, a useful attribute common to annual and perennial Lolium species. Lolium species are cool

INTRODUCTION Protection against herbivory in cool season grasses is often associated with the presence of fungally produced bioactive alkaloids in plant tissue, with these bioactive secondary products acting as a chemical defense. In symbiotic interactions this is commonly referred to as the defensive mutualism hypothesis.1 Epichloë fungal endophytes typically protect their host plants from herbivory by producing different classes of alkaloids. Four main classes of alkaloids are known to be produced by Epichloë spp.2 including ergopeptine alkaloids, mainly ergovaline; indole diterpene alkaloids, lolitrem B but also the epoxy-janthitrems; loline alkaloids, mainly N-acetylloline, N-formylloline, and N-acetylnorloline; and the pyrrolopyrazine alkaloid peramine. Epichloë festucae var. lolii,3 which infects the perennial ryegrass Lolium perenne L., is known to produce several bioactive alkaloids that enhance plant persistence through deterrence of herbivory.4 The range of effects produced by these alkaloids on herbivorous species and the underlying genetics of alkaloid expression have been reviewed extensively, particularly with respect to perennial ryegrass.1,5−11 Several studies have elucidated the biochemical pathways associated with different alkaloid groups,12−16 identifying key gene clusters involved in determining alkaloid expression. The discrepancy in the type and levels of alkaloids found between Epichloë species is directly related to the diversity of alkaloid genes present in each endophyte and the interaction between grass−fungal associations and different environments.11 Alkaloid production can vary with plant maturity,17 in response to seasonal and environmental conditions,18−21 © XXXX American Chemical Society

Received: June 23, 2015 Revised: October 29, 2015 Accepted: November 9, 2015

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DOI: 10.1021/acs.jafc.5b03089 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

glasshouse conditions and segregated for endophyte infection using the leaf sheath peel technique.44,45 Endophyte-infected and noninfected plants were produced successfully in the glasshouse and arranged to maximize pollen exchange between all plants to allow for cross pollination. Due to the absence of wind, pollen exchange between plants was facilitated by grouping them in close proximity and gently shaking the panicles during flowering. This enabled successful propagation of a single haplotype, segregating only by endophyte infection status, for future experimentation with both infected and uninfected seed. Subsequently, both positively and negatively endophyte-infected seeds of this haplotype were pregerminated in sterile plastic Petri dishes lined with Advantec (No. 2) filter paper with 3 mL of deionized water added. Three plants per replicate from each endophyte infection status (noninfected, E−, and infected, E+) were then transplanted into individual pots (20 cm diameter × 20 cm height) lined with polyethylene bags containing a substrate of 50:50 sand:peat mix (Lithuanian Peat Moss, Klasmann−Deilmann GmbH, Germany) with a net weight of 3 kg. Plant nutrients were applied systematically by incorporating 0.22 g of slow release fertilizer (Osmocote) into each pot according to label directions. Gravimetric moisture holding capacity was determined for the sand:peat mix, and all plants were watered to 75% field capacity twice per week. The experiment was arranged in a temperature controlled glasshouse in a randomized complete block design with three replications. Plants were maintained at 15−25 °C day−night temperatures until sampling. Epichloë infection in our living glasshouse-produced plants was determined using the tissue print-immunoblotting technique.46 Each tiller was sampled for alkaloid determination by cutting through the meristematic region and blotting cut tissue onto a nitrocellulose membrane (Whatman Protran 0.45 μm). Membrane paper was later sent to AgResearch, Grasslands Research Centre, New Zealand, for blot development and evaluation. Plant tillers were dissected into different plant parts, and three tillers from separate plants in each replicate were bulked by infection status and plant part to determine the distribution of alkaloids within the plant at flowering (Zadoks Code 68). The sampled plant parts included leaf, immature inflorescence, meristem, and pseudostem tissues (Figure 2). Limited

season grasses considered to be both pasture components and weed threats, depending on the agricultural context. In Australia, Lolium rigidum (Gaud.) is the most commonly encountered annual Lolium spp; it is considered to be a weed of crops, infesting over 12 million ha, and is the most serious and costly weed of annual winter cropping systems in southern Australia. Among major weeds of broadacre crops, annual ryegrass is associated with the largest proportion of crop losses, resulting in an estimated $139.7 million per annum in lost yield.38 In addition, the recent and rapid evolution of herbicide resistance in L. rigidum has led to the recent loss of key weed control strategies.39,40 Despite substantial expenditure on annual ryegrass control, it remains a persistent and increasing threat to Australian agricultural crop production. Epichloë occultans is frequently found in association with annual Lolium species.41 However, compared with the endophytes of important pasture species, Epichloë festucae var. lolii and Epichloë coenophiala, which infect L. perenne and Lolium arundinacea respectively, E. occultans has hitherto been little researched. Unlike other Epichloë/grass symbioses which colonize the intercellular space of all above ground tissue, E. occultans is typically localized in the meristematic region of the plant.15 E. occultans is able to successfully colonize the developing seed in an annual plant host, a feature which is a crucial stage in its life-cycle, as E. occultans is strictly a vertically transmitted asexual fungus lacking the ability to produce reproductive spores. Given that L. rigidum is a serious weed in grain crops across Australia, is highly invasive and resistant to most herbicides, and is widely infected by E. occultans, we hypothesized that E. occultans may be associated with the “weediness” of L. rigidum and through its symbiosis may increase the competitive abilities of L. rigidum. In studies conducted in our laboratory, all Australian annual ryegrass populations surveyed were shown to be positively infected by E. occultans,42 albeit at different levels of infection. In other closely related species of grasses, fungal endophytes have been shown to provide considerable benefits to their plant hosts through production of defense compounds including various alkaloids.43 Currently, the extent of this benefit is unstudied in Australian plant populations of L. rigidum. As the endophyte is exclusively but imperfectly vertically transmitted, this implies a potential ecological advantage for the endophyte symbiosis in L. rigidum. Therefore, the two primary aims of this study were to evaluate (1) the occurrence and distribution of fungally produced alkaloids in L. rigidum tissue and (2) the production of alkaloids in both Australian and international Lolium accessions. In addition, we included a less closely related L. arundinacea species and two varieties of L. perenne, all produced under similar controlled conditions, as standards for comparison with the annual Lolium species. This study represents the first comprehensive study of Epichloë alkaloids in plant tissues of annual ryegrass populations, and will also potentially improve our understanding of the role of alkaloids in plant defense, plant invasion, and symbioses.

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Figure 1. A broad geographic selection of 17 southern Australian annual ryegrass biotypes were chosen for alkaloid analysis based on the available viable endophyte in seed lots tested for endophyte presence. Geographic distribution is determined by the location of collection across southern Australian cropping systems. Bias may be present in this survey due to samples primarily being collected from cropping areas with limited representation from South Australia as few samples were available from HRTS.

MATERIALS AND METHODS

Distribution of Alkaloids within a Single Haplotype of Annual Ryegrass L. rigidum. Seed from a single haplotype (030158) was obtained in 2011−2012 from the Herbicide Resistance Testing Service (HRTS), Charles Sturt University, and comprised both endophyte-infected and noninfected seed lots. In 2010, this specific annual ryegrass haplotype was produced under controlled B


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using a linear gradient profile (eluent A = aqueous 0.1% formic acid, eluent B = acetonitrile; T0 = 5% B, T9 = 40% B, T11 = 90% B, T13 = 90% B, followed by equilibration to initial conditions over the following 8 min). Peramine was quantified by mass spectroscopy (using homoperamine as an internal standard) according to the parameters described in Rasmussen et al.48 A 5 μL injection volume gave a limit of quantitation for this technique of 0.1 μg g−1 DM (1 ppm). Lolitrem B was extracted from plant material using a method modified from Moate et al.49 A sample of lyophilized and ground grass material (50 mg) was extracted for 1 h with 1 mL of 2:1 dichloromethane/methanol. The sample was then centrifuged (5000g, 5 min) and a 500 μL aliquot of the supernatant transferred via a 0.45 μm syringe filter (PVDF) to an HPLC vial for analysis. Separation was achieved with an isocratic flow (1 mL/min, 80% dichloromethane/20% acetonitrile) using a Luna Silica 250 × 2.0 mm (5 μm) column (Phenomenex, Torrance, CA, USA), with the lolitrem B peak detected with a Shimadzu RF-10Axl fluorescence detector (excitation at 260 nm, emission detection at 410 nm). The limit of quantitation of this technique was 0.1 μg g−1 DM (0.1 ppm). Epoxy-janthitrems were detected using a modification of the method from Moate et al.49 A 50 mg sample of lyophilized and ground grass tissue was extracted for 1 h with 1 mL 80% acetone and analyzed by HPLC-fluorescence. Separation was achieved using a 4.6 × 250 mm (5 μm) ODS C18 column (Phenomenex, Torrance, CA, USA) with an isocratic flow of water−acetonitrile (1:19, 1 mL/min). Eluting compounds were detected with an Agilent Series 1100 fluorescence detector (excitation at 333 nm, emission detection at 385 nm). The limit of quantitation for this method was 0.1 μg g−1 DM (0.1 ppm). Indole diterpene intermediates of the lolitrem B biosynthetic pathway and ergot alkaloids (clavines/ergopeptines) of the ergovaline biosynthetic pathway were extracted by a method modified from that of Mace et al.22 Briefly, milled tiller samples were extracted with 500 μL of 75% methanol (containing ergotamine tartrate (Sigma Chemical Co., St. Louis, MO, USA), 0.54 μg mL−1, and homoperamine nitrate (BDG Synthesis, Lower Hutt, NZ), 1.9 μg mL−1, internal standards) for 1 h in the dark. Samples were centrifuged (5000g, 5 min) and the supernatant transferred via a 0.45 μm syringe filter (PVDF) to 2 mL amber HPLC vials. Extracts were analyzed by LC−MS/MS based on existing methods.48 This analysis allowed for direct quantification of peramine, chanoclavine, agrocalvine, elymoclavine, lysergol, lysergic acid, ergovaline, dehydroergovaline, and ergine (including epimers) as well as relative quantitation of lysergyl alanine (and epimer) and a wide range of indole diterpenes. Due to the lack of indole diterpene standards, chromatographic peak areas were normalized to an instrumental standard and sample weight (area/g). This allowed for comparison between samples for the level of production of a given indole diterpene, but did not allow comparison between indole diterepenes, either between samples or within the same sample. Peak integration was conducted using LCQuan 2.7 (Thermo Fisher Scientific Inc., San Jose, CA, USA). Survey of Endophyte Presence and Endophyte Alkaloids. Preliminary examination of endophyte presence in seed was performed by seed squash to determine seed lots that may contain viable endophyte.50 Seeds (1 g) from each seed lot were separately placed in McCartney bottles and soaked in 5% NaOH solution overnight to soften, after which seeds were rinsed with tap water to stop the process. Approximately 15 mL of Garner’s solution (0.32 g of aniline blue, 100 mL of deionized water, and 50 mL of 85% lactic acid) was then added, and bottles were subsequently heated on a laboratory hot plate until boiling, which was maintained for 10 min. Seeds were then squashed onto microscope slides with a coverslip. For consistency with previous taxonomic descriptions, Epichloë spp. infection was confirmed when rarely branched, highly convoluted hyphae, 1 to 2 μm in diameter with nonstaining septa were detected using microscopy.41,42,45 Accessions were selected and imported from the United States Department of Agriculture (USDA) germplasm center (Washington State University, Regional Plant Introduction Station, Washington,

Figure 2. Diagrammatic representations of plant parts that were dissected and bulked by replicate and infection status. seed availability precluded the testing of alkaloids in the original seed lot. Samples with confirmed endophyte status were ground under liquid nitrogen and freeze-dried for subsequent alkaloid determination. Non-endophyte-infected samples were used as negative controls to verify that selected alkaloids were only expressed in infected plant samples and to validate the accuracy of the immunoblotting method. Alkaloid Analysis. Loline alkaloids were measured using a modification of the published gas chromatographic methods.47 A sample of lyophilized and ground grass tissue (50 mg) was additionally ground with a bead ruptor (FastPrep FP120; Savant Instruments Inc., Farmingdale, NY, USA) with 3 × 3 mm stainless steel beads in a 2 mL vial (10 s at 5 m/s). Samples were extracted for 1 h with 50 μL of 40% methanol/5% ammonia and 1 mL of 1,2-dichloroethane (containing 53.7 ng mL−1 4-phenylmorpholine (Sigma Chemical Co., St. Louis, MO, USA) as internal standard) followed by centrifugation at 8000g for 5 min. Supernatant was transferred to a glass GC vial via a 10 μm filter for analysis. The analysis was conducted on a gas chromatography−flame ionization detector (Shimadzu GC2010Plus, Shimadzu Corporation, Japan) equipped with Rtx-5Sil MS capillary column (30 m × 0.25 mm × 0.25 μm film; Restek US, Bellefonte, PA 16823, USA). The limit of quantitation using this technique was 25 μg g−1 DM (25 ppm). Ergovaline was extracted from plant material by using a modified method from Baldauf et al.47 Samples were additionally ground as previously described. Samples were extracted for 1 h with 1 mL of the prepared extraction solvent (50% methanol, 0.54 μg mL−1 ergotamine tartrate (Sigma Chemical Co., St. Louis, MO, USA) as internal standard). The sample was then centrifuged (5000g, 5 min) and a 500 μL aliquot of the supernatant transferred via a 0.45 μm syringe filter (PVDF) to an HPLC vial for analysis. Ergovaline and ergotamine (and their epimers) was analyzed by HPLC using a Gemini-NX C18 150 × 2.0 mm (3 μm) column (Phenomenex, Torrance, CA, USA) with target analytes detected with a Shimadzu RF-10Axl fluorescence detector (excitation at 310 nm, emission detected at 410 nm). Separation was achieved using a linear gradient profile (eluent A = 10 mM ammonium carbonate with 20% acetonitrile (v/v), eluent B = acetonitrile; T0 = 8% B, T9 = 25% B, T7.5 = 60% B, T8 = 83.3% B, T9 = 83.3% B, followed by equilibration to initial conditions over the following 9 min) at a flow rate of 200 μL/min. The limit of quantification of this technique was 0.1 μg g−1 DM (0.1 ppm). Peramine was extracted from plant material using a method modified from Baldauf et al.47 A sample of lyophilized and ground grass tissue (50 mg) was extracted for 1 h with 1 mL of the prepared extraction solvent (50% methanol with 2.064 ng mL−1 homoperamine nitrate (BDG Synthesis, Lower Hutt, NZ) as internal standard14). The sample was then centrifuged (8000g, 5 min) and a 500 μL aliquot of the supernatant transferred via a 0.45 μm syringe filter (PVDF) to an HPLC vial for analysis. Separation was achieved on a Synergi Polar-RP 100 × 2.00 mm (2.5 μm) column (Phenomenex, Torrance, CA, USA) C


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Journal of Agricultural and Food Chemistry USA) and the Margot Forde Forage Germplasm Center (AgResearch, Grasslands, Palmerston North, New Zealand) and also obtained from D. Hume, AgResearch, Grasslands (XTR-AR1, XTR-AR37, Quantum and Concord) in New Zealand. We obtained 37 accessions in total, representing 8 Lolium species (L. arundinacea Darbysh., L. canariense Steud., L. perenne L., L. multif lorum Lam., L. persicum Boiss. & Hohen., L. remotum Shrank, L. rigidum Gaud., and L. temulentum L.). The international samples under evaluation were assembled by selecting a wide geographical range of accessions (n = 77), testing the seed for endophyte presence, and subsequently assessing plants grown from endophyte infected seed lots for endophyte infection status. Accessions were also selected on the basis of collection date, as samples of recent origin were more likely to contain viable endophyte. Only seed from accessions shown to contain endophyte were used for the experimentation described in this study, where viable endophyte presence was subsequently assessed in planta. Differences in endophyte colonization rates between accessions were hypothesized to exist due to inconsistent storage times, collection dates, and seed renewal regimes of the germplasm centers utilized. Australian L. rigidum samples obtained from the HRTS, Charles Sturt University (Table 1, Figure 1), were assigned biotype numbers

Table 2. Source of International Plant and Seed Samples aNalyzed for Epichloë-Produced Alkaloids Including Country of Origin, Species, and Germplasm Centera accession B2806 B2808 B3171 B3836 B3866 B3867 B3868 B3869 B5221 B5237 B5486 B5494 B5505 B5507 B5509 B5511 B5513 BZ1141 BZ4643 Concord PI206691 PI222807 PI229764 PI239782 PI239783 PI239784 PI239792 PI239793 PI250085 PI287857 PI298416 PI302919 PI601579 Quantum XTR-AR1 XTR-AR37

Table 1. Origin of Australian Biotypes Analyzed for EpichloëProduced Alkaloids Including Both Number of Individual Plants Analyzed and the Endophyte Infection Percentage Found in Original Seed Lota accession

location

state

no. of plants

seed E%

120077 120189 120024 120144 12001J 12002J 090030b 120310 120159 100027b 120141 100069b 120311 110050 120022 120099 120347b

Beckom Cowra Narromine Rand West Wyalong Holbrook Curramulka Millicent Epping Forest Boort Donald Hexham Lake Bolac Penhurst Corrigin Cranbrook Hyden

NSW NSW NSW NSW NSW NSW SA SA TAS VIC VIC VIC VIC VIC WA WA WA

3 4 5 4 seed seed 1 6 6 1 8 1 4 6 6 6 3

92 92 92 80 90 88 84 68 76 64 72 88 80 100 90 68 80

species L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.

temulentum remotum multif lorum canariense rigidum rigidum rigidum rigidum multif lorum rigidum remotum persicum rigidum rigidum rigidum rigidum rigidum rigidum temulentum multif lorum temulentum persicum persicum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum arundinacea perenne perenne

origin

source

Germany Denmark New Zealand France Portugal Portugal Spain Portugal Spain Portugal France Iran Portugal Greece unknown Turkey unknown Australia France New Zealand Turkey Iran Iran Portugal Portugal Portugal Greece Greece Egypt Spain Turkey Spain U.S.A New Zealand New Zealand New Zealand

MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC MFGC USDA USDA USDA USDA USDA USDA USDA USDA USDA USDA USDA USDA USDA MFGC MFGC MFGC

a

Accession number corresponds to the CSU Herbicide Resistance Testing Service (HRTS) numbering system except 12001J and 12002J, which were collected by hand and provided by a supplier (Hart Bros Seed, Wagga Wagga) respectively. bBiotypes that were excluded from a bulked sampling due to limited sample size.

All seed lots were directly imported from germplasm centers through an Australian Government Permit to import quarantine materials. Accession numbers correspond to the Margot Forde Germplasm Centre (MFGC) and the United States Department of Agriculture (USDA) germplasm resources information network databases. Accessions labelled XTR-AR1, XTR-AR37, Concord, and Quantum were obtained from D. Hume (Agresearch, Grasslands).

shared with the original database. Accession numbers of international samples correspond to the numbering system utilized by the germplasm center from which they were obtained (Table 2). Additionally, species name and origin of accession are outlined (Table 2). Fifteen Australian biotypes (Table 1) were obtained from Charles Sturt University HRTS based on endophyte viability and provided broad regional geographic spread among samples. Annual ryegrass biotypes were collected regionally from agricultural producers reporting incidence of herbicide resistance and therefore were likely representative of biotypes originating from sites under intensive crop production. Additionally, two other biotypes of annual ryegrass were included in the analysis, one from a commercial source in Holbrook, NSW, used for pasture establishment and the other originating from a naturalized area bordering a cropping paddock in West Wyalong, NSW.

Seeds were pregerminated in sterile plastic 90 mm Petri dishes lined with Advantec (No. 2) filter paper with 3 mL of deionized water added. Ten plants of each accession/biotype (Table 1) were transplanted into individual pots (150 mm diameter) containing a 50:50 mixture of sand and peat moss. Slow release fertilizer (15 g per pot, Osmocote, Scotts Co.) was added to each pot, and plants were watered regularly as needed and maintained in a protected outdoor area at ambient temperatures between May and October 2013. To allow for sufficient biomass production and to provide sufficient time for alkaloids to be expressed, plants were allowed to grow until early flowering stage. To allow for differences in flowering times between accessions utilized, sampling occurred over the month of October. Endophyte infection status was determined at flowering (Z68) in plant tillers using an immunoblot assay.51,52 Commercial phytoscreen Neotyphodium immunoblot test kits for seeds from Agrinostics were used for qualitative determination of Epichloë spp. presence in each

a

D


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Journal of Agricultural and Food Chemistry tiller sampled.53,54 Agrinostics test kits were previously found to be selective for Epichloë spp. infection in L. rigidum samples.55 The immunoblot assay used monoclonal antibodies specific to different epitopes of Epichloë cell wall proteins.56,57 In this study, analytical procedures followed those outlined by the immunoblot manufacturer (cat. #ENDO7973, Phytoscreen greenhouse grow-out tiller endophyte detection kit, Agrinostics Ltd. Co., Watkinsville, GA 30677, USA). Two tillers per infected plant were retained after endophyte presence was confirmed by the previously described immunoblot method. Subsequently, samples were frozen and retained for later selection for chemical analyses if endophyte detection was positive. Experiment 1 showed that only infected plant tillers produced loline alkaloids, and so only infected tillers were utilized for determination of endophyte alkaloids. Therefore, for further experimentation we included individual plant samples consisting of one infected immature inflorescence that was subsequently freeze-dried and then ground sufficiently to pass through a 1 mm sieve. Additionally, where sample size permitted, a bulked sample (Table 1) of remaining plant material from each biotype/accession was prepared to evaluate peramine, ergot alkaloids, and indole diterpenes. Bulked samples were used to provide uniform profiling for assessment of alkaloid production (rather than assessment of alkaloids in individual plant samples) and also allowed for a wider range of samples to be included in analyses due to insufficient yield obtained from individual samples for further analysis and detection. The full indole diterpene and ergot alkaloid biosynthetic pathways were assessed using the LC−MS/MS techniques to achieve an overall picture of the biosynthetic potential of the annual ryegrass associations surveyed. Statistical Analyses. All descriptive statistical analyses were performed using the statistical package R (version 3.1.0; R Foundation for Statistical Computing, Vienna, Austria; URL http://www.Rproject.org/). Standard error of the mean (SEM) was calculated and reported where appropriate. The distribution of alkaloids between plant parts was also subjected to an analysis of variance with replicate fitted as a random component.

Figure 3. Levels of alkaloids found in leaf, inflorescence, pseudostem, and meristem samples in bulked samples (3 plants per sample) of endophyte infected L. rigidum plant samples (n = 24). No alkaloids were detected in noninfected endophyte samples (columns on left of plant part labels) and endophyte-infected leaf samples. Different letters denote significant differences (Tukey; P < 0.05). Bars refer to standard error of means.

alkaloids. Australian biotypes of L. rigidum also typically expressed similar ranges in concentration of lolines to those of international samples; these ranged from nondetectable to 1178 μg g−1 in plant 10, accession PI239792. In comparison the highest concentration in an Australian accession was detected in accession 120311 (plant 5) at 1200 μg g−1. The range and profile of loline alkaloids were similar between Australian and international inflorescence (Z68) samples (Figure 4). Most Lolium species extracts contained NFL and NANL (Figure 6). However, the detectable limit for the lolines was 5 μg g−1, and concentrations lower than this may have remained undetected in our samples. In addition to the production of NANL and NFL, 15 seed accessions also contained NAL at low concentrations. NAL was not detected in any tillers sampled. Lolines were also not detected in seed samples infected with commercial L. perenne endophytes (AR1 and AR37). NANL was the only loline observed in the seed sample of tall fescue cv. Quantum X (infected with commercial endophyte AR542). The highest concentration of total lolines detected in sampled tissue was obtained in extracts of accession BZ4643 (L. temulentum plant 2, 1414 μg g−1), a Lolium spp. from Clermont-Ferrand in France (Figure 5). The highest concentration of total lolines found in any of the seed samples evaluated was 2220 μg g−1 in the Iranian L. persicum accession PI222807 (Figure 5). Peramine was detected in 8 of the Australian L. rigidum samples and 7 of the international annual Lolium samples infected with Epichloë spp. (Table 3 and Table 4). Certain indole diterpenes associated with early steps in the biosynthesis of lolitremanes (see Table 4) were also found in some Australian L. rigidum samples. L. persicum samples contained high levels of such indole diterpenes as paspaline, terpendole E, 13-deoxypaxilline, and terpendole C in comparison with other Lolium spp. (Table 4). However, lolitrem B and other related lolitrems were not detected in any Australian or international accessions. Additionally, specific intermediates and end products of the ergot alkaloid pathway (including chanoclavine,

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RESULTS Distribution of Alkaloids within a Single Haplotype of Annual Ryegrass. Alkaloids were not detected in any nonendophyte-infected tissue extracts observed at flowering (Z68). Ergovaline, lolitrem B, peramine, and epoxy-janthitrems were also not detected in either infected or noninfected samples. In contrast, lolines were detected in endophyte-infected tissues, and subsequent concentrations were compared among plant parts; concentrations were highest in immature inflorescence samples (175−501 ppm), followed by meristem (160−239 ppm) and pseudostem (90−170 ppm) samples. Pseudostem tissues had considerably lower levels of loline alkaloids. Loline alkaloids were not detected in any leaf samples, in clear contrast to inflorescence and stem samples. The largest variation and highest concentration of total lolines was observed in bulked inflorescence samples (Figure 3). Survey of Endophyte Alkaloids. Lolines were positively identified in inflorescence tissue samples of 15 Australian infected plant and seed haplotypes (Figure 4). However, five infected Australian plants produced no detectable loline alkaloids. NFL and NANL were present in all L. rigidum haplotypes infected with Epichoë spp., although NAL was only present in the seed samples tested. An approximate NFL to NANL ratio of 2:1 was observed in all inflorescence tissues sampled; alkaloid concentration was highly correlated with type of loline alkaloid. Significant variation in the amount of loline alkaloids was detected within and between the Australian biotypes tested, with two biotypes identified as producing particularly high (accessions 120311 and 100069) or low (accessions 120141 and 120024) concentrations of loline E


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Figure 5. Levels of total loline alkaloids found in bulked single samples of seed tissue of 2 Australian L. rigidum and 18 international annual Lolium spp. biotypes.

Figure 4. Mean levels of total loline alkaloids found in inflorescence (Z68) tissue of endophyte-infected (A) Australian samples L. rigidum (n = 15) and (B) international samples of Lolium species (n = 16). Bars refer to standard error of means.

Figure 6. Levels of different loline alkaloids found in inflorescence tissue of 62 Australian L. rigidum and 75 international annual Lolium spp. accessions infected with Epichloë species.

Australian and international annual Lolium spp. accessions shown to harbor Epichloë spp. This suggests that production of fungal alkaloids may be regulated by the plant host genetics as well as environmental influences including temperature, drought stress, and herbivore feeding pressure.8,58−60 We also observed the widespread occurrence of peramine and indole diterpene intermediates in glasshouse-grown accessions of both Australian and international Lolium spp. including L. rigidum. For the L. rigidum haplotype 030158, lolines were the only alkaloids detected and were produced, as expected, only in infected plants. Enhanced plant defense and specifically herbivore resistance resulting from the presence of loline alkaloids in seedling and mature plant tissues may be important in explaining the high frequency of E. occultans symbiosis observed in the majority of annual ryegrass populations

agroclavine, elymoclavine, lysergol, lysergic acid, ergovaline, ergine, and lysergol alanine) were not detected in any extracts obtained from the associations evaluated. L. perenne infected with commercial endophyte AR37 was used as a positive control for the production of epoxyjanthitrems under the experimental conditions, and was the only sample in which these alkaloids were detected (Table 4).

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DISCUSSION Loline alkaloids were differentially distributed within infected tissue of the single Australian haplotype of L. rigidum evaluated in glasshouse experimentation (accession 030158) (Figure 3). Despite production under identical conditions, high variation in loline alkaloid production was observed in a wide range of F


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hosts grow with the leaf through intercalary extension and therefore may colonize most aerial regions of a perennial grass host. In contrast, E. occultans, which infects annual hosts, is uniquely concentrated within 1−2 mm of the meristematic regions of the plant.41,42 This suggests that the mechanism of intercalary growth may be absent from E. occultans associations, with growth restricted to the rapidly differentiating tissue of the meristematic region and inflorescence. Perennial hosts may benefit from endophyte presence throughout the plant, thus protecting vegetative growth and ensuring plant survival year on year. Conversely, in annual host plants, the limited expansive range of the endophyte would likely limit its impact on host nutrition, allowing the plant to utilize and focus its resources during a short life-cycle. Localization of the endophyte in L. rigidum results in protection of the important meristematic tissues (pseudostem and inflorescence) and regenerative organs (the seed). Seed dissemination of the endophyte in annual plants is of particular importance to both endophyte and plant to ensure continued survival of the endophyte and potential for enhanced plant defense in young seedlings. It has been shown that the presence of fungally produced alkaloids may reduce seed predation by insects.62 Levels of alkaloids found in the seed coat and embryo of infected plants are often higher in endophytes that are vertically transmitted in comparison with those exhibiting horizontal transmission of endophytes.24,27,28

Table 3. Levels of Major Epichloë Alkaloids (ppm) Found in Inflorescence Tissue of Australian Biotypes of L. rigiduma biotype

lolitrem B

epoxy-janthitrems

peramine

ergovaline

120141 120024 120159 120189 120144 120077 090030 120099 120022 120310 110050 120311 12001J 12002J

nd nd nd nd nd nd nd nd nd nd nd nd nd nd


nd trace 2.5 1.6 nd 2.4 nd 1.5 trace 1.6 3.1 trace 4.6 20.2


a

nd denotes below detection limit (0.03 ppm); trace denotes below limit of quantitation (0.1 ppm).

sampled from a broad geographic distribution across Australia.42 However, the lack of detectable loline alkaloids in some leaf samples may be explained by the specific and restricted growth habit of E. occultans within the plant.41 Christensen et al.61 showed that Epichloë fungi from perennial

Table 4. Levels of Epichloë Alkaloids (ppm) Found in Bulked Accessions of Lolium Speciesa alkaloid accession Quantum B3171 120293 XTR-AR1 XTR-AR37 PI222807 PI229764 120077 120099 120159 120189 120310 PI302919 1100502 B3866 B3867 B5511 BZ1141 H2011 PI239782 PI239783 PI239784 PI250805 PI250085 PI287857 12001J PI206691 BZ4643 PI206691

species L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.

arundinacea multif lorum multif lorum perenne perenne persicum persicum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum rigidum temulentum temulentum temulentum

tissue

peramine

paspaline

Terp-E

Pasp-B

13-Des

paxilline

Terp-C

Jan

seed inflorescence inflorescence seed seed inflorescence inflorescence inflorescence inflorescence inflorescence inflorescence inflorescence inflorescence inflorescence inflorescence seed inflorescence inflorescence seed seed seed seed seed inflorescence inflorescence seed inflorescence inflorescence inflorescence

23.9 4.3 nd 34.2 nd 5.1 4.7 2.4 1.5 2.5 1.6 1.6 nd 3.1 nd nd 2.7 nd 20.2 nd nd nd 1.0 nd nd 4.6 1.2 1.0 5.3

67 194 3 485 103 277 195 nd nd nd nd 4 nd 52 nd 64 8 8 314 116 8 3 48 4 4 328 nd nd 45

nd 33 nd 4 nd 52 61 nd nd nd nd nd nd 9 2 3 nd nd 74 19 nd nd nd nd nd 96 nd nd nd

nd nd nd 4 nd 3 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

nd 27 nd 5 13 246 383 nd nd nd nd nd 3 nd nd 43 nd nd 138 26 nd nd 15 nd nd 130 nd nd 15

nd nd nd nd nd 3.4 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 2.0 nd nd nd

17 570 2 372 nd 1039 1038 nd nd nd nd nd nd 38 5 352 11 10 1102 119 nd 11 110 3.2 32 665 nd nd 505

nd nd nd nd 1269 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

a

Accessions that did not contain these alkaloids were excluded from table. nd denotes below detection limit. Terp-E is terpendole E, Pasp-B is paspaline B, 13-Des is 13-desoxypaxilline, Terp-C is terpendole C, and Jan is epoxy-janthitrem I. G


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in New Zealand,71 although similar effects were not found in E. occultans infected L. multif lorum, presumably due to lower peramine concentrations observed within the plant in this association than in those with the endophyte specific to perennial ryegrass (E. festucae var. lolii). Peramine concentrations in seed tissues assessed in these experiments were higher than that of inflorescence tissue. This may be associated with protection from seed predation although these findings may be limited by the small sample size of seed extracts examined. Peramine presence in Australian L. rigidum haplotypes suggests bioactive potential in Australian plants; however, further experimentation is required to test this hypothesis with a selection of insect pests known to challenge L. rigidum. Limited data exist as to peramine production in E. occultans. Hume et al. (Pers. comm.) analyzed E. occultansinfected Lolium hybridium (L. multif lorum crossed with L. perenne) seed for alkaloid profiles and detected peramine at low levels relative to those in perennial ryegrass. Sugawara et al. (2006) also found peramine (6.3 ppm) in a single collected sample of L. multif lorum. In this study, peramine was found in the host species L. temulentum, L. rigidum (both Australian and international), L. persicum, and L. multif lorum at concentration levels known to be bioactive. The variability in peramine production may be due to host genetic differences, symbiont genetic differences, or an interaction of these with the environment. The bioactivity of the lolines and peramine have been shown to enhance the competitiveness of a host grass plant at similar levels as in this study.5,43,67,72 The widespread detection of peramine and loline alkaloids in geographically diverse populations of annual ryegrass and across several tissue types likely implies an ecological purpose for their production, and further suggests potential protection from invertebrate herbivory. Ergovaline and lolitrem B were not detected in any of the infected samples tested. This is not surprising as ergovaline and lolitrem B have not been identified previously in plants hosting E. occultans.64,73 Kuldau and Bacon (2008) suggest that their presence is associated with reduced vertebrate predation, but we did not observe their presence in any infected samples, congruent with the absence of animal toxicity found in annual ryegrasses. In our study, no ergot alkaloid pathway intermediates or products, such as chanoclavine, agroclavine, elymoclavine, lysergol, lysergic acid, ergovaline, ergine, and lysergol alanine, were detected in the associations tested. We did detect, however, several precursor compounds of interest in the indole diterpene biosynthetic pathway (Table 4), implying that one of the other major alkaloid pathways is partially but not entirely expressed in this association. Similar to the ergovaline, the biosynthetic pathway of indole diterpenes consists of multiple linked genes that are required for the production of lolitrem B. Deleterious mutations of the endophyte genome, with no selection pressure for retention of the complete pathway, over time may have led to the loss of the some of the complex alkaloid biosynthesis mechanisms, thus resulting in the relatively simple profile found in the associations surveyed in this study. Some of these indole diterpene intermediates, such as terpendole C, show minor tremogenic effects against vertebrate predators and may also be bioactive against invertebrate predators.5 Further work is needed to establish whether these intermediates of lolitrem B are also important for plant protection from invertebrates.

This is presumably associated with the benefits of enhanced protection against herbivores provided during the critical periods of seed dissemination and may be suggestive of adaptive evolution leading to enhanced plant defense. Fungal alkaloids which support the protection of seed in annual plants may also be associated with enhanced survival of both fungi and plant. Plant to plant variation in total loline production within a population was significant among various plant accessions when produced under similar glasshouse conditions, and varied by 2− 3-fold in total concentrations assessed. However, mean total loline concentrations varied by up to 100-fold in L. rigidum seed samples, up to 12-fold in Australian samples and up to 60-fold in international samples. Peramine levels showed a 4-fold variation in seed samples and greater than 10-fold variation in tiller samples. In a few cases, plants that tested positive for endophyte infection showed no detectable alkaloids. This may be due to limitations of the immunoassay technique employed for endophyte detection,63 differences in endophyte biomass present resulting in alkaloids expressed below the detectable limits in the analysis, or the absence of production of these specific alkaloids by the particular haplotype of endophyte present. Loline alkaloids were detected in most Epichloë infected accessions/biotypes. However, only N-acetylnorloline (NANL) and N-formylloline (NFL) were detected in plant tissue samples. N-Acetylloline (NAL) was only detected in seed of 12 accessions at low levels, combined with high levels of NANL and NFL. NAL may simply be produced below the limit of detection of the analysis method used or perhaps endophyte detection resulted in a false positive. Tepaske et al. (1993) found that E. occultans infected hosts produced lolines in USDA accessions L. multif lorum (PI410154), L. temulentum (PI249725), L. persicum (PI222807), and L. rigidum (PI250805).64 The latter two accessions were also evaluated in this study and were shown to contain lolines. E. occultans infected Italian ryegrass (L. multif lorum) collected in western Japan contained high concentrations of N-formylloline, ranging from 496 to 1031 ppm,65 and this was also consistent with our findings in several hosts. Loline alkaloids typically demonstrate broad spectrum insecticidal activity, although their mode of action has not been clearly elucidated to date.16 Lolines act as both metabolic toxins and feeding deterrents depending on the species of insect challenging the plant with different effective concentrations depending on the target insect and loline species.66−68 N-Formylloline, the predominant loline chemical species found, is toxic to several different insects from ingestion, topical, and injected applications.69 NFL was active against some insect species well below the concentrations found in most of the loline-expressing accessions tested in this study. From our studies evaluating production of loline alkaloids in diverse Lolium genotypes, we can infer that the concentrations of loline alkaloids observed in stem and inflorescence tissues were well within ranges associated with bioactivity against certain plant-feeding insects. These findings raise intriguing questions regarding the nature and extent of the effects produced by loline alkaloids with different environmental conditions regulating plant and fungal growth. Peramine is the most common class of alkaloids found in Epichloë associations70 and was also found to be present in many of the accessions and haplotypes evaluated in this study. Peramine is a potent feeding deterrent of adult Argentine stem weevil (L. bonariensis), an important pest of perennial ryegrass H


Article

Journal of Agricultural and Food Chemistry This research presents the first report of endophyte alkaloids in a wide range of annual ryegrasses infected with Epichloë endophytes from geographically diverse regions grown in the same controlled environment. Discrete production of loline alkaloids in meristematic areas could be explained by the localization of the endophyte within meristem of the plant host, which is uncommon compared with related perennial grass− Epichloë associations. The variation of alkaloids expressed between and across infected annual ryegrass species is likely due to divergent evolution of symbioses, selecting incomplete or inactive metabolic pathways. The coevolution of endophytes with their hosts is also demonstrated by similar divergences in phylogeny and may also account for the variation in alkaloid profiles.74 Given the widespread production of peramine and loline alkaloids at bioactive concentrations in annual ryegrass and the role alkaloids play in promoting the fitness of other Epichloë−grass associations, it is possible that Epichloë presence may enhance the competitiveness of its plant host, albeit variably within and between populations.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +61 0269334211. Funding

The senior author is grateful to Charles Sturt University for an Australian Postgraduate Award and to the Grains Research and Development Corporation for an associated scholarship. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Anouck de Bonth for immunoblot development and Nelson West for technical assistance. ABBREVIATIONS USED NAL, N-acetylnorloline; NFL, N-formylloline; NANL, Nacetylloline; Terp-E, terpendole E; Pasp-B, paspaline-B; 13Des, 13-desoxypaxilline; Terp-C, terpendole-C; Jan, epoxy janthitrem; HRTS, Herbicide Resistance Testing Service; USDA GRIN, United States Department of Agriculture Germplasm Resources Information Network; EAS, ergot alkaloid biosynthesis

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