Characterisation of novel perennial ryegrass host ... - CSIRO Publishing

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Crop & Pasture Science, 2013, 64, 716–725 http://dx.doi.org/10.1071/CP13067

Characterisation of novel perennial ryegrass host–Neotyphodium endophyte associations P. Tian A,B,C,D,E, T.-N. Le A,E, E. J. Ludlow A,D,E, K. F. Smith B,C,D,E, J. W. Forster A,C,D,E, K. M. Guthridge A,D,E, and G. C. Spangenberg A,C,D,E,F A

Department of Environment and Primary Industries, Biosciences Research Division, AgriBio, The Centre for AgriBioscience, 5 Ring Road, Bundoora, Vic. 3083, Australia. B Department of Environment and Primary Industries, Biosciences Research Division, Hamilton Centre, Mount Napier Road, Hamilton, Vic. 3300, Australia. C La Trobe University, Bundoora, Vic. 3083, Australia. D Molecular Plant Breeding Cooperative Research Centre, Victorian AgriBiosciences Centre, La Trobe Research and Development Park, Bundoora, Vic. 3083, Australia. E Dairy Futures Cooperative Research Centre, AgriBio, the Centre for AgriBioscience, 5 Ring Road, Bundoora, Vic. 3083, Australia. F Corresponding author. Email: [email protected]

Abstract. The temperate pasture grass Lolium perenne L. is commonly found in symbiotic association with the asexual fungal endophyte Neotyphodium lolii. Levels of endophyte colonisation and alkaloid content were evaluated in associations formed by plant genotypes from cv. Bronsyn with the standard endophyte (SE) and five distinct commercial endophyte strains. Bronsyn–SE produced all of the measured alkaloids (lolitrem B, peramine, and ergovaline). Bronsyn–AR1 produced only peramine, while Bronsyn–AR37 produced none of the tested alkaloids. Bronsyn–NEA2, Bronsyn–NEA3, and Bronsyn–NEA6 produced both ergovaline and peramine. Both endophyte strain and host genotype exerted significant effects on alkaloid production. Analysis of endophyte colonisation using qPCR revealed differences between each association. With the exception of Bronsyn–AR1 and Bronsyn–NEA3, host genotype also significantly affected colonisation levels. Phenotypic performance of each association was also assessed, based on measurement of morphological traits under glasshouse conditions in hydroponic culture. Significant variation due to different endophyte and host genotypes was observed. Collectively, these studies confirm that differences in both endophyte and host genotypes contribute to host–endophyte performance in a complex interactive manner. Additional keywords: colonisation, ergovaline, lolitrem B, Lolium, morphogenesis, peramine, symbiosis. Received 18 February 2013, accepted 12 August 2013, published online 4 October 2013

Introduction Perennial ryegrass (Lolium perenne L.) is a globally important forage and turf grass (Jung et al. 1996), which commonly forms symbiotic associations with the fungal endophyte Neotyphodium lolii (Latch, Christensen and Samuels) Glenn, Bacon and Hanlin (Latch et al. 1984). In naturally occurring associations, Neotyphodium endophytes interact with host plants by providing major fitness enhancements and protection from both biotic and abiotic environmental stresses (Bush et al. 1997; Saikkonen et al. 2004). The benefits that endophytes confer on plant health, and the detrimental effects on animal health, are partially due to the production of biologically active alkaloids. Two important classes of compounds for the performance of perennial ryegrass–Neotyphodium endophyte associations are ergot alkaloids and lolitrems, both of which cause neurotoxic effects in grazing vertebrates (Siegel et al. 1987; Prestidge 1993; Schmidt Journal compilation CSIRO 2013

and Osborn 1993; Paterson et al. 1995). Two other alkaloid types (peramine and lolines) are known to be highly active against invertebrates, but have little or no effect on mammalian herbivores (Porter 1995; Bush et al. 1997). Several endophytes have been identified that produce no, or low levels of, anti-mammalian toxins (Fletcher and Easton 1997; Hunt and Newman 2005; van Zijll de Jong et al. 2008a, 2008b) and may therefore be termed ‘animal-safe’. Several studies have reported the advantageous nature of such endophytes over the standard endophyte (SE), also known as wild-type or high endophyte, for animal health (Bluett et al. 2005a, 2005b; di Menna et al. 2012). Endophyte AR1 has been shown to produce peramine, but not lolitrem B and ergovaline (Bultman et al. 2003). However, AR1 does produce several indole-diterpene intermediates, including paspaline, 13-desoxypaxilline, and terpendole C, in lieu of lolitrem B (Young et al. 2009). www.publish.csiro.au/journals/cp

Characterisation of novel grass–endophyte associations

Endophyte AR37, also known as N. lolii Lp14 (Christensen et al. 1993), does not produce lolitrem B, ergovaline, or peramine, but does produce epoxy-janthitrems, a group of indole-diterpenoid alkaloids with structural similarity to lolitrems (Bultman et al. 2003; Rasmussen et al. 2007, 2008; Rasmussen et al. 2009). The presence of AR37 in perennial ryegrass has been shown to deter insect herbivory, but can sporadically cause staggers in some grazing animals such as sheep (Fletcher and Sutherland 2009). Endophytes NEA2 (also known as NEA2A), NEA3, and NEA6 (aka NEA2B), in contrast, produce significant levels of peramine and ergovaline but not lolitrem B, hence providing protection against invertebrate pests without causing ryegrass staggers (van Zijll de Jong et al. 2008a). However, studies of animal toxicosis in Australian and New Zealand ryegrass pastures have focussed mainly on the effect of lolitrem B, and less on that of ergovaline (reviewed in Thom et al. 2012). One study demonstrated that Australian dairy cows that were fed perennial ryegrass silage containing high ergovaline content showed a significant decrease in milk production (Lean 2001). Host–endophyte interactions have been demonstrated to strongly influence endophyte presence, alkaloid production, and host performance (Belesky et al. 1989; Belesky and Fedders 1995; Faeth and Bultman 2002; Faeth and Sullivan 2003). Although endophyte genotypic variation may largely account for qualitative variation in alkaloid production, interactions with the host plant genotype are important for quantitative variation (Easton et al. 2002; Rasmussen et al. 2007). Variation in alkaloid production provides opportunities for selection of associations that retain the advantages of high pasture yield, yet are safe for health and production of grazing mammals. The variable nature of each new perennial ryegrass–endophyte association warrants detailed study before introduction into an agricultural system. Alkaloid production levels should be evaluated for each new association, as these may be affected by the environment (Lane et al. 1997), plant genotype (Easton et al. 2002; Spiering et al. 2005), tissue type, and leaf age, and may also be correlated with endophyte colonisation levels (Ball et al. 1995; Keogh et al. 1996; Easton et al. 2002). For example, the alkaloid levels produced by AR1 and AR37 vary significantly between different perennial ryegrass host cultivars and during growth at different levels of nitrogen supply (Rasmussen et al. 2007). Alkaloid concentrations of NEA2 and NEA6 have also been observed to vary in relation to host genotype (van Zijll de Jong et al. 2008b). Endophyte colonisation, as another measure of compatibility, can be assessed in terms of the effects of environmental (e.g. nitrogen supply Rasmussen et al. 2007) and host factors (Easton et al. 2002). Previous techniques based on hyphal counts and reporter transgenes (Spiering et al. 2005) have not yet permitted broad-scale quantification of endophyte content. However, development of a PCR-based assay to measure in planta endophyte colonisation has enabled efficient screening of large numbers of associations (Young et al. 2005; Rasmussen et al. 2007). A third, and arguably the most important, measure of compatibility is the effect of genetic interaction between endophyte and host genotypes on agronomic performance (Christensen 1995; Saikkonen et al. 2004). For example,

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endophyte-specific effects on drought tolerance are strongly dependent on both host genotype and environmental conditions (Marks and Clay 1996). Several studies have investigated the effects of SE and AR1 on host plant performance (Popay and Baltus 2001; Popay et al. 2003; Pennell et al. 2005). However, equivalent studies of other commercial endophytes (such as NEA2, NEA3, NEA6, and AR37) have been limited (Hume et al. 2007; Moate et al. 2012). This study describes the effects of the endophyte strains SE, AR1, AR37, NEA2, NEA3, and NEA6 on the compatibility of host–endophyte associations, through measurement of alkaloid production levels and endophyte colonisation in soil-borne plants and plant growth characteristics in hydroponic culture. Evidence for both qualitative variation (based largely on endophyte genotypic identity) and quantitative variation (associated with host genotype) was obtained. The implications for molecular breeding of enhanced symbiotic interactions are discussed. Materials and methods Plant materials Seeds of perennial ryegrass cv. Bronsyn containing SE, AR1, AR37, NEA2, NEA3, and NEA6 (Tian et al. 2013), as well endophyte-devoid (E–) seed, were obtained from New Zealand Agriseeds Ltd, Christchurch, New Zealand. To generate these samples, resident endophyte was removed from Bronsyn seed batches by heat treatment at 608C before inoculation, following normal commercial practice. Endophytes SE, NEA2, and NEA6 have been previously named as Standard Toxic (ST) endophyte, NEA2A, and NEA2B, respectively (van Zijll de Jong et al. 2008a, 2008b). Table 1 lists the endophytes used in this study, and their expected alkaloid profiles. Endophyte presence in tillers was confirmed using Phytoscreen Immunoblot Kits (Agrinostics, Watkinsville, GA, USA) following the manufacturer’s instructions. Presence in tillers and unique identity of inoculated endophytes was confirmed by diagnostic simple sequence repeat (SSR) testing (van Zijll de Jong et al. 2008a), along with the absence of endophyte from the E– control plants. Genetic diversity of 34 Bronsyn genotypes Table 1. List of endophytes used in this study and their expected alkaloid profiles IDT, indole-diterpene; ?, based on gene prediction Endophyte strain

Alkaloid profile

Reference

SE

Lolitrem B Ergovaline Peramine IDT intermediates Peramine IDT intermediates? Epoxy-janthitrems Lolitrem B Ergovaline Peramine Ergovaline Peramine Lolitrem B Ergovaline Peramine

Siegel et al. (1990) Christensen et al. (1993)

AR1 AR37 NEA2

NEA3 NEA6

Young et al. (2009) Bultman et al. (2003) Young et al. (2009) Tapper and Lane (2004) van Zijll de Jong et al. (2008b)

van Zijll de Jong et al. (2008a) van Zijll de Jong et al. (2008b)

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was assessed using 60 SSR markers as previously described (Wang et al. 2009). The genetic distances between genotypes were calculated by principal component analysis (PCA) using the GenAlEx6 software (Peakall and Smouse 2006) (see Supplementary Materials Fig. 1 as available on journal’s website). Four host genotypes from each host–endophyte association were randomly selected from within this group for further study. Plant growth Seeds were germinated and seedlings were planted in seedling trays and grown for 2 months under glasshouse conditions (natural lighting and an average controlled temperature of 228C) at Department of Environment and Primary Industries— Hamilton, Victoria. After 3 months’ growth, plants were transplanted into 115-mm-diameter round pots containing Van Schaik’s Bio Gro Compost Potting Mix (Van Schaik’s Pty Ltd, Mount Gambier, S. Aust.). Plants were watered when required, and received Results Plus® liquid fertiliser as per manufacturer’s instructions (SprayGro Ltd, Adelaide, S. Aust.). When all of the plants had grown to a density of >15 tillers, individual plants were split into four clonal replicates and transplanted into pots of the same size, followed by maintenance under the glasshouse conditions as previously described. Measurements of alkaloid production After a further 2 months’ growth in the glasshouse, tissue was sampled in early spring (September–October 2008) from each clonal replicate (4) of each host genotype (4) for each endophyte strain (6), generating 96 experimental units. Leaf blades of the harvested tillers were discarded and the remaining pseudostem component was freeze-dried and analysed for alkaloid content. Concentrations of peramine, lolitrem B, and ergot alkaloids (sum of ergovaline and ergovalinine) were measured using highperformance liquid chromatography (HPLC) (Gallagher et al. 1985; Ball et al. 1995). Measurements of endophyte colonisation The same 96 experimental units for which alkaloid profiles were determined were harvested by selection of the three oldest tillers. Tillers were removed by cutting ~0.5 cm from the base and placed into 96-well collection microtube racks (Qiagen GmbH, Hilden, Germany) and freeze-dried (Genesis 25XL FreezeDryer, VirTis, Canton, MA, USA) for 48 h. DNA extraction was performed with the Qiagen MagAttract DNA Extraction kit (Qiagen GmbH) as per manufacturer’s instructions. A single tungsten carbide bead and 400 mL of Buffer AP1 was added to each sample well and samples were ground using a Retsch MM300 Mixer Mill (Retsch Inc., Newtown, PA, USA) for 1.5 min at 30 Hz twice, changing the orientation of the rack once between each grind. Samples were vortexed at 14 000G for 20 s and centrifuged for 5 min at 6000G. DNA extraction was performed semi-automatically using the Biomek® FX robotic workstation (Beckman Coulter, Foster City, CA, USA), which had been programmed with the MagAttract extraction protocol. DNA concentration was quantified using the Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and samples were diluted to 20 ng/mL with sterile distilled H2O. Endophyte colonisation was measured using quantitative

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PCR (qPCR) of perA, the peramine biosynthesis gene, which is present as a single copy gene within each endophyte used in this study based on genome sequence data (not shown). A 639-bp fragment of the SE perA gene was amplified using the primers 50 -TACGAACTCTCATGGCAGCCG-30 and 50 -CATGTCGTGCATGCAGCGCTC-30 , and cloned into the pGEM-T Easy vector (Promega Corp., Fitchburg, WI, USA) (Supplementary Materials Fig. 2). Plasmid DNA containing the perA gene fragment was serially diluted from 2 106 to 2 copies to create a standard curve dilution for qPCR. Primers for qPCR were designed to amplify a 73-bp fragment within a conserved region of perA across all endophytes, including AR37, which is known to possess a truncated perA gene (Fleetwood et al. 2011). Each qPCR mixture contained 5 mL of 2 SYBR® Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA), 0.2 mL forward primer (10 mM; sequence 50 -AACATCGAGCACT CTCATTGC-30 ), 0.2 mL reverse primer (10 mM; sequence 50 -CCGTTTCCGATGGTGCCATTG-30 ), and 2.6 mL filtersterilised distilled H2O. The master mix (8 mL) was added to a 96-well qPCR plate and 2 mL of each standard curve dilution and 2 mL (40 ng) of each unknown DNA sample, not exceeding 50 ng total gDNA per reaction, was placed into appropriate wells and the plate was pulse-spun (~900G for 20 s). The PCR was performed using a real-time thermocycler (Stratagene MX 3005P; Agilent, Santa Clara, CA, USA) with the following conditions: 1 cycle of 958C for 10 min, and 40 cycles with three steps (958C for 30 s, 628C for 1 min, 728C for 30 s). The dissociation curve kinetics was determined under continuous data acquisition with the following parameters: 958C for 1 min, 608C for 1 min, ramping to 958C in 20 min, and 958C for 1 min. Following qPCR cycle completion, the unknown samples and the standard curve were assessed to ensure that all parameters were within acceptable guidelines. Eppendorf software (Realplex 4; Eppendorf AG, Hamburg, Germany) was used to determine the number of perA gene copies from an unknown sample against the standard curve. To determine copies per ng whole plant genomic DNA, the number of perA copies was divided by the amount of DNA in the reaction. Assessment of morphological characters The experimental design for assessing plant growth characteristics in hydroponic culture is outlined in Fig. 1. For alkaloid measurements, four Bronsyn genotypes were assessed for each endophyte strain, and E– plants were included for comparison. Each association was clonally replicated 12-fold, with the exception of two E– plant genotypes that were clonally replicated 24-fold in order to occupy fully the otherwise empty positions in the experimental design. An incomplete block design was used, with 18 hydroponic tubs as blocks. Initially, three similar-sized tillers per replicate were removed following growth in soil pots under glasshouse conditions. Each replicate was trimmed such that both roots and shoots were 5 cm in length, and they were then placed in hydroponic solution. Each clonal ramet was placed between Growool propagating blocks (Growool Horticultural System Pty Ltd, Kurmond, NSW) with identification tags, and inserted into one of 20 slots within each tub. This process was repeated to obtain the required number of clones. The hydroponic nutrient solution was checked weekly


4 genotypes per Bronsyn endophyte symbiota Bronsyn -SE Bronsyn -AR1 Bronsyn -AR37 Bronsyn -NEA2 Bronsyn -NEA3 Bronsyn -NEA6 Bronsyn -E-


12 replications per Bronsyn genotype endophyte symbiotium

719

5 weeks growth in hydroponic culture Measurements of tiller number and leaf length

3 tillers/replication

10 weeks growth in hydroponic culture 13 weeks growth in hydroponic culture

Measurements of tiller number and leaf length

Destructive harvest

Measurements of tiller number, leaf length, longest root length, leaf area of three fully emerged leaves, dry weight of pseudostem, leaf blades and roots

Fig. 1. Outline of the experimental design for assessing plant growth characteristics of six different Bronsyn–endophyte associations in hydroponic culture. Analyses of endophyte colonisation and alkaloid content were performed on soil-grown plants.

and maintained at pH 5.8–6. The hydroponic solution and Growool were changed monthly, or as required. Plants were maintained under glasshouse conditions, and two morphological traits, tiller number and the longest fully emerged leaf length, were recorded at both 5- and 10-week intervals. Number of tillers was determined solely on the basis of emergence from the parental sheath. Longest fully emerged leaf length was measured from the ligule/collar to leaf tip for those longest leaf blades for which the ligule had emerged from the sheath. At week 13, plants were destructively harvested and further morphological measurements were recorded. Roots, pseudostem, and leaf blades were harvested from each plant, and the substrate was gently washed from the pseudostem and root. Tiller number, and lengths of the longest fully emerged leaf and root, were measured. Root length was measured from the base of the pseudostem to the tip of the longest root. The three longest fully emerged leaf blades were also used to measure leaf area. Leaf area was measured using a LI-3100 area meter (LI-COR Biosciences, Lincoln, NE, USA) with a resolution of 0.1 mm2. Dry weights (g) of pseudostem, leaf blades, and roots were measured after 48 h of oven-drying at 105 28C. Statistical analyses Correlation between alkaloid production and endophyte colonisation levels was measured using GENSTAT Edition 12 (VSN International Ltd, Hemel Hempstead, UK), based on the average of four replicates from each Bronsyn–endophyte

association. The correlation graphs were drawn in Excel 2003 (Microsoft Corp., Redmond, WA, USA). Statistical analysis of derived data was performed using the residual maximum likelihood (REML) model within GENSTAT. The model used for analysis was a mixed model with endophyte strain and host genotype fitted as fixed effects and experimental design factors such as bench + tub/position + replication were fitted as random effects. The least significant difference (l.s.d.) at P = 0.05 was generated to test for effects arising from differences between endophytes and between host genotypes within each association (Supplementary Materials Table 1). Results Variation in endophyte colonisation and alkaloid production Comprehensive analysis of genetic diversity between the Bronsyn genotypes used in this study showed no obvious subpopulations according to association or seed batch (Supplementary Materials Fig. 1). However, analysis of endophyte colonisation using qPCR revealed differences between each association (Fig. 2). Endophytes SE, AR37, and NEA2 exhibited significantly (P < 0.05) higher colonisation levels than AR1, which in turn was greater (P < 0.05) than NEA6. Colonisation of NEA3 was intermediate between that of AR1 and NEA6. With the exception of Bronsyn–AR1 and Bronsyn–NEA3, host genotype exerted a significant (P < 0.05) effect on endophyte colonisation levels (Fig. 2).


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Ergovaline (ppm)

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Peramine (ppm)

Endophyte biomass (copies/ng DNA)

25.0

a

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18.0 16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0

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720

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0.2 0.0

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AR37

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SE

AR1

AR37

NEA2

NEA3

NEA6

Fig. 2. Endophyte colonisation and concentration of lolitrem B, ergovaline, and peramine in different Bronsyn–endophyte associations. For each association, the first four bars on the left show the average of four clonal replications per Bronsyn genotype. The fifth bar on the right, represented by the darker colour tone, shows the overall average of each combination (4 different host plant genotypes each with 4 clonal replications to a total of 16 samples). Capped lines are standard errors of the mean. Means with different letters above bars are significantly different at P = 0.05 (see Supplementary Materials Table 1) between endophytes (letters a–c) or between host genotypes within the same Bronsyn–endophyte association (letters d–f). The units copies/ng DNA denote the number of copies of the perA gene in each ng DNA extracted from healthy plant tillers. Alkaloid concentration was calculated in parts per million (ppm).

Alkaloid analyses revealed that Bronsyn–SE produced all three tested alkaloids, while Bronsyn–NEA2, Bronsyn–NEA3, and Bronsyn–NEA6 produced both ergovaline and peramine, but not lolitrem B (Fig. 2). Bronsyn–AR1 produced only peramine, and Bronsyn–AR37 produced none of the alkaloids tested. Quantitatively, the results indicated that both endophyte strain and host genotype also exerted significant effects on alkaloid production (P < 0.05; Table 2, Fig. 2). First, plants inoculated with SE or AR1 produced peramine at similar levels (10 ppm), which were at least double those of plants inoculated with NEA2, NEA3, or NEA6. Conversely, plants inoculated with NEA6 produced the highest levels of ergovaline (~1 ppm), followed by Bronsyn–NEA3 plants (0.7 ppm). Second, different genotypes of Bronsyn inoculated with the same endophyte displayed significantly (P < 0.05) different alkaloid concentrations, providing direct evidence for host genotype (Gh)–endophyte genotype (Ge) interaction. No evidence was obtained for correlation between endophyte colonisation and alkaloid production across all of the tested associations (see Supplementary Materials Fig. 3 and Supplementary Materials Table 2). However, analysis of individual associations indicated that peramine concentration within Bronsyn–SE and Bronsyn– NEA6 plants was positively correlated (P < 0.05) with endophyte colonisation (Supplementary Materials Table 2). Variation in tiller number and leaf length Assessment of tiller number and length of longest fully emerged leaf after 5, 10, and 13 weeks of hydroponic growth revealed significant differences (P < 0.05) between associations (Fig. 3). Bronsyn–NEA3 propagated significantly (P < 0.05) more tillers at each of the three time points than the other associations, which also differed from one another. After 5 weeks’ growth, Bronsyn–NEA6 and Bronsyn–E– plants propagated significantly

more tillers (P < 0.05) than Bronsyn–AR1, Bronsyn–AR37, and Bronsyn–SE. In contrast, after 10 and 13 weeks of growth, no significant differences were observed between associations, other than the higher tiller number characteristic of Bronsyn– NEA3 plants. Effects of host genotype on tiller number were also observed. Host genotypes within Bronsyn–E–, Bronsyn–AR1, Bronsyn–NEA3, and Bronsyn–NEA6 populations performed significantly (P < 0.05) differently at all time points (Fig. 3), although those of Bronsyn–AR37, Bronsyn–NEA2, and Bronsyn–SE did not vary significantly. Similar results were obtained for the longest fully emerged leaf length character (Fig. 3). After 5 weeks’ growth in hydroponic culture, Bronsyn–NEA2 and Bronsyn–NEA3 plants exhibited significantly greater leaf length (P < 0.05) than Bronsyn–AR37 and Bronsyn–SE. Equivalent results were obtained after 10 weeks, with the addition of Bronsyn–NEA6 to the lower performing category. After 13 weeks, performance of Bronsyn–E–, Bronsyn–AR1, Bronsyn–NEA2, and Bronsyn– NEA3 plants significantly exceeded (P < 0.05) that of Bronsyn– AR37 and Bronsyn–SE. Host genotype-specific differences were again observed. After 5 weeks, host genotypes within Bronsyn–E– and all six associations performed differently (P < 0.05). After 10 and 13 weeks, performance of host genotypes within Bronsyn–E– and all Bronsyn–endophyte associations, with the exception of Bronsyn–NEA3, varied significantly (P < 0.05) (Fig. 3). Variation in additional morphological traits after 13 weeks of hydroponic growth Assessment of root length revealed variation (P < 0.05) between different populations due to both Gh and Ge (Fig. 4). Bronsyn–NEA3 plants exhibited significantly (P < 0.05) greater root length than Bronsyn–SE and Bronsyn–AR37.



Roots from other populations (AR1, NEA2, NEA6, and E–) were intermediate in length. Variation in root length between different host genotypes was also observed (P < 0.05) within each population, with the exception of Bronsyn–AR1. Measurement of leaf area revealed that endophyte genotype exerted significant effects (P < 0.05) on host performance (Fig. 4). Bronsyn–NEA2 and Bronsyn–NEA3 displayed significantly greater (P < 0.05) leaf areas than Bronsyn–SE, Bronsyn–AR37, and Bronsyn–NEA6. Although Bronsyn–E– and Bronsyn–AR1 did not differ significantly from Bronsyn– NEA2 and Bronsyn–NEA3, these four populations displayed significantly greater (P < 0.05) leaf area than Bronsyn–SE and Bronsyn–AR37. Significant differences (P < 0.05) in leaf area due to host genotype were also observed, with the exception of Bronsyn–AR37 and Bronsyn–NEA3. Finally, endophyte-specific effects were also observed for dry weight of roots, leaf blades, and pseudostem (Fig. 4). Bronsyn–NEA3 displayed significantly higher (P < 0.05) values than other associations, as well as Bronsyn–E–.

Bronsyn–NEA2, Bronsyn–NEA6, and Bronsyn–E– had significantly greater (P < 0.05) root and leaf blade dry weight than Bronsyn–SE and Bronsyn–AR37. Bronsyn–E– and Bronsyn–NEA6 also displayed significantly greater (P < 0.05) dry pseudostem weight than Bronsyn–SE and Bronsyn–AR37. Variation between host genotypes was also observed (P < 0.05) for all morphological traits, with the exceptions of both Bronsyn–AR37 and Bronsyn–NEA2 for root dry weight, Bronsyn–AR37 for leaf blade dry weight, and Bronsyn–AR37 and Bronsyn–NEA2 for pseudostem dry weight. Analysis of variance for the REML model showed that both endophyte strain and host genotype had significant effects (P < 0.001) on all of the plant morphological traits measured (Supplementary Materials Table 3). Discussion Variation of endophyte colonisation In the present study, endophyte colonisation, alkaloid biosynthesis, and morphological characters were assessed in order to improve understanding of interactions between host and endophyte in artificially generated associations. Quantitative variation for each trait was observed, revealing complex interactions between endophyte and host genotypes. Although comprehensive studies have not been performed, previous reports of variable endophyte colonisation levels between host genotypes have been obtained (Musgrave 1984; Ball et al. 1995; Cheplick 1998; Tan et al. 2001; Spiering et al. 2005; Rasmussen et al. 2007).

Table 2. Analysis of variance for the REML model describing the effect of Bronsyn host genotype and endophyte strain on alkaloid content and endophyte colonisation Wald statistic, standard Wald parameter calculated under Wald statistic model; d.f., degrees of freedom; Chi pr, level of probability Effects

Wald statistic

d.f.

Chi pr

148 90.18

3 12