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a AgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand
b AgResearch, Grasslands Research Centre, Present address: Northland Polytechnic, Private Bag 9019, Whangarei, New Zealand
* Corresponding author (sydney.easton{at}agresearch.co.nz)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: HPLC, high pressure liquid chromatography • ELISA, enzyme-linked immunosorbent assay • GCA, general combining ability • SCA, specific combining ability • Wr, covariance • Vr, variance • FS, full sibling
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pest resistance and the adverse effects on livestock are ascribed to different alkaloid groups synthesized in planta by the endophyte (Lane et al., 2000). Lolitrem B (Gallagher et al., 1984), an indole-diterpenoid, is a neurotoxic tremorgen, primarily responsible for ryegrass staggers. Ergovaline, an ergopeptide closely associated with tall fescue toxicosis (Yates et al., 1985), is present in significant quantities in endophyte-infected perennial ryegrass (Rowan and Shaw, 1987). Other ergopeptides and lysergyl derivatives are also present (Lane et al., 2000). The presence of ergopeptides in perennial ryegrass depresses serum prolactin levels of grazing livestock, causes heat stress, and exacerbates ryegrass staggers (Fletcher and Easton, 1997). Peramine, a pyrrolopyrazine alkaloid, actively deters feeding by Argentine stem weevil (Listronotus bonariensis Kuschel) (Rowan and Gaynor, 1986), a major pasture pest in New Zealand. Ergopeptides (Ball et al., 1997b; Dymock et al., 1988) and lolitrems (Prestidge and Gallagher, 1988) have also been shown to deter insect predation.
Alkaloid concentrations in ryegrass tissue vary through the year (Ball et al., 1995a; 1991; Woodburn et al., 1993) and in response to environment (Barker et al., 1993; Lane et al., 1997b). Similar variation in time and with conditions has been documented for endophyte-derived alkaloids in tall fescue (Festuca arundinacea Schreb.) infected with Neotyphodium coenophialum (Morgan-Jones & Gams) Glenn, Bacon & Hanlin (Belesky et al., 1988).
Alkaloid concentrations also vary between infected plants growing in the same conditions. Up to five-fold and six-fold variation was measured in lolitrem B and peramine respectively, depending on the stage of the season, among 17 perennial ryegrass plants collected in old New Zealand pastures (Ball et al., 1995b), and this variation was associated with variation in the concentration of fungal mycelium in the plants. Likewise, five-fold variation in peramine concentration and four-fold variation in ergovaline were recorded among eight plants (Ball et al., 1995a). Variation in herbage concentrations of endophyte-derived alkaloids may reflect variation in endophyte strains, in host genotype, or in interaction between the two.
Endophyte strains vary, quantitatively and qualitatively, in their ability to produce alkaloids in planta (Latch, 1989; 1994). Strains can be isolated from their natural hosts and inserted into different host populations (Latch and Christensen, 1985), and maintain the properties observed in the natural host (Davies et al., 1993). Host genotypes infected with the same endophyte strain may also vary in alkaloid concentration. Latch (1994) found 10-fold variation in ergovaline concentration of 19 perennial ryegrass genotypes (from one population) infected with the same endophyte. Variation in endophyte-related properties has been reported in another set of perennial ryegrass plants infected with a common endophyte (Schmid et al., 2000).
Hill and co-workers have studied host plant influence on alkaloid concentrations in endophyte-infected tall fescue tissue (Adcock et al., 1997; Agee and Hill, 1994; Hiatt and Hill, 1997; Hill et al., 1991; Roylance et al., 1994). Ergot alkaloid concentration of a single full-sibling family was intermediate between that of the two parents, and independent of which was the seed parent (Agee and Hill, 1994). However, another experiment with the same material (Roylance et al., 1994) did find different ergopeptine concentrations between the reciprocal crosses, but no differences in peramine levels. Families resulting from mating three different pollen parents with a common seed parent differed significantly in intensity of hyphal infection (Hiatt and Hill, 1997), although all three families were infected with the same endophyte genotype received from the common seed parent. Mean ergot alkaloid concentration also varied between the families, but less consistently, and there was not a consistent relation between this and the intensity of hyphal infection. Inoculation of a common host genotype with different endophyte isolates also produced some variation in ergot alkaloid concentration (Hiatt and Hill, 1997; Hill et al., 1991). Host and endophyte genotypes were all drawn from within one fescue breeding population. Analysis of a four-parent diallel (Adcock et al., 1997), with each parent possibly infected with a different endophyte genotype, showed stronger maternal than paternal effects on ergopeptine concentrations, suggesting that in this material the endophyte genotype influenced variation more than the host. Selection within a tall fescue breeding population for ergovaline concentration was successful over two generations (Adcock et al., 1997), with realized heritability 0.49 and 0.56 for increased and decreased concentration respectively in the first generation, and 0.45 and 0.91 in the second generation.
A diallel set of families derived from perennial ryegrass plants infected with a common endophyte was studied to determine genetic control of host plant effects and their relation with intensity of mycelial infection.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Seed was germinated in April 1994, and 16 seedlings of each side of each cross (now termed "entries") were transplanted to pots. On 14 Oct. 1994 (southern spring), up to eight plants of each entry were assigned to each of two glasshouse units. Ramets of the original parents were also potted and grown with the seedlings. Plants were placed completely randomly within each glasshouse, and rerandomized regularly. Throughout the experiment, plants were supplied with nonlimiting water and nutrients. All plants used in the experiment were confirmed to be infected with endophyte.
One month after being placed in the glasshouses, the plants were trimmed back to 1 cm, thus removing reproductive growth, and allowed to grow again. After 30 d, on 14 Dec. 1994, they were harvested to 1 cm. Herbage from all plants of each entry was bulked, sub-samples were dissected, discarding the leaf laminae, and 5g fresh weight of the leaf sheath ("pseudostem") fraction was frozen and freeze dried. The plants were maintained and regularly trimmed through the summer, and one month's early autumn regrowth was harvested on 28 Mar. 1995 and processed in the same way.
Freeze-dried samples were ground and analyzed by HPLC for peramine and for ergovaline, as described by Barker et al. (1993). After extraction, peramine was detected by UV absorption following separation on a silica HPLC column and compared with a synthetic homoperamine internal standard added to each sample. Ergovaline was determined as the sum of ergovaline and ergovalinine (regarded as a more stable and repeatable measure, Lane, 1999), detected by fluorescence after reversed phase HPLC and compared to an ergotamine internal standard added to each sample.
Freeze-dried milled samples for the first harvest, which had been stored frozen for 3 yr, were analyzed in 1997 for concentration of endophyte mycelium, using a sandwich ELISA. The ELISA protocol was similar to that described by Miles et al. (1998), except that samples were incubated at 30°C rather than 20°C, and samples were extracted for 30 min in phosphate-buffered saline containing 0.5% Tween 20.
Parent data were analyzed by analysis of variance with harvest (fixed), glasshouse (random) and parent (random) as main effects. Parent means were also compared with the mean of their progenies. Data for crosses were analyzed by analysis of variance with harvest (fixed), glasshouse (random) and entry (random) as main effects. Entry mean square was further analyzed in three ways. First, sets of three entries were formed as maternal (or alternatively paternal) half-sibling groups. Second, reciprocal entries (formed from the same mating but harvested from different respective seed parents) were grouped into full-sibling (FS) family pairs. Finally, data were analyzed by the diallel analysis (Griffing, 1956), with both reciprocals of family pairs and without the parent data. Two degrees of freedom from the FS family total were assigned to differences between the diallel groups. Parental data were included in further analysis of the diallel to test hypotheses of gene action (Hayman, 1954). Variance and diallel analyses were also applied to residual ergovaline and peramine data for Harvest 1 after regression on ELISA data.
Heritability coefficients (narrow sense, h2) were calculated from the analysis of variance tables, assuming no epistasis, as additive genetic variance 
divided by the sum of additive and dominance genetic variances 
, variance of interaction with harvest (
2IH, in the cases of ergovaline and peramine) and error variance 
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The 16 parent clones differed significantly in ergovaline and peramine concentration, and in the amount of endophyte mycelium as indicated by ELISA, and this result was consistent over harvests (in the case of the alkaloids) and glasshouses (Table 1). Concentrations of the parent clones ranged 21-fold (16-fold for the 12 parents listed in Table 1), 25-fold and 10-fold for ergovaline and peramine and ELISA, respectively. Ergovaline concentrations were correlated r = 0.89, P < 0.0001) with measurements on the same clones (then young seedlings) 19 months earlier. Mean ergovaline and peramine concentrations of progeny families were 8.4 and 13.9 mg g-1 respectively, for the first harvest and 8.5 and 13.0 mg g-1 respectively, for the second harvest. FS family means (two harvests, two glasshouses, two entries per family) ranged approximately three-fold for both ergovaline and peramine, and two-fold for ELISA (one harvest).
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Variances calculated from the analyses of variance gave narrow sense heritability estimates of 0.70, 0.72, and 0.58, respectively, for ergovaline, peramine, and ELISA.
The FS family mean concentrations of ergovaline and peramine (mean of two harvests) and ELISA were correlated with their mid-parent values (r = 0.79, 0.92, and 0.84 respectively, P < 0.0001 in all cases). The respective regressions of family means on mid-parent values were 0.91, 1.04, and 0.67. For ergovaline concentrations, the coefficient of regression of progeny means on mid-parent values calculated from the original measurements (that is from a different experiment) was 0.64.
Peramine and ergovaline concentrations were correlated, and both were correlated with the amount of endophyte mycelium in the plant (Table 3, P < 0.01 in all cases). Genetically controlled variation for herbage ergovaline and peramine concentrations was in part accounted for as a function of mycelial mass, 41 and 65% respectively. Residuals for plot ergovaline and peramine concentrations for Harvest 1, calculated after regression on plot ELISA values, gave significant ANOVA and diallel GCA terms (Table 5). For ergovaline, the SCA term was significant and the GCA tested against SCA had probability P = 0.11.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The GCA mean squares indicate that a major component of the host genotype effect on amount of mycelium (ELISA) and accumulation of endophyte-derived alkaloids is a simply inherited direct effect of a parent on its progeny (Table 3). For ELISA, GCA was the only significant component. For ergovaline and peramine for one harvest there was a residual SCA component, indicating interactions between the two parents, derived from dominance or epitasis, but this was not so for the two harvests analyzed together.
Extended diallel analysis (Hayman, 1954) indicated that an additive-dominance model satisfactorily accounted for ELISA and peramine data. The Wr/Vr regression departed from unity for ergovaline. Since the departure was not great, and since ergovaline data were similar to peramine and ELISA data in other respects, this does not provide strong evidence for epistasis or other complex gene action. The lack of any tendency of half-sibling family groups with high array means to fall toward one end of the Wr/Vr line indicates that alleles for high values at different loci may be dominant or recessive.
Further evidence that the host influence on intensity of infection and alkaloid concentration is heritable is presented by the regression of progeny on mid-parent values. The regression coefficients were not different from 1.0 for ergovaline and peramine (0.91 and 1.04), and was 0.67 for ELISA, indicating high heritability for the three traits. In the case of ergovaline, a better indication is provided by using parent values from a different trial, measured on the seedling clones in May 1993. This value, 0.64, is an independent estimate of narrow-sense heritability, and is in agreement with the estimate from the analysis of variance (0.70).
Hill and co-workers (Adcock et al., 1997; Agee and Hill, 1994; Hiatt and Hill, 1997; Hill et al., 1991; Roylance et al., 1994) concluded that for tall fescue the host plant genome exercised significant control over the concentrations in herbage of ergopeptides produced by the endophyte fungus. Our data show that control is exercised in perennial ryegrass for a substantial set of inter-related families and for two harvests in different seasons, and that additive heritable elements are the major factors in this control. Further, the control also is exercised over peramine concentration.
The host trait is inherited from both parents, with no difference between reciprocal entries within a FS family pair, and no evidence of specifically maternal effects (Table 2). With the same plants used as seed and as pollen parents, the truly genetic effects, which are preponderant, are distributed between and within the maternal or paternal groups, confounding the mean squares. The absence of maternal effects contrasts with data showing stronger maternal than paternal influence on ergopeptine concentrations in tall fescue (Adcock et al., 1997; Roylance et al., 1994). However, the overall conclusions from the work of Hill and co-workers is that general genetic effects of both parents are more important than specifically maternal effects. Another source of greater variation between maternal than paternal half-sibling groups might be a significant degree of self-fertilization. The absence of any maternal effects in our data with perennial ryegrass also confirms that there was effectively no self-fertilization of the parents.
A plant receives its endophyte symbiont from its seed parent (Hinton and Bacon, 1985; Philipson and Christey, 1986). In this set of material, all parents were infected with a common endophyte strain, so that no variation between maternal groups due to differences in endophyte were expected. It remained possible that differences between the seed parents in their level of compatibility with the endophyte might have been reflected in the transfer of the endophyte to the seed, and the subsequent seedlings. Carryover effects of seed parent management on endophyte-related qualities of seedlings have been reported. Nitrogen status of the seed parent was reflected in intensity of endophyte infection (determined microscopically) of seedlings at least for the first year of their life, and in intensity of infection of seed harvested from the seedlings (Stewart, 1986). Parent plants of the same cultivar may vary in the efficiency (% seedlings infected) with which they transmit an endophyte strain to their progeny (Easton, Latch & Simpson, unpublished data). However, there is no evidence of any effect ascribable to a differential effect of the seed parent (Table 2).
The parent clones varied in ergovaline concentration to a similar extent as reported previously (Latch, 1994; Table 1). There is no evidence that differences first measured in May 1993, two mo after initial infection, have moderated in the ensuing 19 mo, or that they are affected by the physiological stage of the plant. For all three characters, the progeny bulks varied less than the parent clones. Variation would be expected to be less for bulks than for individual plants, but the lower progeny variance was not a symmetrical contraction. The maximum values for parents and progeny were of the same order, whereas the minimum values were lower for parents than for progeny. There may have been a degree of adaptation of the new association of host plant and endophyte. The parents were artificially infected plants with the endophyte strain, whereas the progeny were naturally infected during seed production. Directly infected plants may not reliably indicate the variation to be expected in subsequent generations. However, while the difference between generations in the ratio of maximum and minimum values appears large (20-fold and 3-fold for ergovaline concentration), similar mean values and high mid-parent-progeny correlation coefficients show that the control of variation is largely present within weeks of successful inoculation.
The significant correlation of the three traits measured indicates a degree of common control, interpreted most simply in terms of alkaloid production being in part dependent on the quantity of mycelium present in the host tissue. Lolitrem B and peramine mean annual concentrations in the leaf were correlated with ELISA values among 17 perennial ryegrass plants (r = 0.74; P < 0.001 and r = 0.83; P < 0.0001 for the regrowth and basal material respectively) (Ball et al., 1995b), and ergovaline concentrations of eight plants in spring (but not annual means) were correlated with ELISA (r = 0.810, P < 0.01) (Ball et al., 1995a). No consistent relationship between mycelial mass and alkaloid concentration was observed for tall fescue infected with a common endophyte strain (Hiatt and Hill, 1997). However, the 15 plants studied were derived from only one seed parent and three pollen parents, and the relationship observed in the study reported here might not have been evident in a smaller experiment.
Host control of intensity of endophyte infection and of secondary metabolite production is exercised within the adapted interactive relationship between the endophyte and its host (Schmid et al., 2000). Control is exercised over the timing and extent of fungal growth, and over mycelial branching. Intense metabolic activity of the fungus continues after growth stops, and secondary metabolite production is apparently not related to nutritional crises for the fungus. The nature of the signals exchanged between the organisms is unknown. However, while the host plant and fungal endophyte have co-evolved, the adaptive interaction is robust in artificial associations, such as that reported here, achieved by infecting plants with an endophyte strain isolated from an unrelated and geographically distant grass population.
The correlation between traits, while important, did leave a portion of the variation unaccounted for. Of this, some would be random variation, but departures from linearity can be observed. Figure 1 shows the ergovaline and peramine family (reciprocal pair) mean concentrations for Harvest 1 as functions of ELISA values. While both alkaloids can be seen to increase with ELISA values, there are families with values for ELISA and peramine close to the mean but which are moderately low for ergovaline. Furthermore, the residual values, after regression of ergovaline and peramine concentrations on ELISA values, were themselves amenable to diallel analysis and showed significant GCA effects. Selection for low herbage ergovaline combined with high amounts of mycelium in grass tissue and high herbage peramine can therefore be expected to succeed.
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The ELISA data indicate the mass of mycelium present in the herbage. The two-fold variation among the progeny for this artificial association compares with two- to 10-fold variation, depending on the season, for individual plants growing outside, perhaps infected with different endophyte strains (Ball et al., 1995b). di Menna and Waller (1986) reported plant-to-plant variation in numbers of hyphae observed microscopically, substantially greater than variation between tillers of the same plant, but did not report actual values.
Care is required in comparing the variation between families in alkaloid concentrations with other data. Family concentrations are derived from family bulk samples rather than individual plants, are for an artificial rather than a natural association of plant and endophyte and are for plants grown in a glasshouse. The range in ergovaline and peramine concentrations observed for the progeny was not greatly different (four or five-fold, rather than three-fold) from that determined for field-collected plants, for which the identity of the endophyte was unknown (Ball et al., 1995a; Ball et al., 1995b), suggesting that variation between endophyte genotypes was not a major contributor to the variation in these other studies.
While peramine has been identified as the major factor in protecting endophyte-infected perennial ryegrass from Argentine stem weevil (Rowan and Gaynor, 1986), the presence of ergopeptine alkaloids, including ergovaline, may be important in some situations, and in New Zealand particularly where black beetle (Heteronychus arator F.) is an active pest (Ball et al., 1997b). Total elimination of particular compounds from endophyte-infected pasture can be achieved using endophyte strains lacking competence to produce the compounds in question (Davies et al., 1993; Latch, 1994). The genetic control evident in our data, and the relatively high heritability estimates, indicate that if controlled low levels of certain compounds are required, it may be possible to achieve them by host plant selection.
Endophyte alkaloid concentrations in herbage are artificially high in glasshouse experiments (Lane et al., 1997a). Field concentrations in pasture vary through the year (Ball et al., 1995a) and are affected by soil nitrogen and water status and perhaps other environmental variables (Lane et al., 1997b). Effective development of controlled low alkaloid levels will depend on genetic expression that is stable in varying environmental conditions.
Received for publication November 1, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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