《牧草饲料作物栽培学》课程教学资源（文献资料）Ryegrass Endophyte（Report）An Up-to-Date Review of its Effects.pdf_大学文库

2.0 REVIEW: ENDOPHYTES IN NZ PASTURES 2.1 Introduction Perennial ryegrasses (PRG) is the most widely sown species in NZ pastures and has been intensively researched since the 1920s (Hunt & Easton, 1989). However, as late as 1980, the discoveries that the endophytic fungus, Neotyphodium lolii, caused ryegrass staggers (Fletcher & Harvey, 1981) and protected the plant from Argentine stem weevil (ASW) (Prestidge et al., 1982) have required a total re-examination of all we thought we knew about ryegrass pastures. Endophyte research has also enabled us to revise our understanding of tall fescue. The endophytic fungi in PRG and tall fescue had been described in 1933 (Sampson, 1933) and further studied in the 1940s by NZ scientists (Neill, 1941), and the possibility that they caused livestock disorders was raised and investigated (Cunningham, 1958). However, the experiments failed to make the suggested link, and another 20 years passed before the significance of the endophytes was established. There was a considerable amount of serendipity about the crucial initial discoveries (Easton et al., 2001). Since then, endophyte research has been focused and multidisciplinary. Another feature of the work has been the importance of international contacts, and in particular the close and very useful interchange with research groups in the south east of the USA (Easton et al., 2001). When the endophyte of PRG was first discovered it was named Acremonium lolii, however it was later reclassified as Neotyphodium lolii (Fletcher & Easton pers. comm.) on the basis of the alkaloids produced (Glenn & Bacon, 1997). 2.2 Endophyte in New Zealand Pastures Perennial ryegrass (Lolium perenne) is naturally infected with the endophyte, Neotyphodium lolii, but other endophytic fungi are also regularly found in perennial ryegrass (Latch et al., 1984). The influence of the latter on the pasture-livestock system is unknown and probably small. They are not considered in this discussion, and the word “endophyte” refers here to N. lolii or related species in other grass hosts. Ryegrass collected from old pastures throughout NZ has regularly been found to be infected with N. lolii (Prestidge et al., 1985; Wedderburn et al., 1989; Widdup & Ryan, 1992), as has ryegrass collected in Europe (Lewis et al., 1997). Endophyte was first noted in ryegrass in NZ 60 years ago (Neill, 1940). Other grass genera and species are similarly infected with related endophytes, notably tall fescue (Festuca arundinacea) (Siegel et al., 1987). PAGE 4

New Zealand experience is that perennial ryegrass in most old pastures is normally infected at very high frequency.However,in the south of NZ,infection rates are lower and some old pastures are nearly free 0f50 two vere less cted,but 29 tha ted perenni ryegras naopnyte (Piggot New Zealand pastures have higher levels of infection than those studied overseas.Of 54 onulations collected in north est Spain,15 were free of endophyte (Oliveira Castro 1998).and only 12 populations were 60%or more infected.Similar results were observed with French populations(Ravel et al.,1994).These infection levels are lower than those observed in southern NZ(Widdup Ryan,1992). 2.3 The Effects of Endophvte on Various Aspects of Pasture Yield Production Endophyte is essential to the thrift of perennial ryegrass pastures in NZ because it ryegrass nvertebra ests ASW 1982 (Mortime d tidge ely to d or Hunt.1983) those ent are pr at al rass stems bore less vigo ously into infected plants (Prestidge Gallag er,1988) observed to be dar ed in late summ in swards but was observed in infected swards (Popay&Mainland 1991).Under pressure from ASW,the frequency of endophyte infection increases rapidly as non-infected plants die and are replaced by expansion of infected plants (Hume Brock,1997;Prestidge et al.,1984). ASW has been shown to be an important factor in pasture failure in Canterbury (Goldson 1982)and much of the North Island(Barker et al.,1984b).Yield responses of 50-60%in mixed ryegrass-wnite insectici 1982 1n19 pastu 1991) was estimated a costi (FI it ophyte was shown t pro seed ilit genetic or varietal diffe 1982,and thougn Endophvte also protects perennial n rass from other invertebrates (Prestidge Ball 1993).Black beetle (Heteronychus arator)is intermittently a serious pest in northern NZ (East et al.,1981).Endophyte-free ryegrass can be quickly destroyed by black beetle (Lee et al.,1982),but endophyte deters feeding by the adults,and egg and larval numbers are reduced(Ball Prestidge,1992).Various nematode species,some endo-parasitic. others free-living in the root zone,are found in ryegrass pastures,and there is some evidence that populations are affected by endophyte infection (Eerens et al., 1998b Stewart et al.,1993).There is very little NZ information on the importance of grass in pastures (Watson et al., 1986, Yeates Prestidge, 1986 sture m aly bug (B s poae)is an &Bal,1999 Mealy bug presence is trivial n pasture PAGE 5

New Zealand experience is that perennial ryegrass in most old pastures is normally infected at very high frequency. However, in the south of NZ, infection rates are lower, and some old pastures are nearly free of endophyte (Widdup & Ryan, 1992). Of 50 populations examined, two were less than 20% infected, but 29 populations were more than 60% infected. Naturally occurring hybrid ryegrass may also be infected with the perennial ryegrass endophyte (Piggot et al., 1988). New Zealand pastures have higher levels of infection than those studied overseas. Of 54 populations collected in north-west Spain, 15 were free of endophyte (Oliveira & Castro, 1998), and only 12 populations were 60% or more infected. Similar results were observed with French populations (Ravel et al., 1994). These infection levels are lower than those observed in southern NZ (Widdup & Ryan, 1992). 2.3 The Effects of Endophyte on Various Aspects of Pasture Yield & Production Endophyte is essential to the thrift of perennial ryegrass pastures in NZ because it protects ryegrass from invertebrate pests, notably Argentine stem weevil (ASW) (Listronotus bonariensis) (Mortimer & di Menna, 1983; Prestidge & Ball, 1993; Prestidge et al., 1982). Adult ASW are less likely to feed on infected ryegrass (Barker et al., 1984d; Rowan & Gaynor, 1986). Less eggs and larvae are present in infected swards (Gaynor & Hunt, 1983), those present survive less well (Barker et al., 1984c) and larvae attacking ryegrass stems bore less vigorously into infected plants (Prestidge & Gallagher, 1988). In Manawatu, 70% of tillers were observed to be damaged in late summer in endophyte-free swards but negligible damage was observed in infected swards (Popay & Mainland, 1991). Under pressure from ASW, the frequency of endophyte infection increases rapidly as non-infected plants die and are replaced by expansion of infected plants (Hume & Brock, 1997; Prestidge et al., 1984). ASW has been shown to be an important factor in pasture failure in Canterbury (Goldson, 1982) and much of the North Island (Barker et al., 1984b). Yield responses of 50-60% in mixed ryegrass-white clover swards were achieved by protecting from ASW with insecticide (Kain et al., 1982). In 1991, pasture damage by ASW was estimated as costing NZ $46-$200m annually (Prestidge et al., 1991). Before endophyte was shown to provide protection, it had been noted that different seed lines of perennial ryegrass differed in their vulnerability to attack (Kain et al., 1982), and this was thought to indicate genetic or varietal differences. Endophyte also protects perennial ryegrass from other invertebrates (Prestidge & Ball, 1993). Black beetle (Heteronychus arator) is intermittently a serious pest in northern NZ (East et al., 1981). Endophyte-free ryegrass can be quickly destroyed by black beetle (Lee et al., 1982), but endophyte deters feeding by the adults, and egg and larval numbers are reduced (Ball & Prestidge, 1992). Various nematode species, some endo-parasitic, others free-living in the root zone, are found in ryegrass pastures, and there is some evidence that populations are affected by endophyte infection (Eerens et al., 1998b; Stewart et al., 1993). There is very little NZ information on the importance of grassassociated nematodes in pastures (Watson et al., 1986; Yeates & Prestidge, 1986). Pasture mealy bug (Balanococcus poae) is an insect frequently present in pastures but of unknown importance. It is sensitive to the presence of endophyte (Pearson, 1989; Pennell & Ball, 1999). Mealy bug presence is trivial in pastures intensively infected with PAGE 5

endophyte,but large numbers may build up in endophyte-free pastures in late summer in dryland conditions (C.G.Pennell,unpub.cited by Easton,1999).Serious infestation is associated with poor ryegrass growth and persistence,but cause and effect are difficult to establish. insect that is sensitive to endophyte (Dymock et al., Evid ence that ryegrass shw greater growth (Latch et al.,1985)or ith d fo estoera when 1993 cabinet and glasshouse-based e eriments of a few ks duration have not sho any en dophvte effects on stress tolerance (Barker et al.,1997:Eerens et al.,1998c:Hume et al.,1993 Easton Rolston,unpub.data cited by Easton 1999;Easton Hume,unpub.data cited by Easton 1999).Field experiments are difficult to protect from all invertebrate pressure factors,but a series of row trials run over 2-4 years,where no serious invertebrate pressure was observed,has indicated significant advantage to ryegrass naturally infected with endophyte or infected with certain strains (Hume Latch,unpub.data cited by Easton 1999). Other field experiments have indicated no such effects (Eerens et al., 1998a Widdup Ryan,1992). Likewise,results in France were inconsistent,but endophyte conferred some advantage to ryegra absence of apparent invertebrate attack.in ,1995 n endoph 1993 be less ed ards (F by lives swards clos ser to the g nd further into the leaf sheath horizon.especially after the first few days Similar differences have been docur mented in non-choice grazing experiment by R.H.Watson (cited by Easton,1999). If such differ es in a razino pressure were sustained over time,they could,in the absence of any other factors.lead to a more rapid decline in the endophyte-free sward. Whether due to pest pressure,drought tolerance or any other mechanism,endophyte- infected ryegrass pastures produce more dry matter than endophyte-free pastures. In a recent series of trials at six sites throughout NZ(Popay et al.,1999),endophyte-infected ryegrass consistently out-yielded endophyte- -tree ryegrass. Averaged over three years and the k sites,th difterence was more than 20%in summe and mo e than 0%in autumn.At some sites and in some years,the difference was much greater than this be a 20:Su d for cted pasture 1993).The effect is not alway rens et al..1998a).and is mostly an inv e effect of rven igour (Prestidge Direct inhibitor(allelopathic)effects on white clover have been documented (Sutherland et al..1999).but apparent stimulatory effects have also been noted (Eerens et al.1998d).Livestock may preferentially araze clover in a mixed sward(Milne et al.,1982).This preference is partial (Parsons et al. 1994),and more active avoidance of ryegrass if infected may intensify it.Alternatively,if clover is growing evenly through a grass sward,rather than in a mosaic,endophyte by discouraging close grazing by stock may protect the clover stolons.Poorer clover content in a ryegrass pasture will exacerbate any negative effects of endophyte on livestock performance,and these two factors have been confounded in some experiments PAGE6

endophyte, but large numbers may build up in endophyte-free pastures in late summer in dryland conditions (C.G. Pennell, unpub. cited by Easton, 1999). Serious infestation is associated with poor ryegrass growth and persistence, but cause and effect are difficult to establish. Cutworm (Graphania mutans) is another insect that is sensitive to endophyte (Dymock et al., 1989b). It is found throughout NZ but is of unknown significance. Evidence that ryegrass shows greater growth (Latch et al., 1985) or stress tolerance when infected with endophyte, as has been widely reported for tall fescue (West & Gwinn, 1993), has proved to be inconsistent. Intensive growth cabinet and glasshouse-based experiments of a few weeks duration have not shown any endophyte effects on growth or stress tolerance (Barker et al., 1997; Eerens et al., 1998c; Hume et al., 1993; Easton & Rolston, unpub. data cited by Easton 1999; Easton & Hume, unpub. data cited by Easton, 1999). Field experiments are difficult to protect from all invertebrate pressure factors, but a series of row trials run over 2-4 years, where no serious invertebrate pressure was observed, has indicated significant advantage to ryegrass naturally infected with endophyte or infected with certain strains (Hume & Latch, unpub. data cited by Easton 1999). Other field experiments have indicated no such effects (Eerens et al., 1998a; Widdup & Ryan, 1992). Likewise, results in France were inconsistent, but endophyte conferred some advantage to ryegrass, in the absence of apparent invertebrate attack, in plots at more stressful sites (Ravel et al., 1995). Ryegrass infected with endophyte may be less severely grazed by livestock than uninfected swards (Edwards et al., 1993). Sheep offered a choice, grazed uninfected swards closer to the ground, and further into the leaf sheath horizon, especially after the first few days. Similar differences have been documented in a non-choice grazing experiment by R.H. Watson (cited by Easton, 1999). If such differences in grazing pressure were sustained over time, they could, in the absence of any other factors, lead to a more rapid decline in the endophyte-free sward. Whether due to pest pressure, drought tolerance or any other mechanism, endophyteinfected ryegrass pastures produce more dry matter than endophyte-free pastures. In a recent series of trials at six sites throughout NZ (Popay et al., 1999), endophyte-infected ryegrass consistently out-yielded endophyte-free ryegrass. Averaged over three years and the six sites, the difference was more than 20% in summer, and more than 30% in autumn. At some sites and in some years, the difference was much greater than this. To the list of adverse effects can be added a trend for poorer clover growth in endophyteinfected pasture (Stevens & Hickey, 1990; Sutherland & Hoglund, 1989; Valentine et al., 1993). The effect is not always observed (Eerens et al., 1998a), and is mostly an inverse effect of ryegrass vigour (Prestidge et al., 1992). Direct inhibitor (allelopathic) effects on white clover have been documented (Sutherland et al., 1999), but apparent stimulatory effects have also been noted (Eerens et al., 1998d). Livestock may preferentially graze clover in a mixed sward (Milne et al., 1982). This preference is partial (Parsons et al., 1994), and more active avoidance of ryegrass if infected may intensify it. Alternatively, if clover is growing evenly through a grass sward, rather than in a mosaic, endophyte by discouraging close grazing by stock may protect the clover stolons. Poorer clover content in a ryegrass pasture will exacerbate any negative effects of endophyte on livestock performance, and these two factors have been confounded in some experiments. PAGE 6

However,ryegrass endophyte contributes to livestock productivity,by ensuring a sustained leafy ryegrass pasture in stressful situations.The alternative to endophyte- sp or tropical grassesyasureom poor et al 1996). ophyte-infected es The discovery of the novel AR1 endophyte could completely change this scenario as outlined later (Easton et al..2001). 2.4 Alkaloid Metabolites Produced by Endophyte Before 1980.it was thought likely that a fungus was involved with ryegrass staggers,and research aimed to isolate the chemical compound(s)responsible. The discovery of the role of the endophyte hastened this process,and lolitrem B was isolated and described (Gallagher et al., 981). Several related compounds have been id entified,but lolitrem B is mo dant and perhaps the mo ost poten (Mile em ay Smith 999.1994. et a 1993)a staggers na n the ue to enceoe ins er he ueot0npamnas different.and peramine was isolated and described (Rowan Gay or 1986) arch had identified ergovaline as the primany fescue toxin.and this compound was later found in endophyte-infected PRG(Rowan Shaw 1987).Ergovaline and lolitrem B primarily studied for their effects on livestock,also offer protection against invertebrate pests (Dymock et al.,1989;Ball et al.,1997).Co-evolution of endophyte and host is reflected in the array of compounds occurring in different endophyte-infected grasses (Lane et al.,2000). Endophyte produces a range of alkaloid metabolites (Lane et al.,1999;Lane,1999) Endophyte was sho Bwas shown to be thegent or ryegrafastaggers (hetchen Harvey, 1981 e major ecte caus tor (Gall et a 1981).althc n yegras: re tre D i 9 nd su al 1989: adult Asw Peramine was shown to be the major causal factor(Rowan Ga or 1986).Tall fescue toxicosis is associated with endophyte infection (Hoveland et al.,1980).an d the chief toxin responsible is ergovaline (Yates et al.,1985).This compound is also found in infected ryegrass(Rowan et al.,1990),accounting for reports of heat stress of livestock in northern NZ (Easton et a/.,1996;Sutherland,1984)and perhaps interacting with lolitrem B to exacerbate ryegrass staggers(Fletcher Easton,1997).Ergovaline is the most abundant of a number of lysergyl compounds produced by endophyte,and these may have a range of pharmacological effects.They have been shown to contribute to ryegrass resistance against black eetle (Ball et al.,1997c).Other less-understood compounds are also produced by endophytes in their host grasses (Lane ef al.,2000). 1995: Keogh 1996) ergovaline PAGE 7

However, ryegrass endophyte contributes to livestock productivity, by ensuring a sustained leafy ryegrass pasture in stressful situations. The alternative to endophyteinfected ryegrass in many situations would be bare ground, or pasture dominated by Poa spp or tropical grasses of poor quality (Campbell et al., 1996). Endophyte-infected ryegrass pastures generally provide more digestible dry matter to grazing livestock than endophyte-free pastures. The discovery of the novel AR1 endophyte could completely change this scenario as outlined later (Easton et al., 2001). 2.4 Alkaloid Metabolites Produced by Endophyte Before 1980, it was thought likely that a fungus was involved with ryegrass staggers, and research aimed to isolate the chemical compound(s) responsible. The discovery of the role of the endophyte hastened this process, and lolitrem B was isolated and described (Gallagher et al., 1981). Several related compounds have been identified, but lolitrem B is the most abundant and perhaps the most potent (Miles et al., 1994). Paxilline is similar to lolitrem B and is a tremorgen itself (McLeay & Smith, 1999; Fletcher et al., 1993), but much milder (Easton pers. comm.). Mild staggers has occurred in the absence of lolitrem B (Fletcher et al., 1993) and this may have been due to paxilline or other unidentified neurotoxins. The main compound responsible for protection from ASW was shown to be different, and peramine was isolated and described (Rowan & Gaynor, 1986). USA research had identified ergovaline as the primary fescue toxin, and this compound was later found in endophyte-infected PRG (Rowan & Shaw 1987). Ergovaline and lolitrem B, primarily studied for their effects on livestock, also offer protection against invertebrate pests (Dymock et al., 1989; Ball et al., 1997). Co-evolution of endophyte and host is reflected in the array of compounds occurring in different endophyte-infected grasses (Lane et al., 2000). Endophyte produces a range of alkaloid metabolites (Lane et al., 1999; Lane, 1999). Endophyte was shown to be the agent of ryegrass staggers (Fletcher & Harvey, 1981), and lolitrem B was shown to be the major causal factor (Gallagher et al., 1981), although infected ryegrass may contain a number of similar compounds (Miles et al., 1992), some more tremorgenic than others. Lolitrem B is also active against ASW, deterring larval feeding and reducing larval growth rates and survival (Dymock et al., 1989a; Prestidge & Gallagher, 1988). As mentioned above, endophyte deters feeding by adult ASW. Peramine was shown to be the major causal factor (Rowan & Gaynor, 1986). Tall fescue toxicosis is associated with endophyte infection (Hoveland et al., 1980), and the chief toxin responsible is ergovaline (Yates et al., 1985). This compound is also found in infected ryegrass (Rowan et al., 1990), accounting for reports of heat stress of livestock in northern NZ (Easton et al., 1996; Sutherland, 1984) and perhaps interacting with lolitrem B to exacerbate ryegrass staggers (Fletcher & Easton, 1997). Ergovaline is the most abundant of a number of lysergyl compounds produced by endophyte, and these may have a range of pharmacological effects. They have been shown to contribute to ryegrass resistance against black beetle (Ball et al., 1997c). Other less-understood compounds are also produced by endophytes in their host grasses (Lane et al., 2000). The location of the alkaloid metabolites in the plant reflects in part the location of the endophyte (Ball et al., 1995; Keogh et al., 1996); lolitrem B and ergovaline are found PAGE 7

primarily in the leaf sheath,the true stem,inflorescence and the seed.Ergovaline is particularly elevated in the crown and the inflorescence (Lane et al.,1997a).Peramine moves more freely through the plant shoot,and may have a higher concentration in the leaf blade than the sheath.When the seed germina tes,peramine and,to a lesser extent move through the develop ng see n against inse t attack betore the peramine( st-germination endophyte activity begins to generatef Low amounts of peramine,ergovaline and lolitrem B have been found in root tissue(Ball et al..1997a:Ball et al..1997b).There is evider nce that so me root-feeding inve may be sensitive to endophyte-infection(Ee ens et al.,1998b).but there is no conclusive evidence of important effects(Prestidge Ball,1993). Production of peramine,lolitrem B (and similar compounds)and ergovaline (with other lysergyl compounds)are properties of the fungus,not the plant (Lane et al.,1999). However,the plant does exercise some genetic control over the growth of the endophyte within it and the endophyte production of metabolites(Ball et al.,1995:Latch,1994). may even have additiona 2.5 Variation in Alkaloid Concentrations Concentrations of peramine.eraovaline and lolitrem b vary in parts of the plant as different tissues age Thus,concentrations of lolitrem B are greater in older than in younger leaves,while those of peramine are the reverse(Keogh et al.,1996).Endophyte hyphae extend during the period of growth of a particular leaf,but there appears to be no extra growth of hyphae after leaf growth ceases(Schmid Christenson,1999).Production of beramine. ergovaline and lolitrem B is maintained when the leaf is mature (after endophyte hyphal growth has ceased),until senescence.As senescence approaches, peramine declines in the leaf,while the concentration of lolitrem B remains high. The an concent tions of peramine,ergovaline and lolitrem r reproa Environmental conditions also affect metabolite concentrations (Lane et al 1997b) Water deficit was associated with increased ergovaline and lolitrem B concentrations in both field and growth cabinet.Peramine levels were increased in the field by water stress in only one of two years.Peramine was not measured in the growth cabinet.Ergovaline concentrations were elevated in large and in small plots receiving extra nitrogen.Lolitrem B levels were higher in the high nitrogen large plots,but the reverse effect was observed in a small plot experiment.No consistent effect of temperature was observed in the growth cabinet experiment PAGE8

primarily in the leaf sheath, the true stem, inflorescence and the seed. Ergovaline is particularly elevated in the crown and the inflorescence (Lane et al., 1997a). Peramine moves more freely through the plant shoot, and may have a higher concentration in the leaf blade than the sheath. When the seed germinates, peramine and, to a lesser extent, lolitrem B move through the developing seedling, offering protection against insect attack before the renewed post-germination endophyte activity begins to generate fresh peramine (Ball et al., 1993). Low amounts of peramine, ergovaline and lolitrem B have been found in root tissue (Ball et al., 1997a; Ball et al., 1997b). There is evidence that some root-feeding invertebrates may be sensitive to endophyte-infection (Eerens et al., 1998b), but there is no conclusive evidence of important effects (Prestidge & Ball, 1993). Production of peramine, lolitrem B (and similar compounds) and ergovaline (with other lysergyl compounds) are properties of the fungus, not the plant (Lane et al., 1999). However, the plant does exercise some genetic control over the growth of the endophyte within it and the endophyte production of metabolites (Ball et al., 1995; Latch, 1994). In their physiologic effects on animals, the endotoxins appear to often have a synergistic effect, and may even have additional synergistic effects with Fusaria toxins when consumed by grazing animals (Whalley, 2002). 2.5 Variation in Alkaloid Concentrations Concentrations of peramine, ergovaline and lolitrem B vary in parts of the plant as different tissues age. Thus, concentrations of lolitrem B are greater in older than in younger leaves, while those of peramine are the reverse (Keogh et al., 1996). Endophyte hyphae extend during the period of growth of a particular leaf, but there appears to be no extra growth of hyphae after leaf growth ceases (Schmid & Christenson, 1999). Production of peramine, ergovaline and lolitrem B is maintained when the leaf is mature (after endophyte hyphal growth has ceased), until senescence. As senescence approaches, peramine declines in the leaf, while the concentration of lolitrem B remains high. The mean concentrations of peramine, ergovaline and lolitrem B in pasture vary with time as the structure of the pasture canopy evolves. In particular, levels increase as the proportion of reproductive tissue in the plant increases. Environmental conditions also affect metabolite concentrations (Lane et al., 1997b). Water deficit was associated with increased ergovaline and lolitrem B concentrations in both field and growth cabinet. Peramine levels were increased in the field by water stress, in only one of two years. Peramine was not measured in the growth cabinet. Ergovaline concentrations were elevated in large and in small plots receiving extra nitrogen. Lolitrem B levels were higher in the high nitrogen large plots, but the reverse effect was observed in a small plot experiment. No consistent effect of temperature was observed in the growth cabinet experiment. In a pasture, higher herbage concentrations of lolitrem B and ergovaline have been measured in urine patches than in surrounding areas (Keogh & Clements, 1993). The PAGE 8

importance of this observation is augmented by a tendency of livestock to preferentially graze these areas(Keogh,1986). The combined effects nditio (Ball et al.,1993 Both ergove d to in develop 4 at maximum seedne ad c 9 The levels fall in the post-reproductive regrowth to rise again through the sum r in response to increasin water stress and perhaps temperature,alond with an accumulation of older leaf and under continued grazing.an increase in the proportion of leaf sheath to leaf blade Concentrations fall again in the autumn,but this is the least predictable period.Prolonged water stress and high temperatures in some circumstances may delay falls in alkaloid levels.and the release of mineralized nitrogen when rain ends an extended dry penod (Russell,1991)may provoke a late peak. Ergovaline eaea1weosaU9su2IeC3latuaIo8gkg9o5s2g leaves killed by de leaf in past ure may therefore be a source of bo ns, ma also retain some hay. 2.6 Pest Protection Persistence:A Summary of the Main Events In the trial established at Ruakura to compare stock performance on infected and endophyte-free pasture,the endophyte-free sward was severely damaged by ASW (Prestidge et a/.,1982).Subsequent work established the crucial role played by endophyte in ensuring the persistence of PRG(Table 1)and the reputation of the species as well adapted to NZ.Without endophyte,PRG swards in Waikato,Canterbury and some other districts simply did not persist(Mortimer di Menna,1983;Prestidge Ball 1993;Popay Rowan,1994).Both field plots and glasshouse pot experiments were used in this work. The resistar of e hyte-intected PRG to ASW is ba ed on det 346 eggs an &ng the adult wee nt, 33 o tha al hoot ovides this 6 amine seed coat is translocated after th rough the emergina seedling. insect feeding in the early pe wth(Stew art,1985:Ball et a/..1993). ASW larvae are not deterred from feeding,but growth rates and survival of larvae are lower on endophyte-infected than endophyte-free PRG(Barker et a/.,1984a). ASW is not the only invertebrate pest sensitive to endophyte.Black beetle (Heteronychus arator)is sporadically serious in northern NZ,attacking PRG pastures if they are free of endopnyte,and several other grass species However.infected PRG is not severely attacked (Ball Prestidge,1992). PAGE9

importance of this observation is augmented by a tendency of livestock to preferentially graze these areas (Keogh, 1986). The combined effects of plant development and changing environmental conditions lead to a pattern of change of metabolite levels throughout the year (Ball et al., 1995; di Menna et al., 1992; Easton et al., 1993). Both ergovaline and lolitrem B tend to be in low concentration in early spring, and to increase with rising temperatures and reproductive development, to a first peak at maximum seedhead emergence. The levels fall in the post-reproductive regrowth to rise again through the summer in response to increasing water stress and perhaps temperature, along with an accumulation of older leaf and, under continued grazing, an increase in the proportion of leaf sheath to leaf blade. Concentrations fall again in the autumn, but this is the least predictable period. Prolonged water stress and high temperatures in some circumstances may delay falls in alkaloid levels, and the release of mineralized nitrogen when rain ends an extended dry period (Russell, 1991) may provoke a late peak. Ergovaline and, to a lesser extent, lolitrem B remain in senescent leaves, and also in leaves killed by defoliation or rapid desiccation. Dead leaf in pasture may therefore be a significant source of both toxins, as may hay. There is some evidence that silage may also retain some toxic potential (Clark et al., 1996). 2.6 Pest Protection & Persistence: A Summary of the Main Events In the trial established at Ruakura to compare stock performance on infected and endophyte-free pasture, the endophyte-free sward was severely damaged by ASW (Prestidge et al., 1982). Subsequent work established the crucial role played by endophyte in ensuring the persistence of PRG (Table 1) and the reputation of the species as well adapted to NZ. Without endophyte, PRG swards in Waikato, Canterbury and some other districts simply did not persist (Mortimer & di Menna, 1983; Prestidge & Ball, 1993; Popay & Rowan, 1994). Both field plots and glasshouse pot experiments were used in this work. The resistance of endophyte-infected PRG to ASW is based on deterring the adult weevil from feeding (Barker et al., 1984b). Fewer eggs are laid (Gaynor & Hunt, 1983) so that larval numbers are lower. The compound peramine, produced by the endophyte and translocated through the shoot system, provides this deterrence. Peramine in the seed coat is translocated after germination through the emerging seedling, deterring insect feeding in the early period of growth (Stewart, 1985; Ball et al., 1993). ASW larvae are not deterred from feeding, but growth rates and survival of larvae are lower on endophyte-infected than endophyte-free PRG (Barker et al., 1984a). ASW is not the only invertebrate pest sensitive to endophyte. Black beetle (Heteronychus arator) is sporadically serious in northern NZ, attacking PRG pastures if they are free of endophyte, and several other grass species. However, infected PRG is not severely attacked (Ball & Prestidge, 1992). PAGE 9

Table 1: Major discoveries of endophyte effects on pasture invertebrates. (From Easton et al., 2001). Discovery Reference Ryegrass infected with wild-type endophyte is resistant to Argentine stem weevil (ASW). Prestidge et al. (1982) The ASW feeding deterrent, peramine, is isolated from wild-type-infected ryegrass. Rowan & Gaynor (1986) Lolitrem B affects ASW larvae growth and development. Dymock et al. (1989) Ergovaline deters adult ASW. Popay et al. (1990) Lolitrem-free endophytes give resistance to ASW. Fletcher et al. (1991) Black beetle adults are deterred by wildtype endophyte. Ball & Prestidge (1992) Ergovaline is identified as a major factor in black beetle resistance. Ball et al. (1997) AR1-infected ryegrass is as resistant to ASW as wild-type. Popay et al. (1999) Ryegrass with endophyte, including AR1, is resistant to a pasture mealy bug. Pennell (1998) unpublished data; Popay et al. (2000) Ryegrass with AR1 has some resistance to black beetle. Popay & Baltus (2001) Recent data have revealed the sensitivity to endophyte of the pasture mealy bug (Balanococcus poae), and the severe effects this little-known insect can have on endophyte-free PRG pastures, particularly after a dry summer (Pennell & Ball, 1999; Popay et al., 2001). Other invertebrate research has studied root- and soil-dwelling nematode species (Eerens et al., 1998b; Watson, 1990; Stewart et al., 1993; Watson et al., 1995), several other pasture pests and beneficial soil invertebrates such as earthworms (Prestidge & Marshall 1997; Prestidge et al., 1997). None of these have shown the clear response to endophyte established for ASW, black beetle and pasture mealy bug. Direct enhancement of PRG growth by endophyte was documented by Latch et al. (1985), but other experiments (Barker et al., 1997; Hume et al., 1993) (D.E. Hume, H.S. Easton & M.P. Rolston, unpublished data cited by Easton, 2001) have not shown this. Endophyteinfected PRG competed more aggressively with associated white clover (Sutherland & Hoglund 1989; Stevens & Hickey 1990; Sutherland et al., 1999), and a direct suppressing factor released from grass litter has been suggested. However, most of the effect can be ascribed to greater grass vigour, arising from protection from invertebrate pests and overgrazing (Prestidge et al., 1992). American scientists have identified pests that are sensitive to endophyte, but do not consider this the major factor in the superior persistence of endophyte-infected fescue. Their research has focused on direct effects of the fungus, on grass growth and drought tolerance. In contrast, it is considered in NZ that pest protection is the primary component of superior productivity and persistence of endophyte-infected PRG pastures (Easton et al., 2001). PAGE 10