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a Texas A&M Univ., Texas Agric. Exp. Stn., P.O.B. 1658, Vernon, TX 76385 USA
b USDA-ARS, Appalachian Farming Systems Research Center, 1224 Airport Rd., Beaver, WV 25813 USA
d-malinowski{at}tamu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMechanisms of Biotic Stress...Adaptations and Mechanisms of...Adaptations and Mechanisms of...Manipulating of the Neotyphodium...Symbiosis of Cool-Season Grasses...ConclusionsREFERENCES |
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Abbreviations: DM, dry matter • PWUE, instantaneous water use efficiency • TW/DW, turgid weight:dry weight ratio • WUE, water use efficiency
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMechanisms of Biotic Stress...Adaptations and Mechanisms of...Adaptations and Mechanisms of...Manipulating of the Neotyphodium...Symbiosis of Cool-Season Grasses...ConclusionsREFERENCES |
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Interactions between Neotyphodium spp. endophytes and cool-season grasses have been referred to as defensive mutualism (Clay, 1988). In these associations, endophytes produce a range of alkaloids (Porter, 1994) or stimulate the host grass to synthesize alkaloids and other secondary metabolites (Bush et al., 1997; Ju et al., 1998) that protect the symbiotum from mammalian, insect, and nematode herbivores. Mutualistic symbioses, however, result in a net benefit to each participant. By comparing genetically identical endophyte-infected with noninfected plants, such benefits to the grass host may be shown. In contrast, Neotyphodium spp. endophytes cannot exist without their grass hosts (cultured mycelia excepted); therefore, they are completely dependent on the host grass for protection and supply for nutrients and water. Wilkinson and Schardl (1997) revised the concept of mutualistic symbioses between Neotyphodium spp. endophytes and their grass hosts and defined them as asymptomatic symbioses. In such associations, the loss of sexuality is a fundamental cost in the long term in contrast to associations which have both means of reproduction (called balanced symbioses). Thus, the most important benefit to Neotyphodium spp. endophytes appears to be dissemination through seeds of the host plant. This may, in part, explain an often reported increase in fitness of endophyte-infected grasses. Apparently, maximizing host growth by endophyte is in the ecological interest of both partners. Recently, virus-like particles have been identified in the endophyte Epichloë festucae Leuchtmann, Schardl and Siegel isolated from asymptomatic F. rubra L. plants (Zabalgogeazcoa et al., 1998), suggesting a third component (a mycovirus) of endophyte–grass associations. Although the presence of viral RNA did not induce any phenotypic effects in its fungal host, possible phenotypic effects on the grass host deserve attention in future experiments.
Endophyte-infected grasses express a range of adaptations to biotic (reviewed in Latch, 1993) and abiotic stresses, including drought (reviewed in Bacon, 1993; West, 1994), mineral imbalance (Lyons et al., 1990; Malinowski et al., 1998a, 1999b), and soil acidity (Belesky and Fedders, 1995; Malinowski and Belesky, 1999a). As a result, endophyte-infected grasses are more competitive than noninfected grasses and thrive better than noninfected grasses with limited resources (Marks et al., 1991; Bacon and Hill, 1996; Hill et al., 1998). Recent approaches to endophyte–grass interactions focus on isolated endophyte strains that, in association with grasses, show minimal or no production of alkaloids toxic to livestock yet retain the pest- and drought-resistance benefits of symbiotic plants (Fletcher and Easton, 1997; Funk and White, 1997; Bacon and Hinton, 1998). These associations are crucial for improved livestock performance on fescue and ryegrass. Once alkaloid production has been altered, it is essential to understand mechanisms involved in abiotic stress tolerance of endophyte-infected grasses for their continued improvement and persistence for a range of applications.
We outline the principal research findings and conclusions on abiotic and biotic stress tolerance of Neotyphodium spp. endophyte–grass associations, emphasizing recent research progress on endophyte-induced mechanisms of mineral stress tolerance in tall fescue.
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Mechanisms of Biotic Stress Tolerance |
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TOPABSTRACTINTRODUCTIONMechanisms of Biotic Stress...Adaptations and Mechanisms of...Adaptations and Mechanisms of...Manipulating of the Neotyphodium...Symbiosis of Cool-Season Grasses...ConclusionsREFERENCES |
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Researchers identified several alkaloids responsible for poor performance of animals grazed or fed on endophyte-infected grasses (Porter, 1994). Ergot alkaloids (clavines, lysergic acid and its amides, ergopeptines) are responsible for fescue toxicosis in grazing livestock and are produced by endophytes of tall fescue and perennial ryegrass, N. coenophialum and N. lolli, respectively. When artificially infected with N. coenophialum, meadow fescue can also synthesize ergot alkaloids in contrast to the natural N. uncinatum-meadow fescue association, which does not (Christensen, 1997). Ergot alkaloids were detected in many endophyte-infected grass species other than Lolium spp. and Festuca spp. (Bacon et al., 1986; Miles et al., 1996). Lolitrem (indole diterpene) alkaloids [paxilline, paxitriol(s), lolitriol, lolitrems] were first identified in symbiotic ryegrass plants and related to livestock neurotoxicities known as ryegrass staggers. The ability to produce lolitrems and paxilline has also been shown in vitro for cultures of N. coenophialum and N. uncinatum (Penn et al., 1993), and in vivo in endophyte-infected tall fescue (Garthwaite, 1997). The pyrrolopyrazine alkaloid peramine is produced by endophyte-infected ryegrass, tall fescue, and other grasses (Siegel et al., 1990). Peramine has insect-feeding deterrent properties (Breen, 1994), but no apparent activity against mammalian herbivores (Bush et al., 1997). Loline alkaloids (N-acetylloline, N-formylloline) are synthesized by endophyte-infected grasses (Siegel et al., 1990; Porter, 1994) and they are potent insecticides (Dahlman et al., 1997). In endophyte-infected meadow fescue and tall fescue, the fungal component of the association seems to play a key role in synthesis of loline alkaloids (Wilkinson et al., 1997). Huizing et al. (1991) documented involvement of high temperatures in loline alkaloid production by endophyte-infected perennial ryegrass. Loline alkaloids have negligible effects on livestock compared with ergot or lolitrem alkaloids (Strickland et al., 1996), although their concentrations in endophyte-infected grasses are 100 to 1000 times higher than those of other alkaloids (Siegel et al., 1989). Indole alkaloids (indoleglycerol, indoleacetic acid) are products of tryptophan metabolism of endophyte, plant, and the symbiotum. Their effects on animal performance are not known. Indole alkaloids have the potential to interfere with host plant physiology and synthesis of other secondary metabolites by the symbiotum. Other miscellaneous alkaloids (ergosterol, ergostatetraeneone, ergosterol-peroxide, harman, norharman, halostachine) found in endophyte-infected grasses may be involved in animal toxicities and may act as phytoalexins (antibiotics) as well (Latch, 1993).
Non-alkaloid secondary metabolites occurring in timothy (Phleum pratense L.) infected with E. typhina (Pers. : Fr.) have been classified as sesquiterpenoids, fatty acids, and phenolic compounds (Yoshihara et al., 1985; Koshino et al., 1988, 1989), while flavonoids have been detected in bluegrass (Poa ampla Merr.) infected with N. typhinum (Morgan-Jones and Gams) Glenn, Bacon and Hanlin (Ju et al., 1998). The ecological significance of these compounds in terms of biotic stress tolerance of endophyte-infected grasses is unknown.
Endophytes can indirectly reduce insect-transmitted viral diseases in grasses (West et al., 1990). Endophyte-infected grasses are more resistant than noninfected grasses to soil-borne nematodes (West et al., 1988; Eerens et al., 1997), with resistance probably attributable to alkaloids present in roots (Malinowski et al., 1998b, 1999a). Increased secretion of phenolic-like compounds from roots of infected tall fescue (Malinowski et al., 1998a) could act as feeding deterrents in the rhizosphere, although this remains to be resolved. The reader is referred to Bacon et al. (1997) for a thorough review of endophyte–grass association effects on biotic factors including bacteria, fungi, nematodes, insects, and non-domesticated mammals.
Endophyte-infected grasses, in general, compete successfully with non-infected conspecifics as well as with other plant species and, in time, can dominate a sward (Clay and Holah, 1999). In some cases, the competitive advantage may result from morphological responses such as more massive and more numerous tillers (Hill et al., 1990), greater leaf elongation rate (Eerens et al., 1993; Malinowski et al., 1997b), and altered root architecture (Malinowski et al., 1999b). In others, endophyte-infected grasses may have direct chemical effects (allelopathy) on the competitor. For example, the number of white clover (Trifolium repens L.) plants declined in pastures dominated by endophyte-infected, compared with non-infected, perennial ryegrass (Percival and Duder, 1983; Sutherland and Hoglund, 1989). Seed extracts of endophyte-infected tall fescue inhibited germination of Trifolium spp. (Springer, 1997). Strong suppression of leguminous companion species by endophyte-infected tall fescue in a mixed sward could have significant negative effects on productivity and N economy of a sward. Loline alkaloids may be one such group of endophyte-related secondary metabolites with allelopathic properties. Despite their role as insect deterrants, loline alkaloids were the only group of endophyte-related alkaloids shown to reduce germination rate of monocot and dicot seeds (Petroski et al., 1990); therefore, loline alkaloids may enhance the competitive ability of endophyte-infected grasses by retarding the establishment of competitors in a sward. Recently, allelopathic effects of lolines in endophyte-infected tall fescue-competitor associations have been reported by Bush et al. (1997) and Malinowski et al. (1999a). Malinowski et al. (1999a) observed that loline alkaloid concentration in roots of endophyte-infected tall fescue genotype DN2 was twice that detected in genotype DN11 (1083 µg g-1 and 536 µg g-1, respectively). When both tall fescue genotypes were grown in binary mixtures with red clover (Trifolium pratense L.), the relative yield total (RYT) of the red clover/DN2 tall fescue mixture was less than 1.0 in response to endophyte infection (Fig. 1) . This suggested an allelopathic interaction between these two components of the mixture (Trenbath, 1974). In contrast, the RYT values of red clover binary mixtures with endophyte-infected DN11 and noninfected DN2 and DN11 tall fescue were not significantly different from 1.0, indicating full competition for the same limiting resources (Snaydon and Sattore, 1989). Detailed research with additional tall fescue genotypes is needed to resolve the role of loline alkaloids in allelopathic effects of endophyte-infected grasses on other plant species.
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Adaptations and Mechanisms of Drought Stress Tolerance |
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TOPABSTRACTINTRODUCTIONMechanisms of Biotic Stress...Adaptations and Mechanisms of...Adaptations and Mechanisms of...Manipulating of the Neotyphodium...Symbiosis of Cool-Season Grasses...ConclusionsREFERENCES |
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Drought Avoidance
Drought-avoiding plants possess a variety of morphological and physiological adaptations to maintain favorable water status, either by maintaining an efficient water supply to aboveground organs or conserving water during periods of soil water deficit (Clarke and Durley, 1981). The adaptations may be divided into three basic groups: (i) improved water uptake from the soil by an extensive root system, (ii) reduced transpiration losses, and (iii) water storage in plant tissues.
Extensive Root System
Improved water uptake from the soil may be promoted by a deeper and denser root system (Arraudeau, 1989). In response to endophyte infection, increased root dry matter (DM) has frequently been reported in perennial ryegrass (Latch et al., 1985), tall fescue (De Battista et al., 1990), and meadow fescue (Malinowski et al., 1997a, b). A direct relationship between the size of root system and drought tolerance, however, has not been documented for endophyte-infected grasses. Responses of grass root system to infection with shoot-located endophytes are not easy to quantify because of difficulties in measuring root characteristics. Malinowski et al. (1999b) showed that endophyte infection increased root hair length and decreased root diameter in tall fescue. These traits could increase root surface area for water and mineral acquisition. Because roots are the first plant organs to sense water depletion in the soil, further research on root activity of endophyte-infected grasses should clarify the hypothesis of a water stress signaling system existing in endophyte-infected grasses (West et al., 1994).
Control of Transpiration
Water deficits in plants induce stomatal closure, which may benefit plants in the short term by reducing water loss via transpiration (Turner, 1986). Endophyte presence in shoots was shown to affect stomatal behavior in tall fescue (Elmi and West, 1995; Buck et al., 1997) and meadow fescue (Malinowski et al., 1997a, b), but apparently not in perennial ryegrass (Barker et al., 1997). Stomatal conductance of water-stressed endophyte-infected fescues (Festuca spp.) declined earlier and faster than that of noninfected plants, suggesting more rapid stomatal closing (Elbersen et al., 1994; Elmi and West, 1995). The direct mechanism of this phenomenon is not known. A biochemical signal from the endophyte or an altered hormonal status in the host may predispose leaf blades to respond quickly to early stages of water deficit (Belesky et al., 1987a; West et al., 1994). The hypothesized biochemical signal induced in endophyte–grass associations in response to water deficit may be interpreted as an endophyte-related perturbance in host grass homeostasis. Endophyte, as a xenobiotic component of a grass plant, may induce a permanent internal stress in the host grass. This may precondition or sensitize the plant to drought and other stresses, thereby permitting the plant to exhibit adaptive responses (such as stomata closure) sooner than noninfected conspecifics. Recent research results on symptomless fungal endophytes and their host plants suggest mutual defense responses involving a range of secondary metabolites produced in much higher quantities than observed in plant-fungal pathogen interactions (Schulz et al., 1999). The authors hypothesize that both the pathogen-host and the endophyte-host interactions involve constant mutual antagonisms at least in part based on the secondary metabolites the partners produce. Whereas the pathogen-host interaction is imbalanced and results in a disease, that of the endophyte and its host is a balanced antagonism. Responses suggest that host grasses recognize Neotyphodium spp. endophyte infection by inducing a range of defense mechanisms similar to those against pathogenic fungi. Circumstantial evidence for defense mechanisms of grasses to endophyte infection includes the presence of chitinase (a pathogenesis-related protein) (Roberts et al., 1992), resveratrol (a phenolic compound with antifungal activity correlated with disease resistance) (Powell et al., 1994), and increased concentration of phenolic-like compounds in shoots and roots compared with noninfected grasses (Malinowski et al., 1998a). Novel endophyte–grass associations may be highly incompatible (Koga et al., 1993; Christensen, 1995). This incompatibility may be a result of fungal problems with enzymatic degradation of the middle lamella of host cell walls, which in turn would restrict the nutritional supply within the apoplast of host tissues (Leuchtmann, 1994), also suggesting multifaceted defense responses.
Water Storage in Plant Tissues
Water content of tiller bases of some endophyte–grass associations may be maintained at higher levels than those in noninfected plants during drought (Elbersen and West, 1996; Buck et al., 1997). This phenomenon may be explained by enhanced accumulation of solutes in tissues of endophyte-infected plants compared with noninfected plants, or by reduced leaf conductance and a slow-down of the transpiration stream, or thicker cuticle.
Drought Tolerance
Drought tolerance refers to physiological and biochemical adaptations that enable plant tissues to withstand water deficits (Clarke and Durley, 1981). In most grasses, a combination of drought avoidance and tolerance mechanisms determines the survival potential during drought. Mechanisms of drought tolerance have been divided into three major groups: (i) accumulation and translocation of assimilates, (ii) osmotic adjustment, and (iii) maintenance of cell wall elasticity (Arraudeau, 1989). All these mechanisms can be affected by endophyte infection in grasses.
Accumulation and Translocation of Assimilates
Accumulation of carbohydrates during growth under non-drought conditions and translocation during imposed drought stress may contribute to drought tolerance. In some endophyte–tall fescue associations, endophyte-infected plants had greater concentrations of total nonstructural carbohydrates in tillers than noninfected plants when grown under non-limiting water supply (Hill et al., 1990). Water-stressed endophyte-infected plants of tall fescue genotype CB1 accumulated more glucose and fructose in leaf blades and leaf sheaths than noninfected plants (Richardson et al., 1992). These carbohydrates are osmotically active and may play a role in endophyte-related osmotic adjustment in tall fescue.
Osmotic Adjustment
Accumulation of solutes in tissues in response to drought helps maintain turgor and facilitates physiological and biochemical processes. The following groups of solutes have been particularly considered to contribute to osmotic adjustment in endophyte-infected grasse.
Future research should resolve the role of loline alkaloids in drought tolerance of cool-season grasses. One approach may be a comparison of drought stress responses between endophyte-infected plants producing loline alkaloids and endophyte-infected plants with a disturbed pattern of loline alkaloid synthesis. Loline alkaloids are synthesized from two polyamines, putrescine and spermidine (Bush et al., 1993; Graser and Hartmann, 1997). Spermidine is supposed to be the precursor of the pyrrolizidine ring system of loline alkaloids (Bush et al., 1993). At this time, it is not known if the loline alkaloid precursors putrescine and spermidine are of fungal or host origin. If loline alkaloids were produced from fungal-derived polyamines, a disturbed pattern of polyamine synthesis by endophyte might affect synthesis of loline alkaloids in the symbiotum. This could create a basis for a precise identification of physiological mechanisms of abiotic stress tolerance in endophyte-infected grasses directly related to loline alkaloid action. Certain chemicals have been shown to disturb polyamine synthesis in fungi with negligible effects on plant-synthesized polyamines and plant growth (Elghachtouli et al., 1996; Walters and Macintosh, 1997). For example, 
-difluoromethylornitine (DFMO) is an inhibitor of ornithine decarboxylase, and -difluoromethylarginine (DFMA) inhibits arginine decarboxylase (Walters and Macintosh, 1997). These inhibitors may suppress polyamine synthesis by endophyte and affect loline alkaloid production in the host. If endophyte could synthesize loline alkaloids from grass-originated polyamines, fungal polyamine inhibitors might not affect loline alkaloid synthesis in infected grasses. To date, there is no published information of endophyte effects on polyamine accumulation and involvement of polyamines in drought stress-related accumulation of loline alkaloids in infected grasses.
Maintaining Cell Wall Elasticity
Cellular (tissue) elasticity enables cell size to change in synchrony with water availability (Arraudeau, 1989). White et al. (1992) showed that endophyte infection lowered bulk modulus of tissue elasticity (suggesting less rigid cell walls) and increased turgid weight:dry weight (TW/DW) ratio in water stressed tall fescue. The TW/DW determines water uptake capacity and reflects the ability of cells to absorb water. Higher values of the TW/DW ratio indicate less damaged cell walls. Similar to tall fescue, the TW/DW ratio in meadow fescue leaves was also higher in response to endophyte infection during imposed water stress (Malinowski, 1995). These results suggest that in response to water stress, endophyte may alter cell wall characteristics in a similar way in both grass species. At this time, it is not known which metabolites are directly involved in the process.
Water Use Efficiency
One adaptation to drought stress in plants is efficient water use. Considerable research has been conducted concerning limitations to efficient water use in grass production and improving water use efficiency (WUE) in grasses (reviewed in Johnson and Asay, 1993). The WUE measures the ability to limit water loss while maintaining net carbon uptake in the leaves. Because endophyte-infected grasses can reduce water loss through stomatal closure during soil drying, they may be better able to prolong assimilation or resume growth after drought more rapidly than noninfected grasses (West, 1994). Experimental results are not consistent in support of this hypothesis (Belesky et al., 1987a; Marks and Clay, 1996). Under water non-limiting conditions in a greenhouse, instantaneous water use efficiency (PWUE, ratio of photosynthesis:transpiration rates on unit leaf area basis) calculated from data reported by Richardson et al. (1990) tended to be higher in leaves of endophyte-infected tall fescue genotypes than in noninfected conspecifics. In contrast, PWUE values of drought-stressed endophyte-infected tall fescue genotypes were lower than those of noninfected plants (Bates and Joost, 1990). The latter results showed, however, that noninfected tall fescue plants reduced PWUE more rapidly than endophyte-infected plants as soil water deficit increased, suggesting less efficient adaptation to water stress.
In cool-season grasses and other plant species that fix carbon by the C3 pathway, WUE is related to the stable 13C isotope composition of tissues and can be indirectly estimated from the 13C to 12C ratio in the biomass (Johnson and Bassett, 1991). Plants discriminate against 13C during the process of photosynthesis and the 13C discrimination (
) is described as:
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Theoretically, the gives an integrated estimate of the ratio of leaf intercellular CO2 concentration (Ci) to ambient CO2 concentration (Ca) at constant vapor pressure difference (Johnson and Bassett, 1991). Drought-stressed endophyte-infected grasses have a reduced transpiration rate because of a more rapid stomatal closure than noninfected plants (Elbersen et al., 1994; Elmi and West, 1995). Therefore, one would also expect a reduced carbon fixation rate in endophyte-infected grasses due to a lower Ci/Ca ratio and, in consequence, higher values (Johnson, 1993). Because the is negatively correlated with WUE (Farquhar et al., 1989; Read et al., 1991), endophyte-infected grasses are expected to have lower WUE than noninfected plants under drought. A limited number of experiments on and WUE in endophyte-infected grasses do confirm this hypothesis. In a population of Kentucky-31 tall fescue, Johnson and Tieszen (1993) observed lower mean in noninfected plants compared with endophyte-infected plants in response to drought. Although the authors considered this result as preliminary, it might suggest that endophyte-infected plants had lower WUE than noninfected plants. In other words, endophyte-infected plants might be more conservative than noninfected plants by reducing growth during drought and conserving water in the soil–plant system. Detailed results on WUE in perennial ryegrass confirmed that endophyte-infected plants had lower WUE than noninfected plants in response to drought (Eerens et al., 1998). Interestingly, endophyte-infected plants of perennial ryegrass were less wilted (thus less stressed) than noninfected plants because of improved water status characteristics. Results suggest that endophytes do not directly affect water use efficiency in drought-stressed grasses. Endophytes appear to help maintain physiological and biochemical processes at the cost of reduced growth during drought and facilitate rapid regrowth of host grasses soon after soil water becomes available.
Recovery mechanisms determine the ability of plants to regrow when drought stress is relieved. In endophyte-infected grasses, these mechanisms include a rapid water uptake by roots and the ability of tissues to restore physiological functions. Endophyte effects on growth and water relations in grasses were observed during the weeks following water stress (West et al., 1994; Malinowski et al., 1997b). West et al. (1994) postulated that endophyte effects on growth and drought tolerance may be more important for grass persistence during recovery from drought than during the onset of drought.
Although responses of endophyte-infected grasses to drought have been well documented, the understanding of mechanisms by which endophytes enhance host grass survival during drought is incomplete. Endophyte infection of grasses induces a range of responses to water deficit that are a combination of drought avoidance, tolerance and recovery mechanisms. These responses may or may not be expressed by a particular endophyte–grass association, but the sum of responses of individual endophyte–grass associations determines the ecological success of a given grass population (West, 1994). As a result, the range of adaptation of endophyte-infected cool-season grasses can be extended into areas dominated by warm-season grasses which can help improve pasture productivity (Bouton et al., 1993; Cunningham et al., 1994).
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Adaptations and Mechanisms of Mineral Stress Tolerance |
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TOPABSTRACTINTRODUCTIONMechanisms of Biotic Stress...Adaptations and Mechanisms of...Adaptations and Mechanisms of...Manipulating of the Neotyphodium...Symbiosis of Cool-Season Grasses...ConclusionsREFERENCES |
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Nitrogen
High rates of N fertilizer increased the concentration of ergopeptine alkaloids in controlled-environment (Lyons and Bacon, 1984) and field grown endophyte-infected tall fescue (Belesky et al., 1988). Increasing N fertilization from 134 kg ha-1 to 334 kg ha-1 increased the concentration of total ergopeptine alkaloids by 60% to 80%, depending on the year (Belesky et al., 1988). Using a single clone of Kentucky-31 tall fescue, Arechavaleta et al. (1992) showed that accumulation of ergot alkaloids increased not only in response to N fertilization but was also dependent on N source. In that experiment, ergot alkaloids were not detected at a very low N level in the soil (11 mg pot-1). In contrast, concentrations of ergovaline and total ergopeptides were 0.41 and 0.57 µg mg-1 DM, respectively, at a high N level (220 mg pot-1). The NH+4 form of applied N had a greater effect on accumulation of ergot alkaloids than the NO-3 form. The concentration of loline alkaloids is also affected by N fertilization. Belesky et al. (1987b) showed that greater loline alkaloid concentrations on a unit of endophyte infection rate were associated with increased N availability in the soil.
It is well known that N fertilization increases herbage yield of grasses. Clay (1987) analyzed endophyte effects on DM production of tall fescue and perennial ryegrass seedlings grown under optimal nutrient supply (weekly applied soluble fertilizer 20:20:20 NPK, 1 g L-1). For both species, tiller number and above-ground biomass of endophyte-infected plants were greater than those of noninfected plants. Cheplick et al. (1989) found that DM production by seedlings of tall fescue and perennial ryegrass depended on nutrient availability and endophyte infection. Seedling DM of endophyte-infected ryegrass was greater at intermediate (biweekly applied soluble fertilizer 20:20:20 NPK, 0.15 g L-1) and high (1.5 g L-1) nutrient levels compared with noninfected plants but no differences in plant biomass were observed when no nutrients were added. In contrast, endophyte infection reduced biomass of tall fescue seedlings at low nutrient level but increased plant biomass at high nutrient level. Endophyte-infected adult plants of tall fescue produced more biomass than did noninfected plants, whereas adult ryegrass plants did not respond to endophyte infection, regardless of nutrient availability. On the basis of these results, the authors concluded that endophyte infection might induce an additional metabolic cost to the host plant grown under mineral stress conditions. Plants used by Cheplick et al. (1989) represented a population. As such, they varied in the genetic make up, thus endophyte-related effects on growth were inconsistent. Belesky et al. (1991) showed that endophyte did not affect carbohydrate accumulation in field-grown tall fescue populations grown either at low (134 kg ha-1) or high (336 kg ha-1) N availability. The authors concluded that endophyte was not detrimental to the host grass grown at low soil N level. Working on genetically identical plants, Belesky et al. (1989a) found that four of five tall fescue clones produced more biomass (15% on the average) in response to endophyte infection when grown at optimal N level of 150 mg kg-1 soil and adequate water supply. A detailed study by Arachevaleta et al. (1989) showed that N-use efficiency was greater in endophyte-infected plants. In response to endophyte infection, plants of a tall fescue clone derived from cv. Kentucky-31 grown at low (11 mg pot-1) N level produced a similar herbage DM to noninfected plants grown at high (220 mg pot-1) N level. With increased N supply, the difference between endophyte-infected and noninfected plants became more dramatic as shown by a 100% and a 50% increase in herbage DM, respectively for plants grown at 73 and 220 mg N pot-1.
Proline and amino acid concentrations in Kentucky-31 tall fescue populations (the degree of endophyte infection not determined) depended on soil N availability (Belesky et al., 1982, 1984). Similarly, amino acid concentrations were greater in endophyte-infected than in noninfected tall fescue as N fertilization increased (Belesky et al., 1984). Lyons et al. (1990) observed a greater accumulation of amino acids in leaf sheaths than blades of tall fescue. An endophyte-related increase in total amino acid concentration was significant only at high N level (10 mM ) but no effects were observed at low N level (0.5 mM). They found an endophyte-related increase in sheath NH+4 concentrations and decrease in sheath and blade NO-3 concentrations, which suggested an endophyte effect on nitrogen metabolism in tall fescue. A detailed investigation showed that endophyte-infected plants had greater activity of glutamine synthetase, an enzyme responsible for NH+4 reassimilation, regardless of N availability. This mechanism helped explain a more efficient use of N by endophyte-infected tall fescue as previously shown by Arachevaleta et al. (1989). Alteration of N metabolism by endophyte in tall fescue appears to involve both assimilatory and basic nitrogen metabolism (Lyons et al., 1990) and may be correlated with mechanisms of in vitro N utilization by endophyte mycelium. Kulkarni and Nielsen (1986) showed that a N. coenophialum strain could assimilate NH+4 but not NO-3 as a source of N from the growth medium. In contrast, three out of four N. coenophialum isolates from Kentucky-31 tall fescue analyzed by Ferguson et al. (1993) could utilize NO-3 and all of them could utilize NH+4 as N source. Naffaa et al. (1998b) examined growth of endophytes from 12 grass species of Poaceae and found that all of them (including N. lolii) could assimilate NH+4 but only A. chilense-like endophytes (a-endophytes) could assimilate NO-3. These results suggest a considerable variability in N utilization among endophyte isolates from different grass species. It remains to be resolved if increased N use efficiency in grasses could be directly (fungal enzymes) or indirectly (stimulation of the host enzymes) related to endophyte infection.
Responses of endophyte-infected grasses, other than tall fescue, to N nutrition are less well studied. In a single clone of perennial ryegrass grown in nutrient solutions at low (3 µM NH4NO3) and high (30 µM NH4NO3) N levels, endophyte infection did not affect either DM production or N uptake (Lewis et al., 1996). In an experiment by Ravel et al. (1997) with three clones of perennial ryegrass, endophyte-infected plants had more leaves and tended to have more tillers than noninfected plants when grown at low (2.33 mM) N level in the soil. Dry matter yield, however, was not affected by endophyte, confirming results by Lewis et al. (1996). They suggested that photosynthesis was similar for endophyte-infected and noninfected plants grown under N deficiency. Interestingly, endophyte-infected plants compensated for lower net photosynthesis with an increased number of green leaves compared with noninfected plants. Malinowski (1995) observed no significant effect of endophyte on N use efficiency (DM produced per unit of uptaken N) of meadow fescue grown under nonstressed (daily supply with 7.5 mM N nutrient solution) conditions, regardless of soil water content. Cloned genotypes of Arizona fescue infected with a Neotyphodium spp. endophyte had a greater herbage DM at low soil N level but no responses were observed at high soil N level compared with noninfected plants (Louis and Faeth, 1997). In a subsequent study with grazed populations of Arizona fescue, Schulthess and Faeth (1998) found more N. starri-infected plants at a location with the lowest soil N concentration (about 0.6 mg kg-1) than at a location with the highest soil N concentration (about 2.0 mg kg-1). These results suggest that endophyte benefits in cool-season grasses depend on specific endophyte–host associations and site nutrient availability conditions.
Phosphorus
Phosphorus is involved in ergot alkaloid biosynthesis in Claviceps spp., a fungus related to clavicipitaceous grass endophytes (Robbers, 1984; Flieger et al., 1991). A high P concentration restricted the activity of the first enzyme in the biosynthesis pathway of ergot alkaloids, dimethylallyl tryptophan synthase (DMATase), in in vitro grown Claviceps spp. A similar pathway of ergot alkaloid biosynthesis is proposed for grass endophytes within the Clavicipitaceae (Garner et al., 1993; Glenn and Bacon, 1997). Phosphorus nutrition, therefore, has been postulated to affect ergot alkaloid production and growth of Neotyphodium spp. infected grasses (Porter, 1994).
Detailed studies on P involvement in growth and ergot alkaloid production in endophyte-infected tall fescue were initiated by Azevedo (1992) and Azevedo et al. (1993). The authors investigated two cloned genotypes of Kentucky-31 tall fescue, DN7 and DN11, grown at contrasting soil P levels determined as deficient (0.02 mg kg-1 P) to sufficient (2.0 mg kg-1 P). These tall fescue clones produce low (DN7) and high (DN11) amounts of ergot alkaloids when endophyte-infected, and have been used extensively to study endophyte effects on tall fescue growth and physiology (Belesky et al., 1989a,b; Hill et al., 1990, 1996). Growth and photosynthesis rates of both endophyte-infected tall fescue genotypes were reduced compared to noninfected plants as P availability in the soil increased. Endophyte appeared to be a sink for P (Azevedo, 1992). The production of ergovaline was a function of increased P availability and was not related to specific endophyte strain–tall fescue genotype association (Azevedo et al., 1993). In a subsequent study (Azevedo and Welty, 1995), endophyte hyphae accumulated inorganic P, similar to that found in mycorrhizal fungi (White and Brown, 1979; Lapeyrie et al., 1984). Such P reserves in endophyte hyphae might benefit grass host under low soil P conditions.
Our research group conducted a series of experiments to clarify the role of endophyte in tall fescue tolerance to P deficiency. We grew four tall fescue cloned genotypes (DN2, DN4, DN7, and DN11) infected with endemic endophytes and their noninfected clones in an acidic, high Al content soil at three P levels of 17, 50, and 96 mg kg-1 (Malinowski et al., 1998b). The P levels represented low, medium, and high P fertilization rates appropriate for acidic soils (Wright et al., 1987). Endophyte-infected plants had less root and shoot DM compared with noninfected plants as P availability increased (Fig. 2) . At low and medium P levels, specific root length of endophyte-infected tall fescue was considerably greater than that of noninfected plants suggesting a finer root system. Concentrations of P, magnesium (Mg), and calcium (Ca) in roots and shoots were greater in response to endophyte infection only when plants were grown at low soil P level (Fig. 3) . Concentrations of ergot alkaloids were increased in low ergot alkaloid genotypes DN2 and DN4 as P fertilization increased. In contrast, ergot alkaloid production in high ergot alkaloid genotypes DN7 and DN11 reached a peak at medium soil P level and declined with a further increase in soil P availability. Interestingly, Al concentration in roots was greater than in shoots of endophyte-infected plants compared with noninfected plants, suggesting an Al sequestration mechanism located in/on the roots of endophyte-infected tall fescue. The results show that endophyte infection does not benefit tall fescue growth as P availability in the soil increases, and that manipulating P nutrition may reduce or increase production of ergot alkaloids, depending on particular endophyte–tall fescue associations.
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An endophyte-enhanced release of phenolic-like compounds with Fe3+- reducing activity into the rhizosphere (Malinowski et al., 1998a) may affect Fe uptake by tall fescue. The mechanism of Fe uptake in grasses involves nonproteinogenic amino acid carriers (phytosiderophores) (Takagi, 1976; Marschner, 1986). Recently, an alternative mechanism has been reported for mycorrhiza-infected sorghum [Sorghum bicolor (L.) Moench] (Caris et al., 1998). The authors reported a direct involvement of mycorrhizal fungi in Fe mobilization or uptake and transfer into the host plant. Phytosiderophores exuded by grass roots form highly stable complexes with Fe3+ (Mino et al., 1983) but not with Fe2+ (Bene
et al., 1983). The Fe3+ chelates are transported into root cells where Fe3+ is reduced to Fe2+ and used in biochemical processes. Because endophyte-infected tall fescue had greater Fe3+ reducing activity on root surfaces (Malinowski et al., 1998a) compared with noninfected plants, Fe uptake may be indirectly affected by endophyte. More detailed research is required to resolve the relationship between rhizosphere chemistry and Fe uptake by endophyte-infected tall fescue.
Soil Acidity
The major factors limiting plant growth on acid soils are increased concentrations of exchangable H+, exchangable Al3+, and soluble Mn, decreased concentrations of exchangable Ca2+, Mg2+, K+, and reduced solubility of P and molybdenum (Mo) (Marschner, 1991). Low soil pH and high exchangable Al concentration restrict root growth, N uptake, and N transport within grass plants (Thornton, 1998). Research on endophyte effects on tolerance to acid soils is very limited. Under simulated acid rain, Cheplick (1993) observed a reduced productivity of tall fescue in response to endophyte infection. In contrast, some endophyte-infected tall fescue genotypes (DN2 and DN7) grown in acidic, high exchangable Al content soil produced greater root systems than did noninfected plants while other genotypes (DN4, DN5, and DN11) did not respond to endophyte infection (Belesky and Fedders, 1995). Malinowski and Belesky (1999a) observed that the pH of a limed, acidic soil increased faster as a result of root activity of endophyte-infected tall fescue compared with noninfected plants under P deficient conditions. We observed an increase in the pH of a P-deficient (0.31µg mL-1 P) nutrient solution from 6.0 to 7.5 within 3 d, regardless of endophyte status of tall fescue plants (1998, unpublished data). In contrast, changes in the pH of a P-sufficient (31µg mL-1 P) nutrient solution were dependent on specific endophyte–tall fescue associations. This suggests that rhizosphere chemistry may be affected in different ways depending on endophyte and tall fescue genome interactions.
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Manipulating of the Neotyphodium–Grass Association for Beneficial Uses |
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TOPABSTRACTINTRODUCTIONMechanisms of Biotic Stress...Adaptations and Mechanisms of...Adaptations and Mechanisms of...Manipulating of the Neotyphodium...Symbiosis of Cool-Season Grasses...ConclusionsREFERENCES |
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Symbiosis of Cool-Season Grasses with Neotyphodium spp. Fungal Leaf Endophytes and Environmental Stress Tolerance—An Alternative Strategy to VA Mycorrhiza? |
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TOPABSTRACTINTRODUCTIONMechanisms of Biotic Stress...Adaptations and Mechanisms of...Adaptations and Mechanisms of...Manipulating of the Neotyphodium...Symbiosis of Cool-Season Grasses...ConclusionsREFERENCES |
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