Abiotic stress tolerances (moisture, nutrients) and photosynthesis in endophyte-infected tall fescue

Agriculture, Ecosystems and Environment, 44 ( 1993 ) 123-141

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Elsevier Science Publishers B.V., Amsterdam

Abiotic stress tolerances (moisture, nutrients) and photosynthesis in endophyte-infected tall fescue Charles W. Bacon Toxicology and Mycotoxin Research Unit, Russell Research Center, Athens. GA 30613, USA

ABSTRACT Bacon, C.W., 1993. Abiotic stress tolerances (moisture, nutrients) and photosynthesis in endophytcinfected tall fescue. Agric. Ecosystems Environ., 44:123-141. The ecological significance resulting from the association of each tall fescue (Festuca arundinacea Schreb. ) genotype and its companion fungal endophyte (Acremonium coenophialum Morgan-Jones and Gams) is probably inherent in the nutritional interactions, and the resulting physiological and biochemical requirements of each. The colonization of this grass by the fungus follows the natural sequence of fescue seed germination, seedling and tiller growth. Infected grasses are natural and extensive, therefore tall fescue should be considered a symbiotic plant. The nature of this relationship at the population level is more appropriately described as an obligately biotrophic conjunctive mutualism. The use of this terminology at the population level describes the overall ecological effect; however, allowances must be made for infected tall fescue genotypes within the population that may not show any positive adaptive strategies. Genotypes of this later category may be categorized, possibly only transiently, as obligately neutral symbiotic. Infected genotypes of the earlier category offer adaptations to environmental stresses and may be exploited for these characteristics. Evidence from research is reviewed to indicate that selected genotypes within the population of tall fescue are more tolerant of environmental abiotic stresses than uninfected grasses. Infected tall fescue seed require more moisture to germinate than uninfected seed. Endophyte-infected seedlings require more nutrients than uninfected seedlings. Although infected tall fescue contains less soluble nitrogen which would encourage more predation, strains resulting from the stresses of insect herbivory are prevented because of an accumulation of an insect deterrent, toxins and their synergists. At the morphological level infected grasses show tolerance to water stress by early shedding of older leaves and rolling of younger leaves. Tolerance to water stress is further evidenced by low stomatal conductances and by development of an enhanced osmoregulatory system that produces increased cellular turgot pressure. One genotype of tall fescue contains polyols, some of which are absent in the uninfected clone. Infected tall fescue also shows increased efficiency to low soil nitrogen, possibly due to an increased level of glutamine synthetase which would enhance its competitive ability under low soil nitrogen. There is no endophyte effect of consequence on photosynthesis and associated processes in several genotypes of infected grasses.

Correspondence to: Charles W. Bacon, U S D A / A R S , Toxicology and Mycotoxin Research Unit, Russell Research Agricultural Center, PO Box 5677 Athens, GA 30613, USA.

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INTRODUCTION

The value of an interspecific association, or symbiosis, is measured by how much it increases or decreases the probability of survival of the associates, or symbionts. The value is expected to vary with genetic constitution, ecological settings, and other changing conditions. Thus interspecific associations have profound effects on biological evolution and species formation. Symbioses, or interspecific associations, are long-lasting or permanent (conjunctive) and are not limited to nor synonymous with mutualism. Indeed, according to de Bary (1887), mutualism would be only one subcategory of symbiosis, the others being parasitism and commensalism. Therefore, symbionts may either benefit from, be harmed by, or not be affected by an association. Symbiotic associations may be considered as a continuum of relationships, and there is expected to be some overlap within each of the three subcategories. The degree of overlap depends on the environment. There are thousands of symbioses occurring worldwide, in and between all the major groups of organisms. Of these associations, mutualistic symbioses have been exploited the least for biological control. However, the use of the tall fescue mutualism in the biological management of pests has potential (Saha et al., 1987; Clay, 1988; Cheplick and Clay, 1988) but can be fully achieved only if we completely understand the nature of each specific ecologically functional relationship. Of particular interest is the nature of the interspecific association of tall fescue, Festuca arundinacea (Schreb.), with an endophytic fungus Acremonium coenophialum Morgan-Jones and Gams. This association is conjunctive and begins with the sequence of fescue seed germination which is followed by seedling infection that is initiated and synchronized with growth cycles of tall fescue. Perennation of infection is assured by continued infection of each tiller initial and growth along with matured plant parts, and culminates with growth into florets, and finally back into seeds for dissemination and reinfection (Cole and White, 1985; Hinton and Bacon, 1985). This cyclic association of the fescue endophyte contains several characteristics of a symbiosis (Smith and Douglas, 1987 ), and other characteristics include specificity (M. Siegel, personal communication, 1988), biotrophic nutrition, and possibly genetic and metabolic interactions with the grass (Prestidge et al., 1985; Hardy et al., 1986; Lyons et al., 1986, 1990; Belesky et al., 1987; Arechavaleta et al., 1989 ). The ecological consequence of the tall fescue-endophyte association is a mutualism because reduced cattle and insect herbivory, drought tolerance, and increased growth and tiller number are associated with endophyte-infected tall fescue (Schmidt et al., 1982; Hoveland et al., 1983; Read and Camp, 1986; Bacon et al., 1986; Clay, 1988). These are all positive defensive responses to either harsh abiotic or biotic stresses in the environment. Clay (1988) aptly summarized this association as a defensive mutualism. Since

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there is genetic variation among isolated fescue endophytes (Bacon, 1989) and endophyte-infected plants (Belesky et al., 1987 ), signs of a mutualistic defense reaction indicative of adaptive and competitive strategies will reflect this variation, especially at the population level. This concept is discussed further in the next section of this review. Tall fescue, a temperate perennial grass, is distributed within a broad range of contrasting climatic, edaphic and geographic locations. It is considered that the major factors limiting tall fescue are climatic and geographic, and lesser importance is given to edaphic, pyric or biotic factors (Burns and Chamblee, 1979). These requirements were established before the impact of the endophyte was known; therefore both biotic and abiotic factors may be equally important to the ecological success of tall fescue. Although the extent of infected tall fescue is unknown, toxicity of endophyte-infected tall fescue has been reported from South America (Lopez, 1979), Europe (Latch et al., 1987), Australia (Pulsford, 1950), New Zealand (Latch et al., 1984), and the USA. Within the USA well over 95% of all fescue acres contain plants that are infected, indicating that tall fescue should be considered a symbiotic plant. The distribution and occurrence of this grass may well reflect mutualistic benefits derived from this symbiosis. Contained within this chapter will be results of experimental approaches designed to examine the contention that tall fescue occupies diverse habitats partially because of stress modifying or resistance mechanisms inherent in the symbiotic habit with A. coenophialum. The effects of two abiotic factors, moisture and mineral nutrition will be reviewed relative to performance of endophyte-infected and endophyte-free tall fescue. Further, the effects of these factors on photosynthesis will be reviewed in order to determine if endophyte infection imposes stress at a biochemical level of expression. EXPERIMENTAL CONCEPTS AND DEFINITIONS

In dealing with a mutualistic symbiosis, the definition of stress used for studying and understanding this specific association should be broader and more useful than the usual concept of stress. Thus stress is defined as constitutive and exogenous constraints which limit growth and reproductive performance of the grass within the association. This definition ignores the contribution of the grass even though it is important, and considers the fungus as an important 'internal' environmental factor, but which is nevertheless innately distinct from abiotic factors. Because stress resistance refers to the ability of plants to survive and grow in unfavorable conditions, it is proper that infected genotypes or populations be studied under environmentally stressing conditions. Precise physiological information is best obtained from research under environmentally controlled conditions with infected and uninfected genotypes; this information can then

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be extended to the population level. Not all stress resistance mechanisms are expected to involve avoidance or tolerance; some endophyte-infected genotypes might modify either the stress or the resulting strain. Therefore appropriate experimental design should be taken to account for this variation. The fungus can easily be isolated from grass material and studied in culture. However, such studies are difficult to relate to the in situ situation because isolated fungi probably differ in a number of characteristics such as growth rate, reproduction and biosynthesis of secondary metabolites (Bacon, 1989). Nevertheless, studies of the endophyte under laboratory conditions have indicated that it can synthesize several of the ruminant toxins (Yates et al., 1985; Lyons et al., 1986), insect deterrents (Rowan et al., 1986), and other compounds (Bacon et al., 1986; De Battista et al., 1990a). Thus from such studies the indication is that the fungus is controlling at least one aspect of resistance: herbivory. Research methods on these toxins and other secondary metabolites produced by the isolated endophyte are tedious but details of this approach, as well as media and methods of isolation have been reviewed (Bacon, 1989). The effects of endophyte infection on plant growth, measured as herbage yield, is not only subject to a mutualistic interaction but also to a wide variety of environmental stresses or constraints which may be conveniently placed under either constitutive or exogenous controls. Exogenous controls include not only the qualitative and quantitative na'mre of solar energy, soil nutrients, temperatures, and water, but also soil or atmospheric growth-inhibiting toxins. Constitutive controls are innate qualities of the specific association, and constraints on this include fungal and plant genetic and biochemical differences in combination, as well as their separate qualities. It is apparent from these definitions that specific stress tolerances will not necessarily be present in all endophyte-infected individuals. However, for those infected grasses that do demonstrate similar tolerances to specific stresses, a similarity in the basic biochemical and physiological mechanisms of action is expected. Research on the role of the endophyte in sustaining tall fescue under abiotic stresses is based on performance of endophyte-infected populations compared with non-infected populations (Lyons et al., 1986, 1990; West et al., 1988, 1990; Cheplick et al., 1989; Wilkinson et al., 1989; De Battista et al., 1990b), and on performance of specific endophyte-infected genotypes compared with corresponding non-infected genotypes (Belesky et al., 1987, 1989; Arechavaleta et al., 1989; De Battista et al., 1990b; Hill et al., 1990; Richardson et al., 1990). Such an approach is necessary in order to define the nature of this mutualistic symbiosis. Results of studies based on similar genetic plant material freed of the endophyte, and maintained under similar controlled conditions can only indicate that there is an ecotype capable of surviving under an imposed stress. Although such studies can define that there is an endophyte effect, they might not relate to the population. Currently, it is possi-

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ble to remove endophytes from grasses, and study infected and uninfected ramets (Belesky et al., 1987; Arechavaleta et al., 1989). Experiments designed to determine the effects of genetically identical endophytes entails infecting seedlings with the same endophyte, and removing the endophyte from divisions of these. Unfortunately the effects of identical endophytes on genetically dissimilar or similar plants cannot be studied as this would involve infecting matured plants which is not possible to date. However, the use of callus tissues for starting materials and the subsequent infection of their somatic embryos is a means of achieving infection (Johnson et al., 1986; Kearney et al., 1991 ). The discussions below will consider evidence that endophyte-infected tall fescue has resistance or tolerance to exogenous biotic constraints at both the population and clonal levels. E V I D E N C E F O R A D A P T A T I O N TO D R O U G H T T O L E R A N C E

Read and Camp (1986) were the first to document that ungrazed endophyte-infected tall fescue plants exhibited more persistence under drought conditions when compared with tall fescue plants with a low infection level (Table 1 ). Their data also reflect the variation in seed-sown populations as one of the endophyte-infected plantings within their replicated experiment showed only a 5% loss. Assuming that all abiotic factors within their replication were constant, their results suggest that individuals within this first pasture have the genetic constitution to survive without the endophyte. However, because all infected pastures were established from seed within a randomized experiment, this explanation is difficult to accept. This illustrates the difficulty with interpretations of an endophytic effect at the population level under less than strictly defined conditions. Clay (1987) studied the phenomenon of tall fescue seed germination and obtained data which indicated that endophyte-infected seed germinated approximately 10% higher than uninfected seed. These studies were conducted TABLE I Loss of Kenhy tall fescue grass during a summer drought period ~ Pasture No.

1 2 3

Average infection level 94%

12%

02 11 1

5 74 84

~Data from Read and Camp (1986). 2percentage area loss.

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C.W. BACON

under controlled ideal germination conditions. Bacon studied the effects of fresh (non-refrigerated) fescue seed germination under conditions of drought (C.W. Bacon, unpublished data, 1990). In this experiment, designed to simulate water stress on germination, polyethylene glycol 20 000 was used as the osmotic agent and seeds were matured but freshly harvested. This study showed that at 25°C endophyte-infected seed did not germinate as the osmotic potential increased (Table 2). Because the seed lot used in this experiment was only 80% infected, this difference may have been much larger if 100% infected seeds were used. These data suggest that infected seeds are not as drought tolerant as uninfected seed. There are apparently some additional germination requirements which drought stress inhibits. Cheplick et al. (1989) concluded that there is an interaction between infected seed and nutrient supply. Their data showed that seedlings from infected seed produced less biomass under nutrient-poor conditions which suggested that benefits from infection to tall fescue were apparent only when nutrient availability was in excess (Cheplick and Clay, 1988). The data in Table 2 indicate that there is an increased moisture requirement for infected seed. The increased moisture requirement might be purely physical, reflecting a need for leakage of inhibitory metabolites from endophyte-infected seed, rather than a genuine metabolic use. The early report of allelopathy in tall fescue (Peters, 1968 ), the finding of allelopathic substances (Luu et al., 1989), and the identity and activity of the loline alkaloids as additional allelopathic compounds (Petroski et al., 1990) suggest that these compounds should also be tested as self-inhibitors of tall fescue seed germination. Self-inhibition of seed germination is as common a phenomenon (Evenari, 1949) as allelopathy, and most self-inhibitors are water soluble and easily leached away, allowing seed germination to proceed. West et al. ( 1988, 1990) studied in a more detailed series of experiments TABLE 2 Seven day percentage germination of'Kentucky 31' tall fescue seed on Polyethylene Glycol 20 000 measured at 25 °C and various osmotic potentials ~ Osmotic potential ( - MPa)

0 0.17 0.3 0.4 0.6 0.9 1.7

% Germination Infected

Uninfected

82 81 51 45 30 5 0

81 84 79 85 80 20 0

~Data of Bacon (C.W. Bacon, unpublished data, 1990).

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the effects of endophyte infection on drought tolerance and persistence of ungrazed cultivar 'Kentucky 3 l' tall fescue. Their data indicated that stand density and forage yield were enhanced by endophyte infection in plots subjected to water deficits (West et al., 1989). However, this effect was only observed on the medium and low irrigated treatments; at the high irrigated treatment there was no endophyte advantage. They also reported that endophyte-infected fescue showed decreased green leaf area per tiller, an increase in leaf tissue senescence and shedding, and had a more positive canopy-air temperature differential than uninfected plants (West et al., 1988, 1989 ). Beiesky et al. (1989) determined that one of their genotypes, I l, was characterized by a larger amount of senesced tissue and root dry weight than uninfected plants (Table 3 ). Overall they reported that there was significantly more senesced tissue in infected grasses and grasses receiving adequate water than uninfected water-stressed grasses (Belesky et al., 1989 ). Similar observations of leaf shedding were reported by Arechavaleta et al. ( 1989 ), although leaf rolling was a marked characteristic of the genotype used in this study. Leaf rolling and other leaf morphological changes have been reported in additional genotypes (Belesky et al., 1989; Hill et al., 1990) which would indicate that this phenomenon is widespread in the population of tall fescue. These morphological changes of infected genotypes were compared with uninfected genotypes and it was observed that infected genotypes were characterized by more leaf-modified characteristics which reduce water loss. These alterations may be a mechanism of drought resistance at high tissuewater potential (i.e. short-term deficits). Although data were not presented, Belesky et al. ( 1987 ) suggested that endophyte-infected plants do have higher stomatal resistance than uninfected grasses, and this is another means for regulating water loss. The mechanisms for tolerance to water deficits in endophyte-infected plants TABLE3 Dead leaf and r o r t dry weight o f tall fescue as influenced by endophyte infected ( + ) uninfected ( - ) and water regime' Accession

Dead leaf (g per pot ) - 0 . 0 3 MPa

7 9 11 17

Root (g per pot ) - 1.5 MPa

- 0 . 0 3 MPa

- 1.5 MPa

(+)

(-)

(+)

(-)

(+)

/-)

(+)

(-)

1.1 0.8 2.2 0.8

1.2 0.9 1.7 0.3

0.6 0.5 2.0 0.2

0.9 0.4 1.2 0.4

4.6 4.3 7.6 4.2

4.8 5.0 5.9 1.8

2.9 3.3 3.9 2.3

4.1 3.2 2.8 2.4

'Data o f Belesky et al. ( 1989 ); values represent three replications and water deficit ( - 1.5 M Pa ) was achieved by two 3-day periods and recovery cycles.

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C.W. BACON

are unknown but may well be associated with the ability of infected grasses to develop a lower osmotic potential than uninfected plants. Generally, solute accumulation or osmotic adjustment in grasses results in maintenance ofturgor and continued leaf growth during periods of drought (Hellebust, 1976; Morgan, 1984), although leaf or tiller survival is the ultimate result (Meyer and Boyer, 1972; Hellebust, 1976). West et al. (1990) measured osmotic adjustments of tissue from greenhouse grown endophyte-infected tall fescue in order to determine the response and resistance of endophyte-infected grasses to water stress. Only endophyte-infected grasses were used in this experiment. They measured osmotic adjustment in meristematic and elongating leaf tissues and suggested that osmoregulation in these tissues, may favor survival during drought. They found that not all tissues from endophyte-infected grasses were equally sensitive to development of water stress (Table 4). Young meristematic and elongating leaf tissue adjusted osmotically much more than matured leaf tissue. In another experiment, osmotic adjustment of water-stressed infected and uninfected tall fescue was compared and the results indicated that osmotic adjustments of blade and sheath tissue of infected plants attained 0.55 MPa and 0.52 MPa as opposed to 0.22 MPa and 0.34 MPa in blade and sheath tissue, respectively, of uninfected plants (Elmi et al., 1989). Because osmotic adjustment persisted in young tissue and immature leaf blades, they concluded that osmotic adjustment may be the mechanism of persistence and tiller survival in tall fescue under intermittent drought conditions (Elmi et al., 1989; West et at., 1990). However, even this physiological parameter shows variation. White ( 1989 ) studied osmotic adjustment in two clones of tall fescue and found that relative leaf-water content and leaf-water potential at zero turgor and leaf elongation during moisture deficits were opposite for the two clones. He concluded that in these two clones cell wall elasticity appeared to TABLE 4 Percentage osmotic adjustment of various leaf tissues of endophyte-infected tall fescue after rehydration Leaf tissue

Percentage (%)

Mature blade Mature sheath Immature blade Unemerged immature blade Basal region 2

5.8 8.4 26.4 24.8 37.7

IData of West et al. (1990). Percentages of osmotic adjustment were calculated by comparing leaf osmotic potential of stressed plants after rewatering with that of controls. 2Consisted of mature and immature unemerged sheath, and unemerged immature blade all within a whorl.

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explain differences in turgor maintenance among infected and uninfected plants. Although the reasons for differences in sensitivity to osmotic adjustment in young tissue are not understood, they must reflect a greater concentration of osmotic constituents. Osmotic constituents in many plant species are sugars and amino acids (Hellebust, 1976; Morgan, 1984). The prime candidates for an osmoticum are the polyhydroxy alcohols (polyols). Polyols, specifically glycerol, are major osmotic metabolites of other symbiotic systems which include lichens, mycorrhizae and symbiotic algae (Lewis, 1967; Muscatine, 1967 ). With the exception of glycerol (Gerber et al., 1988 ), polyols are not primary metabolites of vascular plants (Lewis, 1967) but because they are non-metabolizable they accumulate in plants and confer the essential characteristic of being osmotically active, yet compatible, i.e. not interfering with normal plant biochemistry. Polyols are, however, normal metabolites of fungi and an analysis of tall fescue for these compounds indicated that infected grasses contain more polyols than uninfected grasses (Table 5 ) (C.W. Bacon and G. Chapman, unpublished data, 1989). Free polyols are osmotic regulators in vascular plants (Morgan, 1984; Gerber et al., 1988), algae (Van Eck et al., 1989), symbiotic algae (Muscatine, 1967), and fungi (Morgan, 1984); they also function as a cryoprotectant in frogs (Storey and Storey, 1985 ) and insects (Storey and Storey, 1983 ). Specific polyols commonly associated with these functions include glycerol, arabitol, mannitol, and meso-erythritol (Spencer and Spencer, 1978). Because these polyols are normal metabolites of fungi, and most are absent in uninfected plants (Table 5 ), it may be assumed that the endophyte is responsible for their synthesis within the sheath. There is no indication as to whether soil nutrients affect the concentration of polyols. However, in addition to polyols, small nitrogenous compounds such as glycine, proline and betaines have also been implicated in drought resistance of plants (Aspinall and Paleg, 1981; Zuniga et al., 1989). Lyons et al. (1990) reported that there was a significant difference in the proline conTABLE 5 Sugar alcohol content of an endophyte-infected and uninfected genotype of tall fescue 1 Sugar alcohols

Mannitol Inositol Arabitol Glycerol

% Dry weight Infected

Uninfected

0.01 0.01 0.02 0.01

0.00 0.02 Trace 0.00

~C.W. Bacon and G. Chapman, unpublished data, 1989.

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c.w. BACON

tent of infected leaf blades when fertilized with a high nitrate or ammonia nitrogen rate ( l 0 mM). The amounts reported, less than 4 gmol g-1 dry weight, would suggest that this compound is not functioning as an osmoticum. However, these experiments were not conducted under moisture stress. The phenomenon of drought-induced accumulation of proline was discovered on Lolium perenne (Kemble and MacPherson, 1954), a grass now known to be infected with a related endophyte, Acremonium lolii. Most investigators consider that proline accumulation is a symptom of metabolic modifications induced by water stress. Venekamp (1989) further considers proline accumulation a process in which cytosol acidity is reduced under conditions of drought. Additional research is required before it can be concluded that polyols are osmoregulatory and that there is a host-mediated or regulated mechanism for the synthesis of polyols or proline. If there are no regulatory mechanisms, drought tolerance in infected fescue may only be a coincidental consequence of its added polyol content. In addition to drought tolerance, there are also some undocumented observations that endophyte-infected tall fescue is more cold tolerant. Polyols and the basic mechanism for drought tolerance might well relate to cold tolerance (Storey and Storey, 1983, 1985). However, this aspect might be complex as nitrogen rates, harvesting and grazing pressures also effect cold hardiness (Burns and Chamblee, 1979). Another characteristic of a plant's response to drought tolerance is an increased root depth and density. Belesky et al. (1989) concluded that endophyte status and the water deficit regime used had no effect upon shoot-toroot ratio (Table 3 ). In another study of other genotypes, endophyte-infected tall fescue was characterized as having a greater root mass within individual clones than uninfected tall fescue (De Battista et al., 1990b) but again the root-to-shoot ratio was not affected. Drought-stressed endophyte-infected grasses show an increased regrowth rate. When drought-stressed grasses were rewatered, endophyte-infected plants had higher rates of regrowth than uninfected plants (Arechavaleta et al., 1989; West et al., 1990). It is possible that osmotic adjustment which prevents loss ofturgor allows for rapid cell elongation (Meyer and Boyer, 1972 ) producing a faster growth rate. The endophyte might be involved with this aspect because it has produced indole acetic acid (IAA) in culture (De Battista et al., 1990a). Moreover, a higher content of IAA in endophyte-infected tall fescue could be responsible for all growth responses observed. The total concentration of IAA in endophyte-infected plants compared with uninfected plants undergoing regrowth has not been determined. At the other extreme, tall fescue has an ability to survive flooding for at least 112 days (Rogers and Davis, 1973). Plants grown under waterlogged conditions did not differ significantly in yield or dry matter, but showed a

133

ABIOTIC FACTORS AND ENDOPHYTE-INFECTED TALL FESCUE

TABLE 6 Endophyte-free (EF) and endophyte-infected (El) tall fescue leaf blade width (LBW), thickness (LBT), and in vitro dry matter disappearance (IVDMD) as influenced by nitrogen (N) rate when flooded for 90 days I Infection status

N rate ( mg per pot)

LBW (mm)

LBT (mm)

IVDMD (g kg- ~)

EF

11 220 I1 220

10.98c 10.59c 9.18b 8.23a

0.51a 0.46a 0.64b 0.57ab

660 650 630 660

E!

~Data or Arechavaleta et al. ( 1989 ). Values within columns, means followed by same letter are not significantly different at the 0.05 level by LSD.

significant decrease in the percentage of herbage potassium and nitrogen (Rogers and Davis, 1973 ). However, at cool temperatures ( 18 °C ) there was a 31% reduction in yield after 40 days of being waterlogged (Gilbert and Chamblee, 1965 ). An endophyte contribution to these data cannot be given because they predate knowledge of an endophyte relationship. Arechavaleta et al. ( 1989, 1992) re-examined the data of Gilbert and Chamblee ( 1965 ) and also studied ergot alkaloid accumulation pattern along with nitrogen concentrations under waterlogged conditions in one endophyte-infected genotype and its fungus-free tamer grown under greenhouse conditions (Table 6 ). They concluded that after 90 days of being waterlogged, endophyte-infected tall fescue continued to produce ergot alkaloids and produced more dry matter than the uninfected ramet (Arechavaleta et al., 1992 ). Infected grasses grown under waterlogged conditions quickly produced rhizomes above the water surface which was not observed in uninfected grasses (C. Hoveland, unpublished data, 1987). This observation can be interpreted as another indication that infected grasses are tolerant of waterlogged conditions, and suggests that vegetative growth is not impared. E V I D E N C E FOR E F F I C I E N T USE O F M I N E R A L N U T R I T I O N

Tall fescue, like most other plants, responds positively to soil nutrients, especially nitrogen. Here discussion is concerned with how soil nutrients interact directly or indirectly with endophyte-infected tall fescue to reduce or prevent a stressful situation. There have been only a few controlled experiments designed to show responses of endophyte-infected tall fescue to variation in individual soil nutrient requirements. Nutritional experiments conducted, for example, in sand and water culture should define any role of nutrients on the resulting ecological behavior of infected tall fescue. The few studies that do exist are con-

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C.W. BACON

cerned mainly with nitrogen (Lyons et al., 1986, 1990; Arechavaleta et al., 1989; Cheplick et al., 1989) because of the historic role that high levels of soil nitrogen played in exacerbating fescue toxicity. These experiments are important because they indicate that an abiotic factor can influence the degree of toxicity to ruminants. The effect of increased soil nitrogen on toxicity is an example of an indirect effect of an abiotic factor on elevating a stress; in this instance it is reduced herbivory. It is well established that the type and rate of nitrogen applied to endophyte-infected tall fescue determine the quantitative and qualitative accumulation of ergot alkaloids (Tables 7 and 8) (Lyons et al., 1986; Arechavaleta et al., 1992). The direct effects of soil nitrogen on the accumulation of other defensive compounds have not been determined, but since they also contain nitrogen, and are alkaloids, they may also be affected by soil nitrogen. The efficient utilization of low soil nitrogen provides evidence for direct effects on survival within the symbiosis. Efficiency of nitrogen utilization is indicated by one genotype of tall fescue (Table 9) (Arechavaleta et al., 1989). The amounts of dry matter produced by endophyte-free grass at each of the three rates of nitrogen are not significantly different from the amount produced by endophyte-infected tall fescue receiving the low rate of nitrogen. There was a 50% increase in dry matter for endophyte-infected grasses receiving the medium rate of nitrogen. It is not known how common this effect is within the population level. Lyons et al. (1990) examined and compared the activity of several enzymes from several genotypes of infected and uninfected tall fescue plants and concluded that leaf blades of infected plants exhibited substantial increases in the activity of glutamine synthetase regardless of the nitrogen rates or form. Glutamine synthetase is primarily responsible for the reassimilation of ammonia within plants, and the high activity of this enzyme in grasses grown at low rates of nitrogen indicates that there is a much more efficient TABLE 7 Concentration of ergot alkaloids in sheaths of endophyte-infected tall fescue grown at low (0.5 mM ) or high ( 10 mM ) rates of KNO3 or ( NH4 ) 2SO4 fertilizers I Nitrogen treatment

Ergot alkaloid concentration (ILgg- 1) Total

Low KNO3 High KNO3 Low (NH4)2SO4 High (NH4)zSO4 1Data of Lyons et al. ( 1986 ).

Ergopeptide

Sheath

Blade

Sheath

Blade

8.9+_2.3 22.2+ 5.3 9.5_+ 1.9 17.4 + 2.8

2.5+_0.9 3.5+- I.I 3.1 +0.4 3. I +_0.2

1.3+-0.9 2.8+0.2 1.3+_0.6 4.0 _+0.7

0.17+0.10 0.14+-0.07 0.07+0.01 0.47 + 0.07

ABIOTIC FACTORS AND ENDOPHYTE-INFECTED TALL FESCUE

135

TABLE 8 Effects of 90 days flooding and nitrogen (N) rates on ergot alkaloid concentration of endophyteinfected (El) and endophyte-free (EF) tall fescue grown in pot culture I Treatment

Total (/~g g-t dry weight) Ergovaline

Ergopeptide

0.68a 1.10b 2.80

I. 10a 1.50a 4.20b

0.07a

0.14a

0.12a 1.30b

0.20a 2.10b

0.31a 2.00b

0.78a 2.41 b

Waterlogged El before flooding E! after flooding Non-waterlogged controls

Waterlogged and N rates El before flooding El after flooding I I m g N per pot 220 mg N per pot

Non- waterlogged controls II mgN per pot 220 mg N per pot

~Data from Arechavaleta et al. ( 1992 ). Values in each column within an experiment not having the same letter differ significantly (P< 0.05 ). TABLE 9 Efficiency of nitrogen utilization, dry herbage yield and 14 day regrowth height of endophyte-free (EF) and endophyte-infected (El) tall fescue after a prior 40-day exposure period to three soil moisture levels t Infection status

N rate (mg per pot ) ll

73

Prior soil moisture (MPa) 220

Dry herbage yield (g per pot ) EF El

0.28a 0.39ab

0.41b 0.82d

-0.03

-0.05

- 0.5

14.2b 22.3c

10.8a 22.8c

Height (cm) 0.43b 0.68c

I1.2a 16.3b

~Data ofArechavaleta et al. (1989). Means followed by the same letter are not significantly different at 0.05 level by LSD.

means of utilizing nitrogen. Thus the high level of this enzyme improves the nitrogen economy of the plant and increase the competitive ability of infected grasses under conditions of low soil nitrogen. The results from Lyons et al. (1990) and those of Arechavaleta et al. (1989) indicate that the increased nitrogen efficiency by endophyte-infected plants may be a general phenomenon. The total nitrogen content of plants affects the degree of insect herbivory

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(Mattson, 1980). Therefore herbivores feeding on tall fescue low in nitrogen would consume more grass and impose more stress than those feeding on high nitrogen fertilized plants. Lyons et al. (1990) determined that the total free nitrogen (nitrate and ammonium) concentration of leaves of'Kentucky 3 l' tall fescue was decreased by endophyte infection even though the plants were fertilized with a high rate of nitrogen (Table 10). An immediate interpretation of this data is that endophyte-infected grasses are utilizing the products of this free pool of nitrogen for the synthesis of fungus-specific compounds. The interaction of consequence is the fact that grasses fertilized with low nitrogen rates contain low concentrations of nitrogen, but because infected leaves also contain the insect-deterrent peramine, and insect toxins and synergistic compounds (lolines, ergot alkaloids, and perloline) (Siegel et al., 1991 ), insects would consume less infected leaves than uninfected leaves. Under low nitrogen fertilization, the tendency for increased herbivory is prevented, affording infected grasses a competitive edge. The same mechanism is considered to work for infected grasses under high nitrogen fertilizer rates even though they contain less nitrogen than uninfected grasses (Table 10). The presence of insect toxins and deterrents would offset any tendency to overgraze infected grasses due to nitrogen content. The effects of nitrogen on fescue seedlings were studied by Cheplick et al. (1989). They attributed this to less nutrient availability in seeds and seedlings. They also reported that in matured infected plants, low nitrogen significantly affected biomass more than uninfected plants. However, their results from mature plants are in agreement with those of Lyons et al. (1990) and Arechavaleta et al. ( 1989 ). The efficient utilization of nitrogen is apparently a developmental phenomenon, occurring in matured endophyte-infected grasses. TABLE 10 Effects of Acremonium coenophialum on total soluble nitrate-ammonium nitrogen ( N ) concentration in leaves of'Kentucky 31' tall fescue j Infection status

Total N concentration (ug N g-t dry weight)2 Nitrate fertilized

Infected Uninfected

Ammonia fertilized

0.5 mM

10 mM

0.5 mM

10 mM

173 198

7837 I 0161

171 241

1762 4872

1Data modified from Lyon et al. (1990) and represent a summation of nitrate and ammonium concentration of leaf blade and sheath of their original data. 2Values are mean nitrogen concentration.

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PHOTOSYNTHETICEFFICIENCY The endophyte of tall fescue is a biotrophic fungus, deriving its nutrients from the apoplasm. Inherent in the concept of mutualism is the principle that neither organism is harmed. The degree of harm must be examined at both the physiological and morphological level. It is expected that the vital process of photosynthesis, which also affects the competitive ability of plants, should not be negatively affected. Indirect evidence that this is the case was obtained from the preceding discussion of increased yield under drought conditions by endophyte-infected grasses. The effects of endophyte on photosynthesis are limited to one published account (Belesky et al., 1987 ), although additional work is in progress (Richardson et al., 1990). Belesky et al. ( 1987 ) studied photosynthetic activity in five tall fescue genotypes and their uninfected ramet grown under ideal conditions. They concluded that photosynthetic rates were significantly greater in uninfected tall fescue than in infected tall fescue. However, in a subsequent study (Belesky et al., 1989) these same infected clones showed increased growth indicating that this increase in net photosynthetic rate of uninfected ramets may be interpreted as an inefficient utilization of photosynthates; it probably reflects the inability of uninfected plants to closely regulate closing of stomates. The effects of endophyte removal on diurnal cycles of apparent photosynthesis, transpiration, internal CO2 concentrations, stomatal conductance and water-use efficiency were examined in four genotypes of 'Kentucky 31' (Richardson et al., 1990). The results indicated that the four genotypes differed in their apparent photosynthesis throughout the photoperiod but there were no consistent differences between infected and uninfected genotypes. Endophyte-infected plants eventually reached the same photosynthetic rates under increasing irradiance as the uninfected plants. These results differed from the earlier report (Belesky et al., 1987 ) and might reflect the two different methods of culturing plants, determining photosynthesis and analysing data. However, two genotypes appeared to have reduced apparent photosynthesis. Stomatal conductance was also reduced in those genotypes by infection but this effect appeared to depend on low light intensities and photoperiods. The internal CO2 concentrations of all genotypes were variable and probably reflect the contribution from the fungus. Richardson et al. ( 1990 ) were the first to report that transpirational water loss varied, i.e. was not affected in two genotypes but was reduced in the remaining two genotypes. There were no alterations in water-use efficiency by any genotype, infected or uninfected. Generally, plants parasitized by other biotrophic fungi show an increased rate of photosynthesis, indicative of an added sink. The results cited above indicate that there is no sink. Because the endophyte is associated intercellu-

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lady, and does not possess nutrient-absorbing structures, it must utilize apoplasmic nutrients entirely, which consist of sugars and nitrogen compounds that have diffused out of the cytoplasm. The studies of Belesky et al. ( 1987 ) and Richardson et al. ( 1990 ) support the concept that photosynthesis and related processes are just as efficient in infected grasses as they are in uninfected grasses and that the presence of an endophyte does not alter this basic process. However, these experiments used single-leaf measurements. Experiments based on total leaf area differences may lead to differences in total carbon accumulation and competitiveness of whole plants.

CONCLUSION

Within the past decade several significant experimental studies have provided insight into grass-endophyte biology and have suggested possible uses of a fungus-grass relationship as biological controls and tolerance mechanisms. It is evident from the experimental data reviewed here that most aspects of the tall fescue-endophyte association should be viewed as a mutualism. The degree of each mutualistic response varies within the infected population. It is also apparent that because of the great diversity of patterns of response, it is not easy to predict what the response to soil nutrition and moisture will be. In view of the large differences among endophyte-infected tall fescue genotypes, studies of individual genotypes (infected and uninfected) are essential. It would appear that the differences in dry weight yield, indicative of growth, and moisture stress tolerance between uninfected and infected grasses have ecological implications. Further long-term experiments, involving competition between endophyte-infected tall fescue genotypes or populations grown under various moisture or nutrient levels are obviously required before the role of this association in delineating ecotypes can be determined. The abiotic environmental factors discussed in this review are considered major contributors to the biotic responses within an infected grass. There are other determinants which have not been studied to date. Clearly humidity, additional macro- and micronutrients, and temperature-dependent processes as well as solar radiation may be equally important in delineating the success of endophyte-infected grasses. The use of an endophyte to confer or increase a tolerance mechanism to a grass still has merit. Indeed, there may exist within an endophyte-infected tall fescue population other genotypes with differential responses to natural or unnatural factors (e.g. soil or atmospheric toxins) which would extend the utility of endophyte-infected grasses.

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