Crop Science 41:1981-1984 (2001)
© 2001 Crop Science Society of America
NOTES
Endophyte infection can contribute to aluminum tolerance in fine fescues
D. E. Zaurova,
S. Bonosa,
J. A. Murphya,
M. Richardsonc and
F. C. Belanger*,b
a Dep. of Plant Science, Rutgers, The State Univ. of New Jersey, 59 Dudley Rd., New Brunswick, NJ 08901-8520
b Dep. of Plant Pathology and Biotechnology Center for Agric. and the Environment, Rutgers, The State Univ. of New Jersey, 59 Dudley Rd., New Brunswick, NJ 08901-8520
c Dep. of Horticulture, Univ. of Arkansas, Fayetteville, AR 72701
* Corresponding author (belanger{at}aesop.rutgers.edu)
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ABSTRACT
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Thirteen clonal pairs of endophyte-free and endophyte-infected fine fescues were evaluated in a growth chamber study for growth in three sand-soil formulations differing in available aluminum. This set of plants consisted of four Chewings fescue [Festuca rubra L. subsp. fallax (Thuill) Nyman] and two strong creeping red fescue (Festuca rubra L. subsp. rubra) genotypes inoculated with endophytes originating from Chewings fescue, strong creeping red fescue, and Poa ampla Merr. hosts. The results revealed a considerable endophyte-host interaction on plant growth in the different soils. In most cases, the effect of endophyte infection on dry weight was either positive or neutral. In a few cases, endophyte infection had a negative effect on dry weight. In some endophyte-host combinations, the endophyte-infected clone had significantly better growth in the high aluminum soils relative to the endophyte-free clone. These results indicate that endophyte infection alone is not enough to confer aluminum tolerance in fine fescues, but in certain plant-fungus combinations, endophyte infection can contribute to enhanced aluminum tolerance.
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INTRODUCTION
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MANY CULTIVATED and wild grass species are hosts to fungal endophytes of the genus Neotyphodium or the related teleomorphic genus Epichloe (Glenn et al., 1996). These associations are considered to be mutualistic (Clay, 1988), with the plants providing nutrients for the fungi and the fungi synthesizing alkaloids which protect the plants from herbivory. The relationship of endophyte infection and protection from insect and mammalian herbivores via synthesis of toxic alkaloids is well established and has been the subject of numerous studies (Breen, 1994; Bush et al., 1997).
Several studies have indicated endophyte infection can also result in increased plant vigor and confer tolerance to biotic and abiotic stresses, unrelated to the reduction in herbivory. Endophyte infection has been associated with enhanced drought tolerance (Arachevaleta et al., 1989; West, 1994), phosphorus utilization (Malinowski and Belesky, 1999a), and disease resistance (Clarke et al., 2001). The physiological mechanisms which produce these effects are not well understood. Several studies have examined the effect of endophyte-infection on plant growth under different conditions, with varying results depending on plant species, age, and nutrient conditions (Belesky and Fedders, 1995; Cheplick et al., 1989; Malinowski and Belesky, 1999a).
Aluminum phytotoxicity in acidic soils is becoming an increasingly serious problem, both in the USA and worldwide (Carver and Ownby, 1995). Fine fescue species, including Chewings fescue and strong creeping red fescue, are important low maintenance turfgrasses that are tolerant of poor, droughty soils (Funk et al., 1994). Development of fine fescue cultivars with enhanced ability to persist in acidic soils with minimal lime input would be of great value.
This study was stimulated by a previous study screening fine fescue cultivars for differences in aluminum tolerance (Liu et al., 1996). In that study, fine fescue cultivar populations known to be infected with fungal endophytes generally exhibited better growth than cultivars not infected with endophytes when grown in soils of low pH and high aluminum content. This raised the question of whether the observed enhanced aluminum tolerance could be attributable to infection with the fungal endophytes. Since the previous comparisons were between mixed populations of different plant genotypes, it was not possible to draw a conclusion regarding any role of endophyte infection in the observed differences in aluminum tolerance. The objective of this study, therefore, was to compare the growth of endophyte-infected and endophyte-free clonal lines of fine fescues in different soil types with varying levels of available aluminum.
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Materials and Methods
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Endophyte-Free and Endophyte-Infected Clonal Plant Material
Clonal Chewings fescue and strong creeping red fescue plants inoculated with different fungal endophyte genotypes were as described previously (Johnson-Cicalese et al., 2000). Mature tillers of different strong creeping red fescue and Chewings fescue genotypes were inoculated with fungal endophytes by inserting a small piece of fungal mycelium into a slit made at the junction of the root and shoot. By inoculating mature tillers we could also maintain an endophyte-free clone of the same plant genotype. In this set of plants, the same plant genotype was inoculated with different endophyte genotypes and the same endophyte genotype was inoculated into different plant genotypes. Thirteen different clonal endophyte-free and endophyte-infected pairs were evaluated.
Three of the endophytes were E. festucae (Leuchtmann et al., 1994) and were designated as follows: the Rose City endophyte (RC) isolated from a strong creeping red fescue and the Cambridge (CA) and the Delaware (DE) endophytes isolated from Chewings fescue plants. The fourth endophyte was Neotyphodium sp. and was designated the P. ampla endophyte (PA). It was found in ‘Service’, a cultivar of P. ampla released by the Alaska Department of Natural Resources. The endophyte-free fine fescue plants used for inoculation were selected from breeding nurseries and were designated by their nursery identification numbers.
Soil Treatments
The effect of endophyte-infection on growth was evaluated in three different soil formulations with varying levels of available aluminum. The soil used in all three formulations was a mixture (v/v) consisting of 70% coarse silica quartz sand and 30% acid Tatum soil (Clayey, mixed, thermic, typic, Hapludult) from Orange, VA. The acid Tatum soil had a pH of 4.4 (1:1 soil/water ratio), a cation exchange capacity of 13.0 cmol kg-1, and contained 8.82 cmol kg-1 of KCl-extractable Al (69% exchangeable Al). The other cations determined by extraction with 1 M NH4C2H3O2 at pH 7.0 were: 0.36 cmol kg-1 Na, 0.64 cmol kg-1 Mg, 0.21 cmol kg-1 Ca, and 0.32 cmol kg-1 of K. This sand and soil mixture, without any amendments, was designated soil A.
In the formulation designated soil B, the sand and soil mixture was enhanced with fertilizer at 100, 54.5, and 68.5 mg of N, P, and K, respectively, per kg of soil added as NH4NO3 and KH2PO4. The pH of both soils A and B was 4.4, a pH at which the aluminum would be soluble and available.
Soil formulation C contained the same fertilizer amendments as soil B and was also treated with 3500 mg kg-1 of CaCO3 to raise the pH and reduce aluminum availability in the soil. The pH of the limed soil increased to 6.5 within 2 wk after liming and then stabilized. At pH 6.0 limed acid Tatum soil has 0.64 cmol kg-1 exchangeable aluminum (Foy, 1996). Limed and unlimed acid Tatum soil has been used previously to screen for Al tolerance (Foy, 1996; Liu et al., 1996; Foy and Murray, 1998).
The experiments were carried out in a growth chamber with a daylength of 16 h. The light intensity was 600 µmol m-2 s-1 and the relative humidity was 60%. The daily maximum air temperature ranged from 22 to 25°C. The soil was uniformly packed into 4-cm-diam by 21-cm-deep plastic containers (Stuewe & Sons, Inc., Corvallis, OR). A randomized complete block design with three replications was used. During planting, blocked pairs of endophyte-free and endophyte-infected clones were standardized according to fresh weight and number of tiller primordia (Hill et al., 1990). The number of tiller primordia ranged from one to three. The plants had been previously maintained in the greenhouse and were all mature plants of the same age. Each container was watered daily with an equal volume of distilled water at a pH of either 4.5 or 6.5, depending on the soil formulation. The volume of water applied was adjusted from 5 to 15 mL as the plants grew during the course of the experiment. Plants were grown for 90 d and the roots and shoots were harvested. Roots and shoots were oven dried at 60°C and dry weights were determined.
All data were subjected to analysis of variance by the ANOVA procedure of the Statistical Analysis Systems Institute (Cary, NC). Since there were significant endophyte interactions a separate analysis of variance was performed with the main effects of plant-endophyte combination and replication in the model for each soil type.
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Results and Discussion
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Effect of Endophyte Infection on Plant Growth in Soils of Varying Aluminum Availability
Shoot and root dry weights were determined on clonal pairs of endophyte-infected and endophyte-free fine fescues growing in sand-soil mixtures of varying aluminum availability. Analysis of variance of all data revealed significant differences in growth among soil types and significant endophyte x clone interactions (Table 1).
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Table 1. Analysis of variance for root and shoot weights of 19 plant-endophyte combinations grown in three different soil conditions varying in aluminum availability.
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The mean root and shoot masses of the plants grown in the three different soils are shown in Table 2. As expected, the tissue mass of the plants, irrespective of endophyte status, was dependent on the soil type. The poorest soil, soil A, which had no fertilizer and no CaCO3, supported the least growth while soil C, the fertilized and limed soil, supported the most growth.
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Table 2. Mean root and shoot weights of 19 plant-endophyte combinations grown in three different soil conditions varying in aluminum availability.
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When the effect of endophyte status on growth in the different soil types was evaluated, it was clear there was considerable host-fungus interaction (Table 3). In some host-fungus combinations, there was a significant positive effect of endophyte presence on tissue mass, whereas in other combinations there was no effect. For example, in the Chewings fescue 1090 genotype the CA endophyte had a positive effect on growth in all three soil types, but in the 1117 plant genotype the same endophyte genotype had no significant (P = 0.05) effect. In the 1116/PA combination, endophyte infection had some negative effects on growth. In this combination, the plant-fungus interaction appears to be shifted from mutualistic to pathogenic. In the 1117 host genotype, however, the PA endophyte had some positive effects indicating the PA endophyte genotype is not inherently pathogenic in fine fescues.
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Table 3. Effect of endophyte infection on growth of fine fescue clones in different soil conditions varying in aluminum availability.
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In most cases where there was a positive effect of endophyte infection, the effect was seen in both the root and shoot dry weights (for example, the 1090/CA combination). There were, however, some instances where there was a positive effect in only the root or the shoot (for example, the 1139/DL combination).
No general pattern emerged regarding the effect of specific endophyte isolates on plant performance in the different soil types. If there were a general effect of endophyte infection on aluminum tolerance in fine fescues, one would expect greater difference in growth to be observed in the low pH soils. In most cases where there was a positive effect of endophyte presence on growth in the low pH soils, there was also an effect in the high pH soil where aluminum toxicity would not be a factor. This suggests that, in these cases, the positive effect of endophyte infection observed in the low pH soils may be more complex than strictly because of aluminum tolerance. In these cases, a general endophyte-mediated enhanced vigor of the plants, as observed by others (Arechaveleta et al., 1989; Hill et al., 1990; Richardson et al., 1999), may contribute to the observed aluminum tolerance. Only in the case of the 1117 genotype infected with the RC and PA endophytes and the 1090 genotype infected with the DL endophyte was there a positive effect in low pH soil and no effect in the high pH soil. When inoculated into other plant genotypes, however, these same fungal genotypes resulted in different effects on growth. Overall, these results suggest that the positive effect of endophyte presence on growth observed in some plant-fungus combinations, irrespective of soil type, is due to the interaction of the two species and is not attributable to the endophyte alone. When this same set of endophyte-free and endophyte-infected plants were evaluated in relation to a number of field parameters, a similar host-fungus interaction was observed (Johnson-Cicalese et al., 2000).
Some plant-fungus combinations enhanced growth in the high aluminum soils, relative to the respective endophyte-free clone. Although endophyte infection alone is not enough to confer aluminum tolerance in fine fescues, in certain plant-fungus combinations, endophyte infection enhanced aluminum tolerance. The superior performance in high aluminum soils reported for some current endophyte-infected fine fescue cultivars (Liu et al., 1996) may reflect a high preponderance of beneficial individual plant-fungus combinations within those cultivar populations. The particular combinations reported here will be valuable germplasm for future studies aimed at elucidating the mechanism of the observed aluminum tolerance and for inclusion in breeding programs for Al tolerance.
In a previous study of five endophyte-infected and endophyte-free tall fescue (Festuca arundinacea Schreb.) clones grown in limed and unlimed Porters soil (coarse-loamy, mixed, mesic Umbric Dystrochrept), the same range of effects on plant growth was observed, also indicating a plant-fungus interaction (Belesky and Fedders, 1995). In another study, two genotypes of endophyte-infected tall fescue were found to have greater reduction in root and shoot dry matter than the uninfected clones when grown in the presence of Al (Malinowski and Belesky, 1999b). The endophyte-infected plants also had more Al on the surface of the roots and in root tissues than the endophyte-free plants (Malinowski and Belesky, 1999b).
Cheplick et al (1989) reported that endophyte-infected tall fescue seedlings showed a reduction in growth under low nutrient conditions. This was attributed to an endophyte-imposed nutrient drain on the plant. In the case of the fine fescues studied here, in most cases there was either a positive effect of endophyte infection or no effect, even in the poorest soil conditions. In only a few cases was there a negative effect, indicating that in fine fescues endophyte-infection does not generally result in significant competition for nutrients.
In some tall fescue-endophyte associations, the utilization of sparingly available phosphorus was reported to be enhanced relative to endophyte-free clones (Malinowski and Belesky, 1999a). Such a mechanism could be related to the increased dry matter accumulation observed in the poorest soil in some of the fine fescues infected with endophyte.
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Conclusions
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This is the first study to compare such a large number of clonal endophyte-infected and endophyte-free pairs of plants for any growth response. Although there was a wide range of responses, most plant-endophyte combinations had either a clearly positive effect or no effect on plant growth. There were, however, a few combinations in which endophyte presence had a negative effect on growth. As such, no general conclusion can be drawn regarding the effect of fungal endophyte infection on plant growth in the fine fescues. Rather, the effects on growth are specific to the particular combination of plant and fungal genotypes. The results reported here and elsewhere (Johnson-Cicalese et al., 2000; Malinowski and Belesky, 2000) on clonal comparisons of endophyte infected grasses emphasize that it may not be possible to reach universally applicable conclusions regarding some of the physiological responses of grasses infected with fungal endophytes.
This study was designed to determine if endophyte infection was a factor in aluminum tolerance in fine fescues. Our results indicate that, in certain endophyte–plant combinations, endophyte infection can indeed contribute to enhanced aluminum tolerance. These specific endophyte-plant combinations could be utilized in a breeding program specifically designed to develop fine fescue cultivars with enhanced aluminum tolerance.
Received for publication January 18, 2000.
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ABSTRACT
INTRODUCTION
