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Annals of Botany 81 : 483–488, 1998 Encroachment of Endophyte-infected on Endophyte-free Tall Fescue N. S. H I L L*†, D. P. B E L E S K Y‡ and W. C. S T R I N G E R§ * Crop and Soil Sciences Department, Uniersity of Georgia, Athens, GA, USA 30602, ‡ USDA-ARS Appalachian Soil and Water Conseration Lab, Beckley, WV, USA 25802, and § Plant Sciences Department, Clemson Uniersity, Clemson, SC, USA 29631 Received : 3 July 1997 Returned for revision : 8 September 1997 Accepted : 4 December 1997 Persistence of endophyte-free (E®) tall fescue (Festuca arundinacea Schreb.) is erratic. Little information exists as to how fast endophyte (Neotyphodium coenophialum)-infected (E) tall fescue might encroach on E® tall fescue and whether specific conditions might influence the speed of encroachment. Plots of E and E® tall fescue genotypes 7 and 17 were established using a modified Nelder’s design to compare performance of the E forms of the plants in pure and mixed communities at different population densities. The plots were planted at the USDA Southern Piedmont Conservation Research Laboratory in Watkinsville, Georgia, and the University of Georgia Plant Sciences Farm in Bogart, Georgia. Plants were grown over a 5 year period and dry matter yield monitored 1, 3, and 5 years after establishment. Relative crowding coefficients were calculated for each to establish trends of encroachment of the E on the E® plants in the mixed communities. Generally, dry matter yields of E tall fescue were greater than E® tall fescue regardless of whether they were grown in pure or mixed communities. As time progressed, the difference in dry matter yield between E and E® tall fescue grown in mixed communities was greater than that of the pure communities. Relative crowding coefficients increased as time progressed. Relative crowding coefficients at the Watkinsville location were greater after 5 years than those at the Plant Sciences Farm. Therefore, site specific conditions exist which affect the competitiveness of E® tall fescue and degree of encroachment by E tall fescue. Research is needed to identify which biotic, abiotic and management variables exacerbate encroachment of E tall fescue to better define the conditions which best suit E® tall fescue. # 1998 Annals of Botany Company Key words : Tall fescue, endophyte, Neotyphodium coenophialum, Festuca arundinacea, competition, population density. INTRODUCTION One of the better defined mutualistic associations between an endophytic fungus and its plant host is that between Neotyphodium coenophialum (formerly called Acremonium coenophialum) and tall fescue (Festuca arundinacea Schreb.) (Hill, 1994). N. coenophialum increases root and shoot mass, tiller number (Belesky, Stringer and Hill, 1989 ; Hill et al., 1990) and drought resistance (West et al., 1993 ; Hill, Pachon and Bacon, 1996), and provides chemical resistances to insects (Clay, Hardy and Hammond, 1985) and nematodes (West and Gwinn, 1993). The collective effect of the endophyte on physiology and morphology of tall fescue is a population that is adapted over wide climatological and geographical regimes (Hill et al., 1990). Detrimental effects of toxins produced by N. coenophialum make the use of endophyte-free (E®) tall fescue an attractive option for livestock producers. However, removal of the endophyte from the grassland ecosystem risks the loss of tall fescue stands. Persistence of E® tall fescue is irregular. Read and Camp (1986) and Bransby et al. (1988) found E® pastures to be less persistent than endophyte-infected (E) pastures, while Hoveland and coworkers found little or no difference in persistence of E® and E pastures or clipped plots (Hoveland et al., 1983 ; Hoveland, 1994). Others have found that E® pastures can be infested by E seed after * For correspondence. 0305-7364}98}04048306 $25.00}0 passing through the digestive tract of the grazing animal (Shelby and Schmidt, 1991). Once infestation occurs, the frequency of E plants may increase over time (Belesky et al., 1987 ; Shelby and Dalrymple, 1993). Little information exists on how often, how fast, or under what conditions E® stands might convert to E once infestation has occurred. Conceivably, aggressivity of E tall fescue on E® stands could be affected by soil type, local geography and aspect, soil moisture, nematode or insect populations. Therefore, studies involving E and E® tall fescue are needed to assess the competitiveness of the two forms. Previously, we reported on the competitiveness between E and E® forms of two tall fescue genotypes (Hill, Belesky and Stringer, 1991). The original objective of the study was to determine whether E and E® plant genotypes varied in their competitive ability. We reported that competitiveness of E and E® plants varied between genotypes, and that competitiveness appeared to be site specific during the establishment year. The plots were subsequently maintained with the objective of evaluating whether the competitive ability of the E and E® plants remained constant or changed over time, location, or plant genotype. MATERIALS AND METHODS Plant materials were collected from a population of E Kentucky 31 growing on the Simpson Experimental Farm in bo980583 # 1998 Annals of Botany Company 484 Hill et al.—Encroachment of Endophyte-infected on Endophyte-free Tall Fescue T 1. Mean dry matter yield (g per plant) of endophyte-infected (E) and non-infected (E®) plants from two tall fescue genotypes grown at two locations 1988 1992 Genotype Location Watkinsville Infection status E E® Mean LSD (0±05)* Genotype (G) Infection (I) G¬I 7 17 Mean 7 17 Mean 191 149 170 213 200 207 202 175 192 49 126 253 48 151 223 49 — — 36 — — 17 1989 1991 1993 Genotype Plant Sciences E E® Mean LSD (0±05) Genotype (G) Infection (I) G¬I 7 17 Mean 7 17 Mean 7 17 Mean 396 277 337 397 309 352 397 292 460 266 363 628 241 435 544 254 175 82 129 194 87 141 185 85 — — 18 — — 48 n.s. 18 n.s. * Least significant difference at the 0±05 level of probability. Pendleton, South Carolina, USA. Plants were vegetatively propagated and one half of the individuals from each were treated to kill the endophyte (Hill et al., 1990). The plants in this study were at least five vegetative generations beyond endophyte removal to minimize chances of remnant fungicide effects. Plants were vegetatively propagated and grown in 7±5¬10 cm plastic cups in the glasshouse prior to establishment in the field as outlined by Hill et al. (1991). Plots were established at the USDA Southern Piedmont Conservation Laboratory in Watkinsville, Georgia, USA on 4 Oct. 1987. The site was a Cecil sandy clay loam soil. A second site was established on 4 Oct. 1988 at the University of Georgia Plant Sciences Farm, located near Bogart Georgia, USA, approx. 20 km from Watkinsville. The Plant Sciences Farm site was a Pacolet sandy clay loam soil. The soils at the two sites differ in that typical Cecil soils have deeper A horizons (% 36 cm) with a greater water holding capacity (C 2±2 cm) than the Pacolet series (A horizon ! 15 cm, water holding capacity C 0±45 cm). Plots were established in a modified Nelder’s systematic design for spacing experiments (Nelder, 1962). The plots were in a wagon wheel arrangement consisting of 16 spokes emanating from the centre to the edge of the plot. Each wheel was divided into four sections of four spokes each, and two sections randomly assigned to pure stands of E or E® plants. The remaining two sections were assigned to a mixture of E and E® plants within and between spokes. Each wheel was planted with one genotype. Each spoke had 11 plants. The four plants closest to the centre were planted 10 cm apart, the next four distal plants were planted 20 cm apart, and the outside three plants were planted 40 cm apart. Plants within spokes that were at the interface of spacing distances were considered the border of the plot. Hence planting density treatments were 4±7, 5±7, 13, 16, 21, 66 and 102 plants m−# (see schematic in Hill et al., 1991). Three replications of each plant genotype were used, for a total of six wagon wheels. Plots were fertilized with 90, 54 and 81 kg ha−" N, P and K, respectively, immediately after planting and again in February of the following year. In subsequent years the plants were fertilized every February with 100, 59 and 90 kg ha−" N, P and K, respectively. Plots were harvested for yield determinations at physiological maturity of the seed in early June during years 1, 3 and 5 following establishment. Plots were harvested by hand-clipping the forage to a height of 7±5 cm. In alternate years plots were mowed at late anthesis with a rotary mower and the forage removed immediately after harvest. Except for year 1, plots were mowed in February to remove residue from the previous year. Harvested tissue was oven-dried at 65 °C for approx. 7 d. Relative crowding coefficients (RCC) were calculated for all population densities within each year to determine the competitiveness of the endophytic forms of each genotype using eqn (1) (Harper, 1977) : RCC ¯ DMYEIM}DMYNIM DMYEIP}DMYNIP (1) where DMYEIM and DMYNIM are the dry matter yields of E and E® plants, respectively, in mixed stands, and DMYEIP and DMYNIP are the dry matter yields of E and E® plants, respectively, in pure stands. Treatment variables were assigned to a split-strip plot Hill et al.—Encroachment of Endophyte-infected on Endophyte-free Tall Fescue design repeated over time (years), with genotype as the whole plot, a complete factorial of stand type (mixed s. pure) and endophyte infection status as the split, and population density as the strip within each genotype. Since locations were established in different years, data were analysed within each location. Genotypes, infection status, stand type and population density were considered fixed effects, and years a random effect since environmental conditions varied over years. The statistical model was tested with the SAS general linear models program (SAS Institute, Cary, NC). Essentially all interactions between years and other treatment variables were significant, so the data were reanalysed within years and locations using a split-strip-plot model with genotype as the whole plot, a complete factorial between infection status and stand as the split, and population density as the strip within each genotype. The PROC REG procedure of SAS (SAS Institute, Cary, NC) was used to define relationships between dry matter yield or RCCs (dependent variables) and population density. Linear, quadratic and cubic regression models were tested and the equation with significance (P % 0±05) of the highest order regression coefficient (cubic " quadratic " linear) selected to define the relationship between population density and the dependent variables. RESULTS Plants grown at the two highest population densities could not be distinguished from one another after the initial year of growth. Therefore, data from the two highest population 485 1000 E+ 1988 Watkinsville E– 800 600 2 y = 452 – 40.64x + 1.18x R2 = 0.96 Dry matter yield (g) 400 y = 394 – 35.41x + 1.00x2 R2 = 0.96 200 0 1992 Watkinsville 800 600 y = 479 – 35.78x + 1.09x R2 = 0.99 2 400 y = 112 – 5.83x R2 = 0.90 200 0 6 9 12 15 18 Plant density (plants m–2) 21 F. 1. Mean dry matter yield of endophyte-infected (E) and endophyte-free (E®) forms of two tall fescue genotypes at five different population densities grown at Watkinsville, GA in 1988 and 1992. T 2. Mean dry matter yield (g per plant) of endophyte-infected (E) and non-infected (E®) tall fescue plants grown in mixed or pure stands grown at two locations 1988 1992 Infection status Location Watkinsville Stand type Mixture Pure Mean LSD (0±05)* Stand type (S) Infection (I) S¬I E E® Mean E E® Mean 210 193 202 184 164 174 197 179 256 189 126 30 67 151 143 128 n.s. 22 n.s. 1989 — — 17 1991 1993 Infection status Plant Sciences Mixture Pure Mean LSD (0±05) Stand type (S) Infection (I) S¬I E E® Mean E E® Mean E E® Mean 416 378 397 278 306 292 347 342 586 501 544 159 349 254 373 425 222 147 185 54 115 85 138 131 — — 17 * Least significant difference at the 0±05 level of probability. — — 48 — — 26 486 Hill et al.—Encroachment of Endophyte-infected on Endophyte-free Tall Fescue 1000 10 E+ 800 E– 1989 Plant Sciences Farm 8 y = 874 – 68.49x + 1.65x2 R2 = 0.98 400 200 y = 581 – 23.7x 2 R = 0.94 Dry matter yield (g) 0 1991 Plant Sciences Farm 800 y = 1296 – 118.5x + 3.31x2 R2 = 0.97 600 400 200 40 200 6 y = 1.29 – 0.029x R2 = 0.77 1992 Watkinsville 30 y = –13.35 + 2.635x 2 R = 0.97 y = 1.32 + 0.816x 2 R = 0.75 6 9 12 15 18 Plant density (plants m–2) 21 F. 3. Relative crowding coefficients of tall fescue genotypes 7 and 17 at five different population densities grown at Watkinsville, GA in 1988 and 1992. 600 0 0 0 1993 Plant Sciences Farm y = 175 – 7.46x 2 R = 0.80 mean = 2.03 2 10 0 400 4 20 y = 498 – 22.6x 2 R = 0.93 800 Genotype 7 Genotype 17 6 Relative crowding coeffieient 600 1988 Watkinsville y = 373 – 30.1x + 0.91x2 R2 = 0.97 9 12 15 18 –2 Plant density (plants m ) 21 24 F. 2. Mean dry matter yield of endophyte-infected (E) and endophyte-free (E®) forms of two tall fescue genotypes at five different population densities grown at the Plant Sciences Farm near Bogart Georgia in 1989, 1991 and 1993. densities were omitted during all years. In 1990, approximately one third of the samples collected at Watkinsville were destroyed due to power failure during drying and, therefore, no 1990 data will be presented for that location. Dry matter yields varied from year to year, depending largely upon spring rainfall. Growing conditions were uncommonly dry in 1992 and 1993, with pan evaporation exceeding precipitation after 1 April each year (Hill, Cabrera and Agee, 1995). There were significant genotype¬ endophyte infection¬stand type¬population density¬ year interactions for dry matter yield at both the Watkinsville and Plant Sciences Farm locations. To aid interpretation of the five-way interaction, means of the genotype¬infection status, stand type¬infection status, and population density¬infection status interactions were compared within years for each location. Endophyte-infected (E) genotype 7 had a greater dry matter yield than the E® form, but the E and E® genotype 17 did not differ from one another when averaged across population densities and stand type at the Watkinsville location in 1988 (Table 1). The E forms of both tall fescue genotypes had a greater dry matter yield than the E® forms when averaged across population densities and stand type at the Watkinsville location in 1992. The E form of tall fescue genotype 17 had a greater dry matter yield than E genotype 7, but dry matter yields did not differ between the E® genotypes at the Watkinsville location in 1992. Dry matter yield of the E forms for both genotypes were greater than the E® forms at the Plant Sciences Farm in 1989. Dry matter yield of E® genotype 7 was less than E® genotype 17 in that year. Dry matter yield of the E forms of the genotypes were greater than the E® forms at the Plant Sciences Farm, and E genotype 17 was greater than E genotype 7 in both 1991 and 1993. Dry matter yield of the E® forms of the plant genotypes did not differ from one another in either 1991 or 1993 at the Plant Sciences Farm. There was no difference in dry matter yield of E plants when grown in mixtures with E® plants compared to pure stands at Watkinsville in 1988 (Table 2). By 1992, E plants grown in mixtures had a greater dry matter yield than E® plants grown in pure stands at Watkinsville. The E® plants grown in pure stands had a greater yield than E® plants grown in mixtures with E plants in 1992 at Watkinsville. The E plants had a greater dry matter yield when grown in mixtures with E® plants than when grown in pure stands at the Plant Sciences Farm, regardless of year. The E® plants had a greater dry matter yield when grown in pure stands than when grown in mixtures with E plants at the Plant Sciences Farm, regardless of year. Quadratic equations gave best fits when dry matter yield was regressed with population density of E plants at both locations (Figs 1 and 2). Linear equations gave best fits Hill et al.—Encroachment of Endophyte-infected on Endophyte-free Tall Fescue 6 Two important trends occurred among the RCCs. First, RCCs increased with time, especially at the Watkinsville location and secondly, RCCs were greater for genotype 7 at the Watkinsville location, but tended to be greater for genotype 17 at the Plant Sciences Farm. 4 DISCUSSION 10 1989 Plant Sciences Farm Genotype 7 Genotype 17 8 mean = 1.80 2 mean = 1.75 Relative crowding coefficient 487 0 1991 Plant Sciences Farm 8 6 4 mean = 3.61 2 y = 1.94 – 0.047x R2 = 0.66 0 8 1993 Plant Sciences Farm 6 mean = 4.45 4 y = 2.09 – 0.119x R2 = 0.64 2 0 6 9 12 15 18 Plant density (plants m–2) 21 F. 4. Relative crowding coefficients of tall fescue genotypes 7 and 17 at five different population densities grown at the Plant Sciences Farm near Bogart Georgia in 1989, 1991 and 1993. when dry matter yield was regressed with population density of E® plants with the exception of those grown at Watkinsville in 1988. Generally, dry matter yield of E plants was greater than E® plants regardless of population density, year, or location, but yields were most similar between E and E® plants during 1988 at Watkinsville and 1989 at the Plant Sciences Farm, the years the plots were established. Dry matter yield of E and E® tall fescue genotypes was also most similar at the higher population densities in all years when averaged over mixed and pure stands. Relative crowding coefficients were similar among all population densities for genotype 7 at the Watkinsville location in 1988, but genotype 17 showed a slight decrease in RCC as population density increased (Fig. 3). Both genotypes had linear increases in RCCs as population density increased at Watkinsville in 1992. Relative crowding coefficients did not differ within years and among population densities for genotype 17 at the Plant Sciences Farm location (Fig. 4). Relative crowding coefficients were similar for all population densities for genotype 7 during 1989, but showed a linear increase as population density increased in 1991 and 1993. Since rainfall varied between years, it is difficult to interpret a trend for the performance of E and E® during the course of the experiment based upon dry matter yields. Weights of E and E® tall fescue were most similar during the establishment years, but as time progressed E plants grown in mixtures had a greater mass than those grown in pure stands. Also, E® plants grown in mixtures had a smaller mass than those grown in pure stands as time progressed. These data suggest that E plants encroached upon E® plants (Table 2). However, interpretation of these data is confounded since E® plants had a smaller mass than E plants when grown in pure stands as well. Similarly, it is difficult to interpret the competitiveness of the E and E® forms for each genotype using dry matter yield data. There was a genotype¬infection status interaction at the Watkinsville location in 1988 and 1992. The mass of tall fescue genotype 17 was similar regardless of endophyte status in 1988 and E® genotype 17 had approx. 20 % of the mass of the E form in 1992 (Table 1). There were genotype¬infection status interactions for dry matter yield for plants grown at the Plant Sciences Farm in 1989 and 1991, but the endophyte effect was significant only in 1993. Consequently, comparisons of how E and E® plants performed in mixtures relative to performance in pure stands were necessary to properly interpret the data (Hill et al., 1991). Relative crowding coefficients compare the performance of E and E® plants on a relative basis in mixed s. pure stands at each population density (Harper, 1977 ; Hill et al., 1991). An RCC " 1 indicates the E form was more competitive and encroached on the E® form, while the reverse is true if the RCC is ! 1. By definition, E tall fescue genotypes were increasingly aggressive to the E® form as the RCCs increased above 1. Relative crowding coefficients were generally between 1 and 2 for genotype 7 but were 1 or less for genotype 17 in 1988 at the Watkinsville location (Fig. 3). Relative crowding coefficients tended to be lower at the higher population density, suggesting that E® genotype 17 encroached upon the E form. Conversely, the data suggested that the E form of genotype 7 encroached on the E® form in 1988. Relative crowding coefficients in 1992 suggested that the E forms of both genotypes were encroaching on the E® forms at the Watkinsville location, and that encroachment was more pronounced at higher population densities. Relative crowding coefficients were " 1 for both genotypes at the Plant Sciences Farm in all years tested (Fig. 4), indicating that E plants were encroaching on E® plants. Trends in relative crowding coefficients were similar at both locations in that they increased numerically over years. Increases in relative crowding coefficients at the Plant Sciences Farm were more subtle than at the Watkinsville 488 Hill et al.—Encroachment of Endophyte-infected on Endophyte-free Tall Fescue location, but the genotypic ranking of relative crowding coefficients changed among locations. These observations suggest that encroachment of E tall fescue on the E® forms is site and genotype specific. It is not surprising that different genotypes perform differently at the two locations depending upon infection status. The combination of plant genotype and endophyte genotype seemingly increases phenotypic variation in tall fescue plants (Hill et al., 1991). Hence, the interaction of the two symbionts increases plasticity in a tall fescue population (Schmid, 1985 ; Hill et al., 1991), giving tall fescue its broad geographic adaptation. The A horizon of the Cecil soil at Watkinsville was deeper with a higher water holding capacity than the A horizon of the Pacolet at the Plant Sciences Farm, suggesting that Watkinsville would be a more hospitable site for the more drought susceptible E® tall fescue. Indeed, the relative crowding coefficients were approx. 1 or lower during the establishment year suggesting Watkinsville was a more hospitable site (Fig. 3). However, high RCCs 5 years after establishment at the Watkinsville location make it clear that this site was especially susceptible to encroachment by E tall fescue. This phenomenon demonstrates that a more systematic ecological approach towards testing fitness of E or E® tall fescue is needed to establish benchmarks for recommendations concerning utilization of each. Identification of abiotic variables (such as soil type, climatic and fertility variables), biotic variables (such as insect or disease pressures), and intensity, duration and type of defoliation, are likely to lead to a better understanding of the role each plays in encroachment of E on E® tall fescue. There is an abundance of data in which each has been observed as a component ; however, the interactions of each need to be addressed in systems studies to establish whether synergistic or antagonistic interactions exist among those variables relative to the performance of E® tall fescue. Such data could be useful when making recommendations concerning utilization of E or E® tall fescue, and prescribe management variables to reduce the probability of E encroachment. LITERATURE CITED Belesky DP, Robbins JD, Stuedeman JA, Wilkinson SR, Devine OJ. 1987. Fungal endophyte infection-loline derivative alkaloid concentration of grazed tall fescue. Agronomy Journal 79 : 217–220. Belesky DP, Stringer WC, Hill NS. 1989. Influence of endophyte and water regime on tall fescue accessions. I. Growth characteristics. Annals of Botany 63 : 495–503. Bransby DI, Schmidt SP, Griffey W, Eason JT. 1988. Heavy grazing is best for infected fescue. Alabama Agricultural Experiment Station Highlights 35 : 12. Clay K, Hardy TN, Hammond AM. 1985. Fungal endophytes of grasses and their effects on an insect herbivore. Oecologia 66 : 1–6. Harper JL. 1977. Population biology of plants. London : Academic Press. Hill NS. 1994. Ecological relationships of Balansiae-infected graminoids. In : Bacon CW, White JF, eds. Biotechnology of endophytic fungi of grasses. Boca Raton : CRC Press, 59–71. Hill NS, Belesky DP, Stringer WC. 1991. Competitiveness of tall fescue as influenced by Acremonium coenophialum. Crop Science 31 : 185–190. Hill NS, Cabrera ML, Agee CS. 1995. Morphological and climatological predictors of forage quality in tall fescue. Crop Science 35 : 541–549. 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The effects of the fungal endophyte Acremonium coenophialum in tall fescue on animal performance, toxicity and stand maintenance. Agronomy Journal 78 : 848–850. Schmid B. 1985. Clonal growth in grassland perennials : III. Genetic variation and plasticity between and within populations of Bellis perennis and Prunella ulgaris. Journal of Ecology 73 : 819–830. Shelby RA, Dalrymple LW. 1993. Long-term changes of endophyte infection in tall fescue stands. Grass and Forage Science 48 : 356–361. Shelby RA, Schmidt SP. 1991. Survival of the tall fescue endophyte in the digestive tract of cattle and horses. Plant Disease 75 : 776–778. West CP, Gwinn K. 1993. Role of Acremonium coenophialum in drought, pest, and disease tolerances of grasses. In : Hume DE, Latch GCM, Easton HS, ed. Proceedings of the Second International Symposium on Acremonium}grass interactions : Plenary Papers. Palmerston North New Zealand : AgResearch Grasslands, 131–140. West CP, Izekor E, Turner KE, Elmi AA. 1993. Endophyte effects on growth and persistence of tall fescue along a water-supply gradient. Agronomy Journal 85 : 264–270.