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SEED PHYSIOLOGY, PRODUCTION & TECHNOLOGY

Seed Maturity, Germination, and Endophyte Relationships in Tall Fescue

^a Dep. Crop and Soil Sciences, Univ. of Georgia, Athens, GA 30602
^b Large Scale Biologicals, Inc., Owenboro, KY 42301

^* Corresponding author (nhill{at}uga.edu)

	ABSTRACT

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Endophytic fungi were once considered detrimental components of cool season pasture grass seed because of their association with toxic compounds. Development of new cultivars of forage grasses infected with nontoxic endophytes suggests endophytes are an essential component of pasture ecosystems. The process of transmitting endophytes via the seed embryo is poorly understood, and little is known about harvest conditions and endophyte transmission or viability. The objective of this project was to develop methods of predicting seed maturity and determine the effect of harvest maturity on viable endophyte transmission to seedling plants. Sixty panicles of tall fescue (Festuca arundinaceae Shreb.) from fields in Georgia and Oregon were sampled from 15 d after flowering until seed freely shattered (Georgia) or seed fields were swathed (Oregon). All fields had previously tested >90% infection with endophyte before the study. Panicles were staged for maturity by scoring for color of the panicle and seed, as well as seed shatter. Chlorophyll was extracted from a fresh subsample of seed, and dry matter was determined from a second subsample. Chlorophyll was estimated by measuring light absorbance at 540 nm. Seed with varying maturity scores were germinated following different prechill conditions. Regression of chlorophyll and dry matter content (dependent variables) with maturity score (independent variable) suggest chlorophyll content (r² = 0.90) provided a better estimate of maturity than did dry matter (r² = 0.55). The more mature seed had greater mass, germinated faster, had a higher total germination, and produced more vigorous seedlings. Endophyte transmission into the developing seed was similar within different maturity seed, but infection of seedlings increased from 81 to 91% with seed maturity. We conclude that predicting tall fescue seed maturity by chlorophyll analysis provides a better estimate for seed quality when maintaining endophyte viability is of concern in the harvested seed.

Abbreviations: GA-5, ‘Georgia 5’ • LEA, late embryogenesis abundant

	INTRODUCTION

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SEED QUALITY IS traditionally defined as the sum of genetic potential, physical purity, germination, and freedom from seedborne disease (McDonald and Copeland, 1997). Mutualistic endosymbionts such as Neotyphodium spp. fungi (commonly referred to as endophytes) are present in pasture and turf grass species and are passed from one plant generation to the next via seed (Bacon and Siegel, 1988). They express no outward symptoms on the host plant but provide fitness-related traits (drought tolerance and insect resistance) that make their hosts more competitive (Hill et al., 1991, 1998; Clay, 1985; West et al., 1993). Many of these organisms produce mycotoxins that adversely affect grazing livestock (Stuedemann and Hoveland, 1988; Fletcher and Harvey, 1981) and consequently have received considerable negative attention from a seed quality point of view. Thus, efforts were made to remove endophytes from the host species to alleviate toxicosis syndromes of grazing livestock.

New developments in Neotyphodium-related technology led to forage-type grass varieties infected with endophytes that are not toxic to grazing livestock (Bouton et al., 2002). Livestock performance when grazing grasses infected with nontoxic endophytes is improved compared with livestock grazing pastures containing toxins produced by endemic endophytes. However, agronomic performance of pastures containing nontoxic endophytes is superior to that of the endophyte-free forms of the grass cultivars. Consequently, endophytes are now considered essential components of sustainable grass based pasture ecosystems, a radical shift in thinking from just a few years ago.

While the endophyte transmission process has been examined at the cellular and tissue level (Phillipson and Christey, 1986), little is known about the relationship between plant development and endophyte transmission. Endophyte presence in plant tissues varies with season (DiMenna and Waller, 1986), temperature (Ju et al., 2001), and possibly plant development. Interactions among climatic and physiological variables suggest endophyte transmission is likely to be a highly regulated process. Consider then, the seed development and maturation process. Embryos of seed protect cellular components during final dry down by coating membranes with highly hydrophilic late embryogenesis abundant (LEA) proteins to prevent organelle and, thus, embryo death (Galau et al., 1987; Hughes and Galau, 1989). Similar mechanisms occur in yeast [Saccharomyces cerevisiae (Meyen ex E.C. Hansen)] (Garay-Arroyo and Covarrubias, 1999; Garay-Arroyo et al., 2000; Sales et al., 2000). Therefore, it is logical to assume a similar cellular protection mechanism occurs in other fungi exposed to desiccation events. If LEA protein-like desiccation tolerance mechanisms are involved in providing Neotyphodium protection during seed maturation and desiccation, management of the developing crop could be vital to subsequent endophyte viability during the transmission process.

Typically, grass seed producers harvest their crop by swathing when seed dry matter content reaches 550 to 600 g kg^–1 because the majority of seed approaches physiological maturity at this time. However, this truncates the developmental process and induces a rapid desiccation event because of vascular disruption. To date, there are no studies which have examined whether interruption of natural desiccation alters endophyte transmission and viability in the seed. The objective of this research was to develop a reliable method for predicting tall fescue seed maturity and to characterize endophyte transmission as a function of seed maturity.

	MATERIALS AND METHODS

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Field Selection and Panicle Sampling
Four seed fields located within 0.9 km of one another were selected for sampling in Georgia. Each seed field was established at least 4 yr before sampling for this experiment. All fields were fertilized in accordance with University of Georgia soil fertility recommendations for phosphorus and potassium and received adequate lime to maintain soil pH above 6.0. The fields were fertilized with 100 kg N ha^–1 in late February of each year. Two of the seed fields were planted to the tall fescue cultivars Georgia 5 (GA-5) and Jesup, both containing the nontoxic endophyte AR542. The other two fields were planted to wild-type toxic endophyte-infected GA-5 and Jesup. Beginning on approximately 18 May and continuing until approximately 4 June of both 2001 and 2002, sixty panicles were systematically harvested while walking through the seed fields. Panicles were harvested between 1000 and 1300 h of each day by clipping the culm immediately below the basal inflorescence. Sampling was initiated approximately 15 d after pollination and continued on an every-other-day basis until the seed freely shattered from the panicles. The panicles were staged for maturity (see section on staging) immediately after harvest and the seed removed from the panicles by hand. Two subsamples of seed were obtained immediately after harvest, one for chlorophyll extraction and one dried at 60°C in a convection oven overnight for dry matter analysis. The remaining seed were placed in a cloth bag and air dried at room temperature.

Four seed fields were selected in the Willamette Valley of Oregon. The pH, potassium, and phosphorus levels were maintained according to soil test recommendations by Oregon State University. Each field was established at least 2 yr before initiation of this experiment. Each field was fertilized with 96 kg N ha^–1 in early April of each year. The fields were located within 100 km of one another and consisted of two fields of Jesup and two fields of GA-5, all containing the AR542 endophyte. Sixty panicles from each field were sampled as previously described. Samples were taken every other day beginning 25 June 2001 and 2002 until they were swathed for harvest. A final sample was taken from the windrow the day the field was swathed. Distances between seed fields and varying climatic conditions did not permit sampling within a defined time period of each day. Samples were gathered from 1100 h until approximately 1500 h. The panicles were staged for maturity, the seed removed by hand, and a subsample taken for chlorophyll analysis. An additional subsample of seed was dried overnight at 60°C in a convection oven to obtain dry matter. The remaining seed were placed in a cloth bag and dried at room temperature.

Staging Panicles for Maturity and Chlorophyll Analysis
A scoring system was developed to model panicle maturity by examining (i) seed color and (ii) culm color at the top, middle, or bottom of the panicle, and (iii) seed shatter at the top middle and bottom of the panicle. Each panicle was given a numerical value when the entire culm was green (1), the top straw was colored (2), middle was straw colored (3), or the entire culm was straw colored (4), respectively. Seed were assigned a numerical value when all seed were green (1), those on the top of the panicle were straw colored (2), those in the middle of the panicle were straw colored (3), or all seed were straw colored (4), respectively. A shattering score was assigned value when there was no seed shatter (0), or seed on the top (1), middle (2), or bottom (3) of the panicle shattered, respectively. Each score was calculated on a cumulative basis. For example, if a panicle was completely straw colored, it received a score of 10 (i.e., 4 + 3 + 2 + 1). The cumulative scores for the panicle color, seed color, and seed shatter were summed for each panicle. Thus, a minimum score of 2 and a maximum score of 26 was possible for each panicle. Mean maturity scores for all panicles from a sampling date within a seed field were calculated by dividing the total of the summed scores by the number of panicles in the sample.

Chlorophyll was extracted using a 25-mL volume of seed from each sample. The seed was lightly packed into a 50-mL disposable centrifuge tube, and sufficient methanol poured into the tube to cover the seed. The tube was capped and placed in the dark for exactly 60 min. The methanol was decanted into a 15-mL graduated plastic tube and placed in the refrigerator at 4°C until all samples were gathered. Chlorophyll extracts from Oregon were shipped overnight express to Georgia. One hundred and fifty microliters of chlorophyll extract was pipetted into a 96-well polycarbonate microplate, and absorbance measured at 540 nm on a BioTek (Winooski, VT) ELx800 microplate reader.

Predicting Seed Maturity
Dry matter and chlorophyll absorbance values (dependent variables) were independently regressed with seed maturity score (independent variable) to determine which gave the best prediction of seed development. Linear, quadratic, cubic, and quartic equations were tested to determine which gave the best fit to the data. Equations with the highest order regression coefficient with significance at the 0.05 level of probability in the regression model were considered the best fit of the data.

Seed Germination
Seed lots with mean maturity scores approximating 16, 10, 5, and 2 were selected to conduct germination studies. Before germination, 1000 seed were counted and weighed to estimate seed plumpness. Forty-five seed from each lot were placed into CYG growth pouches (Mega International, Minneapolis, MN). The pouches were placed into wire racks and distilled water added. The seed were incubated at 4°C for 0, 3, 5, and 7 d to break dormancy and placed under continuous fluorescent lighting. Seed were examined at 0800 h daily and the number of germinated seed recorded during a 10-d period. Germinated seed were defined as those from which the coleoptiles were protruding from the caryopsis. Germination data for each seed lot and dormancy breaking (chill) treatment were fitted to the following logistic equation:

[1]

where X = any given day from 0 to 10, Y = the percentage of seed that germinated for Day X, y₀ = the predicted y intercept; a = the difference between the maximum germination and y₀ for a given seed lot, x₀ is the calculated time in days necessary to achieve one-half of a, and b = the calculated slope at 1/2 a. Independent y₀, x₀, a, and b values were calculated from the logistic function for seed samples with different maturity scores and chilling treatments.

Endophyte Analysis
Viable endophyte was determined by growing seedling plants from the germination study until they reached the four-leaf stage. The seedling plants were fertilized with 60 mL of a solution containing 150, 300, and 150 mg L^–1 N, P₂O₅, and K₂O, respectively, beginning 10 d after germination, and every 7 d thereafter until seedling plants reached the four-leaf stage. Plants were harvested by cutting the psuedostem immediately above the seed, and a 2- to 3-mm stem cross-section of psuedostem was removed for endophyte analysis. The remaining plant tissue was dried at 60°C overnight to determine seedling weight. Endophyte analysis was conducted using a commercial immunoblot test kit (Agrinostics Ltd. Co., Watkinsville, GA). Percentage endophyte infection was calculated for each harvested seed sample and the seedling plants grown from that seed.

Statistical Analysis
Thousand-seed weight, seedling weight, seed and seedling endophyte infection, plant development score, and chlorophyll absorbance data were evaluated by ANOVA. Years were considered random variables while harvest dates and location (Georgia vs. Oregon) were fixed effects. The experimental design was a split-split plot design with location as the whole plot, years the first split, and harvest dates as the second split. Seed fields served as blocks for each location. Means were separated using a Fisher's protected LSD at the 0.05 level of probability. Analysis of variance was also used to examine the effects of harvest dates, location, and prechilling treatment on the logistical parameters (y₀, x₀, a, and b) from Eq. [1] for the germination study. Since all germinations were conducted at the same time, all treatment effects were considered fixed and the ANOVA was conducted using a completely randomized model. Means for each parameter were separated using a Fisher's protected LSD at the 0.05 level of probability.

	RESULTS

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Panicles from the first harvest had green seed, the stems of the panicles were green, and no seed shattered. Those harvested at the end of the sampling period had primarily straw-colored seed and panicles with seed that freely shattered. Therefore, mean panicle maturity scores for the harvested panicles ranged from 2.0 for panicles harvested first to 22.5 for panicles harvested at the end of the sampling period (Fig. 1) . Ranges of panicle maturity scores were similar across years and locations (data not shown). Absorbance of light at 540 nm measures light in the green range of the spectrum, and therefore is a measure of chlorophyll. The chlorophyll absorbance values of the seed decreased as maturity increased. Thus, there was an inverse relationship between panicle maturity score and chlorophyll absorbance values, and was best described by a cubic regression equation. It is important to keep in mind this regression equation was developed from absorbance and maturity score values from seed fields located in two distinctly different environments (Georgia and Oregon) sampled in two different years. The high coefficient of determination (r² = 0.90) from the regression equation with this diverse population of samples suggests chlorophyll is a good predictor of tall fescue seed maturity. There was a linear relationship between dry matter content and maturity score (Fig. 2) . However, the coefficient of determination was lower (r² = 0.55) than that for the equation for seed maturity when chlorophyll measurements were the independent variable.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Response curve between absorbance values of chlorophyll extracts and maturity scores of tall fescue seed. Data points represent those harvested from two locations (Georgia and Oregon) during a 2-yr period.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Response curve between dry matter content of harvested seed and maturity scores of tall fescue seed. Data points represent those harvested from two locations (Georgia and Oregon) during a 2-yr period.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Logistic response curves for mature seed receiving 7 d of prechilling at 4°C to break dormancy vs. immature seed receiving no prechilling treatment. The curves have the predicted values for germination at time 0 (y0), the mean time for germination to reach one-half of the maximum value (x0), and the predicted maximum germination rate (a) depicted for each.

Endophyte infection frequency of seed was high and similar regardless of the stage of maturity at harvest. However, infection of seedling plants increased with seed maturity (Table 1). Thus, stage of harvest affected endophyte viability.

	DISCUSSION

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Production of quality seed requires timely harvest at physiological maturity for maximum seed vigor. Dry matter content of seed is used as an industry standard for estimating maturity of grass seed, but dessication during seed maturation is rapid (Gallau et al., 1989) and, therefore, is not a precise measure of seed maturity. Seed maturity is not uniform in tall fescue, and flowering of different culms within a plant or culms from different plants may occur across a 14-d period. When environmental variables such as temperature, humidity, and wind speed are factored into estimating seed moisture, it should be no surprise that seed dry matter and seed development are not strongly correlated (Fig. 2). Chlorophyll content, on the other hand, diminishes as seed development progresses in many plant species (Jalink et al., 1998, 1999; Konstantinova et al., 2002) and represents an average maturity of all seed. It appears to be independent of environmental conditions and therefore is highly correlated with seed maturation (Fig. 1). The consistency of maturity prediction across distinct locations and years suggests chlorophyll analysis is a better system for monitoring seed maturity of tall fescue than seed dry matter.

Seed development occurs in three distinct phases: histodifferentiation, reserve deposition, and maturation drying (Bewley and Black, 1995). Complete embryonic cellular division is prioritized during histodifferentiation until a complete, albeit minute, embryo has developed. Cellular expansion occurs concomitantly with reserve deposition and seed enlargement, and abscisic acid accumulates to elicit dormancy within the developing and mature seed. Highly hydrophilic LEA proteins are expressed just before maturation drying to protect organelles from the extremely low water potentials occurring after final dessication (Galau et al., 1987; Hughes and Galau, 1989). Generally, seed which reach physiological maturity before harvest have more germinable seed and yield seedling plants which are more vigorous that those in which the maturation process is truncated. Seed harvested when maturity scores were 2 and 6 had lighter 1000-seed weights (Table 1) while only the seed with the lowest maturity score had reduced germination (Table 2) and mean seedling weights (Table 1).

The life cycle of N. coenophialum is simple in that it resides in vegetative tissue of the plant, invades developing seed heads when the plant enters the reproductive phase, and is passed from one generation of plants to the next via seed (Bacon and Siegel, 1988). The environment in which it lives is largely tempered from the water potentials of ambient conditions by the host plant, but the endophyte must survive the dessication process the same as the embryo of a mature seed. Plants that produce orthodox seed have adapted to stress associated with dessication by accumulating LEA proteins at membrane interfaces (Galau et al., 1987; Hughes and Galau, 1989). The LEA proteins are also widespread among different taxa (Garay-Arroyo et al., 2000) and as many as 12 proteins are involved in dessication tolerance in Saccharomyces cerevisiae (Garay-Arroyo and Covarrubias, 1999; Garay-Arroyo et al., 2000; Sales et al., 2000). Inasmuch as the endophyte must survive seed desiccation for survival, it is highly probable that it also contains LEA proteins to protect vital organelles during extremely low water potentials. Truncating the developmental process would affect endophyte viability. Although seed infection rates were similar regardless of when seed were harvested, seedling endophyte infection rates were lower in seed harvested before maturity, suggesting endophyte mortality occurred (Table 1). Philosophies and economic implications of having endophytes in grasses have changed in recent years from attempting to eliminate endophytes from seed stock to now trying to maintain endophytes in seed stock. Our findings suggest that truncating the process of seed maturity will diminish the infection levels of the resulting seedling plants.

Risk associated with losing endophytes from elite cultivars will undoubtedly diminish the value of the seed. Having a reliable system of monitoring seed development is the first step toward producing seed in which the health and maintenance of nontoxic endophytes will be optimized. Our findings strongly suggest predicting maturity by monitoring seed chlorophyll is superior to dry matter analysis of the developing seed.

Received for publication February 2, 2004.

	REFERENCES

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This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Hill, N. S. Articles by Kittle, B. Search for Related Content PubMed Articles by Hill, N. S. Articles by Kittle, B. Agricola Articles by Hill, N. S. Articles by Kittle, B. Related Collections Forage Management Other Forage Crops Seed Physiology Seed Technology