Use of accelerated aging as a surrogate phenotyping approach to improve endophyte survival during storage of tall fescue seed

Field Crops Research 183 (2015) 43–49

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Use of accelerated aging as a surrogate phenotyping approach to improve endophyte survival during storage of tall fescue seed Ali M. Missaoui a,∗ , Nicholas S. Hill b a Department of Crop and Soil Sciences and Institute of Plant Breeding Genetics and Genomics, University of Georgia, 111 Riverbend Rd., Athens, GA 30602, United States b Department of Crop and Soil Sciences, University of Georgia, 3111 Miller Plant Sciences Bldg., Athens, GA 30602, United States

a r t i c l e

i n f o

Article history: Received 14 April 2015 Received in revised form 8 July 2015 Accepted 14 July 2015 Available online 31 July 2015 Keywords: Tall fescue Endophyte viability Accelerated aging Selection Seed storage Heritability

a b s t r a c t Survival of the endophyte (Neotyphodium coenophialum) during seed storage of tall fescue (Lolium arundinaceum (Schreb.) Darbysh) has commercial value. The objectives of this research were to (1) identify accelerated aging (AA) conditions that compromise endophyte viability without affecting seed germination, (2) Determine whether the response to selection for endophyte persistence is consistent in laboratory and warehouse conditions (3) determine whether selection affected agronomic characteristics of the populations. ‘Jesup’ tall fescue with non-toxic endophyte strain AR542 was incubated for 35-d in sealed food containers with different salt solutions to create varying relative humidities (RH). Seed were sampled and tested for germination and endophyte viability every 7 d to determine which treatment provided the most effective conditions to induce AA. Base populations (C0) of ‘Jesup’ AR542 (Jesup MaxQ) and Jesup AR584 had previously been selected for endophyte survival after 18 months under ambient laboratory conditions to produce C1 populations. Seed from C1 were exposed to accelerated aging conditions, and seedlings with viable endophytes were selected to produce C2AA populations. All six populations (two each of C0, C1, and C2AA) were grown in isolated blocks in the field to produce the seed used in these experiments. Seed from C0 and C1 populations was stored in cloth bags at ∼21 ◦ C in the laboratory and tested for endophyte viability monthly for 22 months. Seed from C2AA populations in addition to C0 and C1 were stored in a warehouse for 27 months and endophyte viability tested every 3 months. Heritability for endophyte survival was calculated for the C1 and C2AA populations. Seventy-five percent RH was effective in inducing accelerated aging of the endophyte without compromising seed health. Heritability estimates for endophyte survival were high in both Jesup AR542 and JesupAR584 for both C1 (0.51-0.62) and C2AA (0.54-0.64). Agronomic characteristics for all populations remained comparable to the original Jesup release (C0) after both cycles of selection (C1 and C2AA). © 2015 Elsevier B.V. All rights reserved.

1. Introduction Tall fescue (Lolium arundinaceum Schreb.) plants like many other grass species harbor a mutualistic endophyte fungus (Neotyphodium coenophialum) that imparts drought tolerance, insect resistance, and disease resistance to the host plant in exchange for habitat and nutrition (Hall et al., 2014; Nagabhyru et al., 2013; Ryan et al., 2014; Young et al., 2014). Neotyphodium endophytes reside within the lower stem and leaf sheath tissues and migrate to the seed head during reproduction resulting in vertical transmission to the next generation through the seed (Gundel et al., 2009; Van der Heijden et al., 2015; Wiewiora et al., 2015). Naturally occurring

∗ Corresponding author. E-mail address: [email protected] (A.M. Missaoui). http://dx.doi.org/10.1016/j.fcr.2015.07.016 0378-4290/© 2015 Elsevier B.V. All rights reserved.

‘wild type’ endophytes present in tall fescue populations such as Kentucky 31 produce ergot alkaloid toxins that have adverse effect on herbivores (Zbib et al., 2014). Scientists have identified novel, non-toxic endophyte strains originating from tall fescue ecotypes from the Mediterranean region and these new strains are being used to replace wild-type toxic endophytes (Latch et al., 2000; et al., 2003; Rolston and Simpson, 2007 Rolston and Simpson, 2007). Seed companies who develop or license cultivars containing novel fungal endophytes have to maintain quality assurances to protect the integrity of the technology. They target, in addition to normal seed certification tests, a presence of off-type toxic endophytes less than 5% in their seed lots, and that seed must express 70 percent viable (live) endophyte at the time of sale (http://grasslandrenewal.org/ qualitycontrol.htm). Because endophytes are maternally transmitted through the seed, and because their mortality precedes seed mortality during storage, the viability of the endophyte in seed

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A.M. Missaoui, N.S. Hill / Field Crops Research 183 (2015) 43–49

Fig. 1. Flowchart representing the various steps involved in the selection for endophyte survival under ambient (C1) and accelerated aging (C2AA) conditions, recombination of the selected progenies, assessment of endophyte viability and calculation of the heritability of the trait.

is an important trait for product quality and standard seed label information required by law (Hill et al., 2005). Numerous variables affect endophyte expression in tall fescue populations. Expression of some fungal traits are altered by plant genetics, while others appear to be regulated by the fungus itself. Genetic interactions between endophytes and their grass hosts control the extent of fungus transmission from one generation to the next (Bouton et al., 2002) as well as ergot alkaloid concentration (Adcock et al., 1997; Roylance et al., 1994; Agee and Hill, 1994; Hill and Roach, 2009). Natural tall fescue populations have broad genetic variation and may host diverse endophyte genotypes. This diversity of both plant and endophyte provides for a plastic population with high stability under changeable ecophysiological conditions, leading to a more sustainable mutualistic association (Clay et al., 1985). When plant and/or endophyte genetic variation becomes limited through the selection process, components of plasticity are lost, making compatibility screening necessary during the cultivar development process (Bouton et al., 2002). Seed storage conditions affect endophyte viability (Siegel et al., 1984; Welty et al., 1987). Increasing either temperature or humidity negatively impacts the fungus viability during seed storage (Welty et al., 1987). The options for selling novel endophyteinfected seed with low endophyte viability are limited and, therefore, does not capitalize on the value added trait because loss of endophyte viability in seed portends a compromised product with lower commercial value. Thus, extending the longevity of the endophyte fungus during seed storage has commercial appeal. In a previous study, Hill and Roach (2009) tested the survival of endophyte AR542 in different tall fescue cultivars and endophytes AR542 and AR584 in a Jesup tall fescue during seed storage at room temperature for 18 months. They found significant differences in endophyte viability during storage depending on the genetic background of the germplasm as well as differences between strains with endophyte AR584 having longer viability than AR542 in Jesup background. Selection for endophyte survival resulted in an increase in endophyte viability. This study confirmed that endophyte survival during seed storage is controlled by both plant and endophyte traits. Plant factors that enhance endophyte survival during storage are heritable and, hence, breeding for increased endophyte viability during seed storage is a desirable, and a feasible target. Unfortunately, real time evaluation of endophyte longevity is a slow process that requires

maintaining seed for 18–24 months at ambient temperatures to identify plants in which the endophyte survived. This constitutes an inefficient selection process which may discourage plant breeders from capitalizing on the opportunity to improve this commercially valuable trait. Therefore, developing a method to speed up the evaluation process would increase the breeder’s ability to manipulate the longevity of endophyte survival in improved germplasm. Finding a means of accelerating the aging process which compromises the endophyte viability without affecting seed germination may increase the selection efficiency to improve endophyte viability during seed storage and this is the main focus of this work. The objectives of this research are to: (1) identify accelerated aging (AA) conditions that maintain seed germination but compromise endophyte viability, (2) Determine whether selection for endophyte persistence following AA is heritable and consistent in laboratory and warehouse conditions, and (3) determine whether selection for endophyte viability resulted in as shift of the agronomic characteristics of the original populations. 2. Materials and methods 2.1. Identify accelerated aging conditions that compromise endophyte viability without affecting seed germination Relative humidity is an important factor to consider during seed storage. Various salt concentrations were used to modify the relative humidity conditions in chambers in which the seed were placed because they maintain constant humidity conditions over the temperatures used in this study (Hedlin and Trofimenkoff, 1963; Wexler and Hasegawa, 1954). One hundred milliliters of saturated solutions consisting of magnesium chloride, magnesium nitrate, sodium chloride, or distilled water were each assigned to three dual chamber Rubbermaid® (Newell Rubbermaid Inc., Atlanta, GA) polyethylene containers (a total of 12 containers). These salt solutions were selected because they provide consistent relative humidities of 33, 52, 76 or 100% over a range of temperatures (Wexler and Hasegawa, 1954; Wexler and Hasegawa, 1954). One hundred mL of salt solution was placed into one side of the polyethylene containers. Seed from a population of ‘Jesup MaxQ’ tall fescue with the non-toxic endophyte AR542 was harvested from a space planted polycross block two months prior to initiation of the experiment. The seed was stored at 10 ◦ C and 18% moisture

A.M. Missaoui, N.S. Hill / Field Crops Research 183 (2015) 43–49

content prior to initiation of the experiment. Approximately 5 g of seed of Jesup with endophyte AR542 was placed into the side of the polyethylene container adjacent to the salt solutions. The lids of the containers were secured and the containers were placed into a laboratory incubator at 30 ◦ C. The plastic containers were tilted by placing a No. 5 rubber stopper under the end of the container to prevent condensation from dropping onto the seed. Seed were incubated for five weeks. Approximately 0.5 g of seed was sampled at 7 d intervals from the polyethylene containers. The chambers were re-sealed with their lids and put back into the incubator until the next sampling date. The retrieved seed were placed into 50-mL beakers, 20 mL of distilled water was added, and the seed placed in a refrigerator at 4 ◦ C for 7 d to break dormancy. Seventy-two seed were planted into 72 cell Daisy trays (Cassco, Montgomery, AL) containing Fafard (Sun Gro Horticulture, Agawam, MA) germination mix. Cell packs were transferred to the greenhouse, watered regularly, and grown under ambient greenhouse temperatures. Seedling emergence was counted 14 d after planting to estimate germination rates for the treatments. Plants were grown for six weeks and 50 randomly selected plants were tested for endophyte viability as described by Hill and Roach (2009). The experimental design was a randomized complete block with a split plot treatment assignment. Humidity treatments served as whole plots and days of incubation as repeated measures within the whole plot. Replications were performed by randomly selecting one of the three polyethylene containers for each humidity treatment and placing it on separate shelves within the incubator. Analysis of variance was conducted using the PROC MIXED subroutine in SAS in which humidity and time of incubation were considered fixed effects and replication a random effect. Two-way interactions occurred for both the germination and endophyte viability data. Data were sorted with effects of humidity tested within each sampling date, and the effect of sampling data tested within humidity treatments using one-way analysis of variance. Means were separated using a Fishers’ protected least significant difference (LSD) at the 0.05 level of probability.

2.2. Determine whether the response to selection for endophyte persistence is consistent in laboratory and warehouse conditions 2.2.1. Selection process Selection for endophyte viability during seed storage was imposed on two base populations (C0) of ‘Jesup’ tall fescue, one with non-toxic endophyte AR542 and the other with the non-toxic endophyte AR584 as outlined by Hill and Roach (2009). Two C1 populations were generated, one with AR542 and the other with AR584. The C0 populations and those selected for endophyte viability during seed storage (C1) were previously planted and were maintained in isolated space-planted plots at the University of Georgia Plant Sciences farm located near Watkinsville, GA (N: 33◦ 52 20.1”, W: 83◦ 32 35.0”). One-thousand seeds from each of the C1 populations were placed in a dual-chamber Rubbermaid containers, one chamber containing 100 mL of saturated NaCl solution and the other with seed from one of the C1 populations. Seeds were exposed to accelerated aging conditions for 28 d at 30 ◦ C and 76% relative humidity. Seventy seedling plants with viable endophyte from the accelerated aging treatments (C2AA) were space-planted in isolated polycross blocks at the University of Georgia Plant Sciences Farm near the C0 and C1 populations. Seed from all populations were harvested at physiological maturity in June 2011. Seed were separated for uniformity of density using a Seedboro (Des Plains, IL) forced-air density gradient and for size uniformity by sieving through a 1.5mm screen (Newark Wire and Cloth Co., Newark, NJ). Panicle pieces containing more than one floret were removed from the sieved seed using tweezers.

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2.2.2. Population evaluation 2.2.2.1. Two experiments were conducted.

– Testing C0 and C1 populations for endophyte viability in ambient laboratory conditions. This experiment was designed to compare the survival of endophytes from C1 vs C0 at ambient temperatures in the laboratory. Seed from the base C0 and C1 selected populations were stored in cloth bags at ambient temperature (∼21 ◦ C) in the laboratory. Endophyte viability was tested using the seedling grow-out method (Hill and Roach, 2009) every month over a 22 month period. – Testing viability of C0, C1, and C2AA populations when stored under warehouse conditions. The second experiment included evaluation of endophyte survival in all three cycles after storage in commercial conditions in a warehouse. This experiment enabled us to compare the magnitude of gain obtained between C0 and C1 based on selection for endophyte survival at ambient temperatures with the gain we obtained between C1 and C2AA based on selection using accelerated aging. Approximately 0.5 g of the seed from the C0, C1 and C2AA populations were also placed into nylon mesh fabric bags (L’eggs Hosing, Hanesbrands Inc., Winston-Salem, NC). A set of six nylon mesh bags containing the C0, C1, and C2AA populations of Jesup with AR542 and Jesup with AR584 were transported to a Pennington Seed Co. (Madison, GA) warehouse in Lebanon, OR and placed within commercial bags of tall fescue seed. The commercial bags of seed were placed on a pallet and stored in warehouse conditions for 27 months. Sets of the selected populations were retrieved every 3 months by randomly selecting one of the seed bags on the pallet and removing the nylon mesh bags. The nylon mesh bags were transported via overnight shipment to Georgia and tested for endophyte viability using the seedling grow out method. A schematic of the selection and evaluation steps for the populations is summarized in Fig. 1.

2.2.3. Calculating heritability Realized heritability was calculated based on the selection differential ratio (Hill and Roach. 2009) (Eq. (1)). Since selection was applied in this experiment, the ratio of the observed selection response (the change in the mean phenotype between generations) to the observed selection differential (the difference in mean phenotype between the parents selected for breeding) and the overall mean in their generation was used to estimate heritability. Since the experiment involved repeated measures at different time points on the same populations, the means over all the measurements were used in the estimation of realized heritability [Eq. (1)]

H2 =

(␹s2 /␹bp2 − 1) (␹s1 /␹bp1 − 1)

(1)

For estimating the heritability of C1 , ␹s2 is the mean percent of viable endophyte in the C1 population over all months, ␹bp2 is the mean percent viable endophyte in the C0 population averaged over all months at the time of progeny evaluation, ␹s1 is the mean level of viable endophyte in the parents of the selected populations (=100), and ␹bp1 is the mean percentage viable endophyte in C0 at the time of selection. For estimating the heritability of C2 , ␹s2 is the mean percent of viable endophyte in the C2 population over all months, ␹bp2 is the mean percent viable endophyte in the C1 population averaged over all months at the time of progeny evaluation, ␹s1 is the mean level of viable endophyte in the parents of C2 (=100), and ␹bp1 is the mean percentage viable endophyte in C1 at the time of selection.

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Table 1 Seedling emergence of the Jesup AR542 base poplation (C0) after exposure to accelerated aging conditions for various lengths of time. Percent relative humidity during storage Days stored

35 . . . % emergence . . .

50

75

100

LSD (0.05)a

0 7 14 21 28 35 LSD (0.05)

98 89 68 56 27 13 8.1

98 96 96 94.5 93.5 93 N.s.

98 97 94.5 94 94 93 N.s.

98 94 91 83.5 47 13.5 8.2

N.s.b N.s. 6.6 6.9 8.7 8.8

a b

Least significant difference at the 0.05 level of probability. Not different at the 0.05 level of probability.

2.3. Statistical analysis Infection percentages were used to conduct pairwise t-tests for all possible combinations of seed lots within each experiment. Regression analysis was used to describe the relationship between months of storage (independent variable) and endophyte infection of the seedling plants (dependent variable). Equations describing first- through sixth-order regression coefficients were calculated and sequentially evaluated for significance of the regression coefficients. The equation with the most complex coefficient having significance was selected to describe response surface.

2.4. Determining whether selection for endophyte viability resulted in as shift of the agronomic traits of the parent populations Seed from the base and selected populations were planted alongside with a ‘Kentucky-31 ecotype, ‘Au-Triumph’, and ‘Rebel III’ tall fescue at The University of Georgia Plant Sciences Farm located near Watkinsville, GA. The ground was prepared with a single pass of a tandem disk harrow. Each plot was 1.8 × 3.2 m and treatments were assigned to a randomized complete block with four replications. ‘The seed were planted on 1 October 2011 at a rate of 25 kg ha−1 using a Hege Equipment Co. (Colwich, KS) no-till plot planter. Plots were fertilized with 50 kg ha−1 of commercial 10–10–10 NPK fertilizer immediately after planting. No yield data was taken in the spring of 2012 due to volunteer annual ryegrass infestation. The plot area was mowed in late May 2012, the biomass removed from the plot area, and an application of pendimethalin (N-(1-ethylpropyl)-3,4-dimethyl2,6-dinitrobenzamine; Prowl, BASF, Florham Park, NJ, USA) was applied at a rate of 1.2 kg ha−1 to control summer annual grass weeds. Pendimethalin was also applied on or about 1 Oct 2012 and 2013 to prevent annual ryegrass infestation during winter months.

Applications of 60 kg ha−1 of N as urea were applied on or near 1 March 2013 and 2014. Agronomic traits (flowering date, leaf length:width ratio, plant height, dry matter yield) were measured in the spring of 2013 and 2014. Flowering date was determined when 50% of the panicles in the plots had exposed anthers from the flower structures. Plant height and flag leaf length:width ratio were measured at 10 locations within the plot at the time of flowering. Flag leaf length:width ratios were determined by first measuring the flag leaf length followed by the leaf width at the midpoint between the collar and the tip of the leaf. A Swift Machine forage harvester (Swift Current, SK) was used to harvest a 0.7 by 3.0 m swath from the center of the plots immediately after the final tall fescue population expressed flowering (approximately 23 April of each year). Fresh weights were recorded, a 400-g subsample dried at 60 ◦ C to establish dry matter content of the forage, and dry matter yield calculated. Data were analyzed using a PROC MIXED model of SAS. Populations were considered fixed effects and year and replications were considered random effects. There were no population x year interactions and data are reported as the means of the two years. Means were separated using a Fishers protected least significance difference test at the 0.05 level of probability.

3. Results and discussion 3.1. Identify accelerated aging conditions that maintain germination but compromise endophyte viability There was an interaction between humidity and length of incubation for 14-d seed emergence of Jesup tall fescue (Table 1). Emergence was negatively impacted within seven days of exposure to 35% relative humidity and decreased with each 7-d increment thereafter. Decreased emergence of seed exposed to 100% relative humidity occurred after 21 days of exposure and decreased with each 7-d increment thereafter. However, there was no effect of

Table 2 Endophyte survival in Jesup AR542 basae population (C0) after exposure to accelerated aging conditions for various lengths of time. The tall fescue seed contained 98% viable endophyte at time of initial exposure. Percent relative humidity during storage Days stored

35 50 % endophyte infection in 6-week old seedling plants

75

100

LSD (0.05)a

0 7 14 21 28 35 LSD (0.05)

98 95.5 93 33 0 0 8.1

98 96 94 67 20 0 6.4

98 93 83.5 35 11 0 8.2

N.s.b N.s. 8.9 9.6 6.6 0.6

a b

Least significant difference at the 0.05 level of probability Not different at the 0.05 level of probability.

99 99 99 98 98 98 N.s.

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Table 3 Realized heritability estimates for endophyte survival when stored for 22 months in ambient laboratory conditions. Storage conditions Population

Selection Cycle

Laboratory . . . heritability . . .

Warehouse

Jesup 542 Jesup 542 Jesup 584 Jesup 584

C1 C2AA C1 C2AA

0.42 – 0.5 –

0.51 0.54 0.62 0.64

incubation time on seed emergence when seed was exposed to 50 or 75% relative humidity. Maintaining germination and emergence rates is important for plant breeding purposes, so the 50 or 76% relative humidity conditions are good candidates for our studies because they compromised endophyte viability without affecting seed germination. Endophyte viability varied depending upon the length of exposure to the relative humidity conditions (Table 2). Endophyte viability was not different among seed exposed to 50% relative humidity, regardless of the length of time seed were exposed during the experiment. However, all other relative humidity treatments had reduced endophyte viability as the length of exposure to the incubation conditions increased. The only treatment in the experiment which maintained germination and emergence rates over time and compromised endophyte viability was the saturated NaCl solutions that maintained 75% relative humidity during the accelerated aging process (Tables 1 and 2). Thus, using saturated NaCl to maintain 75% relative humidity during the accelerated aging treatments was an acceptable method to induce endophyte death without compromising seed health. Therefore, this treatment combination was used to implement subsequent experiments. 3.2. Determine whether the response to selection for endophyte persistence is consistent in laboratory and warehouse conditions The impetus to develop an accelerated aging process to select for endophyte survival in tall fescue seed was to eliminate the need to store seed for extended periods under ambient laboratory conditions. The C1 populations developed by Hill and Roach (2009) were therefore used to test accelerated aging conditions for endophyte survival. Endophyte survival of the C0 and C1 populations of Jesup with AR542 or AR584 endophytes (Fig. 2A) and heritability estimates of those populations (Table 3) were both similar to that reported by Hill and Roach (2009) when stored in laboratory conditions. This provided us confidence that the effect of environments from different seed production years had little if any effect on the endophyte survival characteristics of the Jesup C0 and C1 populations, regardless of endophyte. Warehouse storage of the C0 and C1 populations of Jesup with AR542 or AR584 resulted in greater realized heritability estimates than when the seed was stored in the laboratory (Table 3). However, the C1 population of Jesup with endophyte AR584 had greater realized heritability estimates than C1 population of Jesup with endophyte AR542 when stored in both laboratory and warehouse conditions. The consistency of the heritability estimate trends in laboratory and warehouse storage conditions is additional evidence that seed from the crossing blocks for the C0 and C1 were not affected by environmental influences associated with different production years. The C2AA populations had greater endophyte survival rates than did the C0 or C1 populations when stored in warehouse conditions (Fig. 2A and 2B). Furthermore, the similarity of the realized heritability estimates for the C1 and C2AA populations in Jesup with

Fig. 2. Endophyte viability of Jesup C0 and C1 populations containing AR542 and AR584 endophytes when stored at ambient conditions in the laboratory (A), and of Jesup tall fescue containing either AR542 (B) or AR584 (C) endophye strains when stored in warehouse conditions in Lebanon, OR.

endphyte AR542 (0.51 and 0.54 for laboratory and AA selection, respectively) and in Jesup with AR584 (0.62 and 0.64 for laboratory and AA selection respectively) suggests that selection under AA was as efficient as the more time consuming laboratory selection method. Selection for endophyte survival affected the regression equations that best described the endophyte survival rate over time (Table 4). The C0 and C1 populations of Jesup with AR542 had quadratic equations that best described their endophyte survival response in warehouse conditions, but their linear coefficients differed from one another. The C2AA population of Jesup with AR542 had a cubic equation that best described the response surface. The regression equations that gave the best fit of the data among the Jesup C0, C1, and C2AA populations with AR584 changed in order from quadratic, to cubic, to quartic, respectively. Variation in regression equations that describe the response of endophyte survival to duration of storage is added evidence that each cycle of selection impacted the endophyte survival rate over time. The coefficients of determination of all equations were >0.90, so the equations were used to calculate the number of months required to reduce endophyte viability to 70%, the mini-

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Table 4 Intercepts, linear, quadratic, cubic, and quartic coefficients and coefficients of determination for regression equations describing how endophyte survived in the base and selected populations of Jesup tall fescue when stored in warehouse conditions for 27 months. . . . Regression coefficients . . . Population

Intercept

†

AR542 C0 AR542 C1 AR542 C2AA AR584 C0 AR584 C1 AR584 C2AA a

Linear

−0.234 −0.25 0.264 −0.268 0.223 −1.007

a

92.8 89.9 95 91.3 94.7 86.3

Coefficient of

Quadratic

2.33 3.33a −1.5 3.46 −1.1 5.65

Cubic

Quartic

determination

−0.002

0.91 0.98 0.99 0.94 0.99 0.99

−0.013 −0.012 0.065

Significantly different from one another at the 0.05 level of probability.

Table 5 Calculated number of months for Jesup tall fescue populations to reach 70% viability based on regression equations for endophytes AR542 and AR584. Cycle of selection of seed populations Endophyte

C0 C1 . . .Months of storage to reach 70% endophyte infection . . .

C2AA

AR542 AR584

16 17.45

19.87 21.87

17.75 19.95

mum standard set by the Alliance for Grassland Renewal (http:// grasslandrenewal.org/qualitycontrol.htm). Each cycle of selection increased the numbers of months it takes for endophyte survival to drop from 93% and above to 70% infection (Table 5). It must be kept in mind that endophyte transmission occurs vertically through the seed and, therefore, maternal effects impact endophyte conditions. Maternal effects affect heritability of other endophyte-mediated traits as well. Ergot and pyrrolopyrazine alkaloids are produced only by the endophytes in tall fescue, but the maternal host influences to what extent the endophyte is capable of producing those chemicals (Agee et al., 1994; Roylance et al., 1994; Adcock et al., 1997; Eason et al., 2002; Eason, 2007 Roylance et al., 1994; Adcock et al., 1997; Eason et al., 2002; Eason, 2007). Thus, the results herein are not unusual nor unprecedented. The high heritability estimates regardless of endophyte screening method indicate that much of the endophyte survival variability in the populations is caused by plant genetic factors (Visscher et al., 2006). The significance of this finding is that populations of tall fescue can be screened using the AA method, a much shorter and more efficient system for screening than the laboratory method (28 days vs. 18 month of incubation). 3.3. Determining whether selection for endophyte viability resulted in as shift of the agronomic traits of the parent populations Jesup is a winter dormant European continental type of forage tall fescue. AU-Triumph, Rebel III and KY-31 cultivars were selected

for comparison with the populations of Jesup because they were used in the plant variety protection (PVP) characterization of the original Jesup release (Bouton et al., 1997). They also have diverse genetic backgrounds. AU-Triumph is a winter-active North African Mediterranean type and Rebel III is a winter dormant European continental tall fescue which had been selected for short stature, short and narrow leaves, and dark green color. Flowering date of all Jesup populations were similar, but there appeared to be a modest endophyte effect. Populations with AR542 flowered two days later than populations with endophyte AR584 (Table 5). Flowering date of Jesup with AR542 and AR584 were 4 and 6 days earlier than KY-31, 19 and 21 days later than AU-Triumph, and 10 and 12 days earlier with Rebel II, respectively. All populations of Jesup had similar plant heights as KY-31 but were taller than Rebel III and AU-Triumph. The flag leaf length:width was were similar among all Jesup populations, but less than AU-Triumph or KY-31. Spring dry matter yields were similar among the Jesup and KY-31 populations, but greater than Rebel III and AU-Triumph. These data were consistent with comparisons for the original Jesup tall fescue release and suggest that selection for endophyte fungus viability during seed storage had no impact on the agronomic characteristics of the populations Table 6. Genetic variability among endophtyte fungal populations appears to be less than that of host plants (Eason, 2007). However, there are endophyte traits that are influenced by plant genetics (Agee et al., 1994; Roylance et al., 1994; Adcock et al., 1997; Eason, 2002 Roylance et al., 1994; Adcock et al., 1997), therefore, selection can be imposed on the plant population to optimize these

Table 6 Field results for plant traits used to determine impact of selection for endophyte persistence on agronomic traits. Values are means of 2013 and 2014. Cultivars used are those included as checks in the PVP data comparison with Jesup original release. Cultivar

Endophyte

Selection

Flowering datea

Plant height (cm)

Flag leaf length: width ratio

Yield (MT ha-1 )

AU-Triumph KY-31 Rebel III Jesup Jesup Jesup Jesup Jesup Jesup LSD (P = 0.05)b

nil wild wild 542 584 542 584 542 584

– – – C0 C0 C1 C1 C2AA C2AA

102 124 132 118 120 118 120 118 120 1

122 143 109 147 144 144 144 141 144 9

25 24.1 – 23.04 23.09 22.68 22.73 22.83 23.06 0.58

8.78 9.49 8.11 9.81 9.55 10.01 9.81 10.04 10.01 0.62

a b

Number of days after January 1. Least significant difference at the 0.05 level of probability.

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traits, such as endophyte survival during seed storage (Hill and Roach, 2009). Methods for inducing endophyte death and screening for survival through subjecting the seed to long-term storage under various temperature and humidity conditions are not efficient enough to enable selection in a practical breeding program (Hill and Roach, 2009; Tian et al., 2013). Therefore, finding conditions that expedite the selection process was the impetus for this project. The collective data herein demonstrate that endophyte survival is a highly heritable trait and that exposing seed to 75% relative humidity for 28 d to expedite the aging process resulted in populations with heritability estimates comparable to long-term ambient aging. The method would allow elimination of plant genotypes with poor vertical transmission of endophyte, or had poor endophyte viability under storage conditions early in the selection process. Using the AA process to screen plant/endophyte combinations will increase the allele frequency of genotypes with longer endophyte survival in the breeding population. Furthermore, agronomic traits that were used to characterize the base population for the PVP and variety registration (Bouton et al., 1997) did not change after selection for endophyte viability regardless of the aging process used. Therefore, the AA parameters identified in this manuscript can be used to improve the efficiency of selection for enhanced endophyte survival in host plants without appreciably affecting agronomic traits of the population. References Adcock, R.A., Hill, J.H., Boerma, H.R., Ware, G.O., 1997. Symbiont regulation and reducing ergot alkaloid concentration by breeding endophyte-infected tall fescue. J. Chem. Ecol. 23, 691–704. Agee, C.S., Hill, N.S., 1994. Ergovaline variability in acremonium-infected tall fescue due to environment and plant genotype. Crop Sci. 34, 224–226. Bouton, J.H., Duncan, R.R., Gates, R.N., Hoveland, C.S., Wood, D.T., 1997. Registration of Jesup tall fescue. Crop Sci. 37, 1011–1012. Bouton, J.H., Latch, G.C.M., Hill, N.S., Hoveland, C.S., McCann, M.A., Watson, R.H., Parish, J.A., Hawkins, L.L., Thompson, F.N., 2002. Reinfection of tall fescue cultivars with non-ergot alkaloid producing endophytes. Agron. J. 94, 567–574. Clay, K., Hardy, T.N., Hammond, A.M., 1985. Fungal endophytes of grasses and their effects on an insect herbivore. Oecologia 66, 1–6. Eason, H.S., Latch, G.C.M., Tapper, B.A., Ball, O.J.P., 2002. Ryegrass host genetic control of concentrations of endophyte-derived alkaloids. Crop. Sci. 42, 51–57. Eason, H.S., 2007. Grasses and Neotyphodium endophytes: co-adaptation and adaptive breeding. Euphytica 154, 295–306. Gundel, P.E., Garibaldi, L.A., Tognetti, P.M., Aragon, R., Ghersa, C.M., Omacini, M., 2009. Imperfect vertical transmission of the endophyte Neotyphodium in exotic grasses in grasslands of the flooding pampa. Microb. Ecol. 57, 740–748, http:// dx.doi.org/10.1007/s00248-008-9447-y Hall, S.L., McCulley, R.L., Barney, R.J., Phillips, T.D., 2014. Does fungal endophyte infection improve tall fescue’s growth response to fire and water limitation? PLoS One 9, e86904, http://dx.doi.org/10.1371/journal.pone.0086904

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