Crop Science 41:570-576 (2001)
© 2001 Crop Science Society of America
PLANT GENETIC RESOURCES
Incidence and Diversity of Neotyphodium Fungal Endophytes in Tall Fescue from Morocco, Tunisia, and Sardinia
S.L. Clementa,
L.R. Elbersona,
N.N. Youssefb,
C.M. Davittc and
R.P. Dossd
a USDA-ARS, Plant Germplasm Introduction and Testing Research Unit, Washington State Univ., Pullman, WA 99164-6402
b Department of Plant, Soil, and Entomological Sci., Univ. of Idaho, Moscow, ID 83843
c Electron Microscopy Center, Washington State Univ., Pullman, WA 99164-4210
d USDA-ARS, Horticultural Crops Research Unit, Corvallis, OR 97330
Corresponding author (slclement{at}wsu.edu)
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ABSTRACT
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There is a premium on having Neotyphodium germplasm available for temperate grass improvement programs because these fungal endophytes present opportunities for developing new grass–endophyte combinations for enhanced tolerance to abiotic and biotic stresses. Unfortunately, surveys have revealed a low incidence of Neotyphodium fungi in grass germplasm collections. This research surveyed tall fescue (Festuca arundinacea Schreb.) accessions from a 1994 Australian–U.S. plant-collection trip to Morocco, Tunisia, and Italy (Sardinia) for viable Neotyphodium fungi and determined whether infected accessions harbor different Neotyphodium genotypes. Conidial measurements of isolates cultured on agar and bioassays of the differential survival of bird cherry-oat aphid [Rhopalosiphum padi (L.)] on infected accessions were used to characterize Neotyphodium diversity. A secondary objective determined the consistency of a polymerase chain reaction (PCR) method to detect Neotyphodium fungi in tall fescue. Neotyphodium was detected in 336 of 439 plants (76.5%) distributed among 104 accessions, of which 99 were endophyte-infected. Mean conidial lengths of 42 isolates ranged from 3.91 to 9.91 µm. Most of the isolates (71.4%) had conidia with mean lengths smaller than the lower limit (6.5 µm) characteristic of the tall fescue endophyte N. coenophialum (Morgan-Jones and Gams) Glenn, Bacon, and Hanlin. In aphid assays, all endophyte-free plants were susceptible to R. padi and all but two infected plants were resistant to this aphid. Thus, a Mediterranean plant-collection trip secured diverse Neotyphodium endophytes in tall fescue for storage in seed banks, and a PCR assay detected Neotyphodium in tall fescue plants of diverse geographical origin.
Abbreviations: PCR, polymerase chain reaction • PDA, potato dextrose agar • SEM, scanning electron microscope • WRPIS, Western Regional Plant Introduction Station
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INTRODUCTION
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PLANT EXPLORATION as a primary mechanism for finding and adding new plant germplasm to ex situ repositories is also a means for collecting microbial germplasm in the form of Neotyphodium Glenn, Bacon, and Hanlin (formerly Acremonium) (Glenn et al., 1996) fungal endophytes of temperate grasses for storage in repositories (Clement et al., 1994). For example, plant exploration missions to North Africa and Sardinia during the early 1990s targeted forage grasses and their associated Neotyphodium endophytes for collection and preservation in seed banks (West et al., 1992; Chakroun et al., 1995; Cunningham et al., 1997). These collecting missions aimed to broaden the genetic diversity of forage grasses and Neotyphodium endophytes in germplasm collections.
There is widespread interest in Neotyphodium endophytes because their presence in temperate grasses is linked to enhanced plant fitness, such as greater drought tolerance and resistance to insect and mammalian herbivores (Clement et al., 1994; West, 1994; Bacon et al., 1997; Latch, 1997). Toxicity to insects and mammals is the result of specific metabolites (alkaloids) produced by the endophytes in association with their hosts (Porter, 1994). A way to overcome the detrimental characteristic of mammalian toxicoses associated with endophyte infection is to produce new grass–endophyte associations with naturally occurring strains of Neotyphodium that do not produce mammalian toxins, but do produce the necessary metabolites for insect resistance and other ecological benefits (Rowan and Latch, 1994; Latch, 1997). For such purposes, having a large diversity of Neotyphodium endophytes available is as important as having a diversity of forage grass germplasm on hand for plant breeding.
Although conserved seed might be expected to supply the necessary pool of Neotyphodium and alkaloid diversity for forage grass and turfgrass improvement programs, surveys of seed banks in Europe and the USA revealed a relatively low incidence of endophyte infection among accessions of Festuca (Latch et al., 1987; Springer and Kindler, 1990; Holder et al., 1994; Siegel et al., 1995; Oliveira and Castro, 1997), Lolium (Latch et al., 1987; Wilson et al., 1991a; Siegel et al., 1995; Lewis et al., 1997), and Hordeum (Wilson et al., 1991b) grass species. On the other hand, Neotyphodium endophytes inhabit wild Festuca and Lolium growing in their Palearctic centers of origin (Lewis and Clements, 1986; Riccioni and Piano, 1994; Do Valle Ribeiro et al., 1996; Lewis et al., 1997; Naffaa et al., 1998) and some wild Triticum endemic to Turkey (Marshall et al., 1999). Thus, newly collected seed of wild grasses should harbor diverse endophytes to bolster holdings of this microbial germplasm in seed banks.
The primary objectives of this research were (i) to survey tall fescue accessions collected during a 1994 Australian–U. S. plant-exploration trip to North Africa (Morocco, Tunisia) and Italy (Sardinia) for viable Neotyphodium endophytes and (ii) to survey infected accessions in this new collection for the presence of different Neotyphodium genotypes. A secondary objective determined the consistency of a PCR assay to detect Neotyphodium endophytes in tall fescue of diverse geographical origin.
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MATERIALS AND METHODS
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Plant and Endophyte Material
The senior author arranged to have the seed collected in North Africa and Sardinia and bound for the USA expeditiously processed through the USDA-ARS Plant Quarantine Facility, Beltsville, MD, and shipped to the USDA-ARS Western Regional Plant Introduction Station (WRPIS), Pullman, WA. This was necessary because a lengthy interval between the collection of endophyte-infected tall fescue seed and deposition of the seed in a repository can reduce endophyte viability (Oliveira and Castro, 1997). The collecting trip ended on 30 July 1994 and seed arrived 19 August 1994 in Pullman, WA, where it was placed in the WRPIS seed bank (4°C, 30% relative humidity, -10°C dew point) on 22 August 1994. This seed was transferred to a storage freezer (-88°C) on 20 October 1994. The material included 126 accessions of tall fescue. Seed of each accession was collected from 50 to 100 plants in North Africa and Sardinia, which was bulked before being divided among the participants on the collecting mission and shipped to seed repositories (Cunningham et al., 1997).
For this study, a small sample of the original seed (n = 5) from each of 104 tall fescue accessions (Table 1) was selected. Seeds were placed in water-saturated vermiculite in covered 11- by 11- by 3.5-cm plastic boxes and placed in a seed germinator at 25/15°C and 16 h light/8 h dark. Newly germinated seeds were planted individually in 15-cm pots containing a commercial soil mix from which three to five seedlings grew per accession. Greenhouse-grown (15–30°C; natural lighting) plants received 0.24 g of Peters (20-20-20) fertilizer (Grace-Sierra Horticultural Products Co., Milpitas, CA) in 400 mL of H2O every 2 wk before the endophyte infection status of each 3- to 5-mo-old plant was determined in March and April 1995. Estimates of Neotyphodium infection percentages in this new tall fescue collection could not be improved by examining more accessions or more plants per accession because only small quantities of original seed were available for this study.
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Table 1. Neotyphodium infection status of tall fescue germplasm collected in North Africa and Italy (Sardinia), 1994
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Isolation and Identification of Neotyphodium Fungi
The endophyte status of each plant was determined by isolating Neotyphodium fungi on potato dextrose agar (PDA) with streptomycin sulfate and penicillin G (0.10 g of each per 1 L of PDA). Basal stem sections (1–2 cm) from four young tillers were disinfected in a solution of 0.525% sodium hypochlorite plus 8 drops of Tween-20 (per 100 mL) for 90 s and placed on PDA in petri dishes (100 by 15 mm). The sealed petri dishes were incubated at 22°C ± 2°C on a laboratory bench (diffused fluorescent light, 10–12 h light/12–14 h dark) and examined for mycelial growth from plant tissue at 2- to 3-d intervals for 8 wk. A plant was scored endophyte-free if Neotyphodium mycelia did not appear after 8 wk. This isolation procedure also was necessary to obtain Neotyphodium colonies for scanning electron micrographs of conidia. Although this procedure can lead to the growth of contaminant fungi, thereby making isolation of Neotyphodium fungi difficult (Bacon and White, 1994), we experienced few problems with contaminants in this study. We attribute this to the selection of multiple stem sections from four young tillers per plant, good surface disinfection of samples, the addition of antibiotics to PDA, and the removal of contaminant colonies from plates as they appeared.
In 1996, 42 isolates representing the range of Neotyphodium colony types on PDA were characterized by measuring the lengths and widths of 20 conidia per isolate using a scanning electron microscope (SEM). The lengths and widths of conidia help differentiate Neotyphodium strains and possibly different species in tall fescue (Christensen and Latch, 1991), and conidial size may reflect the genome sizes of Epichloe and Neotyphodium species (Kuldau et al., 1999). Scanning electron micrographs were prepared using mycelial plugs from cultures as described by Clement et al. (1997). Briefly, the plugs were fixed in 2% glutaraldehyde/2% paraformaldehyde (buffered in 0.1 M Pipes buffer), dehydrated in an ethanol series, and critical-point dried. The plugs were then affixed to SEM mounts using double sticky carbon tape and sputter coated with gold.
The identity of Neotyphodium fungi was confirmed with published descriptions of colonies on agar and of conidia (Morgan-Jones and Gams, 1982; Latch et al., 1984; White and Morgan-Jones, 1987). However, we did not group isolates into strains, species, or distinct genotypes because the taxonomic identity of Neotyphodium fungi and other endophytic Clavicipitaceae is an unsettled question (White, 1997; Cabral et al., 1999). Moreover, it would be premature to assign these endophytes to specific species without more research, such as rDNA sequence analysis (Schardl et al., 1991). Herein, isolates are grouped into two conidial morphotypes: (i) those that produce short conidia with mean lengths smaller than the lower limit (6.5 µm) reported by White and Morgan-Jones (1987) for the tall fescue endophyte N. coenophialum, and (ii) those that produce long conidia (mean lengths >6.5 µm) characteristic of N. coenophialum. The use of morphotypes to group Neotyphodium isolates from a single host plant species was used by Schulthess and Faeth (1998).
Aphid Assays
Christensen and Latch (1991) reported that Mediterranean tall fescue populations harboring diverse strains of Neotyphodium endophytes differed in their ability to resist feeding and colonization by the bird cherry-oat aphid, Rhopalosiphum padi (L.) (Homoptera: Aphididae). We conducted three assays to test the hypothesis that any differential response of this aphid to infected plants from North Africa and Sardinia would reflect the presence of different Neotyphodium genotypes in this germplasm.
Aphids were obtained from a laboratory colony reared on ‘Stevens’ wheat, Triticum aestivum L., in a growth chamber at 21°C ± 2°C, 14 h light/10 h dark, and 200 µmol m-2 s-1 photosynthetic photon flux density. This colony was initiated with one R. padi nymph collected on an endophyte-free tall fescue plant in May 1997. Assays were conducted in a separate growth chamber under identical conditions.
Plants that provided experimental material for assays were grown from original seed germinated in a controlled-environmental chamber (25/15°C and 16 h light/8 h dark). Germinated seeds were sown in separate 15-cm pots containing a commercial soil mix and maintained in a greenhouse (natural light; 16–26°C) and fertilized biweekly with 0.24 g of Peters (20-20-20) in 400 mL of H2O. To simplify terminology, these plants will be referred to as source plants. Before each assay, and when source plants were at least 8 wk old, the endophyte infection status of each was determined by isolation on PDA from basal stem sections as described above. Once the infection status of these plants was established, PCR was carried out by extracting DNA from basal stem sections of four tillers of each source plant. Procedures were identical to those of Doss et al. (1998) except that an annealing/reaction temperature of 62°C for 30 s was used instead of 60°C for 1 min. This PCR method uses tub-2 primers specific for detection of Neotyphodium endophytes in tall fescue accessions of diverse geographical origin (Doss et al., 1998). Personnel carrying out PCR were unaware of the endophyte status of each plant.
Each 7-mo-old infected and endophyte-free source plant was split to obtain 10 vegetative propagules, each with two tillers, for rooting in plastic cone-tainers (Ray Leech Cone-tainers, Canby, OR) (40 mm at the top tapering to 28 mm at the bottom) containing the commercial soil mix. Cone-tainers were placed in holding racks positioned over metal trays filled with water in a greenhouse (natural light; 16–26°C). After 3 to 5 d, each clone received 0.006 g of Peters (20-20-20) fertilizer in 10 mL of H2O. Two weeks after transplanting to cone-tainers, each clone was clipped to leave one vigorously growing tiller and allowed to grow for another 7 to 10 d under greenhouse conditions. Five of the most vigorously growing clones of each source plant were then moved to a growth chamber where each was infested with 20 adult apterous R. padi. Aphids were transferred with a camel's-hair brush to the base of each plant. Clear plastic vented tubular cages (350-mm diam by 300-mm height), capped with nylon organdy screen, were tightly inserted into cone-tainers to confine the aphids.
The experimental propagules for assays 1, 2, and 3 were cloned from 10 (6 endophyte-infected, 4 endophyte-free), 7 (5 infected, 2 endophyte-free), and 15 (10 infected, 5 endophyte-free) source plants, respectively. These source plants represented plants of ‘Tribute’ tall fescue (controls), five Sardinia accessions (Assay 1), four Tunisia accessions (Assay 2), and eight Moroccan accessions (Assay 3). Separate Tribute tall fescue plants, one infected with N. coenophialum and one without this endophyte species, provided infected and endophyte-free clones (controls) for each assay. The presence of N. coenophialum in tall fescue adversely affects the survival of R. padi (Latch et al., 1985; Eichenseer and Dahlman, 1992). Infected source plants of North African and Sardinian accessions were selected on the basis of their harboring diverse Neotyphodium genotypes as reflected by variation in the lengths of conidia from agar cultures. In each assay, plants in cone-tainers were arranged in a completely randomized design and the number of live aphids on each plant was recorded after 10 d.
Statistical Analysis
Conidial length and width data and aphid assay data were analyzed by a simple statistical procedure using confidence intervals (Steel and Torrie, 1960). Conidial length means and aphid count means with overlapping confidence intervals (95%) were assumed to be similar. This is equivalent to conducting a t-test to compare specific means (Steel and Torrie, 1960). A nested ANOVA (accessions nested within country, replicates within accession) using the GLM procedure in SAS (SAS Institute, 1987) was performed to test for the effect of location (country) on conidial length.
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RESULTS AND DISCUSSION
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Accession-infection frequencies were high with rates of 85.7% and 98.2% for collections from Tunisia (n = 28 accessions) and Morocco (n = 56), respectively. All 20 of the Sardinian accessions harbored Neotyphodium fungi, as did 15 Sardinian populations examined earlier by Riccioni and Piano (1994). Five accessions, one from Morocco and four from Tunisia, produced Neotyphodium-free plants. The percentage of infected plants varied, with rates of 89.3%, 68.6%, and 57.5% for plants from Morocco (n = 233), Sardinia (n = 86), and Tunisia (n = 120), respectively (Table 1). These plant-infection percentages, and the accession-infection percentages for Tunisia and Morocco that include endophyte-free accessions, should not be considered absolute because they are based on examinations of three to five plants per accession. Moreover, we do not know if the seed that produced the endophyte-free plants originated from an uninfected plant or from an infected plant where endophyte transmission to seed was less than 100%. Our findings are consistent with reports that Neotyphodium endophytes are widely distributed in wild populations of cool-season perennial grasses in Europe. In addition, these results show that Cunningham et al. (1997) fulfilled their objective of collecting Neotyphodium genetic resources for conservation in seed banks.
Populations of Neotyphodium-infected tall fescue were distributed over a wide range of altitudes, rainfall classes, and soil conditions (Cunningham et al., 1997). Alkaline (Morocco, Tunisia) and acid (Sardinia) soils supported infected populations (Table 2).
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Table 2. Characteristics of locations in North Africa and Italy (Sardinia) supporting populations of Neotyphodium-infected tall fescue
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Non-overlap of 95% confidence intervals around the means for conidial lengths of 42 isolates (means from 3.91–9.91 µm) suggests that Neotyphodium endophytes in North African and Sardinian tall fescue are a heterogeneous group (Table 3). Mean conidial widths among the 42 isolates also differed (data not shown). Most (81.3%) of the isolates from 32 Morocco and Tunisia accessions produced short conidia, while most (60%) of the isolates from 10 Sardinian accessions produced long conidia (Table 3). Cunningham (1996) is the source of the Australian accession numbers in Table 3. Overall, 71.4% of the isolates had conidia with mean lengths shorter than the lower limit (6.5 µm) for N. coenophialum. Christensen and Latch (1991) also found wide variation among 18 Neotyphodium isolates (mean conidial lengths ranged from 5.5–11.9 µm) from tall fescue plants of diverse origin (Europe, USA, Australia, New Zealand, and Algeria). However, most of their isolates could be accommodated by the taxon N. coenophialum with conidia 6.5–13 µm (White and Morgan-Jones, 1987).
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Table 3. Variation in conidial lengths of 42 Neotyphodium isolates from selected Festuca arundinacea accessions of diverse origin
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Scanning electron micrographs of conidia (Fig. 1)
illustrate the morphological diversity of Neotyphodium fungi in tall fescue accessions from Morocco, Tunisia, and Sardinia. This diversity is also reflected by the variation in mean conidial lengths of isolates from plants from each of the three countries, where lengths averaged 6.72 (Sardinia), 5.69 (Morocco), and 4.93 µm (Tunisia). These means, while not significantly different (F = 2.93; df = 2, 39; P = 0.065), suggest a greater propensity for isolates from Moroccan and Tunisian tall fescue to produce shorter conidia than isolates from Sardinian tall fescue. Interestingly, the isolates with the shortest conidia (means 5.5–6.0 µm) in Christensen and Latch's (1991) study were from Algerian tall fescue. Thus, the collective evidence suggests that the production of short conidia is a general characteristic of Neotyphodium isolates from North African tall fescue.
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Fig. 1. Scanning electron microscope photomicrographs of conidia of Neotyphodium isolates from tall fescue accessions W6 15860 (Morocco) (A), W6 16139 (Sardinia) (B), W6 16206 (Sardinia) (C), and W6 16079 (Tunisia) (D). Scale bars = 3 µm
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In aphid assays, N. coenophialum-infected controls were resistant to R. padi and endophyte-free controls were susceptible to aphid reproduction and development (Table 4). In assays involving experimental clones from Sardinian (Table 4) and Moroccan (data not shown) accessions, all infected materials were resistant and all endophyte-free materials were susceptible to R. padi. However, non-overlap of 95% confidence intervals of mean aphid counts showed that one endophyte-free accession (16206-1 in Assay 1) was less suitable for R. padi reproduction than other endophyte-free accessions (Table 4). A plant genetic component may be responsible for the low susceptibility of this accession to R. padi. Markedly different results were obtained in the assay involving Tunisian accessions (Assay 2), with R. padi reproduction and development unaffected by Neotyphodium infection in two (16058-1 and 16059-4) out of four infected accessions. Indeed, aphid densities on 16058-1 were similar (based on overlap of 95% confidence intervals) to densities on the two noninfected entries (C9 control and 16079-5) in the assay. Based on non-overlap of 95% confidence intervals, the infected accession 16059-4, while supporting R. padi reproduction, was less suitable as a host than was 16058-1, C9, and 16079-5 (Table 4). These results involving R. padi and Tunisian accessions further substantiate the presence of diverse Neotyphodium endophytes in Mediterranean tall fescue. Although the experimental clones from source plants 16058-1 and 16059-4 were not examined for Neotyphodium infection, it seems apparent they harbored the endophyte because the presence of the fungus in 100% of the tillers (n = 10) of these source plants was verified via isolation on PDA (Clement, unpublished data, 1999).
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Table 4. Means of bird cherry-oat aphid on replicate clones of Neotyphodium-infected and noninfected tall fescue plants of diverse origins
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Contrary to Christensen and Latch's (1991) finding, we detected no correspondence between the small conidial size of Neotyphodium isolates and R. padi resistance (Table 4). Although infected plant samples were not analyzed for alkaloids in this study, it is possible that variation in the type and concentration of alkaloids was responsible for the differential survival of R. padi on infected tall fescue accessions. Support for this hypothesis comes from Christensen et al. (1993), who found that alkaloid profiles varied considerably among natural populations of tall fescue harboring different Neotyphodium strains. Moreover, the survival of R. padi on endophyte-infected tall fescue is correlated with the presence of specific alkaloids (Siegel et al., 1990).
The PCR method confirmed the results from PDA culture. All 6, 5, and 10 of the endophyte-infected source plants in Assays 1, 2 (Fig. 2)
, and 3 (results not shown), respectively, yielded the expected 444 base pair amplification product denoting tall fescue infection with Neotyphodium endophyte. This diagnostic amplification product was not detected with samples from Neotyphodium-free source plants. In the study by Doss et al. (1998), the Sardinian accession 16206 was scored endophyte-free on the basis of the PCR method; however, this accession was correctly identified as infected using the PCR procedure in this study (16206-5 in Fig. 2). The reason for the earlier false negative is unknown.
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Fig. 2. Ethidium bromide-stained agarose gel showing 444 base pair (bp) amplification product obtained after polymerase chain reaction with tubulin-2 primers and DNA from Neotyphodium-infected and Neotyphodium-free plant tissue from Sardinia (panel A) and Tunisia (panel B) tall fescue accessions. Tall fescue accession and plant number designations are indicated above lanes (for example, accession 16153 and plant number 3 as 16153-3). Molecular weight standards are in the leftmost lane. C-16 and C-8 (panel A) and C-16 and C-9 (panel B) represent plants of a commercial tall fescue line (cv. Tribute). A Neotyphodium-specific band is evident for each of the endophyte-infected plants, namely C-16, 16153-3, 16206-5, 16139-5, 16150-5, and 16188-4 in panel A, and C-16, 15978-1, 16058-1, 16059-4, and 16079-7 in panel B. The absence of bands in lanes for C-8, 16206-1, 16150-1, and 16188-5 in panel A, and C-9 and 16079-5 in panel B reflects the endophyte-free status of these plants
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Preserving this new Neotyphodium germplasm is important because it may provide an expanded pool of endophyte and alkaloid diversity needed for developing new endophyte-tall fescue combinations for specific needs such as pest resistance and livestock production. Indeed, as previously mentioned, different alkaloid profiles are likely to be associated with this new collection of Neotyphodium-infected tall fescue. Original and regenerated seed of this collection is stored at -88°C at the WRPIS. Storage at this temperature will likely retain endophyte viability because survival of Neotyphodium fungi in seed of tall fescue stored for 8 yr at 4°C and -196°C has been excellent (Clement, unpublished data, 1998). Storage of endophyte-infected seed at high temperatures (>20°C) and high seed moisture content (>15%) will adversely affect endophyte survival (Welty et al., 1987).
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ACKNOWLEDGMENTS
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We thank V. Nebling and S.-R. Kuy for technical assistance and M. Evans for advice on data analysis. Gratitude is expressed to V. Bradley, F. Dugan, M. Evans, R. Hannan, R.C. Johnson, K. Reed, and N. Robertson for manuscript review. Plant and microbial germplasm used in this research was collected in cooperation with scientists at the Institute National de la Recherche Agronomique de Tunisia (INRAT) in Tunisia, the Institut National de la Recherche Agronomique (INRA) in Morocco, and the Centro di Studio sui Pascoli Mediterranei (CNR) in Sardinia. W. Graves is acknowledged for expediting the introduction of endophyte-infected grasses into the U.S. National Plant Germplasm System.
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NOTES
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Mention of trademark or proprietary product does not constitute a guarantee or warranty by the USDA and does not imply its approval over other suitable products.
Received for publication January 11, 2000.
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