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PLANT GENETIC RESOURCES

Occurrence of Fungal Endophytes in Species of Wild Triticum

^a Texas A&M University Research & Extension Center, 17360 Coit Rd., Dallas, TX 75252-6599 USA
^b Ankara Plant Protection Research Institute, 06172 Yenimahalle, Ankara, Turkey
^c Texas A&M University Research & Extension Center, P.O. Box E, Overton, TX 75684 USA

d-marshall{at}tamu.edu

	ABSTRACT

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Seedborne, nonpathogenic, fungal endophytes are commonly found in symbiotic relationships with many members of the cool-season grass subfamily Pooideae. The beneficial effects on plants possessing fungal endophytes, and the detrimental effects on consumers of fungal endophyte-infected plants are widely known. The objective of our research was to determine if fungal endophytes exist in indigenous, wild Triticum (wheat) species from Turkey. From the Triticum species collected, we found two different fungal endophytes. Fungi identified morphologically as members of the genus Neotyphodium were found in the diploid Triticum species T. dichasians (Zhuk.) Bowden and T. tripsacoides (Jaub. & Spach) Bowden. The second endophyte, an Acremonium species, was found in T. columnare (Zhuk.) Morris & Sears, T. cylindricum Ces., T. monococcum L., T. neglecta Morris & Sears, T. recta Morris & Sears, T. triunciale (L.) Raspail, T. turgidum L. , and T. umbellulatum (Zhuk.) Bowden. No fungal endophytes were found in T. kotschyi (Boiss.) Bowden, T. ovatum (L.) Raspail, T. peregrinum Morris & Sears, T. speltoides (Tausch) Gren. ex Richter, and T. tauschii (Coss.) Schmal., although the number of samples tested was small for some of these species. Both Acremonium endophyte-infected and Acremonium endophyte-free plants of T. triunciale were found to occur at different frequencies at four collection sites on the Anatolian Plateau. Through two selfed generations of the plants, it was found that the Neotyphodium endophyte was transmitted to 100% of the progeny of T. dichasians and T. tripsacoides. However, the Acremonium endophytes were not transmitted in all plants that originally possessed them. We concluded that fungal endophytes of the genera Neotyphodium and Acremonium inhabit some wild wheat species grown indigenously in Turkey. These endophytes may influence the ecology and distribution of Triticum species, and may also serve as a source of biological control agents of pests or abiotic stress factors in wheat.

	INTRODUCTION

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TO ECONOMICALLY FEED an increasing world population, it is important that food production be increased while the cost of producing the food be decreased. The strategic use of naturally occurring organisms to control pest populations and increase production of major crops represents a viable option to host-plant resistance and pesticide-based pest control. One group of biological control agents that provide a source for novel pest control are the mutualistic fungal symbionts belonging to the genus Epichloë (Clay, 1989). These fungi, in association with their host grass plant, produce a range of deterrence to various insects and some plant diseases (Latch, 1993). In addition, improved growth and drought tolerance are characteristic of some plants possessing fungal endophytes (West, 1994). The basis of much of the pest deterrence in grasses possessing endophytes is the production of alkaloids by the endophytes (Siegel et al., 1991). Endophyte-infected grasses have caused toxicity-related problems in livestock, such as cattle, sheep, and horses, that graze on the infected pastures (Hoveland, 1993). Humans ingesting food products derived from endophyte-infected grasses probably would also suffer similar toxicity, because the fungal endophytes involved are relatives of the sclerotial (ergot) forming Clavicipiteae, whose toxicity effects are widely known (Groger, 1972). Thus, if fungal endophytes are to be safe and effective biological control agents of food crops, the means must be found to either eliminate any potentially harmful toxic compounds or to select toxic-specific fungal strains.

The systemic, seedborne, nonpathogenic, fungal endophytes of most interest as biological control agents belong to the genus Neotyphodium Glenn, Bacon, Price, and Hanlin (formerly Acremonium section Albolanosa Morgan-Jones and Gams) (Glenn et al., 1996). These fungi are conidial anamorphs of Epichloë spp. (Persoon:Fries) Tulasne (Schardl and Phillips, 1997). Another group of fungal endophytes of grasses that have been identified are the p-endophytes, which as a group are closely related to each other, and have been found to sometimes coexist in plants with Neotyphodium endophytes (An et al., 1993). However, the biology and ecology of the p-endophytes are relatively unknown. Fungal endophytes belonging to the genus Acremonium, such as A. chilense Morgan-Jones, White, and Piontelli (1990), could represent a third endophytic grouping based on the apparent unrelatedness to the p-endophytes and Neotyphodium.

In the Gramineae family, fungal endophytes have been found in several tribes of the cool-season grass subfamily Pooideae. The most intensively studied are from the Poeae tribe in the genera Festuca, Lolium, and Poa (Bacon, 1995). Other grass tribes having members that possess fungal endophytes include the Bromeae (White, 1987), Stipeae (White and Morgan-Jones, 1987; Bruehl et al., 1994; Kaiser et al., 1996), Meliceae (White, 1987), Aveneae (White, 1994), and Triticeae (Wilson et al., 1991). In the Triticeae tribe, fungal endophytes have been found in Elymus (White, 1987) and Hordeum (Wilson et al., 1991). A member of the Triticeae tribe of paramount importance to global food resources is the genus Triticum, which contains the wild and cultivated forms of wheat. Cultivated wheat has been found to be free of beneficial, seedborne, nonpathogenic fungal endophytes (Marshall, 1991, unpublished data). However, by examining seeds of Triticum species from the USDA National Small Grains Collection, we observed some evidence of fungal hyphae associated with the aleurone cells of some accessions but were never able to find endophytic fungal hyphae in living plants, nor were we able to isolate endophytic-fungal cultures (Marshall, 1991, unpublished data). We then hypothesized that endophytic fungi may be found in freshly collected seed of wild Triticum from indigenous habitats. We selected Turkey as the collection area because more diverse species of naturally occurring Triticum exist within Turkey's borders than any country (Kimber and Feldman, 1987). In the research presented here, we have used the classification of Morris and Sears (1967) that combines the two former genera Triticum and Aegilops, into the single genus Triticum.

	Materials and methods

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Plant Material
Seeds of Triticum species as well as other cereals and grasses were collected by the authors during July 1992 from more than 80 sites in Turkey, mainly on the Anatolian Plateau, ranging from approximately 36°30' to 41°30' N and from 30° to 35° E. At each site, ripe or near-ripe seeds were collected into envelopes, and assigned an accession number. Additional data recorded at each site included collection date, geographic location, altitude, and a general description of the physical environment of the site. Seeds from other indigenous cereals and temperate grasses were also collected at each site. A complete, descriptive listing of the accessions collected can be obtained from the senior author.

Microscopic Detection, Isolation, and Taxonomic Grouping of Endophytic Fungi
Initial screening for the detection of fungal endophytes was done by squashing 15 to 20 seeds of each accession and examining the aleurone layer and adjoining seed coat for fungal hyphae. At first, the seeds were soaked in 5% NaOH for 16 h at room temperature, washed thoroughly in sterile deionized water, and stained for 36 to 48 h in 5% aqueous ethanol, rose bengal (Saha et al., 1988). Later, we found it more expedient to detect endophytic fungal hyphae in squashed seeds by following the sodium hydroxide soaking and water washing with a 60- to 90-s boiling in 0.4% aqueous aniline blue stain. After staining, individual seeds were placed on a microscope slide in a drop of 0.2% aqueous aniline blue stain, squashed under a cover slip, and observed microscopically for fungal hyphae.

In addition to seed examination, leaf sheaths from living plants were also examined for endophytes. Here, seeds were surface sterilized in 50% aqueous sodium hypochlorite (NaOCl) for about 5 min, rinsed three times in sterile water, then planted into steam-sterilized potting medium in a greenhouse. After 4 to 5 wk, a leaf sheath was removed, and an epidermal strip from the inside surface of the sheath was placed in a drop of 0.2% aniline blue on a microscope slide, covered with a slip, passed briefly over a flame, and microscopically observed. Leaf sheaths were examined in a minimum of ten plants from each accession.

For endophyte isolation, several methods of seed decontamination were tested (White et al., 1993; Bacon, 1988; Bacon and White, 1994); however, these techniques were inconsistent for subsequent seed germination or adequate decontamination. A technique that was more effective in removing contaminants while maintaining seed and endophyte viability was to surface sterilize dry seeds by placing them in porous tissue paper and suspending the seeds over a mixture of full-strength bleach (6.25% NaOCl; 100 mL) with 5 mL of HCl in a desiccator placed in a fume hood. This technique produces chlorine gas, to which the seeds were exposed for about 2 to 3 h prior to plating onto Difco malt extract agar. Endophytic fungi were visible from the seed of some species after 6 d, while other endophytes took nearly 3 wk to grow out. Endophytic fungi were also isolated from leaf sheath and stem material. Plants were grown in the greenhouse as mentioned above. After 4 to 5 wk of growth, some sheath and stem material was removed from the plants and surface decontaminated by chlorine gas (as mentioned above), or by the method described by Bacon and White (1994).

Classification of the endophytes into taxons was based on phialide and conidia morphology, and growth in culture. All of the endophytes classified as Neotyphodium had solitary phialides, arising from aerial hyphae, with no basal septa. The conidia were fusiform in shape and >10 mm in length. All those endophytes classified as Acremonium produced small conidia (2–6 mm in length), which were collected in slimy heads.

Multiple Generation Testing
Plants possessing endophytes were designated E+, and those plants lacking endophytes were designated E-. In order to determine if the endophytic fungi observed in the originally collected seeds were transmitted through successive generations, seeds from both E+ and E- plants were collected from the original self-pollinated plants that had been grown in the greenhouse for fungal detection and isolation. Some of the selfed seeds were squashed and examined for fungal endophytes as described above. Other selfed seeds were decontaminated by chlorine gas, germinated on 3% water agar plates, then planted into a sterilized soil mix and placed in a growth chamber set at 10-h days of 23°C and 14-h nights of 18°C for 3 to 4 wk. Leaf sheath samples were taken for endophyte detection and isolation as described above. Plants were vernalized at 3°C for 4 wk, then transferred to the greenhouse until the plants were mature, and selfed for a second generation. Seeds produced from the second generation of selfed plants were processed and examined for endophytes in the same manner as the first generation seeds. All of the species tested over multiple generations were self-pollinated, except for T. tripsacoides, which required cross-fertilization. Here, we allowed four E+ plants from each T. tripsacoides accession to intermate for each of the two generations tested. The multiple generation testing for fungal endophytes in Triticum species was conducted during a 4-yr period of time from 1993 to 1996.

	Results

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Endophyte Occurrence
Of the 787 cereal and grass accessions collected, 411 belonged to the genus Triticum. The remaining accessions belonged to genera of other cereals (Avena, Hordeum, and Secale) and some temperate grasses (Agropyron, Bromus, Dactylis, Digitaria, Festuca, Lolium, Poa, and Taeniathrum). Within Triticum, we collected 16 species, 10 of which were E+ for hyphae detected in seeds and plants (Table 1) . From the growth characteristics of the hyphae in the plants, and subsequently, from fungi isolated from within the plants, we identified two different endophytes. The first was a Neotyphodium species that exhibited convoluted hyphae in association with the seed aleurone cells and in the leaf sheath. Isolates of Neotyphodium were slow growing and produced solitary conidia that were reniform in shape and >10 mm in length. The second type was an Acremonium species that generally had straight-shaped hyphae in seeds and plants. The Acremonium produced cylindrically shaped conidia, which varied from 2 to 6 mm in length, and which collected in slimy heads in culture.

All of the Triticum species collected had some accessions that were endophyte-free (Table 1). However some of the species that were sparsely collected, such as T. columnare, T. dichasians, and T. recta, had high percentages of accessions that were E+ (Table 1). Two of the more numerously collected species, T. cylindricum and T. monococcum, each had about 64% E+ accessions. At all collection sites, we observed no evidence of "choke" symptoms (stromata) on any of the Triticum species in nature. Similarly, no choke symptoms were found on any of the plants grown in the greenhouse.

Endophyte Distribution in Triticum triunciale
The most numerous and widely distributed species collected was T. triunciale. Across all collection sites, we found that 53% of the accessions of T. triunciale were E+ with the Acremonium endophyte. We did not find the Neotyphodium endophyte in T. triunciale (Table 1). At four collection sites, populations of T. triunciale were particularly numerous, and we determined the percent of the plants that contained endophytes at each of the four locations. At Afyon and Gölbai, the total percentage of E+ plants was similar (54% at Afyon and 52% at Gölbai) (Table 2) . However, at Kalecik, we found that 76% of the T. triunciale plants were E+ and only 24% were E-. A shift toward endophyte-free plants occurred at Kaman, where 40% of the plants were E+ and 60% were E- (Table 2).

	Discussion

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This study represents an exploratory survey with a limited sampling of wild Triticum species and may not be representative of a more detailed study of the species. Obviously, the biological, genetic, and environmental history of a location have had their cumulative effects on their present status, and therefore, the status of the seed collected. Thus, an in situ study of endophyte persistence is needed, as it would aid in understanding the role these endophytes play in Triticum ecology, evolution, and diversity. We did not find endophytes in the accessions of collected cultivated wheat (T. aestivum). Possibly, fungal endophytes have inadvertently been removed from cultivated wheat because of selection against adverse human effects, poor seed storage conditions, or the use of wild species principally as pollen donors in crosses. Similarly, Latch (1987), examined Lolium and Festuca grasses from their center of origin in Italy and southern France, and found endophyte infection in 86% of the plants, whereas commercial lines from the same area had just 19% infection. Thus, endophyte-free ryegrass and fescue plants have only become common since the development of modern agricultural practices and the storage of seed prior to planting (Latch, 1987).

Within the tribe Triticeae, seedborne nonpathogenic fungal endophytes of the genus Neotyphodium have been found in the genera Elymus (White, 1987) and Hordeum (Wilson et al., 1991). Our study is the first accounting of the Neotyphodium endophyte in the genus Triticum. Even though some of the Triticum species we observed were represented by few accessions, a total of six diploid, eight tetraploid, and two hexaploid species were examined (Table 1). Neotyphodium endophytes were found only in the diploid species, T. dichasians and T. tripsacoides. The affinity of the Neotyphodium endophytes from T. dichasians and T. tripsacoides to those from other grasses has yet to be elucidated. The diploids T. monococcum and T. umbellulatum contained the Acremonium endophyte, but not Neotyphodium. Two other diploids, T. speltoides and T. tauschii, appeared to be free of fungal endophytes. Within the tetraploids, T. columnare, T. cylindricum, T. neglecta, T. triunciale, and T. turgidum contained the Acremonium endophyte. However, three other tetraploid species, T. kotschyi, T. ovatum, and T. peregrinum were apparently endophyte-free. The only wild hexaploid collected, T. recta contained the Acremonium, but not the Neotyphodium endophyte. This indicates that the genome size of the plant may have an effect on the type of symbiotic association established with fungal endophytes. The genome type may also have an effect. The endophytes were found in all the collected species containing the C genome, whereas plants from S genome species did not contain endophytes (Table 1). An endophyte–wheat genome relationship could lend evidence as to which parent was female when hybridization occurred in the evolution of Triticum polyploids, but more plants would need to be collected and analyzed in order to get a better estimate of the effect of genome type and ploidy level.

Seedborne transmission of the Acremonium endophyte decreased somewhat during two generations of testing. On the average across all species, 84% of the original plants that were infected with the Acremonium endophyte transmitted the endophyte on to the initial progeny. From the initial progeny to the next generation, the Acremonium endophyte was transmitted to an average of 95% of the plants. However, the Neotyphodium endophyte was always transmitted to the progeny of Neotyphodium E+ plants. This may indicate that the relationship of the Acremonium endophyte to the plant host is not as dependent as is the Neotyphodium–Triticum relationship. It could be that the Acremonium endophyte in this study may be endophytic only under certain conditions, but under other circumstances may live outside the plant. We could find no evidence of pathogenesis of either of the endophytes on any of the host species. Thus, the purpose these endophytes have in the plants has yet to be elucidated. In Festuca and Lolium, it was speculated that the cosymbiotic endophytes (Neotyphodium and p-endophytes) may have synergistic activities in biological protection and other aspects of host fitness (An et al., 1993). The role of the p-endophytes in Festuca and Lolium may be less ecologically important to their hosts than the Neotyphodium endophytes because the p-endophytes are not as commonly disseminated by seed as is Neotyphodium (Siegel et al., 1995).

Classification of the endophytes in this study into either Neotyphodium or Acremonium was simply based on morphology of the phialides and conidia, conidial size, and cultural characteristics. Differences between the two groups were definitive and consistent during the 4 yr of this study. More detailed phyletic studies need to be conducted to determine the relationship of these endophytes to other members of the Neotyphodium or Acremonium genera. It is possible the endophytes we have identified as being members of Neotyphodium, may be a new taxonomic grouping.

The finding of nonpathogenic, fungal symbionts in wild wheat relatives under natural, indigenous conditions could lead to new methods and strategies of controlling pests and subsequently increasing yields in cultivated wheat. In addition, the evolution of Triticum species and the domestication of wheat may have been influenced by Neotyphodium and/or Acremonium endophytes. However, much work needs to be done, in particular concerning the relatedness of the Triticum endophytes to those of other grasses, as well as concerning the pest-deterrent effect (if any) of the Triticum endophytes.

	ACKNOWLEDGMENTS

 
Funding was provided by the Texas Agricultural Experiment Station; USDA/CSRS Special Grant No.92-34103-6940; and the Texas Higher Education Coordinating Board, Advanced Technology Progam no. 999902-210. Special thanks to Dr. Hans Braun, CIMMYT, Ankara, Turkey for providing assistance in the collection of the wild Triticum species. Thanks also to Mike Christensen and Gary Latch of AgResearch in New Zealand for advice and assistance on preparation of parts of this paper.

Received for publication July 14, 1998.

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