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NOTES

Cross Species Inoculation of Chewings and Strong Creeping Red Fescues with Fungal Endophytes

Dep. of Plant Pathology and Biotechnology Center for Agric. and the Environment, Rutgers, the State University of New Jersey, 59 Dudley Rd., New Brunswick, NJ 08901-8520 USA

belanger{at}aesop.rutgers.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results and Discussion Conclusions REFERENCES

 
An effective technique was developed to inoculate mature Chewings fescue [Festuca rubra L. subsp. fallax (Thuill) Nyman] and strong creeping red fescue (Festuca rubra L. subsp. rubra) tillers with fungal endophytes (Epichloe festucae Leuchtm., Schardl, & Siegel and Neotyphodium spp.). Six fine fescue genotypes were successfully inoculated with endophytes originating from fine fescues and Poa ampla Merr. Eleven percent of the tillers were successfully inoculated, and all inoculated plants transmitted the endophytes to their offspring. This set of inoculated plants, with various combinations of genotypes and endophyte strains, allowed us to compare the effects of different endophytes on one host, and one endophyte on several hosts. When evaluated in the field, the growth characteristics of these plants were dependent on endophyte and host genotype. Considerable interaction between the genotypes was seen. For example, one host inoculated with the Poa ampla endophyte showed enhanced performance, while another host inoculated with the same endophyte performed poorly.

Abbreviations: CA, Cambridge Epichloe festucae endophyte • DE, Delaware E. festucae endophyte • E-, endophyte free • PA, Poa ampla endophyte • PDA, potato dextrose agar • RC, Rose City E. festucae endophyte

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results and Discussion Conclusions REFERENCES

 
MANY COMMERCIAL TURFGRASS CULTIVARS are naturally infected with fungal endophytes of the genera Neotyphodium (i.e., tall fescue, F. arundinacea Schreb.; perennial ryegrass, Lolium perenne L.) or Epichloe (i.e., fine fescues, Festuca spp.) (Funk and White, 1997). Presence of fungal endophytes results in the synthesis of alkaloids that protect the plants from insect predation (Funk et al., 1983; Breen, 1994). In some associations, drought tolerance (West, 1994), disease resistance (Clarke et al., 2000), and effects on plant morphology (Hill et al., 1990) have also been reported. Because of the benefits conferred by the presence of endophytes, turfgrass breeders generally prefer to develop new cultivars with a high percentage of endophyte infection.

Turfgrass breeding programs rely on evaluation of the endophyte–plant association as a single unit when making plant selections. The genotypes of the two components are not subject to separate evaluations. An alternate breeding strategy has been proposed in which the best endophyte-free plants and the best endophytes are selected independently, and then brought together (Funk and White, 1997). In theory, this approach could result in a superior endophyte–plant association that could be used in a turfgrass breeding program.

There are two major requirements in implementing such an approach: (i) the ability to inoculate a superior plant once it is identified and (ii) the ability to evaluate the endophytes. Currently there are obstacles to both of these requirements.

Seedlings of tall fescue, perennial ryegrass, hard fescue (Festuca brevipila Tracey) and strong creeping red fescue have been successfully inoculated with fungal endophytes (Latch and Christensen, 1985; Christensen et al., 1997). However, this method does not allow for the prior field selection of a superior plant. Callus culture (Johnson et al., 1986) and somatic embryos (Kearney et al., 1991) have been used to inoculate tall fescue, but these methods may result in somaclonal variation. Mature perennial ryegrass tillers (Ravel et al., 1994, as cited by Simpson et al., 1997) and axillary buds from perennial ryegrass and tall fescue meristems (Simpson et al., 1997) have also been inoculated, but the success rate was low and contamination from saprophytic organisms was a problem. Inoculation of plantlets derived from meristems of mature perennial ryegrass and tall fescue plants (O'Sullivan and Latch, 1993) yielded a high percentage of infected plants but required sterile culture of the meristems.

Evaluations of endophytes in culture have indicated that there are differences among endophyte accessions. In culture, endophytes differ in their inhibition of fungal pathogens and morphological characteristics (White and Cole, 1985; Siegel and Latch, 1991; Christensen and Latch, 1991; Christensen et al., 1991). DNA fingerprinting methods have been developed that distinguish endophyte sources (Groppe et al., 1995; White and Huff, 1996; Tredway et al., 1999).

To evaluate an endophyte for its usefulness it is best to evaluate it in its host plant. For example, differences among endophyte-infected plants have been found in the quantity and type of alkaloids produced and in the level of insect resistance (Breen, 1993a, 1993b, 1994; Richardson et al., 1997). The effect of endophyte and host plant were not distinguished in these studies. Agee and Hill (1994) determined that ergovaline production was influenced by plant genotype by evaluating progeny from a cross between a low and high ergovaline-producing tall fescue. Siegel et al. (1990) used artificially inoculated ryegrass and fescue seedlings and found that the same endophyte produced different alkaloids depending on the host, suggesting interaction between the fungus and plant.

Therefore to best compare endophytes, different endophyte genotypes in the same plant genotype should be compared. The endophytes could then be ranked based on evaluation of plant performance. This requires the ability to infect individual tillers from one plant genotype with different fungal isolates.

Here we report for the first time a method for inoculating mature tillers of Chewings fescue and strong creeping red fescue. Our objective was to evaluate the effect different endophytes had on the morphology and physiology of different host genotypes. Chewings and strong creeping red fescues were chosen because of the impact endophytes can have on these important, low maintenance turfgrass species (Saha et al., 1987; Breen, 1993a, 1993b; Funk et al., 1994; Clarke et al., 2000).

	Materials and Methods

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results and Discussion Conclusions REFERENCES

 
Inoculation of Fine Fescue Selections
Four sources of endophyte, originating from diverse regions of the United States, were selected. Three of these endophytes were E. festucae (Leuchtmann et al., 1994) and were designated as follows: the Rose City endophyte (RC) isolated from a strong creeping red fescue and the Cambridge (CA) and the Delaware (DE) endophytes isolated from Chewings fescue plants. These three E. festucae sources have been confirmed by amplified fragment length polymorphisms to represent unique isolates (Tredway et al., 1999). The fourth endophyte was Neotyphodium sp. and was designated the P. ampla endophyte (PA). It was found in `Service', a cultivar of P. ampla released by the Alaska Department of Natural Resources.

Cultures of these endophytes were established from surface-sterilized endophyte-infected seed using a protocol modified from Bacon and White (1994). Seeds were sterilized in a solution of 1.9% (v/v) sodium hypochlorite and 0.1% (v/v) Triton X-100 for 15 min, washed four times with distilled water, and incubated in water overnight to allow any viable microbial spores to germinate. The sodium hypochlorite treatment was repeated the following day and the seeds were rinsed four times with sterile water in a laminar flow cabinet and plated onto potato dextrose agar (PDA) plates.

The plates were incubated under light, which allowed the germinating seedlings to continue to grow. After the seeds germinated it was sometimes necessary to aseptically push the seedling back into the agar surface using sterile forceps. The fungal endophytes emerged from the seedlings in 2 wk. Fungal cultures were maintained by subculturing every 2 to 3 wk. To aid in removing mycelium from the plates, fungi were subcultured on PDA plates overlaid with a piece of cellophane (Type 195PUTSD2, Flexel, Covington, IN).

Fine fescue plants were selected for inoculation based on above average quality in turfgrass performance trials and seed yield capability in spaced plant nurseries. The plants were removed from breeding nurseries and maintained in the greenhouse. Their endophyte-free status was confirmed by microscopic examination of leaf sheath tissue (Saha et al., 1988). Five Chewings fescue genotypes and six strong creeping red fescue genotypes were used (Table 1) .

Field Evaluation
To evaluate the effect of endophyte infection on plant growth, 20 different endophyte-inoculated plants and their endophyte-free (E-) counterparts (Table 2) were planted in a spaced-plant nursery at Adelphia, NJ, on 5 Sept. 1996. The trial was arranged in a randomized complete block design with 10 replications, with two plants from each treatment per replication, and 0.6-m spacing between plants. Maintenance consisted of no irrigation or mowing, and applications of 0.77 kg N 100 m^-2 as NH₄NO₃ on 16 Sept., 7 Oct., and 6 Nov. 1996. Herbicides were applied as follows: DCPA (dimethyl 2,3,5,6-tetrachloro-1,4-benzenedicarboxylate), 2,4-D [(2,4-dichlorophenoxy)acetic acid], and halosulfuron (methyl 5-{[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonylaminosulfonyl}-3-chloro-1-methyl-1-H-pyrazole-4-carboxylate) on 15 September; 2,4-D and dicamba (3,6-dichloro-2-methoxybenzoic acid) on 5 Nov. 1996; 2,4-D, dichlorprop [(±)-2-(2,4-dichlorophenoxy)propanoic acid], and dicamba on 27 March; and DCPA on 11 Apr. 1997, all at recommended rates.

Analysis of variance was calculated for each measurement, using the main effects of plant–endophyte combination and replication in the model. Because we did not have a complete set of inoculated plants (for example, only two of six host plants contained the PA endophyte), it was not valid to test the main effects of endophyte genotype or plant genotype. The means for each plant–endophyte combination were separated using Fisher's protected least significant difference (LSD) test (SAS Institute, 1989).

To evaluate the transmission of endophytes from inoculated plants to their offspring, a sample of 50 seeds from each endophyte–plant combination was microscopically examined (Saha et al., 1988). The remaining seeds harvested from the nursery were used to establish turfgrass evaluation plots (1.1 by 1.7 m) in September 1997. Twenty-five plants were randomly selected from each plot in September 1998 and a tiller from each was microscopically examined to determine the percentage of viable endophyte.

	Results and Discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results and Discussion Conclusions REFERENCES

 
Inoculation of Fine Fescue Selections
The results of the inoculation attempts using various plant and endophyte combinations are shown in Table 1. Some tillers died due to wounding during inoculation. Overall, 37 of 340 surviving inoculated tillers (11%) were successfully infected with an endophyte. All five Chewings fescues and three of the six strong creeping red fescues were successfully inoculated (Table 1). The DE, CA, and RC endophytes were successfully inoculated into both the original host species and the other fescue species. Additionally, two Chewings fescue genotypes were infected with the PA endophyte. These results demonstrate that it is possible to inoculate mature tillers of Chewings and strong creeping red fescues with fungal endophytes originating from different plant species.

There was variability in the success of inoculation among the different plant–endophyte combinations, ranging from 0 to 100% infection (Table 1). Overall, the PA and RC endophytes resulted in higher infection percentages (44 and 15%, respectively) than CA (6%) or DE (4%), yet in some hosts, the PA and RC endophytes resulted in 0% infection. The variation we observed may be due to random error since the inoculation attempts were not replicated. Because of this variability, no definitive statements can be made regarding one host plant, or one endophyte, being more compatible than another.

Field Evaluation
A spaced-plant nursery was established for field evaluation of the effects of the different endophyte sources on the different plant genotypes. Inoculation of Chewings and strong creeping red fescues with fungal endophytes had an impact on many of the plant growth characteristics measured in 1997 (Table 2). When each plant–endophyte combination was compared with its E- counterpart, the effect of endophyte presence appeared to be highly variable, depending on host plant, endophyte, and the parameter measured.

The PA endophyte, for example, had opposite effects in its two hosts, C1117 and C1116. C1117–PA had earlier panicle emergence, and more panicles and seed than C1117–E- (Table 2). C1116–PA, on the other hand, had latter panicle emergence, and fewer panicles and seed than C1116–E-. These differences were also seen in horizontal spread, plant height, and flag leaf length. Inoculation with the RC endophyte also resulted in varied host responses. C1117–RC showed enhanced performance in almost all parameters compared with C1117–E-, while C3188-1–RC performed poorly compared with C3188-1–E- (Table 2).

When the performance of a particular host plant was examined, it varied depending on the endophyte with which it was inoculated. For example, when inoculated with DE and RC, C3188-1 had decreased panicles and seed, but when inoculated with CA, panicle number and seed yield were the same as endophyte-free C3188-1. In some host plants, all of the endophytes had a positive effect. In C1117, CA, PA, and RC all enhanced number of panicles per plant, horizontal spread, and plant height compared with C1117–E- (Table 2).

The presence of choke disease appeared to be more dependent on the host plant than on the endophyte (Table 2). Plant C1117 did not show choke disease symptoms with any of the three endophytes (CA, PA, RC) with which it was inoculated, while C3188-1 exhibited choke stromas with all three of its endophyte associations (DE, CA, RC). On the other hand, four of five plant genotypes inoculated with the RC endophyte showed choke symptoms, suggesting that this particular endophyte may have a stronger influence on choke production. Another example of the endophyte influencing a trait is the CA endophyte increasing seed yield in three of its four hosts. Additional data is needed to see if these observations hold true with time and in additional host plants.

All inoculated plants were able to transmit the endophyte to a high percentage of their seeds (Table 3) . The turf plots established from these seeds contained equally high percentages of endophyte-infected plants (Table 3). The incompatibility and death of the endophyte observed in other inoculation studies (Christensen, 1995) was not a problem in these plants.

	Conclusions

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results and Discussion Conclusions REFERENCES

Field evaluation of the endophyte-free and endophyte-infected clonal lines generated in this study suggested that the same endophyte genotype could produce different effects when inoculated into different plant genotypes and vice versa. These results suggest that there are unpredictable host–endophyte interactive effects that make it difficult to identify a universally superior endophyte genotype. Further testing of this set of plants and the development of additional comparisons may allow for a better understanding of these relationships. The possibility of cross-species inoculation of endophytes into fine fescues will now provide turfgrass breeders access to additional endophyte genotypes for inclusion in their selection process.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge C.R. Funk, for his advice and use of plant materials and endophytes, and R. Bara, G. Zieminski, and G. Zhang (Rutgers, the State University of New Jersey) for technical assistance. Some of this work was conducted as part of New Jersey Agric. Exp. Stn. Project no. 12264, supported by New Jersey Agric. Exp. Stn. funds, other grants, and gifts. Additional support was received from the United States Golf Association, the Golf Course Superintendents Association of America, the New Jersey Turfgrass Association, and National Science Foundation Grant No. IBN 96-04537.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results and Discussion Conclusions REFERENCES

 
Contribution as Publication no. D12264-8-99 of the New Jersey Agric. Exp. Stn.

Received for publication October 29, 1999.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION Materials and Methods Results and Discussion Conclusions REFERENCES

 

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