Crop Science 40:1490-1491 (2000)
© 2000 Crop Science Society of America
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Endophyte Viability in Seedling Tall Fescue Treated with Fungicides
N.S. Hilla and
E. Brownb
a Dep. Crop and Soil Sciences, 3111 Miller Plant Sciences Building, Athens, GA USA
b Dep. Plant Pathology, 2111 Miller Plant Sciences Building, Univ. of Georgia, Athens, GA 30602 USA
nhill{at}arches.uga.edu
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ABSTRACT
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Commercial tall fescue (Festuca arundinacea Shreb.) cultivars containing non-toxic endophytes (Neotyphodium coenophialum Glenn, Bacon, Hanlin) are available. Hence, seed testing objectives have changed to examining seed for viable endophyte. Seedling diseases occur during testing and this study compared effects of fungicides on endophyte infection of seedling plants. Endophyte-infected `Jesup' tall fescue seed were planted into cell packs and grown in the greenhouse. Chloroneb (1,4-dichloro-2,5-dimethoxybenzene), terrazole (5-ethoxy-3-(trichoromethyl)-1,2,4-thiadiazole), and propiconazole (1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxan-2yl]methyl]-1H-1,2,4-triazole) were applied weekly to plants beginning 7 d until 21 d post germination. In a second experiment, seedling plants received applications of Propiconazole at d 7, 14, 21 and all possible combinations. Seedling endophyte infection rates were higher when terrazole or chloroneb (83.1 and 78.1%, respectively) were applied compared with no fungicide (73.5%) or propiconazole (5.0%). Endophyte mortality was reduced but not eliminated when propiconazole application to seedling plants was delayed. Contact fungicides should be used to control seedling diseases when evaluating endophyte viability.
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INTRODUCTION
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FESCUE TOXICOSIS occurs when livestock graze endophyte-infected tall fescue infected with endophytes that produce toxins (Steudemann and Hoveland, 1988). Since the endophyte is passed from one generation to the next only in the seed (Bacon and Siegel, 1988), testing has involved relatively simple histological or immunhistochemical methods to detect endophyte in seed. Recently, non-toxin producing endophytes have been inserted into tall fescue to take advantage of improved agronomic performance of endophyte-infected plants while eliminating deleterious effects on the grazing animal (Bouton, 1998). This has made testing for endophyte more complicated because (i) viability of endophyte must be verified, and (ii) toxin producing capability of the endophyte must be determined. Therefore, seedling plants must be established in the greenhouse and tested for endophyte presence and toxicity.
Seedling damping-off disease commonly occurs when greenhouse-grown tall fescue plants are established to determine viable endophyte content of seed (Hill and Bouton, personal observation). Whether fungicides can be used to control seedling damping-off is unknown since fungicides historically have been evaluated for their potential to eliminate the endophyte from tall fescue (Harvey et al., 1982). Whereas the objective of viability testing for endophyte is to maintain endophyte in the plant, fungicides need to be tested for their benign effects on endophytes in planta. The objective of this study was to evaluate systemic and contact fungicides on endophyte viability in seedling plants.
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Materials and Methods
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Experiment 1. Screening Fungicides for Effect on Viable Endophyte in Seedling Tall Fescue
Seeds of endophyte-infected Jesup tall fescue were planted in cell packs (1 seed per cell; cells 2.5 cm wide by 2.5 cm wide by 5.0 cm deep) in the greenhouse during winter and spring months of 1998–1999. No artificial lighting was provided and day/night greenhouse temperatures were approximately 21/15°C. Chloroneb, terrazole (contact fungicides), and propiconazole (systemic fungicide) were individually mixed in misting applicators at rates of 1.78, 1.68, and 4.00 g active ingredient per liter water, respectively, without adjuvant. Seedling tall fescue plants were sprayed until solutions were dripping from leaves at 7, 14, and 21 d post germination. A control treatment of distilled water was used to compare fungicide effects on endophyte viability. Fungicide treatments were replicated five times, and the study was repeated a second time.
Plants were harvested at the four-leaf stage (approximately 1 wk following the final application of fungicide) by removing the plants from the cell packs, washing the roots in tap water, and excising the roots precisely above the crown of the plant. A 2-mm cross section of psuedostem tissue was analyzed for endophyte using commercial immunoblot test kits (Agrinostics Ltd. Co., Watkinsville, GA). Briefly, a piece of nitrocellulose membrane was placed on a sponge containing extraction buffer, the cross section of psuedostem placed on the membrane with a cut surface down, and incubated overnight at 4°C. The following morning the psuedostem pieces were gently removed from the membrane, and the membrane dried. The membrane was then placed through a series of endophyte-specific antibody solutions and presence of endophyte determined by addition of a chromogenic solution and evaluation of color reaction (Hiatt et al., 1999).
Experiment 2. Effect of Propiconazole on Endophyte Viability in Seedling Tall Fescue
Seeds of endophyte-infected Jesup tall fescue were planted in cell packs (1 seed per cell; cells 2.5 cm wide by 2.5 cm wide by 5.0 cm deep) in the greenhouse during spring of 1999. No artificial lighting was provided and day/night greenhouse temperatures were approximately 21/15°C. Propiconazole was mixed in a misting applicator at a concentration of 4.00 g active ingredient per liter water. Propiconazole was applied by spraying leaves of seedling plants using the following treatment dates: (i) 7 d post germination, (ii) 14 d post germination, (iii) 21 d post germination, (iv) 7 and 14 d post germination, (v) 7 and 21 d post germination, (vi) 14 and 21 d post germination, (vii) 7, 14, and 21 d post germination, and (viii) a water control. The treatments were assigned to a randomized complete block with 3 replications. The study was repeated a second time. Plants were harvested at the four-leaf stage (approximately 7 d following the final application of propiconazole). Roots were excised from the psuedostem stem, and the psuedostem bases analyzed for endophyte presence as described in Exp. 1.
Statistical Analysis
Both experiments were analyzed as randomized complete blocks repeated in time. There were no treatment by time interactions for either experiment, so data were pooled and analyzed as a randomized complete block by the PROC ANOVA subroutine of the SAS Institute (Cary, NC). Means were separated using protected least significant differences at the 0.05 level of probability.
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Results and Discussion
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The fungicides used in these studies are at rates recommended by University of Georgia Cooperative Extension Service for control of seedling diseases in turfgrass. They were selected because two (chloroneb and terrazole) are contact fungicides and one (propiconazole) is a systemic fungicide. The hypothesis was that contact fungicides have less effect on endophyte than a systemic fungicide because of the proximity and physical separation of the endophyte from contact fungicides by plant tissue. Conversely, systemic fungicides are transported through the apoplasm and vascular tissues of plants (Crowdy, 1977). These are plant anatomical features which are inhabited by Neotyphodium spp. (Phillipson and Christy, 1986; Hiatt et al., 1997). Hence, systemic fungicides are more likely to adversely affect endophyte viability than contact fungicides.
Weekly applications of the systemic fungicide, Propiconazole (Exp. 1), virtually eliminated the endophyte from the seedling plants (Table 1)
. Weekly applications of the contact fungicides, terrazole and chloroneb, had no apparent adverse effect on endophyte viability. In fact, terrazole and chloroneb tended to enhance endophyte viability compared with the control treatment, an observation that was consistent when the experiment was repeated in time. Why the contact fungicides increased viability of endophyte is unclear but may be related to disease. Some plants receiving the control treatment had lower leaves that had wilted and were senescing, an indication that seedling diseases were beginning to manifest infection of the plants. Although not measured, plants receiving contact fungicides were slightly taller and appeared more vigorous than control plants receiving water. Constitutive endophytes, such as Neotyphodium coenophialum, are totally dependent upon the plant for nutrition and habitat (Hill, 1994). Even though fluctuations in plant health are likely to have a concomitant effect on endophyte health and well-being, increased endophyte infection rates resulting from applications of contact fungicides were surprising.
Although repeated applications of propiconazole killed the fungus, it is unlikely that repeated applications would be necessary to control seedling disease. Therefore, various combinations of application dates were tested in Exp. 2 to determine whether alternatives to weekly applications might have less of an effect on endophyte viability. Endophyte infection rates were lower than the control treatment regardless of frequency of propiconazole treatment (Table 2)
, a response consistent with other studies in which the fungicide was applied to vegetative tissue (Harvey et al., 1982; Hill et al., 1990) or as a seed treatment (Harvey et al., 1982). However, seedling plants receiving treatments in which propiconazole was applied 7 d post germination had lower endophyte infection rate than when applied at 14 and/or 21 d. These data suggest either entrance of propiconazole into the leaf is reduced as plant age increases or N. coenophialum becomes more tolerant of Propiconazole as the plant develops during the seedling stage. Regardless, this study demonstrates that use of contact fungicides for control of seedling diseases during grow-out studies to determine endophyte viability resulted in higher infection rates than even untreated plants and that systemic fungicides adversely affect endophyte in seedling plants.
Received for publication December 6, 1999.
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REFERENCES
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- Bacon C.W., Siegel M.R. Endophyte parasitism of tall fescue. J. Prod. Agric. 1988;1:45-55.
- Bouton, J.A. 1998. Breeding tall fescue for the lower South. p. 9–11. In L. Bush (ed.). Proceedings of the tall fescue toxicosis workshop. SERAIEG-8. 1–2 Nov., 1998. Nashville, TN. Univ. Kentucky Publ.
- Crowdy S.H. Transolcation. In: Marsh R.W., et al. , ed. Systemic fungicides, 2nd Ed New York: Longman Ltd, 1977:92-114.
- Harvey I.C., Fletcher L.R., Emms L.M. Effects of several fungicides on the Lolium endophyte in ryegrass plants, seeds, and in culture. N.Z. J. Agric. Res. 1982;25:601-606.
- Hiatt E.H., III., Hill N.S., Bouton J.H., Mims C.W. Monoclonal antibodies for detection of Neotyphodium coenophialum. Crop Sci. 1997;37:1265-1269.[Abstract/Free Full Text]
- Hiatt E.H., Hill N.S., Bouton J.H. Tall fescue endophyte detection: Commercial immunoblot test kit compared with microscopic analysis. Crop Sci. 1999;39:796-799.[Abstract/Free Full Text]
- Hill N.S. Ecological relationships of Balansiae-infected grasses. In: Bacon C.W., White J.F., Jr., eds. Biotechnology of endophytic fungi of grasses. Boca Raton, FL: CRC Press, 1994:59-72.
- Hill N.S., Stringer W.C., Rottinghaus G.E., Belesky D.P., Parrott W.A., Pope D.D. Growth, morphological, and chemical component responses of tall fescue to Acremonium coenophialum. Crop Sci. 1990;30:156-161.[Abstract/Free Full Text]
- Phillipson M.N., Christy M.C. The relationship of host and endophyte during flowering, seed formation, and germination of Lolium perenne. N.Z. J. Bot. 1986;24:125-134.
- Steudemann J.A., Hoveland C.S. The fescue endophyte: History and impact on animal agriculture. J. Prod. Agric. 1988;1:39-44.
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ABSTRACT
INTRODUCTION
