IntroductionNeotyophodium coenophialum is a

fungus that grows endophytically within grasses (Fig. 1). It is clear that N.coenophialum can have detrimental effects on some herbivorous insects through the alkaloids it produces (Clement et al. 1994; Faeth and Bultman 2002). It is also becoming evident that variation in plant and fungal genotype can influence alkaloid content (Bouton et al. 2002). We tested if variation in plant cultivar and fungal isolate affected the performance and preference of the bird cherry-oat aphids that were feeding on tall fescue grass.

Methods• Plant and Fungal MaterialTo assess the influence of genetic variation

in the endophyte we used two cultivars of tall fescue: Jesup and Georgia (Bouton et al. 1997). Plants were infected with the "wildtype" strain of N. coenophialum, the most common strain from pastures in Georgia, USA, or strains (AR542 for Georgia and AR502 or AR542 for Jesup) isolated from native, wild populations of tall fescue in Europe. Fungal isolates were cultured from parent plants and then introduced into tall fescue plants using standard inoculation techniques (Christensen et al. 1997). Seeds were then produced from infected host plants with subsequent disinfection via heat treatment (Nott and Latch 1993) of some plants; both E- (uninfected) and E+ (infected) seeds were all from the same maternal line. Plants were grown in 10.2 cm dia pots and fertilized weekly with Peter’s liquid fertilizer (20N:20P:20K) at 300 ppm N.

 • Aphid PerformanceTo assess aphid performance, 8 weeks

after planting, 4 apterous nymphs (2nd and 3rd instars) of R. padi were placed onto each of 25 plants in one of the 7 treatment groups. Aphids were placed on the base of the most recently fully expanded leaf blade of the largest tiller and enclosed in a clip bag (5cm x 2.5cm) made of synthetic fabric (0.5mm mesh). Aphids were confined to clip bags for 4 days while plants were kept on a light stand (14L:10D) in the greenhouse where temperatures varied from 140C during the night to 280C during the day. After 4 days, bags were removed and apterous aphids counted.

Counts of apterous aphids were analyzed by a 1-way ANOVA (PROC GLM, SAS 1985) with fungal isolate as the main effect. Data were square root transformed prior to analyses to meet the assumption of normality. We used Tukey-Kramer multiple comparisons to compare aphid numbers among fungal isolates (SAS 1985).

Abstract- Neotyphodium coenophialum (Ascomycota: Clavicipitaceae) is an endophytic fungus of grasses that produces alkaloids which can have detrimental effects on some insect herbivores. We tested if variation in plant cultivar and fungal isolate affected the performance and preference of the bird cherry-oat aphids. We found that aphid populations grew faster on E- plants in both Jesup and Georgia cultivars (F3,96 = 6.7, p < 0.001; F2,72 = 12.7, p < 0.001, respectively). In

the Georgia cultivar, there was no difference between the reproduction of aphids on plants infected with Wild or 542 isolates; however, in the Jesup cultivar, aphids reproduced faster on plants infected with 502 and 542 cultivars, compared to the wildtype isolate. Aphid preferences mirrored performance, for the most part. In all comparisons involving E- plants, aphids preferred the uninfected plants over those infected with N. coenophialum . In comparing just the fungal isolates, aphids showed no preference among them in both the Georgia and Jesup cultivars. Our data clearly show that R. padi is deterred by N. coenophialum and when given a choice, prefers to feed on plants lacking infection by the endophyte. Our results also show that both fungal isolates in the Georgia cultivar depressed aphid reproduction, while only the wildtype isolate did so in the Jesup cultivar. However, aphids tended to avoid all fungal isolates equally. Thus, all fungal isolates we tested in the Georgia and Jesup cultivars provide some protection from R. padi.

Effects of Plant Cultivar and Fungal Endophyte Isolate on Performance and Preference of

Bird Cherry-Oat AphidT.L. Bultman, T.J. Sullivan, C. Pulas, and G. Bell*

Biology Department

Hope College

*Division of Science

Truman State University

ResultsWe found that aphid populations grew

faster on E- plants in both Jesup and Georgia cultivars (F3,96 = 6.7, p < 0.001; F2,72 = 12.7, p

< 0.001, respectively, Fig. 2). In the Georgia cultivar, there was no difference between the reproduction of aphids on plants infected with Wild or 542 isolates (Fig. 2); however, in the Jesup cultivar, aphids reproduced faster on plants infected with 502 and 542 cultivars, compared to the wildtype isolate (Fig. 2).

Aphid preferences mirrored performance, for the most part. In all comparisons involving E- plants, aphids preferred the uninfected plants over those infected with N. coenophialum (Fig. 3 & 4). In comparing just the fungal isolates, aphids showed no preference among them in both the Georgia (Fig. 3) and Jesup (Fig. 4) cultivars.

• Aphid PreferenceTo assess aphid preference we placed 10 2nd and 3rd instar nymphs (which had been starved for 2 hrs) between two or three 4 cm long cut leaf blades of tall fescue within petri dishes. Combinations of leaf blades within petri dishes were: Jesup uninfected (E-) vs. Jesup Wild (wildtype); Jesup E- vs. Jesup 502; Jesup E- vs. Jesup 542; Jesup Wild vs. Jesup 502 vs. Jesup 542 ; Georgia E- vs. Georgia Wild; Georgia E- vs. Georgia 542; Georgia Wild vs. Georgia 542. Petri dishes were placed in an environmental chamber at 210 C for 4 hrs and then the number of aphids feeding on each blade was recorded. Evidence for preference was determined by a X2 goodness-of-fit test.

Figure 1. Stained squash of tall fescue seed. Plant aleurone cells are stained dark red; fungal hyphae appear as pink spaghetti-like strands.

Figure 2. Number of R. padi aphids within clip bags on leaf blades of tall fescue following 4 days of growth. A. Georgia cultivar - tall fescue. B. Jesup cultivar - tall fescue. E- = grass lacking fungal infection; 542 and Wild are different isolates of N. coenophialum. Histograms with common letters above them are not significantly different as determined by Tukey Kramer multiple comparisons test. Error bars ±1 SEM

Figure 3. Frequency of R. padi aphids on cut leaf blades of Georgia cultivar tall fescue plants when given a choice between plants infected with differing fungal isolates, or uninfected (E-). A. Uninfected vs. infected with wildtype isolate. B. Unifected vs. infected with the 542 isolate. C. Infected with the wildtype isolate vs. the 542 isolate. Statistical results from goodness-of-fit test.

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X 2 = 46.4, p < 0.001

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X 2 = 45.4, p < 0.001

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CX 2 = 2.4, ns

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Figure 4. Frequency of R. padi aphids on cut leaf blades of Jesup cultivar tall fescue plants when given a choice between plants infected with differing fungal isolates, or uninfected (E-). A. Uninfected vs. infected with wildtype isolate. B. Uninfected vs. infected with the 502 isolate. C. Infected with the wildtype isolate vs. the 542 isolate vs. the 502 isolate. D. Comparison among wildtype, 502 and 542 fungal isolates. Statistical results from goodness-of-fit test.

Jesup Aphid E-/W

Obs Exp Obs Exp0

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2=16.5, p<.001

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2=10.1, p<.01

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2=8.6, p<.01

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2=6, p<.1

Conclusions• Our data clearly show that R. padi is deterred by N. coenophialum and when given a choice, prefers to feed on plants lacking infection by the endophyte, as has been shown previously (i.e., Eichenseer et al. 1991). • Our results also show that both fungal isolates in the Georgia cultivar depressed aphid reproduction, while only the wildtype isolate did so in the Jesup cultivar. However, aphids tended to avoid all fungal isolates equally. This result varies somewhat from that of Clement et al. (1996) who found that the Russian wheat aphid (Diuraphis noxia) showed no aversion to endophyte infection in tall fescue . Yet, they did find that D. noxia populations declined on all plants containing a fungal isolate. • Combining our results for aphid performance and preference, it appears that all fungal isolates we tested in the Georgia and Jesup cultivars provide some protection from R. padi

AcknowledgmentsThis work was funded by a NSF grant DEB-9527600 and USDA grant grant 99-35302-8164 to T.L. Bultman. J. Bouton kindly supplied Georgia and Jesup seeds. M. Cortez, J. Librizzi, , M. Nelsen, J. Skoug assisted in poster preparation.

 

Literature CitationsBouton, J.H., R.R. Duncan, R.N. Gates, C.S. Hoveland, and D.T. Wood. 1997.  Registration of 'Jesup' tall fescue. Crop Sci.  37:1011-1012.

Bouton, J.H., G.C.M. Latch, N.S. Hill, C.S. Hoveland, M.A. McCann, R.H. Watson, J.A. Parish,L.L. Hawkins, and F.N. Thompson. 2002. Reinfection of tall fescue cultivars with non- ergot alkaloid-producing endophytes. Agr. J. 94:567-574.

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Clement, S.L., Kaiser, W.J., and Eichenseer, H. 1994. Acremonium endophytes in germplasms of major grasses and their utilization for insect resistance, p. 185-199. In C.W. Bacon and J.F.White, Jr (eds.) Biotechnology of endophytic fungi of grasses. CRC Press, Boca Raton,Florida, USA.

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Latch, G. C.M., Christensen, M.J. and Gaynor, D.L. 1985. Aphid detection of endophyte infection in fall fescue. NZ J. Agr. Res.28: 129-132.

Nott, H.M., and G.C.M. Latch. 1993. A simple method of killing endophyte in ryegrass seed. p. 4-15. In D.E. Hume, G.C.M. Latch, and H.S.

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