increased as the percentage of black cutworm feeding on clippings increased especially on E- tall fescue. This study shows that E+ tall fescue can be

Transcription

1 100 CHAPTER FIVE MOWING HEIGHT AND ERGOT ALKALOIDS ON BLACK CUTWORM HERBIVORY IN ENDOPHYTIC AND NON-ENDOPHYTIC TURF-TYPE TALL FESCUE AND RESULTING PATTERNS IN ANTIOXIDANT METABOLISM Abstract Turfgrasses exposed to abiotic and biotic stresses produce active oxygen species such as superoxide radical (O2), hydrogen peroxide (H2O2), hydroxyl free radical (OH), and singlet oxygen ('02). Plant cells are protected from these species through a complex antioxidant system. Previous research has shown that antioxidant metabolism is affected when subjected to abiotic stresses such as heat, cold, drought, and salinity. Very few studies have evaluated the effect of biotic stresses like insect herbivory on antioxidant metabolism. This greenhouse study consisted of three trials which evaluated the changes in antioxidant metabolism in endophytic (E+) and non-endophytic (E-) turf-type tall fescue (Schedonorus phoenix (Scop.) Holub; formally know as Festuca arundinacea Schreb.) subjected to black cutworm {Agrotis ipsilon Hufiiagel) herbivory at two mowing heights (5 and 9 cm). Twenty-five black cutworm larvae were placed into Petri dishes and fed endophyte-infected (E+) or non-endophyte infected (E-, heat treated) 'DaVinci' turf-type tall fescue clippings daily. Neonate settling response (number feeding on clippings) was measured on day two and mortality was recorded daily. Time affected black cutworm performance during all three trials and endophyte and mowing height affected settling response and mortality at day 7 for trial two only. Correlations matrices showed that ergot alkaloids negatively influenced settling response for all three trials. As for antioxidant metabolism, superoxide dismutase (SOD) activity was found to be correlated to settling response during trial two. Catalase (CAT) activity was found to also be correlated to settling response in trial three. Antioxidant activity of SOD and CAT

2 101 increased as the percentage of black cutworm feeding on clippings increased especially on E- tall fescue. This study shows that E+ tall fescue can be advantageous as a pest management tool to reduce the application of insecticides to control insect feeding.

3 102 Introduction Tall fescue {Schedonorus phoenix (Scop.) Holub; formally know as Festuca arundinacea Schreb.) is frequently infected with the endophytic fungus Neotyphodium coenophialum [Morgan-Jones and Gams] Glenn, Bacon, and Hanlin) that lives in the stem and leaves of the grass plant. Endophytes and the grasses they infect have a mutualistic, symbiotic association with one another in which the plant provides the fungi with water, nutrients, and structural refuge and in return the endophyte provides several benefits such as enhanced competitiveness (Hill et al., 1991, 1998; Malinowski et al., 1999; Marks et al., 1991) more efficient water utilization (Arachevaleta et al., 1989), and enhanced environmental stress tolerance (Elmi and West, 1995; Elbersen and West, 1996; Ravel et al., 1995). More specifically in tall fescue, N. coenophialum increases tillering and root growth, resistance to drought stress (Arachavaleta et al., 1989), fungal pathogens (Gwinn and Gavin, 1992), nematodes (Kimmons et al., 1990), mammalian herbivores (Bacon et al., 1977) and protection against insect herbivory (Rowan, 1993). These benefits have made the use of endophytes in turf an attractive alternative to the chemical inputs required, such as insecticides, to control insects and to enhance the overall turfgrass performance (Funk et al., 1985; Sun et al., 1990; Clarke et al., 2006). Previous research has shown that endophyte-infected grasses are often more vigorous and resistant to herbivory than uninfected grasses (Clay et al., 1985, Bacon et al., 1986; Read and Camp, 1986; Clay, 1990). Previous research conducted on insect herbivory in endophyte-infected grass include: fall armyworms (Clay and Cheplick 1993; Davidson and Potter, 1995; Boning and Bultman, 1996; Braman et al., 2002; Richmond et al., 2004), sod webworms (Murphy et al., 1993; Richmond, 2007), bluegrass webworms, (Richmond and Shetlar, 1999,) billbugs (Murphy et al., 1993; Richmond et al., 2000), black cutworm (Richmond and Shetlar, 2001), chinch bugs (Richmond and Shetlar, 2000; Anderson et al., 2006), and aphids (Davidson and Potter, 1995; de Sassi et al., 2006; Hunt and Newman, 2005; Meister et al., 2005; Krauss et al., 2007). Insect herbivory can alter lighting conditions influencing the canopy environment, nutrient cycling, and water use in plants (Malinowski and Belesky, 2000). The protection against herbivory comes in the form of defensive chemicals called alkaloids which are

4 103 produced by the endophyte or by the grass in response to the endophyte. Alkaloids are toxins which are poisonous or distasteful to many insects which may deter insect feeding (antixenosis), or reduce insect performance (antibiosis) (Braman et al., 2002; Bacon et al., 1997; Breen, 1994) on grasses containing endophytes. Four classes of alkaloids have been associated with endophytes: ergot (clavines, lysergic acid, ergopeptines), lolitrem or indolediterpenes (paxilline, paxitriol(s), lolitriol, lolotrem(s), pyrrolopyrazine (peramine), and saturated aminopyrrolizidines (lolines). All four classes of alkaloids provide resistance to insect herbivory (Porter, 1994; Rowan, 1993) but the most potent are the ergots (particularly ergovaline), peramine and the lolines (Dahlman et al., 1991; Popay and Bonos, 2005; Wilkinson et al., 2000; Siegel and Bush, 1996). As a result, endophyteinfected grasses are more competitive, and thrive better than non-infected grasses with limited resources, due to the presence of alkaloids (Marks et al., 1991; Bacon and Hill, 1996; Hill et al., 1998). However, alkaloid concentrations within endophyte-infected grasses can vary among plant species, plant genotype (Hiatt and Hill, 1997; Rowan and Latch 1993; Roylance et al., 1994), plant parts such as leaf blades and sheaths (Lane et al., 2000; Ball et al., 1997; Siegel and Bush, 1996), and plant age (Lyons et al., 1986). It is unclear how common turfgrass cultural practices, such as mowing height, affect endophyte infection, alkaloid production, and in turn affect insect herbivory. Of all the cultural practices used to maintain turf, none directly affects plant growth and physiology, and the ability to tolerate biotic and abiotic stresses, to the degree that mowing does (Fry and Huang, 2004). Clipping removal has been shown to reduce alkaloid content in tall fescue as a function of nonstructural carbohydrates that were channeled from alkaloid synthesis to production of leaf matter (Belesky and Hill, 1997). Salminen et al., (2003) found that decreased mowing frequency (biweekly vs. weekly mowing) enhances alkaloid production in tall fescue mowed at 5 cm. Small increases in alkaloid content could have a major impact on herbivore performance. Bigelow and Richmond (2006) showed that after a 48 h settling period, newly hatched black cutworm (Agrotis ipsilom, Hufhagel) larvae showed a stronger preference for clippings mowed at 5 cm than 9 cm for three of the four turf-type cultivars in the study including 'Da Vinci'. Black cutworm survival was reduced by the 9 cm mowing height on two of the four

5 104 cultivars during a five-day feeding study. Bigelow and Richmond (2006) also reported that black cutworm larvae survival was higher on endophtye-infected 'Plantation' and '2 nd Millennium' turf-type tall fescue clippings mowed at 5 cm and higher on clippings from '2 nd Millennium' fertilized with urea without a nitrification inhibitor. Abiotic (drought) and biotic (insects and pathogens) stress may result in the production and accumulation of toxic reactive oxygen species (ROS) such as superoxide radicals (O2), singlet oxygen CO2), hydrogen peroxide (H2O2) and hydroxyl radicals (OH) (Sairam and Saxena, 2000). Plants have evolved several mechanisms to prevent damage from ROS. One way for the plant to scavenge and detoxify ROS is by using their enzymatic antioxidant system that includes superoxide dismutase (SOD), catalase, (CAT), peroxidase (POD), and ascorbate peroxidase (APX) (Bowler et al.,1992; Nayyar and Chander, 2004; Zhang and Nan, 2007). Superoxide dismutase is the first line of defense against ROS by dismutating O2" to H2O2 (Bowler et al., 1992) which is then decomposed by POD and CAT. Alterations in plant oxidative enzymes levels have been reported to be among a plant's first response to insect feeding (Felton et al., 1994; Miller et al., 1994; Chaman et al., 2001). Several researchers (Hildebrand et al., 1986; Felton et al., 1994; Dowd and Lagrimini, 1997) have speculated that oxidative enzymes may play a direct role in the plant's response to insect attack by conferring resistance through adversely affecting the development or behavior of the insect as well as enabling the plant to detoxify ROS perhaps produced in response to plant feeding or wounding (Heng-Moss et al., 2006). However, none of these previous studies have used E+ tall fescue as food source which from an ecological perspective has several advantages as a pest management tool to reduce the use of insecticides. Therefore, the first objective of this study was to examine how endophytic (E+) and non-endophytic (E-) turf-type tall fescue mowed at two mowing heights (5 and 9 cm) affect black cutworm performance. The second objective was to investigate oxidative enzyme responses in E+ and E- turf-type tall fescue, maintained at two mowing heights, challenged by black cutworm feeding.

6 105 Materials and Methods This greenhouse study began January, 2007 with the propagation of endophyteinfected (E+) and non-endophyte-infected (E-) 'Da Vinci' turf-type tall fescue. This cultivar was selected based on it's previously reported high, 84%, endophyte seed infection level (Mohr et al., 2002). To eradicate the endophyte, some of the seed was heat treated in a 50 C forced-air oven for two weeks. After which both E+ and E- seed where placed into propagation trays. One seed was placed into each cell filled with potting mix. The trays were fertilized with liquid fertilizer, , every two weeks during propagation to promote tillering. The trays were also watered for five minutes, three times a day. When the plants had reached the three tiller phase, approximately two months, one tiller from each plant was cut and tested for endophyte with a tissue printimmunoblot (TPIB) technique that detects endophyte proteins in the tissue prints of tall fescue (Gwinn et al., 1991) using commercial immunoblot test kits (Agrinostics Ltd. Co., Watkinsville, GA). This TPIB technique was used to verify that the E+ treatment was positive for endophytes and the E- treatment was negative for endophytes, those that were not were discarded. Three plants of either E+ or E- were planted in 18 cm diameter pots containing potting mix. The black cutworm feeding trials begin in May 2007 once the pots containing either the E+ or E- treatment obtained 100 % cover. There were three independent feeding trials: 23 May 2007 through 5 June 2007, 12 June 2007 through 25 June 2007, and 26 June 2007 through 15 July Mowing at either 5 or 9 cm began the first day of each trial and pots were mowed daily and clippings collected from each mowing were used to feed to the black cutworm larvae. Black Cutworm Resistance Black cutworm eggs (Benzon Research, Carlisle, PA) were surface sterilized upon arrival with a 1% bleach solution. Eggs were then triple rinsed with deionized water and kept at room temperature until they hatched. Twenty-five black cutworm larvae were placed into a 90 mm Petri dish lined with moistured filter paper (0.5 ml deionized water). Four clippings and a cotton dental wick (containing 1 ml deionized water) to maintain

7 106 moisture was placed into the Petri dish and larvae were allowed to settle and feed for 24 h. after which, settling response was recorded by counting the number of larvae on the clippings. Fresh clippings were provided at 24 hrs. and every day thereafter. Each day, the number of surviving was recorded and at the end of each trial, surviving larvae from each dish were weighed and mean larval weight was recorded. Alkaloids and Antioxidants Clippings from each pot were taken on day 1 and 7 to determine ergot alkaloid concentration (Hill and Agee, 1994) using a commercial enzyme-linked immunosorbent assay (ELISA) test kit (Agrinostics Ltd. Co., Watkinsville, GA) and antioxidant metabolism. The clippings fed to the black cutworms on day 1 and 7 were also tested for antioxidant metabolism. Samples were placed in liquid N and stored at -80 C until leaf tissue could be ground using liquid N. Protein content was determined using the Bradford assay (Bradford, 1976). Total superoxide dismutase (SOD) activity was measured (Giannopolities and Rise, 1977). The assay medium contains 50 mmphosphate buffer (ph 7.8), 13 mmmethionine, 75 \*Mpnitro blue tetrazolium chloride (NBT), 2uMriboflavin, 0.1 mmedta, and 30 to 40 ul of enzyme extract. A reaction mixture was illuminated under 80 to 90 umol m' 2 s" 1 for 10 min. One unit SOD activity is defined as the amount of enzymes that can cause 50 % inhibition in the rate of NBT reduction (Wang and Jiang, 2007). The activity of catalase (CAT) was determined by an increase in absorbance at 240 nm for 1 min. The assay contained 2.5 ml of 50 mm NaPO 4 buffer (ph 7.0) and 300 ul of 10 x stock peroxide (150 mm). The reaction was initiated by adding 25 to 100 ul of enzyme extract. The activity of peroxidase (POD) was determined by an increase in absorbance at 470 nm for 1 min. The assay contained 50 ul of 20 mmguaiacol, 2.83 ml of 10 mm phosphate buffer (ph 7), and 75 to 100 (il of enzyme extract. The reaction was initiated by adding H 2 O 2 (Kochhar et al, 1979).

8 107 Experimental Design and Statistical Analysis Each treatment was replicated four times and pots were arranged in a randomized complete-block design. All data was subjected to analysis of variance (ANOVA) using Statistica 7.0 (Statsoft Inc., Tulsa, OK, USA). In addition, repeated measures MANOVA was used to determine what effect time had on black cutworm performance (settling response and mortality). Correlation matrices were also used to identify which plant parameters, alkaloids and antioxidants, were associated with overall black cutworm performance. Black cutworm settling response and mortality as well as plant parameter data was initially pooled for all three trials (Table 5-2). If trial was found to be significant (p<0.05) each trial period was ran separately (Table 5-3 through Table 5-5). Results Initially, all three trials were analyzed together using ANOVA to determine the effects endophyte treatment (E+ and E-) and mowing height had on black cutworm performance (settling response and mortality) in which endophyte (E+ or E-) treatment was significant (F=5.6; df=l, 44; p=0.02). Repeated measures MANOVA was also used to determine if a time effect existed which resulted in time x endophyte treatment being significant, F=20.4; df=3, 132; p=0.000, in which black cutworm mortality increased from day 1 to day 14 (Figure 5-1). There was also a time x endophyte treatment x mowing height interaction as well (F=3.1, df=3, 132; p=0.03). Univariate results for settling response and mortality on day 1, 7, and 14 showed that settling response was affected by endophyte treatment (p<0.001). Mortality on day 7 showed an interaction between endophyte and mowing height (p<0.02). Trial for both the ANOVA and repeated measures MANOVA was significant (po.ool). Therefore, each trial was analyzed separately to determine the effects of endophyte treatment, mowing height, and time. For trial one (23 May 2007 through 5 June 2007), only time was significant (F=192.3; df=3, 36; p=0.00) in which black cutworm mortality increased from day 1 to day 14. For trial two (12 June 2007 through 25 June 2007), there was an endophyte treatment effect (F=17.9; df=l, 12; p=0.001). In addition, there was a time effect (F=406.7; df=3, 36; p=0.00), a time x endophyte treatment effect (F=52.2; df=3, 36;

9 108 p=0.00) and a time x endophyte x mowing height interaction (F=5.1; df 3, 36; p=0.005). More specifically, settling response was affected by endophyte treatment (p<0.01) and there was an endophyte treatment and mowing height interaction (p<0.05) (Figure 5-2). On day 7, mortality was affected by endophyte treatment and mowing height (p<0.03) (Figure 5-3). For trial three (26 June 2007 through 15 July 2007), there was a time effect (F=81.4; df=3, 36; p=0.00) and a time by endophyte treatment interaction (F=4.26; df=3, 36; p=0.01). Only settling response for this trial was affected by endophyte treatment (p<0.02). Correlation matrices were run to identify if ergot alkaloids affected black cutworm performance. Additional correlation matrices were also run to distinguish if black cutworm feeding affected antioxidant (SOD, CAT, and POD) activity. Ergot alkaloids, SOD, and CAT were found to be correlated with black cutworm settling response (p<0.05). As alkaloid concentration increased, settling response decreased (r=- 0.76; p-0.004) (Figure 5-4). Analysis of variance showed that alkaloid concentrations were affected by endophyte treatment on day 1 and 7 of all three trials and there was a mowing height effect on day 1 of trial two (Table 5-1). The E+ treatment had higher alkaloid concentrations ( ppm) than the E- treatment (Table 5-1). As for antioxidants, SOD (r=0.98; p=0.02) (Figure 5-5) and CAT (r=0.95, p=0.05) (Figure 5-6) were correlated with black cutworm settling response for trials two and three, respectively (Tables 5-4 and 5-5). By contrast, POD was not correlated in any trial (Table 5-2). The E- clippings had a greater percentage of black cutworms feeding on the leaf tissue causing the SOD and CAT concentrations to increase. Discussion As expected, time had an effect on overall black cutworm performance in which mortality increased as the number of days exposed to E+ or E- clippings increased. Williamson and Potter (1997) indicated that black cutworm larvae are relatively unaffected by the presence of fungal endophytes in plants. In this study, however, settling response in two out of the three trials was affected by endophyte. Previous insect feeding trials conducted in the laboratory with a variety of grass species have shown that

10 109 endophyte infection acts as a feeding deterrent (Hardy et al., 1985, Johnson et al., 1985) and reduced the survival, growth, and even developmental rates of feeding insects (Clay et al., 1985; Latch et al., 1985). In addition, this study showed a mowing height and treatment interaction for both settling response and black cutworm mortality in trail two. Bigelow and Richmond (2006) reported that after a 48 h settling period, black cutworm larvae showed a stronger preference for clippings mowed at 5 cm than 9 cm for three of the four turf-type cultivars in the study including 'Da Vinci'. Black cutworm survival was reduced by the 9 cm mowing height on two of the four cultivars during a five-day feeding study. In this study, black cutworm feeding on E- clippings showed a preference for those clippings mowed at 5 cm. In addition, this study shows that those black cutworms feeding on E+ (approx. 5 to 15%) clippings showed a preference for clippings mowed at 9 cm. Although this study did not include nitrogen treatments, insect growth rates are enhanced by increased nitrogen fertilization, but reduced through higher concentrations of higher toxic alkaloid levels (Krauss et al., 2007). The clippings from the E+, 9 cm mowing height treatment could contain higher concentrations of nitrogen, sugar, protein or amino-n attracting more black cutworms than those clippings from the E+, 5 cm mowing height treatment. Mowing or clipping removal has also been shown to reduce alkaloid content in tall fescue as a function of nonstructural carbohydrates that were channeled from alkaloid synthesis to production of leaf matter (Belesky and Hill, 1997). Salminen et al. (2003) found that decreased mowing frequency (biweekly vs. weekly mowing) enhances alkaloid production in tall fescue mowed at 5 cm. This indicates that small increases in alkaloid content could have a major impact on herbivore performance. In this study, mowing height had very little effect on ergot alkaloid concentrations (Table 5-1), however, alkaloid concentrations have been shown to be the basis of insect resistance (Clay, 1988). Endophyte-mediated alkaloids provide a chemical defense against insect herbivory (Prestidge et al., 1982; Siegel et al., 1987; Murphy et al., 1993; Porter 1994; Justus et al., 1997; Richmond et al., 2004) which may deter insect feeding (antixenosis), or reduce insect performance (antibiosis) (Braman et al., 2002; Bacon et al., 1997; Breen,

11 ). The types and concentrations of alkaloids present in E+ infected plants is likely an important determinant of how insects respond to endophyte-mediated defenses (Richmond and Bigelow, 2008). Some alkaloids are known feeding deterrents capable of eliciting strong behavioral responses from insects (Tanaka et al. 2005, Rowan et al. 1986). In this study, endophyte treatment, E+ or E-, had the greatest effect on ergot alkaloids and black cutworm settling response was negatively correlated with ergot alkaloid concentrations. Although only ergot alkaloids were analyzed in this study, this data suggests that ergot alkaloids are associated with reducing overall black cutworm performance. Insect feeding may also impact other chemical responses, such as antioxidant activity in the plant rather than the endophyte. In this study, we found that SOD and CAT activity increased in the E- clippings in trial two and three, respectively, as settling response increased. Heng-Moss et al. (2006) observed increases in both CAT and POD activity in resistant and susceptible cultivars of buffalograss in response to chinch bug feeding. Chaman et al., (2001) also found an increase in POD activity as aphid infestation increased. In this study, however, we did not observe POD activity in either the E+ or E- tall fescue in response to black cutworm feeding. However, SOD is the first line of defense against ROS by dismutating O2" to H2O2 (Bowler et al., 1992) which was observed in this study. Then H2O2 is decomposed by POD and CAT. Although POD was not observed in this study, CAT was however observed. Recent studies have provided new insights into how levels of ROS are controlled in the plant cells. This has raised questions about the relationship between ROS stress and the production and scavenging of ROS. Traditionally, ROS are considered to be toxic by-products produced when the plant is under stress, which are then disposed of using antioxidants. However, recent research has indicated that ROS may play a central role in the defense of plants against pathogen attack activating plant cell death (Mittler, 2002). Typically, antioxidants scavenge and detoxify ROS to prevent cell damage. Instead, it is believed that antioxidants suppressed (ascorbate peroxidase and CAT) by plant hormones, salicylic acid and nitric oxide, resulting in the over-accumulation of ROS and the activation of plant cell death (Klessig, 2000). The differences in the function of ROS between biotic

12 Ill and abiotic stress might result from the action of hormones, from cross-talk between different signaling pathways, or from differences in the steady-state level of ROS produced during the different stresses (Mittler, 2002). This may explain the differences we observed in antioxidant activity in this study since pathogens and insect herbivory are both considered biotic stresses. The biochemical and physiological function of these oxidative enzymes and their role in the plant's defense response remains unclear. Therefore, future research should further investigate insect-plant interactions to determine what role antioxidants play in plant resistance to insect feeding. Compared to E+ and E-, management practices, such as differences in mowing height, did not have as much of an impact on overall black cutworm performance. Therefore, E+ grasses should be planted wherever insect feeding is a concern as a pest management tool to reduce overall insecticide use.

13 112 References Abernethy, G.A. and M.T. McManus Biochemical response to an imposed water deficit in mature leaf tissue of Festuca arundinacea. Environ. Exp. Bot. 40: Anderson, W.G., T.M. Heng-Moss, and F.P. Baxendale Evaluation of cool- and warm-season grasses for resistance to multiple chinch bug (Hemiptera: Blissidae) Species. J. Econ. Entomol. 99(l): Arachevaleta, M., C.W. Bacon, and C.S. Hoveland Effect of the tall fescue endophyte on plant response to environmental stress. Agron. J. 81: Bacon, C.W. andn.s. Hill Symptomless grass endophytes: Products of coevolutionary symbioses and their role in the ecological adaptation of infected grasses, p In S.C. Redlin and L.M. Carris (ed.) Systematics, ecology and evolution of endophytic fungi of grasses and woody plants. Am. Phytopathol. Soc, St. Paul, MN. Bacon, C.W., J.K. Porter, J. Robbins, and E.S. Luttrell Epichloe typhina from toxic tall fescue grasses. Appl. Environ. Microbiol. 34: Bacon, C.W., M.D. Richardson, and J.F. White, Jr Modification and uses of endophyte enhanced turfgrasses: A role for molecular technology. Crop Sci. 37: Bacon, C.W., P.C. Lyons, J.K.Porter, and J.D. Robbins Ergot toxicity from endophyte-infected grasses: a review. Agron. J. 78: Ball, O.J.P., G.M. Barker, R.A. Prestidge, and D.R. Lauren Distribution and accumulation of the alkaloid peramine in Neotyphodium /o///-infected perennial ryegrass. J. Chem. Ecol. 23: Belesky, D.P. and N.S. Hill Defoliation and leafage influence on ergot alkaloids in tall fescue. Ann. Bot. 79: Bigelow, C.A. and D.S. Richmond Cultivars, cutting heights affect black cutworm feeding. TurfGrass TRENDS, p. 48, 52, 54. Boning, R.A. and T.L. Bultman A test for constitutive and induced resistance by tall fescue {Festuca arundinacea) to an insect herbivore: Impact of the fungal endophyte, Acremonium coenophialum. Am. Midi. Nat. 136:

14 113 Bowler, C, M.V. Montagu, and D. Inze Superoxide dismutase and stress tolerance. Ann. Rev. Plant Physiol. Plant Mol. Biol. 43: Bradford, M.M A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of a protein-dye binding. Anal. Biochem. 72: Braman, S.K., R.R. Duncan, M.C. Engelke, W.W. Hanna, K. Hignight, and D. Rush Grass species and endophyte effects on survival and development of fall armyworm (Lepidoptera: Noctuidae). J. Econ. Entomol. 95(2): Breen, J.P Acremonium endophyte interactions with enhanced plant resistance to insects. Annu. Rev. Entomol. 39: Chaman, M.E., L.J. Corcuera, G.E. Zuniga, L. Caedemil, and V.H. Argandona Induction of soluble and cell wall peroxidases by aphid infestation in barley. J. of Agricultural and Food Chemistry. 49: Clarke, B.B., J.F. White, Jr., R.H. Hurley, M.S. Torres, S. Sun, and D.R. Huff Endophyte-mediated suppression of dollar spot disease in fine fescues. Plant Disease. 90: Clay, K Fungal endophytes of grasses: A defensive mutualism between plants and fungi. Ecology. 69(1): Clay, K Fungal endophytes of grasses. Annu. Rev. Ecol. Syst. 21: Clay K., T.N. Hardy, and A.M. Hammond, Jr Fungal endophytes of grasses and their effects on an insect herbivore. Oecologia. 66:1-6. Clay, K., S. Marks, and G.P. Cheplick Effects of insect herbivory and fungal endophyte infection on competitive interactions among grasses. Ecology 74(6): Dahlman, D.L., H. Eichenseer, and M.R. Siegel Chemical perspectives on endophyte grass interactions and their implications to insect herbivory. In: Barbosa, P., Krischik, V.A., and C.G. Jones (eds), Microbial mediation of plantherbivore interactions. John Wiley and Sons, pp

15 114 Davidson, A.W. and D.A. Potter Response of plant-feeding, predatory, and soilinhibiting invertebrates to Acremonium endophyte and nitrogen fertilization in tall fescue turf. J. Econ. Entomol. 88(2): de Sassi, C, C.B. Miiller, and J. Krauss Fungal plant endosymbionts alter life history and reproductive success of aphid predators. Proc. R. Soc. B. 273: Dowd, P. and L.M. Lagrimini The role of peroxidase in host insect defense. Pp In N. Carozzi and M. Koziel (eds.) Advances in insect control. Taylor & Francis, London. Elbersen, H.W. and C.P. West Growth and water relations of field-grown tall fescue as influenced by drought and endophyte. Grass Forage Sci. 51: Elmi, A.A. and C.P. West Endophyte infection effects on stomatal conductance, osmotic adjustment and drought recovery of tall fescue. New Phytol. 131: Felton, G.W., C.B. Summers, and A.J. Mueller Oxidative responses in soybean foliage to herbivory by bean leaf beetle and three-corned alfalfa leafhopper. J. Chem. Ecol. 20: Fry, J. and B. Huang Applied turfgrass science and physiology. John Wiley and Sons, Inc., Hoboken, NJ. Funk, C.R., P.M. Halisky, S. Ahmad, and R.H. Hurley How endophytes modify turfgrass performance and response to insect pests in turfgrass breeding and evaluation trials. Pages In Proc. Int. Turfgrass Research Conf. 5 th. F. Lemarie, ed. Giannopolities, C.N. and S.K. Rise Superoxide dismutases I. Occurrence in higher plants. Plant Physiol. 59: Gwinn, K.D., M.H. Collins-Shepard, and B.B. Reddick Tissue print-immunoblot, an accurate method for detection of Acremonium coenophialum in tall fescue. Phytopathology. 81: Gwinn, K.D. and A.M. Gavin Relationship between endophyte infestation level of tall fescue seedlots and Rhizoctonia zeae seedling disease. Plant Dis. 76:

16 115 Hardy, T.N., K. Clay, and A.MJ. Hammond Fall armyworm (Lepidoptera: Noctuidae): a laboratory bioassay and larval preference study for the fungal endophyte of perennial ryegrass. J. Econ. Entomol. 78: Heng-Moss, T.M., F.P. Baxendale, T.E. Eickhoff, and R.C. Shearman Physiological and biochemical responses of resistant and susceptible buffalograss to chinch bug feeding. USGA Turfgrass and Environmental Research Online. Hiatt, E.E.I, and N.S. Hill Neotyphodium coenophialum mycelial protein and herbage mass effects on ergot alkaloid concentration in tall fescue. J. Chem. Ecol. 23: Hildebrand, D.F., J.G. Rodriguez, G.C. Brown, K.T. Luu, and C.S. Volden Peroxidative responses to leaves in two soybean genotypes injured by twospotted spider mites (Acari: Tetranychidae). J. Econ. Entomol. 79: Hill, N.S. and C.S. Agee Detection of ergopeptine alkaloids in endophyte-infected tall fescue populations by immunoassay. Crop Sci. 34: Hill, N.S., D.P. Belesky, and W.C. Stringer Competitiveness of tall fescue as influenced by endophyte. Crop Sci. 31: Hill, N.S., D.P. Belesky, and W.C. Stringer Encroachment of endophyte on endophyte-free tall fescue. Ann. Bot. 81: Hunt, M.G. and J.A. Newman Reduced herbivore resistance from a novel grassendophyte association. J. Appl. Ecol. 42: Johnson, M.C., D.L. Dahlman, M.R. Siegel, L.P. Bush, and G.C.M. Latch Insect fedding deterrents in endophyte-infected tall fescue. Appl. Environ. Microbiol. 49: Kimmons, C.A., K.D. Gwinn, and E.C. Nernards Nematode reproduction on endophyte-infected and endophyte free tall fescue. Plant Dis. 74: Klessig, D.F Nitric oxide and salicyclic acid signaling in plant defense. Proc. Natl. Acad. Sci. U.S.A. 97:

17 116 Kochhar, S., V.K. Kochhar, and S.D. Khanduja Changes in the pattern of isoperoxidases during maturation of grape berries cv. Gulabi as effected by ethephon. (2-chloroethyl phosphoric acid). Am. J. Enol. Vitic. 30: Krauss, J., S.A. Hani, L. Bush, R. Husi, L. Bigler, S.A. Power, and C.B Muller Effects of fertilizer, fungal endophytes and plant cultivar on the performance of insect herbivores and their natural enemies. Functional Ecology. 21: Lane, G.A, MJ. Christensen, and CO. Miles Coevolution of fungal endophytes with grasses: The significance of secondary metabolites, pp In C.W. Bacon and J.F. White, Jr. (ed.) Microbial endophytes. Marcel Dekker, Inc., New York, NY. Latch, G.C.M Physiological interactions of endophytic fungi and their hosts. Biotic stress tolerance imparted to grasses by endophytes. Agric. Ecosyt. Environ. 44: Latch, G.C.M., MJ. Christensen, and D.L. Gaynor Aphid detection of endophytic infection in tall fescue. NZ J. Agric. Res. 28: Lyons, P.C, R.D. Plattner, and C.W. Bacon Occurrence of peptide and clavinet ergot alkaloids in tall fescue grass. Science. 232: Malinowski, D.P. and D.P. Belesky Neotyphodium coenophialum-endophyte infection affects the ability of tall fescue to use sparingly available phosphorus. J. Plant Nutr. 22: Malinowski, D.P. and D.P. Belesky Adaptations of endophyte-infected coolseason grasses to environmental stresses: Mechanisms of drought and mineral stress tolerance. Crop Sci: 40: Marks, S., K. Clay, and G.P. Cheplick Effects of fungal endophytes on interspecific and intraspecific competition in the grasses Festuca arundinacea and Lolium perenne. J. Appl. Ecol. 28: Meister, B., J. Krauss, S.A Harri, M.V. Schneider, and C.B. Muller Fungal endosymbionts affect aphid population size by reduction of adult life span and fecundity. Basic Appl. Ecol. 7:

18 117 Miller, H., D.R. Porter, J.D. Burd, D.W. Mornhinweg, and R.L. Burton Physiological effects of Russian wheat aphid (Homoptera:Aphididae) on resistant and susceptible barley. J. Econ. Entomol. 87: Mittler, R Oxidative stress, antioxidants and stress tolerance. TRENDS in Plant Science. Vol.7 No.9, September. Mohr, M.M., W.A. Meyer, and C. Mansue Incidence of Neotyphodium endophyte in seed lots of cultivars and selections of the 2001 National Tall Fescue Test. p. [ ]. In 2002 Rutgers Turfgrass Proceedings. Atlantic City, New Jersey: December 10-12, New Brunswick, NJ. Murphy J.A, S. Sun, and L.L. Betts Endophyte-enhanced resistance to billbug (Coleoptera: Curculionidae), sod webworm (Lepidoptera: Pyralidae), and white grub (Coleoptera: Scarabeidea) in tall fescue. Environ. Entomol. 22(3): Nayyar, H. and S. Chander Protective effects of polyamines against oxidative stress induced by water and cold stress in chickpea. J. Agron. Crop Sci. 190: Popay, A.J. and S.A. Bonos Biotic responses in endophytic grasses, pp In: Neotyphodium in cool season grasses. Eds. Roberts, C.A., C.P.West, D.E. Spiers, Blackwell Publishing, Ames, IA, U.S.A. Porter, J.K Chemical constituents of grass endophytes. pp In C.W. Bacon and J.F. White (ed.) Biotechnology of endophytic fungi of grasses. CRC Press, Boca Raton, FL. Prestidge, R.A., R.P. Pottinger, and G.M. Barker An association of Lolium endophyte with ryegrass assistance to Argentine stem weevil. Proc. 35 th Conf. N.Z. Weed Pest Control Conf., 35: Ravel, C, G. Charmet, and F. Balfourier Influence of the fungal endophyte Acremonium lolii on agronomic traits of perennial ryegrass in France. Grass Forage Sci. 50: Read, J.C. and BJ. Camp The effect of fungal endophyte Acremonium coenophialum in tall fescue on animal performance, toxicity, and stand maintenance. Agron. J. 78:

19 118 Richmond, D.S Mediation of herbivore-natural enemy interactions by Neotyphodium endophytes: The role of insect behavioral response. In: Proceedings of the 6 th International Symposium on Fungal Endophytes of Grasses. Christchurch, New Zealand. March 25-28, Eds. Popay, A.J. and E.R.Thom Richmond, D.S. and C.A. Bigelow (In Review) Variation in Endophyte-Plant Associations Influences Interactions between Black Cutworm (Lepidoptera: Noctuidae) and the Parasitic Nematode Steinemema carpocapsae. Environ. Entomol. Richmond, D.S. and DJ. Shetlar Larval survival and movement of bluegrass webworm in mixed stands of endophytic perennial ryegrass and Kentucky bluegrass. J. Econ. Entomol. 92(6): Richmond, D.S. and DJ. Shetlar Hairy chinch bug (Hemiptera: Lygaeidae) damage, population density, and movement in relation to the incidence of perennial ryegrass infected by Neotyphodium endophytes. J. Econ. Entomol. 93(4): Richmond, D.S. and DJ. Shetlar Black cutworm (Lepidoptera: Noctuidae) larval emigration and biomass in mixtures of endophytic perennial ryegrass and Kentucky bluegrass. J. Econ. Entomol. 94(5): Richmond, D.S., H.D. Niemczyk, and D J. Shetlar Overseeding endophytic perennial ryegrass into stands of Kentucky bluegrass to manage bluegrass billbug (Coleoptera: Curculionidae). J. Econ. Entomol. 93(6): Rowan, D.D Lolitrems, paxilline and peramine: mycotoxins of the ryegrass/endophyte infection. Agric. Ecosyst. Environ. 44: Rowan, D.D., M.B. Hunt, and D.L. Gaynor Peramine, a novel insect feeding deterrent from ryegrass infected with the endophyte Acremonium loliae. J. Chem. Soc. Chem. Comm. 1986:

20 119 Rowan, D.D. and G.M.C. Latch Utilization of endophyte-infected perennial ryegrass for increased insect resistance, pp In C.W. Bacon and J.F. White, Jr. (eds.), Biotechnology of endophytic fungi of grasses. CRC. Boca Raton, FL. Roylance, J.T., N.S. Hill, C.S. Agee Ergovaline and peramine production in endophyte-infected tall fescue: Independent regulation and effects of plant and endophyte genotype. J. Chem. Ecol. 20: Sairam, R.K. and D.C. Saxena Oxidative stress and antioxidants in wheat genotypes :possible mechanism of water stress tolerance. J. Agron. Crop Sci. 184: Salminen, S.O., P.S. Grewal, and M.F. Quigley Does mowing height influence alkaloid production in endophytic tall fescue and perennial ryegrass? J. Chem. Ecol.29(6): Siegel, M.R. and L.P. Bush Defensive chemicals in grass-fungal endophyte associations. Recent Advances in Phytochemistry. 30: Siegel, M.R., G.C.M. Latch, and M.C. Johnson Fungal endophytes of grasses. Annu. Rev. Phytopathol. 25: Sun, S., B.B. Clarke, and C.R. Funk Effects of fertilizer and fungicide applications on choke expression and endophyte transmission in Chewings fescue. Pages in: Proc. Int. Sympos. Acremoniuml'Grass Interact. S.S. Quisenberry and R.E. Joost, eds. Louisiana Agric. Exp. Stn., Baton Rouge, LA. Tanaka, A., B. A. Tapper, A. Popay, E. J. Parker, and B. Scott A symbiosis expressed non-ribosomal peptide synthetase from a mutualistic endophyte of perennial ryegrass confers protection to the symbiotum from insect herbivory. Mol. Microbiol. 57: Wang K. and Y. Jiang Antioxidant responses of Creeping bentgrass roots to waterlogging. Crop Sci. 47: Wilkinson, H.H., M.R. Siegel, J.D. Blankenship, A.C. Mallory, L.P. Bush, and C.L. Schardl Contribution of fungal loline alkaloids to protection from aphids in a grass endophyte mutualism. Mol. Plant-Microbe Interact. 13:

21 120 Williamson, R.C. and D.A. Potter Turfgrass species and endophyte effects on survival, development and deeding preference of black cutworms (Lepidoptera: Noctuidea). J. Econ. Entomol. 90: Zhang, Y.P. and Z.B. Nan Growth and anti-oxidative systems changes in Elymus dahuricus is affected by Neotyphodium endophyte under contrasting water availability. J. Agron. Crop Sci. 193:

22 121 Table 5-1. Ergot alkaloids (ppm) as affected by endophyte treatment (E+ versus E-) and mowing height (5 and 9 cm) for day 1 and 7 for all three trials. Ergot Alkaloids Trial 1 Trial 2 Trial 3 Treatment Mowing Height Day 1 Day 7 Day 1 Day 7 Day 1 Day 7 E+ E- cm a 3.3 b 0.9 c 0.5 c 4.5 a 4.3 a 1.7 b 1.6b 4.8 a 3.6 a 1.8 b 1.1b -ppm 4.0 a 4.0 a 0.9 b 0.7 b 3.4 a 3.7 a 0.5 b 0.6 b 2.5 a 2.9 a 0.7 b 0.5 b ANOVA Height (H) Treatment (T) HxT NS *** NS NS ** NS * *** NS NS ** NS NS *** NS Means in the same column followed by the same letter are not significantly different according to Fisher's protected LSD t-test (P=0.05). *, **, *** and NS refer to significant at the 0.05, 0.01, level and non-significant, respectively. There were three independent feeding trials: 23 May 2007 through 5 June 2007,12 June 2007 through 25 June 2007, and 26 June 2007 through 15 July NS *** NS

23 122 Table 5-2. Black cutworm performance (settling response and mortality) correlations relative to ergot alkaloid, superoxide dismutase (SOD) catalase (CAT), and peroxidase (POD) concentrations on day 1 and 7 for all three trials. Ergot alkaloids Day 1 Day 7 Day 1 SOD Trials 1-3 Day 7 Day 1 CAT Day 7 Day 1 POD Day 7 Trial Settling response Mortality day Mortality day Bold faced correlations indicate significance at p< Not applicable

24 123 Table 5-3. Black cutworm performance (settling response and mortality) correlations relative to antioxidant activity (superoxide dismutase, SOD; catalase, CAT; and peroxidase, POD) on day 1 and 7 for trial 1. Settling response Mortality day 1 Trial 1 SOD CAT POD Day 1 Day 7 Day 1 Day 7 Day 1 Day Mortality day Bold faced correlations indicate significance at p< Not applicable. -r value

25 124 Table 5-4. Black cutworm performance (settling response and mortality) correlations relative to antioxidant activity (superoxide dismutase, SOD; catalase, CAT; and peroxidase, POD) on day 1 and 7 for trial 2. Settling response Mortality day 1 Trial 2 SOD CAT POD Day 1 Day 7 Day 1 Day 7 Day 1 Day Mortality day Bold faced correlations indicate significance at p< Not applicable. -r value

26 125 Table 5-5. Black cutworm performance (settling response and mortality) correlations relative to antioxidant activity (superoxide dismutase, SOD; catalase, CAT; and peroxidase, POD) on day 1 and 7 for trial 3. Settling response Mortality day 1 Trial 3 SOD CAT POD Day 1 Day 7 Day 1 Day 7 Day 1 Day Mortality day Bold faced correlations indicate significance at p< Not applicable. -r value

27 126 D7 Time (days) D14 Figure 5-1. Black cutworm mortality (%) as affected by time on day 1, 7, and 14 for all three trials in 2007.

28 E+ IE- 70 $^ v 60 CO o 50 CO CD D) 40 t J [ ] CD CO c) c> Mowing Height (cm) Figure 5-2. Settling response (%) as affected by endophyte treatment (E+ and E-) and mowing height (5 and 9 cm) for trial two in 2007.

29 T E+ 151 E- 70 ) 65 c O CD 60 [ 1 > Mowing Height (cm) Figure 5-3. Black cutworm mortality (%) as affected by endophyte treatment (E+ and E-) and mowing height (5 and 9 cm) on day 7 for trial two in 2007.

30 [r = , p = E+, 5 cm E-, 5 cm \ E+, 9 cm E-, 9 cm f 40 ico Ergot Alkaloids (ppm) Figure 5-4. Settling response (%) as affected by ergot alkaloids for all three trials in 2007.

31 >< E+, 5 cm \ E-, 5 cm "V^ E+, 9 cm \ E-, 9 cm r = , p = (0 I 60 ID). 40 CO SOD ( units mg' 1 protein) Figure 5-5. Superoxide dismutase (SOD) activity as affected by settling response (%) for trial two in 2007.

32 "^ E+, 5 cm ^'N. E-, 5 cm ^ E+, 9 cm \ E-, 9 cm r = , p = S 60 I I.E 40-4 «i ,-1-1 CAT ( jmol min mg protein) Figure 5-6. Catalase (CAT) activity as affected by settling response (%) for trial three in 2007.