Several insect species were sensitive to N-formylloline, a pyrrolizidine alkaloid found in significant concentrations in tall fescue (Festuca arundinacea L.) infected with Neotyphodium coenophialum (= Acremonium coenophialum) Glenn, Bacon et Hanlin. Topical or injected doses were toxic in a dose dependent manner. Acute doses resulted in quick incapacitation; lower doses caused behavioral changes, uncoordinated movements, tremors, etc. Insects tested included green bug, bird cherry-oat aphid, large milkweed bug, spined soldier bug, American cockroach, house fly, face fly, cat flea and Japanese beetle

Bush, L P

Dahlman, D L

Siegel, M R

English

University of Kentucky

Session 13 - Constraints on Forage and Grassland Production 13-5ABSTRACTSeveral insect species were sensitive to N-formylloline, apyrrolizidine alkaloid found in significant concentrations in tall fescue(Festuca arundinacea L.) infected with Neotyphodium coenophialum(= Acremonium coenophialum) Glenn, Bacon et Hanlin. Topical orinjected doses were toxic in a dose dependent manner. Acute dosesresulted in quick incapacitation; lower doses caused behavioralchanges, uncoordinated movements, tremors, etc. Insects testedincluded green bug, bird cherry-oat aphid, large milkweed bug, spinedsoldier bug, American cockroach, house fly, face fly, cat flea andJapanese beetle.KEYWORDSTall fescue, N-formyl loline, Acremonium coenophialum, insectsAcronyms: EI = endophyte-infectedINTRODUCTIONIt is widely accepted that endophytes are common inhabitants ofgrasses. Forage and turf varieties of endophyte-infected (EI) perennialryegrass and EI tall fescue are resistant to many insects naturallyexposed to EI plant material (Dahlman et al., 1991; Breen, 1994).Here we report on the insecticidal activity of N-formylloline, apyrrolizidine alkaloid found in significant concentrations in EI tallfescue, but not found in endophyte-free plants.MATERIALS AND METHODSTests were conducted with bird cherry-oat aphid, Rophalosiphumpadi (L.); greenbug, Schizaphis graminum (Rondani); large milkweedbug, Oncopeltus fasciatus (Dallas); spined soldier bug, Podisusmaculiventris (Say); American cockroach, Periplaneta americana(L.); insecticide susceptible strains of house fly, Musca domesticaL. and face fly, Musca autumnalis De Geer; cat flea, Ctenocephalidesfelis (Bouché) and Japanese beetle, Popillia japonica Newman.For the test of sprayed plants, 2-3 individual plants of tall fescue(endophyte-free, Mustang variety) were carefully separated intoramets and placed in assay cages described by Siegel et al. (1990).Ramets were sprayed to run off with 1.2 ml and allowed to dry beforeplacement of cages and 50 alate aphids. Trypan blue (0.1%) wassprayed on test ramets using identical conditions. After drying, thedye was washed from the ramet and the concentration estimated byits absorption at 590 nm. We estimate that 2-4% of the total materialwas held on the ramet.The loline preparation contained 180 mg of N-formylloline/ml in3.67 M formic acid. All controls contained the same amounts offormic acid even though dilutions were so large that effects of formicacid were negligible. Loline was provided to milkweed bug nymphsvia cotton pad containing water (Johnson et al., 1985). Amicroapplicator was used for all topical applications and injections.Glass petri dishes (15 cm dia) were treated with acetone solutions ofN-formyl loline prior to placement of individual adult cockroaches.Twenty-five cat flea eggs were placed on 5 cm dia filter paper discswhich had been treated with the acetone solutions of test chemicals.The discs were held in 5 cm dia glass petri dishes at 80% relativehumidity for 30 days, after which the number of adult fleas wasdetermined.RESULTS AND DISCUSSIONA marked increase in mortality of newly hatched milkweed bugswas observed between N-formylloline concentrations of 7.5 and 30µg/ml. The insecticide, methomyl, was 10-fold more toxic than eitheracephate or carbaryl when evaluated using the same experimentalconditions. We estimate that N-formylloline was 30-50 times lesstoxic than methomyl.Topical and injected applications of N-formylloline to adult milkweedbugs produced dose-dependent responses ranging from almostimmediate paralysis to no effect. Intermediate responses were typifiedby spasmodic and uncoordinated movements. Greater than 90% werekilled or incapacitated with topical doses ž2.5 µg within the first 24h (Table 1). Response to injected N-formylloline was less acute eventhough the bugs were rapidly knocked down (Table 1). Perhaps theN-formylloline is either rapidly metabolized and/or sequestered, thusreducing the absolute amount of available toxicant.Schizaphis graminum was more sensitive to N-formylloline than R.padi.  The 3 day LC50 was 9.3 µg /ml for S. graminum while the LC50for R. padi was >37.5 µg/ml (Table 2). Based on our dye solutionprocedure, we estimate the approximate LC50 of N-formylloline forS. graminum and R. padi to be 0.37 and 1.5 µg/ml, respectively.An injection of as little as 0.78 µg N-formylloline/adult (ca. 16 ppm)produced an immediate and predictable response in the spined soldierbug, a predacious homopteran. The insect extended its proboscis,then its wings, tilted from side to side for a few seconds, then fellover. Most were immobile within a few minutes and >90% weredead within 24 h.Adult American cockroaches placed in glass petri dishes coated withN-formylloline responded within 4-6 h with uncoordinated and jerkymovements, ultimately lying on their backs and twitching. Affectedinsects did not recover. Ten µg/cm2 caused 73% mortality at 24 hwhile 5 µg/cm2 gave 27% mortality. No mortality was observed withacetone alone or with 8.88 µg/cm2 formic acid, the amount presentin the highest concentration of N-formylloline used in the test.A dose of 50 µg/cm2 caused 95% mortality with the cat flea but 5µg/cm2 only caused 15% mortality. When filters originally treated atthe higher dose were re-evaluated for residual effects 48 days later,only 15% corrected mortality was obtained. Thus there was nosignificant residual effect.Topical application of 10 and 20 µg N-formylloline/Japanese beetlelarvae (165 and 330 ppm, respectively) caused 60-70% mortalitybut a dose of 5 µg had no effect.Topical applications of 10 µg N-formylloline/house fly (ca. 645 ppm)caused less than 20% mortality after 24 h. A topical dose of 8 µg/face fly caused 56% corrected mortality after 24 h.Several species of insects from different orders were sensitive on N-formylloline and similar symptoms of toxicity were observed. Insectswere quickly incapacitated, although not immediately killed by acutedoses. At lower doses the insects demonstrated various behavioralchanges, uncoordinated movements, tremors, etc.A substantial amount of additional work will be required before themode of action and practical use of N-formylloline will be elucidated.Meanwhile, it should be emphasized that N-formylloline, as well asID NO. 849INSECTICIDAL ACTIVITY OF N-FORMYLLOLINED.L. Dahlman1, M.R. Siegel2 and L.P. Bush31Dept. Entomology, 2Dept. Plant Pathology, 3Dept. AgronomyUniversity of Kentucky, Lexington, KY 40546-0091 USASession 13 - Constraints on Forage and Grassland Production 13-6other toxic alkaloids produced either by the endophyte or the plantin response to endophyte infection, makes a significant contributionto the success of the plant in resisting insect attack. Therefore,elimination of endophyte from potential forage crops to render themmore favorable for large animal consumption may also removesources of insect resistance.REFERENCESAbbott, W.S. 1925. A method for computing the effectiveness of aninsecticide. J. Econ. Entomol. 18: 265-267.Breen, J.P. 1994. Acremonium endophyte interactions with enhancedplant resistance to insects. Ann. Rev. Entomol. 39:401-423.Dahlman, D.L., H. Eichenseer and M.R. Siegel. 1991. Chemicalperspectives on endophyte-grass interactions and their implicationsto insect herbivory. Pages 227-252 in P. Barbosa, V.A. Krischik andC.G. Jones, eds. Microbial mediation of plant-herbivore interactions.Wiley, N.Y.Johnson, M.D., D.L. Dahlman, M.R. Siegel, L.P. Bush, G.C.M.Latch, D.A. Potter and D.R. Varney. 1985. Insect feeding deterrentsin endophyte-infected tall fescue. Appl. & Envir. Microbiol. 49: 568-571.Siegel, M.R., G.C.M. Latch, L.P. Bush, F.F. Fannin, D.D. Rowan,B.A. Tapper, C.W. Bacon and M.C. Johnson. 1990. Fungalendophyte-infected grasses: alkaloid accumulation and aphidresponse. J. Chem. Ecol. 16: 3301-3315.Paper 96-08-115 of the Kentucky Agricultural Experiment Station,Lexington, KY.Table 1One day Oncopeltis fasciatus adult response to topical and injectedapplications of N-formylloline.aTopical Conc. %  Mortalityb Injected Conc. % Mortalityc(µg/bug) (µg/bug)10.00 97.5 25.00 957.50 97.5 12.5 385.00 90.0 6.25 282.50 92.5 3.12 331.00 57.5 1.56 150.50 40.0 1.04 80.25 0 0.78 3 a 40 adult insects/treatment. b Values adjusted for morbidity and mortality in water control(Abbott,1925).   No mortality in control during test. c Values adjusted for mortality in water control (Abbott, 1925).Mortality in water injected controls after 1 day = 2.5%.Table 2Three day mortality of Schizaphis graminum and Rophalosiphumpadi on endophyte-free tall fescue ramets sprayed with N-formylloline.aCorrected Percent MortalitycLoline Conc. (µg/ml)b R. padi S. graminum150.0 87.5 no data75.0 72.5 89.237.5 32.6 71.318.7 13.2 57.49.3 2.5 52.34.7 no data 21.6a   Six replications/treatment, approximately 50 aphids/replicationb   Each plant received 1.2 ml of solution, sprayed to run off, 2-4%of which was estimated to remained on the plant.c   Values adjusted for mortality in control (Abbott, 1925).Mortality in control for S. graminum = 9.9%, for R. padi = 3.5%.

Insecticidal Activity of N-Formylloline

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Insecticidal Activity of N-Formylloline

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