中国科技论文在线 http://www.paper.edu.cn Available online at www.sciencedirect.com Agricultural Sciences in China SCIENCE @DIRECT* 2006, 5(10): 793-797 October 2006

Seasonal Change of Loline in -Infected Meadow Fescue

TONG De-wen[-2,WANG Jin-yil, Brain Patchettz and Ravi Gooneratne2

1 College of Animal Science and Technology, Northwest A&F University, Yangling 712100, P. R. China ZAgriculture and Life Sciences Division, Lincoln Universiiy, P.O. Box 84, Canterbury, New Zealand

Abstract

Lolines are a group of saturated pyrrolizidine alkaloids that possess broad bioactivity against a wide array of herbivorous insects. However, they do not exhibit toxicity to ruminants such as cattle and sheep. In order to study the direct and potential physiological effects on ruminants and the mechanism of insecticidehsectifuge, the distribution of loline alkaloids in endophyte-infected meadow fescue and the seasonal change of the distribution were analyzed. The crowns, roots and leaves of endophyte-infected meadow fescue at its four different growth periods, i.e., spring, summer, early autumn and late autumn, in New Zealand were colleted. After powdering, organic solvent extraction and purification by column chromatography, all loline samples were analyzed by capillary gas chromatography with 4-phenylmorpholine (PM) as an internal standard. The analytic results showed that the loline contents in the roots, crowns and leaves of endophyte-infected meadow fescue vary with seasons. Even within the same season, the distribution of lolines in endophyte-infected meadow fescue varies. During summer, lolines mainly existed in the leaves and roots, but in early autumn, they are produced in the crowns. It was concluded that, lolines were mainly produced in the leaves and roots of endophyte-infected meadow fescue. In gas chromatographic analysis, N-formylloline, the major component of loline alkaloid in the plant, was employed to assay the alkaloids.

Key words: Festuca pratensis Huds., loline alkaloids, gas chromatography

In many temperate pasture grasses infected by INTRODUCTION endophyte, such as tall fescue and meadow fescue in- fected with Acremonium uncinatum, loline alkaloids can Loline alkaloids (Powell and Petroski 1992a, b) are a accumulate to levels up to 2% plant dry matter. The group of pyrrolizidine alkaloids that have an unusual investigation of loline alkaloids in pasture grasses was structure, containing three heterocyclic rings in a first conducted after cattle grazing on Festuca strained arrangement. The loline group of pyrrolizidines arundinacea, in which a development lameness known is comprised of saturated 1-amino pyrrolizidines with as “fescue foot”, and subsequent reports of abdominal various substitutes on the 1-amino group and an oxy- fat necrosis and increased respiration were followed gen bridge between C, and C,. Naturally, lolines occur (Paul et al. 2001). in endophyte-infectedLolium cuneatum (Dannhardt and Loline alkaloids are structurally unique saturated Steindl 1985), (Richard and pyrrolizidines containing an amino substituent at C- 1 Donald 2003), Adenocarpus decorticans (Richard and and an linkage between C-2 and C-7; they do not Donald 2003) and Argyreia mollis (Tofen et al. 1999). appear to occur naturally as N-oxides. Lolines possess

~ ~ ~ Received 14 March, 2006 Accepted 12 September, 2006 TONG De-wen, Associate Professor, Ph D, Tel: +86-2947091192, E-mail: tdw41149nwsuaf.edu.cn

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7 94 TONG De-wen et al. broad bioactivity (Adriana and Alicia 1997) that pro- ergovaline, peramine and lolitrem B, and tall fescue give vides their host plantdgrasses with biological protec- rise to ergovaline, peramine and loline (Fig.2). tion from herbivorous insects. More importantly, they Ergovaline and lolitrem B are toxic to ruminants. do not cause classical pyrrolizidine toxicity since they By employing capillary gas chromatography, the dis- lack the requisite unsaturation between C-1 and C-2 tribution of lolines in the roots, crowns and leaves, and and the esterified acid moiety (TePaske et al. 1993). the fluctuation of the distribution during its four growth Studying on loline distribution in grasses and its bio- periods were analyzed and the results are discussed in logical activity is a very effective approach to improve this paper. the quality of pasture grasses, as well as to protect grasses from insects. The biosynthesis of lolines in MATERIALS AND METHODS grasses was studied by Spiering et al. (2002). Paul et al. (2001) synthesized (+)-loline via a pathway that employed intramolecular [4+2] cycloaddition of an Plant material acy lnitrosodiene. Meadow fescue (Festuca pratensis Huds.) (Justus et Infected meadow fescue (F. pratensis, Fig.3) plants al. 1997), one of the prominent pasture grasses that and seeds were either collected in New Zealand, or were grow under cool, moist conditions, and tolerates wet artificially infected with (Neotyphodiurn and occasionally flooded soils, was infected by the en- uncinaturn) via meristem wounding of 1-week-old seed- dophytic , Neotyphodiurn uncinatum (Petroski lings using the method of Latch and Christensen (Siege1 et al. 1990) in New Zealand and produces its related et al. 1990). Endophytes, isolated through the use of alkaloids. In contrast to the other two main pasture potato dextrose agar, were identified as Neotyphodium grasses (perennial ryegrass and tall fescue) (McLeod uncinaturn (Fig.3). et al. 2001) in New Zealand, endophyte-infected Meadow fescue plants were grown in 10-cm plastic meadow fescue only produced loline alkaloids, such as pots in a greenhouse (winter 17-21°C, summer 24- N-formylloline, N-acetylnorloline, N-methylloline, N- 28°C) and maintained by dividing older plants and acetylloline, norloline and N-formylnorloline (Fig. 1). repotting them in a mixture of one part soil (Maury silt Endophyte-infected perennial ryegrass contains loam) and three parts PRO-MIX BR every four to six

R' Rz

Rt RZ N-R' N-formylloline (1) HC=o CH3 N-acelylnorloline (2) CH$=O H loline (3) CH3 H N-methylloline (4) CH3 CH3 N-acetylloline (5) CH3C=0 CH3 norloline (6) H H N-formylnorloline (7) HC=O H

Fig. 1 Loline alkaloids (saturated pyrrolizidine alkaloids).

Ergovaline Peramine Lolitrem B

Fig. 2 Ergovaline and peramine isolated from perennial ryegrass and tall fescue, and lolitrem B from perennial ryegrass.

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Seasonal Change of Loline Alkaloids in Endophyte-Infected Meadow Fescue 795

Fig. 3 Festuca pratensis and Neotyphodium uncinatum.

months. Most, but not all plants were routinely trimmed twice with 10 mL dichloromethane for 22-24 h. After to reduce excess foliage. Plants were fertilized biweekly removing the solid residues through a Whatman No. 41 in winter and weekly in summer with a dilute solution filter paper, the filtrate was treated with a 3-mL Baker of soluble fertilizer (317 mg L-I of N-P-K). column, which was prepared by 1 mL dichloromethane/ Meadow fescue samples used for the lolines analysis methanol (75:25, v/v). The extract filtrate (1.0 mL) were harvested at four different growth periods, i.e., was applied to the column, and then was eluted with 3 spring, summer, early autumn and late autumn. In or- mL dichloromethane/methane (75:25, v/v), 1 mL der to study the distribution of loline alkaloids in meadow dichloromethane/methanol/ NH,OH (75:25:0.05, v/v), fescue at its different growth periods, we also collected 3 mL methanol/ NH,OH (1005, v/v), respectively. All samples from different parts (i.e., leaf, root and crown) alkaloidal solutions were collected and combined of meadow fescue plants. By conjunction with the together, and then were kept in a refrigerator at 4°C previous labeling methods, the samples used for final prior to gas chromatography analysis. analysis were labeled as MI.,,MI., MIS, M,,, MZr, RH,.c, RH,.,, qr,RH,.c7 RH,.,, RH,.r and RH,.c9 Capillary GC analysis respectively. All the samples were air-dried at ambient tempera- Loline analysis was out in a Model 6890 gas ture and powdered to pass through a OS-mm mesh chromatograph (Hewlett-Packard, Avondale, PA, USA) and were stored at -20°C prior to extraction. equipped with a flame ionization detector. The injector was maintained at a temperature of 220°C and pres- Standards sure 115 kPa. Detector temperature was 250°C. The separation was done in fused-silica capillary wide-bore Standard Festuca pratensis bulk seed was used as a (0.53-mm) HP-1 columns 10-m, with a film thickness reference sample. 4-phenylmorpholine (PM) was pur- of 2.65 pm. Split-vent flow rates were 1 mL 18 s-I for chased from Aldrich Chemical Co., Milwaukee, WI, 5-m column. The initial oven temperature was 100°C. USA and used as an internal standard in gas chromato- After 2 min, a 6°C min-I gradient was applied to reach graphic analysis. 220"C, and this temperature was maintained for 30 min. The carrier gas was helium at 20 mL min-I. Hydrogen Alkaloid extraction flow rate was 30 mL min-', and air was 400 mL min-I. A volume of 1.5 pL of loline solution was injected into Ground meadow fescue material (1 .O g) and the inter- the GC system. The process was repeated three times nal standard, 4-phenylmorpholine (0.3 g) were extracted per sample.

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796 TONG De-wen et al.

RESULTS AND DISCUSSION phyte-infected meadow fescue. Fig. 5 presents the results of the distribution of lolines in endophyte-infected meadow fescue and the change After being infected by the endophyte, Neotyphodium in distribution at different growth periods obtained from uncinatum, meadow fescue will produce loline alkaloids. the gas chromatography analysis. In order to minimize In order to study the distribution of lolines in the plant and the fluctuation of the distribution within its growth periods, infected samples were analyzed by capillary PA:

gas chromatography. A typical gas chromatography 200- analysis is presented in Fig.4. GC analytic results 180- showed that endophyte-infected meadow fescue plants contain loline (l),N-methylloline (2), N-acetylnorloline 160- 4 (3), and N-formylloline (4).Identification of these peaks 140- was based on Powell and Petroski (1992a, b) and our previous GC-MS analysis. 120- 2 3 I ;! r: F 5 From the results above, N-formylloline (4) is the 0 10 15 20 major alkaloid in endophyte-infected meadow fescue. Fig. 4 CapillaryI GC analysis of an extract of endophyte-infected Thus, in the analysis, N-formylloline was used as a GC meadow fescue. S, 4-Phenylmorpholine (PM); 1, loline; 2, N- standard to evaluate the distribution of loline in endo- methylloline; 3, N-acetylnorloline; 4, N-formylloline.

-B- High loline content group -o- High loline content group A B 20~, --cLow loline content group 450 -Low loline content group 400 1 ;i 350 c 3 300 .ll $ 250 : 200 s 150 9 100 20 . 50 0. n Spring Summer Early autumn Late autumn Spring Summer Early autumn Late autumn

= Loline in the crowns = Loline in the leaves D 280 0Loline in the roots ' 220 T 260 200 . 240 180 220 160. $ 200 140. 180 160 120 ' '2 140 100. 3 5 120 80 . . 2 100 60 4 80 40 . 60 20. 2.

_" Spring Summer Early autumn Late autumn Spring Summer Early autumn Late autumn

Fig. 5 Seasonal change of loline alkaloids in endophyte-infected meadow fescue. A, N-formylloline in the crowns of meadow fescue during its growth periods; B. N- formylloline in the leaves of meadow fescue during its growth periods; C, N-formylloline in the rmts of meadow fescue during its growth periods; D, distribution of N- formylloline in meadow fescue during its growth periods.

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Seasonal Change of Loline Alkaloids in Endophyte-Infected Meadow Fescue 797

the analytical error, the GC results were separated into References a high loline content group and low loline content group Adriana C C, Alicia B P. 1997. Alkaloids from endophyte-infected (Fig.5). Festuca argentina. Journal of Ethnopharmacology, 57, 1. Fig.5-A shows the change of N-formylloline in the Dannhardt G, Steindl L. 1985. Alkaloids of temulentum: crowns of meadow fescue at its four growth periods, isolation, identification and pharmacological activity. Planta i.e., spring, summer, early autumn and late autumn, Medica, 3, 212-214. Justus M, Witte L, Hartmann T. 1997. Levels and tissue respectively. As presented in Fig.5, in spring, summer distribution of loline alkaloids in endophyte-infected Festuca and late autumn, the N-formylloline content in the pratensis. Phytochemistry, 44,5 1-57. crowns is very low, almost close to zero, but arrives at McLeod A R, Rey A, Newsham K K, Lewis G C, Wolferstan P. a maximum in early autumn. With the coming of late 2001. Effects of elevated ultraviolet radiation and endophytic autumn, N-formylloline content in the crowns begins fungi on plant growth and insect feeding in Loliumperenne, to decline. However, the maximum content of N- Festuca rubra, F. arundinacea and F. pratensis. Journal of formylloline in the leaves and roots of meadow fescue Photochemistry and Photobiology (B, Biology). Elsevier appears in summer (Fig.5-B, -C). In contrast to the Science S A, Lausanne, Switzerland, 62,97-107. content of N-formylloline in the crowns, the change of Paul R B, Kim S K, Volker K S, James D W. 2001. Asymmetric N-formylloline content in the leaves and roots is con- synthesis of (+)-loline, a from rye grass and tall fescue. Journul of Chemistry Society, 1,18317- 1832 1. siderable with the season. Petroski R J, Dombos D L, Powell R G. 1990. Germination and Fig.5-D exhibits the average distribution of N- growth inhibition of annual ryegrass (Lolium multiflorum L.) formylloline in endophyte-infected meadow fescue. and alfalfa (Medicago sativa L.) by loline alkaloids and From the bar chart, we know that only in early autumn, synthetic N-acylloline derivatives. Journal of Agricultural N-formylloline can be detected in the crown, and the arid Food Chemistry, 38, I7 16- 1718. amount of lolines in the crown within this period is the Powell R G, Petroski R J. 1992a. Alkaloids toxins in endophyte- highest in meadow fescue. The highest content of N- infected grasses. Nature Toxins, 2, 100-102. formylloline in the roots and leaves is in summer. In Powell R G, Petroski R J. 1992b. In the Alkaloids: Chemical and conclusion, N-formylloline mainly occurred in the leaves Biological Perspectives. New York: Springer-Verlag, 8,320- and roots of endophyte-infected meadow fescue during 326. the entire growth period. The content differences of N- Richard S G, Donald R D. 2003. (la,6a, 7a, 7ap)-2,3,5,6,7, 7a-hexahydro- 1,6-epoxy-IH-pyrrolizin-7-01. Acta formylloline in the leaves, roots and crowns indicate Crystallographica (Section E), 1, 1270- 127 1. that the function of the fungal endophyte Neotyphodium Siege1 M R, Latch G C M, Bush L P, Fannin F F, Rowan D D, uncinatum in the different parts of Festuca pratensis Tapper B A, Bacon C W, Johnson M C. 1990. Fungal varies with respect to season not the same during dif- endophyte-infected grass: alkaloids accumulation and ferent seasons. Studies on the mechanism and the bio- response. Journal of Chemical Ecology, 16, 3301. activity to ruminants are underway. Spiering M J, Wilkinson H H, Blankenship J D, Schardl C L. 2002. Expressed sequence tags and genes associated with Acknowledgements loline alkaloid expression by the fungal endophyte Neotyphodium uncinatum. Fungal Genetics and Biology, 36, We are grateful to Brian Patchett, Martin Wellby and 242. Dr. Ravi Gooneratne for the help in loline alkaloid ex- TePaske M R, Powell R G, Clement S L. 1993. Analyses of traction and gas chromatography analysis. This study selected endophyte-infected grasses for the presence of loline- was also partly supported by the National Natural Sci- type and ergot-type alkaloids. Journal of Agricultural and ence Foundation of China (30371067), Fok Ying Tung Food Chemistry, 41,2299-2303. Education Foundation, China (9 1033) and Foundation Tofen B, Kaloga M, Witte L, Hartmann T, Eich E. 1999. for Excellent Talents in Northwest A&F University, Occurrence of loline alkaloids in argyreia mollis China (11241). (). Phytochemistry, 51, 1177.

(Edited by WANG Lu-han)

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