Effects of the Endophyte-Associated Alkaloid Peramine on Southern

Chapter 10 Effects of the Endophyte-Associated Alkaloid Peramine on Southern Armyworm Microsomal Cytochrome P450 1

2

Downloaded by STANFORD UNIV GREEN LIBR on August 2, 2012 | http://pubs.acs.org Publication Date: September 22, 1992 | doi: 10.1021/bk-1992-0505.ch010

E. N. Dubis , L. B. Brattsten , and L. B. Dungan Department of Entomology, Rutgers University, New Brunswick, NJ 08903-0231 Several kinds o f a l k a l o i d s are produced i n p l a n t s c o n t a i n i n g endophytic f u n g i . The a s s o c i a t i o n is m u t u a l i s t i c (1). The fungus d e r i v e s n u t r i e n t s , p r o t e c t i o n , and propagat i o n sometimes without s p o r u l a t i o n and the p l a n t gains p r o t e c t i o n from herbivory and i n some cases increased growth r a t e and drought r e s i s t a n c e (2). The a l k a l o i d s are r e s p o n s i b l e f o r the a n t i h e r b i v o r y e f f e c t s . Some a l k a l o i d s , e.g., peramine, are a n t i f e e d a n t s f o r i n s e c t s , others are t o x i c t o i n s e c t and v e r t e b r a t e herbivores. Peramine i n t e r f e r e s with microsomal cytochrome P450 causing the carbamate i n s e c t i c i d e c a r b a r y l t o be twice as t o x i c as normal t o Spodoptera e r i d a n i a c a t e r pillars.

Endophytic fungi l i v e t h e i r whole l i f e , or a l l of i t except f o r the reproductive stage, inconspicuously i n the t i s s u e s of p l a n t s (3). The e c o l o g i c a l or p h y s i o l o g i c a l e f f e c t s of endophyte-associated a l k a l o i d s on i n s e c t h e r b i vores are not w e l l understood and have only been s t u d i e d r e c e n t l y (1^ 4 - 7 ) . Peramine and l o l i t r e m B are the two major N-heterocyc l i c compounds i n p e r e n n i a l ryegrass, Lolium perenne, c o n t a i n i n g the endophytic fungus Acremonium l o l i i (8, 9 ) . The t o x i c a c t i o n o f the a l k a l o i d s i s unknown as are t h e i r i n t e r a c t i o n s with d e t o x i f y i n g enzymes, i n p a r t i c u -

1

Current address: Warsaw University, Bialystok Branch, Institute of Chemistry, Al. Pilsudskiego 11/4, 15229 Bialystok, Poland Corresponding author

2

0097-6156/92/0505-0125$06.00/0 © 1992 American Chemical Society

In Molecular Mechanisms of Insecticide Resistance; Mullin, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

126

MOLECULAR MECHANISMS OF INSECTICIDE RESISTANCE


l a r , cytochrome P450. T h i s enzyme i s important i n h e r b i vores and i s known t o metabolize a l k a l o i d s and i n s e c t i c i d e s (10). I n t e r a c t i o n s between the a l k a l o i d s and c y t o chrome P450 may a f f e c t the s u i t a b i l i t y of endophytec o n t a i n i n g p l a n t s as food sources f o r herbivores or even the e f f i c a c y of s y n t h e t i c i n s e c t i c i d e s . In t h i s paper, we f i r s t g i v e a summary of the e f f e c t s of endophytes and then, we present data on the i n t e r a c t i o n s between pure peramine and microsomal cytochrome P450 from Spodoptera eridania c a t e r p i l l a r s . Effects

o f endophytes

The e n d o p h y t e / g r a s s c o m p l e x . Ascomycetes i n the family C l a v i c i p i t a c e a e , t r i b e Balansiae i n h a b i t many p l a n t spec i e s as endophytes. As many fungi i n t h i s t r i b e appear t o be completely endophytic, o c c u r r i n g only as i n t e r c e l l u l a r mycelia i n p l a n t t i s s u e s , and s p o r u l a t i o n stages have not been found, c l a s s i f i c a t i o n i s not without controversy (1, 3.) . Many of these fungi appear t o be t r a n s m i t t e d e x c l u s i v e l y by hyphae i n seeds t o the next p l a n t generation. Others form s p o r u l a t i o n bodies known e i t h e r as ergots o r chokes. Ergot- or choke-forming f u n g i may i n h i b i t the seed set o f the host grass (11). Otherwise, these f u n g i are not at a l l p l a n t pathogenic. On the contrary, host grasses seem t o d e r i v e s e v e r a l competitive advantages. Herbivore r e s i s t a n c e i s only one of the b e n e f i t s t o p l a n t s h o s t i n g endophytic f u n g i . A_;_ l o l i i - c o n t a i n i n g L. perenne had s i g n i f i c a n t l y increased shoot and root growth (12.) . A_;_ coenophialum-containing Festuca arundinacea grown under c o n t r o l l e d c o n d i t i o n s had increased r a t e of photos y n t h e s i s , f r e s h weight gain, t i l l e r production during regrowth, and decreased l e a f r o l l during drought, a l l compared t o g e n o t y p i c a l l y i d e n t i c a l , endophyte-free clones grown under equivalent c o n d i t i o n s (2). In a 7-year f i e l d t r i a l , endophyte-containing |\. arundinacea was more r e s i s t a n t t o crabgrass i n v a s i o n and recovered quicker from summer drought s t r e s s than i d e n t i c a l endophyte-free genotypes (13.) • The reasons f o r these s e l e c t i v e advantages are not c l e a r . In the l o l i i / L . perenne complex t h a t produces l o l i t r e m a l k a l o i d s , i n d o l e intermediates may mimic p l a n t growth hormones, some of which are i n d o l e - d e r i v a t i v e s . I t i s c l e a r , however, t h a t the a l k a l o i d s are r e s p o n s i b l e f o r the a n t i - h e r b i v o r y e f f e c t s . Peramine. The l i p o p h i l i c i t y o f the p y r r o l o p y r a z i n e nucleus i n peramine, [3-(1,2-dihydro-2-methyl-l-oxopyrrolo[1, 2-a]pyrazin-3-yl)propyl]guanidine, (1) i n Figure 1, i s o f f s e t by the s t r o n g l y b a s i c guanidino s u b s t i t u e n t on the propyl s i d e chain causing the molecule t o be i o n i z e d a t p h y s i o l o g i c a l pH. The two fused r i n g s c o n s t i t u t e a reson a t i n g system rendering t h i s p a r t of the molecule very stable. L. perenne seeds contain a t l e a s t 1 ppm of peramine (14). The concentrations i n a e r e a l p a r t s of perenne containing l o l i i may be up t o 40 ppm (dry weight) (15) .



NCH,

O

MeOH

j

1

4

4

k

CD

Figure

_. . _,, _ # N H • HBr CHjCHjCHjNHC

IS CO ^

'I

2

3

to

synthesis.

7.22 (br s, 1H), 7.29 (dd, 1H, J=1.5 and J=2.6Hz UV (MeOH) X ^ , 230.6 (e 34794), 283.7 (e 8781)

and J=4.1Hz), 6.99 (ddd, 1H, J=0.7, J=1.5, and J=2.6Hz),

3.26 (t, 2H, J«6.6Hz), 3.4 (s, 3H), 6.65 (dd, 1H, J=2.5

2

2

H NMR (D 0. 400MHz) 8: 1.88 (br q. 2H). 2.64 (t, 2H, J=7.4Hz),

2

I_ NCH;

DMF KN

(7)

CH,CH,CH,a

Y

NCH,

CH CH CH

Y

(8) I. HjNNHj >HO I 2.5% HQ

Peramine HBr, fine white needles, mp. 243-245°C

a

a

2. INIICI

1. CH,NII/rHF

CH,CH CH,a

CHJCHJCHJNHJ

NCH,

p

T

V |

(»)

1. Scheme of peramine

]

a 1

H)

CHJCOCHJ

^^coca, IJJEoJ

(CH SC(»NH)NH ) -H^SO, H,SO,

6

CHJCCHDKCHJJO

Hj8 BrCH,C(CHj),a

#NH.HjS0 - HjO CH,CH,CH^HC sjjj^ ^ *

Y

(10)

(»

CH,C(CH,),CI

9

(3)


128



The t o t a l s y n t h e s i s of peramine has been p u b l i s h e d (1618). I t i s an a n t i f e e d a n t to a few i n s e c t s p e c i e s , notably the Argentine stem weevil ( L i s t r o n o t u s b o n a r i e n s i s ) , and the i n t a c t p y r r o l o p y r a z i n e nucleus i s important f o r the feeding d e t e r r e n t p r o p e r t i e s of the molecule (19). L o l i t r e m B. T h i s i n d o l e a l k a l o i d , a l a r g e , h i g h l y complex and l i p o p h i l i c diterpene (Figure 2) i s neurotoxic, causing tremors and i n c o o r d i n a t i o n i n mice and i s t o x i c to L. b o n a r i e n s i s l a r v a e (8_i_ 20-22). Because of the complexity of the molecule, no s t r u c t u r e / a c t i v i t y s t u d i e s have been performed. The i n t e r a c t i o n s with t a r g e t or d e t o x i f y i n g macromolecules are unknown. The content of l o l i t r e m B i n L perenne leaves and stems i s between 1 and 5 ppm, and the h i g h e s t content i s i n the l e a f sheaths; the l e v e l s i n c r e a s e with the age of p l a n t s and can reach 25 ppm i n l e a f sheaths of 5-week o l d grass (23.) . E f f e c t s o f E n d o p h y t e - C o n t a i n i n g G r a s s e s and A l k a l o i d s on Insects. b o n a r i e n s i s shows a feeding and o v i p o s i t i o n preference f o r endophyte-free perenne when o f f e r e d a choice. T h i s e f f e c t i s r e l a t e d to the a n t i f e e d a n t propert i e s of peramine and the t o x i c i t y of l o l i t r e m B (9_t 24.) . Pure peramine i s not t o x i c to b o n a r i e n s i s l a r v a e (19). Endophyte-containing L^. perenne a l s o deters feeding by one aphid species (Rhopalosiphum maidis) out of four (2J5) and a bluegrass b i l l b u g (26). S i g n i f i c a n t l y reduced feeding and o v i p o s i t i o n by s e v e r a l sod webworm s p e c i e s were a l s o seen on endophyte-containing perenne compared to the endophyte-free clone (27, 28). House c r i c k e t s fed endophyte-containing L^. perenne s u f f e r e d 100% m o r t a l i t y w i t h i n 4 days compared to 20-40% of those fed endophyte-free grass. The e p i t h e l i u m of the crop and p r o v e n t r i c u l u s was destroyed i n dying c r i c k e t s (29), apparently causing septicemia. The e f f e c t looks s i m i l a r to the gross t o x i c e f f e c t seen with s e v e r a l other, u n r e l a t e d t o x i c a n t s , e.g., tannins (30), cyanide (3_1) , or the 5 -endotoxin of B a c i l l u s t h u r i n q i e n s i s (32.), implying i n t e r a c t i o n with a p r o t e i n i n the gut e p i t h e l i u m . Endophyte-containing L^. perenne i s a l s o l e t h a l t o t h i r d and f o u r t h i n s t a r S^. e r i d a n i a larvae (33>) • Endophyte-containing perenne slows down the growth and development of Spodoptera f r u g i p e r d a c a t e r p i l l a r s but has only s l i g h t t o x i c e f f e c t s (34-36), implying e v o l v i n g defense mechanisms i n t h i s n o t o r i o u s l y g r a s s - f e e d i n g s p e c i e s . S i g n i f i c a n t d i f f e r e n c e s i n S_=_ f r u q i p e r d a l a r v a l body weight and food consumption were a l s o seen when l a r v a e fed on an endophyte-containing clone of perenne with a high c o n c e n t r a t i o n of l o l i i mycelia (37.) . Cytochrome P450. Microsomal cytochrome P450 i s of major importance f o r the metabolism of x e n o b i o t i c s i n a l l aerob i c organisms, h e l p i n g herbivorous i n s e c t s t o adapt quickl y to new food sources t h a t c o n t a i n p o t e n t i a l l y t o x i c



10.

DUBIS ET AL.

Effects of Peramine on Microsomal Cytochrome P450

a l l e l o c h e m i c a l s . The v e r s a t i l i t y of t h i s enzyme r e s i d e s mainly i n i t s occurrence i n m u l t i p l e isoenzymic forms, i t s a b i l i t y t o be induced, and i t s a b i l i t y t o o x i d i z e many d i f f e r e n t s u b s t r a t e molecules; the system i s a l s o i n h i b i t ed by s e v e r a l types of a l l e l o c h e m i c a l s . There i s a v a s t l i t e r a t u r e about t h i s enzyme system; see 38-41 f o r recent comprehensive reviews about i n s e c t cytochrome P450. Each organism, perhaps even each t i s s u e , has a s e t of s e v e r a l cytochrome P450 isozymes, some of which accept many d i f f e r e n t types of molecules as s u b s t r a t e s and others of which may be h i g h l y s u b s t r a t e s p e c i f i c . In most cases, each cytochrome P450 i s coded f o r by i t s own gene; 154 d i s t i n c t P450 genes are assigned t o 27 f a m i l i e s (42. 43.) . Ten of them occur i n a l l mammals s t u d i e d t o date and are organized i n t o 18 s u b f a m i l i e s each c o n s i s t i n g of a c l u s t e r of t i g h t l y l i n k e d genes. The expression o f these genes i s r e g u l a t e d by endogenous f a c t o r s such as developmental stage, c e l l - t y p e s p e c i f i c s i g n a l s , and hormones, and by e x t e r n a l f a c t o r s such as inducing chemicals. Most o f the P450 molecular g e n e t i c s has been done with small l a b o r a t o ry mammals, but recent s t u d i e s i n d i c a t e t h a t a m u l t i p l i c i ty s i m i l a r t o t h a t i n mammals a l s o occurs i n i n s e c t s (45, 46). The molecular g e n e t i c s of cytochrome P4 50 i s reviewed i n s e v e r a l recent papers (42, 43, 46-48). Several i n s e c t cytochrome P450 isozymes a s s o c i a t e d with r e s i s t a n c e t o i n s e c t i c i d e s or t o x i c p l a n t a l l e l o c h e m i c a l s have been c h a r a c t e r i z e d . A cytochrome t h a t detoxi f i e s p y r e t h r o i d i n s e c t i c i d e s was i s o l a t e d from a s t r a i n of Musca domestica h i g h l y r e s i s t a n t t o p y r e t h r o i d s (49). Two phenobarbital-induced cytochromes were i s o l a t e d from a s t r a i n of M^ domestica with high r e s i s t a n c e t o organophosphate i n s e c t i c i d e s (50). Two isozymes were i s o l a t e d from Drosophila melanogaster and one was a s s o c i a t e d with r e s i s t a n c e t o phenylurea (51). P a p i l i o polyxenes c a t e r p i l l a r s have a P450 that i s induced s p e c i f i c a l l y by furanocoumarins and d e t o x i f i e s these compounds (52, 53). Cytochrome P450 can be induced by a l a r g e number of compounds (47. 54). Induction can r e s u l t i n more o f the o r i g i n a l a c t i v i t i e s i f a major, n o n - s p e c i f i c isozyme i s induced or i n a new a c t i v i t y i f a s p e c i f i c , o r i g i n a l l y minor isozyme i s induced. There i s some s p e c i f i c i t y i n the inducer a c t i o n . I t was c l e a r e a r l y t h a t p o l y c y c l i c aromati c hydrocarbons (PAHs) induce a form of cytochrome P450 t h a t o x i d i z e s PAHs and not many other types of compounds. Phenobarbital, on the other hand, induces a broad spectrum of a c t i v i t i e s , i n c l u d i n g more of the o r i g i n a l a c t i v i t i e s . Interactions

b e t w e e n p u r e p e r a m i n e and c y t o c h r o m e P450

Peramine S y n t h e s i s . Peramine was synthesized as d e s c r i b e d (18) with minor m o d i f i c a t i o n s (Figure 1). Peramine bromide was used throughout our experiments. C o n t r o l experiments i n c l u d e d an equimolar dose of potassium bromide and showed t h a t there were no e f f e c t s from the bromide i o n .


129

130



G r o s s E f f e c t s o f P e r a m i n e on eridania. U n l i k e whole endophyte-containing k perenne, pure peramine i s not acutely toxic to e r i d a n i a l a r v a e and doesn't i n h i b i t feeding o r growth i n t h i s s p e c i e s . When a d i e t with 0.1% pure peramine was f e d ad l i b i t u m t o e i t h e r t h i r d i n s t a r l a r v a e u n t i l pupation or t o l a s t i n s t a r l a r v a e , development r a t e s were not d i f f e r e n t from those o f the c o n t r o l s (Table 1). I t should be noted t h a t 0.1% i s a very high T a b l e 1. E f f e c t s o f d i e t a r y p e r a m i n e o n 6 t h i n s t a r S. e r i d a n i a l a r v a e F i t n e s s Factor Mortality Feeding r a t e ( g / l a r v a , 3 days) Average weight of l a r v a a f t e r 3 days (mg) Average weight of pupae (mg)

Control 0 5.,9 (0.5)

Peramine 0 5. 9 (0.5)

732

(75)

796

(81)

191

(29)

225

(27)

Groups of 3 0 newly molted 6th i n s t a r l a r v a e were f e d e i t h e r c o n t r o l or 0.1% peramine d i e t (57) ( i n 3 days, each l a r v a ingested 5.6 mg peramine). There are no d i f f e r e n c e s between the means (Student's Tt e s t ) . In another experiment (data not shown), 3rd i n s t a r l a r v a e were s t a r t e d on the same kinds of d i e t s and reared t o pupation, a l s o showing no d i f ferences i n growth or feeding r a t e s . Numbers i n p a r e n t h e s i s are S.E. (N=30). c o n c e n t r a t i o n of peramine; b o n a r i e n s i s stops feeding on d i e t s c o n t a i n i n g 1 ppm (55). The data i n Table 1 show t h a t S. e r i d a n i a i s a good model i n s e c t f o r studying the molecu l a r e f f e c t s of peramine because the experimental i n s e c t s are n e i t h e r s i c k nor s t a r v i n g . M o l e c u l a r E f f e c t s o f Peramine. P r e l i m i n a r y s t u d i e s of peramine metabolism i n v i t r o i n d i c a t e t h a t peramine i s metabolized only s l i g h t l y by cytochrome P450 i n t i s s u e f r a c t i o n s from midguts of S^_ e r i d a n i a o r S_s_ f r u g i p e r d a or l i v e r s from mice, sheep, or c a t t l e (J56) . About 85-90% i s excreted unmetabolized w i t h i n 36 h r s i n l a s t i n s t a r S. e r i d a n i a c a t e r p i l l a r s (5j>) . T h i s i s c o n s i s t e n t with the resonance s t a b i l i t y o f the fused r i n g system and the i o n i z a t i o n a t p h y s i o l o g i c a l pH of peramine. When S^. e r i d a n i a l a s t i n s t a r c a t e r p i l l a r s had f e d on a 0.1% peraminec o n t a i n i n g d i e t f o r three days, there was i n s i g n i f i c a n t i n h i b i t i o n of a l d r i n epoxidation. However, N-demethylation of p-chloro N-methylaniline was 52% of c o n t r o l a c t i v i t y , and O-demethylation of methoxyresorufin was 32% o f c o n t r o l


10. DUBIS ET AL.


T a b l e 2 . E f f e c t s o f d i e t a r y p e r a m i n e on a c t i v i t i e s o f m i d g u t d e f e n s i v e enzymes i n S± e r i d a n i a 6 t h instar larvae


Activity

Control

Cytochrome P-4 50 0. 36 (0. 07) (nmol/mg protein) N-Demethylation 4. 22 (0. 5) (nmol/min, mg protein) O-Demethylation 18. 10 (1. 9) (pmol/min, mg protein) Epoxidation 2. 98 (0. 3) (nmol/min, mg protein) Microsomal Esterases 14. 50 (1. 5) (umol/min, mg protein) S o l u b l e Esterases 56. 01 (5. 5) (umol/min, mg protein) Glutathione Transferase 0. 75 (0. 08) (umol/min, mg protein)

Peramine 0. 48 (0.04) 2. 38 (0.5) 7. 0 (0.8) 2. 46 (0.3) 15. 66 (1.6) 62. 01 (6.5) 0. 72 (0.08)

Groups of 3 0 l a r v a e were fed a c o n t r o l d i e t or a d i e t c o n t a i n i n g 0.1% peramine f o r 3 days beginning immediately a f t e r molting t o the 6th i n s t a r . Midgut microsomes and s o l u b l e f r a c t i o n s were used f o r the assays (62). T o t a l P450 content was estimated with the carbonyl ferrocytochrome d i f f e r e n c e spectrum (63). The s u b s t r a t e f o r N-demethylation was p - c h l o r o N-methylaniline; f o r O-demethylation, methoxyresoruf i n ; f o r epoxidation, a l d r i n ; f o r e s t e r a s e s , 1naphthylacetate; f o r g l u t a t h i o n e t r a n s f e r a s e , 1chloro-2,4-dinitrobenzene (57, 64, 65). The data i n p a r e n t h e s i s are S.E. (N=3). Only the d i f f e r e n c e s i n the N- and O-demethylation data are s i g n i f i c a n t (Student's T - t e s t ) .

a c t i v i t y (Table 2). Peramine has no i n d u c t i v e or i n h i b i t o ry e f f e c t on general esterase or g l u t a t h i o n e t r a n s f e r a s e a c t i v i t i e s (Table 2). The microsomal carbonyl f e r r o c y t o chrome P450 d i f f e r e n c e spectrum was c o n s i s t e n t l y 133% of c o n t r o l ; t h i s i s not a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r ence, whether or not i t i s b i o l o g i c a l l y s i g n i f i c a n t , depends on the P450 isozyme(s) a f f e c t e d by peramine. SDS-PAGE (Figure 3) shows an apparent small i n c r e a s e i n a minor cytochrome P450 band but no apparent decrease i n any of the other cytochrome P450 bands; t h i s i s d i f f i c u l t t o q u a n t i f y without a p u r i f i e d cytochrome P450 f r a c tion. A f t e r feeding s i x t h i n s t a r S^_ e r i d a n i a l a r v a e on a d i e t c o n t a i n i n g 0.1% peramine f o r three days, the L D ^ Q of c a r b a r y l was h a l f of that t o i n s e c t s fed a c o n t r o l d i e t ; c a r b a r y l i s d e t o x i f i e d e x c l u s i v e l y by P450 (6JL) . There were no d i f f e r e n c e s i n the t o x i c i t i e s of f l u v a l i n a t e , a p y r e t h r o i d t h a t can be d e t o x i f i e d by e s t e r a s e h y d r o l y s i s


131


132


F i g u r e 3. SDS-PAGE o f microsomal p r o t e i n from S. e r i d a n i a midguts. The p r o t e i n was from l a r v a e t h a t had fed on a c o n t r o l d i e t ( l e f t l a n e ) , a d i e t c o n t a i n i n g 0.1% peramine (middle l a n e ) , and a d i e t c o n t a i n i n g 0.2% pentamethylbenzene, an inducer o f i n s e c t c y t o chrome P450 (58). Seventy ug o f p r o t e i n from washed microsomes i n 10 mM T r i s with mM EDTA, pH 7.5, were added t o each lane. The running g e l was 8%, and the gel was run a t a constant c u r r e n t o f 20 mAmps f o r the s t a c k i n g g e l and 3 0 mAmps f o r the running g e l , modif i e d from (59). The g e l was s t a i n e d with p u r i f i e d Coomassie b r i l l i a n t blue G-250 according t o (60). The molecular weight standards ( i n the o u t s i d e lanes) were from Biorad (161-0304). The p u t a t i v e P450 bands show estimated molecular masses o f 56, 53.8 (large bands), 53 (increased by pentamethylbenzene and peramine), and 52.7 kD (small bands). In Molecular Mechanisms of Insecticide Resistance; Mullin, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

10. DUBIS ET AL.

Effects of Peramine on Microsomal Cytochrome P4S0

as w e l l as by P450-catalyzed o x i d a t i o n , or c h l o r p y r i f o s , an organophosphate t h a t can a l s o be d e t o x i f i e d by e s t e r ases or g l u t a t h i o n e t r a n s f e r a s e s (Table 3). The t o x i c i t i e s of the three i n s e c t i c i d e s widely used on t u r f are, thus, c o n s i s t e n t with the p a t t e r n of e f f e c t s on the i n s e c t i c i d e - d e t o x i f y i n g enzymes (Table 2 ) .


Table 3. T o x i c i t y of t u r f i n s e c t i c i d e s to 6th S. e r i d a n i a l a r v a e Insecticide

Control Diet LD

Carbaryl Fluvalinate Chlorpyrifos

5 0

LD

9 5

265 420 4.2 10 1.6 3.7

instar

0.1% Peramine Diet LD

5 0

130 4.2 1.5

LD

9 5

230 10 3.7

The t o x i c i t i e s were measured 24 hours a f t e r t o p i c a l a p p l i c a t i o n t o groups of 10 l a r v a e . F i v e i n s e c t i c i d e concentrations were used, each repeated three times. A f t e r treatment the l a r v a e were h e l d a t 22°C and provided with c o n t r o l d i e t . Conclusions. Endophytes have a great p o t e n t i a l f o r safe and s e l e c t i v e i n s e c t c o n t r o l i n areas where t u r f g r a s s e s are used f o r home or urban landscaping. Endophytes may a l s o , i n the f u t u r e , be engineered i n t o crop p l a n t s and combined with a minimized spray a p p l i c a t i o n of s y n t h e t i c i n s e c t i c i d e s . There are c e r t a i n s t r a i n s of A_j_ l o l i i t h a t b i o s y n t h e s i z e peramine but not the l o l i t r e m s (Popay, 1990 personal communication) t h a t may be used i n c o n j u n c t i o n with s y n t h e t i c i n s e c t i c i d e s . V a r i a t i o n s and permutations i n the combinations of endophyte a l k a l o i d s and i n s e c t i c i d e s w i l l help reduce the e v o l u t i o n o f r e s i s t a n c e i n insects. Our research shows i n t e r a c t i o n s of peramine with cytochrome P450. The i n h i b i t o r y e f f e c t s imply t h a t peramine could be a s y n e r g i s t a t l e a s t f o r carbamate i n s e c t i c i d e s . Peramine may a l s o be a s y n e r g i s t f o r c o - o c c u r r i n g , t o x i c l o l i t r e m s or ergot a l k a l o i d s . T h i s would not be a unique case; many p l a n t s i n a d d i t i o n t o Chrysanthemum c i n e r a r i e f o l i u m , "the pyrethrum flower" c o n t a i n l i g n a n s (compounds t h a t i n h i b i t cytochrome P450 by a benzodioxole group) and other s y n e r g i s t s together with one or more t o x i c a n t s , e.g., the parsnip (6J5) and Piperaceae peppers (67) • A c k n o w l e d g m e n t s . We thank N. M. E l b a r r a d , D. A. Berger, and H. P. Young f o r a s s i s t a n c e . T h i s i s paper No. D0811102-92 from the New Jersey A g r i c u l t u r a l Experiment S t a t i o n .


133

134



Literature

Cited

1. Clay, K. Ecology 1988, 69:10-16. 2. Clay, K.; G. P. Cheplick; S. Marks. Oecologia 1989, 80:374-380. 3. S i e g e l , M. R.; G. C. M. Latch; M. C. Johnson. Ann. Rev. Phytopathol. 1987, 25:293-315. 4. Clay, K. Ecology 1990, 71:558-570. 5. Clay, K. In M i c r o b i a l Mediation of Plant-Herbivore I n t e r a c t i o n s , P. Barbosa, V. A. K r i s c h i k and C. G. Jones, eds, Wiley, 1991, pp. 199-226. 6. C a r r o l , G. Ecology 1988, 69:2-9. 7. Dahlman, D. L.; H. Eichenseer; M. R. S i e g e l . In Micro bial Mediation of Plant-Herbivore I n t e r a c t i o n s , Wiley, 1991, pp. 227-252. 8. P r e s t i d g e , R. A.; R. T. Gallagher. Proc. N. Z. Weed and Pest C o n t r o l Conf. 1985, 38:38-40. 9. Rowan, D. D.; D. L. Gaynor. J. Chem. E c o l . 1986, 12:647-658. 10. B r a t t s t e n , L. B. In Molecular Aspects o f I n s e c t - P l a n t A s s o c i a t i o n s . L. B. B r a t t s t e n and S. Ahmad, eds, Plenum, 1986, pp. 211-255. 11. Smith, K. T.; C. W. Bacon; E. S. Luttrell. Phytopat h o l . 1985, 25:293-315. 12. Latch, G. C. M.; M. J. Christensen; D. L. Gaynor. N. Z. A g r i c . Res. 1985, 28:129-132. 13. Funk, C. R.; P. M. H a l i s k y ; S. Ahmad; R. H. Hurley. In Proc. 5th I n t e r n . Turf Res. Conf., Avignon, F. Lemaire ed., INRA, 1985, pp. 137-145. 14. Rowan, D. D.; B. A. Tapper. Nat. Prod. 1989, 52:193-195. 15. S i e g e l , M. R. e t 7 al. Chem. E c o l . 1990, 12:33013315. 16. Brimble, M. A.; D. D. Rowan. J. Chem. Soc. Chem. Commun. 1988, p. 978. 17. Brimble, M. A.; D. D. Rowan. J. Chem. Soc. P e r k i n Trans. 1990, 311-314. 18. Dumas, D. J. J. Org. Chem. 1988, 53:4650-4653. 19. Rowan, D. D.; J. J. Dymock; M. A. Brimble. J. Chem. E c o l . 1990, 16:1683-1695. 20. P r e s t i d g e , R. A.; R. T. Gallagher. E c o l . Entomol. 1988, 13:429-435. 21. Gallagher, R. T.; A. D. Hawkes. E x p e r i e n t i a 1986, 42:823-825. 22. Gallagher, R. T.; A. D. Hawkes; P. S. Steyn; R. V l e g gaar. J. Chem. Soc., Chem. Commun., 1984, 614-616. 23. Gallagher, R. T.; G. S. Smith; J . M. Sprosen. Proc. 4th AAAP Animal Science Cong. Hamilton, 1987, p. 404. 24. Rowan, D. D.; M. B. Hunt; D. L. Gaynor. J. Chem. Soc. Chem. Commun., 1986:935-936. 25. Johnson, M. C. e t 6 al. Appl. Environ. M i c r o b i o l . 1985, 49:568-571. 26. Ahmad, S.;, J. M. Johnson-Cicalese; W. K. Dickson; C. R. Funk. Entomol. exp. a p p l . 1986, 41:3-10. 27. Funk, C. R. e t 7 al. Bio/Technology 1983, 1:189-191.



10.

DUBIS ET AL.


28. Ahmad, S.; C. R. Funk, J. Econ. Entomol. 1983, 76:414-416. 29. Ahmad, S.; S. Govindarajan; C. R. Funk; J. M. Johnson-Cicalese, Entomol. exp. a p p l . 1985, 39:183-190. 30. S t e i n l y , B. A.; M. Berenbaum. Entomol. exp. a p p l . 1985, 39:3-9. 31. B r a t t s t e n , L. B.; J. H. Samuelian; K. Y. Long; S. A. K i n k a i d ; C. K. Evans. E c o l . Entomol. 1983, 8:125-132. 32. Endo, Y.; J. N i s h i i t s u t s u j i - U w o . J. I n v e r t . P a t h o l . 1980, 36:90-103. 33. Ahmad, S.; S. Govindarajan; J . M. Johnson-Cicalese; C. R. Funk. Entomol. exp. a p p l . 1987, 43:287-294. 34. Clay, K.; T. N. Hardy; A. M. Hammond, Jr. Oecologia 1985, 66:1-5. 35. Hardy, T. N.; K. Clay; A. M. Hammond, Jr. J. Econ. Entomol. 1985, 78:571-575. 36. Hardy, T. N.; K. Clay; A. M. Hammond. Environ. Entomol. 1986, 15:1083-1086. 37. Breen, J . P. Endophyte-enhanced i n s e c t r e s i s t a n c e in t u r f g r a s s e s : v a r i a t i o n among endophytes, host s p e c i e s and i n s e c t h e r b i v o r e s . Ph. D. D i s s . , Rutgers U n i v e r s i t y , 1990. 38. Agosin, M., In Comprehensive Insect Biochemistry, Physiology and Pharmacology. G. A. Kerkut and L. I . G i l b e r t , eds, V o l . 12, Pergamon; 1985. pp. 647-712. 39. Hodgson, E. In Comprehensive Insect Biochemistry, Physiology and Pharmacology, G. A. Kerkut and L. I . G i l b e r t , eds, Pergamon, 1985, V o l . 11, pp. 225-321. 40. Ahmad, S.; L. B. B r a t t s t e n ; C. A. M u l l i n ; S. J. Yu. In Molecular Aspects of I n s e c t - P l a n t A s s o c i a t i o n s , L. B. B r a t t s t e n and S. Ahmad, eds, Plenum, 1986, pp. 73-151. 41. B r a t t s t e n , L. B. In Herbivores - t h e i r i n t e r a c t i o n s with secondary p l a n t metabolites; G. A. Rosenthal and M. R. Berenbaum, eds, Academic, V o l . 2, 1992, i n press. 42. Nebert, D. W. e t 12 al. DNA 1989, 8:1-13. 43. Nebert, D. W. e t 13 al. DNA and Cell B i o l . 1991, 10:1-14. 44. Nebert, D. W.; D. R. Nelson; R. Feyereisen. X e n o b i o t i ca 1989, 19:1149-1160. 45. Feyereisen, R. J.; F. Koener; D. E. Farnsworth; D. W. Nebert. Proc. N a t l . Acad. S c i . , USA, 1989, 85:58595863. 46. Nebert, D. W. Crit. Revs T o x i c o l . 1989, 20:153-174. 47. Nebert, D. W.; F. J. Gonzalez. Ann. Rev. Biochem. 1987, 56:945-993. 48. Gibson, C. G. Xenobiotica. 1989, 19:1123-1148. 49. Wheelock, G. D.; J. G. S c o t t . Insect Biochem. 1989, 19:481-488. 50. F i s h e r , C. W.; R. T. Mayer. Arch. Insect Biochem. P h y s i o l . 1984, 1:127-138. 51. Waters, L. C.; C. E. Nix. P e s t i c . Biochem. P h y s i o l . 1988, 30:214-227. 52. Berenbaum, M. R.; M. B. Cohen; M. A. Schuler. In Molecular Insect Science, H. H. Hagedorn, J. G. H i l d e - -


135

136

53. 54. 55.


56. 57. 58. 59. 60.

61. 62. 63. 64. 65.

66. 67.


brand, M. G. Kidwell and J . H. Law, eds, Plenum, 1990, pp. 257-262. Cohen, M. B.; M. R. Berenbaum; M. A. Schuler. J. Chem. E c o l . 1989, 15:2347-2355. Black, S. D.; M. J . Coon. Adv. Enzymol. 1987, 60:3587. Popay, A. J . ; R. T. P r e s t i d g e ; D. D. Rowan; J . J . Dymock. In Proc. I n t e r n . Symp. Acremonium/grass i n t e r a c t i o n s . 1990, pp. 44-47. B r a t t s t e n , L. B.; E. N. Dubis; X. Gu. Abstr. Intern Symp. Acremonium/grass i n t e r a c t . 1990, p. 17. B r a t t s t e n , L. B.; S. L. P r i c e ; C. A. Gunderson. Comp. Biochem. P h y s i o l . 1980, 66C.-231-237. B r a t t s t e n , L. B.; C. F. Wilkinson. P e s t i c . Biochem. P h y s i o l . , 1973, 3:393-407. Cohen, M. B.; M. R. Berenbaum; M. A. Schuler. Insect Biochem. 1990, 20:777-783. Harlow, E.; D. Lane, A n t i b o d i e s . a Laboratory Manual, Cold Spring Harbor Laboratory P u b l i c a t i o n s , Cold Spring Harbor, 1988. Kuhr, R. J . J. A g r i c . Food Chem. 1970, 18:1023-1030. B r a t t s t e n , L. B.; C. A. Gunderson. P e s t i c . Biochem. P h y s i o l . 1981, 16:187-198. Omura, T.; Sato, R. J. B i o l . Chem. 1964, 239:23702385. B r a t t s t e n , L. B.; C. K. Evans; S. B o n e t t i ; L. H. Zalkow. Comp. Biochem. P h y s i o l . 1984, 77C:29-37. B r a t t s t e n , L. B.; C. A. Gunderson; J . T. Fleming; K. N. Nikbahkt. P e s t i c . Biochem. P h y s i o l . 1986, 25:346357. Berenbaum, M. R.; J. J. Neal. Chem. E c o l . 1985, 11:1349-1358. Miyakado, M.; I. Nakayama; N. Ohno; H. Yoshioka. In Natural Products f o r Innovative Pest Management, D. L. Whitehead and W. S. Bowers, eds, Pergamon, 1983, pp. 369-382.

RECEIVED February 25, 1992


Effects of the Endophyte-Associated Alkaloid Peramine on Southern

Recommend Documents