Host Plant Resistance to Pests

14 Natural Insecticides from Cotton (Gossypium) ROBERT D. STIPANOVIC, ALOIS A. BELL, and MAURICE J. LUKEFAHR

Downloaded by PURDUE UNIV on July 6, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch014

N a t i o n a l C o t t o n P a t h o l o g y Research L a b o r a t o r y , U. S . D e p a r t m e n t of A g r i c u l t u r e , A g r i c u l t u r a l Research Service, C o l l e g e Station, TX 77843

Resistance has been defined as the a b i l i t y of a plant to prevent, restrict or retard partially the injurious effects of a pest (1, 2). The mechanisms of resistance are defined as: a) tolerance -- the plant suffers only slight injury despite a pest population that severely damages a susceptible plant (1,2); b) nonpreference -- the plant is unattractive for feeding, reproduction, or shelter (1); c) antibiosis -- the plant adversely affects the biology or ecology of the pest because of toxic chemicals in the plant tissue (1); and d) hypersensitivity -- the plant inactivates and localizes the invading pest by rapid morphological and histochemical changes that cause the invaded tissues to die prematurely (2, 3). A plant breeder may employ any or a l l of these mechanisms in order to obtain the most desirable agronomic characteristics. H i s t o r i c a l l y , plant lines have been improved by performance and observations in the f i e l d . Until recently there was no concerted effort to understand the chemical bases of pest resistance. However, as early as 1906 Cook suggested that "oil glands" in the cotton plant might be involved in resistance to cotton insects (4). Although the term "antibiosis" had not been coined, Cook recognized this mechanism of pest resistance in cotton. Heliothis Resistance in Cotton. In the early 1960s, the resistance of the Heliothis complex (cotton bollworm, H. zea and tobacco budworm, H. virescens) to insecticides was recognized. In 1965 research was initiated to discover new sources of host plant resistance to these pests. Wild race stocks and "backyard" plantings from Mexico, Central and South America, and the Caribbean Islands were investigated. Over 1200 different races of Gossypium hirsutum were tested of which 78 had some resistance. In general, resistance among races increased with increasing numbers of glands and with increasing gossypol content (5^ 6).

197 Hedin; Host Plant Resistance to Pests ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

198

HOST P L A N T RESISTANCE

T O PESTS


Gossypol ( I ) , a d i m e r i c s e s q u i t e r p e n o i d found i n cottonseed pigment glands, was u s u a l l y measured i n leaves or flower buds by the s p e c t r o p h o t o m e t r y method of Smith [7). T h i s procedure i s based on the r e a c t i o n o f a n i l i n e with gossypol, which presumably

I

occurs by a condensation r e a c t i o n o f the amine group o f a n i l i n e with the aldehyde group i n gossypol. A y e l l o w c o l o r i s produced and the e x t i n c t i o n o f l i g h t a t 445 nm i s measured. A n i l i n e i s not a s p e c i f i c reagent f o r g o s s y p o l , because i t w i l l r e a c t with other aromatic aldehydes, g i v i n g products which are a l s o y e l l o w i n c o l o r . Pigment glands are l o c a t e d i n most t i s s u e s o f the c o t t o n p l a n t , i n c l u d i n g f o l i a r p a r t s and the seed (8, 9). They are absent from the seed coat xylem. In the f o l i a r p a r t s , the glands are l o c a t e d below the epidermis and hypodermis. The " o i l " i n these glands i s composed o f low molecular weight, v o l a t i l e terpenoids t h a t s o l u b i l i z e higher molecular weight pigments such as gossypol. C e r t a i n Heliothis-resistant cotton v a r i e t i e s c o n t a i n more pigment glands than s u s c e p t i b l e v a r i e t i e s . The r e s i s t a n t v a r i e t i e s , thus, are c a l l e d high glanded or high gossypol c o t t o n s (5, 6, 10, 11). The t o x i c i t y o f gossypol to Heliothis spp. was demonstrated (9_, ]Vj, and gossypol t h e r e f o r e was considered the a c t i v e com ponent i n the glands o f the c o t t o n flower bud and b o l l . Most commercial v a r i e t i e s o f c o t t o n c o n t a i n 0.5% gossypol i n f l o w e r buds (dry weight), as measured by the method o f Smith (_7). In l a b o r a t o r y t e s t s , 1.2% gossypol s i g n i f i c a n t l y i n h i b i t e d l a r v a l growth and development o f Heliothis (12). Recently, c e r t a i n w i l d or p r i m i t i v e cottons were found t o e x h i b i t more i n s e c t i c i d a l a c t i v i t y than could be accounted f o r by the gossypol c o n c e n t r a t i o n alone. The t o x i c a c t i v i t y was generally a s c r i b e d to " X - f a c t o r s " (13, 14, 15J. The " X - f a c t o r s " have been i d e n t i f i e d as the s e s q u i t e r p e n o i d s , hemigossypolone ( I I ) and hemigossypolone-7-methyl e t h e r ( I I I ) , and the d e r i v e d t e r p e n o i d s , heliocides Η (IV), H (V), H (VI), H (VII), B (VIII), B (IX), B (X) and B ( X I ) . The "H" and "B" d e s i g n a t i o n s i n d i c a t e t h a t these compounds were f i r s t found i n G. hirsutwn and G. barbadense cottons, respectively. χ

3

2

3

h

x

4

Hedin; Host Plant Resistance to Pests ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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D e t e c t i o n , I s o l a t i o n and L o c a t i o n o f the H e l i o c i d e s i n R e s i s t a n t Cottons.

Heliothis

Freeze d r i e d (0.5 gm) whole cotton flower buds are ground i n a blender and e x t r a c t e d with EtOAcrHex (1:3). T h i s e x t r a c t i s spotted d i r e c t l y on S i gel p l a t e s and developed with Et 0:Hex: HC00H (15:84:1). The p l a t e s a r e then sprayed with p h l o r o g l u c i n o l reagent (equal volumes o f 5% p h l o r o g l u c i n o l i n 95% EtOH and cone. HC1) t o detect t e r p e n o i d aldehydes. Hemigossypolone, hemigossy pol one-7-methyl ether and gossypol form magenta t o maroon c o l o r s , and t h e h e l i o c i d e s form orange c o l o r s . S u s c e p t i b l e normally glanded c u l t i v a r s o f G. hirsutum c o n t a i n h e l i o c i d e s H H , H , H , hemigossypolone and gossypol. The r e s i s t a n t high glanded c u l t i v a r s o f G. hirsutum contained t h e same compounds but the c o n c e n t r a t i o n s , p a r t i c u l a r l y o f H and H^, a r e as much as three times higher than t h a t found i n s u s c e p t i b l e normally glanded cotton (16). The f o l i a r p a r t s o f g l a n d l e s s cottons do not c o n t a i n t e r p e n o i d aldehydes. Thus t h e t e r p e n o i d aldehydes appear t o be l o c a t e d s p e c i f i c a l l y i n t h e glands. Unpublished histochemical t e s t s con f i r m t h i s . I t i s o f i n t e r e s t t o note t h a t g l a n d l e s s cottons a r e h i g h l y s u s c e p t i b l e t o Heliothis spp. and many other i n s e c t s (17). G. barbadense cottons form methyl ether d e r i v a t i v e s o f gossypol and i t s b i o s y n t h e t i c precursors i n root bark (18, 19, 20). G. barbadense a l s o s y n t h e s i z e s t e r p e n o i d methyl ethers i n i t s f o l i a r t i s s u e s . For example, the G. barbadense c u l t i v a r Seabrook Sea I s l a n d contained hemigossypolone-7-methyl ether and the methy l a t e d h e l i o c i d e s B and B i n s l i g h t l y higher c o n c e n t r a t i o n s than hemi gossypol one, h e l i o c i d e H and H^. H e l i o c i d e s were prepared i n q u a n t i t y from young b o l l s (2-3 days-old) and b r a c t s o f f i e l d grown p l a n t s . T i s s u e s were l y o p h i l ized and ground t o powder i n a blender. The powder was e x t r a c t e d and h e l i o c i d e s were p u r i f i e d by chromatography as p r e v i o u s l y described (2Ί, 22). 2

l s

2

3

4


x

x

4

x

C h a r a c t e r i z a t i o n o f Cotton

Terpenoids.

S t r u c t u r e Determination. S p e c t r o s c o p i c methods (UV, IR, MS, ^-NMR and C-NMR), X-ray c r y s t a l a n a l y s i s , and s y n t h e s i s were employed i n determining t h e s t r u c t u r e s o f h e l i o c i d e s from c o t t o n . H e l i o c i d e H (V) was the f i r s t C - t e r p e n o i d analyzed (23). I t was s y n t h e s i z e d by a D i e l s - A l d e r r e a c t i o n o f hemigossypôTone and myrcene. The product which c r y s t a l l i z e d was i d e n t i c a l (UV, IR, MS, m.p., mixed m.p.) t o h e l i o c i d e H from pigment glands. The C-NMR data i n d i c a t e d t h a t t h e s i d e chain was l o c a t e d as shown in V. To prove t h i s , t h e dibromide was prepared and submitted to X-ray c r y s t a l a n a l y s i s ( F i g . 1). T h i s u n e q u i v o c a l l y establ i s h e d t h e p o s i t i o n o f the s i d e chain and proved t h a t the c y c l o hexene r i n g was c i s - f u s e d . Because h e l i o c i d e H was s y n t h e s i z e d from hemigossypolone, t h e X-ray c r y s t a l a n l a y s i s a l s o proved t h a t ld

2

25

2

13

2



Figure 1.

The confirmation of the dibromide derivative of heliocide H by x-ray crystal analysis

2

as determined



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hemigossypolone i s a para-napthoquinone with hydroxyls at C6 and C7 (24, 25). The mother l i q u o r s from the D i e l s - A l d e r r e a c t i o n o f hemigossypolone and myrcene y i e l d e d another C 5 - t e r p e n o i d which was i d e n t i c a l (UV, IR, MS, m.p. and mixed m.p.) to h e l i o c i d e H from pigment glands (21). The C-NMR spectrum o f h e l i o c i d e H was very s i m i l a r t o t h a t o f h e l i o c i d e H but i n d i c a t e d a d i f f e r e n c e in the l o c a t i o n o f the s i d e c h a i n . That i s , the major chemical s h i f t changes were at the methylene carbons o f the cyclohexene r i n g , and were i n a d i r e c t i o n commensurate with the s i d e chain l o c a t e d as shown i n s t r u c t u r e VI. H e l i o c i d e s Η χ and H were s y n t h e s i z e d by the D i e l s - A l d e r r e a c t i o n o f hemigossypolone with t r a n s - 3 - o c i m e n e (22). H e l i o c i d e s Hi and Hi+ do not r e v e r t to s t a r t i n g m a t e r i a l a t room temperature 2

3

13

3

2

k

( i . e . , they do not undergo the r e v e r s e D i e l s - A l d e r r e a c t i o n ) . T h e r e f o r e , they are the products of a k i n e t i c a l l y c o n t r o l l e d r e a c t i o n that proceeds through an e n d s - t r a n s i t i o n s t a t e g i v i n g a cis-fused product (26J. Further, trans-1-substituted butadienes and quinones form adducts i n which the diene s u b s t i t u e n t i s trans to the bridgehead s u b s t i t u e n t s on the quinone r i n g (27, 28). Thus h e l i o c i d e s H and H have ois-fused r i n g systems with transoid isopentenyl s i d e chains as shown f o r s t r u c t u r e s IV and VII. 2

k



202


T O PESTS

13

The C-NMR s p e c t r a o f h e l i o c i d e s H and H were compared t o those o f h e l i o c i d e s H and H . On t h e b a s i s o f these comparisons h e l i o c i d e s H i and Hi+ were assigned s t r u c t u r e s IV and V I I , r e s p e c t ively. H e l i o c i d e s B (VIII) and B (XI) are found i n G. barbadense [Seabrook Sea Island 12B2 (SBSI)]. They were synthesized by t h e D i e l s - A l d e r r e a c t i o n o f hemigossypolone-7-methyl ether ( I I I ) and trarcs-3-ocimene (29). The s t r u c t u r e s were assigned by comparing x

2

4

3

x

4

the C-NMR s p e c t r a o f h e l i o c i d e s B i and B with h e l i o c i d e s Hi and Hi+. H e l i o c i d e s B (IX) and B (X) have been s y n t h e s i z e d from hemigossypolone-7-methyl ether ( I I I ) and myrcene. These compounds are a p p a r e n t l y not found i n SBSI, b u t they have been t e n t a t i v e l y i d e n t i f i e d i n F progeny from c r o s s e s o f G. hirsutum and G. 13

4

2

3

2

barbadense ( 3 0 ) .



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*H-NMR Screening Techniques. To breed p l a n t s which c o n t a i n the most e f f e c t i v e mixture o f t e r p e n o i d aldehydes, a r a p i d method o f screening was d e s i r a b l e . We have developed a ^-NMR technique t h a t d i f f e r e n t i a t e s most o f the t e r p e n o i d aldehydes and estimates t h e i r c o n c e n t r a t i o n s i n crude e x t r a c t s . The aldehyde proton o f the quinones, h e l i o c i d e s and gossypol appear i n the region between 6 10 and 12. Examples o f the H-NMR (6 10 t o 12) o f e x t r a c t s from a high gossypol, G. hirsutum cotton (HG-6-1-N) and a G. barbadense cotton (SBSI) are shown i n F i g . 2 and F i g . 3, r e s p e c t i v e l y . Acetone was used as the s o l v e n t because i t gave the best peak r e s o l u t i o n . Pure terpenoids were added t o the crude e x t r a c t s t o determine which compound(s) corresponded t o each peak. H e l i o c i d e s H , H , B , and B could not be r e s o l v e d completely from each other by t h i s technique. However, the other h e l i o c i d e s , quinones and gossypol are a l l r e s o l v e d , and t h e i r c o n c e n t r a t i o n s may be e s t i mated. U n f o r t u n a t e l y , the chemical s h i f t s o f the peak change s l i g h t l y with i n c r e a s e s i n c o n c e n t r a t i o n ; consequently, r e s o l u t i o n i s i n v e r s e l y p r o p o r t i o n a l t o the c o n c e n t r a t i o n . X

2

2

3

3

X

TLC Screening Techniques. The H-NMR method o f screening p l a n t progeny r e q u i r e s about 3 gm o f f r e e z e d r i e d powder. A TLC method t h a t r e q u i r e s a s i n g l e l e a f has been developed (17_). T h i s method uses f r e s h terminal l e a v e s , a l l o w i n g t e r p e n o i d q u a n t i t a t i o n before f l o w e r i n g . A three-man team can screen 240 progeny/day. An e x t r a c t from the l e a f i s spotted on a S i gel p l a t e and developed with Hex:Et 0:HC00H (84:15:1). The s e q u e n t i a l appearance o f the compounds going up the p l a t e are gossypol, hemigossypolone (R^ ca. 0.30), h e l i o c i d e s H , H and H (mixed as one s p o t ) , hemigossypolone-7-methyl e t h e r mixed with h e l i o c i d e Ηχ, h e l i o c i d e s B , B , and Bi+ (mixed as one s p o t ) , and h e l i o c i d e Βχ. The i n d i v i d u a l spots are v i s u a l i z e d by spraying with p h l o r o g l u c i n o l reagent. Gossypol forms rose spots, hemigossypolone and hemigossypolone-7methyl ether form magenta t o maroon c o l o r s , and the h e l i o c i d e s give orange spots. T h i s method separates h e l i o c i d e s H and H from h e l i o c i d e s 2

2

3

4

2

2

3


3

HOST P L A N T RESISTANCE T O PESTS

204


Acetone Solvent

Figure 2. *H NMR (B 10-12) of an ethyl acetateihexane (1:3) extract of freeze dried 3-4-day-old both with bracts from G. hirsutum. G -gossypol; HGQ = hemigossypolone; H H , H , H = heliocide H H , H and Hj>. ly

2

3

k

u

.

—ι— 12

2

s>

10

Acetone Solvent

MHGQ.

Figure 3. 'H NMR (8 10-12) of an ethyl acetate ihexane (1:3) extract of freeze dried 3-4-day-old bolls with bracts from G. barbadense. MG = 6-methyl ether derivatives of gossypol; G = gossypol; HGQ = hemigossy polone, MHGQ = hemigossypolone-! methyl ether, B H B , H = helio cide B H B H . u

u

u

h

ly

ky

MG G HGQi

k

k

ι 12

r

π


r 10

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X

B and B . Thus, the H-NMR and TLC screening techniques, when used together, give good e s t i m a t i o n s o f the h e l i o c i d e s present. 2

3

Occurrence. Hemigossypolone and the h e l i o c i d e s H i , H , H , and H^ occur i n both upland cottons (G. hirsutum) and Egyptian cottons (G. barbadense). Most commercial v a r i e t i e s o f upland cotton which were screened by the TLC technique above contained l a r g e r q u a n t i t i e s o f h e l i o c i d e s H and H than o f h e l i o c i d e s Hi and Ηΐψ. The methyl ether d e r i v a t i v e s , hemigossypolone-7-methyl ether and h e l i o c i d e s B i and Bi+, were found i n the G. barbadense c o t t o n , SBSI, which d i d not c o n t a i n h e l i o c i d e s H , H , B and B . Apparently SBSI does not s y n t h e s i z e myrcene i n the f o l i a r glands and t h e r e f o r e h e l i o c i d e s H , H , B and B are not produced. When F p l a n t s from crosses between G. hirsutum and G. barbadense were screened by the TLC technique, spots corresponding to h e l i o c i d e s H , H , B and B were observed from some progeny, but the i d e n t i t y o f these compounds has not been confirmed. Gossypol i s present i n both s p e c i e s . I t s methyl ether d e r i v a t i v e s , 6-methoxygossypol and 6,6'-dimethoxygossypol, are probably present at l e a s t i n G. barbadense, but t h i s has not been confirmed. 2

2

3

2

2

3

3

2

3

2

3

3


2

2

3

2

3

D i s t r i b u t i o n o f H e l i o c i d e s and Related Compounds i n Plant T i s s u e . E x t r a c t s have been prepared from v a r i o u s t i s s u e s o f g l a n d l e s s and glanded G. hirsutum

and G. barbadense,

and q u a l i t y and

quantity

o f t e r p e n o i d aldehydes were compared by t h i n l a y e r chromatography. Only glanded l e a v e s , stems, and flowers y i e l d e d t e r p e n o i d aldehydes i n d i c a t i n g t h a t the terpenoids are l o c a l i z e d i n the pigment glands. This has been confirmed by the histochemical t e s t s developed by Mace, et a l . (31_, 32, 33). Terpenoid content v a r i e s c o n s i d e r a b l y i n q u a n t i t y and q u a l i t y depending on t i s s u e , age, and whether the p l a n t i s healthy or diseased. Other v a r i a b l e s , such as temperature, s u n l i g h t , and photoperiod, are p r e s e n t l y being i n v e s t i g a t e d . The d i s t r i b u t i o n of terpenoids among parts and t i s s u e s of h e a l t h y , mature p l a n t s o f G. hirsutum and G. barbadense i s shown i n Table I. In pigment glands, the methylated terpenoids occurred i n G. barbadense but not G. hirsutum. J u v e n i l e p l a n t s o f G. hirsutum* however, contained methylated terpenoids i n the epidermis and c o r t e x o f healthy roots and i n parenchyma c e l l s o f diseased s t e l e and hypocotyl (18, 19^, 30, 32). Terpenoid quinones, and t h e i r heli ocide d e r i v a t i v e s , were the primary components i n glands o f stems, l e a v e s , and green parts o f flower buds. N e i t h e r the quinones nor the h e l i o c i d e s were found i n t i s s u e s t h a t lacked c h l o r o p h y l l (e.g. p e t a l s and stamens) or were s h i e l d e d from l i g h t (e.g. embryos, s t e l e , and phloem). In h e a l t h y p l a n t s , a p p r e c i a b l e amounts o f gossypol or i t s methyl ether d e r i v a t i v e s were found o n l y i n embryos, bark (phloem), p e t a l s , and stamens. More than 95% of the terpenoids i n seeds o f G. hirsutum were gossypol and i t s methyl ether d e r i v a t i v e s . In healthy g l a n d l e s s p l a n t s , terpenoids were



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Table I. Major t e r p e n o i d aldehyde components i n pigment glands i n different tissues o f cultivated cotton.


Tissue Seed: Embryo Coat Stems : Cortex Phloem^ Xylem Leaves: Cotyledonary True

Upland Cotton

Egyptian Cotton

{G. hirsutum)

{G. barbadense)

no glands

G, MG, DMG no glands

H , H3, ( H i , Hi+) G no glands

Bi

2

G HGQ, H , H , ( H i , 2

3

9 Bit, H i , Hit G, MG, DMG no glands

G, MG, DMG HGQ, MHGQ, B B^, Hi Hit B i , Bit, H i , Hit

HI+)

l f

9

Petiole Flowers: B r a c t s and c a l y x P e t a l s and stamens Pollen Ovary and stigma

H»

(Hi,

H3,

2

Hi+)

H , H3, ( H i , H4) G no glands HGQ, H , H3, ( H i , Hit) 2

2

Roots : Cortex^ PhloenT Xylem

B i , Bit, H i , Hit G, MG, DMG no glands HGQ, MHGQ, B B , Hi » Hit l9

4

G, MG, DMG G, MG, DMG no glands

G G no glands

A b b r e v i a t i o n s : G=gossypol; MG=6-methoxygossypol; DMG=6,6'-dimethoxygossypol ; H i , H , H , Η , Βχ, and Bi =heliocides Ηχ, H , H3, Hit, i > and B ; HGQ=hemigossypolone; and MHGQ=hemigossypolone7-methyl e t h e r . Parentheses i n d i c a t e t h a t these components were prominent i n o n l y a few c u l t i v a r s . M o s t o f the t e r p e n o i d s i n t h i s t i s s u e were i n t h e epidermis o r phelloderm o f the r o o t r a t h e r than i n glands. ^Glands do not occur i n the phloem o f s e e d l i n g s , but develop as the p l a n t ages. 2

3

4

+

B

4

e


2

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207

Cotton

t o t a l l y absent from a l l t i s s u e s , except the outer c e l l s o f the root bark. We compared the g l a n d u l a r t e r p e n o i d s from leaves o f 24 Gossypium and three Cienfuegosia s p e c i e s . The r e s u l t s f o r the Gossypium s p e c i e s are shown i n Table I I . The t e r p e n o i d quinones and the h e l i o c i d e s were m i s s i n g i n most D genome c o t t o n s , and i n the t h r e e Cienfuegosia s p e c i e s . These compounds were present i n o n l y t r a c e amounts i n A f r i c a n c o t t o n o f the Ε genome. Methylated terpenoids o c c u r r e d i n the B, C, F, AD and AD genomes, but were absent i n the A, D, E, and AD genomes. G. vaimondii, G. davidsonii, and G. klotzsohianum possessed unique t e r p e n o i d s m i s s i n g in the other s p e c i e s . 2

3

2


B i o s y n t h e s i s o f the Terpenoids. H e i n s t e i n , e t a l . have shown t h a t gossypol i s b i o s y n t h e s i z e d from a c e t a t e via the i s o p r e n o i d pathway (34, 35). They i s o l a t e d an enzyme system from homogenates o f c o t t o n r o o t s t h a t s t e r e o s p e c i f i c a l l y i n c o r p o r a t e d s i x molecules o f mevalonate-2- C i n each molecule o f g o s s y p o l . The b i o s y n t h e s i s was via a s p e c i f i c c y c l i z a t i o n o f ois, c i s - f a r n e s y l pyrophosphate. Veech, e t a l . showed t h a t peroxidase c a t a l y z e s the c o u p l i n g o f hemigossypol to form gossypol (36). Thus, cis, c i s - f a r n e s y l pyrophosphate i s a l s o a b i o s y n t h e t i c p r e c u r s o r to hemigossypol. The proposed steps i n t e r p e n o i d b i o s y n t h e s i s are shown i n F i g . 4. Enzymatic reactions appear to be i n v o l v e d i n the formation o f desoxy-6-methoxyhemigossypol (dMHG, F i g . 4) from desoxyhemigossypol (dHG), and i n the o x i d a t i o n o f hemigossypol (HG) or methoxyhemigossypol (MHG) to hemigossypolone (HGQ) and hemigossypolone-7-methyl e t h e r (MHG), r e s p e c t i v e l y . dHG and dMHG spontaneously o x i d i z e to HG and MHG in the presence o f a i r (20), and myrcene and £rarcs-3-ocimene r e a c t with the quinones, HGQ and MHGQ, a t room temperature without c a t a l y s t s to form h e l i o c i d e s (21_, 22, 29J. Thus, an enzyme may not be r e q u i r e d f o r these l a t t e r r e a c t i o n s . ll4

Antibiosis. When c o t t o n s without pigment glands ( g l a n d l e s s ) were d i s c o v e r ed and developed i n the I960's (37J, i t was found t h a t many i n s e c t s which d i d not a t t a c k glanded c o t t o n almost completely destroyed g l a n d l e s s s t r a i n s . These o b s e r v a t i o n s s t i m u l a t e d s t u d i e s on the importance o f pigment glands i n host p l a n t r e s i s t a n c e . A review o f the importance o f pigment glands i n host p l a n t r e s i s t a n c e ha been p u b l i s h e d (17). The t o x i c i t y o f glanded f l o w e r buds to i n s e c t s has been c o r r e l a t e d with t h e i r gossypol content ( 6 ) . However, the gossypol content was determined by the a n i l i n e method. As mentioned p r e v i o u s l y , t h i s i s a n o n s p e c i f i c reagent f o r aromatic aldehydes. It i s now known t h a t many compounds c o n t a i n i n g aromatic aldehyde groups are present i n the glands, and these g i v e r e a c t i o n products



herbaoeum arboreum anomalum sturtianum australe biokii thurberi harknessii armourianum davidsonii klotzsohianum raimondii gossypioides trilobum stooksii somalense inoanum longioalyx hirsutum barbadense tomentosum +

tr tr tr tr tr tr tr tr tr

+

tr tr tr tr

+

-

-

+ +

-

++

tr

+++ ++

-

+++ ++ ++ ++ +++ +

-

+ + +

tr tr tr ++

(f

HG

2

-

++

-

+++ ++ ++ ++

++ + ++ +

4

+

-

-

tr

-

+++

++

tr tr

++

tr

+

+++

-

tr

-

tr

-

tr

+

tr

-

+ ++ +++

2

W

+ ++

+++

x

H

++ + ++ +++ + +++

HGQ d

++ + ++ ++

+

tr

-

-

++

++

-

++

tr

+

tr

-

-

+

-

+ +++

-

Bi

tr

-

tr tr

+ ++

-

-

-

of Terpenoids^ MHGQ

a

MHG

Relative Concentration

Species

3

3

£+++ = l a r g e intense s p o t ; + = small but d i s t i n c t s p o t ; and t r = t r a c e on t h i n l a y e r chromatograms. I d e n t i t i e s and s t r u c t u r e s o f the abbreviated chemicals are given i n Table I . ^Spots c o n t a i n up to three compounds, g o s s y p o l , 6-methoxygossypol, 6,6'-dimethoxygossypol. Spots c o n t a i n up to three compounds, h e l i o c i d e s H , H , and H . ^.Spots c o n t a i n up to three compounds, h e l i o c i d e s B2, B , and B^. •Û = u n i d e n t i f i e d t e r p e n o i d s .

3

2

X

Fi AD AD AD

^

2

8

6

5

3

3

2

2

3

2

G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G. G.

Species

Type

Ai A Bi Ci c C, Di D D -l D -d D -r D D D Ei E

Gossypium

Terpenoid Content o f Pigment Glands i n Young Leaves of Gossypium

Genome

Table I I .


tr tr

-

tr

-

tr

-

tr

++

-

B 2

e

-

+ ++ +++

-

-

U

f


Figure 4.

Proposed pathway for biosynthesis of terpenoid aldehydes in cotton


1

3 ο ο ο

Ο

ce

S:

δ*

I

>

M H

η

§

H

t—»


210

H O S T P L A N T RESISTANCE T O

PESTS

t h a t i n t e r f e r e with the measurement o f gossypol. Thus, previous measurements o f gossypol, e s p e c i a l l y i n green t i s s u e , a r e l a r g e l y erroneous. We a r e attempting t o bioassay a l l o f t h e h e l i o c i d e s , quinones, gossypol, and gossypol-methyl ethers f o r t h e i r t o x i c i t y t o t h e tobacco budworm (#. virescens), pink bollworm [Pectinophora gossypiella), and t h e b o l l weevil (Anthonomus grandis). T h i s work i s incomplete, but the r e s u l t s t o date f o r the tobacco budworm and pink bollworm are given i n Table I I I . S y n e r g i s t i c e f f e c t s o f these compounds a r e being s t u d i e d . The b o l l weevil has been i n s e n s i t i v e t o gossypol, t h e h e l i o c i d e s , and the quinones a t c o n c e n t r a t i o n s up t o 0.2% (net weight). The p r e l i m i n a r y data t h a t we have i n d i c a t e t h a t a l l o f the t e r p e n o i d aldehydes a r e about e q u a l l y t o x i c t o the l a r v a l stage o f the pink bollworm. G. barba dense i s more r e s i s t a n t t o the bollworm (#. zea) than G. hirsutum (35). H e l i o c i d e s i n G. barbadense a r e formed o n l y from ocimene and are more than 50% methylated. H e l i o c i d e s Η χ and Βχ (from ocimene) are more t o x i c t o tobacco budworms than H e l i o c i d e s H o r H (from myrcene). Thus, q u a l i t a t i v e d i f f e r e n c e s i n h e l i o c i d e s between G. hirsutum and G. barbadense may be r e s p o n s i b l e f o r differences i n resistance. 2

3

Genetics o f Methyl Ether Formation i n Glands. We have made p r e l i m i n a r y s t u d i e s o f the g e n e t i c s o f t e r p e n o i d methyl ether formation. Methyl ether formation was determined by e s t i m a t i n g c o n c e n t r a t i o n s o f hemigossypolone and hemigossypolone7-methyl ether. These are major t e r p e n o i d s i n j u v e n i l e l e a v e s , flower buds, and young b o l l s , and a r e precursors t o the h e l i o c i d e s . Three v a r i e t i e s o f G. hirsutum (Acala SJ-1, CAM-1 and Stonev i l l e 213) were crossed r e c i p r o c a l l y with G. barbadense (SBSI). One-half expanded terminal leaves o f the G. hirsutum v a r i e t i e s contained hemigossypolone but no hemigossypolone-7-methyl ether. S i m i l a r leaves o f G. barbadense contained s l i g h t l y more hemi gossypol one-7-methyl ether than hemigossypolone. The segregation o f hemi gossypol one-7-methyl e t h e r i n Fx and F progenies a r e shown in Table IV. Hemigossypolone-7-methyl ether and h e l i o c i d e s Βχ and B always occurred together among more than 2000 F progeny. The r e s u l t s i n d i c a t e t h a t terpenoid methyl ether formation i n pigment glands i n progeny from G. hirsutum X G. barbadense i s c o n t r o l l e d by a s i n g l e r e c e s s i v e locus f o r which we have proposed the symbol tmx (29). While t e r p e n o i d methyl ether formation i n G. hirsutum i s not expressed i n pigment glands (Table IV), i t i s p a r t i a l l y expressed (about 20% o f t h a t i n G. barbadense) i n diseased xylem (33) o r cambium (18). F u r t h e r , n e a r l y 50% o f a l l terpenoids i n j u v e n i l e t i s s u e s o f G. hirsutum [7-day-old h e a l t h y r a d i c a l s (19.) o r 7-dayo l d i n f e c t e d hypocotyls (30)] a r e present as methyl e t h e r s ; t h i s i s t h e same percentage as i n G. barbadense. We conclude t h a t G. hirusutum has a s t r u c t u r a l gene, t h a t gives i t the same b a s i c 2

4

2


14.

sTiPANOvic E T A L .

Table I I I . E D

Natural

Insecticides

a

50

V a l u e s f o r Heliothis

Pectinophora

virescens

ED H.

50

2

3

x


2

3

2.5 Π.2 3.9 4.6 N.E. 10.5 N.E. 0.8

ED50. gossypiella

b

vtresaens

(ymoles/g d i e t ) χ

and

gossypiella.

Compound

Heliocide Η Heliocide H Heliocide H Heliocide B , Heliocide B & B Hemigossypolone Methoxyhemigossypolone Gossypol

211

from Cotton

P.

0

(ymoles/g diet) 0.8 2.4 — —

e

—

1.4 —

1.8

C o n c e n t r a t i o n r e q u i r e d t o reduce l a r v a l growth by 50%. ^Two-day-old l a r v a e were placed on media (Vanderzant-Adkisson d i e t ) at t h e beginning o f the experiment; a f t e r 7 days on amended media, a l l l a r v a e were returned t o r e g u l a r media. Âmended d i e t ingested f o r d u r a t i o n o f l a r v a l stage. Âdded t o the d i e t as a mixture o f h e l i o c i d e s B and B (67% B , 33% B ) . ^Compounds had no a p p r e c i a b l e e f f e c t on i n s e c t growth a t a c o n c e n t r a t i o n o f 2.5ymoles/g d i e t . 2

3

2

3

a

Table IV. Genetics o f Terpenoid Methyl Ether F o r m a t i o n i n Crosses Between V a r i e t i e s o f G. hirsutum (ASJ-1, CAM-1, and S-213) and G. barbadense (SBSI). Fi

Cross

MHGQ

F2

No MHGQ

MHGQ

No MHGQ

Ratio

Number o f P l a n t s ASJ-1 X SBSI SBSI X ASJ-1 CAM-1 X SBSI SBSI X CAM-1 S-213 X SBSI SBSI X S-213

0 0 0 0 0 0

30 30 30 30 30 30

86 124 91 82 71 99

263 263 376 377 355 354

1/3.06 1/2.12 1/4.13 1/4.60 1/5.00 1/3.58

Mean

0

30

92

331

1/3.59

'The presence o f hemigossypolone-7-methyl ether (MHGQ) i n o n e - h a l f expanded terminal leaves was noted as a marker f o r methyl e t h e r formation. Terpenoid methyl ethers o r i g i n a l l y were present o n l y i n t h e G. barbadense parent.


212

H O S T P L A N T RESISTANCE

T O PESTS

p o t e n t i a l f o r methyl ether formation as i s found i n G. barbadense. However, methyl ether formation i s not expressed i n f o l i a r pigment glands o f G. hirsutum because o f the product o f the r e g u l a t o r a l l e l e TMj. The patterns o f terpenoids found i n leaves (Table I I ) suggests t h a t the tmj homozygote occurs u n i v e r s a l l y i n D, E, and AD genome c o t t o n s . X

Search f o r New Compounds Involved i n Host P l a n t Resistance. Table II i n d i c a t e s new compounds observed i n G. davidsonii, and G. raimondii. The s t r u c t u r e and biochemical a c t i v i t y o f these compounds must be s t u d i e d . Crosses o f these species with G. hirsutum o r G. barbadense might lead t o a d i f f e r ent s e r i e s o f compounds t h a t could i n c r e a s e host p l a n t r e s i s t a n c e .


G. klotzsohianum,

Conclusion. Cotton leaves and flower buds c o n t a i n several t o x i c t e r p e n o i d aldehydes. The c o n c e n t r a t i o n o f these compounds vary among races and s p e c i e s . We are p r e s e n t l y i n v e s t i g a t i n g which compound o r mixture o f compounds i s the most e f f e c t i v e i n s e c t i c i d e f o r v a r i o u s cotton pests. The b i o s y n t h e t i c pathway l e a d i n g t o these compounds i s being s t u d i e d . Genes t h a t c o n t r o l c r i t i c a l branches i n t h i s pathway are known, and we are a p p l y i n g t h i s knowledge i n a breeding program aimed a t i n c o r p o r a t i n g e f f e c t i v e n a t u r a l i n s e c t i c i d e s i n t o an a c c e p t a b l e agronomic l i n e . A s p e c i f i c example i s found i n h e l i o c i d e s Hi and Hi+. Our present data i n d i c a t e s t h a t these may be the most t o x i c h e l i o c i d e s in G. hirsutum. They are formed from t:rans-3-ocimene and hemigossypolone. H e l i o c i d e s H and H3, formed from myrcene, a r e l e s s e f f e c t i v e t o x i n s , and t h e i r formation leads t o lower l e v e l s o f i n s e c t i c i d a l a c t i v i t y . SBSI produces trans-3-ocimene, with l i t t l e or no myrcene, because i t produces o n l y h e l i o c i d e s H i , Hi+, B i and Bit. Therefore a s p e c i f i c gene t h a t d i r e c t s the s y n t h e s i s o f trans3-ocimene r a t h e r than myrcene would seem t o be o p e r a t i n g . I f t h i s gene r a t h e r than the gene a l l o w i n g the s y n t h e s i s o f myrcene could be i n c o r p o r a t e d i n t o an acceptable agronomic l i n e , the r e s i s t a n c e to Heliothis should be i n c r e a s e d . Furthermore, the quinones a r e not as t o x i c as h e l i o c i d e s Hi and Hi+. T h e r e f o r e , a v a r i e t y o f cotton t h a t produced l a r g e r q u a n t i t i e s o f trans-3-ocimene a l s o would be d e s i r a b l e because t h i s would i n c r e a s e the c o n c e n t r a t i o n o f h e l i o c i d e s and decrease the c o n c e n t r a t i o n s o f quinones. One aspect t h a t appears t o be beyond g e n e t i c c o n t r o l i s t h e r a t i o o f h e l i o c i d e s Hi t o Htf. The r e a c t i o n o f the quinone with trans-3-ocimene appears t o occur i n a nonenzymatic f a s h i o n and t h i s r a t i o appears t o remain almost constant i n a l l v a r i e t i e s tested. Our understanding o f the s t r u c t u r e s and b i o s y n t h e s i s o f t h e cotton terpenoids i s a l l o w i n g us t o p r e d i c t what can and can n o t be accomplished t o i n c r e a s e host p l a n t r e s i s t a n c e by genetic 2


14.

Natural


Insecticides

from

Cotton

213

manipulation o f the p l a n t . Acknowledgements: We thank J . K. C o r n i s h , G. W. T r i b b l e , M. E. Bearden and J . G. G a r c i a f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . We are g r a t e f u l to Dr. Ron Grigsby f o r high r e s o l u t i o n mass measurements and Dr. Daniel O'Brien f o r C-NMR s t u d i e s t h a t were i n v a l u a b l e i n our structure determinations. 13

LITERATURE CITED

1. Downloaded by PURDUE UNIV on July 6, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0062.ch014

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Painter, R. H., "Insect Resistance in Crop Plants," MacMillan, New York, NY. 1951. B e l l , Α. Α., "Biological Control of Plant Insects and Diseases," F. G. Maxwell and F. S. Harris, Ed. 403-461. The University Press of M i s s i s s i p p i , Jackson, Miss. 1974. Muller, K. O., "Plant Pathology," Vol. 1, J. G. Horsfall and A. E. Diamond, Ed. 469-519. Academic Press, New York, NY. 1959. Cook, O. F., U. S. Dept. Agric. Bur. Plant Ind. Bul. No. 88 (1906) 87. Bottger, G. T., Sheehan, E. T., Lukefahr, M. J . , J. Econ. Entomol. (1964) 57, 283. Lukefahr, M. J . , Bottger, G. T., Maxwell, F., Proc. Beltwide Cotton Prod. Res. Conf., Jan. 11-12, Memphis, Tenn. (1966) 215. Smith, F. H., J. Am. Oil Chem. Soc. (1958) 35, 261. Reeves, R. G., Beasley, J. O., J. Agric. Res. (1935) 51, 935. Stanford, Ε. Ε., Viehoever, Α., J. Agri. Res. (1918) 13, 419. Lukefahr, M. J . , Marlin, D. F., J. Econ. Entomol. (1966) 59, 176. Bottger, G. T., Patana, R., J. Econ. Entomol. (1966) 59, 1166. Lukefahr, M. J . , Shaver, Τ. Ν., Parrott, W. L., Proc. Belt wide Cotton Prod. Res. Conf., Jan. 7-8, New Orleans, LA (1969) 81. Lukefahr, M. J . , Houghtalling, J. E., Cruhm, D. G., J. Econ. Entomol. (1975) 68, 743. Leigh, T. F., Proc. Beltwide Cotton Prod. Res. Conf., Jan. 78, New Orleans, LA (1975) 140. Niles, G. Α., Proc. Beltwide Cotton Prod. Res. Conf., Jan. 57, Las Vegas, Nev. (1976) 168. B e l l , Α. Α., Stipanovic, R. D., Proc. Beltwide Cotton Prod. Res. Conf., Jan. 5-7, Las Vegas, Nev. (1976) 52. B e l l , Α. Α., Stipanovic, R. D., Proc. Beltwide Cotton Prod. Res. Conf., Jan. 10-12, Atlanta, GA (1977) in press. B e l l , Α. Α., Stipanovic, R. D., Howell, C. R., F r y x e l l , P. Α., Phytochem. (1975) 14, 225. Stipanovic, R. D., B e l l , Α. Α., Mace, M. E., Howell, C. R., Phytochem. (1975) 14, 1077.



214


T O PESTS

20. Stipanovic, R. D., Bell, Α. Α., Howell, C. R., Phytochem. (1975) 14, 1809. 21. Stipanovic, R. D., Bell, Α. Α., O'Brien, D. H., Lukefahr, M. J., Phytochem. (1977) in press. 22. Stipanovic, R. D., Bell, Α. Α., O'Brien, D. H., Lukefahr, M. J., J. Agric. Food Chem. (1977) in press. 23. Stipanovic, R. D., Bell, Α. Α., O'Brien, D. H., Lukefahr, M. J., Tetrahedron Letters (1977) 567. 24. Stipanovic, R. D., Bell, Α. Α., Lukefahr, M. J., Gray, J. R., Mabry, T. J., Proc. Beltwide Cotton Prod. Res. Conf., Jan. 57, Las Vegas, Nev. (1976) 91. 25. Gray, J. R., Mabry, R. J., Bell, Α. Α., Stipanovic, R. D., Lukefahr, M. J., J. Chem. Soc., Chem. Comm. (1976) 109. 26. Creiger, R., Becher, P., Chem. Ber. (1957) 90, 2516. 27. Bloom, S. Μ., J. Org. Chem. (1959) 24, 278. 28. Martin, J. G., Hill, R. K., Chem. Rev. (1961) 61, 537. 29. Bell, Α. Α., Stipanovic, R. D., O'Brien, D. H., Lukefahr, M. J., Phytochem. (1977) in press. 30. Unpublished observations. 31. Mace, M. E., Bell, Α. Α., Stipanovic, R. D., Proc. Beltwide Cotton Prod. Res. Conf., Jan. 7-9, Dallas, TX, (1974) 46. 32. Mace, M. E., Bell, Α. Α., Stipanovic, R. D., Phytopathol. (1974) 64, 1297. 33. Mace, M. E., Bell, Α. Α., Beckman, C. H., Can. J. Bot. (1976) 54, 2095. 34. Heinstein, P. F., Smith, F. H., Tove, S. B., J. Biol. Chem. (1962) 237, 2643. 35. Heinstein, P. F., Herman, D. L., Tove, S. B., Smith, F. H., J. Biol. Chem (1970) 245, 4658. 36. Veech, J. Α., Stipanovic, R. D., Bell, Α. Α., J. Chem. Soc., Chem. Comm. (1976) 144. 37. McMichael, S. C., Agron. J. (1960) 52, 385.


Host Plant Resistance to Pests

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