20 Multiple Factors in Cotton Contributing to Resistance to the Tobacco Budworm, Heliothis virescens F. P. A. HEDIN, J. N. JENKINS, D. H. COLLUM, W. H. WHITE, and W. L. PARROTT U.S. Department of Agriculture, Science and Education Administration, Agricultural Research Southern Region, Boll Weevil Research Laboratory, Chemistry Research Unit, Mississippi State,MI39762
Cyanidin-3-ß-glucoside has been shown t o be an important f a c t o r o f r e s i s t a n c e in cotton Gossypium hirsutum L. leaves t o the feeding of tobacco buworm Heliothis v i r e s c e n s (Fab.) in the field. The reported e f f e c t i v e n e s s of gossypol was confirmed, but the condensed tannins (proanthocyanidins) i n t e r m i n a l leaves were not c o r r e l a t e d w i t h r e s i s t a n c e . Paradoxically, these 3 compounds when incorporated in l a b o r a t o r y d i e t s are e q u a l l y t o x i c t o l a r v a e . These f i n d i n g s provide a potential b a s i s f o r a c h i e v i n g i n s e c t r e s i s t a n c e in non-glanded cotton and other crops i n f e s t e d by Heliothis.
Since the o r i g i n a l development of glandless cotton by McMichael ( 1 ) , entomologists and p l a n t breeders have noted that the experimental g l a n d l e s s l i n e s are g e n e r a l l y s u s c e p t i b l e t o c e r t a i n phytophagous i n s e c t s . Bottger e t a l . (2), r e p o r t i n g on the r e l a t i o n s h i p between the gossypol content of cotton p l a n t s and i n s e c t r e s i s t a n c e , noted that s e v e r a l i n s e c t s f e d on a g l a n d l e s s l i n e i n preference t o glanded l i n e s . Jenkins e t a l . (3_) reported increased s u s c e p t i b i l i t y of s e v e r a l g l a n d l e s s l i n e s t o the bollworm ( H e l i o t h i s zea Boddie). Lukefahr e t a l . (4_) showed that the growth of bollworm and tobacco budworm ( H e l i o t h i s v i r e s c e n s F.) l a r v a e increased on d i e t s of g l a n d l e s s cotton l i n e s compared w i t h that on d i e t s of the corresponding glanded l i n e . Lukefahr and M a r t i n (5) incorporated 3 cotton pigments, gossypol, q u e r c e t i n , and r u t i n , i n t o a standard bollworm d i e t whereupon l a r v a l growth was decreased. They suggested that p l a n t breeders might s e l e c t f o r cotton p l a n t s w i t h higher pigment (gossypol and q u e r c e t i n ) content as a mechanism of r e s i s t a n c e . I t was observed that flower buds from c e r t a i n w i l d and p r i m i t i v e cottons showed 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 gossypol, and the a d d i t i o n a l a c t i v i t y was a s c r i b e d t o "X" f a c t o r s ( 6 , 7 ) . The "X" f a c t o r s were i d e n t i f i e d
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as sesquiterpenoid quinones, hemigossypols, and h e l i o c i d e s i n a s e r i e s of i n v e s t i g a t i o n s by t h i s College S t a t i o n , Texas group (8-11). In summary, they i s o l a t e d at l e a s t 15 terpenoid aldehydes and r e l a t e d compounds from glanded cotton p l a n t s , present i n v a r y i n g amounts from d i f f e r e n t l i n e s . They reported that t o x i c i t y (ED50; c o n c e n t r a t i o n to reduce l a r v a l growth by 50%) approached or e q u a l l e d gossypol (0.05%) f o r a number of these compounds when i n c o r p o r a t e d i n d i e t s fed to tobacco budworms. Gossypol was g e n e r a l l y found i n considerably higher concentations than the other terpenoid aldehydes i n the l i n e s i n v e s t i g a t e d . This group (12) a l s o chromatographically i d e n t i f i e d c a t e c h i n , g a l l o c a t e c h i n , q u e r c e t i n , and condensed tannin from V e r t i c i l l i u m w i l t - r e s i s t a n t young cotton leaves i n higher concentrations than from s u s c e p t i b l e l a r g e r l e a v e s . Subsequently, a s e r i e s of i n v e s t i g a t i o n s by a group at the Western Regional Research Center i n C a l i f o r n i a and a s s o c i a t e s have shown condensed cotton tannin to be an a n t i b i o t i c chemical f o r the bollworm, tobacco budworm, and pink bollworm (13, 14, 15). I n i t i a l l y , they sought to i n v e s t i g a t e the "X" f a c t o r that had been described by i t s e x t r a c t i o n from freeze dehydrated t i s s u e w i t h e t h y l ether or acetone. They found that i f f r e e z e dehydrated cotton powder was f i r s t e x t r a c t e d w i t h hexane or a s i m i l a r non-polar solvent system, gossypol and the other t e r penoid aldehydes were i s o l a t e d i n the e x t r a c t . This e x t r a c t when incorporated i n i n s e c t d i e t s decreased l a r v a l growth as prev i o u s l y reported. The r e s i d u a l powder was then e x t r a c t e d w i t h methanol; t h i s e x t r a c t when i n c o r p o r a t e d i n the l a r v a l d i e t depressed growth s e v e r e l y . In f a c t , i n a study where c o n t r o l tobacco budworm l a r v a e weighed 306 mg a f t e r 14 days, l a r v a e fed the hexane e x t r a c t (from 6 g of powder added to 30 g of d i e t ) weighed 56.6 mg, those fed the acetone e x t r a c t weighed 343 mg, and those fed the comparable amount of the ethanol e x t r a c t from the methanolic e x t r a c t weighed 1.1 g. The major a n t i b i o t i c compound i n the methanol s o l u b l e f r a c t i o n was subsequently c h a r a c t e r i z e d as condensed t a n n i n , which when hydrolyzed w i t h HC1 i n n-butanol y i e l d e d c y a n i d i n and d e l p h i n i d i n . By osmotic measurements, the average molecular weight was estimated to be 4850. In r e l a t e d work, Chan et a l . (14) i s o l a t e d the cyclopropen o i d f a t t y a c i d s , the terpene aldehydes i n c l u d i n g gossypol, the f l a v o n o i d s , and the condensed t a n n i n s . When fed to tobacco budworm [ a l s o bollworm and pink bollworm (Pectinophora g o s s y p i e l l a Saunders)] h a t c h l i n g l a r v a e i n d i e t s , the ED50 values (percent of d i e t ) were as f o l l o w s : gossypol, 0.12; hemigossypolone, 0.08; h e l i o c i d e H , 0.12; h e l i o c i d e H , 0.13; c a t e c h i n , 0.13; querc e t i n , 0.05; condensed t a n n i n , 0.15; methyl s t e r c u l a t e , 0.41; and methyl malvalate, 0.49. S t i p a n o v i c (16) obtained s i m i l a r ED50 values f o r the tobacco budworm w i t h gossypol, hemigossypolone, and h e l i o c i d e s and H2. x
nin
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In another t e s t , Chan et a l . (14) analyzed the condensed t a n and gossypol ( w i t h analogs) content of 10 cotton p l a n t p a r t s .
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I n b r i e f , the t a n n i n content of various l e a f t i s s u e s was f a i r l y h i g h w h i l e the gossypol content was r e l a t i v e l y low. In the anthers, c o r o l l a , and c a l y x , the reverse s i t u a t i o n e x i s t e d . When the p l a n t parts were fed t o budworms, they had comparatively l i t t l e t o x i c i t y ( E D - 1.7%) d e s p i t e the r e l a t i v e l y high gossyp o l content. Except f o r the high t o x i c i t y of c o r o l l a t i s s u e (0.15%) which was a t t r i b u t e d to f l a v o n o i d s , the various l e a f t i s s u e s , ( e a r l y feeding s i t e s i n the f i e l d ) were otherwise the most t o x i c (0.27-0.50%). Shaver and P a r r o t t (17) reared bollworm and tobacco budworm l a r v a e on a standard l a r v a l d i e t , and t r a n s f e r r e d them a t 5 ages onto media c o n t a i n i n g 0-0.4% gossypol. The i n f l u e n c e on development increased w i t h l a r v a l age a t the time of t r a n s f e r . Recently Waiss and h i s co-workers (18) incorporated condensed t a n n i n i n d i e t s fed t o the tobacco budworm where the l a r v a e i n i t i a l l y were of d i f f e r e n t ages. The ED5Q values were as f o l l o w s : 1 day, 0.10; 3 day, 0.10-0.15; 5 day, 0.20-0.30; and 7 day, non-toxic. Thus, i t i s evident that l a r v a e a l s o become more t o l e r a n t t o condensed tannin w i t h age. Shaver e t a l . (19) s t u d i e d feeding and l a r v a l growth i n the l a b o r a t o r y of the tobacco budworm on component parts of cotton f l o w e r buds. They found that most of the feeding of 2-4 day l a r vae occurred on the anthers a f t e r they penetrated i n t o the i n t e r i o r of the bud through the p e t a l s . For p e r s p e c t i v e , i t should be noted that females o v i p o s i t on the young t e r m i n a l s and young l e a v e s , so that the h a t c h l i n g l a r v a e feed on these s i t e s before m i g r a t i n g t o the bud. When component parts of the flower bud were incorporated i n t o l a r v a l d i e t , only the p e t a l s ( c o r o l l a ) i n h i b i t e d growth of 3 day l a r v a e . This i n h i b i t i o n occurred both on glanded and g l a n d l e s s l i n e s , so i t can not be a t t r i b u t e d t o the gossypol content alone. L a t e r , Shaver et a l . (20) f e d 3-day-old tobacco budworm l a r v a e l a b o r a t o r y d i e t s i n t o which e t h y l ether e x t r a c t s of flower buds were i n c o r p o r a t e d . They reported that l i n e s w i t h high gossypol and gossypol r e l a t e d compounds, as determined by the a n i l i n e t e s t , reduced l a r v a l weight g a i n s . Bud tannins were not analyzed, nor were they e x t r a c t e d and fed i n t h i s study. Schuster e t a l . (21) i d e n t i f i e d cotton plant r e s i s t a n c e t o the two-spotted s p i d e r mite (Tetranycus u r t i c a Koch) by mass screening s e e d l i n g s . L a t e r Schuster and Lane (22) were able to show that high t a n n i n l i n e s , p a r t i c u l a r l y TX-1055, showed r e s i s t a n c e t o t h i s arachnid and the bollworm. Another t r a i t of cotton a f f e c t i n g l a r v a l growth of tobacco budworm i s p o l l e n c o l o r which ranges from cream t o yellow t o even orange. Hanny e t a l . (23) f e d 1st i n s t a r tobacco budworm l a r v a e whole f r e s h anthers of 5 cream and yellow l i n e s . I n a number of t e s t s conducted during 1977 and 1978, weight gains a f t e r 7 days were 13-15% l e s s on the y e l l o w p o l l e n . Hanny (24) reported the f o l l o w i n g average analyses: gossypol, yellow 0.88%; cream 0.70%; condensed t a n n i n s , y e l l o w 4.79%; cream 5.34%; and f l a v o n o i d s , 5 0
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yellow 0.56%, cream 0.54%. The I s o l a t i o n of 4 gossypetin g l y c o s i d e s (but not gossypin), 8 q u e r c e t i n g l y c o s i d e s , and an anthocyanin were reported. In p r e l i m i n a r y work at t h i s l o c a t i o n (25), gossypol and t a n n i n were n e g a t i v e l y c o r r e l a t e d with weight gains of tobacco budworm l a r v a e f e d i n the f i e l d on plant terminals. This was based on seasonal averages f o r a f i v e c u l t i v a r t e s t . In l a b o r a t o r y t e s t s , the ED50 values f o r s e v e r a l flavonoids and condensed c o t ton tannins were determined and found to be s i m i l a r (0.05-0.15%) to the values reported by Chan et a l . (14) and S t i p a n o v i c ( 1 6 ) . The ED50 values f o r a number of cotton c u l t i v a r s , h i b i s c u s , sorghum, and s i n f o i n , were found to range from 0.03-0.10%, i n d i c a t i v e that the tannin could be b i o l o g i c a l l y s i m i l a r . L a r v a l feeding t e s t s were a l s o performed on a number of chromatographic f r a c t i o n s obtained from solvent e x t r a c t s of cotton terminal t i s s u e . Those containing gossypol, tannins, and f l a v o n o i d s were most t o x i c . From the preceding, i t appears that chemical r e s i s t a n c e i n cotton to H e l i o t h i s i n s e c t s i s due to m u l t i p l e f a c t o r s . D i f f e r e n t l i n e s , each with i n s e c t r e s i s t a n c e , may possess d i f f e r e n t r a t i o s of a n t i b i o t i c compounds. Thus, i t may be p o s s i b l e to increase r e s i s t a n c e by c r o s s i n g l i n e s where each c o n t r i b u t e s genes f o r b i o s y n t h e s i s of d i f f e r e n t a n t i b i o t i c compounds. The tobacco budworm was s e l e c t e d f o r study i n preference to the c o t ton bollworm because i t i s e a s i e r to rear and use i n the l a b o r a t o r y , i s more r e s i s t a n t to i n s e c t i c i d e s i n the f i e l d , and i t i s approximately as s u s c e p t i b l e to cotton c o n s t i t u e n t s incorporated i n laboratory d i e t s ( 1 4 ) . This present study was c a r r i e d out to i d e n t i f y and analyze f o r cotton c o n s t i t u e n t s that were t o x i c i n l a b o r a t o r y feeding t e s t s , and to determine whether there were p o s i t i v e c o r r e l a t i o n s of t h e i r content i n leaves and/or other t i s s u e with f i e l d r e s i s t a n c e . From t h i s information, the generat i o n of l i n e s with m u l t i p l e f a c t o r s f o r r e s i s t a n c e could be i n i tiated. M a t e r i a l s and Methods 1/ Agronomic and entomological p r a c t i c e s . Plants of d i v e r s e cotton l i n e s were grown i n f i e l d p l o t s during the years 1978-1982 on the Plant Science Farm at M i s s i s s i p p i State U n i v e r s i t y . Plants were t r e a t e d f o r b o l l weevils with Guthion, and normal f e r t i l i z e r , h e r b i c i d e , and other c u l t u r a l p r a c t i c e s were a p p l i e d . The f i e l d design was normally a randomized complete block with 4 r e p l i c a t i o n s . Plant m a t e r i a l ( t e r m i n a l s ,
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l a r g e r l e a v e s , squares, b r a c t s , f l o w e r s , and b o l l s ) was c o l l e c t e d a t i n t e r v a l s throughout the season, freeze dehydrated, ground, and stored at -10°C u n t i l used. F i r s t i n s t a r tobacco budworm l a r v a e were r e s t r a i n e d i n 15 cm long d i a l y s i s casings that were s l i p p e d over the t e r m i n a l s , and c o l l e c t e d and weighed a f t e r f i v e days. Adequate numbers and r e p l i c a t i o n s were employed f o r s t a t i s t i c a l e v a l u a t i o n . Laboratory bioassays were performed by p l a c i n g 1st i n s t a r tobacco budworm l a r v a e on a commercial medium (742-A) prepared by Bioserv I n c . , Frenchtown, NJ. Organic s o l v e n t - s o l u b l e t e s t compounds were d i l u t e d i n e i t h e r hexane or e t h a n o l , added to c a s e i n and evaporated to dryness i n a r o t a r y evaporator. Water s o l u b l e compounds were d i s s o l v e d i n water and added i n place of the p r e s c r i b e d water content. The c a s e i n was then incorporated w i t h the remainder of the i n g r e d i e n t s and poured i n t o 10 cm p e t r i p l a tes to g e l . D i e t c y l i n d e r s of 10 mm diameter, 5 mm height were cut w i t h a cork borer and t r a n s f e r r e d i n t o 15 x 45 (1 dram) s h e l l v i a l s . A neonate l a r v a was added and the v i a l s were incubated at 26°C, 60%RH, 12L:12D f o r 5 days at which time the l a r v a e were weighed. A randomized complete block design w i t h 8 r e p l i c a t e s of 5 l a r v a e each was used. Compounds were t e s t e d at 5-8 conc e n t r a t i o n s ranging from 0.006 to 0.6% of the d i e t on a dry weight b a s i s . Chemical analyses and f r a c t i o n a t i o n s . Freeze-dehydrated c o t ton t i s s u e was analyzed f o r condensed tannin (heated n-BuOH-HCl), gossypol ( p h l o r o g l u c i n o l - H C l ) , and anthocyanins-anthocyanidins ( a l e . HC1 at 540 nm). Other analyses performed but not reported here were f o r c a t e c h i n , t o t a l phenols, E ^ ( t a n n i n ) , and a n i l i n e r e a c t i v e terpenes ( g o s s y p o l ) . Freeze dehydrated p l a n t t i s s u e was e x t r a c t e d by Soxhlet f i r s t w i t h cyclohexane/ethyl a c e t a t e / a c e t i c acid:500/500/1 (CHEA) and then w i t h acetone/water:7/3. The CHEA e x t r a c t was chromatographed on a 40 cm s i l i c i c a c i d column w i t h hexane and s o l v e n t s of i n c r e a s i n g p o l a r i t y to y i e l d gossypol and s e v e r a l other components, each of which was formulated f o r l a b o r a t o r y bioassay t e s t i n g . The aqueous acetone f r a c t i o n was chromatographed on a 1 m Sephadex LH-20 column w i t h 70% aqueous methanol, methanol and dimethyl formamide/methanol:10/90 and 25/75 to y i e l d the f l a v o n o i d s , anthocyanin, and condensed t a n n i n . S i m i l a r l y , each was formulated f o r l a b o r a t o r y bioassay t e s t i n g . The i s o l a t e d components were rechromatographed on LH-20, polyamide, and c e l l u l o s e columns as r e q u i r e d to achieve p u r i t y f o r b i o l o g i c a l e v a l u a t i o n and i d e n t i f i c a t i o n work. I d e n t i f i c a t i o n of c o t t o n p l a n t compounds. The i d e n t i t y of gossypol was confirmed by comparison of the s p e c t r a l (NMR, MS) and chromatographic p r o p e r t i e s w i t h an a u t h e n t i c sample. The i d e n t i t y of the cotton l e a f anthocyanin was confirmed by comp a r i s o n of the chromatographic and s p e c t r a l p r o p e r t i e s of the
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i s o l a t e d from cotton f l o w e r s , by *H NMR, and by procedures that we described p r e v i o u s l y (26). The condensed t a n nins (polymeric proanthocyanidins) were c h a r a c t e r i z e d w i t h regard t o t h e i r stereochemistry, s t r u c t u r a l u n i t s , and molecular weight by the procedures of Czochanska et a l . (27). The condensed tann i n s were found t o c o n s i s t of a mixture of r e l a t e d polymers, the molecular weight ranging from 1500-6000, the p r o d e l p h i n i d i n : p r o c y a n i d i n r a t i o from 1.8-3.7, and the stereochemistry of the monomer u n i t s p r i m a r i l y c i s (81-95%). Figure 1 i s a 13C MNR spectrum of a h i g h l y p u r i f i e d c u l t i v a r BJA-592 condensed t a n n i n . The s p e c t r a were obtained w i t h an a c q u i s i t i o n time of 0.2 s e c , a pulse width of 13 u sec, and a 45° f l i p angle. The average molecular weight was deduced from the r a t i o of the s i g n a l s at 72/67 o t o be 4221. chry8anthemln
H i s t o l o g i c a l Examinations. For the h i s t o l o g i c a l h i s t o c h e m i c a l work, f r e s h samples were frozen a t -20C i n a c r y o s t a t ( I n t . Equip. Co., Model CTI) , mounted on specimen h o l d e r s , trimmed, and s l i c e d w i t h wedge shaped knives at -20C t o produce 20-30 um t i s s u e s l i c e s . For t a n n i n s , small pieces of l e a f (or other t i s s u e ) were f i x e d f o r 78h i n FeS04-5% formol s a l i n e , s l i c e d a t 30 ym and picked up on warm (room temp.) s l i des. F e r r i c c h l o r i d e (1%) or 10% KOH were f r e q u e n t l y used as s t a i n i n t e n s i f i e r s . Tannin granules ( c e l l s ) s t a i n e d b l a c k i s h i n unmodified f i x , b l u i s h w i t h FeCl3 modified f i x , and brown t o redbrown i n KOH modified f i x . In unfixed t i s s u e s , gossypol s t a i n e d red w i t h p h l o r o g l u c i n o l - H C l , and l i g n i f i e d elements s t a i n e d l i g h t v i o l e t . To v i s u a l i z e anthocyanins and gossypol, 30 um f r e s h f r o z e n s e c t i o n s were f i x e d over formaldehyde vapors f o r 24h, and t r e a t e d w i t h 5% HC1 o r NaOH t o produce red or green anthocyanin r e a c t i o n s r e s p e c t i v e l y . Gossypol remained yellow w i t h a c i d or base treatment, i n c o n t r a s t to the anthocyanins. Flower p e t a l s were examined under 10-70X m a g n i f i c a t i o n and photographed i n s i t u a f t e r treatment w i t h 5% KOH which s t a i n e d anthocyanins green. P h l o r o g l u c i n o l - H C l t r e a t e d gossypol glands were s t a i n e d red, and f l a v o n o i d s y e l l o w . R e s u l t s and D i s c u s s i o n T o x i c i t y of cotton p l a n t compounds i n d i e t s t o TBW l a r v a e . Table I gives ED5Q values f o r a number of cotton c o n s t i t u e n t s t e s t e d as i n h i b i t o r s of tobacco budworm l a r v a l growth. For comp a r i s o n , values obtained independently by Chan e t a l . (12) and S t i p a n o v i c (unpublished data) using e s s e n t i a l l y i d e n t i c i a l procedues are i n c l u d e d . The ED50 values were q u i t e r e p r o d u c i b l e by each l a b o r a t o r y . Further separation of the tannin by Sephadex LH-20 chromatography gave f r a c t i o n s w i t h average molecular weights ranging from 1500 t o 6000. When bioassayed, the ED5Q values v a r i e d no more than from 0.05 to 0.10%, so i t i s deduced that w i t h i n l i m i t s , the s i z e of the molecule does not
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0.042
—
0.05
0.10
Quercetin
Isoquercitrin
Chrysanthemin
Delphinidin
Cyanidin
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—
0.063
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0.15
Condensed tannin
0.138
0.070
—
0.166
—
—
0.060
0.052
—
0.13
(+)-Catechin
—
N.T.
—
sterculate
0.41
Methyl
—
0.12
H e l i o c i d e Hj
0.29
M i s s . State 0.113
0.10
0.03
Chan et a l . S t i p a n o v i c 0.12 0.05
Hemigossypolone
Constituent Gossypol
2
y=7.91x"*707 r =0.81 P>F=.001
2
y=124.43-540.36x r =0.89 P>F=.001
2
y=105.66-332.89x r =0.71 P>F=.001
2
y=4.49x~»888 r =0.85 P>F-.001
2
y=3.29x"-705 r =0.90 P>F=.001
2
y=2.07x r =0.87 P>F=0.00
2
y=7.06x--562 r - 0 . 9 0 P>F=0.001
Regression equation y=104.66-504.30x+596.12 r=0.68 P>F=0.01
TABLE I I n h i b i t i o n of Tobacco Budworm L a r v a l Growth by Cotton C o n s t i t u e n t s , ED50 as Percent of D i e t
20.
HEDIN
ET AL.
Contributing
Factors in
Cotton
355
a p p r e c i a b l y a f f e c t t o x i c i t y . I n recent years as the chemical bases f o r r e s i s t a n c e were explored, there was the expectation that some i n d i v i d u a l compound or group of r e l a t e d compounds would be found that could account f o r the r e s i s t a n c e of cotton t o t h i s i n s e c t . This has been shown not t o be the case based on d i e t a r y i n c o r p o r a t i o n bioassays, because the t o x i c i t i e s are e s s e n t i a l l y e q u i v a l e n t at the ED50 v a l u e s . However, the growth response becomes c u r v e l i n e a r a t higher l e v e l s . While i t i s p o s s i b l e t o reduce growth t o l e s s than 10% of the c o n t r o l w i t h gossypol and chrysanthemin, i t was impossible t o reduce growth t o l e s s than 20% w i t h some o t h e r s . E f f e c t of gossypol, t a n n i n , and chrysanthemin i n terminals on TBW l a r v a l growth. Figures 2, 3 and 4 give percent conc e n t r a t i o n s of gossypol, chrysanthemin, and tannins i n t e r m i n a l l e a v e s , and a l s o give tobacco budworm l a r v a l weights f o r the 20 c o t t o n l i n e s , 15 glanded and 5 non-glanded. The weights are f o r l a r v a e feeding on i n t a c t p l a n t s i n the f i e l d . The percent cont e n t s of gossypol and chrysanthemin were n e g a t i v e l y c o r r e l a t e d w i t h l a r v a l weights ( r » -.38 and -.40) while the tannins were weakly p o s i t i v e l y c o r r e l a t e d ( r « +.16); i n f a c t , g l a n d l e s s l i n e s that produced l a r g e l a r v a e were as high i n tannin as most of the l i n e s that produced s m a l l l a r v a e . Thus, these s t u d i e s c o r r o borate the work of Hanny e t a l . (23,24) and suggest that the absolute c o n c e n t r a t i o n of the tannins i n the t o t a l t i s s u e does not e x p l a i n the expected t o x i c e f f e c t s of the i n s e c t feeding on i n t a c t t i s s u e . Female tobacco budworm moths o v i p o s i t mainly on the t e r m i n a l l e a v e s , although i n mid- and late-season they ovipos i t s i g n i f i c a n t numbers on the square b r a c t s . Consequently, the i n i t i a l feeding s i t e i s most o f t e n the t e r m i n a l l e a v e s . Our s t u d i e s (25) have shown that the l a r v a e migrate down the p l a n t , feeding on b r a c t s , meristematic ( o l d e r ) t i s s u e buds, flower p e t a l s , and b o l l s i n the process where the content of anthocyanins, gossypol, and tannins i s mostly s i m i l a r . Table I I i s a summary of the averages of t a n n i n s , gossypol, and chrysanthemin i n 18 c u l t i v a r s harvested as 3 r e p l i c a t e s i n August 1981. The values (somewhat lower than i n 1980) show that the 3 c o n s t i t u e n t s are present i n comparable q u a n t i t i e s i n a l l t i s s u e s analyzed except lower i n medium b o l l s . I t i s t o the medium b o l l s that the l a r v a e e v e n t u a l l y migrate, perhaps t o avoid h i g h l e v e l s of a l l e l o c h e m i c s . As the season progresses, t a n n i n i n c r e a s e s i n a l l t i s s u e s sharply while gossypol and chrysanthemin g r a d u a l l y decrease (Figure 5 ) . During the same p e r i o d , the l a r v a l weight gains remained s i m i l a r , however.
356
Figure 3.
PLANT RESISTANCE TO INSECTS
Percent chrysanthemin in cotton terminal leaves and ranked tobacco budworm larval weights for 20 strains (r = 0.3958).
HEDIN ET AL.
Figure 4.
Contributing
357
Factors in Cotton
Percent tannins in cotton terminal leaves and ranked tobacco budworm larval weights for 20 strains (r — 0.1613).
.10 20 27
Figure 5.
I* 11 18 25 1 8 15 22 29 JULY AUGUST
Seasonal trends in chrysanthemin (—), gossypol ( ( ) content during 1981.
—), and tannin
358
PLANT RESISTANCE TO INSECTS
TABLE I I Content of Tannins, Gossypol, and Chrysanthemin i n Cotton P l a n t T i s s u e s ; Seasonal Averages f o r 3 August 1981 R e p l i c a t e s , 18 C u l t i v a r s
Tissue
Terminals Leaves Squares Square b r a c t s Small b o l l s Medium b o l l s Medium b o l l carpels
Tannins, %
6.02 8.10 7.92 6.02 11.71 9.36 17.07
Gossypol, %
Chrysanthemin,%
0.21 0.23 0.50*/ 0.21 0.29 0.04 0.18
0.14 0.18 0.10 0.14 0.11 0.05 0.10
a/ Somewhat high; average of 0.28 i n 1980, 0.23 f o r 16 other l i n e s i n 1981, but 0.47 by E l l i g e r et a l . ( 1 5 ) .
E f f e c t of g o s s y p o l , t a n n i n , and chrysanthemin i n flower p e t a l s on TBW l a r v a l growth and s u r v i v a l . Our recent observations demonstrated that i n the l a b o r a t o r y , l a r v a e fed more s u c c e s s f u l l y on w h i t e , f i r s t day p e t a l s ( i n t a c t ) than on red, second day p e t a l s , and that they fed more s u c c e s s f u l l y on non-glanded p e t a l s than on glanded p e t a l s . Table I I I presents t a n n i n , g o s s y p o l , and chrysanthemin c o n c e n t r a t i o n s of white and red p e t a l s of 2 glanded and 2 non-glanded ( g l a n d l e s s ) l i n e s . Values f o r tobacco budworm 5 day l a r v a l weights and percent s u r v i v a l are a l s o i n c l u d e d . The t a n n i n contents of the non-glanded white p e t a l s were higher than those of the glanded s t r a i n s , but there was l i t t l e d i f f e r e n c e i n the red p e t a l t a n n i n contents. The gossypol contents of the p e t a l s of the glanded l i n e s were much higher than those of the non-glanded l i n e s as could be expected. The chrysanthemin cont e n t s of a l l red p e t a l s were a l s o much higher than those of white p e t a l s as was expected. The weights and s u r v i v a l of larvae feeding on red p e t a l s were c o n s i d e r a b l y l e s s than those f e e d i n g on white p e t a l s . Both percents t a n n i n and chyrsanthemin were higher i n red p e t a l s than i n white p e t a l s (Table I I I ) . Larvae weights and s u r v i v a l were reduced on red p e t a l s when compared w i t h white p e t a l s . Based c o l l e c t i v e l y on data i n Table I I I and Figures 3 and 4, we conclude that chrysanthemin i s more important than t a n n i n f o r the reduced l a r v a l s i z e and s u r v i v a l on g l a n d l e s s (gossypol-low) red p e t a l s . Gossypol and chrysanthemin c o n t r i b u t e to the t o x i c i t y of glanded red p e t a l s , and gossypol t o that of glanded white p e t a l s . We now have p r e l i m i n a r y data that l a r v a e fed leaves and b r a c t s of red cottons gained 20% l e s s ( s t a t i s t i c a l l y s i g n i f i c a n t ) than those fed leaves and b r a c t s from com-
5.40 8.55
3.49 8.75
3.16 6.25
W R
W R
W R
DH 66 (NG)
ST-213 (G)
DH 126 (G) 1.72 2.46
0.52 0.79
0.17 0.13
0.10 0.11
Gossypol, %
0.18 0.65
0.13 0.59
0.07 0.73
0.07 0.67
Chrysanthemin, %
—
—e/
1.52 b 0.41
4.18 a 0.56
4.72 a 0.46
28.0 8.5
31.0 16.5
39.5 7.0
L a r v a l Wt, mgf*/ L a r v a l s u r v i v a l , %
a/ % of dry weight. b/ NG = Nonglanded, G = glanded. cJ W = w h i t e ; f i r s t day flower c o l o r , R = red; second day flower c o l o r . Means of l a r v a e f e d on white p e t a l s not s i g n i f i c a n t l y d i f f e r e n t at .05 l e v e l i f followed by the same l e t t e r . dj Average tobacco budworm weight a f t e r feeding 5 days on p e t a l s , e/ Not f e d .
5.79 8.68
Tannins, %
IS/ R
Petal color
ST-7AGN (NG)b/
Cultivar
TABLE I I I R e l a t i v e E f f e c t s of Cotton Flower P e t a l C o n s t i t u e n t s on Tobacco Budworm Growth and S u r v i v a l ^ /
360
PLANT RESISTANCE TO INSECTS
parable green s t r a i n s . Thus, red c o l o r a t i o n now appears to be a f a c t o r of considerable importance i n i n s e c t feeding of both p e t a l s and leaves. There i s s t i l l a r e s i d u a l m o r t a l i t y of i n s e c t s feeding on white g l a n d l e s s p e t a l s (Table I I I ) . This can be a t t r i b u t e d at l e a s t i n part to the f l a v o n o i d s , some of which we have p r e v i o u s l y i d e n t i f i e d (28) and demonstrated to be t o x i c to t h i s i n s e c t (Table I). H i s t o c h e m i c a l s t u d i e s on l o c a l i z a t i o n of t a n n i n s , gossypol, and anthocyannins i n p l a n t t i s s u e s . We were able to observe by m a g n i f i c a t i o n that tobacco budworm l a r v a e avoided gossypol glands d u r i n g feeding ( F i g u r e 6 ) . Waiss et a l . (29) had made s i m i l a r observations w i t h J*, zea. To determine where the t a n n i n s , gossyp o l , and anthocyanin are l o c a l i z e d i n the p l a n t , some h i s t o l o g i c a l s t u d i e s were c a r r i e d out. Figure 7 shows m a g n i f i c a t i o n s of t i s s u e s l i c e s of a glanded l i n e , DH-126, f i x e d i n FeSo4~5% formal s a l i n e and s t a i n e d w i t h p h l o r o g l u c i n o l - H C l to v i s u a l i z e t a n n i n s . The m i d r i b i s prominent i n the center of Figure 7, and the tannins appear to be concentrated near the surface i n granular form. In g l a n d l e s s l i n e s , t a n n i n i s more d i f f u s e . Figure 8 i s a 5pm p a r a p l a s t s e c t i o n through a c o t t o n l e a f at the gossypol gland s i t e which shows the outer anthocyanin-containing envelope (halo) surrounding the gossypol gland. In f r e s h t i s s u e s e c t i o n s , the outer halo s t a i n s b r i g h t red i n a c i d . The halo when subsequently n e u t r a l i z e d w i t h KOH i s converted to green, v e r i f y i n g that i t i s anthocyanin. I t i s tempting to speculate from t h i s that the biosyntheses of l e a f gossypol and anthocyanin are p l e i o t r o p i c a l l y r e l a t e d because the content of each i s much higher i n glanded than i n non-glanded leaves (Figures 2 and 3 ) . However, the anthocyanin content can be high and gossypol low as i n flower p e t a l s (Table I I I ) . Gossypol and r e l a t e d aldehydes are b i o s y n t h e s i z e d v i a the acetogenic pathway and anthocyanins and other f l a v o n o i d s by the mixed a c e t a t e / s h i k i m i c pathways. Thus each should be able to i n c r e a s e independently i n the p l a n t as we have demonstrated i n glanded and g l a n d l e s s red p e t a l s . This halo e f f e c t may a l s o a l t e r the i n t e r p r e t a t i o n of Figure 6 that shows the apparent avoidance of gossypol glands by tobacco budworm l a r v a e . The presumed feeding deterrence of gossypol may be i n f a c t caused by the anthocyanin h a l o . Although both the anthocyanin and gossypol are t o x i c when ingested, an a n t i f e e d a n t mechanism may be expressed i n t h i s instance. F i g u r e 9 shows a Pima (G. barbadense L.) flower p e t a l t r e a t e d w i t h HC1 to v i s u a l i z e red anthocyanin "granules". Treatment w i t h a l k a l i converted the granule c o l o r to green. The halo surrounding the gossypol gland i n s i d e the outer c a r p e l w a l l of DH-126 buds (Figure 10) i s a l s o anthocyanin. The anthocyanin (chrysanthemin) i n very young S t o n v i l l e - 2 1 3 b o l l s on the day of a n t h e s i s was found by d i s s e c t i n g , e x t r a c t i o n , and TLC a n a l y s i s to be l o c a t e d p r i m a r i l y i n the outer c a r p e l w a l l .
20.
HEDIN ET AL.
Figure 6.
Figure 7.
Contributing
Factors in Cotton
A voidance of gossypol glands by tobacco budworm while feeding.
Cross-section of midrib and adjacent blade tissue of DH-126 leaves showing tannin cells.
361
362
Figure 8.
PLANT RESISTANCE TO INSECTS
Paraplast section (5 m) of a gossypol gland and the anthocyanin envelope.
HEDIN
ET AL.
Figure 9.
Figure 10.
Contributing
Factors in
Cotton
Acid treatment of Pima flower petal to visualize anthocyanin.
Acid visualization of anthocyanin halo in Stoneville 213 bud.
364
PLANT RESISTANCE TO INSECTS
I n summary, the contents of chrysanthemin and gossypol were shown to be n e g a t i v e l y c o r r e l a t e d w i t h tobacco budworm l a r v a l growth i n the f i e l d w h i l e the tannins were s l i g h t l y p o s i t i v e l y correlated. The r e c o g n i t i o n of chrysanthemin as a r e s i s t a n c e f a c t o r provides a b a s i s for developing r e s i s t a n t cotton c u l t i v a r s that are low or devoid of g o s s y p o l , a long sought o b j e c t i v e . It a l s o suggests that anthocyanins i n general may be a b a s i s for s e l e c t i n g f o r r e s i s t a n c e i n other crops to H e l i o t h i s . Literature 1. 2. 3. 4. 5. 6.
7.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
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McMichael, S. C. Agr. J. 1959. 51, 30. B o t t g e r , G. T., Sheehan, E . T. Lukefahr, M . J. J. Econ. Entomol. 1964, 57, 283. J e n k i n s , J. N., Maxwell, F . G., L a f e v e r , H . N . J. Econ. Entomol. 1966. 59, 352. Lukefahr, M . J., Nobel, L. W., Houghtaling, J. E . J. Econ. Entomol. 1966, 59, 817. Lukefahr, M . J., M a r t i n , D. F . J. Econ. Entomol. 1966, 59, 176. Lukefahr, M . J., Shaver, T. N., Cruhm, D. E., Houghtaling, J. E . P r o c . Beltwide Cotton P r o d . Res. C o n f . , 1974, Memphis, TN, p . 93. Bell, A . A., S t i p a n o v i c , R. D., H o w e l l , C. R., Mace, M. E. P r o c . Beltwide Cotton P r o d . Res. Conf. 1974, J a n . 7-9, D a l l a s , TX, 40. Gray, J. R . , Mabry, R. J., Bell, A . A., S t i p a n o v i c , R. D., Lukefahr, M . J. J. C . S. Chem. Comm. 1976. 109. Bell, A . A., S t i p a n o v i c , R. D. H o w e l l , C. R., Fryxell, P . A . Phytochemistry. 1975, 14 Bell, A . A., S t i p a n o v i c , R. D. P r o c . Beltwide Cotton P r o d u c t i o n Res. Conf. 1977, J a n . 10-12, A t l a n t a , GA, 244. S t i p a n o v i c , R. D., Bell, A . A., O ' B r i e n , D. H., Lukefahr, M . J. Tetrahedron L e t t e r s . 1977. 6, 567. H o w e l l , C. R., Bell, A . A., S t i p a n o v i c , R. D. P h y s i o l . P l a n t P a t h o l . 1976. 8, 181. Chan, B . G., Waiss, A . C., Lukefahr, M. J. J. Insect Physiol. 1978. 24, 113. Chan, B . C., Waiss, A . C., B i n d e r , R. G., Elliger, C. A . E n t . Exp. & A p p l . 1978. 24, 94. Elliger, C . A., Chan, B . G., Waiss, A . C. J. Econ. Entomol. 1978. 71, 161. S t i p a n o v i c , R. D . ; personal communication. Shaver, T. W., P a r r o t t , W. L. J. Econ. Entomol. 1970. 63, 1802. Waiss, A . C.; personal communication. Shaver, T. N., G a r c i a , J. A., D i l d a y , R. H . E n v i r o n . Entomol. 1977. 6, 82. Shaver, T. N., D i l d a y , R. H., W i l s o n , F . D. Crop Sci. 1980. 20, 545.
RECEIVED
20.
21. 22. 23. 24. 25.
26. 27. 28. 29.
HEDIN
ET
AL.
Contributing
Factors
in Cotton
365
Schuster, M. F., Maxwell, F. G., J e n k i n s , J. N., P a r r o t t , W. L. J. Econ. Entomol. 1972. 65, 1104. Schuster, M. F., Lane, H. C. Proc. Beltwide Cotton Prod. Res. Conf. 1980. Jan. 6-10, S t . L o u i s , MO, p. 83. Hanny, B. W., B a i l e y , J. C., Meredith, W. R. E n v i r o n . Entomol. 1979. 8, 706. Hanny, B. W. J. Agr. Food Chem. 1980. 28, 504. Hedin, P. A., Collum, D. H., White, W. H., P a r r o t t , W. L., Lane, H. C., J e n k i n s , J. N. P r o c . I n t . Conf. on R e g u l a t i o n of Insect Development and Behavior, Wroclaw Tech. Univ. P r e s s , Wroclaw, Poland. 1980, 1071. Hedin, P. A., Minyard, J. P., Thompson, A. C., S t r u c k , R. E., Frye, J. Phytochemistry. 1967. 6, 1165. Czochanska, Z., Foo, L. P., Newman, R. H., P o r t e r , L. J . J.C.S. P e r k i n I . 1980, 2278. Hedin, P. A., M i l e s , L. R., Thompson, A. C., Minyard, J. P. J. Agr. Food Chem. 1968. 16, 505. Waiss, A. C., Chan, B. G., Elliger, C. A., and Binder, R. G. P r o c . Beltwide Cotton Proc. Res. Conf. 1981. Jan. 4-8. New Orleans, La. p. 61. September 28, 1982
