Endophytic Bacteria for the Delivery of Agrochemicals to Plants

Chapter 9

Endophytic Bacteria for the Delivery of Agrochemicals to Plants Jed W. Fahey

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Crop Genetics International, 7170 Standard Drive, Hanover, MD 21076

A novel delivery system (InCide) utilizing naturally occuring plant endophytic bacteria is being developed for the systemic delivery of agrichemicals in-planta. Host crops are systemically colonized by the endophyte, thus providing an environmentally safe vehicle with which to deliver potent, targeted, biologically-derived agrichemicals to the plants. InCide products, including those having insecticide, fungicide, bactericide, viricide, nematicide and plant growth enhancer activity are designed to be applied as a seed, seedling or young plant treatment only once during the life of a plant. They exploit the biological characteristics of a natural endophytic microbe by systemically colonizing the xylem and achieving sustained levels in their host plants. Because of their in-planta growth, effects on non-target organisms and the environment are minimized and significant advantages are afforded over current externally applied agrichemicals. The first InCide product is an endophyte of corn that has been genetically modified by insertion of a gene from Bacillus thuringiensis encoding for the production of a highly specific insecticidal protein (delta-endotoxin) active against the European Corn Borer. Refinement and scale-up of techniques for inoculation of this product into corn seeds will permit rapid commercialization of this new delivery system. An e x a m i n a t i o n o f p l a n t - a s s o c i a t e d m i c r o b e s w i l l i l l u s t r a t e t h e d i v e r s i t y o f r e l a t i o n s h i p s e x i s t i n g between p l a n t s and " i n t e r n a l l y dwelling" plant-neutral or b e n e f i c i a l microbes. J u s t how some o f these r e l a t i o n s h i p s evolved i s n o tf u l l y understood, but there a r e a v a r i e t y o f s t a b l e , m u t u a l i s t i c o r s y m b i o t i c r e l a t i o n s h i p s between

0097-6156/88/0380-0120$06.00/0 ° 1988 American Chemical Society

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

9. FAIIEY

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Bacterial Delivery ofAgrochemicals

h i g h e r p l a n t s and m i c r o b i a l endophytes. Endophytes a r e microbes which l i v e w i t h i n plants. Maintenance of a l l plant-endophyte r e l a t i o n s h i p s r e q u i r e t h a t a l t h o u g h t h e p l a n t may r e c o g n i z e t h e i n v a d e r , i t does n o t t r i g g e r any o v e r t p a t h o g e n i c response t o t h e microbe or that the microbe s u c c e s s f u l l y avoids the plant's defense r e a c t i o n s and does n o t cause d i s e a s e . Numerous p l a n t - e n d o p h y t e i n t e r a c t i o n s have been i d e n t i f i e d over t h e past century. To d a t e , o n l y t h e R h i z o b i u m s p e c i e s , n o t e v e n t r u e e n d o p h y t e s b u t m i l d p l a n t pathogens o f t h e legume f a m i l y , have been commercialized f o r t h e i r a g r i c u l t u r a l p o t e n t i a l as symbionts.


Naturally Occurring

Endophytes

The n o d u l a t i o n o f l e g u m e s l i k e s o y b e a n , c l o v e r a n d a l f a l f a b y t h e g r a m - p o s i t i v e R h i z o b i u m & B r a d y r h i z o b i u m i s by f a r t h e most thoroughly characterized plant-microbe r e l a t i o n s h i p and t h e o n l y one w h i c h h a s b e e n e x p l o i t e d c o m m e r c i a l l y . Rhizobium species i n symbiotic association with plants are responsible f o r the c o n v e r s i o n o f a s much a s 2 x 1 0 t o n s o f a t m o s p h e r i c N p e r y e a r t o ammonium ( 1 ) . T h i s " f i x e d " n i t r o g e n i s t h e n u t i l i z e d by p l a n t s f o r t h e s y n t h e s i s o f amino a c i d s and p r o t e i n . I n Rhizobium symbioses, t h e b a c t e r i a i n f e c t p l a n t r o o t s and a r e then c o n t a i n e d w i t h i n s p e c i a l i z e d structures called root nodules. A highly i n t e g r a t e d e x p r e s s i o n o f genes f r o m b o t h t h e p l a n t and t h e b a c t e r i a lead t o the formation of a morphologically d i s t i n c t nodule, with i t s own m e r i s t e m a t i c r e g i o n s a n d v a s c u l a r i n n e r v a t i o n . A molecule very s i m i l a r t o the hemoglobin found i n blood i s synthesized t o p r o t e c t t h e o x y g e n - s e n s i t i v e enzyme, n i t r o g e n a s e , a n d t h e b a c t e r i a a c t u a l l y a l t e r t h e i r morphology once e s t a b l i s h e d i n a d e v e l o p i n g nodule. W i t h o u t t h e p r o t e c t i v e environment and carbon supply a f f o r d e d t h e b a c t e r i a by t h e p l a n t , t h e b a c t e r i a w o u l d n o t p e r f o r m the e n e r g y - i n t e n s i v e process of n i t r o g e n r e d u c t i o n . Without the b a c t e r i a ' s s i g n i f i c a n t nitrogen input, the plants would not, i n many c a s e s , be a b l e t o p r o s p e r i n a n o t h e r w i s e n i t r o g e n p o o r environment. This n a t u r a l p l a n t - b a c t e r i a l symbiosis can therefore s a v e f a r m e r s g r o w i n g many l e g u m i n o u s c r o p s f r o m t h e c o n s i d e r a b l e e x p e n s e a s s o c i a t e d w i t h f e r t i l i z e r n i t r o g e n i n p u t a n d may p e r m i t c e r t a i n species t o t h r i v e i n areas which they might otherwise n o t occupy. 7

2

C e r t a i n non-legumes a l s o form r o o t nodules w h i c h a r e responsible f o rnitrogen f i x a t i o n . The m i c r o b e f o u n d i n a l l non-legume r o o t nodules ( a c t i n o r h i z a l p l a n t s ) s t u d i e d t o date i s the actinomycete, F r a n k i a . Most a c t i n o r h i z a l symbyotic a s s o c i a t i o n s a r e f o u n d i n woody p l a n t s i n t h e t e m p e r a t e o r c o l d zones o f t h e Northern Hemisphere. O v e r 178 s p e c i e s i n 20 d i f f e r e n t genera have been documented t o c o n t a i n a c t i n o r h i z a l symbionts ( 2 ) . There a r e two m a j o r m o r p h o l o g i c a l t y p e s o f a c t i n o r h i z a l n o d u l e s : The A l n u s - t y p e o r c o r a l l o i d n o d u l e s a r e s h o r t , s t u b b y , d i c h o t o m o u s l y b r a n c h e d g r o w t h s on l a t e r a l r o o t s . Myrica-type or l o b e d n o d u l e s a r e t h i n , and produce n e g a t i v e l y g e o t r o p i c r o o t l e t s a t t h e i r apex ( 3 ) .


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BIOLOGICALLY ACTIVE NATURAL PRODUCTS


S t e m n o d u l e s , t o o , a r e f o r m e d o n c e r t a i n members o f t h e L e g u m i n o s a e f a m i l y by R h i z o b i u m b a c t e r i a . These n i t r o g e n - f i x i n g symbioses have been documented i n t h r e e genera of p r e d o m i n a n t l y t r o p i c a l or h y d r o p h y t i c p l a n t s Aeschvnomene. S e s b a n i a a n d N e p t u n i a , w h i c h a r e r e p r e s e n t e d by a b o u t 2 0 0 , 70 a n d 15 s p e c i e s r e s p e c t i v e l y ( 4 ) . N i t r o g e n f i x a t i o n by t h e e n d o p h y t e i n s t e m n o d u l e s a n d t h e p r o t e c t i o n o f t h e o x y g e n - s e n s i t i v e m i c r o b i a l enzyme seems t o be a c c o m p l i s h e d v i a a s i m i l a r s t r a t e g e m o f l e g h e m o g l o b i n a n d n o d u l e m o r p h o l o g y t o t h a t a d o p t e d by t h e l e g u m e r o o t n o d u l e s . L e a f n o d u l e s o c c u r p r e d o m i n a n t l y i n t h e f a m i l i e s R u b i a c e a e and M y r s i n a c e a e w h i c h c o n t a i n o v e r 400 n o d u l e - f o r m i n g s p e c i e s ( 5 ) . These f a m i l i e s of p r e d o m i n a n t l y s u b - t r o p i c a l shrubs a r e n o d u l a t e d by a g r a m n e g a t i v e , p l e i o m o r p h i c b a c t e r i u m i n a n a s s o c i a t i o n w h i c h i n a t l e a s t one s p e c i e s ( P s y c h o t r i a b a c t e r i o p h i l a ) h a s b e e n shown t o be o b l i g a t e ( 6 ) . I n a d d i t i o n , one f a m i l y o f m o n o c o t s , D i o s c o r e a c e a e has been i d e n t i f i e d as h a v i n g a s p e c i e s w i t h l e a f n o d u l e s c o l o n i z e d by e n d o p h y t i c b a c t e r i a ( 7 ) . A l t h o u g h t h e n a t u r e of these symbioses are not f u l l y understood, i t i s h y p o t h e s i z e d t h a t the b a c t e r i a have a growth promoting e f f e c t . T h e r e i s a t l e a s t one w e l l - c h a r a c t e r i z e d a s s o c i a t i o n o f a heterocystous, n i t r o g e n - f i x i n g blue green a l g a (cyanobacterium) with a plant. The c y a n o b a c t e r i u m A n a b a e n a s p . d e v e l o p s i n a m u t u a l i s t i c r e l a t i o n s h i p w i t h the water f e r n ( A z o l l a s p . ) . Anabaena i s a f i l a m e n t o u s , p h o t o s y n t h e t i c c y a n o b a c t e r i u m c o n t a i n i n g h e t e r o c y s t s at i n t e r v a l s along a c h a i n of v e g e t a t i v e c e l l s . These h e t e r o c y s t s c o n t a i n t h e r e q u i s i t e enzymes f o r d i - n i t r o g e n fixation. A z o l l a p r o v i d e s "safe harbour" f o r the N - f i x i n g s y m b i o n t w i t h i n c a v i t i e s o r n o d u l e s on t h e u n d e r s i d e s o f i t s leaves. 2

A n o t h e r i m p o r t a n t and e x t r e m e l y w i d e s p r e a d group o f endophytes i s the mycorrhyzal f u n g i . These f u n g i r a m i f y t h r o u g h o u t the r o o t c e l l s o f p l a n t s a n d a r e h y p o t h e s i z e d t o f u n c t i o n by a i d i n g i n t h e m o b i l i z a t i o n o f r e l a t i v e l y i n s o l u b l e s o i l n u t r i e n t s s u c h as p h o s p h a t e and z i n c t o t h e p l a n t . Ascribing a generalized function to these endophytes i s the o b j e c t of c o n s i d e r a b l e c o n t r o v e r s y . E n d o - m y c o r r h y z a e p e n e t r a t e and r a m i f y t h r o u g h o u t c e r t a i n r o o t cells. E c t o - m y c o r r h y z a e a r e t h o s e w h o s e h y p h a e do n o t p e n e t r a t e the r o o t c e l l s , but e i t h e r envelop the r o o t s and/or e n t e r the space between r o o t c e l l s , u s u a l l y o u t s i d e the endodermis. The m a j o r i t y of h i g h e r p l a n t taxa form a s s o c i a t i o n s w i t h mycorrhyzal endophytes. The A c r e m o n i u m - t y p e f u n g i a r e e n d o p h y t e s o f c e r t a i n t y p e s o f grasses. They have been r e s p o n s i b l e f o r s i g n i f i c a n t o u t b r e a k s o f l i v e s t o c k t o x i c i t y i n forage g r a s s e s , however, they are a l s o responsible f o r c o n f e r r i n g b e n e f i c i a l q u a l i t i e s to c e r t a i n of t h e i r hosts. Enhanced p e r f o r m a n c e , enhanced i n s e c t r e s i s t a n c e and i m p r o v e d p e r s i s t a n c e o f L o l i u m and F e s t u c a s p e c i e s have been d o c u m e n t e d i n t u r f g r a s s s t a n d s c o l o n i z e d by s p e c i e s o f A c r e m o n i u m (8).



9. F A H E Y


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And f i n a l l y , t h e r e a r e t h e x y l e m - i n h a b i t i n g b a c t e r i a . These b a c t e r i a a r e n o t u b i q u i t o u s , but c e r t a i n s p e c i e s c a n r e l i a b l y be recovered from t h e i r host plant species i n a pattern which suggests t h a t t h e s e b a c t e r i a may be p a r t o f t h e n o r m a l m i c r o f l o r a o f t h o s e hosts. B a c t e r i a f r o m 13 d i f f e r e n t g e n e r a w e r e i s o l a t e d f r o m t h e x y l e m o f h e a l t h y C i t r u s t r e e s (9) w h i c h were c o l o n i z e d a t l e v e l s o f u p t o 2 x 10* CFU/g. The p r e s e n c e i n C i t r u s o f l a r g e numbers o f d i v e r s e e n d o p h y t i c b a c t e r i a has been c o n f i r m e d i n s u b s e q u e n t s t u d i e s ( Z a b l o t o w i c z , R.M., A l l e l i x , personal c o m m u n i c a t i o n ) . a l t h o u g h t h i s phenomenon a p p e a r s t o be t h e exception a n d n o t t h e r u l e . X y l e m i n h a b i t i n g e n d o p h y t i c b a c t e r i a may b e r e s p o n s i b l e f o r the f r e q u e n t l y observed i n a b i l i t y o f p l a n t t i s s u e culturists to "disinfest" cultures of rigorouslysurface-sterilized explant material. The f a i l u r e o f c e r t a i n s p e c i e s t o grow i n - v i t r o c o u l d i n f a c t be r e l a t e d t o a s t i m u l a t o r y e f f e c t o f a n e n d o p h y t i c m i c r o b e w h i c h c a n n o t be r e p r o d u c e d i n t h e c u l t u r e o f i s o l a t e d , bacteria-free explants. I t i s these xylem-inhabiting endophytes w h i c h we a r e e x p l o i t i n g . Crop

Protection

Today's c r o p p r o t e c t i o n m a r k e t was c r e a t e d a n d i s p r e s e n t l y dominated by the c h e m i c a l i n d u s t r y . B i o l o g i c a l s account f o r only $100 m i l l i o n o f t h i s $14 b i l l i o n m a r k e t . Although synthetic c h e m i c a l s p r e s e n t l y dominate the market, the s a f e t y o f these c h e m i c a l s h a s been c a l l e d i n t o q u e s t i o n . B i o l o g i c a l pesticidesare known t o be s a f e b u t h a v e l a c k e d t h e e f f i c a c y o f s y n t h e t i c chemicals. The s a f e t y i s s u e o f c h e m i c a l s a n d t h e e f f i c a c y p r o b l e m of b i o l o g i c a l p e s t i c i d e s are consequences o f t h e i r e x t e r n a l application. C u r r e n t l y u t i l i z e d methods f o r a p p l i c a t i o n o f e i t h e r chemicals o r b i o l o g i c a l s (e.g. f o l i a r sprays, s o i l a p p l i c a t i o n s o r seed t r e a t m e n t s ) , a l l i n v o l v e p l a c i n g the treatment o u t s i d e t h e p l a n t even i f the treatments themselves have systemic activity. W i t h o u t c o n v e n t i o n a l p e s t c o n t r o l , f o o d p r o d u c t i o n i n t h e U.S. a l o n e c o u l d d r o p b y a s much a s o n e - t h i r d . Despite the presence o f c h e m i c a l p e s t i c i d e s , more t h a n o n e - t h i r d o f t h e w o r l d ' s p o t e n t i a l crop y i e l d i s s t i l l l o s t t o fungal diseases and i n s e c t pests. Some c h e m i c a l p r o d u c t s leave t o x i c r e s i d u e s on the crop, l e a c h i n t o groundwater and are t o x i c t o farm workers. While chemicals a r e e f f e c t i v e a g a i n s t a broad spectrum o f p l a n t p e s t s , they can destroy n a t u r a l pest predators and other b e n e f i c i a l organisms. F o r many p e s t i c i d e s and f o r e x t e r n a l l y d e l i v e r e d b i o l o g i c a l s , m u l t i p l e a p p l i c a t i o n s are r e q u i r e d because the p r o d u c t s are d i l u t e d by r a i n , d i s s i p a t e d by w i n d a n d degraded by s u n l i g h t a n d m i c r o o r g a n i s m s . The e f f e c t i v e n e s s o f c h e m i c a l s may d e c l i n e o v e r t i m e d u e t o t h e development o f r e s i s t a n c e by the t a r g e t p e s t s . T h e r e i s much l e s s r i s k o f target pests developing resistance t o endophyte-delivered a g r i c h e m i c a l s b e c a u s e o f the manner i n w h i c h t h e y a r e e x p o s e d a n d the v a s t l y s m a l l e r q u a n t i t i e s o f a c t i v e i n g r e d i e n t w h i c h thus need t o be p r e s e n t i n t h e c r o p . F i n a l l y , government r e g u l a t i o n s p r o t e c t i n g consumers, workers and the environment are i n c r e a s i n g l y


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restricting are making difficult. biologicals the process

t h e use o f p r e s e n t l y - r e g i s t e r e d c h e m i c a l products and t h e i n t r o d u c t i o n o f new c h e m i c a l p r o d u c t s m o r e The r e g u l a t o r y g r o u n d - r u l e s f o r t h e i n t r o d u c t i o n o f a r e c u r r e n t l y b e i n g w r i t t e n as numerous groups a r e i n o f a t t e m p t i n g t o f i e l d - t e s t and r e g i s t e r such p r o d u c t s .


E n d o p h y t i c B a c t e r i a . A New D e l i v e r y

System

InCide b i o p e s t i c i d e technology i s a system of crop protection i n which biology i s substituted f o rchemistry: Incide products are m i c r o o r g a n i s m s g e n e t i c a l l y e n g i n e e r e d t o be b o t h environmentally safe and e f f i c a c i o u s . InCide products a r e designed t o f u n c t i o n i n t e r n a l l y i n a p l a n t ' s v a s c u l a r system. They i n v o l v e t h e u s e o f n a t u r a l l y - o c c u r i n g endophytes f o r t h e p r o d u c t i o n and d e l i v e r y o f crop p r o t e c t a n t s and growth enhancers. Methods have been developed t o screen, i d e n t i f y , r e c o v e r and c h a r a c t e r i z e endophytic microorganisms. CGI has c o n d u c t e d a n e x t e n s i v e s e a r c h f o r and a n a l y s i s o f endophytes capable o f c o l o n i z i n g t h e major crops and now h a s a l a r g e c o l l e c t i o n o f e n d o p h y t e s . Microorganisms are s e l e c t e d f o r t h e Company's c o l l e c t i o n b a s e d o n a b i l i t y t o l i v e i n s i d e , and i n a b i l i t y t o l i v e o u t s i d e , t h e t a r g e t c r o p s . CGI has i n d e n t i f i e d endophytes capable of c o l o n i z i n g corn, cotton, soybeans, wheat and r i c e as w e l l as o t h e r major c r o p s . InCide p r o d u c t s a r e b e i n g d e v e l o p e d t o s o l v e many o f t h e p r o b l e m s a s s o c i a t e d w i t h e x t e r n a l l y a p p l i e d c h e m i c a l s and b i o l o g i c a l s . There a r e numerous economic, e n v i r o n m e n t a l and t e c h n o l o g i c a l a d v a n t a g e s t o be r e a l i z e d b y u s i n g e n d o p h y t i c b a c t e r i a f o r t h e delivery of agrichemicals to plants. Some o f t h e a d v a n t a g e s o f t h i s technology over conventional d e l i v e r y of p e s t i c i d e s i n c l u d e s : Economic

Advantages.

o Single Application. Seed i n o c u l a t i o n o r i n o c u l a t i o n o f the j u v e n i l e p l a n t s w i t h an a p p r o p r i a t e endophyte r e s u l t s i n colonization of that plant. The e n d o p h y t e s l i v e i n s i d e t h e p l a n t and a r e thus p r o t e c t e d from t h e e x t e r n a l environment. E x t e r n a l l y a p p l i e d c h e m i c a l s and b i o l o g i c a l s a r e u n p r o t e c t e d and o f t e n r e q u i r e m u l t i p l e a p p l i c a t i o n s . o M i n u t e Dosage. S e e d a p p l i c a t i o n o f e n d o p h y t e s c a n be accomplished with only m i l l i g r a m q u a n t i t i e s of bacteria per acre. A f t e r a p p l i c a t i o n , the endophytes m u l t i p l y i n s i d e each p l a n t so t h a t t h e f i n a l m a n u f a c t u r i n g s t e p occurs a f t e r the point of sale. Conventional externally applied chemicals are generally a p p l i e d i n pounds p e r a c r e . o Sustained Potency. Endophytes can t h r i v e and produce t h e desired agrichemical f o rthe duration of the plant's life. E x t e r n a l l y applied products are adversely affected or r e n d e r e d i n e f f e c t i v e by t h e e n v i r o n m e n t a n d by s u b s e q u e n t plant growth.


9.

FAHEY

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Environmental

Advantages.

o Contained A c t i v i t y . E n d o p h y t e s s u r v i v e and f u n c t i o n o n l y w i t h i n the p l a n t w h i c h they p r o t e c t or enhance. E x t e r n a l l y a p p l i e d products are d i s p e r s e d w i d e l y each time they are a p p l i e d .


o P l a n t Dependency. E n d o p h y t e s , by n a t u r e , do n o t s u r v i v e o u t s i d e t h e p l a n t a n d t h e r e f o r e do n o t m u l t i p l y o r spread i n the environment or remain a c t i v e a f t e r harvest. E x t e r n a l l y a p p l i e d m i c r o o r g a n i s m s must s u r v i v e i n t h e e n v i r o n m e n t t o be e f f e c t i v e . o No T o x i c R e s i d u e s . E n d o p h y t e p r o d u c t s c a n be d e s i g n e d t o be e n v i r o n m e n t a l l y s a f e a n d d e g r a d a b l e . Most e x t e r n a l l y a p p l i e d p e s t i c i d e s m u s t r e s i s t d e g r a d a t i o n t o be effective. Technological Advantages. C e r t a i n e n d o p h y t e p r o d u c t s may the f o l l o w i n g t e c h n o l o g i c a l advantages over g e n e t i c a l l y improved p l a n t s : o Rapid Development. Changing the g e n e t i c s of m i c r o o r g a n i s m s i s a more r a p i d and s i m p l e r p r o c e s s changing the g e n e t i c s of p l a n t s .

have

than

o Early Commercialization. Development of endophyte-based p r o d u c t s does not r e q u i r e m u l t i - y e a r p l a n t b r e e d i n g programs. o Wide A p p l i c a b i l i t y . E n d o p h y t e - b a s e i p r o d u c t s c a n be designed to f u n c t i o n i n a wide range of commercially u s e f u l v a r i e t i e s of the t a r g e t e d crop. o Yield. E n d o p h y t e d e l i v e r y has m i n i m a l i m p a c t on h o s t p l a n t p h y s i o l o g y ( e . g . l i t t l e o r no e f f e c t on y i e l d , v i g o u r or q u a l i t y ) . I n many i n s t a n c e s , c h a n g i n g t h e g e n e t i c s of p l a n t s can reduce y i e l d s u b s t a n t i a l l y . o Repeat Sales. Many e n d o p h y t e s a r e n o t s e e d t r a n s m i t t e d and f a r m e r s w i l l n e e d t o p u r c h a s e p r o d u c t s b a s e d on t h e s e organisms each growing season. New p l a n t v a r i e t i e s n o r m a l l y have r e p e a t s a l e s o p p o r t u n i t i e s o n l y when the purchased seed i s a h y b r i d . P r o g r e s s i s underway t o d e v e l o p the I n C i d e d e l i v e r y system f o r the d e l i v e r y of i n s e c t i c i d e s , f u n g i c i d e s , n e m a t i c i d e s , viricides and b a c t e r i c i d e s t o p r o t e c t c o r n , c o t t o n , s o y b e a n s , r i c e and w h e a t , as w e l l as o t h e r v e g e t a b l e , f o r e s t r y , and h o r t i c u l t u r a l c r o p s . We are developing a f a m i l y of g e n e t i c a l l y engineered b i o p e s t i c i d e s u s i n g e n d o p h y t e s t h a t c o l o n i z e t h e m a j o r c r o p s and genes t h a t p r o t e c t against major p l a n t pests.




F i g u r e 1. Lumen o f x y l e m a e r e n c h y m a o f Z e a mays v a r . F R 6 3 2 c o l o n i z e d w i t h endophytic bacterium C l a v i b a c t e r x y l i i subsp. cynodontis. F r e e z e - f r a c t u r e s u r f a c e r e v e a l s a n n u l a r r i n g s on l e f t of plate. M a g n i f i c a t i o n = 4250 X



9. FAHEY


127

The first InCide product under development is an insecticide for the United States and French corn market. We are conducting extensive field and greenhouse trials with our best corn endophyte. These tests are designed to accumulate data for product registration in the United States and France. We are seeking regulatory approval to field test our first recombinant corn insecticide product in the United States and France in the spring of 1988. This product uses the endophyte, Clavibacter xyli subsp. cynodontis. a Coryneform bacterium which was originally isolated from-a non-crop plant and can be reproducibly introduced into corn (Fig. 1). This bacterium has been transformed by inserting a gene encoding for production of an insecticidal protein from the bacterium Bacillus thuringiensis (B.t.). B.t. has experienced decades of safe use as an insecticidal product. B.t. was first sold in France in 1939 and has been a leading biological insecticide in the U.S. since registration in 1961. There are more than one thousand B.t. isolates, each with one or more genes having a specific spectrum of insecticidal activity. Some B.t. genes are active against certain lepidoptera (caterpillars) and others are toxic to certain coleoptera (beetles). For its initial products, the Company purchased the rights to a B.t. gene effective against the caterpillar stage of European Corn Borer. Insect feeding trials have shown that the toxin produced by the B.t. gene is lethal to the European Corn Borer. The wild-type endophyte rapidly colonizes the xylem of inoculated corn plants and achieves average levels of up to 1 x 108 CFU/g fresh weight of tissue. It systemically colonizes the roots, stem, leaves and husks of inoculated plants and can be detected within a week of inoculation but it does not transmit via the seed of colonized plants. Endophytes can be found throughout the plant kingdom. Natural endophytes exist which provide manifold benefits to the plants with which they are associated. We are using the tools of biotechnology to add specific beneficial qualities to carefully chosen endophytes. By selective enhancement, endophytes can be engineered to help solve some of agriculture's most pressing problems using a biological system which has been around for millions of years. Literature Cited 1. 2. 3. 4. 5.

Burris, R.H. In Free-living Systems and Chemical Models. Nitrogen Fixation; Newton, W.E., Orme-Johnson, W.H. Eds.; University Park Press: Baltimore, 1980; Vol. 1, pp 7-16. Moiroud, A. & Gianinazzi-Pearson, V. In Genes Involved in Microbe-Plant Interactions; Verma, D.P.S. & Hohn, T., Eds.; Springer-Verlag: New York, 1984; pp 205-223. Becking, J. In Dinitrogen Fixation; Hardy, R., Silver, W. Eds.; Wiley & Sons Inc.: New York, 1977; Vol. 2, pp 185-275. Legocki, R.P. & Szalay, A.A. In Genes Involved in Microbe-Plant Interactions; Verma, D.P.S. & Hohn, T., Eds.; Springer-Verlag: New York, 1984; pp 225-268 Lersten, N.R. & Horner, H.T. Bot. Rev. 1976, 42, 146-214.


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Lersten, N.R. & Horner, H.T. J. Bacteriol. 1967, 94(6), 2027-2036. 7. Schaede, R. Jarhb. f. wiss. Botanik. 1939, 88, 1-21. 8. Funk, C.R., Halisky, P.M., Ahmad, S. & Hurley, R.H. In Proc. 33rd Short Course for Seedsman. Seed Technology Lab. Mississippi State University. 1985, pp 85-97. 9. Gardner, J.M., Feldman, A.W. & Zablotowicz, R.M. Appl. Environ. Microbiol. 1982, 43(6), 1335-1342. RECEIVED April 12, 1988


6.


Endophytic Bacteria for the Delivery of Agrochemicals to Plants

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