Chapter 10
Microbes and Microbial Products as Herbicides Downloaded from pubs.acs.org by SWINBURNE UNIV OF TECHNOLOGY on 11/26/18. For personal use only.
Biotechnological Approaches to Control of Weeds with Pathogens D. C. Sands , R. V. Miller , and E. J. Ford 1
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Department of Plant Pathology, Montana State University, Baseman, M T 59717 Mycogen Corporation, 3303 McDonald Avenue, Hasten, LA 71270 1
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Plant pathogens have rarely been successfully used as biocontrol agents of weeds. One reason for this is that they are usually not lethal enough at low concentrations. In addition, they are usually not host specific. Our approach has been to mutate lethal broad host-range pathogens to obtain isolates that are still lethal to target hosts, but reduced in host range, survival capacity, or otherwise biologically contained. Two such types of biological containment are presented in a fungus, Sclerotinia sclerotiorum, a lethal pathogen of 40 different weeds. Modern g e n e t i c technology h a s t h e p o t e n t i a l t o advance t h e p r a c t i c e s of medicine, a g r i c u l t u r e , and environmental protection. Potential d a n g e r s o f t h i s new t e c h n o l o g y do e x i s t a n d must b e a v o i d e d . Opponents t o r e l e a s e s o f g e n e t i c a l l y a l t e r e d organisms c i t e examples of the destruction incurred by unintentional releases o f plant pathogens such as t h e Dutch e l m fungus. During the course o f our s t u d i e s , m u t a n t s o f S. sclerotiorum induced with u l t r a v i o l e t i r r a d i a t i o n were o b t a i n e d t h a t e x h i b i t e d i n c r e a s e d h o s t - r a n g e s (Table I) . T h e f a c t t h a t many i n d u c e d m u t a n t s ( c h e m i c a l o r i r r a d i a t i o n ) e x h i b i t e n h a n c e d v i r u l e n c e (1.2) s u p p o r t s t h e u s e o f reasonable caution before r e l e a s i n g c e r t a i n types o f g e n e t i c a l l y modified organisms. Our charge i s t o determine i f i t i s p o s s i b l e t o a v o i d t h e k i n d s o f d i s a s t e r s t h a t o f t e n accompany new t e c h n o l o g i e s v i a containment systems, g e n e t i c a l l y engineered o r otherwise. Natural
Containment
Systems
Containment o f microorganisms w i t h i n a s p e c i f i c niche o r w i t h i n a c e r t a i n a r e a i s n o t a n o v e l phenomenon. Most p l a n t pathogens, f o r i n s t a n c e , a r e g e n e t i c a l l y d e l i m i t e d to a s p e c i f i c n i c h e , such as plant host species or climate. The mechanisms o f h o s t range 0097-6156790/0439-0184506.00/0 © 1990 American Chemical Society
10. SANDS ET AL
Biotechnological Contrai of Weeds with Pathogens
d e l i m i t a t i o n are complex and not understood i n t h e i r e n t i r e t y . N u t r i t i v e f a s t i d i o u s n e s s , absence of t r i g g e r m e t a b o l i t e s , specific b i n d i n g , a n d enzyme p r o d u c t i o n a r e a l l i n v o l v e d . Whatever the mechanisms, the g e n e r a l p r a c t i c e of crop r o t a t i o n ( f o r c i n g h o s t s p e c i f i c pathogens to s u r v i v e i n absence of t h e i r h o s t ) , i s o f t e n an e f f e c t i v e m e t h o d o f c o n t r o l o f many p l a n t p a t h o g e n s . Perhaps these n a t u r a l c a s e s o f s e l f - c o n t a i n m e n t c a n be u s e d as models i n t h e development of containment systems f o r s a f e l y r e l e a s i n g g e n e t i c a l l y a l t e r e d microbes. A Non-Engineered Approach to
Containment
O u r own r e s e a r c h i n v o l v e s d e v e l o p m e n t o f g e n e t i c c o n s t r a i n t s i n broad host-range p l a n t pathogens. T h e p a t h o g e n we w o r k w i t h , Sclerotinia sclerotiorum, a t t a c k s o v e r 40 n o x i o u s broad-leafed w e e d s , a n d a s many c r o p s i n m o s t c o u n t r i e s . By d e l e t i o n m u t a g e n e s i s , we h a v e o b t a i n e d a n a u x o t r o p h i c m u t a n t , A l - p y r , t h a t has p y r i m i d i n e s as an a b s o l u t e growth r e q u i r e m e n t ( 3 ) . This n u t r i t i o n a l r e q u i r e m e n t i s complemented f u l l y by c y t o s i n e s u p p l e m e n t s a n d t o a l e s s e r e x t e n t b y u r a c i l ( F i g . 1) a d d e d t o a m o d i f i e d Czapek s o l u t i o n agar (4). Thymidine supplements were i n e f f e c t i v e i n overcoming the auxotrophy of t h i s mutant. The e f f e c t of c y t o s i n e , concomitantly w i t h the mutant fungus on seven h o s t s u n d e r greenhouse c o n d i t i o n s , i s shown i n F i g . 2. Preliminary e v i d e n c e f r o m l i m i t e d f i e l d t r i a l s i n d i c a t e s t h a t when A l - p y r i s a p p l i e d w i t h an e x t e r n a l source of c y t o s i n e , i t k i l l s p l a n t s i n the t a r g e t a r e a ; b u t t h e fungus f a i l e d t o i n f e c t p l a n t s when e x t e r n a l cytosine i s absent. T a b l e I . Expanded H o s t Ranges
of
S. sclerotiorum Percent K i l l e d
Host Alfalfa Bean* Canada T h i s t l e Clover Dandelion Leafy Spurge* Lentil Lettuce Lupine Poppy Rape Safflower S p o t t e d Knapweed Sunflower
Wildtype Parent 0 0 50 •0 75 0 85 100 0 100 85 100 40
11
B-326 0 70 100 0 75 30 70 85 0 65 85 100 65 100
Mutants Plants
Mutant B-850 100 70 100 60 50 65 100 100 65 100 100 100 85 100
• L e s i o n s d e v e l o p e d w i t h w i l d t y p e p a r e n t b u t d i d n o t succumb to the disease. (Reproduced w i t h p e r m i s s i o n from R e f . 9 . 1986 E n v i r o n m e n t a l P r o t e c t i o n A g e n c y . )
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16 32 CYTOSINE (mg/l)
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F i g u r e 1. R a d i a l g r o w t h r e s p o n s e o f S. eclerotiorum to p y r i m i d i n e supplements added t o m o d i f i e d Czapek s o l u t i o n a g a r . C o l o n y d i a m e t e r (mm), 48 h r a f t e r i n o c u l a t i o n o n t o m o d i f i e d Czapek s o l u t i o n agar. Mean o f t h r e e r e p l i c a t i o n s .
Ν U M Β Ε R Ο F Ρ Ο
τ s
1 -
F i g u r e 2. E f f e c t o f e x t e r n a l c y t o s i n e (50 m g / l ) on v i r u l e n c e o f S. sclerotîorwn Al-pyr. A p p l i e d on c o t t o n plugs to s i t e of i n o c u l a t i o n (PDA c u l t u r e s ) o f S . eclerotioiwi Al-pyr. Number o f p o t s showing d i s e a s e on s e e d l i n g s . Two t o t e n s e e d l i n g s o f e a c h species per pot.
10. SANDS ET AL
Biotechnological Control of Weeds with Pathogens
A n o t h e r S. sclerotiorum mutant o b t a i n e d i n t h i s s t u d y , S L - 1 , cannot form s c l e r o t i a . S c l e r o t i a are morphological s t r u c t u r e s that s e r v e s b o t h as p r e c u r s o r s f o r f r u i t i n g b o d i e s ( a s c o c a r p s ) and f o r dormant s u r v i v a l d u r i n g a d v e r s e c o n d i t i o n s (5). T h i s mutant i s u n a b l e t o make s p o r e s , o r s u r v i v e t h e w i n t e r , o r u n d e r g o s e x u a l r e c o m b i n a t i o n , r e s u l t i n g i n i t s demise d u r i n g the w i n t e r months. S L - 1 may b e s l i g h t l y l e s s v i r u l e n t e v i d e n c e d b y i t s t e n d e n c y t o cause d i s e a s e i n l o w e r p e r c e n t a g e s o f h o s t p l a n t s ( F i g . 3). However t h e a p p a r e n t h o s t r a n g e was u n c h a n g e d i n r e s p e c t t o t h e p l a n t s t e s t e d ( F i g . 3). As the w i l d t y p e fungus i s w i d e l y endemic, the u n l i k e l y occurrence of back mutations to prototrophy or s c l e r o t i a l formation would not s i g n i f i c a n t l y increase r i s k to crops. Neither o f t h e s e m u t a n t s a r e g e n e t i c a l l y e n g i n e e r e d a n d b o t h h a v e met requirements necessary for safe experimental releases. They are p r e s e n t l y b e i n g t e s t e d i n EPA r e v i e w e d s m a l l s c a l e f i e l d t e s t s i n Montana. The v a l u e o f s u c h o r g a n i s m s l i e s i n t h e i r b r o a d h o s t range, l e t h a l i t y , ease o f c u l t u r e , and r e s t r i c t e d d i s s e m i n a t i o n . In t h e f u t u r e , t h e y may b e e n g i n e e r e d w i t h t h e c y t o s i n e r e q u i r e m e n t o r s c l e r o t i a l formation attached behind a host specific promoter. G e n e t i c a l l y - E n g i n e e r e d Containment
Systems
Containment systems, u s e f u l , but often not c r i t i c a l i n none n g i n e e r e d s i t u a t i o n s , may w e l l b e c r i t i c a l p r i o r t o r e l e a s e o f many genetically-engineered microorganisms. To d a t e , t h r e e s t r a t e g i e s have been proposed f o r c o n t a i n i n g these organisms. The f i r s t s t r a t e g y , ( 6 . 7 ) . i n v o l v e s i n c o r p o r a t i o n o f recombinant genes to a s u i c i d e v e c t o r . By m e t h y l a t i o n , t h e s u i c i d e p o r t i o n of the v e c t o r i s turned o f f w h i l e the organism i s i n the presence of a s p e c i f i c substrate (e.g. crude o i l ) . The c a r r i e r m i c r o o r g a n i s m t h e n s e l f d e s t r u c t s once t h e s u b s t r a t e become l i m i t i n g i n the environment. S i m i l a r l y , B e j e t a l . (8) c o n s t r u c t e d a s u i c i d e v e c t o r t h a t i s turned o f f i n the presence of c a r b e n i c i l l i n . N e i t h e r o f t h e s e s y s t e m s e x c l u d e t h e p o s s i b i l i t i e s t h a t r e c o m b i n a n t DNA c o u l d be t r a n s f e r r e d t o o t h e r m i c r o o r g a n i s m s w h i l e i n t h e p r e s e n c e of the necessary s u b s t r a t e , and t h a t a l t e r n a t e organisms might a l l o w p e r s i s t e n c e o f the c l o n e d genes w i t h o u t s e l f - d e s t r u c t i n g . These s u i c i d e s y s t e m s do n o t p r e c l u d e m u t a t i o n s a r o u n d t h e s u i c i d e m o d u l e which c o u l d r e s u l t i n n o n - d e s t r u c t i v e r e t e n t i o n of the cloned genes. T h e s e c o n d s t r a t e g y , p r o p o s e d b y P e t e r S i d e r i u s (9) involves c o - i n d u c t i o n o f a s u i c i d a l gene w i t h t h e gene o f i n t e r e s t ( F i g . 4). T h i s r e s u l t s i n c o n c o m i t a n t d e a t h o f t h e o r g a n i s m w i t h gene expression. T h u s , t h e d e s i r e d gene p r o d u c t i s p r o d u c e d b u t t h e gene is t o t a l l y contained. The m a j o r l i m i t a t i o n t o t h i s p r o p o s e d s y s t e m i s t h a t i t i s n o t a p p l i c a b l e t o a r e a s s u c h as b i o l o g i c a l weed c o n t r o l where a v i a b l e o r g a n i s m i s r e q u i r e d f o r a c t i v i t y . T h e t h i r d s t r a t e g y p r o p o s e d b y o u r l a b o r a t o r y (9) a t t e m p t s t o o v e r c o m e some o f t h e p r o b l e m s i n c u r r e d b y t h e o t h e r t w o s y s t e m s . T h i s s t r a t e g y i n v o l v e s s p l i t t i n g gene e x p r e s s i o n b e t w e e n two o r t h r e e d i f f e r e n t l o c i , u s u a l l y d i f f e r e n t p l a s m i d s o r genomes ( F i g . 5). As a l l l o c i must be p r e s e n t f o r gene e x p r e s s i o n , t h e r i s k o f c o n c u r r e n t t r a n s f e r i s v e r y l o w and t h e gene i s effectively contained.
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8
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s
i
I
S
H*
10. SANDS ET AL
BioUchtological Gmtnd ۤ Weeds with Pathogens VP2
F i g u r e 4 . A s u i c i d e gene e x p r e s s i o n c o n t a i n m e n t model s y s t e m . PI - Promoter o f i n d u c i b l e o p e r o n . VP2 - V i r a l p r o m o t e r 1 . VP2 - V i r a l p r o m o t e r 2 .
INDUCE8
REPRES8E8
DESIRED GENE PRODUCT
LETHAL GENE PRODUCT F i g u r e 5. A m o d e l t r i p l i c a t e s a f e g u a r d c a s s e t t e f o r f u n g i . Note: C a r r i e r s i n c l u d e genomic o r autonomous e l e m e n t s .
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V i r a l promoters, r e s t r i c t i o n nucleases, and host s p e c i f i c p r o m o t e r s may a l l b e b o r r o w e d f r o m p l a n t p a t h o g e n s f o r u s e i n development o f c e l l u l a r s e l f - d e s t r u c t systems ( 6 , 1 0 ) . Reasons
f o r Containment
F r o m o u r o w n a n a l y s i s o f p o t e n t i a l r i s k s , we f i n d i t d i f f i c u l t t o see how a n y p u b l i c i n s t i t u t i o n o r p r i v a t e c o r p o r a t i o n w o u l d c o n s i d e r r e l e a s e o f g e n e t i c a l l y m o d i f i e d m i c r o b e s w i t h o u t i n c o r p o r a t i n g some s o r t o f containment system. F i r s t i s the problem, perceived o r actual, of l i a b i l i t y . S e c o n d l y , t h e y may f i n d t h a t t h e i r p r o d u c t p e r s i s t s too long, i n t e r f e r i n g with future sales and with i n t r o d u c t i o n s o f improved s t r a i n s . F i n a l l y , g o v e r n m e n t a p p r o v a l may t a k e a n i n o r d i n a t e amount o f t i m e r e v i e w i n g t h e r i s k f a c t o r s o f a n o n - c o n t a i n e d system, as compared t o one w i t h a p r o v e n type o f containment. Reasons
A g a i n s t Containment o f Engineered Microbes
Few c o n t a i n m e n t s y s t e m s c u r r e n t l y e x i s t , a n d n o n e h a v e b e e n extensively tested. A requirement f o r containment might hinder research andinvestment i n b i o c o n t r o l agents considered safe b y v i r t u e of host s p e c i f i c i t y . S i n g l e d e l e t i o n s , done b y r e c o m b i n a n t m e t h o d o l o g y , r a t h e r t h a n b y c h e m i c a l m u t a g e n e s i s , a l s o seem t o present low r i s k andshould n o t require containment systems. Conclusions C o n t a i n m e n t s y s t e m s may b e v a l u a b l e a d d e n d a t o r e l e a s e d m i c r o b e g e n e t i c systems t o reduce l i a b i l i t y e x p o s u r e , enhance i n v e s t m e n t , and e x p e d i t e e v a l u a t i o n o f e n v i r o n m e n t a l s a f e t y . Even n o n engineered microbes c a n be e f f e c t i v e l y c o n s t r a i n e d b y auxotrophy o r developmental blocks.
Literature Cited 1. Miller, R. V.; Ford, E. J.; Sands, D. C. Phytopathology 1987, 77, (12):1720 (Abstr.). 2. Simons, M. D. Ann. Rev. Phytopath. 1979, 17, 75-96. 3. Miller, R. V.; Ford, E. J.; Zidack, Ν. K.; Sands, D. C. J. Gen. Micro. 1989, in press. 4. Wong, A. L.; Willetts, H. J. Trans. Brit. Mycol. Soc. 1975, 61, 167-178. 5. Miller, R. V.; Ford, E. J.; Sands, D. C. Can. J. Microbiol 1989, 35:517-520. 6. Cusky, S. M. Appl Environ. Micro. 1989, in press. 7. McCormick, D. Biotechnology 1986, 4, 762. 8. Bej, Asim K.; Perlin, M. H.; Atlas, R. M. App. Environ. Micro. 1988, 54, 2472-2477. 9. Miller, R. V.; Siderius, P. G.; Sands, D. C. 1986. p. 43-64. In Terrestrial Biotechnology: Research Imperatives for 1987. Environmental Research Laboratory, Corvallis, Oregon, Environmental Protection Agency. 10. Molin, S.; Klemm, P.; Poulsen, L. K.; Biehl, H.; Gerdes, K.; Andersson, P. Bio Tech. 1987, 5, 1315-1318. RECEIVED February 2,
1990
