Pest Control with Enhanced Environmental Safety - American

Chapter 8

Altering the Host Range of Mycoherbicides by Genetic Manipulation David C. Sands and R. Vincent Miller

Downloaded by UNIV OF LEEDS on January 15, 2018 | http://pubs.acs.org Publication Date: March 12, 1993 | doi: 10.1021/bk-1993-0524.ch008

Department of Plant Pathology, Montana State University, Bozeman, MT 59717

New strategies for the development of b i o l o g i c a l control agents of weeds with plant patho gens are discussed. The basic premise i s based on using highly virulent broad-host range path ogens that lack the h o s t - s p e c i f i c i t y currently thought necessary for bioherbicides. These pathogens could be rendered safe by conferring biological confinement via auxotrophy or reduced survival a b i l i t y . Additional discus sion i s on possible methods for genetically constructing h o s t - s p e c i f i c i t y based on root exudates that are unique to a target host similar to a "Zip code", or using the host i t s e l f as a source of s e l f destructive genes, which we refer to as the "Pogo theory" from a quote by the comic s t r i p character, "We have found the enemy and it is us". The most widespread and important methods of weed c o n t r o l c u r r e n t l y used are t i l l a g e and chemical h e r b i c i d e s ( 1 ) . The use o f h e r b i c i d e s with documented adverse a f f e c t s on groundwater q u a l i t y , mammalian t o x i c i t y , and human h e a l t h have l e d to* increased i n t e r e s t s i n a l t e r n a t i v e s such as the use o f b i o l o g i c a l c o n t r o l . Despite a few successes though, b i o l o g i c a l c o n t r o l as i t i s now p r a c t i c e d cannot be expected t o completely r e p l a c e the use o f chemical herbicides. Present b i o c o n t r o l agents, developed w i t h e i t h e r the c l a s s i c a l or mycoherbicide approach (2,3), must be h o s t - s p e c i f i c f o r reasons o f environmental s a f e t y and l i a b i l i t y . And t h e r e simply may not be ample p r o s p e c t i v e h o s t - s p e c i f i c b i o c o n t r o l agents f o r each problem weed. Further, economic c o n s t r a i n t s p r e c l u d e t h e use o f h o s t s p e c i f i c agents f o r many s i t e s with m u l t i p l e weed s p e c i e s . Thus, the j u s t i f i c a t i o n s t o attempt t o s h i f t from chemical herbicides t o b i o l o g i c a l control are neither entirely

0097-6156/93/0524-0101$06.00/0 © 1993 American Chemical Society Duke et al.; Pest Control with Enhanced Environmental Safety ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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PEST CONTROL WITH ENHANCED ENVIRONMENTAL SAFETY

c l e a r nor c o n v i n c i n g . Probably the best j u s t i f i c a t i o n f o r b i o c o n t r o l involves the p o t e n t i a l marketing of "pesticide f r e e " c r o p s , b u t p r o d u c t i o n c o s t s w o u l d be h i g h e r , at l e a s t u n t i l b e t t e r b i o c o n t r o l agents are a v a i l a b l e f o r c o n t r o l o f i n s e c t s , w e e d s , a n d f u n g i . A m a r k e t may become e s t a b l i s h e d i f s u f f i c i e n t numbers o f c u s t o m e r s d e s i r e s u c h pesticide-free products.


B i o l o g i c a l Containment Systems I t seems l i k e l y t h a t , a s i n t h e c h e m i c a l h e r b i c i d e i n d u s try, one o r s e v e r a l p a r a d i g m s h i f t s w i l l be necessary b e f o r e any a d v a n c e s a r e made. T h e p r o p o s e d p a r a d i g m s h i f t i s away f r o m t h e c l a s s i c a l a p p r o a c h o f s c r e e n i n g only h o s t - s p e c i f i c e x o t i c o r n a t i v e p e s t s t o one e m p h a s i z i n g g e n e t i c m a n i p u l a t i o n and an e x t e n s i v e knowledge o f p h y s i o l o g y and b i o c h e m i s t r y of host/ p a r a s i t e i n t e r a c t i o n s . Our b a s i c p r o p o s a l i s t h a t c o n t a i n e d b r o a d - h o s t r a n g e p a t h o g e n s m i g h t b e t t e r compete w i t h b r o a d s p e c t r u m h e r b i c i d e s by v i r t u e o f t h e i r use a g a i n s t m u l t i p l e target weeds, r e d u c e d r e s i d u e r i s k , r e d u c e d l i a b i l i t y , r e p e a t e d s a l e s r e q u i r e m e n t s , and p o s s i b l y " p e r c e i v e d " a s p e c t s o f safety.

Broad-Host Range P l a n t Pathogens B r o a d - h o s t r a n g e p a t h o g e n s , w e l l known b y p l a n t p a t h o l o g i s t s , i n c l u d e Sclerotinia sclerotiorum, Pythium ultima,

Sclerotium

r o l f s i i , Phymatotrichum

omnivorum, a n d P s e u d o -

monas solanacearum (4,5). Often s o i l - b o r n e , these patho gens a r e c a p a b l e o f s u r v i v i n g i n the s o i l a f t e r k i l l i n g one p l a n t u n t i l t h e y a t t a c k a n o t h e r s u s c e p t i b l e p l a n t o f t h e same o r d i f f e r e n t s p e c i e s . T h e i r mode o f action i n v o l v e s r o o t o r crown i n v a s i o n , v a s c u l a r p l u g g i n g and wilting, and p r o d u c t i o n o f considerable biomass for survival (4,5). The g e n e t i c a l l y m a n i p u l a t a b l e a s p e c t s o f t h e s e pathogens are t h e i r host range, sexual s t r u c t u r e s , s u r v i v a l , a n d t h e i r a b i l i t y t o s y n t h e s i z e most i f n o t a l l v i t a m i n s , amino a c i d s , f a t t y a c i d s , p u r i n e s a n d p y r i m i dines. As w i t h o t h e r p l a n t pathogens, the mechanisms i n v o l v e d i n h o s t r a n g e r e s t r i c t i o n a r e unknown.

Sclerotinia

sclerotiorum

Model System

We h a v e d e v e l o p e d a s e r i e s o f m u t a n t s t r a i n s o f Sclerotinia sclerotiorum (6,7). T h i s pathogen n o r m a l l y a t t a c k s o v e r 300 g e n e r a o f p l a n t s i n c l u d i n g t h e o v e r 30 s p e c i e s o f weeds ( T a b l e I) ( 8 ) . T h e o b j e c t i v e o f t h i s r e s e a r c h was t o o b t a i n mutants o f t h i s b r o a d - h o s t range fungus t h a t e i t h e r h a v e a r e d u c e d h o s t r a n g e o r a r e i n some o t h e r way useful for genetic manipulation. T h i s fungus s e r v e s as a model system t o demonstrate t h a t b r o a d - h o s t range p a t h o g e n s i n g e n e r a l c a n be made more u s e f u l a s b i o l o g i c a l c o n t r o l agents. We o b t a i n e d a g r o u p o f a u x o t r o p h i c S.

Duke et al.; Pest Control with Enhanced Environmental Safety ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

8. SANDS & MILLER

Table I .

Altering Mycoherbicides by Genetic Manipulation

Common Weed-Hosts o f Sclerotinia

Common Name

S c i e n t i f i c Name Medicago lupulina


Brassica nigra B. rapa L.

sclerotiorum

Black Medic

L.

(L.) W.Koch

Black Field

Mustard Mustard

Orobanche sp. L.

Broomrape

Cirsium arvense L. (Scop.) C. vulgare (Savi) Ten.

Canada T h i s t l e Bull Thistle

Stellaria

Common Chickweed

media (L.) V i l l .

Xanthium strumarium L.

Cocklebur

Portulaca

Common Purslane

oleracea

Sonchus oleraceus

L.

Solidago

Common S o w t h i s t l e

L.

Taraxacum officinale

Wigg.

Goldenrods

sp. L.

Senecio

vulgaris

Cannabis

sativa

Dandelion

Common Groundsel

L. L.

Hemp

Centaurea maculosa L. C. repens L. C. s o l s t i t i a l i s L.

Spotted Knapweed Russian Knapweed Yellowstar T h i s t l e

Chenopodium album L.

Lambs-quarters

Lupinus

Lupines

Urtica

sp. L. dioica

Stinging Nettle

L.

Solanum nigrum L.

Common

Chrysanthemum leucanthemum L.

Oxeye Daisy

Thlaspi

Field

arvense L.

Nightshade

Pennycress

Amaranthus r e t r c f l e x u s L.

Redroot

Conium maculatum L.

Poison Hemlock

AmJbrosia

artemisiifolia

Capsella bursa-pastoris Medik Galinsoga

parviflora

Euphorbia

sp.

Rumex crispus

Pigweed

L.

Common Ragweed

L.

Shepherd's Purse

Cav.

Sma11-flowered Galinosoga Spurges

L.

C u r l y Dock


103

104


sclerotiorum mutants unable t o grow without c e r t a i n n u t r i t i o n a l supplements and a s e r i e s o f s t r a i n s w i t h v a r i o u s degrees o f v i r u l e n c e t h a t were unable t o make the s u r vival/sexual structures called s c l e r o t i a (Table II) . R e s u l t s obtained from f i e l d s i t e s e s t a b l i s h e d t h a t some o f these mutants a r e s t i l l v i r u l e n t , though l e s s so as compared t o t h e wild-type fungus (Figure 1 ) .


Table I I . Current Mutants o f Sclerotinia

Mutant

Phenotype

sclerotiorum

Parental Origin

Al-PYR

Cytosine r e q u i r e d f o r growth

KA2

A2-CYS

Methionine o r c y s t e i n e r e q u i r e d f o r growth

84. IB

A3-PYR

Cytosine r e q u i r e d f o r growth

A-169

A4-ARG

A r g i n i n e r e q u i r e d f o r growth

A-169

A5-LYS

Lysine r e q u i r e d f o r growth

A-169

A6-ARG

A r g i n i n e r e q u i r e d f o r growth

A-169

A8-LEU

Leucine r e q u i r e d f o r growth

A9-ISO/VAL

I s o l e u c i n e and v a l i n e r e q u i r e d f o r normal growth

A-169

A10-ADE

Adenosine r e q u i r e d f o r growth

CM813-2

All-CHOL

Choline r e q u i r e d f o r growth

CM813-2

A13-LYS

Lysine r e q u i r e d f o r growth

CM813-2

A14-ISO/VAL

I s o l e u c i n e and v a l i n e r e q u i r e d f o r normal growth

CM813-2

SL-1

S c l e r o t i a l e s s , low v i r u l e n c e

84. IB

SL-5

Sclerotialess, avirulent

A-9

SL-7

S c l e r o t i a l e s s , moderate virulence

A-169

In t h e f i e l d t r i a l i n Figure 1, the fungus was grown i n shake c u l t u r e s and homogenized mycelium used t o i n o c u l a t e s t e r i l e canola seed imbibed with a p p r o p r i a t e n u t r i e n t s o l u t i o n s . The i n f e s t e d canola was a p p l i e d a t 500, 1000 and 2000 l b / a c . A l b e i t high r a t e s o f a p p l i c a t i o n are used i n t h i s f i e l d t e s t , the r e s u l t s d r a m a t i c a l l y i l l u s t r a t e the p o t e n t i a l of u t i l i z i n g g e n e t i c a l l y d e l i m i t e d , but v i r u l e n t s t r a i n s of broad-host range pathogens i n b i o l o g i c a l c o n t r o l . P r e l i m i n a r y l a b o r a t o r y and greenhouse t e s t s


8. SANDS & MILLER


105


WEED TARGET

F i g u r e 1. E f f i c a c y of 5. sclerotiorum mutants compared t o t h e w i l d - t y p e s t r a i n on s i x t u r f weeds i n f i e l d t r i a l s conducted i n M i s s i s s i p p i . Treatments c o n s i s t e d o f 1000 l b / a c r e fungus i n f e s t e d c a n o l a seed imbibed with the one p e r c e n t t r y p t o n e , 10 mM sucrose, and f o r t h e auxotrophs, 50 a r g i n i n e o r l e u c i n e . Mean of t h r e e r e p l i c a t i o n s o f f i v e f o o t square p l o t s analyzed a f t e r 21 days.


106


i n d i c a t e t h a t formulations can s i g n i f i c a n t l y reduce necessary a p p l i c a t i o n r a t e s . Further, i t i s our contention t h a t these r a t e s a r e t o l e r a b l e as spot treatments i n c e r t a i n home and garden markets. S t r a i n Improvement v i a Mating Auxotrophs o f d i f f e r e n t parent s t r a i n s might be u s e f u l f o r s e v e r a l other reasons. For example, they could be crossed (lys" x arg") t o search f o r g e n e t i c recombinants ( a r g and l y s ) t h a t might have d i f f e r e n t u s e f u l t r a i t s . Secondly, auxotrophs can be t e s t e d f o r v i r u l e n c e i n t h e presence o r absence o f the appropriate n u t r i e n t . One example would be the c y t o s i n e minus s t r a i n s t h a t r e q u i r e e x t e r n a l c y t o s i n e f o r i n f e c t i o n (6) . T h i r d l y , auxotrophs can be used t o f a c i l i t a t e g e n e t i c m o d i f i c a t i o n . The c y t o s i n e p l u s gene can be added by transformation t o a c y t o s i n e minus s t r a i n but on a d i f f e r e n t promoter. I f the promoter only a c t i vates t r a n s c r i p t i o n i n t h e presence o f c e r t a i n p l a n t s , then t h e p r e v i o u s l y broad-host range pathogen would be rendered h o s t - s p e c i f i c . +


+

G e n e t i c a l l y Constructed H o s t - S p e c i f i c i t y The key s i g n a l s t h a t could be used t o a c t i v a t e modified pathogens would probably be compounds t h a t a r e unique t o the t a r g e t host. An example o f a t a r g e t s p e c i f i c compound might be t h e a l k a l o i d c n i c i n found i n and on knapweed leaves (9) . T h i s compound might serve as a s p e c i f i c inducer o f prototropy i n a g e n e t i c a l l y modified pathogen. For instance, without c n i c i n t h e pathogen would r e q u i r e c y t o s i n e whereas i n the presence o f c n i c i n c y t o s i n e would be synthesized. N a t u r a l chemical r e c o g n i t i o n systems may be a v a i l able from pathogens, epiphytes, i n s e c t s , nematodes, o r rhizosphere m i c r o f l o r a a s s o c i a t e d with the t a r g e t host, o r even from the host i t s e l f . For instance, c n i c i n u t i l i z i n g m i c r o f l o r a may be obtained from organisms i s o l a t e d from spotted knapweed i t s e l f , s o i l - b o r n e microorganisms, o r from t h e i n t e s t i n a l f l o r a o f i n s e c t s or nematodes t h a t feed on t h i s p l a n t . These promotor o r r e p r e s s o r systems can be cloned upstream t o the gene r e s t o r i n g prototropy and then i n s e r t e d i n t o t h e pathogen v i a t r a n s f o r m a t i o n . A Z i p Code System f o r Auxotrophy The r o o t exudates o f p l a n t s have been w e l l c h a r a c t e r i z e d and g e n e r a l l y c o n s i s t p r i m a r i l y of c e r t a i n amino a c i d s and sugars (20). P a t h o l o g i s t s have found l i t t l e r e l a t i o n s h i p between r o o t exudate content and h o s t / p a r a s i t e s p e c i f i c i t y (10) . However, as e a r l y as the mid 1950 s, Garber (11,12) demonstrated a r e l a t i o n s h i p between auxotrophic mutants and c u l t i v a r - l e v e l host s e l e c t i v i t y with Erwinia aroideae, precedence f o r a n u t r i t i o n a l l y - b a s e d system f o r c o n f e r r i n g f



8, SANDS & MILLER


107

h o s t - s p e c i f i c i t y . Other f a c t o r s t h a t have been i m p l i c a t e d i n h o s t - s p e c i f i c i t y i n c l u d e l e c t i n b i n d i n g , pathogens p e c i f i c and non-pathogen s p e c i f i c t o x i n s , spore a g g l u t i n a t i n g f a c t o r s , and n o n - s p e c i f i c defense mechanisms (13) . We can determine which f r e e amino a c i d s a r e i n high amounts i n t h e s u b s o i l crown of the t a r g e t p l a n t and, as might be expected, c e r t a i n amino a c i d s a r e not common i n a l l crowns o f a l l p l a n t s p e c i e s . A m u l t i p l e auxotroph c o u l d then be designed t o s p e c i f i c a l l y a t t a c k a c e r t a i n p l a n t based on i t s f r e e amino a c i d p r o f i l e . An example might be Centaurea maculosa, the spotted knapweed which has high amounts of f r e e asparagine, a l a n i n e , a s p a r t a t e , p r o l i n e , and threonine i n i t s crown (Table I I I ) • P r o l i n e i s h i g h l y v a r i a b l e i n p l a n t s p a r t i c u l a r l y i n response t o v a r i o u s s t r e s s e s such as senescence o r drought. But an auxotroph r e q u i r i n g t h e other four amino a c i d s might be able t o i n f e c t the crown of t h i s p l a n t whereas other nont a r g e t hosts might not provide s u f f i c i e n t l e v e l s o f these e s s e n t i a l amino a c i d s . Such " z i p coding" i s an a r t i f i c i a l system t h a t may enable development o f h o s t - s p e c i f i c i t y v i a auxotrophs. A General Strategy f o r F i n d i n g S p e c i f i c i t y or Virulence

Useful

Genes t o Enhance

B i o c o n t r o l agents are not always as s p e c i f i c o r v i r u l e n t ( l e t h a l ) as r e q u i r e d . One p o t e n t i a l source o f genes t o enhance b i o c o n t r o l agents i s from the t a r g e t host i t s e l f . The u n d e r l y i n g presumption i s t h a t most m u l t i c e l l u l a r organisms c o n t a i n numerous intermediary metabolites, hormones, and enzymes, t h a t i f overproduced o r produced a t the wrong time would be l e t h a l . We r e f e r t o t h i s general s t r a t e g y as t h e Pogo Strategy a f t e r t h e Walt K e l l y cartoon c h a r a c t e r who s a i d , "We have found t h e enemy and i t i s us." Examples might i n c l u d e molting hormones i n nematodes and i n s e c t s , i n s e c t egg hatching f a c t o r s , seed germination i n h i b i t o r s o r simulators, p l a n t growth hormones, a l k a l o i d s , o r p l a n t defenses such as p h y t o a l e x i n production (14,15,16,17,18) . The g e n e t i c approach t o t h i s s t r a t e g y would i n v o l v e using cDNA i n a expression v e c t o r and assaying t h e cloned gene products back on t h e host. A f t e r f i n d i n g a c t i v e compounds, these c o u l d be used d i r e c t l y o r t h e i r appropriate genes could be cloned i n t o an appropriate pathogen. Notably, many o f t h e f i n a l products l i s t e d a r e m u l t i g e n i c i n nature but a r e o f t e n under r e g u l a t o r y systems t h a t may be coded by a s i n g l e gene and thus a c c e s s i b l e t o manipulation. Conclusions We expect t o see s e v e r a l s h i f t s i n s t r a t e g y as b i o l o g i c a l c o n t r o l becomes l e s s a c l a s s i c a l hunting and g a t h e r i n g o p e r a t i o n and more i n v o l v e d with g e n e t i c manipulation. As


108


ways t o make broad-host range pathogens s a f e r a r e devised, we may see the use o f b i o l o g i c a l s analogous t o t h a t o f broad spectrum chemical h e r b i c i d e s .

Table I I I .

Amino A c i d P r o f i l e o f Spotted Knapweed 1

Mmol/ml


Amino A c i d Alanine

0.248

Arginine

0.652

Aspartic Acid

0.523

Cystine

0.039

Glutamic A c i d

0.340

Glycine

0.030

Histidine

0.150

Isoleucine

0.092

Leucine

0.041

Lysine

0.056

Methionine Phenylalanine

Not

Tested 0.024

Proline

1.520

Serine

0.342

Threonine

0.327

Tryptophan

0.061

Tyrosine

0.042

Valine

0.214

Asparagine

6.870

Glutamine

1.270

a-Aminobutyric A c i d

0.033

Ethanolamine

0.073

Sarcosine

0.047

/Ltmol/ml o f an e t h a n o l i c e x t r a c t o f crowns.


8. SANDS & MILLER Altering Mycoherbicides by Genetic Manipulation 109

Literature Cited 1. 2. 3.


4. 5. 6. 7. 8.

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