Soilborne Fungi for Biological Control of Weeds - American Chemical

Chapter 15

Soilborne Fungi for Biological Control of Weeds

Downloaded by EAST CAROLINA UNIV on March 8, 2017 | http://pubs.acs.org Publication Date: September 25, 1990 | doi: 10.1021/bk-1990-0439.ch015

Richard W. Jones and Joseph G. Hancock Department of Plant Pathology, 147 Hilgard Hall, University of California—Berkeley, Berkeley, CA 94720

Current methods of biological weed control with plant pathogens rely mainly upon foliar application of host -specific pathogens which infect an established weed population. This contrasts with chemical weed control where application can provide broad-spectrum pre planting, pre-emergence or post-emergence control of potential weed populations. This limitation to the value of mycoherbicides, relative to chemical herbicides, could be overcome with the finding that the saprophytic, soilborne fungus Gliocladium virens Miller, Giddens & Foster can provide the needed broad -spectrum pre-emergence control of weeds. Application of G. virens, cultured on peat moss amended with sucrose and ammonium nitrate, reduced a broad range of weeds by at least 90 percent. Those seedlings which did emerge were severely stunted. Herbicidal activity was correlated with production of the steroidal phytotoxin viridiol. Viridiol caused a severe necrosis of roots but did not affect other tissues. Crop toxicity was avoided by directed application of the mycoherbicide above the root zone of crop seedlings. The use of G. virens and other soilborne fungi for weed control is discussed.

Soilborne fungi remain the l e a s t s t u d i e d organisms f o r the b i o l o g i c a l c o n t r o l o f weeds. T h i s i s s u r p r i s i n g when one c o n s i d e r s that the f i r s t f u l l y registered commercially available microbial h e r b i c i d e w a s t h e s o i l b o r n e f u n g u s Phytophthora palmivora Butl. (1,2). M a r k e t e d a s D e V i n e ( A b b o t t L a b o r a t o r i e s , C h i c a g o I L ) , P . palmivora i s a p p l i e d t o s o i l a r o u n d c i t r u s t r e e s f o r t h e c o n t r o l o f s t r a n g l e r v i n e (Morrenia odorata L i n d l . ) . I t has proven h i g h l y 0097-6156790A)439-0276$06.00A) © 1990 American Chemical Society

Hoagland; Microbes and Microbial Products as Herbicides ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


15. JONES* HANCOCK

SoUbome FungiforBiological Control of Weeds

e f f e c t i v e i n c o n t r o l o f both s e e d l i n g s and mature v i n e s . P e r s i s t e n c e o f t h i s m y c o h e r b i c i d e i s h i g h l i g h t e d b y t h e 95 t o 100 % v i n e c o n t r o l r e p o r t e d 6 years a f t e r a p p l i c a t i o n o f t h e fungus Q ) . S o i l b o r n e f u n g i r e d u c e weed p o p u l a t i o n s t h r o u g h t h e d e c a y o f seeds b e f o r e emergence o r b y c a u s i n g t h e d e a t h o f s e e d l i n g s s h o r t l y a f t e r emergence. S o i l b o r n e f u n g i c a n a l s o cause severe r o o t decay, g i r d l i n g s o i l - l i n e l e s i o n s and i n t e r n a l stem n e c r o s i s , each r e s u l t i n g i n reduced competitive a b i l i t y and decreased reproductive c a p a c i t y o f i n f e c t e d weeds. W h i l e t h i s damage may b e o v e r l o o k e d o n a w e e d , i t i s r e a d i l y d o c u m e n t e d w h e n i t c a u s e s s i m i l a r damage t o a crop. P l a n t p a t h o l o g i s t s a r e w e l l a w a r e o f t h e damage c a u s e d t o crops b y s o i l b o r n e f u n g i , however t h e i r main i n t e r e s t i s p r o t e c t i o n o f t h e c r o p , n o t t h e p o t e n t i a l t o i n f l i c t t h e same t y p e o f damage t o weed s p e c i e s . A w e a l t h o f i n f o r m a t i o n e x i s t s o n t h e e c o l o g y a n d physiology of soilborne phytopathogenic f u n g i . This information can provide a valuable resource f o r s e l e c t i o n and o p t i m i z a t i o n of s o i l b o r n e phytopathogenic f u n g i as mycoherbicides. Weed h o s t s a r e a n i m p o r t a n t r e s e r v o i r f o r p a t h o g e n s o f c u l t i v a t e d p l a n t s p e c i e s , t h u s t h e y a r e l i s t e d i n many s t u d i e s o f s o i l b o r n e crop pathogens. T h i s documentation c a n be a v a l u a b l e s o u r c e o f new m y c o h e r b i c i d e s . I t s h o u l d b e p o s s i b l e t o e m p l o y a c r o p p a t h o g e n f o r c o n t r o l o f a c l o s e l y r e l a t e d weed s p e c i e s as l o n g as t h e crop s p e c i e s i s n o t p r e s e n t . One o f t h e v e r y f e w r e p o r t e d e x a m p l e s where t h i s s t r a t e g y h a s b e e n a p p l i e d was t h e a t t e m p t t o

control yellow starthistle

{Centaurea s o l s t i t i a l is L . ) w i t h

p a t h o g e n s o f t h e c l o s e l y r e l a t e d c r o p s a f f l o w e r (Carthamus tinctorius L.) (4). The u s e o f p h y t o p a t h o g e n i c s o i l b o r n e f u n g i i s o b v i o u s l y l i m i t e d by the p o t e n t i a l f o r exposure o f s u s c e p t i b l e crop species. T h i s l i m i t a t i o n c a n be reduced i f t h e fungus h a s a v e r y l i m i t e d host range, although t h i s l i m i t s i t s value as a broads p e c t r u m weed c o n t r o l . E x p o s u r e c a n o c c u r t h r o u g h t h e s u b s e q u e n t p l a n t i n g o f susceptible crops into mycoherbicide i n f e s t e d s o i l , or by d i s p e r s a l i n t o s u r r o u n d i n g f i e l d s c o n t a i n i n g s u s c e p t i b l e c r o p s . I f crop r o t a t i o n s are p r a c t i c e d over a s u f f i c i e n t l e n g t h o f time i t may b e p o s s i b l e t o p l a n t s u s c e p t i b l e c r o p s , b u t o n l y f o r t h o s e c a s e s where t h e s o i l b o r n e fungus i s known t o be g r e a t l y r e d u c e d i n p o p u l a t i o n i n t h e absence o f a h o s t . The p o s s i b i l i t y t h a t a s o i l b o r n e fungus would be d i s p e r s e d t o s u r r o u n d i n g f i e l d s , w h i l e possible, i s less l i k e l y than the possible d i s p e r s a l of a f o l i a r p a t h o g e n whose p r o p a g u l e s w o u l d b e a v a i l a b l e f o r w i n d a n d w a t e r dispersal. T h e r e a r e numerous a d d i t i o n a l a d v a n t a g e s i n t h e u s e o f s o i l b o r n e f u n g i f o r w e e d c o n t r o l . One a d v a n t a g e o f s o i l b o r n e f u n g i i s t h e i r g r e a t e r independence from e n v i r o n m e n t a l c o n d i t i o n s ( m o i s t u r e , t e m p . ) w h i c h l i m i t t h e u s e o f f o l i a r m y c o h e r b i c i d e s . The extended periods of h i g h r e l a t i v e humidity r e q u i r e d f o r germination and i n f e c t i o n by f o l i a r mycoherbicides s e v e r e l y r e s t r i c t s the p e r i o d o f a p p l i c a t i o n . S o i l b o r n e f u n g i c a n be a p p l i e d a t t h e c o n v e n i e n c e o f t h e grower when weed c o n t r o l i s d e s i r e d . S o i l b o r n e f u n g i c a n a l s o be a p p l i e d i n v a r i o u s g r a n u l a r f o r m u l a t i o n s w h i c h p r o v i d e a s u b s t r a t e w h i c h f a v o r s p r o l i f e r a t i o n o f t h e added mycoherbicide.


111

278

MICROBES AND MICROBIAL PRODUCTS AS HERBICIDES

F u s a r i a as

Mycoherbicides

A l i s t o f s o i l b o r n e p h y t o p a t h o g e n s w h i c h have been s t u d i e d as m y c o h e r b i c i d e s i s f o u n d i n T a b l e I. With the exception of Sclerotinia sclerotiorum ( L i b . ) de B a r y , t e s t e d f o r c o n t r o l o f C a n a d a t h i s t l e [Cirsium arvense ( L . ) S c o p . ] ( 5 ) , a n d P . palmivora, the only s o i l b o r n e p l a n t pathogenic fungi c u r r e n t l y being s t u d i e d as m y c o h e r b i c i d e s i n c r o p s b e l o n g t o t h e genus F u s a r i u m .


Table

I.

Soilborne Phytopathogenic as M y c o h e r b i c i d e s

Fungus

Fusarium

Target

lateritLum

Fusarium oxysporum f . s p . cannabis Fusarium oxysporum f . s p . carthami Fusarium roseum Fusarium solani f . s p . cucurbitae Phytophthora palmivora Rhizoctonia solani Sclerotinia sclerotiorum

Fungi

Studied

Weed

Reference

prickly sida s p u r r e d anoda velvetleaf hemp

(8)

yellow

(4)

starthistle

(2)

hydrilla Texas gourd

(9) (6)

stranglervine water hyacinth Canada t h i s t l e

(1) (10) Q)

Fusarium i s found i n v i r t u a l l y a l l s o i l s on a worldwide b a s i s . S u r v i v a l i n s o i l i s g e n e r a l l y achieved by p r o d u c t i o n of chlamydospores which a r i s e through the t h i c k e n i n g of hyphal or conidial cell walls. S p e c i e s o f F u s a r i u m have b e e n r e p o r t e d as p a r a s i t e s on v i r t u a l l y a l l c u l t i v a t e d crop s p e c i e s . Many f u s a r i a a r e q u i t e h o s t s p e c i f i c , and a r e c l a s s i f i e d i n formae s p e c i a l i s according to t h e i r s p e c i f i c i t y . F o r e x a m p l e , Fusarium solani ( M a r t . ) A p p e l . & W r . f o r m a e s p e c i a l i s ( f . s p . ) cucurbitae, applied f o r c o n t r o l o f T e x a s g o u r d (Cucurbita texana G r a y ) ( 6 ) , i s l i m i t e d to i n f e c t i o n of cucurbits. This specialization provides a p r e d i c t a b l e host range, thus reducing the r i s k of i n f e c t i n g a p l a n t s p e c i e s absent from h o s t - r a n g e s c r e e n i n g . I n the absence of a h o s t , t h e s e s p e c i a l i z e d p a t h o g e n s do n o t s u r v i v e b e y o n d a f e w s e a s o n s so t h e y s h o u l d n o t pose a t h r e a t t o f u t u r e p l a n t i n g s o f susceptible crops. An a d d i t i o n a l b e n e f i t of t h i s s p e c i a l i z a t i o n i s t h a t r e s i s t a n c e g e n e s a r e a v a i l a b l e i n some c r o p s , t h u s a l l o w i n g use of the fungus w i t h otherwise s u s c e p t i b l e c r o p s . The p r e s e n c e o f r e s i s t a n c e i n t h e c r o p s u g g e s t s t h a t r e s i s t a n c e may b e selected f o r i n t h e weed p o p u l a t i o n a f t e r r e p e a t e d a p p l i c a t i o n o f h o s t specific fusarium-based mycoherbicides. F u s a r i u m r e s i s t a n c e was f o u n d d u r i n g i n i t i a l a p p l i c a t i o n o f F. oxysporum S c h l e c h t . f . sp. cannabis N o v i e l l o & S n y d e r f o r c o n t r o l o f i l l i c i t hemp (Cannabis

sativa

L.) (2).


15. JONES & HANCOCK

Sodbome Fungifor Biological Control of Wads

One l i m i t a t i o n i n t h e u s e o f F u s a r i a a s m y c o h e r b i c i d e s h a s b e e n t h e i n a b i l i t y t o c o n t r o l more t h a n one weed s p e c i e s . Unless the t a r g e t weed i s t h e m a j o r component o f t h e c o m p e t i t i v e weed p o p u l a t i o n i t i s uneconomical to apply the mycoherbicide. This l i m i t a t i o n h a s b e e n r e d u c e d s o m e w h a t t h r o u g h u s e o f Fusarium lateritium Nees ex F r . , w h i c h i s e f f e c t i v e a g a i n s t s p u r r e d anoda


[Anoda cristata

( L . ) S c h l e c h t ] , v e l v e t l e a f (Abutilon

theophrasti

M e d i c . ) a n d p r i c k l y s i d a (Sida spinosa L . ) ( £ ) . These weeds a r e , h o w e v e r , a l l members o f t h e M a l v a c a e , t h u s c o n t r o l r e m a i n s c o n f i n e d to c l o s e l y r e l a t e d weeds. I t may b e p o s s i b l e t o c o m b i n e v a r i o u s F u s a r i a t o o b t a i n a b r o a d e r range o f weed c o n t r o l . W h i l e t h e r e i s some p r o m i s e i n t h e u s e o f F u s a r i a a s m y c o h e r b i c i d e s , t h e r e may b e d i f f i c u l t i e s i n r e g i s t r a t i o n a n d c o m m e r c i a l i z a t i o n o f a c r o p p a t h o g e n f o r weed c o n t r o l . Growers, c o n c e r n e d a b o u t t h e p o t e n t i a l f o r damage t o t h e i r own a n d n e i g h b o r i n g f i e l d s may b e w a r y o f m y c o h e r b i c i d e s c l a s s i f i e d a s p l a n t pathogens. Another type of mycoherbicide, employing nonphytopathogenic s o i l b o r n e f u n g i , can provide an a l t e r n a t i v e to the r i s k s o f a p p l y i n g p a t h o g e n i c f u n g i f o r weed c o n t r o l . The n o n p h y t o p a t h o g e n i c f u n g i a c t b y p r o d u c i n g p h y t o t o x i c compounds u n d e r c e r t a i n n u t r i e n t regimes. T h u s , w h i l e t h e y do n o t i n f e c t p l a n t s t h e i r p h y t o t o x i c m e t a b o l i t e s r e s u l t i n s t u n t i n g and death of nearby plants. As p h y t o t o x i c l e v e l s o f s y n t h e s i s occur o n l y w i t h the a d d i t i o n of h i g h l e v e l s of s p e c i f i c n u t r i e n t s the d u r a t i o n of h e r b i c i d a l a c t i v i t y c a n be r e g u l a t e d . I n the absence of the added s u b s t r a t e the fungus e x i s t s i n the s o i l s i m p l y as a s a p r o p h y t e , c a u s i n g no p o t e n t i a l d i s e a s e t h r e a t .

P h v t o t o x i n P r o d u c t i o n b v Gliocladium

virens

B r o a d - s p e c t r u m weed c o n t r o l h a s r e c e n t l y b e e n a c h i e v e d u s i n g t h e n o n - p h y t o p a t h o g e n i c s o i l b o r n e f u n g u s Gliocladium virens (11.12). A p p l i c a t i o n o f the fungus a f t e r c u l t u r i n g on n u t r i e n t - a m e n d e d p e a t r e s u l t e d i n d r a m a t i c r e d u c t i o n s i n s e e d l i n g emergence and d r y w e i g h t s as shown i n T a b l e I I . A l l t e s t seeds were f i r s t p l a n t e d i n s o i l i n w h i c h 16% o f t h e t o t a l s o i l v o l u m e was t h e f u n g u s - p e a t mixture. The m a j o r i t y o f t e s t weed s p e c i e s f a i l e d t o emerge a t t h i s l e v e l , t h e few w h i c h d i d were s e v e r e l y s t u n t e d . Those s p e c i e s w h i c h f a i l e d t o e m e r g e a t t h e 16% c o n c e n t r a t i o n w e r e p l a n t e d i n s o i l i n w h i c h 8.7% o f t h e t o t a l s o i l v o l u m e was t h e f u n g u s - p e a t m i x t u r e . Those f a i l i n g t o emerge a t t h i s c o n c e n t r a t i o n were p l a n t e d a t l o w e r c o n c e n t r a t i o n s u n t i l e m e r g e n c e l e v e l s a t l e a s t 10% t h a t o f the c o n t r o l were a c h i e v e d . An i m p o r t a n t aspect i n a d d i t i o n to the b r o a d - s p e c t r u m n a t u r e o f t h i s mycoherbicide i s t h a t i s posseses pre-emergence a c t i v i t y . The s y s t e m i s s i m i l a r i n many w a y s t o w e e d c o n t r o l p r o v i d e d b y p r e emergence c h e m i c a l h e r b i c i d e s , w h i c h a c c o u n t f o r t h e m a j o r i t y o f herbicide applications. W h i l e most m y c o h e r b i c i d e s cause v a r i o u s degrees o f p h y s i o l o g i c a l s t r e s s to t a r g e t weeds, i t i s o f t e n o n l y a f t e r t h e y h a v e become e s t a b l i s h e d i n t h e c r o p . As the e f f e c t s o f weed c o m p e t i t i o n a r e g e n e r a l l y g r e a t e s t e a r l y i n c r o p s t a n d e s t a b l i s h m e n t , t h i s i s the most e f f e c t i v e t i m e f o r c h e m i c a l o r mycoherbicide a c t i v i t y ( U ) · T h e a c t i v i t y o f G. virens a s a m y c o h e r b i c i d e i s a c h i e v e d t h r o u g h p r o d u c t i o n o f t h e s t e r o i d - l i k e p h y t o t o x i n v i r i d i o l whose s t r u c t u r e i s s h o w n i n F i g u r e 1. V i r i d i o l p r o d u c t i o n i n s o i l was


279


8.7

16.0

« )

Application

II.

rate

Table

of

Safflower Bean Alfalfa Sugarbeet Spinach Black nightshade F i e l d bindweed Annual bluegrass Yellow f o x t a i l Canarygrass C u r l y dock Buckhorn p l a n t a i n

(Z o f

100 100 75 100 79 23 67 40 53 29 20 46

75 69 32 41 44 25 83 71 33 52 89

control)

30.8 189.6 6.3 8.5 8.1 0.9 12.0 1.3 6.8 1.8 0.6 0.9

44.1 31.6 1.4 59.0 16.5 13.9 103.6 15.5 2.2 2.7 30.7

Control

after

16.3 143.1 2.1 1.6 2.3 0.9* 3.7 0.5 1.5 1.2 0.2 0.5

3.4 15.4 0.2 34.6 3.5 0.6 64.1 3.7 0.3 0.1 17.8

Treated

f V

14

77.7 423.3 17.6 25.2 19.5 2.9 27.7 2.2 15.6 2.1 2.3 1.5

150.1 105.4 5.6 176.7 66.7 61.7 92.6 32.8 11.9 12.4 13.7

31.8 257.1 3.2 3.5 4.3 1.3 9.5 0.4 2.5 1.9* 0.9 0.8

41.1 48.4 0.4 86.7 11.4 1.6 36.0 5.4 0.9 0.5 6.8

Shoot Treated

days

Control

Dry weight

species

Root

m i x t u r e on c r o p and weed

Seedling emergence

G. v i r e n s - p e a t

Cotton Sunflower Lettuce Soybean Cucumber Cantaloupe Com Sudangraes Mustard Tomato W i l d Oat

Plant

Effect


Hoagland; Microbes and Microbial Products as Herbicides ACS Symposium Series; American Chemical Society: Washington, DC, 1990. 1.9 1.8 0.5 1.0

0.9 1.0 0.3 0.6

0.1 0.1 0.1 0.6 0.1 2.6 4.1 1.8 3.1

2.7 5.7 2.5 5.3 0.7 1.2 2.4 1.1 1.0

0.7 0.1 0.2 1.7 0.4

(Reproduced

Figure

1.

12.

Society

of

by G l i o c l a d i u m v i r e n s .

1988 Weed S c i e n c e

Viridiol

Copyright

S t e r o i d a l p h y t o t o x i n produced

w i t h p e r m i s s i o n from Ref.

for

dry America.)

t r e a t m e n t means asterisk.

weight

F u n g u e - p e a t m i x t u r e as percentage of t o t a l s o i l volume. V a l u e s r e p r e s e n t t r e a t m e n t l e v e l s at w h i c h a t l e a s t 10% o f s e e d l i n g s e m e r g e d r e l a t i v e t o t h e c o n t r o l . S p e c i e s t h a t f a i l e d t o e m e r g e t o t h i s l e v e l w e r e s e e d e d a t t h e n e x t l o w e r c o n c e n t r a t i o n u n t i l e m e r g e n c e e x c e e d e d 10Z o f c o n t r o l s .

60 62 96 58

Dandelion Bristly oxtongue Groundsel Annual s o w t h i s t l e

2.3

1.6 1.5 1.0 0.9 0.2

^ V a l u e s r e p r e s e n t mean o f i n d i v i d u a l s e e d l i n g s . D i f f e r e n c e s b e t w e e n were a l l s i g n i f i c a n t ( P - 0 . 0 5 ) u s i n g ANOVA-1, e x c e p t where n o t e d by

a

58 71 67 57 69

Spotted catsear Redroot pigweed Common p u r s l a n e Common f i d d l e n e c k Lambsquarter

4.5


282



c l o s e l y r e l a t e d w i t h c o n t r o l o f weed emergence, b o t h l a s t i n g a b o u t two w e e k s . Production of phytotoxic l e v e l s of v i r i d i o l is dependent upon t h e h i g h n u t r i e n t s u b s t r a t e on which t h e fungus is cultured. W h i l e numerous c a r b o n a n d n i t r o g e n s o u r c e s support v i r i d i o l production, optimal l e v e l s are produced w i t h h i g h carbon:nitrogen ratios (14). I t i s i m p o r t a n t t h a t t h e s u b s t r a t e be below pH 7.0 as v i r i d i o l i s u n s t a b l e i n a l k a l i n e c o n d i t i o n s . A p p l i c a t i o n f o r m u l a t i o n s a r e p r e p a r e d b y c u l t u r i n g G. virens o n n u t r i e n t - a m e n d e d peat i n s h a l l o w t r a y s f o r s i x days u n t i l t h e fungus has c o l o n i z e d the p e a t . The f u n g u s - p e a t m i x t u r e i s a i r d r i e d then s t o r e d u n t i l needed. F o r a p p l i c a t i o n the d r i e d mixture i s i n c o r p o r a t e d i n a band t o t h e s o i l between the top o f the crop seed and the s o i l s u r f a c e . Exposure to v i r i d i o l r e s u l t s i n n e c r o s i s and death o f emerging radicles. R o o t t i p s a r e a f f e c t e d i n t h e same m a n n e r , w h e r e a s hypocotyls and f o l i a g e are unaffected. The o r g a n s p e c i f i c i t y o f v i r i d i o l a l l o w s i t t o be a p p l i e d i n t h e presence o f s e n s i t i v e crop species. A p p l i c a t i o n i s s i m p l y d i r e c t e d t o t h e s o i l above t h e crop seed. As the crop seed germinates the emerging r a d i c l e and subsequent roots extend i n t o the s o i l below the r e g i o n o f v i r i d i o l production. V i r i d i o l production occurs only on the peat substrate, i n c o n t a c t w i t h t h e added n u t r i e n t s , thus s y n t h e s i s does n o t occur b e y o n d t h e s u b s t r a t e . We h a v e f o u n d n o e v i d e n c e f o r movement o f v i r i d i o l into the v i r i d i o l - s e n s i t i v e root region of test crop s e e d l i n g s . The r e g i o n between t h e c r o p seed a n d t h e s o i l s u r f a c e i s where weed c o n t r o l c a n be s e e n . A weed s e e d w h i c h g e r m i n a t e s a t a depth greater than that which the fungus-peat mixture i s a p p l i e d w i l l be u n a f f e c t e d . However, t h e m a j o r i t y o f weed s e e d s g e r m i n a t e w i t h i n t h e top few c e n t i m e t e r s o f s o i l t h e r e f o r e a h i g h degree o f weed c o n t r o l c a n be e x p e c t e d . N u m e r o u s a t t r i b u t e s make G. virens a n i d e a l f u n g u s f o r mycoherbicide formulations. I t i s r e a d i l y c u l t u r e d , grows r a p i d l y a n d i s l o n g - l i v e d i n d r i e d p r e p a r a t i o n s . A u n i q u e f e a t u r e o f G. virens i s t h e p r o d u c t i o n o f c h e m i c a l c o m p o u n d s i n a d d i t i o n t o t h e p h y t o t o x i n v i r i d i o l . The two m a i n compounds a r e t h e epipolythiodiketopiperazines g l i o t o x i n and g l i o v i r i n (15,16). T h e s e c o m p o u n d s a r e s t r o n g l y i n h i b i t o r y t o w a r d s Pythium ultimum T r o w a n d Rhizoctonia solani K u h n , t w o s o i l b o r n e f u n g i r e s p o n s i b l e f o r t h e m a j o r i t y o f crop seed and s e e d l i n g decay. A c l o s e r e x a m i n a t i o n o f o t h e r p h y t o t o x i n - p r o d u c i n g s o i l b o r n e f u n g i may y i e l d a d d i t i o n a l m y c o h e r b i c i d e c a n d i d a t e s a s e f f e c t i v e a s G. virens.

Screening for Phytotoxin-producing Soilborne

Fungi

V a r i o u s s o i l b o r n e f u n g i have been r e p o r t e d t o produce p h y t o t o x i n s , some o f w h i c h a r e l i s t e d i n T a b l e I I I . C e r t a i n p h y t o t o x i c compounds a r e known t o h a v e p r o n o u n c e d m y c o t o x i c a c t i v i t y ( e g . moniliformin). We h a v e e x c l u d e d t h o s e f u n g i w h i c h h a v e t h e p o t e n t i a l to c o l o n i z e e d i b l e p a r t s o f crops and produce mycotoxic


15. JONES A HANCOCK

Soilborne Fungifor Biological Control of Weeds

compounds a s t h e y w o u l d o b v i o u s l y b e u n s u i t a b l e f o r a p p l i c a t i o n . V e r y few p h y t o p a t h o g e n i c f u n g i a r e known t o p r o d u c e p h y t o t o x i n s . The m a j o r i t y o f p h y t o t o x i n - p r o d u c i n g s o i l b o r n e f u n g i a r e saprophytes.

Table


Fungus

I I I . Phytotoxin-producing Soilborne

Fungi

Phytotoxin

uncharacterized Actinomycete s p p . desmethoxyviridiol Gliocladium delLquescens viridiol Gliocladium virens phaseolinone Macrophomina phaseolina desmethoxyviridiol Nodulisporium hinnuleum citreoviridin Pénicillium charlesii PC t o x i n Periconia circinata Streptomyces s p . anisomycin h e r b i m y c i n s A,Β Streptomyces saganonensis Streptomyces νiridochromogenes b i a l o p h o s c y c l o c a r b i m i d e A,Β Streptoverticillium sp. p e n t y l pyrones Trichoderma harzianum p e n t y l pyrones Trichoderma viride

Reference

(18,12) (14) (11.12) (24) (21) (26) (22) (28) (2i) (30) (26) (11) (32)

Few s t u d i e s h a v e c e n t e r e d o n t h e i s o l a t i o n o f p h y t o t o x i n producing soilborne fungi. The p h y t o t o x i c a c t i v i t y o f f u n g a l metabolites i s g e n e r a l l y found d u r i n g r o u t i n e screening o f m e t a b o l i t e s f o r o v e r a l l b i o l o g i c a l a c t i v i t y (1Z). Screening of soilborne fungi s p e c i f i c a l l y for the production of phytotoxins s h o u l d y i e l d numerous p r o m i s i n g c a n d i d a t e s f o r u s e a s mycoherbicides. To d a t e , e f f o r t s t o i d e n t i f y p h y t o t o x i n - p r o d u c i n g s t r a i n s h a v e b e e n l a r g e l y l i m i t e d t o Streptomyces a n d Actinomycete

s p p . (18,19).

V a r i o u s methods a r e a v a i l a b l e t o r a p i d l y s c r e e n s o i l b o r n e f u n g i for production of phytotoxic metabolites. One o f t h e s i m p l e s t i n v o l v e s p l a c i n g a f u n g a l c u l t u r e on a n agar p l a t e c o n t a i n i n g seeds o f t h e t a r g e t weed. T h i s i s l i m i t e d t o f u n g a l i s o l a t e s w h i c h do not r a p i d l y colonize the agar p l a t e . Rapid c o l o n i z a t i o n would obscure phytotoxin production. For those fungi o f r e s t r i c t e d c o l o n y s i z e , one r e l i e s o n t h e a b i l i t y o f t h e p h y t o t o x i n t o d i f f u s e from the c o l o n y t o the s u s c e p t i b l e t a r g e t t i s s u e . While t h i s i s a d e q u a t e f o r some c o m p o u n d s , i t may l i m i t t h e d e t e c t i o n o f h y d r o p h o b i c compounds w h i c h d i f f u s e l e s s r e a d i l y t h r o u g h t h e a g a r . An a l t e r n a t i v e t o d i r e c t s c r e e n i n g o f p o t e n t i a l p h y t o t o x i n producing f u n g i i s the t e s t i n g o f crude metabolites produced i n l i q u i d c u l t u r e by the fungi. The m e t a b o l i t e s c a n be p l a c e d i n w e l l s c u t i n t o t h e a g a r n e x t t o t h e t e s t w e e d s e e d o r i t may b e incorporated d i r e c t l y into the agar. T h e m e t a b o l i t e s may a l s o b e a p p l i e d d i r e c t l y t o s e e d s , c a l l i o r s h o o t c u l t u r e s (22) t o t e s t f o r metabolite t o x i c i t y . A l g a e have been used as i n d i c a t o r s o f metabolite phytotoxicity. Chlorella i s often used and i s e f f e c t i v e f o r d e t e c t i o n o f p h o t o s y n t h e t i c i n h i b i t o r s (21)· Chlamydomonas may a l s o prove a u s e f u l a l g a l model f o r d e t e c t i o n o f f u n g a l


283


284


phytotoxins. Chlamydomonas i s s e n s i t i v e t o i n h i b i t o r s o f c e l l d i v i s i o n and enlargement ( 2 2 ) , thus i t c o u l d provide a s e n s i t i v e i n d i c a t o r o f compounds i n h i b i t o r y t o s e e d g e r m i n a t i o n a n d s e e d l i n g growth where r a p i d c e l l d i v i s i o n a n d e n l a r g e m e n t i s o c c u r i n g . I n h i b i t o r s o f seed g e r m i n a t i o n and s e e d l i n g growth would be p a r t i c u l a r i l y desirable a c t i v i t i e s f o r a mycoherbicide as they c o u l d e l i m i n a t e weed c o m p e t i t i o n a t a v e r y e a r l y s t a g e . Cell c u l t u r e s , c a l l u s a n d s h o o t c u l t u r e s may b e u s e d i n m e t a b o l i t e s c r e e n i n g , however most c u l t u r e s a r e c u r r e n t l y d e r i v e d from t o b a c c o a n d i t i s n o t known how d i f f i c u l t i t w o u l d b e t o c u l t u r e c e l l s f r o m v a r i o u s weeds. Another i n t e r e s t i n g source o f t i s s u e f o r p h y t o t o x i c i t y s t u d i e s w o u l d b e Agrobacterium rhizogenes-transformed root c u l t u r e s ( 2 3 ) , as they maintain the c e l l u l a r s t r u c t u r e o f intact roots. F o r b o t h t h e a l g a l a n d t i s s u e c u l t u r e systems i t i s n o t w e l l known i f t h e y a d e q u a t e l y r e p r e s e n t t h e r e s p o n s e o f a c t u a l plants. W h i l e each o f t h e p r e v i o u s l y m e n t i o n e d methods f o r d e t e c t i n g p h y t o t o x i c m e t a b o l i t e s may p r o v e u s e f u l t h e y s h o u l d n o t r e p l a c e t e s t i n g o f t h e a c t u a l seed o r s e e d l i n g o f t h e t a r g e t weed in soil. They may, however, p r o v i d e a u s e f u l system f o r t h e i n i t i a l s c r e e n i n g o f v e r y l a r g e numbers o f f u n g a l i s o l a t e s . Factors Affecting Phytotoxin Production S c r e e n i n g f o r p h y t o t o x i n p r o d u c t i o n s h o u l d be c a r r i e d o u t w i t h as many d i f f e r e n t m e d i a c o m p o s i t i o n s a s p o s s i b l e d u e t o t h e v a r i a b l e l e v e l o f metabolite synthesis on d i f f e r e n t media. The c o m p o s i t i o n of t h e media s i g n i f i c a n t l y a f f e c t s p r o d u c t i o n o f t h e p h y t o t o x i n v i r i d i o l b y G. virens. I n t h i s c a s e , i t was n o t t h e p r e s e n c e o f a s p e c i f i c c a r b o n o r n i t r o g e n source b u t t h e i r r e l a t i v e r a t i o . The pH w a s a l s o c r i t i c a l b e c a u s e v i r i d i o l w a s n o t s t a b l e i n a l k a l i n e solution. Large s c a l e s c r e e n i n g o f l i q u i d media c a n be performed i n disposable microfuge tubes. We h a v e s c r e e n e d n u m e r o u s m e d i a b y i n o c u l a t i n g G. virens c o n i d i a i n t o 1 . 5 m i l l i l i t e r m i c r o f u g e tubes c o n t a i n i n g one m i l l i l i t e r o f medium. Following a five-day i n c u b a t i o n , t h e fungal mycelium i s p e l l e t e d by c e n t r i f u g a t i o n and the supernatant t r a n s f e r r e d t o another tube. Both t h e mycelium and the c u l t u r e f l u i d c a n then be e x t r a c t e d and f r a c t i o n a t e d t o i s o l a t e phytotoxic metabolites. P r o d u c t i o n o f p h y t o t o x i n s s h o u l d be m o n i t o r e d o v e r a n e x t e n d e d t i m e p e r i o d s i n c e m e t a b o l i t e s may b e p r o d u c e d a n d t h e n c o n v e r t e d t o a n o t h e r compound w i t h a d i f f e r e n t l e v e l o r spectrum o f a c t i v i t y . T h i s was t h e c a s e f o r v i r i d i o l production. V i r i d i o l was shown t o b e t h e p r o d u c t o f a C - 3 k e t o r e d u c t i o n o f t h e a n t i b i o t i c compound v i r i d i n , w h i c h i s p r o d u c e d b y G. virens s h o r t l y b e f o r e c o n v e r s i o n t o v i r i d i o l . I t i s p o s s i b l e that a metabolite produced n o t b e p r o d u c e d a t t h e same l e v e l o n a s o l i d f o r m e t a b o l i t e p r o d u c t i o n o n s o l i d medium i s a p p l i c a t i o n o f t h e n u t r i e n t s from t h e l i q u i d s o l i d , p r e f e r a b l y one w h i c h c o u l d be used a s application.

i n liquid culture w i l l substrate. Testing best achieved by medium t o a n i n e r t a carrier for field


15. JONES & HANCOCK


Prospects

Soilborne FungiforBiological Control of Weeds

for Soilborne

Fungi as

Mycoherbicides

To c o m p e t e w i t h t h e c u r r e n t a r s e n a l o f c h e m i c a l h e r b i c i d e s , m y c o h e r b i c i d e s m u s t p r o v i d e a n e f f e c t i v e means f o r c o n t r o l l i n g a b r o a d s p e c t r u m o f weeds, p r e f e r a b l y e a r l y i n weed s e e d l i n g development. Among t h e p h y t o p a t h o g e n i c s o i l b o r n e f u n g i , Fusarium s p p . show t h e g r e a t e s t p r o m i s e , a l t h o u g h t h e i r u s e w i l l r e m a i n l i m i t e d u n l e s s t h e problems p r e v i o u s l y o u t l i n e d c a n be overcome. A m o r e p r o m i s i n g a v e n u e f o r f u t u r e m y c o h e r b i c i d e r e s e a r c h may b e found i n the use of non-phytopathogenic soilborne f u n g i . The n o n p h y t o p a t h o g e n i c f u n g i a c h i e v e weed c o n t r o l t h r o u g h p r o d u c t i o n o f p h y t o t o x i c compounds. P r o d u c t i o n o f t h e s e compounds g e n e r a l l y r e l i e s upon the a v a i l a b i l i t y o f c e r t a i n n u t r i e n t s , thus p h y t o t o x i n s y n t h e s i s c a n be c o n t r o l l e d . P h y t o t o x i c m e t a b o l i t e s o f some s o i l b o r n e f u n g i have a s u f f i c i e n t l y b r o a d spectrum o f a c t i v i t y t o b e e f f e c t i v e i n c o n t r o l o f n u m e r o u s members o f a w e e d c o m m u n i t y . Future progress w i l l b e g i n by s c r e e n i n g a wide range o f s o i l s to i s o l a t e phytotoxin producing f u n g i . S c r e e n i n g methods a r e s i m p l e enough t o accomodate l a r g e s c a l e s c r e e n i n g . Strain s e l e c t i o n a n d i m p r o v e m e n t s h o u l d y i e l d more e f f e c t i v e phytotoxin producers. We h a v e f o u n d t h a t v i r i d i o l p r o d u c t i o n b y G. virens v a r i e d m a n y - f o l d b e t w e e n i s o l a t e s o b t a i n e d f r o m t h e same f i e l d s o i l , t h u s t h e r e i s s t i l l room f o r f u r t h e r enhancement o f t h i s m y c o h e r b i c i d e a f t e r more e x t e n s i v e selections. A f t e r s e l e c t i o n o f o p t i m a l s t r a i n s , f u r t h e r improvements c a n a r i s e through protoplast fusions, genetic crosses or d i r e c t genetic m a n i p u l a t i o n b y gene i n s e r t i o n s . P r o t o p l a s t f u s i o n s may b e t o o unstable f o r general u s e , and genetic crosses are only p o s s i b l e i f the s e x u a l o r teleomorph stage o f the fungus i s a v a i l a b l e . A p p l i c a t i o n o f g e n e t i c e n g i n e e r i n g t e c h n i q u e s may y i e l d t h e b e s t results. While the genetic c o m p o s i t i o n o f s t r a i n s a r i s i n g from p r o t o p l a s t f u s i o n and genetic crosses remain undefined, genetic engineering allows incorporation of defined genetic elements. For t h e m a j o r i t y o f s o i l b o r n e f u n g i , methods f o r d i r e c t g e n e t i c manipulation remain untested. This i s not the case f o r Streptomyces s p p . , i n w h i c h c l o n i n g a n d e x p r e s s i o n t e c h n i q u e s a r e w e l l documented. Gene c l u s t e r s i n v o l v e d i n a n t i b i o t i c s y n t h e s i s and r e s i s t a n c e have been c l o n e d and e x p r e s s e d i n v a r i o u s s p e c i e s o f Streptomyces ( 3 3 , 3 4 ) · I s o l a t i o n o f g e n e c l u s t e r r e s p o n s i b l e f o r p h y t o t o x i n p r o d u c t i o n s h o u l d be no d i f f e r e n t . Multiple phytotoxin s y n t h e s i s g e n e s may b e e x p r e s s e d b y i n s e r t i o n i n t o a s i n g l e species o f Streptomycete t h u s a l l o w i n g a b r o a d e r d e g r e e o f w e e d c o n t r o l . C o n t r o l o f p h y t o t o x i n e x p r e s s i o n might be e n g i n e e r e d by i n s e r t i o n of a promoter responsive to a s p e c i f i c s u b s t r a t e not r e a d i l y a v a i l a b l e i n the s o i l . The a p p l i c a t i o n o f g e n e t i c t e c h n i q u e s t o s t r a i n improvement w i l l r e q u i r e a l o n g - t e r m commitment t o t h e project. For the near term, the p r e v i o u s l y mentioned screening methods s h o u l d p r o v i d e numerous s o i l b o r n e p h y t o t o x i n - p r o d u c i n g f u n g i w h i c h w i l l prove v a l u a b l e as f u t u r e m y c o h e r b i c i d e s a n d be a v a i l a b l e f o r more i m m e d i a t e f i e l d a p p l i c a t i o n .


285

286


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8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.

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