Biochemical Responses of Plants to Pathogens - ACS Publications

Chapter 5

Biochemical Responses of Plants to Pathogens Robert E. Hoagland

Downloaded by UNIV OF ARIZONA on April 10, 2017 | http://pubs.acs.org Publication Date: September 25, 1990 | doi: 10.1021/bk-1990-0439.ch005

Southern Weed Science Laboratory, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 350, Stoneville, MS 38776

Various enzymes have been implicated in host plant resistance to disease. Among the more important of these are chitinase, glucanase, glycosidase, NADPH oxidase, and several enzymes of secondary plant metabolism including phenylalanine ammonia-lyase, peroxidase, and polyphenol oxidase. Studies on enzymatic resistance to pathogen attack of crop species indicate the lack of a universal resistance mechanism(s). That is, specific enzyme activities in various plant species have often been both positively and negatively correlated with infection by various pathogens. There is a great need to examine resistance mechanisms in pathogen-weed situations if the potential manipulation of such interactions for biological control is to be realized. Responses of these enzymes to pathogen attack and the biochemical processes regulated by these enzyme reactions are surveyed. Particular emphasis is on enzymes and constituents of secondary plant metabolism since their involvement in defense has been studied most. Regulation of secondary metabolism by agrochemicals and enzyme inhibitors as related to pathogen infection and recent research on biochemical interactions of some weeds and pathogens are also discussed. P l a n t s defend t h e m s e l v e s from a t t a c k by n e a r l y a l l m i c r o o r g a n i s m s , but some m i c r o o r g a n i s m s ( p l a n t pathogens, I . e . , f u n g i , b a c t e r i a , and v i r u s e s ) have I n f e c t i o n p r o c e s s e s t h a t can evade p l a n t d e f e n s e mechanisms and cause I n f e c t i o n t h a t may l e a d t o p l a n t d e a t h . These pathogens may confound d e f e n s e mechanisms by s u p p r e s s i o n o f t h e mechanism(s) o r by n o t b e i n g r e c o g n i z e d by t h e h o s t p l a n t . Once t h e p l a n t does r e a c t t o I n v a s i o n by a pathogen, s e v e r a l enzyme a c t i v i t i e s a r e u s u a l l y i n c r e a s e d ( m o s t l y enzymes o f secondary p l a n t metabolism) which r e s u l t s i n t h e s y n t h e s i s o f compounds such as p h y t o a l e x i n s , p h e n o l 1 c s , l i g n i n and o t h e r s which a r e known d e f e n s e c h e m i c a l s .

This chapter not subject to U.S. copyright Published 1990 American Chemical Society

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


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MICROBES AND MICROBIAL PRODUCTS AS HERBICIDES

Data from whence knowledge o f t h e s e d e f e n s e mechanisms o r i g i n a t e d have n e a r l y a l l been from s t u d i e s on c r o p p l a n t s and t h e i r r e s p e c t i v e d i s e a s e o r g a n i s m s . There a r e v i r t u a l l y no s t u d i e s o f such mechanisms i n weed-pathogen i n t e r a c t i o n s . T h i s r e p r e s e n t s a s e r i o u s v o i d i n t h e f u l l u t i l i z a t i o n o f b i o l o g i c a l weed c o n t r o l u s i n g b i o h e r b i c i d e s (pathogens o r t h e i r ρhytotoxic p r o d u c t s ) . Because t h e s e d e f e n s e mechanisms d i f f e r w i d e l y depending on p l a n t s p e c i e s and d i s e a s e o r g a n i s m s , and because most p l a n t pathogens have s p e c i f i c o r a v e r y l i m i t e d h o s t r a n g e , t h e r e 1s a need t o examine weed p a t h o g e n - h o s t I n t e r a c t i o n s I n d i v i d u a l l y . Knowledge o f s p e c i f i c weed-pathogen I n t e r a c t i o n s c o u l d p o s s i b l y l e a d t o c h e m i c a l m a n i p u l a t i o n o r c o n t r o l l e d r e g u l a t i o n o f t h e d e f e n s e mechanisms t o I n c r e a s e b i o h e r b i c i d e e f f i c a c y and p r o v i d e b e t t e r weed c o n t r o l . Knowledge o f agrocheraical e f f e c t s on b i o c o n t r o l m i c r o o r g a n i s m s 1s a l s o l a c k i n g . The scope o f t h i s c h a p t e r w i l l be t o examine some o f t h e p l a n t d e f e n s e mechanisms t h a t have been r e p o r t e d i n t h e literature. P a r t i c u l a r emphasis i s p l a c e d on enzymes and p r o d u c t s o f secondary p l a n t m e t a b o l i s m [ i . e . , p h e n y l a l a n i n e ammonia-lyase ( P A L ) , p o l y p h e n y l o x i d a s e (PPO), p e r o x i d a s e (PO) and g e n e r a l and s p e c i f i c p h e n o l i c compounds] and on noted I n s t a n c e s o f c h e m i c a l I n t e r a c t i o n s o f t h e s e enzymes and secondary p l a n t p r o d u c t s w i t h enzyme i n h i b i t o r s , h e r b i c i d e s , and p l a n t growth r e g u l a t o r s . Some Important Enzvmes I m p l i c a t e d i n P l a n t Defense The enzymes most commonly s t u d i e d i n p l a n t d e f e n s e r e a c t i o n s can be d i v i d e d I n t o two major groups ( l y t i c enzymes and enzymes a s s o c i a t e d w i t h p l a n t p h e n o l i c metabolism) and a l e s s e r t h i r d group o f g e n e r a l l y u n r e l a t e d enzymes ( T a b l e Γ ) . D i s c u s s i o n o f some o f t h e s a l i e n t I n f o r m a t i o n r e g a r d i n g t h e r o l e s and a c t i o n o f t h e s e enzymes when p l a n t s a r e i n f e c t e d by pathogens f o l l o w s . I n s t a n c e s where t h e s e enzymes a r e thought n o t t o p l a y r o l e s i n p l a n t d e f e n s e a r e a l s o presented. C h i t i n a s e and 1 . 3 - l - G l u c a n a s e . C h i t l n a s e and l , 3 - / J - g l u c a n a s e a r e l y t i c enzymes t h a t degrade c h i t i n and g l u c a n s , two major components o f f u n g i and f u n g i Imperfect1 c e l l w a l l s . Both enzymes have been i m p l i c a t e d f o r some t i m e as a n t i f u n g a l d e f e n s e p r o t e i n s i n p l a n t s (1-4), a l t h o u g h p a t h o g e n i c f u n g i a l s o produce s i m i l a r enzymes (5). G e n e r a l l y , t h i s I m p l i c a t i o n r e l y s on s e v e r a l p i e c e s o f I n d i r e c t e v i d e n c e : (1) even though c h i t i n a s e has no s u b s t r a t e i n p l a n t s and c a l l o s e ( 1 , 3 - 0 - g l u c a n a s e s u b s t r a t e ) 1s e x t r e m e l y low i n p l a n t s , h i g h a c t i v i t y l e v e l s o f both enzymes commonly o c c u r ( 1 , 1); (2) i s o l a t e d f u n g a l c e l l w a l l s a r e degraded by p u r i f i e d f r a c t i o n s o f t h e s e enzymes (fi-S); (3) pathogen I n f e c t i o n , e l l d t o r s from pathogens, and e t h y l e n e Induce t h e s e enzymes (S» l f l ) ; and (4) a t l e a s t one f u n g a l pathogen produces p r o t e i n a c e o u s i n h i b i t o r s o f 1 , 3 - 0 - g l u c a n a s e (11). F u r t h e r m o r e , p h y s i o l o g i c a l c o n c e n t r a t i o n s o f t h e s e two enzymes a r e i n h i b i t o r y t o t h e growth o f p a t h o g e n i c f u n g i (12)R e c e n t l y c h i t i n a s e and 1 , 3 - 0 - g l u c a n a s e have been shown t o a c t s y n e r g l s t l c a l l y t o I n h i b i t f u n g a l growth (11). When t h e host and pathogen come i n c o n t a c t , each produces l y t i c enzymes as a d e f e n s e mechanism ( F i g u r e 1 ) . P e c t i n a s e and c e l l u l a s e a r e major emzymes o f pathogens t h a t a r e r e l a t e d t o i n f e c t i o n


5. HOAGLAND

Biochemical Responses of Plants to Pathogens

Table I.

89

Some Enzymes o f P l a n t Defense Mechanisms


Reaction L v t i c enzvmes C h i t i n a s e (EC 3 . 2 . 1 . 1 4 )

0 - 1 , 3 Glucanase (EC 3 . 2 . 1 . 3 9 ) Enzymes t h a t m e t a b o l i z e p h e n o l i c s P e r o x i d a s e (PO) (EC 1 . 1 1 . 1 . 7 )

Random h y d r o l y s i s o f 0-1,4acetamido-2-deoxy-D-glucoside l i n k a g e s i n c h i t i n and chitodetrin H y d r o l y s i s o f 1,3-0-D-glucos i d e s i n 1,3-0-D-glucans Donor + H 0 => o x i d i z e d donor + H 0 ; e x . donor: pyrogallol, guaiacol, gallate, o t h e r p h e n o l i c s and some amines 2

2

2

P h e n y l a l a n i n e ammonia-lyase (PAL) (EC 1 . 1 1 . 1 . 7 )

P h e n y l a l a n i n e => i - c i n n a m a t e + NH- "

P o l y p h e n o l o x i d a s e (PPO) (EC 1 . 1 4 . 1 8 . 1 )

L-Dihydroxyphenyl a l a n i n e => "DOPA chrome" (one o f many possible reactions)

0 - g l y c o s i d a s e (EC 3 . 2 . 1 . 2 1 )

p h e n o l i c g l y c o s i d e s =* f u n g i t o x i c aglycones (ex. a r b u t i n hydroquinone)

O t h e r l e s s - s t u d i e d defense enzymes NADPH o x i d a s e "system" Protease

V

20

+

+ NADPH + H =» 2 0 ' + NADP +

2

+

2

H y d r o l y s i s o f amide l i n k a g e s i n p r o t e i n s and p e p t i d e s


90



Phytoalexins Detoxification Glucanases Chitinases Lysozymes •*Oe

\PATHOGEN '

,··»

HOST

Cellulases Pectinases Detoxification Phytotoxins F i g u r e 1. Schematic r e p r e s e n t a t i o n o f l y t i c enzyme a c t i o n i n plant-pathogen I n t e r a c t i o n s . (Redrawn w i t h p e r m i s s i o n from Réf. 1J. C o p y r i g h t 1988 American S o c i e t y P l a n t Physiologists).


5. HOAGLAND

91

Biochemical Responses cfPlants to Pathogens


p r o c e s s e s . Both t h e h o s t and pathogen may a l s o have d e f e n s i v e c o n s t i t u e n t s a g a i n s t each o t h e r ' s enzymes, as i n p l a n t - p r o d u c e d p e c t i n a s e i n h i b i t o r s (14) and f u n g a l - p r o d u c e d 1 , 3 - 0 - g l u c a n a s e i n h i b i t o r s (11)· A n o t h e r analogy i n t h i s m e c h a n i s t i c scheme i s t h e p r o d u c t i o n o f t o x i c c h e m i c a l c o n s t i t u e n t s by t h e h o s t ( p h y t o a l e x i n s ) and pathogen ( p h y t o t o x i n s ) . C h l t l n a s e (15) and 1 , 3 - 0 - g l u c a n a s e (IS) can cause ( p h y t o a l e x l n ) e l i c i t o r r e l e a s e from c e l l w a l l s o f fungi. P e r o x i d a s e ( P 0 1 . P e r o x i d a s e (PO) 1s found 1n both p l a n t s and m i c r o o r g a n i s m s . A l t h o u g h t h e p h y s i o l o i g c a l r o l e o f t h e enzyme i s n o t completely understood, i t c a t a l y z e s several r e a c t i o n s , Including p o l y m e r i z a t i o n o f h y d r o x y d n n a m y l a l c o h o l s which l e a d s t o l i g n i n f o r m a t i o n ; s u b e r i z a t i o n ; o x i d a t i o n o f many mono- and d i p h e n o l s t o t o x i c q u i n o n e s ; and o x i d a t i o n o f 1ndo1eacet1c a d d (IAA) (1Z). PO has been s t u d i e d w i t h r e g a r d t o d i s e a s e r e s i s t a n c e 1n many p l a n t s ( T a b l e I I ) and t h e enzyme I t s e l f 1s t o x i c t o some m i c r o o r g a n i s m s (26)· High I n h e r e n t PO a c t i v i t y i n a p i c a l l e a v e s and r o o t t i p s o f p o t a t o p l a n t s was s t r o n g l y c o r r e l a t e d w i t h r e s i s t a n c e t o P h v t o o h t h o r a i n f e s t a n s ( I S ) , whereas o t h e r l e a v e s , t u b e r p e e l s , and p u l p had low PO a c t i v i t y and were s u s c e p t i b l e ( I S ) . T h i s c o r r e l a t i o n o f r e s i s t a n c e and h i g h PO a c t i v i t y was extended t o e x p l a i n d i f f e r e n c e s 1n s u s c e p t i b i l i t y o f potato v a r i e t i e s . Table I I .

Selected Instances o f Peroxidase A c t i v i t y R e l a t i v e to Disease Resistance

D i s e a s e Organism

Host

Reference

Peroxidase Involved i n Disease Resistance PhYtQPhtPra I n f e s t a n s L. Dhaseolicola CeratPÇYStiS f l m b r i a t a Pseudomonas t a b a c i PlJÇÇlnU qramlnlS f . s p . t r i t i c i TMY PIpTptiU Q o s s v o l n a Xanthpwnas p h a s e o i i v a r . SQ.1ens1S Rididoporus Honosus

potato bean sweet p o t a t o tobacco wheat tobacco cotton

I I , 18 12 2S 21, 22

soybean rubber t r e e

21 21

21 2i 21

P e r o x i d a s e Not I n v o l v e d 1n D i s e a s e R e s i s t a n c e HelniinthQSPQrlMfl carbonum PMCÇIM* araminis

trltlc

CMV Fusarlum PseildPTOnilS solanacearum Pseudpfnpnas syrinqae p v . tpnatp

maize wheat cucumber tomato tobacco

21

tomato

11

22, I f i 21 12 21



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M I C R O B E S A N D MICROBIAL PRODUCTS AS HERBICIDES

C o t t o n b o l l s o f I n t e r m e d i a t e age were more r e s i s t a n t t o D i p l o d i a g o s s v p i n a than young o r o l d b o l l s (25). PO a c t i v i t y was h i g h e r i n t h e s e i n t e r m e d i a t e b o l l s and PO i n c r e a s e d s e v e r a l f o l d when r e s i s t a n t b o l l s were i n o c u l a t e d w i t h t h i s pathogen. More r e c e n t l y , PO was shown t o be i n v o l v e d i n r o o t - r o t ( R i a i d o p o r u s l i o n o s u s and P h e l l i n u s noxium) i n f e c t i o n 1n r u b b e r t r e e s (Hevea b r a s i l i e n s i s ) (2Ζ)· PO a c t i v i t y I n c r e a s e d more r a p i d i l y i n r e s i s t a n t cucumber h y p o c o t y l s t h a n i n s u s c e p t i b l e t i s s u e when I n o c u l a t e d w i t h t h e pathogen C l a d o s p o r l u m cucumerinum E l l 1s & A r t h u r (25). PO Isozyme p a t t e r n s i n I n f e c t e d r e s i s t a n t t i s s u e were s 1 m i l i a r t o wound-Induced isozyme p a t t e r n s . S i m i l a r t y p e s o f s t u d i e s have suggested a d i r e c t r o l e o f PO i n d i s e a s e r e s i s t a n c e . However, some h o s t - p a t h o g e n s t u d i e s I n d i c a t e t h a t PO i s not I n v o l v e d i n h o s t d e f e n s e ( T a b l e I I ) . PO a c t i v i t y was i n c r e a s e d i n both r e s i s t a n t and s u s c e p t i b l e maize p l a n t s I n o c u l a t e d w i t h H e l m i n t h o s p o r l u m carbonum. but t h e I n c r e a s e was g r e a t e r i n s u s c e p t i b l e p l a n t s (28)· Recent s t u d i e s w i t h Pseudomonas solanacearum I n f e c t i o n o f t o b a c c o have c o n c l u d e d t h a t PO I n c r e a s e s a r e not I n v o l v e d i n Induced r e s i s t a n c e (22). P h e n y l a l a n i n e ammonia-lvase ( P A L I . The c o n v e r s i o n o f L - p h e n y l a l a n i n e t o i - c i n n a m a t e and ammonia by PAL r e p r e s e n t s a key branch p o i n t from p r i m a r y t o secondary p l a n t m e t a b o l i s m ( F i g u r e 2 ) . PAL, t h e r e f o r e , i s p i v o t a l f o r r e g u l a t i o n of the s y n t h e s i s o f v a r i o u s p h e n o l i c compounds, i n c l u d i n g f l a v o n o i d s , h y d r o x y c i n n a m a t e s , e t c . , some o f which a r e p h y t o a l e x i n s . The enzyme has wide d i s t r i b u t i o n i n p l a n t s (and some m i c r o o r g a n i s m s ) and I t s a c t i v i t y i n p l a n t s 1s I n c r e a s e d by exposure t o v a r i o u s f a c t o r s , I n c l u d i n g l i g h t , wounding, t e m p e r a t u r e changes, and I n f e c t i o n by pathogens. S t u d i e s on I t s p r o p e r t i e s and b e h a v i o r have been reviewed (25, 2Z). In a v a r i e t y o f p l a n t s , t h e r e 1s o f t e n an I n c r e a s e i n PAL a c t i v i t y a s s o c i a t e d w i t h I n c r e a s e d p r o d u c t i o n o f s p e c i f i c p h e n o l i c compounds e a r l y a f t e r I n o c u l a t i o n and I n f e c t i o n as summarized i n r e v i e w s (28, 23)· PAL a c t i v i t y l e v e l s were I n c r e a s e d s e v e r a l f o l d In r e s i s t a n t v s . s u s c e p t i b l e p o t a t o t u b e r t i s s u e a f t e r i n o c u l a t i o n w i t h Phytoohthora i n f e s t a n s . i n d i c a t i n g a p o s i t i v e d e f e n s e mechanism ( 4 0 ) . Fusarium s o l a n i I n f e c t i o n caused s i m i l a r PAL i n c r e a s e s (41)· PAL i n r e s i s t a n t l u c e r n e i n c r e a s e d r a p i d l y i n response t o i n f e c t i o n by V e r t i c i l ! i u m a l b o - a t r a m . but not in a susceptible variety (42). PAL i n d u c t i o n and p h y t o a l e x i n a c c u m u l a t i o n i s common t o a wide range o f h o s t - p a r a s i t e I n t e r a c t i o n s when p l a n t s a r e t r e a t e d w i t h e l i c i t o r s (42). Studies with several plant c e l l cultures also i n d i c a t e d t h a t PAL a c t i v i t i e s a r e r e a d i l y I n c r e a s e d by e l i c i t o r s

(44)·

O t h e r s t u d i e s do not show a s t r o n g c o r r e l a t i o n o f I n c r e a s e s i n PAL a c t i v i t y and d i s e a s e r e s i s t a n c e . For example, PAL l e v e l s i n c r e a s e d i n both s u s c e p t i b l e and r e s i s t a n t h o s t s d u r i n g d i s e a s e i n t e r a c t i o n s (45-4Z). In o t h e r i n s t a n c e s , PAL a c t i v i t y was s t a b l e a f t e r i n f e c t i o n , even though p h e n o l i c s have been shown t o be i n v o l v e d i n h o s t r e s p o n s e (48, 42). PAL a c t i v i t y I n c r e a s e s a r e a l s o not c o r r e l a t e d w i t h r e s i s t a n c e i n o t h e r s t u d i e s (50-52)· Additionally, PAL a c t i v i t y was shown t o i n c r e a s e i n s u s c e p t i b l e maize t i s s u e t r e a t e d w i t h H e l m i n t h o r s p o r i u m m a v d i s . but not i n r e s i s t a n t maize t r e a t e d w i t h H. mavdis o r 11. carbonum (51). Few s t u d i e s examining enzyme responses i n h o s t d e f e n s e mechanisms have been a p p l i e d t o b i o l o g i c a l c o n t r o l o f weeds w i t h p l a n t


5. HOAGLAND

Biochemical Responses cfPlants to Pathogens

Phosphoenolpyruvate + — Erythrose-4-P

Dehydroshikimate

Shikimate


Gallate Prephenate

/

«4— 5-Enolpyruvylshikimate-3-P

Shikimate-3-P

\

Tyrosine

Phenylalanine

1

I

p-Coumarate

Cinnamate — • o-Coumarate

» Coumarins

Flavonoids Isoflavonoids Procyanidins

Caffeate Ferulate Sinapate

Flavones

/ \ Ar-CH«CH-COSCoA

Styrylpyrones

CO,

Lignans

Ar-CH*CH-CH OH 2

/ Lignans

ι

\

I

Lign

Ar-COSCoA

/

me

Acylation of alkaloids, polyphenols, etc.

C C glycosides, esters, etc. 6

Neoflavonoids F i g u r e 2. B i o s y n t h e t i c pathway o f s h i k i m a t e - and cinnamate-produced p h e n o l i c compounds i n p l a n t s .


94



p a t h o g e n s . R e c e n t l y , however, phenyl propanoic) m e t a b o l i s m has been I m p l i c a t e d i n a weed-pathogen I n t e r a c t i o n [ I . e . ; s i c k l e p o d ( C a s s i a o b t u s i f o H a i and t h e f u n g a l pathogen A l t e r n a r l a c a s s l a e l (54). PAL a c t i v i t y was I n c r e a s e d about 3 - f o l d i n weed s e e d l i n g s c h a l l e n g e d by s p o r e s o f t h i s pathogen ( F i g u r e 3 ) . Enzyme a c t i v i t y I n c r e a s e s g e n e r a l l y preceeded I n c r e a s e d s o l u b l e p h e n o l i c compound p r o d u c t i o n and t h e appearance o f n e c r o t i c l e s i o n s . However, i n u n d a t l v e spore l e v e l s n e c e s s a r y t o k i l l p l a n t s overwhelmed any d e f e n s e r e s p o n s e mediated by PAL o r o t h e r enzymes. P o l y p h e n o l o x i d a s e (PPO). I t has been suggested t h a t PPO p l a y s a r o l e i n d i s e a s e r e s i s t a n c e due t o I t s o x i d a t i o n o f v a r i o u s p h e n o l i c compounds I n t o t o x i c quinones (55). A l t h o u g h PPO a c t i v i t y l e v e l s d i d not d i f f e r 1n l e a v e s o f h e a l t h y a p p l e v a r i e t i e s w i t h a range o f r e s i s t a n c e t o a p p l e s c a b , t h e r e was a s i g n i f i c a n t I n c r e a s e i n a c t i v i t y o f r e s i s t a n t v s . s u s c e p t i b l e v a r i e t i e s when c h a l l e n g e d by t h e d i s e a s e o r g a n i s m (55). PPO was a l s o I n h i b i t o r y t o v i r a l I n f e c t i o n o f cowpea (5Z). PPO a c t i v i t y was h i g h e r i n r e s i s t a n t v s . s u s c e p t i b l e tomato c u l t i v a r s and i n mature v s . young tomato l e a v e s (24)· M e c h a n i c a l I n j u r y I n d u c t i o n o f PPO I n c r e a s e d pathogen r e s i s t a n c e , and r e s i s t a n c e and PPO l e v e l s were d i r e c t l y c o r r e l a t e d i n 20 tomato c u l t i v a r s w i t h a wide range o f r e s i s t a n c e and susceptibility. PPO has been shown t o be Important f o r s y n t h e s i s o f d i h y d r o x y p h e n o l s i n p l a s t i d s (58). However, r e c e n t s t u d i e s I n d i c a t e t h a t PPO 1s n o t I n v o l v e d i n t h e fi-hydroxylation o f p h e n o l i c compounds i n mungbean ( V i a n a radiâta) (52). Some s t u d i e s o f PPO ( c a t e c h a l o x i d a s e ) g e n e r a l l y suggest t h e enzyme I n c r e a s e s a f t e r I n f e c t i o n by v i r u s , b a c t e r i a , o r f u n g i (££). A l t h o u g h PPO may i n some I n s t a n c e s be I n v o l v e d i n p l a n t d e f e n s e , i n many c a s e s t h e r e 1s Inadequate e v i d e n c e t o prove a s i g n i f i c a n t r o l e f o r PPO i n such mechanisms (6S). θ-Glvcosldases. One way p l a n t s a r e p r o t e c t e d from a u t o t o x l c p h e n o l i c l e v e l s 1s by f o r m a t i o n o f p h e n o l i c g l y c o s i d e s . The g l y c o s i d e s a r e u s u a l l y I n a c t i v e and a r e c o m p a r t m e n t a l i z e d , but membrane damage and I n f e c t i o n cause r e l e a s e and h y d r o l y s i s . Host g l y c o s i d a s e s , i f induced by e l i c i t o r s , can r e l e a s e f u n g i t o x i c p h e n o l i c s at the onset o f i n f e c t i o n . /J-Glycosidase i n apple t r e e s has been shown t o be r e s p o n s i b l e f o r r e s i s t a n c e t o a p p l e scab d i s e a s e (51). The enzyme c o n v e r t s p h l o r i d z i n ( n o n - t o x i c g l y c o s i d e ) to phloretin (toxic agiycone). In pear t r e e s , a r b u t i n 1s a major g l y c o s i d e and h y d r o l y s i s by / J - g l y c o s i d a s e y i e l d s t h e a g l y c o n e h y d r o q u l n o n e , which 1s v e r y t o x i c t o E r w i n i a a m v l o v o r a . t h e pathogen c a u s i n g f i r e b l i g h t (52). Tree p a r t s most s u s c e p t i b l e t o t h i s o r g a n i s m had low enzyme l e v e l s , whereas s i t e s o f h i g h enzyme a c t i v i t y were r e s i s t a n t . Oat l e a v e s c o n t a i n a v e n a c o s i d e s A and B, which a r e I n a c t i v e s a p o n i n s t h a t a r e h y d r o l y z e d a f t e r wounding by a 0 - g l u c o s i d a s e s p e c i f i c f o r g l u c o s e a t carbon 26 (52-55). A c t i v a t i o n r e q u i r e s o n l y minutes a n d , t h u s , t h i s 1s an e f f e c t i v e d e f e n s e mechanism. O t h e r s t u d i e s I n d i c a t e t h a t m i c r o b i a l g l y c o s i d a s e s can a l s o c a t a l y z e r e l e a s e o f a n t i f u n g a l p r o d u c t s from c y a n o g e n i c g l y c o s i d e s (66)· S t e r o l s i n f u n g a l c e l l membranes a r e r e c e p t o r s f o r t h e t o x i c a g l y c o n e s (6Z)> and g l y c o s i d a s e i n h i b i t o r s such as 1 , 5 - a l d o n o l a c t o n e s reduce f u n g a l membrane damage (58).



5. HOAGLAND

Biochemical Responses of PlantstoPathogens

Sicklepod infected w/ A cassia*

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Time After T r e a t m e n t Figure 3.

56 (h)

E x t r a c t a b l e PAL a c t i v i t y ( s p e c i f i c a c t i v i t y , u n i t s / m g p r o t e i n ) from s i c k l e pod s e e d l i n g s w i t h ( · ) and w i t h o u t ( O ) A l t e r n a r l a casslae spores. (Reprinted with p e r m i s s i o n from R e f . 54. C o p y r i g h t 1990 Paul Parey Scientific Publications).


95

96



Some p a t h o g e n i c f u n g i a r e r e s i s t a n t t o s a p o n i n s because f u n g a l g l y c o s i d a s e s a r e s e c r e t e d o r a r e c e l l w a l l - b o u n d r a t h e r than membrane-bound. T h i s causes g l y c o s i d e h y d r o l y s i s t o o c c u r a t a d i s t a n c e from s e n s i t i v e membranes and, t h u s , a g l y c o n e s do n o t r e a c h membranes due t o l a c k o f s o l u b i l i t y and t r a n s p o r t . A l t e r n a r l a s o l an1 p o s s e s s e s t h i s system and I t s g l y c o s i d a s e h y d r o l y z e s t o m a t i n e t o t o m a t i d i n e w i t h o u t harm t o t h e fungus (63). NADPH o x i d a s e . T h i s d e f e n s e mechanism has n o t r e c e i v e d much a t t e n t i o n , b u t has been i m p l i c a t e d i n some h o s t - p a t h o g e n i n t e r a c t i o n s . NADPH o x i d a s e i n p o t a t o t u b e r t i s s u e was shown t o be a c t i v a t e d Immediately a f t e r I n v a s i o n by an I n c o m p a t i b l e r a c e o f Phvtophthora I n f e s t a n s . causing simultaneous superoxide p r o d u c t i o n , h y p e r s e n s i t i v e c e l l d e a t h , and p h y t o a l e x i n p r o d u c t i o n (Ζ8-Ζ2)· This enzyme system was n o t a c t i v a t e d by a c o m p a t i b l e £ . I n f e s t a n s r a c e (Zl). T h i s enzyme was a c t i v a t e d i n p o t a t o l e a v e s by both c o m p a t i b l e and I n c o m p a t i b l e r a c e s b e f o r e p e n e t r a t i o n , b u t s u p e r o x i d e was o n l y I n c r e a s e d when p e n e t r a t i o n o c c u r r e d by I n c o m p a t i b l e r a c e s ( Z I ) . A w a t e r - s o l u b l e g l u c a n , found t o be a h y p e r s e n s i t i v i t y - i n h i b i t i n g f a c t o r from c o m p a t i b l e r a c e s , p r e v e n t e d a c t i v a t i o n o f t h e superoxide-generating reaction i n the protoplast (Zl). Digitonin c o u l d a c t i v a t e t h e s u p e r o x i d e - g e n e r a t i n g enzyme system which might enhance r e s i s t a n c e t o £ . i n f e s t a n s (Z4). O t h e r r e s e a r c h w i l l be n e c e s s a r y t o e v a l u a t e t h i s enzyme system as a h o s t d e f e n s e mechanism i n o t h e r h o s t and pathogen s p e c i e s . Proteases. P r o t e o l y t i c enzyme a c t i v i t y ( h y d r o l y s i s o f amide bonds o f p r o t e i n s and p e p t i d e s ) has r e c e i v e d v e r y l i t t l e s t u d y r e l a t e d t o d i s e a s e r e s i s t a n c e . P r o t e o l y t i c a c t i v i t y 1s w i d e s p r e a d i n both h o s t s and p a t h o g e n s . A h i g h c o r r e l a t i o n was found w i t h tomato c u l t i v a r s i n f e c t e d by Pseudomonas s v r i n a e p v . tomato and d i s e a s e s e v e r i t y [15). O t h e r s t u d i e s a l s o suggest p r o t e o l y t i c a c t i v i t y c o r r e l a t i o n s w i t h d i s e a s e (Z6. Z Z ) . E t h y l e n e . E t h y l e n e i s a u b i q u i t o u s p l a n t hormone t h a t r e g u l a t e s many p l a n t growth r e s p o n s e s and t h e a c t i v i t y o f many enzymes (18). Some r e s p o n s e s t o e t h y l e n e o f enzymes I m p l i c a t e d i n d e f e n s e a g a i n s t pathogens I n c l u d e , I n c r e a s e d PAL a c t i v i t y (Z9) and I n c r e a s e d PO (88) and 1 , 3 - 0 - g l u c a n a s e (81). Some p l a n t s enhance e t h y l e n e l e v e l s i n r e s p o n s e t o pathogen a t t a c k (82-84)· Exogenous e t h y l e n e can t r i g g e r e l e v a t e d b i o c h e m i c a l d e f e n s e r e a c t i o n s a g a i n s t v a r i o u s pathogens (86)· T h i s e v i d e n c e l e a d t o t h e h y p o t h e s i s t h a t endogenous s t r e s s - p r o d u c e d e t h y l e n e may be a s i g n a l f o r t h e p l a n t t o a c t i v a t e d e f e n s e systems (S2, 82)· R e c e n t l y , e t h y l e n e I n c r e a s e s i n tomato p l a n t s I n f e c t e d by P h v t o p h t h o r a i n f e s t a n s d u r i n g t h e h y p e r s e n s i t i v e r e s p o n s e have been a t t r i b u t e d p r i m a r i l y t o e l e v a t e d ACC ( 1 - a m i n o c l y c l o p r o p a n e - l - c a r b o x c y l i c a c i d ) s y n t h a s e a c t i v i t y (86)· O t h e r s t u d i e s u s i n g a m i n o e t h o x y v i n y l g l y c i n e (AVG) t o s u p p r e s s e t h y l e n e p r o d u c t i o n i n e l i c i t o r - t r e a t e d soybean ( G l y c i n e max L . ) t i s s u e and i n i n f e c t e d c a n t a l o u p e (Cucumis m e l o l s e e d l i n g s (84) d i d not reduce p h y t o a l e x i n p r o d u c t i o n and o n l y m a r g i n a l l y reduced l e v e l s o f a d e f e n s e g l y c o p r o t e i n , r e s p e c t i v e l y . These l a t t e r r e s u l t s tend t o suggest t h a t endogenous s t r e s s e t h y l e n e p l a y s o n l y a minor r o l e i n p l a n t b i o c h e m i c a l d e f e n s e ; however, exogenous e t h y l e n e had l i t t l e o r no e f f e c t on t h e s e two d e f e n s e r e a c t i o n s . Exogenous e t h y l e n e markedly i n c r e a s e s both c h i t i n a s e and 1 , 3 - 0 - g l u c a n a s e i n some


5. HOAGLAND

Biochemical Responses of Plants to Pathogens

97


p l a n t s (1). O v e r a l l , ethylene l e v e l s are e l e v a t e d i n i n f e c t e d p l a n t s , c a u s i n g e l e v a t e d d e f e n s e enzyme a c t i v i t i e s . T h i s l e a d s t o i n c r e a s e d p r o d u c t i o n o f phenol l e s , p h y t o a l e x i n s , and g l y c o p r o t e i n s which can p r o v i d e r e s i s t a n c e i n some c a s e s (SZ). Problems i n A s s e s s i n g Defense F a c t o r s . Some c a u t i o n s h o u l d be e x e r c i s e d i n a s s e s s i n g some o f t h e s e o v e r a l l r e s u l t s p e r t a i n i n g t o t h e i n v o l v e m e n t o f enzymes i n r e s i s t a n c e mechanisms. Resistance e x p r e s s i o n o f p l a n t s a f t e r pathogen a t t a c k i s a v e r y complex p r o c e s s i n v o l v i n g many c l o s e l y i n t e g r a t e d and r e g u l a t e d f a c t o r s . Many h o s t enzyme s t u d i e s i n v o l v i n g pathogen e f f e c t s e i t h e r were not a t t e m p t i n g t o prove t h e r o l e o f a p a r t i c u l a r enzyme i n d e f e n s e a g a i n s t a pathogen, o r were not p r o p e r l y d e s i g n e d t o a c c o m p l i s h t h a t e n d . F u r t h e r m o r e , t h e d a t a may be f u r t h e r confounded by compartimenta i i z a t i o n ( s u b c e l l u l a r o r p l a n t organ) o f t h e enzyme o r i t s p r o d u c t ( s ) , enzyme I s o l a t i o n and e x t r a c t i o n t e c h n i q u e s , and t h e f a c t t h a t many pathogens p o s s e s s t h e same enzyme systems i m p l i c a t e d i n r e s i s t a n c e . Compounds ( e s p e c i a l l y p h e n o l i c s ) r e l e a s e d by pathogen a c t i o n and by homogenation and e x t r a c t i o n p r o c e d u r e s can a l t e r enzymes a c t i v i t i e s (88-28). Inoculum s i z e and e n v i r o n m e n t a l f a c t o r s a l s o i n f l u e n c e t h e dynamics o f i n f e c t i o n which c a n , i n t u r n , i n f l u e n c e defense response. Isozymes o f t h e s e enzymes may a l s o be numerous [as f o r PO (21)] and may be d i f f e r e n t i a l l y i n d u c e d , not a f f e c t e d by pathogen a c t i o n (22), o r i d e n t i c a l i n r e s i s t a n t and s u s c e p t i b l e c u l t i v a r s . S i n c e v i r u s e s do not have PO and PPO a c t i v i t y , t h e s e pathogens may be u s e f u l t o e v a l u a t e t h e r o l e s o f t h o s e enzymes i n p r i m a r y i n f e c t i o n p r o c e s s e s (21). Enzvme I n h i b i t o r s t o M a n i p u l a t e

Infection

C o n t r o l o f enzymes r e l a t e d t o p l a n t d e f e n s e a g a i n s t pathogens c o u l d be e f f e c t i v e i n r e g u l a t i n g p l a n t d i s e a s e . The use o f s p e c i f i c o r n o n - s p e c i f i c enzyme i n h i b i t o r s ( o r a c t i v a t o r s ) c o u l d r e s u l t i n r e g u l a t e d d i s e a s e i n i t i a t i o n and development t h a t c o u l d promote o r s y n e r g i z e t h e e f f e c t s o f m i c r o b i a l pathogens f o r b i o l o g i c a l c o n t r o l o f weeds. A l t h o u g h some n a t u r a l l y o c c u r r i n g and s y n t h e t i c enzyme r e g u l a t o r s are known, r e l a t i v e l y few s t u d i e s have examined t h e i r p o t e n t i a l f o r d i s e a s e r e g u l a t i o n . Of t h e s e s t u d i e s , most have f o c u s e d on c r o p - p a t h o g e n i n t e r a c t i o n s . I n h i b i t i o n o f PPO. PO. c h i t i n a s e . a l u c a n a s e . and fl-alucosidase. Few s y n t h e t i c o r n a t u r a l I n h i b i t o r s o f t h e s e enzymes have been e l u c i d a t e d . Some n a t u r a l p r o d u c t s such as c u r c u r b i t a c i n I and D can r e p r e s s i n d u c t i o n o f l a c c a s e by t h e p a t h o g e n i c fungus o f cucumber, B o t r v t i s c i n e r e a (84). I t i s not known how t h i s compound i s i n v o l v e d i n r e s i s t a n c e o r i f t h e s e compounds a r e a c t i v e i n h i b i t o r s t h a t c o u l d reduce d e f e n s e i n p l a n t s p e c i e s by c a u s i n g PPO o r PO i n h i b i t i o n . O x a l a t e s a r e n a t u r a l p r o d u c t i n h i b i t o r s o f c h l o r o p l a s t i c PPO (25) and 4 - c h l o r o r e s o r c i n o l has a l s o been used as a PPO i n h i b i t o r (25) in a d v e n t i t i o u s r o o t promotion s t u d i e s (2Z). The s y n t h e t i c compound, d i e t h y l d i t h i o c a r b a m a t e i s a w e l l known PPO i n h i b i t o r (28) t h a t might be e f f e c t i v e i n b l o c k i n g PPO a c t i v i t y i n c a s e s where PPO i s i n v o l v e d i n d e f e n s e . G l y p h o s a t e [ { H p h o s p h o n o m e t h y l ) g l y c i n e ] caused minor e l e v a t i o n o f PPO a c t i v i t y i n p l a n t t i s s u e s (22, 1001. but i t i s not known i f t h i s has p o t e n t i a l t o i n c r e a s e t h e d e f e n s e o f p l a n t s t o



98

MICROBES A N D MICROBIAL PRODUCTS AS HERBICIDES

d i s e a s e . D i r e c t e f f e c t s o f g l y p h o s a t e on pathogens w i t h p o t e n t i a l f o r weed c o n t r o l would be Important t o d e t e r m i n e . Perhaps one o f t h e most I n t e r e s t i n g PPO i n h i b i t o r s 1s t e n t o x i n , a f u n g a l p h y t o t o x i n produced by t h e pathogen A l t e m a H a a l t e r n a t a . which can t o t a l l y e l i m i n a t e PPO a c t i v i t y i n some p l a n t s p e c i e s (lffll) w h i l e e x h i b i t i n g no e f f e c t on fl-hydroxy1 a t e d p h e n o l i c l e v e l s ( 1 0 2 ) . T h i s p h y t o t o x i n does n o t a l t e r i n v i t r o PPO a c t i v i t y ( l u i ) . P h y t o t o x i c i t y caused by t e n t o x i n 1s low even though PPO 1s I n h i b i t e d and c h l o r o s i s 1s Induced w i t h l i t t l e o r no e f f e c t on p l a n t morphogenesis ( 1 0 2 ) . PPO o x i d a t i o n o f p h e n o l i c s can p l a y a r o l e i n p l a n t r e s i s t a n c e t o pathogens (1041. The use o f PPO I n h i b i t o r s t o s t u d y weed-pathogen I n t e r a c t i o n s c o u l d p r o v i d e u s e f u l I n f o r m a t i o n f o r b i o l o g i c a l weed c o n t r o l and f o r d e t e r m i n i n g t h e r o l e o f PPO 1η weed-pathogen I n t e r a c t i o n s . I n h i b i t i o n o f p l a n t c h i t i n a s e s and/or glucanases without I n h i b i t i o n o f s i m i l a r enzymes i n f u n g a l weed pathogens c o u l d d r a m a t i c a l l y reduce p l a n t r e s i s t a n c e t o p a t h o g e n s . R e c e n t l y a s t r e p t o r a y c e t e a n t i b i o t i c , a l l o s a m i d i n , was found t o be a p o t e n t c o m p e t i t i v e I n h i b i t o r o f c h i t i n a s e from Candida a l b i c a n s , a human f u n g a l pathogen ( 1 0 5 ) . The i n s e c t i d d a l p r o p e r t i e s o f t h i s a n t i b i o t i c may a l s o a r i s e from I t s p o t e n t I n h i b i t i o n o f I n s e c t chitinase. Plumbagin, an a n a l o g o f j u g l o n e , 1s r e p o r t e d t o be a c h i t i n a s e I n h i b i t o r ( 1 0 6 . 1 0 7 ) . These compounds have a p p a r e n t l y n o t been t e s t e d i n p l a n t s . V a r i o u s p h e n o l i c compounds have been observed t o I n h i b i t p e r o x i d a s e - c a t a l y z e d IAA o x i d a t i o n (1081 and 0 - g l u c o s i d a s e ( 1 0 2 ) . However, no s y n t h e t i c o r s p e c i f i c I n h i b i t o r s f o r t h e s e enzymes a r e a v a i l a b l e t h a t c o u l d be used t o reduce weed r e s i s t a n c e t o p l a n t pathogens. I n h i b i t i o n o f PAL. PAL has been w i d e l y s t u d i e d , and many o f t h e s e I n v e s t i g a t i o n s have c e n t e r e d on t h e r e g u l a t i o n o f PAL a c t i v i t y by natural or synthetic I n h i b i t o r s . Naturally-occurring phenolic compounds such as i - c i n n a m a t e , g-coumarate, and o t h e r PAL r e g u l a t o r s a c t v i a feedback I n h i b i t i o n ( 1 1 0 ) . D - P h e n y l a l a n i n e and β - f l u o r ο p h e n y l a l a n i n e were a l s o d i s c o v e r e d i n e a r l y r e s e a r c h t o i n h i b i t PAL In v i t r o ( 1 1 1 ) . Some o t h e r i n h i b i t o r s u s e f u l i n s t u d y i n g secondary m e t a b o l i s m v i a PAL i n h i b i t i o n have been found ( F i g u r e 4 ) . Some o f t h e compounds d e p i c t e d h e r e , as w e l l as many o t h e r s w i t h v a r y i n g I n h i b i t o r y a c t i v i t y , have been used o r d i s c o v e r e d i n A m r h e i n ' s l a b o r a t o r y ( 1 1 2 ) . Aminooxyacetate (Α0Α) was used as a t r a n s a m i n a s e I n h i b i t o r i n t h e development o f a PAL a s s a y i n I n t a c t c e l l s and was found t o be a weak I n h i b i t o r o f PAL ( 1 1 3 ) . Α0Α I n h i b i t i o n o f PAL and v a r i o u s t r a n s a m i n a s e s r e s u l t s i n p h y t o t o x i c i t y t o many p l a n t s ; hence, i t i s n o t a v e r y u s e f u l In v i v o i n h i b i t o r i n some c a s e s due t o l a c k o f s p e c i f i c i t y r e s u l t i n g i n pronounced p h y t o t o x l c e f f e c t s ( 1 1 4 ) . In f a c t , Α0Α has been r e g i s t e r e d f o r use as a h e r b i c i d e ( 1 1 5 ) . An a n a l o g o f Α0Α, L-«-am1nooxy-0-phenylprop1on1c a d d (Α0ΡΡ), was found t o be a v e r y p o t e n t and more s p e c i f i c I n h i b i t o r t h a n AOA and c o u l d be used in v i v o w i t h o u t d e l e t e r i o u s e f f e c t s on p l a n t growth (US, 1 1 7 ) . 0-Benzylhydroxylam1ne was a l s o found t o be an e f f e c t i v e PAL i n h i b i t o r ( 1 1 8 ) . A n o t h e r i n h i b i t o r o f PAL, 3 - ( l , 4 - c y c l o h e x a d i e n y l ) - L - a l a n i n e , has a l s o been r i g o r o u s l y s t u d i e d ( 1 1 9 ) . One o f t h e newest and perhaps most u s e f u l PAL i n h i b i t o r s i s ( l - a m i n o - 2 p h e n y l e t h y l ) phosphonic a c i d (APEP) (120) which has potency about



5. HOAGLAND

BiochemkalResponsaofPlantstoPathogtJis

99

equal t o AOPP, but a K* which 1s 10* t i m e s h i g h e r than t h e K4 o f AOPP. Some PAL I n h i b i t o r s ( e s p e c i a l l y AOA and AOPP) have been used t o t e s t t h e r o l e o f PAL 1n d i s e a s e r e s i s t a n c e . These PAL I n h i b i t o r s d i d not b l o c k r e s i s t a n c e e x p r e s s i o n o r g l y c e o l l i n a c c u m u l a t i o n i n soybean t i s s u e t r e a t e d w i t h an I n c o m p a t i b l e r a c e o f P h v t o p h t h o r a meqasoerma f . s p . a l v c i n e a (121. 122). AOA and AOPP reduced p h e n y l p r o p a n o i d f l u x by I n h i b i t i n g PAL i n v i v o , and d e c r e a s e d l o c a l i z a t i o n o f t h e h y p e r s e n s i t i v e r e a c t i o n o f TMV ( t o b a c c o mosaic v i r u s ) i n t o b a c c o ; i . e . , l e s i o n s i z e was I n c r e a s e d and v i r u s p r o g r e s s e d w i t h t h e l e s i o n edge (121, 124). T h i s s u g g e s t s t h a t enhanced PAL a c t i v i t y and p r o d u c t i o n o f p h e n o l i c m e t a b o l i t e s 1s d i r e c t l y i n v o l v e d i n h y p e r s e n s i t i v e r e s i s t a n c e t o v i r a l i n f e c t i o n . AOPP reduced g l y c e o l l i n l e v e l s i n soybean, c a u s i n g I n c r e a s e d s u s c e p t i b i l i t y t o PhYtQPhthQHI meoasperma (125, 125). The PAL I n h i b i t o r APEP a l s o p r e v e n t s g l y c e o l l i n i n d u c t i o n and t r a n s f o r m s an I n c o m p a t i b l e pathogen I n t e r a c t i o n t o a c o m p a t i b l e one, s u g g e s t i n g a r o l e f o r PAL and t o x i c p h e n o l i c compounds i n r e s i s t a n c e (12Z). AOA i n h i b i t e d PAL a c t i v i t y i n v i v o , but not i t s s y n t h e s i s , r e s u l t i n g i n i n c r e a s e d e x t r a c t a b l e PAL a c t i v i t y from maize m e s o c o t y l s which p r e v e n t e d r e s i s t a n c e e x p r e s s i o n . T h i s suggested t h a t PAL 1s I n v o l v e d i n r e s i s t a n c e (128). P h o s p h i t e was found t o I n c r e a s e PAL a c t i v i t y and t o i n h i b i t growth and i n f e c t i v i t y o f P h v t o p h t h o r a c r v o t o a e a on cowpea l e a v e s . P r e t r e a t m e n t o f l e a v e s w i t h AOA, p r i o r t o p h o s p h i t e and £. c r v o t o a e a . lowered PAL, reduced k i e v e t o n e and p h a s e o l l i d i n a c c u m u l a t i o n , and caused s i g n i f i c a n t I n c r e a s e s i n d i s e a s e symptomology (1291. It 1s e v i d e n t from t h e s e s t u d i e s t h a t t h e use o f PAL I n h i b i t o r s can weaken p l a n t d e f e n s e i n some c a s e s , t h e r e b y I n c r e a s i n g d i s e a s e severity. In b i o l o g i c a l weed c o n t r o l , such compounds might be u s e f u l as pathogen s y n e r g i s t s . T h i s c o u l d p r o v i d e b e t t e r weed c o n t r o l w i t h l e s s pathogen i n o c u l u m . A p p a r e n t l y none o f t h e s e PAL i n h i b i t o r s have been t e s t e d f o r t h i s u t i l i t y i n weed c o n t r o l . It i s a l s o p o s s i b l e t h a t more e f f i c i e n t i n h i b i t o r s may be s y n t h e s i z e d i n t h e f u t u r e as knowledge o f PAL and secondary m e t a b o l i s m i n c r e a s e s . X e n o b i o t i c Compounds That A l t e r Secondary P l a n t

Products

X e n o b i o t i c s can a l t e r v a r i o u s m e t a b o l i c pathways i n p l a n t s . Chemical a l t e r a t i o n o f p h e n o l i c compounds f o r a g r i c u l t u r a l purposes has been suggested t o have p o t e n t i a l (130. 1311. S i n c e s e v e r a l major p l a n t enzymes o f secondary m e t a b o l i s m a r e I n v o l v e d i n r e s i s t a n c e t o pathogens, a l t e r a t i o n o f secondary m e t a b o l i s m c o u l d I n f l u e n c e d i s e a s e development. H e r b i c i d e s , p l a n t growth r e g u l a t o r s ( s y n t h e t i c and n a t u r a l ) , and enzyme i n h i b i t o r s such as t h o s e p r e s e n t e d f o r PAL ( F i g . 4) have been r e p o r t e d t o a l t e r secondary p l a n t p r o d u c t s ( T a b l e I I I ) . R e s u l t s here demonstrate t h a t t h e l e v e l s o f many p h e n o l i c d e r i v a t i v e s a r e i n f l u e n c e d by such compounds when a p p l i e d t o a v a r i e t y o f p l a n t species. I f such x e n o b i o t i c s a r e t o be used t o r e g u l a t e l e v e l s o f secondary p r o d u c t s , a p p l i c a t i o n c o n c e n t r a t i o n , u p t a k e , t r a n s l o c a t i o n t o a c t i v e s i t e s , and c o n c e n t r a t i o n a t a c t i v e s i t e s a r e c r i t i c a l f a c t o r s t h a t need t o be d e t e r m i n e d . Lack o f r a p i d m e t a b o l i s m ( o r t h e n a t u r e o f metabolism) o r d e g r a d a t i o n w i t h i n p l a n t s would a l s o be Important.


100

MICROBES AND MICROBIAL PRODUCTS AS HERBICIDES HN 2

Ο

C H g — COOH

Aminooxy acetic acid

HN 9

Ο

CH

à

COOH


α-Aminooxy-f-phenyl propionic acid

o-Benzylhydroxylamine

HN 9

CH

CH:

COOH 3-(1,4-Cyclohexadienyl)alanine

(1-Amino-2-phenylethyl)phosphonic acid Figure 4. Table I I I .

Chemical Herbicides acifluorfen

alachlor

Chemical s t r u c t u r e s o f s e v e r a l PAL i n h i b i t o r s .

E f f e c t s o f S y n t h e t i c and N a t u r a l l y O c c u r r i n g C h e m i c a l s on Secondary P l a n t Product L e v e l s i n I n t a c t , E x c i s e d , o r Cell Culture Tissues Plant spinach soybean bean pea broadbean celery cotton sorghum

Secondary Plant Product f e r u l a t e amine glyceollins glyceofuran phaseollin pisatin medicarpin xanthotoxin hemigossypol anthocyanin lignin

Qualitative Effect + + + + + + + + -


BêL. 122 113 133 133 133 133 J U 134 134 134

5. HOAGLAND Table I I I ,

2,4-D DSMA glyphosate

metribuzin propanil sethoxydim Funaicides WL 28325

soybean sunflower

tobacco sunflower soybean soybean buckwheat tobacco ryegrass various soybean soybean corn rice

P l a n t growth r e g u l a t o r s ethylene sorghum turnip cranberry poinesttia lettuce kinetin Amaranthus s p . IAA soybean Dimethipin soybean PAL i n h i b i t o r s AOPP

mint soybean coleus carrot buckwheat cabbage radish

AOA OBHA

101

(con'tl

chlorsulfuron


Biochemical Responses of PlantstoPathogens

sphagnum moss cucumber tobacco soybean mungbean

anthocyanin β-coumarate caffeate ferulate sinapate scopolin scopolin anthocyanin anthocyanin shikimate shikimate shikimate gallate 4-hydroybenzoate anthocyanin anthocyanin anthocyanin

+ + + + + +

135

+

122 14Q 111 142 142 142 142 122

+

139 144

momilactone A momilactone Β

+

145

anthocyanin anthocyanin anthocyanin anthocyanin flavonoids amaranthin anthocyanin anthocyanin

+ + + + -

146 146 146 146 14Z 143 14a 15£

caffeate glyceollin rosmarinate anthocyanin anthocyanin vanillin (fr. lignin) anthocyanin feruloyl deriv. kaempferol pelargonidin sphagnorubin hydroxycinnamates secondary p h e n o l i c s anthocyanin anthocyanin

-

-

+ + + + +

-

+


116 126 136

126 HZ 138

145

121 125.126 122 152 111 154 i l l 155 155 155 156.157 158 102 114 113


102


X e n o b i o t i c s That A l t e r E x t r a c t a b l e PAL A c t i v i t y . PAL i n h i b i t i o n has been chosen as a s i t e o f a c t i o n f o r h e r b i c i d e d e s i g n ; however, s e v e r a l h e r b i c i d e s and o t h e r c h e m i c a l s were found t o have no s p e c i f i c e f f e c t on PAL ( 1 5 9 ) . Analogous r e s u l t s were o b t a i n e d when h e r b i c i d e s encompassing t h e major h e r b i c i d e c l a s s e s were examined (132, I S f i ) . The h i g h I n c r e a s e o f PAL by g l y p h o s a t e makes t h i s h e r b i c i d e unique i n t h e s e c o r r e l a t i o n s . G e n e r a l l y PAL a c t i v i t y c o r r e l a t e s w e l l w i t h t h e p r o d u c t i o n o f secondary p r o d u c t s , and t h i s has a l s o been demonstrated i n PAL-phenol 1c ( F i g . 5) and P A L - a n t h o c y a n i n ( F i g . 6 ) c o r r e l a t i o n s i n soybean s e e d l i n g s as a f f e c t e d by seventeen h e r b i c i d e s (112)· O t h e r h e r b i c i d e s , f u n g i c i d e s , I n s e c t i c i d e s , and a d j u v a n t s c a n a l t e r secondary p l a n t compound l e v e l s v i a I n d i r e c t e f f e c t s (15fi). Some h e r b i c i d e s have more d r a m a t i c e f f e c t s on e x t r a c t a b l e PAL a c t i v i t y l e v e l s from v a r i o u s p l a n t s and have been examined f o r some I n t e r a c t i o n s w i t h p l a n t s and d i s e a s e organisms ( F i g u r e 7 ) . Because g l y p h o s a t e reduced a r o m a t i c amino a d d ( p h e n y l a l a n i n e , t y r o s i n e , and t r y p t o p h a n ) p o o l s i n Rhizobium iaponicum and duckweed (Lsfflli o i b b a L . ) , and s u p p l e m e n t a t i o n o f t h e s e amino a d d s r e v e r s e d g l y p h o s a t e a c t i o n , I n h i b i t i o n o f t h i s pathway was proposed as t h i s h e r b i c i d e ' s mode o f a c t i o n ( 1 6 1 ) . I n d u c t i o n o f PAL a c t i v i t y c a n a l s o reduce a r o m a t i c amino a d d l e v e l s s u f f i c i e n t l y t o reduce g r o w t h , and r e v e r s a l 1s p o s s i b l e by f e e d i n g p h e n y l a l a n i n e (152). Thus, I n c r e a s e d PAL a c t i v i t y was examined as a p o s s i b l e mode o f a c t i o n o f t h i s h e r b i c i d e ( 1 6 3 ) . E x t r a c t a b l e PAL a c t i v i t y i n p l a n t s was found t o I n c r e a s e d r a m a t i c a l l y a f t e r t r e a t m e n t w i t h g l y p h o s a t e , b u t no i n v i t r o e f f e c t was noted (22» 1 6 3 ) . A l t h o u g h g l y p h o s a t e ' s p r i m a r y mode o f a c t i o n has now been shown t o be I n h i b i t i o n o f 5 - e n o l p y r u v y l s h i k i m a t e - 3 - p h o s p h a t e (EPSP) s y n t h a s e ( 1 6 4 ) . I n c r e a s e d PAL, and reduced EPSP s y n t h a s e , r e s u l t i n g i n p h e n o l i c p r o d u c t a l t e r a t i o n by g l y p h o s a t e c o u l d a f f e c t d i s e a s e development i n weeds o r c r o p p l a n t s or i n g e n e t i c a l l y a l t e r e d g l y p h o s a t e - r e s i s t a n t crop p l a n t s . S u b l e t h a l g l y p h o s a t e c o n c e n t r a t i o n s were found t o b l o c k g l y c e l l o i n a c c u m u l a t i o n and t o l o w e r r e s i s t a n c e o f soybean t o a b a c t e r i a l pathogen ( 1 2 1 . 1 2 2 ) . G l y p h o s a t e has a l s o been examined i n o t h e r i n s t a n c e s f o r i n t e r a c t i o n s o f p l a n t s and d i s e a s e organisms (165-15Ζ)· The h e r b i c i d e 2,4-D ( 2 - 4 - d i c h l o r o p h e n o x y a c e t i c a d d ) has been known f o r some t i m e t o i n h i b i t PAL i n v i v o b u t n o t I n v i t r o (153)· 2,4-D a l s o i n t e r a c t s w i t h IAA t o r e v e r s e g l y p h o s a t e growth I n h i b i t i o n i n t i s s u e c u l t u r e s ( 1 6 9 . 1 7 0 ) . b u t n o t i n i n t a c t p l a n t s (1Z1). E x t r a c t a b l e PAL a c t i v i t y i n soybean s e e d l i n g s was I n h i b i t e d by 2,4-D (171) and 2,4-D lowered PAL a c t i v i t y i n c r e a s e s caused by g l y p h o s a t e ( 1 7 1 ) . The h e r b i c i d e has a l s o been examined f o r i n t e r a c t i o n s o f I n d u c t i o n and s e c r e t i o n o f p a t h o g e n e s i s - r e l a t e d p r o t e i n s i n t o b a c c o suspension c u l t u r e s (172). Adf1uorfen {5-[2-chloro-4-(trif1uoromethyl)phenoxy]-2-n1trob e n z o i c a d d } 1s a d i p h e n y l e t h e r h e r b i c i d e which r e q u i r e s l i g h t and oxygen f o r i t s t o x i c a c t i o n ( 1 7 3 ) . T h i s compound a f f e c t s secondary p l a n t m e t a b o l i s m by i n c r e a s i n g e x t r a c t a b l e PAL a c t i v i t y ( H 2 , 1Z4» 175). F o r i n s t a n c e , i t i n c r e a s e s M - f e r u l o y l - 3 - m e t h o x y t y r a m i n e (FMT) l e v e l s ( 1 3 2 . 1 3 3 . 1 7 6 ) . M o d i f i c a t i o n o f enzymes and p r o d u c t s o f secondary p l a n t metabolism by a c i f l u o r f e n ( 1 3 3 . 174) i s s i m i l a r i n some h o s t - p a t h o g e n i n t e r a c t i o n s ( 1 7 7 ) . A c i f l u o r f e n has been a p p l i e d w i t h t h e weed pathogen C o l l e t o t r i c h u m a l o e o s p o r i d e s ( P e n z . ) S a c c . f . i u s s i a e as a m i x t u r e o r s e q u e n t i a l l y and shown n o t t o i n h i b i t d i s e a s e development on n o r t h e r n j o i n t v e t c h rAeschvnomene v i r o i n i c a ( L . ) B . S . P . ] (123).


Biochemical Responses

Biochemical Responses of Plants to Pathogens - ACS Publications

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