Antifungal Activity of Plant Steroids - ACS Symposium Series (ACS

Chapter 18

Antifungal Activity of Plant Steroids James G. Roddick Downloaded by NORTH CAROLINA STATE UNIV on July 9, 2013 | http://pubs.acs.org Publication Date: December 24, 1987 | doi: 10.1021/bk-1987-0325.ch018

Department of Biological Sciences, University of Exeter, Exeter, United Kingdom

Antifungal steroids in plants are represented mainly by the glycoalkaloids (especially the Solanum type) and the saponins. Both types of compound are strongly fungistatic/fungicidal in vitro apparently due to their ability to complex with membrane sterols and disrupt membrane integrity. The suggestion that the aglycone (rather than the glycoside) is the active moiety is questioned partly on the basis of data from studies on synthetic lipid membranes. Available evidence suggests that glycoalkaloids and saponins are not key factors in the resistance of vegetative organs to fungal infections although they may be of greater significance in reproductive structures and also contribute to the general defences of the plant. Plants produce a wide range of steroids (Table I) and accumulate some i n considerable quantities but our knowledge of the functions of most of these compounds i s meagre (1). Probably the major exception i s the s t e r o l s which are known to be important membrane components {2,3) as well as precursors of other steroids (4). Estrogens, androgens, corticosteroids {5_,6) and brassinosteroids Ç7) may have growth regulatory a c t i v i t y although t h i s i s s t i l l not c e r t a i n . For the remaining groups, no d e f i n i t e role has been established within the plant but the toxic nature of many of these steroids suggests they could have an ecological rather than metabolic function cont r i b u t i n g to plant resistance t o pathogens (especially fungi) and predators (especially insects) (8). This a r t i c l e w i l l review the antifungal a c t i v i t y of plant steroids but i t should be noted that not a l l steroids act i n an i n h i b i t o r y capacity towards fungi. Certain s t e r o l s have a stimulatory e f f e c t on fungal development promoting growth, the d i f f e r e n t i a t i o n of reproductive structures, and f e r t i l i s a t i o n i n species of Pythium and Phytophthora which lack the a b i l i t y to synthesize s t e r o l s (9). This subject i s considered elsewhere i n t h i s volume. Antifungal a c t i v i t y has been demonstrated mainly i n two groups of plant steroids, glycoalkaloids of the Solanum type and saponins. Members of both groups are comprised of a s t e r o i d a l aglycone

0097-6156/87/0325-0286$06.00/0 © 1987 American Chemical Society

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Downloaded by NORTH CAROLINA STATE UNIV on July 9, 2013 | http://pubs.acs.org Publication Date: December 24, 1987 | doi: 10.1021/bk-1987-0325.ch018

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Antifungal Activity of Plant Steroids

attached to one or more carbohydrate moieties. In addition, alka l o i d s contain a basic nitrogen group which renders them much more t o x i c , e s p e c i a l l y to homoiothermic organisms (10). Steroidal sapogenins are usually based on a spirostane skeleton (Figure 1). Various types of Solanum glycoalkaloids e x i s t but those so f a r shown to be fungitoxic i n v a r i a b l y possess a spirosolane- or solanidane-type aglycone (Figure 1). These glycoalkaloids and the monodesmosidic saponins are b i o l o g i c a l l y - a c t i v e amphipathic molecules with an o l i g o saccharide comprising up to f i v e monosaccharides attached at C - 3 ; b i desmosidic saponins have an additional sugar moiety (usually one glucose) at C-26 and are b i o l o g i c a l l y i n a c t i v e . The chemistry of the Solanum alkaloids and saponins has been reviewed by various workers (11-14). Of the two groups, the glycoalkaloids have received more research attention undoubtedly because they are present i n edible parts of the important food plants, potato and tomato, and have caused i l l n e s s and death of humans and livestock on a number of occasions (15). Steroidal saponins are present i n the non-edible parts of some less prominent food plants (e.g. oat) and have not caused serious incidences of poisoning. The b i o l o g i c a l properties of these compounds are d e t a i l e d i n a number of reviews ( 1 3 , 1 4 , 1 6 - 2 0 ) . Table I.

Major Groups of Steroids found i n Plants

Group Sterol Estrogen Androgen Corticosteroid Brassinosteroid Progestogen Withanolide Ecdysteroid Cardiac glycoside Saponin Glycoalkaloid

Example S i t o s t e r o l , stigmasterol E s t r a d i o l , estrone Testosterone, androstenedione 11-deoxycorticosterone Brassinolide Progesterone, pregnenolone Withaferin, nicandrenone Ecdysone, ecdysterone Calotropin Digitonin, avenacosides A and Β Solanine, j ervine

Reference 3, 96 5, 6 5, 98 5, 102 7 97, 98 99 100 101 13, 14

11/ 11

In the following section some of the more important i n v i t r o studies which established the f u n g i t o x i c i t y of glycoalkaloids and saponins are described leading, i n the two subsequent sections, to a c r i t i c a l assessment of how these compounds a f f e c t fungi at the bio chemical l e v e l and what contributions they make to the resistance of plants to fungal pathogens. Fungitoxicity of Glycoalkaloids and Saponins i n v i t r o A large number of studies have been made i n t h i s f i e l d with reports dating from a t least 1933 when the potato glycoalkaloid 'solanine' (Figure 2) was reported to i n h i b i t growth of Cladosporium fulvum (21 ,£2) . (At t h i s time 'solanine' preparations probably contained both potato a l k a l o i d s , α-solanine and α-chaconine, the l a t t e r not being discovered u n t i l 1954). The f i n d i n g around the same time that expressed juice from tomato plants i n h i b i t e d Fusarium oxysporum f.sp. l y c o p e r s i c i (23) subsequently l e d to the discovery of the tomato glycoalkaloid, α-tomatine (Figure 2), the active ingredient of the extracts. Further work (24,25) demonstrated the general t o x i c i t y of



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ECOLOGY AND METABOLISM OF PLANT LIPIDS

Figure 1. Aglycone skeletons of fungitoxic s t e r o i d a l saponins and Solanum glycoalkaloids.



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Figure 2 .

Solanum s t e r o i d a l glycoalkaloids.


289

290


tomatine towards fungi, with F. oxysporum and various human dermatophytic fungi proving p a r t i c u l a r l y s e n s i t i v e . The d i f f e r e n t i a l s u s c e p t i b i l i t y of fungi to tomatine was l a t e r confirmed i n a more comprehensive investigation involving 3 0 species from 1 9 genera ( 2 6 ) . The maximum and minimum concentrations of tomatine to completely i n h i b i t mycelial growth ranged from 8 5 0 mM to 1 3 0 uM, a factor of 6 5 0 0 . An important feature of glycoalkaloid t o x i c i t y was highlighted by McKee ( 2 7 ) when he demonstrated the pH dependence of solanine-, tomatine- and demissine- (Figure 2 ) induced disruption of spores of a number of fungi, including Fusarium caeruleum. Disruption was greatest i n a l k a l i n e conditions, the L D of solanine at pH 5 . 6 being lOOx greater than at pH 7 . 6 . The saponin d i g i t o n i n (Figure 3 ) , although more toxic than these a l k a l o i d s , was r e l a t i v e l y unaffected by pH (Table I I ) . The pH dependence of glycoalkaloid action has since been shown i n other fungi (e.g. 2 8 - 3 0 ) and to be a general e f f e c t . Although d e t a i l s remain uncertain, protonation of the a l k a l o i d produces a form with less b i o l o g i c a l a c t i v i t y whereas d i s s o c i a t i o n i n alkaline conditions y i e l d s the unprotonated, highly active form ( 2 8 ) .


5Q

Table I I . 5 0 ( g/D of Glycoalkaloids and Saponins against Spores of Fusarium caeruleum i n r e l a t i o n to pH. After McKee (_27) . L

D

m

PH Steroid Tomatine Solanine Demissine Digitonin

5.0

27

5.6

7.0 32

7.6

8.0

460

220

6.5 64

2000

1100

260

85

20

11

260

72

22

11

-

8

-

18

-

-

14

-

-

5.9

13

-

8.3 7 8

In the study by McKee ( 2 7 ) , chaconine (Figure 2 ) proved more toxic than solanine with t h e i r common aglycone, solanidine (Figure 2 ) , much less so. S i m i l a r l y , tomatine (a tetraoside) was more e f f e c t i v e than i t s trisaccharide hydrolysis products against Helminthosporium turcicum, Septoria l i n i c o l a and Colletotrichum orbiculare with the aglycone, tomatidine (Figure 2 ) , being l e a s t e f f e c t i v e ( 2 8 ) . The e f f e c t of tomatidine varied considerably with the t e s t organism. The greater t o x i c i t y of glycosides compared with aglycones has also been shown with B o t r y t i s cinerea ( 3 1 ) and Phytophthora cactorum ( 3 2 ) , the l a t t e r authors also demonstrating t h i s feature f o r solasonine/solasodine (Figure 2 ) . Nevertheless, a few reports also e x i s t of tomatidine and solanidine being more fungitoxic than t h e i r respective glycosides ( 3 3 , 3 4 ) . V i r t u a l l y a l l the reports on fungal development i n the presence of glycoalkaloids or saponins describe i n h i b i t o r y e f f e c t s , but recently tomatine was found to stimulate sporulation i n F. oxysporum f.sp. l y c o p e r s i c i even though i t depressed colony growth, spore germination and germ tube growth (_35) . Promotion of reproductive development, however, was not observed i n P. cactorum; on the contrary, various s t e r o i d a l alkaloids of both the Solanum (solanine, tomatine) and Veratrum (jervine, muldamine, Figure 4 ) types, as well as tomatidine, solanidine and solasodine, a l l i n h i b i t e d s i t o s t e r o l induced spore production ( 3 2 ) . However, i n h i b i t i o n of vegetative hyphae by these compounds was not so marked or consistent, and varied with the s i t o s t e r o l content of the medium. McKee ( 2 7 ) also found


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solanine less damaging towards hyphae than towards spores but a t t r i buted t h i s to the a b i l i t y of hyphae to degrade the a l k a l o i d . The d i f f e r e n t response of vegetative hyphae and reproductive structures towards such compounds obviously has important implications for the experimental assessment of f u n g i t o x i c i t y . Probably the most widelyused method has been measurement of colony diameter but the v a l i d i t y of t h i s parameter has recently been questioned (36). Few i n v i t r o studies with fungi make reference to the c e l l u l a r nature of growth impairment. However, the disintegration of Phytophthora infestans zoospores (which lack a c e l l wall) by solanine (27) and the release of amino acids from digitonin-treated Pythium ultimim hyphae (37) , point to damage to l i m i t i n g (and possibly other) membranes. Mode of Fungitoxic Action Almost 60 years ago Fischer (38) and Boas (39) proposed that 'solanine' and d i g i t o n i n caused haemolysis by interacting with steroids i n the erythrocyte membrane, but t h i s work appears to have been f o r gotten for the next 30 years. In that time, the surfactant properties of glycoalkaloids and saponins were elucidated and figured prominentl y i n explanations of membrane l y s i s by these compounds. Consistent with t h i s thinking was the observation that aglycones had lower surface a c t i v i t y and were less disruptive than glycosides. Resurrection of the steroid-binding hypothesis probably dates from 1957 when Schulz and Sander (40) demons rated the formation of 1 : 1 molecular complex i n v i t r o between tomatine and 33-hydroxy steroids such as cholesterol. Since then, a large number of glycoalkaloids and saponins have been shown capable of complexing with various sterols (including the fungal s t e r o l ergosterol) i n v i t r o (41,42). Evidence was presented (28) that the reduced f u n g i t o x i c i t y of hydrolysis products of tomatine (including tomatidine) could not be explained solely on the basis of surfactant properties but was more l i k e l y related to t h e i r i n a b i l i t y to complex with s t e r o l s . I t was further reported (_28,43) that binding to sterols i n v i t r o only occurs to a s i g n i f i c a n t degree with the unprotonated a l k a l o i d i n alkaline conditions (Figure 5). Such a mode of action i s reminiscent of the sterolbinding polyene a n t i b i o t i c s (44), and species of Pythium and Phytophthora which are unaffected by polyenes because t h e i r membranes lack sterols are also r e l a t i v e l y insensitive to tomatine (26) and d i g i t o n i n (31_,45). However, when grown i n the presence of s t e r o l s , these fungi incorporate them into t h e i r membranes, a process which sensitizes them to polyenes, saponins, etc. (37,45). Work by t h i s author (46) indicated that tomatine disrupts isolated organelles i n a similar manner to polyenes such as f i l i p i n and nystatin, causing loss of lysosomal contents and i n h i b i t i o n of chloroplast PS II a c t i v i t y , but having no e f f e c t on the respiratory a c t i v i t y of mitochondria. Nystatin-resistant mutants of Fusarium solani with lower s t e r o l levels were also less sensitive to tomatine (47,48). These mutants were unaffected by 800 ppm tomatine whereas wild-type strains succumbed to 100 ppm tomatine. Crossing experiments between mutants and wildtypes showed that low s t e r o l content and i n s e n s i t i v i t y to tomatine or d i g i t o n i n were always inherited together (49). As with polyenes, the complexing of glycoalkaloids and saponins to membrane sterols i s thought to lead to the formation of pores i n membranes (50^,5^) -




Ν)


glucosidase

R*

R l I x

I

Plant

glucosidase

R"

R'

S t e r o i d a l saponins.

= H

2 6 - D e s g l u c o a v e n a c o s i d e B, R' (active) _„

glycosidase

;glu-

;glu

A v e n a c o s i d e B, (inactive)

N u a t i g e n i n , R» = R" (inactive)

fungal

rham"

= glu

= H

rham^

= glu

= glu

Figure 3, Continued.

2 6 - D e s g l u c o a v e n a c o s i d e A, R' (active)

Plant

A v e n a c o s i d e A, (inactive)

rham

glu-glu-

H

rham'

glu-glu^

glu


;glu'

-glu'

to

I

Co

t


294


Figure 5. E f f e c t of pH on binding of tomatine to cholesterol i n vitro.



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295

Freeze-fracture E.M. studies of natural and synthetic membranes treated with f i l i p i n , d i g i t o n i n and tomatine revealed c h a r a c t e r i s t i c protuberances on the surfaces of membranes (52,_53) but how these structures r e l a t e to the chemical complexes or to the formation of pores i s not understood. Despite the evidence supporting a sterol-binding mechanism, a number of doubts s t i l l remain. For example, no quantitative r e l a t i o n ship could be established between the i n v i t r o sterol-binding capacity of glycoalkaloids and saponins and their haemolytic action (41). Nor has the greater antifungal a c t i v i t y of aglycones than glycosides observed by Wolters (33) and Sinden et a l . (34) yet been s a t i s f a c t o r i l y explained. Also, although less potent than tomatine, tomatidine was s t i l l capable of i n t e r f e r i n g with mycelial growth of H. turcicum, S. l i n i c o l a and (particularly) C. orbiculare (28). The minimum concentrations of the aglycone required to cause complete growth i n h i b i t i o n were respectively 400x, 2000x and only 3x the minimum concentration of tomatine. Tomatidine and solanidine have also been shown to reduce sporulation i n P. cactorum (32). Another proposed weakness of the sterol-binding hypothesis i s the lack of d i r e c t evidence of incorporation of glycoalkaloids or saponins into the fungal mycelium or membrane (36). In evidence, solanine did not influence sporulation i n P. cactorum and, i n t r i t i a t e d form, was not incorporated into mycelium whereas PH]-solanidine was both deleterious to sporulation and incorporated into mycelium. The a b i l i t y of t h i s fungus to hydrolyse solanine to solanidine suggested to the authors that t h e i r findings might be explicable on the basis of a quite d i f f e r e n t hypothesis for mechanism of action which l a i d emphasis on the aglycone rather than the glycoside. This hypothesis has i t s origins i n haemolysis experiments c a r r i e d out by Segal et a l . (54). Following treatment of erythrocytes with d i g i t o n i n , solanine or tomatine, aglycones but not glycosides could be detected i n haemolysed ghosts whereas both aglycones and glycosides were associated with non-haemolysed c e l l s . Aglycones alone were also haemolytic. I t was proposed that the aglycone was the active moiety being released from the glycoside by a membrane glycosidase. Consistent with t h i s claim was the subsequent finding (55) that the addition of gluconolactone or galactonolactone (reputedly s p e c i f i c i n h i b i t o r s of glycosidases) i n h i b i t e d tomatineand (to a lesser extent) digitonin-induced haemolysis. When these experiments were repeated on B. cinerea and Rhizoctonia s o l a n i , e s s e n t i a l l y s i m i l a r r e s u l t s were obtained (56). Thus, i n addition to the requirement for membrane s t e r o l to bind the glycoside, t h i s hypothesis necessitates a second prerequisite i n the form of a membrane glycosidase. How the aglycone brings about l y s i s was not made c l e a r . The glycoside was thus considered to be simply the "water-soluble transport form" (56), but such a r o l e has recently been disputed (57,58). Although there are good grounds for accepting that inactive, non-sterol-binding bidesmosidic saponins (e.g. avenacosides A and B, Figure 3) are enzymically hydrolysed i n damaged c e l l s to active, monodesmosidic saponins (59), a number of problems arise when attempting to explain the t o x i c i t y of monodesmosidic saponins and glycoalkaloids on the basis of a similar hydrolytic a c t i v a t i o n process. The p r i n c i p a l objections come from work on synthetic l i p i d membranes which lack glycosidases. E l f e r i n k (60), for


296



+

example, found that d i g i t o n i n caused s i g n i f i c a n t leakage of K from phosphatidylcholine liposomes only when cholesterol was also present. A similar observation was reported recently by t h i s author (43) using tomatine (Table I I I ) . The extent of liposome disruption was directly related to the concentration of both the s t e r o l and the a l k a l o i d (with a strong interaction between the two) as well as to pH. The aglycone was not active i n t h i s system. In experiments on planar l i p i d b i l a y e r s and monolayers (61), d i g i t o n i n was p a r t i c u l a r l y e f f e c tive i n causing channel-like conductance changes i n membranes containing sterols; i n sterol-free membranes, e f f e c t s could only be achieved with s i g n i f i c a n t l y higher concentrations. Obviously, caution i s essential when attempting to extrapolate from a r t i f i c i a l membranes to c e l l membranes but the s i m i l a r i t i e s i n responses/suscept i b i l i t y of the two systems to glycoalkaloids and saponins i n relation to pH, s t e r o l content, etc. suggest that such comparisons have some validity. Table I I I . E f f e c t of Tomatine on Release of Peroxidase from Liposomes containing d i f f e r e n t Phospholipids and Sterols Sterol ErgoNo CholeStigmaPhospholipid Sterol sterol Treatment sterol sterol 19.2 19.6 Control 19.5 26.0 Phosphatidylcholine 51.4 69.2 15.4 Tomatine 52.0 Sphingomye1in 7.4 48.4 Control 10.6 6.3 50.0 24.9 31.8 Tomatine 29.8 Values are % of liposome peroxidase a c t i v i t y released into supernatant. Liposomes were treated with 150 uM a l k a l o i d at pH 7.2 for 1 hour. Adapted from Roddick and Drysdale (43). Some aspects of the experimentation which support the aglycone hypothesis are also open to doubt. For instance, aglycones were apparently dissolved i n 20-25% DMSO which i s i t s e l f strongly haemol y t i c , and no control data were presented. With tomatidine i n 1% (or less) DMSO and proper controls, no haemolysis could be attributed to either of these components (Roddick, unpublished). High concentrations of sugar lactones can depress pH i n weakly-buffered solutions and thus possibly also the action of pH-dependent glycoalkaloids. The i n a b i l i t y of sugar lactones to i n h i b i t aglycone-induced haemol y s i s could be explained by l y s i s being caused by DMSO, as indicated above. The f a c t that haemolysed erythrocyte ghosts were washed whereas non-haemolysed c e l l s were not could account for the f a i l u r e to detect glycosides i n the former but t h e i r presence i n the l a t t e r . The s i t u a t i o n i s further confused by claims that a fungal membrane glycosidase activates tomatine (56) whereas a fungal wall glycosidase, e f f e c t i n g an i d e n t i c a l hydrolysis, i n h i b i t s tomatine action (62). In neither case was the proposed location of these enzymes established using unequivocal (e.g. fractionation) techniques. However, t h i s i s not to refute the existence or a c t i v i t y o f such glycosidases. Numerous reports exist of the hydrolysis of glycoalkaloids by fungal glycosidases (e.g. 27, 3^, 63-65) but i n virtually every case hydrolysis was viewed as an i n a c t i v a t i o n of a toxic glycoside. Nor do the doubts attaching the aglycone hypothesis necessarily



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mean that membrane d e s t a b i l i z a t i o n by glycoalkaloids and saponins can be explained i n a l l cases by sterol-binding. Many anomalies s t i l l e x i s t i n t h i s area e.g. the lack of a quantitative relationship between s t e r o l binding i n v i t r o and haemolysis (41); the greater d i s ruption of s t e r o l - f r e e than sterol-containing liposomes by the saponin p a r i l l i n (Figure 3) (60). I t may be that d i f f e r e n t membrane-active s t e r o i d a l glycosides operate i n s l i g h t l y , or even markedly, d i f f e r e n t ways. Even so, the weight of evidence points to s t e r o l binding as being an important factor, perhaps q u a l i t a t i v e l y rather than quanti t a t i v e l y , i n the d e s t a b i l i z a t i o n of membranes by the compounds i n question. Interactions, of either a d i r e c t or i n d i r e c t nature, between glycoalkaloids/saponins and membrane phospholipids and/or proteins may also be involved. Opinions have been voiced both i n favour (36,j52) and against (37) such p o s s i b i l i t i e s . Obviously more work i s required i n t h i s f i e l d . Glycoalkaloids and Saponins as Resistance

Factors

The t o x i c i t y of s t e r o i d a l glycoalkaloids and saponins to various p a r a s i t i c fungi i n v i t r o has naturally led to suggestions that these compounds might also operate i n a similar capacity i n the i n t a c t , i n fected plant. Early studies (24,33,66) gave some support to t h i s idea with tomato pathogenic fungi apparently less susceptible to tomatine than non-pathogens. From a more critical investigation (26) under standard conditions with 30 species of fungi, including tomato pathogens, non-pathogens and general saprophytes, a ranking order of increasing s u s c e p t i b i l i t y to tomatine was produced i n which tomato pathogens occupied 14 of the f i r s t 16 places. The p r o b a b i l i t y of such a ranking occurring by chance was calculated as 1 i n 10^. How ever, demonstration of a c o r r e l a t i o n between s u s c e p t i b i l i t y i n v i t r o and pathogenicity i s not i n i t s e l f s u f f i c i e n t evidence that compounds l i k e tomatine play a major role i n resistance to fungal pathogens. Although only one of many factors, d i f f e r e n t i a l s u s c e p t i b i l i t y of fungi to toxic plant metabolites i s undoubtedly not without s i g n i f i c a n c e . A probable explanation for t h i s i n Pythium and Phytophthora spp. i s the lack of membrane s t e r o l s , a supposition which finds support i n the f a c t that growth i n a sterol-containing medium sensi t i z e s these fungi to glycoalkaloids and saponins (37,45). An alterna t i v e explanation i s that these fungi may not assimilate the glycosides (36) although the significance of assimilation has yet to be ascer tained. Detoxification of glycoalkaloids or saponins by extracellular glycosidases which hydrolyse glycosides to aglycones may also con tribute to the r e l a t i v e i n s e n s i t i v i t y of these species (32,67) a l though t h i s mechanism may be more important i n other species of fungi. The leaf-spot fungus Septoria l y c o p e r s i c i , for example, inactivates tomatine by removing one glucose to produce 3 2 ~ (63,64) whereas F. oxysporum f.sp. l y c o p e r s i c i (65) and B. cinerea (31) both remove the whole tetrasaccharide moiety. In A l t e r n a r i a solani (62) and F. caeruleum (27) there i s evidence of a stepwise removal of a l l the monosaccharide units of glycoalkaloids. In damaged oat leaves, the inactive, bidesmosidic saponins avenacosides A and Β are hydrolysed at C-26 by leaf enzymes to y i e l d the active, monodesmosidic derivatives. However on i n f e c t i o n by Helminthosporium avenae, the monodesmosidic saponins are further degraded to the inactive agly cone, nuatigenin, by a fungal glycosidase which removes the carbot o m a t : i

n e



298


hydrate moiety at C - 3 (Figure 3 ) ( 5 9 ) . In potato tubers, the conversion of solanine to solanidine observed i n tissue damaged by I>. infestans or the bacterium Erwinia atroseptica was reported to be due to the release of h o s t - c e l l hydrolases ( 6 8 ) . In infected, but nondamaged, r e s i s t a n t v a r i e t i e s the aglycone was not detectable. The proposal that tomatine might be a factor i n the resistance of tomato v a r i e t i e s to F. oxysporum f.sp. l y c o p e r s i c i ( 2 3 , 6 9 ) was not confirmed by Kern ( 6 6 ) who considered the a l k a l o i d neither sufficientl y i n h i b i t o r y to the fungus nor s u f f i c i e n t l y abundant i n lower stems and roots. Drysdale and coworkers have argued that the tomatine concentration i n these organs i s adequate to i n h i b i t growth and spore germination of F. oxysporum ( 7 0 ) but rule out a primary resistance role for t h i s a l k a l o i d because i t was not detectable i n f l u i d from xylem (where t h i s w i l t fungus i s located) and because i t s l e v e l s i n creased to the same extent i n both r e s i s t a n t and susceptible cultivars following i n f e c t i o n Ç71_) . Others ( 3 5 ) have detected tomatine i n tracheal f l u i d but again did not observe differences i n a l k a l o i d concentration i n r e s i s t a n t and susceptible c u l t i v a r s before or a f t e r i n f e c t i o n . Further evidence against a primary role for tomatine was that the a l k a l o i d stimulated sporulation of F. oxysporum f.sp. l y c o p e r s i c i ( 3 5 ) , a process which the authors point out i s more important i n invasion by t h i s fungus than mycelial growth. On the other hand, some workers have arrived at the opposite conclusion regarding the importance of tomatine i n fusarium w i l t of tomato. Sarhan and Kirâly (12) concluded that i n f e c t i o n was related to s o i l nutrients and fungicide treatments and that resistance was mediated v i a elevated tomatine l e v e l s . A s i m i l a r claim was made from a study of the fungit o x i c i t y of extracts from infected and non-infected tomato plants of r e s i s t a n t and susceptible c u l t i v a r s (13); differences from the conclusions of other workers (e.g. 7 0 ) were explained by v a r i e t a l differences. The above information together with the f a c t that F. oxysporum f.sp. l y c o p e r s i c i i s a fungus of low (or zero) tomatine regions capable of detoxifying t h i s a l k a l o i d ( 6 5 ) suggest that tomatine i s probably not a major factor i n resistance to fusarium w i l t . Claims that tomatine may contribute to resistance to b a c t e r i a l w i l t (Pseudomonas solanacearum) i n roots of Lycopersicon pimpineHifolium ( 7 4 ) require substantiation. Glycoalkaloid l e v e l s i n whole potato tubers tend to be similar to the low l e v e l s found i n roots and stems although the skin and peel of tubers have much higher concentrations Ç 7 5 ) . Nevertheless, glycoalkaloids are not thought to be important i n combatting tuber i n f e c tions by F. caeruleum ( 7 6 ) or R. solani ( 7 7 ) . I t may be s i g n i f i c a n t that F. caeruleum i s able to degrade potato glycoalkaloids ( 2 7 ) . S i m i l a r l y , no correlations were observed between a l k a l o i d l e v e l s i n potato roots/stems and V e r t i c i l l i u m w i l t (V. albo-atro), or between tuber a l k a l o i d s and the b a c t e r i a l disease, common scab (Streptomyces scabies) ( 7 8 ) . Nor are glycoalkaloids apparently involved i n r e s i s tance of potatoes to b a c t e r i a l r i n g rot caused by Corynebacterium sepedonicum ( 7 9 ) . Much work into the role of glycoalkaloids i n potato tuber resistance to the l a t e - b l i g h t fungus (P. infestans) has been done by Kuc and coworkers and has recently been reviewed by Kuc ( 8 0 ) . Early investigations ( 8 1 _ , 8 2 ) pointed to glycoalkaloids as being a possible factor i n tuber resistance but the subsequent demons t r a t i o n that a l k a l o i d accumulation (which increases i n damaged



18. RODDICK


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tubers) i s suppressed by compatible and incompatible races of l>. infestans (83) questioned the involvement of these compounds i n R gene and hypersensitive resistance. A study of t h i s disease i n 15 potato clones (84) also produced no evidence of a glycoalkaloid cont r i b u t i o n to multigenic (field) resistance. Despite these conclusions, Kuc (80) i s of the opinion that tuber glycoalkaloids may s t i l l play a role i n general resistance to disease. In view of t h e i r higher concentrations i n leaves and reproductive structures, i t has been proposed (63) that glycoalkaloids might be more important against l e a f - or f r u i t - i n f e c t i n g fungi. The leaf pathogen C. fulvum was adversely affected by tomatine but i t was not clear whether t h i s fungus, which grows i n i n t e r c e l l u l a r spaces, could cause tomatine leakage from c e l l s (30). In potato leaves, elevated resistance to the early b l i g h t fungus A. solani observed i n continuous l i g h t was not attributable to glycoalkaloids (85). Sinden et a l . (34) concluded likewise for older (120 day) leaves which had a lower alkal o i d content (260 ppm) but suggested that i n younger (30 day) leaves, higher a l k a l o i d levels (1570 ppm) might r e s t r i c t development of A. s o l a n i . In a study of leaf i n f e c t i o n by A. solani and P. infestans in 10 potato c u l t i v a r s , no correlations were apparent between glycoalkaloids and resistance to disease (78) although t h i s might be explained by the age of the plants (approx. 8 weeks) i n the case of A. solani. Tomato f r u i t s are interesting organs for phytopathological studies being s i t e s of tomatine synthesis and high tomatine accumulations (in small, green f r u i t s ) as well as tomatine degradation and low to zero a l k a l o i d levels (in large green to ripe f r u i t s ) (58,86). Of i n t e r e s t i n t h i s respect i s the finding that B. cinerea germ tubes penetrated epidermal c e l l s of green f r u i t s but no further development of the fungus occurred (31). However, the authors expressed doubt that t h i s was due to tomatine as the fungus i s able to degrade t h i s a l k a l o i d and also d i d not resume growth i n ripe f r u i t s , though s t i l l a l i v e . More clear-cut r e s u l t s have been reported for F. solani, low s t e r o l mutants of which caused severe r o t of green tomato f r u i t s whereas wild type strains d i d not (48). Both types were equally aggressive on ripe tomatoes. The authors suggested that tomatine could be a major resistance factor i n f r u i t s (at least to t h i s fungus). Further evidence i n support of t h i s claim was that i n crossing experiments the a b i l i t y to r o t green f r u i t s was inherited along with i n s e n s i t i v i t y to tomatine (49). Ripening tomato f r u i t s show a decline in their resistance to Colletotrichum phomoides (87) but i t i s not known i f t h i s i s related to the concomitant decrease i n f r u i t txmatine. A study of colonisation of tomato f r u i t s (of d i f f e r e n t developmental stages) by the f r u i t r o t pathogens Corticium r o l f s i i , B. cinerea, Monilia fructigena and Gloeosporium fructigenum revealed an order of pathogenicity (as shown) which corresponded to that of i n s e n s i t i v i t y to tomatine i n v i t r o (29). Colonization was explained, not by secret i o n of hydrolytic enzymes, but by a l t e r a t i o n to the pH at the inocul a t i o n s i t e s . The most successful pathogen (C. r o l f s i i ) lowered the pH from 5.7 to 3.6, the least successful (G. fructigenum) increased the pH to 6.4, and the two intermediate pathogens gave intermediate pH changes (to 4.4). The relationship between pH and resistance was explained on the basis of the pH dependence of tomatine t o x i c i t y . In another report, the same author (62) showed that invasion of tomato f r u i t s by various species and races of A l t e r n a r i a , pathogenic and non-



300


pathogenic to tomato, was related, not to pH changes ( a l l test fungi increased t h i s equally), but to t h e i r a b i l i t y to secrete tomatinehydrolysing enzymes. The species/strains which did not produce such enzymes were incapable of successful colonization. The major factors which influence the t o x i c i t y of glycoalkaloids and saponins to fungi i n vivo can thus be i d e n t i f i e d as the l e v e l of glycoside, fungal s t e r o l content, secretion of fungal hydrolases, production of suppressors of glycoside synthesis and a l t e r a t i o n of pH. To t h i s l i s t could be added a number of other factors which may be important i n moderating the above or having a bearing i n t h e i r own r i g h t , but which have been l i t t l e researched. For example, glyco a l k a l o i d l e v e l can be influenced by age (34) and/or developmental stage (86) as well as by v a r i e t y , environmental factors and c u l t u r a l practices (88,89). Consideration should also be given to the i d e n t i t y (and ratios) of compounds present as some are more toxic than others (27,34). In view of the d i f f e r e n t i a l s e n s i t i v i t y of vegetative hyphae and reproductive structures to alkaloids (2Ί_,32) , developmental aspects of the fungal pathogen could be important. A number of com pounds of host o r i g i n liberated along with glycoalkaloids or saponins following damage to t h e i r main storage s i t e , the vacuole (90,91), or to other c e l l compartments are known to enhance, ameliorate or even n u l l i f y t o x i c i t y of these compounds. These include sugars (27), ions such as Na , K and C a (27), s t e r o l s (32) and glycosidases (92-94). +

+

2 +

Conclusions Despite t h e i r potent antifungal action i n v i t r o , s t e r o i d a l glyco alkaloids and saponins do not appear to be key factors i n the r e s i s tance of roots, stems and leaves to fungal disease, although they may play a greater role i n resistance to f r u i t i n f e c t i o n s . Neverthe less, many authors s t i l l believe these compounds play some role i n the general defences of the plant against fungi. The f a c t that many successful fungal parasites have means of degrading or i n a c t i v a t i n g glycoalkaloids or saponins or of suppressing t h e i r synthesis, or do not synthesize t h e i r target molecules, etc. suggests that i n evolu tionary, i f not e c o l o g i c a l , terms these compounds do have a poten t i a l l y protective function. Many p a r a s i t i c fungi are not pathogenic to plants which elaborate glycoalkaloids or saponins (26), a s i t u a t i o n which could be due to the presence of these compounds. In the coevolution of plants and fungal parasites, any equilibrium i s usually a dynamic one and the r e l a t i v e importance of a p a r t i c u l a r protective device i n a plant undoubtedly changes with the evolution of i t s parasites. Such f l e x i b i l i t y necessitates the maintenance of a variety of defence mechanisms only one, or a few, of which may oper ate as the ' f r o n t - l i n e ' system at any one time and/or against any one pathogen, with the others acting i n an a u x i l i a r y or 'back-up' capacity. Thus, although i t may not be possible to demonstrate a primary role for a p a r t i c u l a r compound, i t i s not unreasonable to assume that i t s synthesis and accumulation s t i l l represent an impor tant, and possibly v i t a l , investment i n evolutionary terms. F i n a l l y , i t must be borne i n mind that chemical defences i n plants have evolved i n response to a number of b i o t i c pressures, not least those from herbivores, and p a r t i c u l a r l y insects. The r a p i d i t y with which damage i s caused by feeding i s such that, to be e f f e c t i v e ,


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counter measures must usually be equally rapid and the release o f preformed i n h i b i t o r s , toxins or repellants i s one way of achieving t h i s . The p o s s i b i l i t y therefore that s t e r o i d a l glycoalkaloids and saponins evolved primarily i n response to prédation pressures mainly from insects i s a very r e a l one (95). Even so, the general t o x i c i t y of glycoalkaloids and saponins renders i t u n l i k e l y that any deterrent role played by these compounds would be a highly s p e c i f i c one.

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RECEIVED May 1, 1986 In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Antifungal Activity of Plant Steroids - ACS Symposium Series (ACS

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