Themselves Against

C H A P T E R 6

How Plants Defend

Themselves Against

Pathogens

E A CH P L A NT species is affected by approximately one hundred or more different kinds of pathogens, including fungi, bacteria, viruses, parasitic higher plants, and nematodes. Frequently, a single plant is attacked by hundreds, thousands, and, in the leafspot diseases of large trees, probably by hundreds of thousands or millions of individuals of a single kind of pathogen. Yet, although such plants may suffer dam- a ge to a lesser or greater extent, many survive all these attacks and, not uncommonly, m a n a ge to grow well and to produce appreciable yields.

That plants b e c o me d i s e a s ed can b e readily ascertained by a more or less careful examination of one or a few plants anywhere. Almost every plant s e e ms to have at least a few infections on its foliage, some more perhaps on its stems and fruit and, possibly, on its roots. Why doesn't a pathogen, once it has established an infection, continue to invade and destroy a host plant entirely? A few kinds of pathogens, of course, can, and they do just that. But, of the thousands of species of plant pathogens, only about a h u n d r ed attack each plant species, and of these all but a few cause infections that are very localized in area.

105

Why do they not spread further? And why do s o me of these pathogens attack or cause more severe symptoms on s o me varieties while they either cannot infect others at all or c a u se only light symptoms on them? And why don't all plant pathogenic microorganisms attack each and every plant s p e c i e s?

Whether, of course, a particular plant will b e attacked by a certain pathogen is determined by the genetic constitution of the plant and, probably, of the pathogen. Furthermore, even when a pathogen does attack a plant, the kind and extent of the symptoms that will d e v e l op also d e p e nd on the genetic constitution of the host plant and of the pathogen. It is obvious then that whether a plant is susceptible or re- sistant to infection by a particular pathogen is determined by its genetic m a k e u p, as is the d e g r ee of its resistance or susceptibility and the kind of infection that will develop. But how does the ge- netic m a k e up of a plant determine its resistance or susceptibility to a pathogen?

T h e genetic material of a plant determines the extent of the poten- tialities of its cells and tissues. In most cases, plants and their patho- gens have evolved together, and only the plants that p o s s e s s ed fea- tures that h e l p ed them e s c a pe or limit infections by pathogens survived. Through mutation, hybridization, etc., additional such fea- tures were, from time to time, a d d ed to the plant arsenal. S o me of these features apparently consist of structural characteristics of the plant that act as physical obstacles and make it difficult or impossible for the pathogen to gain entrance into the plant or to spread through it.

Certain of these structural characteristics are present at the surface or in the tissues of the plant regardless of any contact the plant might or might not have had with the pathogen. Certain other structural charac- teristics, however, are absent in the healthy plant but b e g in to form as soon as the plant is attacked by the pathogen and in r e s p o n se to the infection by the pathogen. Whethe r present in advance or formed after infection, the structural, barricade-like features h e lp the plant defend itself against penetration or invasion by the pathogen. Since this de- fense is imparted to the plant by microscopically visible structural changes in its cells and tissues it is usually called structural defense.

T h e formation of structural barriers is, of course, the result of bio- chemical reactions taking place in the cells and tissues of the plant.

Such reactions, however, are not limited to the production of structural changes, but they also produce substances or conditions within the cell which, although invisible, contribute to the defense of the plant in a variety of ways. In s o me cases, substances toxic to the pathogen or conditions inhibiting the growth of the pathogen are present on the

Structural Defense 107

plant naturally, i.e., before the pathogen has e v en c o me into contact with the host. In other cases, these are p r o d u c ed only after the patho- gen has b e g un to infect the host. D e f e n se reactions derived from changes in the physiology or biochemistry of the host plant are usually called physiological or biochemical defense. Although the results of biochemical defense are sometimes a c c o m p a n i ed by visible manifes- tations, the defensive action stems primarily from the p r e s e n ce of chemical substances and the effect of biochemical reactions rather than those of structural barriers.

Different plants, of course, defend themselves against pathogens in different ways. E a ch kind of plant, probably, employs different de- fense mechanisms against each of the various pathogens that attack it.

T h e reaction of a plant to a pathogen, then, apparently d e p e n ds on the kind of pathogen but is also influenced by the environmental condi- tions prevalent during infection since they may influence the physio- logical activity of the plant to a lesser or greater extent.

Although, as we shall see, there are various mechanisms by which plants can defend themselves, pathogens s e em to be , nevertheless, capable of causing a great n u m b er of plant diseases. What, then, is the function of the defense m e c h a n i s m s? It is true that not all defense mechanisms protect the plant from infection, yet it is apparent that many of them, singly or in combination with others, do protect the host plant and either k e e p it free from infection or, at least, limit the extent of the infection. In many resistant varieties their resistance can be attributed directly to one or more of their defense mechanisms. In the susceptible varieties, on the other hand, effective defense mecha- nisms either do not exist or they do not appear soon enough after infection to prevent or limit it. Furthermore, the ability of the patho- gen to produce more virulent strains capable of b y p a s s i ng certain de- fense mechanisms of the plant and the effect of the environment on the physiology of the host must also b e taken into consideration w h en the outcome of any host-pathogen confrontation is examined.

Structural Defense

T h e first line of defense of plants against pathogens is their surface which the pathogen must penetrate if it is to cause infection. Patho- gens enter plants either by penetrating directly through the epidermal cell walls or by penetrating the epidermis through openings which either exist naturally at the epidermis, such as stomata, lenticels, and hydathodes, or appear at the epidermis as wounds created by various

animate or inanimate agents. Further invasion of the pathogen into the host is usually limited to m o v e m e nt of the pathogen b e t w e en or through succulent, thin-walled parenchymatous cells and, in the vas- cular diseases, through the lumen of xylem vessels. Certain structural characteristics of the plant epidermis or its interior may greatly affect the ability of the pathogen to penetrate or to invade a host plant. Such defense structures may either exist before inoculation and infection takes place, or they may b e p r o d u c ed by the host in response to infec- tion by the pathogen.

Preexisting Defense Structures

D e f e n se structures present in the plant, even before the pathogen comes in contact with the plant, include the amount and quality of wax and cuticle that cover the epidermal cells, the structure of the epidermal cell walls, the size, location, and shapes of stomata and lenticels, and the presence in the plant of tissues m a de of thick- walled cells that hinder the advance of the pathogen.

T H E R O LE OF WA X AND C U T I C LE AS BARRIERS TO P E N E T R A T I ON

T h e cuticle, consisting of cutin and waxes (Fig. 5), comprises the outermost covering of the epidermal cells and appears as a noncellu- lar, membranous layer.

Waxes are thought to play a defensive role on leaf and fruit surfaces by forming a hydrophobic surface which acts as a water repellent and thereby prevents the retention of a water drop or formation on the tis- sue of a film of water on which pathogens might b e d e p o s i t ed and germinate (fungi) or multiply (bacteria). A thick mat of hairs on a plant surface may also, conceivably, exert a similar water-repelling effect and may reduce infection.

Cuticle thickness has often b e e n linked to resistance to infection in diseases in which the pathogen enters its host only through direct penetration. This s e e ms to b e even more important in cases in which the pathogen (e.g., fungi) d e p e n ds mainly on mechanical pressure for penetration into its host. In several cases, the cuticle of varieties re- sistant to a pathogen were shown to b e thicker and more resistant to n e e d le puncture than that of susceptible varieties. A thick cuticle, in addition to limiting the entrance of a fungus, may also limit the ability of a fungus to break out of a d i s e a s ed plant and thus reduce the avail- able inoculum. A waxy cuticle may also limit the exudation of nu-

Structural Defense 109

trients and other substances required by the pathogen in the initial stages of infection and so may, indirectly, contribute to the defense of the plant. Cuticle thickness, however, is not always correlated with resistance and many plant varieties with cuticle of considerable thick- ness are easily invaded by directly penetrating pathogens.

T H E S T R U C T U RE OF E P I D E R M AL C E LL W A L LS

T h e thickness and toughness of the outer wall of epidermal cells are apparently important factors in the resistance of s o me plants to certain pathogens. Thick, tough walls of epidermal cells make direct penetra- tion by fungal pathogens difficult or impossible. Plants with such walls are often resistant, although, if the pathogen is introduced be - yond the epidermis of the s a me plants by m e a ns of a wound, the inner tissues of the plant are easily invaded by the pathogen. T h e toughness of outer epidermal cell walls, and therefore their resistance to p e n e - tration, may vary, even w h en they are of the s a me thickness, b e c a u se of lignification, p r e s e n ce of silicic acid, etc., in the epidermis of s o me plants or of s o me cells of these plants but not in others. This s e e ms to b e the case in rice plants, in which the outer walls of most epidermal cells are lignified and are s e l d om invaded by the rice blast fungus Piri- cularia oryzae, while the walls of the motor cells are pectinaceous rather than lignified and they are the main points of penetration by the fungus.

T H E S T R U C T U RE OF N A T U R AL O P E N I N GS

Many pathogenic fungi and bacteria enter plants only through sto- mata. Although the majority of them can force their way through closed stomata, some, like the stem rust of wheat, can enter only w h en stomata are open. T h u s, s o me wheat varieties, in which the stomata open late in the day, are resistant b e c a u se the germ tubes of spores germinating in the night d ew desiccate owing to evaporation of the d e w before the stomata b e g in to open. T h e kind of structure of stomata may also confer resistance to s o me varieties against certain of their pathogens. T h u s the mandarin variety Szinkum is resistant to the cit- rus canker bacterium Pseudomonas citri b e c a u se the stomata of this variety have a very narrow entrance surrounded by broad, elevated lips which prevent water and the bacteria s u s p e n d ed in it from enter- ing the stoma and initiating infection (Fig. 8).

Penetration through lenticels is rather c o m m on in several fungal and bacterial diseases. T h e size and, probably, the internal structure

of lenticels are important as defensive factors against disease. For example, the small lenticels of the fruit of most apple varieties protect them from infection by the apple spot bacterium Pseudomonas papil- losum, while the bacterium readily infects the fruit of the variety Mutsu through its large lenticels. Early formation of cork layers at the b a se of lenticels in many varieties is another effective defensive bar- rier, as indicated by the increased susceptibility of varieties slow in forming such cork layers.

I N T E R N AL S T R U C T U R AL BARRIERS TO P A T H O G EN INVASION T h e thickness and toughness of the cell walls of the tissues b e i ng invaded vary and may sometimes make the advance of the pathogen quite difficult. T h e presence, in particular, of b u n d l es or extended areas of sclerenchyma cells, such as are found in the stems of many cereal crops, may stop the further spread of pathogens like the stem rust fungi. Also, the xylem, b u n d le sheath, and sclerenchyma cells of

F i g. 8. Stomata of S z i n k um mandarin (A) resistant to the citrus canker bacterium (Pseudomonas citri) a nd of a variety (B) s u s c e p t i b le to the pathogen. SP = stomatal pore. (After M c L e a n, 1921.)

Structural Defense

the leaf veins effectively block the spread of s o me fungal, bacterial, and nematode pathogens which thus c a u se the various " a n g u l a r" leaf spots b e c a u se of their spread only into areas between, but not across, veins.

Defense Structures Formed in Response to Infection by the Pathogen

Although s o me pathogens may b e blocked from entering or from invading their host plants by the preformed superficial or internal defense structures, most pathogens m a n a ge to penetrate their hosts and to produce various degrees of infection. E v en after the pathogen has penetrated the preformed defense structures, however, plants exhibiting various d e g r e es of resistance usually respond by forming one or more types of structures that are more or less successful in de- fending the plant from further invasion by the pathogen. T h e common characteristic of these structures is that they are not present in the plant before infection, but their formation d e p e n ds on the irritation of the host by the pathogen. T h e type of defense structures p r o d u c ed may b e nonspecific, i.e., they may b e the s a me for different kinds of pathogens, or they may b e specific, i.e., certain defense structures are p r o d u c ed by a host plant only upon infection by one or a few specific pathogens. S o me of the defense structures formed are the result of differentiation of tissues or deposition of substances in tissues ahead of or around the pathogen and may b e called histological defense structures; others are the result of morphological and, possibly, chem - ical changes in the walls of invaded cells and may b e called cellular defense structures; still others are the result of morphological changes in the cytoplasm of the cells under attack and the process may b e called cytoplasmic defense reaction. Finally, death of the invaded cell may protect the plant from further invasion and this is called necrotic or hypersensitive defense reaction.

H I S T O L O G I C AL D E F E N SE S T R U C T U R ES

D e f e n se structures involving histological changes include the for- mation of cork layers, formation of abscission layers, formation of ty- loses, and deposition of gum.

Formation of Cork Layers

Infection of plants by fungi or bacteria and even by s o me viruses and nematodes frequently induces formation of several layers of cork cells b e y o nd the point of infection (Figs. 9 and 10), apparently as a re-

Structural Defense 113

F i g. 10. Formation of cork layer on potato tuber following infection with Rhizoctonia.

[After G. E. R a m s ey ( 1 9 1 7 ) . / . Agr. Res. 9: 4 2 1 - 4 2 6 .]

suit of stimulation of the host cells by substances secreted by the path- ogen. Cork layer formation in response to infection is more common around infection loci on the stem, roots, and young fruit but is also possible on leaves and other plant organs. T h e cork layer acts as a bar- rier that is usually impenetrable by the pathogen b e c a u se of the thick- ness and strength of the suberized walls of the tightly arranged cork cells. In some plants the cork cells may, in addition, be impregnated with lignin, which makes them even more resistant to penetration.

T h e cork layers not only inhibit the further invasion by the pathogen b e y o nd the initial lesion but also block the spread of any toxic sub- stances that the pathogen may secrete, and thus prevent any d a m a ge to the underlying tissues which could possibly result from such toxic substances. Furthermore, cork layers stop the flow of nutrients and water from the healthy to the infected area and, although this results in the death of s o me yet uninfected cells, it also deprives the pathogen

F i g. 9. Formation of cork layer b e t w e en infected a nd healthy areas of leaf. CL = cork layer; Ç = healthy leaf area; J = infected; Ñ = p h e l l o g e n. (After C u n n i n g h a m, 1928.)

of nourishment and may result in its weakening, inability to sporulate, or death. T h e d e ad tissues, including the pathogen, are thus delimit- ted by the cork layers and either remain in place forming a necrotic lesion (spot) or are p u s h ed outward by the underlying healthy tissues and form scabs that may further b e sloughed off and thus remove the pathogen from the host completely.

T h e effectiveness of the cork layers as defensive barriers d e p e n ds on the s p e ed with which the host can produce them following infec- tion, on the thickness and d e g r ee of impregnation of the cork cell walls with suberin or lignin, and on the properties of the particular pathogen. T h e inability of many pathogens to infect w o u n d ed organs after the latter have b e e n allowed to heal by forming cork layers, as in the case of Rhizopus soft rot of sweet potatoes, or the limitation of the pathogens to small stem, fruit, or leaf lesions, as in the cases of the common scab of potatoes c a u s ed by Streptomyces scabies, the cherry leaf spot c a u s ed by Coccomyces hiemalis, the Helminthosporium canker of pear, the necrotic lesions on Nicotiana glutinosa c a u s ed by tobacco mosaic virus, indicate the effectiveness of the cork layers as defensive structures.

Formation of Abscission Layers

Abscission layers are formed on young, active leaves of stone fruit trees following infection by any of several fungi, bacteria, or viruses.

An abscission layer does not consist of layers of cells of any particular kind, but rather of a gap b e t w e en two circular layers of cells of a leaf surrounding the locus of infection. U p on infection —e.g., of peach leaves by the bacterium Xanthomonas pruni or by the fungus Clado- sporium carpophilum, and of sour cherry trees by the necrotic ring- spot and other viruses —one or two layers of cells surrounding the in- fected spots swell and b e c o me thin walled, while the pectic materials of the middle m a m e l la b e t w e en them are dissolved (Fig. 11). T h e dis- solution of the middle lamella b e t w e en these two layers of cells throughout the thickness of the leaf leaves the inner layer of cells and the lesion they contain completely unsupported. T h e central area, be- ing completely cut off from the rest of the leaf, gradually shrivels, dies, and sloughs off, carrying with it the pathogen. Thus, the plant, by dis- carding the infected area along with a few yet uninfected cells, pro- tects the rest of the leaf tissue from b e c o m i ng invaded by the patho- gen and from b e c o m i ng affected by the toxic secretions of the pathogen. Infections of mature leaves or even young leaves in dry weather by the same pathogens may not result in formation of abscis-

Structural Defense 115 Abscission layer

Abscission layer

F i g. 11. Formation of a b s c i s s i on layer around a d i s e a s ed spot of a Prunus leaf. (After S a m u e l, 1927.)

sion layers but, after the initial swelling, the two layers of cells be - come suberized, and often lignified, and so protect the leaf by a mech - anism similar to cork layer formation rather than through formation of abscission layer. In that case the infected lesion b e c o m es necrotic, but it does not usually slough off.

Formation of Tyloses

Tyloses form in xylem vessels of most plants under various condi- tions of stress and during invasion by most of the vascular pathogens.

Tyloses are overgrowths of the protoplast of adjacent living paren- chymatous cells which protrude into xylem vessels through half- bordered pits (Fig. 12). Tyloses have cellulosic walls and may, by their size and numbers, clog the vessel completely. Tyloses are usually con- sidered to b e one of the factors responsible for d e v e l o p m e nt of the wilt

Healthy area Diseased area

Lignified cells

F i g. 12. D e v e l o p m e nt of tyloses in xylem v e s s e l s. Longitudinal (A) a nd cross-section (B) v i e ws of healthy v e s s e ls (left), a nd of v e s s e ls with tyloses. V e s s e ls on right are c o m p l e t e ly c l o g g ed with tyloses. PP = perforation plate; V = xylem v e s s e l; XP = xylem p a r e n c h y ma cell; T= tylosis.

Structural Defense 117

symptoms through interference with the transport of water in d i s e a s ed plants. T h e s p e ed and the location of appearance of tyloses after infec- tion, however, may determine whether tyloses will play a defensive role against pathogen invasion or whether they will b e detrimental to the health of the plant. T h u s, in s o me diseases, e.g., the sweet potato wilt, c a u s ed by the fungus Fusarium oxysporum f. batatas, tyloses in some varieties form abundantly and quickly ahead of the pathogen while the pathogen is still in the young roots, they block the further advance of the pathogen, and the plants of these varieties remain free of, and, therefore, resistant to this pathogen. Varieties in which few, if any, tyloses form ahead of the pathogen are susceptible to the d i s e a s e, although even in these plants tyloses do d e v e l op but m u ch later, after the pathogen has invaded the tissues. Tyloses are not penetrated by pathogens, and vessels clogged by tyloses a h e ad of the pathogen form an effective, impenetrable barrier to its spread.

Deposition of Gums

Various types of gums are p r o d u c ed by many plants around lesions following infection by pathogens or injury by mechanical means, in- sects, etc. G um secretion is most c o m m on in stone fruit trees infected with fungi (e.g., Valsa), bacteria (e.g., Pseudomonas syringae), or vi- ruses (e.g., Shirofugen cherry infected with necrotic ringspot virus).

In these and in many other diseases, however, the defensive role of gums stems from the fact that they are quickly deposited in the inter- cellular spaces and within the cells surrounding the locus of infection, thus forming an impenetrable barrier which completely encloses the pathogen. This is true, for example, in the silver leaf and black rot dis- eases of apple c a u s ed by the fungi Stereum purpureum and Physalos- pora cydoniae, respectively, in which g um forms in parenchyma and xylem cells as well as intercellular spaces of resistant varieties in ad- vance of the pathogen (Fig. 13), which b e c o m es isolated, starved, and sooner or later dies. G um may b e d e p o s i t ed also around lesions in leaves and limit the enlargement of the lesion as in the case of rice varieties resistant to the rice blast d i s e a s e, c a u s ed by Piriculafia ory- zae, or to Helminthosporium leaf spot. G um deposition in vessels may also play a role in the defense of plants against vascular pathogens, since in some resistant varieties g um deposits appear in the border pits and in the vessels well in advance of the pathogen and may block the spread of the pathogen into adjacent living cells or through the vessels clogged by the tyloses " c e m e n t e d" together with gums.

WP WF

XV

ι

Ì

F i g. 13. G um barrier in a p p le twig infected with Physalospora cydoniae. Ì = m y c e- lium in v e s s e l s; XV = xylem v e s s e l; WF = w o od fiber; WP = w o od p a r e n c h y m a.

(After H e s l e r, 1916.)

C E L L U L AR D E F E N SE S T R U C T U R ES

T h e cellular defense structures involve morphological changes in the cell wall, or derived from the cell wall, of the cell b e i ng invaded.

T h e effectiveness of these structures as defense m e c h a n i s ms s e e ms to be rather limited, however. T wo main types of such structures have b e e n observed in fungal diseases and include swelling of the cell wall and sheathing of the advancing hyphae.

Swelling of the Cell Wall

This form of defense reaction appears as a swelling of the outer wall of epidermal cells during direct penetration and may inhibit host pen- etration and establishment of infection by the pathogen. It may also appear as swelling of the walls of subepidermal cells b e i ng invaded by the pathogen and may limit the spread of the pathogen through the

Structural Defense 119

host as in the case of the c u c u m b er varieties resistant to the scab fun- gus Cladosporium cucumerinum. T h e thickening of the cell wall is often accompanied by deposition of suberin, lignin, or g u m my materi- als in the thickened portion, which further increases its resistance to penetration.

Sheathing of Hyphae

H y p h ae of fungi penetrating a cell wall are often e n v e l o p ed in a sheath formed by the extension of the cell wall inward in a way that surrounds and p r e c e d es the invading hypha (Fig. 14). It is not clear whether the sheath consists of cellulose, " c a l l u s" substances, or other materials and it appears that, at least in s o me cases, it originates from substances deposited around the hypha by the cytoplasm rather than b e i ng formed by the cell wall. H y p h a e, however, generally m a n a ge to penetrate the sheath and invade the cell lumen, although their ad- vance is probably slowed down by the sheath that envelops them.

F i g. 14. F o r m a t i on of sheath around h y p ha penetrating a cell wall. CW= cell wall; Ç = hypha; A = a p p r e s s o r i u m; AH = a d v a n c i ng h y p ha still e n c l o s ed in sheath; HC = h y p ha in c y t o p l a s m; S = sheath.

CYTOPLASMIC D E F E N SE R E A C T I ON

E v en w h en the pathogen has m a n a g ed to penetrate the outer de- fenses of the plant and has gotten through the cell wall of a particular cell, the cytoplasmic contents of cells may b e organized in ways that appear to present an additional —and the last—line of resistance of the cell against the pathogen invader.

F e w cases of such defense reactions are known, and almost all s e em to be associated with diseases c a u s ed by slowly growing, weakly path- ogenic, fungi that induce chronic diseases or nearly symbiotic condi- tions. T h u s, in the outer cells of s o me of the mycorrhiza-like diseases, the cytoplasm invests the c l u mp of hyphae and the nucleus is stretched to the point where it breaks in two. In these cells however, the cytoplasmic reaction is overcome and the protoplast disappears while fungal growth increases. On the other hand, in invaded cells d e e p er in the root the cytoplasm and nucleus enlarge. T h e cytoplasm b e c o m es granular and d e n s e, and various particles or organelle-like structures appear in it. Finally, the mycelium of the fungal pathogen disintegrates into small granular bodies or larger clusterlike conglom- erations, and thus the advance of the invasion stops.

N E C R O T IC D E F E N SE REACTION: D E F E N SE THROUGH HYPERSENSITIVITY

In many host-pathogen combinations, the pathogen may penetrate the cell wall, but as soon as it establishes contact with the protoplast of the cell, the nucleus moves toward the intruding pathogen and soon disintegrates, and brown, resinlike granules form in the cytoplasm, first around the pathogen and then throughout the cytoplasm. A swell- ing of cell membranes m ay also occur simultaneously. As the brown- ing discoloration of the cytoplasm of the plant cell continues and death sets in, the invading hypha begins to degenerate as its nucleus disintegrates into a h o m o g e n e o us mass and its cytoplasm b e c o m es d e n se (Fig. 15). In most cases the hypha does not grow out of such cells and further invasion is stopped.

T h e necrotic or hypersensitive type of defense is very common, par- ticularly in diseases c a u s ed by obligate fungal parasites and by viruses and nematodes. Although the mechanism of such defense s e e ms to b e of a complex, biochemical nature, a simple explanation would be that the necrotic tissue isolates the obligate parasite from the living sub- stance, on which it d e p e n ds absolutely for its nutrition for growth and multiplication, and, therefore, results in its starvation and death. T h e

Structural Defense

1

F i g. 15. S t a g es in the d e v e l o p m e nt of necrotic d e f e n se reaction in cell of a very resist- ant potato variety infected by Phytophthora infestans. Í = n u c l e u s; PS = proto- p l a s m ic strands; Æ = z o o s p o r e; Ç = h y p h a; G = granular material; NC = necrotic cell. [After K. T o m i y a ma (1956). Ann. Phytopathol. Soc. Japan 21: 54-62.]

faster the host cell dies following invasion the more resistant to infec- tion the plant s e e ms to b e .

Selected References

Akai, S. 1959. Histology of d e f e n se in plants. In " P l a nt P a t h o l o g y" (J. G. Horsfall a nd A.

E . D i m o n d, eds.), Vol. I, p p. 3 9 1 - 4 3 4 . A c a d e m ic Press, N e w York.

Brooks, F. T., a nd G. H. Brenchley. 1931. Silver leaf d i s e a s e. V I . / . Pomol. Hort. Set.

9 : 1 - 2 9.

C u n n i n g h a m, H. S. 1928. A study of the histologic c h a n g es i n d u c ed in l e a v es by certain leaf-spotting fungi. Phytopathology 1 8 : 7 1 7 - 7 5 1 .

Hart, H e l e n. 1929. Relation of stomatal b e h a v i o ur to stem-rust resistance in wheat. J.

Agr. Res. 3 9 : 9 2 9 - 9 4 8 .

Hart, H e l e n . 1931. M o r p h o l o g ic a nd p h y s i o l o g ic studies on stem-rust resistance in ce - reals. U.S. Dept. Agr. and Minn. Agr. Expt. Sta. Tech. Bull. 2 6 6 : 7 6 p p.

H e s l e r, L. R. 1916. Black rot, leaf spot, a nd canker of p o m a c e o us fruits. Í. Y. (Cornell) Agr. Expt. Sta. Bull. 3 7 9 : 5 3 - 1 4 8 .

Jhooty, J. S., a nd W. E. M c K e e n. 1965. S t u d i es on p o w d e ry m i l d ew of strawberry c a u s ed b y Sphaerotheca macularis. Phytopathology 5 5 : 2 8 1 - 2 8 5 .

J o n e s, A. P. 1931. T h e histogeny of potato scab. Ann. Appl. Biol. 18:313-333 .

M c L e a n, F. T. 1921 . A study of the structure of the stomata of two s p e c i es of citrus in relation to citrus canker. Bull. Torrey Botan. Club 4 8 : 1 0 1 - 1 0 6 .

Martin, J. T. 1964. Role of cuticle in the d e f e n se against plant d i s e a s e. Ann. Rev. Phyto- pathol. 2 : 8 1 - 1 0 0.

Muller, K. O. 1959. Hypersensitivity. In " P l a nt P a t h o l o g y" (J. G. Horsfall a nd A. E . D i m o n d, eds.), Vol. I, p p. 4 6 9 - 5 1 9 . A c a d e m ic Press, N e w York.

Pierson, C. F., a nd J. C. Walker. 1954. Relation of Cladosporium cucumerinum to sus- c e p t i b le a nd resistant c u c u m b er tissue. Phytopathology 4 4 : 4 5 9 - 4 6 5 .

R o m i n g, R. W., a nd R. M. C a l d w e l l. 1964. Stomatal exclusion of Puccinia recondita by w h e at p e d u n c l es a nd sheaths. Phytopathology 5 4 : 2 1 4 - 2 1 8 .

S a m u e l, G. 1927. On the shot-hole d i s e a se c a u s ed by Cladosporium carpophilum a nd on the " s h o t h o l e" effect. Ann. Botany (London) 4 1 : 3 7 5 - 4 0 4 .

Stanghellini, Ì . E., a nd M. Aragaki. 1966. Relation of p e r i d e rm formation a nd callose deposition to anthracnose resistance in p a p a ya fruit. Phytopathology 5 6 : 4 4 4 - 4 5 0 . Sharvelle, R. 1936. T h e nature of resistance of flax to Melampsora lini. J. Agr. Res.

5 3 : 8 1 - 1 2 7 .

Weimer, J. L., a nd L. L. Harter. 1921 . Wound-cork formation in the s w e et p o t a t o . /. Agr.

Res. 2 1 : 6 3 7 - 6 4 7 .

Wells, J. M. 1963. Anatomical aspects of resistance in sweetpotato to Fusarium wilt, Phytopathology 5 3 : 7 4 7 - 7 4 8 (abstr.).

Wylie, R. B. 1931. Cicatrization of foliage l e a v e s. I I. W o u n d r e s p o n s es of certain broad- l e a v ed e v e r g r e e n s. Botan. Gaz. 9 2 : 2 7 9 - 2 9 5 .

Biochemical D e f e n se

Although preformed or induced structural characteristics of host plants may provide the plant with various degrees of defense against attacking pathogens, it is b e c o m i ng increasingly clear that the resis- tance of a plant against pathogen attacks d e p e n ds not so m u ch on its structural barriers as on the metabolic processes of its cells preceding or following infection. This b e c o m es apparent from the fact that a par- ticular pathogen will not penetrate or infect certain plant varieties al- though no structural barriers of any kind s e em to b e present or to form in these varieties. Similarly, in resistant varieties, in which biochemi- cal defense mechanisms are operating, the growth rate of d i s e a se le- sions soon slows down and, finally, in the a b s e n ce of structural de- fenses, their growth is completely checked. Moreover, many patho- gens which enter nonhost plants naturally, or which are introduced into nonhost plants artificially, fail to cause infection and to induce symptom development, although no apparent visible host structures inhibit them from doing so. T h e se examples suggest that defense mechanisms of a chemical rather than a structural nature are respon- sible for the resistance to infection exhibited by plants against certain pathogens.

Biochemical Defense 123

T h e biochemical defense m e c h a n i s ms may consist of the presence or a b s e n ce of a particular chemical substance or group of substances in a host plant which interferes with the growth and multiplication of the pathogen. Such a condition may exist before the pathogen attacks the plant or it may appear as a reaction of the host following infection by the pathogen. Biochemical defense reactions on the part of the host may b e induced by the activities and secretions of the pathogen and may b e a i m ed at stopping these activities and at inactivating the sub- stances secreted by the pathogen, or they may b e directed toward b y p a s s i ng the injurious effects of the pathogen through alterations in the normal metabolic processes of the plant.

Regardless of how the biochemical defense is expressed, it must b e kept in m i nd that it is always the result of genetic potentialities pres- ent in the plant cells at all times, but which manifest themselves only as an interaction b e t w e en the host and the pathogen. Whethe r pre- formed or induced, the types of biochemical defense mechanisms exhibited by a plant against a certain pathogen are the result of evolu- tionary changes brought about in the host during its coexistence through time with the pathogen and, are, therefore, genetically con- trolled.

Preexisting Biochemical Defense

INHIBITORS R E L E A S ED BY T H E P L A NT IN I TS ENVIRONMENT

Plants, generally, exude substances through the surface of their above-ground parts as well as through the surface of their roots. T h e e x u d ed substances either accumulate on the surface of the plant or- gan, e.g., leaf, or they may diffuse into the moisture surrounding the organ, e.g., water droplets on a leaf or the moist soil environment sur- rounding the root. A m o ng the e x u d ed substances are included most of the substances involved in the cellular metabolism of higher plants such as amino acids, simple sugars, glycosides, organic acids, en- zymes, alkaloids, and inorganic ions.

S o me kinds of plants, however, release, in addition to the above substances, other compounds which s e em to have an inhibitory action against certain pathogens. Fungitoxic exudates on leaves of tomato and sugar beet, e.g., s e em to b e present in sufficient concentrations to inhibit germination of conidia of Botrytis and Cercospora in d ew or rain droplets on these leaves. Similarly, w h en a wax extract from leaves of apple varieties resistant to powdery m i l d ew (Podosphaera

leucotricha) was deposited on leaves of susceptible varieties, and the latter were inoculated with conidia of the fungus, the conidia failed to germinate as did conidia of Botrytis on similarly treated susceptible leaves of Vicia faba. Several other cases of inhibition of infection by substances e x u d ed from leaves are known.

T h e role of exudates from roots or from other underground plant parts in the defense of plants against pathogens has b e e n investigated rather extensively but, in most cases, the results are rather inconclu- sive. In the case of onion s m u d g e, c a u s ed by the fungus Colletotri- chum circinans, resistant varieties generally have red scales and con- tain, in addition to the red pigments, protocatechuic acid and catechol.

In the presence of water drops or soil moisture containing conidia of the fungus, these two fungitoxic substances diffuse into the liquid, inhibit the germination of the conidia and cause them to burst, thus protecting the plant from infection. Both the fungitoxic exudates and the inhibition of infection are missing in the white-scaled, susceptible onion varieties.

Root exudates responsible for the resistance of s o me plants against certain of their pathogens have b e e n implicated in the fusarial wilts of flax and pea. In the first case, resistant flax varieties exude a glucoside which upon breakdown produces hydrocyanide ( H C N ), which is an extremely potent poison to many living organisms, including Fusar- ium. Interestingly, H C N does not affect the growth of another soil fungus, Trichoderma mride, which produces the antibiotic glyotoxin and is antagonistic to many soil fungi, including Fusarium. In the case of the Fusarium wilt of pea, it has b e e n claimed that at least part of the resistance of three p ea varieties, each showing differential resistance to three races of the fungus Fusarium oxysporum f. pisi, is d ue to the fungi toxicity of the root exudates of the plants. This was s u g g e s t ed by observations that conidial germination and mycelial growth of the fungus were d e p r e s s ed or inhibited by extracts from resistant plants while it was stimulated by those from susceptible plants.

INHIBITORS P R E S E NT IN P L A NT C E L LS B E F O RE I N F E C T I ON Many attempts have b e e n m a de to find and identify toxic com- pounds which, by their presence in the resistant varieties and a b s e n ce or smaller concentration in the susceptible ones, could b e a s s i g n ed a defensive role against the particular pathogen. F e w, however, if any, cases in which such compounds were correlated with preinfectional defense against the pathogen have b e e n adequately documented.

T h u s, in the case of the potato scab, c a u s ed by Streptomyces scabies,

Biochemical Defense 125

tubers of resistant varieties contain higher concentrations of chloro- genic acid, which is a phenolic c o m p o u nd toxic to the pathogen, than do tubers of susceptible varieties. T h e concentration of chlorogenic acid in resistant varieties is especially high in tissues through which the pathogen enters (lenticels) and in which it normally grows (outer layers of tuber). T h e high content in chlorogenic acid of roots of cer- tain potato varieties has also b e e n considered as the main mechanism of defense against the Verticillium wilt pathogen, since resistant vari- eties contain more chlorogenic acid in their roots than do susceptible varieties, and since even susceptible varieties are not attacked while young, w h en their roots contain high concentrations of chlorogenic acid, but b e c o me susceptible later, when their content in chlorogenic acid declines.

A substance, possibly the glucoside avenacin, was found to b e pres- ent in oat seedlings and to inhibit in vitro the germination of spores of several species of fungi that do not normally attack oats while, on the contrary, it stimulated the spore germination of species of the same gener a that normally parasitize oats. A variety of other sub- stances contained in plant cells have b e e n s u g g e s t ed as inhibitors of pathogens. A m o ng these are several phenols, oxazolinones, alkaloids, etc., but their role has not yet b e e n fully documented. A rather large number of toxic substances, mostly phenols, has b e e n shown to b e responsible for the natural resistance of w o od to deterioration by mi- croorganisms which can attack the s a me kind of w o od after the naturally occurring fungitoxic substances have b e e n w a s h ed away.

In almost all cases of preformed inhibitors in plant cells, however, other defense mechanisms also s e em to b e involved in the final ex- pression of resistance to infection, so that the role of the preinfectional toxic substances in cells is probably contributory rather than solely responsible for resistance.

D E F E N SE THROUGH D E F I C I E N CY IN N U T R I E N TS E S S E N T I AL F OR T H E P A T H O G EN

Although most nonobligate parasites have a rather w i de host range and can usually attack several plants — sometimes quite different tax- onomically — s o me nonobligate and most obligate parsites, especially a m o ng the fungi, can attack only a few hosts, sometimes only a single variety. This might suggest that parasites in the first group can obtain all the nutrients they n e e d from a variety of plants, possibly b e c a u se they n e e d only a few basic substances and can synthesize the rest from these substances. On the contrary, the host specialization of the

obligate and some nonobligate fungi may b e d ue to the specialized n e e d of these pathogens for a substance that is present, or available in adequate quantities, only in the host(s) they can infect. Thus, the species or varieties of plants that do not produce this substance w o u ld b e resistant to the pathogen that requires it, and, other things b e i ng equal, the less common this substance is in plants the narrower the host range of the pathogen will be . Similarly, if a particular race of a pathogen has lost, through mutation, the ability to synthesize a certain substance not found in a natural host plant, this race will appear non- virulent to this host and the host will b e protected from infection be - cause it lacks that substance.

Only two examples of plant diseases, in which the host is protected by the lack of a substance essential to the pathogen, are known. In the Rhizoctonia diseases of seedlings, infection d e p e n ds on the presence in the susceptible plant of a substance necessary for initiation of a hyphal cushion formation from which the fungus sends into the plant its penetration hyphae. This substance is apparently lacking in resist- ant plants, cushions do not form and infection does not occur. T h e fungus does not normally form hyphal cushions in pure cultures, but will form them when extracts from a susceptible plant are a d d ed to the culture. Addition to the cultures of extracts from resistant plants does not induce cushion formation.

In the apple scab disease, c a u s ed by Venturia inaequalis, certain pathogen mutants were isolated from the wild pathogenic fungus, which, through mutation, lost the ability to synthesize a certain growth factor and, also, the ability to cause infection. When , however, the particular growth factor is sprayed on the a p p le leaves during in- oculation with the mutant, the mutant not only survives, but it also causes infection. T h e advance of the infection though continues only as long as the growth factor is s u p p l i ed to the mutant externally, and the infection stops soon after the application of the growth factor is discontinued.

D E F E N SE THROUGH A B S E N CE OF COMMON A N T I G E NS

It is known that animals produce specific antibodies against foreign proteins (antigens) injected into their system and, thereby, defend themselves against infections by microorganisms. Animals do not, on the other hand, produce antibodies against an injected protein if such a protein is also produced by the animal itself.

T h e r e is no conclusive evidence at present that plants can produce antibodies against invading pathogens such as fungi, bacteria or virus-

Biochemical Defense 127

es, but certain studies indicate that s o me kind of immunological re- s p o n se may also b e operating in plants. In serological work with the flax rust d i s e a s e, c a u s ed by the fungus Melampsora lint, the antigens of four rust races, each differing from the others in one g e n e for viru- lence, were c o m p a r ed with the antigens of four flax varieties, each b e i ng susceptible to one or more of the four rust races and differing from each other in one or more g e n e s for resistance. It was shown in these experiments that a specific antigen in each of the four rust races is commonly shared by only those lines of flax that are susceptible to a particular race. Whe n a given variety does not have an antigen that is present in a particular rust race, the variety is resistant to that race, suggesting that susceptibility and resistance are d ue to the p r e s e n ce or a b s e n ce of the specific rust antigens in the flax lines. Similar results were obtained with antigens of certain varieties of cotton and certain races of the bacterium Xanthomonas malvacearum, the c a u se of angu- lar leaf spot of cotton, and also b e t w e en varieties of sweet potato and races of the black rot fungus Ceratocystis fimbriate.

Although the above experimental results support the possibility of common antigens b e i ng involved in the susceptibility or resistance of plants to infection and d i s e a s e, the occurrence of i m m u ne responses in plants similar to those that occur in animals is questionable, since there is no conclusive e v i d e n ce that antibodies or e v en antibody-like substances are formed in plant cells or tissues in response to the pres- enc e of foreign antigens.

Biochemical Defense Induced by the Attacking Pathogen

BIOCHEMICAL INHIBITORS P R O D U C ED IN P L A N TS IN R E S P O N SE TO INJURY BY T H E P A T H O G EN

Plant cells and tissues respond to injury, whether it b e c a u s ed by a pathogen, mechanical or chemical agent, through a series of biochem- ical reactions which s e em to b e a i m ed at isolating the irritant and at healing the wound. This reaction is often associated with the produc- tion of fungitoxic substances around the site of injury as well as forma- tion of layers of protective tissue such as callus and cork. S o me of the c o m p o u n ds thus p r o d u c ed are present in concentrations high enough to inhibit growth of most nonpathogens of the host. T h e se compounds include s o me common phenolics, such as chlorogenic and caffeic ac- ids, oxidation products of phloretin, hydroquinone, and hydroxytyra- mine, and also the phytoalexins. S o me of these compounds, e.g., chlorogenic and caffeic acids, are widely distributed throughout the

b e n z e n e ring are usually referred to as polyphenols. T h e number and variety of phenolic compounds found in plants is very large and in- cludes the anthocyanins, leucoanthocyanins, anthoxanthines, glyco- sides, sugar esters of phenolic acids, coumarin derivatives, and others.

Phenolic compounds are produced in plants primarily via the shi- kimic acid pathway and the acetic acid pathway. In the latter, pheno- lics are produced by a " h e ad to tail" condensation of acetate units de- rived from the breakdown of sugars during respiration. In the shikimic acid pathway, phosphoenol pyruvate from glycolysis reacts with ery- throse, produced during the pentose pathway, the activity of which is increased in d i s e a s ed plants, and forms dehydroquinic and then shikimic acid. F r om shikimic acid and through various intermediate compounds, some of which are still unknown, phenolic c o m p o u n ds are formed. S o me of the most important phenolic c o m p o u n ds impli- cated in the defense of plants against pathogens are shown, together with a diagrammatic s c h e me of their origin, in S c h e me 2.

Many of the enzymes catalyzing the reactions of biosynthesis of phenolic compounds are already known to occur in plants. S o me of

S c h e me 2. Origin a nd structure of s o me fungitoxic p h e n o l ic c o m p o u n ds synthe- s i z ed via the shikimic acid pathway.

Phenol

plant kingdom, while most others are limited to a narrow host range.

Although such substances are p r o d u c ed in response to injury as well as to infection, they are p r o d u c ed in higher quantities following infec- tion rather than injury, probably b e c a u se of greater physiological stress d ue to the continuous irritation of the infected tissue by the pathogen.

Role of Phenolic Compounds

Nature and Origin of Phenolic Compounds. Phenolic compounds are those which contain one or more aromatic (benzene) rings with one or more phenolic hydroxyl groups. T h e simplest c o m p o u nd is phenol. C o m p o u n ds containing more than one hydroxyl group on a

Carbohydrate

Glycolysis Pentose pathway

Phosphoenol | pyruvate C—OP

CHO

H (TI ^OH Erythrose HCOH phosphate

CH2OP

CH3O.

Phaseolin

HO Ç è \ . OH

U

HO ËÔÔÔÃË CH2—C—COOH Υ º Prephenic acid H ^ ^ C O OH

C H2- C H ( N H2) — C O OH

Tyrosine

Ί

CH2CH2C OH

OH OH Phloretin

C H = C H - C O OH

Coumaric acid

C H = C H — C —Ï

y=y H O ) — / OH HO HO

Chlorogenic acid

CH3O CHS

Scopoletin

Isocoumarin

HO Ï CH2—CH(NH2)—COOH

Phenylalanine

\ C H = C H— COOH

Cinnamic acid

Coumarin

•

Pisatin

t

H O —^

r

HO

CH=CH—COOH

Caffeic acid Umbelliferone

CH=CH—COOH

Ferulic acid HO —

HO

CH,0.

HC ÇÏ -

ÇÏ

L c H

^ C H3

the phenolic enzymes most commonly found in healthy as well as dis- e a s ed plants are the phenol-oxidizing enzymes, known as phenolases, phenoloxidases, polyphenoloxidases, etc. T h e y oxidize various phe- nolic compounds in the p r e s e n ce of oxygen either by adding oxygen to monophenols, thus forming complicated polyphenols such as fla- vonoids, tannins, lignins, etc., or by removing hydrogen and thus forming the various oxidation products of phenolics, quinones, which are generally p i g m e n t ed c o m p o u n ds and play a role in the browning discoloration of plant tissues. Another e n z y me commonly found in plants and capable of oxidizing several phenolics is peroxidase, which removes hydrogen atoms from the phenolic c o m p o u n ds and combines them with the oxygen of the peroxide.

S o me of the phenolics implicated in d i s e a se resistance are found in healthy as well as d i s e a s ed plants, but their synthesis or accumulation seems to b e accelerated following infection. Such c o m p o u n ds may b e called " c o m m o n" phenolic compounds. Certain other phenolics, however, are not present in healthy plants but are p r o d u c ed upon stimulation of a plant by a pathogen or by a mechanical or chemical injury. Such c o m p o u n ds are known as phytoalexins.

"Common" Phenolics. It has often b e e n observed that certain phe- nolic c o m p o u n ds that are toxic to pathogens are produced and accu- mulate at a faster rate after infection in a resistant variety than in a susceptible variety. E x a m p l es of such phenolic compounds are chlorogenic acid in sweet potato, white potato, and carrot infected with the fungus Ceratocystis fimbriata; orthodiphenols and scopole- tin in potato infected with the late blight fungus Phytophthora infes- tans, and in local lesion-forming tobacco plants infected with tobacco mosaic virus; caffeic acid and umbelliferone in sweet potato infected with C. fimbriata; certain steroid glycoalkaloids (á-solanine, a-chal- conine, and solanidine) in potato infected with Helminthosporium carbonum, etc. It appears that most of these compounds are not pres- ent in resistant tissues in concentrations high e n o u gh to inhibit infec- tion but that a continuous flow of phenolic compounds from adjacent tissue to the infection site may take place and thus retard d i s e a se de- velopment. This may also explain the fact that although susceptible plants may have higher final levels of phenols than resistant ones, it is the localization of infection in the resistant plants that is responsible for the higher, per cell, concentration of phenolics at the site of infec- tion of resistant plants and for the inhibition of further infection.

Although s o me of the common phenolics may each reach concentra- tions that could b e toxic to the pathogen, it should b e noted that sev- eral of them appear concurrently in the s a me d i s e a s ed tissue and it is

Biochemical Defense

possible that the c o m b i n ed toxic effect of all fungitoxic phenolics present, rather than that of each one separately, is responsible for the inhibition of infection in resistant varieties.

It is known, on the other hand, that several phenolics affect the physiological processes of the host and may function as hormones important in physiological stress, thus serving a key function in w o u nd healing and d i s e a se resistance that may b e far more important than their effect as inhibitors per se of microbial development. For example, chlorogenic and caffeic acids as well as other phenolics in- hibit indoleacetic acid (IAA) oxidase and stimulate the production of IAA from tryptophan. Chlorogenic acid has also b e e n associated with growth responses in plants, such as proliferation and increased meta- bolic activity of cells adjacent to injury or infection, and in gall forma- tion.

Phytoalexins. Phytoalexins are fungitoxic substances, mostly pheno- lics, produced in many plants as a result of stimulation by micro- organisms, or chemical and mechanical injury, and which inhibit the growth of microorganisms pathogenic to plants. T h e se c o m p o u n ds include: ipomeamarone, orchinol, an isocoumarin, pisatin, and phaseolin. Ipomeamarone is a nonphenolic furanoterpenoid and appears to b e synthesized via the acetate pathway, and the others are synthesized via the skikimic acid pathway.

I p o m e a m a r o ne is p r o d u c ed in sweet potatoes inoculated with the black rot fungus Ceratocystis fimbriate. Its concentration increases substantially in infected tissue and in d i s e a s ed tissue very closely ad-

jacent to infected tissue. I p o m e a m a r o ne levels in the infected tissue often e x c e e d 1.0 %, and it is known that concentrations of e v en 0.1 % of ipomeamarone have a striking inhibitory effect on the fungus. More ipomeamarone is p r o d u c ed in resistant than in susceptible varieties following infection.

Orchinol is p r o d u c ed by Orchis militaris upon infection by Rhizoc- tonia repens and several other fungi. Hircinol, a derivative of orchin- ol, is also p r o d u c ed in Orchis and other plants following infection, and

it appears to have phytoalexin-like action. Another p r e s u m ed phyto- alexin n a m ed rishitin was isolated recently from resistant potato tu- bers inoculated with the late-blight fungus Phytophthora infestans.

Isocoumarin is produced in fungitoxic levels at the site of penetra- tion of carrot roots by the fungus C. fimbriata, a nonpathogen of carrot roots. Isocoumarin strongly inhibits the growth of this fungus in cul- ture, and the cessation of fungal growth in the carrot coincides with the production of isocoumarin at fungitoxic levels in the environment of the fungus. Different other microorganisms also induce formation of isocoumarin in carrot tissue, but its concentration varies from 5 to 342 μg per gram of carrot, d e p e n d i ng upon the organism u s ed for induc- tion.

Pisatin is produced by the e x p o s ed endocarp of detached p ea pods in response to inoculation with many fungi or injury. Whe n different fungi, pathogenic and nonpathogenic to pea, are u s ed for inoculation, they induce production of different concentrations of pisatin. T h e nonpathogenic fungi induce formation of pisatin at concentrations high enough to inhibit their growth. P ea pathogens, if they induce the formation of pisatin, induce it at concentrations much below those that are toxic to the pathogen. Production of pisatin by p ea pods inoculated with Monilinia fructicola, a nonpathogen, is r e d u c ed or d e l a y ed at high temperatures or anaerobic storage, and the p ea pods b e c o me sus- ceptible to this fungus, while storage at normal temperatures and aerobic conditions result in the accumulation of high levels of pisatin and resistance.

Phaseolin is similar to pisatin in chemistry and function. It was iso- lated from detached, o p e n ed b e an pods following inoculation with the nonpathogen Monilinia fructicola.

Phytoalexins in general are not produced by healthy plants but are produced by plants following infection, injury, or at least stimulation by certain fungal, but not bacterial, secretions. F u n gi pathogenic to a particular plant species s e em to stimulate production of generally lower concentration of phytoalexins than nonpathogens and, b e s i d e s, pathogenic fungi s e em to b e less sensitive to the toxicity of the phyto- alexin produced by their host plant than are nonpathogenic fungi. In the case of pisatin production by p ea pods inoculated with the patho- gen Ascochyta pisi, different varieties of p e a produce different concen- trations of pisatin which approximately parallel the resistance of the variety to the pathogen. Whe n the s a me p e a variety is inoculated with different strains of the fungus, the concentration of pisatin produced varies with the fungus strain u s ed for inoculation and it is, approxi-