How Pathogens Attack Plants
T H E I N T A C T, healthy plant is a community of cells built in a fortress- like fashion. T h e plant surfaces that come in contact with the environ- men t either consist of the cellulosic outer cell walls, such as those found in the epidermal cells of roots and in the intercellular spaces of parenchyma, especially leaf parenchyma cells, or, in the aerial parts of plants, consist of a layer of cuticle which covers the epidermal cell walls. Often an additional layer, consisting of waxes, is deposited out- side the cuticle, especially on younger parts of plants (Fig. 5).
Pathogens attack plants b e c a u se during their evolutionary develop- men t they have acquired the ability to live off the substances manufac- tured by the host plants, and some of the pathogens d e p e n d on these substances for survival. Such substances, however, are contained in the protoplast of the plant cells, and, if pathogens are to gain access to them, they must first penetrate the outer barriers formed by the cuticle and/or cell walls. E v en after the outer cell walls have b e e n penetrat- ed, further invasion of the plant by the pathogen will necessitate pen- etration of more cell walls. Furthermore, the plant cell contents are not always found in forms immediately utilizable by the pathogen and 36
Mechanical Forces 37
must be transformed to units which the pathogen can absorb and as- similate. Moreover, the plant, reacting to the p r e s e n ce and activities of the pathogen, produces structures and chemical substances that interfere with the advance or the existence of the pathogen; if the pathogen is to survive and to continue living off the plant, it must b e able to overcome such obstacles. All morphological and physiological properties of plants are the expression of their genetic constitution, and the s a me is true for pathogens. T h e interactions of plants and pathogens, whatever these might b e , are governed, in the final analy- sis, by the genetic potentials of the two organisms. Phenotypic expres- sions of the genetic material of the plant which interfere with infec- tion by the pathogen, must, therefore, b e met by phenotypic characteristics d e v e l o p ed through changes or readjustments in the genetic material of the pathogen.
F r om the above, it b e c o m es evident that for a pathoggrr to infect a plant it must b e able to make its way into and through the plant, obtain nutrients from the plant, and neutralize the defense reactions of the plant. Pathogens accomplish these activities mostly through secre- tions of chemical substances that affect certain components or meta- bolic mechanisms of their hosts. Penetration and invasion, however, s e em to b e a i d ed by, or in s o me cases b e entirely the result of, me- chanical force exerted by certain pathogens on the cell walls of the plant.
MECHANICAL FORCES E X E R T ED BY PATHOGENS ON HOST TISSUES Plant pathogens are, generally, tiny microorganisms which, with the
exception of nematodes, lack a muscular system that w o u ld enable them to d e v e l op a "voluntary" force and apply it on a plant surface. It is c o n c e d ed that viruses can exert no mechanical force whatsoever, and the s a me s e e ms to b e true for bacteria. S o me fungi, parasitic higher plants, and nematodes, however, appear to apply mechanical pressure to the plant surface they are about to penetrate, although the amount of pressure may vary greatly with the d e g r ee of "presoften- i n g" of the plant surface by the enzymatic secretions of the pathogen.
F or fungi and parasitic higher plants to penetrate a plant surface, they must, generally, first adhere to it. Although hyphae and radicles are usually surrounded by mucilaginous substances, their adhesion to the plant s e e ms to b e brought about primarily by the intermolecular forces d e v e l o p i ng b e t w e en the surfaces of plant and pathogen upon
/Cuticle
— Epidermal cells Wax projections
1
-Wax layer^Wax lamellae
^ C u t i n
Cellulose lamellae Pectin lamellae Cellulose layer Plasma membrane Cytoplasm
F i g. 5. S c h e m a t ic representation of the structure a nd composition of the cuticle a nd cell wall of foliar e p i d e r m al cells. [ A d a p t ed from G o o d m a n, Kiraly, a nd Zaitlin (1967). " T he Biochemistry a nd P h y s i o l o gy of Infectious Plant D i s e a s e /' Van Nostrand, Princeton, N e w J e r s e y .]
close contact with each other. After contact is established, the diame- ter of the part of hypha or radicle in contact with the host increases and forms a flattened, bulblike structure called the " a p p r e s s o r i u m ."
This increases the adherent area b e t w e en the two organisms and se- curely fastens the pathogen to the plant. It is from the appressorium that a fine growing point, called the "penetration p e g ," arises and advances into and through the cuticle and/or cell wall. F or the grow- ing point to form b e t w e en the closely adhering surfaces, a loosening of the macromolecules of the wall must take place. If the underlying host wall is soft, penetration occurs easily. Whe n the underlying wall is hard, however, and penetration is difficult, the force of the growing point may b e greater than the adhesion force of the two surfaces and may cause the separation of the appressorial and host walls. T h e pene - tration force exercised by the growing point s e e ms to b e derived from the osmotic pressure of the fungus a p p l i ed on the small diameter of the potential penetration tube, but no direct measurements on this exist to date.
Mechanical Forces
Although penetration of host barriers may, conceivably, b e attained by a fungus wholly by mechanical force, the p r e s e n ce of enzymes se- creted by the pathogen at the penetration site, and the softening or dissolution of the barrier through enzymatic action, may not b e ruled out. E v en in the best-studied cases, such enzymes and the results of their action may have e s c a p ed detection b e c a u se of technical difficul- ties inherent in such a problem.
While the penetration tube is p a s s i ng through the cuticle, it usually attains its smallest diameter and appears threadlike. Following p e n e - tration of the cuticle, the hyphal tube diameter often increases consid- erably and may appear as an inverted funnel; it may form a vesicle or it may vary irregularly. T h e increased diameter of the penetration tube in the pectin-cellulose layers of the cell wall appears to b e the result of either r e d u c ed resistance of these layers, compared to cuti- cle, to the mechanical force of the pathogen, or to greater effects on them by the e n z y me system of the pathogen. T h e penetration tube attains the diameter normal for the hyphae of the particular fungus only after it has p a s s ed through the cell wall.
N e m a t o d es penetrate plant surfaces by m e a ns of their stylet, which is thrust back and forth and exerts mechanical pressure on the cell wall. T h e nematode first adheres to the plant surface by suction which it develops by bringing its fused lips in contact with the plant. After adhesion is accomplished, the nematode brings its body, or at least its forward portion, to a position vertical to the cell wall. T h e nematode then, with its h e ad stationary and fixed to the cell wall, thrusts its sty- let forward while the rear part of its b o dy sways or rotates slowly round and round. After several consecutive thrusts of the stylet, the cell wall is pierced and the stylet or the entire nematode enters the cell. Saliva s e e ms to b e secreted by the nematode after the stylet has penetrated the wall, but it has not b e e n determined whether its en- zymes facilitate stylet penetration through partial dissolution of the cell wall.
O n ce a fungus or nematode has entered a cell it generally secretes increased amounts of enzymes which, presumably, soften or dissolve the opposite cell wall and make its penetration easier. Mechanical force, however, probably is brought to bear in most such penetrations, although to a lesser extent. It should b e noted that in many fungal infections the diameter of the hypha b e c o m es m u ch smaller than the normal whenever it penetrates a cell wall and r e s u m es its normal size once the wall has b e e n penetrated.
Considerable mechanical force is also exerted on host tissues by s o me pathogenic fungi upon formation of their fructifications in the
39
tissues beneath the plant surface. Through increased osmotic pressure the sporophore hyphae as well as fruiting b o d i e s, such as pycnidia and perithecia, p u sh outward and cause the cell walls and the cuticle to expand, b e c o me raised in the form of blisterlike protruberances, and finally break.
Selected References
Akai, S., M. F u k u t o m i, N. Ishida, a nd H. Kunoh, 1967. An anatomical a p p r o a ch to the m e c h a n i sm of fungal infections in plants. In " T he D y n a m ic R o le of M o l e c u l ar C o n- stituents in Plant-Parasite I n t e r a c t i o n" (C. J. Mirocha a nd I. Uritani, eds.), p p. 1-20.
B r u c e, St. Paul, Minnesota.
Brown, W., a nd C. C. Harvey. 1927. S t u d i es in the p h y s i o l o gy of parasitism. 10. Ann.
Botany (London) 41:643-662.
Dickinson, S. 1959. T h e b e h a v i o ur of larvae of Heterodera schachtii on nitrocellulose m e m b r a n e s. Nematologica 4:60-66.
Dickinson, S. 1959. T h e m e c h a n i c al ability to b r e a ch the host barriers. In " P l a nt Pathol- o g y" (J. G. Horsfall, a nd A. E. D i m o n d, eds.), Vol. 2, p p. 2 0 3 - 2 3 2 . A c a d e m ic Press, N e w York.
Kerr, Á., a nd Í . T. Flentje. 1957. H o st infection in Pellicularia filamentosa controlled by c h e m i c al stimuli. Nature 179:204-205.
L e a c h, J. G. 1923. T h e parasitism of Colletotrichum lindemuthianum. Minn. Agr. Expt.
Sta.y Tech. Bull. 14.
Linford, Ì . B. 1937. T h e f e e d i ng of the root-knot n e m a t o de in root t i s s ue a nd nutrient solution. Phytopathology 27:829-835.
Martin, J. T. 1964. R o le of cuticle in the d e f e n se against plant d i s e a s e. Ann. Rev. Phyto- pathol. 2:81-100.
N u s b a u m, C. J., a nd G. W. Keitt. 1938. A cytological study of host-parasite relations of Venturia inaequalis on a p p le l e a v e s . /. Agr. Res. 56:595-618.
Paddock, W. C. 1953. Histological study of s u s c e p t - p a t h o g en relationships b e t w e en Helminthosporium victoriae a nd s e e d l i ng oat leaves. Í.¾. (Cornell) Agr. Expt. Sta.
Mem. 315.
Roberts, M. F., J. T. Martin, a nd O. S. Peries. 1960. S t u d i es on plant cuticle. IV. T h e leaf cuticle in relation to invasion by fungi. Ann. Rept. Agr. Hort. Sta. Long Ashton, Bristol, p p. 102-110.
CHEMICAL WEAPONS OF PATHOGENS Although some pathogens may u se mechanical force to penetrate
plant tissues, the activities of pathogens in plants are largely chemical in nature. Therefore, the effects c a u s ed by pathogens on plants are almost entirely the result of biochemical reactions taking place be -
Chemical Weapons
tween substances secreted by the pathogen and those present in, or produced by, the plant.
T h e main groups of substances secreted by pathogens in plants, and which s e em to b e involved in production of d i s e a s e, either directly or indirectly, include enzymes, toxins, growth regulators, polysaccha- rides, and antibiotics. T h e se groups vary greatly as to their importance in pathogenicity, and their relative importance may b e different from one d i s e a se to another. Thus in some d i s e a s e, e.g., soft rots, enzymes s e em to b e by far the most important, whereas in diseases like crown gall, growth regulators are apparently the main substances involved, and in the Helminthosporium blight of Victoria oats the d i s e a se is primarily the result of a toxin secreted in the plant by the pathogen.
E n z y m e s, toxins, and growth regulators, probably in that order, are considerably more common and more important in plant d i s e a se de- velopment than are polysaccharides and antibiotics. T h e latter, in fact, have b e e n implicated only rarely as factors in plant d i s e a se produc- tion, they appear to b e p r o d u c ed by few pathogens, and w h en present they usually occur along with one or more of the first three groups of substances.
Of the five kinds of plant pathogens, all but the viruses produce enzymes, growth regulators, polysaccharides, and, probably toxins;
antibiotics are known, so far, to b e p r o d u c ed by only fungi and bac- teria. Plant viruses are not known to produce any substances them- selves, but they may induce the host cell to produce either excessive amounts of certain substances already found in healthy host cells or certain substances completely ne w to the host, which may belong to the groups mentioned above.
Pathogens produce these substances in the normal course of their activities, whether growing on the host or on nutrient media, or their production may b e induced upon growth on certain substrates. Un- doubtedly, natural selection has favored the survival of pathogens that are assisted in their parasitism through the production of such sub- stances. T h e presence or the amount of any such substance produced, however, may or may not b e related to the ability of the pathogen to cause disease. Many substances, identical to those produced by patho- gens, are also produced by the healthy host plant. In general, plant pathogenic enzymes disintegrate the structural components of host cells, break down inert food substances in the cell, or affect the proto- plast directly and interfere with its functioning systems; toxins s e em to act directly on the protoplast and interfere with its permeability and its function; growth regulators exert a hormonal effect on the cells and
41
either increase or decrease their ability to divide and enlarge; poly- saccharides s e em to play a role only in the vascular diseases in which they may, passively, interfere with the translocation of water in the plants or they may also b e toxic; antibiotics have b e e n studied least of all and s e em to act in ways resembling those of toxins.
E n z y m es
E n z y m es are protein molecules, approximately 2-100 millimicrons in diameter, which catalyze all the interrelated reactions in a living cell, including the replication of the genetic material, the reactions which supply the structural materials and the energ y for the formation of ne w materials, and also the synthesis of specific proteins, i.e., more enzymes. S o me enzymes also contain nonprotein prosthetic groups (coenzymes). For each kind of chemical reaction that occurs in a cell there is a different enzyme which catalyzes that reaction. It is esti- mated that a typical cell contains several hundred million enzyme molecules belonging to about ten thousand kinds of enzymes. T h e formation of each kind of enzyme is controlled by a gen e or gene s of the genetic material, but its final steps are carried out by the interac- tion of m e s s e n g er R NA and ribosomes with the assistance of appropri- ate specialized enzyme molecules.
Although the enzymes are produced in the cytoplasm, in the nucle- us, and perhaps in mitochondria and at the cytoplasmic m e m b r a n e, they may accumulate in any one of these structures, in the ground substance of the cytoplasm, or in the cell wall; or they may be se- creted. Heterotrophic microorganisms, pathogens in particular, se- crete a number of enzymes with the obvious function of making insol- uble substances available as substrates for growth. Pathogens also secrete enzymes which, along with other secreted substances, affect the activity of various enzymes of the host cells.
E n z y m es function as catalysts of almost all the biochemical reac- tions in which a ne w molecule is synthesized from smaller molecules, radicals, or atoms; altered to a different molecule by the transfer of an atom or radical to a different site on the molecule; or broken down to smaller component molecules. Minute amounts of enzymes are re- quired to carry out any of these reactions. T h e mechanisms by which this catalytic efficiency of the enzymes is achieved are yet unknown.
E n z y m e s, d e p e n d i ng on their function or specificity, are classified as:
1. Oxidoreductases — E n z y m es concerned with oxidation-reduction
Chemical Weapons — Enzymes
processes in plant metabolism through the transfer of electrons form the oxidized to the reduced substance.
2. H y d r o l a s e s - E n z y m es cleaving ester, glycosidic, etc., linkages through the addition of one molecule of water.
3. L y a s es —Enzymes with a degrading action through cleavage of C — C, C—O, or C—Í bonds.
4. Transferases —Enzymes catalyzing the transfer of a group from one substance to another.
5. Isomerases —Enzymes catalyzing an intramolecular rearrange- men t of atoms or groups.
6. L i g a s es —Enzymes catalyzing the joining of two molecules with the aid of energ y released by the cleavage of an energy-rich phosphate bond.
All groups of enzymes are p r o d u c ed in cells regardless of origin.
F e w of the enzymes secreted by microorganisms, however, are known to play a role in d i s e a se development. Almost all of these break down plant substances into smaller molecules which the pathogen may ab- sorb and utilize for growth and energy. Of the above groups of en- zymes, hydrolases s e em to b e by far the most important enzymes in- volved in the production of d i s e a se by plant pathogens. Since plant tissues consist, on the one hand, of structural materials making up the cell walls and their protective or connective layers, and, on the other hand, of substances found within the protoplast, it is convenient to examine separately the enzymes that affect each of these groups of substances.
Enzymatic Degradation of Cell Wall Substances
Most plant pathogens secrete enzymes throughout their existence or upon contact with a substrate. Usually, the first contact of pathogens with their host plants occurs at a plant surface. Such a surface may consist primarily of cellulose which makes up the epidermal cell walls or, on the aerial plant parts, of cellulose plus cuticle. Cuticle, which consists of cutin, is frequently covered with a layer of wax. Protein and lignin may also b e found in epidermal cell walls. Penetration of patho- gens into, and collapse of, parenchymatous tissues is brought about by the breakdown of the cell walls, consisting of cellulose, pectins, and hemicelluloses, and of the m i d d le lamella, consisting primarily of pectins. C o m p l e te plant tissue disintegration involves, in addition, breakdown of lignin. T h e degradation of each of these substances is brought about by the action of one or more sets of enzymes secreted by the pathogen.
43
P r o p o s ed structure of part of a cutin m o l e c u l e. [ A d a p t ed from L i n s k e ns et al. (1965).
In " R e s i d ue R e v i e w s" (F. A. Gunther, ed.). Springer, N e w York.]
C U T IN
C U T I C U L AR WA X
Plant waxes are found as granular or rodlike projections or as contin- uous layers outside or within the cuticle of many aerial plant parts.
Waxes are produced continuously and migrate toward the surface through minute pores originating in the living cells underlying the cuticle (Fig. 5).
T h e cuticular waxes consist of mixtures of long-chain molecules of paraffin hydrocarbons, alcohols, ketones, esters, and acids. Most mole- cules found in wax contain 15-37 carbon atoms with only one chemi- cally reactive group; they are, therefore, highly resistant to chemical changes.
No pathogens are known to date to produce enzymes that can de- grade waxes. Wax layers on plant surfaces are apparently penetrated by fungi and parasitic higher plants by means of mechanical force alone.
Chemical Weapons—Enzymes
Cutin is the main component of the cuticular layer. T h e upper part of the layer is admixed with waxes, while its lower part, in the region where it merges into the outer walls of epidermal cells, cutin is ad- mixed with pectin and cellulose (Fig. 5).
Cutin is a polyester of hydroxylated monocarboxylic acids, each containing chains of 16 to 18 carbon atoms a nd either two of three hydroxyl groups. Through linkage of the hydroxyl groups of one chain with the carboxyl or hydroxyl groups of other chains, a three-dimen- sional polymeric structure is formed a nd results in the production of the cuticular layer. T h e formula on p. 44 gives an idea of the structure and complexity of only a part of the cutin molecule. T h e scarcity of reactive sites suggests at least one reason for the stability of cutin.
T h e r e is e v i d e n ce that at least s o me phytopathogenic fungi produce cutinases, i.e., enzymes that catalyze the breakdown a nd dissolution of cutin. O ne enzyme, cutin esterase, catalyzes the hydrolysis of ester bonds occurring b e t w e en free hydroxyl a nd carboxyl groups of the cutin acids:
R — C =0
ό cutin e s t e r a s e^ _rc qh +q _CRH ( _ Q H )
R— C — C - O— H20 \
Ç II Ï
Another enzyme, carboxy cutin esterase or carboxy cutin peroxidase, catalyzes the hydrolysis of the peroxide groups of cutin:
H ^ - i C H ^ - C H - i C H ^ - C O OH c a r b Q xy ^ H2C H C H2) „ - C H - ( C H2) „ - C O OH Ï peroxidase OH OH
Ï ú æü + 0 = C - ( C H2) „ — C H - C H - ( C H2) „ - C H2OH H2C - ( C H2) „ — C H - C H - ( C H2) „ — C O OH + %C>2
OH OH OH OH OH
T h e result of the catalytic action of the two enzymes is breakdown of the three-dimensional structure of the cuticle a nd release of free hydroxylated monocarboxylic acids. E v en in these cases, however, cutin softening or breakdown is limited to the immediate area b e l ow and around the invading hypha a nd s e e ms to b e associated only with the penetration p h a se of the pathogen. It has b e e n shown also that a p p le leaves infected with the a p p le scab fungus contain less cutin in their cuticle than healthy leaves; this observation indicates that per-
45
haps some of the cutin of affected leaves is broken down by the fun- gus, although the possibility exists that the smaller deposits in the cu- ticle of infected leaves may have b e e n d ue to interruption of the deposition of cutin rather than to the breakdown of cutin already formed.
F i g. 6. S c h e m a t ic representation of structure a nd composition of plant cell walls.
Chemical Weapons—Enzymes
F i g. 7. S c h e m a t ic diagram of the gross structure of c e l l u l o se a nd microfibrils (A), a nd of the a r r a n g e m e nt of c e l l u l o se m o l e c u l es within a microfibril (B). MF = microfi- bril; GS = g r o u nd s u b s t a n ce (pectin, h e m i c e l l u l o s e s, or lignin); AR = a m o r p h o us region of c e l l u l o s e; CR = crystalline region; Ì = m i c e l l e; S CC = s i n g le c e l l u l o se chain (molecule). [ A d a p t ed from H. P. B r o w n, A. J. P a n s h i n g, a nd C. C. Forsaith (1949). " T e x t b o ok of Wood T e c h n o l o g y ," Vol. 1. Mc Graw-Hill, N e w York.]
P E C T IC S U B S T A N C ES
Pectic substances constitute the main components of the m i d d le lamella, i.e., the intercellular c e m e n t which holds in place the cells of plant tissues (Fig. 6), and also a large portion of the primary cell wall, in which they form an amorphous gel filling the spaces b e t w e en the cellulose microfibrils (Fig. 7).
Pectic substances are polysaccharides containing a very high per- centage of galacturonic acid residues. It was thought earlier that pec- tins w e re a polymer of pure a-D-(l,4)-linked galacturonic acid (I) residues, but it is now b e c o m i ng apparent that other sugars are cova- lently attached to the polygalacturonide backbone, and they may e v en form an integral part of the main chain through glycosidic b o n d i ng b e t w e en the galacturonic acid and the sugar. It s e e ms probable that galacturonic acid residues not only m a ke up the backbone of s o me polysaccharides, but may b e found in the side chains of others and, in s o me cases, in both the b a c k b o ne a nd the side chains of polysaccha- rides. It is p o s s i b le then that most of the so-called pectins are polymers
47
of galacturonic acid residues resulting from partial degradation of more complex polysaccharides. Several kinds of pectic substances can b e , rather arbitrarily, distinguished, d e p e n d i ng on the d e g r ee of ester- ification of their carboxyl groups with methanol. Chains of galactu- ronic acid free of methyl groups are called pectic acid (II); when methyl groups are attached to less than 7 5 % of the galacturonic acid carboxyls, the chains are called pectinic acid (III), and chains with more methyl groups would be called pectin. E a ch of these pectic chains may form cross-linkages —for example, with other pectic chains, with cellulose chains, with other sugars — through bonding between the carboxyl groups of the galacturonic acid residues and the hydroxy 1 or carboxyl groups of the other compounds. T h e carboxyl groups of pectic chains may also form ionic bonds with polyvalent ca- tions such as C a2 + and M g2 + and result in rather insoluble pectate salts.
Several enzymes are known to degrade pectic substances. T h e se enzymes may b e produced by many plant pathogens in vitro (in culture), and some have b e e n shown to b e produced by them in vivo (in the d i s e a s ed plant). T h e main groups of pectin-degrading enzymes are described below.
Chemical Weapons —Enzymes
Pectin Methylesterases (PME)
T h e se enzymes remove all or s o me of the esterified methyl groups of the pectin or pectinic acid chains through hydrolysis and yield methanol and pectic acid or less methylated pectinic acids:
ï
+ 2 C H 3 OH
ï
T h e removal of methyl groups has no effect on the length of the pectin chains but it does alter their solubility and it also affects the rate at which they can b e attacked by the chain-splitting enzymes.
Pectin Chain-Splitting Enzymes
T h e se enzymes cleave the pectic chain at the 1-4 glycosidic linkage and release chain portions containing one or more residues of galactu- ronic acid. D e p e n d i ng on the mechanisms of cleavage of the 1-4 linkage, these enzymes are distinguished as:
Pectin glycosidases or polygalacturonases which break the 1-4 gly- cosidic bonds of pectic substances through hydrolysis:
S o me pectin glycosidases (endoenzymes) cause cleavage of the chain at random, while others (exoenzymes) cleave only the terminal 1-4 bond. Among the endo- and the exoenzymes, some attack primarily pectic acid chains and are called polygalacturonases (PG), while oth- ers attack primarily the methylated chains of pectin and pectinic acid and are called polymethylgalacturonases (PMG).
49
Pectin lyases or transeliminases, which break the 1-4 linkages of pectic substances through a transeliminative cleavage, in which cleav- age at C-4 is accompanied by a simultaneous elimination of Ç from C-5:
Transeliminases also occur as endo- and exoenzymes, and among them some show preference for pectic acid (pectic acid transeliminase or P A T E) and others for pectin or pectinic acid (pectin transeliminase or P T E ). T h e exotranseliminases release a dimer rather than a monomer, however.
Many plant pathogens produce pectic enzymes either as a matter of course (constitutively) or in response to a substrate (inductively).
Different pathogens, however, are capable of producing different combinations or only one of the enzymes described above. Moreover, pathogens may produce certain pectic enzymes in vitro, and other enzymes, or different concentrations of the s a me enzymes, in vivo. It should be noted that plant tissues also produce pectic enzymes, most commonly pectin methylesterases, and certain ripening fruits produce endopolygalacturonases. Pectin glycosidases and lyases, however, have only infrequently b e e n reported in healthy plant tissues.
T h e pectin-degrading enzymes have b e e n shown to b e involved in the production of many diseases. Pectic enzymes are p r o d u c ed by germinating spores and apparently, acting together with other patho- gen metabolites, assist in the penetration of the host. Pectin degrada- tion results in weakening of cell walls or tissue maceration which undoubtedly facilitates the inter- or intracellular invasion of the tis- sues by a pathogen. Since many pathogenic fungi and bacteria can grow on nutrient m e d ia containing pectic substances as the only source of carbon, it is obvious that pectic enzymes may provide nu- trients for the pathogen in infected tissues. Pectic enzymes also s e em to b e involved in the induction of vascular plugs and occlusions in the vascular wilt diseases. Cells are usually quickly killed in tissues ma- cerated by pectic enzymes, but the role of these enzymes in the death of cell protoplasts has not yet b e e n elucidated.
Chemical Weapons—Enzymes
Although pectic substances are d e g r a d ed in the p r e s e n ce of pectic enzymes, several factors may modify or inhibit their activities. T h u s, phenolic c o m p o u n ds and/or their oxidation products, found especially in darkened injured tissues, can inactivate pectic as well as other en- zymes. Indoleacetic acid (IAA) also inhibits certain pectic enzymes, presumably by binding the e n z y me to certain reaction products formed as a result of the e n z y me activity. T h e number of calcium bridges b e t w e en carboxyls of adjacent pectic chains s e e ms to influ- enc e the accessibility of the chain to s o me e n z y m es and, therefore, af- fects the d e g r ee of its degradation. On the other hand, the p r e s e n ce of C a2 + s e e ms to stimulate the activity of endotranseliminases. Accessi- bility of pectic substances to pectin-degrading enzymes is also re- d u c ed by the increased deposition of other polysaccharides and lignin in the walls of mature cells.
C E L L U L O SE
Cellulose occurs in all higher plants as the skeletal substance of cell walls in the form of microfibrils (Figs. 6 and 7). Microfibrils are the basic structural unit of the wall even though they account for less than 2 0 % of the wall volume in most meristematic cells. T h e cellulose con- tent of tissues varies from about 1 2 % in the nonwoody tissues of grasses to about 5 0 % in mature w o od tissues to more than 9 0 % in the cotton fibers.
C e l l u l o se is a linear polymer of 0-1,4-linked D-glucose units:
T h e chain lengths of native cellulose vary greatly, the longest chains measuring 3-4 microns (30,000-40,000 A) and having a molecular weight of more than a million. Such chains consist of about 6000-8000 glucose molecules. T h e chain molecules are generally juxtaposed in a way that they form linear, rectangularly s h a p ed crystals within the microfibrils. Sections of several cellulose chains align themselves parallel to each other and form highly crystalline regions of cellulose known as micelles. T h e latter are approximately 600 A long and 5 0 - 2 00 A in diameter. Sections of cellulose molecules outside the
51
micelles are not oriented in any particular way and they form the so- called regions of " a m o r p h o u s" cellulose (Fig. 7). Since cellulose mol- ecules are several times as long as micelles, sections of a single cellu- lose chain may b e found in several micelles and as many regions of amorphous cellulose. T h e spaces b e t w e en microfibrils and b e t w e en micelles or cellulose chains within the microfibrils may b e filled with pectins and hemicellulose and probably s o me lignin at maturation.
Cellulose-degrading enzymes have b e e n shown to b e produced by several phytopathogenic fungi, bacteria, and nematodes, and they are undoubtedly produced by parasitic higher plants. Saprophytic fungi, mainly certain groups of basidiomycetes, and to a smaller degree sap- rophytic bacteria cause the breakdown of most of the cellulose decom- p o s ed in nature. In living plant tissues, however, cellulolytic enzymes secreted by pathogens play a role in the softening and/or disintegra- tion of cell-wall material and microfibrils; they facilitate the penetra- tion and spread of the pathogen in the host and cause the collapse and disintegration of the cellular structure, thereby aiding the pathogen in the production of disease. Cellulolytic enzymes may further partici- pate indirectly in d i s e a se d e v e l o p m e nt by releasing from cellulose chains soluble cellosaccharides which serve as food for the pathogen, and, in the vascular diseases, by liberating into the transpiration stream large molecules from cellulose which interfere with the nor- mal m o v e m e nt of water.
T h e enzymatic breakdown of cellulose results in the final produc- tion of glucose molecules. This is brought about by a series of enzy- matic reactions:
Linear Soluble Native cellulose C* e n z y me > cellulose Cx enzymes > low-molecular
chains HaO cellosaccharides
and glucose
Glucose /3-glucosidase Cellobiose and glucose
T h e Cx enzyme s e e ms to act on cellulose on other than the j8-l,4 linkages, perhaps by cleaving cross-linkages b e t w e en chains of the microfibrils. This results in the loosening of the cellulose chains within the microfibrils, loss of crystallinity, loss of tensile strength,
Chemical Weapons—Enzymes
swelling and greater accessibility of the cellulose chains to the chain- splitting enzymes (Cx).
T h e Cx enzymes hydrolyze the cleavage of the 0-1,4 linkages of the cellulose chains and are sometimes called 0-1,4-glucanases. Cx en- zymes can hydrolyze crystalline cellulose only in the p r e s e n ce of Ci enzymes and only after the forces that hold the cellulose molecules together have b e e n broken or loosened by Q. Although Cx enzymes usually split the cellulose chain at random (endoenzymes) and release soluble, low molecular-weight products containing six or fewer glu- cose residues, they may occasionally act as exoenzymes, removing glucose or cellobiose successively from the e n d of the cellulose chain.
T h e low molecular oligosaccharides are further hydrolyzed by the Cx or other enzymes to cellobiose and glucose, the cellobiose b e i ng finally hydrolyzed to glucose by 0-glucosidases or cellobiase.
H E M I C E L L U L O S ES
H e m i c e l l u l o s es are a mixture of water-insoluble polysaccharides that interpenetrate the cellulose and lignin of plant cell walls. T h e y form an integral part of the cell wall and presumably fulfill a structural function in the plant. H e m i c e l l u l o s es s e em to b e particularly abun- dant in mature and thickened cell walls of wood, grasses, and seeds.
T h e hemicellulose content of various tissues or species may range from 11 to 3 2 %. T h e concentration in cell walls of the individual hex- ose or pentose sugars which make up the hemicellulose molecules also varies considerably: glucose, most of which is found in cellulose, 5 5 - 7 3 %, xylose 9 - 3 9 %, galactose 1-17%, mannose 0.4-16%, arabi- nose less than 3.5%. In addition, there are minor amounts of rham- nose, methyl rhamnose, methyl-D-glucuronic acid, and galacturonic acid. Several molecules of each hexose or pentose sugar are j o i n ed together and form chains to which chains of other sugar molecules are attached. D e p e n d i ng on the sugars involved, these chain skeletons are called xylans, arabinoxylans, glucuronoxylans, arabinoglucu- ronoxylans, arabinogalactans, glucomannans, etc. U p on chemical or enzymatic degradation of hemicelluloses, the simple sugar compo- nents are liberated.
Neither the structure nor the m e c h a n i sm of enzymatic breakdown of hemicellulose has b e e n studied sufficiently yet. Several microor- ganisms, however, both parasitic and saprophytic, produce enzymes, called hemicellulases, that d e g r a de hemicellulose. Hemicellulases apparently convert hemicellulose hydrolytically to its component sugars, mostly pentose and uronides. Cellulolytic enzymes of certain
53
fungi also s e em capable of hydrolyzing directly certain components of hemicelluloses. Degradation of hemicelluloses exposes the e m b e d- d ed cellulose microfibrils and, in the lignified cells, the lignin to the appropriate enzymes, which in turn degrade these substances.
Lignin is found in the m i d d le lamella and the cell wall of xylem vessels and the fibers that strengthen plants. It is also found in epider- mal and occasionally hypodermal cell walls of some plants. T h e lignin content of mature woody plants varies from 15 to 38 % and is second only to cellulose.
Lignin is an amorphous polymeric material that is different from both carbohydrates and proteins in composition and properties. Al- though it is not certain whether lignin is a single c o m p o u nd or a mix- ture of related polymers, it appears that the most common basic struc- tural unit of lignin is a phenylpropanoid:
Lignin is formed by C —C and C—Ï bonds b e t w e en the carbon and/or the hydroxyls of the aromatic ring and of the side chains of different phenylpropanoid or similar units. T h e three-dimensional polymer may consist of up to 100 phenylpropanoid or other aromatic basic units and forms a framework that ramifies through the interfibrillar and intermicellar spaces of the cell wall and b e t w e en cell walls, re- placing pectic substances almost entirely in s o me older tissues. T h e lignin polymer is perhaps more resistant to enzymatic degradation than any other plant substance. This is probably d ue to the fact that although the lignin building blocks (precursors) are formed enzymati- cally, the polymer itself is formed as a result of the nonenzymatic reac- tion of free radicals of some precursors with those of other precursors, producing autocatalytic chain reactions.
It is obvious that enormous amounts of lignin are d e g r a d ed by mi- croorganisms in nature, as is e v i d e n c ed by the decomposition of all annual plants and a large portion of perennial plants that disintegrate annually. It is generally accepted, however, that only a small group of microorganisms are capable of degrading lignin. Actually, only about 500 fungus species, almost all of them basidiomycetes, have b e e n LIGNIN
Chemical Weapons — Enzymes
reported, so far, as b e i ng capable of d e c o m p o s i ng wood. About one- fourth of these (the brown-rot fungi) s e em to c a u se little, if any, degra- dation of lignin. T h e remaining species of this group, the white-rot fungi, have b e e n shown to b e capable of degrading lignin, although few experimental data have b e e n a d v a n c ed in elucidation of the mechanisms of such degradation. Bacteria s e em to b e of no impor- tance in the degradation of lignin.
It appears that the white-rot fungi secrete an extracellular poly- phenol oxidase, possibly laccase, which enables them to utilize lignin.
This enzyme is also capable of catalyzing the oxidation of certain lig- nin-related model compounds associated with native lignin. T h e ac- tual presence and involvement, however, of such an enzyme in lignin degradation has not yet b e e n demonstrated. On the other hand, all types of wood-rotting fungi s e em to produce a " t r a n s m e t h y l a s e" that removes methoxyl groups from lignin, thus " m o d i f y i n g" the lignin and increasing the exposure of the e m b e d d e d cellulose and other polysaccharides to fungal enzymes.
It is as yet not known whether phytopathogenic fungi other than the wood-decaying ones can attack lignin to any extent and whether such ability, if present, influences their ability to cause disease. Also, there is no information regarding the ability of the other kinds of pathogens to degrade lignin. It appears probable, however, that, with the excep- tion of the wood-rotting fungi, the other pathogens produce few or no lignin-degrading enzymes and that the diseases they cause are not d e p e n d e nt on the presence of such enzymes.
C E L L - W A LL P R O T E IN
A small amount of protein appears to b e present in all plant cell walls, in the cutin region of the cuticle, and possibly in the m i d d le lamella. Part of this protein is m a de up of various enzymes or portions of plasmodesmata, etc., but the remaining part is truly structural cell- wall protein. T h e structural wall protein s e e ms to b e similar to other cell proteins in all aspects except that, in addition, it contains hydroxy- proline, an imino acid not found in any other plant protein. Covalent bonds b e t w e en the protein molecules and cell wall polysaccharides, resulting in the formation of mucopolysaccharides, have b e e n sug- gested, as has the involvement of protein in cell wall elongation.
However, nothing definite is known to date as to the origin and func- tion of the cell wall protein, or its importance, if any, in the infection of plants by pathogens.
Pathogens are known to degrade proteins enzymatically, and, since 55
no particular enzyme responsible for the degradation of the cell wall protein is known, it is p r e s u m ed that its breakdown follows the s a me steps found in the degradation of the cytoplasmic proteins; this is dis- c u s s ed below.
Enzymatic Degradation of Substances Contained in Plant Cells S o me fungi, possibly some of the bacteria, and all viruses, live all or part of their lives in association with or inside the living protoplast.
T h e se pathogens obviously derive nutrients from the protoplast. All the other pathogens obtain nutrients from protoplasts after the latter have b e e n killed. S o me of the nutrients, e.g., monosaccharides and amino acids, are probably sufficiently small molecules to b e absorbed by the pathogen directly. S o me of the other plant cell constituents, however, such as starch, proteins, and lipids, can b e utilized only after degradation by the enzymes secreted by the pathogen.
PROTEINS
Plant cells contain innumerable different proteins which play di- verse roles as catalysts of cellular reactions (enzymes) or as structural material. Proteins are formed by the condensation of numerous mole- cules of about twenty different kinds of amino acids. Amino acids in various combinations and s e q u e n c es are b o u nd together by p e p t i de linkages in which the amino group of one amino acid reacts with the carboxyl group of another amino acid, water b e i ng split off in the pro- cess:
R— CH—COOH R—CH—COOH , η I I
R—CH—COiOH + Ç'—NH R—CH— CO—NH + H20
I 1 1 I
NH2 NH,
T h e free amino and carboxyl groups react with carboxyl or amino groups, respectively, of still other amino acids to form long chains (peptides) and cross-linkages b e t w e en chains, producing polypep- tides and finally resulting in a three-dimensional molecule of protein, which may consist of several hundred or thousand amino acids.
All pathogens s e em to b e capable of degrading many kinds of pro- tein molecules. T h e plant pathogenic enzymes involved in protein degradation are similar to those present in higher plants and animals.
Chemical Weapons —Enzymes
Proteolytic enzymes catalyze the hydrolysis of p e p t i de linkages.
D e p e n d i ng on the size of molecules that they can attack, or preferably attack, they are called (a) proteinases, w h en they hydrolyze proteins which they break down to polypeptides; (b) peptidases, when they hydrolyze polypeptides to smaller peptides and amino acids.
T h e distinction b e t w e en proteinases and peptidases, however, is unrealistic since proteinases will also d e g r a de certain peptides, and many peptidases will also d e g r a de proteins. S o me p e p t i d a s es can hydrolyze only one terminal amino acid at a time but can continue doing so until most of a protein is degraded. Peptidases that degrade polypeptide chains by stepwise removal of the N-terminal amino acid are called aminopeptidases, and p e p t i d a s es that d e g r a de peptides and proteins from the C-terminal amino acid are called carboxypeptidases.
T h e e n d products of a complete protein degradation are always simple amino acids, which can b e a b s o r b ed by the pathogen and can b e uti- lized as building blocks for its own proteins.
Considering the paramount importance of proteins as enzymes, constituents of cell m e m b r a n e s, and structural components of plant cells, degradation of host proteins by proteolytic enzymes secreted by pathogens can profoundly affect the organization and function of the host cells. T h e nature and extent of such effects, however, has b e e n little investigated so far, and their significance in d i s e a se develop- men t is not known.
S T A R CH
Starch is one of the main reserve polysaccharides found in plant cells. Starch is synthesized in the chloroplasts and, in nonphotosyn- thetic organs, in the amyloplasts. Starch is a glucose polymer and ex- ists in two forms: amylose, an essentially linear molecule consisting of a - ( 1 ^ 4) glucosidic linkages with molecular weight ranging from 10,000 to 100,000, corresponding to 50 to 500 glucose units; and amylo- pectin, a highly branched molecule of various chain lengths linked by a-( 1—»6) bonds to the main chain. T h e molecular weight of amylopec- tin ranges from 50,000 to 1,000,000, corresponding to 250 to 5000 glu- cose units.
Most pathogens utilize starch, and other reserve polysaccharides, in their metabolic activities. T h e degradation of starch is brought about mainly by the action of two enzymes, á-amylase and /3-amylase, both of which are secreted by most pathogens. A third enzyme, isoamylase, is also known to affect starch breakdown.
β-Amylase. This enzyme degrades both amylose and amylopectin 57
Amy lose
from the nonreducing e n d (the left side in the formulas given above) by causing hydrolysis of the a-(l —> 4) linkage and forming maltose (a disaccharide). /3-Amylase can d e g r a de amylose completely, unless there are chains attached to the amylose by a-(l 6) linkages, at which point the action of /3-amylase stops. Since j3-amylase cannot bypass the branched-chain points, it can d e g r a de only the outer chains of amylopectin, yielding the residual, high molecular weight fractions called dextrins.
α-Amylase. Both amylose and amylopectin are attacked by á-amy- lase at random a-(l —» 4) linkages, yielding at first linear oligosaccha- rides and dextrins. Further hydrolysis produces maltose, glucose, and shorter straight-chain dextrins. Finally, the amylopectin molecule is d e g r a d ed to maltose and glucose and only the branch points [a-(l - » 6) linkages] remain intact.
Chemical Weapons—Enzymes
Isoamylase. This enzyme attacks only a-(l - » 6) linkages of amylo- pectin and produces short-chain amylose-type molecules, which are subsequently attacked by β- or á-amylases.
T h e glucose produced by the amylolytic enzymes is utilized by the pathogens directly. T h e maltoses, however, must b e further broken down, and this is accomplished by the e n z y me maltase, which is pres- ent in most starch-utilizing microorganisms and hydrolyzes maltose into two units of glucose.
L I P I DS
Various types of lipids occur in all plant cells, although some of them may not b e present in all kinds of plant cells. T h e most impor- tant plant lipid types are the neutral lipids, functioning as energ y storage compounds and including the oils and fats found in many cells, especially in s e e d s; the wax lipids, found on most aerial epider- mal cells; the phospholipids and the glycolipids, both of which, along with protein, are the main constituents of all plant cell membranes.
T h e common characteristic of all lipids is that they contain fatty acids that may b e saturated or unsaturated.
Several fungi, bacteria, and nematodes are known to b e capable of degrading lipids. Little is known, however, about the specific en- zymes of pathogens involved in lipid breakdown. Lipolytic enzymes, called lipases, phospholipidases, etc., hydrolyze the liberation of the fatty acids from the lipid molecule. T h e fatty acids are presumably uti- lized by the pathogen directly, and the s a me may b e true for the re- maining moiety of the lipid molecule.
N U C L E IC ACIDS
All cells contain small amounts of both nucleic acids; ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). D NA is found mainly in the nucleus, and in very small amounts in chloroplasts and mitochon- dria. R NA is found throughout the living parts of the cell: in the ground cytoplasm, or in particles such as the ribosomes, mitochondria, nucleolus, and chloroplasts. D N A, of course, carries the genetic code and determines the genetic capabilities of each organism, while R NA in its different forms translates the genetic m e s s a ge of the D NA and interacts with the ribosomes to produce proteins (enzymes) from the amino acids.
Both nucleic acids consist of linear chains of alternating molecules of phosphate and a sugar (ribose for RNA, deoxyribose for DNA). A molecule of a b a se (adenine, cytosine, guanine, and uracil for RNA,
59
and thymine instead of uracil for D N A ), is attached to each sugar mol- ecule of the chain.
Several pathogenic fungi and bacteria are known to d e g r a de nucleic acids, but most of the information on the enzymes involved and the mechanism of nucleic acid degradation has b e e n obtained from stud- ies with bacteria.
Ribonuclease and deoxyribonuclease attack R NA and D N A, respec- tively, and through hydrolysis, yield primarily mononucleotides (units consisting of one b a s e, one sugar, and one phosphate molecule).
T h e nucleotides are further dephosphorylated by the hydrolytic ac- tion of, usually, nonspecific phosphatases, yielding inorganic phos- phate and nucleosides. Nucleotides and nucleosides may b e acted upon by various deaminases which remove the amino groups from the bases.
Degradation of nucleic acids apparently occurs in many d i s e a s ed tissues but its significance in d i s e a se d e v e l o p m e nt is not known.
Selected References
Akazawa, T. 1965. Starch, inulin, a nd other r e s e r ve p o l y s a c c h a r i d e s. In " P l a nt Bio- c h e m i s t r y" (J. B o n n er and J. E. Varner, eds.), p p. 2 5 8 - 2 9 7 . A c a d e m ic Press, N e w York.
A l b e r s h e i m, P. 1965. T h e substructure a nd function of the cell wall. In " P l a nt B i o c h e m- istry" (J. B o n n er a nd J. E. Varner, eds.), p p. 151-188. A c a d e m ic P r e s s. N ew York.
A l b e r s h e i m, P. 1965. B i o g e n e s is of the cell wall. In " P l a nt B i o c h e m i s t r y" (J. B o n n er a nd J. E. Varner, eds.), p p. 2 9 8 - 3 2 2 . A c a d e m ic Press. N e w York.
B a t e m a n, D. F., a nd R. L. Millar. 1966. Pectic e n z y m es in t i s s ue degradation. Ann. Rev.
Phytopathol. 4 : 1 1 9 - 1 4 6 .
Brown, W. 1965. Toxins a nd cell-wall d i s s o l v i ng e n z y m es in relation to plant d i s e a s e.
Ann. Rev. Phytopathol. 3:1-18.
C o w l i n g, Å. B. 1961. C o m p a r a t i ve biochemistry of the d e c ay of s w e e t g um s a p w o od by white-rot a nd brown-rot fungi. U.S. Dept. Agr. Forest Serv. Tech. Bull. 1258, 79 p p.
Dehority, Â. Á., R. R. J o h n s o n, a nd H. R. Conrad. 1962. Digestibility of forage hemicel- lulose a nd pectin by r u m en bacteria in vitro a nd the effect of lignification thereon.
]. Dairy Sci. 4 5 : 5 0 8 - 5 1 2 .
Hancock, J. G., a nd R. L. Millar. 1965. Association of cellulolytic, proteolytic and xylol- ytic e n z y m es with southern anthracnose, spring black stem, a nd StemphyHum leaf spot of alfalfa. Phytopathology 55:346-355.
H e i n e n , W. 1960.
U b e r
d e n e n z y m a t i s c h en C u t i n a b b a u. I. N a q h w e i ss e i n e s " C u t i n a s e"S y s t e m s. Acta Botan. Neerl. 9 : 1 6 7 - 1 9 0 .
H e i n e n , W. 1963. U b er d en e n z y m a t i s c h en C u t i n a b b a u. V. D ie L y se von Peroxydbruk- ten in Cutin durch P e r o x i d a se aus Penicillium spinulosum Thorn. Acta Botan.
Neerl. 12:51.
H o l l e y, R. W. 1965. Protein m e t a b o l i s m. In " P l a nt B i o c h e m i s t r y" (J. B o n n er a nd J. E . Varner, eds.), p p. 3 4 6 - 3 6 0 . A c a d e m ic Press. N e w York.
Chemical Weapons —Microbial Toxins
H u s a i n, Á., a nd A. K e l m a n. 1959. T i s s ue is disintegrated. 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. 1 4 3 - 1 8 8 . A c a d e m ic P r e s s. N e w York.
L a m p o r t, D. T. A. 1965. T h e protein c o m p o n e nt of primary cell walls. Advan. Botan.
Res. 2 : 1 5 1 - 2 1 8 .
L i n s k e n s, H. F., W. H e i n e n , a nd A. L. Stoffers. 1965. C u t i c u la of l e a v es a nd the r e s i d ue p r o b l e m. Residue Rev. 8 : 1 3 6 - 1 7 8 .
M c C l e n d o n, J. H., G. F. S o m e r s, a nd J. W. H e u b e r g e r. 1960. T h e o c c u r r e n ce of a variety of e n z y m es hydrolyzing plant cell-wall p o l y s a c c h a r i d es in a p p le rotted by Botryos- phaeria ribis. Phytopathology 5 0 : 2 5 8 - 2 6 1 .
Martin, J. T. 1964. R o le 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 .
Mirocha, C. J., a nd A. I. Zaki. 1966. Fluctuation in a m o u nt of starch in host plants in- v a d ed by rust a nd m i l d ew fungi. Phytopathology 5 6 : 1 2 2 0 - 1 2 2 4 .
Norkrans, Birgitta. 1963. D e g r a d a t i on of c e l l u l o s e. Ann. Rev. Phytopathol. 1:325-350.
S e m p i o, C. 1959. T h e host is starved. In " P l a nt P a t h o l o g y" (J. G. Horsfall a nd Á. Å.
D i m o n d, eds.). Vol. I, p p. 2 7 7 - 3 1 2 . A c a d e m ic Press. N e w York.
Starr, M. P., a nd F. Moran. 1962. E l i m i n a t i ve split of pectic s u b s t a n c es b y phytopatho- g e n ic soft-rot bacteria. Science 1 3 5 : 9 2 0 - 9 2 1 .
Stumpf, P. K. 1965. L i p id m e t a b o l i s m. In " P l a nt B i o c h e m i s t r y" (J. B o n n er a nd J. Å.
Varner, eds.), p p. 3 2 3 - 3 4 5 . A c a d e m ic Press, N e w York.
Van Etten, H. 1966. E n d o p e p t i d a s es a nd cell wall d e g r a d i ng e n z y m es a s s o c i a t ed with b e an hypocotyls infected with Rhizoctonia solani. M.S. T h e s i s, Cornell Univ., Itha- ca, N e w York. 89 p p.
Varner, J. E. 1965. E n z y m e s. In " P l a nt B i o c h e m i s t r y" (J. B o n n er a nd J. E . Varner, eds.), p p. 14-20. A c a d e m ic Press. N e w York.
Varner, J. E. 1965. E n z y m o l o g y. In " P l a nt B i o c h e m i s t r y" (J. B o n n er a nd J. E . Varner, eds.), p p. 189-212. A c a d e m ic Press, N e w York.
Whitney, H. S., M. S h a w, a nd J. M. Naylor. 1962. T h e physiology of host-parasite rela- tions. X I I. A cytophotometric study of the distribution of D NA a nd R NA in rust-in- fected l e a v e s. Can.]. Botany 4 0 : 1 5 3 3 - 1 5 4 4 .
Microbial Toxins in Plant Disease
L i v i ng plant cells are complex systems in which many interde- p e n d e nt biochemical reactions are taking place concurrently or in a well-defined succession resulting in the intricate and well-organized processes essential for life. Disturbance of any of these metabolic re- actions causes disruption or shift of the physiological processes that sustain the plant and leads to d e v e l o p m e nt of disease. A m o ng the fac- tors inducing such disturbances are metabolites that are produced by plant pathogenic microorganisms and act directly on living host proto- plasts, seriously damaging or killing the cells of the plant. Such metab- olites are called toxins. S o me toxins act as general protoplasmic poisons and affect many species of plants representing different fami-
61
