Plant Diseases Caused toy

C H A P T E R 12

Plant Diseases Caused toy

Viruses

Introduction

Viruses are submicroscopic entities that multiply only inside living cells and have the ability to cause disease. All viruses are parasitic in cells and cause a multitude of diseases to alLforms of living organisms, from single-celled plants or animals to large trees and mammals. S o me viruses attack man and/or animals and cause diseases such as influen- za, polio, rabies, smallpox, and warts; others attack plants; and still others attack microorganisms, e.g., bacteria. T h e total number of vi- ruses known to date is well over a thousand, and ne w viruses are a d d ed to these almost every month. More than half of all known vi- ruses attack and cause diseases of plants. O ne virus may infect one or dozens of different plants, and one plant may b e attacked by one or many different viruses.

Although viruses are agents of d i s e a se and share with other living organisms genetic functions and the ability to reproduce, it is possible to describe them in terms of the properties of chemical molecules. At

395

their simplest, viruses consist of nucleic acid and protein, with the protein w r a p p ed around the nucleic acid. Although viruses can take any of several forms, they are mostly either rod-shaped or polyhedral, or variants of these two basic structures. T h e r e is always only one kind of nucleic acid in each virus and, in most viruses, only one kind of pro- tein. S o me of the larger viruses, however, may have several different proteins, each with a different function.

Viruses do not divide and do not produce any kind of reproductive structures such as spores, but they multiply by inducing host cells to form more virus. Viruses cause d i s e a se not by consuming cells or kill- ing them with toxins, but by upsetting the metabolism of the cells which, in turn, leads to the development by the cell of abnormal sub- stances and conditions injurious to the functions and the life of the cell or the organism.

Characteristics of Plant Viruses

Plant viruses differ greatly from all other plant pathogens not only in size and shape, but also in their chemical constitution and physical structure, methods of infection, multiplication, translocation within the host, dissemination, and the symptoms they produce on the host.

B e c a u se of their small size and transparency of their bodies, viruses cannot even b e v i e w ed and detected by the methods u s ed for other pathogens.

Detection

Whe n a plant d i s e a se is c a u s ed by any of the other pathogens, the pathogen itself (with the possible exception of the ectoparasitic migra- tory nematodes) can almost always b e found in or on the infected tis- sues and can b e examined macroscopically or microscopically. In vi- rus diseases, on the other hand, individual virus particles cannot b e seen with the light microscope, although some virus-containing inclu- sions or crystals may b e seen in virus-infected cells. Examination of sections of cells or of crude sap from virus-infected plants under the electron microscope may or may not reveal viruslike particles. Virus particles are not always easy to find under the electron microscope, and even in the all too rare cases in which such particles are revealed, proof that the particles are a virus, and that this virus causes the partic- ular disease, requires much additional work and time.

It will b e seen below that a few plant symptoms, such as oak leaf

Characteristics of Plant Viruses 397

patterns on leaves and chlorotic or necrotic ring spots, can b e attrib- uted to viruses with s o me d e g r ee of certainty. T h e other symptoms c a u s ed by viruses r e s e m b le those c a u s ed by mutations, nutrient defi- ciencies or toxicities, insect secretions, by other pathogens, and other factors. T h e determination, therefore, that certain plant symptoms are c a u s ed by viruses involves the elimination of every other possible cause of the d i s e a s e, and the transmission of the virus from d i s e a s ed to healthy plants in a way that w o u ld exclude transmission of any of the other causal agents.

T h e present methods for detection of plant viruses are still the s a me that were u s ed to detect viruses more than eighty years ago. T h e se methods involve primarily the transmission of the virus from a dis- e a s ed to a healthy plant by budding, grafting, or by plant sap that has b e e n p a s s ed through bacterial filters to remove any bacteria or other microorganisms present in the sap. Certain other methods of transmis- sion, such as by dodder or insect vectors, are also u s ed to demonstrate the presence of a virus. Reproduction of "virus-like" symptoms after u se of these methods has almost always b e e n sufficient, as proved by s u b s e q u e nt work, for establishing that one or more viruses are present in the infected plant and that the d i s e a se is of viral origin.

Reproduction of symptoms after grafting or mechanical inoculation proves that the symptoms could not b e c a u s ed by mutation or by envi- ronmental conditions, but does not exclude the possibility that bacte- ria, fungi, or toxic substances are involved. T h e a b s e n ce of other path- ogens can b e checked, however, by microscopic examination of the sap of the infected plants, since all pathogens but viruses are visible in the light microscope. T h e differentiation of viruses from toxins and other nonliving agents that can cause physiological disturbances upon transfer to other plants is usually m a de on the basis of serial transmis- sion of the virus from one plant to the next. Any toxic substances that might have b e e n present in the original infected plant w o u ld b e di- luted too greatly to cause symptoms in s u b s e q u e nt transmission tests, and it w o u ld b e concluded that the symptoms were c a u s ed by a virus.

Modern methods of purification, electron microscopy, and particu- larly serology provide still other approaches to the problem of virus detection. Purification of a virus is usually not attempted until it is shown fairly definitely by other means that a particular d i s e a se is c a u s ed by a virus. It could b e used, however, in combination with electron microscopy and other methods for determining conclusively the existence of a virus in the plant. Unfortunately, b e c a u se of inher- ent difficulties of purification, only relatively few of the known viruses have b e e n purified so far.

Serology, on the other hand, provides an excellent tool for both de- tection and identification of a virus in a plant. Serological tests can b e carried out rather rapidly and are u s ed in practice for detection of vi- ruses in mother plants, tubers, etc., that are u s ed for production of vi- rus-free stock. T h e main difficulty with serology is that it can b e u s ed to detect only viruses that have b e e n already studied extensively and for which appropriate antisera are available.

Morphology

Plant viruses come in different shapes and sizes, but for conve- nience they can all b e grouped as either elongate or spherical (Fig.

83).

S o me elongated viruses are short, bacillus-like rods, approximately two to three times as long as they are wide, as in the cases of alfalfa mosaic virus which measures 18 x 58 millimicrons (ðéì), wheat striate mosaic virus (78 x 176 ðéì) , and the Gomphrena virus (90 x 2 4 0 nux).

Others, like tobacco mosaic virus and barley stripe mosaic virus, have the shape of rigid rods with measurements about 15 x 300, and 20 x 130 ðéì , respectively. Finally, most of the elongated viruses appear as long, thin, flexible threads that are usually 10-13 ðéì, wide and range in length from 480 ðéì (white clover mosaic virus) to 1250 ðéì (beet yellows virus). Many of the elongated viruses s e em to occur in parti- cles of differing lengths, and the number given usually represents the length that is more common than any other, and which, in s o me virus- es, is also the arithmetical mean of all particle lengths.

Most, and probably all, spherical viruses are actually polyhedral, ranging in diameter from about 17 ðéì (satellite virus) to 60 ðéì (wound tumor virus). Recently, a polyhedral virus (blue green algal virus LPP-1) 56 ðéì in diameter was discovered that infects algae and that has a tail approximately 14 ðéì long. A tail has also b e e n observed in the tomato spotted wilt virus.

T h e surface of both the elongated and the spherical viruses is not continuous and smooth but consists of a definite number of " b u m p s ."

T h e se b u m ps are the protein subunits, which are spirally arranged in

F i g. 83. Relative s h a p e s, sizes, a nd structures of s o me representative plant viruses. (A) An elongate virus a p p e a r i ng as a flexuous thread. (B) A rigid rod-shaped virus. ( B - l) S i de arrangement of protein subunits (PS) a nd nucleic acid (NA) in viruses A a nd B.

(B-2) C r o ss section v i ew of the s a me viruses. HC = hollow core. (C) A short, bacil- lus-like virus. ( C - l) Cross-section v i ew of s u ch a virus. (D) A polyhedral virus. ( D - l) An icosahedron, r e p r e s e n t i ng the 20-sided symmetry of the protein subunits of the polyhedral virus.

Β-Ι _ß B - 3

c ^C-l

D D-l

A Β

the elongated viruses, and packed on the sides of the polyhedral parti- cles of the spherical viruses. In cross sections, the elongated viruses appear as hollow tubes with the protein subunits forming the outer coat and the nucleic acid, also spirally arranged, e m b e d d e d b e t w e en the inner ends of two successive spirals of the protein subunits. T h e spherical viruses are also hollow, the visible shell consisting of the protein subunits, with the nucleic acid inside the shell and arranged in a yet unknown manner.

S o me of the short, bacillus-like viruses, like potato yellow dwarf virus, and the Gomphrena virus, and, of the polyhedral viruses, to- mato spotted wilt virus, s e em to b e provided with an outer envelope or membrane.

Composition and Structure

E a ch plant virus consists of at least a nucleic acid and a protein.

E v i d e n ce accumulates, however, that some viruses may consist of more than one protein, and also that s o me of them may contain addi- tional chemical compounds, such as polyamines.

T h e proportions of nucleic acid and protein vary with each virus, nucleic acid making up 5 - 4 0 % of the virus and protein making up the remaining 6 0 - 9 5 %. T h e lower nucleic acid and the higher protein percentages are found in the elongated viruses, while the spherical viruses contain higher percentages of nucleic acid and lower percent- ages of proteins. T h e total weight of the nucleoprotein of different vi- rus particles varies from 4.6 million molecular weight units (bromegrass mosaic virus) to 39 million (tobacco mosaic virus) to 73 million (tobacco rattle virus). T h e weight of the nucleic acid alone, however, ranges only b e t w e en 1 and 3 million molecular weight units per virus particle, approximately.

COMPOSITION AND S T R U C T U RE OF VIRAL P R O T E IN

Viral proteins, like all proteins, consist of amino acids. About 2 0 amino acids s e em to combine in various s e q u e n c es to form the various viral proteins. S o me amino acids s e em to b e present in all viral pro- teins while others are present in some, but not in all. T h e s e q u e n ce of amino acids within a protein is presumably dictated by the genetic material, which in viruses is either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and determines the nature of the protein.

T h e protein components of plant viruses do not consist of single peptide chains but are c o m p o s ed instead of repeating subunits rang-

Characteristics of Plant Viruses 401

ing in molecular weight from about 17,000 to 60,000 for the various viruses. T h e amino acid content and s e q u e n ce is constant for the iden- tical protein subunits of a virus, but may vary for different viruses, different strains of the s a me virus, and even for different proteins of the s a me virus particle. T h e content and partial s e q u e n c es of amino acids are known for the proteins of several viruses, but only for the protein of tobacco mosaic virus (TMV) is the complete s e q u e n ce of amino acids known. T h u s, the protein subunit of T MV consists of 158 amino acid residues in a constant s e q u e n c e, b e g i n n i ng with N-acetyl- serine and e n d i ng with threonine. T h e molecular weights of the 158 amino acids of each T MV protein subunit add up to a total of 17,531.

Since each T MV virus particle weighs approximately 39,000,000 mo- lecular weight units, of which 95 % or 37,000,000 is protein, it is easily calculated that the protein of each virus particle consists of approxi- mately 2100 protein subunits.

T h e spatial arrangement of the peptide chain for the formation of the protein subunit is yet unknown, although there is s o me e v i d e n ce that it has a helical configuration and that it must be folded to fit in the space allotted to each protein subunit. In T MV the protein subunit s e e ms to b e a prolate ellipsoid 70 A long (lA = 0.1 τημ) and 2 0 - 2 5 A in diameter, each subunit exhibiting a notch at 40 A radius in which the R NA chain of the complete virus particle is tightly packed. T h e pro- tein subunits are arranged in a helix with a 23 A pitch containing 16%

subunits per turn (or 49 subunits per three turns). T h e central hole of the virus particle down the axis has a diameter of 40 A, while the maxi- m um diameter of the particle is 180 A. E a ch T MV particle consists of approximately 130 helix turns of protein subunits.

In the polyhedral plant viruses the protein subunits appear to b e arranged in structures that show icosahedral symmetry, i.e., the virus particles consist of 60, or s o me multiple of 60, asymmetric subunits arranged in groups of 5 or 6. T h e protein subunits are tightly packed in arrangements that produce 20, or some multiple of 20, facets and form a shell. Within this shell the nucleic acid is folded or otherwise orga- nized, probably in ways related to the structure of the shell, but the exact relationships are as yet unknown.

COMPOSITION AND S T R U C T U RE OF V I R AL N U C L E IC ACID

T h e nucleic acid of most plant viruses s e e ms to consist of ribonu- cleic acid ( R N A) but recently a virus (blue-green algal virus LPP-1) that attacks algae was shown to contain deoxyribonucleic acid ( D N A ).

Both nucleic acids are long-chain polymers consisting of hundreds or,

more often, thousands of units called nucleotides. E a ch nucleotide consists of a ring c o m p o u nd called the b a se attached to a 5-carbon sugar [ribose (I) in RNA, deoxyribose (II) in D N A] which in turn is esterified with phosphoric acid. T h e sugar of one nucleotide may also react with the phosphate of another nucleotide, and this may b e re- peated many times, thus forming the R NA or D NA strand. In viral RNA, one of only four b a s es can b e attached to each ribose molecule.

HOCH.

(i) ^(II)

NH2

C ^XN II I

N / C ^ C H

Adenine

NH2

X ^. Cytosine HC Í

^ N^ ^O O - P — 0 - C H2^ Ox

o^x O"

OH Ï — Ñ—O—CH,

Characteristics of Plant Viruses 403

T h e se b a s es are adenine, guanine, cytosine, and uracil, which are fre- quently indicated by their initials, A, G, C, and U. T h e first two, ade- nine and guanine, are purines, while cytosine and uracil are pyrimi- dines. T h e chemical formulas of the b a s es and one of their possible relative positions in the R NA chain, are shown in (III). D N A, whether in the chromosomes or in D NA viruses, is similar to R NA with two small, but very important differences: the oxygen of the sugar hy- droxyl is missing; and the b a se uracil is replaced by the b a s es thymine (IV), 5-methyluracil (V) or, in s o me cases, by certain other bases.

I

H C^ / CL Ç ^O

(IV) (V)

T h e s e q u e n ce and the frequency of the b a s es on the R NA strand may vary greatly from one R NA to another, but they are fixed within a given R NA and determine its properties. T h u s, T MV R NA s e e ms to contain 1900 molecules of adenine, 1680 of guanine, 1180 of cytosine, and 1740 of uracil, for a total of 6500 nucleotides. T h e s e q u e n ce of b a s es is not yet known for any of the virus RNA's, but recen t discover- ies have revealed the s e q u e n ce of b a s es on certain RNA's found in the normal cell. RNA, whether in R NA viruses or in healthy cells, is usually found as single strands, although rarely it may exist as double- stranded RNA. T h e double-stranded R NA is possible through hydro- gen bonds b e t w e en certain b a s e s; there are two such bonds b e t w e en adenine and uracil, and three bonds b e t w e en cytosine and guanine.

B e c a u se the b a s es of different R NA segments are randomly distribut- ed, b o n d i ng is possible only where complementary b a s es h a p p en to mee t by chance; b o n d i ng is therefore limited and the double structure is weak and unstable.

In chromosomal or viral double-stranded D N A, the s e q u e n ce and frequency of the four b a s es on the one strand determine the s a me on the other strand, b e c a u se during replication each adenine molecule selects and holds the complementary thymine molecule, and each guanine molecule selects and holds the complementary cytosine mol- ecule, and vice versa. This leads to the existence in the D NA mole- cule of equal amounts of adenine and thymine and also equal amounts of guanine and cytosine. T h e perpetual facing of complementary bases all along the D NA molecule results in maximal b o n d i ng b e - tween every opposite pair of b a s es and the double-stranded structure

NHo I II HC.

I Ç

of D NA is most stable. T h e whole D NA molecule is intertwined to form a helical pattern.

With the exception of the R NA of T M V , w h o se simple arrangement in the form of a helix within the notches of the identical protein sub- units has already b e e n mentioned, little is known about the arrange- men t of the nucleic acid in any virus. T h e r e is also very little informa- tion about the functional interrelationships b e t w e en the nucleic acid and the protein components of viruses. S o me plant viruses, e.g., tur- nip yellow mosaic virus, normally produce in the plant empty protein shells along with normal virus particles, and under proper environ- mental conditions purified T MV protein can b e m a de to form rods seemingly identical in appearance with those of complete T MV parti- cles, although they are of random lengths. This would indicate that the nucleic acid does not dictate the arrangement of the protein, al- though it determines the shape of the protein subunit which in turn determines the shape and size of the protein coat. T h e presence of nucleic acid, however, at least in the elongated viruses, s e e ms to play a role in the stabilization of the protein shell and thus determines its length.

The Biological Function of Viral Components —Coding

Although apparently each virus and virus strain produces its own distinct protein coat, the only function of the protein component of all viruses is to provide a protective sheathing for the RNA. Protein pro- tects the R NA from the disintegrating effect of the host enzyme ribo- nuclease (RNase), from destruction by heat, ultraviolet light, and numerous chemical substances. Protein itself has no infectivity, since inoculations with purified protein do not lead to virus synthesis or multiplication, nor to d e v e l o p m e nt of symptoms. In inoculations with intact virus particles (virions) the protein does not s e em to assist or to affect the R NA either in its functions or its composition, since inocula- tions with R NA alone can cause infection and lead to synthesis of ne w R NA and also of ne w protein, both b e i ng identical with the R NA and protein of the original virus. T h e synthesis, composition, and structure of the protein, on the other hand, d e p e nd entirely on the R NA compo- nent. Whe n the R NA of a strain of T MV (A), from which all protein has b e e n removed, is allowed to react in vitro with the protein of another strain of T MV (B), complete virus particles are formed from the R NA of A and the protein of B. Inoculations with this " h y b r i d" virus always lead to infections and symptoms identical with those produced by strain A, and all the ne w virus produced in the plant is identical with

Virus Infection and Virus Synthesis 405

virus A. This indicates clearly the nonparticipation of the protein component in the infection and the multiplication of the virus, and also that R NA alone is responsible for the synthesis and a s s e m b ly of both the R NA and the protein.

Infectivity of viruses is thus strictly the property of their nucleic acid, which in most plant viruses is RNA. T h e capability, however, of the viral R NA to reproduce both itself and its specific protein, indi- cates that the R NA carries the genetic determinants of the viral charac- teristics. T h e expression of each inherited characteristic d e p e n ds on the s e q u e n ce of nucleotides within a certain area (cistron) of the viral R NA which determines the s e q u e n ce of amino acids in a particular protein, either structural or enzyme. This is called coding and s e e ms to b e identical in all living organisms and the viruses.

T h e code consists of coding units called codons. E a ch codon con- sists of three adjacent nucleotides and codes for (determines the posi- tion of) a given amino acid. C o d o ns do not overlap, that is, each triplet of adjacent nucleotides participates in only one codon, yet each codon may code for more than one, but only a certain few, amino acids, and under certain conditions, more than one codon can code for the s a me amino acid.

T h e amount of RNA, then, contained in each virus indicates the approximate length of, and the number of nucleotides in, the viral RNA. This in turn determines the number of codons in each R NA and, therefore, the number of amino acids that can b e c o d ed for. Since the protein subunit of viruses contains relatively few amino acids (158 in TMV), the number of codons utilized for its synthesis is only a fraction of the total n u m b er of codons available (158 out of 2 1 3 0 in TMV). T h e remaining codons are presumably involved in the synthesis of several other proteins, either structural or e n z y m e s, and it is these proteins that are apparently responsible to a large extent for the d i s e a s ed con- ditions p r o d u c ed in many virus infections of plants.

Virus Infection and Virus Synthesis

Infection of a plant with a virus is directly d e p e n d e nt on virus syn- thesis, since infection will not occur unless the virus multiplies within the susceptible host tissue.

Plant viruses enter cells only through wounds m a de mechanically or by vectors or by deposition into an ovule by an infected pollen grain. Following contact of the virus with the cytoplasm of a suscepti- b le plant cell, the virus b e c o m e s, somehow, attached to the cell and cannot b e r e m o v ed by thorough and repeated washing.

Since the active part of the virus, the nucleic acid, is completely enclosed by the protein, it must b e freed from the protein shell in or- der to establish infection. T h e uncoating of the nucleic acid appar- ently takes place during the first hour or so following inoculation, oc- curs in an orderly stepwise manner, and is probably accomplished by the host cell rather than the virus, since no plant virus is known to con- tain or produce by itself any enzymes. T h e fragments of the d e g r a d ed protein coat remain in the cell and probably b e c o me part of the pro- tein pool of tne cell and are utilized in the synthetic processes of the cell.

Once the nucleic acid (RNA) of the virus is freed from the protein coat, its first effect s e e ms to b e the induction of formation by the cell of enzymes called RNA-polymerases, RNA-synthetases, or RNA-repli- cases. T h e se enzymes, in the presence of the viral R NA acting as a template and of the nucleotides that c o m p o se RNA, produce addi- tional RNA. It appears that the first ne w R NA produced is not the viral R NA but a complementary strand that is a mirror i m a ge of the virus, and which, as it is formed, is temporarily connected to the viral strand (Fig. 84). T h u s, the two form a double-stranded R NA that soon sepa- rates to produce the original virus R NA and the complementary (—) strand, the latter then serving as a template for more virus (+ strand) R NA synthesis. If the viral nucleic acid is double-stranded R NA or D N A, the process of replication is probably the same, but the step of formation of the complementary (-) strand is eliminated.

As soon as ne w viral nucleic acid is p r o d u c ed it induces the host cell to produce the protein molecules that will b e the protein subunits and that will form the protein coat of the virus. Apparently, only a part of the viral R NA strand is n e e d e d to participate in the formation of the viral protein. Since each amino acid on the protein subunit molecule is " c o d e d" by three nucleotides of the viral RNA, for T M V , whose R NA consists of 6400 nucleotides and its protein of 158 amino acids, only 474 nucleotides are required to code the arrangement of the amino acids in the protein subunit.

Protein synthesis in healthy cells d e p e n ds on the presence of amino acids and the cooperation of ribosomes, m e s s e n g er RNA, and transfer RNA's. E a ch transfer R NA is specific for one amino acid which it car- ries toward and along the m e s s e n g er RNA. M e s s e n g er RNA, which is produced in the nucleus and reflects part of the D NA code, deter- mines the kind of protein that will b e p r o d u c ed by coding the se- q u e n ce in which the amino acids will b e arranged. T h e ribosomes s e em to travel along the m e s s e n g er R NA and to provide the energ y for the bonding of the prearranged amino acids to form the protein (Fig.

85).

^ RNA ^| ^ J nucleotides wzzm Indicates virus strand ® Virus RNA (parent) <j> Complementary RNA strand (replicative RNA) ® New virus RNA Fig. 84. Hypothetical schematic representation of viral RNA replication.

F i g. 85. S c h e m a t ic representation of the b a s ic functions in a living cell. N o te the fun- damental role of m e s s e n g er R NA in the formation of e n z y m e s.

Proteins (Enzymes^

Photosynthates Inorganic +

^ nutrients >

Messenger RNA

Polysome Ribosomes

Nucleus

DNA

Cell division

RNA-nucleotides

Amino acids

Translocation and Distribution of Viruses in Plants 409

For virus protein synthesis, the viral R NA itself (or the part of the viral R NA coding for the viral protein) plays the role of m e s s e n g er RNA. T h e virus apparently utilizes the amino acids, ribosomes, and transfer RNA's of the host, but it b e c o m es its own blueprint (messenger RNA), and the protein formed is for exclusive u se by the virus as a coat (Fig. 86).

D u r i ng virus synthesis it appears that parts of its nucleic acid also b e c o me involved with synthesis of proteins other than the viral coat protein. S o me of these proteins are probably enzymes, either of the kinds already present in the host cell or entirely new, which may acti- vate or initiate in the cell chemical reactions that, in turn, may affect the physiological functions of the cell.

Whe n ne w virus nucleic acid and virus protein subunits have b e e n produced, the nucleic acid s e e ms to organize the protein subunits around it, and the two are a s s e m b l ed together to form the complete virus particle, the virion.

T h e site or sites of the cell in which virus R NA and protein are syn- thesized and in which these two components are a s s e m b l ed to pro- d u ce the virions have not yet b e e n determined with absolute certainty for any of the plant viruses. Studies with T MV suggest that the virus RNA, after it is freed from the protein coat, moves into the nucleus and possibly the nucleolus, where it replicates itself. T h e ne w virus R NA is then r e l e a s ed into the cytoplasm, where it serves as a m e s s e n g er R NA and, in cooperation with the ribosomes and transfer RNA's, pro- duces the virus protein subunits. T h e a s s e m b ly of virions follows, also in the cytoplasm. In other viruses, e.g., p ea enation mosaic virus, b e an yellow mosaic virus, Gomphrena virus, the synthesis of viral nucleic acid and protein, as well as their a s s e m b ly into virions, s e em to take place in the nucleus, from which the virus particles are then released into the cytoplasm. Although there is e v i d e n ce that both components of some viruses are synthesized and a s s e m b l ed in the cytoplasm alone and that s o me viruses are synthesized e v en inside chloroplasts, ade- quate proof for both of these statements is not yet available.

T h e first intact virions appear in plant cells approximately 10 hours after inoculation. T h e virus particles may exist singly or in groups and may form amorphous or crystalline inclusion bodies within the cell areas (cytoplasm, nucleus, nucleolus) in which they h a p p en to be .

Translocation and Distribution of Viruses in Plants

Following introduction of a virus into a cell, the virus apparently moves, before or after s h e d d i ng its protein coat, toward the sites of

Translocation and Distribution of Viruses in Plants 411 synthesis and a s s e m b ly of its components. This m o v e m e nt is probably passive and d e p e n ds on the protoplasmic streaming of the cell, but other mechanisms may also b e involved. F or viruses w h o se R NA or the entire virion is synthesized in the nucleus, the nucleus as well as the virus may b e moving toward each other. If the virus protein is syn- thesized in the cytoplasm, the R NA then must m o ve out of the nucleus and into the cytoplasm.

After virus reproduction has taken place the virions are usually found aggregated in amorphous or crystalline inclusion bodies or they are dispersed, e.g., in the cytoplasm, the nucleus. Although most plant viruses s e em to b e present primarily in the ground cytoplasm or the endoplasmic reticulum, several have b e e n found in the nucleus, nu- cleolus, and the chloroplasts, either as inclusion bodies or as dis- p e r s ed virions. This makes it obvious that viruses or their R NA move from one part of the cell to another, but how this is accomplished is not known.

For infection of a plant by a virus to take place, the virus must move from one cell to another and must multiply in most, if not all, cells into which it moves. In their m o v e m e nt from cell to cell, viruses are thought to follow the pathways through the p l a s m o d e s m a ta connect- ing adjacent cells, and through which the e n d o p l a s m ic reticulum may connect one cell to another. Viruses, however, do not s e em to move through parenchyma cells unless they infect the cells and multiply in them, thus resulting in continuous and direct cell-to-cell invasion.

T h e rate of cell-to-cell spread of viruses s e e ms to vary with the kind and age of plant cells infected and is greater b e t w e en elongated, young cells than b e t w e en round, older cells. Viruses also s e em to move faster at higher than they do at lower temperatures, probably b e c a u se of the increased protoplasmic streaming and general acceler- ation of all cell activities during higher temperatures. Although var-

Host DNA J W U o r V R Viral RNA ΙËËËËËËËËËËËËËËËÃ

Viral RNA replicase J V X T w Complementary viral RNA Ë Ë Ë . Ribosomal RNA X*»e(J Transfer RNA

Amino acid ^m Viral protein ^{j w}

Fig. 86. S e q u e n ce of e v e n ts in virus infection a nd b i o s y n t h e s i s. Infection b e g i ns with the arrival of virus particle (VP) in a w o u n d ed cell (lower left) a nd is c o m p l e t ed with the production of n u m e r o us n e w virus particles in the cell (lower center left).

CW = cell wall, R = r i b o s o m e, Í = n u c l e u s, ç = n u c l e o l u s, Ñ = p o l y r i b o s o me (polysome), Pp = p o l y p e p t i d e, Ps = protein subunit.

ious rates of virus m o v e m e nt have b e e n proposed by several investi- gators, it appears that in leaf parenchyma cells the virus moves approximately 1 mm, or 8-10 cells, per day.

Although some viruses appear to b e more or less restricted to cell- to-cell movement through primarily parenchyma cells, a large number of viruses are known to b e rapidly transported over long distances through the phloem and a few viruses through the xylem. Transport of viruses in the phloem apparently occurs in the sieve tubes, in which they can m o ve as rapidly as 15 cm in the first 6 minutes. However, there s e e ms to b e a delay in the virus moving from parenchyma cells into phloem cells, since most viruses require 2-5 or more days to move out of an inoculated leaf. Once the virus has entered the phloem, it moves rapidly away from the point of entrance in the phloem toward growing regions (apical meristems) or other regions of food utilization in the plant like tubers and rhizomes (Fig. 87). F or example, when potato virus is introduced into the basal leaves of young potato plants, it moves rapidly up the stem, but when plants already forming tubers are similarly inoculated, the virus does not move upward for more than 30 days while it moves downward into the tubers. This indicates that viruses in the phloem generally move in the same direction and at rates similar to those reported for photosyn- thates and other materials in the phloem. T h e r e is also evidence that viruses m o ve through stems and roots in directions opposite to that of the food transport. Once in the phloem the virus spreads systemically throughout the plant and reenters the parenchyma cells adjacent to the phloem through plasmodesmata.

T wo viruses, alfalfa dwarf virus and phony peach virus, are defi- nitely known to spread in, and to b e closely restricted to, the xylem.

Another virus, southern b e an mosaic virus, also moves through the xylem, although it is not limited to it and can move in parenchyma and for long distances in the phloem. T h e m o v e m e nt from a living cell into a nonliving xylem vessel suggests that the virus may b e able to pass through the p l a s ma m e m b r a ne of the living cell, since no plasmodes- mata are known to connect these two kinds of cells.

T h e form (RNA or complete virions) in which viruses are translo- cated has not yet b e e n determined with certainty. It appears, howev- er, that although it is equally possible for most but the largest diameter polyhedral viruses to move from cell to cell through the plas- m o d e s m a ta either as R NA or as complete virus particles, viruses in the phloem and xylem move mainly or exclusively as complete virions.

T h e distribution of viruses within plants varies with the virus, the plant, and the interaction of the two. T h e d e v e l o p m e nt of local lesion

F ig 87. S c h e m a t ic representation of the direction a nd rate of translocation of a virus in a plant. [ A d a p t ed from G. S a m u el (1934). Ann. Appl. Biol 2 1 : 90-111.]

* 10 days 18 days ^25 days

> 3 days > 4 days 5 days

3 days I day

symptoms has b e e n considered as an indication of the localization of the virus within the necrotic area; although this is probably true in some cases, in several diseases the lesions continue to enlarge and, sometimes, d e v e l o p m e nt of systemic symptoms follows, indicating that the virus continued to spread b e y o nd the lesions.

In systemic virus infections, some phloem- or xylem-translocated viruses s e em to be limited to these tissues and to a few adjacent paren- chyma cells. T h e se include most of the yellows-type diseases (aster yellows, potato leaf roll, cereal yellow dwarf, etc.) and the two xylem- limited viruses causing alfalfa dwarf and phony peach diseases. Vi- ruses causing mosaic-type d i s e a s es are not generally tissue-restricted, although there may be different patterns of localization. Mosaic virus- infected plant cells have b e e n estimated to contain b e t w e en 100,000 and 10,000,000 virus particles per cell. Systemic distribution of s o me viruses is quite thorough and may involve all living cells of a plant.

Other viruses, however, s e em to leave segments or gaps of tissues that are virus-free. S o me viruses invade newly p r o d u c ed apical meriste- matic tissues almost immediately, while in other cases growing points of stems or roots of affected plants remain free of virus.

Of the more than 500 known viruses, all of which invade s o me plant hosts systemically, less than a hundred have b e e n shown to b e trans- mitted by s e ed and in fewer than ten of these does the transmission excee d 5 0 %. It appears, therefore, that most viruses are somehow in- capable of invading developing embryos, perhaps b e c a u se of the lack of plasmodesmatal connections b e t w e en the embryos and the sur- rounding tissues. This may also b e explained by the inability of vi- ruses to survive in the micro- or m e g a s p o re mother cells or embryo sacs, or by the inactivation of the virus during maturation and storage of the seed. In cases in which s e ed transmission of virus occurs, the virus may b e present in the ovule or pollen, or both.

Symptoms C a u s ed by Plant Viruses

Plants infected with viruses may d e v e l op various types of symptoms on all or some of their parts. T h e symptoms may b e either general and nonspecific for the virus, or they may b e quite characteristic of the par- ticular virus or virus group that causes them.

T h e most common and sometimes the only kind of symptoms pro- d u c ed is r e d u c ed growth rate of the plant, resulting in various degrees of dwarfing or stunting of the entire plant. Almost all viral diseases s e em to cause some d e g r ee of reduction in total yield, and the length of life of virus-infected plants is usually shortened. Any or all of these

Symptoms Caused by Plant Viruses 415 effects may b e severe and easily noticeable, or they may b e very slight and easily overlooked.

T h e most obvious symptoms of virus-infected plants are usually those appearing on the foliage, but s o me viruses may cause striking symptoms on the stem, fruit, and roots, with or without symptom de- velopment on the leaves. In almost all virus d i s e a s es of plants occur- ring in the field, the virus is present throughout the plant (systemic infection) and the symptoms p r o d u c ed are called systemic symptoms.

In many plants inoculated artificially with certain viruses, and proba- bly in s o me natural infections, the virus causes the formation of small, usually necrotic lesions only at the points of entry (local infections), and the symptoms are called local symptoms or local lesions. Many viruses may infect certain hosts without ever causing d e v e l o p m e nt of visible symptoms on them. Such viruses are usually called latent vi- ruses, and the hosts are called symptomless carriers. In other cases, however, plants that usually d e v e l op symptoms upon infection with a certain virus may remain temporarily symptomless under certain en- vironmental conditions (e.g., high or low temperature), and such symptoms are called masked. Finally, plants may show acute or se- vere symptoms soon after inoculation that may lead to death of the host; if the host survives the initial shock phase, the symptoms tend to b e c o me milder (chronic symptoms) in the subsequently d e v e l o p i ng parts of the plant, leading to partial or even total recovery. On the other hand, symptoms may progressively increase in severity and may result in gradual (slow) or quick decline of the plant.

T h e most c o m m on types of plant symptoms p r o d u c ed by systemic virus infections are mosaics, yellows, and ringspots.

Mosaics, characterized by light-green, yellow, or white areas inter- m i n g l ed with the normal green of the leaves or fruit, or of whitish areas intermingled with areas of the normal color of flowers or fruit.

D e p e n d i ng on the intensity or particular pattern of discolorations, mosaic-type symptoms may b e described as mottling, streak, ring pat- tern, line pattern, veinclearing, veinbanding, chlorotic spotting, etc.

T h e viruses causing most mosaic diseases are mechanically transmit- ted and usually have aphid vectors in nature, are generally resistant to brief heat treatments, and do not stop flowering or affect the dormancy of b u d s.

Yellows, characterized by uniform discoloration (chlorosis, yellow- ing, bronzing, or reddening) of the foliage without any spotting pat- terns, although s o me veinclearing may b e present. Viruses causing the true yellows d i s e a s es show a tendency to produce virescent flow- ers, to break the dormancy of axillary b u ds and induce cessation of

flowering, to b e leafhopper transmitted, and to b e relatively sensitive to heat treatment.

Ringspots, characterized by the appearance of chlorotic or necrotic rings on the leaves and sometimes also on the fruit and stem. In many ringspot diseases the symptoms, but not the virus, tend to disappear after onset and to reappear under certain environmental conditions.

Most ringspot-causing viruses are not transmitted by either aphids or leafhoppers, but some of them are transmitted by nematodes.

A large number of other less common virus symptoms have b e e n described and include stunt (e.g., corn stunt), dwarf (e.g., barley yel- low dwarf), leaf roll (e.g., potato leaf roll), rosette (e.g., peach rosette), witches' broom (e.g., lilac witches' broom), phloem necrosis (e.g., elm p h l o em necrosis), enation (e.g., p ea enation mosaic), tumors (e.g., w o u nd tumor), rubbery w o od (e.g., apple rubbery wood), pitting of stem (e.g., apple stem pitting), pitting of fruit (e.g., pear stony pit), and flattening and distortion of stem (e.g., apple flat limb). T h e se symp- toms may b e accompanied by other symptoms on other parts of the same plant.

In addition to the macroscopically visible symptoms, several histo- logical and cytological abnormalities appear in virus-infected plants.

In some, certain tissues or cells appear to enlarge, while others remain smaller than normal and may b e c o me discolored, distorted, or ne - crotic. Chloroplasts may b e smaller and fewer in many mosaic- or yel- lows-affected tissues. Intracellular inclusions of various shapes may b e found in cells of virus-infected plants.

Physiology of Virus-Infected Plants

Plant viruses do not contain any enzymes, toxins, or other sub- stances involved in the pathogenicity of other pathogens, and yet cause a variety of deleterious effects on the host. T h e viral nucleic acid (RNA) s e e ms to b e the only determinant of disease, but the mere presence of R NA or complete virions in a plant, even in large quanti- ties, does not s e em to b e sufficient reason for the disease syndrome, since some plants containing much higher concentrations of virus than others may show milder symptoms than the latter or they may b e symptomless carriers. This indicates that viral diseases of plants are not d ue primarily to depletion of metabolites that have b e e n diverted toward synthesis of the virus itself, but to other more indirect effects of the virus on the metabolism of the host. T h e se effects are brought about, possibly, through the virus-induced synthesis of ne w proteins

Physiology of Virus-Infected Plants 417 by the host, s o me of which are biologically active substances (enzymes, toxins, etc.) and can interfere with the normal metabolism of the host. Such interference could lead to alteration of the perme- ability of m e m b r a n e s, activation of host e n z y me systems that may re- sult in toxic e n d products, interruption of metabolic cycles resulting in shifts to other pathways, and accumulation of normally transient me- tabolites. Although the mechanisms by which viruses affect the physi- ology of the plant are not clearly understood yet, considerable infor- mation is available on the virus-induced changes in certain physiological processes and in the metabolism of certain groups of c o m p o u n ds in the infected plant.

Viruses generally are known to cause a decrease in photosynthesis through a decrease in chlorophyll per leaf, in chlorophyll efficiency, and in leaf area per plant. Viruses also cause a decrease in the amount of growth-regulating substances (hormones) in the plant, frequently by inducing an increase in growth-inhibiting substances. A decrease in soluble nitrogen during rapid virus synthesis is rather common in virus diseases of plants, and in the mosaic diseases there is a chronic decrease in the levels of carbohydrates in the plant tissues.

Respiration of plants is generally increased immediately after infec- tion with a virus, but after the initial increase the respiration of plants infected with some viruses remains higher, while with other viruses it b e c o m es lower than that of healthy plants, and with still other viruses it may return to normal. T h e activity of most oxidative enzymes, such as polyphenoloxidase, cytochrome oxidase, and peroxidase s e e ms to b e generally higher in virus-infected than in healthy plants, but a de- crease in enzymatic activity has also b e e n reported for several oxida- tive enzymes in different host-virus systems and in various stages of the s a me disease. Also, contrary to the decrease in carbohydrate levels in mosaic diseases, carbohydrates, and particularly starch, s e em to accumulate in yellows-like d i s e a s e s, such as the curly top of sugar beets.

T h e amounts of nonvirus nitrogenous c o m p o u n ds in d i s e a s ed plants s e em to b e generally lower than those found in healthy plants, proba- bly b e c a u se the virus, which in s o me virus-host systems may account for 3 3 - 6 5 % of the total nitrogen in the plant, is formed at the e x p e n se of the normal levels of nitrogenous compounds in the plant. Whe n the plant, however, is provided with high nitrogen nutrition, the amount of total nitrogen in d i s e a s ed plants may b e higher than that in healthy plants, especially after completion of the phase of rapid virus synthe- sis. A similar trend appears to exist regarding the phosphorylated c o m p o u n d s; there is a higher content in R NA and D NA phosphorus in

infected than in healthy leaves, while the nonnucleic acid phosphorus appears to decrease during virus synthesis but to reach almost normal levels after completion of virus synthesis.

T h e effect of virus infections on the accumulation of other groups of substances, such as organic acids and phenolics, has not yet b e e n de- termined conclusively, but the information available now indicates that organic acids are relatively little affected by virus infections, while at least some phenolics may increase considerably in virus-in- fected plants.

It is obvious from the above that most or all of the functional sys- tems of the plant may b e directly or indirectly affected by virus infec- tion and may in one way or another lead to abnormal accumulations of metabolites. Certain degrees or types of such metabolic derangements can probably b e tolerated by the plant and do not cause any symp- toms, while others probably have a deleterious effect on the host and contribute to symptom development. T h e effect of virus on nitrogen- ous compounds, on growth regulators, and on phenolics, have often b e e n considered to b e the immediate causes of various types of symp- toms, since the first two are so profoundly involved in anything con- cerne d with plant growth and differentiation, and since the oxidized products of phenolics may themselves, b e c a u se of their toxicity, b e responsible for the d e v e l o p m e nt of certain kinds of necrotic symp- toms.

Transmission of Plant Viruses

Plant viruses rarely, if ever, come out of the plant spontaneously.

For this reason, viruses are not disseminated as such by wind or water, and even when they are carried in plant sap or debris they generally do not cause infections unless they come in contact with the contents of a w o u n d ed living cell. Viruses, however, are transmitted from plant to plant in a number of ways such as vegetative propagation; mechani- cally through sap; and by seed, pollen, insects, mites, nematodes, dodder, and fungi.

TRANSMISSION OF V I R U S ES BY V E G E T A T I VE PROPAGATION Wheneve r plants are propagated vegetatively by b u d d i ng or graft- ing, by cuttings, or by the use of tubers, corms, bulbs, or rhizomes, any viruses present in the mother plant from which these organs are taken will almost always b e transmitted to the progeny. Considering that

Transmission of Plant Viruses 419

almost all fruit and many ornamental trees and shrubs are propagated by budding, grafting, or cuttings, and that many field crops, e.g., pota- toes, and most florist's crops are propagated by tubers, corms, or cut- tings, this m e a ns of transmission of viruses is the most important for all these types of crop plants. Transmission of viruses by vegetative propagation not only makes the n e w plants d i s e a s e d, but in the cases of propagation by b u d d i ng or grafting, the p r e s e n ce of a virus in the b ud or graft may result in appreciable reduction of successful b u d or graft unions with the rootstock and, therefore, in poor stands.

Transmission of viruses may also occur through natural root grafts of adjacent plants, particularly trees, the roots of which are often inter- m i n g l ed and in contact with each other. F or several tree viruses, natu- ral root grafts are the only known means of tree-to-tree spread of the virus within established orchards.

M E C H A N I C AL TRANSMISSION OF V I R U S ES THROUGH S AP

Mechanical transmission of plant viruses in nature, by direct trans- fer of sap through contact of one plant with another, is uncommon and relatively unimportant. Such transmission may take place b e t w e en closely s p a c ed plants following a strong wind that could cause the leaves of adjacent plants to rub together and, if w o u n d e d, to exchange some of their sap, and thus transmit any virus present in the sap. Po- tato virus X (PVX) s e e ms to b e one of the viruses most easily transmit- ted that way. Whe n plants are w o u n d ed by man during cultural prac- tices in the field or greenhouse and s o me of the virus-infected sap adhering to the tools, hands, or clothes is accidentally transferred to subsequently w o u n d ed plants, virus transmission through sap may b e rapid and w i d e s p r e ad and, as in the case of T MV on tobacco and to- mato, may result in very serious losses. Virus-infected sap transferred from plant to plant on the mouthparts or b o dy of animals feeding on and moving a m o ng the plants may similarly lead to virus transmission.

T h e greatest importance of mechanical transmission of plant viruses stems from its indispensability in studying almost every facet of the viruses that c a u se plant diseases, since all investigations of virus out- side the host are d e p e n d e nt on the ability to demonstrate and m e a s u re the infectiousness of the material.

F or mechanical transmission of a virus from one plant to another, tissues of the infected plant b e l i e v ed to contain a high concentration of the virus, i.e., young leaves and flower petals, are ground with a mortar and pestle or with s o me other grinder. Breakage of the cells results in release of the virus in the sap. Sometimes a buffer solution,

usually phosphate buffer, is a d d ed for stabilization of the virus. T h e expressed sap is then strained through cheesecloth and is centrifuged at low s p e e ds to remove tissue fragments, or at alternate low and high s p e e ds if concentration or purification of the virus is desired. T h e crude or treated sap is then a p p l i ed to the surface of leaves of young plants which have b e e n previously dusted with an abrasive such as 600-mesh Carborundum. Application of the sap is usually m a de by gently rubbing the leaves with a cheesecloth or gauze p ad d i p p ed in the sap, with the finger, a glass spatula, a painter's brush, or with a small sprayer. In successful inoculations, the virus enters the leaf cells through the wounds m a de by the abrasive or through broken leaf hairs and initiates ne w infections. In local-lesion hosts, symptoms usually appear within 4 to 7 or more days, and the number of local lesions is proportional to the concentration of the virus in the sap. In systemi- cally infected hosts, symptoms usually take 10-14 or more days to develop. Sometimes the s a me plants may first d e v e l op local lesions and then systemic symptoms. In mechanical transmission of viruses, the taxonomic relationship of the donor and receiving (indicator) plants is unimportant, since virus from one kind of plant, whether herbaceous or a tree, may b e transmitted to dozens of unrelated herba- ceous plants (vegetables, flowers, or weeds).

Although viruses are almost always transmitted by b u d d i ng or graft- ing, several viruses, especially many of those causing yellows-type diseases and many viruses of woody plants, have not yet b e e n trans- mitted mechanically. T h e p o s s i b le reasons for this failure s e em to b e that some viruses are not present in high enough concentration in the donor plant, they are unstable in sap or are quickly inactivated by in- hibitory substances released or formed upon grinding of the cells, and also b e c a u se s o me viruses, e.g., those causing yellows-type diseases, apparently require that they b e introduced into specific tissues (phloem) if they are to cause infection.

S E ED TRANSMISSION

F e w er than one hundred viruses have b e e n reported to b e transmit- ted by seed, and, of these, only about 50 have b e e n definitely proved to b e s e ed transmitted. As a rule, however, only a small portion (1-10%) of the seeds derived from virus-infected plants transmit the virus, and the frequency varies with the host-virus combination. In a few cases, e.g., tobacco ringspot virus in soybean, the virus may b e transmitted by almost 1 0 0 % of the s e e ds of infected plants, and in others, s e ed transmission may b e quite high, e.g., 2 8 - 9 4 % in musk-

Transmission of Plant Viruses 421

melon mosaic virus in watermelon, 5 0 - 1 0 0 % in barley stripe mosaic virus in barley. E v en within a species, however, different varieties or plants inoculated at different stages of their growth may vary in the percentages of their s e e ds that transmit the virus.

In most seed-transmitted viruses, the virus s e e ms to come primarily from the ovule of infected plants, but several cases are now known in which the virus in the s e ed s e e ms to b e just as often derived from the pollen that fertilized the flower.

In most, if not all, seed-transmitted viruses, the virus is carried in- ternally rather than as a surface contaminant. Wh y only about 1 0 % of the viruses are seed-transmitted, and why, even a m o ng these, only a small percentage of the s e ed of virus-infected plants carries the virus, is not yet known.

P O L L EN TRANSMISSION

Several host-virus combinations are known in which flowers of healthy plants pollinated with pollen from virus-infected plants pro- d u ce s e ed that carries the virus. Recently, however, it has b e e n shown that virus transmitted by pollen may infect not only the s e ed and the s e e d l i ng that will grow from it, but more important, it can s p r e ad through the fertilized flower and down into the mother plant, which thus b e c o m es infected with the virus. Such plant-to-plant transmis- sion of virus through pollen is known to occur, for example, in stone fruit ringspot virus in s q u a sh and in sour cherry, and in sour cherry yellows virus in sour cherry.

Although pollination of flowers with virus-infected pollen results in considerably lower fruit set than is p r o d u c ed with virus-free pollen, transmission of pollen-carried virus from plant-to-plant is apparently quite rare or it occurs with only a few of the viruses.

I N S E CT TRANSMISSION

Undoubtedly the most c o m m on and economically most important m e a ns of transmission of viruses in the field is by insect vectors.

M e m b e rs of relatively few groups of insects, however, can transmit plant viruses. T h e order Homoptera, which includes both aphids (Aphidae) and leafhoppers (Cicadellidae or J a s s i d a e ), contains by far the largest number and the most important insect vectors of plant vi- ruses. Certain species of several other families of the s a me order also transmit plant viruses, but neither their numbers nor their importance