Ann. Rev. Phytopathol. 1984. 22:361-96 Copyright © 1984 by Annual Reviews Inc. All rights reserved

WALL-LESS PROKARYOTES

OF PLANTS

J. M. Bove

Laboratoire de Biologie Cellulaire et Moleculaire, INRA et Universite de Bordeaux II, Domaine de la Grande Ferrade, 33140 Pont de la Maye, France

MYCOPLASMA-LIKE ORGANISMS AND

SPIROPLASMAS: HISTORICAL ASPECTS AND

DEFINITIONS

The first mention of an association between wall-less prokaryotes and plants was published by Doi et al (53) in Japanese in 1967 in a work entitled, "Mycoplasma or PLT:group-like microorganisms found in the phloem ele­ments of plants infected with mulberry dwarf, potato witches' broom, aster yellows, or paulownia witches' broom."J Similar microorganisms had been seen at about the same time in rice infected with yellow dwarf disease (121). When plants containing the newly discovered microorganisms were given tetracycline treatments, symptom remission occurred as long as the antibiotic application lasted, and the observed microorganisms disappeared temporarily from the phloem of these plants (76, 121). Penicillin treatments had no effect. These results have been amply confirmed (108, 153). Because mycoplasmas were known to be sensitive to tetracycline but not penicillin, whereas the PLT agents were affected by both of these antibiotics (7, 75; see also 50), the microorganisms found by the Japanese workers were considered mycoplasma­like and not chlamydiae-like and have been called mycoplasma-like organisms (MLOs).2 Similar MLOs have since been found to be associated with more than 100 diseases in plants (103).

Mycoplasmas and permanent (irreversible) L-variants (161) of bacteria have

'PLT refers to psittacosis-lymphogranuloma-trachoma; the agents of these diseases are the Chlamydiae, a group of obligate intracellular parasites with a Gram-negative type of cell envelope.

2The trivial lenn mycoplasma in the phrase MLO is used as a synonym for mollicute and does not imply the genus Mycoplasma. The name mollicute was first proposed in 1967 (59).

361 0066-4286/84/0901-0361 $02. 00

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

362 BOVE

the same morphology, ultrastructure, filterability, colony type, and behavior toward tetracycline and penicillin (see 50). The differences between L-variants and mycoplasmas lie in their immunological, biochemical, and genomic prop­erties. These properties cannot be evaluated at the present time, because the agents first discovered by Doi et al have not yet been cultured. Whether these agents are mycoplasma-like or permanent L-variants remains to be seen; in this presentation they will be called MLOs. The term MLO, as used here, refers to pleiomorphic, wall-less prokaryotic microorganisms morphologically and ultrastructurally resembling L-variants or non-helical mycoplasmas and lo­cated in the sieve tubes of plants carrying symptoms such as leaf yellows (hence the name yellows diseases), leaf mottling, leaf dwarfing, flower virescence, flower dwarfing, witches' brooms, internode shortening, stunting, etc. This definition excludes: (a) pathogenic, sieve-tube-restricted mycoplasmas with helical morphology (the spiroplasmas Spiroplasma citri and com stunt spiro­plasma); (b) sieve tube-restricted bacteria-like agents such as the organisms associated with citrus greening (11) or clover club leaf (180) diseases; (c) xylem-confined bacteria such as the Pierce's disease agent (73, 74); and (d) the mollicutes (Acholeplasma sp., Mycoplasma sp., and Spiroplasma sp.) that occur on the surface of plants and flowers (see below).

Being constantly associated with diseased plants but never present in healthy plants, MLOs fulfill the first of Koch's postulates. Since they have never been raised in culture or in a highly purified form, Koch's remaining postulates have not yet been satisfied. There is strong circumstancial evidence that the MLOs are the actual agents of disease, however (96).

Strictly speaking, the only wall-less prokaryotes that have been grown in culture and shown to be agents of diseases in plants are the two helical mollicutes: S. cirri, the causal agent of citrus stubborn disease, and com stunt spiroplasma (CSS). Citrus stubborn disease and com stunt have played an important role in the discovery of spiroplasmas. The work on com stunt has revealed the existence of the helical filaments n�med spiroplasmas as well as their constant association with the disease (51 ). Citrus infected with the stubborn agent was the first plant from which a mollicute was consistently cultured in California (63) and in France (142, 1 43). The work from France was presented at the Symposium on Pathogenic Mycoplasmas at the CIBA Founda­tion in London in January 1972 (144), where E. A. Freundt from Denmark, D. Taylor-Robinson from England, and J. G. Tully from the US agreed to participate in the characterization of the stubborn organism. One unexpected result of this rewarding international cooperative effort was Tully's finding that the citrus agent was helical and motile and resembled in this respect the not-yet cultured com stunt spiroplasma. This was the first link established between citrus stubborn and com stunt, which hitherto were considered completely foreign to each other. Now their comparative study would yield complementary

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

WALL-LESS PROKARYOTES 363

results and pave the road for the discovery of many more representatives of this new, unexpected group of helical, motile mollicutes. Several review articles have dealt with the discovery of MLOs and spiroplasmas (8, 1 0, 14, 24, 50, 96, 146, 1 80).

When it was first described (83, 84), the agent of citrus greening disease was thought to be mycoplasma-like, but researchers soon realized that this was not so ( 1 43). We now know from in situ studies that this as-yet uncultured prokaryote has a cell wall of the Gram-negative type comprised of an outer membrane and a peptidoglycane layer (65, 68). This explains the symptom remission observed with the penicillin treatment of greening-affected citrus plants (5, 1 1 ). The phloem-restricted clover club leaf organism is also a bacterium (180, 1 88). No media for the cultivation of the greening organism or the clover club leaf agent are known at this time. These two organisms being bacteria-like, they will not be discussed here.

The culture and characterization of the two plant pathogenic spiroplasmas, S citri (63, 1 45) and CSS (25, 1 87), have led to the discovery of many more spiroplasmas. Understandably, much more work has been devoted to spiroplas­mas in general than to the as-yet uncultured MLOs. Reviews and articles on spiroplasmas cover their cultivation (17, 24, 88); their description, properties, and host range ( 14, 43, 97,98, 179); their classification and taxonomy (46, 48, 77, 1 72, 1 78, 1 82, 1 84); their cellular and molecular biology ( 14); their interaction with plant, arthropod, and animal hosts ( 170); the ecology of spiroplasma diseases ( 179); the mechanism of spiroplasma pathogenicity (35); the host range of S. citri (20, 1 28); and stubborn disease (70). Other papers embrace broader subjects: mycoplasmas and yellows diseases ( 103); mycoplas­mas, spiroplasmas, and vascular-limited bacteria as plant pathogens (47, 125); mycoplasma infections of plants (9, 10); mycoplasma-plant-insect interrela­tionships ( 101, 131, 1 69, 1 8 1 ); control of vector-borne mycoplasmas (99, 1 08, 1 53); and the evolution of wall-less prokaryotes (95). Finally, Volume 3 of The Mycoplasmas ( 183) is devoted entirely to plant and arthropod MLOs and spiroplasmas and much information on techniques used in the study of molli­cutes can be found in Methods in Mycoplasmology ( 140, 171 ) and in Plant and

Insect Mycoplasma Techniques (37). This article will present recent advances and current research approaches in the study of wall-less prokaryotes of plants. The literature on new MLO diseases ( 106, 1 07, 124) and new epidemics (93) is not covered.

SPIROPLASMA CLASSIFICATION

The taxonomical description of spiroplasmas in Table l1llustrates the progress made in the last ten years in the exploding field of spiroplasmology. Beside the eleven well-defined groups listed there, several other groups or subgroups

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

Table 1 Spiroplasma classification"

Principal DNA homology with (%) Proteins shared with (%) Group or strains G+C Principal

Common or binomial name subgroup (ATCC number) (mol %) S. <itri S. apis S. citri S. apis host Disease incited

Spiroplasma citri complex Citrus stubborn spiTOplasma I-I R8A2(27556) 25-27 100 100 Dicots, leafhoppers Citrus stubborn, Horse-

(S. citri) CI89(27665) radish brittle root Israel

Honeybee spiropl asma 1-2 AS 576(29416) 25-27 65 45 Bees Honeybee spiroplas-BC-3(33219) mosis

Corn stunt spiroplasma 1-3 1-747(29051) 25-27 49 28 Maize, leafhoppers Corn stunt E275(29320) B655(33289)

277F spiroplasrna 1-4 277F(29761 ) 25-27 18 15 Rabbit tick None known Green leaf bug spiroplasma 1-5 LB-12(33649) 25-27 nd 27 Green leaf bug None known Maryland flower spiro- 1-6 M55(33502) (28-29)b 17 26 Flowers None known

plasma ET-I Eristalis fly Cocos spiroplasma 1-7 N525(33287) 25-27 nd 19 Cocos surface None known Periwinkle spiroplasma 1-8 P40 25-27 nd nd Unknown Periwinkle disease Sex ratio organisms II WSRO, NSRO 25-27 Drosophila Sex-ratio trait Spiroplasma floricola III 23-6(29989) 25-27 Rowers, insects Lethargie of beetles.

BNRI(33220) OBMG(33221)

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

Spiroplasma apis complex IV B31(33834) 29--31 100 100 Honeybees May disease FI2 29--31 95 91 Flowers FI6 29--31 89 73 Flowers FI 29--31 83 55 Flowers F2 29--31 85 55 Flowers L89 29--31 89 49 Spittelbug None known PPSI(33450) 29--31 80 52 Flowers None known SR3(33095) 29--3 I nd Flowers None known

.spiropJasma 'mirum V SMCA(29335) 29--3 I Rabbit.tick Suckling mouse cataract GT-48(29334) TP-2(33503)

Ixodes spiroplasma VI Y32(33835) 24-26 Ixodes licks None known

Monobia spiroplasma VII MQ-1(33825) (27-29)b Monobia wasp None known

Syrphid spiroplasma VIII EA-I(33826) 29--31 Eristalis fly None known

Cotinus spiroplasma IX CN·5(33827) 29--31 Cotinus beetle None known

Mosquilo spiroplasma X AES-1 25-27 Aedes mosquito None known

Monobia spiroplasma XI MQ-4 25-27 Monobia wasp None known

aBased on (12,31,77,116,117, 17la, 184, P. Carle-Junca, J. M. Bove, unpublished data) bStill under investigation

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

366 HOVE

presently are being studied, and many more spiroplasmas probably remain to be discovered. Flowers and plant surfaces have proven to be rich sources of spiroplasmas (see below), and new spiroplasmas have recently been found in diverse insect hosts (31). It is likely that many of these microorganisms are inhabitants of the intestines of insects and are transmitted from insect to insect by contamination of the plant surface (31). However, until now only two spiroplasmas have been discovered to be pathogenic to plants, S. citri (sub­group 1-1) and CSS (Subgroup 1-3).

Following a serological analysis of the then-current spiroplasma strains (48),

Junca et al proposed the first extensive classification of these organisms in 1980 (77) and included five groups, I-V. This scheme was subsequently revised to include several newly discovered spiroplasmas (184), bringing the number of groups to eight. Table 1, showing eleven groups, is the most recent version of the spiroplasma classification and incorporates new data (12,31, 115-117,

182). It is also the result of a truly international collaborative effort. In Table 1,

spiroplasma groups and subgroups are defined not only on the basis of serolog­ical distinctions (182, 184), but also on recognized characteristics of spiroplas­mal DNA, particularly the guanine plus cytosine (G + C) content and the DNA-DNA hybridization percentages (15, 77), as well as on spiroplasma protein patterns as defined by one- and two-dimensional polyacrylamide gel electrophoresis (36, 117-120). All the spiroplasmas in Table 1 have been cultivated on artificial media except the Drosophila spiroplasmas (group II). The latter are also unique among spiroplasmas in that they are transmitted vertically through the egg. Some of the spiroplasmas are agents that had been grown previously on a variety of substrates but were misidentified as to their microbial nature. The 277F spiroplasma (subgroup 1-4) from rabbit ticks (18)

was first grown in 1968 on an artificial medium and described as a spirochete (130). Two other isolates from rabbit ticks (SMCA and GT-48 of group V) were first grown in the chick embryo and described as a virus (27) and as a mycoplasma (190) before they were recognized as spiroplasmas (173, 174).

However, most newly discovered spiroplasmas represent previously unknown agents that have been isolated primarily from insects or from the surfaces of plants and flowers. Clark (30) was the first to find spiroplasmas at high titers in the hemolymph of honey bees. This critical observation led to the search for spiroplasmas on the surfaces of flowers visited by foraging bees. Indeed, many such "flower" spiroplasmas have since been found, especially in the United States and France (see below). However, as important as the flower habitat has been in the discovery of new spiroplasmas, it seems apparent now that this habitat is only an occasional secondary result of the colonization of insects by these organisms (31).

A detailed description of the various spiroplasmas in Table 1 is beyond the scope of this review. However, since the spiroplasmas of group I contain the

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

WALL-LESS PROKARYOTES 367

organisms pathogenic to plants, they deserve further attention. Plant-surface spiroplasmas will be examined further below.

SPIROPLASMAS OF GROUP I

Origin and Host

Subgroup 1-1 of the spiroplasma classification is devoted to S. elm and subgroup 1-3 to CSS. Both of these spiroplasmas are confined to the phloem and are regarded as intracellular pathogens. Both organisms are transmitted by leafhopper vectors, with spiroplasma replication occurring within insect host tissues. Although S. citri causes an economically important disease in citrus (stubborn disease) and is associated with (45, 135) and responsible for (60) brittle root of horseradish , many other plants can be infected with the organism, and a number of plant hosts are susceptible to experimental infections (20, 128). In these infections, the disease is frequently more severe than in citrus infections, but S. citri has never been found to cause flower virescence or phyllody. In a few instances where S. citri has been isolated from plants showing virescence, mixed infections have been found (see below).

CSS (subgroup 1-3) has a much smaller host range than S. citri. In addition to com (Zea mays L.) and Mexican teosinte (Z. mays L. mexieana), Z. perennis and Z. dipioperennis are susceptible to CSS (122). In addition, CSS has been transmitted experimentally by the leafhopper Euseelidius variegatus to peri­winkle, broad bean (Viciafaba), and rye grass (Lotium sp.) (102). In the field, CSS is transmitted by Dalbulus maidis and D. elimatus. Other leafhoppers are experimental vectors (122). Com stunt is widespread through South, Central, and North America and has been reported in Jamaica by Eden-Green. It has never been reported in Europe and the Mediterranean area. In addition to CSS, com can also be infected by an MLO, the agent associated with maize bushy stunt (122).

The first spiroplasmas identified in honey bees (30) were those assigned to subgroup 1-2. These organisms are pathogenic to the bee, and additional spiroplasmas identical to the bee isolates were later recovered from flower surfaces (41, 48). However, in France a well-known disease of honey bees (May disease) was found to be associated with group IV spiroplasmas (115, 116). Thus, spiroplasmas from two distinct groups are now clearly documented as bee pathogens.

Subgroup 1-4 is represented at this point by a single spiroplasma (strain 277F) (18). Although this organism was recovered from rabbit ticks, additional isolates will be required to clearly establish the true host origin. Subgroup 1-5 spiroplasmas also contain a single representative (strain LB-12), recovered from the green leaf bug (Trigonotylus ruficornis) in Taiwan (87). Spiroplasmas in subgroup 1-6 represent three organisms recovered from the surface of flowers

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

368 BOVE

( 185). Subgroup 1-7 contains two isolates from coconut palms (56, 58). In contrast to the intracellular location of S. citri and CSS, flower and plant spiroplasmas in subgroups 1-6 and 1-7 have been found only on plant surfaces. At this time, there is no evidence that these new subgroup members are associated with any plant disease. Finally, a new sieve-tube-restricted spiro­plasma (see below), only the third such organism after S. citri and CSS, is tentatively classified in subgroup 1-8.

Genome Size and Guanine Plus Cytosine Content

S. citri has been found to have a genome size close to 109 daltons (85, 145). Similar values have been found for group III and group IV spiroplasmas.

The guanine plus cytosine (G + C) content of the DNA of strains within the group 1 complex has been examined extensively ( 1 5, 77, 85). The DNAs of most group I spiroplasmas, including those of subgroups 1-5 and 1-7 (P. Carle-Junca, unpublished observation) have been found to have 26 ± I mole percent G + C. The DNA of spiroplasma M55 (subgroup 1-6) was found to have a somewhat higher G + C content and needs further work.

Relatedness of Group I Spiroplasmas

The spiroplasmas of the first three subgroups in Table 1 show significant heterologous crossing in all serological tests, including growth inhibition, deformation, metabolism inhibition, and ELISA ( 1 82, 184). Spiroplasma 277F (subgroup 1-4) clearly crossreacts in'a number of serological procedures with the com stunt spiroplasma E275 (subgroup 1-3) and with spiroplasmas of subgroups 1-5, 1-6, and 1-7 (184). Little or no serological cross has been observed between 277F and strains of eitherS. citri (subgroup 1- 1 ) or honey bee spiroplasmas (subgroup 1-2).

DNA-DNA hybridization, EcoRI restriction enzyme analysis of DNA, and comparisons of spiroplasma protein maps support the serological results ( 12).

Classification and Pathogenicity

S. citri (subgroup 1- 1 ), the honey bee spiroplasma (subgroup 1-2), and CSS (subgroup 1-3) are aU clearly pathogenic organisms (Table 1). Since a number of susceptible and diseased hosts are readily available within each of these subgroups, a respectable collection of individual spiroplasma strains have become available for intragroup comparisons. Such comparisons have shown remarkable homogeneity within representatives of subgroups 1- 1,1-2, and 1-3. Thus, an individual spiroplasma strain within each of these subgroups can be specifically identified with a variety of techniques. This is possible despite the fact that these organisms show intergroup relatedness as measured by serology, DNA hybridization, and cell protein analysis. Subgroup 1- 1 spiroplasma strains have received a specific taxonomic designation (S. citri). Since it is of con-

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

WALL-LESS PROKARYOTES 369

siderable practical importance to distinguish between pathogenic spiroplasmas, and because differences in DNA-DNA homology are within acceptable levels for the erection of prokaryote species designations, Latin binomials for the honey bee spiroplasmas (subgroup 1-2) and com stunt spiroplasmas (subgroup 1-3) appear appropriate (12, 182). Assigning them such names amounts to elevating the spiroplasmas of subgroups 1-2 and 1-3 to the rank of species, as

was proposed earlier (46).

SPIROPLASMA CITRI: A PATHOGEN AND A MODEL

For many reasons, S. cirri is probably the most studied of all the spiroplasmas. It was the first cultured mollicute of plant origin; moreover, cultures became available as early as 1970 and have been freely distributed for international collaboration. It is the causal agent of citrus stubborn disease, one of the major diseases of citrus in the Old as well as in the New World; it is also the cause of brittle root disease of horse radish in the eastern United States. It infects many plant species, and it is a model for biological and biochemical studies. The discovery of S. cirri plasmids opens the way to recombinant DNA technology and spiroplasma genetics. In the following paragraphs, emphasis will be put on recent results.

Growth and Division

S. citri grows from short elementary helices into longer parental helices that divide by constriction to produce elementary helices again (66). Membrane synthesis during growth and division can occur a priori over the entire surface of the organism or at specific growing zones. Researchers have investigated the question by pulse labeling the growing zones of the membrane with tritiated amino acids and by using quantitative high-resolution autoradiography to visualize the growing zones on the helices (67). The data obtained in this way suggest that S. citri helices grow and divide according to the scheme in Figure 1.

This growth model is very similar to the unit cell model described for Escherichia coli (54). In this model, the organisms grow at one site when they are less than a certain critical length, and at two sites when they are more than this length. In the case of S. citri. the elementary helix can be compared to the unit cell defined for E. coli.

In most species of mollicutes, DNA replication and cell division are not closely coordinated; however, in Mycoplasma gallisepticum cell division by binary fission is synchronous with DNA replication (132). In the case of S.

citri, the elongation observed around the constriction zone may play a role in the segregation of DNA strands in the two newly formed elementary helices.

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

370 HOVE

� ----------------� ..... lhour

Figure I Scheme for spiroplasma growth. The growing zones are represented by dark areas. For

simplicity's sake. the helical morphology of the organism is not shown. An elementary helix with one blunt end and one tapered end (a) develops into an elementary helix with two blunt ends (b).

Growth starts at one end. as represented by the dark area on the organism (e). At the same time. parental helices ready to divide (A) grow around the constriction (B) to yield two elementary helices with one blunt end and one labeled. tapered end (C). This tapered end may tum into a blunt end (C/). In agreement with the radioactive labeling data. the percentage of elementary helices with two blunt ends labeled at one end is 63%. while the percentage of helices with a labeled tapered end is 37%. The presence of elementary or parental helices labeled at two sites is explained on the right

side of this figure. It implies that an elementary helix already labeled at one end grows into a four-tum parental helix and then becomes labeled in the middle at the place where a constriction

will occur. This constriction will lead to the formation of two elementary helices. one with labels at

both ends and one with a label at only one end.

Enzymes for DNA Replication and Transcription

S. citri has been shown to contain three DNA polymerases: ScA, ScH, and ScC (22, 23). These three enzymes have different elution properties on diethylami­noethyl cellulose . ScA is not inhibited by N-ethylmaleimide (NEM), but both ScB and ScC are NEM-sensitive. Ethanol slightly stimulates ScA, whereas SeB and ScC are inhibited. Other comparative properties of the three enzymes have been studied. The honey bee spiroplasma BC3 (subgroup 1-2) and the flower spiroplasma BNR I (group III) also contain three DNA polymerases. Spiroplasma Be3 is serologically related to S. citri, the two sprioplasmas being members of group I (Table I ) . Interestingly, each of the three DNA polymerases of strain HC3 seems to be similar to the respective enzyme of S. citri. However. flower spiroplasma BNR I . unrelated to S. citri, contains DNA polymerases showing some differences from those of S. citri. In any case, spiroplasmas have three DNA polymerases and in this respect they do not differ from bacteria.

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

WALL-LESS PROKARYOTES 371

The situation is quite different with DNA-dependent RNA-polymerase. It is well known that the antibiotic rifampicin inhibits bacterial growth. In E. coli, rifampicin inhibits RNA transcription by binding to the B subunit of RNA­polymerase. In contrast to test results on Eubacteria, none of the mollicutes tested was inhibited by rifampicin; those examinated were Mycoplasma

mycoides, Ureaplasma urealyticum, Acholeplasma laidlawii (see also 38), S. citri, S. apis, and honey bee spiroplasma B88 (subgroup 1-2). Furthermore, the spiroplasma RNA-polymerases were purified to the stage where the enzyme required added DNA for activity and were tested for rifampicin sensitivity. While as little as 0.01 mg of the antibiotic inhibited 50% of the activity of theE. coli enzyme, concentrations as high as 100 mg were required for 50% inhibi­tion of the spiroplasma RNA-polymerases (64). These results show that the spiroplasma enzymes are essentially insensitive to rifampicin. In preliminary experiments (A. Gadeau, C. Mouches, J. M. Bove, unpublished data), the subunit structure of the RNA-polymerase of spiroplasma B88 was compared with that of E. coli and H alohacterium halobium, a member of the Archaebac­teria. The spiroplasma enzyme with four major subunits ( 1 30 kd, 1 17 kd, 49 kd, and 40 kd) had a structure resembling neither that of the Eubacteria nor of the Archaebacteria. The two large subunits ( 1 30 kd and 1 17 kd) were definitely smaller than the two large subunits of E. coli ( 160 and 155 kd for subunits Band B' respectively).

In conclusion, these results suggest that all mollicutes might be insensitive to rifampicin. This property would distinguish them from the Eubacteria but not from the Archaebacteria. According to recent hypotheses (see 95), mollicutes repres.ent degenerate eubacterial forms and, instead of being primitive ances­tors of prokaryotes, can be regarded as the most highly evolved of the prokary­otes. During this evolution, the property of RNA-polymerase to bind rifampi­cin probably has been lost.

Ribosomal RNA Operons

The eubacterial chromosome is known to carry several repeats of the ribosomal RNA (rRNA) operon. E. coli has seven and Bacillus subtilis at least ten rRNA operons; H. halobium, an Archaebacterium, has only one. The number of rRNA operons in the DNA of spiroplasmas and other mollicutes has been examined recently ( 1 39). First the DNA was digested with restriction endonu­cleases. Then the fragmented DNA was electrophoresed in agarose-gels, trans­ferred to nitrocellulose filters, and hybridized with a variety of DNA probes containing portions of one of the rRNA operons of E. coli. Analysis of the hybridization bands showed that spiroplasmas as well as all other mollie utes tested carry only one or two rRNA operons and that rRNA genes in molli­cutes are linked together in the same fashion as in E. coli: that is, 1 6 S-23 S-5 S. The data also imply a marked nucleotide sequence homology along

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

372 BOVE

the rRNA operon of E. coli and the RNA operons of the various mollicutes ( 139).

Presence of S. Citri Plasmid Sequences in Other Spiroplasmas

Plasmids play a significant role in the biology of microorganisms in the functions for which they code (pathogenicity, antibiotic resistance, etc) and in their dissemination of genetic information. Many spiroplasmas, including several S. citri strains, contain plasmids of between 2 and 50 kilobase pairs (kbp) (3, 14, 138). However, so far all spiroplasma plasmids are cryptic, i.e. none has been associated with a given phenotypic character. Also, the ex­perimental transfer of a plasmid from one spiroplasma to another has not yet been achieved and, more generally, transfer of genetic material between mollicutes has not yet been described.

Two plasmids, pMHI with 7 kbp and 26 mol% G + C, and pM41 with 8 kbp and 22 mol% G + C, have been purified from S. citri strains MH and M4 respectively. The MH strain was cultured from a stubborn-affected sweet orange tree growing in Morocco, strain M4 from a naturally infected periwin­kle also from Morocco. On the basis of G + C content and restriction enzyme mapping, the two plasmids are different. Using recombinant DNA technology, the linearized pMHI plasmid was introduced into E. coli plasmid vector pBR328 and was cloned in E. coli ( 1 10). This is the first time that a mollicute plasmid has been cloned in a bacterium. However, when used in similar experiments, plasmid pM41 could not be cloned. This plasmid is identical to plasmid pH 2000, which has been isolated from S. citri strain ASPI (3). Cloning experiments with pH 2000 have also failed. The reasons for these failures are unclear.

Radioactive probes of plasmids pMH 1 and pM41 were used to investigate the occurrence of these plasmid sequences in other spiroplasmas. Plasmid pM41 from S. citri strain M4 was present in three additional S. citri strains (ASP!, Iran, Arizona) but was not present in several others, including strain MH, the source of pM HI. It also occurred in the following spiroplasmas: strain 275 of the CSS, tick spiroplasma 277 F, and Cocos spiroplasma N525 (113). Plasmid pMHl was found as a free 7-kbp plasmid only in S. citri strain MH. Interestingly, however, the pMH 1 probe hybridized strongly with high molecu­lar weight DNA of several S. citri strains and of strains of spiroplasmas other than S. citri. These results suggest that pMHI sequences might be integrated into the genome of these spiroplasma strains (111, 112).

Expression in E. Coli of the Spiralin Cistron of S. Citri

Spiralin or protein 39 ( 120) is the major membrane protein of S. citri (186, 189) and is strongly antigenic. Rabbit antibodies against whole S. citri cells react strongly with spiralin ( 1 17). Several S. citri-specific monoclonal antibody

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

WALL-LESS PROKARYOTES 373

(MCA)-producing clones have also been obtained; they have all been directed against spiralin (117).

ELISA (29, 148) offers a convenient and sensitive technique for detecting S. citri proteins. It has been used to screen E. coli clones that were transformed with restricted S. citri DNA fragments inserted into E. coli plasmid vector pBR328 (C. Mouches, T. Candresse, C. Saillard, G. Barroso, J. M. Bove, unpublished data). One E. coli transformant reacted strongly in ELISA with S. citri polyclonal antiserum. The same transformant also reacted positively to monospecific antiserum against spiralin. The spiralin from E. coli has an electrophoretic mobility slightly smaller than that from S. citri. This result suggests that spiralin occurs in E. coli essentially as a preprotein, as do the results of tryptic digests. The restriction fragment coding for spiralin in E. coli measures 6.5 kbp and is much larger than required for spiralin expression. A strong promoter is probably present in the sequences flanking the spiralin cistron. Even though spiralin is specific to S. citri, the spiralin restriction fragment hybridizes with the DNA of all spiroplasmas tested. The flanking sequences are probably involved in this hybridization. These results show that at least one gene from S. citri, the spiralin gene, can be expressed in a bacterium. This opens the way to physical mapping of S. citri genes on the spiroplasma genome.

S. Citri Viruses in Plants and Their Effect on Symptom Expression

There are three S. citri viruses. SpVl (89, 90) is a unenveloped rod 230--280 nm by 10-- 1 5 nm with circular, single-stranded DNA (J. Renaudin, J. M. Bove, unpublished data). SpV2 is a long-tailed polyhedron ch;:rracteristic of type B bacteriophages (32, 34). SpV3 is a short-tailed polyheqral virus with double-stranded linear DNA; it is peculiar in that it emerges from infected cells in buds of membranes (32, 33). Many S. citri isolates are infected with both SpVI and SpV3, and these viruses are often seen in primocultures of S. citri from citrus. SpV2 is seen much more rarely; it is specific to S. citri. However, Sp V 1 and Sp V3 have been seen in the cultures of many spiroplasmas other than S. citri. Some S. citri cultures contain all three viruses.

Recently, it was reported for the first time that viruses can be produced by spiroplasmas within infected host plants ( 1 , 2). In these experiments, two strains of S. citri have been used. One, SP-A, is severe; once infected, plants die of lethal wilting within 12 weeks. The other .strain, SPV3, produces unusually mild symptoms and the plants live. Electron microscopy revealed that not only viruses similar to SpV3 (2) but also to SpVl and SpV2 ( 1 ) were produced in a periwinkle infected with the mild SPV3 strain of S. citri. Sap filtrates from this plant contained virus particles that gave rise to plaques on lawns of the lethal SP-A strain of S. citri. Periwinkle plants grafted first with

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

374 BOVE

the lethal strain and subsequently with the mild, virus-producing strain de­veloped only mild symptoms and did not die. The dual-infected plants con­tained relatively low concentrations of spiroplasmas and high titers of virus; the spiroplasmas were distorted and swollen.

These experiments suggest that spiroplasma viruses play a role in preventing the usual lethal effect of the severe strain of S. citri. They probably exert this action by decreasing the number and viability of the spiroplasmas within the plant. Dual infections of periwinkles with S. citri and MLOs also prevent lethal wilting (see below).

A fourth spiroplasma virus, SpV4, was discovered recently in strain B63 of the honey bee spiroplasmas of subgroup 1-2. This strain was cultured from a

honey bee that was captured from Morocco. The virus is isometric and has a diameter of 27 nm; its single-stranded DNA is circular and has a molecular weight of 1.5-1.7 x 106 d. SpV4 forms clear plaques on lawns of spiroplasma G 1 (subgroup 1-2); it infects only spiroplasmas of subgroup 1-2 (J. Renaudin, M. C. Pascarel, J. M. Bove, unpublished data). Transfection of spiroplasma Gl with SpV4 DNA has been achieved (M. C. Pascarel, J. Renaudin, J. M. Bove, unpublished data). Viruses similar to SpV4 but specific to S. citri are being sought in S. citri isolates from stubborn-affected trees.

Interactions of Insect Cells in Culture with S. Citri and Other Spiroplasmas

Insects are primary hosts for many mollicutes (31, 181), but little is known about the interactions between mycoplasmas and the cells, tissues, or organs of the insect hosts. Techniques and insect cell lines are now available to study these interactions in vitro. One Drosophila (Dm-I) and two leafhopper (AS-2 and AC-20) cell lines have been used to study the attachment and pathogenicity of spiroplasmas and mycoplasmas (162). Cytopathic effects have been observed with most of the spiroplasmas, leading to death of the insect cells. With dark field and DNA staining microscopy, adsorption of spiroplasmas to AS-2 and AC-20 cells has been detected. While spiroplasmas 277F (subgroup 1-4) and B88 (subgroup 1-2) adsorbed heavily, S. citri and CSS showed slight adsorption. In addition, non-helical forms of S. citri and other spiroplasmas within the leafhopper cells were shown to be present. The identity of the non-helical forms of S. citri was confirmed serologically by indirect immuno­fluorescence. Intracellular location and non-helical morphology of spiroplas­mas in insect cells corresponded with in vivo findings, Le. that in leafhopper tissues S. citri and CSS are not helical and reside inside the cells (92, 168).

In AS-2 and AC-20.cuItures the titers measured with B88, 277F, BNRI, and PPS-l were similar to the ones in the fresh LB cell culture medium. CSS did not mUltiply. However, S. citri reached nearly two logs higher titers in AS-2 and AC-20 cultures than in fresh LB medium.

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

WALL-LESS PROKARYOTES 375

S. citri was also shown to grow to higher titers in "conditioned" LB medium, in which AS-2 cells were grown for three days and then were removed by filtration, than in fresh LB medium alone. This and similar experiments suggest that the conditioned medium contains a cell-derived factor that stimulates the growth of S. citri. Studies along these lines might offer an approach to reinvestigating the culture of MLOs.

NATURAL TRANSMISSION OF S. CITRI AND THE DISCOVERY OF A

NEW SIEVE- TUBE-RESTRICTED SPIROPLASMA

In California, the natural spread of S. citri and its implications for the control of stubborn disease have received much attention (19). Vector transmission of S.

citri occurs mainly in the summer months and affects essentially young trees. The amount of spread can be particularly high in certain years; in these cases, stubborn epidemics result (70). Three leafhoppers have been shown in Califor­nia to be vectors of S. citri: Neoaliturus (ex Circulifer) tenellus, Scaphytopius nitridus, and Scaphytopius acutus delongi (129). Obviously, the range of plants found to be naturally infected with S. citri depends on the nature of the leafhopper vectors prevalent in a given area. N. tenellus. the sugar beet leafhopper, seems to be one of the active S. citri vectors in California; it also

transmits curly top virus. Transmission of S. citr; by N. tene/lus (91) and the relationship between the spiroplasma and the vector (92) have been carefully studied.

S. citri can be cultured from N. tene/lus leafhoppers collected not only in California but also in southcentral Washington, several hundred miles north of the citrus-growing area of California ( 129). N. tenellus has a wide distribution, being known in Central and North America from Mexico to Canada, in Florida and the West Indies, in South Africa, in the Mediterranean Basin and the Near East. Cruciferous plants are hosts of the leafhopper (20), and this factor might explain the large number of cruciferous plants found to be naturally infected with S. citri in California. In this connection, an interesting development has occurred on the East Coast of the US. The causal agent of the long-known brittle root disease of horseradish, a cruciferous plant, has recently been identified as S. citri, and one of the vectors is N. tenellus (60). Another vector is Macrosteles fascifrons, common in horseradish fields in Illinois ( 126).

In Morocco, Syria, and Iraq, to mention only a few countries, citrus stubborn disease is widespread and in certain orchards a high proportion of trees are contaminated. The wide distribution of stubborn disease in the Mediterranean area and in the Near and Middle East is probably because diseased bud wood is used in these areas. A second reason is natural spread, for which experimental evidence is now available (123). For instance, in Morocco 24% of 264 peri-

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

376 BOVE

winkle plants near a sweet orange orchard were exposed to natural infection from July to October 1980. They were contaminated with S. citri. as demon­strated by lethal wilting as well as by ELISA and culturing of the spiroplasma (9). In Syria, a great number of young sweet orange trees propagated from stubborn-free budwood were found infected with S. citri (16). These studies have been greatly facilitated by the fact that it is possible to detect S. citri using ELISA (29, 147-149) and culture of the organism. When mature citrus leaves from the summer flush of growth are collected in October, a high proportion of stubborn trees are found positive by both ELISA and culture of S. cirri (J. M. Bove, A. Fos, M. Gamier, C. Saillard, J. C. Vignault, unpublished data). Having an S. citri positive ELISA reaction on a sample from which a spiroplas­rna culture also has been obtained is already serological evidence that the cultured spiroplasma is S. citri.

The leafhoppers involved in the natural spread of S. citri in the Mediterra­nean have not yet been identified. N. tenellus , the California vector, is known to be present in Algeria, Tunisia, Libya, Egypt, Spain, and Morocco (61, 62). In several surveys of leafhoppers in Morocco, Turkey, and Syria beginning in 1 978 (A. Fos, J. Bonfils, J. M. Bove, C. Saillard, J. C. Vignault, unpublished data), we foundN. tenellus in very few instances and in very small numbers. N. haematoceps. a closely related species, was present in somewhat higher num­bers and was even one of the dominant leafhopper species caught in yellow traps placed in periwinkle beds near a stubborn-infected Valencia late sweet orange orchard in Morocco (A. Fos, unpublished data; 9). It is interesting to note that as early as 1953 Frazier (61) also found N. haematoceps to be the dominant form of Neoaliturus. From these data it seems that N. haematoceps is a good vector candidate for the natural spread of S. citri in the Mediterranean area. In the Adana region of Turkey, where citrus stubborn is a serious problem, N. haematoceps. also named N. opacipennis. is a well-known leafhopper species; it is the supposed vector of beet curly top virus in Turkey. The case for its involvement in S. citri transmission has received strong support from work carried out in nearby Syria in October 1983 (1. M. Bove, A. Fos, M. Gamier, C. Saillard, J. C. Vignault, unpublished data). We found large populations of N. haematoceps, unmixed with other leafhopper species, in one particular biotope on Cheiranthus cheiri and Matthiola annua plants near Afamia in the Ghab region, a major sugar beet-producing region in Syria. Spiroplasma isolates were cultured from several samples of 10--15 insects from this group and identified as S. citri. Several samples of five insects also tested positively for S. citri in the ELISA test. In 1978, S. citri had been cultured from one sample ofN. haematoceps in Morocco (13). Experimental transmissions of S. citri by N. haematoceps is the next step.

Spread of S. citri in Syria was studied by exposing periwinkle plants to natural infection in several locations during the summer of 1983 (1. M. Bove,

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

WALL-LESS PROKARYOTES 377

A. Fos, M. Garnier, C. Saillard, J. C. Vignault, unpublished data). Symp­tomatic periwinkles were analyzed for S. citri infection with ELISA and culture techniques. Helical and non-helical organisms in their sieve tubes were ex­amined by electron microscopy. While some periwinkles showed a positive reaction to ELISA and gave a spiroplasma isolate identified as S. citri, other periwinkles yielded a spiroplasma culture but reacted negatively to ELISA for S. citri. The spiroplasma cultured from the ELISA-negative periwinkles, named P40, was examined for serological relatedness to other spiroplasmas using ELISA and metabolism inhibition.

Spiroplasma P40 was found to be related to group I spiroplasmas and more

particularly to spiroplasma LB12 (subgroup 1-5); we found no relationships with spiroplasmas of any other groups (C. Saillard, J. C. Vignault, J. M. Bove, R . F. Whitcomb, J. G . Tully, unpublished data). Sieve tubes of the periwinkles

from which spiroplasma P40 was cultured were filled with helical organisms. These periwinkles, when examined with ELISA using an antiserum against spiroplasma P40, showed a strong positive reaction; under the same conditions S. citri-infected periwinkles had a negative reaction. These results show that spiroplasma P40 and the helical organism seen in the symptomatic Syrian periwinkles are the same. Hence, spiroplasma P40 is a sieve-tube-restricted organism, not a surface contaminant. It is also highly likely that spiroplasma P40 is a plant pathogen, although its pathogenicity will have to be confirmed by leafhopper transmissions of pure P40 culture to periwinkles. It is interesting to note that the P40 spiroplasma, as well as the two previously known sieve-tube­restricted p'athogenic spiroplasmas, S. citri and the CSS, tum out to be mem­bers of serogroup I spiroplasmas, P40 being confined to the new subgroup 1-8. Finally, the periwinkles from which spiroplasma P40 was cultured became infected as a result of natural transmission, very probably via leafhopper vectors. The original diseased host on which the insect vector acquired the P40 agent remains to be identified.

MLO AND THE PRESENCE OF MOLLICUTES

ON THE SURFACE OF PLANTS AND FLOWERS

In recent years one of the most interesting developments in the field of mollicute study has been finding that plants can carry true acholeplasmas, mycoplasmas, and spiroplasmas on their surfaces. These mollicutes are orphans in that they are not associated with diseases of the plants on which they are found. It may be, however, that they are not orphans in hosts other than plants, as will be shown below for three flower spiroplasmas. The presence of these orphan mollicutes also offers a possible explanation for earlier reports' that some groups of workers occasionally were able to cultivate mollicutes from diseased plants carrying sieve-tube MLOs while many others were not

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

378 HOVE

(69; see also 50). In some cases, the cultured mollicute might have been a contamination from ingredients used to prepare the growth medium; serum especially has been shown to be a source of contamination. But in other cases it is likely that the cultured mollicute could have been a surface organism.

Spiroplasmas

Clark (30) discovered that a seasonal disease of honey bees occurring in May and June is due to a spiroplasma (Table 1, subgroup 1-2). He thought that the bees became contaminated while foraging on flowers and as a result started to look for spiroplasmas on such flowers. He was able to isolate spiroplasmas BNRI and OBMG from tulip tree and magnolia flowers respectively (Table 1 , serogroup III). but these were unrelated to the honey bee spiroplasmas. Many more flower spiroplasmas since have been discovered (42, 109, 114, 185,). They fall into five groups or subgroups: 1-2, 1-6, 1-7, III, and IV.

SPIROPLASMAS OF SUBGROUP 1-2 The spiroplasmas of subgroup 1-2 have been cultured from two sources, infected honey bees and the surfaces of flowers. Some were isolated in the eastern United States from the flowers of Bidens pilosa (strains GI, G2) and Tilia americana (strain BW) (41, 44). Spiroplasmas of subgroup 1-2 also occur in France (114) on flowers (strain F3) and in honey bees (strains B24, B25, B27. B29, B62) and in California on flowers (136). The flower isolates from the United States and France were shown to be pathogenic in the honey bee. It is the first exampie of an orphan spiroplasma with no pathological role on the plant host that is pathogenic in an insect host.

SPIROPLASMAS OF SUBGROUP 1-6 Whitcomb (185) has recently isolated spiroplasmas from flowers in Maryland. Serology (185) and protein analysis (12) show these isolates to be somewhat related to one of the members of group I, the rabbit tick spiroplasma 277F, hence the tentative classification of these spiroplasmas in group I, subgroup 6.

SPIROPLASMAS OF SUBGROUP 1-7 Spiroplasmas of subgroup 1-7 were iso­lated from both healthy and lethal yellowing-diseased tissues of coconut palms (56, 58). They are serologically related to spiroplasmas 277F and BC3, and they represent a new subgroup (1-7) within the S. citri group (Table 1) (12).

SPIROPLASMAS OF GROUP III The spiroplasmas first isolated by Clark from tulip trees and magnolia flowers belong to serogroup III. Davis (40) has also cultured such spiroplasmas from tulip tree flowers and has proposed the name Spiroplasma floricola for these agents (49).

An interesting development has arisen from the work of Giannotti & Vago on

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

WALL-LESS PROKARYOTES 379

the "lethargie" disease of cockchafer grubs (Melolontha melolontha) ( 1 7S). They were able to culture a spiroplasma from affected grubs and reproduced the disease by reinjecting the spiroplasma into normal grubs. Further investigation showed that the lethargy spiroplasma seems serologically identical to the flower spiroplasmas of serogroup III (J. G. Tully, J. M. Bove, J , Giannotti, unpublished results) ! This is the second example of a spiroplasma harmless on its plant host but pathogenic in its insect host.

SPIROPLASMAS OF GROUP IV While the flower spiroplasmas of groups 1-2, 1-6, 1-7, and III have DNA with 25-27 mole% (G + C), those of serogroup IV have a G + C content of 29-3 1 %. The first spiroplasma of this group was isolated from the surface of powder puff (Calliandra haematocephala) flowers in Florida (the PPS I strain) ( 109) and tulip tree flowers in Connecticut (the SR3 strain) (40). These early flower isolations were followed by many others not only from the Central United States (185) but also from continental France (strains P I , P2, P4, PI2, F 1 6) and Corsica (strain P2S) (77, 1 14 , 176). Group IV spiroplasmas were also obtained from honey bees in Morocco (strain B 13) and in California ( 1 36) and from froghoppers (Cercopidae) in Corsica (strain L89). Extensive serological work ( 1 17, I71a) as well as two-dimensional protein analysis on polyacrylamide gels ( 1 17) have been devoted to many of these spiroplasmas and have greatly contributed to the classification of these organisms in a new, distinctive group, group IV ,

A new development concerning the group IV spiroplasmas has occurred recently ( 1 14, 1 15, 1 16), Honey bees affected by a disorder resembling the classical May disease in southwestern Prance were found to contain numerous spiroplasmas in their digestive tract and hemolymph. Two strains (B31 and B39) were cultured and shown to be pathogenic to the honey bee. They were characterized and found to be related to group IV members. Spiroplasmas nearly identical to the B3l and B39 strains were also recovered from the surfaces of flowers collected within the area visited by bees from the diseased hives. The name Spiroplasma apis has been proposed for the group IV spiro­plasmas ( 1 16). S. apis is the third example of a spiroplasma harmless on its plant host but pathogenic in its insect host.

Acholeplasma and Mycoplasma Species

Non-helical mollicutes have been cultured from tulip tree (39), melaleuca and jasmin ( 109), and Lactuca perennis (c. Saillard, J. C. Vignault, unpublished data) flowers. Presumed acholeplasmas related by serology but unrelated to any described species have been isolated from powder puff (PP2 strain), lemon (Ll strain), and grapefruit flowers in Florida ( 1 OS, 109); the G + C content of these acholeplasmas is 27%, lower than that of several known acholeplasmas, which is 32%. These three strains form a cluster , and the Ll strain (ATCC 33453) has

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

380 BOVE

been chosen as a type strain for the group. This group has been shown to be unrelated in DNA-DNA homology comparisons with the eight currently recog­nized acholeplasma species ( 1 63). Several presumed acholeplasmas have also been cultured from flowers collected in the Central United States ( 1 85). These strains were serologically related to the Florida PP2 organism. All these isolates are tentatively identified as new strains of an unnamed mollicute species that appears to be widely distributed geographically on plant surfaces (185).

Known species of Acholeplasma and Mycoplasma have also been isolated. A. occu/i, A. axanthum, A . laidlawii, and M. verecundum have been cultured from such vegetables as endive , cabbage, and broccoli purchased at supermar­kets ( 160). The strains of A. axanthum isolated from coconut palms in Jamaica (57) are probably surface organisms and not the causal MLOs of coconut lethal yellowing, since McCoy has never been able to isolate A . axanthum from diseased palms in Horida (R. E. McCoy, personal communication).

Similarly, a mollicute has been cultured ( 177) from aborted seeds of apples affected by proliferation, a disease characterized by the presence of MLOs in the sieve tubes. The triply cloned organism grows in simple media and requires cholesterol (J . G . Tully, unpublished data) . It is probably a member of the genus Mycoplasma. but no serological relationships with any of the known Mycoplasma species have been detected. Hence, the apple mycoplasma seems to be a new species. This coulc\ be considered an argument in favor of its involvement in proliferation disease, but there are at least two arguments against it: (a) only one apple isolate of the organism has been obtained so far, in spite of several isolation attempts, and (b) optimum growth temperature is close to 43°C, a temperature apparently too high to explain symptom expression in apple trees in the field.

LACK OF EVIDENCE FOR RELATEDNESS OF MLOs

TO S . CITRI AND OTHER KNOWN MOLLICUTES

In their attempts to culture MLOs, researchers have sometimes obtained mollicutes from MLO-infected plants. Cultures of A. laidlawii have been recovered from periwinkles infected with the phyllQdy-virescence MLO (69). The antiserum against thi� A. laidlawii strain showed a strong positive reaction to antigens of the cultured organism during ELISA testing but a negative reaction to extracts of rhe phyllody periwinkle. Similar experiments ( 147) showed the stolbur MLO in periwinkles to be unrelated to a bovine mycoplas­ma (Leach serogroup 7) recovered from the stolbur-affected periwinkles. In addition, ELISA showed that neither phyl�ody nor stolbur MLOs were related

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

WALL-LESS PROKARYOTES 381

to A. axanthum, A. modicum, A. granularum, and A. occuli (147). Finally, 25 MLO strains found in periwinkles or other host plants showed negative ELISA results with antisera againSt S. citri and A . laidlawii (52).

ELISA has also been widely used in the search for serological relationships between MLOs and S. citri. When carried out with S. citri antiserum, ELISA showed strong positive reactions toward crude homogenates of S. citri-infected periwinkles (4, 28, 29, 147-149), citrus (16, 71 , 148, 149), and leafhoppers (4, 71, 147, 149). However, the reaction was negative not oilly with healthy plant material but also with extracts of periwinkles infected with the following MLOs: phyllody-virescence, stolbur-flower dwarfing, apple proliferation (Seemuller strain), virescence transmitted by dodder (Cuscuta campestris) from Opuntia tuna monstrosa to periwinkle, and chloraniy (149; c . Saillard, unpublished data). MLOs from apple, cabbage, gladiolus, hydrangea, primula, and European aster yellows, clover phyllody (Dutch and English strains) (28), and American aster yellows (104) also elicited a negative reaction. Hence, ELISA detected no serological relationship between MLOs and S. citri in particular .

Another approach (164) also failed to detect a relationship between MLOs and S. citri or Spiroplasma in general. Spiroplasmas contain long flexible fibrils composed of a single protein of molecular weight 55,000 ( 166). This fibril proteiri is a Spiroplasma-specific antigen ( 165). It has been detected in all spiroplasmas tested, including the poorly helical ASP-l strain, but has not been found in several Mycoplasma spp. and Acholeplasma spp. that were examined. Hence, a technique was developed (164) with which the fibril antigen can be detected in diseased plants. This technique involves gel electrophoresis of detergent-solubilized plant material, blot transfer of the separated proteins to nitrocellulose filters, and, finally, serological detection of the antigens. The sensitivity of the technique is comparable to that of ELISA. By this method the fibril antigen has been detected in S. citri- and CSS-infected periwinkles but not in periwinkles infected with MLOs of clover phyllody, clover disease, cabbage disease, European aster yellows, primula disease, hydrangea disease, or apple disease. The test was also negative with the cactus Opuntia tuna monstrosa. The number of MLOs per gram of tissue ( 1010) was well within the detection limits of the method.

In conclusion, none of the MLOs tested in the above studies showed any relationship to Spiroplasma or Acholeplasma. Among these MLOs were those associated with O. tuna monstrosa, aster yellows, and decline-affected pear trees. It is therefore surprising that other workers have been able to culture S. citri from plants infected with precisely these three MLOs. One reason for these contradictory results might be the fact that some plant infections are caused by a combination of an MLO and S. citri.

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

382 BOYE

DOUBLE INFECTIONS OF PLANTS WITH AN MLO AND

S. CITRI

Oldfield ( 127) was the first to report the occurrence of double infections of S.

citri and MLO in brassicaceous plants and periwinkles in California. He showed that the leafhopper N. tenellus, an active vector of S. citri, can also transmit a virescence MLO. In fact, field-collected N. tenellus leafhoppers, when caged with periwinkles, transmitted the following agents:

1 . S. citri alone. In this case, the infected plants showed severe chlorosis, S.

citri could be cultured from them, and they died within 3-5 months. 2. A virescence MLO alone. In this case, the infected plants showed viresc­

ence symptoms, S. citri could not be isolated, and electron microscopy detected only MLOs in the sieve tubes. The plants lived.

3. S. citri plus a virescence MLO. In this case, the plants showed virescence symptoms but no severe chlorosis, S. citri could be isolated, and the plants lived. Furthermore, the leafhopper Scaphytopius nitridus selectively trans­mitted S. citri from the doubly infected donor periwinkles to healthy periwinkles. The latter developed severe chlorosis but no virescence and died prematurely, in contrast to the donor periwinkles, which lived.

Further confirmation of the occurrence of double infections comes from the work of Kloepper & Garrot (78) in California. These workers have isolated S. citri from peach and cherry trees affected by typical symptoms of X-disease. However, the number of trees from which S. citri could be isolated was very small: 6 out of 111 peach trees tested and 16 out of 459 cherry trees examined. The trees from which no S. citri culture was obtained contained only non­helical MLOs in their sieve tubes, while 4 cherry trees from which S. citri was isolated contained both MLOs and filamentous forms that resembled spiroplas­mas. These results were confirmed and extended in later experiments (79). S. citri was isolated from only a small percentage of X- diseased cherry samples, and the spiroplasma was also found present in symptomless trees. These results support the conclusion that the etiological agent of X -disease is not S. citri and that spiroplasmas are distinct from the MLOs. The association of S. citri with X-diseased trees is probably a chance coinfection with S. citri that also occurs in symptomless trees and does not contribute to the symptoms of X-disease (79).

A spiroplasma serologically indistinguishable from S. citri has been cultured from pear trees showing pear decline ( 133). While these data were first interpreted to mean that S. citri was the causal agent of pear decline, in later experiments S. citri was isolated in only 4 of 330 isolation attempts. On the basis of this result and others, it was eventually concluded that the isolated spiroplasma was not the causal agent of pear decline and that S. citri may occur as part of a mixed infection with the pear decline agent in the same plants ( 137).

S. citri has been isolated from lettuce with aster yellows (A Y) symptoms (8 1,

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

WALL-LESS PROKARYOTES 383

94, 1 00, 1 33) and has been implicitely considered the causal agent of aster yellows . Final success in cultivating the "aster yellows mycoplasmas" was attributed essentially to the choice of source material, diseased lettuce plants (98). Kloepper & Garrot (78) also tried to isolate S. citri from A V-affected plants. They were unable to culture S. citri from 14 A V-affected lettuce plants and 48 A Y -affected plantago plants; however, 4 out of 53 A Y -affected peri­winkles yielded an S. citri culture . Plantago plants showing an atypical form of A Y were also tested for S. citri isolation; in this case, S. citri was consistently cultured, with an isolation frequency greater than 90%. Here again, mixed infections explain the results. The 14 lettuce plants , the 48 plantagos, and the 53 periwinkles showing typical AY (virescence) symptoms were all infected with the A Y MLO, but 4 of the periwinkles were doubly infected and carried S. citri in addition to the MLO, a situation very similar to the one that Oldfield observed in Southern California (see above) . The plantago plants with atypical symptoms of A Y that consistently yielded S. citri were probably infected with S. citri alone. Hence, their symptoms were those of an S. citri infection and not those associated with the typical AY MLO. This conclusion is further sup­ported by the fact that S. citri is serologically unrelated to the MLO found in AY-affected plants ( 158).

Thus, it appears that the "Aster yellows spiroplasma" (98), S. citri, is irrelevant to aster yellows disease. The aster yellows controversy has been thoroughly discussed in two recent reviews ( 156, 179), and additional argu­ments against a spiroplasmal etiology for A Y have been presented. Contamina­tion in the laboratory or in the greenhouse from S. citri-infected leafhoppers must also be considered.

In conclusion, active natural transmission of S. citri and MLOs by insect vectors results not only in plants infected with S. citri or MLOs alone, but also in double infections, where S. citri and MLOs are present in the same plant.

Single and double infections can be effected experimentally in periwinkle by graft inoculations ( 150). In single infections , periwinkle plants show character­istic symptoms when inoculated with phyllody-virescence, stolbur-flower dwarfing, or chloranty MLOs, for example . Infected with S. citri, they show lethal wilting six weeks after inoculation and exposure to temperatures close to 32°C, the temperatures at which S. citri grows best. In double infections, when S. citri and an MLO were inoculated at the same time, the periwinkles showed symptoms of MLO infection but no lethal wilting, even though they were kept at 32°C. S. citri was present in these plants; it could be cultured from them and was detected by ELISA. Similar results were obtained when the periwinkles were first infected with the MLO, and later with S. citri, at a time (four weeks post-inoculation) when MLO symptoms had already appeared. Plants that received S. citri first and an MLO four weeks thereafter developed lethal wilting, but after twelve weeks rather than six weeks in the absence of the MLO.

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

384 BOYE

There is good reason to think that the severity of symptoms shown by an S. citri-infected plant increases with the number of S. citri helices present in that plant. This number is influenced by temperature. In plants grown at 32°C, S. citri helices are much more numerous than in those exposed at lower tempera­tures (84). In doubly infected periwinkles, where the presence of MLOs has prevented lethal wilting due to S. citri, electron microscopy is able to detect many plieomorphic MLOs but few S. citri helices in the sieve tubes, even though ELISA and/or culture has detected S. citri. In California, it has been found that field-infected broccoli plants contain a lower spiroplasma population in double infections than in single ones (80). It thus seems that MLO multi­plication somehow interfers with the multiplication of S.citri. In this way, S. citri concentration is prevented from developing sufficiently to induce lethal Wilting.

Some of the plants doubly infected with graft inoculation were indistinguish­able from periwinkles naturally infected in Morocco, Turkey, and Syria ( 1 50) . On the basis of their symptom expression, both the naturally and the ex­perimentally infected periwinkles seemed to be infected by an MLO only, yet ELISA and/or culture could detect the presence of S. citri.

The last example to be discussed concerns the ornamental cactus Opuntia tuna monstrosa from which S. citri has been cultured (26, 82) . This spiroplas­rna has been assumed to be the causal agent of the "monstrosa condition" of O.

tuna, even though, as Christi!lnsen and co-workers pointed out (26) , electron microscopy of O. tuna monstrosa material has never revealed the helical organisms. On the other hand, ELISA failed to detect S. citri in O. tuna

monstrosa material (52) and we obtained the same result (C. Saillard, J. M. Bove, unpublished data). Furthermore, we were never able to culture S. citri

from monstrosa cactus material (J. C. Vignault, J. M. Bove, unpublished data; 164). Finally, a virescence-inducing MLO was transmitted from O. tuna monstrosa to periwinkle by dodder (Cuscuta campestris) (c. Saillard, J. M. Bove, unpublished data), and S. citri could not be detected in the periwinkles by ELISA or culture. Therefore, the monstrosa cactus used in this research was infected with a virescence-inducing MLO, but its infection with S. citri is very doubtful. Further studies are definitely required to elucidate the etiology of the monstrosa condition of O. tuna. One prerequisite in these studies should be to use the same plant material. Some cactus might have a mixed infection (MLO + S. citri) , others a single infection (MLO).

PURIFICATION OF MLOs: AN APPROACH TO THEIR

CHARACTERIZATION

So far, no MLOs have been cultured. As an alternative to their cultivation, several groups of workers have attempted their purification (4, 2 1 , 28, 86, 15 1 ,

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

WALL-LESS PROKARYOTES 385

157, 1 58) . Even though the partially purified MLO preparations have been used to study the ultrastructure of MLOs ( 152), their lipid composition ( 154), and their proteins (159), major efforts have been directed toward the production of antisera (21 , 28, 1 55 , 1 58). Immunosera were first used in double diffusion tests ( 155); more recently immunosorbent electron microscopy (IEM) (21 , 1 58) and especially ELISA (28) have become the prderred techniques. This work takes advantage of previous experience in the use of ELISA to detect S. citri in plants and insects (29, 147-149) and in the use of plants infected with S. citri (4, 28) or the com stunt spiroplasma (55, 134) as models or positive control systems. MLO preparations from affected plants are, at the present stage, not yet free of contaminating host plant antigens; the antisera obtained from them often show high anti-plant activity. This activity can be reduced by extensively cross-absorbing the antisera with antigens from healthy plants (28). In addition, a new indirect ELISA test using immunoglobulin F (ab ' h fragments in the coating step, whole antiserum as the second antibody, and immunoglobulin Fc fragments in the conjugate has been developed (6) and applied to the detection of MLO antigens (28). Considerable improvement in limiting background reactions due to anti-plant activity was achieved by diluting the already CroSS­absorbed whole antiserum used as the second antibody in an extract of healthy plants (28). Another way of decreasing background reaction is to prepare the MLO antigens needed to raise the antiserum from a host other than those to be tested (28). One application of this approach is to use MLO-infected leafhop­pers to prepare the antigens for immunizing and applying the antiserum to the detection of the MLO in plants. In this way, the agent ofJlavescence doree from infected Vicia Jaba plants was seen with IEM using antibodies raised in the leafhopper Euscelidius variegatus (21) . This elegant approach can now be applied to determining the serological relationships between various MLOs. This is an important problem, and the first results along these lines have already appeared.

Antisera have been obtained from periwinkle plants infected with either S. citri or the clover phyllody MLO and used in the new indirect ELISA men­tioned above (28). The antibodies were able to specifically detect their homolo­gous antigens. The clover phyllody antiserum reacted to extracts of plants infected with the clover phyllody MLO whether the infected plant was peri­winkle or clover, or whether the clover phyllody strain was from England or the Netherlands; it also reacted to strawberry plants affected by green petal disease. Interestingly, clover phyllody and strawberry green petal diseases have been reported to share a common etiology on the basis of transmission experiments; now, the first direct serological confirmation of this relationship has been obtained (28). Finally, the clover phyllody antiserum did not react with MLO from apple, cabbage, dahlia, gladiolus, hydrangea, or primula. Similar studies involving ELISA and IEM have been carried out with antisera against MLOs from aster (Callistephus chinensis) plants infected with the A Y MLO ( 1 58).

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

386 BOYE

Until now, attempts to characterize MLOs were essentially based on the reactions they induced in their plant or insect hosts. The above data clearly indicate the feasibility of investigating serological specificity and relationships among the MLOs themselves, without having to culture the organism in vitro (28).

The production of an antiserum would not require live organisms, nor would highly purified MLO preparations be needed, if ways could be found to reduce background reactions, as indicated above. However, for certain studies, such as morphology and etiology examinations, it might be necessary to preserve the structure of the MLOs and to use live, unfixed organisms. With these objec­tives in mind, a technique has been developed in which the sieve elements of periwinkle plants infected with S. citri, MLOs, or the bacterial clover c;lub leaf agent were separated from other cell types by partial digestion of the plant tissues with cellulase and macerozyme (86). The microorganisms present in the sieve elements from diseased plants could be seen clearly through the cell walls when viewed through dark field microscopy. In sieve elements from plants infected with pear decline, aster yellows, or tomato big bud agent, no helical microorganisms were seen, but pleiomorphic bodies of predominantly filamentous form were present. The sieve elements could be ruptured by osmotic shock, and this provided another way to studying MLO morphology further. All MLOs observed in this way had the same morphology. Similar results were obtained previously using scanning electron microscopy (SEM) of MLOs after the freeze fracture of aster plants affected with either clover phyllody or aster yellows (72). Only a few MLOs have been examined with SEM, but the results obtained do not invalidate the conclusion drawn from traditional transmission electron microscopy: namely, that the MLOs associ­ated with one disease are morphologically indistinguishable from those associ­ated with another.

While the partially purified MLO preparations have been mainly used to produce antisera for serological investigations of MLOs, they have not yet been employed to study biochemical or biophysical properties, probably because such studies require a purification level higher than that for antisera production. Efforts along these lines should prove fruitful, however. MLO DNA can be extracted from purified MLO preparations and analyzed by techniques that have already been developed for genuine mycoplasmas. One of these tech­niques is a restriction enzyme analysis of the DNA. This technique ( 14, 15) was originally developed to study MLO DNA, but it was never applied to MLO DNA because MLO purification techniques had not yet been able to produce MLO DNA pure enough to be analyzed. However, it has been found most useful for the characterization of Acholeplasma, Mycoplasma, and Spiroplas­rna species ( 15 , 141) . In another approach, the DNA from one MLO can be radioactively labeled by nick translation and used for DNA-DNA hybridization

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

WALL-LESS PROKARYOTES 387

with unlabeled DNA from other MLOs. The determination of G + C content does not even require extraction of the DNA. The partially purified MLO preparation can be submitted directly to equilibrium centrifugation in cesium chloride; the G + C content can be calculated from the buoyant density of the DNA band. This technique has been used to determine the G + C content of the unculturable Drosophila spiroplasma (77).

The techniques developed for one- or two-dimensional analyses of myco­

plasma proteins on polyacrylamide gels (36, 1 1 7 , 1 1 9 , 1 67) have not been sufficiently adapted to purified MLO preparations. With the very sensitive protein staining methods now available, only limited amounts of proteins are required. In this way, MLO-specific protein spots can be identified on the gels and used to prepare highly specific rabbit or guinea pig antisera. Such mono­specific antibodies have been prepared against single S. citri proteins ( 1 1 7) and they stand as witnesses for the feasability of this approach.

Monoclonal antibodies (MCAs) can also provide monospecific immuno­globulins against individual proteins. MCAs have been prepared against S. citri ( 1 1 7) , and both S. citri-specific and non-specific MCA secreting hybridoma clones have been obtained. All the specific MCAs were directed against one single S. citri protein: spiralin, the main membrane protein of S. citri. The nonspecific MCAs gave positive ELISA results not only with S. citri but also with spiroplasmas other than S. citri ( 1 1 7). Application of the hybridoma technique to MLOs seems highly promising in that only partially purified MLO preparations are required.

In spite of much circumstantial evidence concerning their pathogenic role in yellows diseases, MLOs have not yet been strictly shown to be the causal agents of the diseases in which they are seen. Some of the techniques presented in this section might eventually lead to preparations of live MLOs pure enough not only for biochemical , biophysical, and serological characterizations, but for the fulfillment of Koch's postulates as well.

CONCLUSION

Three types of wall-less prokaryotes are associated with plants: the sieve-tube­specific, yellows disease-associated MLOs; the pathogenic spiroplasmas, also sieve-tube-specific; and the mollicutes (Spiroplasma, Acholeplasma, Myco­plasma) found on the surfaces of plants and flowers. Among the agents associated with or responsible for diseases in plants, only the spiroplasmas have been cultured. The plant pathogenic spiroplasmas may not be limited to S. citri and CSS. Other spiroplasmas might yet be found to be plant pathogens; spiroplasma P4Q is one such candidate. MLOs have not yet been cultured and their true nature remains unknown. Techniques for their purification are being developed and progress has already been made toward understanding their

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

388 BOVE

serological character and relationships. More and more evidence accumulates to indicate that spiroplasmas, especially S. citri. do not derive from MLOs. Double infections of plants with both S. citri and an MLO are well documented and offer at least one explanation of why S. citri occasionally can be cultured from MLO-infected plants, such as periwinkles with aster yellows, pear trees with decline, and O. tuna with the monstrosa condition.

The well-known presence of mollicutes on the surface of both healthy and diseased plants now makes understandable why mycoplasmas can occasionally be cultured from MLO-infected hosts. The isolation of S. citri and Acholeplas­rna sp. from healthy and lethal yellowing-affected coco tissues is a good

illustration of this point. The culturable spiroplasmas, especially S. citri, have lent themselves to

numerous studies on their biological, biochemical, and immunological prop­erties. The cloning of a spiroplasma plasmid and the expression of a spiroplas­rna gene in E. coli open the way to sp�roplasma genetics based on recombinant DNA technology. Transfection of spiroplasmas with viral DNA shows that these organisms can be transformed and that viral DNA might become a vector for cloning in mollicutes. Host-parasite models using spiroplasma-insect systems, at the level of the whole organism or at the level of the cell, might throw light on mycoplasma pathogenicity, a still poorly understood phenom­enon.

Finally, the close interrelationship between the mycoplasmas and their plant and insect hosts makes the ecology of the wall-less prokaryotes of plants a fascinating subject.

Literature Cited

1 . Alivizatos, A. S . , Markham, P. G . , Townsend, R . 1982. Spirophlsma viruses in plants. 4th Inti. Congr. Inti. Org. Mycoplasmol. (10M). Tokyo. 1-7 September. Abstr. P-74. 1 24 pp.

2. Alivazatos. A. S . , Townsend. R . , Mark­ham, P. G. 1982. Effects of i'nfection with a spiroplasma virus on the symp­toms produced by Spiroplasma citri. Ann. Appl. Bioi. 101 :85-91

3. Archer, D. B . , Best, J . , Barber, C. 1 98 1 . Isolation and restriction mapping of a spiroplasma plasmid. J. Gen. Microbiol. 1 26:51 1-14

4. Archer, D. B . , Townsend, R., Mark­ham, P. G. 1982. Detection of Spiroplas­rna citri in plants and insect hosts by ELISA. Plant Pathol. 3 1 :299--306

5. Aubert, B . , Bove, J. M. 1980. Effect of penicillin or tetracycline injections of cit­rus trees affected by greening disease under field conditions in Reunion Island. In Proc . 8th Conj. IOCV, ed. E. C. Cala-

van, S. M. Garnsey, L. W. Timmer, pp. 103-8. Riverside, Calif. : IOCV, Dept. Plant Patho! . , Univ. Calif.

6. Barbara, D. J . , Clark, M. F. 1982. A simple indirect ELISA using F (ab' h fragments o f immunoglobulin. 1 . Gen. Viral. 58:3 15-22

7. Barker, L. F. 1 968. Determination of antibiotic susceptibility of rickettsiae and chlamydiae in BSC-l cell structures. Antimicrob. Ag. Chemother. pp. 425-28

8. Bove, J. M . 1975. Les mycoplasmes. La recherche. 6:210-20

9. Bove, J . M . 1 98 1 . Mycoplasma infec­tions of plants. Israel J. Med. Sci. 17:572-85

10. Bove, J. M. 1982. Les spiroplasmes, nouveaux mycoplasmes pathogenes des vegetaux et des animaux. In Les Mala­dies des Plantes (llIe joumees fran!raises d'etudes d'informations). ACTA, 149 rue de Bercy, 75595 Paris cedex 12. Paris, Octobre 1979, pp. 379--427

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

1 1 . Bove, J. M . , Bonnet, P . , Gamier, M . , Aubert, B . 1980. Penicillin and tetracy­cline treatments of greening disease­affected citrus plants in the glasshouse, and the bacterial nature of the procaryote associated with greening. See Ref. 5 , pp. 9 1-102

12. Bove, J. M . , Mouches, C . , Carle-Junca, P . , Degorce-Dumas, J . R . , Tully, J . G . , Whitcomb, R. F . 1984. Spiroplasmas of group I: the Spiroplasma cirri cluster. Yale J. Bioi. Med. In press

1 3 . Bove, J. M . , Moutous, G . , Saillard, C . , Fos, A . , Bonfils, J . , Vignault, J . e. , Nhami, A . , Abassi, M . , Kabbage, K . , Hafidi , B . , Mouches, C . , Viennot­Bourgin, G. 1979. Mise en evidence de Spiroplasma citri, 1 'agent causal de la maladie du "stubborn" des agrumes dans 7 cicadelles du Maroc. C. R. Acad. Sci. Paris, ser. D. 288:335-38

14. Bove. J. M . , Saillard, C. 1 979. Cell biol­ogy of spiroplasmas. In The Mycoplas­mas: 1I1. Plant and Insect Mycoplasmas. ed. R. F. Whitcomb, J. G . Tully, pp. 83-153. New York: Academic

15 . Bove, J. M . , Saillard, C . , Junca, P. , De­gorce-Dumas, J. R . , Ricard, B . , Nhami, A . , Whitcomb, R. F . , Williamson, D . , Tully, J. G . 1982. Guanine-plus-cytosine content, hybridization percentages, and BeoRI restriction enzyme profiles of spi­roplasmal DNA. Rev. In/. Dis. 4:S I29-36

16. Bove, J. M . , Saillard, e., Vignault, J . C . , Fos, A . 1 984. Citrus stubborn disease in Irak and Syria: Correlation between symptom expression and detection of Spiroplasma citri by culture and ELISA. Proc. 9th Conf. IOCV, Argentina. River­side, Calif. : IOCV, Dept. Plant Pathol. , Univ. Calif. In press

1 7 . Bove, J. M . , Whitcomb, R . F . , McCoy, R. E. 1 983. Culture techniques for spiro­plasmas from plants. See Ref. 17 1 , pp. 225-34

1 8 . Brinton, L. P . , Burgdorfer, W. 1976. Cellular and subcellular organization of the 277F agent, a spiroplasma from rabbit tick (Haemaphysalis leporispalutris (Acari: ixodidael)}. Inti. J. Syst. Bacter­iol. 26:554-60

19. Calavan, E. C . , Ka1oostian, G . H . , Old­field, G. N . , Nauer, E. M . , Gumpf, D. J. 1977. Natural spread of Spiroplasma citri by insect vectors and its implications for control of stubborn disease of citrus. Proc. 1m. Soc. Citriculture, 3:900---2

20. Calavan, E. C . , Oldfield, G. N. 1979. Symptomatology of spiroplasmal plant diseases. See Ref. 14, pp. 37-64

2 1 . Caudwell , A. , Meignoz, R . , Kuszala,

WALL-LESS PROKARYOTES 389

C . , Larrue, J . , Fleury, A., Boudon, E. 1982. Purification serologique et observation ultra-microscopique de l'a­gent pathogene (MLO) de la flavescence don<:e de la vigne dans les extraits liquides de !eves (Vicia !aba L.) malades. C. R. Seances Soc. Bioi. 1 76(5):723-29

22. Charron, A . , Bebear, c. , Brun, G., Yot, P . , Latrille, J . , Bove, J. M . 1979. Separation and partial characterization of two deoxyribonucleic acid polymerases from Spiroplasma citri. J. Bacteriol. 140(3):763-68

23. Charron, A . , Castroviejo, M . , Bebear, e., Latrille, I . , Bove, I. M. 1 982. A third DNA polymerase from Spiropiasma citri and two other spiroplasmas. J. Bacteriol. 149(3): 1 138-41

24. Chen, T. A . , Davis, R. E. 1979. Cultiva­tion of spiroplasmas. See Ref. 1 4, pp. 65-82

25. Chen, T. A . , Liao, C. H. 1 975. Com stunt spiroplasma: Isolation, cultivation and proof of pathogenicity. Science 1 88:1015-17

26. Christiansen, C . , Freundt, E. A . , Mara­morosch, K. 1980. Identity of cactus and lettuce spiroplasmas with Spiroplasma cirri as determined by DNA-DNA hy­bridization. Curro Microbiol. 4:353-56

27. Clark, H. F . , Rorke, L. B. 1 979. Spiro­plasmas of tick origin and their pathogenicity. See Ref. 14, pp. 1 55-74

28. Clark, M. F . , Barbara, D. J . , Davies, D. L. 1 983. Production and characteristics of antisera to Spiroplasma citri and clo­ver phyllody-associated antigens derived from plants. Ann. Appl. Bioi. 103:251-59

29. Clark, M. F. , FJegg, C. L . , Bar-Joseph, M . , Rottem, S. 1978. The detection of Spiroplasma citri by enzyme-linked im­munosorbent assay (ELISA). Phyto­pathol. 92:332-37

30. Clark, T. B. 1977. Spiroplasma sp. : A new pathogen in honey bees. J. Inver­febr. Palhol. 29: 1 1 2-13

31 . Clark, T. B , 1982. Spiroplasmas: Di­versity of arthropod reservoirs and host­parasite relationships . Science 217:57-59

32. Cole, R . M . 1979. Mycoplasma and spir­oplasma viruses: Ultrastructure. In The Mycoplasmas: I. Cell Biology, ed. M. F. Barile, S._ Razin, pp. 385-410. New York: Academic

33. Cole, R. M . , Mitchell, W. O . , Garon, C. F. 1977. Spiroplasmavirus citri 3: Propa­gation, purification, proteins and nucleic acid. Science. 198:1262-63

34. Cole, R. M . , Tully, J . G . , Popkin, T. J . ,

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

390 HOVE

Bove, J. M. 1973. Morphology, ultra­structure and bacteriophage infection of the helical mycoplasma-like organism (Spiroplasma citri gen. nov . , sp. nov.) cultured from "stubborn" disease of cit­rus. J. Bacteriol. 1 15(1):367-86

35. Daniels. M. J. 1983. Mechanisms of spiroplasma pathogenicity. Ann. Rev. Phytopathol. 2 1 :29-43

36. Daniels. M. J . , Archer, D. B . , Stephens. M. A . , Townsend, R . , Longland. J . M . , Best, J . 1980. Comparison of spiroplas­mas by polyacrylamide gel elec­trophoresis of cell proteins. Curro Micro­bioi. 4:377-80

37. Daniels, M. J . , Markham, P. G . , eds. 1982. Plant and Insect Mycoplasma Techniques. London: Croom Helm 369 pp.

38. Das, J . , ManiIoff, 1. 1976. Replication of mycoplasmavirus MVL5 1 . IV. Inhibi­tion of viral synthesis by rifampin. J. Virol. 18(3):969-76

39. Davis, R. E. 1977. Cell wall-free pro­karyotes harbored in flowers. Proc. 3rd Meet. Int. Counc. Lethal Yellowing. Palm Beach County. Florida, Oct.-Nov. 1977. (Abstr.)

40. Davis, R. E. 1978. Spiroplasma associ­ated with flowers of the tulip tree. (Liriodendron tulipifera). Can. J. Micro­bioi. 24:954-59

4 1 . Davis, R. E. 1978. Spiroplasmas from flowers of Bidens pilosa L. and honey bees in Florida: Relationship to honey bee spiroplasma AS576 from Maryland. Phytopathol. News. 12(7):PO-7 (Abstr.)

42. Davis, R. E. 1978. Spiroplasmas: Helical cell wall-free prokaryotes in diverse habi­tats. Proc. ROC US Coop. Sci. Sem. Mycoplasma Diseases of Plants. NSC Symposium Series 1 , pp. 59-64

43 . Davis, R . E. 1979. Spiroplasmas: Newly recognized arthropod-borne pathogens. In Leafhopper Vectors aiuI Plant Disease Agents. ed. K. Maramorosch. K. F. Har­ris, pp. 451-84. New York: Academic

44. Davis, R. E. 198 1 . The enigma of the flower spiroplasmas. In · Mycoplasma Diseases of Trees and Shrubs. ed. K. Maramorosch, S. P. Raychaudhuri, pp. 259-79. New York: Academic '

45. Davis, R. E. , Fletcher, J. 1983. Spiro­plasma citri in Maryland: Isolation from field-grown plants of horseradish (Armoracia rusticana) with brittle root symptoms. Plant Dis. 67:900-3

46. Davis. R. E . • Lee. L M . 1982. Compara­tive properties of spiroplasmas and emerging taxonomic concepts: A propos­al. Rev. Inf. Dis. 4:S122-28

47. Davis, R. E . , Lee, I. M. 1982. Pathogenicity of spiroplasmas, myco-

plasmaIike organisms. and vascular­limited fastidious walled bacteria. In Phytopathogenic Prokaryotes, ed. M . Mount, G. Lacy, 1 :491-5 1 3 . New York: Academic

48. DaviS, R. E. , Lee, L M . • Basciano, L. K. 1979. Spiroplasmas: Serological group­ing of strains associated with plants and insects . Can. J. Microbial. 25:861-6

49. Davis, R. E . , Lee, I. M . , Worley, J . F. 198 1 . Spiropiasma jloricoia, a new spe­cies isolated from surfaces of flowers of the tulip tree Liriodendron tulipifera L.

Int. J. Syst. Bact. 3 1(4):456-64 50. Davis, R. E . , Whitcomb, R. F. 197 1 .

Mycoplasmas, rickettsiae, and chlamy­diae: Possible relation to yellows diseases and other disorders of plants and insects. Ann. Rev. Phytopathol. 9: 1 19-44

5 1 . Davis, R. E . , Worley, 1. F. , Whitcomb, R. F . , Ishijima, T. , Steere, R. L. 1972. Helical filaments produced by a myco­plasma-like organism associated with corn stunt disease. Science 176:521-3

52. De Leeuw, G. T. N . , Polak-Vogelzang, A. A . , Hagenaars, A. M. 1983. Absence of serological relationship between several mycoplasma-like organisni "strains", Spiroplasma citri. com stunt spiroplasma and Acholeplasma laidlawii. determined by the enzyme-linked im­munosorbent assay (ELISA). Phytapath. Z. 107:3 1-36

53. Doi, Y .• Teranaka, M . , Yora, K . , Asuyana, H . 1967. Mycoplasma or P.L.T. group-like microorganisms found in the phloem elements of plants infected with mulberry dwarf potato witches' broom, aster yellows or paxlownia witch­es' broom. Ann. Phytopathol. Soc. Jpn. 33:259-66

54. Donachie, W . D . , Begg, K . 1. 1970. Growth of the bacterial cell. Nature 227 : 1220-24

55. Eden-Green, S. 1. 1982. Detection of com stunt spiroplasma in vivo by ELISA using antisera to extracts from infected corn plants (Zea mays). Plant Pathol. 3 1 :289-97

56. Eden-Green, S . J . , Archer, D . B . , Tully, 1. G . • Waters, H. 1983. Further studies on a spiroplasma isolated from coconut palms in Jamaica. Ann. Appl. Bioi. 102:127-34

57. Eden-Green, S. J . , Tully, J. G. 1979. Isolation of Achaleplasma spp from coconut palms affected by lethal yellow­ing disease in Jamaica. Curro Microbial. 2:3 1 1

58. Eden-Green, S . J . , Waters, H . 198 1 . Isolation and preliminary characteriza­tion of a spiroplasma from coconut palms

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

in Jamaica. J. Gen. Microbiol. 124:263-70

59. Edward, D. G . , Freundt, E. A. 1967. Proposal for Mollicutes as name of the class established for the order Mycoplas­matales. Int. J. Syst. Bacteriol. 17:267-8

60. Fletcher, 1. , Schultz, G. A . , Davis, R. E . , Eastman, C. E . , Goodman, R. M. 198 1 . Brittle root disease of horseradish: Evidence for an etiological role of Spiro­plasl1Ul citri. Phytopathol. 7 1 : 1073-80

6 1 . Frazier, N. W. 1953. A survey of the mediterranean region of the beet leafhop­per. J. Econ. Entomol. 46:55 1-55

62. Freitag, J. G . , Frazier, N. W . , Huffaker, C. B . 1955. Cross breeding beet leafhop­pers from California and French Moroc­co. J. Econ. Entomol. 48:341-42

63. Fudl-Allah, A. F.-S. A. , Calavan, E. C . , Igwegbe, E. C. K. 1972. Culture of a mycoplasma-like organism associated with stubborn disease of citrus. Phyto­pathol. 62:729-31

64. Gadeau, A . , Mouches, C . , Bove, J. M. 1982. Resistance of mollicutes to rifam­picine. See Ref. 1 , Abst. P-1 27. 1 77 pp.

65. Garnier, M . , Bove, J. M. 1983. Trans­mission of the organism associated with citrus greening disease from sweet orange to periwinkle (Vinca rosea) by dodder (Cuscuta campestris) . Phyto­pathol. 73: 1 358-63

66. Garnier, M . , Clerc, M . , Bove, J. M. 198 1 . Growth and division of spiroplas­mas: Morphology of S. citri during growth cycle in liquid medium. J. Bac­teriol. 147(2):642-52

67. Garnier, M . , Clerc, M . , Bove, J. M. 1984. Growth and division of Spiroplas­ma citri: Elongation of elementary helices. J. Bacteriol. 158:23-28

68. Gamier, M . , Danel, N . , Bove, J. M. 1984. Etiology of citrus greening dis­ease. Ann. Microbiol. (Inst. Pasteur) 1 35A: 169-79

69. Giannotti , J. 1974. Cultures de molli­cutes 11 partir de plantes atteintes de jaunisse et d'insectes vecteurs . Colloq. Inst. Natl. Sante Med. Les mycoplasmesl mycoplasmas 33:99-106

70. Gumpf, D. J . , Calavan, E. C. 198 1 . Stubborn disease of citrus. See Ref. 44, pp. 97-134

7 1 . Hafidi, B . , Bencheqroun, N . , Fisher, H. J . , Van der Veken, J . 1979. Application de la technique immunoenzymatique "ELISA" pour la detection de Spiroplas­I1Ul citri, agent de la maladie du stubborn des agrumes dans les orangeraies maro­caines. Parasitica 35: 141-5 1

72. Haggis, G. H . , Sinha, R. C. 1968. Scan­ning electron microscopy of mycoplas-

WALL-LESS PROKARYOTES 391

malike organisms after freeze fracture of plant tissues affected with clover phyl­lody and aster yellows. Phytopathol. 68:677-80

73. Hopkins, D. L. 1977. Diseases caused by leafhopper-borne, rickettsia-like bacteri­a. Ann. Rev. Phytopathol. 15 :277-94

74. Hopkins, D. L. 1983. Gram-negative, xylem-limited bacteria in plant disease. Phytopathol. 73:347-50

75. Hurst, E. W. 1962. Experimental che­motherapy of infection with agents of the psittacosis-lymphogranuloma-trachoma group. Ann. NY Acad. Sci. 98:275-78

76. Ishiie, T . , Doi, Y . , Yora, K . , Asuyama, H. 1967. Suppressive effects of antibiot­ics of tetracycline group on symptom de­velopment of mulberry dwarf disease. Ann. Phytopathol. Soc. Japan . 33:267-75 (In Japanese)

77. Junca, P . , Saillard, C , Tully, J. G. , Gar­cia-Jurado, 0., Degorce-Dumas, J. R . , Mouches, C . , Vignault, 1. C , Vogel, R. , McCoy, R . , Whitcomb, R. , Williamson, D . , Latrille, J . , Bove, J. M. 1980. Carac­terisation de spiroplasmes isoles d'in­sectes et de fleurs de France Continen­tale, de Corse et du Maroc. Proposition pour une classification des spiroplasmes. C. R. Acad. Sci. Paris. 290: 1 209-12

78. Kloepper, J . W . , Garrot, D. G. 1980. Relation of in vivo morphology to isola­tion of plant spiroplasmas. Curro Micro­bioi. , 4:365-70

79. Kloepper, J. W . , Garrott, D. G. 1983. Evidence for a mixed infection of spiro­plasmas and nonhelical mycoplasmalike organisms in cherry with X-disease. Phy­topathol. 73:357-60

80. Kloepper, J. W . , Garrott, D. G . , Old­field, G. N. 1982. Quantification of plant pathogenic spiroplasmas from infected plants. Phytopathol. 72:577-8 1

8 1 . Kondo, F . , Maramorosch, K . , McIn­tosch, A. H . , Varney, E. H. 1977. Aster yellows spiroplasmas: Isolation and cul­tivation in vitro. Proc. Am. Phytopathol. Soc. 4: 190

82. Kondo, F . , McIntosh, A. H . , Padhi, S . B. , Maramorosch, K. 1976. A spiroplas­rna isolated from the ornamental cactus Opuntia tuna monstrosa. Proc. Soc. Gen. Microbiol. 3:154

83. Lafleche, D . , Bove, J. M. 1970. Struc­tures de type mycoplasme dans les feuilles d'orangers atteints de la maladie du greening. C. R. Acad. Sci. Paris. 270: 1915-17

84. Lafleche, D . , Bove, J. M. 1970. Myco­plasmes dans les agrumes atteints de greening, de stubborn ou de maladies similaires. Fruits 25:455-65

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

392 BOVE

85. Lee, I. M . , Davis, R. E. 1980. DNA homology among diverse spiropiasma strains representing several serological groups. Can. 1. Microbiol. 26: 1 356--63

86. Lee, I. M . , Davis, R. E. 1983. Phloem­limited prokaryotes in sieve elements iso­lated by enzyme treatment of diseased plant tissues. Phytopathol. 73: 1 540-43

87 . Lei, J. D . , Su, H. J . , Chen, T. A. 1979. Spiroplasmas isolated from green leaf bug Trigonotylus ruficornis Geoffroy. See Ref. 42, pp. 89-98

88. Liao, C. H. , Chen, T. A. 1 982. Media and methods for culture of spiroplasmas. See Ref. 37, pp. 1 75-200

89. Liss, A . , Cole, R. M . 1 98 1 . Spiroplas­mavirus group I: Isolation, growth, and properties. Curro Microbial. 5:357-62

90. Liss, A . , Cole, R . M . 1982. Spiroplas­mal viruses: Group I characteristics. Rev. InJ. Dis . 4:S 1 15-9

9 1 . Liu, H. Y . , Gumpf, D. J . , Oldfield, G. N., Calavan, E. C. 1983. Transmission of Spiroplasma citri by Circulifer tenel­Ius. Phytopathol. 73:582-85

92. Liu, H . Y . , Gympf, D. J . , Oldfield, G. N. , Calavan, E. C . 1983. The rela­tionship of Spiroplasma citri and Circu­lifer tenellus. Phytopathol. 73:585-90

93. Lovisolo, 0. , Lisa, V . , D'Agostino, G. , Loche, P . , Fresu, B. 1982. Grave malat­tia del pomodoro associata a micoplasmi, in Sardegna. In Aui Giornate Fitopatho­logiche-5upplemento, pp. 35-41 . Bo­logna: Coop. Lib. Univ. Edit.

94. Lowe, S. K . , Raju, B . C. 1978. The morphology of spiroplasmas associated with aster yellows and pear decline dis­ease: A comparative study by negative staining in vitro. Phytopathol. News. 1 2 : 2 1 6

95. Maniloff, J. 1983. Evolution o f wall-less prokaryotes. Ann. Rev. Microbiol. 37: 477-99

96. Maramorosch, K. 1976. Plant mycoplas­ma diseases. In Encyclopedia of Plant Physiology. New Series. Vol. 4. Phys­iological Plant Pathology, ed. R. Heite­fuss, P. H. Williams, pp. 1 50-7 1 . New York: Springer-Verlag

97. Maramorosch, K. 1 980. Spiroplasmas of plants and animals: An overview. India: Nat. Acad. Sci. Gold Jub. Comm. Vol. 20 pp.

98. Maramorosch, K. 1 98 1 . Spiroplasmas: Agents of animal and plant diseases. Bioscience 3 1 (5):374-80

99. Maramorosch, K. 1 982. Control of vec­tor-borne mycoplasmas. In Pathogens, Vectors and Plant Diseases, ed. K. Har­ris, K. Maramorosch, pp. 265-95. New York: Academic

100. Maramorosch, K. , Kondo, F. 1978. As­ter yellows spiroplasma: Infectivity and association with a rod-shaped virus: Zbl. Bakt. Hyg. , I. Abt. Orig. A24 1 : 196 (Abstr.)

1 0 1 . Markham, P. 1 982. The "yellows" plant diseases: Plant hosts and their interaction with the pathogens. See Ref. 37, pp. 82-1 00

102. Markham, P. G . , Townsend, R . , Plas­kilt, K . , Saglio, P. 1977. Transmission of corn stunt to dicotyledonous plants. Plant Dis. Rep. 6 1 :342-45

103. McCoy, R. E. 1 979. Mycoplasmas and yellows diseases. See Ref. 14, pp. 229-64

104. McCoy, R. E . , Basham, H. G. 1 982. Lack of serological relationship between the aster yellows agent and Spiroplasma citri determined by ELISA assay. See Ref. 1 , Abst. P-70. 120 pp.

105. McCoy, R. E . , Basham, G. H . , Tully, J . G. , Rose, D. 1982. Isolation o f a new Acholeplasma from flowers in Florida. Rev . lnf. Dis. 4:5274

1 06. McCoy, R. E . , Mi11er, M. E . , Williams, D. S. 1 980. Lethal yellowing in Texas phoenix palms. Principes 24(4): 1 79-80

107. McCoy , R . E . , Tsai, J . H . , Norris, R . C . 1983. Pigeon pea witches' broom i n Flor­ida. Plant Dis 67:443-45

108. McCoy, R. E . , Williams, D. S. 1 982. Chemical treatment for control of plant mycoplasma diseases. See Ref. 37, pp. 1 52-73

109. McCoy, R. E. , Williams, D . S . , Tho­mas, D. L. 1978. Isolation of mycoplas­mas from flowers. See Ref. 42, pp. 75-8 1

1 10. Mouches, c . , Barroso, G . , Bove, J . M. 1983. Characterization and molecular cloning in Escherichia coli of a plasmid from the mollicute Spiroplasma citri. 1. Bacteriol. 1 56(2):952-55

1 1 1 . Mouches, c . , Barroso, G . , Gadeau, A . , Bove, J. M . 1984. Plasmids and recom­binant DNA from Spirop/asma citri and other spiroplasmas. See Ref. 1 6

1 1 2 . Mouches, c . , Barroso, G. , Gadeau, A. , Bove, J. M. 1984. Characterization of two cryptic plasmids from Spiroplasma citri and occurrence of their DNA se­quences among various spiroplasmas. Ann. Microbiol. (lnst. Pasteur) 1 35A: 17-24

1 1 3 . Mouches , C . , Bove, J . M. 1984. A plas­mid from S. citri strain M4 hybridizes with extrachromosomal DNAs from other spiroplasmas, including corn stunt spiroplasma E275, tick spiroplasma 277F and coco spiroplasma N525. YaleJ. Bioi. Med. In prcss

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

1 14 . Mouches, c . , Bove, J. M . , Albisetti, J . 1984. Pathogenicity of Spiroplasma apis and other spiroplasmas for honey bees in southwestern France. Ann. Microbial. (lnst. Pasteur) 135A: 1 5 1-55

1 15 . Mouches, c . , Bove, J . M . , Albisetti, J . , Clark, T. B . , Tully, J . G. 1982. A spiro­plasma of serogroup IV causes a May­disease like disorder of honeybees in southwestern France. Microb. Eco!. 8: 387-99

1 16. Mouches, C . , Bove, J. M . , Tully, J. G . , Rose, D. L. , McCoy, R. E . , Carle­Junca, P . , Garnier, M . , Saillard, C. 1983. Spiroplasma apis, a new species from the honey-bee Apis mellifera. Ann. Microbial. (Inst. Pasteur) 1 34A:383-97

1 17 . Mouches, C . , Candresse, T . , McGarrity, G. J . , Bove, J. M. 1984. Analysis of spiroplasma proteins: Contribution to the taxonomy of group IV spiroplasmas and the characterization of spiroplasma pro­tein antigens. Yale J. Bioi. Med. In press

1 1 8 . Mouches, c . , Duthil, P . , Vignault, J. c . , Protopapadakis, F . , Nhami, A . , Tully, J . G., Bove, J . M. 1980. Characterization of spiroplasmas by polyacrylamide gel analysis of their proteins and enzymes. See Ref. 5, pp. 1 34-44

1 19 . Mouches, C . , Menara, A . , Tully, J. G . , Bove, J . M . 1982. Polyacrylamide gel analysis of spiroplasmal proteins and its contribution to the taxonomy of spiro­plasmas. Rev . Inf. Dis. 4:S 1 41-47

1 20. Mouches, C . , Vignault, J. C . , Tully, J . G . , Whitcomb, R. F. , Bove, J . M. 1 979. Characterization of spiroplasmas by onc­and two-dimensional protein analysis on polyacrylamide slab gel. Curro Micro­bial. 2:69-74

1 2 1 . Nasu, S . , Sugiura, M . , Wakimoto, T . , Iida, T. T . 1967. O n the etiologic agent of rice yellow dwarf disease. Ann. Phyto­pathol. Soc. Japan. 33:343-44. In Japanese

122. Nault, L. 1980. Maize bushy stunt and corn stunt: A comparison of disease symptoms, pathogen host ranges and vectors . Am. Phytopathol. Soc. 70(7): 659-62

123. Nhami, A. , Bove, J. M . , Bove, C . , Mon­sion, M . , Garnier, M. Saillard, C . , Moutous, G . , Fos, A. 1980. Natural transmission of Spiroplasma citri to peri­winkles in Morocco. See Ref. 5 , pp. 1 53-61

1 24. Nienhaus, F. 1 979. Li4'�hen-Degenera­tion durch Rickettsien-Ahnliche Bakte­rien. Allgemeine Forstzeitschrift, 6

1 25 . Nienhaus, F. , Sikora, R. A. 1979. Myco­plasmas, spiroplasmas, and rickettsia-

WALL-LESS PROKARYOTES 393

like organisms as plant pathogens. Ann. Rev. Phytopathol. 17:37-58

1 26. O'Hayer, K. W. , Schultz, G. A . , East­man, C. E . , Fletcher, J . , Goodman, R. M . 1982. Transmission of Spiroplasma citri by the aster leafhopper Macrosteles

fascifrons (Homoptera: cicadellidae) . Ann. Appl. Bioi . 102:3 1 1- 1 8

1 27 . Oldfield, G. N. 1982. A virescence agent transmitted by Circulifer tenetlus (Baker); aspects of its plant host range and association with Spiroplasma citri. Rev. Inf. Dis. 4:S248

1 28 . Oldfield, G. N . , Calavan, E. C. 1 98 1 . Spiroplasma citri: Non rutaceous hosts. In Description and Illustration of Virus and Virus-Like Diseases of Citrus: A Col­lection of Color Slides Vol. IV. ed. J . Bove, R. Vogel. Paris: IRFA, 6 rue Gem!ral Clergerie, 75 1 16 Paris, France

1 29. Oldfield, G. N . , Kaloostian, G. H . 1978. Vectors and host range of the citrus stub­born disease pathogen, Spiroplasma cit­rio See Ref. 42, pp. 1 19-25

1 30. Pickens, E. G . , Gerloff, R. F . , Burgdor­fer, W. 1968. Spirochetes on the rabbit tick Haemaphysalis leporispalustris (Packard) . I: Isolation and preliminary characterization. J. Bacterial. 95:291-99

1 3 1 . Purcell, A. H. 1982. Insect vector rela­tionships with procaryotic plant path­ogens. Ann. Rev. Phytopathol. 20:397-4 1 7

1 32. Quinlan, D. C . , Maniloff, J . 1972. Mem­brane association of the deoxyribonucleic acid growing point region in Mycoplasma gallisepticum. J. Bacterial. 1 12: 1 375

1 33 . Raju, B. c. , Nyland, G. 1978. Effects of different media on the growth and mor­phology of three newly isolated plant spiroplasmas. Phytopathol. News. 12: 216 (Abstr. 4 1 8)

1 34. Raju, B. C . , Nyland, G. 1 98 1 . Enzymc­linked immunosorbent assay for the de­tection of corn stunt spiroplasma in plant and insect tissues. Current Microbial. 5 : 1 01-4

135 . Raju, B. c . , Nyland, G . , Backus, E. A . , McLean, D. L. 1 98 1 . Association o f a spiroplasma with brittle root of horserad­ish. Phytopathol. 7 1 (1 0): 1067-72

136. Raju, B . C . , Nyland, G . , Meikle, T . , Purcell, A. H . 1 98 1 . Helical, motile mycoplasmas associated with flowers and honey bees in California. Can. J. Microbiol. 27(2):249-53

1 37. Raju, B. C . , Nyland, G . , Purcell, A. H. 1983 . Current status of the etiology of pear decline. Phytopathol. 73:350-53

1 38 . Ranhand, J. M . , Mitchell , W. O . , Pop­kin, T. J . . Cole. R. M. 1980. Covalently closed circular deoxyribonucleic acids in

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

394 BOVE

spiroplasmas. J. Bacteriol. 143: 1 194-9

139. Razin, S . , Glaser, G . , Amikam, D . 1 984. Molecular and biological features of mollicutes (Mycoplasmas). Ann. Mi­crobiol. (lnst. Pasteur) . 1 35A9-15

140. Razin, S. , Tully, J. G., eds. 1 983. Methods in Mycoplasmology, Mycoplas­ma Characterization. Vol. I. New York: Academic. 504 pp.

1 4 1 . Razin, S . , Tully, J. G . , Rose, D. L . , Barile, M. F. 1 983. DNA cleavage pat­terns as indicators of genotypic heter­ogeneity among strains of Acholeplasma and Mycoplasma species. J. Gen. Micro­bioi. 1 29: 1935-44

142. Saglio, P. , Lafleche, D . , Bonissol, C . , Bove, J. M. 1 97 1 . Culture in vitro des mycoplasmes associes au stubborn des agrumes et leur observation au micro­scope eiectronique. C. R. Acad. Sci. 272: 1 387-90

143. SagJio, P . , Lafleche, D . , Bonissol, C . , Bove , J . M. 197 1 . Isolement, culture et observation au microscope electronique des structures de type mycoplasme as so­ciees a la maladie du stubborn des agrumes et leur comparaison avec les structures observees dans Ie cas de la maladie du greening des agrumes. Phys­iol. VCg. 9:569-82

144. Saglio, P. , Lafleche, D . , L'Hospital, M . , Dupont, G . , Bove, J. M. 1972. Isolation and growth of citrus mycoplas­mas . In Pathogenic Mycoplasmas: A ClBA Foundation Symposium, pp. 1 87-203. Amsterdam: Elsevier-Excerpta Medica

1 45 . Saglio, P . , L'Hospital, M . , Lafleche, D . , Dupont, G . , Bove, J. M . , Tully, J. G . , Freundt, E. A. 1 973. Spiroplasma citri gen. and sp. n . : A mycoplasma-like organism associated with stubborn dis­ease of citrus. Inti. J. Syst. Bacteriol. 23: 191-204

146. Saglio, P., Whitcomb, R. F. 1 979. Di­versity of wall-less prokaryotes in plant vascular tissue, fungi, and invertebrate animals. See Ref. 14, pp. 1-36

147. Saillard, C. 198 1 . Caracterisation et tax­onomie des spiroplasmes. These de Doc­teur de 3e cycle, N" d'ordre: 78. Univer­site de Bordeaux II, Mai 198 1

148. Saillard, C . , Dunez, J. , Garcia-Jurado, 0 . , Nhami, A . , Bove, J. M. 1978. De­tection de Spiroplasma citri dans les agrumes et les pervenches par la tech­nique immunoenzymatique ELISA. C. R. Acad. Sci. Paris 286: 1 245-8

149. Saillard, C . , Garcia-Jurado, 0 . , Bove, J . M . , Vignault, J. c . , Moutous, G . , Fos, A . , Bonfils , J . , Nhami, A . , Vogel, R . , Viennot-Bourgin, G . 1980. Application

of ELISA to the detection of Spiroplasma citri in plants and insects. See Ref. 5 , pp. 145-52

ISO. Saillard, C . , Vignault, J. c . , Fos, A . , Bove, J . M. 1 984. Spiroplasma citri induced lethal wilting of periwinkles is prevented by prior or simultaneous in­fection of the periwinkle by an MLO. Ann. Microbiol. (Inst. Pasteur) . 1 35A: 163-68

1 5 1 . Sinha, R. C. 1 974. Purification of my co­plasma-like organisms from China aster plants affected with clover phyllody. Phytopathol. 64(8): 1 156-58

152. Sinha, R. C. 1976. Ultrastructure of mycoplasma-like organisms purified from clover phyllody-affected plants. J. Ultrastr. Res. 54: 183-89

153. Sinha, R. C. 1979. Chemotherapy of mycoplasmal plant diseases . See Ref. 14, pp. 309-35

154. Sinha, R. C. 1979. Lipid composition of mycoplasma-like organisms purified from clover phyllody and aster yellows­affected plants. Phytopathol. Z. 96: 1 32-39

1 55 . Sinha, R. C. 1 979. Purification and serology of mycoplasma-like organisms from aster yellow-infected plants. Can. J. Plant Pathot. 1 :65-120

156. Sinha, R. C. 1984. The aster yellows controversy: Current status. Yale J. Bioi. Med. In press

157. Sinha, R. C. 1983. Physical techniques for purification of mycoplasmas from plant tissues. See Ref. 1 7 , pp. 243-47

158. Sinha, R. C . , Benhamou, N. 1 983. De­tection of mycoplasma-like organism­antigens from aster yellows diseased plants by two serological methods. Phy­topathol. 73: 1 199-202

159. Sinha, R. C . , Madhosingh, C. 1980. Pro­teins of mycoplasma-like organisms puri­fied from clover phyllody and aster yel­lows-affected plants. Phytopathol. Z. 99:294-300

160. Somerson, N. N . , Kocka, J. M . , Del Giudice, R . , Rose, D. L . , 1982. Isolation of acholeplasmas and a mycoplasma from vegetables . Appl. Environ. Micro­bioi. 43:412- 1 7

1 6 1 . Sonnenwirth, A. c . , McGee, Z . A . , Davis, B . D . 1980. Other pathogenic microorganisms; L-phase variants (L­forms). In Microbiology, ed. B . D . Davis, R. Dulbecco, H. N. Eisen, H. S . Ginsberg. Hagerstown, Maryland: Har­per & Row. 803 pp. 3rd ed.

162. Steiner, T . , McGarrity, G. J . , Bove, J. M . , Philips, D. M . , Garnier, M. 1984. Insect cell cultures to study attachment and pathogenicity of spiroplasmas and

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

mycoplasmas. Ann. Microbiol. (Inst. Pasteur). 1 35A:47-53

1 63 . Stephens, E. B . • Aulakh, G. S . , McCoy. R. E .• Rose. D. L . . Tully. J. G . . Barile, M. F. 1 98 1 . Lack of genetic relatedness among some animal and plant achole­plasmas by nucleic acid hybridization. Curro Microbiol. 5:367-70

1 64 . Townsend. R. 1 983. Mycoplasma-like organisms from plants with "yellows" diseases lack a spiroplasma-specific anti­gen. J. Gen. M icrobiol. 1 29: 1959-64

165. Townsend. R . . Archer. D . B . 1 983. Fi­bril protein in an antigen specific to Spir­oplasma. J. Gen. Microbiol. 1 29: 199-200

.

1 66. Townsend. R . , Archer. D. B . • Plaskitt. K. A. 1980. Purification and preliminary characterization of spiroplasma fibrils. J. Bacteriol. 142:694-700

1 67 . Townsend. R . , Daniels, M. J. 1982. Physicochemical characterization of plant and insect mycoplasmas. See Ref. 37, pp. 268-306

1 68. Townsend, R . , Markham. P. G . , Plas­kitt, A. K. 1 977. Multiplication and mor­phology of Spiroplasma citri in the leafhopper Euscelis plebejus. Ann. Appl. Bioi. 87:307-1 3

1 69. Tsai, J . H . 1 979. Vector transmission of mycoplasmal agents of plant diseases. See Ref. 14, pp. 265-307

1 70. Tully, J. G. 1 982. Interaction of spiro­plasmas with plant, arthropod and animal hosts. Rev. Inf. Dis. 4:S I 93-99

1 7 1 . Tully, J. G . , Razin, S . , eds. 1983. Methods in Mycoplasmology. Diagnostic Mycoplasmology. Vol. II. New York! London: Academic. 440 pp.

1 7 1a. Tully, J . G . , Rose, D. L. Garcia­Jurado, 0. , Vignault, J. c . , Sailhird, c . , Bove , J . M . , McCoy, R. E . , Williamson, D. L. 1 980. Serological analysis ofa new group of spiroplasmas. Curro Microbiol. 3:369-72

1 72. Tully, J. G . , Whitcomb, R. F. 1979. The genus Spiroplasma. In The Prokaryotes (Springer-Verlag Handbook), ed. M . P. Starr, H. Stolp, H . G. Truper, A . Balows, H. G . Schlegel. Berlin: Springer-Verlag

1 73 . Tully, J. G . , Whitcomb, R. F. , Clark, H. F . , Williams<,m, D . L. 1 977. Pathogenic mycoplasmas: Cultivation and vertebrate pathogenicity of a new spiroplasma. Sci­ence. 1 95:892-94

1 74. Tully, J . G . , Whitcomb, R. F. , William­son, D. L. , Clark, H. F. 1 976. Suckling mouse cataract agent is a helical wall-free prokaryote (spiroplasma) pathogenic for vertebrates . Nature 259(5539): 1 17-20

1 75. Vago, C . , Giannotti, J . , Meynadier, G . , Morel, G . 1 974. Recherches sur les pro-

WALL-LESS PROKARYOTES 395

caryotes intracellulaires chez les inver­tebres. Colloq. Inst. Natl. Sante Rech. MM. Les mycoplasmeslmycoplasmas. 33:265-72

176. Vignault, J. C . , Bove, J. M . , Saillard, C. , Vogel. R . , Farro, A . , Venegas, L. , Stemmer, W . • Aoki. S . • McCoy. R . • AI­Beldawi. A. S .• Larue, M . , Tuzcu. 0. , Ozsan. M . , Nhami, A . , Abassi. M . •

Bonfils, J . • Moutous. G . , Fos. A . •

Poutiers. F . • Viennot-Bourgin. G. 1980. Mise en culture de spiroplasmes 11 partir de materiel vegetal et d' insectes prove­nant de pays circummediterraneens el du Proche-Orient. C. R. Acad. Sci. Paris 290:775-80

177. Vignaull. J. c . , Saillard. c . . Tully, J . G . • Mouches, c . , Bove. J . M . . Bern­hard, R. 1982. Attempts 10 culture and characterize mycoplasma-like organisms from fruit affected with apple prolifera­tion disease. Rev. In/. Dis. 4:S248

178. Whitcomb. R. F. 1 980. The genus Spiro­plasma. Ann. Rev. Microbiol. 34:677-709

179. Whitcomb. R. F. 1 98 1 . The biology of spiroplasmas. Ann. Rev. Entomol. 26: 397-425

1 80. Whitcomb. R. F. , Black. L. M . 1982. Plant and arthropod mycoplasmas: A his­torical perspective. See Ref. 37. pp. 40-8 1

1 8 1 . Whitcomb. R. F . • Bove. J . M . 1 983. Mycoplasma-plant-insect interrelation­ships. In Methods in Mycoplasmology. Mycoplasma Characterization. Volume I, ed. S. Razin. J. G. Tully, pp. 2 1 -25 . New York: Academic

1 82. Whitcomb. R. F . • Clark. T. B . • Tully. J . G . • Chen. T. A . , Bove. J . M. 1 984. Serological classification of spiroplas­mas: Current status. Yale J. Bioi. Med. In press

1 83. Whitcomb. R. F . • Tully. J. G. 1 979. The Mycoplasmas: III. Plant and Insect Mycoplasmas. New York: Academic. 35 1 pp.

1 84. Whitcomb. R. F .• Tully. J. G .• Clark. T. B . • Williamson. D . L. , Bove. J . M. 1 982. Revised serological classification of spiroplasmas, new provisional groups. and recommendations for serotyping of isolates. Curro Microbiol. 7:291-96

185. Whitcomb. R. F., Tully, J . G . , Rose. D. L.. Stephens, E. B., Smith, A . • McCoy. R. E .• Barile. M. F. 1 982. Wall-less pro­karyotes from fall flowers in central United Slales and Maryland. Curro Mic­robiol. 7:285-90

1 86. Whitcomb. R. F . , Tully. J. G . • Wro­blewski, H. 1 984. Spiralin: Major mem­brane protein specific for subgroup I-I

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.

396 BOVE

spiroplasmas. Curro Microbiol. 9:7- 1 2 187. Williamson, D . L . , Whitcomb, R . F.

1 975. Plant mycoplasmas: A cultivable spiroplasma causes com stunt disease. Science 1 88: 1018--20

1 88. Windsor, I. M . , Black, L. M. 1 973. Re­mission of symptoms of clover club leaf following treatment with penicillin. Phy­topathol. 64:44-46

1 89. Wroblewski, H . , Johansson, K. E . , Hjer-

ten, S. 1 977. Purification and character­ization of spiralin, the main protein of Spiroplasma citri membrane. Biochim .

Biophys. Acta 465:275-89 190. Zeigel, R . F . , Clark, H. F. 1974. Elec­

tron microscopy of the suckling mouse cataract agent: A noncultivable animal pathogen possibly related to mycoplas­ma. Infect. Immun. 9:430-43

Ann

u. R

ev. P

hyto

path

ol. 1

984.

22:3

61-3

96. D

ownl

oade

d fr

om a

rjou

rnal

s.an

nual

revi

ews.

org

by U

NIV

ER

SIT

Y O

F FL

OR

IDA

- S

mat

hers

Lib

rary

on

11/0

4/09

. For

per

sona

l use

onl

y.