Spiroplasma Citri: Fifteen Years of Research

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Spiroplasma citri: Fifteen Years of Research

J. M. Bove Dedicated to Richard Guillierme*

I-HISTORICAL SIGNIFICANCE mas, molecular and cellular biology of OF SPIROPLASMA CITRI spiroplasmas, spiroplasma pathogenicity, ecology of Spiroplasma citri, It is now well recognized that the biology and ecology of Spiroplasma agent of citrus stubborn disease was kunkelii. Volume IV of IOCV's Virus the first mollicute of plant origin to and Virus-like diseases of citrus (7) have been cultured (19, 33) and for also covers isolation, cultivation and which Koch's postulates were fulfilled characterization of S. citri. Stubborn (25). The serological, biological and disease has been reviewed (24). biochemical characterizations of the Methods in Mycoplasmology offers in citrus agent revealed it to be a new two volumes the techniques used in mollicute, one with helical morphol- the study of mollicutes including the ogy and motility (34), hence the name spiroplasmas (30, 37). These proceed- Spiroplasma citri, adopted from ings also cover epidemiology of S. Davis et al. (14, 15) who had given citri in the Old World (4) and spiro- the trivial name spiroplasma to helical plasma gene structure and expression filaments seen in corn stunt infected plants. These "helices" were cultured (5). and shown to be the agent of corn stunt disease in 1975 (9,44); the agent 11-MAJOR PROPERTIES is now called S~iro~lasmakunkelii OF SPIROPLASMA CITRl (40). The first bre;kthrough in the study of yellows diseases came in 1967 Spiroplasma citri is a mollicute with the discovery of mollicute-like (42). Mollicutes are prokaryotes that organisms (MLO) in plants (17). Cul- have evolved by degenerative evolu- ture of the stubborn agent was the tion from low guanine plus cytosine next important advance since it not (G + C) Gram positive bacteria. only offered a model for the study of While the bacterial cell has a pep- plant mollicutes, but also revealed the tidoglycane-containing cell wall, S. existence of a whole new world of citri, as a mollicute, has no cell wall microorganisms: the spiroplasmas, of and, hence, no peptidoglycane. which more than 30 different species Penicillin inhibits one of the last steps or serogroups are known today (42). in peptidoglycane biosynthesis. S. Many reviews on S. citri and other citri, with no peptidoglycane, is in- spiroplasmas have appeared (2, 12, sensitive to penicillin, but not to tet- 13, 39, 42). Be it enough to mention racycline and erythromycine, antibio- volume V of the Mycoplasmas (43), a tics that inhibit protein biosynthesis. revised edition of volume I11 (41), en- On solid medium, S. citri produces tirely devoted to plant and insect mol- fried egg-shaped (umbonate) colonies. licutes with chapters on the following Under certain environmental condi- topics: the genus Spiroplasma, tions, bacterial forms can be obtained spiroplasma-insect host relationships, which lack cell walls (bacterial L vari- nutrition and cultivation of spiroplas- ants). They also show umbonate colonies. Thus. this articular colonv morphology is related to the absenie *Director General of Institut de Recherches of cell wall. sur les Fruits et Agrumes (IRFA) from 1942 to The various mollicutes that were 1973. known in animals before the discov- Diseases Induced by Procaryotic Pathogens ery of the spiroplasmas were acids from acetate. The type of fatty pleiomorphic and did not have a de- acids in the S. dtri membrane de- fined shape. Therefore, the helical pends on the fatty acid composition of morphology of S. citri was entirely the growth medium. S. citri derives unexpected. In addition, the helical its energy for growth from the fer- spiroplasma cells are motile, with mentation of glucose to organic acids, flexional and twitching movements, and the hydrolysis of arginine to yield and often show an apparent rotatory C02, NH, and ATP. The production motility. Flagella, periplasmic fibrils, of acid from glucose during S. citri or other organelles of locomotion are growth can be conveniently followed not present, but intracellular fibrils by the color-shift from red to yellow have been demonstrated. Motility of of the phenol red pH-indicator added S. citri is revealed in the colony mor- to the culture medium. Oxygen is not phology in that the umbonate colonies required for growth. S. citri is a facul- are diffuse, often with satellite col- tative anaerobe. The stubborn agent onies developing from foci adjacent to grows best in a narrow range of tem- the initial site of colony development. peratures centered around 32 C. This Poorly helical, nonmotile variants of explains why stubborn disease is well S. citri are known; their colonies expressed in hot climates, but not in exhibit a typical umbonate appear- those where temperatures above 30 ance with sharp margins and without C are rarely approached. satellite colonies. Growth and division of S. citri has In liquid medium of low viscosity, attracted much attention (21,22). The spiroplasmas do not achieve appreci- smallest helical spiroplasma cell, or able translational motility. Only when helix, was found to be a one- to two- the viscosity is increased by incorpo- turn helix (elementary helix). Such ration of agar or methylcellulose in cells increase in length into parental the medium can the spiroplasmas use helices which divide by constriction, their motility to move along (12b). liberating elementary helices. The Spiroplasmas display chemotaxis i. e., most frequently dividing parental they are attracted by certain sub- helix is one with approximately four stances and repelled by others (12b). turns, yielding two elementary Chemotaxis may be involved in the helices. At the unfavorable tempera- movement of S. citri in the phloem ture of 37 C, the number of short elements. Mutants have recently helices decreases drastically in favor been obtained that are devoid of of long helices with very few constric- motility but have retained their heli- tions, showing that the organism can cal morphology (Cohen & Williamson, still elongate, but not divide; division personal communication). This shows resumes at 32 C. During elongation that helical morphology and motility of an elementary helix, growth seems are genetically independent. to start at one end of the helix (polar Mollicutes of the genus Mycop- growth). lasma, Ureaplasm or Anaerop- In vitro, during log-phase growth, lasm require sterol for growth; all S. citri cells are found to be helical. those of the genus Acholeplasma or The situation is less simple when the Asteroleplasm do not. S. citri and spiroplasmas are studied in the tis- all other spiroplasmas need sterol in sues or cells of their hosts. In S. citri- their growth medium. The sterol infected plants, helical forms can al- (cholesterol) is generally supplied by ways be seen in the sieve tubes, but addition of horse or foetal calf serum nonhelical forms seem to exist too. In (10-20%) in the culture medium. The plants naturally infected with both a serum also supplies fatty acids. These MLO and S. citri (I), the symptoms are required for growth as the spiro- are generally those induced by the plasmas are unable to synthesize fatty MLO and in the sieve tubes the MLO Tenth ZOCV Conference cells outnumber the S. citri cells; the principal vector (4). For both vectors, latter might be difficult to see by elec- Salsola kali L (Russian thistle or tron microscopy. However, their tumbleweed), a Chenopodiaceae, is a presence can be detected by culturing major host plant. The discovery of the spiroplasma from such plants periwinkles naturally infected with S. (MLOs have not yet been obtained in citri in Arizona, California and culture). In contrast to the situation Morocco showed S. citri is not limited in culture media and in plants, the to citrus in nature. For instance, in morphology of S. citri is not helical in California, several species in the leafhoppers. No helical forms are seen Brassicaceae family are infected with either in the hemolymph or in any S. citri (27). In Illinois, brittle-root other tissues and all spiroplasma cells disease of horseradish is the second appear roughly spherical. S. citri can crop disease found to be caused by S. be shown to absorb to, and to be pres- citri (18). ent in cultured leafhopper cells. The S. citri is serologically related to identity of the non-helical forms in the S. melliferum a spiroplasma patho- insect cells as S. citri was de- genic to honeybees, S. kunkelii, the monstrated by immunofluorescence; corn stunt agent, and S. phoeniceum, and their viability demonstrated by a new plant pathogen recently discov- incorporation of 3H-thymidine into ered in periwinkles in Syria (35). The DNA (23). At this time, the factors DNAs of these four spiroplasmas that induce a helical spiroplasma cell show also appreciable degrees of to become nonhelical and vice versa homology (ca 50%). In spite of these are not known. They will probably be close relationships, the four spiroplas- better understood when the mecha- mas can easily be distinguished one nisms by which spiroplasmas achieve from the other by several techniques their helical morphology and motility and they produce specific diseases. have been elucidated. For these reasons, and in spite of In the early 1970s, fulfillment of their relationships with S. citri, the Koch's postulates required that S. corn stunt agent, the honeybee citri, after having been obtained in spiroplasma and the new periwinkle pure culture, be transmitted back to pathogen have eventually received citrus. Euscelis plebejus, a leafhop- species names: S. kunkelii (40), S. per vector of a number of European mell$erum (11) and S. phoeniceum yellows diseases assumed to be (35), respectively. In the spiroplasma caused by mycoplasmas, was origi- classification (38), based on serologi- nally chosen to transmit S. citri to cal and molecular genetic data, S. plants. A micro-injection technique citri and the three other spiroplasma was used to infect the leafhoppers species are classified in group I, the with cultured spiroplasmas. Success- S. citri group, and represent four of a ful transmission of S. citri to white total eight subgroups. clover by injected leafhoppers pro- Much work has been devoted to vided proof of pathogenicity of S. citri molecular genetic data for S. citri and opened the way to positive trans- and other spiroplasmas (2, 3). The mission to citrus in 1974 (25). Since Guanine plus Cytosine (G + C) con- then, several other leafhopper species tent of the DNA of S. citri and all have been used to experimentally other Group I spiroplasmas is 26 mole transmit S. citri to numerous plant %. Other spiroplasmas (S. apis, group species. Natural leafhopper vectors of IV, S. mirum, group V) have DNA S. citri have been identified. In with 30% G + C. However, the G + Arizona and California, Neoaliturus C content of spiroplasmas is not (Circulifer) tenellus seems to be the either 26% or 30%, but ranges from major vector (28, 29). In the Old 24% to 30% with several intermediate World, N. haematoceps, more abun- values. Even so, the G + C content dant than N. tenellus, is probably the of spiroplasmas is low in agreement Diseases Induced by Procaryotic Pathogens with the low G + C mole % of molli- strain to the other, certain strains cutes as a whole (24 to 35%). having more than one plasmid. At this While different species of a given time, however, nothing is known genus may have different G + C con- about the role of the spiroplasma plas- tents, all species of the same genus mids. They are cryptic in that no have the same genome size. S. citri, phenotypic trait has been associated as well as all other spiroplasmas, have with any of them. The only four well- a genome size of lo9 d. Acholeplasma characterized plasmids are all from S. spp. also have a genome of lo9 d, but citri strains. Plasmids pIJ2000 from Mycoplasma spp. and Ureaplasrna strain ASP-1 (cultured from citrus) spp. have genomes twice as small (5 and pM41 from strain M4 (cultured x lo8 d). from periwinkle) are very similar if S. citri has three DNA poly- not identical. Plasmid pMHl from merases and resembles in this respect strain MH (cultured from citrus) and the Eubacteria which have also three pRAl from an early passage of strain DNA synthesizing enzymes. Interest- R8A.2 (cultured from citrus) could be ingly, Mycoplasma spp., with half as cloned in E. coli and shown to be much DNA than Spiroplasma spp. clearly different from one another, seem to have only one DNA and from pM41 or pIJ2000. polymerase. The DNA of plasmid pM41 or Eubacteria have only one DNA- pIJ2000 hybridized with plasmid dependent RNA polymerase. The so- DNA of S. citri strains other than the called "core-enzyme" contains two homologous parent strains M4 and large subunits (p and p') and two ASP-1 (but not with all S. citri strains copies of a small subunit (a), the gen- tested), as well as with strains of eral structure being PP'a2. In the spiroplasma species other than S. "holoenzyme", a sigma factor (a)is as- citri. This shows that identical or sociated with the core enzyme and is similar plasmid sequences occur in responsible for promoter recognition. different spiroplasma strains or S. citri and other spiroplasmas have a species. In contrast to these results, RNA polymerase of the same struc- plasmid pMHl was only detected in ture as Eubacteria, namely pP'a2 the parent S. citri strain MH. It did (20). The "general" sigma factor used not hybridize with extrachromosomal by the spiroplasma enzyme has the (plasmid) DNA of any other spirop- same molecular weight as that of the lasma tested. However, most in- Gram positive B. subtilis enzme, terestingly, it hybridized with namely ca 42,000 d; the Gram nega- chromosomal DNA of several, but tive E. coli general a factor measures not all S. citri strains, as well as 70,000 d. This finding is in agreement strains of S. kunkelii, S. mellifemm with the phylogenetic origin of the and others. These results show that mollicutes, seen as having arisen from DNA sequences present as extrac- low G + C Gram positive bacteria hromosomal plasmid DNA in some (45). spiroplasmas can be integrated in the chromosomal DNA of others. Similar 111- S. CITRI PLASMIDS results have been obtained with S. AND VIRUSES citri plasmid pRA1. This plasmid was found in early passages of S. citri Besides chromosomal (genomic) strain R8A2. With increasing passage DNA, spiroplasmas and especially S. numbers, plasmid sequences pro- citri also have extrachromosomal gressively disappeared as extra- DNA: plasmids andlor viral DNA (3). chromosomal DNA, but were re- Work in several laboratories has covered as chromosomal sequences. shown the wide occurrence of plas- Only part of the early plasmid se- mids in spiroplasmas. With S. citri, quences are integrated in the the plasmid content varies from one chromosomal DNA. The precise na- Tenth ZOCV Conference ture of these integrated sequences is having shown no detectable virus pro- being actively studied. So is the true duction for several passages. How- nature of "plasmid" pRA1. It is not ever, this phenomenon is explainable. unlikely that this so-called "plasmid" It was found that SpV3 can lysogenise could in fact be a double-stranded cir- S. citri, and in addition, all S. citri cular replicative form of a SpV1-type strains tested contained in their virus (see below). chromosome a cryptic (partial) form Viral DNA sequences have also of the viral genome (16). During been found to be integrated in the lysogeny integration of a complete chromosomal DNA of spiroplasmas. viral genome in the host chromosome Four spiroplasma viruses are known occurred adjacent to the cryptic at this time (3). SpVl is a nonlytic, genome. Sequences of SpVl DNA are rod-shaped virus (ca. 230 by 15 nm) also present in the chromosome of all with single-stranded, circular DNA of S. citri strains tested (31). It is not 8.0-8.4 kilobase (kb) infecting S. citri yet known, however, whether a full and many other spiroplasmas. SpV2 viral SpVl genome or only part of it is a polyhedron (55 by 50 nm) with a is integrated in the S. citri chromo- long noncontractile tail (ca. 80 by 7 some. nm); DNA is probably double- Finally, it has recently been stranded (ds). The virus seems to be shown that certain sequences of plas- specific to S. citri, and it has not yet mid pRAl are present, not only in the been propagated experimentally. S. citri genome (see above), but also SpV3 is a polyhedron (ca. 40 by 36 in the DNA of SpV3 (3) and that of nm) with a short tail (ca. 15 by 7); SpVl (Renaudin and Bove, unpub- DNA is double-stranded and linear. lished). Identification of the se- The amount of DNA varies with virus quences that are shared by the DNAs type from 16 to 30 megadaltons (Md). of S. citri SpV3 and SpV1, and plas- Many spiroplasma species, including mid pRAl is in progress. As men- S. citri, are infected with a SpV3-type tioned above, the plasmid nature of virus. Finally, SpV4 is isometric (27 pRAI is being reexamined. nm diameter) with single-stranded circular DNA (4421 bases); the virus IV-GENE STRUCTURE is specific to S. melli$erum. Release AND EXPRESSION of SpV4 virions is lytic while SpVl and SpV3 infection is not cytocidal. The techniques of DNA cloning SpV1- and SpV3-type viruses are and sequencing have recently been the most common viruses in primary applied to S. citri and the related hon- isolations of S. citri. For instance, eybee spiroplasma (S. mell$erum). when 12 Moroccan isolates were Two systems have been studied in examined in the electron microscope particular and have provided, for the seven showed the presence of SpV1, first time, information on the struc- one revealed SpV3, and one had both ture and expression of spiroplasma SpVl and SpV3 virions. In 17 isolates genes (5). The first system concerns from Syria, five and four showed, re- the gene for spiralin, the major mem- spectively, SpVl and SpV3 virions, brane protein of S. citri; the second whereas four had a mixed infection. system deals with spiroplasma virus However, the number of S. citri iso- spv4. lates infected with SpVl and/or SpV3 The spiralin gene was cloned in E. might be higher as indicated by these coli as part of a 4.5 kb Hind111 frag- figures. Indeed, SpVl and SpV3, as ment of S. citri DNA inserted in plas- well as SpV2, have shown a peculiar mid vector pBR328. Production of behaviour ever since they were dis- spiralin in the transformed E. coli covered. They are known to occur clone was demonstrated unambigu- spontaneously in spiroplasma cultures ously (26). This result showed that a Diseases Induced by Procaryotic Pathogens mollicute gene could be expressed in is not a termination codon in the a bacterium, even though the spiralin spiroplasmas. This result taken to- gene has remained, up to now, the gether with that concerning expres- only mollicute gene that could be fully sion of spiralin in E. coli indicates expressed in E. coli. The reason for that UGA codes for tryptophan. this unique situation is now clear and In the so-called "universal" code, resides in the fact that, in the spiro- tryptophan is coded by only one plasmas, the codon UGA is not a ter- codon, UGG. The use of UGA as a mination codon, as in bacteria, but tryptophan codon in the spiroplasmas codes for tryptophan. Therefore, a is only a special case of a more general UGA codon specifying "tryptophan" finding concerning the codon usage in in spiroplasmas, will be read as a stop the spiroplasmas as deduced from the codon in bacteria and will result in capsid protein gene of SpV4. For premature termination of protein syn- those amino acids specified by several thesis. Only spiroplasma genes with- codons, the codons commencing or out UGA codons will be fully expres- terminating by A or T are much more sed in E. coli. This is probably the frequently used than those beginning case with the spiralin gene since or finishing by C or G. In the capsid spiralin contains no tryptophan. The protein there are nine UGA codons gene for spiralin has now been fully for one UGG codon. This special sequenced and, as expected, contains codon usage, favoring A and T over C no UGA codon. and G, reflects the low G + C content The fact that UGA codes for tryp- of the spiroplasmas. . tophan in spiroplasmas was suggested In addition to the ORF for the cap- by the results of Yamao et al. (46), sid protein, the SpV4 genome con- who showed for the first time that tains eight other ORFs, beginning UGA codes for tryptophan in Myco- with ATG or GTG and terminating plasma capricolum. Experimental with TAA or TAG. Each ORF posses- proof for a similar situation in spiro- ses, six to ten nucleotides upstream plasmas comes from the study of the to the initiation codon, a large Shine- genome of spiroplasma virus SpV4 Dalgarno ribosome binding site, com- (32). The double-stranded replicative plementary to the 3'OH end of spiro- form (RF) of this single-stranded cir- plasma1 16 S ribosomal RNA (rRNA). cular DNA virus was cloned in E. Three promoters for initiation of coli. The cloned RF was shown to be transcription have been identified. infectious by transfection of the The conserved sequences of these spiroplasmas. Next, the full sequence promoters (-10 and 35regions) are of the RF was determined. Knowing very similar to the consensus se- the sequence of the SpV4 genome, quences recognized by the B. subtilis search for the open reading frame RNA polymerase functioning with (ORF) of the capsid protein and for the "general" Sigma factor u42or the other ORFs was undertaken. No E. coli enzyme, with u70. Northern ORF large enough to fit the 60,000 d blot analysis of the viral mRNAs, S1 capsid protein could be found when all mapping, and insertion of the restric- three termination codons were taken tion fragments containing the promot- into account. Only when UAA and ers into plasmids for promoter iden- UAG, but not UGA, were used did tification have shown the three pro- the capsid protein ORF (ORF1) be- moters to be functional. Finally an in- come evident. In addition, the amino verted repeat sequence leading to a acid sequence of the N terminal end typical rho factor independent ter- of the capsid protein was found identi- minator hairpin on the mRNA, has cal to the amino sequence predicted been identified and shown to be func- from the base sequence of ORF1. This tional in transcription termination. established ORFl as the gene for the It was indicated above that the capsid protein and showed that UGA spiralin gene is expressed in E. coli. Tenth ZOCV Conference

In these experiments it could be con- S. citri (subgroup I-1), S. kunkelii cluded that the spiralin gene was ex- (subgroup I-3), and S. phoeniceum pressed from its own promoter. The (subgroup 1-8) are all members of sequence of the spiralin gene has in- group I. S. melli$erum, or honeybee deed shown that there is a typical spiroplasma, is also in group I (sub- bacterial promoter seven nucleotides group 1-2). Other named spiroplasmas upstream to the AUG initiation codon are S. jloricola (group 111) which is and a typical rho factor independent found on flower surfaces; S. apis terminator 27 nucleotides down- (group IV), a honeybee pathogen also stream to the UAA termination frequently found on flowers; and S. codon. This means that the bacterial mirum (group V) which produces a RNA polymerase is able to recognise cataract in experimentally inoculated spiroplasmal promoters and that newborn mice. S. culicicola, S. these must therefore be similar to sabaudiense and S. taiwaniense have bacterial promoters. This is precisely rece~tlybeen obtained from mos- the case. Also, since the spiroplasmal quitoes. Many other insects have enzyme uses promoters of the bacte- yielded spiroplasmas (10). Of the 30 rial type, it is likely to have a struc- spiroplasma types in the 23 groups of ture similar to the bacterial enzyme. the classification, 20 are of insect ori- That is indeed so (20). Having a bacte- gin, three from ticks, four from plant rial structure, it is not surprising that surfaces and three are plant patho- the spiroplasmal enzyme is also able gens. S. phoeniceum, the third plant to recognize terminator hairpins of pathogenic spiroplasma, was only dis- the bacterial type. covered in 1983 (35). Spiroplasmas in- In conclusion, this is the first time volved in natural diseases of man and that regulatory sequences such as animals have not yet been found. Shine-Dalgarno sequences, promoters and terminators have been iden- VI-FUTURE PROSPECTS tified in the spiroplasmas and shown Extensive work in Syria and Iraq to be very similar to those existing in (6) has shown the usefulness of bacteria and especially in Gram posi- ELISA and the culture assay for the tive bacteria. detection of S. citri in citrus. How- V- S. CITRI AND THE ever, these techniques are not yet DISCOVERY OF MY OTHER sensitive enough to reliably detect the SPIROPLASMAS stubborn agent in symptomless trees. ELISA could be rendered more sensi- Today, 16 yr after the first culture tive by introducing an avidin-biotin of S. citri in 1970, more than 30 differ- step. Preliminary results show that ent spiroplasmas are known and prob- techniques based on DNA hybridiza- ably many more are still to be discov- tion might also have a future. DNA ered. Spiroplasmas are classified on probes are required for DNA hybridi- the basis of their serological relation- zation. In the case of S. citri these ships, the G + C content of their probes can be derived from total S. DNA, one and two dimensional citri DNA, cloned restriction frag- analysis of their proteins on polyac- ments of S. citri DNA, plasmid DNA rylamide gels, DNA-DNA hybridiza- or viral DNA. For instance, the gene tion, and biological properties. They for spiralin, a protein specific of S. fall into 23 serologically unrelated citri is available as a cloned restric- groups (38). Group I contains S. citri tion fragment (26) and can be used as and seven other spiroplasmas which a S. citri specific probe. On the con- share serological relatedness and trary, probes based on ribosomal DNA homology with S. citri or other DNA genes are able to detect any group I members. Interestingly, the spiroplasma species. Such wide- three plant pathogenic spiroplasmas: range, unspecific probes can also be Diseases Induced by Procaryotic Pathogens developed from SpVl and SpV3 viral and the culture assay and these tech- DNA since it has been shown that niques have greatly contributed to viral DNA sequences are integrated the identification of the leafhopper in the spiroplasma genome (16, 31). vectors of S. citri in the old and the Classical genetics of spiroplasmas new world. and mollicutes is almost a virgin field. Considering the diversity of the Pursuit of mollicute genetics has been spiroplasmas, the need for classifica- greatly set back by a paucity of mu- tion became obvious and was one of tants and the apparent difficulty of the first incentives that prompted obtaining in vivo recombination (con- study of the spiroplasma genome. jugation). Fortunately, techniques for Today, data on genome size, guanine introduction of new genetic informa- plus cytosine content of DNA, and tion into spiroplasma cells by trans- homology between spiroplasmal formation and transfection seem to DNAs have become indispensable as offer promising alternatives. The de- adjuncts to serological analysis. This velopment of plasmid or viral vectors molecular genetic approach to classifi- for transport of genes from spiro- cation has greatly benefited from the plasma to spiroplasma or between development of new or improved bacteria and spiroplasmas is now a techniques; these include, for exam- subject of intense research and will ple, restriction enzyme analysis of greatly contribute to an understand- DNA, protein mapping by two-di- ing of such basic problems as helicity, mensional polyacrylamide gel elec- motility and contractility of the trophoresis, and more recently, clon- spiroplasmas, their relationships with ing and sequencing of DNA. Several plants and insects, the mechanism of spiroplasmal DNAs have been cloned their pathogenicity, and probably and sequenced. This work has not many other problems yet to be discov- only increased our basic knowledge of ered. the spiroplasmas, but also offers ex- It is hoped that this knowledge citing prospects for improved detec- will eventually lead to an efficient tion of spiroplasmas by DNA probes. control of stubborn disease through Recent work on the molecular genetic manipulations of the spiro- biology of the spiroplasmas has also plasma, the host plant or the insect contributed to mollicute phylogeny. vector. As the smallest and simplest self-re- plicating procaryotes, their origin, VII-CONCLUSIONS genealogical relationships and place in the evolutionary scheme have been Fifteen years of research on S. the subject of many debates. Today, citri and other spiroplasmas has the mollicutes are seen as a coherent yielded proof for the mollicute etiol- phylogenetic group that arose by de- ogy of certain diseases of plants generative evolution from low G + C including citrus stubborn disease; Gram positive bacteria and, more has unveiled a new and unsuspected specifically, certain Clostridia (45). group of mollicutes, the spiroplasmas; This view has recently been has documented their worldwide dis- strengthened when it was shown that tribution and their close association DNA transcription and mRNA trans- with insects and arthropods. Spiro- lation in S. citri and S. mell~erumin- plasma ecology and their pathogeni- volve RNA polymerases, promotors, city for plants, insects and mammals terminators and ribosome binding has also been determined. S. citri has sites very similar to those in bacteria, been a model for the study of the yet even if certain features are charac- noncultured mycoplasmalike organ- teristic of the spiroplasmas and prob- ism (MLOs) of plants. The successful ably other mollicutes, such as the use culture of S. citri has led to improved of UGA as a tryptophan codon and not detection techniques such as ELISA a termination codon. 282 Tenth IOCV Conference

The IOCV conference in Japan in we still study viruses in relation to 1969 was the last such meeting where stubborn: the viruses that infect S. the stubborn agent was considered a citri. How ironical would it be if it virus. Today, we know that the turns out that these viruses are in- agent, S. citri, is one of the most volved in pathogenicity! evolved procaryotic organisms, but

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