Biologicals 38 (2010) 193–203

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Biologicals

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Review and phytoplasmas: Microbes associated with plant hosts

Gail E. Gasparich*

Department of Biological Sciences, Towson University, 8000 York Road, Towson, MD 21252, USA article info abstract

Article history: This review will focus on two distinct genera, and ‘Candidatus Phytoplasma,’ within the class Received 4 November 2009 (which also includes the genus Mycoplasma, a concern for animal-based cell culture). As Accepted 12 November 2009 members of the Mollicutes, both are cell wall-less microbes which have a characteristic small size (1–2 mM in diameter) and small genome size (530 Kb–2220 Kb). These two genera contain microbes Keywords: which have a dual host cycle in which they can replicate in their leafhopper or psyllid insect vectors as Spiroplasmas well as in the sieve tubes of their plant hosts. Major distinctions between the two genera are that most Phytoplasmas spiroplasmas are cultivable in nutrient rich media, possess a very characteristic helical morphology, and Phytopathogenic Plant host are motile, while the phytoplasmas remain recalcitrant to cultivation attempts to date and exhibit Microoganism a pleiomorphic or filamentous shape. This review article will provide a historical over view of their discovery, a brief review of taxonomical characteristics, diversity, host interactions (with a focus on plant hosts), phylogeny, and current detection and elimination techniques. Ó 2009 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

1. Overview and rationale host interactions (with a focus on plant hosts), phylogeny, and current detection and elimination techniques. Spiroplasmas and phytoplasmas belong to two genera of under- studied microbes whose presence in plants, from which extracts are 2. Historical perspective on discovery made to supplement the growth of animal cells in vitro,isofgreat interest to the pharmaceutical industry. The industry is moving away 2.1. Spiroplasmas from animal serum as a supplement because of the risk of animal virus contamination. Plant extracts seem to be an acceptable alter- The term spiroplasma was first used in 1973 [1] to describe the native, but microbial contamination remains a concern. uncultivated helical found to be associated with corn stunt This review will focus on two distinct genera, Spiroplasma and disease [2,3]. In France and California, similar microbes were ‘Candidatus Phytoplasma’, within the class Mollicutes (which determined to be the causative agent for citrus stubborn disease, includes the genus Mycoplasma, a concern for animal-based cell but originally described as mycoplasmalike organisms [4,5]. This culture). As members of the Mollicutes, both are cell wall-less organism was the first spiroplasma of plant origin to be cultivated microbes which have a characteristic small size (1–2 mM in diam- and Spiroplasma was elevated to the generic name for this group of eter) and small genome size (530 Kb–2220 Kb). These two genera microbes [6] and added to the Approved List of Bacterial Names in contain microbes which have a dual host cycle in which they can 1983 [7]. replicate in their leafhopper or psyllid insect vectors as well as in Several previously observed microorganisms were later deter- the sieve tubes of their plant hosts. Major distinctions between the mined to belong to the genus Spiroplasma. These included the two genera are that most spiroplasmas are cultivable in nutrient causative agent for the sex-ratio disease in Drosophila [8,9]; Spi- rich media, possess a very characteristic helical morphology, and roplasma mirum, a rabbit tick isolate, [10,11] originally thought to be are motile, while the phytoplasmas remain recalcitrant to cultiva- a virus due to its filterability; and Spiroplasma strain 277F, tion attempts to date and exhibit a pleiomorphic or filamentous cultivated in 1968, which was thought to be a spirochete due to its shape. This review article will provide a historical over view of their helical morphology [12]. The corn stunt agent, Spiroplasma kunkelii, discovery, a brief review of taxonomical characteristics, diversity, was finally cultivated in 1975 [13,14]. Within ten years of their discovery, T. B. Clark [15] showed that spiroplasmas were widely found associated with arthropod hosts. * Tel.: þ1 410 704 4515; fax: þ1 410 704 2405. While exploring potential reservoirs for these organisms, several E-mail address: [email protected] Spiroplasma strains were isolated from plant surfaces, including

1045-1056/$36.00 Ó 2009 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biologicals.2009.11.007 194 G.E. Gasparich / Biologicals 38 (2010) 193–203

flowers. The first floral spiroplasma, Spiroplasma floricola,was characterization of a novel species involves numerous phenotypic, described in 1981 [16]. genotypic, serological and biochemical tests [64]. Several hundred new Spiroplasma species have been identified and many have been fully described. Isolates have been made from 3.2. Phytoplasmas a wide range of arthropods (including crustaceans) and plant hosts from a wide geographic range. Several of these spiroplasmas have Phytoplasmas are cell-wall-less, non-helical, uncultivable been shown to use both plant phloem and insect host habitats [17], prokaryotes associated with diseases in more than a thousand plant however insects are the major reservoir for spiroplasmas [15,18] species [23,26,71–80]. The phytoplasmas are spread by leafhopper and plant surfaces are important in horizontal transmission among and planthopper insect vectors to host plants, requiring an ability insect hosts [19–21]. to adapt to both insect and plant habitats. During their transmission cycle, phytoplasmas cross the insect midgut lining, circulate and 2.2. Phytoplasmas reproduce in the hemolymph, and invade and multiply in insect tissues including salivary glands, where phytoplasmas are inte- Since the early 1900s, Yellows diseases have been described for grated into saliva and injected into the plant phloem during feeding many plant species [22], but were originally assumed to be caused [75,81]. by viruses because the disease agent was filterable, unable to be The inability to cultivate phytoplasmas has limited the use of cultivated, and symptomology was similar to those known to be traditional microbial taxonomic methods. Early taxonomic classi- caused by viruses. In 1967 Japanese scientists were able to observe fication of phytoplasmas was based on biological properties, such microbes in the phloem of plants with Yellows diseases that were as the symptoms induced in plants, the plant host range, and similar in morphology to mycoplasmas known to cause animal and identification of the insect vector(s) if known [82–85]. However, human diseases [23]. Due to the resemblance, these plant- problems with the use of these biological properties are that two or associated microbes were referred to as mycoplasmalike organisms more phytoplasmas can cause similar symptomology in the same (or MLOs). The term MLO was used until 1994 when it was replaced plant in different geographical regions and the same phytoplasma with the trivial name ‘‘phytoplasma’’ [24,25]. Although they remain can cause different diseases in different plant species. A molecular- uncultivable, phytoplasmas have been identified as the causative based approach was developed in the 1980s [86,87] and is now agent for a variety of diseases in over a thousand plant species [26]. used to detect various phytoplasmas associated with plants and Phytoplasma infection can, in some cases, lead to desirable insects and study their genetic interrelationships. ornamental flora as in the case of poinsettias, but in most cases they cause millions of dollars of economic loss globally every year in 4. Diversity and host interactions important crop plants (e.g. rice, corn, potato, soybean), fruit trees (e.g. peach, pear, cherry, apple, citrus, coconut), ornamental plants One commonality between all phytoplasmas and plant-infect- (e.g. hydrangeas, coneflowers), and other trees and bushes (e.g. ing Spiroplasma strains is that they have a dual host transmission elm, mulberry) [27–43]. cycle. They are acquired by insect vectors (leafhoppers or psyllids) that feed on infected plants. To be transmitted to a plant, the 3. Taxonomic characteristics mollicutes need to multiply in the insect midgut, cross the midgut lining, multiply in the hemolymph, and subsequently infect the 3.1. Spiroplasmas salivary glands where they mix with saliva and are injected into a plant as the insect feeds on the phloem. Such a cycle takes 15–20 Taxonomically, the genus Spiroplasma is characterized as days. a group of motile, helical, wall-less procaryotes with genomes ranging in size from 780 to 2220 kbp [44,45] that are associated 4.1. Spiroplasmas primarily with insects, and less frequently, with ticks, plants and crustaceans [46–48]. The helical forms are usually 100–200 nm in Spiroplasmas display a broad range of diversity based on host diameter and 3–5 mm in length. Colonies on solid media are microhabitats with some spiroplasmas exhibiting strict host and/or 0.1–4.0 mm and are frequently diffuse at the borders due to their geographic ranges, while others are generalists. They are able to motility. Fimbriae and pili on the cell surfaces of insect- and plant- invade a diverse range of arthropod gut lumen, and some species pathogenic spiroplasmas are believed to be involved in host-cell are able to cross the midgut cell lining to invade the arthropod attachment [49,50], but not locomotion. Spiroplasmas are faculta- hemolymph and subsequently other organs and tissues. Spi- tively anaerobic with doubling times of 0.7–36.7 h and temperature roplasmas are transmitted among insect hosts through defecation for growth ranges between 5 and 41 C depending upon species and/or regurgitation of fluids on plant surfaces, or are introduced [51]. They are chemoorganotrophic and ferment glucose; have into the phloem by plant-sucking insects. Spiroplasmas also have variable ability to hydrolyze arginine; and cannot hydrolyze urea. been found in association with ticks, plants and higher order Originally, the species concept in spiroplasmas was based on invertebrates. Although, most spiroplasmas appear to be DNA/DNA reassociation [52–56]. However, DNA/DNA reassociation commensals, there are documented cases of mutualism or patho- proved difficult to standardize [57–61], resulting in the use of genicity associated with spiroplasma infections. serology for defining species [48,62–64]. In this classification scheme, ‘‘groups’’ are putative species and defined as organisms 4.1.1. Spiroplasmas and insect hosts with negligible DNA/DNA homology with other groups, but Spiroplasmas are most often associated with insects [15], moderate to high levels of homology within the group. Thirty-four including Coleoptera, Diptera, Hemiptera, Homoptera, Hymenop- groups were defined in 1998 [48]. Four additional groups (XXXV- tera, Lepidoptera, and Odonata. Hackett et al. [88] surveyed a wide XXXVIII) were recently proposed [65] and more are anticipated variety of insect orders and was able to obtain spiroplasma isolates [66]. Subgroups have been defined as groups of spiroplasma strains from 6 orders and 14 insect families. The six main orders of insects with an intermediate level of DNA/DNA homology [67]. Subgroups serving as Spiroplasma spp. hosts include: 1) Hymenoptera (from have been described for group I [62,68], group VIII [69], and group honeybees, bees, and vespid wasps); 2) Coleoptera (from a variety XVI [70] with 9, 3 and 3 subgroups, respectively. Complete of beetles including the green June beetle, soldier beetle, cucumber G.E. Gasparich / Biologicals 38 (2010) 193–203 195 beetle, Colorado Potato beetle, scarabaeid beetle, firefly beetles); 3) different energy sources is required. The impact of the glucose and Diptera (from syrphid flies, mosquitoes, tabanids, and fruit flies); 4) fructose utilization on plant pathogenicity was explored in S. citri. Lepidoptera (from butterflies and moths in both larval and adult Inactivation of the fructose operon was found to reduce pathoge- stages); 5) Homoptera (from leafhoppers); and, 6) Hemiptera (from nicity [118,119]. A transposon-mediated mutant of S. citri, desig- the green leaf bug) [17,89,90]. nated GMT553, was able to reproduce in periwinkle plants Most Spiroplasma–host interactions appear to be commensal equivalent to the wild type when introduced by leafhoppers, but without any apparent adverse effect on the host. Spiroplasma infected plants did not produce any symptoms [118,120–122]. pathogenicity in an insect host is generally linked to its ability to Fructose use by spiroplasmas could impair sucrose loading into the invade the hemolymph and potentially other host tissues beyond sieve tubes and result in carbohydrate accumulation in source the midgut. The use of immunofluorescence confocal laser leaves and depletion in sink tissues [115]. However, the inability to scanning microscopy has revealed the presence of S. kunkelii in the use glucose, as examined through the use of a glucose phospho- midgut, filter chamber, Malpighian tubules, hindgut, fat tissues, transferase permease mutant (GII3-glc1), resulted in severe hemocytes, muscle, trachea and salivary glands of leafhopper hosts symptoms in the infected plant equivalent to those observed for the [91]. There is also some evidence for mutualistic interactions. For wild-type strain [123]. Glucose accumulation induces stunting and example, the infection of Dalbulus maidis by S. kunkelii enhances repression of photosynthesis genes in Arabidopsis thaliana which the leafhopper’s ability to survive cold winter periods without its are the symptoms observed in periwinkle plants infected by wild- plant host available [92]. type S. citri [123]. Once thought to be a genetic mutation, the sex-ratio trait in Transmission of S. citri by leafhopper vectors must involve Drosophila was shown by Poulson and Sakaguchi [8] to be induced adherence to and invasion of insect host cells. S. citri surface protein by Spiroplasma poulsonii [93]. These sex-ratio organisms are P89 was shown to mediate adhesion of spiroplasmas to cells of the transmitted transovarially and preferentially kill male progeny. vector Circulifer tenellus and was designated SARP1 (spiroplasma A number of other spiroplasmas in a variety of insect hosts, adhesion-related protein 1) [124,125]. Electron microscopic studies including beetles and butterflies, have been identified that also of leafhopper midguts [50] have demonstrated the attachment of cause sex-ratio distortions [94–102]. S. kunkelii to the cell membrane by a similar tip structure. This S. kunkelii homolog of SARP1 [126], designated S. citri adhesion- 4.1.2. Tick and spider hosts related protein (ScARP), was not detected in DNA from non-trans- Three Spiroplasma species have been isolated from ticks. Two of missible strains [127]. these, S. mirum and S. sp. 277F, are from the rabbit tick Haema- physalis leporispalustris [10]. The third species, Spiroplasma ixodetis 4.1.4. Spiroplasmas and higher order invertebrates was isolated from Ixodes pacificus ticks [103]. There is no evidence Recent Spiroplasma isolates from higher order invertebrates are that any of these spiroplasmas are transmitted to vertebrate hosts beginning to change our understanding of the host range. Spi- of the ticks. A broad survey for in 16 spider families roplasma spp. have been isolated in both freshwater and saltwater using 16S rRNA sequence analysis revealed that 6 families crustaceans. Spiroplasma penaei (strain SHRIMP) was isolated from contained spiroplasmas, including Agelenidae, Araneidae, Gna- the hemolymph of Pacific White shrimp (Penaeus vannamei) after phosidae, Linyphiidae, Lycosidae and Tetragnathidae [104]. high mortalities were observed in an aquaculture pond in Columbia, South America [128]. Chinese Mitten Crab (Eriocheir 4.1.3. Spiroplasmas and plant hosts sinensis) reared in aquaculture ponds in China became infected Fulfillment of Koch’s postulates has been reported for three with tremor disease. The causative agent was determined to be phytopathogenic spiroplasmas: Spiroplasma citri in citrus [4,105]; a Spiroplasma [129]. The same organism was recovered from red S. kunkelii in corn [14,106,107]; and Spiroplasma phoeniceum in aster swamp crayfish (Procambarus clarkia) co-reared with the infected [68]. Each of these spiroplasmas is maintained in both plant phloem Chinese Mitten Crab [130], as well as from shrimp [131]. A working and insect host habitats. No pathological effects are associated with hypothesis is that the infected aquaculture systems were exposed multiplication of these spiroplasmas in their usual insect host and to Spiroplasma-infected insects leading to transmission of the an infected host can serve as a transmission vector throughout its organisms to the new crustacean hosts. These recent isolations life. Infected plants can exhibit a range of symptoms including expand the potential Spiroplasma-host range and may influence stunting, leaf yellowing, sterility, fruit size reduction and deforma- future biodiversity studies to include aquatic environments. tions, flower malformations, and short internodes. Many of these symptoms are similar to those observed in phytoplasma-infected 4.1.5. Spiroplasmas and vertebrate pathogenicity plants. S. citri has a broad host range causing citrus stubborn disease Thus far, pathogenic spiroplasmas have very limited ability to in citrus [5,108,109], brittle root in horseradish [110,111], a disease in infect vertebrate cells or immunocompromised vertebrates. Many periwinkle [112,113], and carrot purple leaf disease [114]. S. citri has Spiroplasma isolates from insect and plant habitats have the ability been the focus of recent work to elucidate the molecular mecha- to grow at 37 C allowing for the possibility of replication within nisms responsible for spiroplasma pathogenicity in the plant host a vertebrate host. Only one characterized Spiroplasma sp., S. mirum, [115].TwoS. citri genes have been identified that appear to be has been shown to experimentally infect immunocompromised involved in insect transmissibility, the Sc76 (putative ABC trans- vertebrates (chick embryos, suckling mice and rats) [10,132,133]. porter) gene and the spiralin gene. Inactivation of Sc76 results in Recent reports have also suggested that a Spiroplasma sp. may be a 30-fold decrease in the number of spiroplasmas in the salivary associated with transmissible spongiform encephalopathies of glands and subsequently delivered by the insect during feeding humans and animals [134,135]; however, studies have failed to [116]. Spiralin is the major surface antigen and most abundant amplify Spiroplasma or other bacterial 16S rDNA from scrapie membrane protein found in S. citri. Although not essential for infected hamsters [136]. pathogenicity, a spiralin-deficient mutant showed a 100-fold decrease in transmission from the insect to the host plant [117]. 4.1.6. Spiroplasma host specificity and diversity The plant-pathogenic spiroplasmas grow on glucose and fruc- Attempts have been made to estimate taxonomic diversity of tose in the plant sieve tubes and then switch to trehalose in the spiroplasmas based upon the observation that one new Spiroplasma leafhopper hemolymph. Therefore, rapid adaptation between sp. had been found for every 10 species of insects examined in 196 G.E. Gasparich / Biologicals 38 (2010) 193–203 a large survey of insect families [88]. Since there are an estimated several Spiroplasma groups have been isolated only from flowers, 10 million insect species this quickly becomes an incredibly spe- and strains of several other spiroplasmas have been isolated from ciose microbial genera. However, more recent isolations have both insects and flowers, which suggests that these flower isolates indicated that the rate of isolation of novel isolates will decrease as are deposited by visiting arthropods [15,88]. more and more Spiroplasmas spp. are identified and characterized. Results from collection sites on a North American latitudinal 4.2. Phytoplasma distribution gradient from Nova Scotia to the Tropics suggest that the percentage carriage in insects varies geographically, with a prob- Phytoplasmas are phloem-limited plant pathogens that are able peak of diversity in the Tropics [137]. This general trend in spread by sap-sucking insect vectors belonging to the families Cica- diversity has already been observed with spiroplasmas associated dellidea (leafhoppers) and Fulgoridea (planthoppers) [81,154–157]. with the tabanid (Diptera: Tabanidae) flies. Insects feed on phloem tissues, where they acquire phytoplasmas The Tabanidae have been a rich source of Spiroplasma spp. and transmit them from plant to plant. Phytoplasmas may over- isolations primarily from horseflies and deerflies [138] and argu- winter in infected vectors, as well as in perennial plants that serve as ably the most well studied of the Spiroplasma insect hosts. In reservoirs for phytoplasmas. Additionally, phytoplasmas can be a typical sampling from the tropics (Costa Rican highlands), 12 spread by vegetative propagation through cuttings, storage tubers, isolations were made from 13 attempts from a single species of rhizomes, or bulbs [87]. Phytoplasmas that cause many ornamental horse fly [139,140]. Initial serological analyses indicated that these and fruit tree diseases are spread by vegetative propagation and isolates did not belong to the same group. Additional diversity also grafting. comes from the observation that a single tabanid fly host had been Phytoplasmas have been associated with diseases in over infected with two or more Spiroplasma species [141]. a thousand plant species belonging to 98 families and with Unfortunately, there are no broad studies that have experi- numerous homopterous insect vectors, primarily belonging to the mentally tested or environmentally surveyed host specificity for family Cicadellidea (leafhoppers). Geographically, phytoplasmas spiroplasmas; there are only a few discrete cases. Some examples have a global distribution with reports from at least 85 nations [26]. below demonstrate the range of host specificity. Spiroplasma lep- The natural host ranges of phytoplasmas in insect vectors and tinotarsae has very strict host specificity for the Colorado potato plants vary with the phytoplasma strain [26,81,155]. Experimen- beetle [17,142,143]. In contrast, Spiroplasma diabroticae was isolated tally, some phytoplasmas can be transmitted by polyphagous from a corn rootworm beetle host but was able to experimentally vectors to a wide range of host plants. The range of plant species infect the Colorado potato beetle [17]. In general, tabanid-associ- that can be infected by a given phytoplasma in nature is deter- ated spiroplasmas are believed to have a broader host range due to mined by the number of insect vector species capable of transmission on plant surfaces that are frequented by other Spi- transmitting the phytoplasma and by the feeding behaviors of roplasma-harboring insects. For example, Spiroplasma gladiatoris these vectors [158]. Mixed phytoplasma infections in a single plant was isolated from 11 Tabanus species and often occurred in mixed are found in nature and can be experimentally generated infections with other spiroplasmas [138,144]. Common feeding [42,159–164]. Co-infection also provides opportunities for the sites would allow for efficient horizontal transmission of spi- exchange of genetic information which may also contribute to the roplasmas among hosts and explain why some insect hosts might evolution of new strains. have multiple spiroplasmas [17,145]. To a large extent, plant host Recent studies indicate that phytoplasmas are more diverse specificity is dictated by the insect vector. An insect with a very than originally thought and are not distributed uniformly over all narrow host range will limit infection to its feeding range of plants, continents [165,166]. Many seem to be restricted to one continent whereas a generalist may visit many plant surfaces involved in or a specific geographical region. For example, phytoplasmas in the Spiroplasma transmission. ash yellows group are found on the American continent or western hemisphere, whereas the rice yellow dwarf group is restricted to 4.1.7. Spiroplasma distribution Southeast Asia [71]. Geographical isolation of some phytoplasmas Although spiroplasmas have been identified from hosts in seems to be correlated with the distribution of their host plants and Africa, Asia, Australia, Europe, South America and North America, the insect vectors that are native in the particular region. Aster studies suggest biodiversity may be greatest in warm climates [65]. yellows and X-disease phytoplasmas are examples of two of the Spiroplasma spp. distribution will likely be limited by host bioge- most diverse phytoplasmas known to date [165,167]. Phytoplasmas ography in which some spiroplasmas have discrete geographic in the aster yellows groups can be transmitted by over 30 insect distributions, while others are more broadly distributed [146].In vectors into more than 200 plant species from 45 families distrib- addition to host range, other factors such as host specificity and uted globally, whereas members of the X-disease group can be host overwintering strategies may contribute to Spiroplasma ranges transmitted by 14 insect vectors into more than 60 plant species [65]. from 13 families distributed on three continents [71]. Spiroplasmas have been isolated from the guts of tabanids A major gap in knowledge of phytoplasma ecology is the lack of (Diptera:Tabanidae) worldwide [66,138,141,147–152]. Based on the information about the insect vectors which plays a major role in large number of tabanid-associated Spiroplasma isolations in North determining the phytoplasma ecological niche(s) and for under- America, geographical ranges for some species have begun to standing the epidemiology of many phytoplasma-associated emerge. Some species, like Spiroplasma chrysopicola, have a wide diseases. Most phytoplasmas have been found as a direct result of range of distribution, while others, like S. gladiatoris, have a more the pathology they cause, but an extensive survey of ‘‘healthy’’ limited geographic distribution. Additional studies surveying plants may provide more detailed information about the distribu- tabanid flies in Australia, Costa Rica, and Ecuador resulted in 4 new tion of phytoplasmas which do not cause obvious pathology. serogroups from Australia, 15 new serogroups from Costa Rica and 5 new serogroups from Ecuador. In this survey, Costa Rica and U.S. 4.2.1. Phytoplasma disease samples had four serogroups in common, however, serogroups Plants infected by phytoplasmas exhibit an array of symptoms from Australia or Ecuador were confined to those regions [151]. suggestive of imbalances in plant hormones and/or growth regu- Flowers and other plant surfaces represent a major site where lators [26,76,87,168] including virescence (green flowers), phyllody spiroplasmas are transmitted to insects [19,20,153]. Members of (floral parts change to leafy structures), sterility, proliferation of G.E. Gasparich / Biologicals 38 (2010) 193–203 197 shoots (witches’-broom appearance), elongation of internodes, clade contains three serogroup VIII strains all isolated from tabanid stunting, leaf or shoot discoloration, and leaf curling. The etiology flies. The mirum clade contains the tick Spiroplasma S. mirum, and is of phytoplasma-induced disease is made more difficult by the fact always basal to the other Citri-Chrysopicola-Mirum component that similar symptoms can be induced by different types of phy- clades. toplasmas, whereas different types of symptoms can be induced by The majority of the isolates examined fall into the Apis clade, closely related phytoplasmas [169,170]. In general, phytoplasma which is comprised of 14 composite clades [179]. The Apis clade infections are detrimental to plants, although some plant species resulted from a major split that occurred after the divergence appear to be tolerant or resistant to phytoplasmal infection. that led to the Citri-Chrysopicola-Mirum clade. It contains two Originally, it was thought that the disease symptoms induced by basal clades (the lampyridicola-leptinotarsae and sabaudiense- phytoplasma infection were a direct result of depleted nutrients alleghenense-TIUS-1 clades). The S. lampyridicola clade consists from the plant, but this has been shown to not be the case for many of two members which are both beetle specialists associated diseases. Several published differential gene expression studies with the Colorado Potato Beetle and firefly beetles, respectively indicated that virescence- or phyllody-inducing phytoplasmas alter and appear to be transmitted on leaf surfaces. The sabaudiense- plant hormone levels which in turn caused the observed symptoms alleghenense-TIUS-1 clade is difficult to interpret from an [168,171,172]. Recently specific phytoplasma genes have been ecological perspective as the three strains in this clade were shown to produce proteins that are excreted by the bacteria (e.g. isolated from a mosquito, a tiphiid wasp, and a scorpionfly. The SAP 11 from AY-WB and TENGU protein from OY phytoplasmas) remaining 12 Apis clades contain 21 closely related Spiroplasma [173,174] and accumulate in plant tissues located some distance strains. Several of the clades contain a single isolate (taiwanense, from phytoplasma-infected cells, but which directly impact plant clarkii, culicicola, diminutum, and monobiae). The apis-mon- gene expression. A. thaliana genetically engineered with SAP 11 tanense clade, the litorale-turnicum-corruscae clade, and the produces a phenotype similar to a plant with a phytoplasma helicoides-gladiatoris-BARC1901 clade all contain strains isolated infection [173]. from tabanid flies. The chinense-velocicrescens clade and the BIUS-1-W115 clade contain strains isolated from flower surfaces 5. Phylogeny with no insect host yet identified. Clade CB-1-Ar 1357 is comprised of three strains isolated from flower surfaces, can- Phylogenetic analyses [175–179] indicate that the Mollicutes tharid beetles and mosquitoes. The floricola-diabroticae clade arose monophyletically from a gram-positive, low G þ C content contained strains that were either isolated from flower surfaces Clostridial lineage of the Eubacteria [175]. Studies involving or from beetles that frequent flowers [151]. biochemical pathways [180], metabolism [181,182], and rRNA gene The Ixodetis clade consists of a single species, S. ixodetis, which homologies [183] suggest that Mollicute evolution proceded first always appears at the root of the Spiroplasma clade [103]. S. ixodetis with a divergence of the Acholeplasmas and Anaeroplasmas. The appears phenotypically to be the transitional species prior to Spi- Spiroplasma branch [183] and ‘Ca. Phytoplasma’ branch split [177] roplasma speciation with some members exhibiting a classic hel- from the Acholeplasma branch and the Mycoplasma and Ureaplasma icity and others being more filamentous. The genome is 2220 kbp in branches appear to have evolved from the Spiroplasma branch after size which is the largest for the Spiroplasma genus. The evolu- further genome reduction events. tionary distance of S. ixodetis from other Spiroplasma spp. is significant [179]. 5.1. Spiroplasma phylogeny 5.2. Phytoplasma phylogeny The general placement of the genus Spiroplasma in relation to other Eubacteria is well established from several studies using 16S Phytoplasma serological and genomic groups were based origi- rRNA gene sequences with multiple phylogenetic algorithms [179]. nally on reactions to specific antibodies and DNA probes The order Entomoplasmotales was shown to contain four major [26,74,158,161,186–192]. Recent studies on evolution of and clades: the Mycoides-Entomoplasmataceae clade, the Apis clade, phylogenetic relationships within the phytoplasma clade have been the Citri-Chrysopicola-Mirum clade, and the Ixodetis clade [179]. based on phylogenetic analyses of conserved gene sequences, The Mycoides-Enomoplasmataceae clade consists of the non- especially the 16S rRNA gene sequence [162,169,193–200] and helical species of the mycoides group [184] as well as the Ento- multilocus sequence analysis [201]. moplasma and Mesoplasma species. The remaining three clades Recent genome level analyses have revealed some aspects of contain all of the Spiroplasma species. The Apis clade, a sister clade genome organization and led to the development of a theory about to the Mycoides-Entomoplasmataceae clade, contains a large the evolutionary origin of phytoplasmas. Jomantiene [202,203] number of species from diverse insect hosts. The other two Spi- proposed that repeated and targeted mobile element attacks roplasma clades are the monospecific Ixodetis clade (group VI) and formed unique regions (sequence-variable mosaics) in the nascent the Citri-Chrysopicola-Mirum clade. phytoplasma genome, carrying in genes that conveyed properties The Citri-Chrysopicola-Mirum clade is composed of three critical for a new ecological adaptation. The mobile elements component clades: (i) the Citri-Poulsonii clade, (ii) the chrys- forming the sequence-variable mosaics were identified as opicola-syrphidicola-TAAS-1 clade and (iii) the mirum clade. The prophages; thus, the sequence-variable mosaics in phytoplasma Citri-Poulsonii clade contains nine serogroup I spiroplasmas and genomes were recognized as prophage-based pathogenicity the serogroup II species S. poulsonii [128,179]. Members of this islands. Formation of these genomic structures was proposed to clade represent diverse host associations, including honeybees, have triggered evolution of the phytoplasma clade [204]. A study ticks, leafhoppers, plant/flower surfaces, Drosophila and shrimp showing decay of folate biosynthesis genes in phytoplasmas [46,93,128] and is of special interest due to the pathogenicity of suggests ongoing host adaptation to parasitism of their hosts [205]. some members. Diseases include citrus stubborn (S. citri) [5], honey Completion of genome sequencing of four phytoplasma strains bee spiroplasmosis (Spiroplasma melliferum) [185], corn stunt [206–209] should also provide a better understanding of genome (S. kunkelii) [49], periwinkle disease (S. phoeniceum) [68], sex-ratio organization and evolution. disease in Drosophila (S. poulsonii) [93], and the shrimp aquaculture Phylogenetic studies based on 16S rRNA and other house- disease (S. penaei) [128]. The chrysopicola-syrphidicola-TAAS-1 keeping genes have readily placed phytoplasmas in the class of 198 G.E. Gasparich / Biologicals 38 (2010) 193–203

Mollicutes [71,166,177,198,209–212]. The taxonomic use of the sensitive to erythromycin and tetracycline. Elimination strategies ‘‘Candidatus’’ designation for use with uncultivable microbes [213] tend to focus on control of the insect vectors and removal of was proposed, leading to the genus-level provisional taxon ‘Can- infected plants to prevent the spread of infection. Pesticide appli- didatus Phytoplasma.’ [214]. Guidelines for naming new taxa within cation for control of the leafhopper hosts has generally not been the genus ‘Ca. Phytoplasma’ included that a designated species effective to control the spread of the spiroplasmas. Treatment of description should refer to a single, unique 16S rRNA gene sequence infected plants with tetracycline has been tried and leads to of greater than 1200 bp and share less than 97.5% sequence simi- a cessation of disease symptoms and decreased transmission by larity to that of any previously described ‘Ca. Phytoplasma’ species leafhopper hosts [226]. unless the phytoplasma under consideration clearly represented an ecologically separated population [214]. Additional information 6.3. Phytoplasma detection including plant and insect host information, if known, should be included in the species description. Early methods of diagnosis were dependent on characterization In parallel to ‘Candidatus’ species assignment, phytoplasmas are of plant symptoms and observation of phytoplasmas in ultrathin classified into groups and subgroups based on their genetic relat- sections of diseased plants [227]. The development of molecular edness. Two major phytoplasma group-classification schemes probes and PCR primers for phytoplasmas (universal and group- based on 16S rRNA gene sequences have been developed. One specific based on 16S rRNA gene, 16S–23S intergenic spacer region, method is based on phylogenetic analysis of 16S rRNA gene rp gene and elongation factor EF-Tu) has increased diagnostic sequences [29,166,177,210,215] and has proposed 28 ‘Ca. Phyto- ability [83,186,195,213,228–237]. More recent development of 16S plasma’ spp. groups. However, this phylogeny-based scheme does rRNA-based diagnostic tools include the use of an oligonucleotide not allow for subgroup-level classification. The second scheme is microarray that can detect all phytoplasmas that were tested [238] based on restriction fragment length polymorphism (RFLP) analysis and quantitative real-time PCR for the detection and quantification of PCR-amplified 16S rRNA gene fragments [216,217]. The PCR-RFLP of phytoplasmas [239–242]. profiling approach is based on a defined set of 17 restriction enzymes [162,200]. PCR-RFLP analysis, the current method of 6.4. Phytoplasma disease control strategies choice, identifies phytoplasma groups consistent with the 16S rRNA gene sequence-based phylogeny and provides better differentiation It is very difficult to control phytoplasma diseases. Use of of phytoplasma subgroups. This method has defined 29 groups and disease-resistant host plants (when available) and pesticide appli- more than 40 subgroups [71,162,177,218–220]. This number has cation to contol insect vectors have been used with limited success expanded to 30 groups and over 100 subgroups through the use of [243–245]. Applications of tetracycline into infected plants has had computer-simulated RFLP analysis [199–223]. iPhyclassifier, an limited success, is too costly and may lead to the development of internet-based phytoplasma research tool, provides RFLP virtual gel resistance, not to mention potential entry into the human food analysis, taxonomic assignment, and group and subgroup classifi- supply. Genetically engineered host plants are being developed to cation [223]. deter insect vector feeding or interfere with phytoplasma replica- tion [246,247]. 6. Detection and elimination strategies 7. Concluding remarks Early and rapid detection is the best strategy to avoid large batch contamination problems. Rapid diagnostics have been developed to The use of plant extracts to supplement the growth of animal be effective for identification of spiroplasmas and phytoplasmas. It cell cultures in vitro appears to be a safer alternative to the use of is important to note that spiroplasmas and phystoplasmas share animal serum, which may contain viruses, prions and/or myco- some insect hosts and there have been reports indicating co- plasmas leading to contaminated end products. However, the infection of the same plant host with both microbes [76,157]. presence of filterable plant-pathogenic phytoplasmas and spi- roplasmas raises some concerns. Many of these concerns can be 6.1. Spiroplasma detection addressed by an understanding of distribution, diversity, host plants, insect vectors, transmission cycles, issues of co-infection Initial detection strategies involved cultivation followed by and diseases symptoms. Rapid molecular diagnostic screening is microscopy and/or serological analyses, but the development of available and use of greenhouse or growth chamber generated PCR primers has decreased detection times significantly [224]. Two materials should reduce the incidence of infected plants and allow Spiroplasma proteins, spiralin and fibrillin, have been used to for the benefits of plant-derived extracts to be fully exploited. develop Spiroplasma-specific PCR primers [225, Gasparich, unpublished]. Additionally, several PCR-based commercial detec- Acknowledgements tion kits have been developed to detect mycoplasma contamination in cell culture, including LookOut Mycoplasma PCR Detection Kit I would like to thank all my colleagues for assistance in the (Sigma–Aldrich, St. Louis, MO), MycoSolutions Detection Kit preparation of this review article with special thanks to Ing-Ming (AppliChem, LLC, Boca Raton, FL), EZ-PCR Mycoplasma Test Kit (Life Lee. Technologies, Delhi, India) and MicroSEQ Mycoplasma Detection Assay (Applied Biosystems, Carlsbad, CA). Although primarily References designed for Mycoplasma species detection, these kits utilize proprietary PCR primers that also detect Spiroplasma species. [1] Davis RE, Worley JF. Spiroplasma: motile helical microorganism associated with corn stunt disease. Phytopathology 1973;63:403–8. [2] Davis RE, Whitcomb RF, Chen TA, Granados RR. Current status of the aeti- 6.2. Spiroplasma disease control strategies ology of corn stunt disease. In: Elliott K, Birch Jr J, editors. Pathogenic mycoplasmas. North-Holland, Amsterdam: Elsevier-Excerpta Medica; 1972. Despite their lack of a cell wall, spiroplasmas can survive in the p. 205–14. [3] Davis RE, Worley JF, Whitcomb RF, Ishijima T, Steere RL. Helical filaments lyophilized state for decades. They also are resistant to several produced by a mycoplasma-like organism associated with corn stunt disease. antibiotics, including penicillin, ampicillin and rifampin. They are Science 1972;176:521–3. G.E. Gasparich / Biologicals 38 (2010) 193–203 199

[4] Calavan EC, Bove´ JM. Molecular and cellular biology of spiroplasmas. In: [36] Bonnet F, Saillard C, Kollar A, Seemuller E, Dosba F, Bove´ JM. Molecular Whitcomb RF, Tully JE, editors. The mycoplasmas, vol. 5. New York: Academic probes for the apple proliferation MLO. Zentralbl Bakteriol Suppl Press; 1989. p. 425–85. 1990;20:908–9. [5] Saglio P, L’hospital M, Lafle`che D, Dupont G, Bove´ JM, Tully JG, et al. Spi- [37] Poggi-Pollini C, Giunchedi L, Seemuller E, Filippini G, Vindimian G. Etiological roplasma citri gen. and sp. n.: a mycoplasma-like organism associated with studies of apple rubbery wood disease. Acta Hortic 1995;385:503–5. ‘‘stubborn’’ disease of citrus. Int J Syst Bacteriol 1973;23:191–204. [38] Zreik L, Carle P, Bove´ JM, Garnier M. Characterization of the mycoplasmalike [6] Skripal IG. On improvement in the systematics of the class Mollicutes and the organism associated with witches’-broom disease of lime and proposition of establishment in the order Mycoplasmatales of a new family Spi- a Candidatus taxon for the organism, ‘‘ Candidatus Phytoplasma aurantifolia’’ roplasmataceae fam. nova. Mikrobiologii Zhurnal (Kiev) 1974;36:462–7. Int J Syst Bacteriol 1995;45:449–53. [7] Skripal IG. Revival of the name Spiroplasmataceae fam. nova., nom. rev., [39] Harrison NA, Richardson PA. Comparative investigation of MLOs associated omitted from the 1980 approved lists of bacterial names. Int J Syst Bacteriol with Caribbean and African coconut lethal decline diseases by DNA hybrid- 1983;33:408. ization and PCR assays. Plant Dis 1994;78:507–11. [8] Poulson DF, Sakaguchi B. Nature of ‘‘sex-ratio’’ agent in Drosophila. Science [40] Sawayanagi T, Horikoshi N, Kanehira T, Shinohara M, Bertaccini A, Cousin M- 1961;133:1489–90. T, et al. ‘Candidatus Phytoplasma japonicum’, a new phytoplasma taxon [9] Sakagouchi B, Poulson DF. Distribution of ‘‘sex-ratio’’ agent in tissues of associated with Japanese Hydrangea phyllody. Int J Syst Bacteriol Drosophila willistoni. Genetics 1961;46:1665–76. 1999;49:1275–85. [10] Tully JG, Whitcomb RF, Rose DL, Bove´ JM. Spiroplasma mirum, a new species [41] Stanosz GR, Heimann MF, Lee I-M. Purple coneflower is a host of the aster from the rabbit tick (Haemaphysalis leporispalustris). Int J Syst Bacteriol yellows phytoplasma. Plant Dis 1997;81:424. 1982;32:92–100. [42] Lee I-M, Bertaccini A, Vibio M, Gundersen DE, Davis RE, Mittempergher L, [11] Clark HF. Suckling mouse cataract agent. J Infect Dis 1964;114:476–87. et al. Detection and characterization of phytoplasmas associated with disease [12] Pickens EG, Gerloff RK, Burgdorfer W. Spirochete from the rabbit tick, Hae- in Ulmus and Rubus in northern and central Italy. Phytopathol Mediterr maphysalis leporispalustris (Packard). J Bacteriol 1968;95:291–9. 1995;34:174–83. [13] Liao CH, Chen TA. Culture of corn stunt Spiroplasma in a simple medium. [43] Marcone C, Ragozzino A, Seemuller E. Identification and characterization of Phytopathology 1977;67:802–7. the phytoplasma associated with elm yellows in southern Italy and its [14] Williamson DL, Whitcomb RF. Plant Mycoplasmas: a cultivable Spiroplasma relatedness to other phytoplasmas of the elm yellows group. Eur J For Pathol causes corn stunt disease. Science 1975;188:1018–20. 1997;27:45–54. [15] Clark TB. Spiroplasmas: diversity of arthropod reservoirs and host-parasite [44] Carle P, Laigret F, Tully JG, Bove´ JM. Heterogeneity of genome sizes within the relationships. Science 1982;217:57–9. genus Spiroplasma. Int J Syst Bacteriol 1995;45:178–81. [16] Davis RE, Lee IM, Worley JF. Spiroplasma floricola, a new species isolated from [45] Williamson DL, Adams JR, Whitcomb RF, Tully JG, Carle P, Konai M, et al. surfaces of flowers of the tulip tree, Liriodendron tulipifera L. Int J Syst Bac- Spiroplasma platyhelix sp. nov., a new mollicute with unusual morphology teriol 1981;31:456–64. and genome size from the dragonfly Pachydiplax longipennis. Int J Bacteriol [17] Hackett KJ, Clark TB. Ecology of spiroplasmas. In: Whitcomb RF, Tully JG, 1997;47:763–6. editors. The mycoplasmas, vol. 5. New York: Academic Press; 1989. p. [46] Williamson DL, Tully JG, Whitcomb RF. The genus Spiroplasma. In: 113–200. Whitcomb RF, Tully JG, editors. The mycoplasmas, vol. 5. San Diego: [18] Clark TB. Spiroplasma sp., a new pathogen in honey bees. J Invertebr Pathol Academic Press; 1989. p. 71–111. 1977;29:112–3. [47] Tully JG, Whitcomb RF. The genus Spiroplasma. In: Starr MP, Stolp H, [19] Davis RE. Spiroplasma associated with flowers of the tulip tree (Liriodendron Truper HG, Balows A, Schlegel HG, editors. The prokaryotes. New York: tulipifera L.). Can J Microbiol 1978;24:954–9. Springer; 1990. p. 1960–80. [20] McCoy RE, Williams DS, Thomas DL. Isolation of mycoplasmas from flowers. [48] Williamson DL, Whitcomb RF, Tully JG, Gasparich GE, Rose DL, Carle P, et al. In: Proceedings of the US-ROC plant mycoplasma seminar. Taipei, Taiwan: Revised group classification of the genus Spiroplasma. Int J Syst Bacteriol National Science Council; 1979. p. 75–81. 1998;48:1–12. [21] Clark TB, Henegar RB, Rosen L, Hackett KJ, Whitcomb RF, Lowry JE, et al. New [49] O¨ zbek E, Miller SA, Meulia T, Hogenhout SA. Infection and replication sites Spiroplasmas from insects and flowers: isolation, ecology, and host associa- of Spiroplasma kunkelii (Class Mollicutes) in midgut and Malpighian tion. Isr J Med Sci 1987;23:687–90. tubules of the leafhopper Dalbulus maidis. J Invertebr Pathol 2003;82: [22] Kunkel LO. Studies on aster yellows. Am J Bot 1926;23:646–705. 167–75. [23] Doi YM, Teranaka M, Yora K, Asuyama H. Mycoplasma or PLT-group-like [50] Ammar E, Fulton D, Bai XD, Meulia T, Hogenhout SA. An attachment tip and microorganisms found in the phloem elements of plants infected with pili-like structures in insect- and plant-pathogenic spiroplasmas of the class mulberry dwarf, potato witches’ broom, aster yellows, or paulownia witches’ Mollicutes. Arch Microbiol 2004;81:97–105. broom. Ann Phytopathol Soc Jpn 1967;33:259–66. [51] Konai M, Clark EA, Camp M, Koeh AL, Whitcomb RF. Temperature ranges, [24] ICSBSTM. Minutes of the interim meetings, 1 and 2 August 1992, Ames, Iowa. growth optima, and growth rates of Spiroplasma (Spiroplasmataceae, class Int J Syst Bacteriol 1993;43:394–7. Mollicutes) species. Curr Microbiol 1996;32:314–9. [25] ICSBSTM. Minutes of the interim meetings. 12 and 18 July 1996, Orlando, [52] Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandler O, Krichevsky MI, Florida. Int J Syst Bacterol 1997;47:911–4. et al. Report of the ad hoc committee on reconciliation of approaches to [26] McCoy RE, Caudwell A, Chang CJ, Chen T-A, Chiykowski LN, Cousin NT, et al. bacterial systematics. Int J Syst Bacteriol 1987;37:463–4. Plant diseases associated with mycoplasmalike organisms. In: Whitcomb RF, [53] Johnson JL. Similarity analysis of DNAs. In: Gerhardt P, Murray RGE, Tully JG, editors. The mycoplasmas. New York: Academic Press; 1989. p. Wood WA, Krieg NR, editors. Methods in general and molecular bacteriology. 545–60. Washington: Amer Soc Microbiol; 1994. p. 656–82. [27] Nakashima K, Kato S, Iwanami S, Murata N. DNA probes reveal relatedness of [54] ICSB. Revised minimum standards for description of new species of the class rice yellow dwarf mycoplasmalike organisms (MLOs) and distinguish them Mollicutes (division ). Int J Syst Bacteriol 1995;45:605–12. from other MLOs. Appl Environ Microbiol 1993;59:1206–12. [55] Rossello´ -Mora R, Amann R. The species concept for prokaryotes. FEMS [28] Davis MJ, Tsai JH, Cox RL, McDaniel LL, Harrison NA. Cloning of chromosomal Microbiol Rev 2001;25:39–67. and extrachromosomal DNA of the mycoplasma-like organism that causes [56] Stackebrandt E, Frederiksen W, Garrity GM, Grimont PA, Kampfer P, maize bushy stunt disease. Mol Plant-Microbe Interact 1988;1:295–302. Maiden MC, et al. Report of the ad hoc committee for the re-evaluation of [29] Schneider B, Cousin MT, Klinkong S, Seemuller E. Taxonomic relatedness and the species definition in bacteriology. Int J Syst Evol Microbiol 2002;52: phylogenetic positions of phytoplasmas associated with diseases of faba 1043–7. bean, sunhemp, sesame, soybean, and eggplant. Z Pflanzenkr Pflanzenschutz [57] Bove´ JM, Saillard C. Cell biology of spiroplasmas. In: Whitcomb RF, Tully JG, 1995;102:225–32. editors. The mycoplasmas, vol. 3. New York: Academic Press; 1979. p. [30] Ahrens U, Lorenz KH, Seemuller E. Genetic diversity among mycoplasmalike 83–153. organisms associated with stone fruit diseases. Mol Plant-Microbe Interact [58] Christiansen C, Askaa G, Freundt EA, Whitcomb RF. Nucleic-acid hybridiza- 1993;6:686–91. tion experiments with Spiroplasma citri and the corn stunt and suckling [31] Davies DL, Clark MF. Production and characterization of polyclonal and mouse cataract spiroplasmas. Curr Microbiol 1979;2:323–6. monoclonal antibodies against peach yellow leafroll MLO-associated anti- [59] Lee IM, Davis RE. DNA homology among diverse Spiroplasma strains repre- gens. Acta Hortic 1992;383:275–83. senting several serological groups. Can J Microbiol 1980;26:1356–63. [32] Davies DL, Barbara DJ, Clark MF. The detection of MLOs associated with pear [60] Rahimian H, Gumpf DJ. Deoxyribonucleic acid relationship between Spi- decline in pear trees and pear psyllids by polymerase chain rection. Acta roplasma citri and the corn stunt Spiroplasma. Int J Syst Bacteriol Hortic 1995;386:484–8. 1980;30:605–8. [33] Schneider B, Gibb KS. Detection of phytoplasma in decline pears in southern [61] Liao CH, Chen TA. Deoxyribonucleic acid hybridization between Spiroplasma Australia. Plant Dis 1997;81:254–8. citri and the corn stunt spiroplasma. Curr Microbiol 1981;5:83–6. [34] Lee I-M, Zhu S, Gudersen DE, Zhang C, Hadidi A. Detection and identification [62] Junca P, Saillard C, Tully J, Garcia-Jurado O, Degorce-Dumas JR, Mouches C, of a new phytoplasma associated with cherry lethal yellows in China. Phy- et al. Caracte´rization de spiroplasmes isolate´ d’ insectes et fleurs de France topathol 1995;85:1179. continentale, de Corse et du Maroc. Proposition pour une classification des [35] Jarausch W, Saillard C, Dosba F, Bove´ JM. Differentiation of mycoplasmalike spiroplasmes. Compt Rend Acad Sci Paris Ser 1980;290:1209–12. organisms (MLOs) in European fruit trees by PCR using specific primers [63] Tully JG, Rose DL, Clark E, Carle P, Bove´ JM, Henegar RB, et al. Revised group derived from the sequence of a chromosomal fragment of the apple prolif- classification of the genus Spiroplasma (class Mollicutes), with proposed new eration MLO. Appl Environ Microbiol 1994;60:2916–23. groups XII to XXIII. Int J Syst Bacteriol 1987;37:357–64. 200 G.E. Gasparich / Biologicals 38 (2010) 193–203

[64] Brown DR, Whitcomb RF, Bradbury JM. Revised minimal standards for [92] Moya-Raygoza G, Palomera-Avalos V, Galaviz-Mejia C. Field overwintering description of new species of the class Mollicutes (division Tenericutes). Int J biology of Spiroplasma kunkelii (Mycoplasmatales: Spiroplasmataceae) and Syst Evol Microbiol 2007;57:2703–19. its vector Dalbulus maidis (Hemiptera: Cicadellidae). Ann Appl Biol [65] Whitcomb RF, Tully JG, Gasparich GE, Regassa LB, Williamson DL, French FE. 2007;151:373–9. Spiroplasma species in the Costa Rican highlands: implications for biogeog- [93] Williamson DL, Sakaguchi B, Hackett KJ, Whitcomb RF, Tully JG, Carle P, et al. raphy and biodiversity. Biodivers and Conserv 2007;16:3877–94. Spiroplasma poulsonii sp. nov., a new species associated with male-lethality [66] Jandhyam H, Bates CR, Young TE, Beatti L, Gasparich GE, French FE, et al. in Drosophila willistoni, a neotropical species of fruit fly. Int J Syst Bacteriol Global Spiroplasma biodiversity in a single host. China:Abstracts of 17th 1999;49:611. Congress of International Organization for Mycoplasmology; 2008. p. 130. [94] Hurst GD, Graf von der Schulenburg JH, Majerus TM, Bertrand D, Zakharov IA, [67] ICSB. Minutes of the interim meeting. 30 August and 6 September 1982. Baungaard J, et al. Invasion of one insect species, Adalia bipunctata, by two Tokyo, Japan. Int J Syst Bacteriol 1984;34:361–5. different male-killing bacteria. Insect Mol Biol 1999;8:133–9. [68] Saillard C, Vignault JC, Bove´ JM, Raie A, Tully JG, Williamson DL, et al. Spi- [95] Hurst GD, Jiggins FM. Male-killing bacteria in insects: mechanisms, inci- roplasma phoeniceum sp. nov., a new plant-pathogenic species from Syria. Int dence, and implications. Emerg Infect Dis 2000;6:329–36. J Syst Bacteriol 1987;37:106–15. [96] Nakamura K, Ueno H, Miura K. Prevalence of inherited male-killing micro- [69] Gasparich GE, Saillard C, Clark EA, Konai M, French FE, Tully JG, et al. Sero- organisms in Japanese populations of ladybird beetle Harmonia axyridis logic and genomic relatedness of group VIII and group XVII spiroplasmas and (Coleoptera:Coccinellidae). Ann Ent Soc Am 2005;98:96–9. subdivision of Spiroplasma group VIII into subgroups. Int J Syst Bacteriol [97] Montenegro H, Solferini VN, Klaczko LB, Hurst GDD. Male-killing Spi- 1993;43:338–41. roplasma naturally infecting . Insect Mol Biol [70] Abalain-Colloc ML, Williamson DL, Carle P, Abalain JH, Bonnet F, Tully JG, 2005;14:281–7. et al. Division of group XVI spiroplasmas into subgroups. Int J Syst Bacteriol [98] Montenegro H, Hatadani LM, Medeiros HF, Klaczko LB. Male killing in three 1993;43:342–6. species of the tripunctata radiation of Drosophila (Diptera: Drosophilidae). [71] Lee IM, Davis RE, Gundersen-Rindal DE. Phytoplasma: phytopathogenic J Zoo Syst Evol Res 2006;44:130–5. mollicutes. Annu Rev Microbiol 2000;54:221–55. [99] Tinsley MC, Majerus ME. A new male-killing parasitism: Spiroplasma bacteria [72] Bertaccini A. Phytoplasmas: diversity, , and epidemiology. Front infect the ladybird beetle Anisosticta novemdecimpunctata (Coleopter- Biosci 2007;12:673–89. a:Coccinellidae). Parasitology 2006;132:757–65. [73] Hogenhout SA, Oshima K, Ammar el- D, Kakizawa S, Kingdom HN, Namba S. [100] Sokolova MI, Zinkevich NS, Zakharov IA. Bacteria in ovarioles of females from Phytoplasmas: bacteria that manipulate plants and insects. Mol Plant Pathol maleless families of ladybird beetles Adalia bipunctata L. (Coleopter- 2008;9:403–23. a:Coccinellidae) naturally infected with Rickettsia, Wolbachia, and Spi- [74] Marcone C, Neimark A, Ragozzino A, Lauer U, Seemuller E. Chromosome sizes roplasma. J Invertebr Pathol 2002;79:72–9. of phytoplasmas composing major phylogenetic groups and subgroups. [101] Mateos M, Castrezana SJ, Nankivell BJ, Estes AM, Markow TA, Moran NA. Phytopathol 1999;89:805–10. Heritable endosymbionts of Drosophila. Genetics 2006;174:363–76. [75] Seemu¨ ller E, Garnier M, Schneider B. Mycoplasmas of plants and insects. In: [102] Pool JE, Wong A, Aquadro CF. Finding of male-killing Spiroplasma infecting Razin S, Herrmann R, editors. Molecular biology and pathology of myco- Drosophila melanogaster in Africa implies transatlantic migration of this plasmas. London: Kluwer Academic/Plenum Publishers; 2002. p. 91–116. . Heredity 2006;97:27–32. [76] Chang CJ. Pathogenicity of aster yellows phytoplasma and Spiroplasma citri [103] Tully JG, Rose DL, Yunker CE, Carle P, Bove´ JM, Williamson DL, et al. Spi- on periwinkle. Phytopathol 1998;88:1347–50. roplasma ixodetis sp. nov., a new species from Ixodes pacificus ticks collected [77] Lepka P, Stitt M, Moll E, Seemu¨ ller E. Effect of phytoplasmal infection on in Oregon. Int J Syst Bacteriol 1995;45:23–8. concentration and translocation of carbohydrates and amino acids in peri- [104] Goodacre SL, Martin OY, Thomas CFG, Hewitt GM. Wolbachia and other winkle and tobacco. Physiol Mol Plant Pathol 1999;55:59–68. endosymbiont infections in spiders. Mol Ecol 2006;15:517–27. [78] Bertamini M, Grando MS, Muthuchelian K, Nedunchezhian N. Effect of [105] Markham PG, Townsend R, Bar-Joseph M, Daniels MJ, Plaskitt A, Meddins BM. phytoplasmal infection on photosystem II efficiency and thylakoid Spiroplasmas are the causal agents of citrus little-leaf disease. Ann Appl Biol membrane protein changes in field grown apple (Malus pumila) leaves. 1974;78:49–57. Physiol Mol Plant Pathol 2002;61:349–56. [106] Chen TA, Liao CH. Corn stunt Spiroplasma: isolation, cultivation, and proof of [79] Bertamini M, Nedunchezhian N, Tomasi F, Grando MS. Phytoplasma [Stolbur- pathogenicity. Science 1975;188:1015–7. subgroup (Bois Noir-BN)] infection inhibits photosynthetic pigments, ribu- [107] Nault LR, Bradfute OE. Corn stunt: involvement of a complex of leaf- lose-1, 5-bisphosphate carboxylase and photosynthetic activities in field hopper-borne pathogens. In: Maramorosch K, Harris KF, editors. Leaf- grown grapevine (Vitis vinifera L. cv.Chardonnay) leaves. Physiol Mol Plant hopper vectors and plant disease agents. New York: Academic Press; 1979. Pathol 2002;61:357–66. p. 561–86. [80] Curkovic´-Perica M, Lepedus H, Seruga-Music´ M. Effect of indole-3-butyric [108] Fudl-Allah AESA, Calavan EC, Igwegbe ECKE. Culture of a mycoplasmalike acid on phytoplasmas in infected Catharanthus roseus shoots grown in vitro. organism associated with stubborn disease of citrus. Phytopathology FEMS Microbiol Lett 2007;268:171–7. 1972;62:729–31. [81] Tsai JH. Vector transmission of mycoplasmal agents of plant diseases. In: [109] Raju BC, Nyland G, Backus EA, McLean DL. Association of a Spiroplasma with Whitcomb RF, Tully JG, editors. The mycoplasmas, vol. 3. New York: brittle root of horseradish. Phytopathology 1981;71:1067–72. Academic Press; 1979. p. 265–307. [110] Fletcher J, Schultz GA, Davis RE, Eastman EC, Goodman RM. Brittle root [82] Chiykowski LN, Sinha RC. Differentiation of MLO disease by means of disease of horseradish: evidence for an etiological role of Spiroplasma citri. symptomatology and vector transmission. Zentralbl Bakteriol Hyg Suppl Phytopathology 1981;71:1073–80. 1989;20:280–7. [111] Davis RE, Fletcher J. Spiroplasma citri in Maryland: isolation from field-grown [83] Clark MF, Morton A, Buss SL. Preparation of MLO immunogens from plants plants of horseradish (Armoracia rusticana) with brittle root symptoms. Plant and a comparison of polyclonal and monoclonal antibodies made against Dis 1983;67:900–3. primula yellows MLO-associated antigens. Ann Appl Biol 1989;114: [112] Granett AL, Blue RL, Harjung MK, Calavan EC, Gumpf DG. Occurrence of 111–24. Spiroplasma citri in periwinkle in California. Calif Agric 1976;30:18–9. [84] Errampalli D, Fletcher J, Claypool PL. Incidence of yellows in carrot and [113] Kaloostian GH, Oldfield GN, Pierce HD, Calavan EC, Granett AL, Rana GL, et al. lettuce and characterization of mycoplasmalike organism isolates in Okla- Leafhopper-natural citrus stubborn disease? Calif Agric 1975;29:14–5. homa. Plant Dis 1991;75:579–84. [114] Lee I-M, Bottner KD, Munyaneza JE, Davis RE, Crosslin JM, du Toit LJ, et al. [85] Shiomi T, Sugiura M. Grouping of mycoplasma-like organisms transmitted by Carrot purple leaf: a new spiroplasmal disease associated with carrots in the leafhopper vector, Macrosteles orientalis Virvaste, based on host range. Washington State. Plant Dis 2006;90:989–93. Ann Phytopathol Soc Jpn 1984;50:149–57. [115] Bove´ JM, Renaudin J, Saillard C, Foissac X, Garnier M. Spiroplasma citri, a plant [86] Chen T-A, Lei JD, Lin CP. Detection and identification of plant and insect pathogenic mollicute: relationships with its two hosts, the plant and the mollicutes. In: Whitcomb RF, Tully JG, editors. The mycoplasmas, vol. 5. New leafhopper vector. Annu Rev Phytopathol 2003;41:483–500. York: Academic Press; 1992. p. 393–424. [116] Boutareaud A, Danet JL, Garnier M, Saillard C. Disruption of a gene predicted [87] Lee I-M, Davis RE. Mycoplasmas which infect plants and insects. In: to encode a solute binding protein of an ABC transporter reduces trans- Maniloff J, McElhansey RN, Finch LR, Baseman JB, editors. Mycoplasmas: mission of Spiroplasma citri by the leafhopper Circulifer haematoceps. Appl molecular biology and pathogenesis. Washington, DC: American Society for Environ Microbiol 2004;70:3960–7. Microbiology; 1992. p. 379–90. [117] Duret S, Berho N, Danet JL, Garnier M, Renaudin J. Spiralin is not essential for [88] Hackett KJ, Whitcomb RF, Henegar RB, Wagner AG, Clark EA, Tully JG, et al. helicity, motility, or pathogenicity but is required for efficient transmission of Mollicute diversity in arthropod hosts. Zentbl Bakteriol Suppl 1990;20:441–54. Spiroplasma citri by its leafhopper vector Circulifer haematoceps. Appl Environ [89] Gasparich GE. Spiroplasmas: evolution, adaptation and diversity. Front Biosci Microbiol 2003;69:6225–34. 2002;7:619–40. [118] Gaurivaud P, Laigret F, Garnier M, Bove´ JM. Fructose utilization and patho- [90] Hackett KJ, Whitcomb RF, Tully JG, Lloyd JE, Anderson JJ, Clark TB, et al. genicity of Spiroplasma citri: characterization of the fructose operon. Gene Lampyridae (Coleoptera): a plethora of mollicute associations. Microb Ecol 2000;252:61–9. 1992;23:181–93. [119] Gaurivaud P, Laigret F, Garnier M, Bove´ JM. Characterization of fruR as [91] Ammar E, Hogenhout SA. Use of immunofluorescence confocal laser scan- a putative activator of the fructose operon of Spiroplasma citri. FEMS Micro- ning microscopy to study distribution of the bacterium corn stunt Spi- biol 2001;198:73–8. roplasma in vector leafhoppers (Hemiptera: Cicadellidae) and in host plants. [120] Foissac X, Danet JL, Saillard C, Gaurivaud P, Laigret F, Pare´ C, et al. Muta- Ann Entomol Soc Am 2005;98:820–6. genesis by insertion of Tn4001 into the genome of Spiroplasma citri: G.E. Gasparich / Biologicals 38 (2010) 193–203 201

characterization of mutants affected in plant pathogenicity and transmission [149] Le Goff FM, Humphery-Smith I, Leclerq M, Chastel C. Spiroplasmas from to the plant by the leafhopper vector Circulifer haematoceps. Mol Plant- European Tabanidae. Med Vet Entomol 1991;5:143–4. Microbe Interact 1997;10:454–61. [150] Le Goff F, Marjolet M, Humphery-Smith I, Leclercq M, Helias C, Suplisson F, [121] Foissac X, Saillard C, Bove´ JM. Random insertion of transposon Tn4001 in the et al. Tabanid spiroplasmas from France: characterization, ecology and genome of Spiroplasma citri strain GII3. Plasmid 1997;37:80–6. experimental study. Ann Parasitol Hum Comp 1993;68:150–3. [122] Gaurivaud P, Danet JL, Laigret F, Garnier M, Bove´ JM. Fructose utilization and [151] Regassa LB, Gasparich GE. Spiroplasmas: evolutionary relationships and phytopathogenicity of Spiroplasma citri. Mol Plant-Microbe Interact biodiversity. Front Biosci 2006;11:2983–3002. 2000;13:1145–55. [152] Vazeille-Falcoz M, He´lias C, Le Goff F, Rodhain F, Chastel C. Three spi- [123] Andre A, Maucourt M, Moing A, Rolin D, Renaudin J. Sugar import and roplasmas isolated from Haematopota sp. (Diptera:Tabanidae) in France. J phytopathogenicity of Spiroplasma citri: glucose and fructose play distinct Med Entomol 1997;34:238–41. roles. Mol Plant Microbe Interact 2005;18:33–42. [153] Clark TB. Honey bee spiroplasmosis. A new problem for beekeepers. Am Bee J [124] Yu J, Wayadande AC, Fletcher J. Spiroplasma citri surface protein P89 impli- 1978;118:18. cated in adhesion to cells of the vector Circulifer tenellus. Phytopathology [154] Banttari EE, Zeyen RJ. Interactions of mycoplasmalike organisms and viruses 2000;90:716–22. in dually infected leafhoppers, planthoppers, and plants. In: Maramorosch K, [125] Berg M, Melcher U, Fletcher J. Characterization of Spiroplasma citri adhesion Harris KF, editors. Leafhopper vectors and plant disease agents. New York: related protein SARP1, which contains a domain of a novel family designated Academic Press; 1979. p. 327–47. sarpin. Gene 2001;275:57–64. [155] Brcak J. Leafhopper and planthopper vectors of plant disease agents in [126] Davis RE, Dally EL, Jomantiene R, Zhao Y, Roe B, Lin S, et al. Cryptic plasmid central and southern Europe. In: Maramorosch K, Harris KF, editors. Leaf- pSKU146 from the wall-less plant pathogen Spiroplasma kunkelii encodes an hopper vectors and plant disease agents. New York: Academic Press; 1979. p. adhesin and components of a type IV translocation-related conjugation 97–154. system. Plasmid 2005;53:179–90. [156] Grylls NE. Leafhopper vectors and the plant disease agents they transmit in [127] Berho N, Duret D, Renaudin J. Absence of plasmids encoding adhesion- Australia. In: Maramorosch K, Harris KF, editors. Leafhopper vectors and related proteins in non- insect-transmissible strains of Spiroplasma citri. plant disease agents. New York: Academic Press; 1979. p. 179–213. Microbiology 2006;152:873–86. [157] Nielson MW. Taxonomic relationships of leafhopper vectors of plant patho- [128] Nunan LM, Lightner DV, Oduori MA, Gasparich GE. Spiroplasma penaei sp. gens. In: Maramorosch K, Harris KF, editors. Leafhopper vectors and plant nov., associated with mortalities in Penaeus vannamei, Pacific white shrimp. disease agents. New York: Academic Press; 1979. p. 3–27. Int J Syst Evol Microbiol 2005;55:2317–22. [158] Lee I-M, Gundersen DE, Davis RE, Chiykowski LN. Identification and analysis [129] Wang W, Wen B, Gasparich GE, Zhu N, Rong L, Chen J, et al. A Spiroplasma of a genomic strain cluster of mycoplasmalike organisms associated with associated with tremor disease in the Chinese mitten crab (Eriocheir sinensis). Canadian peach (eastern) X-disease, western X-disease, and clover yellow Microbiology 2004;150:3035–40. edge. J Bacteriol 1992;174:6694–8. [130] Wang W, Gu W, Ding Z, Ren Y, Chen J, Hou Y. A novel Spiroplasma pathogen [159] Alma A, Davis RE, Vibio M, Danielli A, Bosco D, Arzone A, et al. Mixed causing systemic infection in the crayfish Procambarus clarkii (Crustacea:- infection of grapevines in northern Italy by phytoplasmas including 16S rRNA Decapod), in China. FEMS Microbiol Lett 2005;249:131–7. RFLP subgroup 16SrI-B strains previously unreported in this host. Plant Dis [131] Bi K, Huang H, Gu W, Wang J, Wang W. Phylogenetic analysis of spiroplasmas 1996;80:418–21. from three freshwater crustaceans (Eriocheir sinensis, Procambarus clarkia and [160] Bianco PA, Davis RE, Prince JP, Lee I-M, Gundersen DE, Fortusini A, et al. Penaeus vannamei) in China. J Invertebr Pathol 2008;99:57–65. Double and single infections by aster yellows and elm yellows MLOs in [132] Burgdorfer W, Brinton LP, Hughes LE. Isolation and characterization of grapevines with symptoms characteristic of flavescence dor´ee. Riv Patol Veg symbiotes from the Rocky Mountain wood tick, Dermacentro andersoni.J 1993;3:69–82. Invertebr Pathol 1975;22:424–34. [161] Lee I-M, Davis RE, Sinclair WA, DeWitt ND, Conti M. Genetic relatedness of [133] Tully JG, Whitcomb RF, Wiliamson DL, Clark HF. Suckling mouse cataract mycoplasmalike organisms detected in Ulmus spp. in USA and Italy by means agent is a helical wall-free prokaryote (Spiroplasma) pathogenic for verte- of DNA probes and polymerase chain reactions. Phytopathology brates. Nature 1976;259:117–20. 1993;83:829–33. [134] Bastian FO, Foster JW. Spiroplasma sp. 16S rDNA in Creutzfeldt-Jakob disease [162] Lee I-M, Gundersen-Rindal DE, Davis RE, Bartoszyk IM. Revised classification and scrapie as shown by PCR and DNA sequence analysis. J Neuropath Exp scheme of phytoplasmas based on RFLP analyses of 16SrRNA and ribosomal Neur 2001;60:613–20. protein gene sequences. Int J Syst Bacteriol 1998;48:1153–69. [135] Bastian FO, Dash S, Garry RF. Linking chronic wasting disease to scrapie by [163] Loi N, Carraro L, Musetti R, Pertot I, Osler R. Dodder transmission of two comparison of Spiroplasma mirum ribosomal DNA sequences. Exp Mol Pathol different MLOs from plum trees affected by a ‘‘leptonecrosis’’ Acta Hortic 2004;77:49–56. 1995;386:465–70. [136] Alexeeva I, Elliott EJ, Rollins S, Gasparich GE, Lazar J, Rohwer RG. Absence of [164] Marcone C, Ragozzino A, Schneider B, Lauer U, Smart CD, Seemuller E. Spiroplasma or other bacterial 16s rRNA genes in brain tissue of hamsters Genetic characterization and classification of two phytoplasmas associated with scrapie. J Clin Microbiol 2006;44:91–7. with spartium witches’-broom disease. Plant Dis 1996;80:365–71. [137] Wedincamp J, French FE, Whitcomb RF, Henegar RB. Spiroplasmas and [165] Lee I-M, Gundersen DE, Hammond RW, Davis RE. Use of mycoplasmalike entomoplasmas (Procaryotae:Mollicutes) associated with fireflies (Lamp- organism (MLO) group-specific oligonucleotide primers for nested-PCR yridae:Coleoptera) and tabanids (Diptera:Tabanidae). J Invert Pathol assays to detect mixed-MLO infections in a single host plant. Phytopathology 1996;68:183–6. 1994;84:559–66. [138] Whitcomb RF, French FE, Tully JG, Carle P, Henegar RB, Hackett KJ, et al. [166] Seemuller E, Marcone C, Lauer U, Ragozzino A, Goschl M. Current status Spiroplasma species, groups and subgroups from North American Tabanidae. of molecular classification of the phytoplasmas. J Plant Pathol 1998;80: Curr Micobiol 1997;35:287–93. 3–26. [139] Wedincamp Jr J, French FE, Whitcomb RF. A new Spiroplasma (Entomo- [167] Gundersen DE, Lee I-M, Schaff DA, Harrison NA, Chang CJ, Davis RE, et al. plasmatales: Spiroplasmataceae) record for Georgia and attempted hori- Genomic diversity and differentiation among phytoplasma strains in 16S zontal transmission via predation. Ent News 1997;108:209–12. rRNA group I (aster yellows and related phytoplasmas) and III (X-disease and [140] Moulder RW, French FE, Whitcomb RF, Henegar R. Spiroplasma carriage in related phytoplasmas). Int J Syst Bacteriol 1996;4 6:64–75. two spring tabanid flies. IOM Lett 1996;4:200–1. [168] Chang CJ, Lee I-M. Pathogenesis of diseases associated with mycoplasmalike [141] French FE, Whitcomb RF, Tully JG, Carle P, Bove´ JM, Henegar RB, et al. Spi- organisms. In: Singh US, Singh RP, Kohmoto K, editors. Pathogenesis and host roplasma linolae sp. nov., from the horsefly Tabanus lineola (Diptera: Taba- specificity in plant diseases. New York: Elsevier; 1995. p. 237–46. nidae). Int J Syst Bacteriol 1997;47:1078–81. [169] Davis RE, Sinclair WA. Phytoplasma identity and disease etiology. Phytopa- [142] Hackett KJ, Whitcomb RF, Clark TB, Henegar RB, Lynn DE, Wagner AG, et al. thology 1998;88:1372–6. Spiroplasma leptinotarsae sp. nov., from the Colarado potato beetle Lep- [170] Martini M, Vibio M, Sfalanga A, Bertaccini A. Molecular and ecological tinotarsa decemlineata. Int J Syst Bacteriol 1996;46:906–11. diversity of phytoplasmas belonging to the elm yellows group in Italy [143] Klein M, Hackett KJ, Braverman Y, Goldberg A, Khanbegyan Y, Chizon- towards their tentative epidemiology. 12th Int Org Mycoplasmol Conf, Syd- Ginzburg A. The fate of spiroplasmas fed to no-host insects. IOM Lett ney, Australia 1998. p. 130. 1994;3:525–6. [171] Davey JE, Van Staden J, DeLeeuw GTN. Endogenous cytokinin levels and [144] Whitcomb RF, French FE, Tully JG, Gasparich GE, Bove´ JM, Carle P, et al. development of flower virescence in Catharanthus roseus infected with Tabanid Spiroplasma serovars. IOM Lett 1992;2:115. mycoplasmas. Plant Pathol 1981;19:193–9. [145] Wedincamp Jr J, French FE, Whitcomb RF, Henegar RB. Laboratory infection [172] Smart CD, Kirkpatrick BC. Identification of host plant genes whose and release of Spiroplasmas (: Spiroplasmataceae) from expression is altered upon aster yellows phytoplasma infection. IOM Lett horse flies (Diptera: Tabanidae). J Entomol Sci 1997;32:398–402. 1996;4:274. [146] Whitcomb RF, Hackett KJ, Tully JG, Clark EA, French FE, Henegar RB, et al. [173] Hogenhout SA, van der Hoorn RA, Terauchi R, Kamoun S. Emerging concepts Tabanid spiroplasmas as a model for mollicute biogeography. Zbl Bakt Suppl in effector biology of plant-associated organisms. Mol Plant Microbe Interact 1990;20:931–3. 2009;22:115–22. [147] French FE, Whitcomb RF, Tully JG, Hackett KJ, Clark EA, Henegar RB, et al. [174] Hoshi A, Oshima K, Kakizawa S, Ishii Y, Ozeki J, Hashimoto M, et al. A unique Tabanid spiroplasmas of the southeast USA: new groups and correlation with virulence factor for proliferation and dwarfism in plants identified from host life history strategy. Zbl Bakteriol Suppl 1990;20:919–22. a phytopathogenic bacterium. Proc Natl Acad Sci U S A 2009;106:6416–21. [148] French FE, Whitcomb RF, Tully JG, Williamson DL, Henegar RB. Spiroplasmas [175] Woese CR, Maniloff J, Zablen LB. Phylogenetic analysis of the mycoplasmas. of Tabanus lineola. IOM Lett 1996;4:211–2. Proc Natl Acad Sci U S A 1980;77:494–8. 202 G.E. Gasparich / Biologicals 38 (2010) 193–203

[176] Weisburg WG, Tully JG, Rose DL, Petzel JP, Oyaizu H, Yang D, et al. A phylo- [203] Wei W, Davis RE, Jomantiene R, Zhao Y. Ancient, recurrent phage attacks and genetic analysis of the mycoplasmas: basis for their classification. J Bacteriol recombination shaped dynamic sequence-variable mosaics at the root of the 1989;171:6455–67. phytoplasma genome evolution. PNAS 2008;105:11827–32. [177] Gundersen DE, Lee I-M, Rehner SA, Davis RE, Kingsbury DT. Phylogeny of [204] Davis RE, Jomantiene R, Zhao Y. Lineage-specific decay of folate biosynthesis mycoplasmalike organisms (phytoplasmas): a basis for their classification. J genes suggests ongoing host adaptation in phytoplasmas. DNA Cell Biol Bacteriol 1994;176:5244–54. 2005;24:832–40. [178] Maniloff J. Phylogeny of mycoplasmas. In: Maniloff J, McElhaney RN, Finch LR, [205] Oshima K, Kakizawa S, Nishigawa H, Jung H-Y, Wei W, Suzuki S, et al. Baseman JB, editors. Mycoplasmas: molecular biology and pathogenesis. Reductive evolution suggested from the complete genome sequence of Washington: Am Soc Microbiol; 1992. p. 549–59. a plant pathogenic phytoplasma. Nat Genet 2004;36:27–9. [179] Gasparich GE, Whitcomb RF, Dodge D, French FE, Glass J, Williamson DL. The [206] Bai X, Zhang J, Ewing A, Miller SA, Radek AJ, Shevchenko DV, et al. Living with genus Spiroplasma and its non-helical descendants: phylogenetic classifica- genome instability: the adaptation of phytoplasmas to diverse environments tion, correlation with phenotype and roots of the Mycoplasma mycoides clade. of their insect and plant hosts. J Bacteriol 2006;188:3682–96. Int J Syst Evol Microbiol 2004;54:893–918. [207] Kube M, Schneider B, Kuhl H, Dandekar T, Heitmann K, Migdoll AM, et al. The [180] Maniloff J. Evolution of wall-less prokaryotes. Annu Rev Microbiol linear chromosome of the plantpathogenic mycoplasma ‘Candidatus Phyto- 1983;37:477–99. plasma mali’. BMC Genomics 2008;9:306. [181] Desantis D, Tryon VV, Pollack JD. Metabolism of the mollicutes: the Embden- [208] Tran-Nguyen LT, Kube M, Schneider B, Reinhardt R, Gibb KB. Comparative Meyerhoff- Parnas pathway and the hexose monophosphate shunt. J Gen genome analysis of ‘ Candidatus Phytoplasma australiense’ (subgroup tuf- Microbiol 1989;135:683–91. Australia I; rp-A) and ‘Ca. Phytoplasma asteris’ strains OY-M and AY-WB. J [182] Pollack JD, Williams MV, Banzon J, Jones MA, Harvey L, Tully JG. Comparative Bacteriol 2008;190:3979–91. metabolism of Mesoplasma, Entomoplasma, mycoplasma and Acholeplasma. [209] Lim PO, Sears BB. 16S rRNA sequence indicates that plant-pathogenic Int J Syst Bacteriol 1996;46:885–90. mycoplasmalike organisms are evolutionarily distinct from animal myco- [183] Rogers MJ, Simmons J, Walker RT, Weisburg WG, Woese CR, Tanner RJ, et al. plasmas. J Bacteriol 1989;171:5901–6. Construction of the mycoplasma evolutionary tree from 5S rRNA sequence [210] Namba S, Oyaizu H, Kato S, Iwanami S, Tsuchizaki T. Phylogenetic diversity of data. Proc Natl Acad Sci 1985;82:1160–4. phytopathogenic organisms. Int J Syst Bacteriol 1993;43:461–7. [184] Johansson K-E, Pettersson B. Taxonomy of mollicutes. In: Razin S, Herrman R, [211] Zhao Y, Davis RE, Lee IM. Phylogenetic positions of ‘ Candidatus Phytoplasma editors. Molecular biology and pathogenicity of Mycoplasmas. London: asteris’ and Spiroplasma kunkelii as inferred from multiple sets of concate- Kluwer Academic/Plenum; 2002. p. 1–p30. nated core housekeeping proteins. Int J Syst Evol Microbiol 2005;55:2131–41. [185] Clark TB, Whitcomb RF, Tully JG, Mouches C, Saillard C, Bove´ JM, et al. Spi- [212] Hodgetts J, Boonham N, Mumford R, Harrison N, Dickinson M. Phytoplasma roplasma melliferum, a new species from the honeybee (Apis mellifera). Int J phylogenetics based on analysis of secA and 23S rRNA gene sequences for Syst Bacteriol 1985;35:296–308. improved resolution of candidate species of ‘Candidatus Phytoplasma’. Int J [186] Lin CP, Chen T-A. Monoclonal antibodies against the aster yellows agent. Syst Evol Microbiol 2008;58:1826–37. Science 1985;227:1233–5. [213] Murray RGE, Schleifer KH. Taxonomic notes: a proposal for recording the [187] Lee I-M, Davis RE. Detection and investigation of genetic relatedness among properties of putative taxa of prokaryotes. Int J Syst Bacteriol 1994;44: aster yellows and other mycoplasmalike organisms by using cloned DNA and 174–6. RNA probes. Mol Plant Microbe Interact 1988;1:303–10. [214] IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy [188] Harrison NA, Bourne CM, Cox RL, Tsai JH, Richardson PA. DNA probes for Group. ‘Candidatus Phytoplasma’, a taxon for the wall-less, non-helical detection of mycoplasmalike organisms associated with lethal yellowing prokaryotes that colonize plant phloem and insects. Int J Syst Evol Microbiol disease of palms in Florida. Phytopathology 1992;82:216–24. 2004;54:1243–55. [189] Kuske CR, Kirkpatrick BC, Davis MJ, Seemuller E. DNA hybridization between [215] Kuske CR, Kirkpatrick BC. Phylogenetic relationships between the western western aster yellows mycoplasmalike organism plasmids and extrachro- aster yellows mycoplasmalike organisms and other prokaryotes established mosomal DNA from other plant pathogenic mycoplasmalike organisms. Mol by 16S rRNA gene sequence. Int J Syst Bacteriol 1992;42:226–33. Plant-Microbe Interact 1991;4:75–80. [216] Lee I-M, Hammond RW, Davis RE, Gundersen DE. Universal amplification and [190] Lee I-M, Davis RE, Chen T-A, Chiykowski LN, Fletcher J, Hiruki C, et al. A analysis of pathogen 16S rDNA for classification and identification of myco- genotype-based system for identification and classification of mycoplasma- plasmalike organisms. Phytopathology 1993;83:834–42. like organisms (MLOs) in the aster yellows MLO strain cluster. Phytopa- [217] Schneider B, Aherns U, Kirkpatrick BC, Seemu¨ ller E. Classification of plant- thology 1992;82:977–86. pathogenic mycoplasma-like organisms using restriction-site analysis of PCR [191] Kison H, Schneider B, Seemuller E. Restriction fragment length poly- amplified16S rDNA. J Gen Microbiol 1993;139:519–27. morphisms within the apple proliferation mycoplasmalike organism. J Phy- [218] Lee I-M, Martini M, Marcone C, Zhu SF. Classification of phytoplasma strains topathol 1994;141:395–401. in the elm yellows group (16SrV) and proposition of ‘Candidatus Phytoplasma [192] Schneider B, Seemuller E. Presence of two sets of ribosomal genes in ulmi’ for the phytoplasma associated with elm yellows. Int J Syst EvolMi- phytopathogenic mollicutes. Appl Environ Microbiol 1994;141:173–85. crobiol 2004;54:337–47. [193] Kirkpatrick BC, Fisher GF, Fraser JD, Purcell AH. Epidemiological and phylo- [219] Lee I-M, Gundersen DE, Davis RE, Bottner KD, Marcone C, Seemu¨ ller E. genetic studies on western-X disease mycoplasmalike organisms. In: ‘Candidatus Phytoplasma asteris’, a novel phytoplasma taxon associated with Stanek GH, Casell GH, Tully JG, Whitcomb RF, editors. Recent advances in aster yellows and related diseases. Int J Syst Evol Microbiol 2004;54: mycoplasmology. Germany: Gustav Fischer Verlag; 1990. p. 288–97. 1037–48. [194] Deng S, Hiruki C. Amplification of 16S rRNA genes from culturable and [220] Al-Saady NA, Khan AJ, Calari A, Al-Subhi AM, Bertaccini A. ‘Candidatus Phy- nonculturable mollicutes. J Microb Methods 1991;14:53–61. toplasma omanense’, associated with witches’-broom of Cassia italic (Mill.) [195] Smart CD, Schneider B, Blomquist CL, Guerra LJ, Harrison NA, Ahrens U, et al. Spreng in Oman. Int J Syst Evol Microbiol 2008;58:461–6. Phytoplasma-specific PCR primers based on sequences of 16S-23S rRNAs- [221] Cai H, Wei W, Davis RE, Chen H, Zhao Y. Genetic diversity among phyto- pacer region. Appl Environ Microbiol 1996;62:2988–93. plasmas infecting Opuntia spp.: virtual RFLP analysis identifies new [196] Jomantiene R, Davis RE, Maas J, Dally EL. Classification of new phytoplasmas subgroups in the peanut witches’-broom phytoplasma group. Int J Syst Evol associated with diseases of strawberry in Florida based on analysis of 16S rRNA Microbiol 2008;58:1448–57. and ribosomal protein gene operon sequences. Int J Syst Bacteriol [222] Zhao Y, Sun Q, Wei W, Davis RE, Wu W, Liu Q. ‘Candidatus phytoplasma 1998;48:269–77. tamaricis’, a novel taxon discovered in witches’-broom diseased salt cedar [197] Shao J, Jomantiene R, Dally EL, Zhao Y, Lee I-M, Nuss DL, et al. Phylogeny and (Tamarix chinesis Lour.). Int J Syst Evol Microbiol 2009;59:2496–504. characterization of phytoplasmal NusA and use of the nusA gene in detection [223] Zhao Y, Wei W, Lee I-M, Shao J, Suo X, Davis RE. Construction of an interactive of group 16SrI strains. J Plant Pathol 2006;88:193–201. online phytoplasma classification tool, iPhyClassifier, and its application in [198] Martini M, Lee I-M, Bottner KD, Zhao Y, Botti S, Bertaccini A, et al. Ribosomal analysis of the peach X-disease phytoplasma group (16SrIII). Int J Syst Evol protein gene- based phylogeny for finer differentiation and classification of Microbiol 2009;59:2582–93. phytoplasmas. Int J Syst Evol Microbiol 2007;57:2037–51. [224] Rangel B, Krueger RR, Lee RF. Current research on Spiroplasma citri in Cal- [199] Wei W, Davis RE, Lee I-M, Zhao Y. Computer-simulated RFLP analysis of 16S ifornia. Proc 16th Conf IOCV, Riverside, CA 2005. p. 439–41. rRNA genes: identification of ten new phytoplasma groups. Int J Sys Evol [225] Foissac X, Bove´ JM, Saillard C. Sequence analysis of Spiroplasma phoeniceum Microbiol 2007;57:1855–67. and Spiroplasma kunkelii spiralin genes and comparison with other spiralin [200] Wei W, Lee I-M, Davis RE, Suo X, Zhao Y. Automated RFLP pattern compar- genes. Curr Microbiol 1997;35:240–3. ison and similarity coefficient calculation for rapid delineation of new and [226] Granados RR. Maize viruses and vectors. In: Maramorosch K, editor. Viruses, distinct phytoplasma 16Sr subgroup lineages. Int J Syst Evol Microbiol vectors and vegetation. New York: lnterscience Publishers; 1969. p. 327–59. 2008;58:2368–77. [227] Haggis GH, Sinha RC. Scanning electron microscopy of mycoplasmalike [201] Arnaud G, Malembic-Maher S, Salar P, Bonnet P, Maixner M, Marcone C, et al. organisms after freeze fracture of plant tissues affected with clover phyllody Multilocus sequence typing confirms the close genetic interrelatedness of and aster yellows. Phytopathology 1978;68:677–80. three distinct Flavescence dore´e phytoplasma strain clusters and group [228] Ahrens U, Seemuller E. Detection of DNA of plant pathogenic mycoplasmalike 16SrV phytoplasmas infecting grapevine and alder in Europe. Appl Environ organisms by a polymerase chain reaction that amplifies a sequence of the Microbiol 2007;73:4001–10. 16S rRNA gene. Phytopathology 1992;82:828–32. [202] Jomantiene R, Zhao Y, Davis RE. Sequence-variable mosaics: composites of [229] Daire X, Boudon-Padieu E, Berville A, Schneider B, Caudwell A. Cloned DNA recurrent transposition characterizing the genomes of phylogenetically probes for detection of grapevine flavescence dor´ee mycoplasma-like diverse phytoplasmas. DNA Cell Biol 2007;26:557–64. organism (MLO). Ann Appl Biol 1992;121:95–103. G.E. Gasparich / Biologicals 38 (2010) 193–203 203

[230] Davis RE, Dally EL, Bertaccini A, Lee IM, Credi RC, Barba M. Restriction [239] Christensen NM, Nicolaisen M, Hansen M, Schulz A. Distribution of phyto- fragment length polymorphism analyses and dot hybridization distiguish plasmas in infected plants as revealed by real-time PCR and bioimaging. Mol mycoplasmalike organisms associated with flavescence dor´ee and southern Plant Microbe Interact 2004;17:1175–84. European grapevine yellows disease in Italy. Phytopathology 1993;83:772–6. [240] Torres E, Bertolini E, Cambra M, Monto´ nC,Martı´nMP.Real-timePCR [231] Davis RE, Lee I-M. Cluster-specific polymerase chain reaction amplification of for simultaneous and quantitative detection of quarantine phytoplasmas 16S rDNA sequences for detection and identification of mycoplasmalike from apple proliferation (16SrX) group. Mol Cell Probes 2005;19: organisms. Phytopathology 1993;83:1008–11. 334–40. [232] Firrao G, Gobbi E, Locci R. Use of polymerase chain reaction to produce [241] Hren M, Boben J, Rotter A, Kralj P, Gruden K, Ravnikar M. Real-time PCR oligonucleotide probes for mycoplasmalike organisms. Phytopathology detection systems for Flavescence dore´e and Bois noir phytoplasmas in 1993;83:602–7. grapevine: comparison with conventional PCR detection and application in [233] Gundersen DE, Lee I-M. Ultrasensitive detection of phytoplasmas by nested- diagnostics. Plant Pathol 2007;56:785–96. PCR assays using two universal primer pairs. Phytopathol Mediterr [242] Wei W, Kakizawa S, Suzuki S, Jung HY, Nishigawa H, Miyata S, et al. In planta 1996;35:114–51. dynamic analysis of onion yellows phytoplasma using localized inoculation [234] Lee I-M, Davis RE, Hiruki C. Genetic relatedness among clover proliferation by insect transmission. Phytopathology 2004;94:244–50. mycoplasmalike organisms (MLOs) and other MLOs investigated by nucleic [243] Carraro L, Loi N, Ermacora P, Osler R. High tolerance of European plum acid hybridization and restriction fragment length polymorphism analyses. varieties to plum leptonecrosis. Eur J Plant Pathol 1998;104:141–5. Appl Environ Microbiol 1991;57:3565–9. [244] Sinclair WA, Whilow TH, Griffiths HM. Heritable tolerance of ash yellows [235] Lim PO, Sears BB. DNA sequence of the ribosomal protein genes rpl2 and phytoplasma in green ash. Can J For Res 1997;27:1928–35. rps19 from a plant-pathogenic mycoplasmalike organism. FEMS Microbiol [245] Thomas PE, Mink GI. Tomato hybrids with nonspecific immunity to viral Lett 1991;84:71–4. and mycoplasma pathogens of potato and tomato. HortScience [236] Lorenz KH, Schneider B, Ahrens U, Seemuller E. Detection of the apple 1998;33:764–5. proliferation and pear decline phytoplasmas by PCR amplification of ribo- [246] Chen YD, Chen T-A. Expression of engineered antibodies in plants: a possible somal and nonribosomal DNA. Phytopathology 1995;85:771–6. tool for Spiroplasma and phytoplasma disease control. Phytopathology [237] Barros TSL, Davis RE, Resende RO, Dally EL. Design of a polymerase chain reaction 1998;88:1367–71. for specific detection of corn stunt Spiroplasma. Plant Dis 2001;85:475–80. [247] Le Gall F, Bove´ JM, Garnier M. Engineering of a single-chain variable fragment [238] Nicolaisen M, Bertaccini A. An oligonucleotide microarray-based assay for (scFv) antibody specific for the stolbur phytoplasma (mollicute) and its identification of phytoplasma 16S ribosomal groups. Plant Pathol expression in Escherichia coli and tobacco plants. Appl Environ Microbiol 2007;56:332–6. 1998;64:4566–72.