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The possibility to grow on artificial media phytoplasmas isolated from micropropagated infected periwinkle shoots and from field-infected tissues is a recent achievement. Isolation and cultivation together with biochemical and biological characterization of some phytoplasmas was consistently obtained. Colonies of phytoplasmas belonging to diverse ribosomal groups were obtained such as those related to a ‘Candidatus Phytoplasma pruni’- related isolate from cassava plants with frog skin diseased, ‘Ca. P. asteris’ and ‘Ca. P. solani’ from diverse infected host plants such as grapevine and coconut palms. Antisera production and their preliminary diagnostic use was also achieved for some of these isolates. Further phytoplasmas were also isolated in liquid and solid media from date palm and citrus tissues. Seed transmission in corn of aster yellows phytoplasmas was confirmed by isolation from seedlings of viable phytoplasma cells.

The genus ‘Candidatus Phytoplasma’ was proposed to accommodate cell wall-less bacteria that are molecularly and biochemically incompletely characterized, and colonize plant phloem and insect vector tissues. This provisional classification is highly relevant due to its application in epidemiological and ecological studies, mainly aimed at keeping the severe phytoplasma plant diseases under control worldwide. Given the increasing discovery of molecular diversity within the genus ‘Ca. Phytoplasma’, the proposed guidelines were revised and clarified to accommodate those ‘Ca. Phytoplasma’ species strains sharing >98.65 % sequence identity of their full or nearly full 16S rRNA gene sequences, obtained with at least twofold coverage of the sequence, compared with those of the reference strain of such species. Strains sharing 98.65 % with other strain(s) within the same ‘Ca. Phytoplasma’ species should be considered related strains to that ‘Ca. Phytoplasma’ species. The guidelines herein, keep the original published reference strains. However, to improve ‘Ca. Phytoplasma’ species assignment, complementary strains are suggested as an alternative to the reference strains. This will be implemented when only a partial 16S rRNA gene and/or a few other genes have been sequenced, or the strain is no longer available for further molecular characterization. Lists of ‘Ca. Phytoplasma’ species and alternative reference strains described are reported. For new ‘Ca. Phytoplasma’ species that will be assigned with identity ≥98.65 % of their 16S rRNA gene sequences, a threshold of 95 % genome-wide average nucleotide identity is suggested. When the whole genome sequences are unavailable, two among conserved housekeeping genes could be used. There are 49 officially published ‘Candidatus Phytoplasma’ species, including ‘Ca. P. cocostanzaniae’ and ‘Ca. P. palmae’ described in this manuscript.

The considerable economic losses in citrus associated with ‘Candidatus Liberibacter’ and ‘Candidatus Phytoplasma’ presence have alerted all producing regions of the world. In Chile, none of these bacteria have been reported in citrus species. During the years 2017 and 2019, 258 samples presenting symptoms similar to those associated with the presence of these bacteria were examined. No detection of ‘Ca. Liberibacter’ associated with “huanglongbing” disease was obtained in the tested samples; therefore, this quarantine pest is maintained as absent in Chile. However, 14 plants resulted positive for phytoplasmas enclosed in subgroups 16SrV-A (12 plants) and 16SrXIII-F (2 plants). Although they have been found in other plant species, this is the first report of these phytoplasmas in citrus worldwide.

The considerable economic losses in citrus associated with ‘Candidatus Liberibacter’ and ‘Candidatus Phytoplasma’ presence have alerted all producing regions of the world. In Chile, none of these bacteria have been reported in citrus species. During the years 2017 and 2019, 258 samples presenting symptoms similar to those associated with the presence of these bacteria were examined. No detection of ‘Ca. Liberibacter’ associated with “huanglongbing” disease was obtained in the tested samples; therefore, this quarantine pest is maintained as absent in Chile. However, 14 plants resulted positive for phytoplasmas enclosed in subgroups 16SrV-A (12 plants) and 16SrXIII-F (2 plants). Although they have been found in other plant species, this is the first report of these phytoplasmas in citrus worldwide.

Phytoplasma ability to infect a new generation of insects by transovarial transmission was demonstrated in some insect vector/plant host combinations mainly by molecular evidence coupled with biological assays. Scaphoideus titanus was the first one in which phytoplasma detection in eggs, newly hatched nymphs and adults (reared on phytoplasma-free Vicia faba seedlings) was demonstrated. This kind of transmission was proved also for mulberry dwarf phytoplasmas and for the agent of white leaf disease of sugarcane, transmitted respectively by Hishimonoides sellatiformis and Matsumuratettix hiroglyphicus. Recently Cacopsylla pruni, vector of ‘Candidatus Phytoplasma prunorum’ and Cacopsylla picta one of the main insect vectors of ‘Candidatus Phytoplasma mali’ were also shown to have phytoplasma transovarial transmission, therefore this kind of transmission should be taken into consideration when epidemiological studies are performed on phytoplasma-associated diseases. The fact that the insect is not only the vector, but also a reservoir of the phytoplasma has implications for disease management, increasing the difficulty of disease control. Up to now only a few phytoplasma ribosomal groups such as 16SrI, 16SrX and 16SrXI have been demonstrated to be transferred transovarially in their insect vectors, very likely those capable of better adaptation to both plant and insect environments. Therefore, it can be speculated that only strains of phytoplasmas with specific genetic characteristics have become transovarially transmissible and probably only after a long host–parasite relationship.

Two genera of the Fabaceae family showed phytoplasmas symptoms in different locations in Europe i.e. Germany and Italy. Spartium junceum L. (Spanish broom) is a deciduous shrub with dark green, round stems and alternate leaves; inflorescences are terminal clusters of several bright yellow somewhat fragrant flowers. This ornamental shrub is frequently spontaneously growing especially in southern Italy where it is affected by spartium witches’ broom (SpaWB) disease, characterized by proliferation of axillary buds and stem fasciation. Two different phytoplasmas have been associated to SpaWB: ‘Candidatus Phytoplasma spartii’ (group 16SrX-D) and a phytoplasma belonging to elm yellows group (16SrV-C). Both were reported associated with SpaWB in Italy while only ‘Ca. P. spartii’ was reported in Spain. In the spring of 2011, typical SpaWB symptoms were observed in a plant up to 2 m tall growing in the city of Ercolano (Campania region, Italy). A similar symptomatology was observed in a group of shrubs of Cytisus scoparius (L) syn. Sarothamnus scoparius, better known as common broom or scotch broom growing in Dahlem botanical garden in Berlin (Germany). This is a perennial shrub native to western and central Europe, but it is considered invasive plant in areas such as North America and New Zealand. Symptomatic and asymptomatic samples were collected in both cases: five samples of C. scoparius and two of S. junceum were analysed for phytoplasma presence by nested-PCR assays employing primer pairs P1A/P7A followed by F1/B6 and R16F2n/R2, phytoplasma identification was achieved by RFLP analyses with Tru1I on the two latter amplicons. Further confirmation of phytoplasma identity was achieved by nested-PCR assays with primers specific for phytoplasma groups 16SrI, 16SrV and 16SrX. All symptomatic samples produced amplicons of the expected lengths and no product was amplified from asymptomatic plants and using 16SrV specific primers. Identification and classification of phytoplasmas allow to detect ‘Ca. P. spartii’ subgroup 16SrX-D and ‘Ca. P. asteris’ subgroup 16SrI-B in both genera. In some of the samples of C. scoparius also stolbur phytoplasmas were identified. Further phytoplasma characterization was carried out on tuf gene using a cocktail primers mix that was able to amplify phytoplasmas identified as ‘Ca. P. asteris’ in S. junceum and phytoplasmas showing two different Tru1I profiles in C. scoparius from Germany that are not present in any published RFLP profile on this gene. Direct amplicon sequencing is in progress in order to verify possible affiliation to ‘Ca. P. spartii’ group since the only available sequences of this gene on phytoplasmas are deposited in Qbank since they were obtained from the Qbol EU project.

Two of the most common leafhoppers present in Chile are the Cicadellidae Paratanus exitiosus and Bergallia valdiviana. They commonly occur in vineyards of central Chile, including some vineyards infected by phytoplasmas. The present study demonstrates that P. exitiosus and B. valdiviana can transmit 16SrIII-J phytoplasmas to grapevine and periwinkle plants. This provides improved understanding of the 16SrIII-J phytoplasma epidemiology in Chilean vineyards. EEA Mendoza Fil: Quiroga, Nicolás. Universidad de Chile. Facultad de Ciencias Agronómicas. Departamento de Sanidad Vegetal; Chile Fil: Longone, Maria Valeria. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Mendoza. Laboratorio de Fitopatología; Argentina Fil: González, Ximena. Universidad de Chile. Facultad de Ciencias Agronómicas. Departamento de Sanidad Vegetal; Chile Fil: Zamorano, Alan. Universidad de Chile. Facultad de Ciencias Agronómicas. Departamento de Sanidad Vegetal; Chile Fil: Pino, Ana María. Universidad de Chile. Facultad de Ciencias Agronómicas. Departamento de Sanidad Vegetal; Chile Fil: Picciau, Luca. Università di Torino. Dipartimento di Scienze Agrarie, Forestali e Alimentari (DISAFA); Italia Fil: Alma, Alberto. Università di Torino. Dipartimento di Scienze Agrarie, Forestali e Alimentari (DISAFA); Italia Fil: Paltrinieri, Samanta. Università di Bologna. Alma Mater Studiorum. Department of Agricultural and Food Sciences (DISTAL), Plant Pathology and Entomology; Italia Fil: Contaldo, Nicoletta. Università di Bologna. Alma Mater Studiorum. Department of Agricultural and Food Sciences (DISTAL), Plant Pathology and Entomology; Italia Fil: Bertaccini, Assunta. Università di Bologna. Alma Mater Studiorum. Department of Agricultural and Food Sciences (DISTAL), Plant Pathology and Entomology; Italia Fil: Fiore, Nicola. Universidad de Chile. Facultad de Ciencias Agronómicas. Departamento de Sanidad Vegetal; Chile

Phytoplasma classification established using 16S ribosomal groups and ‘Candidatus Phytoplasma' taxon are mainly based on the 16S rDNA properties and do not always provide molecular distinction of the closely related strains such as those in the aster yellows group (16SrI or ‘Candidatus Phytoplasma asteris'-related strains). Moreover, because of the highly conserved nature of the 16S rRNA gene, and of the not uncommon presence of 16S rDNA interoperon sequence heterogeneity, more variable single copy genes, such as ribosomal protein (rp), secY and tuf, were shown to be suitable for differentiation of closely related phytoplasma strains. Specific amplification of fragments containing phytoplasma groEL allowed studying its variability in 27 ‘Candidatus Phytoplasma asteris'-related strains belonging to different 16SrI subgroups, of which 11 strains were not studied before and 8 more were not studied on other genes than 16S rDNA. The restriction fragment length polymorphism (RFLP) analyses of the amplified fragments confirmed differentiation among 16SrI-A, I-B, I-C, I-F and I-P subgroups, and showed further differentiation in strains assigned to 16SrI-A, 16SrI-B and 16SrI-C subgroups. However, analyses of groEL gene failed to discriminate strains in subgroups 16SrI-L and 16SrI-M (described on the basis of 16S rDNA interoperon sequence heterogeneity) from strains in subgroup 16SrI-B. On the contrary, the 16SrI unclassified strain ca2006/5 from carrot (showing interoperon sequence heterogeneity) was differentiable on both rp and groEL genes from the strains in subgroup 16SrI-B. These results indicate that interoperon sequence heterogeneity of strains AY2192, PRIVA (16SrI-L), AVUT (16SrI-M) and ca2006/5 resulted in multigenic changes - one evolutionary step further - only in the latter case. Phylogenetic analyses carried out on groEL are in agreement with 16Sr, rp and secY based phylogenies, and confirmed the differentiation obtained by RFLP analyses on groEL amplicons.

In several growing areas of Northern Italy cherry is affected by a severe decline that leads plants to die in two-weeks/one month maximum. From the beginning of the summer the affected plants show leaves of smaller size, with chlorosis, reddening, curling aspect and premature fall; young branches also show some lack of lignifications. Preliminary tests excluded presence of several biotic or abiotic agents; to verify the possible phytoplasma association with the disease nucleic acid extraction was performed from leaf midribs as well as from phloem scrapes of trunks from declining trees. Molecular analyses carried out on samples from declining cherry plants collected in different orchards from 2002 to 2004 allowed identification of phytoplasmas belonging to different ribosomal subgroups such as 16SrV-B; 16SrIII-B and 16SrXII-A (stolbur). Since the area monitored was limited and phytoplasma detection was achieved in both hilly and sandy soil locations on plants usually older than 5–6 years, it appears possible that infection was related to the health status of propagation material employed for plantations.

The molecular identification and characterization of phytoplasmas infecting grapevine in four locations of different grape-growing regions of Serbia is reported. Samples from symptomatic and asymptomatic grapevine plants were collected during 2002 and 2003. Detection and identification of phytoplasmas were done by the restriction fragment length polymorphism analysis (RFLP) of polymerase chain reaction (PCR) amplified 16S rDNA. Grapevine yellows were associated with three molecularly distinguishable phytoplasmas. Flavescence dorée phytoplasmas (elm yellows group - 16SrV-C subgroup) were present only in the Župa Aleksandrovac region; in other surveyed regions Bois noir phytoplasmas (stolbur group - 16SrXII-A subgroup) were detected; a mixed infection of European stone fruit yellows (apple proliferation group - 16SrX-B subgroup) and Bois noir phytoplasmas was identified in one sample. A finer molecular characterization by the RFLP analysis on rpS3 and SecY genes of Flavescence dorée phytoplasmas from Župa Aleksandrovac allowed to verify that the Serbian genotype is indistinguishable from a strain from the Veneto region, Italy. Characterization performed on tuf gene of Bois noir phytoplasmas showed lack of amplification of samples from Erdevik. The identity of HpaII profiles on the tuf gene PCR products was observed between samples from Palić and Radmilovac, this pattern appears indistinguishable from one of the two profiles obtained in samples from the Italian grapevines used as reference strains.

The clear differentiation among ‘Candidatus Phytoplasma asteris’-related strains using Amp-N1/C1 primers amplifying the amp gene was observed in samples classified into different 16SrI subgroups (16SrI-A, I-B, I-C, I-L, and I-M). Samples were maintained in periwinkle or collected from field infected plants and insects; in 27 out of the 31 strains employed the full amp gene was also sequenced. Carrot with yellows symptoms and Macrosteles sexnotatus from the same field in Serbia (16SrI-A, 16SrI-B, and 16Sr-I subgroup not identified); poplar with witches’ broom collected in Serbia (16SrI-P); corn and oil palm samples (16SrI-B) from Colombia showing respectively stunt and decline symptoms were also employed. The amp gene could not be amplified in 16SrI-F and I-P strains; in 16SrI-B, I-L, I-M and in the strain from carrot in a not identified subgroup a 740 bp amplicon was obtained; strains in subgroups 16SrI-A, and I-C produced a 650 bp amplicon. RFLP analyses with TruI and Tsp509I enzymes distinguished up to four different profiles in amplicons from16SrI-B, I-L I-M strains, two profiles in 16SrI-A, while an unique profile was present in 16SrI-C strains. Among the field collected samples the M. sexnotatus sample showed a profile different from those obtained in strains from carrots, but referable to one of those of 16SrI-B phytoplasmas in periwinkle. RFLP profiles of corn and oil palm strains were identical to each others and to the one of 16Sr-I strain carrot. The full sequence of amp gene obtained was also studied with translated nucleotide query using NCBI/BLAST/blastx program. Preliminary results indicate that strains in subgroups 16SrI-A, I-C and I-F and strains DAY, AVUT and PrG in 16SrI-B are the less homologous to strains available in GenBank. The phylogenetic analyses of translated sequences confirmed these results, and showed a further differentiation of strains GLAWC, and PRIVA from the majority of 16SrI-B strains sequenced, and the I-P strain from populus was very different from all the others. Further research could clarify possible relationship between amp gene, plant host and insect vector towards molecular epidemiology study of ‘Ca. P asteris’-related phytoplasmas.

Periwinkle shoots infected with stolbur phytoplasma strain CH1 (group 16SrXII), dodder transmitted from grapevine infected with “bois noir” disease in Italy, were employed for phytoplasma growth trials in axenic medium. Sterile 1-2 mm stems from two different shoots of two years in vitro growing cultures, were cut longitudinally with razor blades under sterile conditions, immersed in 2 tubes each containing 2 ml Mycoplasma Experience liquid medium (a medium suitable for a wide range of mycoplasmas containing pig serum, a broth base and yeast extract) and maintained in an incubator at 26°C for 7 days. About 300 µl of the medium containing the cut shoots were then transferred to new tubes containing 2 ml of fresh medium. Twenty days after the transfer, PCR assays were carried out using 1 µl medium, denaturated by boiling, as template. The R16F2/R2 primers specific for phytoplasma 16S ribosomal gene were employed for 35 cycles at 47°C annealing temperature. RFLP analyses with TruI carried out on the 4 amplicons of the expected 1.2 kb length obtained with the 4 tubes inoculated showed the presence of 16SrXII phytoplasma DNA. The tubes of the first transfer were then incubated under the same conditions for 9 months when acid colour-changes were seen. Aliquots of 100 µl from the 4 tubes were transferred to 4 tubes of fresh medium which all gave acid colour changes after incubation for 3 days, but PCR testing for phytoplasmas was negative. However, after 10 days’ incubation, PCR testing was positive for the presence of phytoplasma DNA. PCR amplification was carried out using as template 1 µl of the pellets obtained from full speed centrifugation of 100 µl medium, resuspended in 10 µl of sterile distilled water. Primers employed for these latter amplifications were R16(I)F1/R1, specific for phytoplasma groups 16SrI, II and XII, and were used under published conditions; RFLP analyses confirmed that 16SrXII phytoplasmas were present in the broth media after transfers. Amplification of about 600 bp in the 16Sr of phytoplasmas followed by direct sequencing confirmed RFLP results. Further experiments are in progress.

Phytoplasmas of the stolbur group infect a wide range of wild and cultivated plants in several areas worldwide. In Europe they are transmitted by polyphagous planthoppers of the Cixiidae family. Based on actual and in silico RFLP analyses of 16S rDNA nucleotide sequences subgroups have been described in the stolbur group. In vineyards, grapevine-affecting stolbur phytoplasmas are associated with bois noir (BN) disease and are transmitted by Hyalesthes obsoletus Signoret, but in wine-growing areas where H. obsoletus is absent, the presence of stolbur phytoplasmas could implies the existence of alternative vectors. Taxonomy of stolbur phytoplasmas on 16S rDNA gene is still poorly studied, and from virtual RFLP analyses there is growing evidence of a discrete amount of variability suggesting delineation of a number of subgroups. Selecting 16Sr DNA sequences from 26 stolbur phytoplasmas infecting diverse plant species such as grapevine, potatoes, corn, and rhododendron in different areas worldwide, and sharing 99% sequence identity, made it possible to distinguish at least 6 different clusters, and many subclusters, based upon nucleotide sequences within the ca. 1,240 bp; actual RFLP patterns confirmed the presence of nine new subgroups in grapevine plants with BN from Northern and Central Italy, Hungary, Serbia and Iran. Key enzymes for distinguishing among these subgroups were AluI, BfaI, BstUI, together with MboII, FauI, Tsp509I, and Hpy188I. However when compared to its closest relatives, stolbur phytoplasma 16Sr DNA is 97.6% identical to the 16Sr DNA of ‘Candidatus Phytoplasma australiense’, 96.5% and 95.3% identical to ‘Ca. P. graminis’ and ‘Ca. P. caricae’, respectively. Due to its 16S identity with ‘Ca. P. australiense’, the designation of stolbur phytoplasma as a ‘Candidatus’ species will be not possible without a comparative analysis of non ribosomal loci. Biological complexity of stolbur phytoplasmas, indicated by the existence of numerous herbaceous hosts and diverse insect vectors, has stimulated studies on molecular markers of stolbur phytoplasma strains. Characterization of stolbur phytoplasmas in Italian vineyards was performed by multilocus sequence analysis of tuf, hlyC, trxA-truB, cbiQ-glyA, and rplS-csdB genes based on PCR-RFLP assays. Tuf gene was selected since two tuf gene sequence variants of stolbur phytoplasmas were found consistently associated with different herbaceous hosts and natural ecologies in Italian vineyards. For each of the other genes it was possible to define two distinct SNP lineages: hlyC SNP genetic lineages were consistent with those identified on the basis of tuf gene sequences; SNP lineages of trxA-truB, cbiQ-glyA, and rplS-csdB were not consistent with tuf-hlyC SNP lineages. Several SNPs of tuf, hlyC, trxA-truB, cbiQ-glyA, and rplS-csdB genes were positioned within recognition sites of restriction enzymes, and were employed for multilocus sequence analyses that grouped the stolbur phytoplasma strains from grapevines in six SNP genetic lineages. Intriguingly, distribution patterns indicated a different prevalence of these SNP lineages in the geographic areas investigated. Furthermore, the finding that the host specificities of stolbur phytoplasma lineages delineated based on hlyC and tuf genes, points to their possible involvement in the interaction of phytoplasmas with specific hosts. Moreover genotyping of the SecY locus revealed 27 stolbur genotypes in the Euro-Mediterranean areas, that formed two major clusters which are congruent with the two tuf groups. The interaction between phytoplasma membrane proteins and host proteins may influence biological and ecological properties of phytoplasmas such as specificity of plants host and insect vectors or symptom induction in plants. Such phytoplasma proteins can be highly variable; therefore their study should present opportunities for further development of phytoplasma genomic markers. Recently, PCR-sequencing and PCR-RFLP genotyping ...

Pistachio (Pistacia vera L.) is one of the important economic crops in Iran and the reach of high quality with low production cost is an important task for the Iranian pistachio industry. Orchards are mainly obtained by seedlings while also grafting is employed and mainly the Ohadi variety on Badami variety as rootstock is used. Northeastern part of Iran (Khorassan province) is one of the important regions for growing pistachio; this species is known to be tolerant to salts, however Iranian pistachio plantations are on sodic soils and irrigated with low quality, saline water resulting over the recent years in reduction of yields. Decline symptoms such as yellowing of the leaves and reduced vigour in some of the branches were observed in orchard where poor production yield is also reported. Nucleic acid samples from 10 pistachio plants grafted on the Badami rootstok and showing decline symptoms were collected in two different locations Feizabab and Bardaskan (South of Khorassan province) in Iran during spring 2009. Molecular analyses were carried out to verify phytoplasma presence and preliminary identification was achieved by PCR/RFLP analyses on R16F2/R2 amplicons obtained after nested PCR on P1/P7 amplicons. Nine out of the 10 samples were positive in nested-PCR and phytoplasma detected could be affiliated to ribosomal groups 16SrI (‘Candidatus Phytoplasma asteris’ – related), 16SrII (‘Ca. P. aurantifoliae’ - related), 16SrIX (‘Ca. P. foenicium’ - related) and 16SrXII (stolbur-related). Preliminary surveys allow to verify presence of potential phytoplasma vectors such as psyllids and leafhoppers. In particular pistachio psylla (Agonoscena pistaciae) and pistachio leafhopper (Idiocerus stali Fieb) were abundantly present in affected fields. Researches are in progress to clarify epidemiological aspect of the phytoplasma presence and spreading in these Iranian pistachio cultivations.

Climate change, expansion of the EU and the increase of international trade may facilitate the spread of phytoplasma associated diseases, therefore a quick and handily system for correct plant pathogen identification is in great demand. DNA barcoding has arisen as a robust and standardised approach to species identification. QBOL, a new project funded by EU FP7 aims to create a barcode database for all quarantine plant pathogens and to make it available for plant health diagnosticians. Phytoplasma ‘barcoding’ has been performed for many years, particularly using the 16S rDNA, but also other genes such as secY, secA, tuf and ribosomal proteins; however most of these regions span more than 1 kb and/or primers are not generic, which make them impractical for routine barcoding of unknown phytoplasmas. Available sequences of elongation factor Tu (Tuf) and 16S genes were explored for selecting regions suitable for phytoplasma DNA barcoding to develop robust markers of a size that can easily be sequenced (400-600 bp) and that can be obtained from most, if not all phytoplasma ribosomal groups and/or ‘Candidatus Phytoplasma’ species using generic primers. A number of phytoplasma strains maintained in periwinkle and field collected were used for PCR amplification with newly developed primers for Tuf and 16S regions and then sequenced. The 5’ end of the Tuf and the 5’ end of 16S genes were used for barcoding. Sequences of approximately 450 bp for Tuf and 625 bp for the 16S gene were obtained from 60 and 40 phytoplasma strains respectively, belonging to 10 different 16Sr groups. Using these sequences as barcodes it was possible to identify the phytoplasmas into ‘Candidatus species’ or 16Sr groups. The obtained sequences will be available in the newly developed QBOL database.

In contrast to mycoplasmas, which cause an array of disorders in animals and humans, phytoplasmas resisted all attempts to culture them in cell-free media. Despite reduced genome size in comparison to their ancestors, they retain an independent metabolism that allows them to survive in environments as diverse as plant phloem and insect haemolymph. This versatility is a unique property among microbes, shared only with some animal- or plant-infecting viruses and a few other microorganisms such as the causal agent of malaria. Very recently the proof that phytoplasmas can now be grown on laboratory media was provided employing specific commercially available media. Several key points were important to achieve this result: plant tissue selected for isolation must contain viable phytoplasmas; release of phytoplasmas must be achieved from the sieve-tubes in the presence of sieve-tube sap coagulation mechanisms to prevent loss on trauma, the incubation period must be of sufficient time to allow evidence of phytoplasma growth to manifest itself. Inadequacy on any of these points would result in failure so there are many reasons, apart from a suitable medium, that could be responsible for previous failures. Besides the initial seven phytoplasmas, six additional strains were grown: ‘Candidatus Phytoplasma trifolii’ (strain PWB, ribosomal group 16SrVI-A), ‘Ca. P. aurantifolia’ (strain WBDL, ribosomal group 16SrII-B), ‘Ca. P. pruni’ (strain CX, ribosomal group 16SrIII-A), ‘Ca. P asteris’ (strain KVE, ribosomal group 16SrI-C), ‘Ca. P. phoenicium’ (strain PEY, ribosomal group 16SrIX-C), and ‘Ca. P. prunorum’ (strain PLNV6, ribosomal group 16SrX-B). Additional types of phytoplasmas are in the initial isolation stage, but it appears that all the available phytoplasma strains can be cultured. Contrary to the prevailing dogma in plant pathology, therefore, phytoplasmas, like mycoplasmas, can indeed be grown independently from the host(s). The results will produce a more detailed knowledge about basic mechanisms that regulate the survival of phytoplasmas, which are among the smallest known living organisms, but can induce among the most severe epidemics in agriculture.

“…we must make an attempt to species definition. In doing so we are confronted by the paradoxical incongruity of trying to establish a fixed stage in the evolutionary stream. If there is evolution in the true sense of the word, as against catastrophism or creation, we should find all kind of species – incipient species, mature species, incipient genera, as well as all intermediate conditions” (Ernst Mayer, 1942, Systematics and the origin of species). This is the sentence opening R.F. Withcomb presentation for his EKN award in 1994, just twenty years ago, when I attended my first IOM meeting. Since then the study of phytopathogenic mycoplasmas (or phytoplasmas) was mainly devoted toward their classification based on molecular dissecting of their ribosomal DNA and of other conserved genes. The availability of a robust and quite exhaustive classification system allowed recently to also developing barcodes capable to identify them. However this research could end in a sterile play, producing shopping lists of genotypes, if the knowledge of phytoplasma biology is not accompanying their taxonomy. We gained a lot of information from full genome sequencing on putative biochemical pathways, all showing that phytoplasmas are very special microorganisms because they lack a lot for relevant features: cell wall, mobility, key enzymes and pathways. What they have is a small efficient chromosome and tricky metabolisms, allowing them to a trans kingdom life of interaction that often increase activity of their hosts enhancing insect fitness, plant shoot production, changing shape and colour of flowers. For some of them it looks also that they are preparing to become relevant permanent cell hosts. However they are still far from loosing independence and freedom as they can also act as very dangerous pathogens for many relevant agricultural crops. Biology represents still the much unknown part of phytopathogenic mycoplasmas; but recently as a small step toward opening the study of their biology was achieved, and the confirmation of the huge amount of molecular information gained in the last twenty years of research is waiting. Only the knowledge of their day by day biology will help in defining feasible solutions to reduce phytoplasma impact on worldwide agriculture suggesting the best management strategy. Just we need to remember that to win the enemy we must know him very well.

Phytoplasma axenic cultivation was recently achieved using as a source micropropagated phytoplasma infected periwinkle shoots from the collection established more than twenty years ago. Following these results, further work was carried out to verify the possibility of achieving axenic phytoplasma cultivation from field collected phytoplasma-infected samples. Shoots showing typical symptoms of phytoplasma infection, together with asymptomatic ones of the same species, were employed. After phytoplasma identification by PCR/RFLP analyses, midribs stripped from fresh leaves were selected for phytoplasma cultivation. From each sample two midribs were surface sterilized for 1 min in 1% NaClO; ends were then discarded and two half midribs per sample were used for tube inoculation following reported procedures. Uninoculated tubes and tubes inoculated with midribs from healthy shoots were also processed under the same conditions. Colonies were obtained only from tubes inoculated with symptomatic plant material and purified by filtering three times through 0.8 µm filters. Several purified colonies were separately collected from three plates per strain, and subjected to nucleic acid extraction by DNeasy Plant Minikit (Qiagen, Germany). At the same time nucleic acid was also extracted from the corresponding tubes containing pure cultures by a phenol/chloroform based method. Phytoplasma identification was carried out by specific PCR assays on 16S rDNA gene with general and group specific phytoplasma primers. Identification of detected phytoplasmas was done using RFLP analyses with appropriate restriction enzymes (in accord with phytoplasma isolated), and allowed the confirmation of phytoplasma nucleic acid presence. The profiles obtained by restriction of amplicons from liquid cultures and from purified colonies were identical to the ones of the original strains. The results of this study clearly confirm that phytoplasmas can grow independently from the plant host(s) and can also be isolated from field infected plants, with little modification to the procedure employed for phytoplasma isolation from strains maintained in micropropagated periwinkle collection.

Turnera ulmifolia, the yellow alder, is a widely distributed species in Brazil where, besides ornamental, it is used for its medicinal properties. Plants of T. ulmifolia exhibiting witches’ broom growths and yellowing, that are symptoms typically associated with phytoplasma presence, have been observed in the location of Penedo, state of Rio de Janeiro. Symptomatic samples from T. ulmifolia were collected and after total nucleic acid extraction, universal primer pairs were used to prime amplification of phytoplasma 16S rDNA sequences, spacer region and beginning of the 23S rDNA. Expected length amplicons of 1.5 kb (F1/B6 primers) and 1.2 kb (R16F2/R2 primers) were obtained from all symptomatic samples tested after nested PCR on P1/P7 amplicons. RFLP analyses were carried out with Tru1I on F1/B6 and R16F2/R2 amplicons; and with TaqI, and AluI on R16F2/R2 amplicons. RFLP patterns were compared with those of phytoplasma reference strains on same size amplicons and allow to identify the detected phytoplasmas as belonging to ribosomal group 16SrXIII. Additional amplifications for molecular characterization of T. ulmifolia phytoplasmas, with primers rp(I)F1/rp(I)R1A amplifying rplV (rpl22) and rpsC (rps3) genes were then performed. The amplification of this gene resulted in the expected 1.2 kb amplicons and the RFLP profile obtained after Tru1I digestion was clearly different from any of those available in the literature for the same gene, indicating that this phytoplasma may represent a new strain in 16SrXIII group.

Phytoplasmas were detected and identified in some tomato cultivation areas in Mauritius. Symptoms most frequently observed were abnormal shoot proliferation, stunting, reduced leaf and fruit size and shortened internodes. In field-grown tomatoes the incidence of abnormalities rarely exceeded 10%, but under hydroponics up to 100% incidence has been recorded. Two different phytoplasmas were identified by PCR/RFLP analyses. Field-grown tomatoes were infected with a phytoplasma belonging to ribosomal subgroup 16SrI-C and the hydroponically-grown tomatoes were infected with a phytoplasma belonging to ribosomal group 16SrV. Further studies need to be done in order to determine their occurrence, incidence, characterization, host range and mode of transmission, so that eventually the most effective method to control phytoplasma diseases will be determined.

Phytoplasmas are prokaryotes characterized by small genomes (530-1,350 kbp) and by a limited number of metabolic pathways. To investigate molecular mechanisms involved in pathogenesis, the differential display technique was applied to identify plant genes whose transcription was significantly modified in leaves of Catharanthus roseus infected by ‘Candidatus Phytoplasma pyri’ phytoplasma. Out of the sixteen genes identified, eleven were up-regulated by phytoplasma presence, while five were downregulated. Identified genes are mainly involved in plant defence/stress responses, signal transduction, protein metabolism and transport, transcriptional regulation, and plant cell wall structure.