Subgroup 16SrXII-A Research Articles

First Report of 'Candidatus Phytoplasma solani' (16SrXII-A) Associated with Fox Nut (Euryale ferox Salisb.) Stolbur Disease in India.

Fox nut also known as Gorgon nut, Makhana (Euryale ferox Salisb.) is a high value aquatic crop belonging to the family Nymphaeaceae. In India, it is generally grown in flood prone areas of North Bihar, lower Assam, parts of West Bengal, Odisha, Manipur and Tripura (Jana et al., 2018). India contributes nearly 70-80% of the global fox nut production. During September 2021, a phytoplasma like symptom was noticed on fox nut leaves at Basudeopur Farm of Research Centre for Makhana, Darbhanga, Bihar, India (23° 9' N and 65° 53' E). The characteristic symptom was that some portion of leaf lamina deformed along the veins with wrinkled and raised overgrowth or hypertrophy. The veins were thickened and reddened in the infected leaf area. The infection occurs in petiole as well as in flower stalk. The disease incidence was found as high as 30% which caused severe yield loss which was calculated to be 40% in that particular field. Total of 20 sampled fox nut plants, 10 symptomatic and 10 asymptomatic ones, were collected and tested for the presence of phytoplasma. A nested PCR assay using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (J. Jović et al. 2011) amplified the expected ~1.2-kb 16S rDNA fragment in all 10 symptomatic samples. No amplification was detected from asymptomatic samples. One of the ten 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced and was deposited in NCBI under the Accession no.OL873590. BLAST analysis showed that the sequence of the PCR 16S rDNA product was 100% identical to several GenBank sequences of Ca. P. solani (16SrXII Stolbur group) viz. KF907506. Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup. Identification of 'Ca. P. solani' was conducted following the STOL11 stolbur-specific protocol (Radonjić et al. 2009). Sequencing of tuf gene (Elongation factor Tu) was performed by using tuf marker genes (Cvrković et al. 2014) from 10 symptomatic and 10 asymptomatic samples. Sequence of the amplified gene (896 bp) was deposited in GenBank under Accession number OM174272. The presence of 'Ca. P. solani' was detected in all symptomatic samples, while all control plants tested negative. The RFLP analysis of tuf gene nested PCR products using HpaII endonuclease (Fermentas) revealed uniform tuf-b type in all positive samples. Nucleotide blast analyses showed that the tuf gene was 100% identical to STOL11 strain of C. P. solani subgroup 16SrXII-A (Accession No JQ797670). For developing a suitable management strategy, identification of the vector is essential. Leaf hoppers visiting the infected plants as well as nearby crop fields will be tested for presence of the phytoplasma. To the best of our knowledge, this is the first report of Candidatus Phytoplasma solani' (16SrXII-A) infecting Fox nut (Euryale ferox Salisb.) in India. References Cvrković et al. 2014. Plant Pathol. 63:42. https://doi.org/10.1111/ppa.12080 Jana, B. R., et al. 2018. Int. J. Curr. Microbiol. App. Sci. 7(12): 578-587. https://doi.org/10.20546/ijcmas.2018.712.072 Jović, J. et al. 2011. B. Insectol. 64:S83. ISI Radonjić, S. et al. 2009. J. Phytopathol. 157:682. https://doi.org/10.1111/j.1439- 0434.2009.01560.

First report of a 16SrXII‐A phytoplasma strain associated with Camelthorn witches’ broom disease in Iran

Camelthorn (Alhagi maurorum, Fabaceae) is an important weed in Iran but in recent years it has also been planted as a crop to provide fodder. In 2021, camelthorn witches’ broom disease was observed in the Seneiee district in Abarkooh county (Yazd province, Iran). The main disease symptoms were little leaf, internode shortening, yellowing and witches’ broom (Fig. 1). The observed disease incidence was up to 10%. Total DNA was extracted from 0.2 g of fresh leaves from both 14 diseased and five asymptomatic plants. DNA samples were tested for phytoplasmas by direct PCR primed by P1/P7 (Lee et al., 1998) and nested PCR using primers P1/P7 and R16F2n/R16R2 (Gundersen & Lee, 1996). PCR amplicons of c. 1.8 and 1.25 Kb respectively were obtained from all 14 diseased Alhagi maurorum plants but not from the symptomless plants. Four R16F2n/R16R2 primed amplicons were sequenced directly. The sequences were identical and a consensus sequence of 1,245 bp was deposited in GenBank (Accession No. OP020342). Sequence comparison by BLAST analysis showed the highest sequence identity with phytoplasma members of the stolbur group (16SrXII). Phylogenetic analysis using the neighbour-joining method (MEGA 7.0) confirmed that the camelthorn phytoplasma clustered within the group 16SrXII phytoplasma clade, closer to strains affiliated to the 16SrXII-A subgroup than to other strains in the same group (Fig. 2). Computer-simulated analysis with 17 restriction endonucleases using iPhyClassifier (https://plantpathology.ba.ars.usda.gov/) showed that the RFLP pattern derived from the camelthorn phytoplasma 16S rRNA gene was identical to “Ca. Phytoplasma solani”, a 16SrXII-A strain (AF248959). This is the first report of a 16SrXII-A phytoplasma associated with camelthorn witches’ broom disease in Iran and to our knowledge, in the world. Previously a 16SrII subgroup C phytoplasma strain was reported to be associated with camelthorn witches’ broom disease in Iran (Esmaeilzadeh-Hosseini & Salehi, 2021). Since 16SrXII-A subgroup phytoplasmas have been detected previously in grapevine in Fars and Lorestan (Salehi et al., 2014) and Yazd (Salehi et al., 2016) provinces, it is suggested that camelthorn can serve as a reservoir for 16SrXII-A phytoplasmas.

‘Candidatus Phytoplasma solani’ (Subgroup 16SrXII-A) Associated with Nicotiana tabacum Leaf Abnormality in Turkey

Tobacco (Nicotiana tabacum) is among the agricultural products with the highest added value in Turkey. Although frequently associated with its negative effects on human health, it also provides important contributions to the Turkish economy with the employment it creates in rural areas and continues to be a strategic product. Many postgraduate theses and studies related to the sociological and economic importance of the production of this plant, which is of great importance for our country, have been carried out. However, there are very limited studies on plant diseases in tobacco production areas in Turkey. Phytoplasma is one of the important plant pathogens that cause yield loss in tobacco. Since available data on phytoplasma diseases on tobacco was very scarce worldwide, field surveys to collect samples showing phytoplasma infection-like symptoms such as yellowish color changes, leaf blisters, proliferation, dwarfism, and other physical abnormalities were carried out in Çanakkale and Balıkesir provinces of Turkey from June to August 2021. The presence of phytoplasmas in six samples was confirmed by 16S ribosomal DNA amplification by nested-PCR using universal phytoplasma primer sets, which also suggested the pathogen associated with the symptoms on tobacco. According to phylogenetic study and virtual-RFLP analysis using AluI and MseI endonuclease enzymes, the six Turkish tobacco phytoplasma strains all belong to group 16SrXII and have more than 99% nucleotide sequence identity with some members of ‘Candidatus Phytoplasma solani’ of the taxonomic subgroup ‘stolbur’ (16SrXII-A). Genetic distances analysis indicated that group 16SrI was more closely related to 16SrXII than 16SrVI, in agreement with the groups clustering in the phylogenetic tree. Neutrality tests found that 16SrI and 16SrXII groups are experiencing expanding or bottleneck selections, probably due to new mutations in the 16S rRNA gene fragment. Meanwhile, 16SrVI populations are shown to be undergoing balancing selections, indicating that its isolates have evolved for a long time.

Phytoplasma diseases of vegetable crops in Russia

Phytoplasma DNA was detected in 72 samples of vegetable crops collected in eight regions/territories of the Russian Federation, including the Republic of Crimea. The analyzed plants belonged to 13 species (Armoracia rusticana, Artemisia dracunculus, Capsicum annuum, Conundrum sátivum, Cucumis melo, Cucurbita maxima, Daucus carota var.sativus, Melissa officinalis, Petroselinum crispum, Phaseolus vulgaris, Solanum lycopersicum, Solanum melongena and Vicia faba), to 7 families (Apiacea, Asteracea, Brassicaceae, Cucurbitaceae, Fabaceae, Lamiaceae and Solanaceae). The belonging of phytoplasma to a group/subgroup was established by restriction fragment length polymorphism (RFLP) analysis of amplicons obtained in nested polymerase chain reaction (PCR). We identified phytoplasmas of four groups most characteristic of the Russian Federation: Aster yellows - 16SrI, X-disease - 16SrIII, Clover proliferation - 16SrVI and Stolbur - 16SrXII. All phytoplasmas isolated from plants collected in the southern regions of the Russian Federation (Astrakhan and Rostov regions, Krasnodar Territory, and the Republic of Crimea) belonged to stolbur group, subgroup 16SrXII-A, like most phytoplasmas from plants of the Samara region. Phytoplasmas of the 16SrVI group were found in plants from the Moscow, Samara, and Novosibirsk regions, the 16SrIII group - in plants from the Vologda and Moscow regions, and the 16SrI group - only in samples from the Moscow region.

Association of a phytoplasma from ribosomal subgroup 16SrXII‐A with walnut witches’‐broom in Iran

Walnut trees displaying symptoms similar to those caused by phytoplasmas, including foliar chlorosis, yellowing and shortening of internodes, were found in a fruit orchard in the north of Fars province, Iran in 2020 (Fig. 1). Samples were collected and analysed by PCR to detect and identify possible phytoplasma(s) associated with diseased plants. Four diseased and four asymptomatic walnut trees were sampled and analysed for the presence of phytoplasma. Total DNA was extracted from 0.1 g of midribs using the CTAB method. Nested PCR was performed using universal phytoplasma primers P1/P7 (Deng & Hiruki, 1991) in the first round, and R16F2n/R16R2 (Smart et al., 1996) in the second. PCR products of the expected size, c. 1.2 kb, were obtained from all diseased plants but not from the symptomless ones. The nucleotide sequence was determined directly and submitted to GenBank (Accession No. MZ005313). A BLAST search showed that the phytoplasma associated with walnut witches’-broom had the highest sequence identity (99.9%) with Iranian alfalfa witches’-broom phytoplasma (KT763373), a member of the ribosomal subgroup 16SrXII-A (‘Candidatus Phytoplasma solani’). Virtual RFLP patterns obtained with iPhyclassifier (https://plantpathology.ba.ars.usda.gov/cgibin/resource/iphyclassifier.cgi) supported the sequence analysis results. Walnut witches’-broom phytoplasma was first reported in Iowa, USA and was associated with the group 16SrIII (‘Ca. P. pruni’) (Yun & Harrington 2011). In Iran, the 16SrXII phytoplasma group has been reported in alfalfa, almond, aubergine, grapevine, peach, plum and tomato (Siampour et al., 2019). It has also been identified from Cannabis sativa (Sichani et al., 2011) and Salix babylonica (Zamharir & Taheri, 2017). In Fars province, walnut orchards are adjacent to stone fruit orchards and vineyards, so it is likely that this phytoplasma has been transferred from such crops to walnut trees. Since large areas of walnut are grown in Iran, this phytoplasma may be a serious threat to walnut cultivation. To the best of our knowledge, this is the first report of the association of a 16SrXII-A related phytoplasma with walnut witches’-broom globally. The authors would like to thank Dr. Sadegh Sadeghi and Mr. Mohsen Amiri for great help in the field.

Molecular detection and identification of a ‘Candidatus Phytoplasma solani’-related strain associated with pumpkin witches’ broom in Xinjiang, China

Pumpkin plants showing symptoms of witches’ broom (PuWB) were observed in Xinjiang Uyghur autonomous region, China, in September 2018. A phytoplasma was detected in symptomatic plants by PCR amplifying portions of the 16S ribosomal and tuf genes. In addition, the phylogeny based on these genes sequencing indicated that the PuWB strain clusters with ‘Candidatus Phytoplasma solani’ (subgroup 16SrXII-A). Furthermore, based on in silico and in vitro restriction fragment length polymorphism analyses, the PuWB phytoplasma was confirmed as a ‘Ca. P. solani’-related strain. This was the first record of the occurrence of phytoplasma presence in pumpkins in China, and the first record of 16SrXII phytoplasma infecting pumpkins in the world.

Natural phytoplasma infections on fruit, vegetable and weed plants at the same agroecosystem and their molecular properties

The phytoplasma associated diseases are an emerging threat to fruit and vegetable crops leading severe yield losses worldwide. Pear (Pyrus communis L.) trees, with symptoms of severe reddening, dwarfing and shoot proliferation were observed in pear orchards of Malatya province of Turkey. Tomato (Solanum lycopersicum L.) plants grown nearby the symptomatic pear orchard displaying leaf rolling, severe flower sterility and purple leaves were observed at the same agroecosystem. To verify the presence and diversity of phytoplasmas, symptomatic pears and tomatoes were sampled and weeds nearby the symptomatic plants were collected. Total plant DNA was purified from midrib of collected leaves using a commercial kit. The DNA samples were analyzed by nested polymerase chain reaction (PCR) using universal primer pairs to amplify 16S rDNA fragments. The phytoplasmas detected in collected samples were differed according to the host. Here we detected and characterized ‘Candidatus Phytoplasma pyri’ belonging to apple proliferation group (subgroup 16SrX-C) from a pear tree, ‘Candidatus Phytoplasma trifolii’ belonging to clover proliferation group (subgroup 16SrVI-A) from a weed (Amaranthus retroflexus) and ‘Candidatus Phytoplasma solani’ belonging to the stolbur phytoplasma group (subgroup 16SrXII-A) from a tomato plant. Direct sequencing of PCR products verified the phytoplasmal nature of the infections. The occurrence of ‘Ca. P. trifolii’ on A. retroflexus is the first report for the world. The irregular presence of the phytoplasmas in fruit and vegetable crops and weeds indicates continuous spread of the phytoplasmas threatening the new crops and new horizons.

Biochemical Changes in Leaves of Vitis vinifera cv. Sangiovese Infected by Bois Noir Phytoplasma.

Bois noir is a disease associated with the presence of phytoplasma ‘Candidatus Phytoplasma solani’ belonging to the Stolbur group (subgroup 16SrXII-A), which has a heavy economic impact on grapevines. This study focused on the changes induced by phytoplasma in terms of the profile and amount of secondary metabolites synthesized in the phenylpropanoid pathway in leaves of Vitis vinifera L. red-berried cultivar Sangiovese. Metabolic alterations were assessed according to the disease progression through measurements of soluble sugars, chlorophyll, and phenolic compounds produced by plant hosts, in response to disease on symptomatic and asymptomatic Bois noir-positive plants. Significant differences were revealed in the amount of soluble sugars, chlorophyll, and accumulation/reduction of some compounds synthesized in the phenylpropanoid pathway of Bois noir-positive and negative grapevine leaves. Our results showed a marked increase in phenolic and flavonoid production and a parallel decrease in lignin content in Bois noir-positive compared to negative leaves. Interestingly, some parameters (chlorophyll a, soluble sugars, total phenolic or flavonoids content, proanthocyanidins, quercetin) differed between Bois noir-positive and negative leaves regardless of symptoms, indicating measurable biochemical changes in asymptomatic leaves. Our grapevine cultivar Sangiovese results highlighted an extensive modulation of the phenylpropanoid biosynthetic pathway as a defense mechanism activated by the host plant in response to Bois noir disease.

Molecular characterization of stolbur phytoplasmas in pepper and tomato from Bulgaria

Tomato and pepper are the main vegetable crops cultivated in Bulgaria. Phytoplasma diseases, mainly stolbur, are important plant diseases for these crops in Bulgaria. The goal of the present paper was to verify association of phytoplasmas with the observed disease symptoms in tomato and pepper and to identify the phytoplasmas detected using RFLP analysis of conserved genes and other uncharacterised phytoplasma chromosomal regions. The presence of phytoplasmas was confirmed in all the samples of tomato and pepper showing typical stolbur symptoms. A phytoplasm sample, which caused severe symptoms, showed the same pattern as the reference strain Mol, while all other phytoplasmic reference strains showed different polymorphisms. RFLP profiles were found useful in distinguishing phytoplasmas in stolbur subgroup (16SrXII-A) in natural plant hosts.

Molecular identification of phytoplasmas in Russian vineyards

Phytosanitary surveys of vineyards in the Krasnodar region were carried out for phytoplasma detection. Symptomatic samples were tested by nested PCR. According to the restriction fragment length polymorphism analysis the identified phytoplasma belongs to the “stolbur” subgroup 16SrXII-A.

Detection and characterisation of phytoplasma strains associated with field bindweed witches’ broom disease in Iran

During 2013–2015 surveys in Fars, Lorestan and Yazd provinces (Iran), a field bindweed witches’ broom (FBWB) disease was observed. The main symptoms were reduction of leaves size, yellowing, internode shortening, witches’ broom and stunting. The agent of FBWB was dodder transmitted to periwinkle plants inducing phytoplasma-type symptoms. Amplifications of nearly 1.8 and 1.2 kbp were, respectively, obtained from 15 symptomatic bindweed plants and 28 symptomatic dodder-inoculated periwinkles. Virtual RFLP analyses showed that the phytoplasma detected belonged to 16SrXII-A subgroup, and it was the same in all the samples examined; phylogenetic analyses confirmed it as a ‘Candidatus Phytoplasma solani’-related strain. This is the first report of 16SrXII-A phytoplasmas presence in bindweed plants showing witches’ broom symptoms in Fars, Lorestan and Yazd provinces. As a perennial widespread weed, it may act as a 16SrXII-A phytoplasma source for alfalfa, grapevine, Sophora alopecuroides, tomato, hemp and Japanese spindle reported diseases in these Iranian provinces.

Diverse phytoplasmas associated with leguminous crops in Russia

A large scale survey of diseased legume plants (mainly clover and alfalfa in the Fabaceae family) was conducted from 2009 to 2013 in four Economic Regions of Russia, Northern (Arkhangelsk and Vologda oblast), Central (Moscow oblast), Volga (Samara oblast) and West Siberian (Novosibirsk oblast). The majority of infected clover plants exhibited symptoms typical of clover phyllody (CPh), clover yellow edge (CYE), or clover proliferation (CP), and infected alfalfa plants exhibited symptoms typical of alfalfa witches’-broom (AWB). Of 161 symptomatic plants from 22 different legume species, 103 tested positive for phytoplasma infection. Phytoplasmas belonging to four groups and six subgroups were detected, of which 31.1% were group 16SrI, with the majority belonging to subgroup 16SrI-C- (causal agent of CPh disease), two belonging to 16SrI-B and two group 16SrI phytoplasmas not identified to the subgroup level;47.6% were group 16SrIII, with the majority belonging to subgroup 16SrIII-B or 16SrIII-B variant (causal agent of CYE disease), and one strain belonging to16SrIII-F; 8.7% were subgroup 16SrVI-A (causal agent of CP and AWB diseases); 9.7% were subgroup 16SrXII-A (causal agent of AWB disease); and 2.9% were mixed infected with subgroups 16SrIII-B and 16SrI-C. The predominant phytoplasma species detected varied by region. In the Northern and Central Regions, the majority of the phytoplasmas detected belonged to subgroups 16SrI-C and 16SrIII-B. In the West Siberian and the Volga Regions, the phytoplasmas predominately detected belonged to subgroups 16SrVI-A and 16SrXII-A, respectively. Subgroup 16SrIII-F was detected in a single plant in the West Siberian Region and a mixed infection of 16SrIII-B and 16SrI-C was detected in three plants, one in the Northern Region and two in the Central Region. Eleven species of insects of the order Hemiptera, suborder Auchenorrhyncha, were collected from leguminous plants in the Moscow oblast of the Central Region. Euscelis incisus and Aphrodes bicinctus were the most prevalent species and may be potential phytoplasma vectors in the Central Region.

THE FIRST REPORT OF A 16SrXII-A PHYTOPLASMA ASSOCIATED WITH TOMATO BIG BUD DISEASE IN IRAN

In 2013 surveys, up to 7.5% incidence of big bud disease was observed in tomato fields of Kazerun area (Fars province, Iran). The main disease symptoms were big bud, little leaf, flower virescence, phyllody, proliferation and sterility. To investigate the phytoplasma presence, total DNAs extracted from four symptomatic and four symptomless tomato plants were tested by nested PCR using P1/P7 primer pair followed by R16F2n/R16R2 primers (Lee et al., 1998). Amplicons of ca. 1.8 and 1.2 kb, respectively were amplified in samples of symptomatic plants but not of symptomless ones. Four P1/P7 amplicons from symptomatic tomato plants were cloned and sequenced. The obtained 16S rDNA sequences showed 100% sequence identity with each other and a representative of these sequences deposited in GenBank (Accession No. KX098490). BLAST search using full length 16S rRNA gene sequence revealed that Kazerun tomato big bud (KTBB) sequence showed 100% identity with a 'Candidatus Phytoplasma solani' strain (AF248959), representative of 16SrXII-A subgroup. Computer-simulated restriction analysis using iPhyClassifier showed that the RFLP profile of KTBB 16S rDNA F2nR2 fragment was identical (similarity coefficient 1.00) to the reference pattern of 16SrXII-A (AF248959). Phylogenetic analysis revealed that KTBB phytoplasma clustered with16SrXII group phytoplasmas closest to 16SrXII-A subgroup reference strain (AF248959). To our knowledge this is the first report of a 16SrXII-A phytoplasma associated with TBB disease in Iran. 16SrXII- related phytoplasmas have been previously reported from grapevine (Salehi et al., 2014) and Cannabis sativa (Vali Sichani et al., 2011) in Iran.

Molecular Identification and Diversity of ‘Candidatus Phytoplasma solani’ Associated with Red-leaf Disease ofSalvia miltiorrhizain China

Reddening disease has recently been threatening Salvia miltiorrhiza in China, ranging from 30 to 50%. The main symptoms observed, such as plant stunting, inflorescence malformation, leaf reddening, fibrous roots browning, skin blackening and eventually root rot, are typically associated with phytoplasma infection. The presence of phytoplasmas was demonstrated through phytoplasma-specific PCR, with the expected amplification (1.8 kb) from symptomatic S. miltiorrhiza plants from Shangluo, Shangzhou and Luonan fields in Shaanxi Province of China. The sequences of 16S rRNA, tuf, secY and vmp1 genes amplified from LN-1 phytoplasma shared the closest homologies of 99%, 100%, 99% and 98% with those of the reference strain Candidatus Phytoplasma solani (subgroup 16SrXII-A), respectively. The phylogenetic trees showed that LN-1 phytoplasma clustered with the members of 16SrXII-A group, including Ca. P. solani. Computer-simulated restriction fragment length polymorphism analysis further supported this classification. Diversity analysis showed that all ‘Ca. P. solani’ strains identified from the three different regions examined shared 100% identical 16S rRNA, tuf, secY and vmp1 nucleotide sequences. To the best of our knowledge, this is the first report of phytoplasma infecting the medicinal plant of S. miltiorrhiza. The results demonstrate that ‘Ca. P. solani’ is the presumptive aetiological agent of S. miltiorrhiza reddening disease in China.

Molecular detection of “stolbur” phytoplasma in celery plants in Romania

In September 2014, several plants of celery showing symptoms of reddish and yellowish leaves were found in a house garden located in Southern Romania (Giurgiu County). After sampling and molecular analyses carried out by nested PCR assays on phytoplasma ribosomal gene, amplicons were obtained from all symptomatic plants, but not from those that were asymptomatic. The restriction fragment length polymorphism (RFLP) analysis, with RsaI and Tru1I restriction enzymes, was carried out on the phytoplasma DNAs amplified in nested PCR with the primer pair R16F2n/R2. The phytoplasmas from all symptomatic celery plants showed identical RFLP profiles, which corresponded to the one of the “stolbur” phytoplasma, that belongs to 16SrXII-A subgroup (‘Candidatus Phytoplasma solani’). This represents the first report on molecular detection of a phytoplasma infecting celery plants in Romania.

Molecular identification and phylogenetic analysis of phytoplasmas associated with alfalfa witches’ broom diseases in the western areas of Iran

During 2014–2015 phytoplasmas associated with alfalfa witches’ broom (AWB) in the western areas of Iran were characterized by molecular analyses studies. Polymerase chain reaction was carried out using direct and nested as says to amplify the 16S ribosomal gene for phytoplasma identification. RFLP analysis of R16F2n/R16R2 amplicons with selected restriction enzymes and phylogenetic studies showed that AWB associated phytoplasma strains from Salafchegan (Qom province), Arak (Markazi province), Tabriz (East Azerbaijan province), Dehgolan and Qhorveh (Kordestan province), Eghlid (Fars province), Mahidasht (Kermanshah province), Dehdasht (Kohgiluyeh and Boyer-Ahmad province), Borujerd (Lorestan province), Chenar-e Mahmudi and Shahrekian (Chaharmahal and Bakhtiari province) belong to 16SrXII-A subgroup (“stolbur”), while AWB associated phytoplasmas from Jowkar (Hamedan province) belong to 16SrII-D subgroup. Disease incidence was variable in the different areas and the highest infection rate (14.7%) was observed in Chenar-e Mahmudi (Chaharmahal and Bakhtiari province). The highly AWB affected areas were generally located in the warmer parts of each region in sparse fields. This is the first report and characterization of 16SrXII-A phytoplasmas associated with alfalfa witches’ broom diseases in Iran and occurrence of 16SrII-D in western areas of Iran.

Occurrence and identification of grapevine phytoplasmas in main viticultural regions of Turkey

Intensive surveys were conducted in the main viticultural areas of Turkey in 2009-2010. Two hundred eighty nine symptomatic and 20 non-symptomatic plant samples were collected and subjected to nucleic acid extraction followed by PCR, nested PCR and RFLP analyses to detect phytoplasma presence and for their identification. The incidence rate of phytoplasma infection was 18.33% and the majority of the symptomatic grapevines were infected with grapevine yellows phytoplasmas belonging to 16SrXII-A subgroup (“bois noir”). Phytoplasmas of 16SrV group, aster yellows (16SrI-B) and pigeon pea witches’ broom (16SrIX) groups were also detected in the surveyed vineyards. Phytoplasma-associated infections were present more on wine grapevine cultivars (73.6%), such as Alphonse Lavallee, Alicante Bouschet, Chardonnay, Shiraz, Cabernet Sauvignon, Merlot, Sauvignon Blanc and Pinot Noir, compared to table grapes (26.4%), such as Bogazkere, Sirfani, Tahannebi and Emir.

First Report of 16SrXII-A Subgroup Phytoplasma (Stolbur) Associated with Reddening ofOenothera biennisin Serbia.

First Report of 16SrXII-A Subgroup Phytoplasma (Stolbur) Associated with Reddening of<i>Oenothera biennis</i>in Serbia.

First Report of Candidatus Phytoplasma solani Associated with Potato Plants in Greece.

In August 2013, potato plants (Solanum tuberosum) cv. Banba displaying symptoms resembling those caused by Candidatus Phytoplasma solani (potato stolbur phytoplasma) were observed in a 2-ha field in the area of the Peripheral Unit of Drama (northern Greece). The plants were 10 weeks old and their symptoms included reddening and upward rolling of leaflets, reduced size of leaves, shortened internodes, and aerial tuber formation. Incidence of affected plants was estimated to be 40% in the field. Four symptomatic potato plants were collected for laboratory testing of possible phytoplasma infection. From each of these four plants, total DNA was extracted from mid veins of reddish leaflets from apical shoot parts and of leaflets emerging from aerial tubers, using a phytoplasma enrichment procedure (1). A nested PCR using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (3) amplified the expected ~1.2-kb 16S rDNA fragment in all four symptomatic potato plants. No amplification was observed with DNA similarly extracted from leaflets of asymptomatic potato plants of the same variety collected from an apparently healthy crop. One of the four 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced by Beckman Coulter Genomics (United Kingdom). At least twofold coverage per base position of the cloned PCR product was achieved. BLAST analysis showed that the obtained sequence of the PCR 16S rDNA product was: i) 100% identical to several GenBank sequences of Ca. P. solani strains, including strains detected previously in Greece infecting tomato (GenBank Accession No. JX311953) and Datura stramonium (HE598778 and HE598779), and ii) 99.7% similar to that of the Ca. P. solani reference strain STOL11 (AF248959). Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup (AF248959). The sequence of this PCR product was deposited in NCBI GenBank database under the accession no. KJ810575. The presence of the stolbur phytoplasma in all four symptomatic potato plants examined was further confirmed by nested PCR using the stolbur-specific STOL11 primers (3) targeting non-ribosomal DNA. Based on the observed symptoms in the field and laboratory molecular examinations, we concluded that the potato plants were infected by a Ca. P. solani related strain. The stolbur disease has been previously reported in Greece affecting tomato (2,5) and varieties of D. stramonium (4). To our knowledge, this is the first report of a Ca. P. solani related strain infecting a potato crop in Greece. As northern Greece is a center of potato production, the source of this pathogen is to be investigated.

Lietuvoje aptikti vabzdžiai, galimi fitoplazmų ‘Candidatus Phytoplasma asteris’ ir ‘Ca. Phytoplasma pruni’ giminingų kamienų pernešėjai

Phytoplasma strains affiliated with groups 16SrI, 16SrIII, 16SrV and 16SrXII have been found in Lithuania, but their insect vectors in the country have not been determined. ‘Candidatus Phytoplasma asteris’ and ‘Ca. Phytoplasma pruni’-related phytoplasma strains were identified in five leafhopper species and three spittlebug species occurring in Lithuania. The occurrence in Lithuania of Anaceratagallia ribauti, reported as a vector of stolbur phytoplasma (‘Ca. Phytoplasma solani’, subgroup 16SrXII-A) elsewhere in Europe, provokes the question of whether this phytoplasma may be present but yet undetected in Lithuania. The finding of subgroup 16SrI-C strains in A. ribauti, Aphrodes sp., Macrosteles sexnotatus and Euscelis incisus; 16SrI-B strains – in Aphrophora alni, Aphrodes sp. and Cicadella viridis; 16SrIII-B strains – in A. ribauti; and 16SrIII-P – in Aphrodes sp. and E. incisus is consistent with the hypothesis that these leafhopper species are vectors of the respective phytoplasmas in Lithuania. Transmission trials were initiated based on these results. Results thus far have revealed that Lithuanian biotype of E. incisus is capable of vectoring subgroup 16SrI-C phytoplasma strains that are found in Lithuania. The presence of diverse phytoplasmas in three spittlebug species, Aphrophora alni, Lepyronia coleopterata and Philaenus spumarius, indicates that these xylem feeders actually ingested phloem sieve cell contents during feeding on phytoplasmainfected plants, bringing into focus the question of whether some of such xylem feeders might act as occasional vectors of phytoplasmas.