Detection and Molecular Characterization of a 16SrII‐A* Phytoplasma in Grapefruit (Citrus paradisi) with Huanglongbing‐like Symptoms in China

In the year 2010, in a survey in Guangxi Province, China, to detect and characterize phytoplasmas in a huanglongbing (HLB)‐infected grapefruit (Citrus paradisi) orchard, 87 leaf samples with symptoms of blotchy mottle were collected from symptomatic grapefruit trees, and 320 leaf samples from symptomless trees adjacent to the symptomatic trees. Nested polymerase chain reaction (PCR) using universal phytoplasma primer set P1/P7 followed by primer set fU5/rU3 identified 7 (8.0%) positive samples from symptomatic samples but none from symptomless samples. Of the 87 symptomatic samples, 77 (88.5%) were positive for ‘Candidatus Liberibacter asiaticus’ and 5 for both phytoplasma and ‘Ca. L. asiaticus’. Sequence analysis indicated that seven 881‐bp amplicons, amplified by nested phytoplasma primer sets P1/P7 and fU5/rU3, shared 100.0% sequence identity with each other. Genome walking was then performed based on the 881 bp known sequences, and 5111 bp of upstream and downstream sequences were obtained. The total 5992 bp sequences contained a complete rRNA operon, composed of a 16S rRNA gene, a tRNA^Ile gene, a 23S rRNA gene and a 5S rRNA gene followed by eight tRNA genes. Phylogenetic analysis and virtual restriction fragment length polymorphism analysis confirmed the phytoplasma was a variant (16SrII‐A*) of phytoplasma subgroup 16SrII‐A. As phytoplasmas were only detected in blotchy‐mottle leaves, the 16SrII‐A* phytoplasma identified was related to HLB‐like symptoms.     

Molecular Identification of a 16SrII‐A Group‐Related Phytoplasma Associated with Cinnamon Yellow Leaf Disease in China

Chinese cinnamon (Cinnamomum cassia Presl), an evergreen tree native to China, is a multifaceted medicinal plant. The stem bark of cinnamon is used worldwide in traditional and modern medicines and is one of the most popular cooking spices. In recent years, cinnamon with pronounced yellow leaf symptoms has been observed in their natural habitat in Hainan, China. Phytoplasmas were detected from symptomatic cinnamon trees via polymerase chain reaction using phytoplasma universal primers P1/P7 followed by R16F2n/R16R2. No amplification products were obtained from templates of asymptomatic cinnamon trees. These results indicated a direct association between phytoplasma infection and the cinnamon yellow leaf (CYL) disease. Sequence analysis of the CYL phytoplasma 16S rRNA gene determined that CYL phytoplasma is a ‘Candidatus Phytoplasma australasiae’‐related strain. Furthermore, virtual restriction fragment length polymorphism pattern analysis and phylogenetic studies showed that CYL phytoplasma belongs to the peanut witches’‐broom (16SrII) group, subgroup A. This is the first report of a 16SrII group phytoplasma infecting cinnamon under natural conditions.     

Molecular Identification of a ‘Candidatus Phytoplasma ziziphi’‐related Strain Infecting Amaranth (Amaranthus retroflexus L.) in China

Amaranth (Amaranthus retroflexus L.) is a common weed that grows vigorously in orchards, roadside verges, fields, woods and scrubland in China. In 2009, phytoplasma disease surveys were made in orchards in Beijing, China, and stem/leaf tissues were collected from asymptomatic amaranths. Direct PCR using universal phytoplasma primers P1/P7 detected 16S rRNA gene sequences in every DNA sample extracted from the symptomless amaranths. Sequence alignment and phylogenetic analyses of the 16S rRNA gene determined that the amaranth phytoplasma strain was related to ‘Candidatus Phytoplasma ziziphi’. Furthermore, virtual RFLP pattern analysis showed that the amaranth phytoplasma belonged to the 16SrV‐B subgroup. This is the first report of symptomless plants containing a ‘Candidatus Phytoplasma ziziphi’‐related strain.     

Aetiology of Opuntia ficus-indica malformations and stunting disease

The phytoplasma aetiology of epidemic stunting of cladodes and stunted growth observed in a cactus pear plantation in Carlentini (Sicily, Italy) was investigated with graft inoculation trials and PCR/restriction fragment length polymorphism analyses of 16S ribosomal RNA (rRNA) gene and S3 ribosomal protein gene. After sequencing of the 1525‐bp from 16S rRNA gene of both naturally infected and graft‐inoculated symptomatic cactus pear samples, phytoplasma TS belonging to ribosomal subgroup 16SrII‐C were identified as aetiological agents of this worldwide spread disease.     

Detection and Characterization of Phytoplasma Associated with Big Bud Disease of Tomato in China

Symptomatic tomato plants exhibiting big bud, proliferation and small leaves of lateral shoots, purplish top leaves, phyllody, enlarged pistils, hypertrophic calyxes and small and polygonal fruit were collected in Yunnan Province of China. Pleomorphic phytoplasma‐like bodies were observed in the phloem sieve tube elements of symptomatic plants by transmission electron microscopy. The presence of phytoplasma in collected samples was further analysed and identified by PCR and virtual computer‐simulated restriction fragment length polymorphism (virtual RFLP). A 1.2 kb product was amplified by PCR with universal primers R16F2n/R16R2. Sequence comparisons revealed that the tested strains shared 99% 16S rRNA gene sequence similarity with members of ‘Candidatus Phytoplasma aurantifolia’ (16SrII group). Phylogenetic and virtual RFLP analysis of the 16S rRNA gene sequences confirmed that the phytoplasma is a member of the 16SrII group. This is the first report of 16SrII group phytoplasma infecting tomato in China.     

Occurrence and Characterization of a 16SrII‐D Subgroup Phytoplasma Associated with Parsley Witches’ Broom Disease in Iran

During 2010–2013 surveys for the presence of phytoplasma diseases in Yazd province (Iran), a parsley witches’ broom (PrWB) disease was observed. Characteristic symptoms were excessive development of short spindly shoots from crown buds, little leaf, yellowing, witches’ broom, stunting, flower virescence and phyllody. The disease causative agent was dodder transmitted from symptomatic parsley to periwinkle and from periwinkle to periwinkle by grafting inducing phytoplasma‐type symptoms. Expected length DNA fragments of nearly 1800 and 1250 bp were, respectively, amplified from naturally infected parsley and experimentally inoculated periwinkle plants in direct polymerase chain reaction (PCR) using phytoplasma primer pair P1/P7 or nested PCR using the same primer pair followed by R16F2n/R16R2 primers. Restriction fragment length polymorphism and phylogenetic analyses of 16S rRNA gene sequences showed that the phytoplasma associated with PrWB disease in Yazd province belong to 16SrII‐D phytoplasma subgroup. This is the first report of association of a 16SrII‐related phytoplasma with PrWB disease in Iran.     

Molecular Characterization and Potential Insect Vector of a Phytoplasma Associated with Garden Beet Witches' Broom in Yazd, Iran

In 2002, garden beet witches’ broom (GBWB) phytoplasma was detected for the first time in garden beet plants (Beta vulgaris L. ssp. esculenta) in Yazd, Iran. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphic (RFLP) analysis of PCR‐amplified phytoplasma 16S rDNA were employed for the detection and identification of the phytoplasma associated with garden beet. A phytoplasma belonging to subgroup 16SrII‐E, in the peanut witches’ broom group (16SrII), was detected in infected plants. Asymptomatic plant samples and the negative control yielded no amplification. The result of analysis of the nucleotide sequence of a 1428 bp fragment of 16S rDNA gene from GBWB phytoplasma (GenBank accession number DQ302722 ) was basically consistent with the classification based on RFLP analysis, in which GBWB phytoplasma clustered with phytoplasmas of the 16SrII‐E subgroup. A search for a natural phytoplasma vector was conducted in Yazd in 2004, in an area where garden beet crops had been affected since 2002. The associated phytoplasma was detected in one leafhopper species, Orosius albicinctus, commonly present in this region. The leafhopper O. albicinctus was used in transmission tests to determine its vector status for the phytoplasma associated with GBWB. Two of eight plants that had been fed on by O. albicinctus, showed mild symptoms of GBWB including stunting and reddening of midveins. A phytoplasma was detected in the two symptomatic test plants by PCR using universal primers and it was identified by RFLP as the GBWB phytoplasma. This finding suggests O. albicinctus is a vector of the GBWB phytoplasma.     

Characterization of 16SrII-D subgroup associated phytoplasmas in new host plants in Egypt

Samples of three plant species displaying phytoplasma symptoms were collected from Kafrelsheikh and Al-Gharbia governorates during 2014. Witches’ broom and virescence symptoms were observed in periwinkle (Catharanthus roseus). Onion (Allium cepa) plants showed yellowing, streaks and twisting and Opuntia abjecta with proliferation and cylindrical of cladodes. Total DNA was extracted from symptomatic and asymptomatic plants, and phytoplasma were detected in all 12 symptomatic plants collected through direct and nested PCR assays with primers P1/P7 and R16F2n/R16R2. The results of phylogenetic analysis revealed that the phytoplasma isolates belong to 16SrII group. With a nucleotide identity greater than 98.7% with three members of 16SrII group, Papaya yellow crinkle, Y10097; “Ca. P. aurantifolia”, U15442; and peanut witches’ broom, Al33765, the strains identified in this study are “Ca. P. aurantifolia”-related strains. Virtual RFLP analysis of the 16S rRNA gene sequences with 17 restriction enzymes confirmed that the phytoplasma isolates belong to the “Candidatus Phytoplasma australasia” 16SrII-D subgroup. To the best of our knowledge, periwinkle, onion and Opuntia abjecta are considered new hosts for 16SrII group in Egypt.     

Detection and Identification of a 16SrII Group Phytoplasma Causing Clover Little Leaf Disease in Iran

White clover plants showing little leaf and leaf reddening symptoms were observed in Isfahan Province in central Iran. Restriction fragment length polymorphism analyses of nested PCR‐amplified fragments from Iranian clover little leaf phytoplasma isolates and representative phytoplasmas from other phytoplasma groups using AluI, CfoI, KpnI and RsaI restriction enzymes indicated that the clover phytoplasma isolates are related to the peanut WB group. Sequence analyses of partial 16S rRNA fragments showed that Iranian clover little leaf phytoplasma has 99% similarity with soybean witches'‐broom phytoplasma, a member of the peanut WB (16SrII) phytoplasma group. This is the first report of clover infection with a phytoplasma related to the 16SrII group.     

Detection and molecular characterization of phytoplasmas associated with vegetable and alfalfa crops in Qassim region

During a survey conducted in Qassim province, Saudi Arabia, in the year in 2015, 120 samples of carrot (Daucus carota subsp. sativus), onion (Allium cepa), faba bean (Vicia faba), green mustard (Brassica juncea) and alfalfa (Medicago sativa) plants displaying symptoms reminiscent of phytoplasma diseases were collected and tested for phytoplasma infection. Phytoplasma-specific PCR products were only amplified from symptomatic plants by nested-PCR. Disease incidence ranged from 3.14% in alfalfa crop fields 1 year after cultivation to 77.48% in 3-year-old fields. In the five carrot fields sampled in this study, the incidence changed from 3.2% to 100% after 7 months of cultivation. Phylogenetic analysis revealed that all Qassim phytoplasma isolates belong to the 16SrII group. Most of them shared 100% identity with papaya yellow crinkle (16SrII-D Y10097). The results from phylogenetic and virtual restriction fragment length polymorphism analyses of the 16S rRNA gene sequence confirmed that the phytoplasma of Qassim isolates under study is a member of 16SrII-D subgroup. To the best of my knowledge, the onion and green mustard are considered new hosts for the 16SrII group; therefore, this is the first report on the association of phytoplasma with diseases of faba bean, onion, carrot, mustard and alfalfa in Qassim province, Saudi Arabia.     

Phytoplasma from little leaf disease affected sweetpotato in Western Australia: detection and phylogeny

Symptoms of leaf and stem chlorosis and plant stunting were common in sweetpotato plants (Ipomoea batatas) in farmers’ fields in two widely separated locations, Kununurra and Broome, in the tropical Kimberley region in the state of Western Australia in 2003 and 2004. In the glasshouse, progeny plants developed similar symptoms characteristic of phytoplasma infection, consisting of chlorosis and a stunted, bushy appearance as a result of proliferation of axillary shoots. The same symptoms were reproduced in the African sweetpotato cv. Tanzania grafted with scions from the plant Aus1 with symptoms and in which no viruses were detected. PCR amplification with phytoplasma‐specific primers and sequencing of the 16S‐23S rRNA gene region from two plants with symptoms, Aus1 (Broome) and Aus142A (Kununurra), revealed highly identical sequences. Phylogenetic analysis of the 16S rRNA gene sequences obtained from previously described sweetpotato phytoplasma and inclusion of other selected phytoplasma for comparison indicated that Aus1 and Aus142A belonged to the Candidatus Phytoplasma aurantifolia species (16SrII). The 16S genes of Aus1 and Aus142A were almost identical to those of sweet potato little leaf (SPLL‐V4) phytoplasma from Australia (99.3%–99.4%) but different from those of the sweetpotato phytoplasma from Taiwan (95.5%–95.6%) and Uganda (SPLL‐UG, 90.0%–90.1%). Phylogenetically, Aus1, Aus142A and a phytoplasma previously described from sweetpotato in the Northern Territory of Australia formed a group distinctly different from other isolates within Ca. Phytoplasma aurantifolia species. These findings indicate that novel isolates of the 16SrII‐type phytoplasma pose a potential threat to sustainable sweetpotato production in northern Australia.     

A new phytoplasma associated with little leaf disease in azalea: multilocus sequence characterization reveals a distinct lineage within the aster yellows phytoplasma group

An azalea little leaf (AzLL) disease characterised by abnormally small leaves, yellowing and witches'‐broom growth symptoms was observed in suburban Kunming, southwest China. Transmission electron microscopic observations of single‐membrane‐bound, ovoid to spherical bodies in phloem sieve elements of diseased plants and detection of phytoplasma‐characteristic 16S rRNA gene sequence in DNA samples from diseased plants provided evidence linking the disease to infection by a phytoplasma. Results from restriction fragment length polymorphism, phylogenetic and comparative structural analyses of multiple genetic loci containing 16S rRNA, rpsS, rplV, rpsC and secY genes indicated that the AzLL phytoplasma represented a distinct, new 16Sr subgroup lineage, designated as 16SrI‐T, in the aster yellows phytoplasma group. The genotyping also revealed that the AzLL phytoplasma represented new rp and secY gene lineages [rp(I)‐P and secY(I)‐O, respectively]. Phylogenetic analyses of secY and rp gene sequences allowed clearer distinctions between AzLL and closely related strains than did analysis of 16S rDNA.     

Sequence Analysis of 16S rRNA and secA Genes Confirms the Association of 16SrI‐B Subgroup Phytoplasma with Oil Palm (Elaeis guineensis Jacq.) Stunting Disease in India

During January 2010, severe stunting symptoms were observed in clonally propagated oil palm (Elaeis guineensis Jacq.) in West Godavari district, Andhra Pradesh, India. Leaf samples of symptomatic oil palms were collected, and the presence of phytoplasma was confirmed by nested polymerase chain reaction (PCR) using universal phytoplasma‐specific primer pairs P1/P7 followed by R16F2n/R16R2 for amplification of the 16S rRNA gene and semi‐nested PCR using universal phytoplasma‐specific primer pairs SecAfor1/SecArev3 followed by SecAfor2/SecArev3 for amplification of a part of the secA gene. Sequencing and BLAST analysis of the ～1.25 kb and ～480 bp of 16S rDNA and secA gene fragments indicated that the phytoplasma associated with oil palm stunting (OPS) disease was identical to 16SrI aster yellows group phytoplasma. Further characterization of the phytoplasma by in silico restriction enzyme digestion of 16S rDNA and virtual gel plotting of sequenced 16S rDNA of ～1.25 kb using iPhyClassifier online tool indicated that OPS phytoplasma is a member of 16SrI‐B subgroup and is a ‘Candidatus Phytoplasma asteris’‐related strain. Phylogenetic analysis of 16S rDNA and secA of OPS phytoplasma also grouped it with 16SrI‐B. This is the first report of association of phytoplasma of the 16SrI‐B subgroup phytoplasma with oil palm in the world.     

Detection and identification of ‘Candidatus Phytoplasma asteris’ in Brassica rapa subsp. pekinensis in Poland

Severe growth abnormalities, including leaf yellowing, sprout proliferation and flower virescence and phyllody, were found on Brassica rapa subsp. pekinensis plants in Poland. The presence of phytoplasma in naturally infected plants was demonstrated by polymerase chain reaction assay employing phytoplasma universal P1/P7 followed by R16F2n/R16R2 primer pairs. The detected phytoplasma was identified using restriction fragment length polymorphism analysis (RFLP) of the 16S rRNA gene fragment with AluI, HhaI, MseI and RsaI endonucleases. After enzymatic digestion, all tested samples showed restriction pattern similar to that of ‘Candidatus phytoplasma asteris’. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Sequences of the 16S rDNA gene fragment of analysed phytoplasma isolates were nearly identical. They revealed high nucleotide sequence identity (>98%) with corresponding sequences of other phytoplasma isolates from subgroup 16SrI‐B, and they were classified as members of ‘Candidatus phytoplasma asteris’. This is the first report of the natural occurrence of phytoplasma‐associated disease in plants of Chinese cabbage.     

Identification of ‘Candidatus Phytoplasma asteris' (16SrI‐B) Causing Witches' Broom Disease of Mallotus japonicus in Korea

Mallotus japonicus with witches' broom disease were observed in Jeollabuk‐do, Korea. A phytoplasma from the infected leaves was identified, based on the 16S rDNA, 16S‐23S intergenic spacer region, and fragment of rp operon and tuf gene sequences. The 16S rDNA sequences exhibited maximum (99.7%) similarity with Iranian lettuce phytoplasma, the rp operon sequences exhibited 100% similarity with Goldenrain stunt phytoplasma, and the tuf gene sequences exhibited 99.8% similarity with Japanese spurge yellows phytoplasma. Results of the sequence analysis and phylogenetic studies confirmed that the phytoplasma associated with M. japonicus in Korea was an isolate of Aster Yellows group (subgroup16SrI‐B).     

Molecular Characterization of Aster Yellows Subgroup 16SrI‐B Phytoplasma in Verbena × hybrida

Symptoms resembling phytoplasma disease were observed on Verbena × hybrida in Alanya, Turkey, during October 2013. Infected plants were growing as perennials in a flower border and showed symptoms of discoloured flowers, poor flower clusters, inflorescences with a small number of developed flowers and thickened fruit stalks. Electron microscopy examination of the ultra‐thin sections revealed polymorphic bodies in the phloem tissue of leaf midribs. The phytoplasma aetiology of this disease was confirmed by polymerase chain reaction of the 16S rRNA gene, the 16–23S rRNA intergenic spacer region and the start of the 23S rRNA gene using universal phytoplasma‐specific primer pair P1A/P7A, two ribosomal protein (rp) genes (rpl22 and rps3) (the group‐specific primer pair rp(I)F1A/rp(I)R1A) and the Tuf gene (group‐specific fTufAy/rTufAy primers) generating amplicons of 1.8 kbp, 1.2 kbp and 940 bp, respectively. Comparison of the amplified sequences with those available in GenBank allowed classification of the phytoplasma into aster yellows subgroups 16SrI‐B, rpI‐B and tufI‐B. This is the first report about molecular detection and identification of natural infection of the genus Verbena by phytoplasma and occurrence of the aster yellows group phytoplasma on an ornamental plant in Turkey.     

Molecular Identification and Diversity of ‘Candidatus Phytoplasma solani’ Associated with Red‐leaf Disease of Salvia miltiorrhiza in 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 Characterization of a Phytoplasma Associated with Potato Witches’‐broom Disease in Iran

Potato plants showing symptoms suggestive of potato witches’‐broom disease including witches’‐broom, little leaf, stunting, yellowing and swollen shoots formation in tubers were observed in the central Iran. For phytoplasma detection, Polymerase Chain Reaction (PCR) and nested PCR assays were performed using phytoplasma universal primer pair P1/P7, followed by primer pair R16F2n/R16R2. Random fragment length polymorphism analysis of potato phytoplasma isolates collected from different production areas using the CfoI restriction enzyme indicated that potato witches’‐broom phytoplasma isolate (PoWB) is genetically different from phytoplasmas associated with potato purple top disease in Iran. Sequence analysis of the partial 16S rRNA gene amplified by nested PCR indicated that ‘Candidatus Phytoplasma trifolii’ is associated with potato witches’‐broom disease in Iran. This is the first report of potato witches’‐broom disease in Iran.     

Multilocus sequence analysis of phytoplasma strains of 16SrII group in Iran and their comparison with related strains

Phytoplasmas of the group 16SrII (peanut witches'‐broom group) are among the most important phytoplasmas identified in Iran. These phytoplasmas are so diverse that they have been classified within 23 subgroups, among which phytoplasmas of subgroups 16SrII‐B, ‐C and ‐D have been recognised in Iran. In this study, we used multilocus sequence analysis as a tool to find the extent of genetic diversity and phylogeny of representative phytoplasmas of 16SrII in Iran in comparison to reference phytoplasma strains characterised elsewhere. The genes used were 16S rRNA, secY, rplV‐rpsC, imp and a hypothetical protein (inmp). Analysis of this study showed that phytoplasmas of 16SrII could be resolved into at least three main phylogenetic lineages. One lineage comprised phytoplasmas of the subgroups 16SrII‐A and II‐D, another included strains of subgroups 16SrII‐B and II‐C and the third lineage comprised phytoplasmas belonging to 16SrII‐E. The significance of host adaptation and geographical distribution in relation to the genetic diversity of these phytoplasmas is discussed. Among five different genetic loci used in this study, imp gene displayed the highest genetic diversity, hence considered as the most powerful genetic tool for differentiation of closely related phytoplasmas.     

Detection, Identification and Molecular Characterization of a Phytoplasma Associated with Arabian Jasmine (Jasminum sambac L.) Witches' Broom in Oman

Arabian jasmine (Jasminum sambac L.) plants showing witches’ broom (WB) symptoms were found in two regions in the Sultanate of Oman. Polymerase chain reaction (PCR) amplification of the 16S rRNA gene and the 16S–23S spacer region utilizing phytoplasma‐specific universal and designed primer pairs, and transmission electron microscopy of phytoplasma‐like structures in phloem elements confirmed phytoplasma infection in the symptomatic plants. PCR products primed with the P1/P7 primer pair were 1804 bp for jasmine witches’ broom (JasWB) and 1805 bp for alfalfa (Medicago sativa L.) witches’ broom (AlfWB). Actual and putative restriction fragment length polymorphic analysis indicated that jasmine and AlfWB phytoplasmas were molecularly indistinguishable from each other and closely related to papaya yellow crinkle (PYC), as well as being distinct from lime WB (LWB) and Omani alfalfa WB (OmAlfWB) phytoplasmas. A sequence homology search of JasWB and AlfWB showed 99.8% similarity with PYC from New Zealand and 99.6% similarity with each other (JasWB/AlfWB). The jasmine and AlfWB phytoplasmas were also shown to be related to the peanut WB group (16SrII) of 16S rRNA groups based on a phylogenetic tree generated from phytoplasma strains primed with the P1/P7 primer pair and representing the 15 phytoplasma groups.