Group 16SrI Research Articles

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.

Identification of 16SrIX-C phytoplasmas in Argyranthemum frutescens in Italy

Phytoplasmas are cell wall-less microorganisms associated with plant diseases worldwide. Many important food, vegetable and fruits crops as well as ornamental plants can be severely affected by these pathogens, with significant economic impacts. Phytoplasma diseases of ornamentals have been described worldwide in a wide range of plant genera, and 11 different 16Sr groups have been identified. In Italy, many ornamental plant species belonging to several botanical families have been found to be infected by phytoplasmas, classified into the ribosomal groups 16SrI, 16SrII, 16SrV and 16SrXII. During a survey carried out in commercial gardens in Rome, some marguerite daisy (Argyranthemum frutescens) plants showing symptoms of phytoplasma-like disease, were collected and submitted to molecular analyses. Cloning and sequencing of the portion of the 16S rRNA gene followed by BLAST analysis, real and virtual restriction fragment length polymorphism anlaysis with Alu I and Rsa I, allowed assignment of the detected phytoplasma to the 16SrIX-C group ( Picris echioides yellows, PEY).

First Report of a 16Sr I-B Phytoplasma Associated with Phyllody and Stem Fasciation of Flax (Linum usitatissimum) in India.

Flax or linseed is grown as a fiber or oilseed crop in tropical and temperate regions. It is commercially cultivated in many countries of the world including Canada, China, India, the United States, Ethiopia, Pakistan, Russia, Poland, and Argentina (1). In December 2013, symptoms suggestive of phytoplasma infection were noticed on flax in different experimental fields of Central Research Institute for Jute and Allied Fibres (CRIJAF) research farm, Barrackpore, India, and the incidence was less than 2%. Because incidence of phytoplasma diseases are increasing worldwide, occurrence of a phytoplasma in a new geographical area poses an imminent threat. The infected plants showed floral virescence, phyllody, and stem fasciation (flattened stem). Floral malformation was very conspicuous with abnormal structures replacing normal flowers. All the floral parts, including petals, turned into green leaves. Total DNA was extracted from leaf mid veins of three symptomatic and three asymptomatic plants using a DNeasy Plant Mini Kit (Qiagen). PCR was carried out with the phytoplasma-specific universal P1/P7 primer set followed by nested primer pair R16F2n/R16R2 (2), resulting in DNA amplicons that were 1.8 kb and 1.2 kb, respectively, in all symptomatic samples tested. No amplification was observed with DNA from symptomless samples. This suggested association of a phytoplasma with the disease. The five purified nested PCR products were cloned in a pGEM-T Easy vector (Promega) and sequenced. One of the sequences that proved to be identical to the others was deposited in GenBank (Accession No. KJ417660). The consensus sequence was analyzed by NCBI BLAST and found to share 99% similarity with the 16Sr DNA sequence of the 'Candidatus Phytoplasma asteris' reference strain (GenBank HQ828108), which belongs to 16SrI group. The phylogenetic tree based on 16SrDNA sequence of phytoplasmas belonging to group 16SrI and other distinct phytoplasma groups also showed that the phytoplasma clustered with members of group 16SrI (3). The nested PCR product of R16F2n/R16R2 was digested using restriction enzymes AluI, BfaI, BstU, HhaI, HpaI, KpnI, MseI, and RsaI. The RFLP patterns were compared with those of known phytoplasma strains (2) and they matched the patterns for aster yellows subgroup B (16Sr I-B). Subsequently, the iPhyClassifier 16Sr group/subgroup classification based on similarity (4) analyses showed that the studied strain had 16SrDNA sequences in the 16SrI-B group with a similarity coefficient of 1.00. To the best of our knowledge, this is the first report of 16SrI-B phytoplasma associated with flax in India.

A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group

A novel phytoplasma was detected in a cherry plum (Prunus cerasifera Ehrh) tree that mainly showed yellow leaf symptom. The tree was growing in an orchard located in Yangling District, Shaanxi Province, China. The leaves started as chlorotic and yellowing along leaf minor veins and leaf tips. Chlorosis rapidly developed to inter-veinal areas with the whole leaf becoming pale yellow in about 1-4 wk. Large numbers of phytoplasma-like bodies (PLBs) were seen under transmission electron microscopy. The majority of the PLBs was spherical or elliptical vesicles, with diameters in range of 0.1-0.6 µm, and distributed in the phloem cells of the infected tissues. A 1 246-bp 16S ribosomal RNA (rRNA) gene fragment was amplified from DNA samples extracted from the yellow leaf tissues using two phytoplasma universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2. Phylogenetic analysis using the 16S rRNA gene sequence suggested that the phytoplasma associated with the yellow leaf symptoms belongs to a novel subclade in the aster yellows (AY) group (16SrI group). Virtual and actual restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene fragment revealed that the phytoplasma was distinguishable from all existing 19 subgroups in the AY group (16SrI) by four restriction sites, Hinf I, Mse I, Sau3A I and Taq I. The similarity coefficients of comparing the RFLP pattern of the 16S rRNA gene fragment of this phytoplasma to each of the 19 reported subgroups ranged from 0.73 to 0.87, which indicates the phytoplasma associated with the cherry plum yellow leaf (CPYL) symptoms is probably a distinct and novel subgroup lineage in the AY group (16SrI). In addition, the novel phytoplasma was experimentally transmitted to periwinkle (Catharanthus roseus) plants from the tree with CPYL symptoms and then back to a healthy 1-yr-old cherry plum tree via dodder (Cuscuta odorata) connections.

First Report of 'Candidatus Phytoplasma solani' and 'Ca. P. convolvuli' Associated with Grapevine Bois Noir and Bindweed Yellows, Respectively, in Georgia.

A survey carried out in Georgian vineyards, located in the Khaketi region, in September 2013, showed the presence of vines of the cultivar Chardonnay with typical grapevine yellows (GY) symptoms including leaf discoloration and curling, berry shriveling, and irregular maturation of wood. In the same vineyards, bindweed (Convolvulus arvensis L.) plants showing shoot proliferation and leaf yellowing were found, suggesting the involvement of phytoplasmas in the disease etiology. Total DNA was extracted by a CTAB method from leaf veins of 18 symptomatic and two asymptomatic grapevines, and from four symptomatic and two asymptomatic bindweeds, and analyzed by PCR assays. Moreover, DNA extracted from 'Candidatus Phytoplasma asteris' strain SAY (group 16SrI), 'Ca. P. solani' strain STOL (group 16SrXII), and 'Ca. P. ulmi' strain EY1 (group 16SrV) were used as positive controls. DNA extracted from healthy periwinkle and a reaction mixture without template were employed as negative controls. Nested PCRs targeting the 16S rDNA, carried out using the primer pairs P1/P7 followed by R16F2n/R16R2 (1), produced a band of the expected size (1,250 nt) in all the symptomatic grapevine and bindweed plants, and in the positive controls. No amplification was observed with DNA from asymptomatic plants nor the negative controls. PCR products were sequenced by a commercial sequencing service (Primm, Milan, Italy). The 16S rDNA nucleotide sequences of phytoplasmas identified in all grapevines and in two bindweed samples shared >99.5% sequence identity with 'Ca. P. solani' reference strain STOL (GenBank Accession No. AF248959), and carried identical STOL-unique signature sequence and distinguishing sequence blocks (3). Moreover, nucleotide sequences of phytoplasmas identified in the other two bindweed samples shared >99.6% sequence identity with 'Ca. P. convolvuli' reference strain BY-S57/11 (JN833705) (2). RFLP and phylogenetic analyses confirmed the affiliation of the phytoplasma strains identified in grapevine and bindweed plants in Georgia to the species 'Ca. P. solani' (subgroup 16SrXII-A) and 'Ca. P. convolvuli' (subgroup 16SrXII-H). Representative 16S rDNA nucleotide sequences were deposited in NCBI GenBank website with accession nos. KF996535 and KF996536 ('Ca. P. solani' from grapevine and bindweed, respectively), and KF996537 ('Ca. P. convolvuli'). Future studies will focus on investigating the spread and impact of 'Ca. P. solani'-associated bois noir (BN) in Georgia. In particular, the identification of 'Ca. P. solani' in bindweeds suggested the presence of the insect Hyalesthes obsoletus, a polyphagous cixiidae responsible for BN phytoplasma transmission in vineyards in Europe. Accurate surveys and molecular analyses will be performed for identifying the insect vector(s) of the BN associated phytoplasma strains in Georgia. Additional studies will be performed to study the spread and impact of 'Ca. P. convolvuli,' identified only in Italy, Germany, Serbia, and Bosnia and Herzegovina (2), throughout the Caucasian countries.

The First Report of Stolbur Phytoplasma Associated with Phyllody of Calendula officinalis in Serbia.

Pot marigold (Calendula officinalis L.) is native to southern Europe. Compounds of marigold flowers exhibit anti-inflammatory, anti-tumor-promoting, and cytotoxic activities (4). In Serbia, pot marigold is cultivated as an important medicinal and ornamental plant. Typical phyllody, virescence, proliferation of axillary buds, and witches' broom symptoms were sporadically observed in 2011 in Pancevo plantation, Serbia (44°51'49″ N, 20°39'33″ E, 80 m above sea level). Until 2013, the number of uniformly distributed affected pot marigold plants reached 20% in the field. Due to the lack of seed production, profitability of the cultivation was seriously affected. Leaf samples from 10 symptomatic and 4 symptomless marigold plants were collected and total nucleic acid was extracted from midrib tissue (3). Direct PCR and nested PCR were carried out with primer pairs P1/16S-SR and R16F2n/R16R2n, respectively (3). Amplicons 1.5 and 1.2 kb in length, specific for the 16S rRNA gene, were amplified in all symptomatic plants. No PCR products were obtained when DNA isolated from symptomless plants was used. Restriction fragment length polymorphism (RFLP) patterns of the 1.2-kb fragments of 16S rDNA were determined by digestion with four endonucleases separately (TruI1, AluI, HpaII, and HhaI) and compared with those of Stolbur (Stol), Aster Yellows (AY), Flavescence dorée-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas (2). RFLP patterns from all symptomatic pot marigold plants were identical to the Stol pattern, indicating Stolbur phytoplasma presence in affected plants. The 1.2-kb amplicon of representative Nv8 strain was sequenced and the data were submitted to GenBank (accession no. KJ174507). BLASTn analysis of the sequence was compared with sequences available in GenBank, showing 100% identity with 16S rRNA gene of strains from Paeonia tenuifolia (KF614623) and corn (JQ730750) from Serbia, and peach (KF263684) from Iran. All of these are members of the 16SrXII 'Candidatus Phytoplasma solani' group, subgroup A (Stolbur). Phytoplasmas belonging to aster yellows (16SrI) (Italy and Canada) and peanut witches' broom related phytoplasma (16SrII) group (Iran) have been identified in diseased pot marigold plants (1). To our knowledge, this is the first report of natural infection of pot marigold by Stolbur phytoplasma in Serbia.

First report of ‘Candidatus Phytoplasma asteris’ (16SrI group) causing stunt of tomato in Cuba

Tomato (Solanum lycopersicum) is an important vegetable in Cuba and worldwide. In 2009, a disease suspected to be associated with phytoplasmas was observed in tomato fields in Mayabeque, in the locality of San Jose in the western region of the country. Symptoms most frequently displayed were shortened internodes and stunting, chlorosis and reduced leaf size in approximately 40% of tomato plants but others included abnormal shoot proliferation and reduced fruit size. Leaf samples of plants with symptoms were collected and total DNA was extracted (Doyle & Doyle, 1990) and used as template in a nested PCR assay to amplify the phytoplasma 16S rDNA gene with primer pairs R16F2n/R2 (Gundersen & Lee, 1996) and fU5/rU3 (Lorenz et al., 1995). Products of the expected size (approximately 1240 and 880 bp respectively) were amplified for fifteen out of forty-five (15/45) symptom-bearing plants, but not from the symptomless plants. Restriction profiles after digestion of amplicons with HpaII, HaeIII and Sau3AI endonucleases (data not shown) were all identical to each other and to those of reference phytoplasma strains related to 'Candidatus Phytoplasma asteris' (16SrI group)(Lee et al., 1998). A selected PCR product was cloned into the pGEM-T Easy Vector (Promega) and three clones were further sequenced. Sequence alignment with ClustalW method demonstrated that the clones from Cuban tomato phytoplasma were indistinguishable. One of the sequences was deposited in GenBank (JN383913) and compared by BLASTn with available DNA sequences. According to the results Cuban tomato phytoplasma showed the highest sequence similarity (99%) with Potato phytoplasma Islamabad (FJ178388), a strain related to 'Candidatus Phytoplasma asteris' (Aster yellows group (16SrI)). Phylogenetic analysis revealed that Cuban tomato phytoplasma isolate clustered with 16SrI phytoplasmas being also related to the phytoplasma reported by Arocha et al. (2007) in sweet pepper (Capsicum sp.) in Cuba (Fig. 1).

First Report of Phytoplasmas Groups 16SrI and 16SrXV in Crotalaria juncea in Brazil.

Sunn hemp (Crotalaria juncea L., Fabaceae) is widely used as a cover crop in sugar cane and citrus plantations in Brazil. C. juncea has been reported in São Paulo State (SPS) by Wulff et al. (3) as a host of the phytoplasma associated with symptoms of huanglongbing (HLB) in citrus, a member of group 16SrIX, that induces witches'-broom in sunn hemp (3). In studying the distribution of group 16SrIX phytoplasma in C. juncea in SPS, we identified this species as a new host of two phytoplasmas. Sunn hemp fields were inspected for symptoms usually associated with phytoplasma infections, such as leaf yellowing, shoot proliferation, witches'-brooms, and virescence. Ninety-nine plant samples were collected and DNA was extracted with the CTAB protocol from stems. Nested PCR was carried out with primers P1/P7, followed by amplification with primers fU3/rU5 (2), both sets being universal for phytoplasma. Asymptomatic sunn hemp samples were used as negative controls and were negative in PCR reactions. PCR products were directly sequenced with primers P1/P7 and fU3/rU5 and phytoplasma identification was conducted with BLASTn and in silico RFLP analysis for delineation of subgroups (4). Plants showing leaf yellowing (three plants; Catanduva County), shoot proliferation (one plant; Ibirá County), or witches'-brooms (one plant; Promissão County) symptoms were found to be infected with the 16SrI phytoplasma group, subgroup S. The 16S rDNA sequence (GenBank Accession No. KF878383) showed 99% identity (E value 0.0) with Candidatus Phytoplasma asteris, Onion yellows phytoplasma OY-M (AP006628), Mulberry yellow dwarf phytoplasma (GQ249410), and Ash witches'-broom phytoplasma (AY566302), among other phytoplasmas from the same group. Sunn hemp plants with shoot proliferation (three plants) carried the 16SrXV phytoplasma group, subgroup A, found in Ibirá (two plants) and Catanduva (one plant) counties, SPS. This sequence (GenBank Accession No. KF878382) displayed 99% identity (E value 0.0) with Ca. P. brasiliense, Hibiscus witches'-broom phytoplasma (AF147708), Guazuma ulmifolia witches'-broom phytoplasma (HQ258882, HQ258883), and Cauliflower stunt phytoplasma (JN818845). Both phytoplasma groups described in this report, 16SrI and 16SrXV, were collected in May 2010 and both have limited geographic distribution and occurred at low incidence. Phytoplasma of group 16SrI (Ca. P. asteris) was identified in C. spectabilis in India (1). To our knowledge, this is the first report of phytoplasmas groups 16SrI and 16SrXV in sunn hemp.

First Report of a 16SrI Group Phytoplasma Associated with Roselle (Hibiscus sabdariffa) Wrinkled Leaves and Phyllody Disorder in Taiwan

Roselle (Hibiscus sabdariffa L.), an annual plant with acidic taste, has been used for making juice, jelly, and other baking additives in Taiwan. In September 2013, symptoms including phyllody and wrinkled leaves were observed on roselle plants in a field in Tantsu Township, Taichung County, Taiwan. Incidence of the infected plants was estimated to be greater than 80% within a single field. A phytoplasma was recently reported as the causal agent of roselle phyllody and reddening of leaves in India and classified as a group 16SrV-D strain (1). Samples including stems, flowers, and leaves were collected from four symptomatic and one asymptomatic roselle plants from the field. Transmission electron microscopy revealed clusters of phytoplasma cells ranging from 400 to 750 nm in diameter only in phloem sieve elements of petioles and stems of symptomatic plants. These cells were not observed in asymptomatic plants. Total DNA was extracted from plant tissues (100 mg each) including stems, petioles, and mid veins of leaves by a modified CTAB method (2). Analyses by a nested PCR assay using universal primer pairs P1/P7 followed by R16F2n/R16R2 were performed to detect putative phytoplasma (1). Each primer pair amplified a single PCR product 1.8 kb and 1.2 kb long, respectively, only from tissues of the four symptomatic plants. The nested PCR products (1.2 kb) amplified from three independent symptomatic plants were cloned separately and sequenced by automatic DNA sequencing method with ABI3730 DNA Analyzer (Applied Biosystems) at the Biotechnology Center, National Chung Hsing University, Taichung, Taiwan (GenBank Accession Nos. KF923397, KF923398, and KF923399). BLAST analysis of the sequences revealed that they shared 99.8% sequence identity with those of 16SrI group phytoplasma strains, e.g., garlic yellows phytoplasma, torenia yellows phytoplasma, and periwinkle leaf yellowing phytoplasma (AB750363, FJ437568, and GU361754). Moreover, i PhyClassifier software (3) was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile for the sequences derived from the symptomatic roselle samples. The 16S rDNA sequences shared 99.6% identity with those of the 'Candidatus Phytoplasma asteris' reference strain (M30790) and the RFLP patterns were identical to that of the 16SrI group. However, this strain may represent a new subgroup because the shared similarity coefficient was only 0.94, which is within the values set for a new subgroup (3). Taken together, these results indicate the phytoplasma infecting roselle in Taiwan is a 'Ca. P. asteris'-related strain belonging to the 16SrI group. To our knowledge, this is the first report of a 16SrI group phytoplasma causing wrinkled leaves and phyllody on roselle in Taiwan. The occurrence of phytoplasma on roselle could have direct implication for the bakery and juice industries in Taiwan.

Phytoplasma associated with Chinese tallow tree yellowing disease in China represents a new 16SrIII subgroup

SummaryRecently, samples of the Chinese tallow tree (Sapium sebiferum) displaying yellowing symptoms were collected from a grove in Tai'an, Shandong Province, China. The association of phytoplasma with yellowing disease was ascertained using nested polymerase chain reaction (PCR) of the 16S rRNA gene by using the phytoplasma‐specific universal primer pair P1/P7, followed by R16F2n/R16R2 as nested primers, and rp genes primed using rpL2F3/rp(I)R1A followed by rp(III)‐FN/rp(I)R1A. The sequence and phylogenetic analyses of the 16S rRNA gene and rp genes revealed that the phytoplasma associated with the Chinese tallow tree belonged to the 16SrIII group (the X‐disease group). Computer‐simulated and gel‐based restriction fragment length polymorphism (RFLP) analyses revealed that the RFLP patterns were different from the reference patterns of all previously established 16SrIII subgroups, with the maximum similarity coefficient exceeding the threshold for delineation of a new subgroup RFLP pattern type within the 16SrIII group. Thus, the phytoplasma associated with the Chinese tallow tree yellowing disease, designated as ‘CTTY’, represents a new subgroup (16SrIII‐Y). This study shows the Chinese tallow tree as a new host of phytoplasma belonging to the 16SrIII group in China and worldwide.

Characterization of a 16SrI-B Subgroup Phytoplasma Associated with Sugarcane Leaf Yellows Disease in India

Leaf mid-rib yellowing along with yellowing of adjoining lamina and development of irregular yellow patches on leaf lamina was observed on two sugarcane varieties (CoLk 94184, CoSe 92423) during 2013–2014 crop season. Phytoplasma association with leaf yellows and leaf lamina with yellow patches symptomatic samples was confirmed through PCR and nested PCR assays using phytoplasma specific primers P1/P6 followed by R16F2n/R16R2. Sequencing and BLASTn analysis of ~1.2 kb product of 16Sr RNA gene fragments of the two phytoplasmas shared 99 and 100 % sequence identity with strains of ‘Candidatus Phytoplasma asteris’ (16SrI). Further characterization through in silico restriction enzyme digestion of 16Sr DNA using iPhyClassifier online tool assigned both the strains into subgroup 16SrI-B and phylogenetic analyses confirmed that both the phytoplasmas were closely related to 16SrI group. This is the first report of association of 16SrI-B subgroup phytoplasma with sugarcane leaf yellows disease in India.

Occurrence of ‘Candidatus Phytoplasma asteris’ in citrus showing Huanglongbing symptoms in Mexico

Huanglongbing (HLB), one of the most devastating citrus diseases in the world, was detected in Mexico in 2009. Currently, HLB is associated with the bacteria Candidatus Liberibacter spp., although several phytoplasmas have been found from trees showing HLB-like symptoms in Brazil and China. The aim of this study was thus to determine if, in addition to ‘Ca. L. asiaticus’ (CLas), phytoplasma species are also associated with HLB-like symptoms in citrus groves of Mexico. Citrus plants exhibiting symptoms such as diffuse chlorosis, blotchy mottle and vein yellowing were collected in the Mexican States of Nayarit, Colima and Sinaloa between August 2011 and September 2012. Samples were then evaluated for phytoplasmas and CLas by PCR, using primers that respectively target the genes for the 16S ribosomal RNA and 50S ribosomal protein of the β operon (rplA-rplJ). Out of 86 HLB-symptomatic citrus plants, 54 were positive for CLas, 20 were positive for phytoplasmas, 7 were found in mixed infections with both pathogens and 19 samples were negative for CLas and phytoplasmas. Actual and virtual RFLP analyses of the 16S rDNA sequences enabled us to classify two HLB phytoplasma strains as members of the aster yellows group (16SrI) ‘Candidatus Phytoplasma asteris’, which was confirmed by phylogenetic analysis. The HLB phytoplasma strain identified from Nayarit (HLBpc-Nay-IB) belongs to subgroup B (16SrI-B), and the strains identified from Colima (HLBpc-Col-IS) and Sinaloa (HLBpc-Sin-IS) belong to subgroup S (16SrI-S). The partial ‘Ca. L. asiaticus’ rplA-rplJ gene sequences were 100% identical to the ‘Ca. L. asiaticus’ strains isolated from several countries affected by HLB. These results confirm the association of ‘Candidatus Phytoplasma asteris’ with HLB-like symptoms in citrus groves in Mexico. Nonetheless, further studies are required to fully describe the ‘Ca. L. asiaticus’ and ‘Ca. P. asteris’ interactions in citrus, which will greatly assist the design of efficient management strategies.

Identification of potential leafhoppers vectors of phytoplasmas (16SrIII group) associated with broccoli stunt disease in Brazil

Recently, disease in broccoli plants (Brassica oleracea var. italica) was associated with three distinct phytoplasmas in Brazil. The disease named broccoli stunt (BS) has caused significant economic losses in Sao Paulo State. Group 16SrIII phytoplasmas is the most common group that have been associated with BS. The BS disease is still poorly understood, and the lack of information about the vectors further impairs its management. In this study, leafhoppers belonging to 18 different species were collected from weeds thriving near broccoli fields that were affected by group 16SrIII phytoplasmas. Specific primers revealed the presence of group 16SrIII phytoplasmas in five leafhoppers: Empoasca spp., Agallia albidula Uhler, Agalliana sticticollis (Stal), Planicephalus flavicosta (Stal), and Atanus nitidus (Linnavuori). The identity of the phytoplasmas was confirmed through DNA sequencing analysis. The leafhoppers were infected by phytoplasmas of the 16SrIII group (proposed “Candidatus Phytoplasma pruni” species) and are phylogenetically related to the broccoli stunt phytoplasma (BSP) strains detected in the study area and, thus, are considered potential vectors of group 16SrIII phytoplasmas to broccoli plants.

First Report of Elm Yellows Subgroup 16SrV-B Phytoplasma as the Cause of Rose Balsam Phyllody in China.

Rose balsam (Impatiens balsamina L.) is an ornamental species frequently cultivated in China and the red flower is often used as nail polish in rural regions. The phytoplasmas previously reported with rose balsam phyllody in China have been classified as aster yellows group (16SrI) (1). In August 2012, some rose balsams were observed with typical phytoplasma symptoms in Handan City, Hebei Province, China, with an incidence of about 70% in the fields. The flowers turned green and petals fascicled. The new leaves wrinkled and deformed and internodes shortened. Infected plants were stunted, matured prematurely, and failed to produce seeds. To confirm phytoplasma infection, 100 mg of plant tissue (leaves, petals) was collected from five symptomatic and four asymptomatic plants and total DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method (2). The 16S rDNA gene was amplified by nested PCR using primer pair P1/P7 followed by R16F2n/R16R2 (3). No amplicons were generated with DNA from asymptomatic samples, but amplicons of approximately 1.2 kb were obtained with DNA from five symptomatic samples. The amplified products were purified with aTIANgel midi purification kit (Tiangen, Beijing) and sequenced at the Sangon Biotech facility (Shanghai, China). The sequences of the amplicons were 100% identical and deposited in NCBI GenBank (Accession No. KC993832). The 16S rDNA gene sequence from this phytoplasma was 99% similar to Jujube witches broom phytoplasma (JQ675716), Puna chicory flat stem phytoplasma (JN582266), Plum yellows phytoplasma (FJ459914), and other elm yellows group phytoplasmas by BLAST search of the NCBI database. Restriction fragment length polymorphism (RFLP) analyses were carried out by digesting the 1.2-kb R16F2n/R16R2 nested PCR product with restriction enzymes AluI, RsaI, HhaI, HpaI, Eco RI, TaqI, HaeIII, HinfI, and KpnI (Takara, Dalian). The 16S rDNA RFLP patterns matched that of Jujube witches broom phytoplasma (JWB, subgroup 16SrV-B) (4). Nucleotide sequences of rose balsam phyllody were analyzed by iPhyClassifier software, which revealed that it had maximum similarity to the reference pattern of 16Sr group V, subgroup B (AB052876). All samples were detected with transmission electron microscopy. The results showed phytoplasma-like cells in phloem sieve element of symptomatic plants, while no phytoplasma-like cells were observed in healthy phloem tissues. The phytoplasma cells ranged from 230 to 470 nm in diameter and were ellipsoidal or orbicular with visible membranes. Combining the RFLP pattern and sequence analysis by iPhyClassifier, we classified the phytoplasma causing rose balsam phyllody into subgroup 16SrV-B. To our knowledge, this is the first report of 16SrV-B group phytoplasmas infecting rose balsam in China.

Detection and Molecular Identification of Aster Yellows Phytoplasma in Date Palm in Egypt

AbstractPhytoplasma‐like symptoms were detected in date palm trees (Phoenix dactylifera L.) in Al‐Giza Governorate in Egypt. Symptoms varied from leaf chlorotic streaks, stunting and marked reduction in fruit and stalk sizes. Direct and nested PCR of symptomatic samples using P1/P7 and R16F2n/R16R2n primers, respectively, of the 16S rRNA gene, resulted in a DNA amplification product of c. 1.3 kbp. Symptomless samples collected from the same location and the healthy control produced no product upon amplification. Products were cloned into TOPO TA vector for sequencing. Data generated were deposited in the GenBank (Accession KF826615). A BLAST search showed that the sequence of the 16SrRNA gene shared ‘Candidatus Phytoplasma asteris’ (16SrI group) with other isolates. Phylogenetic analysis revealed that the isolate clustered with the date palm phytoplasma causing Al‐Wijam disease in Saudi Arabia.

First Report of a 16SrI (Aster Yellows) Group Phytoplasma on Garlic (Allium sativum) in the United States.

During the growing season of 2012, 35 garlic plant samples were submitted to the University of Minnesota Plant Disease Clinic for disease diagnosis. Samples originated from multiple counties throughout Minnesota as well as Iowa, Wisconsin, and South Dakota. Symptoms first appeared at the time plants were starting to produce scapes. Symptoms included leaf discoloration that varied from yellow to purple, plant stunting, and leaf tip necrosis. In severe cases, the plants wilted and died. Bulbs of affected plants ranged from being soft and small to almost normal-looking. Symptoms were similar to those associated with phytoplasma infection in other plants. Total genomic DNA was extracted from 30 symptomatic samples and five asymptomatic leaf samples using a Qiagen DNeasy Plant Mini Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions, and used with the universal phytoplasma primers P1/P7 in a direct PCR assay, and with P1/AYint in a nested PCR assay (2) to yield amplicons of 1.8 and 1.6 kb, respectively. Asymptomatic plants did not produce amplicons. Garlic cultivars displaying a range of symptoms tested positive for the presence of phytoplasma. These cultivars included: Susanville, Middle Eastern, Music, Ajo Rojo, Spanish Roja, Inchelium Red, Silver White, Asian Tempest, Chesnok Red, and Purple Glazer. The P1/P7 PCR products of 1,830 bp were purified using the PureLink PCR Purification kit (Life Technologies, Carlsbad, CA), and cloned in a pGem T-Easy vector system (Promega, Madison, WI). Sequences from a clone from each of Wisconsin, Iowa, and Minnesota were deposited in GenBank under the accession numbers KC000005, KC000006, and KC000007, respectively. A BLASTn similarity search revealed that the Wisconsin and Iowa isolates shared 99% homology to the sequences of 16SrI-A group phytoplasmas, aster yellows phytoplasma (AY389827), and aconitum proliferation phytoplasma (AF510323). The Minnesota isolate had 99% sequence homology to a 16SrI-B group phytoplasma, mulberry yellow dwarf phytoplasma (GQ249410). Also, the iPhyClassifier 16Sr group/subgroup classification based on similarity (3) analyses showed that the Wisconsin and Iowa phytoplasma isolates had 16S rDNA sequences in the 16SrI-A group with similarity coefficients of 0.97 and 1.00, respectively, to aster yellows witches'-broom phytoplasma AYWB (NC_007716). The same analysis revealed that the Minnesota phytoplasma isolate 16S rDNA sequence grouped with the 16SrI-B group onion yellows phytoplasma (NC_005303) with a similarity coefficient of 1.0. A phylogenic tree was deduced by the neighbor joining algorithm, clustering together the Iowa, Minnesota, and Wisconsin isolate sequences with a 16SrI group phytoplasma. Aster yellows phytoplasma has been reported in North America, but only in Canada (1). This is the first documented occurrence of 16SrI aster yellows group phytoplasma in garlic in the United States. The spring of 2012 was unusually warm, and high leafhopper pressure was observed throughout the Midwest; above average numbers of many ornamental crops and small grains were infected with phytoplasma. These events may have contributed to the phytoplasma infection in garlic.

First Report of Sweet Cherry Virescence Disease in China and Its Association with Infection by a 'Candidatus Phytoplasma ziziphi'-Related Strain.

Sweet cherry (Prunus avium L.) is a deciduous tree originating in the Black Sea/Caspian Sea region where Asia and Europe converge. Being highly valued for its timber and fruit, sweet cherry has been cultivated and naturalized on all continents. Over the past decade, the area of sweet cherry cultivation increased rapidly in China and has reached 140,000 ha. In April 2013, sweet cherry trees (cv. Summit) exhibiting floral virescence symptoms were observed in two orchards located in suburban Taian, Shandong Province, China. The diseased trees developed flowers having white petals with green veins or abnormal floral structures having cupped, green petals. The affected flowers failed to set fruit. A month following the first appearance of the virescence symptoms, the diseased trees became wilted and eventually died. Leaf and stem samples were collected from nine symptomatic and two nearby symptomless trees. Total DNA was extracted from each sample using the Plant Quick DNA Extract Kit (TianGen, Beijing, China). Nested-PCR was carried out using phytoplasma-universal primer pairs P1/P7 and R16F2n/R16R2 (1). All PCR assays with DNA templates from symptomatic samples yielded an amplicon of 1.25 kb, corresponding to the full-length F2nR2 region of phytoplasmal 16S rDNA. No amplicon was generated in PCRs containing DNA templates from symptomless plants. The amplicons were cloned into plasmid vector pMD18-T (TaKaRa, Dalian, China) and sequenced. The obtained sequences were nearly identical, and a representative sequence was deposited into GenBank (Accession No. KF268424). An analysis of the sequence through the iPhyClassifier (4) revealed that the sweet cherry virescence (SCV) disease was associated with infection by a phytoplasma closely related to the reference strain of 'Candidatus Phytoplasma ziziphi.' The 16S rDNA F2nR2 region of the SCV phytoplasma shared 99.8% nucleotide sequence identity with that of 'Candidatus Phytoplasma ziziphi' reference strain (Accession No. AB052876). A computer-simulated restriction fragment length polymorphism (RFLP) analysis of the SCV phytoplasma 16S rDNA F2nR2 sequence with a set 17 restriction enzymes (3) resulted in a collective RFLP profile identical to the reference pattern of the elm yellows phytoplasma group, subgroup B (16SrV-B). Phytoplasmal diseases of sweet cherry were reported previously in Europe and the etiological agents were phytoplasmas of other groups, including the aster yellows group (16SrI), the X-disease group (16SrIII), and the apple proliferation group (16SrX) (2). To our knowledge, this is the first report of a phytoplasmal disease of sweet cherry in China, and the SCV phytoplasma is a new member of the subgroup 16SrV-B. Presence of 16SrV-B phytoplasmas and their etiological association with various plant diseases in China have been reported previously; affected host plants included jujube, hemp fiber, paper mulberry, Chinese cherry, plum, apricot, red barberry, clover, dianthus, elm, and sunshine tree. Our identification of the SCV phytoplasma expands the known plant host range of the 16SrV-B phytoplasma lineage. The impact of the SCV phytoplasma in the regional ecosystem and in sweet cherry production is being assessed.

Isolation and comparative analysis of potC GENE of the abc-transport system from coconut root wilt and sugarcane grassy shoot (16SrXI group) phytoplasmas

Root wilt disease of coconut and grassy shoot diseases of sugarcane are diseases associated with phytoplasmas of the 16SrXI group. The present study aimed at characterizing and comparing the potC gene of the coconut root wilt (RWD) and sugarcane grassy shoot (SCGS) phytoplasmas with that of aster yellows (AY) phytoplasma. We isolated the full length potC gene of ATP-Binding Cassette (ABC) transport system from RWD, SCGS, AY phytoplasmas and a partial gene segment of Brinjal little leaf (BLL) phytoplasma using PCR. The full length potC gene, partial sequence of potB and potD genes were sequenced and characterized. Comparative molecular analysis of the potC gene from RWD, SCGS showed that both sequences had 99% nucleotide identity with each other and 70% with AY phytoplasma. Phylogenetic analysis revealed that the potC gene of coconut and sugarcane phytoplasmas clustered together and that AY phytoplasma clustered with 16SrI group phytoplasmas. The phylogeny of potC genes followed the 16S rRNA-based taxonomic classification of phytoplasmas so that it can be considered as a potential candidate for phylogenetic studies. Bioinformatics analysis of putative PotC proteins revealed that phytoplasmas have conserved motifs which are absent in other bacteria. However, the PotC proteins of phytoplasma and bacteria have one common domain (ABC transporter intergral membrane type-1 domain) and six transmembrane helices.

Identification and molecular characterization of a phytoplasma associated with sunflower in Argentina

Sunflower (Helianthus annuus L.) plants showing capitulum with virescence, phyllody and flower malformation, shortened internodes and abnormal branches were found in a field in Pedro Luro (Buenos Aires province, Argentina). Pleomorphic bodies resembling phytoplasmas were observed in sieve tube elements of symptomatic plants but not in healthy ones. DNA from all symptomatic sunflower plants analysed yielded, in direct PCR with phytoplasma universal primers P1/P7 and R16F2n/R2, fragments of expected size 1.8 kb and 1.2 kb, respectively. The phytoplasma associated with the disease, was named Sunflower Phyllody (SunPhy). Real and putative RFLP of the 16S rDNA showed the affiliation of SunPhy to 16SrIII (X-disease group), subgroup J. The 16S rDNA sequence from SunPhy showed the highest identity (99 %) with 16SrIII members and the phylogenetic tree confirmed a closer relationship to subgroup J of the 16SIII ribosomal group. This is the first report of a phytoplasma related to the 16SrIII group affecting sunflower.

Detection of Putative Recombination Events in the 16S Ribosomal RNA Gene of Some Members of Candidatus phytoplasma

Recombination plays a major evolutionary role by creating genetic diversity and provides the potential to find rapid adaptation to new environmental conditions. We sought the occurrence of possible recombination events in the 16S ribosomal RNA gene of 60 accessions belonging to the group 16SrI of Candidatus phytoplasma (aster yellows phytoplasma). Three bioinformatic programs were used (TOPALI v2.5, RECCO and RDP package). All the three programs indicated the presence of putative recombination signals in aligned sequences. Recombination events located in the 16S ribosomal RNA gene revealed the presence of four recombining accessions gathering sugarcane grassy shoot phytoplasmas (JF928001, DQ459439, EF614269 and JN223446).