Phytoplasma Group Research Articles

The development of monoclonal antibodies to the secA protein of Cape St. Paul wilt disease phytoplasma and their evaluation as a diagnostic tool.

Partial recombinant secA proteins were produced from six different phytoplasma isolates representing five 16Sr groups and the expressed, purified recombinant (partial secA) protein from Cape St. Paul wilt disease phytoplasma (CSPWD, 16SrXXII) was used to immunise mice. Monoclonal antibodies (mAbs) were selected by screening hybridoma supernatants for binding to the recombinant proteins. To characterise the binding to proteins from different phytoplasmas, the antibodies were screened by ELISA and western blotting, and epitope mapping was undertaken. Eight different mAbs with varying degrees of specificity against recombinant proteins from different phytoplasma groups were selected. Western blotting revealed that the mAbs bind to proteins in infected plant material, two of which were specific for phytoplasmas. ELISA testing of infected material, however, gave negative results suggesting that either secA was not expressed at sufficiently high levels, or conformational changes of the reagents adversely affected detection. This work has shown that the phytoplasma secA gene is not a suitable antibody target for routine detection, but has illustrated proof of principle for the methodology.

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.

Analyses of the 16S-23S intergenic region of the phytoplasma causing the sugarcane white leaf disease in Yunnan Province, China

Sugarcane white leaf (SCWL) is an important disease caused by a phytoplasma. In Baoshan, Yunnan, China, SCWL was firstly observed in 2012, and has extended its area of occurrence to 600 hm2 . Up to 52% of the plants may become diseased in a field and even complete loss of cane yield may result in the heavily infected fields, posing a serious threat to Yunnan sugar industry. To ascertain the causal agent of suspected SCWL disease in Yunnan, nested PCR using two sets of phytoplasma primer pairs (MOLX/MLOY and P1/ P2) was used to successfully amplify a genomic region of the 16S ribosomal DNA (16S rDNA) from 36 suspected SCWL samples. On the basis of sequencing, phylogenetic analysis and nucleotide alignments of 17 nested PCR products randomly selected from positive samples, identical fragments of 210bp in length were obtained that could be clustered into the 6Sr group XI (Rice Yellow Dwarf group) and shared 100% identity with the 16S-23S intergenic spacer region (ISR) of a member of this group, the SCWL phytoplasma (GenBank: HQ917068), and 99.52% with Sugarcane grassy shoot phytoplasma, of the same group. These results indicate that the SCWL disease in Baoshan is caused by a phytoplasma of the 6Sr group XI.

First Report of a 16SrV-C Phytoplasma Causing Little Leaf and Bunchy Top of Tossa Jute (Corchorus olitorius) in India.

Jute is the most important phloem fiber crop of the world, and is mainly grown in the South East Asian countries of India, Bangladesh, Nepal, China, Indonesia, Thailand, and Myanmar, and few South American countries. The fiber is used in making sacks, ropes, bags, carpets, shoes, geo-textiles, and home decorations. There are two kinds of jute: tossa jute (Corchorus olitorius L.) and white jute (C. capsularis). In June 2012, symptoms suggestive of phytoplasma infection (little leaf and bunchy top) were noticed on tossa jute in different experimental fields of the CRIJAF research farm, Barrackpore, India, and the incidence of the disease varied from 5 to 20%. The infected plants showed profuse lateral branching with a bushy appearance. In many plants, branching at the apical portion developed a bunchy top symptom with tufts of smaller leaves. Leafy stem was also common in many plants with main stems covered with numerous little leaves. Total DNA was extracted from leaf midveins of 15 symptomatic and 5 asymptomatic plants by using an improved salt concentration and simple sodium acetate CTAB method (1). PCR was carried out with universal P1/P7 primer set followed by nested primer pair R16F2n/R16R2 (3), resulting in DNA amplicons that were 1.8 kb and 1.2 kb, respectively, in all symptomatic samples tested. Phytoplasma was not detected in symptomless samples. The five purified nested products were cloned in a pGEM-T Easy vector (Promega) and sequenced. One of the sequences that proved to be identical was deposited in GenBank (Accession No. KF501045). The consensus sequence was analyzed by NCBI BLAST and found to share 99% similarity with the 16Sr DNA sequence of the alder yellows phytoplasma reference strain (GenBank Accession No. AY028789), which belongs to the 16SrV group. The phylogenetic tree based on the 16SrDNA sequence of phytoplasmas belonging to group 16SrV and other distinct phytoplasma groups also showed that the phytoplasma clustered with members of subgroup 16SrV (4). Subsequently, in silico RFLP analysis of the nested PCR product with the pDRAW32 program using AluI and TruI restriction site used for 16SrV subgroups A, B, C, D, and E indicated that the 16SrV Corchorus strain belonged to subgroup C. RFLP patterns from all symptomatic C. olitorius samples were identical to the 16SrV-C pattern (2). The vector species transmitting the concerned phytoplasma in C. olitorius still needs to be identified. The leaf hopper, Amrasca biguttula biguttula, may be a potential vector as it is often noticed in jute fields. To the best of our knowledge, this is the first report of 16SrV-C phytoplasma associated with tossa jute (C. olitorius) in India. Initiative has to be taken to manage this disease; otherwise, branching of the main stems would badly affect the fiber quality as well as yield.

Association of ‘Candidatus Phytoplasma cynodontis’ with the yellow leaf disease of ivy gourd in Thailand

Ivy gourd (Coccinia grandis) is a small climbing perennial plant in the family Cucurbitaceae. The disease symptom of yellow leaves was observed frequently on ivy gourd in Hatyai, Songkhla, Thailand, during 2013. Infected tissue samples were assayed by nested PCR with the universal primer pair P1/P7, followed by R16F2n/R2 to amplify the 16S rDNA fragment. The approximately 1.2 kb PCR products that were observed in symptomatic leaves had the highest nucleotide sequence similarity (99 %) with the 16SrXIV group phytoplasmas. To the author’s knowledge, this is the first report of ivy gourd as a host of ‘Candidatus Phytoplasma cynodontis’ in Thailand or elsewhere.

Identification and molecular characterization of 16SrV group phytoplasmas with flowering cherry in China

SummaryFlowering cherry (Prunus serrulata) trees showing symptoms of excessive axillary growth, stunting, short internodes and decline were observed in Yantai City, Shandong province, China. The samples with the flowering cherry decline were designated as FcD‐YT phytoplasma. Sequencing results, restriction fragment length polymorphism (RFLP) and phylogenetic analysis of sequences from the 1248‐bp R16F2n⁄R16R2 products and partial ribosomal protein operon genes revealed the FcD‐YT phytoplasma as a member of subgroup 16SrV‐B, rpV‐C. Two RFLP patterns were observed in recombinant colonies from the cloned 16S rDNA gene with endonucleases HpaII and MseI. To our knowledge, this is the first report of 16SrV group phytoplasma declining flowering cherry trees in China.

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.

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.

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.

New host record of a ‘Candidatus Phytoplasma asteris’-related strain infecting peach in India

Phytoplasma-like symptoms such as general decline, leaf reddening, yellowing, shortening of internodes and witches broom were observed in peach trees in the orchards of Meerut, India during May–August 2010–13. Leaf samples from symptomatic peach trees were collected and the presence of phytoplasma was confirmed by amplification of the 16S rRNA gene and secA gene by phytoplasma specific primers. Sequencing and BLAST analysis of ∼1.25 kb of the 16S rDNA gene and ∼840 bp of non-ribosomal secA gene fragments indicated that phytoplasma associated with peach disease was identical to the 16SrI Aster yellows group phytoplasma. Phylogenetic analysis of 16S rDNA peach phytoplasma also grouped it with 16SrI-A. This is the first report of association of a phytoplasma of 16SrI-A subgroup with peach trees in India.

Molecular identification, characterization and transmission of phytoplasmas associated with sesame phyllody in Turkey

Phyllody is a destructive disease of sesame in Turkey. The disease has been causing significant economic losses by stunting the plants and altering their floral parts into leafy structures with no capsule and hence no seeds in sesame fields of the country. This research was undertaken to examine symptomatology, etiology, taxonomy and transmission of two recently discovered phyllody phytoplasmas infecting sesame in Turkey. Direct and nested PCR amplifications of 16S rRNA gene with the phytoplasma-specific universal primers P1/P7 and R16F2n/R2, respectively were employed for identification of the phytoplasmas associated with sesame phyllody. Phytoplasma-specific PCR amplicons of 1.8 kb and 1.2 kb were amplified only from symptomatic sesame plants and insect vector samples. Sequencing of the PCR amplicons and computer simulated restriction fragment length polymorphism analysis allowed classification of the phytoplasmas with pigeon pea witches’-broom (16SrIX-C) and peanut witches’-broom (16SrII-D) groups. The sequence homology and phylogenetic analyses further confirmed this classification. Among the insects collected from the sesame fields, the leafhopper Orosius orientalis Matsumara (Syn: O. albicinctus Distant) was the only vector proven to transmit the sesame phyllody phytoplasmas from diseased to healthy sesame plants in transmission assays. The results demonstrated that the 16SrIX-C and 16SrII-D group phytoplasmas were the agent of sesame phyllody and O. orientalis was the vector insect of the disease in Turkey.

Natural occurrence of 16SrI group phytoplasmas on purslane (Portulaca oleraceaL.), a medicinal weed species in India

Portulaca oleracea L., an exotic weed with high medicinal value displaying typical phytoplasma associated symptoms, such as little leaf and yellowing were observed in sugarcane fields at Gorakhpur located in the state of Uttar Pradesh, India during 2013. Molecular analysis using nested-PCR with primer pairs P1/P7 and R16F2n/R16R2, evidenced that the phytoplasma found in all symptomatic plants. The nested PCR amplified product was sequenced. The approximately 1.2 kb PCR products that were observed in symptomatic Portulaca leaves had the highest nucleotide sequence similarity (99%) with members of ‘Ca. P. asteris’ group phytoplasma. Based on the BLAST analysis, the phytoplasma was classified as a member of the group 16SrI. Phylogenetic analysis also showed this phytoplasma as closely related to the reference strain for the group 16SrI. The P. oleracea phytoplasma was therefore assigned as 16SrI group and is the first report in India. Representatives of 16SrI phytoplasmas have been described in several cultivated and weed species in India and P. oleracea is an additional host species for this phytoplasma.

Molecular confirmation and interrelationship of phytoplasmas associated with diseases of palms in South India

Suspected phytoplasma symptoms of yellowing of inner whorl of leaves and rotting of spear in oil palm, yellowing of outer whorl of leaves in arecanut and flaccidity, yellowing and necrosis of crown leaves in coconut were observed in Kerala and Karnataka states of India. Phytoplasma presence in symptomatic oil palm, arecanut and coconut was confirmed by nested PCR assays with phytoplasma specific universal primers. 16Sr DNA sequence analysis and phylogenetic relationship studies confirmed the presence of 16SrXI group phytoplasmas in spear rot disease of oil palm from Kollam and Wayanad districts and in yellow leaf disease of arecanut from Wayanad district of Kerala. However, 16SrXIV group phytoplasmas were identified from root (wilt) disease of coconut from Kollam district of Kerala and yellow leaf disease of arecanut from Sullia of Dakshina Kannada district of Karnataka. This is the first report of association of 16SrXIV group phytoplasma with root (wilt) disease of coconut and yellow leaf disease of arecanut. These studies indicated the possible involvement of more than one group of phytoplasmas (16SrXI and 16SrXIV) in palm diseases in South India.

Molecular characterization based on spermidine/putrescine ABC transporter gene of sugarcane grassy shoot (16SrXI), coconut root wilt (16SrXI), aster yellows (16SrI) and brinjal little leaf (16SrVI) phytoplasmas

In the present study a molecular characterization of spermidine/putrescine ABC transporter gene fragments from four different phytoplasmas, namely coconut root wilt (16SrXI), sugarcane grassy shoot (16SrXI), aster yellows (16srI) and brinjal little leaf (16SrVI) was carried out based on DNA sequencing and in silico methods. Part of the ABC transporter potC and potB gene was employed for actual and virtual RFLP analysis. In a dendrogram, coconut root wilt and sugarcane grassy shoot phytoplasmas clustered together, aster yellows clustered with 16SrI group phytoplasmas and brinjal little leaf phytoplasma was separately placed suggesting that clustering based on this gene is congruent with 16S rDNA based phytoplasma differentiation.

Quantitative phenotyping of X-disease resistance in chokecherry using real-time PCR

A quantitative real-time SYBR Green PCR (qPCR) assay has been developed to detect and quantify X-disease phytoplasmas in chokecherry. An X-disease phytoplasma-specific and high sensitivity primer pair was designed based on the 16S rRNA gene sequence of X-disease phytoplasmas. This primer pair was specific to the 16SrIII group (X-disease) phytoplasmas. The qPCR method can quantify phytoplasmas from a DNA mix (a mix of both chokecherry and X-disease phytoplasma DNA) at as low as 0.001ng, 10-fold lower than conventional PCR using the same primer pair. A significant correlation between the copy number of phytoplasmas and visual phenotypic rating scores of X-disease resistance in chokecherry plants was observed. Disease resistant chokecherries had a significantly lower titer of X-disease phytoplasmas than susceptible plants. This suggests that the qPCR assay provides a more objective tool to phenotype phytoplasma disease severity, particularly for early evaluation of host resistance; therefore, this method will facilitate quantitative phenotyping of disease resistance and has great potential in enhancing plant breeding.

Molecular characterization identifies 16SrXI-B group phytoplasma (‘Candidatus Phytoplasma oryzae’-related strain) associated with root wilt disease of coconut in India

The root wilt disease of coconut is a major threat to coconut cultivation in southern India. Here we report the species assignment and 16Sr sub group classification of phytoplasma associated with coconut root wilt disease. Leaf samples were collected from root wilt symptomatic palms in Kayankulam district of Kerala. The phytoplasma 16S rRNA gene was amplified using three sets of primers namely, 1F7/7R3-1F7/7R2, 3Fwd/3Rev-3Fwd/5Rev, and P1/P7-R16F2n/R16R2 producing amplicons of 490, 1300, and 1250bp respectively. Partial secA gene sequence of 480bp from root wilt disease phytoplasma was amplified using primers cocsf/cocsr. Sequence characterization and phylogenetic analysis of 16S rRNA and secA genes of root wilt disease phytoplasma grouped it with the rice yellow dwarf group phytoplasmas and identified coconut RWD phytoplasma as ‘Candidatus Phytoplasma oryzae’ related strain. Further, in silico restriction digestion study of phytoplasmal 16S rRNA gene region between primers R16F2n/R16R2 was performed for 16Sr group/sub group classification. The root wilt disease phytoplasma grouped with the 16SrXI group members. In the sub group classification it produced identical restriction profile as the sugarcane white leaf phytoplasma and arecanut yellow leaf disease phytoplasma, and hence, it was placed in the 16SrXI-B sub group.

First Report of Two Distinct Phytoplasma Species, 'Candidatus Phytoplasma cynodontis' and 'Candidatus Phytoplasma asteris,' Simultaneously Associated with Yellow Decline of Wodyetia bifurcata (Foxtail Palm) in Malaysia.

The foxtail palm (Wodyetia bifurcata), an Australian native species, is an adaptable and fast-growing landscape tree. The foxtail palm is most commonly used in landscaping in Malaysia. Coconut yellow decline (CYD) is the major disease of coconut associated with 16SrXIV phytoplasma group in Malaysia (1). Symptoms consistent with CYD, such as severe chlorosis, stunting, general decline, and death were observed in foxtail palms from the state of Selangor in Malaysia, indicating putative phytoplasma infection. Symptomatic trees loses their green and vivid appearance as a decorative and landscape ornament. To determine the presence of phytoplasma, samples were collected from the fronds of 12 symptomatic and four asymptomatic palms in September 2012, and total DNA was extracted using the CTAB method (3). Phytoplasma DNA was detected in eight symptomatic palms using nested PCR with universal phytoplasma 16S rDNA primer pairs, P1/P7 followed by R16F2n/R16R2 (2). Amplicons (1.2 kb in length) were generated from symptomatic foxtail palms but not from symptomless plants. Phytoplasma 16S rDNAs were cloned using a TOPO TA cloning kit (Invitrogen). Several white colonies from rDNA PCR products amplified from one sample with R16F2n/R16R2 were sequenced. Phytoplasma 16S rDNA gene sequences from single symptomatic foxtail palms showed 99% homology with a phytoplasma that causes Bermuda grass white leaf (AF248961) and coconut yellow decline (EU636906), which are both members of the 16SrXIV 'Candidatus Phytoplasma cynodontis' group. The sequences also showed 99% sequence identity with the onion yellows phytoplasma, OY-M strain, (NR074811), from the 'Candidatus Phytoplasma asteris' 16SrI-B subgroup. Sequences were deposited in the NCBI GenBank database (Accession Nos. KC751560 and KC751561). Restriction fragment length polymorphism (RFLP) analysis was done on nested PCR products produced with the primer pair R16F2n/R16R2. Amplified products were digested separately with AluI, HhaI, RsaI, and EcoRI restriction enzymes based on manufacturer's specifications. RFLP analysis of 16S rRNA gene sequences from symptomatic plants revealed two distinct profiles belonging to groups 16SrXIV and 16SrI with majority of the 16SrXIV group. RFLP results independently corroborated the findings from DNA sequencing. Additional virtual patterns were obtained by iPhyclassifier software (4). Actual and virtual patterns yielded identical profiles, similar to the reference patterns for the 16SrXIV-A and 16SrI-B subgroups. Both the sequence and RFLP results indicated that symptoms in infected foxtail palms were associated with two distinct phytoplasma species in Malaysia. These phytoplasmas, which are members of two different taxonomic groups, were found in symptomatic palms. Our results revealed that popular evergreen foxtail palms are susceptible to and severely affected by phytoplasma. To our knowledge, this is the first report of a mixed infection of a single host, Wodyetia bifurcata, by two different phytoplasma species, Candidatus Phytoplasma cynodontis and Candidatus Phytoplasma asteris, in Malaysia.

Molecular identification of 16S rII phytoplasma group in commercial pistachio cultivars in Iran

Evaluation of phytoplasmas infection was conducted in the pistachio-growing areas of Iran (Rafsanjan in Kerman province) in early autumn of 2011. A total of 30 pistachio trees collected from a pistachio orchard in Rafsanjan showing Psylla damage symptoms and 10 samples with different abnormal symptoms from miscellaneous orchards were tested for the presence of phytoplasma. By using nested PCR with primer pairs P1/P7 and internal primer sets R16F2N/R16R2 and fU3-rU5, amplified fragment of expected size was observed in some trees with deformation and yellowing symptoms. On the basis of nucleotide sequence analysis of 16S rDNA amplified by PCR, this phytoplasma was classified in group 16S rII. In addition, we observed false positive reaction in three trees of Ahmadaghaei cultivar by using primer sets R16F2N/R16R2 and sequence analysis of ~1250bp PCR product indicated that amplified fragment was related to Schinus terebinthifolius; a species of flowering plant in the Anacardiaceae family.

First report of Rice orange leaf disease phytoplasma (16 SrI) in rice (Oryza sativa) in India

Rice(Oryza sativa L.,)isone ofthemostimportant cerealcropsworldwide.Inthisstudy,phytoplasmashavebeen detected in symptomatic leaves of four varieties of rice using nested-PCR with primer pairs P1/P7 and R16F2n/R16R2, which amplified a 1.2 kb fragment of the 16S rRNA gene. The nucleotide sequence analysis of the fragment had 100 % identity among the sequences from the four varieties (GenBank Accession numbers: JX290545, JX290546, JX290547 and JX290548) and 99 % nucleotide sequence identity with 16S rRNA gene sequences of the Rice orange leaf disease phytoplasmas from the Philippines and Thailand, Maize bushy stunt phytoplasma and Wheat blue dwarf phytoplasma belonging to the 'Candidatus Phytoplasma asteris' (16SrI) group. This is the first report of 16SrI group phytoplasmas infecting rice in India.

Grapevine yellows affecting the Croatian indigenous grapevine cultivar Grk

Abstract - The grapevine cultivar Grk, a close relative of Crljenak kaštelanski/Zinfandel, is grown exclusively in southern Croatia. Grapevine yellows-like symptoms were observed on vines in the vineyards in Lumbarda (southern Croatia) and in propagated grapevines near Zadar and Zagreb. The majority of the detected phytoplasma isolates belonged to the 16SrI group. However, RFLP pattern and R16F2n/R2 fragment sequence assigned one isolate to the 16SrIII group. Thus far, on cv. Grk, phytoplasmas belonging to three different groups have been detected: 16SrI, 16SrIII, and 16SrXII, which was confirmed previously. Aside from the 16SrI, 16SrV and 16SrXII phytoplasma groups previously found on grapevines in Croatia, the finding of 16SrIII group, which is not common on grapevines in Europe, adds to the diversity of phytoplasmas in a very small geographic region.