First Report on the Association of a 16SrII-A Phytoplasma with Sesame (Sesamum indicum) Exhibiting Abnormal Stem Curling and Phyllody in Taiwan

Sesame (Sesamum indicum L.), an annual plant, is grown as an oilseed crop and the seeds are used in bakery products in Taiwan. In June 2013, plants exhibiting symptoms including phyllody and abnormal stem curling were observed in sesame fields in Pitou Township, Changhua County, Taiwan. Incidence of infected plants was estimated to be greater than 90% within a single field. Phytoplasmas associated with sesame exhibiting phyllody, witches'-broom, or virescence have been classified as strains of 16SrI-B in Myanmar (GenBank Accession No. AB558132), 16SrII-A in Thailand (JN006075), 16SrII-D in Oman (EU072505) and India (KF429486), 16SrIV-C in Iran (JF508515), and 16SrVI-A (KF156894) and 16SrIX (KC139791) in Turkey (1). Three symptomatic and four asymptomatic plants were uprooted and transplanted in a greenhouse for further study. Transmission electron microscopy (TEM) revealed clusters of phytoplasma cells ranging from 300 to 800 nm in diameter only in phloem sieve elements of stems of three symptomatic and two asymptomatic plants. Comparable tissues from two other symptomless plants were devoid of phytoplasma cells. Total DNA was extracted with a modified CTAB method (2) from plant tissues (100 mg each) including stem, leaf, petiole, and root from the same plants used for TEM work. Analyses by a nested PCR using universal primer pairs P1/P7 (5′-AAGAGTTTGATCCTGGCTCAGGATT/5′-CGTCCTTCATCGGCTCTT) followed by R16F2n/R16R2 (5′-GAAACGACTGCTAAGACTGG/5′-TGACGGGCGGTGTGTACAAACCCCG) were performed to detect putative phytoplasma DNA (3). Each primer pair amplified a single PCR product of either 1.8 or 1.2 kb, respectively, only from the three symptomatic and two asymptomatic plant tissues that had phytoplasma cells in their sieve elements. It is likely that these two asymptomatic plants were in the early stage of infection before symptoms became noticeable. The nested PCR products (1.2 kb) amplified from the symptomatic plants were cloned separately and sequenced (GenBank Accession Nos. KF923391, KF923392, and KF923393). BLAST analysis of the sequences revealed that they shared 99.2% sequence identity with strains reported from India and Thailand (KF429486 and JN006075), which were classified to the 16SrII-D and 16SrII-A subgroups, respectively. Moreover, iPhyClassifier software (4) was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile. The 16S rDNA sequences shared 99.4% identity with that of the ‘Candidatus Phytoplasma australasiae’ (Y10097) and the RFLP patterns were identical to that of the 16SrII-A subgroup, indicating the Taiwanese strain is a ‘Ca. P. australasiae’-related strain. To our knowledge, this is the first report of a 16SrII-A subgroup phytoplasma causing phyllody and abnormal stem curling on sesame in Taiwan. The occurrence of phytoplasma on sesame could have direct implications for the cultivation of this economically important oilseed plant and the bakery industry in Taiwan. References: (1) M. Catal et al. Plant Dis. 97:835, 2013. (2) T. M. Fulton et al. Plant Mol. Biol. Rep. 13:207, 1995. (3) D. E. Gundersen and I. M. Lee. Phytopathol. Mediterr. 35:144, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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First Report of a 16SrII-A Subgroup Phytoplasma Associated with Purple Coneflower (Echinacea purpurea) Witches'-Broom Disease in Taiwan

Plant Disease ◽

10.1094/pdis-10-11-0888 ◽

2012 ◽

Vol 96 (4) ◽

pp. 582-582 ◽

Cited By ~ 8

Author(s):

Y.-W. Tseng ◽

W.-L. Deng ◽

C.-J. Chang ◽

C.-C. Su ◽

C.-L. Chen ◽

...

Keyword(s):

Medicinal Plant ◽

Nested Pcr ◽

Echinacea Purpurea ◽

Flowering Plant ◽

Diseased Plant ◽

Universal Primer ◽

Tissue Samples ◽

First Report ◽

Rdna Sequences ◽

Purple Coneflower

Purple coneflower (Echinacea purpurea), widely grown as an ornamental and medicinal plant, is a perennial flowering plant that is native to eastern North America. In July 2011, symptoms indicative of phytoplasma disease, including floral virescence, phyllody, and witches'-broom (WB), were observed to be affecting plants in coneflower fields in Wufeng, Taichung City, Taiwan. Incidence of infected plants was estimated to be greater than 90% within a single field. Phytoplasmas previously associated with purple coneflower WB disease have all been classified as aster yellows group (16SrI) strains (GenBank Accession Nos. EU333395, AY394856, EU416172, and EF546778) except for pale purple coneflower (Echinacea pallida) WB in Australia, which was identified as a subgroup 16SrII-D member (2). Three diseased plants were uprooted and transplanted in a greenhouse for further study. Transmission electron microscopy revealed clusters of phytoplasma cells ranging from 170 to 490 nm in diameter in phloem sieve elements of virescent and phylloid flowers and stems from diseased plants. Comparable tissues from symptomless plants were devoid of phytoplasma. Total DNA was extracted from plant tissue samples (50 to 100 mg each) including stems, leaves, and flowers by a modified CTAB method (1) from three symptomatic plants as well as from three asymptomatic coneflower plants seedlings. Analyses by a nested PCR using universal primer pairs P1/P7 followed by R16F2n/R16R2 were performed to detect putative phytoplasma (2). Each primer pair amplified a single PCR product of either 1.8 or 1.2 kb, respectively, from diseased plant tissues only. The nested PCR products (1.2 kb) amplified from phylloid flowers of the three diseased plants were cloned separately and sequenced (GenBank Accession Nos. JN885460, JN885461, and JN885462). Blast analysis of the sequences revealed a 99.7 to 99.8% sequence identity with those of Echinacea WB phytoplasma strain EWB5 and EWB6 (GenBank Accession Nos. JF340076 and JF340080), which reportedly belonged to the 16SrII-D subgroup (2). Moreover, iPhyClassifier software (3) was used to perform sequence comparison and generate the virtual restriction fragment length polymorphism (RFLP) profile. The 16S rDNA sequences share a 99.4 to 99.5% similarity with that of the ‘Candidatus Phytoplasma australasiae’ reference strain (Y10097) and the RFLP patterns are identical to that of the 16SrII-A subgroup. Taken together, these results indicated that the phytoplasma infecting purple coneflower in Taiwan is a ‘Ca. Phytoplasma australasiae’-related strain and belongs to the 16SrII-A subgroup. To our knowledge, this is the first report of a 16SrII-A subgroup phytoplasma causing WB disease on purple coneflower in Taiwan. The occurrence of phytoplasma on purple coneflower could have direct implication for the economically important ornamental, medicinal plant, and floral industry in Taiwan, especially to the growers and breeders that eagerly promote the purple coneflower industry. References: (1) T. M. Fulton et al. Plant Mol. Biol. Rep. 13:207, 1995. (2) T. L. Pearce et al. Plant Dis. 95:773, 2011. (3) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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

Plant Disease ◽

10.1094/pdis-12-13-1256-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 991-991 ◽

Cited By ~ 4

Author(s):

Y.-W. Tseng ◽

C.-J. Chang ◽

J.-W. Chen ◽

W.-L. Deng ◽

F.-J. Jan

Keyword(s):

Nested Pcr ◽

Hibiscus Sabdariffa ◽

Universal Primer ◽

First Report ◽

Rdna Sequences ◽

Transmission Electron ◽

Pcr Product ◽

Ctab Method ◽

Pcr Products ◽

Single Field

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. References: (1) C. Biswas et al. Phytoparasitica 41:539, 2013. (2) I. Echevarría-Machado et al. Mol. Biotechnol. 31:129, 2005. (3) W. Wei et al. Int. J. Syst. Evol. Microbiol. 57:1855, 2007.

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First Report of a Group 16SrI Phytoplasma Associated with Amaranthus hypochondriacus Cladodes in China

Plant Disease ◽

10.1094/pdis-03-11-0219 ◽

2011 ◽

Vol 95 (7) ◽

pp. 871-871

Author(s):

J. Y. Long ◽

Y. H. Chen ◽

J. R. Xia

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Universal Primer ◽

First Report ◽

Amaranthus Hypochondriacus ◽

Rdna Sequences ◽

16S Rdna Sequences ◽

Ctab Method ◽

Pcr Products ◽

Blastn Analysis

Amaranthus spp. are cultivated worldwide as leafy vegetable, cereal, and ornamentals. In China, stems and leaves of Amaranthus hypochondriacus L. are used as a vegetable (2). In July 2010, sporadic amaranth plants exhibiting symptoms of cladodes and spica proliferation were observed in a vegetable garden near Foshan, Guangdong, China. Stem samples were collected from two symptomatic and two asymptomatic plants. Total DNA was extracted with a modified cetyltrimethylammonium bromide (CTAB) method (1). Nested PCR with a combination of phytoplasma-specific universal primer pairs (P1/P7 and R16F2n/R16R2) amplified 16S rDNA sequences with the expected size of 1.2 kb from all samples of symptomatic amaranth plants, but not from the asymptomatic plants (3). Nested PCR products yielded identical AluI, HhaI, HpaII, HaeIII, KpnI, MseI, RsaI, Sau3AI, and TaqI restriction fragment length polymorphism (RFLP) profiles with chinaberry witches'-broom phytoplasma (16SrI-B subgroup), but different from peanut witches'-broom phytoplasma (16SrII group), jujube witches'-broom phytoplasma (16SrV group), and paulownia witches'-broom phytoplasma (16SrI-D subgroup). Nested PCR products were purified, cloned in pMD18-T Simple Vector (TaKaRa, Dalian, China), and sequenced. The 16S rDNA sequences were identical and deposited in GenBank (Accession No. JF323034). GenBank BLASTn analysis indicated that the amaranth extracts showed as high as 99% sequence identity with the members of 16SrI group phytoplasmas, including those associated with arecanut yellow leaf disease (FJ998269) and aster yellow AY-27 (HM467127). A polygenetic tree was constructed using MEGA 4.0 based on the 16S rDNA sequences of amaranth cladode phytoplasma and other phytoplasmas belonging to 16SrI phytoplasma group. In phylogenetic analysis, the sequences clustered on a single branch with members of 16SrI-B subgroup in the tree. Therefore, the phytoplasma was classified in subgroup 16SrI-B. To our knowledge, this is the first report of a subgroup 16SrI-B phytoplasma associated with diseased A. hypochondriacus in China. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) M. Costea et al. Econ. Bot. 57:646, 2003. (3) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

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First Report of Pear Decline Phytoplasmas on Pear in Serbia

Plant Disease ◽

10.1094/pd-89-0774c ◽

2005 ◽

Vol 89 (7) ◽

pp. 774-774 ◽

Cited By ~ 3

Author(s):

B. Duduk ◽

M. Ivanović ◽

A. Obradović ◽

S. Paltrinieri ◽

A. Bertaccini

Keyword(s):

Nested Pcr ◽

Restriction Enzymes ◽

Diagnostic Procedures ◽

Nested Polymerase Chain Reaction ◽

Universal Primer ◽

First Report ◽

Taxonomy Group ◽

Pyrus Communis L ◽

Polymerase Chain ◽

Symptomatic Plant

During August of 2004, pear (Pyrus communis L.) plants with typical symptoms of pear decline (PD) were observed in orchards in central Serbia. The affected plants showed premature reddening and upward rolling of leaves that often showed down-turned petioles. In some cases, premature defoliation was observed. Although a similar decline of pear was observed earlier, until now, the causal agent had not been identified. DNA was extracted with a chloroform/phenol procedure from fresh leaf midribs and branch phloem scrapes of four symptomatic and one asymptomatic pear plants separately. A nested polymerase chain reaction assay (PCR) was used for phytoplasma detection (first PCR round with P1/P7 (4) phytoplasma universal primer pair, followed by nested PCR with group 16SrX specific primers f01/r01) (3). With these primers, the expected products from phloem scrapes and midrib extracts of symptomatic plant samples were obtained. Restriction fragment length polymorphism (RFLP) analyses of the f01/r01 amplicon, with RsaI and SspI restriction enzymes, discriminating among 16SrX subgroup phytoplasmas, showed profiles corresponding to those of the apple proliferation phytoplasma group, 16SrX-C subgroup, “Candidatus Phytoplasma pyri” (2). A 1,155-bp sequence of 16S rDNA gene for one of the PA2f/r (1) amplicons obtained in nested PCR on P1/P7 products from one of the leaf midrib samples was deposited in GenBank (Accession No. AY949984); both strands of the fragment were sequenced with the Big Dye Terminator reaction kit (Applied Biosystems, Foster City, CA). The sequences were analyzed with the Chromas 1.55 DNA sequencing software (Technelysium, Queensland, Australia) and aligned with BLAST software ( http://www.ncbi.nlm.nih.gov ). The blast search showed 100% homology of this sequence with that of PD strain Y16392, confirming the identity with PD of the phytoplasma detected. To our knowledge, this is the first report of pear decline phytoplasmas in Serbia. References: (1) M. Heinrich et al. Plant Mol. Biol. Rep. 19:169, 2001. (2) IRPCM Phytoplasma/Spiroplasma Working Team-Phytoplasma Taxonomy Group. Int. J. Syst. Evol. Microbiol. 54:1243, 2004. (3) K.-H. Lorenz et al. Phytopathology 85:771, 1995. (4) Schneider et al. Pages 369–380 in: Molecular and Diagnostic Procedures in Mycoplasmology. Vol I. S. Razin and J. G. Tully, eds. The American Phytopathological Society, 1995.

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First Report of a Group 16SrI-B Phytoplasma Associated with Gardenia jasminoides in China

Plant Disease ◽

10.1094/pdis-03-12-0301-pdn ◽

2012 ◽

Vol 96 (10) ◽

pp. 1576-1576 ◽

Cited By ~ 3

Author(s):

X. C. Sun ◽

W. J. Zhao

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Consensus Sequence ◽

Rrna Gene ◽

Reference Pattern ◽

Green Belt ◽

First Report ◽

Rdna Sequences ◽

Gardenia Jasminoides ◽

16S Rdna Sequences

Gardenia jasminoides J. Ellis, (also known as common gardenia, cape jasmine, or cape jessamine) is a fragrant flowering evergreen tropical plant, a favorite in gardens worldwide. G. jasminoides were found with small, seriously yellowed leaves, stunted growth, and witches'-broom in a green belt on the Southwest University campus in October 2011. The incidence was lower than 2%. In another green belt, G. jasminoides with only slightly yellowing leaves were found. The incidence was about 5%. Five months later, most seriously yellowed leaves withered. However, no withered leaf was observed among the slightly yellowing leaves. Leaf samples from each symptomatic plant, together with asymptomatic plants from the same belt, were collected for total DNA extraction using a modified cetyltrimethylammoniumbromide method (1). The resulting DNA extracts were analyzed by a nested PCR assay using the phytoplasma 16S rRNA gene primer pairs R16mF2/R16mR1 followed by R16F2n/R16R2 (2). DNA fragments of 1.2 kb that corresponded to 16S rDNA were amplified only from the DNA samples of the five plants with the symptoms mentioned above. The purified nested PCR products were cloned in pGEM-T Easy Vector (Promega) and then sequenced. The resulting 16S rDNA sequences were found to be identical (GenBank Accession No. JQ675713). The consensus sequence was analyzed by the iPhyClassifier online tool ( http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi ) and found to share 99.4% similarity with the 16S rDNA sequence of the ‘Candidatus Phytoplasma asteris’ reference strain (GenBank Accession No. M30790) that belongs to the 16SrI-B subgroup (3). The virtual RFLP pattern of the G. jasminoides phytoplasma 16S rDNA gene sequence showed maximum similarity to the reference pattern of NC005303 (similarity coefficient of 1.0). The phylogenetic tree based on the 16S rDNA sequences of phytoplasmas belonging to group 16SrI and other distinct phytoplasma groups also showed that our sequences clustered with members of subgroup 16SrI-B. Subsequently, the presence of the phytoplasmas in symptomatic plants was also confirmed by transmission electron microscopy. Taken together, the phytoplasma was classified as a member of subgroup 16SrI-B. To our knowledge, this is the first report of a subgroup 16SrI-B phytoplasma associated with diseased G. jasminoides in China. G. jasminoides yellowing is often considered to result from nutrient deficiency (especially iron compounds). However, our findings showed that a phytoplasma can cause G. jasminoides yellowing, which should be considered in the control of leaves yellowing. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) Y. Zhao, et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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First Report of ‘Candidatus Phytoplasma asteris’-Related Strain Associated with Peach Rosette in Canada

Plant Disease ◽

10.1094/pdis-94-7-0916c ◽

2010 ◽

Vol 94 (7) ◽

pp. 916-916 ◽

Cited By ~ 6

Author(s):

S. Zunnoon-Khan ◽

R. Michelutti ◽

Y. Arocha-Rosete ◽

J. Scott ◽

W. Crosby ◽

...

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

The United States ◽

Polymorphism Analysis ◽

Plant Host ◽

First Report ◽

Rdna Sequences ◽

16S Rdna Sequences ◽

Pcr Products ◽

Short Internodes

Prunus persica (L.) Bastch (family Rosaceae) is currently represented by 83 accessions at the Canadian Clonal Genebank. Approximately 3,200 ha are devoted to peach cultivation in Canada where Ontario Province accounts for 82% of the national production. The clonal peach accessions, also located in Ontario, are monitored routinely for symptoms of phytoplasma infection, including rosette-like symptoms (3) that are characterized by new shoots with very short internodes, loss of older shoot leaves leaving only bunches of young leaves on the tips of naked shoots, and flowers that rarely set fruit. From June to August 2009, peach accessions PRU0382 and PRU0445 showed typical peach rosette symptoms, while another 14 accessions exhibited either short internodes or no symptoms. Leaf midrib samples were collected from 16 peach accessions, including 17 symptomatic (from which 8 corresponded to accession PRU0382, 6 for PRU0445, 1 for PRU0335, 1 for PRU0179, and 1 for PRU0451) and 16 asymptomatic (from which 5 corresponded to a representative of each accession PRU0382, PRU0445, PRU0335, PRU0179, and PRU0451 and 11 to other peach accessions). Total DNA was extracted (DNeasy Plant Extraction Mini Kit, QIAGEN, Valencia, CA) from 100 mg of each sample and used as a template in a nested PCR with phytoplasma universal primers R16mF2/R1 (1) and fU5/rU3 (2). Nested PCR products of the expected size (~880 bp) were obtained from all symptomatic samples (14 of 14) of accessions PRU0382 (peach-almond cv. Kando from the Czech Republic) and PRU0445 (peach cv. HW271 from Canada) only. All other plants with or without symptoms yielded no PCR products. Amplicons were purified (Wizard PCR Clean-up, Promega, Madison, WI), cloned in pGEM-T Easy Vector (Promega), and sequenced (Robarts Institute, London, Canada). The resulting 16S rDNA sequences were identical; one of each was archived in GenBank as Accession No. GU223904. BLAST analysis determined that the P. persica phytoplasma sequence shared 99% identity with 16S rDNA sequences of ‘Candidatus Phytoplasma asteris’-related strains. This relationship was also supported by restriction fragment length polymorphism analysis (RFLP) of rDNA amplicons using AluI, RsaI, and MseI endonucleases that yielded fragment profiles indicative of phytoplasmas belonging to group 16SrI (Aster Yellows), subgroup B (16SrI-B). Among phytoplasma diseases, those attributed to group 16SrI strains are most numerous and affect the widest plant host range. They include peach rosette in the United States and Europe (3) as well as diseases of various horticultural crops in Canada, including grapevine (4). To our knowledge, this is the first report of a subgroup 16SrI-B phytoplasma affecting peach in Canada. Early detection of phytoplasmas by PCR in accessions with both European and Canadian origins underscores the importance of prompt identification of infected plants for subsequent thermotherapy treatment to maintain the health of the collection and prevent further disease spread. References: (1) D. E Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:1441, 1996. (2) K. H. Lorenz et al. Phytopathology 85:771, 1995. (3) C. Marcone et al. Acta Hortic. 386:471, 1995. (4) C. Y. Olivier et al. Plant Dis. 93:669, 2009.

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First Report of a 16SrIX Group (‘Candidatus Phytoplasma phoenicium’-Related) Phytoplasma Associated with a Chrysanthemum Disease

Plant Disease ◽

10.1094/pdis-01-13-0050-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1110-1110 ◽

Cited By ~ 4

Author(s):

H. Bayat ◽

J. Th. J. Verhoeven ◽

M. Botermans ◽

D. Peters ◽

A. Hassani-Mehraban

Keyword(s):

Nested Pcr ◽

Ornamental Plants ◽

Rflp Analysis ◽

Chrysanthemum Morifolium ◽

Rrna Gene ◽

Universal Primer ◽

First Report ◽

Production Region ◽

Lactuca Serriola ◽

Plant Pathol

In November 2010, approximately 2% of the chrysanthemum (Chrysanthemum morifolium) cv. Paniz plants showed numerous small leaves in the top and stunting in a field collection of the National Research Center of Ornamental Plants in Mahallat, Iran. Next to these plants, some plants of the same collection showed leaves with a reddish and/or chlorotic discoloration around the veins. The observed symptoms were believed to represent infection by a phytoplasma and/or a viroid. Two plants with each type of the symptoms were individually analyzed. Using a total RNA extract from diseased leaves, RT-PCR with primer pairs targeting all known pospiviroids, including Chrysanthemum stunt viroid (CSVd) (3), were negative. Purified DNA was examined for the highly conserved phytoplasma 16S rRNA gene by nested-PCR using the universal primer sets P1/P7 and R16F2n/R16R2 (2). Fragments of 1.2 kb, obtained only from the plants with the small leaves and stunting, were sequenced and one of these sequences, which were identical, was deposited in GenBank (Accession No. KC176800). BLAST analysis of the chrysanthemum phytoplasma sequence exhibited 99% identity to Candidatus Phytoplasma phoenicium (Ca. P. phoenicium) species of the 16SrIX group. Subsequently, in silico RFLP analysis of the nested PCR product with the pDRAW32 program using AluI and TaqI restriction sites used for 16SrIX subgroups A, B, C, D, and E indicated that the 16SrIX chrysanthemum isolate belonged to subgroup D (1). Recently, based on GenBank sequences, several strains of Ca. P. phoenicium have been isolated and identified from diverse host species like Lactuca serriola, L. sativa, Solanum lycopersicon, Sonchus sp. [16SrIX-E], Carthamus tinctorius, and Prunus amygdalus [16SrIX-B] (4) in Iran. The vector species transmitting Ca. P. phoenicium to C. morifolium still needs to be identified. The leafhopper Neoaliturus fenestratus may be a potential vector as it is an often encountered efficient transmitter vector of 16SrIX group phytoplasmas in Iran (2). Next to the susceptibility of chrysanthemum to members of aster yellows, stolbur, and Ca. P. aurantifolia phytoplasma groups, this is, to our knowledge, the first report of a 16SrIX group member infecting chrysanthemum. The detection of this phytoplasma in chrysanthemum can form a new threat to this crop and other ornamentals in the Mahallat flower production region. References: (1) R. E. Davis et al. New Dis. Rep. 20:35, 2010. (2) M. Salehi et al. Plant Pathol. 56:669, 2007. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) M. G. Zamharir. Afr. J. Microbiol. Res. 5:6013, 2011.

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First Report of a “Candidatus Phytoplasma australiense”-Related Strain in Lucerne (Medicago sativa) in Australia

Plant Disease ◽

10.1094/pd-91-0111a ◽

2007 ◽

Vol 91 (1) ◽

pp. 111-111 ◽

Cited By ~ 7

Author(s):

M. A. Getachew ◽

A. Mitchell ◽

G. M. Gurr ◽

M. J. Fletcher ◽

L. J. Pilkington ◽

...

Keyword(s):

Medicago Sativa ◽

Nested Pcr ◽

The United States ◽

Plant Sample ◽

Related Strain ◽

Universal Primer ◽

Separate Species ◽

First Report ◽

Pcr Products ◽

Plant Pathol

Australian lucerne yellows (ALuY), a phytoplasma-associated disease, is a major problem in Australia that causes the pasture seed industry millions of dollars in losses annually (3). Samples were collected from lucerne (Medicago sativa L.) plants exhibiting symptoms indicative of ALuY (4) in a seed lucerne paddock (cv CW 5558) at Griffith, southwestern New South Wales (NSW), Australia, in November 2005 and again in January 2006. Samples were kept at 4°C and processed within 36 h of collection. Total DNA was extracted from approximately 0.3 g of leaf midribs and petioles of each plant sample and used as template in a nested PCR assay with phytoplasma universal primer pairs P1/P7 and fU5/m23sr. PCR products resulting from the first amplification were diluted (1:30) with sterile distilled water (SDW) before reamplification with fU5/m23sr. DNA of Australian tomato big bud (TBB) phytoplasma and SDW were used as positive and negative assay controls, respectively. Ten of fifteen plant samples collected in November tested positive for phytoplasma DNA. Restriction digestion profiles of nested PCR amplicons with HpaII endonuclease were the same for all symptomatic plants but differed from the control. Phytoplasma identity was determined by sequencing two nested PCR products that yielded identical sequences. One was deposited in the GenBank database (Accession No. DQ786394). BLAST analysis of the latter sequence revealed a >99.6% similarity with “Candidatus Phytoplasma australiense” (L76865) and related strains papaya dieback (Y10095), phormium yellow leaf (U43570), strawberry green petal (AJ243044), and strawberry lethal yellows (AJ243045). Direct PCR with primers FP 5′-GCATGTCGCGGTGAATAC-3′ and RY 5′-TGAGCTATAGGCCCTTAATC-3′ designed to specifically amplify DNA of “Ca. P. australiense” detected the phytoplasma in 8 of 40 samples collected in January. Whether this phytoplasma is the etiological agent solely responsible for ALuY is currently under investigation. “Ca. P. asteris” and stolbur group (16SrXII) phytoplasmas have been reported in lucerne in the United States (2) and Italy (1), respectively. Within the stolbur group 16SrXII, “Ca. P. australiense” and stolbur phytoplasma are regarded as separate species and both are distinct from “Ca. P. asteris”, a group 16SrI strain. To our knowledge, this is the first report of a “Ca. P. australiense” related strain in lucerne. References: (1) C. Marzachi et al. J. Plant Pathol. 82:201, 2000. (2) R. D. Peters et al. Plant Dis. 83:488, 1999. (3) L. J. Pilkington et al. Australas. Plant Pathol. 28:253, 1999. (4) L. J. Pilkington et al. First report of a phytoplasma associated with ‘Australian lucerne yellows’ disease. New Disease Report. Online publication at http://www.bspp.org.uk/ndr/jan2002/2001-46.asp .

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An Emerging Potato Purple Top Disease Associated with a New 16SrIII Group Phytoplasma in Montana

Plant Disease ◽

10.1094/pdis-93-9-0970b ◽

2009 ◽

Vol 93 (9) ◽

pp. 970-970 ◽

Cited By ~ 7

Author(s):

I.-M. Lee ◽

K. D. Bottner ◽

M. Sun

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Restriction Enzymes ◽

The United States ◽

Disease Spread ◽

Potential Threat ◽

Universal Primer ◽

Rdna Sequences ◽

16S Rdna Sequences ◽

Potato Purple Top

Potato purple top (PPT) is a devastating disease that occurs in the United States, Canada, Mexico, Russia, and elsewhere causing great economic loss to the potato industry through substantially reduced tuber yield and quality. Chips and fries processed from infected tubers often develop brown discoloration, greatly reducing their marketability. At least seven distinct phytoplasma strains belonging to five different phytoplasma groups (16SrI, 16SrII, 16SrVI, 16SrXII, and 16SrXVIII) have been reported to cause purple top and related symptoms in potato (3). During an unusual drought in 2007, a newly emerging potato disease with extensive yellowish or reddish purple discoloration of terminal shoots and leaves, similar to PPT symptoms, was observed in isolated potato fields in Montana where over 50% of plants exhibited symptoms. Shoot tissues were collected from three symptomatic plants and 17 tubers randomly collected from 17 other symptomatic plants. The tubers were cold treated to induce sprouting and then planted in the greenhouse. All tubers produced plants of which seven exhibited PPT symptoms including severe stunting. Total nucleic acid was extracted from leaf veinal tissue, stolons, or tubers of 10 symptomatic and 10 asymptomatic plants (both field-collected and greenhouse samples) as previously described (3). A nested-PCR assay, using universal primer pair P1/16S-SR followed by R16F2n/R16R2n, was performed as previously described (2,3) to detect phytoplasmas in these samples. Phytoplasma strains were detected in all symptomatic plants. Restriction fragment length polymorphism (RFLP) analyses of nested-PCR products (approximately 1.2 kb) with seven key restriction enzymes (AluI, MseI, HhaI, Tsp509I, HpaII, RsaI, and BfaI) indicated that all samples contained a very similar or identical phytoplasma most closely related to reference strain MW1 (belonging to subgroup 16SrIII-F) (1). Analysis of cloned 16S rDNA sequences confirmed the identity of this new phytoplasma and sequences of three representative PPT-MT strains were deposited in GenBank with Accession Nos. FJ226074–FJ226076. Computer-simulated RFLP analyses of 1.2-kb 16S rDNA sequences of this new phytoplasma and representative members in the peach X-disease phytoplasma group (16SrIII) available in GenBank indicated the strain is distinct and represents a new subgroup, 16SrIII-M (4). This study also indicated that the phytoplasma is tuber transmissible since approximately 35% of plants produced from infected tubers collected in this study developed symptoms. Transmission via infected tubers may pose a potential threat for disease spread by planting uncertified seed potatoes. To our knowledge, this is the first report of 16SrIII group phytoplasmas-associated diseases in potato. A phytoplasma closely related to the PPT-MT strains has recently been detected in potato seedlings exhibiting purple top, rosette, and stunting in Alaska (GenBank Accession No. FJ376628). References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 56:1593, 2006. (4) W. Wei et al. Int. J. Syst. Evol. Microbiol. 58:2368, 2008.

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First Report of 16SrII-D Phytoplasma ‘Candidatus Phytoplasma aurantifolia’ Associated with Mung Bean Phyllody in Andhra Pradesh, India

Plant Disease ◽

10.1094/pdis-04-14-0428-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1424-1424 ◽

Cited By ~ 4

Author(s):

N. Ragimekula ◽

K. Chittem ◽

V. N. Nagabudi ◽

L. E. del Río Mendoza

Keyword(s):

Nested Pcr ◽

Mung Bean ◽

Andhra Pradesh ◽

Reference Strain ◽

Indian Subcontinent ◽

Extraction Procedure ◽

Direct Pcr ◽

Universal Primer ◽

First Report ◽

Longitudinal Splitting

Mung bean (Vigna radiata (L.) R. Wilczek) is an important edible legume grown in Asia, particularly in the Indian subcontinent, where it is used for human and animal consumption. In September 2013, 10% of a group of 90 mung bean breeding lines in experimental plots of S. V. Agricultural College, Tirupati, Andhra Pradesh, India, exhibited symptoms typical of a phytoplasma infection, including stunting, extensive proliferation of branches, reduction in leaf size, phyllody, and longitudinal splitting of green pods followed by germination of green seeds producing small plants. These symptoms have been associated with mung bean phyllody (1,3). While the severity of infection varied within each line, on average, 20% of symptomatic plants did not produce seeds at all. Leaf samples from two symptomatic plants and one asymptomatic plant were collected and DNA was extracted from leaves following a CTAB DNA extraction procedure (2). Direct PCR and nested PCR assay was performed using phytoplasma 16S rRNA universal primer pairs P1/P7 and R16F2n/R16R2 (4). Both of the symptomatic samples produced 1.8 kb and 1.2 kb size amplicons after direct and nested PCR cycles, respectively. No amplicons were produced with DNA from the asymptomatic sample. Nested PCR products (1.2 kb) from the two symptomatic samples corresponding to the F2nR2 region of 16S rDNA were directly sequenced on an ABI 3730 XL automated sequencer at McLab sequencing services (McLab, CA). Both samples were 100% identical and the representative sequence was designated as APMBP and deposited in GenBank with the accession number KF811205. BLAST analysis revealed a 100% sequence identity with 16SrII group ‘Candidatus Phytoplasma aurantifolia’ phytoplasmas that include sesame phyllody phytoplasmas isolated from sesame and tomato in India (KF429485 and JX104335, respectively). Subgroup identification was performed using the iPhyClassifier online tool (5). The samples were identified as 16SrII-D subgroup phytoplasma based on their 100% identity to the reference strain (GenBank Accession No. Y10097) and virtual RFLP profiles. Phylogenetic analysis based on the F2nR2 sequences with the representative sequences were placed the APMBP in a single distinct cluster with the 16SrII-D reference strain Y10097. Although occurrence of phyllody on mung bean in India was first reported in 1988 (3), the report was based on the appearance of symptoms. This pathogen was recently reported as associated with mung bean phyllody in Pakistan (1). To our knowledge, this is the first report of ‘Ca. P. aurantifolia’ strain infecting mung bean in India. Phytoplasmas belonging to subgroup 16SrII-D are known to have a wide host range, including chickpea, peanuts, sesame, and tomato, which are commonly grown in this region. Mung bean plants infected early failed to produce normal seeds indicating of the potential of this 16SrII-D phytoplasma to become a production constraint for mung bean and other host crops in the area. References: (1) K. P. Akhtar et al. Plant Pathol. 59:399, 2010. (2) J. J. Doyle and J. L. Doyle. Phytochem. Bull. 19:11, 1987. (3) P. Lakshmanan et al. Curr. Sci. 57:809, 1988. (4) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (5) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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