First Report of Clover Proliferation Group Phytoplasmas (16SrVI-A) Associated with Purple Top Diseased Potatoes (Solanum tuberosum) in China

Phytoplasma diseases on potatoes are not well understood and have gone largely undetected in China. During the growing seasons of 2005 through 2010, potato disease surveys were conducted in seed and commercial fields in Yunnan Province. Samples were also harvested from three seed potato production areas in the Inner Mongolia Autonomous Region in 2007 and 2010. Disease incidence in these fields ranged from 15 to 85%. Plants displayed symptoms of branch proliferation, aerial tuber formation, upward rolling yellowish and purplish apical leaves, and extremely short stolen or chain tubers (irregular-shaped tubers). Total DNA from 250 samples was extracted from the leaves, stems, and roots of symptomatic and asymptomatic plants. A nested PCR was performed by using primer pair P1/P7 followed by R16F2n/R16R2 to detect the presence of phytoplasmas (1,3). An approximate 1.2-kb PCR product was amplified from symptomatic plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns were analyzed by digesting the 1.2-kb amplicon singly with restriction enzymes AluI, BfaI, MseI, HhaI, HinfI, HpaII, KpnI, RsaI, and TaqI. The RFLP patterns of 120 of the 250 samples matched patterns of the clover proliferation (CP) group (16SrVI) subgroup A (16SrVI-A) phytoplasma (1). In addition, the nested PCR product of P1A/P7A (2) following P1/P7 amplification was cloned and sequenced (GenBank Accession No. HQ609490). Nucleotide sequences were analyzed by iPhyClassifier software (4), confirming the relationship of this phytoplasma to ‘Candidatus Phytoplasma trifolii’ with RFLP patterns identical to group 16SrVI-A. To our knowledge, this is the first report of the CP group phytoplasmas associated with purple top diseased potatoes in China. 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) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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Molecular Characterization of Stolbur Group Subgroup E (16SrXII-E) Phytoplasma Associated with Potatoes in China

Plant Disease ◽

10.1094/pdis-01-12-0102-pdn ◽

2012 ◽

Vol 96 (9) ◽

pp. 1372-1372 ◽

Cited By ~ 6

Author(s):

M. Cheng ◽

J. Dong ◽

L. Zhang ◽

P. J. Laski ◽

Z. Zhang ◽

...

Keyword(s):

Molecular Characterization ◽

Nested Pcr ◽

Inner Mongolia ◽

Disease Incidence ◽

Restriction Enzymes ◽

Potato Disease ◽

Growing Seasons ◽

Pcr Product ◽

Disease Surveys

Phytoplasmas have been reported from more than 70 plant species in China, most of which are from woody plants and very few are from potatoes (Solanum tuberosum). During the growing seasons of 2005 through 2011, potato disease surveys were conducted in seed and commercial fields in Yunnan Province and Inner Mongolia. Potato plants displayed symptoms of curled, yellowish and purplish leaves, shortened internodes, aerial tuber formation, and few small malformed underground tubers. Although the location of the fields surveyed each year varied, the disease seems to have become increasingly prevalent. In Yunnan, disease incidence was 5 to 24% in 2005 and 15 to 100% in 2010 and 2011. In Inner Mongolia, disease incidence in seed potato fields was 5 to 15% in 2006 and 25 to 50% in 2011. Total DNA was extracted from the leaves, stems, and roots of symptomatic and asymptomatic plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instruction. A nested PCR was performed by using primer pair P1/P7 followed by R16F2n/R16R2 to detect the presence of phytoplasmas (1,3). An approximate 1.25-kb PCR product was amplified from symptomatic plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns were analyzed by digesting the 1.2-kb amplicon singly with restriction enzymes AluI, BfaI, HhaI, HpaI, KpnI, MseI, and TaqI. Comparing the RFLP patterns of samples with previously published phytoplasma strains, the phytoplasmas matched patterns of the stolbur group, subgroup E (16SrXII-E) (1). In addition, the PCR product from P1/P7, diluted 1:30, was amplified by using primer pair P1A/P7A (2). The nested PCR product was cloned into pCR8/GW/TOPO vector (Invitrogen, Carlsbad, CA) and sequenced by the Core Lab of the University of Alaska Fairbanks and GENEWIZ (South Plainfield, NJ). Nucleotide sequences (GenBank Accession No. EU293841) were analyzed by iPhyClassifier software (4), confirming the relationship of this phytoplasma to ‘Candidatus Phytoplasma fragariae’ with RFLP patterns identical to group 16SrXII-E. To our knowledge, this is the first molecular characterization of the stolbur group phytoplasmas associated with potato disease in China. The potato is becoming increasingly important in China. The impacts of stolbur on potato yield losses, disease distributions, and insect vectors are currently under investigation. 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) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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First Report of Aster Yellows (16SrI-B) Associated with Potatoes in Alaska

Plant Disease ◽

10.1094/pdis-12-10-0913 ◽

2011 ◽

Vol 95 (6) ◽

pp. 767-767

Author(s):

J. H. McBeath ◽

P. J. Laski ◽

M. Cheng

Keyword(s):

Nested Pcr ◽

Disease Transmission ◽

Sequence Data ◽

Restriction Enzymes ◽

Reference Pattern ◽

Aster Yellows ◽

First Report ◽

Group I ◽

Pcr Product ◽

Pcr Products

During a disease survey conducted in 2009 in Alaska, one potato plant (Solanum tuberosum) with symptoms characteristic of aster yellows, such as apical leaves rolling inward, leaves turning yellow or purple, and presence of aerial tubers, was found in a commercial field. Total DNA was extracted from leaves, stems, and roots of the symptomatic and symptomless plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the instructions of the manufacturer. A nested PCR was carried out with the first round primer pair P1/P7 followed by second round primer pair R16F2n/R16R2 (1,3). An approximate 1.2-kb PCR product was amplified from the symptomatic plant, but not symptomless plants. The PCR products from R16F2n/R16R2 were digested using restriction enzymes AluI, BfaI, BstUI, HhaI, HpaI, KpnI, MseI, and RsaI. The restriction fragment length polymorphism (RFLP) patterns were compared with those from known phytoplasma strains (1) and they matched the patterns for aster yellows subgroup B (16SrI-B). After P1/P7 amplification, the nested PCR product of primer pair P1A/16S-SR (2) was purified with a MiniElute Gel Extraction kit (Qiagen), sequenced by GENEWIZ (South Plainfield, NJ), and the sequence data analyzed by iPhyClassifier software (4). The results indicated that the sequence (GenBank Accession No. HQ599231) had 99.65% similarity to ‘Candidatus Phytoplasma asteris’ reference strain (GenBank Accession No. M30790). The RFLP similarity was identical (coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank Accession No. NC 005303). To our knowledge, this is the first report on the molecular identification of aster yellows phytoplasma associated with potatoes in Alaska. The source of the phytoplasma and pathway of disease transmission is currently under investigation. 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) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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First Report of Pomegranate Wilt Caused by Ceratocystis fimbriata in Yunnan, China

Plant Disease ◽

10.1094/pdis.2003.87.9.1150b ◽

2003 ◽

Vol 87 (9) ◽

pp. 1150-1150 ◽

Cited By ~ 17

Author(s):

Q. Huang ◽

Y. Y. Zhu ◽

H. R. Chen ◽

Y. Y. Wang ◽

Y. L. Liu ◽

...

Keyword(s):

Yunnan Province ◽

Disease Incidence ◽

Punica Granatum ◽

Ceratocystis Fimbriata ◽

First Report ◽

Initial Symptoms ◽

Disease Surveys ◽

Sterile Medium ◽

Visible Symptom ◽

Production Areas

Pomegranate (Punica granatum Linn.) is an important fruit crop in Yunnan Province, China. Recently, older pomegranate bushes in Mengzi County, Yunnan began dying. Initial symptoms were yellowing and wilting of leaves on one to several branches, followed by sudden death of the bush within 3 to 4 weeks. Roots of diseased bushes appeared brown to black, and irregularly shaped lesions were observed when the bark was removed. A species of Ceratocystis was consistently isolated from discolored roots, stem, and branch tissues from wilted bushes on potato dextrose agar (PDA) and was identified as Ceratocystis fimbriata Ellis & Halst, based on the morphology of perithecia, ascospores, conidia, and conidiophores. Perithecia were black with a globose base (130 to 300 μm) and a long neck (450 to 800 μm). Ascospores exuded from the apex of the perithecium neck in a long coil and were small, hyaline, and hat-shaped (3.8 to 5.0 μm long × 2.3 to 4.0 μm wide). Conidiophores were septate and hyaline to dark greenish brown. Hyaline conidia, 8 to 17 μm long × 6 to 15 μm wide, were usually produced in chains of 10 or more. Thick-walled endoconidia were globose to oval, olive brown, and 8 to 20 μm in diameter. Because of the increasing occurrence of the disease, surveys of the main pomegranate production areas in Mengzi County, including Xinan, Duofale, Caoba, Hongzhai, and Shilipu townships, were conducted from 10 to 20 August 2002. The disease was detected in 17 of 50 plantings surveyed. Disease was more severe in older plantings than in younger plantings. Disease incidence was 1% in 1- to 5-year-old bushes, 3.6% in 6- to 10-year-old bushes, and 6% in bushes more than 10 years old. Scolytid beetles were occasionally found on bushes, but we were unable to isolate the fungus from them as has been reported (1). Inoculations with an isolate of C. fimbrata were made by inserting mycelium with perithecia from 12-day-old cultures growing on PDA into root wounds made with a sterile scalpel on five pomegranate plants and then covering the wounds with Parafilm. Sterile medium was placed in an equal number of wounded bushes to serve as controls. Fourteen days later, symptoms began to appear in two bushes, and 5 days later, all bushes exhibited symptoms. No symptoms were observed on control bushes. The first visible symptom was a small area of blackened tissue near the point of inoculation. Lesions expanded slowly, but they expanded more rapidly upward than downward. The fungus was reisolated on PDA from roots of all artificially inoculated bushes. C. fimbriata has been previously reported as the cause of pomegranate wilt in India (2); however, to our knowledge, this is the first report of C. fimbriata on pomegranate in China. Because environmental conditions which favor the pathogen (temperatures ranging from 18 to 30°C and frequent rains) typically occur in many areas during late spring and summer, the disease has the potential to seriously impact pomegranate production in China. References: (1) Y. M. Somasekhara. Plant Dis. 83:400, 1999. (2) Y. M. Somasekhara, et al. Res. Crops 1(1):63, 2000.

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First Report of a Group 16SrII Phytoplasma Infecting Chickpea in Oman

Plant Disease ◽

10.1094/pd-90-0973c ◽

2006 ◽

Vol 90 (7) ◽

pp. 973-973 ◽

Cited By ~ 8

Author(s):

N. A. Al-Saady ◽

A. M. Al-Subhi ◽

A. Al-Nabhani ◽

A. J. Khan

Keyword(s):

Nested Pcr ◽

Animal Feed ◽

Restriction Enzymes ◽

Universal Primers ◽

Polymorphism Analysis ◽

Disease Symptoms ◽

First Report ◽

Pcr Products ◽

Polymerase Chain ◽

Group 2

Chickpea (Cicer arietinum), locally known as “Dungo”, is grown for legume and animal feed mainly in the interior region of Oman. During February 2006, survey samples of chickpea leaves from plants showing yellows disease symptoms that included phyllody and little leaf were collected from the Nizwa Region (175 km south of Muscat). Total nucleic acid was extracted from asymptomatic and symptomatic chickpea leaves using a cetyltrimethylammoniumbromide method with modifications (3). All leaf samples from eight symptomatic plants consistently tested positive using a polymerase chain reaction assay (PCR) with phytoplasma universal primers (P1/P7) that amplify a 1.8-kb phytoplasma rDNA product and followed by nested PCR with R16F2n/R16R2 primers yielding a product of 1.2 kb (2). No PCR products were evident when DNA extracted from healthy plants was used as template. Restriction fragment length polymorphism analysis of nested PCR products by separate digestion with Tru9I, HaeIII, HpaII, AluI, TaqI, HhaI, and RsaI restriction enzymes revealed that a phytoplasma belonging to group 16SrII peanut witches'-broom group (2) was associated with chickpea phyllody and little leaf disease in Oman. Restriction profiles of chickpea phytoplasma were identical with those of alfalfa witches'-broom phytoplasma, a known subgroup 16SrII-B strain (3). To our knowledge, this is the first report of phytoplasma infecting chickpea crops in Oman. References: (1) A. J. Khan et al. Phytopathology, 92:1038, 2002. (2). I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998 (3) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA. 81:8014, 1984.

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First Report of a Group 16SrII Phytoplasma Associated with Witches'-Broom of Eggplant in Oman

Plant Disease ◽

10.1094/pdis-10-10-0761 ◽

2011 ◽

Vol 95 (3) ◽

pp. 360-360 ◽

Cited By ~ 9

Author(s):

A. M. Al-Subhi ◽

N. A. Al-Saady ◽

A. J. Khan ◽

M. L. Deadman

Keyword(s):

Nested Pcr ◽

Solanum Melongena ◽

Positive Control ◽

Rrna Gene ◽

Direct Pcr ◽

First Report ◽

Pcr Product ◽

Pcr Products ◽

Broom Disease ◽

Phytoplasma Infection

Eggplant (Solanum melongena L.) belongs to the family Solanaceae and is an important vegetable cash crop grown in most parts of Oman. In February 2010, plants showing phyllody symptoms and proliferation of shoots resembling those caused by phytoplasma infection were observed at Khasab, 500 km north of Muscat. Total genomic DNA was extracted from healthy and two symptomatic plants with a modified (CTAB) buffer method (2) and analyzed by direct and nested PCR with universal phytoplasma 16S rDNA primers P1/P7 and R16F2n/ R16R2, respectively. PCR amplifications from all infected plants yielded an expected product of 1.8 kb with P1/P7 primers and a 1.2-kb fragment with nested PCR, while no products were evident with DNA from healthy plants. Restriction fragment length polymorphism (RFLP) profiles of the 1.2-kb nested PCR products of two eggplant phyllody phytoplasma and five phytoplasma control strains belonging to different groups used as positive control were generated with the restriction endonucleases RsaI, AluI, Tru9I, T-HB8I, and HpaII. The eggplant phytoplasma DNA yielded patterns similar to alfalfa witches'-broom phytoplasma (GenBank Accession No. AF438413) belonging to subgroup 16SrII-D, which has been recorded in Oman (1). The DNA sequence of the 1.8-kb direct PCR product was deposited in GenBank (Accession No. HQ423156). Sequence homology results using BLAST revealed that the eggplant phyllody phytoplasma shared >99% sequence identity with Scaevola witches'-broom phytoplasma (Accession No. AB257291.1), eggplant phyllody phytoplasma (Accession No. FN257482.1), and alfalfa witches'-broom phytoplasma (Accession No. AY169323). The RFLP and BLAST results of 16S rRNA gene sequences confirm that eggplant phyllody phytoplasma is similar to the alfalfa phytoplasma belonging to subgroup 16SrII-D. To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group causing witches'-broom disease on eggplant in Oman. References: (1) A. J. Khan et al. Phytopathology 92:1038, 2002. (2) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA, 81:8014, 1984.

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First Report of an Aster Yellows Phytoplasma Associated with Cabbage in Southern Texas

Plant Disease ◽

10.1094/pdis.2001.85.4.447b ◽

2001 ◽

Vol 85 (4) ◽

pp. 447-447 ◽

Cited By ~ 6

Author(s):

I.-M. Lee ◽

R. A. Dane ◽

M. C. Black ◽

Noel Troxclair

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Restriction Enzymes ◽

Diagnostic Procedures ◽

Acid Extraction ◽

Insect Vector ◽

Aster Yellows ◽

First Report ◽

Pcr Products ◽

Aster Yellows Phytoplasma

In early spring 2000 carrot crops in southwestern Texas were severely infected by an outbreak of phyllody associated with aster yellows phytoplasma. Cabbage crops that had been planted adjacent to these carrot fields began to display previously unobserved symptoms characteristic of phytoplasma infection. Symptoms included purple discoloration in leaf veins and at the outer edges of leaves on cabbage heads. Proliferation of sprouts also occurred at the base of the stem and between leaf layers of some plants, and sprouts sometimes continued to proliferate on extended stems. About 5% of cabbage plants in the field exhibited these symptoms. Two symptomless and four symptomatic cabbage heads were collected in early April from one cabbage field. Veinal tissues were stripped from each sample and used for total nucleic acid extraction. To obtain specific and sufficient amount of PCR products for analysis, nested PCR was performed by using primer pairs (first with P1/P7 followed by R16F2n/R16R2) (1,2) universal for phytoplasma detection. A specific 16S rDNA fragment (about 1.2 kb) was strongly amplified from the four symptomatic but not from the two asymptomatic samples. The nested PCR products obtained from the four symptomatic samples were then analyzed by restriction fragment length polymorphism (RFLP) using the restriction enzymes MseI, HhaI, and HpaII, and the RFLP patterns were compared to the published patterns of known phytoplasmas (1). The resulting RFLP patterns were identical to those of a phytoplasma belonging to subgroup B of the aster yellows phytoplasma group (16SrI). These RFLP patterns were also evident in putative restriction sites observed in a 1.5 kbp nucleotide sequence of the 16S rDNA. This is the first report of aster yellows phytoplasma associated disease symptoms in cabbage in Texas. The occurrence of cabbage proliferation coincided with the presence of high populations of the insect vector, aster leafhopper. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) B. Schneider et al. 1995. Molecular and Diagnostic Procedures in Mycoplasmology, Vol. I. Academic Press, San Diego, CA.

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First Report on the Molecular Identification of Phytoplasma (16SrII-D) Associated with Witches' Broom of Kalmegh (Andrographis paniculata) in India

Plant Disease ◽

10.1094/pdis-08-14-0854-pdn ◽

2015 ◽

Vol 99 (1) ◽

pp. 155-155 ◽

Cited By ~ 3

Author(s):

S. T. Saeed ◽

A. Khan ◽

A. Samad

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Disease Incidence ◽

Antioxidant Properties ◽

First Record ◽

Andrographis Paniculata ◽

Medicinal And Aromatic Plants ◽

First Report ◽

16S Rdna Sequence Analysis ◽

Virtual Rflp

Andrographis paniculata (family Acanthaceae), also known as “King of Bitters” or Kalmegh, is an important medicinal plant used for the treatment of various diseases. It has antimicrobial, antiviral, anti-inflammatory, hepatoprotective, antidiabetic, antihyperglycemic, and antioxidant properties (1). During June 2014, while performing a routine survey of the commercial trial fields of Kalmegh at Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India, typical phytoplasma disease symptoms such as virescence, proliferation, and witches' broom along with little leaf and stunted growth were observed. The disease incidence was estimated to be approximately 7 to 10%. To ascertain the presence of phytoplasma, 16 samples of leaves were collected from nine different field sites, and total genomic DNA was extracted from the symptomatic and symptomless Kalmegh plants by the CTAB method. Direct and nested PCR assays were performed targeting the 16S rDNA using generic phytoplasma primer pairs P1/P6 followed by R16F2n/R16R2 (2). Resulting bands of the expected size (1.5 kb and 1.2 kb, respectively) were amplified from symptomatic plants. No amplification was observed with DNA from asymptomatic plant samples. The purified nested PCR products were cloned into E. coli DH5α, using the pGEM-T Easy vector (Promega, United States) and sequenced with primers M13For/M13Rev using an automatic sequencer (ABI Prism, Perkin Elmer) at CIMAP. The sequence was analyzed by BLASTn and found to share 99% similarity with Echinacea witches'-broom phytoplasma and Sesame phyllody phytoplasma strain (GenBank Accession Nos. JF340080 and KF612966, respectively), which belong to the 16SrII-D group. The sequence was deposited in NCBI as GenBank Accession No. KM359410. A phylogenetic tree using MEGA v5.0 (4) was constructed with 16S rDNA; consensus sequences of phytoplasmas belonging to distinct groups revealed that the present phytoplasma clustered with the 16SrII group. iPhyClassifier software was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile (5). On the basis of iPhyClassifier, the 16S rDNA sequence analysis of our isolate showed 99.2% similarity with that of the ‘Candidatus Phytoplasma australasiae’ reference strain (GenBank Accession No. Y10097), which belongs to 16Sr group II. The virtual RFLP pattern of F2n/R2 fragment was most similar to the 16SrII-D subgroup (similarity coefficient of 0.91) but showed a difference in profile with HpaI, HhaI, and MseI enzymes. Several bacterial/fungal and viral diseases have been reported on A. paniculata (3); however, to our knowledge, this is the first report of witches' broom disease in India and the first record of a 16SrII-D group phytoplasma on Kalmegh. Its presence in Kalmegh is of great significance due to its commercial interest. References: (1) S. Akbar. Altern. Med. Rev. 16:1, 2011. (2) D. E. Gundersen and M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) A. Khan and A. Samad. Plant Dis. 98:698, 2014. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (5) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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Sugarcane Mosaic Distribution, Incidence, Increase, and Spatial Pattern in Louisiana

Plant Disease ◽

10.1094/pdis-01-19-0099-re ◽

2019 ◽

Vol 103 (8) ◽

pp. 2051-2056

Author(s):

J. L. Rice ◽

J. W. Hoy ◽

M. P. Grisham

Keyword(s):

Spatial Pattern ◽

Disease Incidence ◽

Aphid Transmission ◽

Field Surveys ◽

Reverse Transcription Pcr ◽

Single Leaf ◽

Disease Surveys ◽

Low Incidence ◽

Mosaic Distribution ◽

Production Areas

Sugarcane mosaic is a historically important disease in Louisiana currently caused by sorghum mosaic virus (SrMV). Successful breeding for resistance reduced the disease to low incidence in commercial cultivars. However, mosaic was detected in experimental clone evaluations at multiple locations, leading to uncertainty concerning the current distribution and incidence in the state. Field surveys were conducted from 2016 to 2018 in breeding program yield trials and experimental clone seed cane increase fields. Mosaic symptomatic plants were observed in a newly released cultivar, HoCP 09-804, in three of five production areas, with incidences ranging from 0 to 10%. Mosaic also was observed in nine additional experimental clones. Single leaf samples were tested for SrMV using reverse transcription PCR. All symptomatic samples and a low percentage (0.3%) of asymptomatic samples tested positive for SrMV, confirming that it continues to be the causal species. Runs analysis detected aggregation of infected plants within at least 70% of rows in 94% of surveyed fields. The spatial pattern and geographical distribution of disease incidence suggested that infected seed cane was the source of the disease. Surveys conducted in the same fields of HoCP 09-804 through two subsequent crops detected disease incidence increases in some fields and decreases in the others in first ratoon, but observed incidence was lower compared with plant cane in all fields in second ratoon. The results indicated that disease increase owing to aphid transmission did not occur under the prevailing conditions.

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Molecular Characterization of ‘Candidatus Phytoplasma asteris’ Associated with Aster Yellows-Diseased Potatoes in China

Plant Disease ◽

10.1094/pdis-12-10-0914 ◽

2011 ◽

Vol 95 (6) ◽

pp. 777-777 ◽

Cited By ~ 3

Author(s):

M. Cheng ◽

J. Dong ◽

P. J. Laski ◽

Z. Zhang ◽

J. H. McBeath

Keyword(s):

Nested Pcr ◽

Inner Mongolia ◽

Restriction Enzymes ◽

Molecular Characteristics ◽

Reference Pattern ◽

Aster Yellows ◽

Potato Plants ◽

Group I ◽

Pcr Product ◽

Aster Yellows Phytoplasma

Potatoes (Solanum tuberosum) are one of the most important crops in China following rice, wheat, and corn. Aster yellows phytoplasma appeared to be widespread in China; it was found to cause diseases on alfalfa, oranges, peaches, periwinkles, bamboo (1), and cactus (4). However, scant information of this pathogen on potatoes is available except for a few short reports published during the 1950s. During the potato disease surveys conducted from 2005 to 2010 in Yunnan and Inner Mongolia, 10 to 35% of potato plants exhibited symptoms of yellowing or purpling of apical leaves, with the top leaves rolling inward and aerial tubers formation. Total DNA was extracted from midveins of leaves and roots of 125 diseased and asymptomatic plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. A nested PCR was carried out with the first round primer pair P1/P7 followed by the second round primer pair R16F2n/R16R2 (2,3). A PCR product of approximately 1.2 kb was amplified from diseased plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns were analyzed by digesting a 1.2-kb product using restriction enzymes AluI, BfaI, BstUI, HhaI, HpaI, KpnI, MseI, and RsaI. Comparing the RFLP patterns with previously published phytoplasma strains (2), aster yellows phytoplasma found on potato plants in Yunnan and Inner Mongolia belong to group I, subgroup B (16SrI-B). The PCR product from P1/P7, diluted 1:30, was amplified by using primer pair P1A/P7A (3) and P1A/16S-SR (3). The nested-PCR products from P1A/P7A and P1A/16S-SR were cloned into pCR8/GW/TOPO vector (Invitrogen, Carlsbad, CA) and sequenced by the Core Lab of the University of Alaska–Fairbanks and GENEWIZ (South Plainfield, NJ). The nucleotide sequence (GenBank Accession No. HQ599228) was analyzed by iPhyClassifier software and had 99.53% sequence identity to the reference strain (GenBank Accession No. M30790) for ‘Candidatus Phytoplasma asteris’. The RFLP similarity is identical (coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank Accession No. NC_005303). To our knowledge, this is the first report revealing the molecular characteristics of a phytoplasma associated with aster yellows-diseased potatoes in China. References: (1) H. Cai et al. Plant Prot. 31:38, 2005. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (4) W. Wei et al. Plant Dis. 91:461, 2007.

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

Plant Disease ◽

10.1094/pdis-05-14-0488-pdn ◽

2014 ◽

Vol 98 (12) ◽

pp. 1739-1739 ◽

Cited By ~ 3

Author(s):

M. C. Holeva ◽

P. E. Glynos ◽

C. D. Karafla ◽

E. M. Koutsioumari ◽

K. B. Simoglou ◽

...

Keyword(s):

16S Rdna ◽

Nested Pcr ◽

Pathogenic Bacteria ◽

Related Strain ◽

Northern Greece ◽

First Report ◽

Potato Plants ◽

Pcr Product ◽

16S Rdna Pcr ◽

Stolbur Phytoplasma

In August 2013, potato plants (Solanum tuberosum) cv. Banba displaying symptoms resembling those caused by Candidatus Phytoplasma solani (potato stolbur phytoplasma) were observed in a 2-ha field in the area of the Peripheral Unit of Drama (northern Greece). The plants were 10 weeks old and their symptoms included reddening and upward rolling of leaflets, reduced size of leaves, shortened internodes, and aerial tuber formation. Incidence of affected plants was estimated to be 40% in the field. Four symptomatic potato plants were collected for laboratory testing of possible phytoplasma infection. From each of these four plants, total DNA was extracted from mid veins of reddish leaflets from apical shoot parts and of leaflets emerging from aerial tubers, using a phytoplasma enrichment procedure (1). A nested PCR using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (3) amplified the expected ~1.2-kb 16S rDNA fragment in all four symptomatic potato plants. No amplification was observed with DNA similarly extracted from leaflets of asymptomatic potato plants of the same variety collected from an apparently healthy crop. One of the four 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced by Beckman Coulter Genomics (United Kingdom). At least twofold coverage per base position of the cloned PCR product was achieved. BLAST analysis showed that the obtained sequence of the PCR 16S rDNA product was: i) 100% identical to several GenBank sequences of Ca. P. solani strains, including strains detected previously in Greece infecting tomato (GenBank Accession No. JX311953) and Datura stramonium (HE598778 and HE598779), and ii) 99.7% similar to that of the Ca. P. solani reference strain STOL11 (AF248959). Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup (AF248959). The sequence of this PCR product was deposited in NCBI GenBank database under the accession no. KJ810575. The presence of the stolbur phytoplasma in all four symptomatic potato plants examined was further confirmed by nested PCR using the stolbur-specific STOL11 primers (3) targeting non-ribosomal DNA. Based on the observed symptoms in the field and laboratory molecular examinations, we concluded that the potato plants were infected by a Ca. P. solani related strain. The stolbur disease has been previously reported in Greece affecting tomato (2,5) and varieties of D. stramonium (4). To our knowledge, this is the first report of a Ca. P. solani related strain infecting a potato crop in Greece. As northern Greece is a center of potato production, the source of this pathogen is to be investigated. References: (1) U. Ahrens and E. Seemuller. Phytopathology 82:828, 1992. (2) A. S. Alivizatos. Pages 945-950 in: Proceedings of the 7th International Conference of Plant Pathogenic Bacteria. Academiai Kiado, Budapest, Hungary, 1989. (3) J. Jović et al. Bull. Insectol. 64:S83, 2011. (4) L. Lotos et al. J. Plant Pathol. 95:447, 2013. (5) E. Vellios and F. Lioliopoulou. Bull. Insectol. 60:157, 2007.

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