First Report of Flavescence Dorée-Related Phytoplasma Affecting Grapevines in Croatia

Flavescence dorée (FD) and Bois noir (BN) phytoplasmas are principal grapevine yellows (GY) agents in the wider Euro-Mediterranean Region. While BN phytoplasma belongs to the ribosomal subgroup 16SrXII-A, the FD agents belong either to the ribosomal subgroups 16SrV-C or -D. During the official GY survey in 2009, 40 symptomatic grapevines (Vitis vinifera L.) were sampled throughout grapevine-growing regions in Croatia. Typical GY symptoms of leaf yellowing or reddening were evident on white and red varieties, respectively. Leaf rolling as well as irregular lignification of the shoots and withering of clusters were also observed. Phloem tissue from cuttings and leaf veins from mature vines were sampled for total DNA extraction and amplification of phytoplasma 16S rRNA gene by using generic primers P1/P7 in a direct PCR assay followed by a nested PCR using primer pair R16F2n/R2 (2). Phytoplasma ribosomal group affiliation was determined by restriction fragment length polymorphism (RFLP) analysis of the nested PCR products with enzyme Tru1I (Fermentas, Vilnius, Lithuania). These initial findings were validated and augmented by a triplex real-time PCR assay targeting the nonribosomal map gene. This assay enables simultaneous detection of BN and FD (16SrV-C and -D) phytoplasmas in grapevine (3). Assay results revealed the majority of GY positive vines (19 of 40) contained BN phytoplasma which is widespread. For the first time in Croatia, two red variety samples, Pinot Noir and Plemenka Crvena, from the vicinity of Ozalj (Vivodina) and Zagreb (Brezje), respectively, were found to harbor FD-related phytoplasmas. Fragments amplified by P1/P7 primers from latter samples were cloned and sequenced. Sequence analyses using online interactive tool iPhyClassifier (4) revealed that the phytoplasma under study from Pinot Noir sample (GenBank Accession No. HQ712064) is a member of 16SrV-C subgroup and shares 99.87% similarity with 16S rDNA sequence of the reference strain (GenBank Accession No. AF176319). The sequence from the Plemenka Crvena sample (GenBank Accession No. HQ712065) shares 99.54% similarity with the reference strain and has the most similar virtual RFLP pattern to the one of the 16SrV-C subgroup (GenBank Accession No. AY197642). These findings are currently limited to vineyards in northwestern Croatia. Even so, the presence of FD principal cicadellid vector Scaphoideus titanus in the country and the occurrence and distribution of FD in neighboring countries (1,2) are factors indicating that the spread of FD in Croatia is highly probable. References: (1) L. Filippin et al. Plant Pathol. 58:826, 2009. (2) S. Kuzmanović et al. Vitis 47:105, 2008. (3) C. Pelletier et al. Vitis 48:87, 2009. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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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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Overestimation of Streptococcus mutans prevalence by nested PCR detection of the 16S rRNA gene

Journal of Medical Microbiology ◽

10.1099/jmm.0.46280-0 ◽

2006 ◽

Vol 55 (1) ◽

pp. 109-113 ◽

Cited By ~ 14

Author(s):

Ali Al-Ahmad ◽

Thorsten Mathias Auschill ◽

Gabriele Braun ◽

Elmar Hellwig ◽

Nicole Birgit Arweiler

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Streptococcus Mutans ◽

Nested Pcr ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Direct Pcr ◽

Specific Primers ◽

The 16S Rrna Gene

This study was carried out in order to compare two PCR-based methods in the detection of Streptococcus mutans. The first PCR method was based on primers for the 16S rRNA gene and the second method was based on specific primers that targeted the glucosyltransferase gene (gtfB). Each PCR was performed with eight different streptococci from the viridans group, five other streptococci and 17 different non-streptococcal bacterial strains. Direct use of the S. mutans 16S rRNA gene-specific primers revealed that Streptococcus gordonii and Streptococcus infantis were also detected. After amplifying the 16S rRNA gene with universal primers and subsequently performing nested PCR, the S. mutans-specific nested primers based on the 16S rRNA gene detected all tested streptococci. There was no cross-reaction of the gtfB primers after direct PCR. Our results indicate that direct PCR and nested PCR based on 16S rRNA genes can reveal false-positive results for oral streptococci and lead to an overestimation of the prevalence of S. mutans with regards to its role as the most prevalent causative agent of dental caries.

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Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-PCR assay

Journal of Medical Virology ◽

10.1002/jmv.10255 ◽

2002 ◽

Vol 69 (1) ◽

pp. 132-144 ◽

Cited By ~ 165

Author(s):

M.T. Coiras ◽

P. Pérez-Breña ◽

M.L. García ◽

I. Casas

Keyword(s):

Respiratory Syncytial Virus ◽

Reverse Transcription ◽

Nested Pcr ◽

Influenza A ◽

Simultaneous Detection ◽

Clinical Samples ◽

Pcr Assay ◽

Syncytial Virus

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Identification and Epidemic Distribution of Two Flavescence Dorée—Related Phytoplasmas in Veneto (Italy)

Plant Disease ◽

10.1094/pdis.1999.83.10.925 ◽

1999 ◽

Vol 83 (10) ◽

pp. 925-930 ◽

Cited By ~ 56

Author(s):

Marta Martini ◽

Ermanno Murari ◽

Nicola Mori ◽

Assunta Bertaccini

Keyword(s):

16S Rdna ◽

Fragment Length Polymorphism ◽

The European Union ◽

Direct Pcr ◽

Nested Polymerase Chain Reaction ◽

Flavescence Dorée ◽

Scaphoideus Titanus ◽

Polymerase Chain ◽

Elm Yellows ◽

Infected Grapevine

Grapevine yellows associated with phytoplasmas of the elm yellows group (16SrV), better known as flavescence dorée (FD), is a serious quarantine problem in some important grapevine growing regions in the European Union. A survey was carried out in 1997 to 1998 in Veneto region (Italy) where a serious outbreak of FD was in progress. Phytoplasma identification by nested polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analyses on 275 grapevine samples and on batches of Scaphoideus titanus was carried out. RFLP analyses of the 16S rDNA/spacer region with TaqI detected the presence of two distinct elm yellows phytoplasma subgroups designated 16SrV-C and 16SrV-D in 77 FD-infected grapevine samples. Only phytoplasmas of the 16SrV-D subgroup were detected in S. titanus. In 1997, the two phytoplasma subgroups appeared to be located in two diverse geographic areas; but in 1998, the 16SrV-D type also was detected in the provinces where in 1997 only 16SrV-C type was identified. The sequencing of a 400-bp fragment at the 3′ end of 16S rDNA plus spacer region allowed a specific primer construction that was successfully employed for detection of both FD types in grapevine by direct PCR.

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Nested PCR Assay for Detection of Granulocytic Ehrlichiae

Journal of Clinical Microbiology ◽

10.1128/jcm.36.4.1090-1095.1998 ◽

1998 ◽

Vol 36 (4) ◽

pp. 1090-1095 ◽

Cited By ~ 237

Author(s):

Robert F. Massung ◽

Kim Slater ◽

Jessica H. Owens ◽

William L. Nicholson ◽

Thomas N. Mather ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Nested Pcr ◽

Limit Of Detection ◽

Rrna Gene ◽

Pcr Assay ◽

Serum Samples ◽

Blood Specimens ◽

The 16S Rrna Gene ◽

Edta Blood

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted fromIxodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.

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First Report of an Elm Yellows Subgroup 16SrV-C Phytoplasma Infecting Grapevine in Serbia

Plant Disease ◽

10.1094/pdis.2003.87.5.599c ◽

2003 ◽

Vol 87 (5) ◽

pp. 599-599 ◽

Cited By ~ 7

Author(s):

B. Duduk ◽

M. Ivanovic ◽

N. Dukic ◽

S. Botti ◽

A. Bertaccini

Keyword(s):

Leaf Roll ◽

Restriction Enzymes ◽

Stem Bark ◽

Rrna Gene ◽

Universal Primer ◽

First Report ◽

Flavescence Dorée ◽

Scaphoideus Titanus ◽

Two Samples ◽

Elm Yellows

During a 2002 survey in Serbia, samples of grapevine (Vitis vinifera) were collected from plants showing typical phytoplasma-like symptoms: leaf roll, leaf redness, vein chlorosis and necrosis, and absence of lignification. The material was collected from one viticultural region (Zupa Aleksandrovac), where the disease was recorded in 2000 and showed an increasing percentage of symptomatic plants every year. Total nucleic acid was extracted separately from leaf midveins and stem bark collected from 10 symptomatic and 2 asymptomatic plants. Phytoplasma infection was detected using polymerase chain reaction (PCR) assays with universal primer pair P1/P7 for the amplification of phytoplasma 16S rRNA gene, and primer pair FD9f2/FD9r followed by FD9f3/FD9r2 in nested PCR for specific amplification of the FD9 nonribosomal DNA fragment of the EY-group (1). Phytoplasmas were detected in 9 of 10 midvein extracts from symptomatic grapevines (three of cv. Plovdina, two of cv. Smederevka, and four of cv. Gamé). Also, 6 of 10 bark preparations representing stem collections from the same plants were positive (two samples of cv. Plovdina, both samples of cv. Smederevka, and two samples of cv. Gamé). Both collections of midveins and bark tissues from asymptomatic plants were negative. Fragments amplified with universal P1/P7 primers (16S-23S rDNA) were analyzed by restriction fragment length polymorphism with TruI and TaqI restriction enzymes. The phytoplasmas produced identical restriction profiles to those of 16SrV Elm Yellows group and 16SrV-C Flavescence doreé subgroup (2). To our knowledge, this is the first report of phytoplasma infecting grapevines in Serbia, and the first survey in progress to verify the presence of Scaphoideus titanus to determine if this grapevine yellows could be defined as Flavescence dorée. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) M. Martini et al. Mol. Cell. Probes 16:197, 2002.

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