scholarly journals First Report of a 16SrIII-L Phytoplasma Associated with Frogskin Disease in Cassava (Manihot esculenta Crantz) in Brazil

Plant Disease ◽  
2014 ◽  
Vol 98 (1) ◽  
pp. 153-153 ◽  
Author(s):  
S. A. S. Oliveira ◽  
E. F. M. Abreu ◽  
T. S. Araújo ◽  
E. J. Oliveira ◽  
E. C. Andrade ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Manihot Esculenta ◽  
Dsrna Virus ◽  
Raw Material ◽  
Rrna Gene ◽  
Manihot Esculenta Crantz ◽  
First Report ◽  
Ctab Method

Cassava (Manihot esculenta Crantz) is a major staple crop in developing countries and a large source of raw material for industrial purposes as flour, starch, and ethanol. In July 2012, 24 cassava genotypes (corresponding to 1.85% of the accessions) with typical symptoms of frogskin disease (CFSD) were observed in one of the maintenance areas of the Brazilian Cassava Germplasm (located at Embrapa Cassava & Fruits, Cruz das Almas, Bahia State, Brazil). All diseased plants were asymptomatic on the aboveground parts (leaves and stem). However, for accessions BGM 880, BGM 1094, BGM 1100, BGM 1212, BGM 1218, and BGM 1526, all roots showed a woody appearance, thickened cork-like peel with opaque aspect, and coalescent lip-like slits in a honeycomb pattern. Based on literature description, two pathogens could be associated with CFSD: a dsRNA virus (belonging to family Reoviridae) and a 16SrIII-L phytoplasma (1). To investigate the presence of phytoplasma associated with the CFSD symptoms, total DNA was extracted from 0.5 g of root tissue collected from both symptomatic and asymptomatic roots by scratching the secondary vessel at the center of the cassava root with a CTAB method. The nested PCR was carried out using phytoplasma-specific primer set P1/Tint followed by R16F2n/R16R2, targeting the 16S rRNA gene sequence of 1.2 kb in length, for the final reaction (4). No phytoplasma was detected in asymptomatic cassava roots that were sampled from the same field. A posterior extraction of total RNA was made but no dsRNA was noticed on the agarose gel, and reaction of RT-PCR with specific primers (2) had no amplification. In order to characterize the strains, the 1.2-kb amplicon was digested with BamHI, MseI, MspI, KpnI, and TaqI endonucleases. The resulting patterns indicated that the symptomatic accessions were infected with a phytoplasma belonging to the 16SrIII group, sharing similarities with pseudo gel mapping from the reference strain of Peach X-Disease Phytoplasma (GenBank Accession No. L33733). Nested PCR products from accessions BGM 1526 and BGM 1212 were purified and sequenced by Macrogen, (Seoul, South Korea) in both directions, manually edited, and the consensus sequences were deposited in the NCBI database (GenBank Accession Nos. KF019184 and KF019185). Phylogenetic studies were conducted based on maximum parsimony, neighbor-joining, and maximum likelihood analysis for 16S rRNA. The phytoplasma 16S rRNA gene sequences from both strains had 99% identity (P < 0.0001) with the 16SrIII-L CFSD phytoplasma (EU346761 and AY737647), described by Alvarez et al. (1) infecting cassava in Colombia. To our knowledge, this is the first report of a phytoplasma associated with Cassava Frogskin Disease in Brazil, where only the dsRNA virus was recognized as causing this symptom (3). This is not likely to be an isolated case, and possibly more cassava plants are infected with this phytoplasma in Brazil. Due to the difficulties to observe the symptoms at the field level, this could be an emerging disease in that country. References: (1) E. Alvarez et al. Plant. Dis. 93:1139, 2009. (2) L. A. Calvert et al. J. Phytopathol. 156:647, 2008. (3) L. S. Poltroniere et al. Comun. Tec., Belem-PA. 006:2p, 1999. (4) C. D. Smart et al. Appl. Environ. Microb. 62:2988, 1996.

scholarly journals First Report of Blueberry Reddening Disease in Serbia Associated with 16SrXII-A (Stolbur) Phytoplasma

Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1653-1653 ◽  
Author(s):  
M. Starović ◽  
S. Kojic ◽  
S. T. Kuzmanovic ◽  
S. D. Stojanovic ◽  
S. Pavlovic ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Vaccinium Corymbosum ◽  
23S Rrna ◽  
Rrna Gene ◽  
Restriction Enzyme Digestion ◽  
First Report ◽  
Stolbur Phytoplasma ◽  
The 16S Rrna Gene

Blueberries (Vaccinium corymbosum) are among the healthiest fruits due to their high antioxidant content. The total growing area of blueberries in Serbia ranges from 80 to 90 ha. A phytoplasma-like disease was observed for the first time during July 2009 in three blueberry cultivars (Bluecrop, Duke, and Spartan) grown in central Serbia, locality Kopljare (44°20′10.9″ N, 20°38′39.3″ E). Symptoms of yellowing and reddening were observed on the upper leaves and proliferating shoots, similar to those already described on blueberries (4). There was uneven ripening of the fruits on affected plants. Incidence of affected plants within a single field was estimated to be greater than 20% in 2009 and 50% in 2010. Blueberry leaves, together with petioles, were collected during two seasons, 2009 and 2010, and six samples from diseased plants and one from symptomless plants from each cultivar, resulting in 42 samples in total. For phytoplasma detection, total DNA was extracted from the veins of symptomatic and asymptomatic leaves of V. corymbosum using the protocol of Angelini et al. (1). Universal oligonucleotide primers P1/P7 were used to amplify a 1.8-kb DNA fragment containing the 16S rRNA gene, the 16S-23S spacer region, and the 5′ end of the 23S rRNA gene. Subsequently, a 1.2-kb fragment of the 16S rRNA gene was amplified by nested PCR with the R16F2n/R16R2 primers. Reactions were performed in a volume of 50 μl using Dream Taq Green master mix (Thermo Scientific, Lithuania). PCR reaction conditions were as reported (3), except for R16F2n/R2 primers set (annealing for 30 s at 58°C). PCR products were obtained only from the DNA of symptomatic plants. Fragments of 1.2 kb were further characterized by the PCR-RFLP analysis, using AluI, HpaII, HhaI, and Tru1I restriction enzymes (Thermo Scientific, Lithuania), as recommended by the manufacturer. The products of restriction enzyme digestion were separated by electrophoresis on 2.5% agarose gel. All R16F2n/R2 amplicons showed identical RFLP patterns corresponding to the profile of the Stolbur phytoplasma (subgroup 16SrXII-A). The results were confirmed by sequencing the nested PCR product from the representative strain Br1. The sequence was deposited in NCBI GenBank database under accession number KC960486. Phylogenetic analysis showed maximal similarities with SH1 isolate from Vitis vinifera, Jordan (KC835139.1), Bushehr (Iran) eggplant big bud phytoplasma (JX483703.1), BA strain isolated from insect in Italy (JQ868436.1), and also with several plants from Serbia: Arnica montana L. (JX891383.1), corn (JQ730750.1), Hypericum perforatum (JQ033928.1), tobacco (JQ730740.1), etc. In conclusion, our results demonstrate that leaf discoloration of V. corymbosum was associated with a phytoplasma belonging to the 16SrXII-A subgroup. The wild European blueberry (Vaccinium myrtillus L.) is already detected as a host plant of 16SrIII-F phytoplasma in Germany, North America, and Lithuania (4). The main vector of the Stolbur phytoplasma, Hyalesthes obsoletus Signoret, was already detected in Serbia (2). The first report of Stolbur phytoplasma occurrence on blueberry in Serbia is significant for the management of the pathogen spreading in blueberry fields. Since the cultivation of blueberry has a great economic potential in the region, it is important to identify emerging disease concerns in order to ensure sustainable production. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) J. Jović et al. Phytopathology 99:1053, 2009. (3) S. Pavlovic et al. J. Med. Plants Res. 6:906, 2012. (4) D. Valiunas et al. J. Plant Pathol. 86:135, 2004.

scholarly journals First Report of Stolbur Phytoplasma Associated with Maize Redness Disease of Maize in Bosnia and Herzegovina

Plant Disease ◽  
2014 ◽  
Vol 98 (3) ◽  
pp. 418-418 ◽  
Author(s):  
M. Kovačević ◽  
Z. Đurić ◽  
J. Jović ◽  
G. Perković ◽  
B. Lolić ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bosnia And Herzegovina ◽  
Nested Pcr ◽  
Wheat Crop ◽  
Rrna Gene ◽  
First Report ◽  
Tuf Gene ◽  
Maize Plants ◽  
Stolbur Phytoplasma

Maize redness (MR), caused by stolbur phytoplasma (16SrXII-A, ‘Candidatus phytoplasma solani’) and vectored by the cixiid planthopper Reptalus panzeri (Löw), is a severe and emerging disease of maize in southeastern Europe (2). Symptoms of MR include midrib, leaf, and stalk reddening, followed by desiccation of the entire plant, abnormal ear development, and incomplete kernel set. MR may cause significant economic losses (2). During 2010, 2011, and 2012, the presence of MR-like symptoms on maize accompanied by significant yield losses were frequently observed in maize fields in the Semberija region of northeastern Bosnia and Herzegovina. From mid-June to early July, potential vectors were collected using mouth-aspirators from maize plants in fields at three locations in the Semberija region where MR-like symptoms were previously observed. At the end of July, symptomatic maize plants were collected from six fields in the same region for phytoplasma identification. In addition, we sampled asymptomatic johnsongrass (Sorghum halepense L.), bindweed (Convolvulus arvensis L.), and volunteer wheat (Triticum aestivum L.) in areas adjacent to maize fields with MR-like symptoms, as potential phytoplasma reservoirs (2,3). A total of 49 plants (38 maize, 6 johnsongrass, 3 bindweed, and 2 wheat) and 43 R. panzeri were tested for the presence of stolbur phytoplasma. Leaves of four maize seedlings, grown in insect-proof greenhouse conditions, were used as controls. Total DNA was extracted from roots of each plant and R. panzeri using the CTAB methods (2). Initial phytoplasma detection was conducted on 16S rRNA gene using nested PCR assay with phytoplasma universal primers P1/P7 and F2n/R2 (4). Subsequently, all phytoplasma positive samples were retested employing stolbur-specific Stol11 protocol with the f2r/f3r2 primer set (1). Molecular characterization of identified phytoplasmas was performed by PCR-RFLP analysis of the tuf gene (3) and by sequence analyses of the 16S rRNA nested PCR products (GenBank Accession No. KC852868). All samples that tested positive on 16S rRNA gene using phytoplasma generic primers gave positive reaction in assays with stolbur-specific primers. Stolbur phytoplasma was identified in 36 of 49 plant samples (34 of 38 symptomatic maize plants and in 2 of 6 johnsongrass) and in 2 of 43 R. panzeri individuals. None of the control plants, bindweed, or wheat samples were positive for the presence of any phytoplasma. Tuf gene RFLP analyses enabled affiliation of all isolates to the stolbur type tuf-b. Comparison of the 16S rRNA sequence using BLAST analyses further confirmed identification of the phytoplasmas as being ‘Candidatus phytoplasma solani.’ The obtained sequence showed 100% identity with ‘Candidatus phytoplasma solani’ from corn in Serbia (JQ730750). These data clearly demonstrated association of stolbur phytoplasma with MR symptoms on maize in Semberija, which represents the first report of the MR disease and stolbur phytoplasma in maize, R. panzeri, and johnsongrass in Bosnia and Herzegovina. In the Semberija region, maize-wheat crop rotation is a traditional practice, which is a key factor for MR occurrence and persistence (2). References: (1) D. Clair et al. Vitis 42:151, 2003. (2) J. Jović et al. Phytopathology 99:1053, 2009. (3) M. Langer and M. Maixner. Vitis 43, 191, 2004. (4) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

scholarly journals First report of Candidatus Phytoplasma cynodontis (16SrXIV-A subgroup) associated with cauliflower phyllody and flat stem in India

Plant Disease ◽  
2021 ◽  
Author(s):  
A. Sajeena ◽  
Govind P. Rao ◽  
Deepu Mathew ◽  
Jacob John ◽  
M. K. Dhanya ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Brassica Oleracea ◽  
Nested Pcr ◽  
Disease Incidence ◽  
Rrna Gene ◽  
Research Station ◽  
First Report ◽  
Pcr Products ◽  
Seca Gene

Symptoms of suspected phytoplasma infection were observed in cauliflower (Brassica oleracea var. botrytis) (cultivar NS60N) at Integrated Farming System Research Station, Trivandrum, Kerala, India (08o28’28”N, 76o57’47”E) in April-2021. The disease incidence was recorded up to 10% in different fields. The disease manifested as stunting, phyllody, floral malformation and flattening of stem (Fig.1A,B). Ten symptomatic and five asymptomatic plants were assayed for the presence of phytoplasma using nested PCR assays performed with P1/P7 and R16F2n/R16R2 primer pairs for 16S rRNA gene and SecAfor1/ SecArev3 and SecAfor2/ SecArev3 for secA gene (Deng and Hiruki 1991; Gundersen and Lee 1996; Hodgetts et al. 2008). The expected amplicons of ~1.25 kb and ~480 bp were consistently amplified in all the symptomatic cauliflower samples with the phytoplasma specific universal 16S rRNA and secA gene specific primers. Nested PCR products (~1.2 kb and 480 bp) amplified from cauliflower was cloned in EcoRI restriction sites of pGEM-T Easy vector (Promega, USA). The cloned nested PCR products were directly sequenced (16S rRNA gene: Acc. Nos. MZ196223, MZ196224; secA gene: MZ215721, MZ215722) in both forward and reverse directions which showed 99.77% sequence identity with Candidatus Phytoplasma cynodontis reference strain (Acc. No. AJ550984). Further analyses of the 16S rRNA and secA genes based phylogenetic tree (Fig. 2A and B) and the iPhyClassifier-based virtual RFLP analysis of 16Sr RNA gene study demonstrated that the phytoplasma-associated with cauliflower phyllody & flat stem disease (CaPP) belonged to 16SrXIV-A subgroup with a similarity coefficient of 1.0. No amplicon was observed from any of the asymptomatic cauliflower plants with the specific tested primers of both the genes. Earlier association of 16SrXV-A subgroup (Candidatus Phytoplasma brasiliense) and 16Sr III-J subgroup in Brazil (Canale and Badendo, 2013; Rappussi et al. 2012), 16SrII-A (Candidatus Phytoplasma aurantifolia) subgroup in China (Cai et al. 2016) and 16SrVI-A (Candidatus Phytoplasma trifolii) subgroup in Iran (Salehi 2007) were reported in cauliflower. Another species of cabbage, Brassica oleracea var. capitata L. was reported as host of Ca. P. trifloii (16Sr VI-D subgroup) from north India (Gopala et al. 2018). To our knowledge, this is the first report of a ‘Candidatus Phytoplasma cynodontis’, 16SrXIV-A subgroup related phytoplasma strain associated with cauliflower phyllody and flat stem in the world. The results described in this report confirm that the 16SrXIV-A phytoplasma, a widely distributed strain associated with sugarcane, wheat, grasses, sapota and many ornamentals in India (Rao 2021), has also infected cauliflower. This is not only the first instance of cauliflower phyllody disease found in India, but also the first instance of CaPP disease caused by 16SrXIV-A subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as cauliflower is the one of the most common vegetable crop grown all over India.

scholarly journals First Report of Group 16SrXII-A Phytoplasma Causing Stolbur Disease in Saponaria officinalis Plants in Serbia

Plant Disease ◽  
2013 ◽  
Vol 97 (3) ◽  
pp. 420-420 ◽  
Author(s):  
D. Josic ◽  
M. Starovic ◽  
S. Stojanovic ◽  
T. Popovic ◽  
N. Dolovac ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Gene Sequence ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
First Report ◽  
Stolbur Phytoplasma ◽  
Saponaria Officinalis

Saponaria officinalis L. (Caryophyllaceae; also known as bouncingbet or soapwort) is a perennial medicinal plant important for the pharmaceutical industry and used as an expectorant, alterative, laxative, and ointment for some skin diseases and arthritic conditions. S. officinalis plants with typical symptoms (23% in 2011 and 47% in 2012) of phytoplasma infection were observed in Pancevo plantation, Serbia. The symptoms appeared in May with leaves changing color from green to brown with severe reddening and necrosis. Severely diseased plants died. The infected plants had a significant reduction in biomass and quality. To investigate the presence of phytoplasma, total DNA was extracted from 10 symptomatic and four asymptomatic plants by a CTAB method. The nested PCR was carried out using phytoplasma-specific primer set P1/16S-SR followed by R16F2n/R16R2, targeting the 16S rRNA gene sequence of 1.5 and 1.2 kb in length, respectively. The amplicons of expected size were obtained from the symptomatic plants, but not from the asymptomatic plants. To obtain restriction fragment length polymorphism (RFLP) patterns, the R16F2n/R2 amplicons were digested with AluI, TruI1, HpaII, and HhaI endonucleases. The resulting patterns indicated that seven plants were infected by a Stolbur phytoplasma belonging to the 16SrXII-A subgroup, since it had the identical RFLP pattern as the STOL reference strain. The 1.2 kb nested PCR products of representative isolate Sap7 were purified using PCR purification kit (Fermentas, Vilnius, Lithuania) according to the recommended protocol and sequenced using facilities of IMGGI SeqService, Belgrade, Serbia. The obtained sequence was deposited in the NCBI database (GenBank Accession No. JX866951). The phytoplasma 16S rRNA gene sequence from Sap7 had a sequence identity of 97% with GenBank accessions GQ273961.1 (‘Euonymus japonicus’ phytoplasma), JX311953.1 (Candidatus Phytoplasma solani clone 5043), JQ412100.1 (Iranian alfalfa phytoplasma M21), and JN561702.1 (‘Convolvulus arvensis’ stolbur phytoplasma clone P1/P7-Conv2/2010-Bg). To our knowledge, this is the first report of a natural infection of S. officinalis by 16SrXII-A subgroup (Stolbur) phytoplasma in Serbia. As cited by Lee et al. (1), the 16SrI-M subgroup phytoplasma in S. officinalis sample was already detected in Lithuania by Valiunas (2). The identification of phytoplasma in the Pancevo plantation caused the intensification of our biological control tests and efforts to reduce the ecological and economic impacts of these phytoplasmas. References: (1) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (2) D. Valiunas. PhD thesis, Institute of Botany, Vilnius, Lithuania, 2003.

scholarly journals 16S rRNA Gene Sequence Analysis ofPhotobacterium damselae and Nested PCR Method for Rapid Detection of the Causative Agent of Fish Pasteurellosis

1999 ◽  
Vol 65 (7) ◽  
pp. 2942-2946 ◽  
Author(s):  
Carlos R. Osorio ◽  
Matthew D. Collins ◽  
Alicia E. Toranzo ◽  
Juan L. Barja ◽  
Jesús L. Romalde
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Causative Agent ◽  
Nested Pcr ◽  
Gene Sequence ◽  
16S Rrna Gene Sequence ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Photobacterium Damselae

ABSTRACT The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified asPhotobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species asPhotobacterium damselae subsp. damselae(formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.

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

2006 ◽  
Vol 55 (1) ◽  
pp. 109-113 ◽  
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.

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

Plant Disease ◽  
2014 ◽  
Vol 98 (8) ◽  
pp. 1152-1152 ◽  
Author(s):  
S. Pavlovic ◽  
M. Starovic ◽  
S. Stojanovic ◽  
G. Aleksic ◽  
S. Kojic ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Natural Infection ◽  
Cytotoxic Activities ◽  
Rrna Gene ◽  
Calendula Officinalis ◽  
Aster Yellows ◽  
First Report ◽  
Pot Marigold ◽  
Stolbur Phytoplasma

Pot marigold (Calendula officinalis L.) is native to southern Europe. Compounds of marigold flowers exhibit anti-inflammatory, anti-tumor-promoting, and cytotoxic activities (4). In Serbia, pot marigold is cultivated as an important medicinal and ornamental plant. Typical phyllody, virescence, proliferation of axillary buds, and witches' broom symptoms were sporadically observed in 2011 in Pancevo plantation, Serbia (44°51′49″ N, 20°39′33″ E, 80 m above sea level). Until 2013, the number of uniformly distributed affected pot marigold plants reached 20% in the field. Due to the lack of seed production, profitability of the cultivation was seriously affected. Leaf samples from 10 symptomatic and 4 symptomless marigold plants were collected and total nucleic acid was extracted from midrib tissue (3). Direct PCR and nested PCR were carried out with primer pairs P1/16S-SR and R16F2n/R16R2n, respectively (3). Amplicons 1.5 and 1.2 kb in length, specific for the 16S rRNA gene, were amplified in all symptomatic plants. No PCR products were obtained when DNA isolated from symptomless plants was used. Restriction fragment length polymorphism (RFLP) patterns of the 1.2-kb fragments of 16S rDNA were determined by digestion with four endonucleases separately (TruI1, AluI, HpaII, and HhaI) and compared with those of Stolbur (Stol), Aster Yellows (AY), Flavescence dorée-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas (2). RFLP patterns from all symptomatic pot marigold plants were identical to the Stol pattern, indicating Stolbur phytoplasma presence in affected plants. The 1.2-kb amplicon of representative Nv8 strain was sequenced and the data were submitted to GenBank (accession no. KJ174507). BLASTn analysis of the sequence was compared with sequences available in GenBank, showing 100% identity with 16S rRNA gene of strains from Paeonia tenuifolia (KF614623) and corn (JQ730750) from Serbia, and peach (KF263684) from Iran. All of these are members of the 16SrXII ‘Candidatus Phytoplasma solani’ group, subgroup A (Stolbur). Phytoplasmas belonging to aster yellows (16SrI) (Italy and Canada) and peanut witches' broom related phytoplasma (16SrII) group (Iran) have been identified in diseased pot marigold plants (1). To our knowledge, this is the first report of natural infection of pot marigold by Stolbur phytoplasma in Serbia. References: (1) S. A. Esmailzadeh-Hosseini et al. Bull. Insectol. 64:S109, 2011. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) J. P. Prince. Phytopathology 83:1130, 1993. (4) M. Ukiya et al. J. Nat. Prod. 69:1692, 2006.

scholarly journals Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

2005 ◽  
Vol 71 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Anne-Ga�lle Le Bourhis ◽  
Katiana Saunier ◽  
Jo�l Dor� ◽  
Jean-Philippe Carlier ◽  
Jean-Fran�ois Chamba ◽  
...  
Keyword(s):  
Temperature Gradient ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis ◽  
Clostridial Species ◽  
The 16S Rrna Gene

ABSTRACT A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.

scholarly journals Analytical Sensitivity, Reproducibility of Results, and Clinical Performance of Five PCR Assays for Detecting Chlamydia pneumoniae DNA in Peripheral Blood Mononuclear Cells

2000 ◽  
Vol 38 (7) ◽  
pp. 2622-2627 ◽  
Author(s):  
J. B. Mahony ◽  
S. Chong ◽  
B. K. Coombes ◽  
M. Smieja ◽  
A. Petrich
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Chlamydia Pneumoniae ◽  
Mononuclear Cells ◽  
Rrna Gene ◽  
Analytical Sensitivity ◽  
Infected Cells ◽  
Pcr Assays ◽  
The 16S Rrna Gene

Chlamydia pneumoniae has been associated with atherosclerosis and coronary artery disease (CAD), and its DNA has been detected in atheromatous lesions of the aorta, carotid, and coronary arteries by a variety of PCR assays. The objective of this study was to compare the performances of five published PCR assays in the detection of C. pneumoniae in peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease. The assays included two conventional PCRs, one targeting a cloned PstI fragment and one targeting the 16S rRNA gene; two nested PCRs, one targeting the 16S rRNA gene and one targeting ompA; and a touchdown enzyme time release (TETR) PCR, targeting the 16S rRNA gene. All PCRs had similar analytical sensitivities and detected a minimum of 0.005 inclusion-forming units (IFU) of C. pneumoniae; the ompA nested PCR and the TETR PCR were slightly more sensitive and detected 0.001 IFU. Assay reproducibility was examined by testing 10 replicates of C. pneumoniae DNA by each assay. All five assays showed excellent reproducibility at high levels of DNA, with scores of 10 out of 10 for 0.01 IFU, but exhibited decreased reproducibility for smaller numbers of C. pneumoniae IFU for all tests. Pairwise comparison of test results indicated that there was a significant difference between tests (Cochran Q = 32.0, P < 0.001), with thePstI fragment (P < 0.001) and 16S rRNA (P = 0.002) assays having lower reproducibility than the nested ompA and TETR assays. To further analyze assay sensitivity, C. pneumoniae-infected U-937 mononuclear cells were added to whole blood, and extracted mononuclear-cell DNA was tested by each assay. All five assays showed similar sensitivities, detecting 15 infected cells; three assays detected 3 infected cells, while all assays were negative at the next dilution (1.5 infected cells). A striking difference in performance of the five assays was seen, however, when PBMCs from CAD patients were tested for C. pneumoniae DNA. The ompA nested PCR detected C. pneumoniae DNA in 11 of 148 (7.4%) specimens, the 16S rRNA nested PCR detected 2 positives among the 148 specimens (1.4%) (P < 0.001), and the other 3 assays detected no positive specimens (P < 0.001, compared with theompA assay). These results indicate that analytical sensitivity alone does not predict the ability of an assay to detectC. pneumoniae in whole-blood-derived PBMCs. Before standardized assays can be used in wide-scale epidemiological studies, further characterization of these assays will be required to improve our understanding of their performance in the detection of C. pneumoniae in clinical material.

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