scholarly journals First Report of Witches'-Broom Disease in a Cannabis spp. in China and Its Association with a Phytoplasma of Elm Yellows Group (16SrV)

Plant Disease ◽  
2007 ◽  
Vol 91 (2) ◽  
pp. 227-227 ◽  
Author(s):  
Y. Zhao ◽  
Q. Sun ◽  
R. E. Davis ◽  
I.-M. Lee ◽  
Q. Liu
Keyword(s):  
Nucleotide Sequence ◽  
16S Rdna ◽  
Geographic Location ◽  
Full Length ◽  
23S Rrna ◽  
Nucleotide Sequence Analysis ◽  
Rrna Gene ◽  
Hemp Fiber ◽  
First Report ◽  
Elm Yellows

Hemp fiber plants (Cannabis spp.) spread naturally in almost every climate zone in China and have a long history of cultivation in the country (1). While hemp stalks provide high-quality fibers for making ropes, clothes, and paper products, hemp seeds are a rich source of edible oil. During the summer of 2004, a disease characterized by witches'-broom symptoms was observed in wild hemp fiber plants growing in suburban Taian, Shandong, China. The diseased plants developed clusters of highly proliferating branches with much shortened internodes and leaves on the affected branches were significantly reduced in size. Phytoplasma infection was suspected in this hemp fiber witches'-broom (HFWB) disease because of the typical symptoms and because of its geographic location where other phytoplasmal diseases such as jujube witches'-broom (JWB), paulownia witches'-broom (PaWB), paper mulberry witches'-broom (PMWB), and Chinese wingnut witches'-broom (CWWB) diseases were previously reported (3,4). Total DNA was extracted from leaves of four diseased and four nearby healthy looking hemp fiber plants. Nested PCR were carried out on the DNA samples using phytoplasma universal 16S rDNA primers (P1A/16S-SR and R16F2n/R16R2) (2). Results revealed that all examined diseased plants were infected by phytoplasma, whereas nearby healthy looking plants were phytoplasma free. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 1.25-kb 16S rDNA R16F2n/R16R2 fragment indicated that the phytoplasma associated with HFWB disease belongs to subgroup 16SrV-B of the elm yellows (EY) phytoplasma group. Nucleotide sequence analysis of the cloned HFWB phytoplasma partial rRNA operon (GenBank Accession No. EF029092), spanning a near full-length 16S rRNA gene and a partial 16S-23S rRNA intergenic spacer, suggested that HFWB phytoplasma is most closely related to JWB and PMWB phytoplasmas, both members of subgroup16SrV-B. To further characterize the HFWB phytoplasma, a genomic segment covering full-length ribosomal protein genes rplV and rpsC was PCR-amplified using primer pair rp(V)F1A/rp(V)R1A (2), cloned, and sequenced (GenBank Accession No. EF029093). The nucleotide sequence of the HFWB phytoplasma rplV and rpsC locus is nearly identical (99.9%) to that of JWB phytoplasma. To our knowledge, this is the first report of a phytoplasmal disease in Cannabis spp. Since HFWB and JWB phytoplasmas share extremely high sequence identity and share the same eco-geographic location, further investigation is warranted to determine whether these two phytoplasmas are actually one species that can infect both plants, an issue having important implications in managing both diseases. References: (1) S. Hong and R. C. Clarke. J. Int. Hemp Assoc. 3:55, 1996. (2) I. M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (3) Q. Liu et al. Plant Dis. 88:770, 2004. (4) Q. Liu et al. Plant Dis. 89:529, 2005.

scholarly journals First Report of Witches'-Broom Disease of Broussonetia papyrifera and Its Association with a Phytoplasma of Elm Yellows Group (16SrV)

Plant Disease ◽  
2004 ◽  
Vol 88 (7) ◽  
pp. 770-770 ◽  
Author(s):  
Qingzhong Liu ◽  
Tianqi Wu ◽  
Robert E. Davis ◽  
Yan Zhao
Keyword(s):  
16S Rdna ◽  
Biological Diversity ◽  
23S Rrna ◽  
Nucleotide Sequence Analysis ◽  
Rrna Gene ◽  
Natural Ecosystems ◽  
Broussonetia Papyrifera ◽  
First Report ◽  
Broom Disease ◽  
Elm Yellows

Broussonetia papyrifera, commonly known as paper mulberry, is an ornamental tree that is native to northeastern Asia. Because of its fast-growing nature and tolerance of dust, smoke, and high temperatures, paper mulberry is an important component of the biological diversity in natural ecosystems as well as a favorable shade tree in the region. In September of 2003, a disease characterized by pronounced witches'-broom symptoms was observed in paper mulberry trees growing near a jujube (Ziziphus jujuba) orchard and in home gardens located in Taian, Shandong, China. The diseased trees developed dense clusters of highly proliferating branches with shortened internodes. Leaves on the affected branches were chlorotic and greatly reduced in size. Phytoplasma infection was first suspected in this paper mulberry witches'-broom (PMWB) disease because the disease occurred in an area where other phytoplasmal diseases, including jujube witches'-broom (JWB) disease and paulownia witches'-broom (PaWB) disease, are common (4). Results from nested polymerase chain reactions (PCR), performed using phytoplasma-universal 16S rDNA primers (P1/P7 and R16F2n/R16R2) (1,2,3), revealed that all seven diseased trees tested contained phytoplasma, whereas PCR assay of comparable leaf samples from three nearby symptomless paper mulberry trees were negative. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA indicated that all diseased trees contained the same phytoplasma and that the PMWB phytoplasma belongs to the subgroup B of the elm yellows (EY) phytoplasma group (16SrV-B). Nucleotide sequence analysis of the cloned PMWB phytoplasma partial rRNA operon (GenBank Accession No. AY576685), spanning a near full-length 16S rRNA gene, a 16S–23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene, suggested that PMWB phytoplasma is most closely related to JWB phytoplasma, a member of the subgroup16SrV-B. To our knowledge, this is the first report of a paper mulberry witches'-broom disease and the first report of its association with a phytoplasma. Further work is underway to determine whether the PMWB phytoplasma is distinct from previously characterized phytoplasmas included in group 16SrV and to assess impacts of the phytoplasma on the ecosystems in the region. References: (1) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) S. Zhu et al. Acta Hortic. 472:701, 1998.

scholarly journals First Report of a Natural Infection of Opuntia sp. by a ‘Candidatus Phytoplasma asteris’-Related Phytoplasma in China

Plant Disease ◽  
2007 ◽  
Vol 91 (4) ◽  
pp. 461-461 ◽  
Author(s):  
W. Wei ◽  
H. Cai ◽  
H. Chen ◽  
R. E. Davis ◽  
Y. Zhao
Keyword(s):  
16S Rdna ◽  
Natural Infection ◽  
Geographical Location ◽  
Rflp Analysis ◽  
23S Rrna ◽  
Nucleotide Sequence Analysis ◽  
Rrna Gene ◽  
First Report ◽  
Aesthetic Appearance ◽  
Geographical Regions

Cacti (Opuntia spp.) are perennial, evergreen, succulent plants native to arid areas of the Americas. Because of their aesthetic appearance, many cacti have been cultivated and introduced to other parts of the world as ornamentals. Cacti are susceptible to phytoplasma infections and develop witches'-broom (WB) disease. Currently, all reported cactus WB cases are associated with infections by phytoplasmas in the peanut witches'-broom group (16SrII) (1,2,4). During a phytoplasma diversity survey carried out during 2004 in Yunnan, China, we collected 29 malformed and 14 healthy-looking naturally occurring cactus plants from 14 locations representing five geographical regions. Each of the 29 malformed plants exhibited stunted growth and possessed clusters of highly proliferating cladodia, typical symptoms of cactus WB disease. Nested-PCR was carried out on the DNA samples extracted from young cladodia of these plants using phytoplasma-universal 16S rDNA primers P1A/P7A and R16F2n/R16R2 (3). Results revealed that all 29 diseased plants that were examined were infected by phytoplasmas, whereas all 14 healthy-looking plants were negative for phytoplasmas. Subsequent restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 1.25-kb 16S rDNA fragments indicated that 28 diseased plants were infected by a phytoplasma of group 16SrII, whereas one plant (from Suan Village) was infected by a ‘Candidatus Phytoplasma asteris’-related (group 16SrI) phytoplasma designated as strain YN26. Nucleotide sequence analysis of the strain YN26 partial rRNA operon (GenBank Accession No. EF190970), covering a near full-length 16S rRNA gene, a 16S-23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene, suggested that this phytoplasma is most closely related to an ash witches'-broom phytoplasma (GenBank Accession No. AY566302, 99.7% identity) and an epilobium phyllody phytoplasma (GenBank Accession No. AY101386, 99.7% identity), both members of subgroup16SrI-B. This YN26-infected cactus plant was transferred to a greenhouse and maintained for more than 2 years, during which time DNA samples were extracted and tested two additional times. The same 16S rDNA RFLP pattern type was consistently obtained in these tests, confirming that the plant remained infected by the 16SrI phytoplasma. To our knowledge, this is the first report of a natural infection of a cactus species by a group 16SrI phytoplasma. Since this 16SrI-cactus WB phytoplasma was found in the same geographical location where 16SrII-cactus WB phytoplasma was detected both in this and a previous study (1), the findings raised the question whether 16SrI- and 16SrII-cactus WB phytoplasmas have overlapping geo- and bioecological niches. References: (1) H. Cai et al. Plant Pathol. 51:394, 2002. (2) E. Choueiri et al. Plant Dis. 89:1129, 2005. (3) I. M. Lee et al. Int. J. Syst. Evol. Microbiol 54:337, 2004. (4) N. Leyva-Lopez et al. Phytopathology (Abstr.) 89(suppl):S45, 1999.

scholarly journals Molecular Identification of a New Phytoplasma Strain Associated with the First Observation of Jujube Witches'-Broom Disease in Northeastern China

Plant Disease ◽  
2007 ◽  
Vol 91 (10) ◽  
pp. 1364-1364 ◽  
Author(s):  
W. Wei ◽  
H. Jiang ◽  
Y. Yang ◽  
Y. Wang ◽  
R. E. Davis ◽  
...  
Keyword(s):  
Sequence Data ◽  
Northeastern China ◽  
Full Length ◽  
Central China ◽  
Liaoning Province ◽  
Deciduous Tree ◽  
23S Rrna ◽  
Rrna Gene ◽  
Hemp Fiber ◽  
Phytoplasma Strain

Jujube (Zizyphus jujuba Mill.) is a deciduous tree that is native to northern Africa and Syria. Because of its tolerance to a broad range of climatic conditions, jujube has attained a wide natural distribution from southeastern Europe to eastern Asia (3). Jujube has a long history of cultivation, especially in Asia, for its valuable medicinal properties, strong wood, and nutritious fruits. Jujube trees are susceptible to phytoplasma infections and develop jujube witches'-broom (JWB) disease. To date, JWB diseases have been reported in Korea, Japan, and central China (1,4). In this communication, we describe a new phytoplasma strain associated with the first observation of JWB disease in northeastern China. In the summer of 2006, six jujube trees exhibiting pronounced witches'-broom symptoms were observed in suburban Dalian, Liaoning Province. The trees developed dense clusters of highly proliferating branches with shortened internodes. Leaves on the affected branches were chlorotic and significantly reduced in size. A DNA segment characteristic of phytoplasma rRNA partial operons was amplified from DNA samples extracted from leaves of all diseased trees in polymerase chain reactions (PCR) using phytoplasma-universal primer pair P1/P7 (2). No PCR product was obtained from DNA samples extracted from two symptomless jujube trees in the same region. The PCR-amplified DNA segment, spanning a near full-length 16S rRNA gene, a 16S-23S rRNA intergenic spacer, a tRNA-Ile gene, and a partial 23S rRNA gene was cloned and sequenced to achieve 4× coverage per base position in sequencing both strands (GenBank Accession No. EF661852). Results from analysis of the sequence data indicated that the six jujube trees were infected by a phytoplasma of elm yellows group (16SrV), to which other reported JWB phytoplasma strains belong. However, the JWB phytoplasma strain identified in the current study, hereby designated as JWB-DL, displayed sequence variations within the partial rRNA operon compared with those of other JWB strains (GenBank Accession Nos. AY072722, AF305240, and AY197661), indicating that JWB-DL is a distinct strain. To further characterize the JWB-DL phytoplasma, a genomic segment covering full-length ribosomal protein genes rplV and rpsC was PCR-amplified using primer pair rp(V)F2A/rpR1 (2), cloned, and sequenced (GenBank Accession No. EF661581). The nucleotide sequence of the JWB-DL phytoplasma rplV-rpsC locus is identical to that of hemp fiber witches'-broom phytoplasma (GenBank Accession No. EF029093) rather than to those of JWB phytoplasma strains described previously. To our knowledge, this is the first report of a JWB disease in northeastern China, and JWB-DL represents a new, distinct ‘Candidatus Phytoplasma ziziphi’-related strain. References: (1) H.-Y. Jung et al. Int. J. Syst. Evol. Microbiol. 53:1037, 2003. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (3) W. H. Outlaw et al. Econ. Bot. 56:198, 2002. (4) J. B. Tian et al. Hortic. Sci 35:1274, 2000.

scholarly journals First Report of Witches'-Broom Disease of Chinese Wingnut in China and its Association with a Phytoplasma of Aster Yellows Group (16SrI)

Plant Disease ◽  
2005 ◽  
Vol 89 (5) ◽  
pp. 529-529 ◽  
Author(s):  
Q. Liu ◽  
Q. Sun ◽  
T. Wu ◽  
R. E. Davis ◽  
Y. Zhao
Keyword(s):  
16S Rdna ◽  
Biological Diversity ◽  
Nucleotide Sequence Analysis ◽  
Soil Drought ◽  
Rrna Gene ◽  
Polymorphism Analysis ◽  
First Report ◽  
Broom Disease ◽  
The Impact ◽  
Chinese Wingnut

Pterocarya stenoptera C. DC., commonly known as Chinese wingnut, is a fast-growing deciduous tree with tough bark and attractive foliage. Because of its tolerance of compact and nutritionally poor soil, drought, and heat, Chinese wingnut is an important component of the biological diversity in natural ecosystems and is a favorable shade tree in China. Chinese wingnut has also been used as a rootstock for walnuts because of its high resistance to soilborne Phytophthora spp. In the spring of 2004, a disease characterized by witches'-broom symptoms was observed affecting Chinese wingnut trees growing in suburban Taian, Shandong, China. The diseased trees developed dense clusters of highly proliferating branches with shortened internodes, leaves on the affected branches were significantly smaller, and some branches and twigs suffered dieback. Phytoplasma infection was suspected as the cause of this Chinese wingnut witches'-broom (CWWB) disease because the disease occurred in an area where phytoplasmal diseases, such as paulownia witches'-broom (PaWB) and jujube witches'-broom (JWB), are common (3). Nested polymerase chain reactions (PCR) were performed on DNA samples extracted from leaves of six diseased trees using phytoplasma-universal 16S rDNA primers (R16mF2/R16mR1 and R16F2n/ R16R2) (1,2). Results revealed that all diseased trees examined were infected by phytoplasma, whereas PCR assays of leaf samples from two nearby symptomless Chinese wingnut trees were negative. Subsequent restriction fragment length polymorphism analysis of the PCR-amplified 16S rDNA indicated that all diseased trees contained the same phytoplasma and that the CWWB phytoplasma belongs to subgroup B of the “Candidatus Phytoplasma asteris” (AY) group (16SrI). Nucleotide sequence analysis of a 16S rRNA gene cloned from CWWB phytoplasma (GenBank Accession No. AY831966) suggested that this phytoplasma is closely related to, but distinct from, PaWB phytoplasma, another member of group16SrI. To our knowledge, this is the first report of Chinese wingnut witches'-broom disease and of its association with a phytoplasma. Further work is being undertaken to examine the ecological and evolutionary relationship between CWWB phytoplasma and other phytoplasmas in the region and to assess the impact of CWWB on walnut rootstock selection. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (3) S. Zhu et al. Acta Hortic. 472:701, 1998.

Phylogenetic placement of unculturedCeanothusmicrosymbionts using 16S rRNA gene sequences

1999 ◽  
Vol 77 (9) ◽  
pp. 1208-1213 ◽  
Author(s):  
Nancy J Ritchie ◽  
David D Myrold
Keyword(s):  
16S Rdna ◽  
Phylogenetic Trees ◽  
Full Length ◽  
Likelihood Method ◽  
Rrna Gene ◽  
Consensus Sequences ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
Polymerase Chain ◽  
Ceanothus Americanus

Full-length 16S rDNA sequences were amplified directly from the nodules of Ceanothus americanus L. and Ceanothus thyrsiflorus Eschsch. using the polymerase chain reaction. Sequences were determined using an automated sequencer, compared against those in GenBank, and assembled into consensus sequences. The sequences were aligned with other full-length Frankia 16S rDNA sequences available from the data base. Phylogenetic trees were obtained using three different algorithms: neighbor joining, parsimony, and the maximum-likelihood method. All three methods showed that these Ceanothus L. microsymbionts were most closely related to the microsymbiont associated with Dryas drummondii Richardson ex Hook. Lvs. rather than Frankia isolated from the Elaeagnaceae.Key words: Frankia, Ceanothus, 16S rDNA.

scholarly journals First Report of Kudzu mosaic virus on Pueraria montana (Kudzu) in China

Plant Disease ◽  
2013 ◽  
Vol 97 (1) ◽  
pp. 148-148 ◽  
Author(s):  
J. Zhang ◽  
Z. J. Wu
Keyword(s):  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Mosaic Virus ◽  
Nucleotide Sequence Identity ◽  
Southern China ◽  
Full Length ◽  
Yellow Vein ◽  
Mosaic Symptom ◽  
First Report ◽  
Sequence Identity

Kudzu (Pueraria montana), a weed widely distributed in southern China, is common in the Fuzhou region of Fujian Province, where many plants show yellow vein mosaic disease. In September 2008, four leaf samples from different plants exhibiting yellow vein mosaic symptom were collected in suburban district of Fuzhou (25°15′ N, 118°08′ E). Whitefly (Bemisia tabaci) infestation was also observed in this region. Total DNA was extracted from all samples using a CTAB method (4). Universal primers (PA/PB) were used to amplify part of the intergenic region and coat protein gene of DNA-A of begomoviruses (1). An amplicon of approximately 500 bp was obtained from all four samples and then sequenced. Comparison of 500-bp fragments (GenBank Accession Nos. FJ539016-18 and FJ539014) revealed the presence of the same virus (98.8 to 99.4%). A pair of back-to-back primers (Yg3FL-F: 5′-GGATCCTTTGTTGAACGCCTTTCC-3′/Yg3FL-R: 5′-GGATCCCACATGTTTAAAGTAAAGC-3′) were designed to amplify the full-length DNA-A from the Chinese isolate identified as Yg3. Sequence analysis showed that full-length DNA-A of Yg3 isolate comprised 2,729 nucleotides (GenBank Accession No. FJ539014) and shared the highest nucleotide sequence identity (91.9%) with Kudzu mosaic virus (KuMV, GenBank Accession No. DQ641690) from Vietnam. To further test the association of DNA-B fragments with the four samples from southern China, rolling circle amplification (RCA) was performed (3). When RCA products were digested with Sph I, approximately 2.7 kb was obtained from all samples. Yg3 isolate was chosen to be sequenced. Sequence analysis showed that full-length DNA-B of Yg3 isolate comprised 2,677 nucleotides (GenBank Accession No. FJ539015) and shared the highest nucleotide sequence identity (76.8%) with KuMV DNA-B (GenBank Accession No. DQ641691) from Vietnam. Based on the current convention of begomovirus species demarcation of <89% sequence identity cut-off criterion (2), Yg3 was identified as an isolate of KuMV. To our knowledge, this is the first report of association of KuMV with yellow vein mosaic symptom of kudzu in China. References: (1). D. Deng et al. Annals Appl. Biol. 125:327, 1994. (2). C. M. Fauquet et al. Arch. Virol. 148:405, 2003. (3). D. Haible et al. J. Virol. Methods 135:9, 2006. (4). Y. Xie et al. Chinese Sci. Bull. 47:197, 2002.

scholarly journals rDNA analyses of planktonic heterocystous cyanobacteria, including members of the genera Anabaenopsis and Cyanospira The GenBank accession numbers of the 16S rDNA gene sequences reported in this paper are AY038032–AY038037.

Microbiology ◽  
2002 ◽  
Vol 148 (2) ◽  
pp. 481-496 ◽  
Author(s):  
Isabelle Iteman ◽  
Rosmarie Rippka ◽  
Nicole Tandeau de Marsac ◽  
Michael Herdman
Keyword(s):  
16S Rrna ◽  
16S Rdna ◽  
Genomic Sequence ◽  
Sequence Similarity ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Phylogenetic Position ◽  
Rrna Gene ◽  
Heterocystous Cyanobacteria

The taxonomic coherence and phylogenetic relationships of 11 planktonic heterocystous cyanobacterial isolates were examined by investigating two areas of the rRNA operon, the 16S rRNA gene (rrnS) and the internal transcribed spacer (ITS) located between the 16S rRNA and 23S rRNA genes. The rrnS sequences were determined for five strains, including representatives of Anabaena flos-aquae, Aphanizomenon flos-aquae, Nodularia sp. and two alkaliphilic planktonic members of the genera Anabaenopsis and Cyanospira, whose phylogenetic position was previously unknown. Comparison of the data with those previously published for individual groups of planktonic heterocystous cyanobacteria showed that, with the exception of members assigned to the genus Cylindrospermopsis, all the planktonic strains form a distinct subclade within the monophyletic clade of heterocystous cyanobacteria. Within this subclade five different phylogenetic clusters were distinguished. The phylogenetic groupings of Anabaena and Aphanizomenon strains within three of these clusters were not always consistent with their generic or specific assignments based on classical morphological definitions, and the high degree of sequence similarity between strains of Anabaenopsis and Cyanospira suggests that they may be assignable to a single genus. Ribotyping and additional studies performed on PCR amplicons of the 16S rDNA or the ITS for the 11 planktonic heterocystous strains demonstrated that they all contain multiple rrn operons and ITS regions of variable size. Finally, evidence is provided for intra-genomic sequence heterogeneity of the 16S rRNA genes within most of the individual isolates.

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 Establishment and Assessment of An Amplicon Sequencing Method Targeting The 16S-ITS-23S rRNA Operon For Analysis of The Equine Gut Microbiome

2021 ◽  
Author(s):  
Yuta Kinoshita ◽  
Hidekazu NIWA ◽  
Eri UCHIDA-FUJII ◽  
Toshio NUKADA
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Full Length ◽  
Rrna Operon ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
23S Rrna ◽  
Rrna Gene ◽  
Fecal Samples

Abstract Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had >90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The rRNA operon amplicon sequencing of equine fecal samples underestimated compositional percentages of bacterial strains containing unlinked rRNA genes by a third to almost a half, but unlinked rRNA genes had a limited effect on the overall results. The rRNA operon amplicon sequencing with the A519F + U2428R primer set was able to reflect archaeal genomes, whereas full-length 16S rRNA with 27F + 1492R could not. Therefore, we conclude that amplicon sequencing targeting the rRNA operon captures more detailed variations of bacterial and archaeal microbiota.

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