scholarly journals Identification of phytoplasma strains associated with witches’ broom and yellowing in Ziziphus jujube nurseries in Iran

2020 ◽  
Vol 59 (1) ◽  
pp. 55-61
Author(s):  
Ghobad BABAEI ◽  
Seyyed Alireza ESMAEILZADEH-HOSSEINI ◽  
Mahbobeh ZANDIAN ◽  
Vahid NIKBAKHT
Keyword(s):  
Restriction Analysis ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Reference Pattern ◽  
Consensus Sequences ◽  
Rdna Sequences ◽  
Rflp Profile ◽  
Ziziphus Jujube ◽  
East Of Iran ◽  
Virtual Rflp

Phytoplasma symptoms, including proliferation, witches’ broom, leaf rolling and yellowing, were observed in jujube (Ziziphus jujube) nurseries in the East of Iran. Total nucleic acid was extracted from symptomatic and symptomless plants, and was tested for phytoplasma presence using nested PCR. Amplicons of about 1.8 kb (primer pair P1/P7) and 1.25 kb (R16F2n/R16R2) were obtained from all symptomatic plants but not from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 amplicons using KpnI, HaeIII, RsaI, AluI, HpaII, HhaI, TaqI, MseI, BfaI and ThaI restriction enzymes showed two RFLP patterns referable to 16SrI and 16SrVI phytoplasma groups. The consensus sequences of Z. jujube yellowing and witches’ broom of six samples correspond to ‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma trifolii’-related strains. Two R16F2n/R16R2 16S rDNA sequences representative of each RFLP profile, one each from witches’ broom (accession number MK379605) and yellowing (MK379604) host symptoms, were submitted to the GenBank. Phylogenetic analysis confirmed that the phytoplasma strains associated with jujube yellowing clustered within the 16SrI phytoplasma clade, and those associated with witches’ broom clustered within the 16SrVI clade. Restriction analysis confirmed that virtual RFLP patterns of the jujube yellowing and witches’ broom phytoplasma strains were identical to the reference pattern of 16SrI-B and 16SrVI-A. This is the first report of these phytoplasma strains associations with witches’ broom and yellowing in jujube plants.

scholarly journals A novel subgroup 16SrVII-D phytoplasma identified in association with erigeron witches' broom

2015 ◽  
Vol 65 (Pt_8) ◽  
pp. 2761-2765 ◽  
Author(s):  
Daniela Flôres ◽  
Ana Paula de Oliveira Amaral Mello ◽  
Thays Benites Camargo Pereira ◽  
Jorge Alberto Marques Rezende ◽  
Ivan Paulo Bedendo
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Rrna Gene ◽  
Phytoplasma Strain ◽  
Total Dna ◽  
Virtual Rflp ◽  
The 16S Rrna Gene

Erigeron sp. plants showing symptoms of witches' broom and stunting were found near orchards of passion fruit in São Paulo state, Brazil. These symptoms were indicative of infection by phytoplasmas. Thus, the aim of this study was to detect and identify possible phytoplasmas associated with diseased plants. Total DNA was extracted from symptomatic and asymptomatic plants and used in nested PCR conducted with the primer pairs P1/Tint and R16F2n/16R2. Amplification of genomic fragments of 1.2 kb from the 16S rRNA gene confirmed the presence of phytoplasma in all symptomatic samples. The sequence identity scores between the 16S rRNA gene of the phytoplasma strain identified in the current study and those of previously reported ‘Candidatus Phytoplasma fraxini’-related strains ranged from 98 % to 99 % indicating the phytoplasma to be a strain affiliated with ‘Candidatus Phytoplasma fraxini’. The results from a phylogenetic analysis and virtual RFLP analysis of the 16S rRNA gene sequence with 17 restriction enzymes revealed that the phytoplasma strain belongs to the ash yellows phytoplasma group (16SrVII); the similarity coefficient of RFLP patterns further suggested that the phytoplasma represents a novel subgroup, designated 16SrVII-D. The representative of this new subgroup was named EboWB phytoplasma (Erigeron bonariensis Witches' Broom).

scholarly journals Plasmid profiles, restriction fragment length polymorphisms and O-serotypes amongVibrio anguillarumisolates

1996 ◽  
Vol 117 (3) ◽  
pp. 471-478 ◽  
Author(s):  
K. Pedersen ◽  
T. Tiainen ◽  
J. L. Larsen
Keyword(s):  
Restriction Fragment ◽  
Fragment Length ◽  
Restriction Analysis ◽  
Vibrio Anguillarum ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Plasmid Profile ◽  
Length Polymorphisms ◽  
Environmental Bacteria ◽  
Restriction Fragment Length

SummaryA total of 279Vibrio anguillarumstrains were serotyped and examined for plasmid content. Plasmids were subjected to digestion with restriction enzymes. Most strains belonged to serogroup O1 (39%) and O2 (16%). In total 164 strains (53%) carried plasmids. Of the O1 and O2 isolates, 92% and 30%, respectively, carried one or more plasmids. Restriction fragment length polymorphism (RFLP) analysis of plasmid DNA indicated that plasmids belonged to several groups. Each group seemed to be restricted to a single O-serovar. The largest group was the pJM1-like plasmids among most serovar O1 strains. Most of these plasmids were about 67 kb like the pJM1 plasmid, but various derivatives ranged from 26–77 kb. RFLP studies of the 67 kb plasmids revealed 17 different restriction patterns. Some patterns were dominant among European strains whereas others were dominant among North American strains. The results confirmed the applicability of O-serotyping together with plasmid profile and restriction analysis of plasmids for typing ofV. anguillarum. They also indicated that plasmids among strains which belonged to the traditional fish pathogenic serogroups, O1 and O2, showed more homology than did strains from most other serogroups, that were usually non-pathogenic, environmental bacteria.

scholarly journals ‘Candidatus Phytoplasma brasiliense’ (16SrXV-A Subgroup) Associated with Cauliflower Displaying Stunt Symptoms in Brazil

Plant Disease ◽  
2013 ◽  
Vol 97 (3) ◽  
pp. 419-419 ◽  
Author(s):  
M. C. Canale ◽  
I. P. Bedendo
Keyword(s):  
Nested Pcr ◽  
Sequence Similarity ◽  
Rflp Analysis ◽  
The United States ◽  
Rrna Gene ◽  
New Host ◽  
Rdna Sequences ◽  
Pcr Products ◽  
Virtual Rflp ◽  
In The Beginning

Cauliflower stunt, caused by a phytoplasma of the group 16SrIII-J, was reported in the beginning of 2012 and has occurred with high incidences of infected plants (up to 90%) in crops located in the state of São Paulo in the southeast region of Brazil (3). Diseased plants exhibit general stunting, malformation of inflorescence, reddening leaves, and vessel necrosis (3). Further investigations with plants displaying identical symptoms collected in Nova Bassano, state of Rio Grande do Sul, Brazilian south region, have revealed the presence of a phytoplasma distinct from 16SrIII-J subgroup. Four symptomatic plus four asymptomatic samples were assayed from a field, and the presence of phytoplasma was evidenced by nested PCR assays performed with primers P1/Tint followed by R16F2n/16R2 in three affected plants, which amplified genomic fragments of 1.2 kb from the 16S rRNA gene. No amplification occurred in non-affected samples. Nested PCR products analyzed by conventional RFLP (2) using the enzymes AluI, RsaI, KpnI, HpaII, MseI, HhaI, MboI, and BstUI pointed to the presence of a phytoplasma belonging to group 16SrXV-A in all three phytoplasma-positive samples. Virtual RFLP analysis based on restriction patterns, derived from in silico digestion with 17 endonucleases (4), confirmed the previous results obtained from those samples by conventional RFLP. The 16S rDNA sequences of this phytoplasma identified in cauliflower (GenBank Accession No. JN818845) shared 99% sequence similarity with the reference phytoplasma for subgroup 16SrXV-A (Hibiscus witches'-broom phytoplasma, AF147708), designated ‘Candidatus Phytoplasma brasiliense.’ Analysis of putative restriction sites showed excellent identity between the phytoplasma studied here and the reference phytoplasma. In addition, the arrangement of branches of a phylogenetic tree constructed with phytoplasmas representing diverse 16Sr groups and subgroups supported that the phytoplasma found in cauliflower is closed related to the representative of the subgroup 16SrXV-A. Association of distinct phytoplasmas with the same kind of disease is not rare and the present pathosystem constitutes a new example. Members of this subgroup have been described almost exclusively in Brazil and previously reported in Sida sp., periwinkle, and hibiscus (1). In some European countries, as well as in the United States and Canada, phytoplasmas belonging to group 16SrI has been associated with this type of disease, which has been reported for various species of the genus Brassica, as published in previous works (3). However, a representative of the group 16SrVI was described in infected plants in Iran (3). Although the 16SrIII-J phytoplasma is currently the most important agent of cauliflower stunt in Brazil, and members of 16SrI are prevalent in other countries, this study revealed that a 16Sr XV-A phytoplasma may be also associated with this important disease of brassicas. Besides, the findings here reported expand the natural host range, including cauliflower as new host for phytoplasmas affiliated with 16SrXV-A. References: (1) B. Eckstein et al. Plant Dis. 95:363, 2009. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) M. C. C. Rappussi et al. Eur. J. Plant. Pathol. 133:829, 2012. (4) Wei et al. Int. J. Syst. Evol. Microbiol. 57:1855, 2007.

scholarly journals Isolation and characterization of Listeria monocytogenes from environmental and clinical sources by culture and PCR-RFLP methods

2019 ◽  
Author(s):  
Hossein Meghdadi ◽  
Azar Dokht Khosravi ◽  
Ahmad Farajzadeh Sheikh ◽  
Ameneh Alami ◽  
Nerssy Nassirabady
Keyword(s):  
Listeria Monocytogenes ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Clinical Samples ◽  
Biochemical Tests ◽  
Clinical Complications ◽  
Isolation And Characterization ◽  
Rflp Profile ◽  
Pcr Products

Background and Objectives: Due to the widespread distribution of Listeria monocytogenes in environmental and animal sources and serious clinical complications in human, this study was aimed to isolate L. monocytogenes from water and clin- ical specimens by culture and PCR methods and to investigate the presence of hlyA and inlA virulence genes. Materials and Methods: Water and clinical samples of vaginal and fecal were screened for the presence of L. monocyto- genes by phenotypic and standard biochemical tests. PCR amplification was performed on extracted DNA using primers based on the hlyA and inlA genes. A 733-bp fragment of inlA gene was used for investigation of polymorphism using RFLP analysis. Results: In total, 45 phenotypically and molecularly confirmed L. monocytogenes strains were isolated from different sourc- es including 30 (16.7%) from water, 9 (11.3%) from vaginal swabs and 6 (7.5%) from fecal samples. RFLP analysis of PCR products using AluI and Tsp509I restriction enzymes, generated two profiles with 8 to 10 bands ranging in size from 15 to 210 bp. The majority of water and clinical isolates were classified in profile 2. Conclusion: We demonstrated 45 L. monocytogenes isolates from tested water and clinical samples by phenotypic and mo- lecular tests. The majority of the isolates were classified in the same RFLP profile, showing the water as a potential source of clinical complications in patients in the region of study.

scholarly journals First Report of an Aster Yellows Subgroup 16SrI-B Phytoplasma Infecting Chayote in Costa Rica

Plant Disease ◽  
2002 ◽  
Vol 86 (3) ◽  
pp. 330-330 ◽  
Author(s):  
W. Villalobos ◽  
L. Moreira ◽  
C. Rivera ◽  
K. D. Bottner ◽  
I.-M. Lee
Keyword(s):  
Costa Rica ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Potential Threat ◽  
Nested Polymerase Chain Reaction ◽  
Aster Yellows ◽  
First Report ◽  
Rdna Sequences ◽  
Pcr Products ◽  
Production Area

An outbreak of a witches' broom disease affected approximately 20% of plants in several chayote (Sechium edule (Jacq.) Schwartz) fields in the commercial production area of the Ujarrás Valley, Cartago Province, Costa Rica during 2000 and 2001. Affected chayote plants exhibited symptoms, including basal proliferation with severe foliage reduction, aborted flowers, and deformed fruits, suggestive of phytoplasmal infection. Two other symptomatic cucurbit species growing near the chayote fields were also identified. These species were tacaco plants (S. tacaco (Pitt.) C. Jeffrey), an edible cucurbit for domestic marketing in Costa Rica, showing severe size reduction of leaves and fruits, and Rytidostylis carthaginensis (Jacq.) Kuntze, a weed in chayote and tacaco fields, exhibiting abnormal tendril proliferation. Plants were analyzed for phytoplasma infection by a nested polymerase chain reaction (PCR) assay, using the universal rRNA primer pair P1/P7 followed by R16F2n/R16R2 (2). Phytoplasmas were detected in all symptomatic samples (18 chayote, 6 tacaco, and 3 weed) tested but were undetectable in all asymptomatic samples (10 chayote, 6 tacaco, and 2 weed). Restriction fragment length polymorphism (RFLP) analysis of PCR products (16S rDNA sequences) by separate digestion with eight restriction enzymes (RsaI, HhaI, KpnI, BfaI, HaeIII, HpaII, AluI, MseI) revealed that a phytoplasma belonging to subgroup 16SrI-B in the aster yellows phytoplasma group (16SrI) was associated with chayote witches' broom (CWB). The same or very similar phytoplasmas were found in both symptomatic tacaco and R. carthaginensis plants. Phylogenetic analysis of 16SrDNA sequences also confirmed the CWB phytoplasma to be most similar to members of subgroup 16SrI-B. Similar diseases in chayote and other cucurbits have been reported in Brazil (3), Taiwan (1), and Mexico (4). The CWB phytoplasma differs from the phytoplasma (16SrIII-J subgroup) associated with chayote in Brazil. The identities of phytoplasmas associated with cucurbits in Taiwan and Mexico are unknown. The occurrence of an aster yellows group phytoplasma in chayote may pose a potential threat to continued production and exportation of this cash crop. To our knowledge, this is the first report of 16SrI-B subgroup phytoplasmas in naturally infected cucurbits in Costa Rica. References: (1) T. G. Chou et al. Plant Dis. Rep. 60:378, 1976. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) H. G. Montano et al. Plant Dis. 84:429, 2000. (4) E. Olivas. Rev. Fitopatol. (Lima) 13:14, 1978.

The gp63 gene locus, a target for genetic characterization of Leishmania belonging to subgenus Viannia

Parasitology ◽  
1998 ◽  
Vol 117 (1) ◽  
pp. 1-13 ◽  
Author(s):  
K. VICTOIR ◽  
A. L. BAÑULS ◽  
J. AREVALO ◽  
A. LLANOS-CUENTAS ◽  
R. HAMERS ◽  
...  
Keyword(s):  
Gene Locus ◽  
Genetic Characterization ◽  
Restriction Analysis ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Rflp ◽  
Natural Isolates ◽  
Phenotype Diversity

In the present study the gp63 gene locus was used as a target for genetic characterization of Leishmania parasites by 2 methods: (i) RFLP analysis with several restriction enzymes (gp63–RFLP), and (ii) intra-genic PCR amplification coupled with restriction analysis (PCR–RFLP). Both methods were applied to a large number of natural isolates belonging to 4 species of the subgenus Viannia, namely L. (V.) braziliensis, L. (V.) peruviana, L. (V.) guyanensis and L. (V.) lainsoni: reference stocks of subgenus Leishmania were included as outgroups. Multilocus isoenzyme typing (MLEE) was used as a reference. On the one hand gp63–RFLP evidenced an extensive polymorphism and revealed specific markers for subgenus, species and geographical populations: congruence with MLEE was demonstrated statistically. The particular interest of gp63–RFLP was illustrated by infra-specific polymorphism, because of the possible relationship with phenotype diversity. On the other hand intra-genic amplification was less resolutive than gp63–RFLP, but also allowed discrimination of the 2 subgenera (PCR alone) and all the species tested in the subgenus Viannia (PCR–RFLP). PCR–RFLP presents an important operational advantage as it allows genetic characterization of minute amounts of parasites, using Leishmania specific primers. The polymorphism revealed by gp63–RFLP and PCR–RFLP illustrates the very high genomic and genetic plasticity of gp63 genes.

ITS-RFLP analysis, an efficient tool for differentiation of Bursaphelenchus species

Nematology ◽  
2009 ◽  
Vol 11 (5) ◽  
pp. 649-668 ◽  
Author(s):  
Wolfgang Burgermeister ◽  
Helen Braasch ◽  
Kai Metge ◽  
Jianfeng Gu ◽  
Thomas Schröder ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Restriction Analysis ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Its Sequence ◽  
Additional Species ◽  
New Information ◽  
Specific Fragment ◽  
Species Specific ◽  
Different Origins

Abstract Restriction analysis of amplified ribosomal ITS sequences has provided species-specific fragment patterns for nematodes of several genera, including Bursaphelenchus. We used restriction enzymes RsaI, HaeIII, MspI, HinfI and AluI to produce ITS-RFLP reference profiles of 44 Bursaphelenchus species, including two intraspecific types in each of B. mucronatus and B. leoni. In addition, reference profiles of Aphelenchoides stammeri and Ruehmaphelenchus asiaticus were produced. Reference profiles of six species are shown here for the first time. Identical ITS-RFLP patterns were usually obtained from different isolates and from individual specimens of the same species. However, in the case of B. 'corneolus', B. lini, B. singaporensis and B. sexdentati, additional bands in the patterns of certain isolates or individual nematodes were observed which may be explained by ITS sequence microheterogeneity, i.e., the presence of ITS sequence variants within the number of rDNA tandem repeats. Since these 'extra' bands appeared only with one out of the five restriction enzymes employed, they did not seriously impair identification of species based on the overall reference patterns. ITS-RFLP analysis has proved valuable for differentiation of the pathogenic pine wood nematode, B. xylophilus, from related species. In many recent descriptions of new Bursaphelenchus species, ITS-RFLP profiles have been used as additional species identification criteria. Comparison of profiles from isolates of many different origins has provided new information on intraspecific types or genetically distinct provenances of several Bursaphelenchus species.

scholarly journals Detection of XIIA phytoplasma group on cultivar Zupljanka in Zupa vineyard region by RFLP analysis of 16s rDNA sequences

Genetika ◽  
2010 ◽  
Vol 42 (1) ◽  
pp. 145-153
Author(s):  
Dragana Josic ◽  
Slobodan Kuzmanovic ◽  
Sasa Stojanovic ◽  
Goran Aleksic ◽  
Snezana Pavlovic ◽  
...  
Keyword(s):  
16S Rdna ◽  
Direct Sequencing ◽  
Intergenic Spacer ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Intergenic Spacer Region ◽  
Region Detection ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
Stolbur Phytoplasma

'Bois noir' (BN) is an important grapevine disease associated with phytoplasmas belonging to ribosomal subgroup 16SrXII-A. Phytoplasmas cause diseases in several hundred plant species. The number of infected cultivars is growing each year and it is important to follow the spreading of the phytoplasma in the different regions and identify which strains are present in specific regions on specific cultivars. Phytoplasmas are identified and classified based on direct sequencing of phytoplasma 16S rDNA or the 16S to 23S intergenic spacer region, but this approach is not always practical when a large number of unknown phytoplasmas is to be analyzed. Classification by RFLP analysis has provided a simple and rapid method that can be used to differentiate and identify a large number of unclarified phytoplasmas. Our objective was to investigate presence of phytoplasmas of 16SrXII-A group (Stolbur) in Zupa vineyard region. Detection was based on RFLP analysis of 16s rDNA sequences using four restriction enzymes: Tru1I, AluI, KpnI and TaqI. We identified phytoplasmas of XIIA group on two of three investigated cultivars (Zupljanka and Frankovka, but not on Plovdina) in the Zupa vineyard regions (Gornje Rataje and Tules locality). This is the first report of Stolbur phytoplasma on cv. Zupljanka in Zupa region.

Molecular identification of fungi isolated from stem tissue of Upland cotton (Gossypium hirsutum)

2005 ◽  
Vol 53 (6) ◽  
pp. 571 ◽  
Author(s):  
L. Augusto Becerra-LopezLavalle ◽  
Jennifer A. Saleeba ◽  
Bruce R. Lyon
Keyword(s):  
Gossypium Hirsutum ◽  
Restriction Analysis ◽  
Rflp Analysis ◽  
Molecular Techniques ◽  
Internal Transcribed Spacers ◽  
Pcr Analysis ◽  
Rrna Gene ◽  
5.8S Rrna Gene ◽  
Rdna Sequences ◽  
Rdna Sequencing

Molecular techniques such as restriction fragment length polymorphism (RFLP) analysis, random amplification of polymorphic DNA (RAPD) fingerprinting, and DNA sequencing and database comparison, were employed to identify fungi isolated from field-grown cotton plants (Gossypium hirsutum L.). DNA fragments of between 510 and 590 bp, representing the two rDNA (rDNA) internal transcribed spacers (ITS1 and ITS2) and the intervening 5.8S rRNA gene, were amplified from the fungi with eukaryotic consensus primers. Subsequent digestion with the restriction endonucleases AluI, CfoI, HaeIII, HinfI and HpaII enabled the allotment of all 57 isolates to 13 different groups. Restriction analysis was supported by RAPD–PCR analysis of multiple isolates and rDNA sequencing of representative fungi from each group. Sequence alignment and comparison with rDNA sequences of other fungi available in GenBank allowed for putative identification of three different taxa of Fusarium, two taxa each of Cladosporium, Diaporthe and Nectria, and one taxon each of Alternaria, Ampelomyces, Bartalinia, Phaeosphaeria and Rhizoctonia. Many of the stem-colonising fungi identified in this study are either pathogenic on cotton or have elsewhere been found to act as biocontrol agents.

Evolution of the chloroplast genome and polymorphic ITS regions in Allium subg. Melanocrommyum

Genome ◽  
1999 ◽  
Vol 42 (2) ◽  
pp. 237-247 ◽  
Author(s):  
Ted HM Mes ◽  
Reinhard M Fritsch ◽  
Sven Pollner ◽  
Konrad Bachmann
Keyword(s):  
Concerted Evolution ◽  
Morphological Evolution ◽  
Restriction Analysis ◽  
Nuclear Genome ◽  
Restriction Enzymes ◽  
Border Region ◽  
Chloroplast Genomes ◽  
Central Asian ◽  
Its Regions ◽  
Chloroplast Haplotypes

Relationships based on PCR-RFLPs of non-coding regions of cpDNA indicate that some of the largest subgenera of the genus Allium and five of the largest sections of the Central Asian subg. Melanocrommyum are artificial. Internested synapomorphic mutations without homoplasy were found only in the chloroplast genomes of plants of subg. Melanocrommyum that occur in the border region of Tajikistan, Uzbekistan, Afghanistan, and Kyrgyzstan. Eighteen of 49 plants surveyed were polymorphic for their ITS regions. Even plants that had identical chloroplast genomes were polymorphic for nuclear ribosomal regions. These individuals had markedly different frequencies of ITS variants that were detected with various restriction enzymes. The geographic partitioning of chloroplast haplotypes and the fact that the ITS variants could not be ordered hierarchically can readily be envisioned to result from gene flow. Processes such as concerted evolution and parallel morphological evolution may also be partly responsible for the disconcordance of mutations in the chloroplast and nuclear genome. However, the chimeric nature of the nuclear ribosomal regions indicates that concerted evolution is not the dominating process in Allium subg. Melanocrommyum.Key words: polymorphic, phylogeny, restriction analysis.

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