PCR Amplification of Ribosomal DNA for Species Identification in the Plant Pathogen Genus Phytophthora

1998 ◽  
Vol 64 (3) ◽  
pp. 948-954 ◽  
Author(s):  
Jean B. Ristaino ◽  
Michael Madritch ◽  
Carol L. Trout ◽  
Gregory Parra
Keyword(s):  
Ribosomal Dna ◽  
Plant Pathogen ◽  
Pcr Amplification ◽  
Its Region ◽  
Restriction Enzymes ◽  
Internal Transcribed Spacers ◽  
Important Species ◽  
Major Species ◽  
Field Samples ◽  
Taxonomic Groups

ABSTRACT We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genusPhytophthora. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymesRsaI, MspI, and HaeIII. Restriction fragment patterns were similar after digestions with RsaI for the following species: P. capsici andP. citricola; P. infestans,P. cactorum, and P. mirabilis;P. fragariae, P. cinnamomi, andP. megasperma from peach; P. palmivora, P. citrophthora, P. erythroseptica, and P. cryptogea; andP. megasperma from raspberry and P. sojae. Restriction digests with MspI separatedP. capsici from P. citricola and separated P. cactorum from P. infestans and P. mirabilis. Restriction digests with HaeIII separated P. citrophthorafrom P. cryptogea, P. cinnamomi fromP. fragariae and P. megasperma on peach, P. palmivora from P. citrophthora, and P. megasperma on raspberry from P. sojae. P. infestans and P. mirabilis digests were identical and P. cryptogea and P. erythroseptica digests were identical with all restriction enzymes tested. A unique DNA sequence from the ITS region I in P. capsici was used to develop a primer called PCAP. The PCAP primer was used in PCRs with ITS 1 and amplified only isolates of P. capsici,P. citricola, and P. citrophthoraand not 13 other species in the genus. Restriction digests withMspI separated P. capsici from the other two species. PCR was superior to traditional isolation methods for detection of P. capsici in infected bell pepper tissue in field samples. The techniques described will provide a powerful tool for identification of the major species in the genusPhytophthora.

scholarly journals Molecular authentication of Euphorbia schimperiana Scheele using internal transcribed spacer sequences of nuclear ribosomal DNA

2021 ◽  
Vol 28 (1) ◽  
pp. 125-130
Author(s):  
Mesfer M Alqahtani ◽  
M Ajmal Ali ◽  
M Oliur Rahman ◽  
Fahad M Al Hemaid ◽  
Sidanand V Kambhar ◽  
...  
Keyword(s):  
Ribosomal Dna ◽  
Molecular Phylogenetics ◽  
Taxonomic Status ◽  
Its Region ◽  
Internal Transcribed Spacers ◽  
Nuclear Ribosomal Dna ◽  
Universal Primers ◽  
Important Species ◽  
Discrimination Ability ◽  
Plant Taxon

The Internal Transcribed Spacers (ITS) sequences of nuclear ribosomal DNA (nrDNA) are commonly used in plant molecular phylogenetics for the molecular based taxonomic identification and DNA barcoding because of shorter length and easy to amplify by using the universal primers, and further has discrimination ability to distinguish the taxon at lower taxonomic level. The present molecular phylogenetic analysis of ITS nrDNA sequences focuses to determine the taxonomic status of an unresolved medicinally important species Euphorbia schimperiana Scheele of the family Euphorbiaceae reported from Saudi Arabia. The combined length of the entire ITS region in E. schimperiana is 644 nucleotides. The study reveals that E. schimperiana shows a close proximity with the members of the subgenus Esula. Bangladesh J. Plant Taxon. 28(1): 125-130, 2021 (June)

Identification of cyst forming nematodes of the genus Heterodera (Nematoda: Heteroderidae) based on the ribosomal DNA-RFLP

Nematology ◽  
2000 ◽  
Vol 2 (2) ◽  
pp. 153-164 ◽  
Author(s):  
Sergei Subbotin ◽  
Lieven Waeyenberge ◽  
Maurice Moens
Keyword(s):  
Ribosomal Dna ◽  
Sibling Species ◽  
Its Region ◽  
Restriction Enzymes ◽  
Nematode Species ◽  
Valid Species ◽  
Important Species ◽  
Its Regions ◽  
Unidentified Species ◽  
Species Specific

Abstract Amplified ITS region products of rDNA from 25 valid species and one unidentified species from the genus Heterodera and from Meloidodera alni were digested by 26 restriction enzymes. A combination of seven enzymes clearly separated the agriculturally most important species from each other and from their sibling species. Species specific digestion profiles of ITS regions and a table with approximate sizes of digested fragments for several identification enzymes are given. Heterogeneity of ITS regions was revealed for some cyst forming nematode species. Des fragments amplifiés de la région de l’ITS du rDNA de 25 espèces valides et d’une espèce non identifiée du genre Heterodera et de Meloidodera alni ont été soumis à une digestion par 26 enzymes de restriction. La combinaison de sept enzymes a permis une séparation nette des espèces les plus importantes en agriculture, tant les unes par rapport aux autres que par rapport aux espèces jumelles. Sont donnés les profils spécifiques de digestion des régions de l’ITS et un tableau regroupant les tailles approximatives des fragments digérés pour plusieurs enzymes d’identification. L’hétérogénéité des régions de l’ITS a été révélée chez quelques espèces de nématodes à kyste.

scholarly journals Specific Polymerase Chain Reaction Identification of Venturia nashicola Using Internally Transcribed Spacer Region in the Ribosomal DNA

2001 ◽  
Vol 91 (9) ◽  
pp. 900-904 ◽  
Author(s):  
Bruno Le Cam ◽  
Martine Devaux ◽  
Luciana Parisi
Keyword(s):  
Polymerase Chain Reaction ◽  
Ribosomal Dna ◽  
Related Species ◽  
Its Region ◽  
Restriction Enzymes ◽  
Oligonucleotide Primer ◽  
Sequence Information ◽  
Chain Reaction ◽  
Venturia Nashicola ◽  
Polymerase Chain

A technique based on the polymerase chain reaction (PCR) was developed for the identification of Venturia nashicola using nucleotide sequence information of the ribosomal DNA region. The complete internal transcribed spacer (ITS) region of V. nashicola strains and phylo-genetically related species was amplified with the two universal ITS1 and ITS4 primers, sequenced, and digested with five restriction enzymes. The alignment of nucleotide sequences and analyses of digestion patterns indicated constant polymorphisms between V. nashicola and related species at nucleotides 126 and 127, which overlapped a TaqI restriction site. An oligonucleotide primer named A126 was designed for identifying this variable region. A primer set (A126 and ITS4) that allowed the amplification of a 391-bp DNA fragment within the ITS region by PCR was specific to V. nashicola when it was checked against fungal genomic DNAs of related fungi. This primer set was a good candidate for a species-specific reagent in a procedure for identification of V. nashicola by PCR.

scholarly journals Conidia as a Substrate for Internal Transcribed Spacer-Based PCR Identification of Members of the Leptosphaeria maculans Species Complex

1998 ◽  
Vol 88 (11) ◽  
pp. 1210-1217 ◽  
Author(s):  
M. H. Balesdent ◽  
M. Jedryczka ◽  
L. Jain ◽  
E. Mendes-Pereira ◽  
J. Bertrandy ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Species Complex ◽  
Large Scale ◽  
Restriction Site ◽  
Leptosphaeria Maculans ◽  
Pcr Amplification ◽  
Its Region ◽  
Restriction Enzymes ◽  
Restriction Site Polymorphism ◽  
Routine Identification

The blackleg disease of oilseed rape is caused by an ascomycete species complex termed Leptosphaeria maculans (anamorph Phoma lingam). L. maculans isolates collected worldwide were gathered in the International Blackleg of Crucifers Network (IBCN) collection. Representative IBCN isolates, along with one P. nigrificans isolate, were further analyzed using polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) region. ITS size polymorphism discriminated three groups: (i) P. nigrificans, (ii) Tox+ and ‘Lepidium’ isolates, and (iii) NA1, NA2, NA3, ‘Thlaspi’, and ‘Erysimum’ isolates. Digestion of the ITS region with 19 selected endonucleases showed restriction site polymorphism between the different subgroups: digestion with RsaI could discriminate Tox+ from ‘Lepidium’ isolates, whereas digestion with four enzymes, i.e., HaeIII, EcoRII, RsaI, and AluI, was needed to discriminate between NA1, NA2, NA3, ‘Thlaspi’, and ‘Erysimum’ isolates. No restriction site polymorphism was observed between isolates within the ‘Thlaspi’, Tox+, NA1, and NA2 subgroups. Direct amplification of the ITS region could be achieved using intact conidia, collected either in axenic cultures or on leaf lesions, with only a 4-min 95°C denaturation step prior to PCR reaction. A routine identification protocol requiring no DNA extraction and a sequential use of a few restriction enzymes following PCR has been used successfully for large-scale identification of French field isolates.

Phylogeny of Sisymbrium (Brassicaceae) based on ITS sequences of nuclear ribosomal DNA

2002 ◽  
Vol 80 (9) ◽  
pp. 1002-1017 ◽  
Author(s):  
Suzanne I Warwick ◽  
Ihsan A Al-Shehbaz ◽  
Robert A Price ◽  
Connie Sauder
Keyword(s):  
Ribosomal Dna ◽  
New World ◽  
Sequence Data ◽  
Its Region ◽  
Internal Transcribed Spacers ◽  
Nuclear Ribosomal Dna ◽  
Maximum Parsimony Analysis ◽  
Rrna Gene ◽  
Old World ◽  
5.8S Rrna Gene

The genus Sisymbrium as currently circumscribed includes about 94 species disjunctly distributed in the Old (41 spp.) and the New World (53 spp.). Sisymbrium has been variously delimited, with several segregate genera proposed (subtribe Sisymbriinae) primarily for the new World taxa, including Schoenocrambe, Coelophragmus, and Mostacillastrum. Using sequence data from the internal transcribed spacers of nuclear ribosomal DNA and the 5.8S rRNA gene (collectively, ITS region), we examined the evolutionary relationships of Old and New World Sisymbrium species with its segregate genera and the validity of O.E. Schulz's classical sectional treatment of Sisymbrium. Sequence data were obtained from 33 Sisymbrium species, representing all 14 sections and two Sisymbrium species formerly assigned to segregate genera Coelophragmus and Mostacillastrum (subtribe Sisymbriinae), and two putative Sisymbrium species currently assigned to Neotorularia. Sequence data were also obtained from 26 taxa from segregate or related genera includingSchoenocrambe, Werdermannia (subtribe Sisymbriinae), eight genera in the Thelypodieae, Sibara (tribe Arabideae) and Pringlea (tribe Pringleeae), four members of the tribe Brassiceae, and three other Neotorularia species. Results from maximum parsimony analysis showed a polyphyletic origin for Sisymbrium and did not correspond well to Schulz's sectional classification. Sisymbrium species were split into three major clades: Old World Sisymbrium (including Neotorularia aculeolata, Neotorularia afghanica, and the type species of Schoenocrambe, Schoenocrambe linifolia, the sole New World member of this Old World clade); New World Sisymbrium (along with the remaining New World taxa) and designated as the New World Thelypodieae alliance; and the tribe Brassiceae ( including Sisymbrium supinum and Sisymbrium thellungii).Key words: Sisymbrium, Schoenocrambe, ITS, Thelypodieae, taxonomy, Brassicaceae.

Phylogenetic relationships in Dermocybe and related Cortinarius taxa based on nuclear ribosomal DNA internal transcribed spacers

1997 ◽  
Vol 75 (4) ◽  
pp. 519-532 ◽  
Author(s):  
Y. J. Liu ◽  
S. O. Rogers ◽  
Y. J. Liu ◽  
J. F. Ammirati
Keyword(s):  
Ribosomal Dna ◽  
Phylogenetic Relationships ◽  
Phylogenetic Trees ◽  
Sequence Data ◽  
Molecular Data ◽  
Internal Transcribed Spacers ◽  
Nuclear Ribosomal Dna ◽  
Herbarium Specimens ◽  
Data Reorganization ◽  
Taxonomic Groups

The genus Cortinarius Fr. (Cortinariaceae, Agaricales) is divided into four or more subgenera. Dermocybe (Fr.) Sacc. has been recognized as either a subgenus of Cortinarius or a separate genus, distinguished in part by the presence of various anthraquinonic pigments. Nucleotide sequences of ribosomal DNA 5.8S and internal transcribed spacers were used to investigate the phylogenetic relationships among species of Dermocybe and selected taxa from subgenera of Cortinarius. Sequence data from 47 herbarium specimens representing 31 taxa (28 species plus 3 varieties) of Dermocybe and Cortinarius were analyzed using parsimony, maximum likelihood, and neighbor joining. In general, molecular data support the morphological groupings of the taxa, although they more closely correspond to biochemical (anthraquinone and other) analyses. Phylogenetic trees showed that, while the sections Dermocybe and Malicoriae are monophyletic, and the concolorous or almost concolorous red species (section Sanguineae, such as D. sanguinea and relatives) together formed a coherent clade, the subgenus Dermocybe sensu lato itself is polyphyletic. Cortinarius californicus clusters with taxa in Cortinarius, subgenus Telamonia, section Armillati. Dermocybe olivaceopicta is more closely related to other subgenera of Cortinarius than to Dermocybe. Within the genus Cortinarius, certain of the subgenera may actually represent coherent genera. Of the subgenera examined, Telamonia, Phlegmacium, and possibly Sericeocybe appear to represent well defined taxonomic groupings. However, current assignments of taxa within Leprocybe and Myxacium were inconsistent with the molecular data. Reorganization of some taxa and taxonomic groups is suggested. Key words: Dermocybe, Cortinarius, molecular phylogeny, rDNA, ITS1, ITS2.

scholarly journals Genetic diversity among isolates of stemphylium solani from cotton

2001 ◽  
Vol 26 (4) ◽  
pp. 703-709 ◽  
Author(s):  
Y.R. MEHTA
Keyword(s):  
Genetic Diversity ◽  
Genetic Difference ◽  
Random Amplified Polymorphic Dna ◽  
Its Region ◽  
Restriction Enzymes ◽  
Geographic Region ◽  
Leaf Blight ◽  
Internal Transcribed Spacers ◽  
Pcr Product ◽  
Specific Variation

The fungus Stemphylium solani causes leaf blight of tomato (Lycopersicon esculentum) in Brazil. In recent years, severe epidemics of a new leaf blight of cotton (Gossipium hyrsutum) caused by S. solani occurred in three major cotton-growing Brazilian states (PR, MT and GO). Molecular analysis was performed to assess the genetic diversity among the S. solani isolates from cotton, and to verify their relationship with representative S. solani isolates from tomato. Random amplified polymorphic DNA (RAPD) markers and internal transcribed spacers of ribosomal DNA (rDNA) were used to compare 33 monosporic isolates of S. solani (28 from cotton and five from tomato). An isolate of Alternaria macrospora from cotton was also used for comparison. RAPD analysis showed the presence of polymorphism between the genera and the species. The A. macrospora and the S. solani isolates from cotton and tomato were distinct from each other, and fell into separate groups. Variation by geographic region was observed for the tomato isolates but not for the cotton isolates. Amplifications of the ITS region using the primer pair ITS4/ITS5 resulted in a single PCR product of approximately 600 bp for all the isolates. Similarly, when amplified fragments were digested with eight restriction enzymes, identical banding patterns were observed for all the isolates. Hence, rDNA analysis revealed no inter-generic or intra-specific variation. The genetic difference observed between the cotton and the tomato isolates provides evidence that S. solani attacking cotton in Brazil belongs to a distinct genotype.

scholarly journals PCR-Based Strategy To Detect and Identify Species of Phaeoacremonium Causing Grapevine Diseases

2007 ◽  
Vol 73 (9) ◽  
pp. 2911-2918 ◽  
Author(s):  
Angeles Aroca ◽  
Rosa Raposo
Keyword(s):  
Morphological Characteristics ◽  
Pcr Amplification ◽  
Its Region ◽  
Restriction Enzymes ◽  
Effective Measure ◽  
Petri Disease ◽  
Specific Pcr ◽  
Plant Nursery ◽  
Species Specific ◽  
Single Amplicon

ABSTRACT Species of Phaeoacremonium (especially Phaeoacremonium aleophilum) are associated with two severe diseases in grapevines, Petri disease in young plants and Esca disease in adult plants. Phaeoacremonium species grow slowly on culture medium, and it is difficult to identify these species on the basis of morphological characteristics. Primers Pm1 and Pm2 were designed in the ribosomal DNA internal transcribed spacer (ITS) regions ITS1 and ITS2, respectively. They yielded a single amplicon of 415 bp for nine species of Phaeoacremonium that may occur in grapevines. A nested PCR (using general fungal primers ITS1F/ITS4 in the primary reaction) was developed to detect Phaeoacremonium directly in grapevine wood. Molecular detection was more sensitive than the traditional method of culturing in growth medium was. Identification of Phaeoacremonium species was achieved by digesting the PCR-amplified fragment with the restriction enzymes BssKI, EcoO109I, and HhaI. It was possible to distinguish these species by their restriction fragment length polymorphism patterns, except for Phaeoacremonium viticola and Phaeoacremonium angustius, which had 100% similarity in their ITS region sequences. A species-specific PCR amplification of the partial β-tubulin gene using the primer pair Pbr4_1/T1 and Pbr8/T1 was necessary to differentiate P. angustius from P. viticola, respectively. An easy and fast protocol was developed to detect and identify species of Phaeoacremonium in a few hours. Primers defined here can be used in a plant nursery sanitation program to produce plants free of Phaeoacremonium spp. Use of healthy grapevine plants in new plantations is the most effective measure to manage Petri disease.

Sign in / Sign up

Export Citation Format

Share Document