scholarly journals Diagnosis of potato rot nematode Ditylenchus destructor using PCR-RFLP

2020 ◽  
Vol 15 (4) ◽  
pp. 353-362
Author(s):  
Niloufar Mahmoudi ◽  
Elena N. Pakina ◽  
Liudmila A. Limantceva ◽  
Anton V. Ivanov
Keyword(s):  
Dna Sequences ◽  
Molecular Taxonomy ◽  
Parasitic Nematodes ◽  
Its2 Region ◽  
Rrna Gene ◽  
Rdna Sequences ◽  
Pcr Product ◽  
Pcr Rflp ◽  
Its Rrna ◽  
Ditylenchus Destructor

During an investigation of nematodes in the Moscow region of Russia in 2019, a known species Ditylenchus destructor was recovered from tubers of potato plants. The genus Destructor is one of the most problematic genera of plant-parasitic nematodes. The numerous species reported for this genus have been cited from various sources. Due to the morphological similarity of many species and the lack of separation characteristics, the identification of D. destructor is difficult. Molecular taxonomy and phylogeny were used to confirm the identification. In the current study, PCR-RFLP illustrative models for the amplification of the ITS-rRNA gene were provided with two enzymes that could recognize D. destructor in potato tubers. Analysis of the rDNA sequences spanning both ITS1-ITS2 regions was carried out on the collected populations. The digestion of the PCR product of the ITS1-5.8S-ITS2 region with the enzyme TaqI produced three fragments; 100, 190, 550, and with Tru1I, two fragments were produced; 300 and 480 bp. The obtained DNA sequences were compared with those DNA sequences deposited in GenBank of populations isolated in other countries. The results showed no distinction between populations isolated from different host plant species, including populations found in the Russian Federation. New sequences from ITS-rRNA were deposited in the GenBank under accession number MN122076, MN658597, MN658599, MN658637, MN658638.

scholarly journals Molecular differentiation of four Reptalus species (Hemiptera: Cixiidae)

2010 ◽  
Vol 100 (5) ◽  
pp. 551-558 ◽  
Author(s):  
S. Bertin ◽  
L. Picciau ◽  
Z. Ács ◽  
A. Alma ◽  
D. Bosco
Keyword(s):  
Control Strategies ◽  
Species Discrimination ◽  
Morphological Description ◽  
Its2 Region ◽  
Pcr Product ◽  
Pcr Rflp ◽  
Gene Coi ◽  
Phytoplasma Infection ◽  
Species Specific ◽  
Dna Pcr

AbstractThe cixiid species Reptalus quinquecostatus, R. cuspidatus, R. panzeri and R. melanochaetus are widely distributed in Europe and are receiving growing attention because of their potential role as phytoplasma vectors. Identifying the Reptalus species is restricted to a few specialist entomologists and relies on the morphology of the male genitalia, hampering the identification of juveniles and females. This study provides the tools for species discrimination by integrating the morphological description, which is primarily for the genus identification, with new molecular assays, based on both ribosomal and mitochondrial DNA. PCR-RFLP assays carried out on the mitochondrial cytochrome oxidase I gene (COI) with AluI provided species-specific profiles for the four Reptalus species. Amplification of a ribosomal internal transcribed spacer (ITS2) region produced species-specific fragments of different sizes for R. quinquecostatus, R. melanochaetus, R. cuspidatus and R. panzeri. The digestion of the ITS2 PCR product with TaqI allowed the discrimination of these latter two species. This molecular identification key ensures reliable results and can be successfully applied not only to adults, but also to the nymphs feeding on the roots. The identification of the nymphs (i) extends the collection period of these monovoltine species to the whole year (adults are present for a short summer period) and (ii) allows the unambiguous identification of their actual host plants because nymphs are steady on the root system while adults tend to disperse onto other plants. Fast and reliable identification of the Reptalus species provides useful help in monitoring activities and, therefore, in designing rational control strategies to protect crops from phytoplasma infection.

Synonymy of Afenestrata with Heterodera supported by phylogenetics with molecular and morphological characterisation of H. koreana comb. n. and H. orientalis comb. n. (Tylenchida: Heteroderidae)

Nematology ◽  
2008 ◽  
Vol 10 (5) ◽  
pp. 611-632 ◽  
Author(s):  
Manuel Mundo-Ocampo ◽  
Alberto Troccoli ◽  
Sergei A. Subbotin ◽  
Julio Del Cid ◽  
James G. Baldwin ◽  
...  
Keyword(s):  
Morphological Identification ◽  
Rrna Gene ◽  
Similar Species ◽  
28S Rrna ◽  
Diagnostic Pcr ◽  
Second Stage ◽  
Pcr Rflp ◽  
Identification Of Species ◽  
Its Rrna ◽  
Labial Disc

Abstract Phylogenetic analysis of five gene fragments: ITS-rRNA, D2 and D3 of 28S rRNA, 18S rRNA, Hsp90 and actin, of Heterodera species and two representative Afenestrata species, A. koreana and A. orientalis, form a clade with H. cynodontis, H. bifenestra and an unidentified Heterodera sp. infecting grasses. Based on these results and the consideration that the key diagnostic characters of Afenestrata are convergent and do not define a clade, synonymisation of Afenestrata with Heterodera is proposed. The following new combinations are made: H. africana comb. n., H. axonopi comb. n., H. koreana comb. n., and H. orientalis comb. n. Furthermore, H. (= Afenestrata) sacchari is renamed as H. saccharophila nom. nov. to avoid homonymy. All these species, together with H. bamboosi, are regarded as members of a paraphyletic ‘Afenestrata group’ within Heterodera. Whilst recognised as artificial, the Afenestrata group is nevertheless an aid to discussion about these similar species. Morphological and molecular characterisation of populations of H. koreana comb. n. from Florida and H. orientalis comb. n. from Florida and Guatemala verify the identification of these populations as valid representatives for molecular studies of the species. Light and SEM observations also provide new detail and a broader understanding of the morphological range of both species. These include a longer stylet for females of H. koreana comb. n. and H. orientalis comb. n. than reported in the original descriptions. In addition, previously unreported tuberculate ridges are noted on the surface of vulval lips of H. orientalis comb. n. The lip region of second-stage juveniles of H. koreana comb. n. and H. orientalis comb. n. both include fused adjacent submedian lips that also fuse with the labial disc and the second lip annulus. The ITS-rRNA gene sequences of H. orientalis comb. n. populations from Florida and Guatemala were similar to those from the Russian type locality. Diagnostic PCR-RFLP of ITS-rRNA profiles with six enzymes for H. orientalis comb. n. and H. koreana comb. n. are given. A key for the morphological identification of species of the Afenestrata group is provided.

scholarly journals Geographic Distribution and Genetic Diversity of Ceanothus-Infective FrankiaStrains

1999 ◽  
Vol 65 (4) ◽  
pp. 1378-1383 ◽  
Author(s):  
Nancy J. Ritchie ◽  
David D. Myrold
Keyword(s):  
Root Nodules ◽  
Intergenic Spacer ◽  
Taxonomic Diversity ◽  
Molecular Techniques ◽  
23S Rrna ◽  
Rrna Gene ◽  
Sample Collection ◽  
Pcr Product ◽  
Pcr Rflp ◽  
23S Rrna Gene

ABSTRACT Little is known about Ceanothus-infectiveFrankia strains because no Frankia strains that can reinfect the host plants have been isolated fromCeonothus spp. Therefore, we studied the diversity of theCeonothus-infective Frankia strains by using molecular techniques. Frankia strains inhabiting root nodules of nine Ceanothus species were characterized. The Ceanothus species used represent the taxonomic diversity and geographic range of the genus; therefore, the breadth of the diversity of Frankia strains that infectCeanothus spp. was studied. DNA was amplified directly from nodular material by using the PCR. The amplified region included the 3′ end of the 16S rRNA gene, the intergenic spacer, and a large portion of the 23S rRNA gene. A series of restriction enzyme digestions of the PCR product allowed us to identify PCR-restriction fragment length polymorphism (RFLP) groups among theCeanothus-infective Frankia strains tested. Twelve different enzymes were used, which resulted in four different PCR-RFLP groups. The groups did not follow the taxonomic lines of the Ceanothus host species. Instead, theFrankia strains present were related to the sample collection locales.

Sequence-based Identification and Classification of Fungi.

2021 ◽  
pp. 198-216
Author(s):  
Andrew M. Borman ◽  
Elizabeth M. Johnson
Keyword(s):  
Dna Sequences ◽  
Its Region ◽  
Pcr Primers ◽  
Taxonomic Resolution ◽  
Its2 Region ◽  
Rrna Gene ◽  
5.8S Rrna Gene ◽  
Fungal Identification ◽  
Identification Of Fungi ◽  
Fungal Dna

Abstract This book chapter describes the advantages and limitations of the ITS Region as a universal barcode for fungal identification. The ITS region offers several practical advantages as a universal fungal barcode region. The region encompasses segments that permit resolution at different taxonomic levels as it includes the highly conserved 5.8S rRNA gene, the moderately rapidly evolving ITS2 region and the rapidly evolving ITS1 region, flanked by the highly conserved SSU and LSU genes which permit design of PCR primers that are almost panfungal. Over the last two decades the sequence-based identification of fungi has certainly come of age. The ITS region is universally accepted as the primary fungal barcoding region owing to the high barcode gap with the locus for many groups of fungi. Since the species-resolution power of ITS is poor for certain groups of fungi, and higher-level taxonomic resolution is greater with proteincoding genes, the TEF1α locus has been proposed as the universal secondary barcode region. In addition, the historical problems surrounding the reliability of fungal DNA sequences in centralized repositories are slowly being resolved by the development of an increasing number of publicly accessible, curated databases.

Morphological and molecular diversity of Radopholus on coffee in Vietnam and description of R. daklakensis sp. n. from Robusta coffee

Nematology ◽  
2012 ◽  
Vol 14 (1) ◽  
pp. 65-83 ◽  
Author(s):  
Phap Q. Trinh ◽  
Lieven Waeyenberge ◽  
Chau N. Nguyen ◽  
Maurice Moens
Keyword(s):  
Molecular Diversity ◽  
Tail Length ◽  
Parasitic Nematodes ◽  
Rrna Gene ◽  
28S Rrna ◽  
Plant Parasitic Nematodes ◽  
Robusta Coffee ◽  
Rdna Sequences ◽  
Stylet Length ◽  
A New Species

Four isolates of Radopholus arabocoffeae and one isolate of R. duriophilus were collected during a survey of plant-parasitic nematodes on coffee in Vietnam. Radopholus species only occurred in the Western Highland region. Information on the degree of variability in morphometrics, and morphological and molecular characters amongst and within these species are reported. The survey also yielded a Radopholus isolate collected from Robusta coffee roots and soil in Dak Lak, which is herein described as a new species, R. daklakensis sp. n. Within the genus, R. daklakensis sp. n. is most similar to R. arabocoffeae and R. duriophilus in morphology and morphometrics. It differs from R. arabocoffeae by the stylet knobs always directed posteriorly and the conical tail shape with rounded terminus in females. Radopholus daklakensis sp. n. differs from R. duriophilus by the stylet knobs, which are always directed posteriorly vs rounded, and dorsal knob sometimes projected anteriorly, and sperm rod-shaped vs oval and kidney-shaped. Radopholus daklakensis sp. n. differs from R. similis by the absence of a postrectal intestinal sac, four incisures of the lateral fields terminating far posterior to the position of the phasmid, the bursa in male reaching one-third of tail to half, never reaching tail terminus and smaller stylet length in females. The canonical discriminant analysis separated the Radopholus species by characters such as lip height, stylet length, b, b′, c, V and ratio tail length/stylet length. Phylogenetic analysis of Radopholus species using ITS-rDNA sequences or sequences from the D2-D3 expansion region of the 28S rRNA gene also confirm R. daklakensis sp. n. to be distinct.

Survey of slug-parasitic nematodes in East and West Flanders, Belgium and description ofAngiostoma gandavensisn. sp. (Nematoda: Angiostomidae) from arionid slugs

2019 ◽  
Vol 94 ◽  
Author(s):  
P.R. Singh ◽  
M. Couvreur ◽  
W. Decraemer ◽  
W. Bert
Keyword(s):  
New Species ◽  
Light Microscopy ◽  
Dna Sequences ◽  
Morphological Characters ◽  
Molecular Data ◽  
Parasitic Nematodes ◽  
Rdna Sequences ◽  
18S Rdna Sequences ◽  
East And West ◽  
Tail Tip

AbstractA survey for slug-associated nematodes in five locations of East and West Flanders in Belgium revealed the presence of one new and six known slug-parasitic nematodes,Agfa flexilis(Dujardin, 1845),Alloionema appendiculatum(Schneider, 1859),Angiostoma dentiferum(Mengert, 1953),Angiostoma limacis(Dujardin, 1845),Angiostoma norvegicum(Rosset al., 2017) andPhasmarhabditis hermaphrodita(Schneider, 1859).Angiostoma norvegicumandP.hermaphroditaare recorded for the first time in Belgium. The six known species are documented by light microscopy (LM) microphotographs and informative DNA sequences.Angiostoma gandavensisn. sp. (Angiostomatidae), discovered from arionid slugs, is described based on light microscopy, scanning electron microscopy (SEM) and molecular data. Based on analyses of D2D3 expansion segment of 28S and 18S rDNA sequences, this new species is found to be related toA.limacis,A.norvegicum,A.margaretae(Rosset al., 2011) andA.milacis(Ivanova and Wilson, 2009). The new species can be distinguished from these others based on morphological characters such as the distinctive mucronate structures at the tail tip of both sexes, presence of lateral ala, reflexed female ovaries and the number and arrangement pattern of male genital papillae.

Variations in ribosomal DNA sequences and phylogeny of Globodera parasitising solanaceous plants

Nematology ◽  
2000 ◽  
Vol 2 (6) ◽  
pp. 591-604 ◽  
Author(s):  
Sergei Subbotin ◽  
Roland Perry ◽  
Andrew Warry ◽  
Paul Halford
Keyword(s):  
Phylogenetic Analysis ◽  
Dna Sequences ◽  
Its Region ◽  
Globodera Rostochiensis ◽  
Its2 Region ◽  
Restriction Patterns ◽  
Rdna Sequences ◽  
Pcr Products ◽  
Solanaceous Plants ◽  
Its Pcr

AbstractThe D3 expansion region of the 28S gene and the ITS1-5.8S-ITS2 region of rDNA sequences from Globodera rostochiensis, G. pallida, G. tabacum tabacum, G. tabacum virginiae and G. tabacum solanacearum have been aligned and compared. There are no nucleotide differences in the D3 region sequences between G. rostochiensis and G. pallida. Sequence analysis and RFLPs of ITS-PCR products showed that several haplotypes are present in the genomes of G. rostochiensis and G. pallida populations. Restriction patterns of PCR products for eight enzymes for differentiation of these two species are given. Phylogenetic analysis of 41 ITS region sequences obtained from populations and species of the subfamily Punctoderinae revealed four distinct main clades within Globodera parasitising solanaceous plants: G. rostochiensis, G. tabacum, G. pallida and an undescribed Globodera sp. from South America. The utility of RFLP profiles and sequences of the rDNA are discussed for diagnostics and phylogeny of Globodera . Variabilité dans les séquences de l'ADN ribosomal et phylogénie des Globodera parasitant les Solanacées - La région d'expansion D3 du gène 28S et la région ITS1-5.8S-ITS2 des séquences d'ADNr de Globodera rostochiensis, G. pallida, G. tabacum tabacum, G. tabacum virginiae et G. tabacum solanacearum ont été alignées et comparées. Il n'y a pas de différence dans les nucléotides de la séquence de la région D3 entre G.rostochiensis et G.pallida. L'analyse séquentielle et les RFLP des produits du ITS-PCR montrent que plusieurs haplotypes sont présents dans les génomes des populations de G. rostochiensis et G. pallida. Les profils de restriction des produits du PCR de huit enzymes choisies pour la différenciation de ces deux espèces sont donnés. L'analyse phylogénique séquentielle de 41 regions ITS de populations et espèces appartenant à la sous-famille des Punctonerinae a révélé l'existence de quatre clades chez les Globodera parasitant les Solanacées: G. rostochiensis, G. tabacum, G. pallida et une espèce non décrite provenant d'Amérique du Sud. L'utilité pour le diagnostic et la phylogénie des Globodera des profils de RFLP et des séquences d'ADNr est discutée.

Length variation and repetitive sequences of Internal Transcribed Spacer of ribosomal RNA gene, diagnostics and relationships of populations of potato rot nematode, Ditylenchus destructor Thorne, 1945 (Tylenchida: Anguinidae)

Nematology ◽  
2011 ◽  
Vol 13 (7) ◽  
pp. 773-785 ◽  
Author(s):  
Sergei A. Subbotin ◽  
Abbas Mohammad Deimi ◽  
Jingwu Zheng ◽  
Vladimir N. Chizhov
Keyword(s):  
Phylogenetic Analyses ◽  
Repetitive Sequences ◽  
Repetitive Elements ◽  
Rrna Gene ◽  
Length Variation ◽  
28S Rrna ◽  
Gene Sequences ◽  
Its Rrna Gene ◽  
Its Rrna ◽  
Ditylenchus Destructor

Abstract Seventy-eight ITS rRNA gene sequences obtained from the potato rot nematode, Ditylenchus destructor, collected across the world from different hosts were compared and analysed. The ITS rRNA gene sequences showed significant length variation between populations. The differences in this rRNA fragment length were due to the presence of repetitive elements in the ITS1, which were characterised by relatively higher rates of substitution changes. Reconstruction of secondary structure for the ITS1 revealed that these minisatellites formed a stem structure. Phylogenetic analyses of ITS rRNA and D2-D3 expansion segments of 28S rRNA gene sequences showed that all studied populations clustered in two major clades: a group of populations having the ITS sequences with the repetitive elements and a group of populations without the repetitive elements in the ITS. We propose to distinguish seven ITS rRNA haplotypes within potato rot nematode populations. PCR-ITS-RFLP diagnostic profiles are presented for these ITS haplotypes and usefulness of recently developed PCR methods with species-specific primers for D. destructor are analysed and discussed.

scholarly journals First Detection of Ditylenchus destructor Parasitizing Maize in Northeast China

Life ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1303
Author(s):  
Fengjuan Pan ◽  
Feng Li ◽  
Yanzhi Mao ◽  
Dan Liu ◽  
Aoshuang Chen ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Economic Losses ◽  
Morphological Identification ◽  
Rrna Gene ◽  
Heilongjiang Province ◽  
Maize Seedlings ◽  
Wide Range ◽  
Its Rrna ◽  
Ditylenchus Destructor ◽  
Plant Parasitic

Maize is one of the most important crops in the world. Heilongjiang province has the largest maize area in China. Plant-parasitic nematodes are important agricultural pests, which cause huge economic losses every year and have attracted global attention. Potato rot nematode Ditylenchus destructor is a plant-parasitic nematode with a wide range of hosts and strong survival ability in different environments, which brings risks to agricultural production. In 2020, D. destructor was detected in seven maize fields in Heilongjiang province. Morphological identification and molecular approach were used to characterize the isolated D. destructor. The observed morphological and morphometric characteristics were highly similar and consistent with the existing description. The DNA sequencing on the D2/D3 region of the ribosomal DNA 28S and the phylogenetic analysis showed that D. destructor population obtained from maize and other isolates infesting carrot, sweet potato, and potato were in subclade I supported by a 96% bootstrap value. Additionally, the phylogenetic analysis of the ITS rRNA gene sequence further indicated that this D. destructor population from maize clustered in a clade I group and belonged to ITS rRNA haplotype C. An inoculation experiment revealed that D. destructor was pathogenic on the maize seedlings in pots and caused the disease symptoms in the stem base of maize seedlings. This is the first report of D. destructor causing stem rot of maize in Heilongjiang province, and contributes additional information on disease control and safe production of maize in the region.

Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

2015 ◽  
Vol 41 (1) ◽  
pp. 51-58
Author(s):  
Mohammad Shamimul Alam ◽  
Hawa Jahan ◽  
Rowshan Ara Begum ◽  
Reza M Shahjahan
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fish Larva ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Valuable Insight ◽  
Multiple Sequence ◽  
Pcr Rflp ◽  
Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

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