scholarly journals Authentication of the Herbal Medicine Angelicae Dahuricae Radix Using an ITS Sequence-Based Multiplex SCAR Assay

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2134 ◽  
Author(s):  
Pureum Noh ◽  
Wook Kim ◽  
Sungyu Yang ◽  
Inkyu Park ◽  
Byeong Moon
Keyword(s):  
Herbal Medicines ◽  
Morphological Characteristics ◽  
Pcr Amplification ◽  
Its Region ◽  
Sequencing Analysis ◽  
Accurate Identification ◽  
Republic Of China ◽  
Sequence Characterized Amplified Region ◽  
Angelicae Dahuricae Radix ◽  
Species Specific

The accurate identification of plant species is of great concern for the quality control of herbal medicines. The Korean Pharmacopoeia and the Pharmacopoeia of the People’s Republic of China define Angelicae Dahuricae Radix (Baek-Ji in Korean and Bai-zhi in Chinese) as the dried roots of Angelica dahurica or A. dahurica var. formosana belonging to the family Apiaceae. Discrimination among Angelica species on the basis of morphological characteristics is difficult due to their extremely polymorphic traits and controversial taxonomic history. Furthermore, dried roots processed for medicinal applications are indistinguishable using conventional methods. DNA barcoding is a useful and reliable method for the identification of species. In this study, we sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes in A. dahurica, A. dahurica var. formosana, and the related species A. anomala and A. japonica. Using these sequences, we designed species-specific primers, and developed and optimized a multiplex sequence-characterized amplified region (SCAR) assay that can simply and rapidly identify respective species, and verify the contamination of adulterant depending on the polymerase chain reaction (PCR) amplification without sequencing analysis in a single PCR reaction. This assay successfully identified commercial samples of Angelicae Dahuricae Radix collected from Korean and Chinese herbal markets, and distinguished them from adulterants. This multiplex SCAR assay shows a great potential in reducing the time and cost involved in the identification of genuine Angelicae Dahuricae Radix and adulterant contamination.

ISSR-Derived Species-Specific SCAR Marker for Rapid and Accurate Authentication of Ocimum tenuiflorum L.

Planta Medica ◽  
2017 ◽  
Vol 84 (02) ◽  
pp. 117-122 ◽  
Author(s):  
Amit Kumar ◽  
Vereena Rodrigues ◽  
Priyanka Mishra ◽  
Kuppusamy Baskaran ◽  
Ashutosh Shukla ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
High Volume ◽  
Inter Simple Sequence Repeat ◽  
Rapid Identification ◽  
Medicinal Species ◽  
Accurate Identification ◽  
Sequence Characterized Amplified Region ◽  
Medicinal Value ◽  
Ocimum Tenuiflorum ◽  
Species Specific

Abstract Ocimum tenuiflorum has been widely used in traditional medicine and has high medicinal value. High volume trade of this potential medicinal plant species led to unscrupulous adulteration of both crude drugs as well as formulations. Morphology-based authentication is difficult in cases of incomplete or damaged samples and in dried herbal materials. In such cases, PCR-based molecular methods may aid in accurate identification. The present study aimed at developing species-specific DNA marker(s) for the authentication of O. tenuiflorum. A species-specific amplicon (279 bp) generated through an inter-simple sequence repeat marker (UBC 835) in all individuals of O. tenuiflorum was cloned, sequenced, and a primer pair was developed (designated as CIM-OT-835F/CIM-OT-835R). The newly developed sequence characterized amplified region marker was validated through PCR amplification in all available seven species of Ocimum, and its specificity for O. tenuiflorum was confirmed with the consistent generation of an amplicon of 177 bp. The developed marker can be used for accurate and rapid identification of the species for certification purposes and will be useful in quality control of medicinal preparations containing this important medicinal species.

scholarly journals Development of Species/Genus specific Primers for Identification of Three Trichoderma Species and for Detection of Trichoderma Genus

2021 ◽  
Author(s):  
You Zhou ◽  
Jun Wang ◽  
Laying Yang ◽  
Lijia Guo ◽  
Shuting He ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Elongation Factor ◽  
Pcr Amplification ◽  
Its Region ◽  
Control Agent ◽  
Plant Diseases ◽  
Specific Primers ◽  
Accurate Identification ◽  
Trichoderma Species ◽  
Species Specific

Abstract Trichoderma is a widely used bio-control agent, and has excellent ability to antagonize plant pathogens and promoting plant growth. It has been successfully used to control various plant diseases. The premise of using Trichoderma is accurate identification of it. In this study, four sets of species/genus-specific primers were designed based on the translation elongation factor 1-alpha (tef1) gene and internal transcribed spacer (ITS) region, in order to identify Trichoderma harzianum, T. koningiopsis, and T.virens, and detect the genus of Trichoderma. Here, the rapid, simple, and reliable PCR methods were development using the species-specific primers EHarF2/EHarR2, EKoisF/EKoisR, and EVireF/EVireR to produce 253 bp (tef1 gene), 255 bp (tef1 gene), and 263 bp (tef1 gene) DNA bands to identify T. harzianum, T. koningiopsis, and T. virens, respectively. The genus-specific primers ITricF/ITricR produces a single DNA band in the range of 103–113 bp (ITS region) to distinguish Trichoderma from other genera fungi, and the size of the band is related to the species of Trichoderma. In addition, ITricF/ITricR could use for real-time PCR amplification for detection the quantity of Trichoderma spp..

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.

scholarly journals Detection of Pythium ultimum Using Polymerase Chain Reaction with Species-Specific Primers

Plant Disease ◽  
1997 ◽  
Vol 81 (10) ◽  
pp. 1155-1160 ◽  
Author(s):  
K. Kageyama ◽  
A. Ohyama ◽  
M. Hyakumachi
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Transcribed Spacer ◽  
Pcr Amplification ◽  
Its Region ◽  
Pythium Ultimum ◽  
Pythium Species ◽  
Chain Reaction ◽  
Specific Primers ◽  
Polymerase Chain ◽  
Species Specific

This study was conducted to sequence the rDNA internal transcribed spacer (ITS) region of Pythium ultimum and Pythium group HS, design species-specific primers for polymerase chain reaction (PCR), and detect P. ultimum from diseased seedlings using PCR. The sequence of the ITS region of P. ultimum was identical with that of Pythium group HS. The results support the reports that the HS group is an asexual strain of P. ultimum. Using PCR, the primer pair K1+K3, designed on portions of the sequence of the ITS region, amplified isolates of P. ultimum and the HS group but not isolates of 20 other Pythium species. DNA extracts from damped-off seedlings were not amplified, but a 10-fold dilution of the extracts with Tris-EDTA (TE) buffer diluted the inhibitors and allowed PCR amplification. The primer pair used detected P. ultimum from a single diseased seedling.

scholarly journals Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma

2015 ◽  
Vol 53 (10) ◽  
pp. 3280-3285 ◽  
Author(s):  
Sarah A. Ahmed ◽  
Wendy W. J. van de Sande ◽  
Marie Desnos-Ollivier ◽  
Ahmed H. Fahal ◽  
Najwa A. Mhmoud ◽  
...  
Keyword(s):  
Morphological Characteristics ◽  
High Specificity ◽  
Its Region ◽  
Molecular Techniques ◽  
Isothermal Amplification ◽  
Content Type ◽  
Fungal Dna ◽  
Madurella Mycetomatis ◽  
Phenotypic Methods ◽  
Species Specific

Appropriate diagnosis and treatment of eumycetoma may vary significantly depending on the causative agent. To date, the most common fungus causing mycetoma worldwide isMadurella mycetomatis. This species fails to express any recognizable morphological characteristics, and reliable identification can therefore only be achieved with the application of molecular techniques. Recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) are proposed as alternatives to phenotypic methods. Species-specific primers were developed to target the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region ofM. mycetomatis. Both isothermal amplification techniques showed high specificity and sufficient sensitivity to amplify fungal DNA and proved to be appropriate for detection ofM. mycetomatis. Diagnostic performance of the techniques was assessed in comparison to conventional PCR using biopsy specimens from eumycetoma patients. RPA is reliable and easy to operate and has the potential to be implemented in areas where mycetoma is endemic. The techniques may be expanded to detect fungal DNA from environmental samples.

scholarly journals First Report of Bursaphelenchus pinophilus on Korean pine (Pinus koraiensis)

Plant Disease ◽  
2009 ◽  
Vol 93 (12) ◽  
pp. 1354-1354 ◽  
Author(s):  
H. Han ◽  
Y.-J. Chung ◽  
S.-C. Shin
Keyword(s):  
Morphological Characteristics ◽  
Pine Wilt Disease ◽  
Its Region ◽  
Wilt Disease ◽  
Pinus Koraiensis ◽  
Molecular Diagnostic ◽  
Accurate Identification ◽  
Causal Organism ◽  
First Report ◽  
Pine Wilt

The genus Bursaphelenchus Fuchs, 1937 contains approximately 90 species (3) that are morphologically similar. Pine wood nematode, Bursaphelenchus xylophilus (4) Nickle, 1970, is the causal organism of pine wilt disease and accurate identification is essential for diagnosis of the disease. In Korea, pine wilt disease was first reported in 1988 and devastated 6,800 ha of pine forest through 2008. For a survey of trees with pine wilt disease, wood samples were taken randomly from dead Pinus koraiensis in Namyangju, Gyeonggi Province in Korea. The extracted nematodes from dead trees were maintained in culture on Botrytis cinerea and morphological characteristics were observed with an inverted light microscope (Leica DE/DMI 3000B). Identification of Bursaphelenchus spp. based on morphological characteristics is difficult, especially for identification of juveniles that carry few morphological features for species identification. The internal transcribed spacer (ITS) region in ribosomal DNA provides useful molecular diagnostic markers for this genus (1). The nematodes were provisionally identified as Bursaphelenchus pinophilus based on the characteristic long and arcuate body shape, male spicule with distinctive rostrum and small cucullus, female vulval flap, and mucronate conical tail. Other Bursaphelenchus spp. with vulval flaps and spicules with cucullus are B. xylophius, B. mucronatus, B. abruptus, and B. pinophilus. For molecular diagnosis, DNA was extracted from more than 30 individual nematodes with a DNeasy Kit (Qiagen, Valencia, CA) and ITS regions 1, 2, and 5.8S in rDNA were amplified by PCR (US/PTC-0220; Bio Rad, Hercules, CA). The ITS-restriction fragment length polymorphism pattern was consistent with that of B. pinophilus (2). The ITS rDNA sequence of B. pinophilus from Korean pines had a 98% sequence homology to that of B. pinophilus in GenBank (Accession No. AM160664). The pathogenicity of B. pinophilus has not been determined. To our knowledge, this is the first report of B. pinophilus on P. koraiensis, but it was previously reported from Poland, Germany, and Portugal on P. sylvestris and P. pinaster (1). References: (1) H. Braasch. EPPO Bull. 31:127, 2001. (2) W. Burgermeister et al. Russ. J. Nematol. 13:29, 2005. (3) R. Sriwati et al. Nematology 10:1, 2008. (4) G. Steiner and E. M. Buhrer. J. Agric. Res. 48:946, 1934.

scholarly journals A New Accurate Genotyping HRM Method for Alternaria Species Related to Fruit Rot Diseases of Apple and Pomegranate

2016 ◽  
Vol 4 (3) ◽  
pp. 159-165 ◽  
Author(s):  
Antonios Zambounis ◽  
Aliki Xanthopoulou ◽  
George Karaoglanidis ◽  
Athanasios Tsaftaris ◽  
Panagiotis Madesis
Keyword(s):  
Morphological Characteristics ◽  
Curve Analysis ◽  
Fruit Rot ◽  
Nucleotide Polymorphisms ◽  
Postharvest Diseases ◽  
Accurate Identification ◽  
Complex Species ◽  
Alternaria Species ◽  
Species Specific ◽  
Pomegranate Fruit

Alternaria core rot and Alternaria black heart rot of apple and pomegranate fruit, respectively, are major pre- and postharvest diseases worldwide. However, it is very difficult to differentiate the rot related Alternaria species in the Alternaria complex as they are not always correlate to species-groups based upon morphological characteristics and due to the limited genetic variation these species exhibit among each other. Therefore, it is crucial to exploit novel assays towards the accurate identification and differentiation of these Alternaria species. We have developed, a real-time PCR assay [using species specific primers targeting the endopolygalacturonase (EndoPG) gene] combined with a high-resolution melting (HRM) curve analysis for discrimination of the 14 single nucleotide polymorphisms (SNPs)-based Alternaria haplotypes, which were assigned based on the aligned sequence profiles of 138 Alternaria spp. strains previously isolated from apple and pomegranate rotted fruit. This analysis specifically generated 14 unique HRM curve haplotype profiles among the Alternaria complex species tested. The results showed that HRM curve analysis allows the rapid and adequate identification and genotyping of the three Alternaria species (A. alternata, A. tenuissima and A. arborescens) responsible mostly for the apple and pomegranate fruit rot diseases.

scholarly journals Molecular Identification and Characterisation of Aspergillus Flavus Isolates Originating from Serbian Wheat Grains

2020 ◽  
Vol 49 (4) ◽  
pp. 382-389
Author(s):  
J. Krulj ◽  
N. Ćurčıć ◽  
A. Bočarov Stančıć ◽  
J. Kojıć ◽  
L. Pezo ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Molecular Genetic ◽  
Pcr Amplification ◽  
Its Region ◽  
Holistic Approach ◽  
Accurate Identification ◽  
Essential Point ◽  
Climate Conditions ◽  
Wheat Grains ◽  
Pcr Rflp

During previous years, regarding the shifts in climate conditions in temperate region, such as occurrence of high temperatures and prolonged drought, increased occurrence frequencies of Aspergillus flavus and aflatoxins in cereal grains were recorded. A reliable and accurate identification of the fungi is of great importance for evaluating the microbiological risks of contamination. The essential point of the present investigation was molecular characterisation and identification of A. flavus isolates originating from common wheat and spelt grains collected after harvest during the period of three years (2015–2017) in Northern Serbia. A holistic approach that included PCR amplification of two DNA genomic regions and PCR-RFLP assay followed by fragment length analysis, provided complete and comprehensive characterisation of A. flavus isolated from wheat grains. The presented results indicate that there was no difference among the tested Aspergillus isolates on the molecular–genetic level. All 38 strains were identified as A. flavus by sequencing of combined ITS region and β-tubulin gene fragments (acc. no.: MH582473 to MH582510). PCR-RFLP method in combination with a Lab-on-a-chip (LoaC) electrophoresis can be successfully used to rapidly identify A. flavus isolates.

scholarly journals First Report of Anthracnose on Fruits of Duchesnea indica Caused by Colletotrichum acutatum in Northwestern Argentina

Plant Disease ◽  
2012 ◽  
Vol 96 (5) ◽  
pp. 765-765
Author(s):  
E. B. Sir ◽  
M. E. Arias ◽  
J. Racedo ◽  
A. Castagnaro ◽  
J. C. Díaz Ricci
Keyword(s):  
White Light ◽  
Morphological Characteristics ◽  
Pcr Amplification ◽  
Aerial Mycelium ◽  
Colletotrichum Acutatum ◽  
Fragaria Ananassa ◽  
Northwestern Argentina ◽  
First Report ◽  
Duchesnea Indica ◽  
Species Specific

Duchesnea indica (Andrews) Focke, a cosmopolitan wild species related to the cultivated strawberry that is widely distributed in northwestern Argentina, grows in close proximity to strawberry crops and has proven to be almost immune to Colletotrichum spp. isolated from diseased strawberry plants (1), hence it has never been considered a phytopathological risk. During a field survey of “La Heladera” (27°01′45″S, 65°39′20″W), Tafí del Valle (Tucumán, Argentina) from November 2009 to November 2010, a genotype of D. indica showing fruits with dark brown, necrotic, irregular, circular lesions of 5 to 20 mm in diameter were collected. Setose acervuli were observed on the center of the fruit lesions. Pathogens were obtained from 10 diseased fruit collected at random, and four fungal isolates were isolated per fruit on potato dextrose agar (PDA). To reduce the number of samples for evaluation, two isolates per fruit that were exhibiting stable but distinctive morphological features were chosen to continue the studies. Isolates were characterized by morphological, molecular, and phytopathological criteria. After 10 days of incubation on PDA medium at 28°C with continuous white light, colonies exhibited a gray, aerial mycelium, whereas the reverse of the colony is a pale maroon with a radial, pale salmon color. Masses of salmon-colored conidia formed in the center of the colonies. Conidia were hyaline, one celled, fusiform, tapered to a point at both ends, and measured 14.8 to 17.3 × 4.5 to 7.4 μm (n = 100). Setae were scarce and sclerotia were absent. All morphological characteristics that were observed indicated that the isolates were C. acutatum (3). To fulfill Koch's postulates and verify the pathogenicity on commercial varieties of strawberry, six healthy plants of D. indica and Fragaria × ananassa cv. Camarosa with mature fruits were used to test each isolate. Four plants were spray inoculated with conidial suspensions of the virulent isolates (1.5 × 106 conidia/ml) and two with sterile distilled water as controls. Both treatments were maintained under white light (2,000 lux, 12 h per day) at 28°C and 70% relative humidity. Nine days after the inoculation, dark brown lesions and salmon-colored masses of conidia were observed only in inoculated fruits of both genotypes. The fungus isolated from diseased fruits and the conidia that were produced were identical to the isolates used to inoculate the plants. To confirm pathogen identity, PCR amplification with the species-specific pair of primer CaInt2/ITS4 (4) were carried out using fungal total DNA from the original isolates and isolates obtained from inoculated fruits. An amplification product of approximately 490 bp, which is specific for C. acutatum, was observed in all DNA samples (4). Although C. acutatum has already been reported in Fragaria × ananassa in Argentina (2), to our knowledge, this is the first report of C. acutaum causing anthracnose in D. indica species. This result is relevant since this species grows close to strawberry fields and can be an alternative host and potential vector of the anthracnose disease agent. References: (1) M. E. Arias. Frutillas Silvestres y Especies Relacionadas con la Cultivada. EDUNT, Argentina, 2007. (2) C. J. Ramallo et al. Plant Dis. 84:706. 2000. (3) B. J. Smith and L. L. Black. Plant Dis. 74:69, 1990. (4) S. Sreenivasaprasad et al. Plant Pathol. 45:650, 1996.

scholarly journals First Report of Leaf Spot Caused by Alternaria alternata on Chinese Dwarf Banana in China

Plant Disease ◽  
2014 ◽  
Vol 98 (5) ◽  
pp. 691-691 ◽  
Author(s):  
B. Z. Fu ◽  
Z. H. Zhang ◽  
L. H. Wang ◽  
G. Y. Li ◽  
J. Z. Zhang ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Leaf Spot ◽  
Morphological Characteristics ◽  
Pcr Amplification ◽  
Its Region ◽  
Leaf Spot Disease ◽  
First Report ◽  
Spot Disease ◽  
Rdna Its ◽  
Its Sequence Analysis

The Chinese dwarf banana (Ensete lasiocarpum) is one of the ornamental bananas that belongs to Musaceae family. The plant is native to the southwestern China, where it grows semi-wild in the mountains between 1,500 and 2,500 m above sea level. During July 2011, a leaf spot disease on this plant was observed in the campus and parks in Kunming, Yunnan Province. The incidence level was about 22%, mainly on the old leaves. The leaf symptoms were irregular spots with gray to off-white centers surrounded by dark brown margins, and usually also surrounded by chlorotic halos. Leaf tissues (3 × 5 mm), cut from the margins of lesions, were surface-disinfected (95% ethanol for 3 min, 0.1% HgCl2 for 2 min, rinsed three times with sterile water), plated on potato sucrose agar (PSA), and incubated at 26°C under natural lights. The same fungus was consistently isolated from the diseased leaves. Colonies of white-to-dark gray mycelia formed on PSA that were black on the underside. The colonies were further identified as Alternaria sp. based on the dark brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (2). Conidia were 5.26 to 30.26 μm long and 3.95 to 15.79 μm wide, averaging 10.21 (±3.17) × 20.02 (±5.75) μm (n = 50), with a beak length of 0 to 7.89 μm, and had 3 to 8 transverse and 0 to 3 longitudinal septa. PCR amplification was carried out by utilizing universal rDNA-ITS primer pair ITS4/ITS5 (1). The ITS region of isolate DY1 (GenBank Accession No. KF516556) was 572 bp in length. BLAST search revealed 99% identity with two Alternaria alternata isolates (JF440581.1 and GQ121322.2). Phylogenetic analysis (MEGA 5.1) using the neighbor-joining algorithm placed the isolate in a well-supported cluster with other A. alternata isolates. The pathogen was identified as A. alternate (Fr.:Fr.) Keissler based on the morphological characteristics and rDNA-ITS sequence analysis. To confirm pathogenicity, Koch's postulates were performed on detached leaves of E. lasiocarpum inoculated with mycelial plugs with ddH2O and agar plugs as a control. Leaf spots identical to those observed in the field developed in 9 days on the inoculated leaves but not on the control. The inoculation assay used three leaves, totaling 72 spots for control and 36 spots for inoculation. The experiments were repeated once. A. alternata was consistently re-isolated from the inoculated leaves. The symptom developed easier with wounds. To our knowledge, this is the first report of E. lasiocarpum leaf spot disease caused by A. alternata in China and the world. References: (1) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (2) T. Y. Zhang. Flora Fungorum Sinicorum, Vol. 16: Alternaria. Science Press, Beijing, China, 2003.

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