Application of eDNA method in the detection of Cordulegaster (Insecta: Odonata) species

The aim of this pilot study was to investigate the potential of eDNA techniques to detect the presence of the two dragonfly species Cordulegaster heros and Cordulegaster bidentata. Both species are classified as “near threatened” according to the IUCN Red List and are strictly protected in several countries. Monitoring these species with traditional sampling methods is often difficult, time-consuming and invasive. In this pilot study, we first collected tissue samples from C. heros and C. bidentata to sequence the traditional DNA-barcode gene fragment COI. We then collected further dragonfly COI sequences from BOLD to design species-specific primers. This, however, was impossible given the enormous variability of COI. Therefore, we refrained from species-specific eDNA assays and followed eDNA metabarcoding protocol using universal (BF2/BF2) and a newly designed dragonfly specific primer. For the evaluation of the method, we took water samples from places where Cordulegaster specimens are known to occur. After the extraction, we used two sequential PCR steps for obtaining the desired amplicon (two-step PCR) using universal primers in the first step, and group (dragonfly) specific primers or universal primers. Amplicons were sequenced on an Illumina MiSeq platform and then analysed the data with the JAMP pipeline. With the newly designed primers and we could effectively detect the targeted dragonfly species from tissue samples, and also from filtered environmental samples. The detection of the species with the traditional method is time consuming and involves the destruction of the specimens. In comparison, with the eDNA method we could easily detect these near threatherned odonates and other dragonfly species in a non-invasive way.

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First Report of Anthracnose Caused by Colletotrichum gloeosporioides in Pumpkin in Trinidad

Plant Disease ◽

10.1094/pdis-94-8-1062a ◽

2010 ◽

Vol 94 (8) ◽

pp. 1062-1062

Author(s):

S. N. Rampersad

Keyword(s):

Colletotrichum Gloeosporioides ◽

Phylogenetic Analyses ◽

Pcr Amplification ◽

Positive Control ◽

Universal Primers ◽

Conidial Suspension ◽

Specific Primers ◽

First Report ◽

Main Production ◽

Species Specific

In Trinidad, pumpkin (Cucurbita pepo L. and C. moschata L.) is extensively grown for local and international export markets. In November 2008, symptoms of foliar chlorosis and necrosis were observed in 15 commercial pumpkin fields located in the main production areas of St. George East, Caroni, Victoria, and St. Patrick counties. Severely infected plants were unable to support fruit maturation, which resulted in yield loss. The pathogen was isolated from surface-sterilized tissues of symptomatic plants. Colonies on potato dextrose agar (PDA) were white to cream with gray spore masses in the center. Conidia were hyaline, cylindrical with rounded ends, aseptate, and measured 12.5 to 16.5 μm × 3.5 to 5.0 μm. PCR amplification was carried out with ITS4/5 universal primers (4) and species-specific primers, CgInt/ITS4 (1), using a positive control of Colletotrichum gloeosporioides (courtesy of D. Perez-Brito). Species-specific primers generated a single amplicon, ~450 bp long, which corresponded with the positive control. The ITS1 region (1) of pumpkin isolates (GenBank No. GU320190) was 100% identical to cognate sequences of C. gloeosporioides isolates (GenBank Nos. AY841136 and FJ624257). Phylogenetic analyses (MEGA 4 – Molecular Evolutionary Genetic Analysis Software version 4 for Windows) using the neighbor-joining (NJ) algorithm placed the pumpkin isolates in a well-supported cluster (>90% bootstrap value based on 1,000 replicates) with other C. gloeosporioides isolates. The tree was rooted with C. crassipes (GenBank No. AJ536230). The pathogen was similar to C. gloeosporioides (Penz.) Penz. & Sacc. (3). In pathogenicity tests, six plants (cv. Jamaican squash) for each of five isolates were spray inoculated to runoff with a conidial suspension (1.0 × 106 conidia/ml). Negative controls were sprayed with sterile distilled water. In repeated tests, plants were symptomatic of infection 7 days postinoculation. There were no symptoms on control plants. Koch's postulates were fulfilled with the reisolation of the pathogen from symptomatic leaf tissues. Anthracnose is a serious threat to cucurbit production; however, infection is not common in pumpkin and squash (2). To my knowledge, this is the first report of C. gloeosporioides causing widespread anthracnose infection in pumpkin in Trinidad. References: (1) A. E. Brown et al. Phytopathology 86:523, 1996. (2) G. Kelly. Acta Hortic. (ISHS) 731:479, 2007. (3) B. C. Sutton. Page 1 in: Colletotrichum: Biology, Pathology and Control. CAB International. Wallingford, UK, 1992. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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New Species-Specific Primers for Molecular Diagnosis of Bactrocera minax and Bactrocera tsuneonis (Diptera: Tephritidae) in China Based on DNA Barcodes

Insects ◽

10.3390/insects10120447 ◽

2019 ◽

Vol 10 (12) ◽

pp. 447 ◽

Cited By ~ 1

Author(s):

Linyu Zheng ◽

Yue Zhang ◽

Wenzhao Yang ◽

Yiying Zeng ◽

Fan Jiang ◽

...

Keyword(s):

Cytochrome C ◽

Cytochrome C Oxidase ◽

Dna Barcode ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Specific Primers ◽

Oxidase Subunit ◽

Preimaginal Stages ◽

Specific Fragment ◽

Species Specific

Tephritidae fruit flies (Diptera: Tephritidae) are regarded as important damage-causing species due to their ability to cause great economic losses in fruit and vegetable crops. Bactrocera minax and Bactrocera tsuneonis are two sibling species of the subgenus Tetradacus of Bactrocera that are distributed across a limited area of China, but have caused serious impacts. They share similar morphological characteristics. These characteristics can only be observed in the female adult individuals. The differences between them cannot be observed in preimaginal stages. Thus, it is difficult to distinguish them in preimaginal stages morphologically. In this study, we used molecular diagnostic methods based on cytochrome c oxidase subunit I and species-specific markers to identify these two species and improve upon the false-positive results of previous species-detection primers. DNA barcode sequences were obtained from 900 individuals of B. minax and 63 individuals of B. tsuneonis. Based on these 658 bp DNA barcode sequences of the cytochrome c oxidase subunit I gene, we successfully designed the species-specific primers for B. minax and B. tsuneonis. The size of the B. minax specific fragment was 422 bp and the size of the B. tsuneonis specific fragment was 456 bp. A series of PCR trials ensured the specificity of these two pairs of primers. Sensitivity assay results demonstrated that the detection limit for the DNA template concentration was 0.1~1 ng/μL for these two species. In this study, we established a more reliable, rapid, and low-cost molecular identification method for all life stages of B. minax and B. tsuneonis. Species-specific PCR can be applied in plant quarantine, monitoring and control of B. minax and B. tsuneonis.

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Identification of Penicillium expansum causing postharvest blue mold decay of apple fruit

Pesticidi i fitomedicina ◽

10.2298/pif1404257v ◽

2014 ◽

Vol 29 (4) ◽

pp. 257-266 ◽

Cited By ~ 10

Author(s):

Ivana Vico ◽

Natasa Duduk ◽

Miljan Vasic ◽

Milica Nikolic

Keyword(s):

Apple Fruit ◽

Penicillium Expansum ◽

Universal Primers ◽

Economic Losses ◽

Blue Mold ◽

Tubulin Gene ◽

Specific Primers ◽

Molecular Features ◽

Species Specific ◽

Storage Facilities

Penicillium expansum (Link) Thom. is one of the most important postharvest pathogens of apple fruit worldwide. It causes blue mold, a decay that can lead to significant economic losses during storage, which can also impact fruit destined for processing due to the production of carcinogenic mycotoxin patulin. Apple fruit cvs. Idared, Golden Delicious and Braeburn with blue mold symptoms were collected from five storage facilities in Serbia and nine fungal isolates were obtained. Pathogenicity of the isolates was tested and proven by artificial inoculation of healthy apples cv. Idared. In order to identify the causal agents of decay, morphological and molecular methods were used. Colony morphology and microscopic features were observed on differential media, and isolates were tested for the production of cyclopiazonic acid. Molecular analysis included PCR amplification with species specific primers for P. expansum based on polygalacturonase gene (Pepg1), universal primers for internal transcribed spacer rDNA region and primers based on ?-tubulin gene. All isolates formed compact blue green colonies with characteristic earthy odor. Conidiophores were terverticillate with smooth septate stipes and conidia were smooth, globose to subglobose, born in colums. The average size of conidia was 3.38 ? 0.49 (SD) x 3 ? 0.36 (SD) ?m. Using species specific primers PEF/PER the texpected amplicons of ~404 bp were obtained in all nine tested isolates and PCR conducted with the Bt-LEVUp4/ Bt-LEV-Lo1 and universal ITS1/ITS4 primer pairs generated amplicons of the expected sizes of ~800 bp and ~600 bp, respectively. MegaBlast analyses of the 2X consensus of nucleotide sequences of the isolate JP1 partial ?-tubulin gene and ITS region showed 99-100% and 100% similarity with several P. expansum sequences of corresponding regions of this species deposited in GenBank. Based on morphological and molecular features, the isolates obtained from decayed apple fruit collected in several storage facilities in Serbia were identified as P. expansum.

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Two-Step PCR-Based Assay for Identification of Bacterial Etiology of Otitis Media with Effusion in Infected Lebanese Children

Journal of Clinical Microbiology ◽

10.1128/jcm.36.5.1185-1188.1998 ◽

1998 ◽

Vol 36 (5) ◽

pp. 1185-1188 ◽

Cited By ~ 34

Author(s):

Ghassan M. Matar ◽

Nada Sidani ◽

Michel Fayad ◽

Usamah Hadi

Keyword(s):

Otitis Media ◽

Otitis Media With Effusion ◽

Bacterial Species ◽

Cost Effective ◽

Universal Primers ◽

Rrna Gene ◽

Specific Primer ◽

Specific Primers ◽

Bacterial Dna ◽

Species Specific

We developed and evaluated a two-step PCR-based assay with universal primers and genus- or species-specific primers for the detection of the most prevalent bacterial etiologies of otitis media with effusion (OME) in children from Lebanese hospitals. These etiologies included Haemophilus, Streptococcus, and Moraxella (Branhamella)catarrhalis, which were detected in middle-ear effusion (MEE) samples taken from children with OME. A total of 47 MEE samples were aspirated from 36 patients during insertion of a tympanostomy tube performed particularly for OME. The duration of effusion in all patients was ≥2 months. DNA was extracted from MEE samples, and PCR was initially done with DNA extracts by using the universal primers RW01 and DG74, which flank an ∼370-bp fragment found in the 16S rRNA gene of all bacterial species. For the identification of specific bacteria, we used in three separate reaction mixtures the following genus- or species-specific primers: (i) aHaemophilus-specific probe (probe RDR125) as a primer along with DG74, (ii) a Streptococcus-specific primer (primer STR1; designed by us) along with DG74, and (iii) an M. catarrhalis-specific primer pair (primer pair MCA1-MCA2). Thirty-five MEE samples (74.5%) gave the expected 370-bp band, indicating the presence of bacterial DNA in the tested samples. Of the 35 PCR-positive samples tested, 33 (94.3%) were positive forHaemophilus, 3 (8.6%) were positive forStreptococcus, and 10 (28.6%) were positive for M. catarrhalis. Ten samples (28.6%) exhibited a mixed infection and were positive for both Haemophilus and M. catarrhalis. Culture was simultaneously performed for all 47 MEE samples. Ten of the 47 MEE samples (21.3%) exhibited bacterial growth. These 10 were PCR positive for bacterial DNA. The remaining 25 PCR-positive samples were negative by culture, thus showing about 53% discordance between PCR results and those of culture. The PCR assay proved to be more sensitive than culture, more rapid, less cumbersome, and more cost-effective than the available PCR-Southern hybridization-based assays.

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Rapid Identification of Candida albicansand Other Human Pathogenic Yeasts by Using Short Oligonucleotides in a PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.36.6.1634-1641.1998 ◽

1998 ◽

Vol 36 (6) ◽

pp. 1634-1641 ◽

Cited By ~ 65

Author(s):

B. M. Mannarelli ◽

C. P. Kurtzman

Keyword(s):

Large Subunit ◽

Extraction Procedure ◽

Variable Region ◽

Small Subunit ◽

Universal Primers ◽

Rrna Gene ◽

Chromosomal Dna ◽

Specific Primers ◽

Dna Fragment ◽

Species Specific

A PCR system that can quickly and accurately identify 14 species of human pathogenic yeasts was developed. The procedure distinguished between nine species of a closely related clade, Lodderomyces elongisporus, Candida parapsilosis, a newCandida sp., C. sojae, C. tropicalis, C. maltosa, C. viswanathii,C. albicans, and C. dubliniensis and between another five more divergent species, Pichia guilliermondii,C. glabrata, C. zeylanoides, C. haemulonii, and C. haemulonii type II. A rapid DNA extraction procedure that yields purified DNA in about 1 h is also described. The system uses uniform conditions with four primers for each reaction, two 40- to 50-mer universal primers that serve as a positive control and two 23- to 30-mer species-specific primers. Species-specific primers were derived from a 600-nucleotide variable region (D1/D2) at the 5′ end of the large-subunit (26S) ribosomal DNA gene and were generally designed to use mismatches at the 3′ end. Universal primers were developed from conserved nucleotide sequences in the small-subunit (18S) rRNA gene. In this system, a control 1,200- to 1,300-base DNA fragment was produced in all reactions and a smaller 114- to 336-base DNA fragment was produced if the chromosomal DNA from the target species was present. The PCR procedure is rapid and easy to interpret and may be used with mixed cultures.

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DNA Barcode-Based PCR-RFLP and Diagnostic PCR for Authentication of Jinqian Baihua She (Bungarus Parvus)

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2015/402820 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 4

Author(s):

Xiaolei Li ◽

Weiping Zeng ◽

Jing Liao ◽

Zhenbiao Liang ◽

Shuhua Huang ◽

...

Keyword(s):

Fragment Length Polymorphism ◽

Dna Barcode ◽

Specific Primers ◽

Diagnostic Pcr ◽

Pcr Rflp ◽

Restriction Sites ◽

Specific Restriction ◽

Polymerase Chain ◽

Coi Sequences ◽

Species Specific

We established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and diagnostic PCR based on cytochrome C oxidase subunit I (COI) barcodes ofBungarus multicinctus, genuine Jinqian Baihua She (JBS), and adulterant snake species. The PCR-RFLP system utilizes the specific restriction sites ofSpeI andBstEII in the COI sequence ofB. multicinctusto allow its cleavage into 3 fragments (120 bp, 230 bp, and 340 bp); the COI sequences of the adulterants do not contain these restriction sites and therefore remained intact after digestion withSpeI andBstEII (except for that ofZaocys dhumnades, which could be cleaved into a 120 bp and a 570 bp fragment). For diagnostic PCR, a pair of species-specific primers (COI37 and COI337) was designed to amplify a specific 300 bp amplicon from the genomic DNA ofB. multicinctus; no such amplicons were found in other allied species. We tested the two methods using 11 commercial JBS samples, and the results demonstrated that barcode-based PCR-RFLP and diagnostic PCR both allowed effective and accurate authentication of JBS.

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DNA Barcode Reveals the Bycatch of Endangered Batoids Species in the Southwest Atlantic: Implications for Sustainable Fisheries Management and Conservation Efforts

Genes ◽

10.3390/genes10040304 ◽

2019 ◽

Vol 10 (4) ◽

pp. 304 ◽

Cited By ~ 6

Author(s):

Bruno Lopes da Silva Ferrette ◽

Rodrigo Rodrigues Domingues ◽

Matheus Marcos Rotundo ◽

Marina Provetti Miranda ◽

Ingrid Vasconcellos Bunholi ◽

...

Keyword(s):

Fishery Management ◽

Dna Barcode ◽

Iucn Red List ◽

Small Scale ◽

Marine Biodiversity ◽

Artisanal Fisheries ◽

Trade Restrictions ◽

Southwest Atlantic ◽

Elasmobranch Species ◽

Species Specific

Today, elasmobranchs are one the most threatened vertebrate groups worldwide. In fact, at least 90% of elasmobranch species are listed in the International Union for Conservation of Nature (IUCN) Red List, while more than 40% are data-deficient. Although these vertebrates are mainly affected by unsustainable fishery activities, bycatch is also one of the major threats to sharks and batoids worldwide, and represents a challenge for both sustainable fishery management and for biodiversity and conservational efforts. Thus, in this study, DNA barcode methodology was used to identify the bycatch composition of batoid species from small-scale industrial fisheries in the southwest Atlantic and artisanal fisheries from southeast Brazil. A total of 228 individuals belonging to four Chondrichthyes orders, seven families, and at least 17 distinct batoid species were sequenced; among these individuals, 131 belonged to species protected in Brazil, 101 to globally threatened species, and some to species with trade restrictions provided by Appendix II of the Convention on International Trade in Endangered Species (CITES). These results highlight the impacts on marine biodiversity of bycatch by small-scale industrial and unmanaged artisanal fisheries from the southwest Atlantic, and support the implementation of DNA-based methodologies for species-specific identification in data-poor fisheries as a powerful tool for improving the quality of fisheries’ catch statistics and for keeping precise bycatch records.

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Transstadial Transmission of Pythium in Bradysia impatiens and Lack of Adult Vectoring Capacity

Phytopathology ◽

10.1094/phyto-04-10-0117 ◽

2010 ◽

Vol 100 (12) ◽

pp. 1307-1314 ◽

Cited By ~ 7

Author(s):

Sarah E. Braun ◽

Louela A. Castrillo ◽

John P. Sanderson ◽

Margery L. Daughtrey ◽

Stephen P. Wraight

Keyword(s):

Pathogenic Fungi ◽

Specific Primers ◽

Tissue Samples ◽

Aerial Dispersal ◽

Fungus Gnats ◽

Fungus Gnat ◽

Transstadial Transmission ◽

Polymerase Chain ◽

Species Specific ◽

Potential Interactions

Fungus gnats have been shown to transmit a variety of plant-pathogenic fungi that produce aerial dispersal stages. However, few studies have examined potential interactions between fungus gnats and oomycetes, including Pythium spp. A series of laboratory experiments were conducted to determine whether fungus gnat adults are vectors of several common greenhouse Pythium spp., including Pythium aphanidermatum, P. irregulare, and P. ultimum. An additional objective was to determine whether P. aphanidermatum can be maintained transstadially in the gut of a fungus gnat larva through the pupal stadium to be transmitted by the subsequent adult. Adult fungus gnats did not pick up infectious Pythium propagules from diseased plants and transmit them to healthy plants in any experiment. Species-specific primers and a probe for real-time polymerase chain reaction were developed to detect the presence of P. aphanidermatum DNA in fungus gnat tissue samples. P. aphanidermatum DNA was detectable in the larval and pupal stages; however, none was detected in adult fungus gnats. These results are in agreement with previous studies that have suggested that adult fungus gnats are unlikely vectors of Pythium spp.

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