A SYBR Green qPCR Method for Detecting and Quantifying Spores of Colletotrichum acutatum and C. gloeosporioides Species Complexes Causing Ripe Rot of Grape

2021 ◽  
Author(s):  
Scott Cosseboom ◽  
Mengjun Hu
Keyword(s):  
Colletotrichum Gloeosporioides ◽  
Cost Effective ◽  
Sybr Green ◽  
Colletotrichum Acutatum ◽  
Quantitative Real Time Pcr ◽  
Specific Primers ◽  
Green Method ◽  
Species Complexes ◽  
Qpcr Method ◽  
Spore Trapping

Four species belonging to Colletotrichum acutatum and Colletotrichum gloeosporioides species complexes, including C. aenigma, C. fioriniae, C. fructicola, and C. nymphaeae, were identified from ripe rot symptomatic fruit from Mid-Atlantic vineyards. A quantitative real-time PCR (qPCR) SYBR Green method was developed to detect and quantify conidia of the two Colletotrichum complexes to better understand the distribution and the extent of Colletotrichum inoculum. Primers were designed to specifically target the β-tubulin gene of the C. acutatum complex or the C. gloeosporioides complex and were found to be specific to the desired complex, not amplifying DNA of other prevalent fungi from vineyards. The sensitivity of the primers was also assessed, and the C. acutatum complex-specific and C. gloeosporioides complex-specific primers were able to quantify as little as 50 C. fioriniae conidia and 100 C. aenigma conidia, respectively. Therefore, this would be a cost-effective, specific, and sensitive detection and quantification method for these two destructive species complexes in vineyards and many other crops through spore trapping applications.

scholarly journals SYBR-Green-Based Quantitative Real-Time PCR for Discriminating Between Closely Related Angiostrongylus Cantonensis and A. Malaysisensis

2020 ◽  
Author(s):  
Wallop Jakkul ◽  
Kittipong Chaisiri ◽  
Naowarat Saralamba ◽  
Yanin Limpanont ◽  
Sirilak Dusitsittipon ◽  
...  
Keyword(s):  
Intermediate Host ◽  
Real Time ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Eosinophilic Meningitis ◽  
Quantitative Real Time Pcr ◽  
The Third ◽  
Snail Intermediate Host ◽  
Third Stage ◽  
Qpcr Method

Abstract Background: Angiostrongylus cantonensis is a well-known pathogen causing human angiostrongyliasis eosinophilic meningitis. Humans, as accidental hosts, are infected by eating undercooked snails containing third-stage larvae. A. malaysiensis is closely related to A. cantonensis and has been described as a potential human pathogen. Recently, the two species have been reported to have overlapping distributions in the same endemic area, particularly in the Indochina region. Because of their similar morphological characteristics, misidentification often occurs, particularly of the third-stage larva in the snail intermediate host. Methods: We designed species-specific primers to mitochondrial cytochrome b, which was used as a genetic marker. SYBR-green quantitative real-time PCR (qPCR) was employed to quantitatively detect and identify the third-stage larvae and tissue debris in the cerebrospinal fluid (CSF) of a patient, and to quantify third-stage larvae in the snail Achatina fulica collected from the field.Results: The newly designed primers were highly specific and sensitive, even when using conventional PCR. SYBR green qPCR quantitatively detected around 10−4 ng of genomic DNA from one larva and facilitated the specific detection and identification of parasitic genetic material from the CSF of a patient with angiostrongyliasis. The method also estimated the number of larvae in A. fulica and revealed that the primary source of Angiostrongylus infection in the King Rama IX public park study area was A. malaysiensis; although, the two Angiostrongylus species each infected 10% of the snails. Conclusions: Our SYBR green qPCR method is a useful and inexpensive technique for parasite identification and has sufficient sensitivity and specificity to detect a single larva and simultaneously discriminate between A. cantonensis and A. malaysiensis. The number of larvae infecting or co-infecting the snail intermediate host can also be estimated. In future research, this qPCR method could be employed in a molecular survey of A. cantonensis and A. malaysiensis occurrence within intermediate and definitive hosts. The technique should also be applied in a study analyzing CSF specimens from patients with eosinophilic meningitis to assess the usefulness of the method for clinical diagnosis.

scholarly journals Antifungal Activity of Propyl Disulfide from Neem (Azadirachta indica) in Vapor and Agar Diffusion Assays against Anthracnose Pathogens (Colletotrichum gloeosporioides and Colletotrichum acutatum) in Mango Fruit

2021 ◽  
Vol 9 (4) ◽  
pp. 839
Author(s):  
Muhammad Rafiullah Khan ◽  
Vanee Chonhenchob ◽  
Chongxing Huang ◽  
Panitee Suwanamornlert
Keyword(s):  
Antifungal Activity ◽  
Mycelial Growth ◽  
Colletotrichum Gloeosporioides ◽  
Colletotrichum Acutatum ◽  
Model Parameters ◽  
Pathogenicity Test ◽  
Agar Diffusion ◽  
Wide Range ◽  
Significant Difference ◽  
Diffusion Assay

Microorganisms causing anthracnose diseases have a medium to a high level of resistance to the existing fungicides. This study aimed to investigate neem plant extract (propyl disulfide, PD) as an alternative to the current fungicides against mango’s anthracnose. Microorganisms were isolated from decayed mango and identified as Colletotrichum gloeosporioides and Colletotrichum acutatum. Next, a pathogenicity test was conducted and after fulfilling Koch’s postulates, fungi were reisolated from these symptomatic fruits and we thus obtained pure cultures. Then, different concentrations of PD were used against these fungi in vapor and agar diffusion assays. Ethanol and distilled water were served as control treatments. PD significantly (p ≤ 0.05) inhibited more of the mycelial growth of these fungi than both controls. The antifungal activity of PD increased with increasing concentrations. The vapor diffusion assay was more effective in inhibiting the mycelial growth of these fungi than the agar diffusion assay. A good fit (R2, 0.950) of the experimental data in the Gompertz growth model and a significant difference in the model parameters, i.e., lag phase (λ), stationary phase (A) and mycelial growth rate, further showed the antifungal efficacy of PD. Therefore, PD could be the best antimicrobial compound against a wide range of microorganisms.

scholarly journals Identification of Echinococcus granulosus Genotypes G1 and G3 by SNPs Genotyping Assays

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 125
Author(s):  
Piero Bonelli ◽  
Silvia Dei Giudici ◽  
Angela Peruzzu ◽  
Lorena Mura ◽  
Cinzia Santucciu ◽  
...  
Keyword(s):  
Echinococcus Granulosus ◽  
Animal Species ◽  
Cost Effective ◽  
Sensu Stricto ◽  
Sybr Green ◽  
Hydatid Cysts ◽  
Genotype Identification ◽  
Host Selectivity ◽  
Nad5 Gene ◽  
Echinococcus Granulosus Sensu Lato

Echinococcus granulosus sensu lato (s.l.) is the causative agent of cystic echinococcosis in animals and humans. Different E. granulosuss.l. genotypes exhibit great diversity in their life cycle, host selectivity and pathogenicity. For this reason, the study of genetic variation within Echinococcus species is of importance for their epidemiological implication. We employed two SNP genotyping technologies to distinguish G1 and G3 E. granulosus sensu stricto (s.s.). genotypes. The genotypes of DNA samples (n = 28) extracted from hydatid cysts of different animal species were identified by amplification and sequencing of a fragment of the mitochondrial nad5 gene. Two SYBR green and three TaqMan real time PCR assays were developed for targeting of three nad5 informative positions (SNP758, 1123, and 1380) known to be able to discriminate G1 from G3. Genotyping by SYBR Green PCR based on cycle threshold (Ct) with melting temperature (Tm) analysis and performed on SNP1123 and SNP1380 failed to identify one DNA sample. TaqMan assays for SNP758, 1123 and 1380 effectively confirmed genotype identification obtained by Sanger sequencing. Our results demonstrated that the combination of the three Taqman assays developed in this study represents a valuable and cost effective tool alternative to DNA sequencing for E. granulosus s.s. genotyping.

scholarly journals First Report of Colletotrichum acutatum on Tomato and Apple Fruits in the Czech Republic

Plant Disease ◽  
2012 ◽  
Vol 96 (5) ◽  
pp. 769-769 ◽  
Author(s):  
J. Víchová ◽  
B. Staňková ◽  
R. Pokorný
Keyword(s):  
Czech Republic ◽  
Tomato Fruit ◽  
Colletotrichum Acutatum ◽  
Distilled Water ◽  
Species Determination ◽  
Specific Primers ◽  
The Czech Republic ◽  
First Report ◽  
Pcr Products ◽  
Apple Fruits

Apple (Malus domestica Borkh.) is a fruit traditionally grown in the Czech Republic, and tomatoes (Solanum lycopersicum Mill.), too, are widely raised in this region. Colletotrichum acutatum J. H. Simmonds is a polyphagous fungal plant pathogen. Earlier, this pathogen caused disease on strawberry in the Czech Republic (2), and now it has become an important pathogen on safflower (4). During the 2010 harvest, anthracnose symptoms were noticed on the fruits of apple and tomato. Infected apples fruits (localities Velká Bíteš and Znojmo) and tomatoes (localities Velká Bíteš and Žabčice) were collected. Typical symptoms on fruit surfaces were round, brown, shriveled and sunken spots, 1.2 to 2.0 cm, with orange conidial masses appearing on the spots. A fungus was isolated from each host on potato dextrose agar and cultured at 25 ± 2°C for 10 days. Mycelium was superficial, partly immersed, and white to gray with occurrence of orange conidial masses. Conidia of the tomato and apple isolates were colorless and fusiform. The size of conidia from the apple and tomato isolates, respectively, ranged from 11 to 15 × 2.5 to 3.5 μm and 11 to 16 × 2.5 to 4 μm. Morphological characteristics suggested that the isolated fungi was a Colletotrichum sp. To fulfill Koch's postulates, healthy tomato and apple fruits were disinfected with 3% sodium hypochlorite for 2 min and rinsed in sterile distilled water. Fruits were pinpricked with a sterile needle and 10 μl of a spore suspension (1 × 105 conidia ml–1) was inoculated by pipetting into the wound. Control fruits were treated with sterile distilled water. The fruits were transferred to a growth cabinet and maintained at a temperature of 25 ± 2°C, relative humidity of 70 ± 5%, and a photoperiod of 12 h. Similar disease symptoms as in the collected fruits were observed on tomato fruits at 7 days and apple fruits at 20 days after inoculation, while no symptoms appeared on control fruits. The pathogen was reisolated from infected fruits. Species determination of the isolates was confirmed by PCR. Specific primers designed in region ITS1, the 5.8S RNA gene, and region ITS2 of the pathogen DNA were selected. Specific primers CaInt2 and ITS4 were used to identify C. acutatum (3), and primers CgInt and ITS4 were used to determine C. gloeosporioides isolate CCM 177 (1), which was used as a control. Our isolates yielded PCR products (490 bp) only with primers designed for C. acutatum. The C. gloeosporioides isolate yielded a PCR product (450 bp) only with CgInt and ITS4 primers. PCR products were sequenced and identified with the BLAST program. The sequence of the tomato fruit isolate (Accession No. JN676199) and apple fruit isolate (Accession No. JN676198) matched with 100% similarity to the C. acutatum sequences in GenBank. The control isolate of C. gloeosporioides matched 100% to sequences AJ749682 and AJ749692. To our knowledge, this is the first report of C. acutatum on tomato and apple fruits in the Czech Republic. This pathogen can endanger the production and storage of apples and tomatoes in this region. References: (1) P. R. Mills et al. FEMS Microbiol. Lett. 98:137, 1992. (2) D. Novotný et al. Plant Dis. 91:1516, 2007. (3) S. Sreenivasaprasad et al. Plant Pathol. 45:650, 1996. (4) J. Víchová et al. Plant Dis. 95:79, 2011.

scholarly journals Comparative evaluation of cost effective extraction free molecular technique for detection of SARS-CoV-2 with reference to standard VTM based RT-qPCR method

2021 ◽  
Author(s):  
Megh Singh Dhakad ◽  
Sanjib Gogoi ◽  
Ansu Kumari ◽  
Aashish Kumar Singh ◽  
Manoj B. Jais ◽  
...  
Keyword(s):  
Detection Method ◽  
Cost Effective ◽  
Molecular Technique ◽  
Rt Pcr ◽  
Index Method ◽  
Resource Limited ◽  
Qpcr Method ◽  
Kappa Value ◽  
The Cost ◽  
Higher Sensitivity

Background and Objectives: The entire globe is undergoing an unprecedented challenge of COVID-19. Considering the need of rapid and accurate diagnostic tests for SARS-CoV-2, this study was planned to evaluate the cost effective extraction free RT-PCR technique in comparison to the standard VTM based RT-qPCR method. Materials and Methods: Paired swabs from nasopharynx and oropharynx were collected for SARS-CoV-2 testing, from 211 adult patients (≥18 years) in VTM and plain sterile tubes (dry swabs). These samples were processed and RT-qPCR was carried out as per standard protocols. Results: 54.5% of the patients were females and 45.5% were males with sex ratio 1:1.19 (M: F). 38.86% were symptomatic, of which fever (86.59%), cough (79.23%) and breathlessness (46.34%) were the most common symptoms. The positivity by VTM based method and index method was 31.27% and 13.27% respectively. Of the 27 inconclusive results from index method, 37.04% were positive, 48.15% were negative by VTM based method. However, in 40 inconclusive results by VTM based method, 90% were negative and rest remained inconclusive by index method. The sensitivity and specificity of the index method were 39.39% and 85.71% respectively. The overall agreement between VTM based method and index method was 49.59% with estimated Kappa value of 0.19. Conclusion: VTM based method showed higher sensitivity compared to the index method. The higher positivity by VTM based method, suggests that VTM based method could plausibly be a better detection method of SARS-CoV-2. Still, the index method might add value in a resource limited setups for detection of SARS-CoV-2.  

The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR

2006 ◽  
Vol 65 (3) ◽  
pp. 476-487 ◽  
Author(s):  
Miguel A. Providenti ◽  
Jason M. O'Brien ◽  
Robyn J. Ewing ◽  
E. Suzanne Paterson ◽  
Myron L. Smith
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Sybr Green ◽  
Quantitative Real Time Pcr ◽  
Genetic Elements

scholarly journals Identification and Characterization of Benomyl-Resistant and -Sensitive Populations of Colletotrichum gloeosporioides from Statice (Limonium spp.)

2006 ◽  
Vol 96 (5) ◽  
pp. 542-548 ◽  
Author(s):  
Marcel Maymon ◽  
Aida Zveibil ◽  
Shimon Pivonia ◽  
Dror Minz ◽  
Stanley Freeman
Keyword(s):  
Colletotrichum Gloeosporioides ◽  
Sequence Analyses ◽  
Specific Primers ◽  
Tubulin Genes ◽  
Colletotrichum Spp ◽  
Polymerase Chain ◽  
Species Specific ◽  
Identification And Characterization ◽  
Propagation Material

Sixty-four isolates of Colletotrichum gloeosporioides were isolated from infected Limonium spp. cultivated in 12 different locations in Israel. All isolates were identified as belonging to the C. gloeosporioides complex by species-specific primers. Of these isolates, 46 were resistant to benomyl at 10 μg/ml and 18 were sensitive to this concentration of fungicide. Based on arbitrarily primed polymerase chain reaction of all isolates and internal transcribed spacer-1 sequence analyses of 12 selected isolates, the benomyl-resistant and -sensitive populations belong to two distinct genotypes. Sequence analyses of the β-tubulin genes, TUB1 and TUB2, of five sensitive and five resistant representative isolates of C. gloeosporioides from Limonium spp. revealed that the benomyl-resistant isolates had an alanine substitute instead of a glutamic acid at position 198 in TUB2. All data suggest that the resistant and sensitive genotypes are two independent and separate populations. Because all Limonium plant propagation material is imported from various geographic regions worldwide, and benomyl is not applied to this crop or for the control of Colletotrichum spp. in Israel, it is presumed that plants are bearing quiescent infections from the points of origin prior to arrival.

A PCR-based method for the identification of the roots of 10 co-occurring grassland species in mesocosm experiments

Botany ◽  
2008 ◽  
Vol 86 (5) ◽  
pp. 485-490 ◽  
Author(s):  
Gordon G. McNickle ◽  
J. F. Cahill ◽  
M. K. Deyholos
Keyword(s):  
Cost Effective ◽  
Ecosystem Functions ◽  
Specific Primers ◽  
False Negatives ◽  
Dna Templates ◽  
Grassland Species ◽  
Target Dna ◽  
Mesocosm Experiments ◽  
Species Specific ◽  
Template Dna

An understanding of factors influencing the distribution of plant roots is intimately linked to our understanding of basic ecosystem functions such as nutrient flux and productivity. However, it is not usually possible to measure root distributions because it is difficult to identify the roots of different species when they are grown in mixture. This is because the roots of most species are not visually distinguishable. We designed a simple, PCR-based method for the identification of roots in mesocosm experiments, which we have applied to 10 co-occurring grassland species. Species-specific primers based on ITS sequences from GenBank were evaluated in PCR assays using either homogeneous or heterogeneous DNA templates, as well as DNA extracted from mixed-root samples from multiple combinations of species. The species-specific primers reported here produced accurate identifications, free from both false negatives and false positives, in 100% of our assays. We also evaluated the sensitivity of our system and demonstrated detection of species when they comprised as little as 0.05 ng of target DNA mixed in a total of 2.5 ng of multi-species template DNA. Our PCR-based method for root identification in mesocosms is more cost effective, and simpler to apply than previously described methods.

scholarly journals Mushroom Emergence Detected by Combining Spore Trapping with Molecular Techniques

2017 ◽  
Vol 83 (13) ◽  
Author(s):  
Carles Castaño ◽  
Jonàs Oliva ◽  
Juan Martínez de Aragón ◽  
Josu G. Alday ◽  
Javier Parladé ◽  
...  
Keyword(s):  
Real Time ◽  
Fruiting Body ◽  
Real Time Pcr ◽  
Fungal Species ◽  
Fruiting Bodies ◽  
Quantitative Real Time Pcr ◽  
Mushroom Production ◽  
Spore Abundance ◽  
Spore Trapping ◽  
Species Specific

ABSTRACT Obtaining reliable and representative mushroom production data requires time-consuming sampling schemes. In this paper, we assessed a simple methodology to detect mushroom emergence by trapping the fungal spores of the fruiting body community in plots where mushroom production was determined weekly. We compared the performance of filter paper traps with that of funnel traps and combined these spore trapping methods with species-specific quantitative real-time PCR and Illumina MiSeq to determine the spore abundance. Significantly more MiSeq proportional reads were generated for both ectomycorrhizal and saprotrophic fungal species using filter traps than were obtained using funnel traps. The spores of 37 fungal species that produced fruiting bodies in the study plots were identified. Spore community composition changed considerably over time due to the emergence of ephemeral fruiting bodies and rapid spore deposition (lasting from 1 to 2 weeks), which occurred in the absence of rainfall events. For many species, the emergence of epigeous fruiting bodies was followed by a peak in the relative abundance of their airborne spores. There were significant positive relationships between fruiting body yields and spore abundance in time for five of seven fungal species. There was no relationship between fruiting body yields and their spore abundance at plot level, indicating that some of the spores captured in each plot were arriving from the surrounding areas. Differences in fungal detection capacity by spore trapping may indicate different dispersal ability between fungal species. Further research can help to identify the spore rain patterns for most common fungal species. IMPORTANCE Mushroom monitoring represents a serious challenge in economic and logistical terms because sampling approaches demand extensive field work at both the spatial and temporal scales. In addition, the identification of fungal taxa depends on the expertise of experienced fungal taxonomists. Similarly, the study of fungal dispersal has been constrained by technological limitations, especially because the morphological identification of spores is a challenging and time-consuming task. Here, we demonstrate that spores from ectomycorrhizal and saprotrophic fungal species can be identified using simple spore traps together with either MiSeq fungus-specific amplicon sequencing or species-specific quantitative real-time PCR. In addition, the proposed methodology can be used to characterize the airborne fungal community and to detect mushroom emergence in forest ecosystems.

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