Real-Time PCR Detection of the Onion Downy Mildew Pathogen Peronospora destructor From Symptomless Onion Seedlings and Soils

Plant Disease ◽  
2021 ◽  
pp. PDIS-05-20-1095
Author(s):  
Kazuki Fujiwara ◽  
Hiroyoshi Inoue ◽  
Ryoichi Sonoda ◽  
Yutaka Iwamoto ◽  
Motoaki Kusaba ◽  
...  
Keyword(s):  
Disease Management ◽  
Real Time ◽  
Downy Mildew ◽  
Real Time Pcr ◽  
Management Strategies ◽  
Leaf Tissue ◽  
Asymptomatic Infection ◽  
Inoculum Sources ◽  
Peronospora Destructor ◽  
Pathogen Dna

An outbreak of downy mildew disease of onion, caused by Peronospora destructor, in Japan in 2016 necessitated a reevaluation of the primary inoculum sources to optimize disease management. Detection of the P. destructor pathogen in plants with asymptomatic infection and in soil would guide the application of fungicides according to the extent of infection before disease development. Here, we detected P. destructor in both plants and soil using newly developed primer sets (Pd ITS and Pd ITS 614) by both conventional and real-time PCR. Validation by real-time PCR with Pd ITS 614 showed that P. destructor DNA was amplified from symptomless seedlings at 3.7 × 102 to 1.0 × 100 conidium cells/50 mg leaf tissue, suggesting the detection of asymptomatic infection. Real-time PCR with Pd ITS amplified pathogen DNA from field soils at 1.6 × 103 to 8.3 × 101 oospore cells/g of soil. This real-time PCR assay provides a useful tool for identifying and quantifying inoculum sources, which may be the foundation of the design of integrated disease management strategies.

scholarly journals Detection and Characterization of Carboxylic Acid Amide-Resistant Plasmopara viticola in China Using a TaqMan-MGB Real-Time PCR

Plant Disease ◽  
2020 ◽  
Vol 104 (9) ◽  
pp. 2338-2345
Author(s):  
Xiaoqing Huang ◽  
Xina Wang ◽  
Fanfang Kong ◽  
Theo van der Lee ◽  
Zhongyue Wang ◽  
...  
Keyword(s):  
Carboxylic Acid ◽  
Real Time ◽  
Downy Mildew ◽  
Real Time Pcr ◽  
Fungicide Resistance ◽  
Management Strategies ◽  
Acid Amide ◽  
Plasmopara Viticola ◽  
Carboxylic Acid Amide ◽  
Caa Fungicides

Grape production is increasing globally and so are problems with downy mildew, one of the main constraints in grape production. Downy mildew on grape is caused by Plasmopara viticola, an obligate biotrophic pathogen belonging to the oomycetes. Control of the disease is usually performed by fungicide applications, of which carboxylic acid amide (CAA) fungicides represent one of the most widely used groups of fungicides. Our previous research showed that the extensive application of CAA fungicides can result in fungicide resistance and in China, CAA-resistant isolates of P. viticola were collected from the field in 2014. To monitor the distribution and spread of CAA fungicide resistance, we developed a TaqMan-minor groove binder (MGB) real-time PCR-based method designed on a functional mutation in the PvCesA3 gene that allows efficient identification of CAA fungicide resistant and sensitive genotypes. The assay was validated on 50 isolates using Sanger sequencing and fungicide bioassays and exploited in a comprehensive survey comprising 2,227 single-sporangiophore isolates from eight major grapevine regions in China. We demonstrate that CAA fungicide resistance in P. viticola is widespread in China. On average, 53.3% of the isolates were found to be resistant, but marked differences were found between locations with percentages of resistant isolates varying from 0.3 to 96.6%. Furthermore, the frequency of CAA-resistant isolates was found to be significantly correlated with the exposure to CAA fungicides (P < 0.05). We further discussed the possibilities to apply the TaqMan-MGB real-time PCR assay to assess the frequency of fungicide-resistant P. viticola isolates in each region or vineyard, which would facilitate the correct choice of fungicide for grape downy mildew and resistance management strategies.

scholarly journals Real-Time PCR Quantification of Peronospora arborescens, the Opium Poppy Downy Mildew Pathogen, in Seed Stocks and Symptomless Infected Plants

Plant Disease ◽  
2011 ◽  
Vol 95 (2) ◽  
pp. 143-152 ◽  
Author(s):  
Miguel Montes-Borrego ◽  
Francisco J. Muñoz-Ledesma ◽  
Rafael M. Jiménez-Díaz ◽  
Blanca B. Landa
Keyword(s):  
Real Time ◽  
Downy Mildew ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Opium Poppy ◽  
Chain Reaction ◽  
Commercial Seed ◽  
Polymerase Chain ◽  
Pathogen Dna ◽  
Accurate Quantification

In this study, we developed a reliable, quick, and accurate quantitative polymerase chain reaction (qPCR) assay based on the MIQE (Minimum Information for publication of Quantitative Real-Time PCR Experiments) guidelines for the quantification of Peronospora arborescens in infected downy mildew–symptomless opium poppy (Papaver somniferum) tissues and commercial seed stocks. The protocol was highly reproducible and allowed accurate quantification of pathogen DNA up to 10 fg in different plant DNA backgrounds without losing specificity and efficiency. Moreover, to further overcome difficulties conferred by the strict biotrophy of this pathogen, we developed dilution series of DNA extracted from a plasmid with the target pathogen DNA as a cloned insert. This facilitated the demonstration of the robustness of the protocol in different laboratories with different qPCR equipment and reagents, which may help in its use on a broad scale. Finally, we validated the usefulness of the qPCR protocol for quarantine purposes and downy mildew resistance screening by quantifying P. arborescens in complex, naturally infested opium poppy samples. Thus, a pathogen biomass of 0.0003 to 0.007‰ or of 0.110 to 5,557 ppm was quantified in symptomless capsules in commercial seed stocks, or in stem samples from symptomless opium poppy plants systemically infected by the pathogen, respectively.

Real Time PCR detection of Macrobrachium rosenbergii (de Man, 1879) larvae with emphasis to their ecology

2021 ◽  
Vol 34 ◽  
pp. 12
Author(s):  
Mahadevan Harikrishnan ◽  
Deepak Jose ◽  
B. Nidhin ◽  
K.P. Anilkumar
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Monsoon Season ◽  
Management Strategies ◽  
Macrobrachium Rosenbergii ◽  
Freshwater Prawn ◽  
Larval Abundance ◽  
Larval Stages ◽  
Species Specific ◽  
De Man

Species specific identification of early larval stages of many decapod crustaceans sampled from plankton collections remains cumbersome owing to lack of distinguishable characteristics, where DNA based molecular methods provide accurate results without taxonomic ambiguities. In the present study, an attempt was made to detect temporal occurrence of early zoea of freshwater prawn Macrobrachium rosenbergii (de Man) using real-time PCR assays in polyhaline, mesohaline and oligohaline areas of a tropical positive estuary, the Vembanad lake (S. India). High caridean larval abundance could be recorded in polyhaline areas in all seasons while it could be recorded in monsoon season in mesohaline and oligohaline areas. 113 DNA isolations were successfully made from morphologically identified taxonomic units (MOTU) and SYBR Green based RT-PCR amplifications using designed primer for M. rosenbergii yielded positive detections in 38 samples (34%) representing all seasons in all three zones. Positive detections could be recorded in all months except May in mesohaline areas and differed significantly (F = 17.2 p < 0.01) with the same in polyhaline and oligohaline areas. The present results of molecular detection of M. rosenbergii larvae extend confirmation of its breeding ground in Vembanad lake where appropriate management strategies could be enforced for stock conservation of this species.

scholarly journals Occurrence of Lily mottle virus on Lilium in Italy

Plant Disease ◽  
2012 ◽  
Vol 96 (5) ◽  
pp. 771-771 ◽  
Author(s):  
D. Rizzo ◽  
L. Stefani ◽  
M. Paoli ◽  
S. Lazzereschi ◽  
B. Nesi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leaf Tissue ◽  
Flowering Plant ◽  
Mottle Virus ◽  
Rt Pcr ◽  
Cdna Synthesis ◽  
Vein Clearing ◽  
Cp Gene ◽  
Necrotic Spots

Lily mottle virus (LMoV), a member of the genus Potyvirus, is one of the main viruses infecting lily. Symptoms on lily differ according to the susceptibility and sensitivity of different cultivars and hybrids. They range from leaf mottle or mosaic, vein clearing, chlorotic and yellow streaking, leaf curling, and necrotic spots, to milder forms of leaf symptoms. Plants may even be symptomless at some stages of growth. A varietal collection of Lilium from the early 1990s is held in Pistoia Province (Tuscany, Italy) and is composed of Asian hybrids obtained from intraspecific breeding of commercial cultivars. During a survey conducted from May to June 2010, several plants showing vein clearing, leaf mottle, leaf mosaic, and reddish brownish necrotic spots were observed. Leaf samples from 60 symptomatic or symptomless lily plants, belonging to 20 cultivars, were collected and tested for the presence of LMoV. Samples were assayed by double-antibody sandwich (DAS)-ELISA and eight of them, belonging to four different cultivars, tested positive. Total RNA was extracted from 2 g of leaf tissue of every collected sample according to the protocol described earlier (3) and cDNA synthesis was performed with an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Samples were tested by reverse transcription (RT)-PCR and real-time PCR assays using primers LMoV1 (5′-GCAAATGAGACACTCAATGCTG-3′) and LMoV2 (5′-CGTGCGTGAAGTAACTTCATAG-3′) designed to amplify 651 bp of the coat protein (CP) gene of LMoV (1). Results obtained with RT-PCR and real-time PCR exactly matched those achieved with ELISA assay, and the eight positive samples showed amplicons of the expected size. PCR products from five infected samples were directly sequenced from both directions and submitted in GenBank (Accessions Nos. JQ655106 to JQ655110). Our isolates share more than 99% nucleotide identity among each other. Comparison with other LMoV-CP gene sequences present in GenBank showed nucleotide identities ranging from 93 to 94% with LMoV isolates from South Korea (GenBank Accession Nos. GQ150683 to GQ150686), China (GenBank Accession Nos. EU348826, AJ748256, AJ564636, and AJ564637), Australia (GenBank Accession No. JN127341), and Japan (GenBank Accession No. AB570195). To our knowledge, this is the first report of LMoV on Lilium in Italy where this virus was already reported to infect escarole (2). Considering the economic importance of Lilium production as a flowering plant in Pistoia Province, and in several other areas of Italy, the report of LMoV present on lilies suggests the use of healthy propagation material and the adoption of preventive measures to avoid its diffusion. References: (1) J.-H. Lim et al. Korean J. Microbiol. 45:251, 2009. (2) V. Lisa et al. Plant Dis. 86:329, 2002. (3) D. J. MacKenzie et al. Plant Dis. 81:222, 1997.

Quantification of the aflatoxin biocontrol strain Aspergillus flavus AF36 in soil, and nuts and leaves of pistachio by real-time PCR

Plant Disease ◽  
2020 ◽  
Author(s):  
MARÍA TERESA GARCÍA LOPEZ ◽  
Yong Luo ◽  
Alejandro Ortega-Beltran ◽  
Ramon Jaime ◽  
Juan Moral Moral ◽  
...  
Keyword(s):  
Real Time ◽  
Aspergillus Flavus ◽  
Real Time Pcr ◽  
Management Strategies ◽  
Cost Savings ◽  
Aflatoxin Contamination ◽  
Conidial Suspension ◽  
Toxigenic Strains ◽  
Important Time ◽  
Biocontrol Strain

The species Aspergillus flavus and A. parasiticus are commonly found in the soils of nut-growing areas in California. Several isolates can produce aflatoxins that occasionally contaminate nut kernels conditioning their sale. The strain AF36 of A. flavus, which does not produce aflatoxins, is registered as a biocontrol agent for use in almond, pistachio, and fig crops in California. After application in the orchards, AF36 displaces aflatoxin-producing Aspergillus spp. and thus reduces aflatoxin contamination. Vegetative compatibility assays (VCA) have traditionally been used to track AF36 in soils and crops where it has been applied. However, VCA is labor-intensive and time-consuming. Here, we developed a quantitative real-time PCR (qPCR) protocol to quantify proportions of AF36 accurately and efficiently in different substrates. Specific primers to target AF36 and toxigenic strains of A. flavus and A. parasiticus were designed based on sequence of aflC, a gene essential for aflatoxin biosynthesis. Standard curves were generated to calculate proportions of AF36 based on threshold values (Cq). Verification assays using pure DNA and conidial suspension mixtures demonstrated a significant relationship by regression analysis between known and qPCR-measured AF36 proportions in DNA (R2 = 0.974; P < 0.001) and conidia mixtures (R2 = 0.950; P < 0.001). The tests conducted by qPCR in pistachio leaves, nuts, and soil samples demonstrated the usefulness of the qPCR method to precisely quantify proportions of AF36 in diverse substrates, ensuring important time and cost savings. The outputs of the current study will serve to design better aflatoxin management strategies for pistachio and other crops.

scholarly journals Development and Evaluation of a TaqMan Real-Time PCR Assay for Fusarium oxysporum f. sp. spinaciae

Plant Disease ◽  
2013 ◽  
Vol 97 (7) ◽  
pp. 927-937 ◽  
Author(s):  
P. A. Okubara ◽  
L. A. Harrison ◽  
E. W. Gatch ◽  
G. Vandemark ◽  
K. L. Schroeder ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Real Time ◽  
Seed Production ◽  
Fusarium Wilt ◽  
Real Time Pcr ◽  
Soil Samples ◽  
Soilborne Pathogen ◽  
Pcr Assay ◽  
The Real ◽  
Pathogen Dna

Fusarium oxysporum f. sp. spinaciae, causal agent of spinach Fusarium wilt, is an important soilborne pathogen in many areas of the world where spinach is grown. The pathogen is persistent in acid soils of maritime western Oregon and Washington, the only region of the United States suitable for commercial spinach seed production. A TaqMan real-time polymerase chain reaction (PCR) assay was developed for rapid identification and quantification of the pathogen, based on sequencing the intergenic spacer (IGS) region of rDNA of isolates of the pathogen. A guanine single-nucleotide polymorphism (G SNP) was detected in the IGS sequences of 36 geographically diverse isolates of F. oxysporum f. sp. spinaciae but not in the sequences of 64 isolates representing other formae speciales and 33 isolates representing other fungal species or genera. The SNP was used to develop a probe for a real-time PCR assay. The real-time PCR assay detected F. oxysporum f. sp. spinaciae at 3–14,056 CFU/g of soil in 82 soil samples collected over 3 years from naturally infested spinach seed production sites in western Washington, although a reliable detection limit of the assay was determined to be 11 CFU/g of soil. A significant (P < 0.05), positive correlation between enumeration of F. oxysporum on Komada's agar and quantification of the pathogen using the TaqMan assay was observed in a comparison of 82 soil samples. Correlations between pathogen DNA levels, Fusarium wilt severity ratings, and spinach biomass were significantly positive for one set of naturally infested soils but not between pathogen DNA levels, wilt incidence ratings, and spinach biomass for other soil samples, suggesting that soilborne pathogen population is not the sole determinant of spinach Fusarium wilt incidence or severity. The presence of the G SNP detected in one isolate of each of F. oxysporum ff. spp. lageneriae, lilii, melongenae, and raphani and reaction of the real-time PCR assay with 16 of 22 nonpathogenic isolates of F. oxysporum associated with spinach plants or soil in which spinach had been grown potentially limits the application of this assay. Nonetheless, because all isolates of F. oxysporum f. sp. spinaciae tested positive with the real-time PCR assay, the assay may provide a valuable means of screening for resistance to Fusarium wilt by quantifying development of the pathogen in spinach plants inoculated with the pathogen.

scholarly journals Detection of the Downy Mildew Pathogen on Seed of Basil Following Field Infection in Southern New Jersey

2018 ◽  
Vol 28 (5) ◽  
pp. 637-641 ◽  
Author(s):  
Christian A. Wyenandt ◽  
Lisa R. Maimone ◽  
Kathryn Homa ◽  
Angela M. Madeiras ◽  
Robert L. Wick ◽  
...  
Keyword(s):  
New Jersey ◽  
Real Time ◽  
Downy Mildew ◽  
Real Time Pcr ◽  
Pathogen Detection ◽  
Agricultural Research ◽  
Pcr Analysis ◽  
Breeding Lines ◽  
Seed Collection ◽  
Seed Harvest

Different basils (Ocimum sp.) and cultivars (28 in 2009 and 32 in 2010) were evaluated for susceptibility to basil downy mildew (Peronospora belbahrii) at the Rutgers Agricultural Research and Extension Center near Bridgeton in southern New Jersey. At the end of each growing season, seed was collected from individual plants and stored for potential downy mildew pathogen detection using real-time polymerase chain reaction (PCR) analysis. Most of the basil cultivars and breeding lines were showing symptoms of basil downy mildew infection at the time of seed collection before the first frost near the end of the production season. Symptoms of basil downy mildew were present on 25 of the 28 (89%) basil lines evaluated in 2009 and 26 of 32 (81%) basil lines tested in 2010 at the time of seed harvest, with sporulation evident on the abaxial surface of infected leaves. Real-time PCR analysis of seed collected from various infected plants detected P. belbahrii on seed of 14 of 25 (56%) basil lines tested in 2009 and 8 of 32 (25%) tested in 2010. Importantly, P. belbahrii was not only detected on seed of sweet basil (Ocimum basilicum) phenotypes but also on seed of ‘Spice’ basil (Ocimum americanum) in 2009 and ‘Sweet Dani Lemon Basil’ basil (Ocimum citriodorum), ‘Holy Red and Green’ basil [Ocimum tenuiflorum (form. sanctum)], ‘Lime’ basil (O. americanum), and again on ‘Spice’ basil in 2010 where no symptoms (i.e., no chlorosis or sporulation) were present on the leaves when seed were collected. This work demonstrates that basil seed, regardless of basil species and whether symptoms are visible on foliage of the basil plant or the plant is immune or resistant to downy mildew, can test positive for the presence of P. belbahrii using a real-time PCR assay following exposure of plants to the pathogen during the natural development of downy mildew under field conditions.

Evaluation of Three Real-Time PCR Methods for Detection of Salmonella from Cloves

2017 ◽  
Vol 80 (6) ◽  
pp. 982-989 ◽  
Author(s):  
Aparna Tatavarthy ◽  
Laila Ali ◽  
Vikas Gill ◽  
Lijun Hu ◽  
Xiaohong Deng ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Real Time Pcr ◽  
Extraction Methods ◽  
Pcr Assay ◽  
Wide Range ◽  
The Mean ◽  
Salmonella Serotypes ◽  
Pathogen Dna ◽  
Dna Extraction Methods

ABSTRACTThe purpose of the study was to evaluate three real-time PCR platforms for rapid detection of Salmonella from cloves and to compare three different DNA extraction methods. Six trials were conducted with two clove cultivars, Ceylon and Madagascar, and three Salmonella serotypes, Montevideo, Typhimurium, and Weltevreden. Each trial consisted of 20 test portions. The preenrichment cultures were used to perform PCR for comparison of the effectiveness of U.S. Food and Drug Administration, Pacific Regional Laboratory Southwest (FDA-PRLSW), Applied Biosystems Inc. (ABI) MicroSEQ, and GeneDisc platforms for detection of Salmonella. Three DNA extraction methods were used: standard extraction method for each PCR platform, boil preparation, and LyseNow food pathogen DNA extraction cards. The results from real-time PCR correlated well with FDA Bacteriological Analytical Manual culture assay results, with a wide range of cycle threshold (CT) values among the three PCR platforms for intended positive samples. The mean CT values for MicroSEQ (16.36 ± 2.78) were significantly lower than for PRLSW (20.37 ± 3.45) and GeneDisc (23.88 ± 2.90) (P &lt; 0.0001). Pairwise comparisons between PCR platforms using different DNA extraction methods indicate that the CT values are inversely proportional to the relative DNA quantity (RDQ) yields by different platform-extraction combinations. The pairing of MicroSEQ and boil preparation generated the highest RDQ of 120 and the lowest average CT value of 14.48, whereas the pairing of GeneDisc and LyseNow generated the lowest RDQ of 0.18 and the highest average CT of 25.97. Boil preparation yielded higher RDQ than the other extraction methods for all three PCR platforms. Although the MicroSEQ platform generated the lowest CT values, its sensitivity was compromised by narrow separations between the positive and negative samples. The PRLSW platform generated the best segregation between positive and negative groups and is less likely to produce false results. In conclusion, FDA-PRLSW was the most efficient PCR assay for Salmonella detection, and boil preparation was the best method for DNA extraction.

scholarly journals A Real-Time PCR Assay for Detection and Quantification of Verticillium dahliae in Spinach Seed

2012 ◽  
Vol 102 (4) ◽  
pp. 443-451 ◽  
Author(s):  
Dechassa Duressa ◽  
Gilda Rauscher ◽  
Steven T. Koike ◽  
Beiquan Mou ◽  
Ryan J. Hayes ◽  
...  
Keyword(s):  
Real Time ◽  
Verticillium Wilt ◽  
Verticillium Dahliae ◽  
Real Time Pcr ◽  
The United States ◽  
Infected Seed ◽  
Polymerase Chain ◽  
Reliable Quantification ◽  
Pathogen Dna ◽  
Detection And Quantification

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.

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