scholarly journals A Comparison of Real-Time PCR Protocols for the Quantitative Monitoring of Asymptomatic Olive Infections by Verticillium dahliae Pathotypes

2013 ◽  
Vol 103 (10) ◽  
pp. 1058-1068 ◽  
Author(s):  
D. Gramaje ◽  
V. Pérez-Serrano ◽  
M. Montes-Borrego ◽  
J. A. Navas-Cortés ◽  
R. M. Jiménez-Díaz ◽  
...  
Keyword(s):  
Real Time ◽  
Verticillium Wilt ◽  
Verticillium Dahliae ◽  
Target Gene ◽  
Quantitative Polymerase Chain Reaction ◽  
Intergenic Spacer ◽  
In Planta ◽  
Development Of Resistance ◽  
Planting Material ◽  
Detection And Quantification

Early, specific, and accurate in planta detection and quantification of Verticillium dahliae are essential to prevent the spread of Verticillium wilt in olive using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, specificity, and efficiency of eight real-time quantitative polymerase chain reaction protocols published since 2002 for the specific detection and quantification of V. dahliae in various host plant species and in soil, using a background of DNAs extracted from olive roots, stems, and leaves. Results showed that some of those protocols were not specific for V. dahliae or were inhibited when using backgrounds other than water. Ranking of protocols according to a weighted score system placed protocols TAQ (based on intergenic spacer ribosomal DNA target gene) and SYBR-4 (based on the β-tubulin 2 target gene) first in sensitivity and efficiency for the quantification of V. dahliae DNA in small amounts and different types of olive tissues (root and stem) tested. Use of TAQ and SYBR-4 protocols allowed accurate quantification of V. dahliae DNA regardless of the background DNA, with a detection limit being fixed at a cycle threshold of 36 (≈18 fg for SYBR-4 and 15 fg for TAQ) of V. dahliae. The amount of DNA from defoliating (D) and nondefoliating (ND) V. dahliae pathotypes was monitored in Verticillium wilt-resistant ‘Frantoio’ olive using the TAQ and SYBR-4 protocols. In the infection bioassay, higher amounts of D V. dahliae DNA were measured in olive stems, whereas the average amount of fungal DNA in roots was higher for ND-infected plants than D-infected ones. Overall, V. dahliae DNA amounts in all olive tissues tested tended to slightly decrease or remain stable by the end of the experiment (35 days after inoculation). The SYBR-4 and TAQ protocols further enabled detection of V. dahliae in tissues of symptomless plants, suggesting that both techniques can be useful for implementing certification schemes of pathogen-free planting material as well as helpful tools in breeding resistance to V. dahliae in olive.

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.

scholarly journals Multiplex Real-Time Quantitative PCR to Detect and Quantify Verticillium dahliae Colonization in Potato Lines that Differ in Response to Verticillium Wilt

2007 ◽  
Vol 97 (7) ◽  
pp. 865-872 ◽  
Author(s):  
Z. K. Atallah ◽  
J. Bae ◽  
S. H. Jansky ◽  
D. I. Rouse ◽  
W. R. Stevenson
Keyword(s):  
Real Time ◽  
Verticillium Wilt ◽  
Verticillium Dahliae ◽  
Quantitative Polymerase Chain Reaction ◽  
Potato Production ◽  
Potato Early Dying ◽  
Breeding Lines ◽  
Real Time Quantitative Pcr ◽  
Polymerase Chain ◽  
Early Dying

Potato early dying (PED), also known as Verticillium wilt, caused by Verticillium dahliae, is a seasonal yield-limiting disease of potato worldwide, and PED-resistant cultivars currently represent only a small percentage of potato production. In this study, we developed a real-time quantitative polymerase chain reaction (Q-PCR) approach to detect and quantify V. dahliae. The efficiency of the designed primer pair VertBt-F/VertBt-R, derived from the sequence of the β-tubulin gene, was greater than 95% in monoplex Q-PCR and duplex (using Plexor technology) procedures with primers PotAct-F/PotAct-R, obtained from the sequence of the actin gene, designed for potato. As few as 148 fg of V. dahliae DNA were detected and quantified, which is equivalent to five nuclei. Q-PCR detected V. dahliae in naturally infected air-dried potato stems and fresh stems of inoculated plants. Spearman correlations indicated a high correlation (upward of 80%) between V. dahliae quantifications using Q-PCR and the currently used plating assays. Moreover, Q-PCR substantially reduced the variability compared with that observed in the plating assay, and allowed for the detection of V. dahliae in 10% of stem samples found to be pathogen free on the culture medium. The described Q-PCR approach should provide breeders with a more sensitive and less variable alternative to time-consuming plating assays to distinguish response of breeding lines to colonization by V. dahliae.

scholarly journals Real-Time Quantitative Expression Studies of the Zearalenone Biosynthetic Gene Cluster in Fusarium graminearum

2009 ◽  
Vol 99 (2) ◽  
pp. 176-184 ◽  
Author(s):  
Erik Lysøe ◽  
Karen R. Bone ◽  
Sonja S. Klemsdal
Keyword(s):  
Real Time ◽  
Gene Cluster ◽  
Polyketide Synthase ◽  
Quantitative Polymerase Chain Reaction ◽  
Alcohol Oxidase ◽  
Infection Process ◽  
Β Subunit ◽  
In Planta ◽  
Expression Study ◽  
Fusarium Spp

The estrogenic mycotoxin zearalenone (ZON) produced by some Fusarium spp. causes reproductive problems and hyperestrogenic syndromes in mammals. In an effort to elucidate the molecular pathways of ZON production, we present a comparative real-time quantitative polymerase chain reaction expression study of seven contiguous genes in the ZON biosynthetic cluster on sterile rice and during wheat and oat infection. Under ZON production on rice, the polyketide synthase (PKS) genes PKS4 and PKS13, alcohol oxidase FG12056 gene, and transcriptional regulator FG02398 gene showed similarly upregulated patterns, whereas the nonribosomal peptide synthetase (NPS) FG02394, the K+ channel β subunit FG12015, and the protein kinase FG02399 displayed a variant pattern. During the same time period under wheat infection when no ZON was produced, the PKS genes and the NPS were downregulated relative to rice whereas the K+ channel β subunit gene FG12015 was markedly upregulated, suggesting that it may play a role in the infection process. This is the first expression study of ZON biosynthetic genes in planta. The results give insight into the regulation and activities of the ZON gene cluster under different experimental systems and suggest a connection between ZON and a K+ channel that could reveal a novel function for ZON in Fusarium spp.

Development of real-time PCR (TaqMan®) assays for the detection and quantification of Botrytis cinerea in planta

2005 ◽  
Vol 43 (9) ◽  
pp. 890-899 ◽  
Author(s):  
M. Belen Suarez ◽  
Kathryn Walsh ◽  
Neil Boonham ◽  
Tim O’Neill ◽  
Simon Pearson ◽  
...  
Keyword(s):  
Real Time ◽  
Botrytis Cinerea ◽  
Real Time Pcr ◽  
In Planta ◽  
Detection And Quantification

scholarly journals Verticillium dahliae effector VDAL protects MYB6 from degradation by interacting with PUB25/26 E3 ligases for enhancing Verticillium wilt resistance in Arabidopsis

2021 ◽  
Author(s):  
Zhizhong Gong ◽  
Junsheng Qi ◽  
Aifang Ma ◽  
Dingpeng Zhang ◽  
Guangxing Wang ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Verticillium Wilt ◽  
Verticillium Dahliae ◽  
Plant Resistance ◽  
Plant Disease Resistance ◽  
Plant Disease ◽  
Effector Proteins ◽  
In Planta ◽  
E3 Ligases

Verticillium wilt is a severe plant disease, increasing the plant resistance to this disease is a critical challenge worldwide. Here, we report that the Verticillium dahliae (V. dahliae)-secreted Aspf2-like protein VDAL causes leaf wilting when applied to cotton leaves in vitro, but enhances the resistance to V. dahliae when overexpressed in Arabidopsis or cotton. VDAL interacts with Arabidopsis E3 ligases PUB25 and PUB26 (PUBs) and is ubiquitinated by PUBs in vitro. However, VDAL is not degraded by PUBs in planta. Besides, the pub25 pub26 shows higher resistance to V. dahliae than the wild type. PUBs interact with the transcription factor MYB6 in a yeast two-hybrid screen. MYB6 promotes plant resistance to Verticillium wilt while PUBs ubiquitinate MYB6 and mediate its degradation. VDAL competes with MYB6 for binding to PUBs, and the role of VDAL in increasing wilt disease depends on MYB6. These results suggest that plants evolute a strategy to utilize the invaded effector protein VDAL to resist the V. dahliae infection without causing a hypersensitive response. This study provides the molecular mechanism for plants increasing disease resistance when overexpressing some effector proteins, and may promote searching for more genes from pathogenic fungi or bacteria to engineer plant disease resistance.

scholarly journals Detection and Quantification of Airborne Claviceps purpurea sensu lato Ascospores from Hirst-Type Spore Traps using Real-Time Quantitative PCR

Plant Disease ◽  
2018 ◽  
Vol 102 (12) ◽  
pp. 2487-2493 ◽  
Author(s):  
Jeremiah K.S. Dung ◽  
Jeness C. Scott ◽  
Qunkang Cheng ◽  
Stephen C. Alderman ◽  
Navneet Kaur ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Management Approach ◽  
Conidial Suspension ◽  
Claviceps Purpurea ◽  
Spore Trap ◽  
Grass Seed ◽  
Wide Range ◽  
Detection And Quantification

The U.S. Pacific Northwest states of Oregon and Washington are major producers of cool-season grass seed. Ergot, caused by fungi in the Claviceps purpurea sensu lato group, is an important seed replacement disease of grass worldwide. Microscopic methods that are currently used to quantify airborne Claviceps ascospores captured by spore traps are not currently rapid enough to allow for detecting and reporting of spore numbers in a timely manner, hindering growers from using this information to help manage ergot. We developed a SYBR Green real-time quantitative polymerase chain reaction (qPCR)-based assay for fast and efficient detection and quantification of C. purpurea sensu lato ascospores from Hirst-type spore traps. Species-specificity of the qPCR assay was confirmed against 41 C. purpurea sensu lato isolates collected from six hosts and six other Claviceps spp. Significant relationships were observed between cycle threshold (Ct) values and standard curves of serial dilutions of DNA ranging from 1 pg to 10 ng (R2 = –0.99; P = 0.0002) and DNA extracted from a conidial suspension representing 8 to 80,000 conidia (R2 = –0.99; P = 0.0004). Ct values from qPCR were significantly correlated with results from microscopic examination of spore trap samples from the field (r = –0.68; P < 0.0001) and the procedure was able to detect a single ascospore from spore trap tape samples. The qPCR procedure developed in this study provided a means for quantifying airborne Claviceps ascospores that was highly specific and useful over a wide range of spore densities, and could be performed in a matter of hours instead of days. The qPCR assay developed in this study could be part of an integrated pest management approach to help grass seed growers make risk-based fungicide application decisions for ergot management in grass grown for seed.

scholarly journals Development of an Assay for Rapid Detection and Quantification of Verticillium dahliae in Soil

2012 ◽  
Vol 102 (3) ◽  
pp. 331-343 ◽  
Author(s):  
Guillaume J. Bilodeau ◽  
Steven T. Koike ◽  
Pedro Uribe ◽  
Frank N. Martin
Keyword(s):  
Real Time ◽  
Verticillium Dahliae ◽  
Internal Control ◽  
Real Time Pcr ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Single Copy ◽  
Taqman Assay ◽  
Wide Range ◽  
Pathogen Populations

Verticillium dahliae is responsible for Verticillium wilt on a wide range of hosts, including strawberry, on which low soil inoculum densities can cause significant crop loss. Determination of inoculum density is currently done by soil plating but this can take 6 to 8 weeks to complete and delay the grower's ability to make planting decisions. To provide a faster means for estimating pathogen populations in the soil, a multiplexed TaqMan real-time polymerase chain reaction (PCR) assay based on the ribosomal DNA (rDNA) intergenic spacer (IGS) was developed for V. dahliae. The assay was specific for V. dahliae and included an internal control for evaluation of inhibition due to the presence of PCR inhibitors in DNA extracted from soil samples. An excellent correlation was observed in regression analysis (R2 = 0.96) between real-time PCR results and inoculum densities determined by soil plating in a range of field soils with pathogen densities as low as 1 to 2 microsclerotia/g of soil. Variation in copy number of the rDNA was also evaluated among isolates by SYBR Green real-time PCR amplification of the V. dahliae-specific amplicon compared with amplification of several single-copy genes and was estimated to range from ≈24 to 73 copies per haploid genome, which translated into possible differences in results among isolates of ≈1.8 cycle thresholds. Analysis of the variation in results of V. dahliae quantification among extractions of the same soil sample indicated that assaying four replicate DNA extractions for each field sample would provide accurate results. A TaqMan assay also was developed to help identify colonies of V. tricorpus on soil plates.

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