Molecular Methods to Detect and Quantify Botryosphaeriaceae Inocula Associated With Grapevine Dieback in Australia

Billones‐Baaijens, Regina; Úrbez-Torres, J. R.; Liu, Meifang; Ayres, Matthew; Sosnowski, Mark; Savocchia, Sandra

doi:10.1094/pdis-11-17-1854-re

Cited by 32 publications

(16 citation statements)

References 32 publications

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“…The use of the highly sensitive qPCR arises as an extremely useful tool for studying various agents of infectious. In plant pathology, this technology is increasingly being used for studying various causal agents of plant diseases, including viruses (Poojari et al, 2016; Malandraki et al, 2017; Abdullah et al, 2018; Baaijens et al, 2018; Campos et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Establishment of a Sensitive qPCR Methodology for Detection of the Olive-Infecting Viruses in Portuguese and Tunisian Orchards

et al. 2019

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Sensitive detection of viruses in olive orchards is actually of main importance since these pathogenic agents cannot be treated, their dissemination is quite easy, and they can have eventual negative effects on olive oil quality. The work presented here describes the development and application of a new SYBR ® Green-based real-time quantitative PCR (qPCR) analysis for specific and reliable quantification of highly spread olive tree viruses: Olive latent virus 1 (OLV-1) , Tobacco necrosis virus D (TNV-D), Olive mild mosaic virus (OMMV), and Olive leaf yellowing-associated virus (OLYaV). qPCR methodology revealed high specificity and sensitivity, estimated in the range of 0.8–8 copies of the virus genome, for the studied viruses. For validation of the method, total RNA and double strand RNA (dsRNA) from naturally infected trees were used. In a first trial, dsRNAs from trees of cv. “Galega vulgar” from a Portuguese orchard, were subjected to qPCR and from the 30 samples tested, 26 were TNV-D and/or OMMV-positive and 25 were OLV-1 positive. In a second trial, total RNA from trees of different cultivars from Tunisian orchards, were here tested by qPCR and all viruses were detected. From the 33 samples studied, the most prevalent virus detected in Tunisia orchards was OLV-1 (31 samples diagnosed), followed by OLYaV (20 samples diagnosed), and finally the combination in last TNV-D and/or OMMV (12 samples diagnosed). In both trials, qPCR demonstrated to be effective and sensitive, even when using total RNA as template. qPCR through the use of a SYBR ® Green methodology enabled, for the first time, a reliable, sensitive, and reproducible estimation of virus accumulation in infected olive trees, in which viruses are usually in low titres, that will allow gaining new insights in virus biology essential for disease control and give an important contribution for establishment of sanitary certification of olive propagative material.

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Section: Discussionmentioning

confidence: 99%

Establishment of a Sensitive qPCR Methodology for Detection of the Olive-Infecting Viruses in Portuguese and Tunisian Orchards

et al. 2019

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“…This current study also represents the first study to use a combination of conventional plant pathology and molecular techniques to detect and quantify Botryosphaeriaceae DNA from artificially inoculated and naturally infected vines. The qPCR primers and probe used in this study [46] are not species-specific, therefore they cannot distinguish the Botryosphaeriaceae pathogen at the species level. However, the isolation of pathogens from naturally infected wood allowed the identification of Botryosphaeriaceae spp.…”

Section: Discussionmentioning

confidence: 99%

“…The qPCR assay using Botryosphaeriaceae multi-species primers and hydrolysis probe developed by Billones-Baaijens et al [46] were used to detect and quantify Botryosphaeriaceae spp. from artificially inoculated and naturally infected vines.…”

Section: Quantification Of Botryosphaeriaceae Spp From Wood Samples By Qpcrmentioning

confidence: 99%

“…To determine the amount of pathogen DNA that was amplified by each qPCR assay, previously developed standard curves [46] were imported in the Rotor-Gene Q software (Version 2.3.1). The standard (Bot-Btub gBlock, 500 pg) that was included in each qPCR assay was used to calibrate the imported standard curve and the resulting regression equations were used to quantify the number of Botryosphaeriaceae β-tubulin gene copies in each reaction as previously described by Billones-Baaijens et al [46] following the MIQE guidelines [66]. To calculate the number of copies of the Botryosphaeriaceae β-tubulin gene in each wood sample, the following formula was used:…”

Section: Quantification Of Botryosphaeriaceae Spp From Wood Samples By Qpcrmentioning

confidence: 99%

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Production of Phytotoxic Metabolites by Botryosphaeriaceae in Naturally Infected and Artificially Inoculated Grapevines

Reveglia

Billones‐Baaijens

Niem

et al. 2021

Plants

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Grapevine trunk diseases (GTDs) are considered a serious problem to viticulture worldwide. Several GTD fungal pathogens produce phytotoxic metabolites (PMs) that were hypothesized to migrate to the foliage where they cause distinct symptoms. The role of PMs in the expression of Botryosphaeria dieback (BD) symptoms in naturally infected and artificially inoculated wood using molecular and analytical chemistry techniques was investigated. Wood samples from field vines naturally infected with BD and one-year-old vines inoculated with Diplodia seriata, Spencermartinsia viticola and Dothiorella vidmadera were analysed by cultural isolations, quantitative PCR (qPCR) and targeted LC-MS/MS to detect three PMs: (R)-mellein, protocatechuic acid and spencertoxin. (R)-mellein was detected in symptomatic naturally infected wood and vines artificially inoculated with D. seriata but was absent in all non-symptomatic wood. The amount of (R)-mellein detected was correlated with the amount of pathogen DNA detected by qPCR. Protocatechuic acid and spencertoxin were absent in all inoculated wood samples. (R)-mellein may be produced by the pathogen during infection to break down the wood, however it was not translocated into other parts of the vine. The foliar symptoms previously reported in vineyards may be due to a combination of PMs produced and climatic and physiological factors that require further investigation.

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“…For example, F. euwallaceae from PSHB and F. kuroshium from KSHB were previously found to be completely identical at the ITS region (Na et al 2018). A target sequence that consists of a single-copy gene per haploid genome and has more polymorphic regions, such as the btubulin (TUB2) gene, has been shown to provide more reliable quantification of fungal DNA from different samples types in multiplex qPCR reaction conditions (Atallah et al 2007;Billones-Baaijens et al 2018;Pouzoulet et al 2013). The ability to detect and quantify fungal DNA using qPCR has been previously developed and applied to detect and quantify plant pathogens from plant tissue (Li et al 2006(Li et al , 2009Oliveira et al 2002;Qu et al 2011).…”

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confidence: 99%

Probe-Based Multiplex Real-Time PCR as a Diagnostic Tool to Distinguish Distinct Fungal Symbionts Associated With Euwallacea kuroshio and Euwallacea whitfordiodendrus in California

et al. 2020

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California has been invaded by two distinct Euwallacea spp. that vector unique plant pathogenic symbiotic fungi on multiple hosts and cause Fusarium dieback. The objective of this study was to develop multiplex real-time quantitative PCR assays using hydrolysis probes targeting the β-tubulin gene to detect, distinguish, and quantify fungi associated with the polyphagous shot hole borer (PSHB; Euwallacea whitfordiodendrus, Fusarium euwallaceae, Graphium euwallaceae, and Paracremonium pembeum) as well as the Kuroshio shot hole borer (KSHB; Euwallacea kuroshio, Fusarium kuroshium, and Graphium kuroshium) from various sample types. Absolute quantification reaction efficiencies ranged from 88.2 to 104.3%, with a coefficient of determination >0.992 and a limit of detection of 100 copies µl−1 for all targets across both assays. Qualitative detection using the real-time assays on artificially inoculated avocado shoot extracts showed more sensitivity compared with conventional fungal isolation from wood. All symbiotic fungi, except P. pembeum, from PSHB and KSHB female heads were detectable and quantified. Field samples from symptomatic Platanus racemosa, Populus spp., and Salix spp. across 17 of 26 city parks were positively identified as PSHB and KSHB through detection of their symbiotic fungi, and both were found occurring together on five trees from three different park locations. The molecular assays presented here can be utilized to accurately identify fungi associated with these invasive pests in California.

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Molecular Methods to Detect and Quantify Botryosphaeriaceae Inocula Associated With Grapevine Dieback in Australia

Cited by 32 publications

References 32 publications

Establishment of a Sensitive qPCR Methodology for Detection of the Olive-Infecting Viruses in Portuguese and Tunisian Orchards

Establishment of a Sensitive qPCR Methodology for Detection of the Olive-Infecting Viruses in Portuguese and Tunisian Orchards

Production of Phytotoxic Metabolites by Botryosphaeriaceae in Naturally Infected and Artificially Inoculated Grapevines

Probe-Based Multiplex Real-Time PCR as a Diagnostic Tool to Distinguish Distinct Fungal Symbionts Associated With Euwallacea kuroshio and Euwallacea whitfordiodendrus in California

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