Detection of Thielaviopsis basicola in soil with real-time quantitative PCR assays

Huang, Junli; Kang, Zhenhui

doi:10.1016/j.micres.2009.09.001

Cited by 24 publications

(18 citation statements)

References 17 publications

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“…These tools would also be valuable in future studies on the epidemiology and control of black‐foot disease of grapevines. Moreover, with the qPCR assay, it is possible to detect soilborne pathogens in soil samples in 4–5 h, avoiding laborious post‐PCR gel electrophoresis and reducing contamination which could be associated with nested PCRs (Huang & Kang, ; Huang et al ., ). Quantitative real‐time PCR is a tool with great value for rapid, specific and sensitive detection and diagnosis of black‐foot pathogens in soil.…”

Section: Discussionmentioning

confidence: 99%

Detection and quantification of Ilyonectria spp. associated with black‐foot disease of grapevine in nursery soils using multiplex nested PCR and quantitative PCR

2013

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Three nursery fields and three rootstock mother fields from commercial nurseries located in Comunidad Valenciana region (central-eastern Spain) were surveyed in July 2011 to detect the presence and to quantify Ilyonectria spp. in the soil. In each field, ten soil samples were taken randomly with a soil probe at a depth of 10-30 cm, and 10-20 cm from the base of the plant. Three replicate subsamples (10 g each) were taken from each soil sample. DNA was extracted and a multiplex nested PCR with species-specific primer pairs (Mac1/MaPa2, Lir1/Lir2 and Pau1/MaPa2) was used to identify the species present. Among the 180 soil DNA samples analysed, Ilyonectria spp. were detected in 172 of them. Ilyonectria macrodidyma complex was the most frequently detected, being identified in 141 samples from all the fields evaluated. However, I. liriodendri was detected in only 16 samples, but was present in all open-root field nurseries and in two rootstock mother fields. In addition, quantitative PCR (qPCR) assays were done to assess the levels of I. liriodendri and I. macrodidyma-complex DNA in the soil samples. Detection of Ilyonectria spp. DNA using qPCR correlated with the fields found positive with the nested multiplex PCR. DNA concentrations of Ilyonectria spp. ranged from 0Á004 to 1904Á8 pg lL À1 . In general, samples from rootstock mother fields showed the highest DNA concentrations. The ability to detect and quantify Ilyonectria spp. genomic DNA in soil samples from nursery fields and rootstock mother fields confirms soils from both field types as important inoculum sources for black-foot pathogens.

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Section: Discussionmentioning

confidence: 99%

Detection and quantification of Ilyonectria spp. associated with black‐foot disease of grapevine in nursery soils using multiplex nested PCR and quantitative PCR

2013

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“…Excitedly, foliar application of nMgO as nanoscale fertilizers or light absorption promoters significantly promoted the growth of several crops (Imada et al, 2016;Cai et al, 2018a). Typically, tobacco black shank and black root rot are widespread in Chongqing and significantly decrease tobacco quality and yield (Zhang et al, 2003;Huang and Kang, 2010). In addition, despite the high toxicity of nMgO on several phytopathogens, direct evidence for their role in the successful control of pathogen infection in vivo is still limited.…”

Section: Introductionmentioning

confidence: 99%

Comparative Study on the Fungicidal Activity of Metallic MgO Nanoparticles and Macroscale MgO Against Soilborne Fungal Phytopathogens

Chen

et al. 2020

Front. Microbiol.

134

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Engineered nanoparticles have provided a basis for innovative agricultural applications, specifically in plant disease management. In this interdisciplinary study, by conducting comparison studies using macroscale magnesium oxide (mMgO), we evaluated the fungicidal activity of MgO nanoparticles (nMgO) against soilborne Phytophthora nicotianae and Thielaviopsis basicola for the first time under laboratory and greenhouse conditions. In vitro studies revealed that nMgO could inhibit fungal growth and spore germination and impede sporangium development more efficiently than could macroscale equivalents. Indispensably, direct contact interactions between nanoparticles and fungal cells or nanoparticle adsorption thereof were found, subsequently provoking cell morphological changes by scanning electron microscopy/energy-dispersive spectrometry (SEM/EDS) and transmission electron microscopy (TEM). In addition, the disturbance of the zeta potential and accumulation of various modes of oxidative stress in nMgO-exposed fungal cells accounted for the underlying antifungal mechanism. In the greenhouse, approximately 36.58 and 42.35% decreases in tobacco black shank and black root rot disease, respectively, could testify to the efficiency by which 500 µg/ml of nMgO suppressed fungal invasion through root irrigation (the final control efficiency reached 50.20 and 62.10%, respectively) when compared with that of untreated controls or mMgO. This study will extend our understanding of nanoparticles potentially being adopted as an effective strategy for preventing diversified fungal infections in agricultural fields.

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“…Although this is not an improvement on the standard PCR it does have the advantage of allowing the pathogen levels to be quantified and this may be important for experiments investigating natural inoculum levels or pathogen quantity within wood. Further optimization of the qPCR and use of other more sensitive probe‐based detection technologies, such as TaqMan probes, may improve the detection sensitivity to levels such as the 0·1 pg recently described by Huang & Kang (2010) for Thielaviopsis basicola . However, as TaqMan probes are most optimal for amplicons <100 bp, transfer to a probe‐based system would only incorporate one of the two multi‐species primers and this may be insufficient to exclude amplification of non‐target species.…”

Section: Discussionmentioning

confidence: 99%

Detection of botryosphaeriaceous species in environmental samples using a multi‐species primer pair

et al. 2011

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Botryosphaeriaceous species are significant grapevine trunk pathogens worldwide, which can be difficult to identify to species level using conventional morphological methods. This study developed and optimized a quick, reliable molecular identification method that could facilitate investigations into the epidemiology of these diseases in vineyards. The multi-species primers, BOT100F and BOT472R, amplified a 371-372 bp portion of the rRNA gene region from the six botryosphaeriaceous species commonly found in New Zealand vineyards. In silico analysis indicated that they would amplify DNA from six of the 12 lineages of the Botryosphaeriaceae, including all of the main species pathogenic to grapevines. A detection sensitivity of 1 and 0AE1 pg DNA in standard and nested PCR, respectively, was achieved and this was calculated as equivalent to 2AE5 conidia. Validation of the primers for environmental samples showed that their specificity was not compromized by the presence of competing DNA templates extracted from wood and soil. Single stranded conformational polymorphism (SSCP) analysis of the amplicons could resolve Neofusicoccum australe, N. luteum, Diplodia mutila and D. seriata, but did not differentiate between N. parvum and N. ribis. The optimized PCR-SSCP was used to identify botryosphaeriaceous species present in rainwater traps collected over 1 year in a vineyard known to contain infected vines. It could detect multiple species in individual samples and demonstrated differences in the dispersal patterns of conidia from different species. Given the specificity and sensitivity of this method it could prove useful in epidemiology studies involving the numerous botryosphaeriaceous species that infect a wide range of host species.

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Detection of Thielaviopsis basicola in soil with real-time quantitative PCR assays

Cited by 24 publications

References 17 publications

Detection and quantification of Ilyonectria spp. associated with black‐foot disease of grapevine in nursery soils using multiplex nested PCR and quantitative PCR

Detection and quantification of Ilyonectria spp. associated with black‐foot disease of grapevine in nursery soils using multiplex nested PCR and quantitative PCR

Comparative Study on the Fungicidal Activity of Metallic MgO Nanoparticles and Macroscale MgO Against Soilborne Fungal Phytopathogens

Detection of botryosphaeriaceous species in environmental samples using a multi‐species primer pair

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