Disease risk assessment of sugar beet root rot using quantitative real-time PCR analysis of Aphanomyces cochlioides in naturally infested soil samples

Almquist, Charlotta; Persson, Lars Erik; Olsson, Åsa; Sundström, Jens F.; Jönsson, Anders

doi:10.1007/s10658-016-0862-5

Cited by 18 publications

(29 citation statements)

References 31 publications

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“…The species-specific primers, listed in Table 2 , were used to analyze the level of natural infestation in 429 soil samples from two Swedish regions with intensive farming. The soil samples had all high clay content, representing characteristics as in soil mixture number four analyzed by Almquist et al ( 2016 ). A study showing that the limit of DNA detection was lowest in samples with high clay content using similar soil handling and DNA extraction methodology as presented here.…”

Section: Resultsmentioning

confidence: 93%

Detection of Verticillium species in Swedish soils using real-time PCR

et al. 2017

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Verticillium species are soilborne plant pathogens, responsible for big yield losses worldwide. Here, we report improved procedures to generate DNA from Verticillium species imbedded in farm soils. Using new genomic sequence information, primers for V. dahliae, V. albo-atrum, V. tricorpus, and V. longisporum were designed. In a survey of 429 samples from intensively farmed soil of two Swedish regions, only V. dahliae and V. longisporum were identified. A bias towards V. longisporum (40%) was seen in the south, whereas V. dahliae was more frequent in the western region (19%). Analyses of soil and leaf samples from 20 sugar beet fields, where foliar wilting had been observed, revealed V. dahliae DNA in all leaf and soil samples and V. longisporum in 18 soil samples, illustrating host choice and longevity of the V. longisporum microsclerotia. This study demonstrates the applicability of new molecular diagnostic tools that are important for growers of variable crops.

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Section: Resultsmentioning

confidence: 93%

Detection of Verticillium species in Swedish soils using real-time PCR

et al. 2017

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“…The detection limit for the P. tracheiphilum multiplexed qPCR assay was established as 10 oospores/g of dry soil, with coefficients of variation less than 2%. In comparison, the detection limit was determined to be between 1 and 50 oospores/g of soil (depending on soil clay content) for Aphanomyces cochlioides (Almquist et al 2016), 10 oospores/g of soil for A. euteiches (Gangneux et al 2014), and 20 oospores/g of soil for Phytophthora infestans (Hussain et al 2005).…”

Section: Discussionmentioning

confidence: 99%

“…These results suggest that the development of a risk index based on inoculum potential measured by real-time qPCR would be possible. The estimation of inoculum potential by real-time qPCR was proven to be successful for several soilborne diseases such as those caused by A. euteiches (Gangneux et al 2014;Sauvage et al 2007), A. cochlioides (Almquist et al 2016), and Plasmodiophora brassicae (Wallenhammar et al 2012).…”

Section: Discussionmentioning

confidence: 99%

Detection and Quantification of Pythium tracheiphilum in Soil by Multiplex Real-Time qPCR

et al. 2019

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In Canada, head lettuce (Lactuca sativa capitata) is extensively produced in the muck soils of southwestern Québec. However, yields are increasingly affected by various soilborne pathogens, including Pythium spp., which cause wilt and damping off. In a survey conducted in Québec muck soils in 2010 and 2011, Pythium tracheiphilum Matta was identified as the predominant Pythium sp. in the root of head lettuce showing Pythium stunt symptoms. Therefore, to improve risk assessment and help further understanding of disease epidemiology, a specific and sensitive real-time quantitative polymerase chain reaction (qPCR) assay based on TaqMan-minor groove binder (MGB) technology was developed for P. tracheiphilum. The PCR primers along with a TaqMan-MGB probe were designed from the ribosomal internal transcribed spacer 2 region. A 100-bp product was amplified by PCR from all P. tracheiphilum isolates tested while no PCR product was obtained from 38 other Pythium spp. or from a selection of additional lettuce pathogens tested. In addition to P. tracheiphilum, the assay was multiplexed with an internal control allowing for the individual validation of each PCR. In artificially infested soils, the sensitivity of the qPCR assay was established as 10 oospores/g of dry soil. P. tracheiphilum was not detected in soils in which lettuce has never been grown; however, inoculum ranged from 0 to more than 200,000 oospores/g of dry soil in commercial lettuce fields. Also, disease incidence was positively correlated with inoculum concentration (r = 0.764). The results suggest that inoculum concentration should be considered when making Pythium stunt management decisions. The developed qPCR assay will facilitate reliable detection and quantification of P. tracheiphilum from field soil.

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“…Because of the limitations posed by these methods, real-time qPCR has become the method of choice for detection of phytopathogenic fungi, bacteria and oomyctes in diverse plant tissues (reviewed in Sanzani et al 2014 ) and other environmental samples such as soil (e.g., Almquist et al 2016 , Zhu et al 2016 ). Recently, qPCR was used to quantify growth patterns of Hymenoscyphus fraxineus (T. Kowalski) Baral et al, the causative agent of ash dieback, in stems of Fraxinus excelsior trees, demonstrating the utility of this technology to understand pathogen invasion of the host tree ( Matsiakh et al 2016 ).…”

Section: Discussionmentioning

confidence: 99%

A novel application of RNase H2-dependent quantitative PCR for detection and quantification of Grosmannia clavigera, a mountain pine beetle fungal symbiont, in environmental samples

et al. 2018

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Mountain pine beetle (Dendroctonus ponderosae Hopkins; MPB) is an economically and ecologically important pest of pine species in western North America. Mountain pine beetles form complex multipartite relationships with microbial partners, including the ophiostomoid fungi Grosmannia clavigera (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Ophiostoma montium (Rumbold) von Arx, Grosmannia aurea (Robinson-Jeffrey and Davidson) Zipfel, de Beer and Wingfield, Leptographium longiclavatum (Lee, Kim, and Breuil) and Leptographium terebrantis (Barras and Perry). These fungi are vectored by MPB to new pine hosts, where the fungi overcome host defenses to grow into the sapwood. A tree’s relative susceptibility to these fungi is conventionally assessed by measuring lesions that develop in response to fungal inoculation. However, these lesions represent a symptom of infection, representing both fungal growth and tree defense capacity. In order to more objectively assess fungal virulence and host tree susceptibility in studies of host–pathogen interactions, a reliable, consistent, sensitive method is required to accurately identify and quantify MPB-associated fungal symbionts in planta. We have adapted RNase H2-dependent PCR, a technique originally designed for rare allele discrimination, to develop a novel RNase H2-dependent quantitative PCR (rh-qPCR) assay that shows greater specificity and sensitivity than previously published PCR-based methods to quantify MPB fungal symbionts in pine xylem and MPB whole beetles. Two sets of assay probes were designed: one that amplifies a broad range of ophiostomoid species, and a second that amplifies G. clavigera but not other MPB-associated ophiostomoid species. Using these primers to quantify G. clavigera in pine stems, we provide evidence that lesion length does not accurately reflect the extent of fungal colonization along the stem nor the quantity of fungal growth within this colonized portion of stem. The sensitivity, specificity, reproducibility, cost effectiveness and high-throughput potential of the rh-qPCR assay makes the technology suitable for identification and quantification of a wide array of pathogenic and beneficial microbes that form associations with plants and other organisms, even when the microbial partner is present in low abundance.

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Disease risk assessment of sugar beet root rot using quantitative real-time PCR analysis of Aphanomyces cochlioides in naturally infested soil samples

Cited by 18 publications

References 31 publications

Detection of Verticillium species in Swedish soils using real-time PCR

Detection of Verticillium species in Swedish soils using real-time PCR

Detection and Quantification of Pythium tracheiphilum in Soil by Multiplex Real-Time qPCR

A novel application of RNase H2-dependent quantitative PCR for detection and quantification of Grosmannia clavigera, a mountain pine beetle fungal symbiont, in environmental samples

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