Quantitative real-time PCR effectively detects and quantifies colonization of sclerotia of Sclerotinia sclerotiorum by Trichoderma spp.

Kim, Tae Gwan; Knudsen, G. R.

doi:10.1016/j.apsoil.2008.03.013

Cited by 32 publications

(27 citation statements)

References 42 publications

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“…As reported in literature, other primers have been described as successful for the detection of S. sclerotiorum for ascospores and in plant tissues infected by the pathogen (Yanni et al, 2009;Kim and Knudsen, 2008;Rogers et al, 2009). In this work these primers were tested in soybeans seeds inoculated with S. sclerotiorum but without success.…”

Section: Specificity Of the Primersmentioning

confidence: 99%

Detection of Sclerotinia sclerotiorum in soybean seeds by conventional and quantitative PCR techniques

Botelho

Barrocas²,

Machado

et al. 2015

J. Seed Sci.

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-Sclerotinia sclerotiorum, the etiological agent of the "white mould" in soybean, is responsible for severe losses in this crop and soil contamination. The introduction and dissemination of the disease can made through the use of seed lots contaminated with sclerotia and by seeds infected by mycelium. Therefore, seed health quality is one aspect to be monitored by means of health testing before to sowing time. In this study conventional and quantitative PCR techniques were used to assess their viability to detect S. sclerotiorum in artificially and naturally infected soybean seed samples. For that, seeds were inoculated by osmotic conditioning technique for 0, 24, 48 and 72 hours of contact of the seed with the fungal colony and mixed with healthy seeds generating incidence levels of 1, 2, 10, 20 and 100% for each incubation time. The cPCR was sensitive to detect S. sclerotiorum in samples with at least incidence 1% inoculated for 72 hours while the qPCR detected the pathogen in all incidence/inoculum potential combinations. The conventional PCR was able to detect 0.25% of the incidence of S. sclerotiorum in soybean seed lots naturally infected added a preincubation step.Index terms: seed healthy, molecular detection, seed borne pathogen, white mould.Detecção de Sclerotinia sclerotiorum em sementes de soja pelas técnicas de PCR convencional e quantitativo RESUMO -Sclerotinia sclerotiorum causador do "mofo branco" na cultura da soja, é responsável por redução da produção e contaminação do solo. A introdução e disseminação da doença podem se dar pelo uso de sementes contaminadas com escleródios e por sementes infectadas pelo micélio. A qualidade das sementes deve ser monitorada por testes de sanidade antes da semeadura. Neste estudo foram utilizadas técnicas de PCR convencional e quantitativo para avaliar a viabilidade de uso das mesmas para a detecção de S. sclerotiorum em amostras de sementes de soja artificialmente e naturalmente infectadas. Para isso, as sementes foram inoculadas usando a técnica do condicionamento osmótico por 0, 24, 48 e 72 horas de contato das sementes com a colônia do fungo e misturadas à sementes sadias gerando incidência de 1, 2, 10, 20 e 100% para cada tempo de incubação. A técnica de cPCR foi sensível para detectar S. sclerotiorum em amostras com, no mínimo, incidência de 1% inoculadas por 72 horas, enquanto que com o qPCR foi possível detectar o patógeno em todas as combinações incidência/potencial de inóculo. A PCR convencional foi capaz de detectar 0,25% de incidência de S. sclerotiorum em lotes de sementes de soja naturalmente infectados adicionando-se uma etapa de pré-incubação das sementes.Termos para indexação: sanidade de sementes, detecção molecular, patógeno de sementes, mofo branco.

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Section: Specificity Of the Primersmentioning

confidence: 99%

Detection of Sclerotinia sclerotiorum in soybean seeds by conventional and quantitative PCR techniques

Botelho

Barrocas²,

Machado

et al. 2015

J. Seed Sci.

View full text Add to dashboard Cite

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“…F. graminearum-specific (Fg16 N-F/R) and T. harzianumspecific (TGP4-F/R) primer sets used in this study were proposed by Nicholson et al (1998) and Kim and Knudsen (2008), respectively. The SmyITS primer set was designed based on ITS regions.…”

Section: Pcr Primers and Primer Designingmentioning

confidence: 99%

“…The finalized real-time PCR conditions for SmyITS-F/R primer set were (1) 3 min at 94°C, (2) 30 cycles of 30 s at 94°C, 30 s at 66°C, and 1 min at 72°C, and (3) 10 min at 72°C. For TGP4-F/R and Fg16 N-F/R primer sets, real-time PCR conditions were outlined in the study by Kim and Knudsen (2008) and Nicholson et al (1998), respectively. Real-time PCR amplifications of genomic DNAs (for S. mycoparasitica, T. harzianum, and F. graminearum 3-ADON) and total DNA extracted from spring wheat roots harvested at the mid-seedling growth (Zadok's growth stage 13) (Zadoks et al 1974) were carried out in MiniOpticon (Bio-Rad, Mississauga, ON, Canada).…”

Section: Validation Of Sphaerodes-specific Primer Setsmentioning

confidence: 99%

qPCR quantification of Sphaerodes mycoparasitica biotrophic mycoparasite interaction with Fusarium graminearum: in vitro and in planta assays

Vujanovic

Goh

2012

Arch Microbiol

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“…gene sequences allowed quantification of sclerotia of S. sclerotiorum by Trichoderma spp. in nonsterile soil [48]. The fact that T. harzianum strain ThzID1-M3 is phenotypically different (in that it expresses GFP) from other soil fungi (including other Trichoderma strains) and that also the corresponding novel gene is well characterized provided another avenue of ecological investigation, since the GFP gene (which is of jellyfish origin) provided a unique sequence to design a polymerase chain reaction (PCR) primer/probe set to exclusively amplify that sequence from ThzID1-M3 [49].…”

Section: Complex Soil Microbial Interactionsmentioning

confidence: 99%

Ecological Complexity and the Success of Fungal Biological Control Agents

Knudsen

Dandurand

2014

Advances in Agriculture

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Fungal biological control agents against plant pathogens, especially those in soil, operate within physically, biologically, and spatially complex systems by means of a variety of trophic and nontrophic interspecific interactions. However, the biocontrol agents themselves are also subject to the same types of interactions, which may reduce or in some cases enhance their efficacy against target plant pathogens. Characterization of these ecologically complex systems is challenging, but a number of tools are available to help unravel this complexity. Several of these tools are described here, including the use of molecular biology to generate biocontrol agents with useful marker genes and then to quantify these agents in natural systems, epifluorescence and confocal laser scanning microscopy to observe their presence and activity in situ, and spatial statistics and computer simulation modeling to evaluate and predict these activities in heterogeneous soil habitats.

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Quantitative real-time PCR effectively detects and quantifies colonization of sclerotia of Sclerotinia sclerotiorum by Trichoderma spp.

Cited by 32 publications

References 42 publications

Detection of Sclerotinia sclerotiorum in soybean seeds by conventional and quantitative PCR techniques

Detection of Sclerotinia sclerotiorum in soybean seeds by conventional and quantitative PCR techniques

qPCR quantification of Sphaerodes mycoparasitica biotrophic mycoparasite interaction with Fusarium graminearum: in vitro and in planta assays

Ecological Complexity and the Success of Fungal Biological Control Agents

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