Rapid PCR-based assay for Sclerotinia sclerotiorum detection on soybean seeds

Grabicoski, Edilaine Maurícia Gelinski; Filho, David de Souza Jaccoud; Pileggi, Marcos; Henneberg, Luciane; Pierre, Marcelo Luiz Cunha; Vrisman, Claudio; Dabul, Audrei Nisio Gebieluca

doi:10.1590/0103-9016-2013-0395

Cited by 6 publications

(2 citation statements)

References 22 publications

(25 reference statements)

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“…Using primers developed by Freeman, Ward, Calderon, and McCartney (), S. sclerotiorum was detected in artificially inoculated soybean seeds, up to an incidence of 10% by conventional PCR and up to 1% by qPCR (Botelho et al, ). Until now, the minimum limit detected for this pathogen in soybean seeds was an incidence of 0.25% for conventional PCR, using the same primers (Grabicoski et al, ). In this study, using the TaqMan system proposed by Chen et al (), we obtained a higher sensitivity for the detection of the pathogen, up to an incidence level of 0.0625%, with good repeatability.…”

Section: Discussionmentioning

confidence: 99%

“…The molecular detection of Phomopsis spp. and S. sclerotiorum was done by the seed‐soaking method, in which 400 inoculated seeds, at the above‐mentioned ratios, were incubated at 20°C in closed bottles with 400 ml sterile water for 4 hr (Grabicoski et al, ). From the soaking solution, 1 ml aliquots was stored at −20°C and subjected to qPCR, under the same conditions as described above, separately for each pathogen.…”

Section: Methodsmentioning

confidence: 99%

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Quick and accurate detection of Sclerotiniasclerotiorum and Phomopsis spp. in soybean seeds using qPCR and seed‐soaking method

Ramiro

Ciampi‐Guillardi

Caldas

et al. 2019

Journal of Phytopathology

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Seed‐borne pathogenic fungi can cause serious damage to soybean crops by reducing the germination, vigour and emergence of the seeds. Special attention should be paid to pathogen detection in seeds to prevent its introduction in disease‐free areas. Considering the importance of rapid and successful diagnosis of seed‐borne pathogenic fungi in soybeans, this study evaluated a method to detect Sclerotinia sclerotiorum and Phomopsis spp. in seeds using quantitative polymerase chain reaction (qPCR). Naturally infested samples were subjected to detection using qPCR and blotter test, and the findings were compared. Using soybean seeds soaked in water, both pathogens were detected at an infestation level up a 0.0625% (one infected seed out of 1,599 healthy seeds) by qPCR. This technique allowed the detection of 300 fg of S. sclerotiorum and 30 fg of Phomopsis spp. DNA in the seed samples. Phomopsis spp. was detected in 40.7% of the evaluated seed batches (81 batches) and S. sclerotiorum was detected in 32.1% of the evaluated batches, although most of the seeds had low infestation levels. It was up to 28.5 times more efficient to use qPCR rather than blotter test to detect pathogens with a low incidence of occurrence in soybean seeds. If routinely used to test healthy seeds, qPCR would contribute to reducing soybean losses due to diseases as well as decreasing the costs required to control those diseases.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Quick and accurate detection of Sclerotiniasclerotiorum and Phomopsis spp. in soybean seeds using qPCR and seed‐soaking method

Ramiro

Ciampi‐Guillardi

Caldas

et al. 2019

Journal of Phytopathology

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Edaphic variables influence soil bacterial structure under successive fertilization of Paulownia plantation substituting native vegetation

Liu

Zwieten

et al. 2021

J Soils Sediments

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Multiplex qPCR Assay for Direct Detection and Quantification of Colletotrichum truncatum, Corynespora cassiicola, and Sclerotinia sclerotiorum in Soybean Seeds

et al. 2020

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Precise diagnosis of plant diseases is one of the most effective tools to minimize yield losses. Colletotrichum truncatum, Corynespora cassiicola, and Sclerotinia sclerotiorum are common soilborne pathogens that affect soybeans all over the world. We developed a multiplex quantitative real-time polymerase chain reaction (qPCR) assay to simultaneously detect and quantify the three pathogens in soybean seeds and to survey their occurrence in the main soybean production areas in Brazil. Species-specific primers and probes for C. truncatum and C. cassiicola were designed based on GAPDH and TEF1 genes, respectively, to be combined with qPCR detection of S. sclerotiorum previously reported. The multiplex qPCR assay was successful in the simultaneous detection of C. truncatum, C. cassiicola, and S. sclerotiorum, along with a host internal control. The four pathogens were detected and quantified in artificially and naturally infested soybean seeds, even in the lowest incidence level tested of 0.0625% or 1 infected seed out of 1,599 healthy ones. From 81 seed samples tested, C. truncatum was the most frequently detected pathogen and with higher incidence levels (0.25 to 0.125%), followed by S. sclerotiorum and C. cassiicola, both with lower incidence levels (0.125 to 0.0625%). Together, the results evidenced the high sensitivity of the multiplex qPCR assay, indicating its usefulness for a quick and reliable diagnosis of soybean diseases in seeds.

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Rapid PCR-based assay for Sclerotinia sclerotiorum detection on soybean seeds

Cited by 6 publications

References 22 publications

Quick and accurate detection of Sclerotiniasclerotiorum and Phomopsis spp. in soybean seeds using qPCR and seed‐soaking method

Quick and accurate detection of Sclerotiniasclerotiorum and Phomopsis spp. in soybean seeds using qPCR and seed‐soaking method

Edaphic variables influence soil bacterial structure under successive fertilization of Paulownia plantation substituting native vegetation

Multiplex qPCR Assay for Direct Detection and Quantification of Colletotrichum truncatum, Corynespora cassiicola, and Sclerotinia sclerotiorum in Soybean Seeds

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