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

Botelho, Luana da Silva; Barrocas, Ellen Noly; Machado, José da Cruz; Martins, Rayana de Sá

doi:10.1590/2317-1545v37n1141460

Cited by 11 publications

(11 citation statements)

References 22 publications

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“…These results reinforce what was reported in other pathosystems, such as Sclerotinia sclerotiorum and Colletotrichum truncatum in soybean (Botelho et al, 2013;Machado et al, 2001) and Fusarium oxysporum f. sp. phaseoli in dry edible bean seeds (Costa et al, 2003).…”

Section: Physiological Performance Of the Inoculated Seeds And Quantisupporting

confidence: 90%

“…Machado (1988) affirms that the damage brought about by the presence of pathogens may be related to the inoculum potential, as well as to the location of the pathogens in the seed. Studies using the technique of inoculation by water restriction show the direct relation between the inoculum potentials and the damage brought about in the seeds (Costa et al, 2003;Botelho et al, 2013;Siqueira et al, 2014) According to the data obtained in the qPCR for S. maydis, the relative efficiency of the curve was 0.79, which was determined by the linear regression equation with correlation coefficient (R²) of 0.99 (Figure 2A). From the standard curve and the Ct values obtained in the reaction, it was possible to quantify the DNA present in the inoculated seeds.…”

Section: Physiological Performance Of the Inoculated Seeds And Quantimentioning

confidence: 99%

“…Physiological quality is related to the ability of seeds to exercise their vital functions and express their potential, and is characterized by germination, dormancy, and vigor. Therefore, the effects of pathogens on seed quality are generally reflected in a decrease in germination percentage, reduced stand, increase in abnormal plants, reduction in seedling vigor, and, consequently, decreased yield (Toledo et al, 2009, Botelho et al, 2013.…”

Section: Introductionmentioning

confidence: 99%

“…The most common method used for detection of necrotrophic fungi in general has been the blotter test. However, currently, the real-time polymerase chain reaction (qPCR) technique has been an important tool to assist in these methods because of its precision and specificity (Konstantinova et al, 2002;Gao et al, 2004;Barros et al, 2008;Duressa et al, 2012;Botelho et al, 2015;Sousa et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of inoculum potential of pathogens in seeds: relation to physiological quality and DNA quantification by qPCR

et al. 2017

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-Given what is already known in regard to seed health and the availability of molecular methods for detection of the pathogens Stenocarpella maydis and Stenocarpella macrospora in maize seeds, Colletotrichum gossypii var. cephalosporioides in cotton seeds, and Corynespora cassiicola in soybean seeds, the aim of this study was to evaluate seed vigor according to different inoculum potentials. The fungus isolates were inoculated on seeds by the technique of water restriction, through which different inoculum potentials are obtained, corresponding to times of seed exposure of 0, 24, 48, and 96 hours for maize and cotton seeds, and 0, 36, 108, and 144 hours for soybean seeds. The seeds were subjected to germination, electrical conductivity, health, and qPCR tests. Results of the blotter test showed that in most pathosystems, there was a higher incidence of the fungi with an increase in inoculum potential. A decrease in germination percentage was observed in all species as inoculum potential increased, as well as further degradation of seed membranes. The qPCR test confirmed that the most damaged seeds in the tests had higher presence of the pathogens.Index terms: seed pathology, fungi, maize, soybean, cotton.Avaliação do potencial de inóculo de patógenos em sementes: sua relação com a qualidade fisiológica e quantificação do DNA por qPCR RESUMO -Diante do que já se conhece em sanidade de sementes e tendo-se em mãos métodos moleculares para a detecção dos fungos Stenocarpella maydis, Stenocarpella macrospora em sementes de milho, Colletotrichum gossypii var. cephalosporioides em algodão e Corynespora cassiicola em soja, objetivou-se neste estudo avaliar o vigor das sementes em função dos diferentes potenciais de inóculo. Os isolados dos fungos foram inoculados nas sementes por meio da técnica de restrição hídrica pela qual se obtém diferentes potenciais de inóculo, que foram representados por P0, P24, P48 e P96 e P0, P36, P108 e P144, que correspondem à exposição das sementes aos períodos de tempo de 0, 24, 48, 96 horas (milho e algodão) e nos tempos 0, 36, 108 e 144 horas (soja), respectivamente. As sementes foram submetidas aos testes de germinação, condutividade elétrica, sanidade e qPCR. Pelo blotter test, na maioria dos patossistemas, houve maior incidência do fungo com o aumento do potencial de inóculo. Foi observada uma queda na porcentagem de germinação de todas as espécies com o aumento do potencial de inóculo, assim como maior degradação das membranas das sementes. A qPCR confirmou que nas sementes mais prejudicadas havia maior quantidade de inóculo dos patógenos.Termos para indexação: patologia de sementes, fungos, milho, soja, algodão.

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Section: Physiological Performance Of the Inoculated Seeds And Quantisupporting

confidence: 90%

Section: Physiological Performance Of the Inoculated Seeds And Quantimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Evaluation of inoculum potential of pathogens in seeds: relation to physiological quality and DNA quantification by qPCR

et al. 2017

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“…Through the polymerase chain reaction (PCR) technique, conventional and/or quantitative, pathogens were successfully detected in seeds by molecular means in the pathosystems Fusarium oxysporum f. sp. phaseoli in common bean seeds (Sousa et al, 2015), Colletotrichum lindemuthianum in common bean seeds (Chen et al, 2007(Chen et al, , 2013, Sclerotinia sclerotiorum in soybean seeds (Botelho et al, 2015), Rhynchosporium secalis in barley seeds (Ríos et al, 2007), Peronospora variabilis in quinoa (Testen et al, 2014), and Macrophomina phaseolina in slender amaranth (Biswas et al, 2013), among others.…”

Section: Introductionmentioning

confidence: 99%

Conventional PCR for detection of Corynespora cassiicola in soybean seeds

Sousa

Siqueira²,

Machado³

2016

J. Seed Sci.

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-The fungus Corynespora cassiicola, causal agent of target spot in soybeans, can be transmitted by soybean seeds and as of that point cause severe damage. This disease may be diagnosed at an early stage by seed testing, but knowledge in this area is insufficient. Because of that and increased attack by the disease in soybean areas in Brazil, further studies are required. The aim of this study was to evaluate the use of conventional PCR in detecting C. cassiicola in soybean seeds. The GA4-F/GA4-R primers described in the literature were tested for their specificity and sensitivity for detection of C. cassiicola in pure culture and in soybean seeds. Uninoculated and inoculated seed samples were used with different incidence levels -100%, 10%, 1%, 0.5%, 0.25%, and 0% of preestablished inoculum potentials, P0, P1, P2, and P3. Detection of C. cassiicola in P1 inoculum potential was observed in samples with incidence levels of 10% to 100%. In the P3 potential, detection of the pathogen was successful in samples at the low level of 0.25%.Index terms: seed pathology, target spot, electrophoresis.Uso de PCR convencional para a detecção de Corynespora cassiicola em sementes de soja RESUMO -O fungo Corynespora cassiicola, agente causal da mancha alvo em soja, pode ser transmitido por sementes de soja e a partir daí causar danos severos. A diagnose precoce desta doença pode ser realizada por meio de testes de sementes, cujo conhecimento é insuficiente. Por esta razão e pelo aumento da área de soja atacada pela referida doença no Brasil, estudos mais aprofundados são necessários. Neste trabalho, o objetivo foi avaliar o uso da técnica PCR convencional na detecção de C. cassiicola em amostras de sementes de soja. Os primers GA4-F/GA4-R, relatados na literatura, foram testados para verificar a sua especificidade e sensibilidade para detecção de C. cassiicola em cultura pura e em sementes de soja. Foram utilizadas amostras de sementes, inoculadas e não inoculadas, com diferentes níveis de incidência, de 100%, 10%, 1%, 0,5%, 0,25% e 0%, por potenciais de inóculo preestabelecidos, P0, P1, P2 e P3. A detecção de C. cassiicola no potencial de inóculo P1, foi observada em amostras com níveis de incidência de 10% a 100%. No potencial P3, a detecção do patógeno foi bem-sucedida em amostras com nível mínimo de 0,25%.Termos para indexação: patologia de sementes, mancha alvo, eletroforese.

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qPCR-based detection of Colletotrichum truncatum in soybean seeds

Júnior

Resende

Pozza

et al. 2020

Trop. plant pathol.

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Soybean seed infected with Colletotrichum truncatum is an important source of primary inoculum for anthracnose epidemics. Based on differences in the GAPDH gene sequences of Colletotrichum species, one pair of species-specific primers, CtruncF1/ CtruncR1, was designed to accurately detect C. truncatum in soybean seed samples. The primers amplified only a single PCR band of 211 bp from C. truncatum. SYBR Green qPCR using these primers enabled the detection of DNA of the target fungus in inoculated soybean seeds and in naturally infested seeds. The sensitivity of the method was 0.000253 ng/μL of C. truncatum DNA template, with an efficiency of 1.78 and a Ct of 30.09. These species-specific primers may be useful for certification of soybean seeds aimed at avoiding introduction of the pathogen into soybean-producing regions.

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

Cited by 11 publications

References 22 publications

Evaluation of inoculum potential of pathogens in seeds: relation to physiological quality and DNA quantification by qPCR

Evaluation of inoculum potential of pathogens in seeds: relation to physiological quality and DNA quantification by qPCR

Conventional PCR for detection of Corynespora cassiicola in soybean seeds

qPCR-based detection of Colletotrichum truncatum in soybean seeds

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