Resumo -O objetivo deste trabalho foi avaliar a eficiência dos métodos de detecção de Sclerotinia sclerotiorum em sementes de soja. Foram realizados experimentos durante três safras, tendo-se utilizado amostras de sementes de soja contaminadas artificial e naturalmente, com incidência conhecida da doença nas amostras e no campo. Os métodos de incubação utilizados foram: papel de filtro, rolo de papel e meio Neon-S. Observou-se aumento da detecção do patógeno nas sementes infectadas artificialmente, à proporção que aumentou a incidência na amostra. Os métodos podem não detectar o patógeno S. sclerotiorum em sementes de soja naturalmente contaminadas. Os níveis de incidência do patógeno em sementes de soja interferem na sensibilidade dos métodos de detecção, e não há relação entre a incidência no campo e na semente colhida.Termos para indexação: meio Neon-S, método do papel de filtro, método do rolo de papel, mofo-branco. Incidence of Sclerotinia sclerotiorum on soybean seeds and sensitivity of detection testsAbstract -The objective of this work was to evaluate the efficiency of detection methods of Sclerotinia sclerotiorum on soybean seeds. Experiments were carried out during three crop years, using samples of soybean seeds artificially and naturally contaminated, with known pathogen incidence on samples and on the field. The incubation methods used were: filter paper, paper roll, and Neon-S medium. An increase in pathogen detection on artificially contaminated seeds was observed, as the incidence in the sample increased. The methods may not detect the pathogen S. sclerotiorum on naturally contaminated soybean seeds. The levels of pathogen incidence on soybean seeds interfere in the sensitivity of the detection methods, and there is no relationship between the incidence on the field and on the harvested seed.

-Most soybean pathogens are seed transmitted, deserving emphasis the fungus Sclerotinia sclerotiorum, which has been presenting worrying levels of field incidence in some soybean cropping areas in several Brazilian states. The objective of this study was to verify the efficiency of different methods for detecting S. sclerotiorum on soybean seeds artificially infected in the laboratory and from field production areas with a historical disease incidence. Seed samples of seven different cultivars collected from naturally infested fields, and one seed sample artificially inoculated in the laboratory were used. The following detection methods recommended in the literature were compared: Blotter test at 7 o C, 14 o C, and 21 o C; Rolled Paper; and Neon-S. Results demonstrated that these methods showed no repeatability and had a low sensitivity for detecting the pathogen in seeds from areas with disease incidence. They were effective, however, for its detection on artificially inoculated seeds. In the Blotter test method at 7 o C, there was a lower incidence of other fungi considered undesirable during seed analysis.Index terms: Glycine max, seed health, white mold, seed quality. Eficiência de métodos para detecção de

Sclerotinia sclerotiorum is one of the most devastating and cosmopolitan plant pathogens. Rapid detection of S. sclerotiorum can provide growers an advantage in knowing what control measures should be taken to minimize crop damage and financial losses caused by it. Loop-mediated isothermal amplification (LAMP) is a fast, sensitive, and specific nucleic acid amplification method that does not require a thermal cycler. This study aimed to develop a LAMP-based assay for the specific detection of S. sclerotiorum (Ss-LAMP). A real-time quantitative LAMP reaction (Ss-qLAMP) and a calcein ion indicator-based LAMP reaction (Ss-cLAMP) were designed, optimized, and tested on fungi, plant, and soil samples. The Ss-LAMP reactions were very specific and sensitive. Applying the artificially inoculated soil samples with DNA purified by five protocols in the Ss-qLAMP reaction, it was possible to detect and quantify the pathogen DNA, regardless of the extraction protocol. Naturally infected soybean tissues had the pathogen detected by Ss-cLAMP directly in the reaction tube with no DNA extraction requirement. The assays should be applicable for many types of samples, such as soil, spore traps, and plant tissues from several crops, with no requirement for DNA extraction. The Ss-LAMP reactions took less than 1 h to complete, and they can be made directly in the field with real-time quantitative results (Ss-qLAMP) or qualitative naked-eye visual results (Ss-cLAMP). Results were obtained with 10 pg of DNA or 10 ng of crude mycelium, suggesting a detection limit close to a single DNA copy. Ss-LAMP reactions will allow rapid and accurate diagnosis of S. sclerotiorum and assist in pathogen management and control.

Caused by Sclerotinia sclerotiorum, white mold is an important seed-transmitted disease of soybean (Glycine max). Incubation-based methods available for the detection and quantification of seed-borne inoculum such as the blotter test, paper roll and Neon-S assay are time-consuming, laborious, and not always sensitive. In this study, we developed and evaluated a molecular assay for the detection of S. sclerotiorum in soybean seeds using a species-specific PCR (polymerase chain reaction) primer set and seed soaking (without DNA extraction) for up to 72 h. The PCR products were amplified in all the samples infected with the pathogen, but not in the other samples of plant material or the other seed-borne fungi DNA.The minimum amount of DNA detected was 10 pg, or one artificially infested seed in a 400-seed sample (0.25 % fungal incidence) and one naturally infected seed in a 300-seed sample (0.33 % incidence). The PCR-based assay was rapid (< 9 h), did not require DNA extraction and was very sensitive.

White mould (Sclerotinia sclerotiorum) is a damaging disease of soybean crops in Brazil. Within-field spatial analyses of white mould disease and inoculum, as well as yield components at the field level may provide insights into their relationship, but this information is lacking in the country. During three years, spatially explicit within-field data on white mould and soybean stand and yield were measured at three fields, with sizes ranging from 4 to 12 ha, located in the Campos Gerais region of Paraná State, Brazil and all naturally infested with sclerotia. Crop stand, white mould incidence and severity, soybean yield and sclerotia collected at harvest were assessed in quadrats of 7.2 m 2 distant from each other by 8 to 50 m. Soilborne sclerotia were counted in four quadrats (0.25 m 2 and 0.05 m depth) at each sampling point. The semivariograms fitted the pure nugget effect, linear, spherical, and exponential models. The pure nugget model best fitted sclerotia data, suggesting a random distribution. Significant and positive associations were found between disease incidence and sclerotia amount at harvest, which were both negative associated with yield.