Real-Time Quantitative and Ion-Metal Indicator LAMP-Based Assays for Rapid Detection of Sclerotinia sclerotiorum.

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.

Different methods of assessing susceptibility of soybean genotypes to white mold / Diferentes métodos de avaliação da suscetibilidade de genótipos de soja ao mofo branco

White mold caused by the fungus Sclerotinia sclerotiorum is an important disease in relation to soybean. The use of less susceptible genotypes can be a productive strategy in the management of this disease, and the development of an appropriate methodology for soybean inoculation is useful for the differentiation of disease-resistant genotypes. The present study aimed to assess the susceptibility of 77 soybean genotypes based on their reaction to oxalic acid, as well as to determine correlations between three traditional disease assay methods (detached leaf, non-wounded stem and straw tests) and the results of the oxalic acid assay. Oxalic acid susceptibility was assessed by using a wilting score scale. For the other methods, the severity of disease symptoms was assessed. To compare methodologies, the values obtained for the genotypes using each method were categorized into classes, and a severity index was used to represent individuals within each class. All the methods used were efficient for the differentiation of soybean genotypes in terms of susceptibility to S. sclerotiorum; however, the behavior of the genotypes depended on the inoculation method adopted. Even though no significant relationship was identified between the severities of the damage resulting from the methodologies, the rankings acquired from the methods strongly agreed. The oxalic acid method was the most rapid, the least laborious, and was the cheapest compared with the other methods that were used.

O mofo branco causado pelo fungo Sclerotinia sclerotiorum é uma doença importante na cultura da soja. A utilização de genótipos menos suscetíveis pode ser uma estratégia interessante no manejo desta doença, e o desenvolvimento de um método de inoculação adequado para a soja é de grande valia para a discriminação de genótipos resistentes. O presente estudo objetivou avaliar a suscetibilidade de 77 genótipos de soja baseado na reação destes ao ácido oxálico, bem como através de três métodos tradicionais (folha destacada, inoculação na haste sem ferimento e utilizando ponteiras de micropipeta), a fim de verificar os resultados obtidos com o teste do ácido oxállico e correlações entre métodos. A suscetibilidade dos genótipos ao ácido oxálico foi avaliada através de uma chave de escores de murchamento. Para os demais métodos, a severidade de ataque do patógeno foi avaliada. Para permitir a comparação entre metodologias, os resultados obtidos para os genótipos foram organizados em classes, e um índice de severidade foi utilizado para representar os individuos de cada classe. Todos os métodos foram eficientes na diferenciação de genótipos de soja quanto à suscetibilidade à S. sclerotiorum, entretanto, o comportamento dos genótipos variou conforme o método empregado. Não foi observada relação entre as severidades e escores de murchamento obtidos com as metodologias, porém, os ranqueamentos dos genótipos obtidos dos diferentes métodos apresentaram forte correlação. O método baseado na reação ao ácido oxálico foi o mais rápido, menos trabalhoso e de menor custo dentre os métodos utilizandos neste trabalho.