Quantification and conceptual validation of the inoculum potential of Sclerotinia sclerotiorum in soybean and bean seeds

Abstract: The fungus Sclerotinia sclerotiorum, the causal agent of white mold, is widespread throughout the world. The disease is considered to be one of the major diseases of soybean and bean crops in Brazil. The pathogen S. sclerotiorum is spread by soybean and bean seeds both in the form of sclerotia and dormant mycelium inside the seeds. The objective of this work was to evaluate the relationship between different potentials of S. sclerotiorum in soybean and bean seeds and the performance of these seeds, as well as to verify the localization and quantification of the inoculum of the pathogen in the seeds inoculated by Real-time PCR (qPCR), validating the term inoculum potential. Soybean and bean seeds were inoculated with the fungus by the osmotic conditioning method based on the exposure of the seeds to the fungus for periods of 24 h, 48 h, 72 h, and 96 h. Molecular analysis was carried out by means of qPCR in whole seeds and dissected in the integument, cotyledon and embryonic axis. The results showed that the effects of S. sclerotiorum on seed germination and vigor were progressive and proportional to the increases in inoculum potentials, since there was more severe damage to the seeds and consequently to the emerged plants at the highest potential (P96). The inoculum of the pathogen was found in all parts of the evaluated seeds, even at its lowest inoculum potential (P24), with an increasing DNA concentration, and the integument obtained a greater amount of DNA than the embryo, in comparison.

摘要：引发白霉病的核盘菌（Sclerotinia sclerotiorum）在全球范围内广泛分布。该病害被认定为巴西大豆与菜豆种植的主要病害之一。核盘菌可借助大豆和菜豆种子进行传播，传播载体包括种子外部的菌核以及种子内部的休眠菌丝体。本研究的核心目标为：探究大豆、菜豆种子携带的核盘菌不同接种体势（inoculum potential）与种子性能之间的关联；同时通过实时荧光定量PCR（qPCR）技术，对接种病原菌的种子内病原菌接种体的定位与定量方法进行验证，以确证该专业术语的内涵合理性。本研究采用渗透调节接种法，将大豆与菜豆种子分别暴露于病原菌环境中24 h、48 h、72 h及96 h，完成病原菌接种。随后分别对完整种子、种皮、子叶及胚轴进行解剖分离，通过qPCR开展分子检测分析。研究结果表明：核盘菌对种子萌发率与活力的影响随接种体势提升呈渐进式且正比例增长；当接种体势达到最高水平（P96）时，种子及后续萌发的幼苗受到的损害最为严重。此外，即便在最低接种体势（P24）处理组中，病原菌接种体也可在被测种子的所有组织中被检出，且病原菌DNA浓度随接种体势提升持续升高；相较而言，种皮中的DNA含量显著高于胚部组织。