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

Sclerotinia sclerotiorum, the etiological agent of the "white mould" in soybean, is responsible for severe losses in this crop and soil contamination. The introduction and dissemination of the disease can made through the use of seed lots contaminated with sclerotia and by seeds infected by mycelium. Therefore, seed health quality is one aspect to be monitored by means of health testing before to sowing time. In this study conventional and quantitative PCR techniques were used to assess their viability to detect S. sclerotiorum in artificially and naturally infected soybean seed samples. For that, seeds were inoculated by osmotic conditioning technique for 0, 24, 48 and 72 hours of contact of the seed with the fungal colony and mixed with healthy seeds generating incidence levels of 1, 2, 10, 20 and 100% for each incubation time. The cPCR was sensitive to detect S. sclerotiorum in samples with at least incidence 1% inoculated for 72 hours while the qPCR detected the pathogen in all incidence/inoculum potential combinations. The conventional PCR was able to detect 0.25% of the incidence of S. sclerotiorum in soybean seed lots naturally infected added a preincubation step.核盘菌（Sclerotinia sclerotiorum）是引发大豆"白霉病"的病原菌，不仅会造成该作物严重减产，还会导致土壤污染。该病害的传入与传播可通过携带菌核的种子批以及被菌丝侵染的种子实现。因此，种子健康质量是播种前需通过健康检测进行监控的关键指标之一。本研究采用常规PCR（conventional PCR，cPCR）与实时荧光定量PCR（quantitative PCR，qPCR）技术，评估二者在人工接种及自然侵染的大豆种子样本中检测核盘菌的能力。为此，本研究采用渗透调节接种法，使种子与真菌菌落分别接触0、24、48、72小时，随后将接种后的种子与健康种子混合，针对每个培养时长分别设置1%、2%、10%、20%及100%的接种发病率水平。常规PCR仅能在接种时长72小时、发病率至少为1%的样本中检测出核盘菌，而实时荧光定量PCR则可在所有发病率/接种量组合的样本中检出该病原菌。经预培养步骤处理后，常规PCR可在自然侵染的大豆种子批中检出发病率仅为0.25%的核盘菌。