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This study was conducted to develop a self-assembled direct competitive enzyme-linked immunosorbent assay (dcELISA) kit for the detection of deoxynivalenol (DON) in food and feed grains. Based on the preparation of anti-DON monoclonal antibodies, we established a standard curve with dcELISA and optimized the detection conditions. The performance of the kit was evaluated by comparison with high-performance liquid chromatography (HPLC). The minimum detection limit of DON with the kit was 0.62 ng/mL, the linear range was from 1.0 to 113.24 ng/mL and the half-maximal inhibition concentration (IC50) was 6.61 ng/mL in the working buffer; there was a limit of detection (LOD) of 62 ng/g, and the detection range was from 100 to 11324 ng/g in authentic agricultural samples. We examined four samples of wheat bran, wheat flour, corn flour and corn for DON recovery. The average recovery was in the range of 77.1% to 107.0%, and the relative standard deviation (RSD) ranged from 4.2% to 11.9%. In addition, the kit has the advantages of high specificity, good stability, a long effective life and negligible sample matrix interference. Finally, wheat samples from farms in the six provinces of Henan, Anhui, Hebei, Shandong, Jiangsu and Gansu in China were analyzed by the kit. A total of 30 samples were randomly checked (five samples in each province), and the results were in good agreement with the standardized HPLC method. These tests showed that the dcELISA kit had good performance and met relevant technical requirements, and it had the characteristics of accuracy, reliability, convenience and high-throughput screening for DON detection. Therefore, the developed kit is suitable for rapid screening of DON in marketed products. [ABSTRACT FROM AUTHOR]

Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum (Lib.) De Bary is a devastating disease infecting hundreds of plant species. It also restricts the yield, quality, and safe production of rapeseed (Brassica napus) worldwide. However, the lack of resistance sources and genes to S. sclerotiorum has greatly restricted rapeseed SSR-resistance breeding. In this study, a previously identified GDSL motif-containing lipase gene, Brassica napus GDSL LIPASE-LIKE 1 (BnaC07.GLIP1), encoding a protein localized to the intercellular space, was characterized as functioning in plant immunity to S. sclerotiorum. The BnaC07.GLIP1 promoter is S. sclerotiorum-inducible and the expression of BnaC07.GLIP1 is substantially enhanced after S. sclerotiorum infection. Arabidopsis (Arabidopsis thaliana) heterologously expressing and rapeseed lines overexpressing BnaC07.GLIP1 showed enhanced resistance to S. sclerotiorum, whereas RNAi suppression and CRISPR/Cas9 knockout B. napus lines were hyper-susceptible to S. sclerotiorum. Moreover, BnaC07.GLIP1 affected the lipid composition and induced the production of phospholipid molecules, such as phosphatidylethanolamine, phosphatidylcholine and phosphatidic acid, which were correlated with decreased levels of reactive oxygen species (ROS) and enhanced expression of defense-related genes. A B. napus bZIP44 transcription factor specifically binds the CGTCA motif of the BnaC07.GLIP1 promoter to positively regulate its expression. BnbZIP44 responded to S. sclerotiorum infection, and its heterologous expression inhibited ROS accumulation, thereby enhancing S. sclerotiorum resistance in Arabidopsis. Thus, BnaC07.GLIP1 functions downstream of BnbZIP44 and is involved in S. sclerotiorum resistance by modulating the production of phospholipid molecules and ROS homeostasis in B. napus, providing insights into the potential roles and functional mechanisms of BnaC07.GLIP1 in plant immunity and for improving rapeseed SSR disease-resistance breeding., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)