Vitazyme biostimulant increases specialty crop production and contains active brassinosteroids; a spectrofluorometric, chemical (LCMS), and bioassay determination.

Biostimulants are agricultural inputs that enhance the ability of plants to absorb and utilize nutrients more efficiently through natural processes. There exists a gap in understanding how various biostimulant fertilizers act in improving crop yields. This knowledge is critical to help growers make educated decisions to optimize their costs and use of different fertilizers to improve food quality, production, and sustainably. Vitazyme, a commercially available biostimulant, is stated to contain phytohormone brassinosteroids (BR) and other plant growth promoting substances. In this study, Vitazyme was tested for its influence on promoting yield of greenhouse-grown specialty crops, and for the presence and activity of BR. Treatment with Vitazyme significantly increased broccoli weight by 46% and number of tomatoes by 52%. Overall, there was a positive trend in all crops tested, with increased number of pea pods, and total harvested weight of daikon radish, plum purple radish, kale, peas, and tomato. These trials are continuing over multiple seasons to establish Vitazyme efficacy in specialty crop production. Detection and quantification of brassinosteroids poses several challenges due to their low concentrations and over 70 known BR family members. Previously, derivatization of BR with a fluorescent label followed by spectrofluorometric analysis has been shown to be effective in detecting BR. However, sugars carry similar geminal hydroxyl groups as BR, are derivatized similarly and interfere in spectrofluorometric assays. We modified the procedures and show that fluorescence profiles of derivatized sugar are distinct from derivatized BR standards, and that this new method can indeed be used to detect total BR in agricultural inputs. These spectrofluorometric assays were used to detect total BR, followed by mass spectrometric analysis to identify and quantify 28-homobrassinolide as one of the BR in Vitazyme. To confirm that this complex fertilizer mixture contains active BR, a bioassay was developed using Arabidopsis mutant de-etiolated2 (det2), defective in BR biosynthesis. Application of Vitazyme recovered the short growth phenotype in dark-grown det2 mutant, confirming that Vitazyme contains BR activity. This study provides a framework of utilizing multiple approaches to investigate and advance our knowledge of how complex fertilizer mixtures act in improving crop production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-025-07934-4.