Collins, Daniel (2022) A HPLC-ESI-MS/MS Study of Hydroxybenzoic Acids and Related Derivatives in Commercial Seaweed Biostimulants and their Plant Growth Bioactivity. PhD thesis, Victoria University.

Abstract

The rapidly growing world population, increasing severity of climate change, and constantly evolving environmental pressures have drawn into question whether current agricultural practices can meet the growing food demands healthily, equitably and sustainably. This has resulted in the rising popularity of natural biostimulants, particularly seaweed extracts, to increase crop productivity in an eco-friendly and safe manner. To better understand the complex modes of action underpinning the well-reported benefits of seaweed biostimulants to crops, their phytochemical composition requires further characterisation. Hydroxybenzoic acids, a subclass of phenolic acids, are an important class of phytochemicals and the aim of this study was to characterise their profile in commercial seaweed biostimulants. This work used modern analytical technologies to investigate salicylic acid and other benzoic acid derivatives in a commercial seaweed biostimulant, and then assessed the biological activity of the monohydroxybenzoic acids using plant growth assays. Qualitative HPLC-ESI-MS/MS methods were developed for the analysis of hydroxybenzoic acids and related derivatives. The various benzoic acid derivatives investigated include monohydroxybenzoic acids, dihydroxybenzoic acids, trihydroxybenzoic acids, methoxylated hydroxybenzoic acids, methoxylated benzoic acids, and an amino substituted benzoic acid. The HPLC-ESI-MS/MS methods for the analysis of the various derivatives were then employed to investigate the presence of these compounds in the commercial seaweed biostimulant. The compounds found to be present were the monohydroxybenzoic acids, 2,3- and 3,4-dihydroxybenzoic acid, syringic acid, and anthranilic acid. A HPLC-ESI-MS/MS method for the analysis of the monohydroxybenzoic acids was optimised and partially validated for the quantification of salicylic acid and its isomers in a commercial seaweed biostimulant. Sample preparation employed acidified acetonitrile partitioning of the seaweed biostimulant before mixed-mode solid-phase extraction. The three isomers were successfully separated using a reversed-phase biphenyl stationary phase with a methanol/water mobile phase acidified with formic acid. The MS/MS detection employed the characteristic MRM transition of m/z 137  93 of the monohydroxybenzoic acids. The concentrations of 2-, 3- and 4-hydroxybenzoic acid in a commercial seaweed biostimulant were found to be 137, 3409, and 1748 μg/L, respectively. Tomato seedling plant growth bioassays were conducted to investigate the biological effects of salicylic acid and its isomers on plant growth. Fresh and dry root and shoot weight data along with longest root length data were assessed to evaluate the biological effects of the various treatments on tomato seedling growth. It was found that a significant increase in root growth was observed when the commercial seaweed biostimulant was fortified with a combination of the three monohydroxybenzoic acids, using dosages that correlate to the concentrations determined in the seaweed biostimulant in this study.

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