A New Method for Discovering Plant Biostimulants

Abstract

Structurally well-defined compounds have advantages for quality control in plant biostimulant production and application processes. Humic acid (HA) is a biostimulant that significantly affects plant growth, and soil-dwelling Protaetia brevitarsis larva (PBLs) can rapidly convert agricultural waste into HA. In this study, we use PBLs as a model to investigate HA formation and screen for structurally well-defined HA-related plant biostimulant compounds. Dephasing magic angle spinning nuclear magnetic resonance (¹³C DD-MAS NMR) analysis indicated HA structural changes during PBL digestion; metabolic profiling detected seven HA-related aromatic ring-containing compounds. A total of six compounds that significantly stimulate plant growth were identified through plant experiments, and all six compounds demonstrate the ability to enhance seed germination. It is noteworthy that piperic acid exhibits a remarkable promotion of root growth in plants, a finding reported for the first time in this study. Thus, this study not only provides insights into the insect-mediated transformation of HA but also illustrates a new method for discovering structurally well-defined plant biostimulant compounds.

Keywords: Protaetia brevitarsis; biostimulant; humic acid; intestinal compound; piperic acid.

Figure 1

Method flow chart of discovering well-defined plant biostimulant compounds. The changes to HA structure in midgut inclusions (MI-HA), hindgut inclusions (HI-HA) and frass (FR-HA) (A). The view of the digestive tract of PBLs, showing relative locations of different compartments (B). Metabolomic analysis of midgut inclusions and hindgut inclusions showed that these compounds were distributed in both midgut and hindgut, with 93 compounds more distributed in the midgut and 53 compounds more distributed in hindgut (C). From the detected compounds, 7 compounds with benzene rings were selected for the seed germination experiment. Among them, 6 compounds could promote seed germination (D). The stimulating effects of 6 compounds on plant growth were further verified by plant growth experiments (E).

Figure 2

¹³C dipolar, dephasing magic angle spinning nuclear magnetic resonance spectra (¹³C DDMAS NMR spectra) of HA of midgut inclusions (MI-HA), hindgut inclusions (HI-HA) and the frass (FR-HA) of PBLs fed fermented corn stalks.

Figure 3

Seven benzene ring-containing compounds were selected in this study. “+” indicates a higher concentration in this gut.

Figure 4

Seed germination index (GI) of seven compounds. GI > 1.0 indicates that the compound can promote seed germination.

Figure 5

A summary of the promoting effects of six compounds on plants. SL indicates stem length; AGFW indicates aboveground fresh weight; AGDW indicates aboveground dry weight; RL indicates root length; FRW indicates fresh root weight; RDW indicates root dry weight. Each compound was administered at concentrations of 20, 40 and 60 μM. The different colours of the boxes represent the percentage increase in the assessed variable in the treatment group compared to the control group. *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 6

Functional verification results of 6 compounds promoting plant growth. (a–c) represent the effects of 6 compounds on aboveground dry weight, root dry weight and the relative chlorophyll content, respectively. *** p < 0.001, ** p < 0.01, * p < 0.05. CK means the control treatment.