Identification and Phytotoxicity Assessment of Phenolic Compounds in Chrysanthemoides monilifera subsp. monilifera (Boneseed)

Abstract

Chrysanthemoides monilifera subsp. monilifera (boneseed), a weed of national significance in Australia, threatens indigenous species and crop production through allelopathy. We aimed to identify phenolic compounds produced by boneseed and to assess their phytotoxicity on native species. Phenolic compounds in water and methanol extracts, and in decomposed litter-mediated soil leachate were identified using HPLC, and phytotoxicity of identified phenolics was assessed (repeatedly) through a standard germination bioassay on native Isotoma axillaris. The impact of boneseed litter on native Xerochrysum bracteatum was evaluated using field soil in a greenhouse. Collectively, we found the highest quantity of phenolic compounds in boneseed litter followed by leaf, root and stem. Quantity varied with extraction media. The rank of phenolics concentration in boneseed was in the order of ferulic acid > phloridzin > catechin > p-coumaric acid and they inhibited germination of I. axillaris with the rank of ferulic acid > catechin > phloridzin > p-coumaric acid. Synergistic effects were more severe compared to individual phenolics. The litter-mediated soil leachate (collected after15 days) exhibited strong phytotoxicity to I. axillaris despite the level of phenolic compounds in the decomposed leachate being decreased significantly compared with their initial level. This suggests the presence of other unidentified allelochemicals that individually or synergistically contributed to the phytotoxicity. Further, the dose response phytotoxic impacts exhibited by the boneseed litter-mediated soil to native X. bracteatum in a more naturalistic greenhouse experiment might ensure the potential allelopathy of other chemical compounds in the boneseed invasion. The reduction of leaf relative water content and chlorophyll level in X. bracteatum suggest possible mechanisms underpinning plant growth inhibition caused by boneseed litter allelopathy. The presence of a substantial quantity of free proline in the target species also suggests that the plant was in a stressed condition due to litter allelopathy. These findings are important for better understanding the invasive potential of boneseed and in devising control strategies.

Fig 1. Concentrations of phenolic compounds in boneseed ((a) methanol extract and (b) water extract).

The x-axis denotes the types of phenolic compounds e.g., catechin (Cat), p-coumaric acid (PCA), ferulic acid (FA) and phloridzin (Phlo). Values on y-axis denote the concentration of phenolic compounds in boneseed organs in μg/ mg. White, light ash, deep ash and black colours (filled) in the columns represent leaf, stem, root and litter extracts respectively. The error bar indicates the standard error. The letters a and b above the columns depict the significant increase and decrease of phenolics in methanol extract respectively compared with water extract while columns without letters indicate non-significant variation.

Fig 2. HPLC output.

Chromatograms of standard phenolic compounds mixer (1 = Catechin, 2 = P-coumaric acid, 3 = ferulic acid, 4 = Phloridzin) and boneseed tissue (leaf, stem, root and litter) extracts. X-axis represents the retention time (min) and y-axis represents the unit (mAu).

Fig 3. Concentrations of phenolic compounds in decomposed boneseed litter-mediated soil leachate.

The x-axis denotes the types of phenolic compounds e.g., catechin (Cat), p-coumaric acid (PCA), ferulic acid (FA) and phloridzin (Phlo). Values on y-axis denote the concentration of phenolic compounds in boneseed organs in μg/ mg. The solid line denotes the phenolics in non-decomposed litter leachate while dotted line represents in decomposed leachate. The error bar indicates the standard error. The letter b on top of markers depicts the significant decrease of phenolic compounds in decomposed leachate compared with non-decomposed leachate while blank means non-significant variation.

Fig 4. Impact of litter-mediated soil leachate on I. axillaris.

The x-axis denotes the types of parameters e.g., total germination (TG), speed of germination (SpG), speed of accumulated germination (SpAG), soefficient of rate of germination (CRG), hypocotyl length (HL), radicle length (RL), hypocotyl weight (HW) and radicle weight (RW). Values on y-axis denote the units of these parameters. Data presented as average while the error bars indicate the standard errors. No filled columns, light ash colour columns and deep ash colour columns represent the impact exhibited by control, soil leachate only and litter-mediated soil leachate respectively. The letter b and c depicts significant decrease of those parameters in I. axillaris exposed to decomposed litter-mediated soil leachate compared with soil alone leachate and control respectively.

Fig 5. Number of X. bracteatum seedlings died due to phytotoxicity by boneseed litter-mediated soil.

The x-axis denotes the treatment types (control and doses of boneseed litter (L)). Values on y-axis denote the number of X. bracteatum seedlings died after one week. The error bar indicates the standard error. The letter a indicates significant increase compared with control.

Fig 6. Effect of boneseed litter on biometric parameters of X. bracteatum.

The x-axis denotes the doses of litter (L) in g/100gm soil. The y-axis denotes shoot length, root length, shoot dry weight, root dry weight and number of leaf of X. bracteatum (% of control). The error bar indicates the standard error.

Fig 7. Effect of boneseed litter on biochemical parameters of X. bracteatum.

The x-axis denotes the doses of litter (L) in g/100gm soil. The y-axis represents leaf relative water content (LRWC), chlorophyll a, chlorophyll b, total chlorophyll and proline content of X. bracteatum leaf. Data presented as % of control. The error bar indicates the standard error.