Terry Haig

Laboratory screening for allelopathic potential of wheat (Triticum aestivum) accessions against annual ryegrass (Lolium rigidum)

A new screening bioassay, the ‘equal-compartment-agar-method (ECAM)’, was developed and employed to evaluate 92 wheat cultivars for their allelopathic activity on the inhibition of root growth of annual ryegrass (L. rigidum). Results showed that the allelopathic activity of wheat was associated with the sowing time of ryegrass seeds, the number of wheat seedlings, agar quantities, and agar concentrations. The addition of activated charcoal into the agar medium significantly alleviated wheat allelopathic inhibition on the root growth of ryegrass, indicating that wheat allelopathic activity is chemically driven. There were significant differences between wheat cultivars in their allelopathic potential at the seedling stage on the inhibition of root elongation of annual ryegrass, varying from 23.98% to 90.91%. Re-screening of 22 selected wheat accessions showed that the allelopathic potential of wheat cultivars is consistent between different years under the same experimental conditions. This newly developed screening bioassay successfully separated the allelopathic effect from the competitive effect between wheat and ryegrass plants, and enabled the constant release and accumulation of allelochemicals from living wheat seedlings into the growth medium to affect the growth of ryegrass. The influence of microorganisms was also avoided because of the sterile conditions. The present study describes this new bioassay suitable for the efficient screening of a large number of wheat cultivars under laboratory conditions.

Evaluation of seedling allelopathy in 453 wheat (Triticum aestivum) accessions against annual ryegrass (Lolium rigidum) by the equal-compartment-agar method.

Allelopathy has been receiving world-wide attention for its potential in integrated weed management. A newly developed screening bioassay, the ‘equal-compartment-agar method’ (ECAM), was used to evaluate seedling allelopathy against annual ryegrass in a collection of 453 wheat accessions originating from 50 countries. Significant differences in allelopathic potential were found in this worldwide collection, inhibiting root growth of ryegrass from 9.7% to 90.9%. Wheat seedling allelopathy also varied significantly with accessions from different countries. Wheat allelopathic activity was normally distributed within the collection, indicating the involvement of multiple genes conferring the allelopathic trait. Of the 453 wheat accessions screened, 2 distinct groups were identified. Condor-derivatives were more allelopathic than Pavon-derivatives, with an average inhibition of root growth of ryegrass by 76% and 46%, respectively. Research was further extended to investigate the near isogenic lines derived from Hartog (Pavon-derivative) and Janz (Condor-derivative). Hartog and its backcrossed lines were less allelopathic than Janz and its backcrossed lines, inhibiting root length of ryegrass by 45% and 81%, respectively. These results strongly indicate that wheat allelopathic activity might also be controlled by major genes, depending on the particular populations. The present study demonstrates that there is a considerable genetic variation of allelopathic activity in wheat germplasm. It is possible to breed for cultivars with enhanced allelopathic activity for weed suppression.

Distribution and exudation of allelochemicals in wheat Triticum aestivum.

Wheat allelopathy has potential for weed suppression. Allelochemicals were identified in wheat seedlings, and they were exuded from seedlings into agar growth medium. p-Hydroxybenzoic, trans-p-coumaric, cis-p-coumaric, syringic, vanillic, trans-ferulic, and cis-ferulic acids and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) were identified in both the shoots and roots of 17-day-old wheat seedlings and their associated agar growth medium. Wheat accessions with previously identified allelopathic activity tended to contain higher levels of allelochemicals than poorly allelopathic ones. The allelopathic compounds present in the shoots generally also were identified in the roots and in the agar medium. Allelochemicals were distributed differentially in wheat, with roots normally containing higher levels of allelochemicals than the shoots. When the eight allelochemicals were grouped into benzoic acid and cinnamic acid derivatives, DIMBOA, total coumaric, and total ferulic acids, the amount of each group of allelochemicals was correlated between the roots and the shoots. Most of the allelochemicals identified in the shoots and roots could be exuded by the living roots of wheat seedling into the agar growth medium. However, the amounts of allelochemicals in the agar growth medium were not proportional to those in the roots. Results suggest that wheat plants may retain allelochemicals once synthesized. The presence of allelochemicals in the agar growth medium demonstrated that wheat seedlings were able to synthesize and to exude phytotoxic compounds through their root system that could inhibit the root growth of annual ryegrass.

Identification and Quantitation of Compounds in a Series of Allelopathic and Non-Allelopathic Rice Root Exudates

An investigation of the chemical basis for rice allelopathy to the rice weed arrowhead (Sagittaria montevidensis) was undertaken using GC/MS and GC/MS/MS techniques. Twenty-five compounds were isolated and identified from the root exudates of both allelopathic and non-allelopathic rice varieties. Phenolics, phenylalkanoic acids, and indoles were among the chemical classes identified. Two indoles previously unreported in rice were detected in the exudates, 5-hydroxy-2-indolecarboxylic acid and 5-hydroxyindole-3-acetic acid. Several other compounds identified in this study have not previously been reported in rice root exudates, namely mercaptoacetic acid, 4-hydroxyphenylacetic acid, and 4-vinylphenol. The levels of 15 compounds present in the exudates were quantified using GC/MS/MS. Six of the seven most abundant compounds were phenolic acids. Significant differences exist between the allelopathic and non-allelopathic cultivars in their production of three of these six compounds. Greater amounts of trans-ferulic acid, p-hydroxybenzoic acid, and caffeic acid were detected in the exudates of allelopathic cultivars. The seventh compound, abietic acid, was significantly higher in the non-allelopathic cultivars.

Evaluation of putative allelochemicals in rice root exudates for their role in the suppression of arrowhead root growth

In previous studies, 15 putative allelopathic compounds detected in rice root exudates were quantified by GC/MS/MS. In this study, multiple regression analysis on these compounds determined that five selected phenolics, namely caffeic, p-hydroxybenzoic, vanillic, syringic, and p-coumaric acids, from rice exudates were best correlated with the observed allelopathic effect on arrowhead (Sagittaria montevidensis) root growth. Despite this positive association, determination of the phenolic acid dose-response curve established that the amount quantified in the exudates was much lower than the required threshold concentration for arrowhead inhibition. A similar dose-response curve resulted from a combination of all 15 quantified compounds. Significant differences between the amounts of trans-ferulic acid, abietic acid, and an indole also existed between allelopathic and non-allelopathic rice cultivars. The potential roles of these three compounds in rice allelopathy were examined by chemoassay. Overall, neither the addition of trans-ferulic acid nor 5-hydroxyindole-3-acetic acid to the phenolic mix significantly contributed to phytotoxicity, although at higher concentrations, trans-ferulic acid appeared to act antagonistically to the phytotoxic effects of the phenolic mix. The addition of abietic acid also decreased the inhibitory effect of the phenolic mix. These studies indicate that the compounds quantified are not directly responsible for the observed allelopathic response. It is possible that the amount of phenolic acids may be indirectly related to the chemicals finally responsible for the observed allelopathic effect.

Simultaneous determination of phenolic acids and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one in wheat (Triticum aestivum L.) by gas chromatography-tandem mass spectrometry.

A procedure using gas chromatography and tandem mass spectrometry (GC-MS-MS) has been developed for the identification and quantification of some allelochemicals in wheat (Triticum aestivum L.). The quantities of allelochemicals in wheat shoots ranged from 2.9 to 110 mg per kilogram of dry shoot residues. Compared with gas chromatography-mass spectrometry (GC-MS), the GC-MS-MS technique significantly increased instrument selectivity and sensitivity, thereby providing more reliable quantitation results in the determination of the phytotoxic compounds examined during this allelopathy research.

Phytotoxicity of Vulpia Residues: IV. Dynamics of Allelochemicals During Decomposition of Vulpia Residues and Their Corresponding Phytotoxicity

The behavior and dynamics of 20 identified allelochemicals in Vulpia residues were collectively and individually monitored, and their kinetic phytotoxicity was assessed. The total content of the identified allelochemicals in decaying Vulpia residues increased from 0.31 mg/g to 1.24 mg/g dry residue over a 21–day decomposition period, while the total phenolic content increased from 1.86 mg/g to 2.16 mg/g dry residue. This corresponded to a phytotoxicity increase from 42% to 82% of radical inhibition. Allelochemicals changed in composition and quantity over the duration of the residue decomposition. Addition of soil to the residues reduced the total allelochemical contents extracted and altered the dynamic pattern. In the same period, the total content of allelochemicals declined from 0.061 mg/g to 0.046 mg/g residue + soil, with the total phenolics dropped from 0.20 mg/g to 0.11 mg/g residue + soil, corresponding to a radical length increase from 53% to 109% of control. Only 14 of the identified allelochemicals were detected in the mix of soil and residues, in contrast to 20 present in the residues alone. The implications of these findings are discussed.

Phytotoxicity of vulpia residues: II. Separation, identification, and quantitation of allelochemicals from Vulpia myuros.

Phytotoxicity-based extraction and fractionation were employed to separate and purify the allelochemicals from an aqueous extract of vulpia (Vulpia myuros) residues. Further analyses, identification, and quantitation of these allelochemicals were conducted by gas chromatography–mass spectrometry on the most toxic fraction. Twenty-one compounds were identified in the ether fraction from the vulpia residue aqueous extract. They were catechol, hydroquinone, 3,4-dimethoxyphenol, pyrogallol, coniferyl alcohol, and benzoic, succinic, hydrocinnamic, salicylic, protocatechuic, vanillic, gentisic, syringic, p-hydroxybenzoic, α-hydroxybenzenepropanoic, p-hydroxyphenylacetic, p-hydroxybenzenepropanoic, hydroferulic, p-coumaric, hydrocaffeic, and ferulic acids. A chromatographic internal standard method with multiple-point calibration graphs was used to quantify the identified compounds. Quantities in the vulpia residues ranged from 1.36 to 81.0 μg/g dry residue, in total accounting for 0.05% of the dry weight residue. The combined syringic and hydroferulic acids were present in the largest amount, 140.11 μg/g residue. Vanillic, succinic, p-hydroxybenzenepropanoic, and salicylic acids were next in amount, ranging from 37.24 to 81.24 μg/g residue. Catechol, hydrocinnamic acid, and hydroquinone were present in the smallest quantities, ranging from 1.36 to 1.82 μg/g residue. The remainder of the compounds were intermediate in amount, ranging from 2.33 to 18.1 μg/g residue.

Phytotoxicity of vulpia residues. I. Investigation of aqueous extracts

Phytotoxic properties of vulpia (V. myuros) residue extracts on wheat (Triticum aestivum L. cv. Vulcan) were examined in the laboratory. Vulpia residues contained water-soluble materials that were toxic to germination and to coleoptile and root growth of wheat. There were strong correlations between extract concentration and toxicity, between extraction times and toxicity, and between extract pH and phytotoxicity. The phytotoxicity was residue rate dependent. There was no significant correlation between the phytotoxicity of residue extracts and the electrical conductivity of aqueous extracts. Milled residues were more toxic than chopped residues. The toxicity of vulpia residues increased as their decomposition proceeded, reaching a peak phytotoxicity after decomposition for 40 days and remaining potent for up to 60 days, gradually declining thereafter. Radicle elongation of wheat was the most sensitive indicator, germination was the least sensitive, and coleoptile growth was intermediate. The phytoxic effects of residue extracts on seed germination had two aspects: germination delay and inhibition. The relative magnitude of each aspect depended upon the potency of the extracts. With a strong phytotoxic potential, inhibition of germination was dominant over seed germination, whereas with a weak toxic level, delay was dominant over seed germination.

Screening rice varieties for allelopathic potential against arrowhead (Sagittaria montevidensis), an aquatic weed infesting Australian Riverina rice crops

Twenty-eight rice varieties with different countries of origin, maturity and stage of improvement were screened in the laboratory for allelopathic potential against arrowhead. Initial rice-density experiments established appropriate bioassay parameters and demonstrated that arrowhead response to rice root exudates follows a typical dose-response curve. Results from the equal compartment agar method (ECAM) bioassay showed that a range of allelopathic potential exists in rice germplasm. This work, together with other published studies suggests such potential to be species specific. In this study, the degree of root inhibition ranged from 26.6 to 99.7%. The potential allelopathic effect of arrowhead on rice seedlings was determined to be negligible.