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Using allelopathy to search for new natural herbicides from plants

Tim Haig, Jim Pratley, Min An, Terry Haig and Shane Hildebrand

The EH Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga 2678, Australia.


Allelopathy has been used as the basis for identifying plant species which may contain phytotoxic chemicals. Suspected allelopathic species were collected for evaluation of novel herbicidal solutions for non-resistant (NR) and multiple herbicide resistant (MHR) biotypes of annual ryegrass (Lolium rigidum Gaud). Sourced whole plants and plant parts, were extracted with water, and then phytotoxically screened in NR annual ryegrass seedling petri dish bioassays. The 100 most phytotoxic plant extracts, at nominal 25% extract strength, ranged from as low as 11.3 % to 100 % average root inhibition. The most potent species extract (#180), at 5 % strength (1.4 mg dried biological material/mL), was more effective in inhibiting NR ryegrass root growth than a 10-4M (0.04 mg/mL) concentration of commercial herbicide triasulfuron. The potential of crude plant extracts for use as viable natural herbicides is also being investigated in a long-term stability (shelf-life) study. Current monitoring under different storage conditions has shown that extract #180 phytotoxicity towards annual ryegrass has, thus far generally increased, at 25 C over a 32 day period of a continuing 8 month study. Extract #180 (at 5 % strength) also inhibited the root growth of a MHR ryegrass biotype by 98.5 %.

Media summary

For the purpose of discovering new natural herbicides, allelopathic plants were phytotoxically screened for their herbicidal potential against the cereal crop weed, annual ryegrass.

Key words

Herbicide resistance (HR), chemoassay, triasulfuron, phytotoxic screening, natural herbicides


The overuse of synthetic herbicides for weed control over the last five decades has resulted in growing public concern over their impacts upon human health, the environment, and the evolution of herbicide resistant (HR) weeds (Vyvyan 2002). Globally, there are an estimated 297 resistant biotypes and 179 resistant species (108 dicots and 71 monocots) (Heap 2005). In Australia, approximately 4000 farms are experiencing some form of HR (Pratley et al. 1999), while in southern New South Wales, the HR problem is particularly serious with one third of paddocks experiencing resistance with respect to the cereal crop weed annual ryegrass (L. rigidum Gaud.). Widespread repeated use of synthetic herbicides has produced biotypes of annual ryegrass resistant to six major herbicide classes (Wu et al. 2003).

Natural compounds from plants offer excellent potential for new herbicidal solutions, or lead compounds for new herbicides (Duke et al. 2000; Vyvyan 2002). This is due to their likely environmentally benign characteristics (Singh et al. 2003), while the possibility of novel compounds provides the chance of unique modes of herbicidal attack (the potential to overcome HR). Many phytotoxic plant compounds also play a role in allelopathy - their herbicidal impacts exhibited in nature. This knowledge greatly aids the selection of source plants for phytotoxicity investigations, an example of which includes the phytotoxic triketone, leptospermone, isolated from allelopathic Callistemon citrinus, whose lead structure was modified to produce the commercial herbicide mesotrione (Mitchell et al. 2001).

In an attempt to discover novel natural herbicides for the NR and MHR L. rigidum problem, this project began with a wide-ranging survey of suspected allelopathic plants. Plant extract phytotoxicity bioassays against NR L. rigidum seedlings were conducted to screen for the most phytotoxic plant species. Crude plant extracts were treated as potential new herbicides, thus prompting a long-term stability study, with extract phytotoxicity being monitored over time. Glasshouse trials, extract testing against other cereal crop weed species, and actual herbicidal compound isolation and identification will be undertaken at a later date.


Plant material

Literature and word-of-mouth surveys were conducted to collect a wide range of suspected allelopathic plants for screening. Plants included Australian natives, noxious weeds and crops, encompassing 45 families, 123 genera, and 151 species. 133 species were tested. MHR ryegrass seeds, resistant to herbicide groups A and M, were obtained from The EH Graham Centre, CSU, Wagga Wagga, Australia.

Aqueous extract preparation

Collected plants were washed with water to remove debris, dehydrated at 40C for 72 hours, and milled into fine powder through a 1mm sieve. Whole plant specimens or, in some cases, specific plant organs were initially sampled. Ten grams of the residue powders were separately placed into glass bottles and extracted in 100 mL deionised water for 3 days at 20C in the dark. The resulting mixtures were then strained through 4 layers of muslin cloth, vacuum-filtered, and stored in a freezer for later use. These filtrates were designated as 100 % strength (nominal). Later bioassay screening required 100 % extract dilution to 50 and 25 % nominal concentrations.

Bioassay screening of plant extracts

NR L. rigidum seeds were pre-germinated by adding 10 mL of deionised water to 2 g of seed within filter paper-lined 9 cm petri dishes, and incubated in the dark at 25C for 2 days. Ryegrass bioassays were undertaken using 4 mL of 0 (deionised water control), 25, 50 and 100 % strength solutions (for 3 days at 25C in the dark), using 15 seedlings per petri dish. After 3 days incubation, seedling root and shoot lengths were measured. Data were reported as the means of triplicates. The most phytotoxic extracts were bioassayed again at lower (10, 5, 1, and 0.1 %) concentrations to establish their minimal level of herbicidal activity.

Triasulfuron chemoassay

Reference standard commercial herbicide triasulfuron was used to make 6 different standard aqueous concentrations of: 10-3; 10-4; 10-5; 10-6; 10-7; and 10-8 M. Each concentration and a deionised water control were bioassayed in triplicate against NR annual ryegrass under the same laboratory bioassay conditions used for the plant extracts.

Stability testing of extracts

One hundred percent strength solutions, of the top extract (#180), were placed in opaque, sealed glass containers in 4 and 25C incubators. After 2, 4, 8, 16, and 32 days, the extracts from each incubator were diluted to 1 % strength and bioassayed in triplicate against NR ryegrass seedlings.

Results and discussion

Based upon the 25 % extract solutions, the 100 most herbicidal extracts inhibited the root growth of NR annual ryegrass by 11.3 to 100 % (Figure 1). Three extracts chosen for further testing because of their potent inhibition (#180, #221, and #34), resulted in 100, 99.9, and 97.2 % root inhibition respectively. Samples #180, #221 and #34 were also tested at lower concentrations (Figure 2), ranging from 0.1 to 10 % extract strength. Extract #180 was markedly more herbicidal than both #221 and #34 against NR annual ryegrass seedlings across the 1 to 10 % extract range.

Extract #180’s strong result against NR annual ryegrass, prompted a comparison to be made between phytotoxic activity of extract #180, and that of the commercial sulfonylurea herbicide, triasulfuron (Figure 3). For a benchmark comparison, a 5% strength extract of #180 (determined to have 1.4 mg dried biological material/mL of solution), had a stronger phytotoxic impact upon annual ryegrass root growth (80.2 % inhibition) than 10-03 M (0.4 mg/mL) of triasufuron (62.1 % inhibition). Chemical isolation and compound identification of #180’s most herbicidal fractions remains to be undertaken.

Figure 1. The top 100 phytotoxic plant extracts against non-resistant annual ryegrass.

Figure 2. Comparison of the three plant extracts for percent mean root inhibition of non-resistant annual ryegrass as affected by different concentrations.

Figure 3. Comparison between effect of extract #180 (1 and 5 % strength) and triasulfuron against NR annual ryegrass root growth.

Plant extracts (compound mixtures) potentially possess multiple phytotoxic compounds and hence multiple modes of simultaneous herbicidal attack, making it more difficult for weeds to develop HR. It is for this reason that herbicidal activity of this research’s most potent plant extracts is being examined. Only minimal research has been undertaken to explore the suitability of crude plant extracts as viable natural herbicides (Duke et al. 2002).

To assess their phytotoxic viability over time, plant extracts are monitored in an ongoing, long-term stability (shelf-life) study under varying environmental conditions (Figure 4). Measurements revealed #180’s phytotoxicity towards NR ryegrass increased by 21 %, over a 32 day period, when stored at 25C in the dark. Herbicidal activity increased only slightly under the cooler 4C over 16 days, until later declining 18 % by day 32. Due to probable chemical transformations in storage, extract phytotoxicities towards ryegrass will be further monitored over time, so as to gauge a suitable shelf-life for this biological extract for potential use as a natural herbicide.

Figure 4. Mean percent root inhibition of non-resistant annual ryegrass as affected by 1 % extract #180 at 4C and 25C in the dark

MHR bioassay work is proceeding, with interim results revealing that a 5 % concentration of #180 inhibited the root growth of a resistant biotype of annual ryegrass by 98.5 % (Table 1).

Table 1. Mean percent root inhibition of class A and M resistant annual ryegrass as affected by extract #180 at different concentrations.

Extract #180 concentration

0 % (control)

0.1 %

1 %

5 %

10 %

25 %

50 %

100 %

Mean % root inhibition









A wider range of bioassay post-emergent testing is to occur with other cereal crop weeds, while glasshouse trials, chromatographic fractionation and spectroscopic compound identification is to be undertaken. It is hoped that this research methodology using allelopathy as an indicator will provide an alternative natural solution for combating Australian cereal crop weeds such as MHR annual ryegrass.


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