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Phytotoxic effects of lippia (Phyla canescens) on germinating seeds

Andrew T. Daley1, Daniel K.Y. Tan1 and Hanwen Wu2

1 Faculty of Agriculture, Food and Natural Resources, The University of Sydney, Sydney, NSW 2006, Australia, www.agric.usyd.edu.au Email tand@agric.usyd.edu.au
2
Queensland Department of Primary Industries and Fisheries, Toowoomba, QLD 4350, Australia, www.qld.gov.au
Email Hanwen.Wu@dpi.qld.gov.au

Abstract

Lippia (Phyla canescens) is a perennial herb (Verbenaceae) of South American origin and is a serious environmental and pastoral weed of the inland river systems of New South Wales and Southern Queensland in Australia. Cultivation and establishment of competitive crops and pastures is an important tool for managing lippia infested country. Allelochemicals are believed to be released by lippia that prevent seed germination and establishment. This work quantifies the allelopathic effects of aqueous lippia extracts on seed germination of lettuce (Lactuca sativa), radish (Raphanus sativa), oats (Avena sativa), ryegrass (Lolium multiflorum), Namoi woolly pod vetch (Vicia villosa), subterranean clover (Trifolium subterraneum) and sorghum (Sorghum bicolor). Seeds of bioassayed species were germinated at 20 ▒ 1 C in petri dishes containing aqueous extracts of lippia leaves and/or stems at a range of concentrations (0, 4, 8, 12, 30, 40 and 50% w/v) with osmotic potentials of 0.2, 21, 30, 33, 69, 90 and 91 m0sm/kg, respectively. Germinated seeds were counted daily and germination was modelled using the Inverse Gaussian Distribution function, and the time at which 50% germination (T50) occurred was estimated. There were no significant differences between the lippia stem and leaf extracts on germination inhibition of radish and oats. Germination probability decreased exponentially with increasing rates of lippia extract in all species tested except sorghum. Bioassayed species responded differentially to lippia extracts. Lippia extract concentrations12% w/v reduced the germination probability of vetch and subterranean clover by more than 80% and 25%, respectively, which suggests that legumes should not be sown immediately into soil previously infested by lippia. Germination probability of ryegrass, oats, radish and lettuce were only slightly reduced (<20%) by extract concentrations12% w/v.

Media summary

Aqueous extracts of the pasture weed, lippia (Phyla canescens) inhibited seed germination of certain pasture and crop species tested by University of Sydney researchers.

Key Words

Lippia, Phyla canescens, allelopathy, seed germination

Introduction

Lippia (Phyla canescens) is a perennial herb (Verbenaceae) of South American origin, and is a serious environmental and pastoral weed of the inland river systems of south-eastern Australia. It is a major threat to watercourse, floodplain and native pasture areas and affects at least 800,000 ha of floodplain grazing country (Dellow et al. 2001). Lippia is currently estimated to be present over an area in excess of 5.3 million ha in the Murray-Darling Basin (Earl 2003). The presence of monoterpenes and sesquiterpenes were reported in the closely related phyla weed (Phyla nodiflora); both terpenes are known to be allelopathic, and suppress growth of other plants and inhibit seed germination (Elakovich 1985; 1987). Extracts of phyla weed suppressed the growth of lettuce seedlings, while monoterpenes are known to inhibit radish seed germination (Elakovich 1987). Dense swards of lippia are often surrounded by a strip of bare ground up to 1 m wide which suggests the presence of allelopathic chemicals released into the soil by lippia (McCosker 1994). A number of pasture failures on the Darling Downs in Queensland have been blamed on the allelopathic effect of lippia (Lucy et al. 1995). These occurred where pasture seed was sown soon after the cultivation of a lippia dominant pasture. Lippia has also been observed to suppress the growth of irrigated lucerne (Medicago sativa) in southern Queensland (Lucy et al. 1995). One possibility of managing this weed is to cultivate it and sow crops or pastures that can out-compete lippia. However, it is not known whether seed germination is inhibited by allelochemicals thought to be present in lippia residue. We conducted germination studies to assess the potential risks due to allelopathy from lippia when establishing crop and pasture species. The objective of this study was to test the hypothesis that aqueous extracts of lippia inhibits seed germination of selected crop and pasture species. The relationship between the concentration of lippia extracts and germination probability (germination percentage), and time (h) to 50% germination (T50) were estimated.

Methods

Plant collection

Mature lippia plants were collected in January 2004 from the banks of the Namoi River at “Cadarga” farm (latitude 30░16’S, longitude 149░40’E) in the Narrabri Shire of northwest New South Wales. At the time of collection, plants were flowering and growing vigorously.

Preparation of lippia extracts

Lippia plants were separated into leaves and stems with roots and flowers removed in Experiment 1 (see below). For Experiment 2, the stems and leaves were combined as there were no significant differences in seed germination probability due to plant part (stems and leaves) in Experiment 1. Fresh lippia tissues were soaked in distilled water for 24 h at 24░C in a lighted room to give the required concentrations (see below) (Tawara and Turk 2003a, b). After soaking, solutions were filtered through 4 layers of cheese cloth and the filtrate was centrifuged (1500 g) for 4 h. The supernatant was filtered again using a 0.2 mm filter to give the final extract. Distilled water was used as the control (Tawara and Turk 2003a, b).

Experiment 1

Experiment 1 was a completely randomised factorial design with 3 factors and 4 replicates. The first factor was plant species [radish (Raphanus sativa), oats (Avena sativa)]. The second was lippia plant part (stems, leaves) and the third factor was lippia extract concentration (0, 4, 8, 12 and 30% w/v, i.e. g per 100 mL distilled water). Osmotic potentials were only measured for lippia extract concentrations in Experiment 2.

Experiment 2

Experiment 2 was a completely randomised factorial design with 2 factors and 4 replicates. The first factor was plant species [lettuce (Lactuca sativa), sorghum (Sorghum bicolor), ryegrass (Lolium multiflorum), subterranean clover (Trifolium subterraneum) and Namoi woolly pod vetch (Vicia villosa)]. The second factor was lippia extract concentration (0, 4, 8, 12, 30, 40 and 50% w/v, i.e. g per 100 mL distilled water). The osmotic potentials measured using an osmometer were 0.2, 21, 30, 33, 69, 90 and 91 m0sm/kg for the 0, 4, 8, 12, 30, 40 and 50% w/v lippia extracts, respectively. These osmotic potentials were below 100 m0sm/kg, and were low enough not to affect seed germination and seedling growth of species such as lucerne (Medicago sativa), ryegrass (Lolium multiflorum) and cotton (Gossypium hirsutum) (Wanjura and Buxton 1972a,b; Smith 1989).

Seed bioassays (Experiments 1 and 2)

Fifty seeds were placed on filter paper in each 9 cm petri dish (experimental unit), and 6 mL of aqueous lippia extract was added. The petri dishes were sealed with parafilm to prevent moisture loss and contamination. Seeds were germinated in incubators with temperatures maintained at 20 ▒ 1 C and exposed to constant light conditions by means of 2 standard fluorescent light tubes (at least 40 Ámol/m2.s for 24 h/day). Seeds were germinated for between 10 and 14 days and checked for germination on a 12 hourly basis for the first 4 days, then on a daily basis for the remaining 6-10 days. Seed germination was defined as when the radicle or shoot had extended 1 mm beyond the seed coat or caryopsis, respectively (Steinmaus et al. 2000). Recording ceased when there was no change in seed germination counts for more than 3 days.

Final germination count data were logit transformed [Y= ln {P/(1-P)}, where P is the probability of germination occurring] to achieve constant variance and analysed using binary logistic regression in Genstat 7.2«. The predicted outcomes for germination probability were fitted using exponential regressions with the equation, Y = A + B*eln(R)*X and constraints: R>1. Time (h) to 50% germination (T50) was derived using the GCUMDISTRIBUTION procedure of Genstat 7.2« (Butler and Brain 1989; O’Neill et al. 2004). This GCUMDISTRIBUTION procedure incorporates the relatively new Inverse Gaussian Distribution function which is able to describe the time (T) to germination by taking into account a possible ‘lag’ phase, which is the period before germination commences (O’Neill et al. 2004). Logistic regressions were used to model the change in T50 over different lippia extract concentrations.

Results

Experiment 1

There were no differences between lippia stem and leaf extracts on the germination probability of oats and radish (P>0.05). Hence, all analyses in Experiment 1 used pooled means of both stem and leaf extracts. There was an exponential decrease in germination probability of oats and radish with increasing rates of lippia extract concentration (Fig. 1a). However, reductions greater than 20% in germination probability were only observed at lippia extract concentrations >12% w/v. T50 initially increased exponentially with increasing extract concentrations, but tended to level out at concentrations >12% w/v (Fig. 1b). Oats was slightly more sensitive to lippia extracts than radish.

Fig. 1. (a) Germination probability fitted to an exponential regression, and (b) time (h) to 50% germination (T50) fitted to a logistic regression for radish and oats in different concentrations of aqueous lippia extract (0, 4, 8, 12 and 30% w/v) in Experiment 1.

Experiment 2

There was no consistent effect on germination probability or T50 for sorghum seed in the extract concentrations tested (Figs. 2a and 2b). Germination probability decreased exponentially with increasing rates of lippia extract in all the other species tested (Fig. 2a). Differential responses to lippia extracts were found among the species tested. Germination probability of vetch and subterranean clover was reduced by >80% and 25%, respectively at 12% w/v lippia extract (Figs. 2a and 3). At 12% w/v lippia extract concentration, germination probability of ryegrass and lettuce were only slightly reduced (<20%). T50 for vetch and subterranean clover were also delayed at extract concentrations >30% w/v (Fig. 2b).

Fig. 2. (a) Germination probability fitted to an exponential function, and (b) time (h) to 50% germination (T50) fitted to a logistic regression for lettuce, ryegrass, Namoi woolly pod vetch, subterranean clover and sorghum in different concentrations of aqueous lippia extract (0, 4, 8, 12, 30, 40 and 50% w/v) in Experiment 2.

Fig. 3. Germination of subterranean clover seeds 20 days after sowing in (a) 0, (b) 4, (c) 8, (d) 12, (e) 30, (f) 40 and (g) 50% w/v aqueous lippia extract at 20 ▒ 1 C in Experiment 2.

Conclusion

Lippia is difficult to control and has been suspected of having allelopathic effects that inhibit seed germination of other plant species. It is difficult to isolate allelopathic effects in the field since there may be confounding effects of allelopathy and plant competition. This study provides the first laboratory-based evidence of the phytotoxic effects of lippia aqueous extracts on a range of crop and pasture species. Both leaves and stems of lippia inhibited seed germination to some extent for all species tested except sorghum. However, germination probabilities of ryegrass, oats, radish and lettuce were only slightly reduced (<20%) by high extract concentrations (≥ 12% w/v), which are possibly higher than those typically experienced in the field. More biologically relevant extract concentrations (viz. 12% w/v) have reduced germination probability of vetch and subterranean clover by >80% and 25%, respectively, which suggests that legumes should not be sown immediately after the cultivation of lippia dominant pasture. This is consistent with the observation of lippia-related growth suppression in another legume, lucerne in southern Queensland (Lucy et al. 1995). Hence, this study suggests that aqueous lippia extracts can have allelopathic effects and inhibit seed germination of certain crop and pasture species such as legumes.

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