Allelopathy of Sage and White Wormwood on Purslane Germination and Seedling Growth
Alireza PIRZAD
1),Vahid GHASEMIAN
2),Reza DARVISHZADEH
1),Mohammad SEDGHI
3),Abbas HASSANI
4),Andrea ONOFRI
5)1)Urmia University, Faculty of Agriculture, Department of Agronomy and Plant Breeding, Urmia, Iran; [email protected], [email protected]
2)Tarbiat Modarres University, Faculty of Agriculture, Iran; [email protected]
3)University of Mohaghegh Ardabili, Faculty of Agriculture, Department of Agronomy and Plant Breeding, Ardabil, Iran; [email protected]
4) Urmia University, Institute of Biotechnology, Department of Medicinal and Industrial Plants, Urmia, Iran; [email protected]
5)University of Perugia, Department of Agricultural and Environmental Sciences, Perugia, Italy; [email protected]
Abstract
A bioassay run was carried out in the incubator to evaluate possible allelopathic effects of water extracts of sage and white wormwood on germination and seedling growth of purslane. Results showed that the type of extract and its concentration (0, 5, 10, 15 and 20%) significantly influenced final germination percentage, germination rate and index, root and shoot length, root/shoot ratio, fresh and dry weight of seedling of purslane. The interaction between these two experimental factors was always significant, producing different results according to the different combination levels. The statistical comparison of means indicated that the maximum germination percentage and germination rate (respectively 68% and 11.4% d-1) were obtained from the untreated control (0% extract), while the lowest values for the same two characters occurred with 15% of sage (respectively 45% and 6.4% d-1) and 20% of white wormwood (38% and 7.5% d-1).
Concerning germination indexes, the highest (55) and the lowest (32) values were observed respectively on the control and with 15% of sage extract or 20% of white wormwood extract. The longest (4.5 cm) and the shortest (1.3 cm) root of purslane were obtained with 5%
of white wormwood and 20% of sage extracts, respectively. Changes in shoot length with sage and white wormwood extracts were similar to those in root length, even though shoot length was less affected by the concentration of extracts. Root and shoot length changes brought to maximum (22.0) and minimum (8.9) values for the root/shoot length ratio, respectively with 5% of white wormwood and 20% of sage extracts. The maximum fresh (2.111 g) and dry (0.338 g) seedling weight of purslane were obtained from untreated control, producing the same seedling weight with 5% of sage, 5 and 10% of white wormwood extract. While the minimum fresh (0.692 g) and dry (0.111 g) seedling weight were obtained from 15% of white wormwood extract, so that there is no significant differences between this value and the seedling weight produced under 10, 15 and 20% of sage and 20% of white wormwood extract.
Keywords: Arthemisia sieberi, germination, Portulaca oleracea, purslane, sage, Salvia officinalis, seedling Introduction
Allelopathy interactions are primarily based on the ability of certain plant species to produce secondary chemical compounds, that exert some sort of biological ef- fects on other organisms, many of which are still unknown (Waller, 2004). Allelopathic compounds are released into the environment through root exudation, leaching by dews and rains and volatilization from decaying plant tissues (Rice, 1984). In most cases, these compounds inhibit the germination or growth of neighboring plants, although sometimes they may show a stimulating effect (Ebana et al., 1981). Inhibitory effects of allelophatic plants may be exploited within an integrated weed management sys- tem to modify crop-weed interactions and lower the need for chemical means of weed control; indeed, yield losses caused by weeds are well documented in many studies and it has been shown that these losses may frequently be high-
er than those caused by pests and diseases (Culter, 1988;
Steinsiek et al., 1982).
This present study considered a very widespread spe- cies, i.e. Portulaca oleracea L. (purslane). Although it is characterized by several ecological types, some of which are occasionally used as vegetables in human dietary (Mi- yanishi and Cavers, 1980), this plant species is mainly re- garded as a drought hardy weed, colonizing waste places and bare areas, but also thriving in moist and fertile soils. Overall, purslane is considered as a serious threat to cultivated fields, throughout tropical, subtropical and temperate areas, attaining this status more because of its very widespread importance, than by being amongst the top few weeds in any one country. Indeed, purslane was ranked 9th of the world’s worst weeds, being recorded in 45 crops in 81 countries (Holm et al., 1977), it showed a rating of 10 in Southeast Asia, while it was ranked 6th in the Pacific and 49th in Australia (Waterhouse, 1993a, Received 18 June 2009; accepted 29 August 2009
oil of Artemisia sieberi from Iran were found to be cam- phor (44-49.3%), 1,8-cineole (11.1-19%), bornyl acetate (5.8%) and camphene (5%) (Weyerstahl et al., 1993). In other studies, thirty-one compounds were identified, in- cluding β-thujone (19.8%), camphor (19.5%), α-thujone (10.6%), verbenole (9.7%), ρ-mentha-1,5-dien-8-ol (6.4%) and 1,8-cineole (5.7%) (Ghorbani-Ghouzhdi et al., 2008). Considering A. sieberi from the Kerman prov- ince of Iran, the main components of essential oil were (a) in non-grazed sites were: 1,8-cineol (29.9%), myrcene (14.1%); (b) in moderate grazed site: myrcene (15.9%), 1,8 cineol (15.1%), Eudesm-7(11)-en-4-ol (11.1%); (c) in heavy grazed site: 4-tepinyl acetate (23.3%), davanone (21.9%), p-cymene (19%) (Bagheri et al., 2007).
Recently, the interest in the application of essential oils to control plants and post harvest pathogens has increased and their potential role in food preservation has been exploited (Lanciotti et al., 2004; Vazquez et al., 2001).
However, S. officinalis and A. sieberi can also play an al- lelopathic role, because their components might exert an inhibitory effect on seed germination and seedling growth of P. oleracea, even though no data is available in literature with this respect.
The aim of this study was to evaluate the allelopathic potential of water extracts obtained from S. officinalis and A. sieberi, with respect to the above mentioned weed spe- cies (P. oleracea).
Materials and methods
Aerial parts of S. officinalis L. and A. sieberi were col- lected from the Urmia region (Iran), were air dried at room temperature (20-25°C) and were ground by an elec- tric grinder. Water extracts were prepared at concentra- tions of 0, 5, 10, 15 and 20%. To prepare 20, 15, 10, and 5% of extract 40, 30, 20, and 10 g dry matter of both spe- cies soaked in 30°C distilled water during 48 hours, so that it produced 200 ml extract, respectively.
Seeds of Portulaca oleracea were collected at the Re- search Farm of the University of Urmia (1320 m above sea level, 37°32’ N, 45°5’ E), Iran. They were sterilised with sodium hypochloride (10%) and washed by distilled wa- ter. For each treatment, one hundred seeds were randomly placed in four Petri dishes with 9 cm diameter, lined with two Whatman discs with filter paper No. 1 at the bottom.
Three ml of deionized water were added to each dish at the beginning of the experiment, by using a pipette. Consid- ering water extract type (S. officinalis and A. sieberi) and concentration (0, 5, 10, 15 and 20%), the experiment was organised according to a two - factor factorial, randomised complete block design with four replicates.
Germination assays were conducted in an incuba- tor at constant temperature of 25°C in darkness, for six days. Germinated seeds from individual Petri dishes were counted and removed every day at an interval of 24 hours.
A seed was considered as germinated when radicle protru- b). In Southeast Asia, it is particularly important in many
upland crops, including vegetables, rice, maize, sorghum, groundnuts and sugarcane.
Purslane, as an important weed (Duke, 1987), is a fleshy annual herb, reproducing by seeds, or by stem-fragments rooting when lying on moist soil. The stems are succulent, often reddish and 0.2 to 0.5 m in length. The leaves are alternate and frequently clustered at the end of branches;
flowers are yellow, sessile, self-pollinated and occur either singly or several together in the above mentioned leaf clus- ters. Flowers open on sunny mornings and produce numer- ous (up to 243000 per plant), tiny (0.5 mm diameter) and black seeds (Waterhouse, 1994). These latter are spread by wind, water, as contaminants of crop seeds and by birds, surviving passage through the digestive tract. They also survive burial for long periods and germinate best above 30°C, but poorly below 24°C (Waterhouse, 1994).
Though it does not compete well with other weeds, it is often successful because it establishes rapidly after soil dis- turbance and may flower and set its seeds before being out competed by taller plants. The succulent leaves and stems are rich in oxalates and nitrates and have been implicated in livestock deaths (Miyanishi and Cavers, 1980).
Two plant genera with potential allelopathic effects were considered in this study, i.e. Salvia and Artemisia.
Concerning the first one, fifty-eight species of Salvia (sage; family of Lamiaceae) are found in Iran, seventeen of which are endemic (Mozaffarian, 1996). Several of these species have been reported to produce secondary metabo- lites with medicinal usage (Hitokoto et al., 1980). Among these, Salvia officinalis L. is very widespread; it is a semi- woody shrub reaching a height of 60 cm and originates from the Mediterranean regions of North Africa, Spain and the Balkans. It has been grown as a medicinal and cu- linary herb for thousands of years and it can now be found everywhere in gardens (D’Antunono et al., 2002). A com- parison of the composition of the Iranian sage oils dur- ing the different developmental stages revealed that 1,8- cineole (15.3-22%), α-thujone (9.1-25.1%) and β-pinene (7.1-16.4%) were the principal components (Mirjalili et al., 2006).
Artemisia (family of Asteraceae) is the largest and most widely distributed genus among the approximately 60 be- longing to the tribe of Anthemideae. This genus comprises a variable number of species, ranging from 200 to over 400, which are predominantly distributed in the northern regions of the world, within the 0-50 cm precipitation area (Tan et al., 1998). Thirty-four species of Artemisia are re- ported in Iran and some of them are endemic. In Iranian folk medicine, some species of Artemisia are used for their various medicinal properties and local people used aerial parts of these plants for their antiviral and spasmolytic ef- fects (Ramezani et al., 2004).
Within this genus, Artemisia sieberi Bess. (white wormwood) is widely distributed in desert areas of Iran (Mozafarian, 1988). The main components of essential
sion was more than 2 mm length. The final germination percentage, average germination rate (n. per day) and ger- mination index were measured for each Petri dish as fol- lows (Ellis and Roberts, 1981; Maguire, 1962):
Germination Rate = ΣXn / Yn Germination Index = Σ (Xn/Yn)
in which, Xn was percentage of germinated seeds at Yn days.
Fifteen germinated seedlings of P. oleracea from each Petri dish were grown in light during day and darkness dur- ing night under treatment conditions (different concen- trations of extracts) until cotyledonal leaves fully opened and then root length, shoot length, seedling fresh and dry weight were measured.
Statistical analysis: All data were submitted to ANO- VA. A graphical inspection of residuals showed that the basic assumptions for linear models were not severely vio- lated and that stabilizing transformations were not needed for any of the measured variables. As regression models could not be successfully fitted into this dataset, means were separated by using a protected Least Significant Dif- ference (LSD), at p = 0.05. All the analyses were performed by using the statistical package MSTATC software.
Results and discussion
Results of analyses showed that the effects of extract type (sage and white wormwood), concentration of ex- tract (0, 5, 10, 15 and 20%) and their interaction on final percentage germination, germination rate and index, root and shoot length, root/shoot length ratio, seedling fresh and dry weight were significant (Tab. 1).
Mean comparisons indicated that the maximum germi- nation percent (68%) was obtained on the control (0% ex- tract) treatment. Application of 5% sage and white worm- wood extracts produced final germination percentages of 56 and 61%, respectively. At higher concentrations, final germination percentages decreased progressively, down to minimum values of 45 and 38%, respectively at 15% sage and 20% of white wormwood (Fig. 1-I).
The germination rate of P. oleracea under different con- centrations of both sage and white wormwood extracts showed the same trend as final germination percentage (Fig. 1-II), i.e. the maximum and minimum germination rates were obtained respectively on the control treatment (11.4% d-1) and with either 15% of sage (6.4% d-1) or 20%
of white wormwood (7.5% d-1) extracts.
Considering the germination index, the highest value was observed in the control treatment (55.1) that was not significantly different from that observed with extracts at concentrations of 5 and 10% of sage and 5, 10 and 15% of white wormwood. On the other hand, the lowest germina- tion index (31.7) was obtained with 15% of sage that was not significantly different from that obtained with 20% of sage and white wormwood (Fig. 1-III).
The highest root length of P. oleracea (4.5 cm) was ob- tained from extracts at 5% concentration of white worm- wood that was significantly higher than the value obtained from control treatment. Increasing the concentration of white wormwood led to shorter purslane roots and such an effect was on average higher with sage extracts., so that the shortest root (1.3 cm) was obtained with 20% of sage extract (Fig. 2-I).
Changes in shoot length were on average smaller than those in root length, and significant differences were only observed between plants treated with 5% white worm- wood or sage and plants treated with 10% sage (Fig. 2-II).
Trends in root and shoot lengths led to maximum (22.0) and minimum (8.9) root/shoot length ratios with 5% of white wormwood and 20% of sage, respectively.
Variations in root/shoot length ratio followed closely vari- ations in root length, thus this latter variable appeared to affect the above ratio more than shoot length (Fig. 2-III).
The maximum fresh (2.1 g) and dry (0.39 g) weight of purslane seedlings were obtained from the control treat- Fig. 1. Means comparisons of interaction effects between extract origin (sage and white wormwood) and concentration on ger- mination percent (I), rate (II) and index (III) of Portulaca olera- cea seeds. The same letters show non significant differences
Fig. 2. Means comparisons of interaction effects between extract origin (sage and white wormwood) and concentration on root length (I), shoot length (II), root/shoot length (III), seedling fresh weight (VI) and seedling dry weight (V) of Portulaca oleracea.
The same letters show non significant differences
Tab. 1. Analysis of variance (MS) of sage and white wormwood water extracts effect on germination and seedling growth of purslane Source of
Variation df Germination Length Seedling
Percent Rate Index Root Shoot Root/Shoot Fresh weight Dry weight
Replication 3 0.004ns 0.004ns 0.001ns 0.500** 0.0003ns 0.009ns 0.001ns 0.001ns Extract (A) 1 0.025** 0.025** 0.030** 3.469** 0.002ns 0.029* 0.014* 0.010**
Concentration (B) 4 0.047** 0.047** 0.056** 5.493** 0.002ns 0.104** 0.088** 0.066**
A×B 4 0.009** 0.009** 0.013** 1.633** 0.004** 0.024* 0.029** 0.021**
Error 27 0.002 0.002 0.002 0.095 0.001 0.006 0.002 0.001
C.V. (%) 2.53 4.61 2.41 11.01 17.45 6.53 12.29 16.58
ns, *, **: non- significant and significant at P<0.05 and P<0.01, respectively; df: Degrees of Freedom
ment, while the weight of seedlings obtained with 5% of sage and 5 and 10% of white wormwood were not signifi- cantly affected. On the contrary, the minimum fresh (0.69 mg) and dry (0.11 mg) seedling weight were obtained with 15% and 20% of white wormwood and 10, 15 and 20% of sage, with no significant differences among these extract types and concentrations (Fig. 2-IV and Fig. 2-V).
In conclusion, results show that the effect of extract type and concentration strongly interacted and led to different effects according to the different combination of these two experimental factors. Seed germination of purslane was ei- ther inhibited or slowed down by the application of little amounts of sage and white wormwood extracts and these effects were greater with sage than with white wormwood.
On the contrary, seedling growth of purslane was slightly stimulated by the lowest concentration of sage (5%), while it was inhibited by higher concentration values. Also in this case, the effect of sage appeared to be stronger than that of white wormwood.
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