A bioassay assessment of safflower allelopathy using equal compartment agar methods

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1 Journal of AGROBIOLOGY J Agrobiol 30(2): , 2013 DOI /agro ISSN (printed) ISSN (on-line) ORIGINAL ARTICLE A bioassay assessment of safflower allelopathy using equal compartment agar methods Mohammad Yousefi Davood, Hassan Karimmojeni, Mohammad Mehdi Khodaee, Mohammad Reza Sabzalian Isfahan University of Technology, Faculty of Agriculture, Department of Agronomy and Plant Breeding, Isfahan, Iran Received: 10 th January 2014 Revised: 17 th December 2014 Published online: 30 th March 2015 Abstract Allelopathy is the study of the interaction of plants with one another through their metabolites. The allelopathic effects of plants can stimulate or inhibit seedling growth and germination. In the first part of this study, the allelochemical donor was wild safflower [Isfahan 1 accession (Carthamus oxycantha)] and the receiver was cultivated safflower (Carthamus tinctorius, Padideh cultivar). In the second part, the former was receiver and the latter was the donor. The experiments had completely randomized designs with three replications. The first factor was the time duration of exposure to exudates, consisting of 3.5, 7 and 10 days, the second factor was the seeding densities of the donor, consisting of groups of 4, 8, 12 and 16 seedlings planted in 400 ml glass beakers. The time duration of exposure of the donor and receiver co-growth was 10 days. The findings showed that the general growth of the parameters under study in the cultivated safflower was stimulated by the wild safflower allelopathy. The maximum stimulation was seen in 12 seedlings density and 5 days exposure to wild safflower. The second experiment findings established clearly that in the same density and the same time duration, the cultivated safflower seedlings inhibited the germination parameters of the wild safflower. Key words: safflower; Carthamus tinctorius L.; Carthamus oxycantha; allelochemicals; donor; receiver; germination indices; density and time; stimulation; inhibition Hassan Karimmojeni, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Isfahan University of Technology, Isfahan, Iran kmojeni@cc.iut.ac.ir INTRODUCTION Global concern for the use of herbicides, their residues in the soil and plant biomass as well as their hazardous impacts on the environment have led plant scientists to look for proper ecologic approaches to the protection of plants against weeds. The allelopathic potential in integrated 97

2 weed management is well documented (An et al. 1997, Abdul et al. 2012). Allelopathy refers to the useful or hazardous effects of one plant on another through direct or indirect release of chemicals into the environment (Rice 1984, Inderjit et al. 2005). Chemicals released from plants that have allelopathic property are called allelochemicals. They are mostly classified as secondary plant metabolites and are produced as offshoots of the primary metabolic pathways of the plants. Allelochemicals are present in all parts of the plant, including leaves, root, stem, flowers and even pollen grains (Rice 1984). Allelochemicals are also released into the environment upon foliar leaching, plant residue decomposition, root exudation and volatile emission (Rice 1984). Among these modes of release, the role of the root in direct exposure to the soil and diffusion of allelochemicals into the environment are more prominent than others. The studies show that the reaction to allelochemicals may be dependent on their concentration. Allelochemicals that inhibit the growth of some species at certain concentrations might in fact stimulate the growth of the same or different species at different concentrations (Narwal 1994). If the allelopathic interactions are to be used in weed management programs, the knowledge of an effective concentration on a given species response is essential. It is likely that the toxicity of allelochemicals is a function of both concentration (static availability at a given point in time) and flux rate (dynamic availability based on the total amount of chemicals moving in and out of the system over a period of time (Williamson and Weidenhamer 1990). Wu et al. (2000a) found that wheat-seedling allelopathy of annual ryegrass (Lolium rigidum) as a receiver was dependent upon the seedling density of the donor wheat plants and that the inhibition increased with wheat-seedling density. So it is essential to make use of the co-growth of two bioassay species during the experimental period (Wu et al. 2001). Oilseed crops are grown throughout the world for production of oil and play an important role in the agricultural sector. Safflower (Carthamus tinctorius L.) is an oilseed multipurpose crop, which has been grown for many centuries throughout the world, especially in Iran, for its high quality oil, and is rich in unsaturated fatty acids (with the proportion of linoleic acid about 78% of total fatty acids), and red and orange pigments (particularly carthamin) extracted from its flowers. In recent years, due to the adaptability of safflower to varied growth conditions, especially to arid and semi-arid climates, with a high yield potential and increasing demand for oil, the attention paid to, and the interest shown in, safflower cultivation, has increased. Since weeds are among the most important factors in reduction of the yield and quality of crops and safflower is not an exception, the need for weed management under these conditions is very necessary. The growth of safflower seedlings in the early stages of development is slow and therefore they are sensitive to the damage caused by weeds. If weeds in safflower fields are well controlled, the damage caused in later stages would be significantly reduced. Wild safflower Carthamus oxycantha has been recognized as a damaging weed in Iranian crop systems causing damage each year in two stages of the growth of crop plants. The allelopathy of crops can be applied for the suppression of weeds, for example, in the vegetative and post-plantation stages and can also be used for the inhibition of weeds in the vegetative growth stages. In the post-harvest stage, plant residues can be used to inhibit weeds, especially during the period of the following crop establishment (Wu et al. 2000b). Recently, a new laboratory bioassay method the Equal-Compartment-Agar Method (ECAM) has been developed for the assessment of wheat seedling allelopathy on ryegrass. There is limited information on the safflower allelopathy potential. Therefore, this study has been carried out for the determination of the highest allelopathic potential of cultivated safflower versus wild safflower by finding the appropriate concentration and the allelochemical flux rate using ECAM. MATERIALS AND METHODS General trends The experiment was conducted at the seed laboratory of the Faculty of Agriculture of Isfahan University of Technology, from September 2010 to April The study consisted of two separate experiments for assessment of the allelopathic potentials of cultivated and wild safflowers and their interaction using ECAM. The wild safflower seeds Isfahan 1 accession, were collected and mixed from seeds of 30 to 50 separate plants at road-sides ending at the university. In the accession, two types of seed with white and dark brown colour were notable. 98

3 The two groups were separated from one another and seeds with a dark brown colour were selected for the experiment. The genotype of the cultivated safflower Padideh Cultivar was selected randomly from the Iranian safflower Germ plasm. To sterilise the surfaces, Isfahan 1 genotype was soaked in 70% ethanol and Tween-80 solution for 150 seconds followed by rinsing 4 times in sterilised distilled water. The Padideh cultivar was treated by soaking in 2.5% hypochlorite solution for 15 min followed by rinsing 5 times in sterilised distilled water. The sterilised seeds of both cultivated and wild safflower genotypes were then incubated in the dark at a temperature of 25 C for 60 hours and 48 hours, respectively. Germinated seeds were used for the bioassay experiments. All devices used for bioassay experiments had already been autoclaved. Experiment 1. Determination of the maximum allelopathic activity of Isfahan 1 (a wild safflower accession) and its effect on cultivated safflower (Padideh cultivar) The purpose of this experiment was to determine in which density of wild safflower and precisely when, release of the allelochemical would lead to the maximum allelopathic activity of the cultivated safflower Padideh cultivar. ECAM (The Equal Compartment Agar Method) was used for the purpose. The pre-germinated Isfahan 1 seeds were selected uniformly and were sown in 4 densities of 4, 8, 12 and 16 seedlings on the agar surface. Before planting the receivers, four treatments were carried out, after 3, 5, 7 and 10 days, on one-half of a glass beaker 400 ml that was filled with 40 ml of 0.4 % water agar. A piece of autoclaved paperboard was inserted into the middle of the beaker with a distance of 0.5 cm above the agar surface. Thus, the whole space above the agar was divided into two compartments and the competition between airborne organs of the donor and the receiver species was prevented while their roots were in contact with each other in the agar. Then the beakers were wrapped in pre-autoclaved pieces of aluminum foil and were placed in a controlled growth cabinet with a daily light/darkness cycle of 16 h/8 h and constant temperature of 25 C. After the growth of the Isfahan 1 seedlings for 3, 5, 7 and 10 days, ten pre-germinated seeds of Padideh cultivar were sown on the other half of the agar surface in 3 rows. Then the beakers were wrapped in aluminum foil again, were returned to the growth cabinet and grew together as donors and receivers of the allelochemical for 10 days. At the end of the experiment, the average root and stem lengths, the dry weight of roots and stems and the lengths of the largest root and stem of the cultivated safflower seedlings Padideh cultivar were measured. The growth of 10 seedlings of Padideh cultivar in the absence of the allelochemical donor Isfahan 1 accession was considered as control. Experiment 2. Determination of the maximum allelopathic activity of the cultivated safflower on the wild safflower The purpose of this experiment was to find the type of allelopathic effect inhibition or stimulation of the cultivated safflower on the wild safflower and its maximum allelopathy. The conditions of this experiment were like those of the first one, except that the allelochemical donor species was the cultivated safflower Padideh cultivar and the allelochemical receiver was the wild safflower Isfahan 1 accession. The experimental design and the statistical analysis The experiments were conducted in the form of a completely randomised design with three replications. The experimental data were analysed by the ANOVA test using SAS 9.1.The means were compared using MSTATC and SAS9.1 and LSD test at a confidence level of 5%. The inhibition/stimulation percent ratio was found using the following formula (Ahn et al. 2005). Inhibition/stimulation ratio = RESULTS AND DISCUSSION [(control-treated)] 100 control The findings showed that the culture of wild safflower seedlings, Isfahan 1 accession, in different densities had no significant effect on the mean root length, dry stem weight, dry root weight and the length of the largest root of the cultivated safflower, Padideh cultivar, but its effect on the mean stem length and the length of the largest stem was significant (Table 1) P<0.01. The time passed for 3, 5, 7, and 10 days of Isfahan 1 culture, showed a significant effect only on the mean stem length (P<0.05), while on the length of the largest stem in Padideh cultivar, that effect was significant at P<

4 Table 1. Results of the effects of time and density analysis for Isfahan 1 allelochemical releasing on Padideh cultivar experimental treatments Average of shoot length Average of root length Dry weight of shoot (g) Dry weight of root (g) Longest shoot Longest root F P-value F P-value F P-value F P-value F P-value F P-value Density 6.57 ** NS NS NS ** NS Time 3.83 * NS NS NS ** NS Time and density interaction * NS NS NS ** NS ** Significantly different at P<0.01; * significantly different at P<0.05; NS, insignificantly different P>0.05. The time density interaction was significant for the mean stem length of the Padideh cultivar at 0.05 significance level, and its highest stem length at 0.01 significance level, but no significant interaction was seen on other parameters of the Padideh cultivar under study. Allelochemicals of the wild safflower, Isfahan 1 accession, in different densities and times had no significant effect on the parameters of the roots of the cultivated safflower, Padideh cultivar (Table 1). With an increase in the density of Isfahan 1 accession seedlings, from 4 to 12 plants per beaker, the mean stem length and the length of the largest stem of Padideh cultivar increased compared with the control (Fig. 1). Also, there was no significant difference between the cultures of 16 and 4 plants per beaker and the control. These findings show that the presence of 12 seedlings of Isfahan 1 accession in co-growth with the Padideh cultivar led to the maximum allelopathic activity of the allelochemical donor of stimulating type. Amini et al. (2009) have reported that ryegrass inhibited the growth of wheat roots. That effect increased significantly with an increase in ryegrass density to 20 seedlings per beaker, while the increase in the ryegrass density to more than 20 seedlings per beaker had no significant effect on the wheat root growth. In this experiment, the results might be explained by the competition between wild safflower species for the limited space available and reduction in the allelopathic activity of Isfahan 1 accession. Some authors have reported that an increase in density might have led to the competition between weed species (Karimmojeni et al. 2010), with the passing of time, of co-growth of the donor and the receiver of allelochemical, the growth of the airborne organ of the Padideh Padideh shoot length Fig. 1. Allelopathic effect of Isfahan 1 genotype density on longest Shoot and average of shoot length of Padideh cultivar. Means within the chart followed by the same letter are not significantly different at the 5% level as determined by Least Significant Differences. Control has shown by

5 cultivar has been stimulated by allelochemicals of Isfahan 1 accession (Fig. 2). On average, stem length and the length of the largest stem of the Padideh cultivar during the initial 3 days of joint growth changed slowly; after 5 days it reached the maximum growth level, 10 days after jointgrowth, too. It led to a high stimulation, which showed no significant difference as compared with that after 5 days. Choosing the 5th day as the day of maximum allelopathic activity of Isfahan 1 accession could be explained as follows: the apparent state of seedlings of the donor and the receiver; even the growth media have a better and healthier state relative to the 10th day. Accumulation of allelochemicals of Isfahan 1 accession in the growth media on days 3 and 7 was not as high as that of day 5. As reported by Amini et al. (2009), the increase in ryegrass growth up to 6 days led to the highest inhibitory effect on the growth of roots and stems in wheat, while the ryegrass growth beyond 6 days reduced inhibition of the growth of wheat root and stem by ryegrass. Huang et al. (2003) found that the concentration of allelopathic compounds between days 6 and 8 of growth was highest and then decreased. It seems that this was due to the limited half-life of the allelochemical in the agar growth media. For example, Macias et al. (2002) found that DIMBOA (2,4-dihydroxy-7-methoxy- 1,4-benzoxazin-3-one) had a half-life of 5.3 h at 28 C for ph of 6.75 and it then disintegrated into the stable compound MBOA (6-methoxybenzoxazolin- 2-one), which still had an inhibitory potential, but of lower intensity. The findings of the interaction of density time of releasing allelochemicals of Isfahan 1 accession support the individual effects of time and density. The treatments of 4 seedlings/3 days and 16 seedlings/10 days of Isfahan 1 accession growth reduced very slightly the mean stem length of the Padideh cultivar, with no significant difference at 5% level versus the control (Fig. 3). It seems that the 4D3T [4 seedlings Density and 3 days Time] treatment led to the situation where Isfahan 1 accession failed to produce enough allelochemicals in the growth media to influence significantly the seedlings of the Padideh cultivar. Since the maximum effects of allelochemicals of Isfahan 1 accession in different treatments were of a stimulatory type, treatments 12D5T, 8D10T and 12D3T showed a significant difference in the mean length of the shoot of the Padideh cultivar and the highest stimulation. Evaluating the individual and mutual effects of time and density treatments, one concludes that the presence of 12 seedlings of wild safflower, Isfahan 1 accession, with a co-growth for 5 days, led to the maximum stimulatory effect on seedlings of cultivated safflower, Padideh cultivar, while 16D10T failed to show the maximum allelopathic potential of Isfahan 1 accession. A probable explanation is a re-uptake of allelochemicals by the donor as shown by Friebe et al. (1995) on wheat Triticum aestivum and Kobayashi et al. (1996) on Agropyron repens. The ANOVA findings of the data of Experiment 2 show that allelochemicals of the Padideh cultivar affected the mean root and stem length and the length of the largest stem of Isfahan 1 accession. The individual effects of experimental treatments of the density of donor seedlings and the time of release were significant Padideh shoot length Fig. 2. Effect of Isfahan 1 growing period s time on longest shoot and average of shoot length of Padideh cultivar. Means within the chart followed by the same letter are not significantly different at the 5% level as determined by Least Significant Differences. 101

6 Padideh shoot length Fig. 3. Interaction effects of density and growing days of Isfahan 1 genotype planting on Padideh s seedlings. Means within the chart followed by the same letter are not significantly different at the 5% level as determined by Least Significant Differences. The growth of receiver (10 seedlings of Padide cultivar) without donor (Isfahan 1) presence was considered as control. D and T mean density and time, respectively. Table 2. Results of the effects of time and density analysis for Padideh s allelochemical on Isfahan 1 genotype s experimental treatments Average of shoot length Average of root length Dry weight of shoot (g) Dry weight of root (g) Longest shoot Longest root F P-value F P-value F P-value F P-value F P-value F P-value Density * 2.23 NS 1.04 NS NS ** NS Time ** NS 1.03 NS 1.66 NS 10.2 ** NS Time and density interaction ** ** 1.31 NS NS ** NS ** Significantly different at P<0.01; * significantly different at P<0.05; NS, insignificantly different P>0.05. only for the largest stem (Table 2). That is why the interaction of experimental treatments on the mean root and stem lengths and the length of the largest stem were significant for all three parameters at P<0.01. In this study, it seems that, due to the paucity of biomasses of seedlings of the Padideh cultivar and Isfahan 1 accession, the parameters of dry weight of their roots and stems failed to prove the effects of allelochemicals on the biomass. Comparison of the means of different culture densities of the Padideh cultivar shows that the effect of allelochemicals on the stem of Isfahan 1 accession was inhibitory as is seen in a density of 12 seedlings while there was no significant difference between different densities and the control (Fig. 4). Times of release of allelochemicals of the Padideh cultivar showed two types of inhibitory and stimulatory effect on Isfahan 1 accession. Seven days after culture, the Padideh cultivar stimulated the mean stem length and the length of the largest stem of Isfahan 1 accession, while at other times of the growth, its effect was of inhibitory type. Five days after the culture, the donor had more inhibitory effect while no significant difference has been observed from days 3 to 10 (Fig. 5). Examination of the interaction of experimental treatments gives us a clearer view of the reactions of seedlings of Isfahan 1 acession under allelochemicals of the Padideh cultivar. As may be seen in Fig. 6, seven days after the culture of the Padideh cultivar, in all densities, relative 102

7 to the control culture of Isfahan 1 accession in the absence of the allelochemical donor, the stem growth of Isfahan 1 was increased. However, the remaining interactive treatments reduced the receiver s stem growth and the highest inhibitory effect was shown after the 5-day treatment with a density of 12 seedlings per beaker. Isfahan 1 shoot length Fig. 4. Allelopathic effect of Padideh cultivar density on longest Shoot and average of shoot length of Isfahan 1 genotype. Means within the chart followed by the same letter are not significantly different at the 5% level as determined by Least Significant Differences. Control has shown by 0. Isfahan 1 genotype shoot length Fig. 5. Effect of Padideh cultivated growing period s time on longest shoot and average of shoot length of Isfahan 1 genotype. Means within the chart followed by the same letter are not significantly different at the 5% level as determined by Least Significant Differences. The most likely explanation is that there are chemical reactions of allelochemicals of both the donor and the receiver, which have remained unclear to the authors, and which make their interpretation difficult. It might be that on day 5, accumulation of the donor allelochemicals was so high that it reduced the growth of the receiver stem while two days later, perhaps due to reuptake by the donor, it led to a stimulation of the receiver stem growth. Concerning the growth of root of Isfahan 1 accession, one could say that according to the comparison of means (Fig. 6), except for treatments 4D7T and 8D10T that were of a stimulatory type, other treatments reduced the root growth as compared with the control. It is interesting that the 12D7T treatment showed 103

8 Isfahan 1 Shoot shoot Lenght length a b c d de de def def efg efg efg fg gh gh gh gh h Interaction of Padideh Density and Growing Days Average of Shoot Length Isfahan 1 shoot length Isfahan 1 root length Fig. 6. Interaction effects of density and growing days of Padideh cultivar planting on Isfahan 1genotype seedlings. Means within the chart followed by the same letter are not significantly different at the 5% level as determined by Least Significant Differences. The growth of receiver (10 seedlings of Isfahan 1 genotype) without donor (Padideh cultivar) presence was considered as control. D and T mean density and time, respectively. 104

9 a maximum inhibitory effect, but it was not significantly different from 8D3T, 12D5T and 4D10T treatments. Generally, an analysis of the experiment reveals that in the laboratory setting, the allelopathy of the cultivated safflower, Padideh cultivar, had the potential to reduce the growth of the root and the stem of the wild safflower, Isfahan 1 accession. Of course, stimulating behaviours, too, were seen among the experimental variants. However, the most frequent effects were of the inhibitory type, since the 12D5T treatments reduced the stem and root growth of Isfahan 1 accession by 31.3 and 30.3 %, respectively. Their contribution to the parameters under study was higher than that of other treatments. Thus, one can conclude that 12 seedlings and 5 days release of allelochemicals of the Padideh cultivar have had the highest inhibitory effect on seedlings of Isfahan 1 accession. Examination of data obtained from the experiment shows that allelochemicals produced by wild safflower, Isfahan 1 accession, in different densities and times of growth, had no significant effect on the roots of cultivated safflower, Padideh cultivar, in joint growth in glass beakers, while they stimulated the stem of the Padideh cultivar. The maximum stimulatory effect was seen in the culture of 12 seedlings and 5 days release of allelochemicals of Isfahan 1 accession. The process was reversed in the second experiment. The growth of seedlings of Isfahan 1 accession under the allelopathy of the Padideh cultivar was of an inhibitory type for most of the time. According to a comparison of the means of treatment of 12 seedlings, 5 days culture of the Padideh cultivar led to the highest reduction in root and stem growth of Isfahan 1 accession. The purpose of this experiment was mostly an examination of intra-species allelopathic behaviours. It was proved that the allelopathic potential varied both between plant species, and types. The findings of this study could be used in screening of different cultivars and genotypes of the species so that one could use the most allelopathic genotype to be introduced for further research. REFERENCES Abdul A, Matloob A, Tanweer A, Bismillah Khan M (2012): Naturally occurring phytotoxins in allelopathic plants help reduce herbicide dose in wheat. Natur Prod Lett 26: Ahn JK, Hahn SJ, Kim JT, Khanh TD, Chung IM (2005): Evaluation of allelopathic potential among rice (Oryza sativa L.) germplasm for control of Echinochloa crus-galli P. Beauv in the field. Crop Prot 24: Amini R, An M, Pratley J, Azimi S (2009): Allelopathic assessment of annual ryegrass Lolium rigidum: Bioassays. Allelopathy J 24: An M, Pratley JE, Haig T, Jellett P (1997): Genotypic variation of plant species to the allelopathic effects of vulpia residues. Austral J Exp Agric 37: Friebe A, Schulz M, Kuck P, Schnabl H (1995): Phytotoxins from shoot extracts and root exudates of Agropyron repens seedlings. Phytochemistry 38: Huang Z, Haig T, Wu H, An M, Pratley J (2003): Correlation between phytotoxicity on annual ryegrass (Lolium rigidum) and production dynamic of allelochemicals within root exudates of an allelopathic wheat. J Chem Ecol 29: Inderjit, Leslie A, Weston LA, Duke SO (2005): Challenges, achievements and opportunities in allelopathy research. J Plant Interact 1: Karimmojeni H, Rahimian Mashhadi H, Alizade HM, Cousens RD, Beheshtian Mesgaran M (2010): Interference between Zea mays and Xanthum strumarium or Datura stramonium. Weed Res 50: Kobayashi A, Kim MJ, Kawazu K (1996): Uptake and exudation of phenolic compounds by wheat and antimicrobial components of the root exudate. Z Naturforsch 51(C): Macias FA, Marin D, Castellano D, Velasco RF, Oliveros-Bastidas A, Chinchilla D, Molinill MG (2002): Synthesis and bioactivity evaluation of hydroxamic acids: Proceedings of 3rd World Congress on Allelopathy, Tsukuba, Japan, Agust 26 30, pp Narwal SS (1994): Allelopathy in Crop Production. Sci. Publ., Jodhpurs, India. Rice EL (1984): Allelopathy. 2nd ed. Academic Press, Orlando. Williamson GB, Weidenhamer JD (1990): Bacterial degradation of juglone: evidence against allelopathy? J Chem Ecol 16: Wu H, Pratley D, Lemerle T, Haig B (2000a): Laboratory screening for allelopathic potential of wheat (Triticum aestivum) accessions against annual ryegrass (Lolium rigidum). Austral J Agr Res 51:

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