Evaluation of Allelopathic Effects of Some Plants Using Sandwich Method

Document Type : Research Article


1 Gorgan University of Agricultural Sciences and Natural Resources

2 Islamic Azad University


Introduction: Widespread use of herbicides in agriculture has led to loss of natural habitats, increase in pollution, generating herbicide-resistant weeds, risk of food poisoning and lower food quality. So new approaches are required in management, production, and utilization of the existing agricultural practices for sustainable agriculture. There are several techniques in this regard including allelopathy. Use of allelopathic compounds is a new approach for lowering undesirable effects of herbicides on the environment and fighting weeds resistant to herbicides. Recently allelopathy knowledge has been developed because of innovative methods and collaboration among scientists and achievements in suitable bioassays. Nowadays, old methods such as petri dishes or activated charcoal method have been replaced by new methods such as dish pack, sandwich, rhizosphere, and plant box. Researchers have indicated that many problems might be solved in allelopathy studies through these methods. Sandwich method is considered as one of the alternative methods for the old ones and is a very useful tool for screening the allelopathic effect of leaf litter under laboratory conditions. This method is a less time-consuming bioassay method and could be applied to screen more samples. Besides, allelopathic properties of many plants are evaluated using fewer plant samples through this method.
Materials and Methods: In this study, using a sandwich method, allelopathic properties of 55 plant species (59 samples) from 27 families were studied. The majority of plants were collected in 2016 from the different regions of South Khorasan province, Iran. The samples authenticated. Then, different parts of plants used for laboratory studies at Agricultural Researches Institute in Birjand University, Iran. Lettuce (Lactuca sativa L. var Great Lake 366) was used as an index because of its seeds sensitivity in chemical compounds, and allelopathic properties on the growth of seedlings of these lettuce varieties were evaluated. To investigate the allelopathic activity, an experiment was conducted as a completely randomized design with 3 replications. In order to assess the allelopathic activity of the selected plants, multi-dishes were used with 6 holes. Each hole had 3.5 cm diameter (Nunc Company, Japan). Ten milligrams of dried samples were placed in all three wells in the upper row and fifty milligrams was placed in rest of three lower wells of the six-well multi-dish plate. For the preparation of the growth medium, commercially available agar was applied. The medium was prepared as 0.5 % (w/v) and autoclaved at 115 °C for 20 min. 5 mL of autoclaved agar was added to each well of the multi-dish plate containing plant samples. After gelatinizing the agar within 30–45 min at room temperature, 5 mL agar was added again to all wells as the second layer and left at room temperature for gelatinized again. This made a sandwich of dried leaves by two layers of agar. Five lettuce seeds were put on the agar surface of the wells and all treatments were replicated three times. Each side of the prepared multi-dishes was then sealed by parafilm and wrapped in aluminum foil to protect them from light penetration and then were placed in an incubator (25°C). After 3 days, length of radicle and hypocotyl were recorded and monitored compared to control samples. Agar medium without plant samples was used as the control.
Results and Discussion: Growth of radicle and hypocotyl of lettuce seedling was present in the form of either inhibition (positive value) or promotion (negative value). The results showed that among the studied plants, Mentha spicata, Nepeta cataria, and Nepeta glomerulosa herbs highly affected growth inhibitory on radicle (96.4 %) and hypocotyl (94.8 %) of lettuce seedlings. Moreover, leaves of Leptorhabdus parviflora and Allium oschanini seeds strongly showed allelopathic effects by inhibiting the growth of radicle (91.0 %) and hypocotyl (85.2 %) of lettuce. Viola tricolor flowers plant had allelopathic effect as a deterrent growth only on radicle growth (90.0 %) and did not have significant effects on hypocotyl. In the current research, the introductory screening was done on 55 plants to recognize the suspected plants containing allelopathic compounds. However, further research is needed on these plants to figure out their effects on weeds.
Conclusions: The plants introduced in this research are mainly contain compounds such as Carvone, Limonene, α-Pinen, β-Pinen, 1,8 cineol, Thymol, Anethol, Camphor, Nepetalactone, β-caryophyllene and Geraniol. The results suggested that introduced plants may fulfill the organic farming and production of naturally originated herbicides. Therefore, more attention is needed for the future research focusing on finding the allelopathic responsible compounds by GC-MS and NMR techniques for using in organic agriculture. It is also necessary to investigate the ability of herbivorous of these compounds specifically.


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