The allelopathic effects of juglone containing nuts Abstract Allelopathy is the inhibition of the growth of

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1 The allelopathic effects of juglone containing nuts Catherine Qin, Manavi Nagai, Whitney Hagins, and Reginald Hobbs* Department of Science AP Biology Lexington High School, 251 Waltham Street, Lexington, MA *Correspondence: Abstract Allelopathy is the inhibition of the growth of a plant by the allelochemicals, produced by a nearby plant, into their shared environment. It is a form of chemical warfare used by plants to defend their territory and resources, thus reducing interspecific competition 1. Although some allelopathic plants produce agents that may benefit rather than harm surrounding species, the majority release chemicals that are toxic to others. To explore this biological phenomenon, the effects of allelopathic nuts of the family Juglandaceae, containing the novel allelopathic agent juglone, were tested on the germination of plants sensitive to allelopathy and those shown to be resistant. Based on previous studies examining allelopathy, it is expected that sensitive plants treated with this agent would cause negative affects on germination, while the seeds that are known to be resistant, would undergo relatively normal germination. The results of these experiments are generally consistent with this model of allelopathic toxicity and resistance. Allelopathy is a unique phenomenon in nature that is relatively new to the scientific community, and the results of this experiment are not only relevant to basic botanical research, but also have real world applications. Introduction Allelochemicals can be found in all parts of the plant, the greatest amounts are most often located in the roots and leaves. Plants release their allelochemicals in several different ways: volatilization, leaching, or exudation. In volatilization, the toxic chemicals are released in the form of a gas from the leaves and then are absorbed by another plant, causing it to die. In leaching, the chemicals that are stored in the plant s leaves seep into the soil, either by the littering and decomposition of the leaves, or via runoff rain, fog, or dew that comes into contact with the leaves. In exudation, the chemicals are released into the soil through the plant s roots 1. Allelochemicals can act on plants in a variety of ways: inhibiting germination by disrupting cell division, interfering with mechanisms of energy transfer such as respiration, and limiting water and nutrient uptake 1. The overall effect is to severely impede the growth of the plant. Examples of plant species that exhibit allelopathy include many trees (sugar maple, eucalyptus, and oak), shrubs, (sumac, rhododendron, and elderberry), agricultural crops (tobacco and rice), and various grasses and ferns 1. Each releases its own type of allelochemical. In this study, the vegetable seeds examined that are known to be sensitive to allelopathic toxins were pecan (Carya illinoinensis), English walnut (Juglans regia), black walnut (Juglans nigra), tomato (Solanum lycopersicum), cabbage (Brassica oleracea), and Wisconsin Fast Plants (Brassica rapa). The seeds that are known to be resistant to allelopathic toxins, bean (Phaseolus vulgaris) and corn (Zea mays)were compared with the sensitive plants, in order to test the phenomena of allelopathy. One of the most well-known allelopathic chemical toxins is known as juglone. Juglone negatively affects the growth of many plants, including pine, birch, blueberry, and tomato, often times killing very sensitive species. The allelopathic properties of Juglans nigra, Juglans regia, and Carya illinoinensis are due to the production of juglone 3. For our experiment, we decided to use the fruit the actual nut of each nut tree, as opposed to the roots, leaves, or branches because nuts are the easiest to obtain and handle. Previous research has shown that tomato and cabbage are sensitive to black walnut, while vegetables such as bean and corn do not seem to be 4. Hence, we decided to use tomato, cabbage, bean, and corn as our test subjects. In addition Wisconsin Fast Plant (Brassica rapa) were tested for allelopathic sensitivity/resistance, because it is closely related to cabbage. Various studies have been written on the allelopathic effects of certain plants, though none have had the same focus and scope of experimentation as ours. A study by Shibu Jose and Andrew R. Gillespie in 1998, showed the effects of juglone on the growth and physiology of corn versus soybean. The findings

2 Catherine Qin, Manavi Nagai, Whitney Hagins, and Reginald Hobbs Page 2 of 5 of the study were that although both crops were sensitive to juglone, soybean showed more of a negative impact than corn 5. These results led to the hypothesis that the germination of corn would be relatively unaffected by nut allelochemicals at the concentrations that we would be testing. In another study, Allelopathic effects of Squash (Cucurbita pepo L. cv. Scarlette) on certain common weed species in Jordan (Qasem, J.R., et al., 2010), the allelopathic effects of squash on several different weed species was investigated. The findings of the study showed that even 1ml of squash extract caused reduced germination and growth in all of the weeds, and these effects were amplified with increased concentrations of squash extract 6. Although the focus of this study was on the allelopathic effects of squash on weeds as opposed to nuts on vegetable seeds, they used a similar experimental design: using a variety of test subjects, and testing various concentrations of the extract. Another study, Phytotoxic effects of Parthenium hysterophorus residues on three Brassica species (Singh, H.P., et al., 2005), the phytotoxic effects of Parthenium hysterophorus, was investigated on three different Brassica species. The findings of the study were that P. hysterophorus does in fact negatively impact the early growth of the three crops by allelopathy, and that there was a direct correlation between the degree of inhibition of growth and the amount of P. hysterophorus residue in the soil 7. Other studies have investigated the allelopathic effects of a variety of other plants, such as sour orange, red maple, eucalyptus, mango, Tree of Heaven, rye, wheat, and broccoli; each of these allelopathic plants interfered with the growth of surrounding plants. In the current experiment, the allelopathic effects of Juglone, contained in the nuts of the Juglandaceae family, was tested on the germination of garden vegetables seeds. The relevance of our experiments to real world applications is obvious. In agriculture, gardening, and landscaping, the growth of various plants may be affected when they are exposed to allelochemicals produced by these nuts. Knowing this, susceptible plants should be grown far away from where nut trees are growing. However, plants that are not as sensitive to allelochemicals can be planted in proximity to the nut trees and grow mostly unaffected. The allelopathic effects of these nuts on certain plants can also be used to an advantage; trees of the Juglandaceae family can be used as natural herbicides on weeds and other unwanted plant growth. Materials and Methods Approximately nuts of each nut species (pecan, English walnut, and black walnut) were ground up into a fine powder. A solution with a ratio of 7g nut powder: 50ml water was refrigerated for 24 hours, and then filtered to remove insoluble material. 2g of nut powder was placed into the corresponding small petri dishes for that nut, on top of 3 wet paper towels, and covered with one wet paper towel. 4g of nut powder was placed into large petri dishes, again on top of 3 wet paper towels, and covered by one paper towel. 4.5ml of each nut solution was pipetted into the small petri dishes and 7ml of each nut solution was pipetted into the large petri dishes. For the controls, 4.5ml or 7ml of water was used instead of nut solution. 10 vegetable seeds(tomato, cabbage, Brassica rapa, bean, and corn) were then placed individually into each of their corresponding petri dishes. To the small petri dishes 0.5ml of water was added each day and 1.5ml of water was added to the large petri dishes. The dishes were kept at room temperature in the dark in a covered box, and were only taking out for observation. The seeds in each dish were watered until ~100% of the control seeds germinated. The percentage of seeds germinated of both the control and treated seeds for each vegetable were recorded daily and then averaged and graphed after three trials. Results The data shows that tomato seed germination was severely slowed down by the allelochemicals (Figure 1), and that the black walnut was extremely detrimental to its germination. Brassica rapa (Figure 2) and cabbage seeds (Figure 3) also showed intense susceptible to juglone containing nuts/nut extracts; the cabbage seeds took until Day 11 to completely germinate. Bean seeds are only resistant to a certain extent to the allelochemicals - germination was slightly inhibited (Figure 4), whereas corn germination seems to be enhanced by juglone (Figure 5). English walnut and pecan treated seeds also germinated at a slower rate than in the original procedure when exposed to juglone extracts (all figures).

3 Catherine Qin, Manavi Nagai, Whitney Hagins, and Reginald Hobbs Page 3 of 5 Figure 1. The Effects of Various Allelopathic Nuts on the Germination of Tomato Seeds: The black walnut treated tomato seeds underwent almost no germination. The English walnut and pecan treated seeds also germinated at a slower rate. Figure 2. The Effects of Various Allelopathic Nuts on the Germination of Cabbage Seeds: The germination of cabbage seeds were negatively impacted by allelochemicals; these seeds took until Day 11 to completely germinate. Figure 3. The Effects of Various Allelopathic Nuts on the Germination of B. rapa Seeds: In Brassica rapa seeds, germination was inhibited by juglone containing nuts/nut extracts. This was most prevalent in the black walnut treated Brassica rapa seeds - they never finished germinating.

4 Catherine Qin, Manavi Nagai, Whitney Hagins, and Reginald Hobbs Page 4 of 5 Figure 4. The Effects of Various Allelopathic Nuts on the Germination of Bean Seeds: Germination in bean seeds was slightly inhibited, contrary to our hypothesis that bean would be resistant. This is shown in the graph by the slightly lower rates of germination of all of the treated seeds, as compared to the control. However, due to our small sample size, it is not known if this slight inhibition is significant. Figure 5. The Effects of Various Allelopathic Nuts on the Germination of Corn Seeds: The germination of the treated corn seeds was actually enhanced by juglone. This data suggests that the allelochemicals of these nuts may actually be helping corn to germinated faster, as opposed to negatively impacting the seeds. Discussion This study supported our predictions that allelopathic sensitive plants would show decreased germination, and that allelopathic resistant plants would not. We showed that tomato, cabbage, B. rapa, and bean showed negatively affected germination in the presence of nut allelochemicals, while corn germination became more enhanced. Tomato and B. rapa germination were the most negatively affected, followed by cabbage, though to a lesser extent. Our result that corn seed germination was not negatively affected but rather enhanced by the presence of nut allelochemicals is surprising, but concurs with our hypothesis. The results obtained from this study showed correlations with the results of past work conducted in the field of allelopathy. Experiments conducted by Shibu Jose and Andrew R. Gillespie in 1998, showed that corn was less negatively affected by juglone than soybeans 5. This supports our conclusion that corn is more tolerant of nut allelochemicals than other vegetable varieties. Our result that cabbage and B. rapa seeds showed significant inhibition in germination by the presence of nut allelochemicals correlated with the results of the study conducted by Harminder Pal Singh, et. al. in 2005; they found that the residues of the allelopathic plant P. hysterophorus severely reduce the growth of Brassica species, including cabbage and B. rapa 7. Another study whose results correlated with ours was conducted by Jamal R. Qasem and Nabil N. Issa in 2010, which found

5 Catherine Qin, Manavi Nagai, Whitney Hagins, and Reginald Hobbs Page 5 of 5 that allelopathic effects on plants are amplified with increased concentrations of allelochemicals 6. In general, the results of our experiment seem to agree with the previous research that has been done in this field. Therefore, after experimentation and data analysis, we can conclude that the results of our experiment support our hypothesis, and concur with past work conducted in the field of allelopathy. References 1 Allelopathy: Chemical warfare among plants. Trees and turf: Are they compatible? University of Minnesota. nd. 2 Brown, C. Allelopathic plants: Nature s weed killers.earth friendly gardening. 2006, November Appleton, B., Berrier, R., Harris, R., Alleman, D., & Swanson, L. Trees for problem landscape sites - the walnut tree. Allelopathic effects and tolerant plants. 2009, May 1. 4 Smith, C. W. Walnut tree allelopathy. New Mexico State University. 2004, October 9. 5 Jose, S., & Gillespie, A. R. Allelopathy in black walnut (Juglans nigral.) alley cropping. II. Effects of juglone on hydroponically grown corn (Zea maysl.) and soybean (Glycine maxl. Merr.) growth and physiology. Plant and Soil, 1998, 203, Qasem, J. R., & Issa, N. N. Allelopathic effects of squash (Cucurbita pepo L. cv. scarlette) on certain common weed species in Jordan. The Regional Institute Singh, H. P., Batish, D. R., Pandher, J. K., & Kohli, R. K. Phytotoxic effects of Parthenium hysterophorus residues on three Brassica species. Weed Biology and Management, 2005, 5, Ferguson, J. J., & Rathinasabapathi, B. Allelopathy: How plants suppress other plants. Horticultural Sciences Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida