Department of Biological Sciences, York College Don’t plant beans with onions: the effect of allelopathy on a natural, living fertilizer Johanna Slankard Department of Biological Sciences, York College RESULTS INTRODUCTION Allelopathy is the negative effect of chemicals released by one plant species on the growth, survival, and reproduction of other organisms (Inderjit and Callaway, 2003). The concept of allelopathy guides the principle of companion planting in garden settings. There are many observational principles about the companions, allies, and enemies in the garden community (Weinman 2011). One such adage recommends that, legumes should not be planted with onions (Allium cepa L.), as they do not grow well together (Cornell Cooperative Extension 2011, Weinman 2011). However, there is no scientific literature to support this common garden principle and onions are known to possess allelopathic properties such as antimicrobial activity (Cowan 1999). Could allelopathy challenge the nitrogen fixing symbiont of legumes? Rhizobium sp. is an intracellular bacterial symbiont associated with leguminous plants, in a mutualistic relationship. Blue Lake bush beans (Phaseolus vulgaris L. var. ‘Blue Lake’) are one such legume and a common garden bean. Any legume will associate with Rhizobia and nodules will form within the root of the plant. Development of nodules results in nitrogen fixation for the specific host and in return, the host provides the energy and environment for the process to occur (Vance 1979). Rhizobium sp. and other bacteria that are gram negative are found in the soil will make up the soil bacterial community which can be assessed using micro titer plates (BiOLOG EcoPlate™ 2007). When plant species are grown in an environment different from their respective soil community, the outcome of the growth of the plant can vary and this feedback can be either positive or negative (Bever 1994, Bever 2002). The ideas of allelopathy and feedback could provide a possible explanation for the observational principle behind not growing legumes and onions together in the garden. RESULTS AND DISCUSSION The biomass of beans was not inhibited by the allelopathic effects of onions. Indeed differences in bean biomass are more likely to be attributed to differences in pot volume. This data does not support Rhizobia growth being inhibited by the presence of onions. In fact, significantly more nodule biomass was observed on beans when planted closer to onions. Nodule growth and senescence is dependent on the time of reproduction (Vance 1979) . Differences in germination time influenced the age of plants at harvest and most likely affected the nodule biomass upon harvest (Figure 5). Higher soil nitrogen concentrations were found around beans alone compared to other treatments and controls. This may have to do with Rhizobia not being present to fix the nitrogen in the first place but since all of the seeds were inoculated, this is most likely not the case. Another possibility is that the nodules were present prior to harvesting but due to differences in the vegetative and reproductive stage of each plant, the Rhizobia biomass was lessened (Fernández-Luqueñ et al 2012). No difference was observed in the soil communities among beans alone, onions alone, and beans and onions planted together. HYPOTHESES The growth of beans will be negatively affected when grown with onions. There will be less Rhizobia (number of nodules and fresh weight) associated with beans when grown with onions. Lower soil concentrations of NO32-, NO22-, PO43- will be present in soil surrounding beans when grown with onions. Microbial functional richness, evenness, and diversity will be lower when beans are grown with onions. Harvest plants for biomass, counting and measuring Rhizobia growth, microbial soil community analysis, and soil nutrient analysis. Control: Nothing Planted Bean inoculated with Rhizobia Onion Bean inoculated with Rhizobia and Onion; 55 cm apart Bean inoculated with Rhizobia and Onion; 25 cm apart FUTURE STUDIES The differences in this study were due to difficulty in establishing the inoculated seed of the bean in the presence of onions (Figure 5), so a logical future study would examine the influence of onions on seed germination. Allelopathic effects of onion on bean seed germination may be a more plausible reason for the garden principle behind beans and onions being garden enemies. METHODS LITERATURE CITED Bever, J.D. 1994. Feedback between plants and their soil communities in and old field community. Ecology. 75(7):1965-1977. Bever, J.D. 2002. Negative feedback within a mutualism: host-specific growth of mycorrhizal fungi reduces plant benefit. Proceedings of the Royal Society of London 269:2595-2601. Available from: http://rspb.royalsocietypublishing.org/content/269/1509/2595.short. Accessed on: February 6, 2012 BiOLOG EcoPlate™. 2007. Microbial community analysis. Biolog, Inc. Available from: http://www.biolog.com/pdf/milit/00A_012_EcoPlate_Sell_Sheet.pdf. Accessed on: March 6, 2012. Cornell Cooperative Extension. 2011. Companion Planting. Available from: http://counties.cce.cornell.edu/chemung/agriculture/publications/companion-planting.pdf. Accessed on: July 7, 2011. Cowan, M.M. 1999. Plant Products as Antimicrobial Agents. Clinical Microbiology Reviews 12(4):564–582. Fernández-Luqueñ, F, Cabrera-Lazaro, G, Méndez-Bautista, J, López-Valdez, F, and Dendooven, L. 2012. Symbiotic nitrogen fixation in nodules from ten common bean cultivars as a reliable estimator of yield during the early stages. African Journal of Agricultural Research. Available from: http://www.academicjournals.org/ajar/PDF/pdf2012/5%20Mar/Fern%C3%A1ndez-Luque%C3%B1o%20et%20al.pdf Accessed on: March 25, 2012. Inderjit and Callaway, R.M. 2003. Experimental designs for the study of alleopathy. Plant and Soil 256: 1-11. Vance, C.P, Heichel, G.H, Barnes, D.K. Bryan, J.W, and Johnson, L. E. 1979. Nitrogen Fixation, Nodule Development, and Vegetative Regrowth of Alfalfa (Medicago sativa L.) following Harvest. Plant Physiology 64:1-8. Accessed on: December 12, 2011. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC426044/pdf/plntphys00541-0074.pdf Weinman, T. 2011. Companion Planting. NDSU Cass County Extension. Available from: http://www.ag.ndsu.edu/hort/info/vegetables/companion.htm. Accessed on: July 7, 2011. Figure 1. Biomass in dry weight (g) of root and shoot for each treatment. Purely for visual representation so no statistical analysis is done. Figure 2. Average nodule fresh weight (g) at time of harvest for each treatment. *(Kruskal-Wallace T=15.24, df=2, p=0.0005) Figure 3. Soil nitrate concentration as a qualitative analysis. *(Kruskal-Wallace T=25.59, df=4, p<0.0001) Figure 4. Microbial functional diversity for each treatment. *(One-way ANOVA F=11.32, df=4, p<0.0001) Figure 5. The successful establishment of beans for each treatment in a percent of total planted (total n=18) to germinate and grow. C: Control; B: Bean inoculated with Rhizobia; O: Onion; L: Bean inoculated with Rhizobia 55 cm away from onion; (L): Onion 55 cm away from L; S: Bean inoculated with Rhizobia 25cm away from onion; (S): Onion 25 cm away from S ACKNOWLEDGEMENTS I would like to thank Dr. K. Kleiner, PhD, for all his insights and advise in coming up with this experimental design and for answering my many questions during the operation of this thesis. I would also like to thank J. Kennedy for helping with data collection.