THE FLORIDA STATE UNIVERSITY

COLLEGE OF ARTS AND SCIENCES

ASSOCIATIONAL EFFECTS: VARIATION IN PLANT-

HERBIVORE INTERACTION AT DIFFERENT DISTANCES OF

HETEROSPECIFIC PLANTS

By

KYLE H. SPELLS

A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for graduation with Honors in the Major

Degree Awarded:

Fall, 2016

Associational Effects: Variation in Plant-Herbivore Interaction at Different Distances of Heterospecific Plants

Kyle H. Spells, Brian D. Inouye, Nora Underwood Department of Biological Science, Florida State University, Tallahassee, FL, 32306, U.S.A.

Abstract: Certain plants are known to influence pest attack on other plants in a phenomenon termed “associational effects” (AE). AE has long been exploited in agriculture by means of companion planting or intercropping, where one or more species in a polyculture of plants confers indirect benefits to another. Many plants in these types of interactions emit volatile compounds that effectively repel insect pests from themselves and surrounding plants (Held, Gonsiska & Potter 2003). Giant red mustard greens have been shown to be an effective neighbor plant using this mechanism, significantly reducing whitefly oviposition on focal collard greens in a greenhouse experiment (Legaspi 2010). However, collards planted in a field 2.4m – 12.2m away from a central mustard plot did not significantly affect attraction or oviposition of whiteflies in the same study. Given these inconsistent results, it is possible that collards planted in the field experiment were too far from the mustard plot to experience repellent effects. The goal of this study was to to determine if AE on collard greens from mustard greens depend on the distance between plants. To quantify AE, we planted giant red mustard greens (Brassica juncea) 10cm – 70cm from collard greens (Brassica oleracea var. acephala) at discrete distances and measured plant damage and various pest abundances on collard greens. Contrary to our expectations, we found a marginally significant negative relationship between plant damage and neighbor distance. We also found a significant negative relationship between whitefly (Aleyrodidae) abundance and neighbor distance, indicating associational susceptibility of collard greens from nearby mustard greens. Our analyses may have been limited by a below-average pest abundance during data collection. Despite previous studies indicating otherwise, mustard greens may not be an effective companion crop for reducing pest attack in the field.

Introduction

The presence of heterospecific plant neighbors has long been understood to influence patterns of attack by insect pests. Volatiles produced by neighbor plants are known to function as a chemical defense against herbivore attack via olfactory cues, resulting in differences in focal plant attack (Held, Gonsiska & Potter 2003). Associational resistance, a term coined by Tahvanainen and Root (1972), is used to describe the phenomenon of neighbor plants reducing pest attack on focal plants. Conversely, associational susceptibility occurs when neighbor plants increase pest attack on focal plants, as described by Letourneau (1995). These terms are referred to collectively as associational effects (abbreviated AE, reviewed in Barbosa et al. 2009, Underwood et al. 2014). AE have long been used as a mechanism of companion planting in agriculture to maximize crop yield by minimizing pest attack. Figure 1. Associational resistance from neighbor Companion planting is used in agriculture as a plant represented as hypothetical sigmoidal form of integrated pest management in operations function of neighbor plant distance. Focal plant like cabbage farming, where planting glossy collard damage represented as inverse of associational greens (Brassica oleracea var. acephala) as a neighbor effects. crop has been shown to reduce diamondback moth Underwood et al. unpublished), 2.4 – 12.2m may be oviposition on focal cabbage crop (Brassica oleracea too far for AE to be present. Determining the var. capitata) in favor of neighboring collards, thus distance at which AE are maximized for mustards reducing plant damage and demonstrating could provide insight to pest population dynamics associational resistance (Badenes-Perez et al. 2004, and be useful for developing more effective integrated Mitchell et al. 2000). Giant red mustards have also pest management methods. been shown to reduce whitefly abundance and The focus of this experiment was to oviposition on collard greens when placed 10cm away determine the spatial pattern of AE from neighbor (Legaspi 2010). Moreover, a study conducted by mustard greens on focal collard greens. Planting Underwood et al. (unpublished) found that mustard mustard greens (B. juncea) at various distances from greens planted 20cm from collards significantly collard greens (B. oleracea) was expected to yield reduced damage from whiteflies. variation in pest abundance and plant damage among There are a number of studies showing that treatments. AE from neighbor plants can vary with the density of focal plants and density of neighbor plants where pest Methods attack is either increased or decreased (Kim and Underwood 2015, Rand and Louda 2004, Singer and This experiment was conducted from Wee 2005, Hambäck and Englund 2005). However, September 2015 to January 2016. 180 collards and There are only a few studies showing how AE vary 144 mustards were germinated in the Florida State with neighbor distance. Of these few studies, many University on-campus greenhouse at the King Life have found that AE decreased with neighbor plant Sciences building in early September 2015. Two distance from focal plant (Dagremond, Pardini and weeks following germination, the plants were thinned Knight 2010; Karban 2007; Russell and Louda 2007; to a single plant per insert, reserving the largest plant Rand and Louda 2004). However, there are no for the experiment. conclusive studies manipulating neighbor plant Two weeks following thinning, after four true distance using mustard greens. Legaspi (2010) planted leaves had developed, plants were transplanted to an collard greens 2.4 - 12.2m from a central mustard outdoor 30-meter by 12-meter plot at FSU’s satellite plot, but found that whitefly attraction and Mission Road Research Facility in mid-October. oviposition were not significantly affected. Given the Using a transect tape, planting began one meter away previous evidence that mustard greens confer from the edge of the plot. An experimental unit associational resistance of collards (Lagaspi 2010, consisted of two focal collard greens 20cm apart, and

Figure 2. Depiction of experimental setup of mustards planted at various distances from collards in increments of 10 cm (d). Distances not to scale. Not depicted: other replicates within each row at 1m apart. two neighboring mustard greens planted on either Orthene® 50% acephate fire ant bait was side of the collards at 10, 20, 30, 40, or 70cm away administered to the base of plants where fire ants had from either side of the collards (Figure 2). Collards built mounds. These were appropriately noted for any with mustards 70cm away were used as a control confounding effects on plant damage from pests where minimal to no AE were expected. There were a during analysis. total of 75 experimental units within 7 blocks and 11 In this study, associational resistance was columns, consisting of 15 replicates per treatment. inferred as the inverse relationship to focal plant Each unit was spaced one meter with a distance of 1.5 damage (Figure 1) and pest abundance. The first meters between each block. The first series of round of data collection took place in late November, replicates were placed in consecutive order from and the second round five weeks later in early 10cm to 70cm (distance from neighbor to focal January; these are referred to “early” and “late” survey plant). For the remainder of the replicates, distance data hereafter. Two surveys were conducted to between collards and mustards was randomized observe changes in pest abundance and percent within each block. damage as plants mature. For each focal collard plant, After crops were planted, they were the percent damage and number of aphids thoroughly watered every other day until (Aphididae) were estimated categorically on each leaf. establishment around a month later. After plants were Other common pests like whiteflies (Aleyrodidae) and established they were only watered as needed. Fire leaf miners were counted individually. Leaf miner ants were a common pest in this plot that killed tunnels were counted as a measure of abundance. Any multiple replicate collard plants. To remedy this, in other insects present were also noted. Total number mid-November, one tablespoon of Ortho® of leaves and longest leaf were measured as estimates

Figure 3a-f. Analysis of data collected on 11/28/15 Figure 4a-f. Analysis of data collected on 1/4/16. A- and 11/29/15. A-e are linear models of e are linear models of measurements. F is logistic measurements. F is logistic regression of pest regression of pest abundance. P-values displayed in abundance. P-values displayed in plot. plot. of plant growth. For the neighbor mustard plants, Discussion total number of leaves and longest leaf were also measured. For the second round of collection, plants had 20-30 leaves. To reduce time needed for a full Contrary to expectations, our findings suggest survey, every other leaf on focal plants was measured that there exists associational susceptibility from for the percent damage and pest abundance, so that neighbor mustard greens. Although marginally data were collected from the full range of leaf ages significant (p=0.0518, df = 102), plant damage from and sizes. pest attack may increase as mustards are planted closer to collards (Figure 3a). Moreover, we found Statistical analyses that average whitefly abundance also increased as neighbor distance decreased (p = 0.0186, df = 102, Linear regression was used to assess focal Figure 4d), also suggesting associational susceptibility. plant growth, average plant damage, and pest These findings seemingly contradict a abundances per leaf as a function of neighbor plant previous greenhouse experiment where whitefly distance. Covariates of plant growth were included in oviposition on collards decreased in the presence of a separate analysis to determine if plant size also mustards 10cm away (Legaspi 2010). Although influenced damage and pest abundance. Logistic distance was not manipulated in that experiment, one regression was used to assess probability of pest would expect whitefly oviposition to increase on focal presence at various neighbor distances. Replicates plants as neighbor plant distance increases if administered fire ant bait were compared to replicates associational resistance was present. The author, without use of bait to determine if poison confounds however, acknowledged that the effect of plant pest presence and abundance. All analyses were done volatiles may have been artificially increased by in R v3.2.3 (R Core Team 2015). conducting the experiment in a greenhouse. In the same study, a field experiment resulted in no significant difference in whitefly oviposition or Results attraction to focal plants (including collards) at various distances of 2.4m - 12.2m from a central The main results from the early and late mustard plot. surveys are shown in Figures 3 and 4, respectively. Previous studies suggest that neighbor plant There was a marginally significant negative density increases intensity and frequency of focal relationship between average damage and neighbor plant attack (Rand and Louda 2004, Kim and distance for the early survey (p = 0.0518, df = 102, Underwood 2015). Although mustard damage and Figure 3a), but it was not significant for the late pest abundance was not assessed in our study, survey (p=0.5490, df=102, Figure 4a). Although plant perhaps mustards attracted plant pests like whiteflies size significantly increased with greater spacing in the at closer distances due to visual cues like higher second survey (Figure 4b.), the inclusion of neighbor apparent treatment density, resulting in greater plant size as a covariate did not alter the significance abundance on collards. in any of the regression analyses (results not shown). Some pests may actually be attracted to A significant negative relationship was found for mustards in the presence of alternative plant hosts. A whitefly abundance and neighbor distance for the late study conducted by Bender, Morrison & Frisbie survey (p=0.0186, df=102, Figure 4d), but it was not (1999) found that cabbage did not exhibit significant significant in the early survey (p=0.3580, df=102, associational resistance to lepidopteran pests when Figure 3d). No significant relationship was found intercropped indian mustard (B. juncea). They also between neighbor plant distance and average aphid or found that hemipteran pests in fact preferred leaf miner abundance for either of the surveys (Figure mustards over cabbage. The inconsistencies between 3c, e; Figure 4c, e). No significant relationship was the findings in this study and those of previous study found between neighbor plant distance and pest further contribute to the uncertain nature of mustard abundance for either survey (Figure 3f,4f). We found greens as an effective companion crop. no significant difference between plant damage with A potential limitation to this study is that AE or without fire ant bait (results not shown). may only be detectable when pest presence reaches a certain threshold. In fall 2013, Underwood et al. (unpublished) found that mustards caused significant associational resistance for collard pests. Whitefly and aphid abundances were approximately 5 and 3 times mustard plants. Determining the effectiveness of higher, respectively in that study. The fact that there these companion crops and optimal planting distance were many fewer pests in this study may have resulted may prove to contribute greater understanding of pest in less plant damage than was necessary to exhibit population dynamics and the development of more stronger effects. effective integrative pest managements methods. Another potential limitation that may have contributed to inconsistent results was that whitefly Acknowledgements larvae and eggs were counted in the second survey, but not the first. Considering whitefly egg and larvae I thank Dr. Brian Inouye for directing this abundances seemed to far outweigh adult abundance, senior honors thesis and providing guidance and not including them in the first survey may have led to feedback throughout the entirety of the project, Dr. their insignificant relationship with neighbor distance. Nora Underwood for her guidance with initiating the To further evaluate the effectiveness and project as well as her helpful edits, Dr. Stephanie Pau optimal placement of mustards as a companion crop, for agreeing to serve as a defense committee member a similar, multi-season and multi-year study should be on short notice and her helpful feedback, and Theresa conducted to account for differences in pest Jepsen for directing FSU’s Mission Road greenhouse community and stochastic variation in pest and providing useful field advice. I also appreciate the abundances among years. Moreover, alternative hard work of J. Faylo, B.D. Inouye, A. Merwin, D. companion crops should be assessed at various McNutt, J. Mutz, M. Nichols, J. Ogilvie and M. Smith distances. Findings in a study by Sarker, Rahman & with data collection in the field. Das (2007) suggest that intercropped garlic and onions significantly reduce aphid abundance on

Literature Cited

Badenes-Perez, F.R., A.M. Shelton, B.A. Nault. 2004. Evaluating Letourneau D. K. 1995. Associational susceptibility: effects of Trap Crops for Diamondback Moth, Plutella cropping pattern and fertilizer on Malawian bean fly xylostella (Lepidoptera: Plutellidae). Journal of levels. Ecological Applications 5:823– 829. Economic Entomology. 97(4):1365-1372. Mitchell, E. R., G. Hu, and D. Johanowicz. 2000. Management Barbosa, P., J. Hines, I. Kaplan, H. Martinson, A. Szczepaniec, of diamondback moth (Lepidoptera: Plutellidae) in and Z. Szendrei. 2009. Associational resistance and cabbage using collards as a trap crop. Hortscience associational susceptibility: having right or wrong 35:875- 879. neighbors. Annual Review of Ecology and Systematics Rand, T.A., S.M. Louda. 2004. Exotic weed invasion increases 40:1-20. the susceptibility of native plants attack by a biocontrol Bender, D.A., W.P. Morrison, R.E. Frisbie. 1999. Intercropping herbivore. Ecology 85(6):1548-1554. Cabbage and Indian Mustard for Potential Control of Russell, F. L., S. M. Louda, et al. 2007. Variation in herbivore- Lepidopterous and Other Insects. HortScience mediated indirect effects of an invasive plant on a 34(2):275-279. native plant. Ecology 88:413-423. Dangremond, E.M., E.A. Pardini, T.M. Knight. 2010. Apparent R Core Team. 2015. R: A language and environment for competition with an invasive plant hastens the statistical computing. R Foundation for Statistical extinction of an endangered lupine. Ecology Computing, Vienna, Austria. URL http://www.R- 91(8):2261-2271. project.org/. Held, D.W., P. Gonsiska, D.A. Potter. 2003. Evaluating Sarker, P.K., M.M. Rahman, B.C. Das. 2007. Effect of companion planting and non-host masking odors for Intercropping of Mustard with Onion and Garlic on protecting roses from the Japanese beetle (Coleoptera: Aphid Population and Yield. Journal of Bio-Science Scarabaeidae). Journal of Economic Entomology 15:35-40. 96(1):81-87. Singer M.C., B. Wee. 2005. Spatial pattern in checker- spot Hambäck P.A., G. Englund. 2005. Patch area, population density butterfly: host plant association at local, meta- and the scaling of migration rates: the resource population and regional scales. Annales Zoologici concentration hypothesis revisited. Ecology Letters Fennici 42:347–361. 8:1057–1065. Tahvanainen, J. O. and R. B. Root. 1972. The influence of Kim, T.N., N. Underwood. 2015. Plant neighborhood effects on vegetational diversity on the population ecology of a herbivory: damage is both density and frequency specialized herbivore, Phyllotreta cruciferae dependent. Ecology 96(5):1431-1437. (Coleoptera: Chrysomelidae). Oecologia 10:321-346. Legaspi J.C. 2010. A Preliminary Investigation of Giant Red Underwood, N., B.D. Inouye and P.A. Hambäck. 2014. A Mustard (Brassica juncea) as a Deterrent of Silverleaf conceptual framework for associational effects: when Whitefly Oviposition. Journal of Entomological do neighbors matter and how would we know? Science. 45(3):262-271. Quarterly Review of Biology 89(1):1-19.