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Home , Quercus agrifolia, Umbellularia

Effects of volatile compounds in bay ( californica) on vegetation growth and insect herbivory

Ashley Adornato1, Hannah Gerber2, Sarah Haas3, Jennifer Perez3

1University of California, ; 2University of California, Davis; 3University of California, Irvine

Secondary metabolites are volatile compounds produced by that can be used as defense mechanisms for reducing stressors such as herbivory and competition. , roots, and stems of California bay trees (Umbellularia californica) get their notable aroma from secondary metabolites called monoterpenes, which are allelopathic constituents in many other plants, such as . In this study, we investigated the potential allelopathic and anti-herbivory effects of California bay trees by examining their understory composition, germination rates of grass seeds in the presence of bay solution, and insect herbivory rates on bay and neighboring trees. We found less vegetation underneath the canopy of bay trees when compared to surrounding areas. Grass seeds also showed decreased germination rates when exposed to the bay leaf solution in comparison to water. Bay trees experienced less insect herbivory than their neighbors. These results suggest that bay trees utilize their secondary metabolites as allelochemicals in their understory and as a direct defense against insect herbivory.

Keywords: Umbellularia californica, secondary metabolites, allelopathy, understory composition, insect herbivory

INTRODUCTION defense mechanism is aimed at reducing resource competition within the Many plants produce secondary community. Allelopathic plants limit the metabolites which are volatile chemical growth and succession of surrounding compounds that can be used as unique plants, effectively reducing their population defensive mechanisms for reducing size by reducing their ability to compete in stressors like herbivory and competition an ecosystem (Kruse et al. 2000). By (Lambers et al. 2008). Secondary reducing the competitive ability of metabolites play an integral role in how surrounding plants and influencing plants interact with their surrounding succession, allelopathic plants are a driving environment. These compounds are factor in plant community composition. referred to as allelopathic compounds Other secondary metabolites are used for (allelochemicals), which are released by more direct defense by using chemicals plants into the surrounding soil through within their leaves and that deter their roots and leaf litter. This interference herbivory. Plant-herbivore interactions play

CEC Research | https://doi.org/10.21973/N38Q1D Fall 2020 1/8

a key role in ecosystem-dynamics, with characteristics, uses volatile monoterpenes plant defenses being an important as a defense mechanism by inhibiting the component of these interactions (Stamp growth and germination of neighboring 2008). The chemical defense mechanisms plants (Zhang 2016). Eucalyptus also uses produced by plants impact how herbivores monoterpenes in their leaves to deter make their food choices which have generalist herbivores, such as possums cascading effects throughout the (Loney et al. 2006). The monoterpenes ecosystems. Overall, these volatile found in eucalyptus trees have been found compounds produced by plants have in the leaf litter of California bay trees widespread effects on their surrounding (Umbellularia californica) which happen to communities. make up most of the bay trees’ main Secondary metabolites are found in many chemical components (Wood et al. 1995). common products that we consume in our California Bay trees (Umbellularia daily life such as , tea, and cannabis californica) are evergreen trees found (Ashok and Upadhyaya 2012, Flores- throughout Oregon and California. They are Sanchez and Verpoorte 2008). Some of the most commonly found in the Klamath, most widely studied secondary metabolites, Transverse, and Coastal mountain ranges, such as tannins, brassinosteroids, and along with the Sierra Foothills. Bay trees monoterpenes, are used as a defense can vary in height from a 2 m to a 30 mechanism against herbivory and plant m , but most often are within the 12 m competition. Tannins are one of the main to 24 m range. Bay trees get their notable secondary metabolites used in plants to aroma from volatile monoterpenes, which deter insect herbivory by preventing proper are chemical constituents of the plant's food digestion (Barbehenn and Constabel essential oils within their leaves and roots 2011, Lambers 2008). A past study revealed (Goralka and Langenheim 1996). To the that the apical half of white tree best of our knowledge there have only been (Quercus alba) produced higher two studies that have tested the potential concentrations of tannin in response to uses of chemical defense by bay trees. A lab weevil consumption (Steele 1993). Another study found that cucumber seeds highly studied secondary metabolite is germinated in the presence of bay leaf brassinosteroids, which is a hormone extract germinated at just 17% of the known to increase plants resistance to control rate, showing a clear inhibition of insect feeding. In a lab study, it was shown germination (Tinnin and Kirkpatrick 1985). that Lotus japonicus, a wild legume, In the same study, it was found that the experienced lower rates of herbivory from presence of ground leaf litter from bay thrips when it was genetically mutated to trees negatively affected the root growth express higher levels of brassinosteroids rate of Douglas fir (Pseudotsuga menziesii) (Miyaji et al. 2014). Monoterpenes are seedlings. This study suggest that bay trees another secondary metabolite that acts as have allelopathic effects on neighboring toxins toward bacteria in the soil that are plants but there has yet to be a study done important for nitrification and germination on how that allelopathy is expressed in of plants (Wood et al. 1995). Eucalyptus, their understory. In addition to using which is well known for its allelopathic monoterpenes as an allelopathic defense,

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they may also use these compounds to METHODS deter herbivory. Mature bay leaves were shown to contain concentrations of 2.1 Study Site monoterpenes that are effective in deterring herbivory of black-tailed deer Our study was conducted from November (Odocoileos hemionus columbianus, Goralka 5th to 11th, 2020, at Hastings Natural and Langenheim 1996). Again, this study History Reservation (36°12ʹ30″N, suggests that bay trees may use chemical 121°33ʹ30″W). The reserve is located in the defense to combat stress caused by deer foothills of the Santa Lucia Mountains in herbivory. While this study provides Monterey county. Hastings is comprised of information on mammal herbivory, there many habitats, including annual and have been no studies done on how the perennial grasslands, oak woodlands, monoterpenes in bay leaves affect insect , and running streams. Along with herbivory. Although there have been blue oak (), valley oak (Q. studies that suggest bay trees exhibit lobata), coast (Q. agrifolia), chemical defenses by inhibiting the growth sycamore (Platanus occidentalis), and black of Douglas firs and deterring mammal walnut trees (Juglans nigra), California bays herbivory, there have not been (Umbellularia californica) are one of the comprehensive studies on how these many trees that inhabit the reserve. chemical defenses influence their 2.2 Tree Canopy and Vegetation surrounding community. Our study aims to further understand the Every accessible bay tree was effect of volatile monoterpenes found in opportunistically chosen on sections of bay leaf on understory composition, plant Carmel Valley Road (36°22'41.5524"N, germination, and insect herbivory. We 121°33'49.3416"W) and Martin Road hypothesized that bay trees will have a (36°22'43.0284"N, 121°33'45.2484"W). lower percent cover of vegetation These roads were selected because of the underneath their canopy when compared abundance of bay trees in the area. Some to surrounding vegetation. In addition, we bay trees were inaccessible for predict that grass seeds germinated in the measurements due to dense poison oak presence of bay leaf solution will show communities and steep hills. In total, we lower rates of germination when compared sampled 60 bay trees. to control seeds. Finally, we predict that To quantify the size of each bay tree, we there will be less insect herbivory on bay measured canopy width using a distance trees when compared to neighboring trees. gun when the canopy was at its widest. To By studying these components we hope to examine whether there was an effect of bay better understand how these volatile tree canopy cover on understory monoterpenes are expressed by bay trees vegetation, we took visual percent of the and what influence that has on their ground cover to quantify vegetation using a community composition. 0.5 m x 0.5 m quadrat at a random orientation halfway between the trunk and the canopy edge and 2 m away from the

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canopy edge. For analysis, we quantified diluted bay leaf solution. The paper towels the canopy effect by subtracting the were compressed to get rid of any excess percent cover of vegetation under the liquid. We enveloped 123 grass seeds in the canopy from the percent cover of control paper towels and 128 seeds in our vegetation away from the canopy. treatment paper towels. We waited five days for them to germinate and counted 2.3 Seed Germination each seed that germinated within that time frame. A seed was considered germinated if To test the effect of bay tree a radicle had broken through the seed coat. allelochemicals on germination, we used Scotts® Turf Builder® Grass Seed Sun & 2.4 Insect Herbivory Shade Mix®, which consists of a blend of Festuca, Lolium, and Poa for their quick In order to test the effects of bay tree germination rate of 5–10 days. We created chemical defenses on insect herbivory, we a concentrated solution by taking 69 compared the percent of herbivory on bay randomly chosen bay leaves from two trees to its neighboring trees, which were branches on a mature tree. These leaves trees closest to the bay tree being sampled. were then blended in 474 mL of water. A For each of our 60 bay trees, we randomly diluted bay leaf solution was made by selected ten leaves that were within reach taking 237 mL of water and adding in 2 mL and counted how many showed signs of of the concentrated solution. We diluted insect herbivory. We did the same for two the solution until it accurately reflected the closest neighboring trees of different strength of aroma on the bay leaves to species, totaling 120 trees sampled (Table mimic the concentrations found in nature. 1). We then compared the percent of To germinate our seeds we used damp herbivory on bay trees to the average paper towels, with our control soaked in percent of herbivory between the two plain water and our treatment soaked in neighboring trees.

Table 1. Percent of h erbivory on each tree species. Table shows the average herbivory of California bay trees, bolded as the focal tree, and each neighboring tree species recorded.

Common Tree Scientific Name Number of Individuals Average Herbivorized Species Name Sampled Leaves California bay Umbellularia californica 60 25 Coast Live Oak 69 45 Pacific Madrone Arbutus menziesii 13 68 Holly Ilex 12 42 Laurel Oak Quercus imbricaria 8 53 Toyon arbutifolia 8 71 Red Alder Alnus rubra 3 47 Kermes Oak Quercus coccifera 2 50 American Sycamore Platanus occidentalis 2 50 Unidentified Species 3 27

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2.5 Statistical Analysis

JMP Pro 15 software was used for all of our statistical analyses. A paired t-test was used to measure the difference between vegetation percent cover underneath the canopy and away the canopy. In order to test if there was any effect of canopy size on relative canopy effect, we used a linear regression. A χ2 test was used to measure the effects of each treatment on grass seed germination. A paired t-test was used to measure the difference between bay trees and the average of its two closest neighbors. Figure 1. Percent of ground vegetation cover under RESULTS and away California bay tree (Umbellularia californica) canopies. Bar graph shows the average 3.1 Tree Canopy and Vegetation percent of ground vegetation cover with under and away of the canopy on the y-axis. Under the canopy there was less vegetation cover. Error bars represent On average, 30% less vegetative cover ± 1 standard deviation from the mean. n= 60, t=9.7 was found under bay tree canopies than p≤0.0001. away from their canopies (n = 60, t = 9.7, p < 0.0001, Figure 1). Mixed grasses seemed to be most inhibited by bay allelochemicals as grass density was rich away from the bay canopies and sparse under the canopies. Poison oak appeared most resistant to bay allelochemicals, as its density was only slightly less under the canopy when compared to away the canopy. We found no correlation between canopy width and relative canopy effect (n = 60, R2 = 0.0002, p = 0.92).

3.2 Grass Seed Germination Figure 2. Percent of herbivory on California bay tree (Umbellularia californica) and neighboring trees. Bay leaf solutions reduced germination by Bar graph shows the average percent of herbivory 10.8% compared to controls (n = 251, χ2= on the x-axis with bay and neighboring trees on the 3.63, p = 0.056). In total, 77% of our control y-axis. Neighboring trees experienced more seeds germinated while 66% of our seeds herbivory than bay trees. Error bars represent ± 1 standard deviation from the mean. n = 60, t = 9.8, p treated with bay extract germinated. ≤ 0.0001.

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3.3 Insect Herbivory exhibit allelopathic qualities (Kruse et. al 2000). Bay trees showed less herbivory in Although we found that overall vegetative comparison to their neighboring trees (n = cover was significantly lower under bay tree 60, t = 9.8, p < 0.0001, Figure 2). Bay tree canopies than away from bay trees, it is leaves on average showed signs of possible that the absence of certain plant herbivory 25% of the time while species may indirectly benefit species that neighboring trees, depending on the can tolerate the allelochemicals of bay species, ranged from 27%-71% (Table 1). trees. For example, a recent study done on kikuyu grass (Pennisetum clandestinum) DISCUSSION showed that while allelochemicals significantly suppressed the growth of Our study aimed to investigate how kikuyu grasses, coniferous tree growth was chemical defenses used by bay trees might indirectly stimulated as nutrients that affect surrounding vegetative and insect would ordinarily be consumed by kikuyu communities. We answered these questions grasses were left available for consumption with three main studies on vegetative cover by conifers (Chou 1999). As we observed surrounding bay trees, grass seed that mixed grasses appeared intolerant to germination when exposed to bay bay allelochemicals while poison oak was monoterpenes, and insect herbivory on bay resilient, future studies could investigate if and surrounding tree leaves. the decrease of intolerant grasses further 4.1 Vegetative Cover stimulates poison oak growth under bay canopies. Our prediction that vegetative cover found under the canopy of bay trees would 4.2 Grass Seed Germination be more sparse than vegetative cover found Our prediction that bay tree away from bay trees was supported. This allelochemicals would have negative effects supports the hypothesis that bay trees on surrounding vegetation was also exhibit allelopathy by inhibiting the growth supported by our germination studies in the and germination of plants in their lab. Grass seeds immersed in bay solution understories. Canopy size did not affect this germinated at a lower percent than the pattern, which implies that juvenile trees control seeds. Our findings align with the are just as effective in deterring growth results of a recent study which found that around their canopies as mature bay trees. bay monoterpenes, a potential This finding concurs with the results of a allelochemical of bay trees, also had study done on eucalyptus trees, which adverse effects on the germination of found that juvenile eucalyptus trees are cucumber seeds (Tinnin and Kirkpatrick equally as effective in producing 1985). As prevention or slowing of allelochemicals as mature trees (Loney et al. germination of nearby seeds is also a 2006). Because a sparsely vegetated defining characteristic of allelopathic plants, understory is a defining characteristic of these findings again suggest that bay trees many allelopathic plants, this finding display allelopathic characteristics. supports the hypothesis that bay trees

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Although our study found that seeds on bay trees or explored any alternative immersed in a low concentration bay effects that herbivory might have. For solution germinated 10.8% less than control example, a recent study discovered that bay seeds, future studies could test the effects trees with higher rates of insect herbivory of different concentrations of bay solution were less likely to spread sudden oak death on germination rates. Experiments like this disease to surrounding (Wininger and would mimic how concentration of Rank 2017). Therefore, understanding both allelochemicals may vary at different the positive and negative effects that distances from the tree and could uncover herbivory can have on plant communities changes in plant community composition would be the next step in better along this gradient. Future research could understanding the interactions between also investigate if seeds of invasive species bay trees and their ecosystems. of plants germinate at a lower rate than Plants all around the world use secondary seeds of native plants when exposed to bay metabolites like tannins, brassinosteroids, tree allelochemicals. Studies on eucalyptus monoterpenes, and many more as chemical trees have shown that plant communities in defenses against competition and different parts of the world that have not herbivory. In our study, we observed that co-evolved with eucalyptus allelochemicals the California Bay tree may also use experience more adverse effects than plant monoterpenes as a chemical defense. These communities that have co-evolved with chemical defenses shape plant communities these toxins (Becerra et al. 2018). If bay and herbivore interactions which have allelochemicals are found to have more cascading effects throughout the adverse effects on invasive species than on ecosystem. By better understanding how native species, this information could be California bay trees use chemical defense integrated into management practices for we can better understand their role in the invasive species. ecosystem.

4.3 Insect Herbivory ACKNOWLEDGMENTS

Our prediction that bay tree This work was performed at the monoterpenes could be effective in University of California’s Hastings Natural deterring insect herbivory was supported as History Reservation, doi:10.21973/N33Q0G. well. We found less insect herbivory on bay tree leaves than on the leaves of REFERENCES surrounding trees. This suggests that Ashok, P. K., & Upadhyaya, K. 2012. Tannins are another function of the monoterpenes astringent. Journal of pharmacognosy and found in bay tree leaves could be to prevent phytochemistry 1(3):45–50. insect herbivory, which would protect the tree’s ability to conduct essential processes Barbehenn, R. V., & Constabel, C. P. 2011. Tannins in like . Although we plant–herbivore interactions. Phytochemistry 72(13):1551–1565. determined that bay trees experience less herbivory than surrounding trees, we did Becerra, P. I., Catford, J. A., Luce McLeod, M., not examine specifically which insects feed Andonian, K., Aschehoug, E. T., Montesinos, D., &

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