TAGETES PATULA AS A COMPANION PLANT IN EAST TENNESSEE PHASEOLUS

VULGARIS CULTIVATION

A Report of a Senior Study

by

Hannah Elizabeth Cummings

Major: Biology

Maryville College

Fall, 2015

Date approved______, by______

Faculty Supervisor

Date approved______, by______

Division Chair

ABSTRACT

Phaseolus vulgaris, common green beans, are prolific throughout East Tennessee but experience damage as a result of Epilachna varivestis herbivory. A solution to the problem of destruction of crops may be found in companion plants known to naturally repel insect pests.

This study examined if marigolds (Tagetes patula) could have an allelopathic effect on E. varivestis. Three different varieties of P. vulgaris, Kentucky Wonder, Blue Lake, and Black

Valentine, were planted without companion plants (controls) or with T. patula to observe whether beans planted with marigolds would be more resistant to E. varivestis than those without. Two endpoints were quantified: bean number and leaf damage. The Blue Lake variety showed a response to being companion planted with T. patula and demonstrated a significantly greater harvest (p=.01) than the control group. Leaf area damaged by pests in both Black Valentine and Blue Lake varieties of bean plants was not influenced by the companion plants. Due to the lack of difference in leaf damage, it is proposed that T. patula did not offer aromatic pest-repellant properties against E. varivestis but instead offered microbial benefits against harmful fungi and root-knot nematodes damaging the roots. Thus,

Tagetes patula is a viable option to increase yields of Phaseolus vulgaris in East Tennessee, but the beneficial mechanism of action is unknown.

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TABLE OF CONTENTS

CHAPTER I ...... 1!

INTRODUCTION ...... 1!

General Uses for Companion Plants ...... 1!

Economic Benefits of Companion Plants ...... 6!

Ecological Benefits of Companion Plants ...... 7!

The Evolution of Companion Plants ...... 8!

Tagetes patula, French Marigolds ...... 9!

Phaseolus vulgaris, Green Bean ...... 10!

Epilachna varivestis or the Mexican bean beetle ...... 10!

Purpose of Study ...... 12!

CHAPTER II ...... 13!

MATERIALS AND METHODS ...... 13!

Experimental Crops and Origins ...... 13!

Experiment Plot Locations and Layout ...... 14!

Soil Testing and Preparation ...... 16! iv

Plant and Seed Purchases and Planting ...... 17!

Leaf Damage Analysis and Bean Harvest Data Collection ...... 17!

Statistical Analysis ...... 21!

CHAPTER III ...... 22!

RESULTS ...... 22!

Soil Analysis and Bean Harvest ...... 22!

Leaf Damage ...... 25!

CHAPTER IV ...... 27!

DISCUSSION ...... 27!

Bean Harvest ...... 27!

Leaf Damage ...... 28!

Allelopathic Influence ...... 29!

Summary ...... 30!

REFERENCES ...... 34!

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LIST OF FIGURES

Figure Page

1 Mexican bean beetle adult and larvae 11

2 Garden plot layout at Maryville College 15

3 Garden plot layout at Blackberry Farm 16

4 Ideal bean harvest length 20

5 Average bean harvest 25

6 Average percent leaf damage of bean plants 26

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LIST OF TABLES

Table Page

1 Beneficial companion plant and crop combinations 4

2 Average soil results 23

3 Green bean and marigold sprout dates 23

4 Green bean harvest totals 24

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ACKNOWLEDGEMENTS

I would like to thank Dr. Drew Crain for the countless hours he spent assisting me in my research and reviewing my thesis document as well as his patience and encouragement. I would also like to thank John Coykendall and Michael Washburn of Blackberry Farm for mentoring me throughout the entire season of my data collection process. I would like to thank Blackberry Farm for hosting one of my study’s plot sites. Lastly, I would like to thank my uncle Joseph D. Gorman whose generosity has allowed me the opportunity to pursue a higher education.

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CHAPTER I

INTRODUCTION

General Uses for Companion Plants

Companion plantings--the planting of two or more species of plants in the same garden or crop system to improve the growth or productivity of one or more of the species-- have been utilized by farmers for thousands of years (Beyfuss et al. 1994). They can provide a pest-repellent (repellent plant), beneficial-insect-attractant (banker plant), or predator- attractant (insectary plant) function (Parolin et al. 2012). Ancient civilizations noticed how crops interacted with each other and often devised ways of increasing crop yields. For instance, native American peoples planted corn, squash, and beans together as companion crops to improve the growth of each plant (Beyfuss et al. 1994). Greek and Roman farmers noticed that cabbages, laurels, and radishes were detrimental to grapevines (Flowerdew

2012). Oaks were considered detrimental to olives, walnut trees sterilized the ground, and lupins were noticed for their weed suppressing ability (Flowerdew 2012). In the first century

A.D., Pliny the Elder promoted planting chickpeas with cabbages and planting turnips with

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vetches to keep away caterpillars (Flowerdew 2012). Indeed, agricultural practices utilizing companion plants is perhaps as old as human history.

The reason that modern civilizations often do not use companion plants on a large scale is due to the ease and efficiency of monocultures. Monoculture allows large amounts of one type of crop to be grown at once, and for the use of specialized machinery which makes harvesting more efficient and less costly. However, one drawback to monoculture is that it allows disease and specialized insect pests to spread rapidly through the species of crop grown thereby decimating it. Planting multiple types of crops within the same crop system has been shown to limit the spread of disease and pests thereby lessening the need for pesticides and making a more sustainable farming system (Balmer et al. 2014). It is argued that inter-planted crops or secondary plants are less appealing to insect pests and may even be more difficult for pests to find (George et al. 2013, Parker et al. 2013). In contrast to monoculture, diverse landscapes of plants increase biodiversity and provide havens for insect predators and parasitoids of the pests that plague crops (Parker et al. 2013).

Pest damage in modern crops is a complex topic that includes more issues than the practice of monoculture. The ecology and biology of crop plants often differ from wild populations. For instance, specialist and generalist herbivores and their parasitoids develop poorly on wild populations of cabbage as compared to domesticated species, and levels of glucosinolates are higher in plants from wild populations (Gols et al. 2008). It has been proposed that programs using domesticated varieties of plants have possibly disrupted plant defense strategies (Evans 1993). The issue of widespread insect herbivory of crops is perhaps furthered by farmers’ practice of planting the most palatable, domesticated varieties

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of plants in large quantities unimpeded by other crops or plants. Utilizing wild varieties of crops along with the added benefit of a naturally pest-repellant companion plant in the effort to reduce insect herbivory of crops warrants future studies. Current companion plant research is surprisingly sparse, but the outcomes of several combinations of companion plants and crops have been studied (Table 1). The studies presented mostly showed that the presence of a companion plant either increased parasitoid density, decreased insect pest density, or minimized herbivory of the host plant.

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Table 1. Beneficial companion plant and crop combinations.

Companion Host Plant Effect Reference Plant Provided alternate food- source for and Coffee Plant Inga subnuda minimized Rezende et al. (Coffea subsp. damage of 2014 arabica) Luschnathiana Leucoptera coffeella and Hypothenemus hampei Prevented Corn Flowers attack by Ditner et al. (Centaurea) spiders and 2013 ground beetles Bishop's Flower (Ammi majus) Cornflower (Centaurea cyanus) Minmized Cabbage Buckwheat attack by Géneau et al. (Brassica (Fagopyrum Mamestra 2012 oleracea) esculentum) brassicae Candytuft (Iberis amara) Common Vetch (Vicia sativa) Disrupted Clover female cabbage Morley et al. (Trifolium root flies from 2005 subterraneum) landing on host plant Cucumber Minimized (Cucumis Buckwheat attack by Platt et al. 1999, sativus) (Fagopyrum cucumber 2005 Squash esculentum) beetles (Cucurbita)

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Table 1 (continued). Beneficial companion plant and crop combinations.

Trapped Common Buckwheat various insect Scarratt et al. Grape Vine (Fagopyrum pests found in 2008 (Vitis vinifera) esculentum) vineyards French Marigold (Tagetes patula) Tomato (Solanum Basil (Ocimum Minimized Tringovska et al. lycopersicum: basilicum) attack by root- 2015 variety Lettuce knot nematodes Dimerosa) (Lactuca sativa) White Mustard (Sinapsis alba) Drew Colorado Potato Wild Tomatoes Potato Beetles Thiery et al. (Solanum (Lycopersicon away from host 1987 tuberosum) hirsutum) plant

Pineland Attracted Threeseed predator Mercury Coleomegilla Sweet Corn maculata (Acalypha Seagraves et al. (Zea mays resulting in ostryifolia) 2009 subsp. mays) oviposition resulting in Tomato increased (Solanum predator density lycopersicum) Rue (Ruta graveolens) Failed attempt Zonal to discourage Geranium the Japanese Rose (Rosa) (Pelargonium beetle from Held et al. 2003 x hortorum) finding and Garlic Chives attacking rose (Allium bushes scheonparum)

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Table 1 (continued). Beneficial companion plant and crop combinations.

Used to attract the parasitoid Diadegma Broccoli Buckwheat semiclausum to (Brassica Lavandero et al. (Fagopyrum increase oleracea var. 2004 esculentum) parasitism rates Italica) of Plutella xylostella (diamondback moth) in broccoli fields

Economic Benefits of Companion Plants

Utilizing companion plants has the potential to provide large economic gain for organic gardeners. Flowering plants with suitable habitat and nectar available for parasitoids increases the likelihood of predation of herbivorous insect pests. The predation of insect pests potentially decreases the damage to valuable crops. The benefits of companion planting are not easily recognized in monoculture. Often, pesticides are used in their place.

Unfortunately, the use of pesticides does not always benefit the crops or the farmer.

In East Africa and North America alike, where pesticides are often used to increase yields, an increasingly common problem is pest populations that are becoming adapted to pesticides

(Muli et al. 2014; Parker et al. 2013). Pesticide resistance can evolve from an insect pest population being exposed to pesticides or pesticide combinations over multiple seasons or years, thereby allowing the survivors from the population to reproduce. Thus, over numerous generations, insect populations become less susceptible to pesticide effects (Sumerford et al.

2013). When pest populations become resistant to pesticides the crops are no longer protected at a large expense monetarily. More importantly, pesticide use often has the side 6

effect of killing important pollinators such as honeybees. Pollination services provided globally by insect pollinators exceed $200 billion (Muli et al. 2014). Therefore, the use of pesticides is not only an issue of economics but of ecological importance, and alternatives to contemporary pesticide use in agriculture is warranted (Horrigan 2002).

Ecological Benefits of Companion Plants

Health implications and potential for decreased biodiversity are issues that are linked to pesticide use. Vulnerable species, species whose populations are in decline, and humans all have the potential to be negatively impacted by pesticides. This is most clearly illustrated in important pollinators such as bee populations.

Bee populations are in decline in North America and East Africa, and pesticides are proving to be a major contributor to their declining numbers (Muli et al. 2014). The pesticides used are decreasing numbers in bee populations which limits pollination thereby further decreasing yields (Muli et al. 2014). Further problems include pesticide run-off often reaching streams and rivers where it impacts wildlife in aquatic communities (Parolin et al.

2014). Human exposure to pesticides is a further danger that could be lessened or eliminated if pest-repellent plants were used instead. Annually, 25 million agricultural workers in developing countries are exposed to organophosphorous pesticides (Jeyaratnam 1990). Farm workers responsible for spraying pesticides in developing countries often use ineffective practices that lead to significant exposure to these agents (Kishi et al. 1995). In addition, farmers exhibited more pesticide poisoning symptoms during periods when pesticides were being used on crops (Kishi et al. 1995). The United Nations reported that 2 million people worldwide experience pesticide-related illness and 10 thousand people die a pesticide-related

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death annually (Quijano 1993). The effects of pesticide poisoning range from symptoms as mild as eye irritation or as severe as cancer and reproductive disorders (Yassi et al. 2004).

Furthermore, only 20% of the world’s agrochemicals are used in the world’s developing countries, but those countries suffer nearly 99% of deaths related to pesticide poisoning

(Jeyaratnam and Chia 1994). Some of the most hazardous pesticides to use--such as organophosphorus pesticides--are unfortunately among the most effective pesticides available yet cause nearly 73% of pesticide related poisonings in humans (Dewan and Sayed

1998).

The harmful effects of pesticide use are a large scale issue that could be remedied by incorporating companion plants into crop systems. Crop systems not only provide food and income but could potentially provide habitat to useful organisms such as pollinators when utilized properly. Flowering plants are a category of companion plants that show particular promise in agriculture.

Planting flowering plants as trap plants alongside crops is useful because the nectar from the flowers provides an alternate food source for parasitoids such as wasps. Plants with extrafloral nectaries may be even more suitable for crop protection than flowering plants because they excrete higher rates of nectar and produce nectar over more extended periods than floral nectaries (Wackers 2005; Pacini and Nepi 2007).

The Evolution of Companion Plants

Insects and plants have a mutualistic relationship: the insect (such as a pollinator) provides pollination services to the plant while the flower of the plant provides a valuable food source. This mutualistic relationship is an example of coevolution. Coevolution in

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plants is a type of extreme mutualism that resulted in many species of insects and plants evolving together (Cain et al. 2011). This mutual reliance by some species of plants is so important that they can only be pollinated by the highly specialized insects that coevolved with them (Cain et al. 2011). Furthermore, certain species of parasitoids specialize in parasitizing a limited number of herbivorous insect hosts in a symbiotic relationship resulting from coevolution (Bilodeau et al. 2013).

Similarly, companion plants have a mutualistic relationship with their host plant.

Clover, for instance, provides a mutualistic relationship with broccoli when the two grow near each other. Lepidopteran herbivory is reduced when broccoli is grown in close proximity to clover (Hooks et al. 2004). Humans have long observed that such plant-plant mutualistic relationships are likely to increase crop yields. Examples include the aforementioned planting of corn, beans, and squash by Native Americans. Much of the information farmers’ use today regarding companion plant-crop pairings involves popular gardening books, the advice of other farmers, or their own instincts (Parker et al. 2013).

One popular contemporary companion plant is Marigolds of the family Asteraceae.

Marigolds are an example of a plant that use allelopathic properties to defend itself

(Balicevic et al. 2014). Allelopathy in plants is the beneficial or harmful influence of one organism on the other through allelochemicals (Rice 1984; Reigosa et al. 2006).

Tagetes patula, French Marigolds

Marigold (Calendula officinalis L.) of the Asteraceae family is a biennial or annual plant that produces yellow or orange flowers (Paradikovic et al. 2013, Erhatic et al. 2014).

Marigolds show allelopathic properties against other plants by secreting 3-O-monoglucoside

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through the roots of the plant (Ruszkowski et al. 2004). Marigolds are used for cosmetic, pharmaceutical, medical, and ornamental purposes (Siljes et al. 1992; Cromack and Smith

1998). French, African, and Mexican marigold varieties originated in Central America

(Flowerdew 2012). In addition to farmers’ beliefs that marigolds protect against Mexican bean beetles, French marigolds have been shown to minimize attack by root-knot nematodes

(Tringovska et al. 2015).

Phaseolus vulgaris, Green Bean

Phaseolus vulgaris, or the common bean--specifically the green bean which is known by names such as snap beans and string beans--in its wild form most likely originated in

South America (Duke 1983). Green beans are now cultivated in temperate and tropical regions worldwide and developed into both bush and twining forms (Duke 1983). Bush varieties grow in clumps about 1 meter tall and twining varieties grow about 4 meters tall with the support of a trellis (Duke 1983). Pods of P. vulgaris green bean variety are harvested while still green and immature and are a nutritious, relatively low-cost food rich in vitamin C

(Duke 1983). The insect pest commonly known as the Mexican bean beetle (Epilachna varivestis) is prolific throughout the Southeastern United States and perhaps the biggest inhibitor of P. vulgaris productivity wreaking devastation on bean crops in Eastern

Tennessee (Howard 1924).

Epilachna varivestis or the Mexican bean beetle

Epilachna varivestis, or the Mexican bean beetle, is in the family Coccinellidae in the order Coleoptera (see Figure 1). It is an herbivorous insect pest indigenous to Southern

Mexico whose habitat ranges from Southern Canada on the North American continent to

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Guatemala on the South American continent (Sanchez-Arroyo 1997). The beetle was first identified in the Southeastern United States in 1920 at the Alabama Experiment Station from

Blocton and Birmingham, Alabama and had been identified in the Southwestern United

States nearly 71 years previously (Howard 1924).

A B Photo Credit: Drew Crain

Figure 1. The Mexican bean beetle (Epilachna varivestis) shown in its adult (A) and larval

(B) stage.

The Mexican bean beetle reached eastern Tennessee by 1921 (Howard 1924), and today is the most injurious pest of P. vulgaris (Hale [date unknown]). Female E. varivestis feed on young bean plants for 1 to 2 weeks then lay 500 to 600 eggs in groups of 40 to 75 eggs at a time on the underside of the bean plants’ leaves. After the larvae hatch, they feed voraciously on the leaves of the plant for 2 to 5 weeks (Sanchez-Arroyo 1997). Bean plants such as P. vulgaris and Phaseolus lunatus are the preferred hosts among Mexican bean beetles with both adults and larvae feeding upon flowers, leaves, and bean pods of plants

(Sanchez-Arroyo 1997). The leaves of the plant, however, usually receive the most damage.

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E. varivestis has at least 17 species of known predators each of which feed on either Mexican bean beetle eggs, larvae, or pupae. The two parasitoids that demonstrate the ability to control large populations of Mexican bean beetles are Paradexodes epilachnae and Pediobius foveolatus which are tachinid fly and eulophid wasp species, respectively (Sanchez-Arroyo

1997). The relationship between the Mexican bean beetle and the green bean illustrate the detrimental economic effects that result when a specialized insect pest voraciously feeds upon a crop.

Purpose of Study

The purpose of this study is to determine whether aromatic, pest-repellant companion plants such as French marigolds will produce a measurable benefit by repelling pests and increasing productivity of bush bean P. vulgaris plants in East Tennessee gardens. It is hypothesized that French marigolds will produce a measurable increase on bush bean productivity. The goal of this study is to provide valuable data to assist East Tennessee farmers in their efforts to grow bush bean crops in a sustainable way that would minimize damage from the insect pest Mexican bean beetles while eliminating or reducing the need for additional pesticides.

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CHAPTER II

MATERIALS AND METHODS

Experimental Crops and Origins

Three different varieties of Phaseolus vulgaris, or green beans, were studied in the field with French marigolds Tagetes patula: Kentucky Wonder, Blue Lake, and Black

Valentine. The Black Valentine beans were used to monitor the success of a heirloom variety compared to the commercial varieties Kentucky Wonder and Blue Lake in resisting damage by the Mexican Bean Beetle (Epilachna varivestis). Half of all varieties were companion- planted with French marigolds to observe whether beans planted with marigolds would be more resistant to Mexican Bean Beetles than those without.

The Kentucky Wonder variety was purchased from the Knoxville Seed Company in

Knoxville, Tn., whereas the Blue Lake variety was purchased from the AgCentral Farmers

Co-Op in Maryville, Tennessee. The Black Valentine beans came from the personal seed collection of heirloom seed collector John Coykendall of Knoxville, Tennessee. The Black

Valentine beans used in this experiment were obtained by Coykendall in St. Landry Parish,

Louisiana from a local farmer in 1994.

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Experiment Plot Locations and Layout

Two locations were used as experimental garden plots. Maryville College in

Maryville, Tn. (35°45'4.95"N, 83°57'39.32"W) hosted a rectangular garden plot subdivided into 14 subplots (see Figure 2). Three subplots were used for each variety of green bean used in the study, and an additional subplot was used which contained two different varieties. In

East Tennessee, prevailing winds blow north-west, and thus the experimental marigolds were placed on the southeast side of the garden in order to minimize contamination between experimental and control subplots. Blackberry Farm in Walland, Tn. ( 35°41'39.32"N,

83°51'53.85"W) was the site of a second experimental plot consisting of a row 10 meters long surrounded by a variety of other vegetables in the farm’s vegetable garden (Figure 3).

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Figure 2. The layout of Black Valentine and Blue Lake plant groups as well as the dimensions of the plot and location of non-experimental plants on the Maryville College campus in Maryville, Tennessee. Orange rings indicate that experimental beans were surrounded by marigolds.

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Figure 3. The overall layout of Black Valentine and Kentucky Wonder plant groups at

Blackberry Farm in Walland, Tennessee.

Soil Testing and Preparation

Four soil samples from Maryville College and 2 soil samples from Blackberry Farm were collected during April 3 and April 1 respectively. The samples were collected from the

4 corners of the plot at Maryville College and at two locations approximately 6 meters apart.

Soil samples were collected using a steel soil sample probe to a depth of 15.24 cm, dried on disposable aluminum cake pans, and were sent to the Soil, Plant and Pest Center in Nashville,

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Tennessee for testing. The garden plots at Maryville College and Blackberry Farm were tilled using a tiller attachment on a tractor. No compost or additives were used in either soil plot.

Plant and Seed Purchases and Planting

To mimic conditions in a typical domestic vegetable garden, a variety of vegetable plants were planted in close proximity to both experimental and control plots. On May 5, non-experimental garden plants were purchased from Home Depot (Maryville, TN). Bonnie

Plants varieties of tomatoes, cucumbers, cantaloupe, squash, and basal were planted on May

5, 2015. Both experimental and control plants were equidistant from these vegetables (see

Figure 2).

On May 22, Kentucky Wonder and Black Valentine bush beans were planted at the

Blackberry Farm plot. On May 27, Black Valentine and Blue Lake bush beans were planted in the Maryville College plot. For both plots, the same planting method was used: a shallow trench of approximately 13 centimeters was made using a hoe, 4 bean seeds were dropped into the trench approximately 120 centimeters apart, the seeds were thoroughly soaked with water, and the seeds were lightly covered with approximately 8 centimeters of dry soil.

French marigolds from Bell Horticultural Company used at the Maryville College plot were purchased from Home Depot, Maryville, TN., whereas the French marigolds used at the Blackberry Farm plot were an heirloom variety obtained by John Coykendall from a stock begun at Blackberry Farm in 1994.

Leaf Damage Analysis and Bean Harvest Data Collection

Two endpoints were quantified: leaf damage and bean number. Leaf damage was assessed by collecting leaves from each bean group according to its plot location and whether

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it had or had not been companion-planted with French marigolds. To avoid bias when choosing representative leaves to sample, only leaves on the North side were selected. Three total representative leaves were collected from each bean group: a leaf from the lowest growth level of the plant, a leaf from the growth level between the topmost and bottommost growth level, and a leaf from the topmost growth level of the plant. The representative leaves from each plant were collected at different levels to include leaves at varying stages of development and leaf damage. The amount of damage to the leaves was analyzed using

ImageJ software obtained from the National Institute of Health.

Leaf analysis using ImageJ followed the following protocol: leaves were laid flat on a sheet of white paper with a ruler marked in centimeters. A picture of each representative leaf for each plant group—the leaf from the middle tier—was uploaded into ImageJ. To set the scale of the image, the line tool was used to draw a line between 1 cm on the ruler to set the scale of the image (approximately 184 pixels per centimeter in most pictures). The image was then cropped to show only the leaf in the image using the rectangular selection tool and the crop function under “image”. The image was converted to 8-bit and the threshold was adjusted so that the leaf was black in contrast to the white background. The rectangle tool was used to crop the image to a 2.54 x 2.54 cm square from the center of the leaf. The area of the leaf and percent damaged was found using the “analyze particles” tool.

The number of beans harvested was recorded by picking all beans longer than 5.0 cm on the set picking date (Figure 4). During the data collection period of the first harvest, beans were picked and organized into 4 separate, general groups: Black Valentine without marigolds, Black Valentine with marigolds, Blue Lake with marigolds, and Blue Lake

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without marigolds. The first harvest of beans was harvested at Maryville College and at

Blackberry Farm on July 24 and August 3, respectively. For this harvest, the number of beans on individual plants was not documented; only total number was gathered. A second harvest of beans occurred only at Maryville College on August 9, and there was no second harvest at Blackberry Farm. For this second harvest, the number of green beans on individual plants was documented.

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Photo Credit: Drew Crain

Figure 4. An example of a green bean over 5 cm long that is an ideal maturity for canning.

The beans shown are from the Blue Lake variety at the Maryville College campus in

Maryville, Tennessee.

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Statistical Analysis

Bean harvest data collected were analyzed using an unpaired t-test in Microsoft Excel to analyze like varieties of beans planted with marigolds and without marigolds. Similarly, the results obtained from the leaf damage data were analyzed using t-tests to compare like varieties planted with and without marigolds against each other.

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CHAPTER III

RESULTS

Soil Analysis and Bean Harvest

All soil samples were shown to have adequate levels of primary and secondary nutrients (Table 2). The Blackberry Farm plot samples varied greatly in pH, phosphorous, and calcium. All Maryville College samples had high levels of all primary nutrients but had a higher pH than the optimal range of 6.1-6.5. Individual sampling site soil sample results are listed in Appendix A.

The sprout dates of green bean plants and the bloom dates of marigold plants occurred in May and June (Table 3). The total number of beans collected at Maryville

College and Blackberry Farm for Harvests 1 and 2 showed that Black Valentine with marigolds initially produced the greatest harvest (Table 4). For Harvest 2, the Blue Lake variety showed a response to being companion planted with marigolds and demonstrated a significantly greater harvest (p=.01) than the control group (Figure 5). Black Valentine harvest totals were significantly less than Blue Lake harvest totals (p-value = 0.000000064).

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Table 2. The average soil test results of each garden plot at Maryville College in Maryville,

Tennessee and Blackberry Farm in Walland, Tennessee.

Plot Site Water pH Phosphorous Potassium Calcium Magnesium Blackberry 6.2 34 171 1403 181 Farm Maryville 7.3 95 340 3400 433 College

Table 3. The sprout dates of green beans and marigolds at Blackberry Farm in Walland

Tennessee and Maryville College in Maryville, Tennessee.

First Marigold Marigold Date Plot Site Recorded Planting Bloom Planted Sprout Date Dates Dates

Maryville 27-May-15 2-Jun-15 5-Jun-15 12-Jun-15 College

Blackberry Farm 22-May-15 26-May-15 6-Jun-15 22-Jul-15

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Table 4. Green Bean Harvest Totals during July and August 2015 at Maryville College in Maryville, Tennessee and Blackberry

Farm in Walland, Tennessee.

Maryville College Blackberry Farm Beans w/ Marigolds Beans w/o Marigolds Beans w/ Marigolds Beans w/o Marigolds Harvest 1 Blue Lake Beans 640 643 X X Black Valentine Beans 1127 674 162 112 Kentucky Wonder X X 29 8 Harvest 2 Blue Lake Beans 869 628 X X Black Valentine Beans 298 355 X X Kentucky Wonder X X X X

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70

60 * 50

40

30

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Number.of.Beans.Harvested 10

0 Black.Valentines. Black.Valentine. Blue.Lake. Blue.Lake.Control w/Marigolds Control w/Marigolds

Figure 5. The mean number of beans harvested (+ 1 SE) from Maryville College in

Maryville, Tennessee for Harvest 2. * indicates significant difference at p<0.01.

Leaf Damage

Although bean harvest was significantly increased in the presence of marigolds in the commercial variety, leaf area damaged by pests in both varieties of bean plants was not influenced by the companion plants (Figure 6). Blue Lake plant leaf data suggested more damage on average when planted with marigolds than without marigolds, but this was not significantly different (p-value = 0.052) in leaf area damaged when companion planted with marigolds. Likewise, Black Valentine bean plants did not show a significant difference in leaf area damage (p-value=0.64) when planted with marigolds. Therefore, although the heirloom variety showed little difference, the commercial variety showed a trend toward increased leaf damage when planted with marigolds.

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25.00

20.00

15.00

10.00

Leaf.Area.Damaged.(%) 5.00

0.00 Black.Valentine. Black.Valentine. Blue.Lake. Blue.Lake.Control. w/Marigold Control w/Marigold

Figure 6. The average leaf area percent damage of the middle leaves in the center square inch (+ 1 SE) of all bean plants planted with and without marigolds from Maryville College in Maryville, Tennessee.

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CHAPTER IV

DISCUSSION

It was hypothesized that green beans would produce more beans per plant when planted with the aromatic pest-repellant French marigold. The results support this hypothesis only in the commercial variety of green bean; the heirloom variety did not show a significant response to companion planting. Leaf damage was not influenced by the companion plants.

Due to the lack of difference in leaf damage, it is proposed that the marigolds did not offer aromatic pest-repellant properties against Mexican bean beetles but instead offered microbial benefits against harmful fungi and root-knot nematodes affecting the roots.

Bean Harvest

Total bean harvest was greatest in the commercial variety. Although the heirloom variety at Maryville College companion planted with marigolds initially had a much higher yield, producing nearly twice as many beans as the commercial variety with marigolds or the heirloom variety without marigolds, by the second harvest, the heirloom variety with marigolds had a yield that was nearly a quarter of its initial production. The commercial variety showed much more stable numbers of beans produced from the first and second harvest. Although the heirloom variety initially showed an explosive response to being

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companion planted with marigolds, the commercial variety ultimately showed a significant response to the marigolds and produced more beans overall.

Leaf Damage

Neither the heirloom nor commercial variety showed significantly reduced leaf damage when planted without marigolds. Blue Lake bean leaves with marigolds showed a greater amount of damage compared to those that were not companion planted. Indeed, there was a trend toward significantly greater leaf damage in commercial beans planted with marigolds. The same plants that produced the greater harvest of beans also had the most amount of leaf damage when comparing the commercial variety with and without marigolds.

Mexican bean beetles were even observed sitting on the leaves of the marigolds although not consuming them (personal observation). The marigolds seem to have no effect on the amount of leaf damage. This suggest that there was no observable aromatic allelopathic pest-repellant effect produced by the marigolds for the benefit of the bean plants.

The method used to calculate percent of leaf area damaged was not a technique available in the current available literature, and this study is perhaps one of the first to use

ImageJ analysis as a means to quantify leaf damage. This method of analysis holds promise for future studies examining leaf damage because it is a free program widely available through the National Institute of Health and is a powerful image analysis due to ability to measure the area of even complex objects such as leaves.

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Allelopathic Influence

Marigolds contain a variety of allelopathic compounds that produce bactericidal, insecticidal, and fungicidal effects (Mares et al., 2004), but the exact mechanism of marigold’s beneficial influence on particular crops is unknown. It is proposed that the marigolds positively affected the number of beans in a way independent of inhibiting leaf damage. Legumes when companion planted with marigolds have experienced decreased root damage and a higher crop yield than the control in previous studies (Adekunle 2011). Indeed, marigolds increase harvest yields of root vegetables like potatoes and carrots when inter- planted with the crops (Korthals et al., 2013). The presence of T. patula can initiate a decrease in Pratylenchus penetrans populations by 90% with effects lasting up to 2 crop cycles (Pudasaini et al., 2006). Marigold’s deep roots provide better nematode suppression than chemical nematicides (Pudasaini et al., 2006). When nematodes penetrate the roots of marigolds coming into contact with thiophenes, the reactive oxygen ultimately kills the nematodes (Gommers et al., 1980). Marigolds have deep, extensive root systems that reach farther than chemical nematicides. (Pudasaini et al., 2006). Although neither of the experimental sites in the present study had soil examined for high concentrations of nematodes or fungus, these properties of the French marigolds may have been beneficial enough to explain the significantly greater harvest in Blue Lake bush beans during the experiment. Future studies should examine the impact of planting green beans and marigolds on soil bacterial, fungal, and nematode diversity.

29

Summary

The average number of green beans harvested was higher in both varieties when companion planted with marigolds, and the Blue Lake bush bean variety showed the greatest response to companion planting. When planted with marigolds, the Blue Lake variety produced the most beans overall. According to the results shown, companion planting with

French marigolds could have a measurable effect on the amount harvested for crops such as green beans. Because the plants did not show a significant difference in leaf damage between control and varieties planted with marigolds, it is proposed that the marigolds benefited the plants in some other way. If the marigolds did not provide significantly greater protection from P. vulgaris’s main pest the Mexican bean beetle, then perhaps it benefited the plants by reducing harmful fungus in the soil or root-knot nematodes.

T. patula seems to be a viable option to increase yields of P. vulgaris although perhaps not for the reason originally hypothesized. The heirloom Black Valentine variety did not show a significantly greater bean harvest when companion planted with T. patula, but the commercial Blue Lake variety did. Neither variety showed a significant decrease in leaf damage when companion-planted bean plants were compared against non-companion- planted plants, but the commercial variety with marigolds showed a trend toward significantly greater damage than the non-marigold plants—directly contradicting the hypothesis that plants planted with marigolds would have less damage than those without marigolds. Therefore, the increase in bean harvest yields can perhaps be explained by a direct benefit to the roots and surrounding soil of bean plants. Future studies should examine leaf damage and bean harvest analyzed as well as soil and root samples to observe whether the

30

plants companion planted with marigolds are benefitting from antimicrobial or nematicidal effects.

31

APPENDIX

.. Water&pH& Phosphorous& Potassium& Calcium& Magnesium&

BBF1. 6.9. 57. 196. 2255. 253. BBF2. 5.4. 10. 146. 551. 108. MC1. 7.2. 162. 336. 3721. 439. MC2. 7.2. 45. 369. 3026. 443. MC3. 7.4. 40. 338. 3277. 443. MC4. 7.3. 131. 315. 3574. 407.

33

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