The Effect of Legume Nitrogen Fixation on Corn Growth Rate/Yield in the Common Garden Mary M. Mortenson Metropolitan State College of Denver, Earth & Atmospheric Science Department December 9, 2011

Keywords: biological nitrogen fixation (BNF); rhizobia bacteria; nodulation; legumes; nitrogenase; urban (common) garden

ABSTRACT Nitrogen is an essential nutrient for soil and agricultural crops. All cultivated crops, except for legumes, require the soil to provide large amounts of nitrogen. The objective of this study was to determine whether growing legumes (fm. Leguminosae) along side corn (fm. Poaceae) in a common urban garden will have an effect on corn growth rate and yield due to nitrogen fixation nodules found on legume roots. Selecting plants that matured at the same rate ensured that the plant yield was not affected by different harvest rates and/or maturity of fruit. In Plot#1 and Plot #2 popcorn (Zea mays everta) was planted and in Plot #2, the common field bean, (Phaseolus vulgaris L.) was planted between the rows of popcorn. Soil tests were preformed for N, P, K and pH; soil texture and moisture levels. Data collection included earthworm count, stalk with ear count, cob length, and grain yield.

Results of higher levels of earthworm activity, moisture

retention, ear count and grain yield in Plot #2 indicated an increase level of nitrogen available for optimal plant growth and seed production. This study has demonstrated that use of legumes with its symbiotic relationship with Rhizobium, a free-living soil bacterium, is a practical option to inorganic fertilizers for the urban garden.

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INTRODUCTION

The objective of this study was to determine whether growing the common field bean along side popcorn in a common home garden will have an effect on the corn plant growth rate and increased grain yield due to the symbiotic relationship between Rhizobia bacteria and its ability to fix nitrogen within the nodules formed on legume roots. The family Leguminosae is named for its fruit, which in most species is a legume (the technical term for bean pod) with 16,000 species (Dimmitt, 2011). Some common varieties are soybeans, chickpeas, field beans, and alfalfa. There are many complex interactions between soil microorganisms and soil functions, such as the breakdown of organic matter, supplying necessary nutrients and executing the biogeochemical cycles (Mukerji et al., 2006). Nitrogen fixation, the breaking of the nitrogen bond, occurs in three different processes: atmospheric, industrial, and with microbial bacteria. Being a primary nutrient, nitrogen is frequently in short supply which can effect crop production; by growing legumes in rotation with a cereal crop has shown increased biomass and plant yield (Cheruiyot et al., 2001). Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by the enzyme nitrogenase, the only known organic factor capable of breaking the powerful triple bond of the nitrogen molecule (de Camp, 2008; Mulongoy, n.d.). For gardens and agricultural systems to remain productive and sustainable it is necessary to restore the nutrients in the soil which are lost either by plant uptake or crop harvesting (Peoples et al., 1995). Nitrogen is an essential nutrient for both the soil and cultivated crops. It is generally the most deficient nutrient in many soils universally and is the most commonly supplied plant nutrient with the use of added fertilizer. All cultivated crops,

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except for legumes, require the soil to provide relatively large amount of nitrogen (Peoples et al., 1995). Supplying nitrogen through BFN with legumes crops can create healthier soil, lower crop expenses and provide agricultural sustainability by limiting Nfertilizers (Cheruiyot et al., 2001; Peoples et al., 1995). Rhizobia bacteria have the unique ability to establish a N2-fixing symbiosis on legume roots which occurs in the soil rhizosphere (Mukerji et al., 2006). These nitrogenfixing microorganisms are specific in these symbiotic relationships: a specific rhizobia bacteria species will only infect a specific species of legume that are native to the environment in which the symbiosis occurs (Cheruiyot et al., 2001; Howard Tan, 2000; Mukerji et al., 2006; Martinez et al., 1985).

Plants species are selective to which

microorganisms are harmful or beneficial to their growth (Denison & Kiers, 2004). Rhizobium phaseoli is the symbiont of Phaseolus vulgaris, the common bean, found originally in Mesoamerican countries (Martinez et al., 1985). The root of a plant helps provide support, allows for water/nutrient intake and performs specialized roles; the subsurface environment is an active area where chemical attractants and repellants are discharged by the roots (Walker, et.al., 2003; Denison & Kiers, 2004). The rhizosphere, the immediate soil surrounding the root, is influenced by the compounds of amino acids, sugars and vitamins which the roots exude. This zone depends on the soil type, the host plant and soil environmental factors including soil moisture, pH, temperature and humidity. The rhizosphere provides a specialized niche where microbes live and influence crop health and yield and is the zone of the symbiotic relationship between the legume and the microbe (Mukerji et al., 2006).

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Rhizobium, a free-living soil bacterium “infects” the legume root at a weakened area (Lindemann & Glover, 2008). This intercellular infection develops into a root nodule where the enzyme nitrogenase fixes the nitrogen from the soil. This enzymatic process of combining the nitrogen molecule with three hydrogen molecules to form an ammonia molecule (N2 + 3H2 → 2NH3) which is absorbed by the plant, requires a great amount of energy in the form of ATP (deCamp, 2008). However, nitrogenase is susceptible to destruction by oxygen and must live in anaerobic conditions. Leghemoglobin, a redcolored, oxygen-binding protein provided by the plant, ensures the proper function of nitrogenase for reducing N2 to ammonia (Arnoys, 2011; Barak et. al, 2005; Lindemann & Glover, 2008). Without this protein, the nitrogen fixation process would fail. According to Lindemann & Glover (2008), nodules appear on the roots 2-3 weeks after germination and are located in masses near the top of the root zone. Nodules on annual legumes, such as Phaselous vulgaris, are short lived and will discontinue when the plant enters its seed production stage; this explains why nodules are not seen on mature plants. The small, immature nodule is white or gray inside and as it develops turn a reddish color indicating nitrogen fixation has started and the leghemoglobin protein is present (Lindemann & Glover, 2008; Martinez et al., 1985). The plant provides the necessary nutrients and energy for the bacteria in exchange for the nitrogen nutrient (Lindemann & Glover, 2008; Denison & Kiers, 2004; Brajesh, 2009). A large proportion of nitrogen accrued by the legume during the growing season is removed with the harvested seed and the vegetative residue (Peoples et al., 1995; Cheruiyot et al., 2001; Lindemann & Glover, 2008). When the legume dies and decomposes, the nitrogen is available for the non-legume plants; therefore, turning in the legume residue is necessary

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for the nitrogen to be released back into the soil and to benefit future crops (Cheruiyot et al., 2001). Historically, since the time of the Romans and among indigenous cultures, planting legumes along side a cereal crop, especially maize, has been a common practice for centuries (Formiga, 2011; Martinez et al., 1985). Corn, the common name for maize, is one of the main cereal grains grown throughout the world for food and biofuel. The 2011 U.S. corn crop is now forecast at 12.497 billion bushels, 50 million larger than the 2010 crop (Good, 2011). Rotating legume and corn crops is a beneficial and a sustainable way to produce increased crop yields while providing the necessary plant nutrient nitrogen. Grain legumes have been shown to increase succeeding maize yields in semi-arid regions (Odhiambo, 2011; Cheruiyot et al., 2001). Biological nitrogen fixation (BNF) within the nitrogen cycle has lead to this study of the fascinating ability of the microscopic rhizobia bacteria and its symbiotic relationship with the legume and to the sustainable prospects for future crop management. Increased awareness of food product additives, organic vs. inorganic and reduction of food budgets, urban gardening is becoming a viable alternative for a healthy lifestyle. Often urban soils can be unhealthy or contaminated and unable to support growth. The question is how effective can the urban gardens be with the use of human-made organic soils, no-till methods and domestic composting. Studies have shown that growing corn and legumes together can increase crop yield on an agricultural basis (Cheruiyot et al., 2001; Peoples et al., 1995; Martinez et al., 1985; Francis, et. al., 1986), but can this same methodology increase production in the common home garden? If planting legumes (Phaseolus vulgaris L.) alongside popcorn (Zea mays everta) in the same garden plot increases the

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nitrogen levels in the soil, then the growth rate and yield would be greater than in the garden plot with only corn planted. Studies of biological nitrogen fixation, the species of the rhizobia bacteria and different soil testing methods were researched to better understand the processes occurring in the soil in relation to the nutrients, the bacteria and utilization of nitrogen.

LITERATURE REVIEW

Nitrogen, a key nutrient in productive plant growth, is essential for a healthy garden crop. Biological nitrogen fixation (BNF) is necessary for providing organic nitrogen to the soil for positive crop yield. With the increase of the use of inorganic N fertilizers, BNF has lost its popularity on the big scale. However, with the emphasis on renewable resources and new wave of interest in organic produce, BNF has been considered to play a major role in providing nitrogen for agricultural crops. According to a study by Peoples, et. al. (1995) the main problem facing farmers is that most N is lost from the soil with the harvest of the plant. Farmers also need necessary BNF management skills in order to regulate and/or rotate their crops accordingly for the best yield. It is difficult to measure nitrogen fixing microorganisms due to many different soil environmental factors, i.e., legume-grain rotations have been studied only for a single season; without subsequent rotations, N in legume residues can change to mineral forms; and high nitrate levels can inhibit nitrogen fixation potential. This study concluded rotating cereal crops with legumes improved the levels of soil nitrogen and was beneficial to decreasing soil infertility; also, the use of green manures, mulch and leaf drop contributed to positive N levels in the soil. However, supplemental

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N fertilizers might need to be applied in high-yielding cereal crops to ensure optimal nutrition. A rhizobia bacterium, the key player in BNF, ensures nitrogen is available to agricultural and garden crops with its symbiotic relationship with the legume. Specificity of the Rhizobium species in association to legume species determines the productivity of the nitrogen fixing nodule formed on the legume root. In a study by Martinez et. al., (1985) reviews the nitrogen fixation gene coding sequences found on the plasmids of the R. phaseoli strain that help differentiate the Phaseolus vulgaris (common bean) species from the other species of Phaseolus. The study concluded although some strains of Rhizobium nodulate more than one host, R. phaseoli is more specific, only successfully nodulating Phaseolus vulgaris. Not all strains of rhizobia are able to fix nitrogen. As explained by Dennison & Kiers, (2004), legumes cannot exclude non-nodulating strains, if they are closely related to their symbiotic partner. Many studies have been conducted throughout the world on the benefits of growing cereal crops alongside or in rotation with legumes. The findings are valuable to both developing countries affected declining soil fertility and raising fertilizer costs and to the urban gardener. A study by Cheruiyot et.al (2001) in Kenya, Africa examined the effects of growing a legume cover crop during the short rain season to produce an organic fertilizer for the maize crop the following season. This study concluded that growing legumes in seasonal rotation with maize, increased crop production considerably without the use of inorganic fertilizers. According to the study by Francis, et. al. (1986), research has been conducted in temperate climate zones, by various methods of strip cropping with corn and legumes to

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maximize crops yields and nitrogen levels in the soil. This is also advantageous to the small farmer or urban gardener by reducing cost output while maintaining a positive yield. The study concluded a marked improvement of grain yield for both the legume and corn crops as compared to sole cropping.

Other benfeits included reduction in

erosion and nutrient loss; cut in costs; and increase of soil nitrogen, thus reducing the need for added N fertilizers. Strip crop rotation year to year showed a positive grain yield. OBJECTIVES The objective of this study is to determine whether growing the common field bean, (Phaseolus vulgaris L.) along side popcorn in a common home garden will have an effect on the corn plant growth rate and increased grain yield due to the symbiotic relationship between common soil bacterium, Rhizobium and its ability to fix nitrogen within the nodules formed on legume roots. The sub-objective will include how earthworm activity correlates with nutrient levels in the soil.

STUDY AREA This study was conducted from March 19 – October 18, 2011 in two home garden plots in unincorporated Arapahoe County, Colorado (N39.61881°; W104.98259°) at an elevation of 5,462 ft, as shown in Figure 1. This location was chosen for a garden due to the western exposure of the sun in the afternoon and void of trees and building shadows. Placed within the lawn, the plots used are in a raised-bed form of gardening. This increases the soil water and air flow and allows for plants to grow without having the soil compacted when working in the plots; these plots have been cultivated for 8 years.

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Control Plot#1 was approximately 10 feet from a building structure and received sunlight during all daylight hours. East of the plot the lawn was dry, ant infested and with crab grass; Plot# 2 was approximately 16 feet from a building structure, received partial sunlight in the morning hours due to a nearby tree and increased sunlight in afternoon and early evening hours. West of the plot the lawn was stable. Both plots were watered by two in-ground lawn sprinklers on a regular every-other-day schedule. The soils are well drained loamy sand texture classified as an aridisol with a granular structure. The home garden used for this study, due to limited space, has benefited by growing the legume and the corn plants together producing good harvest yields for both crops.

Figure 1. Site location.

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METHODS Experimental design and soil testing procedures.

The design for this study

incorporated two garden plots of the same size, 4’x9’3”, spaced 6’8” apart and each facing in a N-S direction; throughout this study the plots will be labeled Control Plot #1 and Plot #2 . Each plot was prepared for planting using the same methods of tilling, composting, planting and watering. In both plots, popcorn (Zea mays everta) was planted and harvested after the ears were completely dried on the stalk. In Plot #2, the common field bean, (Phaseolus vulgaris L.) was planted between the rows of popcorn and harvested when the pods were dry. Selecting plants that matured at the same rate ensured that the plant yield was not affected by different harvest rates and/or maturity of fruit. The plots were mulched prior to this study in the fall, October 2010, with maple tree leaves. The ground was tilled with a garden fork in mid-March 2011, turning in any remaining leaves and to enhance composting. EKO brand, Certified Colorado compost was added to the soil two inches thick and worked into the soil. This compost contained forest products, recycled wood products composted with poultry manure and DPW (dried poultry waste). DPW is commonly used for fertilizer and livestock feed and can be a source of primary nutrients for the soil. There were no additional fertilizers applied to either plot. The plants chosen for this study were Japanese hulless popcorn (Zea mays everta) distributed from the Livingston Seed, Co. and purchased at a local feed store. This is a very old variety of popcorn that has not lost its appeal to home gardeners; petite stalks producing two to three short ears per plant with the kernels arranged irregularly on cob.

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Each ounce is approximately 170 seeds and will reach maturity within 95-100 days. These popcorn seeds were planted in both plots. A common field bean species, Midnight Black Turtle (Phaseolus vulgaris L.) seed was ordered online from Johnny’s Selected Seeds, a company in Maine and was grown as a shell bean. A shell bean can be considered as any bean grown for the bean itself—the seed—rather than the pod. The best variety of this Latin favorite, Midnight is an improved, upright-growing, black bean strain. The tall bush keeps the pods off the ground. Turtle Beans are small black beans, about the size of pea beans and are among the least allergenic of all bean types; 3/4-oz package of 100 seeds. Maturity 85-105 days. These seeds were planted in Plot #2. The plots were planted on May 7, 2011, warm at 85o F and sunny. The rows were furrowed in the E-W direction in the garden boxes. In both plots, Japanese hulless popcorn was planted at the recommended spacing allowing for 7 short rows with 5 seeds each; Plot #2 Midnight Black Turtle beans were planted in between the rows of corn at the recommended spacing allowing for 6 short rows with 15 seeds each. Both plots were hand watered and covered with rabbit fencing to keep out unwanted visitors until plants were sturdy enough to withstand intruders. Scheduled watering was programmed by the lawn sprinklers at the rate of 20 minutes each evening on an every other day basis for most of the growing season. Each plot received water from two different sprinklers. Data collection included earthworm count, soil testing, and harvest yield rates. Soil testing included assessments for pH (acidity or alkalinity), and the primary nutrients:

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nitrogen N for healthy green biomass; phosphorous P for genetic and seed development; and potassium K for carbohydrates, plant protein and strength of the plant. Earthworms were counted from three spades of moist soil, approx. 6 inches deep and chosen randomly within each plot. The worms were tossed back into the plot after counting. This test was performed on each plot separately, twice in early spring (March & April) and once in the fall (October). Mechanical sieve measurements for determining soil texture were performed on approx 14 oz of sun-dried soil samples from each plot retrieved on March 19, 2011. The soil in these plots for this experiment is of a Loamy Sand soil texture, described as a dryloose, single grained; gritty, no or very weak aggregates: moist - gritty; forms easily crumbled ball; does not ribbon; wet - lacks stickiness; may show faint clay staining. Individual grains can be both seen and felt under all moisture conditions. The soil in both plots has a granular structure. Gravitational water measurements were conducted for each plot after a particular watering day late September and for three tests within the next five days. For each test three - 3”x2” samples were taken from each plot and thoroughly mixed.

Two

tablespoons of moist soil were taken from each mixture, weighed and then dried to calculate the soil moisture content. The soil was dried in the microwave to ensure quick and complete drying. The soil was tested seven times from March until October using the Rapitest Soil Test Kit purchased from a local garden center. The soil removed for the earthworm count was thoroughly mixed together by hand and used for the soil testing. Rubber gloves were worn to keep bodily oils and chemicals away from the soil. Small stones and large debris

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was removed. The testing matrix included vigorously mixing 1 part soil with 5 parts water in a clean container and allowing it to settle at least 8 hours. A small amount of water from each container was removed by an eye dropper and placed in each testing comparator along with the particular nutrient indicator capsule and shook; the results were read after 10 minutes. To test for pH, a small amount of soil was put in its comparator and water added with the pH indicator capsule and shook; the results were read after 10 minutes. The test values for the primary nutrients are shown in Table 1. The pH values followed the pH scale. All tests were performed on each plot separately. Soil samples from each test were bagged, labeled and placed in the freezer for future testing. Table 1: Soil Test parameters: Test Levels 4 3 2 1 0

Test Description Surplus Sufficient Adequate Deficient Depleted

Data were collected from each plot and included the following response variables: mature corn stalks with the number of ears on each stalk and graphed per each row. The ears were harvested on October 11, 2011 when dried on the stalk. After further drying, the cobs were shelled and the grain weighed (g) and the length (cm) of the cobs recorded. This was done for each plot separately. As additional data the shell beans stand was counted, the dry pods harvested, shelled and weighed (g) from Plot #2 in mid September.

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Data analysis: To analyze the data in this control-impact study, a 2 sample t-test was used, assuming both population distributions are normal and sample sizes nx and ny are large, with population x as the impact (Plot #2) and population y as the control (Plot #1). Tests were performed for stalk count, ear count, cob length and grain yield.

The

hypothesis was tested with the null hypothesis Ho = μx – μy = 0 and alternative hypothesis Ha = μx - μy > 0; reject Ho if p-value < α (0.05) or t is in the rejection region. Boxplots and basic descriptive statistics performed and analyzed. Statistical analyses and graphs were produced using MINI-TAB software and Microsoft Excel.

RESULTS Garden management treatment effects on soil, primary nutrients, and soil moisture. The soil texture triangle was used to classify the soil as determined by particle percentages shown in Fig. 2. Compost amendment primary nutrient levels are listed in Table 2. Nitrogen peaked April 23, 2011 after the addition of compost, but averaged at deficient throughout the study as shown in Fig. 3A; phosphorous levels were deficient and potassium levels were sufficient as shown in Fig. 3B and Fig. 3C respectively. The pH of the soil was neutral (7.0) in both plots. Earthworm population was higher in Plot #2 than Plot #1 (Fig. 4). Soil moisture was higher in Plot #2 than Plot #1 (Fig. 5).

Growth rate and grain yield for popcorn crop. The common garden crops for growing season 2011 are shown in Table 3. The garden plot diagram, including growth rates are displayed in Fig. 5. The intercrop rows (3, 4 and 5) produced 42% of the stalks and 37% of the ears for Plot #1 and produced 65% of the stalks and 63% of the ears for Plot #2. For the two plots combined, Plot #2 produced 48% of the total stalks and 53% of the total

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ears over Plot #1 with 51% of the stalks and 46% of the ears. Stalk and ear growth was highest in row #3 for both plots (Fig. 7 and Fig. 8). Germination and mature stalk counts per row were higher in Plot #1 (μy=3; n=21) than Plot #2 (μx2.7; n=20) (Fig. 7) with growth rates 60% and 57% respectively. Ear counts per row were higher in Plot #2 (μx=4.7; n=33) than in Plot #1 (μy=4.1; n=29) (Fig. 7) with growth rates per stalk 1.65 and 1.38, respectively.

Harvested cob lengths between plots showed no significant

variance (Fig. 9). The grain yield from Plot #2 (165.66±4.8g; n=8) increased on average 17.55g over Plot #1 (148.11±4.5g; n=8) during the growing season 2011 (two-sample ttest: t-value=2.67, DF=13, p-value = 0.019; Fig. 10).

Percentage in Soil

Loamy Sand Soil Classification 100.00% 80.00% 60.00%

Plot #1

40.00%

Plot #2

20.00% 0.00% Sand

Silt

Clay

Texture Class

Figure 2. Soil classification.

Table 2. Analysis of the primary nutrients found in the dried poultry waste (DPW) EKO brand, Certified Colorado compost: Moisture Type %Nitrogen % Phosphorous % Potassium 75% (fresh) 1.13 0.74 0.63 35% (moist) 2.36 1.31 0.98 10% (dry) 3.84 2.01 1.42 (Bell, 1990)

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A

Nitrogen (N)

Test Values

4 3 Plot #1

2

Plot #2

1 0 20-Mar 23-Apr

4-Jun

20-Jul 23-Aug 18-Sep 18-Oct

Test Dates 2011

B

Phosphorous (P)

Test Values

4 3 Plot #1

2

Plot #2

1 0 20-Mar 23-Apr

4-Jun

20-Jul 23-Aug 18-Sep 18-Oct

Test Dates 2011

C

Potassium (K)

Test Values

4 3 Plot #1

2

Plot #2

1 0 20-Mar 23-Apr

4-Jun

20-Jul 23-Aug 18-Sep 18-Oct

Test Dates 2011

Figure 3. Primary nutrients levels throughout 2011 growing season. (A) Nitrogen; (B) Phosphorous; (C) Potassium. 16

Earthworm Count

Earthworm Activity 100 80 60

Plot #1

40

Plot #2

20 0 March

April

October

Growing Season 2011

Figure 4. Earthworm population.

Soil Water Percentage

Gravitational Water in Soil 40.00% 30.00%

Plot #1

20.00%

Plot #2

10.00% 0.00% Oct 1 - 67º Oct 3 - 65º Oct 5 - 63º Date & Aveage Daily Temperature F

Figure 5. Gravitation water measurements in soil after scheduled irrigation event.

Table 3. Common garden crop growth rates and grain yields for 2011 growing season. Garden Crop popcorn -1 popcorn -2 common field bean - 2

# Rows 7 7 6

# Planted Seeds 35 35 90

Maturity Rate (%) 60 57 71

Grain Yield (g) 1,026.70 1,325.30 233

1 = Plot #1 2 = Plot #2

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Figure 6. Diagram of garden plots including stalk and ear counts, growth rates and grain yields. (Letters A-E represent mature stalks; Arabic numbers represent the ear count per stalk; the letter ‘x’ represents seeds that did not germinate or plants that did not mature.) Stalk Count per Row Plot #1

Stalk Count

5

Plot #2

4 3 2 1 0 1

2

3

4

5

6

7

Row Number

Figure 7. Japanese hulless popcorn mature stalk counts per row.

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Ear Count per Row

Ear Count

10 8 6

Plot #1

4

Plot #2

2 0 1

2

3

4

5

6

7

Row Number

Figure 8. Japanese hulless popcorn mature ear count per row.

Boxplot of Cob Lengths 20.0

Cob Length (cm)

17.5 15.0 12.5 10.0 7.5 5.0 Plot 2

Plot 1

Figure 9. Boxplots of harvested cob lengths of Japanese hulless popcorn.

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Grain Yield 180

Weight of grain (g)

170 160

150 140 130

120 plot 2

plot 1

Figure 10. Plot #2 popcorn grain yield was higher than the grain yield in Plot #1 for the 2011 growing season.

DISCUSSION The rhizobia bacteria species, Rhizobium phaseoli is the key player in biological nitrogen fixation (BNF) of Phaselous vulgaris, forming nodules to utilize nitrogen for plant growth and grain yield (Martinez et al., 1985). Field beans develop best in a sandy loam soil with good drainage, moderate organic matter and not easily compacted (Hardman, 1990); the raised garden beds used in this study were ideal for optimal legume growth. Nitrogen is the most desired and needed nutrient for healthy crops and increased harvest production. Inorganic nitrogen was not added to the soil during this study. The legumes were planted as the main source of nitrogen available for popcorn plant uptake. Maize and legume crops desire a slightly acidic soil (pH 5.8-6.5); the pH 7.0 in this growing season did not have a negative affect on plant growth nor grain yield. Earthworms have shown to improve soil structure and fertility, plant growth and health, increase moisture retention, and even suppress weed growth (Ingram, n.d.).

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Earthworm activity and decomposition of organic matter are related to nitrogen availability in the soil. Nitrogen-fixing bacteria and nitrogenase, the enzyme responsible for nitrogen fixation, can reside in the gut and casts of the earthworm, thus increasing the level of nitrogen available in the soil (Simek & Pizl, 1989). The maple leaf mulch applied to the garden plots in the previous fall (2010) had an effect on the earthworm count and the increased grain yield in Plot #2. Plot #1 was located in the yard where the sun was more intense and the surrounding lawn was weed and ant infested; Plot #2 was partially shaded surrounded by more fertile lawn. These differences can influence the belowground environment including the rootroot, root-insect and root-microbe communications in the rhizosphere (Walker et.al., 2003). Compounds in the legume root exudates are responsible for the forming of the nitrogen-fixing nodules on the plant root.

With possible competition and

miscommunication between plant species, root exudates and microbes, the production of each plot could vary. This difference was manifested in the water retention of the soil, as well as the earthworm count and subsequent higher grain yield in Plot #2. According to Alley et.al (2009), soils with greater water holding capacity have increased grain yields. Biological nitrogen fixation (BNF) occurs in the nodules formed on the legume root by the enzyme nitrogenase. The common field bean, Phaselous vulgaris, is a poor fixer and often will require more N than supplied by fixation; however, without nodulation, additional N fertilizers will not produce a higher grain yield (Lindemann & Glover, 2008). Nodules were seen on two different occasions in this study; first, on a few plants that were destroyed in Plot #2 by the resident dog and second, from young legume plants which had sprouted from dropped mature seed later in the growing season. The nodules

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measured 3-5mm in diameter. The largest specimen displayed red coloring indicating that nitrogen fixation was occurring in the nodule (Lindemann & Glover, 2008). The demand for nitrogen is three-fold in the legume-corn crop production: for nitrogen fixation, plant growth and seed (grain) production (Peoples et. al., 1995). The maize plant requires ample levels of nitrogen for optimal growth; without a doubt, BNF improves the nitrogen in the soil (Peoples et. al., 1995). The greatest uptake occurs a month before the plant tassels and silks appear on the cob and is very influential in fruit sizing, i.e., ear count. Significant amount of N is moved from the leaf biomass to grain production (Alley et .al., 2009). This was evident with the sufficient nitrogen level in April due to added compost and legume nodulation in early summer to deficient level in July, when both plots tasseled, to depleted level late in the growing season. A slight increase of nitrogen in the soil, late in the season, was due to the self-sprouting of a small second crop of legumes. It was on these legumes that the nodules were found. Future cereal crops would benefit if the plant residue from the current legumes were cultivated into the soil rather than the grain harvested in a crop rotation method (Peoples et. al., 1995; Cheruiyot et.al., 2001, Odhiambo, 2011). Due to the small growing area in these garden plots, the practice of growing legumes and corn together has been a necessity to utilize the garden to its fullest. As seen in Plot #2, benefits to growing these crops in close association

included maximum yield

production, decreased weed growth, increased water retention and contribution of N fixation in the nodules of the legume; this resulted in an increase of corn grain yield and a steady yield rate for the legume (Francis et. al., 1986). This is a common agricultural practice in Latin American farming systems with 60% of the corn and 80% of the

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legumes grown together, increasing crops yields over sole cropping (Francis et. al., 1986). The intercrop rows had the greatest growth rates; however, Plot #2 showed the total greatest growth in both ear count and grain yield. Using a level of significance α = 0.05 and with the p-value = 0.019 obtained by the two sample t-test, we can reject H0. There is statistically significant evidence that there is greater grain yield in Plot#2 than in Plot #1. Shown in the diagram of the garden plots (Fig. 5), the mature stalk growth rate was not higher than 60% due to very cold and wet weather conditions during the normal germination period. Total biomass data was not collected because of windstorms that destroyed the dry stalks; therefore growth rates were determined by ear count per stalk, grain yield and cob length. CONCLUSION Significant amounts of biological fixed nitrogen can be achieved in many levels of agricultural and gardening ecosystems. It is evident from this field study that growing the common field bean, (Phaseolus vulgaris L.) along side popcorn (Zea mays everta), in a common home garden increased ear growth rate and grain yield due to the symbiotic relationship between rhizobia bacteria and its ability to fix nitrogen within the nodules formed on legume roots. There is a need for further study of the effects of weather and climate, garden crop rotation, and planting a fall cover crop and its influence on nodulation of the legume and the ability to provide optimal nitrogen for future crops. Improved soil testing procedures are needed for more concise nutrient level statistics.

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ACKNOWLEDGEMENTS Soil testing equipment was loaned by Metropolitan State College of Denver Earth and Atmospheric Science department for a few of the tests performed in this study.

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REFERENCES Alley MM, Martz Jr. ME, Davis PH, & Hammons JL. 2009. “Nitrogen and phosphorous fertilization of corn.” Virginia Cooperative Extension, Virginia Tech, and Virginia State University. http://pubs.ext.vt.edu/424/424-027/424-027.html, November 2011.

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