Nutrient Availability and Changes in Microbial Communities in Flooded Rice Production in South Florida

NUTRIENT AVAILABILITY AND CHANGES IN MICROBIAL COMMUNITIES IN FLOODED RICE PRODUCTION IN SOUTH FLORIDA

RACHELLE BERGER

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2020

To my family and children

ACKNOWLEDGMENTS

I would like to give my give the biggest thank you to my advisors, Dr. Samira

Daroub and Dr. Willm Martens-Habbena, your patience, understanding, caring disposition, and continuous support have allowed me to complete this thesis. I would like to thank all my Dr. Mabry McCray and Dr. Sarah Strauss (committee members) for their guidance and consistent support. I would like to thank everyone at the UF

Everglades Research and Education Center (EREC), UF Fort Lauderdale Research and

Education Center (FLREC), and Gainesville for contributing to my research, education, and career path. I would also like to thank the UF Soil and Water Sciences Department for the incredible opportunities, professors, ad staff. The past three years in graduate school have been some of the best memories of my life. Thank you, Johnny Mosley for helping me navigate through the EAA, you have been a great human compass and soil sampler! Thank you, Tim, for your help with maps and experimental design. Thank you

Viviana, Maryory, and Irina for your help and support in the EREC lab. I would like to thank all the farmers in the EAA that made this research possible and allowing us to sample on your land. Thank you to the Rice Council of the EAA for your financial support. Thank you, Argonne National Laboratory in Chicago, for sequencing my samples. Thank you to Mike Sisk who has encouraged me and made sure I made every deadline since my undergraduate degree. Thank you to the entire Soil and Water

Graduate Student Association and all graduate school association I have had the pleasure of working with. Thank you, Dr. Allan Bacon, for encouraging me to learn more about soil classification and pedology. It was a pleasure to serve at the Vice President of Distance Education for Soil and Water Science Graduate students as well as

President of the Everglades Research and Education Center Student Association. It

was so moving to be able to enhance our centers community growth and support, especially during COVID19. Thank you to Seemanti, Claire, Andy, and Kelly at the UF

FLREC, you all have been so helpful and amazing to work with at your laboratory.

Thank you to everyone in Gainesville. Thank you to all my professors in the Soil and

Water Sciences Department! Thank you to anyone and everyone who has supported me in any way.

Additionally, thank you to my entire family, without which I would not have the support needed to achieve my goals. Thank you to my beautiful children (Ava and

Isaac),sister (Alyssa) , and my amazing friends especially, Tiffany, Alyssa T., Chelsea, and Mike. Thank you to my incredible grandparents who have survived the Holocaust.

Your willingness to survive, never give up has given me a better outlook on life and a passion to become the best version of myself.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 9

ABSTRACT ...... 11

CHAPTER

1 INTRODUCTION ...... 13

The Everglades Agricultural Area (EAA) ...... 13 Histosols in the EAA ...... 14 Subsidence of the Organic Soils in the EAA ...... 15 Best management practices (BMPs) ...... 16 Phosphorus Runoff and Water Quality Issues ...... 17 Flooded Rice Cultivation Benefits in the EAA ...... 18 Soil Chemistry of Flooded Soils ...... 19 Microbial Communities in Flooded Soils ...... 21 Project Hypothesis ...... 23 Project Statement and Objectives ...... 23 Methods and Materials...... 24

2 SOIL NUTRIENT AVAILABILITY FOLLOWING FLOODED RICE PRODUCTION IN SOUTH FLORIDA ...... 31

Introduction of Chapter 2 ...... 31 Subsidence of Histosols ...... 31 Flooded Rice Production ...... 32 Nutrients and Rice Production in Histosols ...... 34 Hypothesis and Objectives ...... 38 Methods and Materials...... 38 Soil Sampling and Analysis ...... 38 Nutrient Analyses ...... 40 Statistical Analysis ...... 40 Results and Discussion...... 41 Soil Properties ...... 41 Total C and N ...... 42 Nutrients ...... 43 Macronutrients and Si ...... 43 Micronutrients ...... 45 Soil Properties ...... 46 Macronutrients ...... 47

Micronutrients ...... 50 Conclusions ...... 55

3 IMPACT OF THE CULTIVATION OF FLOODED RICE ON SOIL MICROBIAL COMMUNITIES ...... 69

Introduction of Chapter 3 ...... 69 Denitrification ...... 73 Fermenting Bacteria ...... 75 Hypotheses and Objectives ...... 76 Methods ...... 77 Soil Sampling and Analysis ...... 77 DNA Extractions ...... 78 Statistical analyses ...... 80 Results and Discussion...... 80 Comparative analysis of selected metabolic groups of microbes ...... 81 Sulfate reducing bacteria (SRB) ...... 84 Methanogens ...... 84 Ammonia-oxidizing Thaumarchaeota ...... 89 Conclusion ...... 90

APPENDIX: FARM REGIONS AND MICROBIAL GROUPS ...... 96

LIST OF REFERENCES ...... 97

BIOGRAPHICAL SKETCH ...... 107

LIST OF TABLES

Table page

1-1 Histosol soil series found in the Everglades Agricultural Area...... 26

2-1 Location of the 21 fields sampled in various farm regions...... 56

2-2 Average values of soil properties, macronutrients, micronutrients, and beneficial element (Si) for shallow and deep muck soils located in the EAA before the cultivation of flooded rice...... 57

2-3 Statistical results using the Wilcoxon test for differences in soil properties and nutrients before and after the cultivation flooded rice in the EAA...... 58

2-4 Differences in soil properties and nutrients as impacted by flooded rice cultivation and depth of soils in the organic soils in the EAA (<20 inches)...... 59

2-5 Differences in soil properties and nutrients as impacted by flooded rice cultivation and depth of soils in the organic soils in the EAA (>20 inches)...... 59

A-1 Sample number, Farm Region and Farm plot shown for the 28 soil samples analyzed in the EAA...... 96

A-2 This table shows the 5 groups we observed in our statistics using a paired t- test and non-parametric testing...... 96

LIST OF FIGURES

Figure page

1-1 Subsidence post located at the Everglades Research and Education Center in Belle Glade, Florida...... 27

1-2 Map of the Everglade Agricultural Area (EAA). The image shows the agriculture grown on the muck soil in the EAA...... 28

1-3 Map of the 21 Farm Plot Soil Sampling Locations in the EAA...... 28

1-4 Flooded Rice field located in the EAA. Soil samples were taken before rice fields were flooded...... 29

1-5 Rice field after drainage and ready for harvest in the EAA. Post-rice soil samples were taken during this time...... 30

1-6 Flooded fallow field located in the EAA. Farmers flood the field fallow as a crop rotation in the summer...... 30

2-1 Soil Properties pre and post flooded rice production (pH, Total C, Total Carbon, Total N [ Total Nitrogen, and OM, organic matter])...... 60

2-2 Macronutrients and beneficial element pre and post flooded rice (Ca, K, Mg, Pw, Pm3, and Si)...... 61

2-3 Micronutrients pre and post flooded rice (Cu, Fe, Mn, and Zn)...... 62

2-4 Soil Properties pre and post flooded rice in shallow soils: (pH, Total C, Total N and OM)...... 63

2-5 Soil Properties pre and post flooded rice in deep soils: (pH, Total C, Total N)...... 64

2-6 Macronutrients and beneficial element (Si) pre and post flooded rice in shallow soils: (Ca, K, Mg, Pw, Pm3, and Si)...... 65

2-7 Macronutrients and beneficial element (Si) pre and post flooded rice in deep soils (Ca, K, Mg, Pw, Pm3, and Si)...... 66

2-8 Micronutrients pre and post flooded rice in shallow soils (Cu, Fe, Mn, and Zn)...... 67

2-9 Micronutrients pre and post flooded rice in deep soils (Cu, Fe, Mn, and Zn). .... 68

3-1 Upper panel: Cross-section through a drained rice microcosm. The rice was cultivated for 90 days under flooded conditions in the greenhouse...... 72

3-2 Oxidation–reduction couples of various electron acceptors arranged from the strongest oxidants ...... 73

3-3 Relative abundance of Clostridia...... 83

3-4: Relative abundance of methane-oxidizing Rokubacteria...... 88

3-5: Principal coordinate analysis of EAA soil microbial communities based on weighted Unifrac distance matrix of 16S rRNA gene sequences...... 92

3-6 Pre- and post-treatment comparison of genus-level microbial taxa in conventionally managed muck soils in the EAA...... 93

3-7 Pre- and post-treatment comparison of genus-level microbial taxa in conventionally managed sandy muck soils in the EAA...... 94

3-8 Pre- and post-treatment comparison of genus-level microbial taxa in organically managed muck soils in the EAA...... 95

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

NUTRIENT AVAILABILITY AND CHANGES IN MICROBIAL COMMUNITIES IN FLOODED RICE PRODUCTION IN SOUTH FLORIDA By

Rachelle Berger

December 2020

Chair: Samira H. Daroub Co-Chair: Willm Martens-Habbena Major: Soil and Water Sciences

Production of flooded rice in Histosols as a crop rotation has the potential to shift soil microbial community dynamics, lower redox potential, and change the chemistry of nutrients. Histosols in the Everglades Agricultural Area (EAA) have undergone substantial subsidence resulting in shallower soils. Growing flooded rice as a crop rotation can induce positive effects on water management, environmental quality, and benefit the succeeding crops with increased nutrient availability (i.e., sugarcane and vegetables). Cycling water through flooded rice fields gives farmers an opportunity to reduce discharging water off their farms, and in turn reduces phosphorus loading.

Microbial processes predominantly control the oxidation of soil carbon and the changes in availability of soil macronutrients and micronutrients. This study was conducted to determine effects of flooded rice on soil properties, nutrient availability, and changes in microbial community relative abundance. Organic soils were sampled from 28 farm plots with different O horizon depth twice during summer season. The effects of flooded rice on soil properties and nutrients were investigated from 21 farm plots (out of the 28 plots) before and after flooded rice cultivation in the EAA. Statistical analyses were

conducted to elucidate the impact of flooded rice in shallow and deep soils on soil properties and nutrient availability. Flooded rice affected total carbon content, water extractable P, silicon (Si), Potassium (K), and Iron (Fe). Concentrations of K and Si in the soil significantly decreased after flooded rice production due to the uptake of these nutrients by the rice crop. On average K decreased from 64.7 kg/ha to 40.2 kg/ha, and

Si decreased on average from 99.6 kg/ha to 57.6 kg/ha. We observed an increase in soil Fe post flooding possibly due to lower redox values. The increase in Fe averaged from 421 mg/kg to 651 mg/kg. Some differences in nutrient availability were also found between shallow and deep soils.

We performed high-throughput sequencing of 16s rRNA gene amplicon to observe bacterial and archaeal communities in all 28 plots. Principal Coordinate

Analysis showed microbial communities at the genus level changed between pre- and post-flooding in all soils and smallest changes were observed in fallow soils. We focused on Thaumarchaeota, Clostridia, Rokubacteria, Sulfate Reducing Bacteria, and

Methanogens. Thaumarchaeota showed highest abundance in these soils and decreased significantly in flooded soils. Nutrient and microbial community data suggest that most soils sampled were under anoxic conditions. However, sulfate reduction and methanogenesis were not significant as data imply that the redox potential did not drop below 200mV. These results suggest that production of flooded rice may not enhance methane production and might have the potential to reduce ammonia oxidation in the

EAA organic soils.

CHAPTER 1 INTRODUCTION

The Everglades Agricultural Area (EAA)

The Everglades Agricultural Area (EAA) is a 296,000-ha region comprised of organic soils south of Lake Okeechobee. Much of the nation’s sugarcane and winter crops are farmed in this region and provide over a billion dollars to Florida’s economy.

Flooded rice is grown as a crop rotation in the summer months. Organic soils in the

EAA are classified as the Histosol soil order based on the high organic matter composition and absence of frigid conditions in the region (Rice et al., 2005). A frigid soil temperature regime indicates a mean annual soil temperature > 8C which is typical where most Histosols form. South Florida weather has subtropical climate and has hyperthermic soil temperature regimes.

These organic soils formed over a 4400-year period from decomposed hydrophytic vegetation that accumulated under anaerobic inundated conditions

(McDowell et al., 1969). The EAA experienced a vast agricultural boom in the 1920s, and since most of the EAA has been farmed for as long as 100 years, the soils have undergone considerable changes (Wright and Hanlon, 2009). These changes include a loss in soil depth (subsidence) due to organic matter oxidation, a change in soil series due to loss of depth in the O horizon and changes in soil properties. The climate in this region is warm and most of the rainy season occurs in the summer months (around

June- October). Due to high crop production in the area and a warm tropical climate; the

EAA is susceptible to erosion and surface runoff from agriculture.

Histosols in the EAA

Over 4,400 years were required for the organic soils of the EAA to form from decaying remains of sawgrass (Cladium jamaciense Crantz) and other marsh plants accumulated under flooded conditions (McCray et al., 2016). Histosols can be classified into four different suborders, including Folists, Fibrists, Hemists, and Saprists. The

Histosol soil order includes most of the world’s organic soils, with the formative element

“Hist” (from Greek histos, tissue) indicating the presence of organically derived soil material (Rice et al., 2005). The organic soils of the EAA lack strong evidence of horizon development and are thus all categorized as Haplosaprists. Histosols are also referred to as “muck” in the EAA which indicates its organic matter is highly decomposed.

Histosols in the EAA are classified into seven soil series with each series primarily describing a different O horizon depth. They also vary with inorganic mineral content and underlying materials below the O horizon. The seven-soil series that describe Histosols in the EAA are: Okeechobee, Okeelanta, Pahokee, Lauderhill,

Dania, Terra Ceia, and Torry (Rice et al., 2005) (Table 1-1). According to Rice et. al

2005, the seven-soil series are classified according to the depth of the O horizon above the bedrock as follows; Torry >51 inches (130cm), Terra Ceia >51inches (130cm),

Okeechobee >51inches (130cm), Pahokee 36-51 inches (91-130cm), Lauderhill 20-36 inches (51-91cm), Dania <20 inches ( <51cm), and Okeelanta 16-50 inches (41-

127cm). The underlying parent material (bedrock) for the seven soils series in the EAA is limestone except for Okeelanta which is sand. Additionally, all seven-soil series have a mineral content of less than 35% except for Torry which has more than 35% mineral content (Table 2-1). The Torry series is distinctively different from the other series and

is important to produce rice. With higher mineral content and a deeper soil profile, rice can grow more optimally at these conditions.

Over time, soil mineralization and subsequent soil subsidence (decline in soil thickness) have resulted in a declining percentage of soils classified as Terra Ceia, and

Pahokee soils, as they transition into the shallower Lauderhill and Dania soils (Table 1-

1). Additionally, if soils continue to subside over time, it is likely that the highly decomposed material will eventually transition into new soil series (Rice et al., 2005).

Underneath the O horizon of most of the organic soils in the EAA is limestone bedrock (calcium carbonate). Most Everglades soils have a moderate pH, especially when compared to other peat soils around the world that normally have low pH (Snyder,

1994). The pH of pear soils varies although, usually have a pH between 3 and 5.5. Due to subsidence, tillage and mixing with calcium carbonate limestone bedrock, the pH of most soils in the EAA range between 6- 8. Soil pH has increased as tillage operations incorporate more calcium carbonate into the topsoil (Wright and Snyder, 2009).

Subsidence of the Organic Soils in the EAA

The steady state of soil organic matter is determined by biological, chemical, and physical soil properties that control microbial activity (Tate, 1987; Stevenson, 1986;

Cole et al., 1987). Subsidence was observed as soon as the Everglades were drained in the early 1900s to remove water from soil to better support crop production (Wright and Snyder, 2009). Histosols in the EAA are subject to high rates of subsidence mainly because of microbial oxidation of organic carbon due to warm temperatures year-round.

Historically, EAA Histosols’ subsidence rate was at 2.5 cm per year (1 in) since it has been documented in the 1920s. In more recent studies scientists have re-measured

surface elevation along base subsidence lines in 1997 and concluded that the average subsidence rate has decreased to 1.45 cm (0.57 inch) and speculated that maintenance of higher water table in recent years was one of the major reasons for the reduction of the subsidence rate (Shih et al., 1998).When soils are flooded, the oxidation of organic matter slows down due to lack of oxygen, and this can reduce subsidence rates.

Anaerobic decomposition of organic matter is more complex and less energetically favorable than aerobic decomposition (Inglett et al., 2005). A major factor influencing the decline in the subsidence rates over time has been improved water management due to mandatory Best Management Practices (BMPs) implemented throughout the basin, as well as soils becoming shallower. Implementing BMPs is essential to farmers in the EAA region to protect the Everglades while ensuring agricultural sustainability.

Many farmers and agricultural producers in the area apply rice crop rotation as a summer cover crop to replenish nutrients in the soil and achieve higher crop yields.

Since rice is grown under flooded conditions, it has the potential to reduce subsidence rates (Figure 1-1). Flooded fallow or flooded rice both have the potential to slow soil subsidence by slowing the oxidation of soil carbon, and loss to the atmosphere as CO2

(Morton and Snyder, 1976).

Best management practices (BMPs)

Prior to construction of extensive public/private system of canals through the northern Everglades, the EAA was flooded most of the year during the rainy season (6-

8 months) (Snyder and Davidson, 1994). The growing season is between March-

October, around 7-8 months out of the year depending on weather. Successful farming in the EAA requires artificially draining the organic soils by using high volume discharge

pumps and extensive canal systems. Due to the drainage of the EAA for agriculture, environmental concerns about P runoff into the Everglades ecosystem and land subsidence have arisen (Daroub et al., 2009; Glaz et al.,2004; Rice et al., 2002).

Phosphorus Runoff and Water Quality Issues

Concerns related to water quality out of the EAA mandated the implementation of BMPs to improve water quality from the EAA per the Everglades Forever Act of 1994

(Daroub et al., 2011). Best management practices in the EAA are mainly designed to improve drainage water quality related to P from the basin while maintaining economical farm production systems. Best Management Practices related to water management include rainfall retention, rainfall detention, maintaining water tables as high as possible without jeopardizing crop yields, and growing crops that are tolerant to high water table levels (Anderson and Flaig, 1995, Daroub et al., 2011). According to Qian S. et al.,

1997, wetlands with elevated levels of P runoff have been known to leading cause of agricultural ecosystem changes like eutrophication and harmful algal blooms. Other

BMPs to reduce P discharge from the EAA often include strategies to reduce quantities and rates of pumping water from agricultural fields (Rice et al., 2002). All these BMPs are believed to have had a positive impact in reducing oxidation rates from the organic soils of the EAA and improving soil health. For example, higher water tables have been observed to cause decreased rates of microbial activity and soil organic matter oxidation (Morris et al., 2004).

Flooded rice production is used as a BMP (crop rotation) to enhance soil fertility and potentially reduce subsidence rates in the EAA. Best management practices like crop rotation are vital to increase soil health and beneficial nutrients and elements in the

soil. Flooded rice has shown benefits to increase the fertility of the soil and promote a higher quality of soil health. Rice in the EAA is produced under flooded conditions, which is likely to slow organic matter oxidation and may reduce rates of subsidence.

Flooded Rice Cultivation Benefits in the EAA

Rice (Oryza sativa L) is one of the world’s major food crops, covering about 11% of global farmland. Rice is capable of growing well in waterlogged and submerged soils because of its well-developed aerenchyma system that facilitates aeration of the roots and the rhizosphere, thus alleviating most of the stresses experienced by upland plants under low oxygen (Setter et al., 1997; Jackson and Ram, 2003). Rice plants under flooded conditions have the capability to respire and survive under anaerobic conditions which would be toxic to other macrophytes (Inglett et al., 2005).

Rice used as a crop rotation has the potential to increase succeeding crop yields, soil nutrient availability, and possibly change the microbial community within the soil due to flooding the soils. The total value of rice in south Florida row-crop rotation far exceeds its monetary return in the world marketplace (Schueneman et al., 2001). Since rice is grown in the rainy season and is always flooded, soil and water conservation and increased habitat for wildlife result (Schueneman et al., 2001). Consequently, flooded rice is expected to produce significantly less methane than flooded fallow fields due to the increase in soil redox potential. Conserving the soil and improving soil structure are challenging to put monetary value on but reason enough to utilize flooded rice as a crop rotation after sugarcane in the EAA (Schueneman et al., 2000).

Soil nitrogen and phosphorus are both sensitive to oxygen content in soil. Under flooded conditions, nitrogen may be lost as a gas, either as N2 or N2O. Prolonged

flooding under rice production may increase minerals such as manganese (Mn), iron

(Fe), and copper (Cu) to the soil. These micronutrients may be responsible for post-rice soil health benefits. Rice production may influence macronutrients nitrogen (N), phosphorus (P), potassium (K), and silicon (Si). Alterations in these soil nutrients before and after the rice production will lead to insights into the mechanisms of increased soil fertility.

Soil Chemistry of Flooded Soils

Soil biological, chemical, and physical changes occur during the time a soil is flooded. Anaerobic soils occur when oxygen consumption of soil biota exceeds the diffusion in the soil profile (Inglett et al., 2005). Diffusion is a natural process that occurs when gasses are distributed throughout the soil profile and occurs as a movement of molecules from an area of higher concentration to an area of lower concentration

(occurs in liquids and gases as their particles move from place to place). When a soil is saturated, atmospheric oxygen is reduced and facultative and anaerobic microorganisms use oxidized compounds as electron acceptors for respiration which results in converting them to reduced forms (i.e., Iron) (Pezeshki & DeLaune, 2012).

The reduction and processes associated influence plant survival and growth under flooded conditions (Pezeshki & DeLaune, 2012).

A chain of reactions occurs when the soil is flooded which can lead to reduced soil redox potential. The reactions include biological, chemical, and physical processes that have significant implications for wetland plants (e.g., flooded rice). Physical properties can include a restriction of atmospheric gas diffusion in the soil which leads to depleted soil oxygen and accumulation of carbon dioxide (Alvarez and Snyder, 1984).

The reduced oxygen leads to a reduction in soil oxidation reduction potential (Eh) which is followed by a chain of soil chemical changes (Alvarez and Snyder, 1984). The processes followed by soil flooding are denitrification, reduction of iron, manganese, and sulfate as well as a changing soil pH (Alvarez and Snyder, 1984, Ponnamperuma,

1972).

Oxidation-Reduction or redox conditions and pH have a pronounced effect on solubility of micronutrients, specifically Fe and Mn, and hence their availability to rice.

The most important chemical change that takes a place when a soil is submerged is the reduction of Fe and the accompanying increase in its solubility (Ponnamperuma, 1972).

Iron (III) oxide compounds are reduced to Fe2+ into the solution. However, Fe deficiencies may occur in EAA Histosols until the rice is flooded and redox potential is reduced. Drill-seeded rice in certain Histosols in the EAA germinated well, but after a few weeks it became chlorotic and grew poorly due to Fe deficiency. After flooding, chlorotic seedlings generally assumed normal color (Snyder and Jones, 1988). In some areas in the EAA, Fe is drilled with the seed to prevent this seedling chlorosis condition

(Snyder and Jones, 1988).

The main transformations of manganese in submerged soils are the reduction of

2+ Mn (IV) oxides to Mn (II), an increase in the concentration of water-soluble Mn . Mn can precipitate as manganous carbonate and undergo reoxidation in the oxygenated interfaces in the soil (Ponnamperuma, 1972). Manganese (Mn) deficiency was determined to limit the growth of rice seedlings drill planted in several drained Histosols in the EAA having pH values ranging from 6.9 to 7.7. Similar to Fe, application of approximately 15 kg Mn ha-1 as MnSO4 with the seed prevents the deficiency and

provided near-maximum grain yields (Snyder et al., 1990). Expansion and contraction of clay particles as they become moist and then dry can shift and crack the soil mass and create aggregates or break them apart. Calcium, magnesium, iron, and aluminum stabilize aggregates via the formation of organic matter – clay bridges. In contrast, aggregate stability decreases with increasing amounts of exchangeable sodium.

Dispersion is promoted when too many sodium ions accumulate between soil particles.

Water Soluble P also increases upon flooding of soils. The increase in concentration of water-soluble P when acid soils are submerged is from (a) hydrolysis of Fe(III) and Al phosphates, (b) release of P held by anion exchange on clay and hydrous oxides of Fe(III) and Al , and (c) reduction of Fe(III) to Fe(II) with liberation of sorbed and chemically bonded P (Ponnamperuma, 1972).

Microbial Communities in Flooded Soils

Soil microorganisms drive plant growth, biogeochemical processes, organic matter, and nutrient availability (Visser, S., & Parkinson, D.,1992). Having knowledge of soil microbial community diversity before and after the cultivation of rice can provide a deeper understanding of the shift in microbial activity in flooded communities and benefit farmers in the region. Scientific understanding of microbial biogeography is particularly weak for soil bacteria, even though the diversity and composition of soil bacteria communities is thought to have a direct influence on a wide range of ecosystem processes (Fierer N. ,2006).

There are 40 million bacterial cells found in a gram of soil alone (Alcamo I. E.,

2003). It has often been stated that there are more microbes within one gram of soil then there are human beings on this planet (Alcamo I. E., 2003). Variations to beneficial

microbial communities or the effects on soil borne plant pathogens, due to soil flooding and rice cultivation, are still unknown on EAA Histosols.

Paddy soils develop in a unique management system that controls redox-driven processes affecting mineral transformation and microbially mediated turnover of organic matter (Kogel-Knabner et al., 2010)

The aerobic metabolism of fungi with its high energy release enables them to carry on vigorous decomposition of organic matter (Ponnamperuma et al., 1972). When soils are flooded, these fungi will be replaced by anaerobic bacteria and the bacteria will operate at a lower energy level and decompose organic matter much slower. The reduction of iron is a consequence of the anaerobic metabolism that occurs under flooded conditions and appears to be chiefly a chemical reduction by bacterial metabolites (Ponnamperuma et al., 1972).

Organic soils subside when they are drained continuously, primarily because of aerobic microbial activity (Morris et al., 2004). When beneficial microorganisms are present in soil; there may be an increase in plant available nutrients, higher soil quality, and a decrease risk of plant disease. Insights into microbial community composition and the factors that determine them may improve our understanding of biogeochemical processes, food web dynamics, biodegradation processes, and overall soil quality

(Drenovsky, 2014). Flooding reduces soil oxygen levels and selects for facultative and obligate anaerobic microorganisms, whereas soil desiccation lowers microbial activity

(Drenovsky, 2014). Therefore, by determining the abundance of microbial communities and the effects these communities have on long-term soil health following the

production of rice, we can determine if utilizing rice in crop rotation is beneficial for farmers in the EAA region.

Project Hypothesis

We hypothesize that the microbial community will change in species composition after the cultivation of flooded rice due to flooded conditions. We also suspect that nutrient availability will be impacted after soils have been flooded for the summer. The purpose of this research project is to help determine if the benefits of planting flooded rice outweighs flooding the fallow fields during the summer.

During flooding and rice cultivation, a reduction of carbon dioxide gas production was observed in a previous research conducted in the EAA; we would like to determine if this due to limited oxygen diffusion, or because we reduced the number and type of microorganisms that oxidize organic carbon in the presence of oxygen (heterotrophic bacteria processes).

To test this hypothesis, we collected, analyzed, and observed the correlations nutrient availability and the relative abundance of microbial communities within the

Histosol soils of the EAA following the cultivation of flooded rice. We took field samples from 21 different field plots in the spring before rotation into flooded rice and after the rice growing season, to evaluate soil microbial shifts.

Project Statement and Objectives

The purpose of this project is to investigate the impact of flooding (pre- and post- rice cultivation) on 21 farm plots within 7 farm regions located in the Everglades

Agricultural Area (See Fig.1-4). Using nutrient analyses, Illumina MiSeq microbial sequencing and Qiime II bioinformatic taxonomic profiling, we will be able to determine

the shifts in nutrient availability, microbial community relative abundance, and the alpha and beta diversity in the microbes within the soil samples.

The objectives of this research were to:

1. Design and set up experiment, organize and chose soil sampling locations with farmers in the region, test nutrients and observe microbial communities (archaea, bacteria, and fungi) before and after flooded rice cultivation.

2. Determine the differences in macronutrients, micronutrients, and soil properties between shallow and deep soils and pre- and post-flooded rice cultivation

3. Evaluate soil microbial shifts after the cultivation of flooded rice in 21 field plots located in the EAA and determine if flooded rice cultivation change microbial communities and nutrients available to the soil.

4. Determine if microbial groups such as Methanogens, Clostridia, Sulfate-reducing bacteria and Methylomirabilis-related Rokubacteria (strict anaerobic microbes) changed before and after the flooded rice cultivation.

Methods and Materials

Experimental Design Overview. This experiment was conducted in the EAA at the University of Florida (UF) Everglades Research and Education Center (EREC) located in Belle Glade, Florida and at the UF Fort Lauderdale Research and Education

(FLREC) located in Fort Lauderdale, Florida. There were three main components of this experiment: soil sampling, nutrient analyses (chapter 2), and microbial analyses

(chapter 3).

We sampled muck soils from 28 farm plots with various O horizon depths located in several farm regions (Shelton Area, East Area, West Organic Area, Southern Ranch

Lease, Roth Farms, King Ranch Farms, and the Manley Area) in the EAA between

March 2018 and September 2018 (See Fig.1-4). Soils were taken from the same

location twice during the summer season and 21 plots were pre- and post- flooded rice production. Soil samples were analyzed for soil properties and nutrient content: total N, available P, total C, K, Mn, Cu, Fe, Zn, Si, Ca, and Mg), pH and organic matter content before planting and flooding and after harvesting. Nutrient analyses will be discussed in further detail in Chapter 2. The experimental design, nutrient analyses, organic matter content, and soil sampling were all conducted at EREC.

The DNA extractions, plating for sequencing (16sr RNA), and Total Nitrogen and

Total Carbon percentages were performed at FLREC. Soils sampled were extracted for microbial soil DNA. Soil DNA samples were plated on skirted PCR plate. The 16S rRNA sequencing was performed at Argonne National Laboratory located in Chicago, Illinois.

Once the 16S rRNA Illumina MiSeq sequencing was received, taxonomic observations were finished using QIIME II bioinformatics programming at FLREC.

Table 1-1: Histosol soil series found in the Everglades Agricultural Area. Soil series Soil name Mineral Thickness Underlying Percentage of content of organic material EAA organic material† soils 1978‡ 1988§ % cm % % (inches) Torry euic, >35 >130 limestone 7 7.1 hyperthermic (>51) Typic Haplosaprist Terra Ceia euic, <35 >130 limestone 37.9 9.5 hyperthermic (>51) Typic Haplosaprist Okeechobee euic, <35 >130 limestone 2.6 2.6 hyperthermic (>51) Hemic Haplosaprist Pahokee euic, <35 91-130 limestone 43.9 27.4 hyperthermic (36-51) Lithic Haplosaprist Lauderhill euic, <35 51-91 limestone 4.7 39.6 hyperthermic (20-36) Lithic Haplosaprist Dania euic, <35 <51 (<20) limestone 0.2 10.2 hyperthermic, shallow Lithic Haplosaprist Okeelanta sandy or sandy- <35 41-127 sand 3.6 3.6 skeletal, silicious, (16-50) euic, hyperthermic Terric Haplosaprist Adapted from: Rice et. al., 2005.

Figure 1-1: Subsidence post located at the Everglades Research and Education Center in Belle Glade, Florida. This post was first put in the ground and driven to bedrock in 1924 and the soil has subsided 1 inch per year to approximately ½ inch per year. The decrease in subsidence occurred due to Best Management Practices (BMPs) by the locals in the area. (Picture taken in September 2019 by Rachelle Berger).

Figure 1-2: Map of the Everglade Agricultural Area (EAA). The image shows the agriculture grown on the muck soil in the EAA. (http://miami.cbslocal.com/the- everglades-where-politics-money-race-collide/).

Figure 1-3: Map of the 21 Farm Plot Soil Sampling Locations in the EAA made with Google Earth. (Image courtesy of Johnny Mosley). 28

Figure 1-4. Flooded Rice field located in the EAA. Soil samples were taken before rice fields were flooded. (Picture taken by Rachelle Berger in summer 2018.)

Figure 1-5. Rice field after drainage and ready for harvest in the EAA. Post-rice soil samples were taken during this time. (Picture taken by Rachelle Berger in summer 2018).

Figure 1-6. Flooded fallow field located in the EAA. Farmers flood the field fallow as a crop rotation in the summer. (Picture taken by Rachelle Berger in summer 2018). 30

CHAPTER 2 SOIL NUTRIENT AVAILABILITY FOLLOWING FLOODED RICE PRODUCTION IN SOUTH FLORIDA

Introduction of Chapter 2

The Everglades Agricultural Area (EAA) consists of approximately 296,000 ha of fertile organic soils in south Florida. The EAA, with 146,000 ha under production, provides 40% of the nation’s winter vegetables and 23% of the nation’s sugar (Glaz et al., 2008) and has increased acreage of flooded rice production. Rice has increased from 4000 to 25000 acres between 2008 and 2018 (Bhadha et al., 2019). Following drainage of the former sawgrass marsh in the early 1900s for agricultural production, the EAA has experienced substantial subsidence due to organic matter oxidation of the organic soils (Order: Histosol). Presently, after three years of sugarcane growth, farmers in the EAA will rotate flooded rice, flooded fallow, or dry fallow during the summer months. Flooded fallow and rice fields both have the potential to reduce soil subsidence by slowing the oxidation of soil organic matter and release of carbon dioxide

(CO2) to the atmosphere. In addition, there are benefits of the flooded rice production to the next crop in the rotation due to increased availability of certain nutrients after flooding the organic soils.

Subsidence of Histosols

Histosols are subject to high rates of microbial oxidation of organic carbon when drained, especially in south Florida with the subtropical climate and high temperatures almost year-round. Most organic soils in the EAA contain more than 80% organic matter content and are susceptible to soil loss via oxidation, a process commonly known as

‘soil subsidence’ in the region (Rice et a.l, 2005). Subsidence was observed as soon as

the Everglades were drained in the early 1900s to remove water from soil to better support crop production (Wright and Snyder, 2009).

Some EAA fields had as much as 3 m (~9 ft) of soil above the limestone bedrock when they were first drained and used for agriculture (Rice et al., 2005, Shih et al,

1998). Historically it is estimated that soil subsidence caused loss of depth in EAA

Histosols at the rate of about 2.5 cm yr-1 before 1978 (Shih et al., 1978). From 1978 until the most recent survey in 1997, the rate of soil loss declined to 1.4 cm yr-1 (Shih et al., 1998, Morris et al., 2004). Depth of soil to bedrock varies, but a small number of sugarcane fields now have less than 40 cm of soil (Shih et al., 1998).

Agricultural producers in the EAA often flood fallow land to control insects and nematodes (Cherry, 1984, Hall and Cherry, 1993). It has been well documented that the subsidence rate is closely aligned with water table depth, as organic matter decomposition is impaired by flooded conditions (Stephens and Johnson, 1951; Snyder et al., 1978). It is estimated for every centimeter of rainfall, the water in the soil profile of

EAA Histosols rises about 10 cm (Glaz et al., 2002). The average rainfall in south

Florida is 54 inches (137 cm) yearly with 66% of the rain falling between June and

December during the rainy season (Ali et al., 2000). Discharging water from farmers’ fields occurs mostly during these hot summer months. Crop production practices such as crop rotation using flooded rice in the summer is considered a BMP leading to less water discharge and less P loads (Daroub et al., 2011).

Flooded Rice Production

Rice is unique in being capable of growing well in waterlogged and submerged soils because of its well-developed aerenchyma system that facilitates aeration of the

roots and the rhizosphere, thus alleviating most of the stresses experienced under low oxygen (Fageria, 2014). Flooded rice production has the potential to slow soil subsidence by slowing the oxidation of soil carbon, and loss to the atmosphere as CO2

(Porter et al., 1992). Rice grown in the EAA has increased from approximately 4000 acres in rotation with sugarcane in 2008 to about 25000 acres in 2018 (Bhadha et al,

2019). From this rotation, both economic and agronomic benefits have been observed

(Alvarez and Snyder, 1984; Snyder et al., 1986a). Sugarcane benefits from flooded rice production with increased availability of nutrients including phosphorus (P) and iron (Fe)

(McCray et al., 2012). Other effects of flooded rice soils are increase in pH of acid soils and a decrease in pH of alkaline soils, decrease in redox potential and increase in electrical conductivity as well as increase in the concentration of phosphate, calcium, magnesium, molybdenum, silica and decrease in concentration and availability of Zn,

Cu and S (Ponnamperuma, 1984).

Rice in the EAA is often grown commercially in rotation with sugarcane and vegetables (Tootoonchi et al., 2018) and from 2008 to 2015, rice production has increased more than 80% (Bhadha et al., 2016). Various studies in the EAA have shown that sugarcane following the production of flooded rice produces more sugarcane per acre than the same variety grown after another crop or after fallow

(Schueneman et al., 2001). Additionally, Alvarez and Snyder (1984) compared sugarcane yields from 82 fields where rice had been the preceding crop versus 36 fields where sugarcane followed a fallow period. An average yield increase of 17.7 tons of cane per hectare (ha) resulted when the sugarcane was preceded by rice. A grower could add years to the productive life of his EAA farm with frequent rice crops in rotation

with sugarcane or vegetables (Schueneman et al., 2001). Moreover, flooded rice effectively stops subsidence of muck soil during the hot summer months, the time of the year when the rate of subsidence is the greatest (Snyder et al., 1978).

Nutrients and Rice Production in Histosols

Farmers in the EAA rely on BMPs for efficient practices to manage nutrients on their farms for environmentally friendly and economical cropping systems (Daroub et al,

2011). In addition to management factors, climate change, shallow soil depths and crop rotations all impact success of farming in the EAA. There are many factors that affect soil nutrient availability in EAA organic soils. Some of these factors are soil depth, pH, reduction- oxidation potential (redox) and organic matter content.

Histosols in the EAA contain 22 g N kg-1 dry soil and are characterized by high aerobic N mineralization rates. Approximately 686 kg N/ha were mineralized for each centimeter of Pahokee muck lost due to microbial oxidation (Terry,1980). Nitrogen fertilization of rice grown on Everglades Histosols is not recommended (Snyder,1993) due to the high mineralization rate and availability of N to the rice crop. This mineralization does not stop with flooded conditions, but nitrification is reduced. Hanlon et. al (1997) found that total soluble N from the surface 15-cm of drained soil columns

-1 -1 ranged from 217 to 509 kg-ha yr , with 50 to 67% released as nitrate-nitrogen (NO3-N).

In contrast, total soluble N released from flooded soils ranged from 168 to 345 kg-ha-1yr-

1 , with less than 3% released as NO3-N. In addition, compared to mineral soils, a drained organic soil can produce a larger amount of N2O (Terry et al.,1981) due to denitrification reactions. In a previous experiment using lysimeters conducted at the

Everglades Research and Education Center (EREC), increased soil fertility in Histosols

were observed following rice production rather than following flooded fallow in the EAA

(Cooper et. al. data not yet published). They observed significant loss of soil nitrogen during rice production (partially from plant uptake), without high production of N2O, directing towards loss of as N2 gas.

Similarly, P is released from organic matter oxidation and no P fertilization is done on flooded rice in the EAA (Jones et al.,1994). Diaz et al. (1993) conducted column incubation experiments for one year and found P mineralization rate ranged from 5.60 to 72.0 kg P ha-1 yr-1 for drained soils and from 36.0 to 87.9 kg P ha-1 yr-1 for flooded soils from the EAA. Generally, P release rates declined with time, but results indicated that higher P release occurs under periodic flooding and draining. Phosphorus availability is highly impacted by pH with maximum availability found between pH 6-7. In mineral high pH soils, P reacts with Ca (and Mg) at high pH soils forming Ca phosphate compounds that become less soluble with time. Low pH mineral soils also have P availability due to precipitation of P with Fe and Al as well as sorption reactions of P on surfaces of Al oxides and Fe oxides (Weil and Brady, 2017). These reactions may not be prevalent in highly organic soils like the EAA due to lack of high content of inorganic materials, especially the oxides. Castillo and Wright (2008) found that organic P comprised 50% of total P using only four sites in the EAA. The Ca-bound P fraction represented the greatest proportion of total P (41%) in the sugarcane site. However, the data is limited regarding the exact nature of P in EAA soils. In addition, when soils are flooded, increased P in the water column is due to the reduction of Fe phosphate compounds and release of both Fe and P into the solution. However, this oxygen

sensitive process in flooded rice cultivation has not been studied in the EAA and may play a role in phosphorus cycling in the region.

Research in the EAA has indicated that rice grown on organic soils in the EAA does not respond to K fertilization. This is not a common method for rice grown in other regions due to the organic soils’ native to the EAA region. Therefore, growers do not fertilize flooded rice with K (Jones et al, 1994). However, the organic soils in the EAA do not have much mineral K compounds due to lack of inorganic matter and application of

K fertilizers are needed for other crops. Potassium fertilization is done frequently for sugarcane crops which possibly carries over to the rice crop in rotation.

Silica (Si) may be considered as one of the most important nutrients for rice production increasing grain yield, improving disease resistance, and tolerance to abiotic stresses like salt stress and drought stress (Datnoff et al., 2001). Silica is an important element to organic soils as they are devoid of Si due to low inorganic matter content.

The response to rice to Si fertilization is affected by many factors, among which concentration of plant-available Si, soil pH, and N application levels are most vital (Li et al., 1999). Soil pH can greatly affect rice responses to Si fertilization by its impact on the solubility of Si in the fertilizer (Datnoff et al., 2001). Previous research conducted on

Histosols has shown Si to be a functional element for either flooded or upland rice

(Datnoff et al., 2001). Benefits of adequate Si concentrations include an increase in rice yield, prevention of Fe and Mn toxicity, and a better uptake of P. On mineral soils with low pH, P which is complexed with Al or Fe may become plant-available with addition of

Si, thereby increasing yields. Although, EAA Histosols are not limiting in P for rice, so Si does not increase P availability (Deren et al., 1994).

Availability of most micronutrients in soil is affected by soil pH and reactions with other nutrients (Wright et al., 2009). In addition, redox potential impacts the availability of Fe and Mn due to reduction reactions in the soils. Although micronutrients differ somewhat in the response to pH, all show decreased availability with increasing pH at values commonly observed in most muck soils (Wright et al., 2009). Micronutrients’ availability of Fe, Zn, Cu and Mn generally increases in soils and are readily available to crops with a decrease in soil pH (Weil and Brady, 2017). In addition, Mn and Fe availability increases with in a decrease in redox potential in the soil due to reduction of

Fe oxides and Mn oxides and released of Fe2+ and Mn2+ into the soil solution (Weil and

Brady, 2017, Inglett, 2015). Research on micronutrients started early in the organic soils in the EAA due to failure of early vegetable crops because of micronutrient deficiencies

(Schueneman and Sanchez, 1994). Copper, Mn, Zn and B were identified as important micronutrients for certain vegetable crops like sugarcane or sweetcorn. Early work showed every crop in the EAA responded to Cu fertilization (Allison et al., 1950).

Copper is strongly complexed by organic matter in soils rendering it unavailable (Weil and Brady, 2017). Generally, after the initial application of Cu to virgin mucks, no further response was observed for most crops (Schueneman and Sanchez, 1994). Zinc is converted to unavailable forms (mainly oxides) at high pH and need to be applied routinely to susceptible crops. Manganese is another micronutrient found to be deficient in virgin organic soils (Allison, 1950) and is recommended to be applied annually for vegetable production in the EAA. Rice in the EAA shows seedling chlorosis condition due to Fe deficiency and often Fe is drilled with the seed (Snyder and Jones, 1988).

In summary, flooded rice production during the summer months in the EAA has the potential to increase succeeding crop yields, soil nutrient availability (P, Fe, Mn) and shift the microbial community within the soil. In this chapter, we discuss the impact of flooded rice production on soil properties and nutrients’ availability.

Hypothesis and Objectives

Chapter 2 focuses on how soil properties, nutrient availability of macronutrients

(P, K, Ca, Mg), micronutrients (Cu, Fe, Mn, and Zn), and beneficial element (Si) changed after the cultivation of flooded rice sampled from 21 farm locations with various soil depths located throughout the EAA. We hypothesize that soil properties and nutrients will vary with soils of different depth of the O horizon. We also hypothesize that

Mehlich-3 extractable P, water extractable P, Mn and Fe will increase after flooded rice cultivation.

This chapter has three objectives:

Objective 1: Determine the differences in soil properties and nutrients in shallow and deep soils in selected series of EAA soils. Objective 2: Determine impact of flooded rice production on nutrients availability and soil properties (pre- and post-flooded rice). Objective 3: Determine impact of flooded rice production on nutrients and soil properties in shallow and deep soils.

Methods and Materials

Soil Sampling and Analysis

We sampled organic soils (order: Histosols) from 21 farm plots located in various farm regions in the EAA between March 2018 and September 2018. Locations, depth, and soil series are described in Table 2-2. The soil series mainly varies in depth of the

O horizon, percent of organic matter, and underlying parent materials. According to

GPS coordinates taken at each soil sampling location we observed Lauderhill, Pahokee,

Terra Ceia soil series using the web soil survey (WSS) in the 21-farm plot study of flooded rice used a crop rotation. However, from soil depth measurements, we assigned different soil series including the Dania series for some samples as they became shallower (Table 2-2).

Soils were sampled from each plot consisting of two sample points on each opposite ends of the farm labeled “A” and “B”. We used 70% ethanol and a sterile paper towel to clean the small stainless-steel trowel shovel used to sample the soils between each soil sampled to ensure sterilization. Soil samples were taken at two different dates from the same farm plot; once after the pre-crop has been harvested and before rice has been planted prior to flooding (pre-rice), and secondly after the rice has been grown and field drained from flooding (post-rice). The soil samples were placed in a sterile plastic bag and directly into a cooler containing ice before returning to the EREC laboratory. In the laboratory, soil samples were homogenized thoroughly in the sterile plastic bag to obtain a uniform sample and stored in the 4 degree C refrigerator.

Soils were sampled (pre-and post-rice cultivation) at a depth of 6 inches (~ 15 cm) in shallow and deep soils. We defined shallow soils in our study as having a depth of equal or less than 20 inches (51 cm) and deep soils as having a depth higher than 20 inches (>51 cm) with a total of 12 deep soils and 9 shallow soils. Soil samples were analyzed for pH, total N and C and organic matter content, macronutrients (P, K, Ca,

Mg), micronutrients (Mn, Cu, Fe, Zn), and Si content.

Nutrient Analyses

Total N and total C were analyzed in soil chemistry lab at the Ft Lauderdale

Research and Education Center with a C: N analyzer (CN628 LECO Corporation, Saint

Joseph, Michigan, USA). All other analyses were performed at the Everglades

Research and Education Center Soil Testing Laboratory. pH was measured in a 1:2 soil: water solution (Thomas, 1996). Soils were extracted with a Mehlich-3 solution using 2.5 ml of soil and 25 ml of solution. Mehlich solution was analyzed for Ca, Mg, and K with Atomic absorption spectrometry (PerkinElmer Model Optima 5300 ICP).

Mehlich 3-extractable P (M3P) was determined using AQ2 discrete analyzer. Mehlich 3 extract was also analyzed for micronutrients with ICP (inductively coupled plasma)

(Agilent 5110 ICP-OES). 10 ml of soil was extracted with 25 ml 0.5 N acetic acid for

Silicon content (Snyder, 2001). In addition, water-extractable P (Pw) (4 ml of soil and 50 ml of DI water) was determined with a probe colorimeter. Phosphorus was determined calorimetrically according to the method of Murphy and Riley (1962)

Statistical Analysis

Data is presented in box plots to show the distribution of the data. To test the difference in soil properties and nutrients due to the cultivation flooded rice (pre- and post-flooded rice) for objective 2, we used the Wilcoxon signed rank test using R software. We tested both the “A and B” samples separately before averaging the two samples made on the same plot. The Wilcoxon signed-rank test is a non-parametric statistical test used to compare two paired groups to assess whether their population mean ranks differ (i.e., it is a paired difference test).

The same Wilcoxon signed rank test was also used for objective 3 to determine if there was an impact of soil depth (shallow soils vs deep soils) after rice cultivation on soil properties and nutrients.

Results and Discussion

Objective 1: Determine the differences in soil properties and nutrients in shallow and deep soils in selected series of EAA soils.

Soil Properties

There were no statistical differences in pH, organic matter content, total C or total

N between the shallow and deep soils in our study (Table 2-2). The pH values for the shallow muck soils were 6.94 and for deep muck soils 7.13 and there was no significant difference. Calcium carbonate (in the limestone bedrock) can potentially raise pH in the organic soils especially in shallow soils. Diaz et. al, 1992 reported pH values ranging from 4.5 to 6.3 in four EAA soil series sampled in 1987. Soil pH was relatively uniform within the fields, with most of the variability occurring close to the roads and ditches. A common practice in the EAA that increases soil variability in pH is the regular cleaning of ditches and canals, and the subsequent dumping and spread of the spoils to one side of the field (Diaz et al., 1992).

Organic matter content (OM) means were similar between shallow and deep soils averaging 74.5% in shallow soils and 72.0 % in deep soils with no statistically significant differences. The Histosols in the EAA have a much higher OM content.

Organic matter content in the soils we sampled in the EAA farm regions ranged between 37.3 -81.7%. The lower OM soils ((Manley - Blumberg are called transitional soils (sandy organic soils) with a higher content of sand and lower OM content.

Interestingly, the highest OM content of 81.8% was found in a shallow Dania soil (48 cm) (Table 2-1), apparently illustrating no clear relationship between depth of the soil and OM content. According to Wright and Hanlon 2009, soils in the EAA vary greatly and should not be considered as a single type and the depth of the organic material generally decreases with distance from Lake Okeechobee. The soils we sampled located towards the southeast, south, and east of Lake Okeechobee (See map Figure

1-4)

Total C and N

Total N content was statistically similar in both depths (2.48 and 2.46% in shallow and deep soils, respectively) (Table 2-2). Total C content were also statistically not significant in both soil depths (39.4 and 37.9% in sallow and deep soils, respectively).

Nitrogen released with organic matter can be taken up by the plant, leached in drainage water or released into atmosphere as NOx gases if denitrification occurs (Weil and

Brady, 2017). Soluble N in soil solution in EAA organic soils was found to be predominantly in the NO3 form over 12-month sampling period due to the abundance of ammonia oxidizing archaea (Zhalnina et al., 2014). Wright and Hanlon (2013) compared two sites with Dania series, uncultivated site and site that has been in sugarcane production for a long time. In the uncultivated Dania soil, more organic carbon and nitrogen were found in larger aggregates compared to aggregates in the sugarcane sites. Thus, tillage was negatively affecting the number and size of aggregates, as well as the organic carbon and nitrogen contained within those aggregates (Wright and Hanlon, 2013).

Nutrients

Macronutrients and Si

Our results showed no statistical differences in macronutrients (P, K, Ca) except for Mg between shallow and deep soils in the farm regions sampled in the EAA in this study (Table 2-2). Earlier reports of nutritional disorders of rice grown on Histosols have focused on N, S, K, Fe, Cu, Mn, and Zn (Snyder, 1986). Macronutrient fertilization is not required for rice production on the organic Histosols of the Florida Everglades (Snyder and Jones, 1989). As mentioned earlier, flooded rice in EAA Histosols benefit from N and P mineralized and have no N fertilizers added to them. Mehlich 3-P levels were adequate and were at 43.7 and 57.0 kg ha-1 in shallow and deep soils, respectively with no statistical difference. Organic soils in Florida has suggested that the Mehlich 3 extractant performs satisfactorily for P extraction over a wider pH range than water only

(McCray et al., 2012). Available P to the rice crop is probably a combination of P mineralization and P added to the crop rotation before the rice crop. McCray et. al

(2010) recommended maximum P recommendation for sugarcane grown on Florida

Histosols be maintained at 36 kg P ha 1, with no P fertilizers added previously to the rice rotation. In addition, rice grown in the area does not respond to K fertilization (Snyder and Jones, 1989). Potassium fertilization of sugarcane is a common practice on the organic soils of the EAA. It is necessary to maintain relatively large quantities of plant available K to meet the sugarcane crop demand. Based on soil testing, K is applied between 0 and 238 kg ha-1 (Coale, 1994). Magnesium was higher in the shallow soils at

1137 kg ha-1 compared to 820 kg ha-1 for the deep soils, but no differences were found with Ca content.

Silicon, after oxygen, is the most abundant element in the earth's crust, with soils containing approximately 32% Si by weight (Lindsay, 1979). There is much less Si in the rootzone of plants growing in Everglades Histosols than in most mineral soils

(Snyder et al., 1986b). The Torry series which has 35% mineral materials provides considerable Si to rice (Snyder 1993), but not the other soil series in the EAA. Rice grown in all soil series except Torry provide suboptimal amounts of Si to rice and can result in lower yields and become more susceptible to diseases (Snyder et al., 1986).

Silicon content was 75 mg kg-1 (26 mg L-1 assuming a bulk density of 0.35 Mg m-3) in the shallow soils and 103 mg kg-1 (31 mgL-1) in the deep muck in our study showing no significance difference between depths. Silicon fertilizers are routinely added to sugarcane and rice according to soil test recommendations.

Silicon levels in our study are found to be optimum for rice production.

Korndörfer et al., 2001 determined Si rates to correct deficiency and obtain optimum rice yield were 1500, 1120 and 0 kg ha-1 for low ( <6 mgL-1), medium (6 to 24 mg L-1) and high (> 24 mg L-1 ) level of soil Si, respectively. Optimal Si content in the rice plant was found to be around 3 % (Korndörfer et al., 2001). Silicon has been reported to benefit rice in a number of ways (6): (i) improvement in efficiency of sun-light use and increase in photosynthetic activity, (ii) reduction in transpiration and improvement in water use efficiency, (iii) increased mechanical strength of cells and reduction in lodging, (iv) increased resistance to certain insects and diseases, (v) reduction in accumulation of toxic concentrations of Mn and other heavy metals, (vi) improvement in rice plant nutrition (Snyder, 1986).

Micronutrients

Iron, Cu, and Mn were significantly different in the shallow and deep soils sampled (Table 2-2). Interestingly, the shallow soils showed a significantly higher Mn and Cu concentration compared to the deep soils. Mn concentration was at 32.4 mg/kg in the shallow soils while the deeper soils had a concentration of 16.20 mg/kg (p-value is 0.011). Diaz et. al (1992) found concentration of Mn ranging from 28-65 mg kg-1 in

Okeelanta, Lauderhill, and Pahokee soil series. According to Diaz et al, 1992, micronutrient variability among the soil series was considerable and Mn was the micronutrient that required the largest number of samples in most of the soil’s series studied. In certain soils having pH values near or above 7, apparent Mn deficiencies of rice have been observed, even though most soils in the EAA have been fertilized with

Mn for many years (Snyder et. al, 1990). Manganese deficiency was determined to limit the growth of rice seedlings in several drained Histosols with pH near or higher than neutral (7.0) (Snyder et al, 1990, Snyder, 1993). Zinc was also slightly but significantly higher in shallow soils at 36.02 mg kg-1 compared to deep soils at 32.50 mg kg-1.

Iron (Fe) concentration means were significantly higher in the deep soils at 504 mg kg-1 than the shallow soils at 386 mg kg-1 (Table 2-2) Diaz et. al (1992) found much higher values in their three-soil series (Okeelanta, Lauderhill, and Pahokee) ranging from 2201 to 4965 mg kg-1, but these were extracted with Mehlich-1 solution. We used

Mehlich-3 in our study. Several research studies using vegetables and organic soils were conducted in Florida have suggested that the Mehlich 3 extractant performs satisfactorily for P extraction over a wider pH range than water (Hochmuth et al., 2009), and Mehlich 3 could also be used for extraction of other nutrients (Mehlich, 1984). No

differences were found with Cu concentrations between shallow and deep soils. Copper means were 13.09 and 13.47 mg kg-1 in shallow and deep soils, respectively. Diaz et al., (1992) reported similar values.

Comparing soils with long term cultivation of sugarcane to uncultivated soils, sugarcane soils had 74, 72, and 94% significantly greater Zn, Mn, and Cu than uncultivated soils from 0–30 cm deep (Wright and Mylavarapu, 2010).

In summary, few differences were found in soil properties and nutrient content between shallow and deep soils sampled, mainly with Fe, Zn, Mn, and Mg. We chose an arbitrary 20 inches to delineate differences in soil depth. We can also speculate that fertilization regimes are similar in the fields sampled following UF IFAS recommendations.

Objective 2: Determine impact of flooded rice production on nutrients availability and soil properties

Soil Properties

Soil properties (pH, total C, total N and OM %) were measured pre- and post- flooded rice cultivation in the 21 field plots. The results are presented in boxplots that show the median values and distribution of the data (Figures 2-1, 2-2, 2-3) and statistical analyses in Table 2-3.

Our study found no significant changes in pH between pre- and post-flooding fields (Figure 2-1). The median pH of the plots pre flooding was close to neutrality

(7.04), and no significant changes in pH were observed post flooding. After flooding soils, pH of acid soils tends to increase and pH of alkaline soils tends to decrease, with pH in both types of soils moving to neutrality. Increase in pH of acid soils depends on

3+ 2 the activities of oxidants (such as NO3, Fe , Mn +) and proton consumption during reduction of these oxidants under flooded conditions. Organic soils in the EAA are close to neutrality therefore, changes in pH were not expected. In alkaline soils, pH is controlled (and generally lowered) by the accumulation of dissolved CO2 and organic acids (Inglett et al., 2005).

Both organic matter and Total C content saw small but significant decreases post flooding (Figure 2-1 & Table 2-3). We can speculate the release of Dissolved organic

Carbon (DOC) under anaerobic condition may partially explain reduction of total C content. In a two-year lysimeter study with rice with different water managements in the

EAA, Tootoonchi et. al (2018) found DOC concentrations in outflow water ranging from

- 21 to 44 mg L 1. In flooded soils, when O2 becomes less available and soil redox potential becomes more reduced, slow organic matter decomposition may result in accumulation of DOC (D'Angelo & Reddy, 1994. Moorberg et al., 2015) and affect drainage water quality.

Macronutrients

We measured differences in macronutrients (Ca, K, Mg, Pm, Pw) and Si due to flooded rice production (Figure 2-2). The impact of flooded rice production was variable among these elements. Three elements (K, Pw and Si) showed a significant decrease,

Ca and Pm3 content did not change significantly, while Mg showed an increase after flooded rice production.

There were no differences in Ca or Pm3 content after flooded rice cultivation.

The mean Ca content was 7963 kg ha-1 pre flooding and 10134 kg ha-1 after flooding, but the increase was not significant (Table 2-3). As mentioned earlier, the Histosols in

the EAA have a limestone bedrock, and the Ca content reflects the cultivation of these soils and the mixing of the limestone with the upper soil layers (Snyder, 2005). In addition, irrigation water from Lake Okeechobee is another source of Ca and Mg in addition to other minerals.

The median (or mean) value of Pm3 was at 59.4 kg ha-1 pre flooding and did not change much after flooding. Growers in the EAA do not add any P fertilization to flooded rice, however, the rice benefits from the P fertilizers added to the previous crop or in previous years as well as from P release from organic matter and possible mineral materials in the soil upon flooding. Although there was no increase in Pm3 after flooding, P released could have been taken up by the plant or leached in drainage water. Phosphorus sorption capacity of organic soils depends on Fe and Al oxides in these soils (Janardhnan and Daroub, 2010), and reduction of Fe oxides is expected to release P into the soil solution.

Water-extractable P (Pw) showed a decrease after flooded rice production.

Water-extractable P (Pw) content decreased from 5.68 to 3.87 kg ha-1. This reflects uptake of P by rice plant and possibly discharge of P in drainage water. Pw extracts soluble P in water and indicates available P to plants. This test is used primarily for short term crops like vegetables and rice, while Pm3 is more suited for long term availability of P and is more appropriate for long term crops like sugarcane which grows for 12 months.

Potassium and Si showed a decrease after flooded rice production. We suspect plant uptake was a major pathway for the decrease, in addition to possible losses of these elements in drainage water. Potassium showed a decrease from 109.8 kg ha-1

for pre flooded rice to 62.0 kg ha-1 post flooded rice production. The response of upland and lowland rice to K is not well documented. Data related to long term K experiments with rice are lacking and short-term experiments do not provide a real picture of the response of rice to K fertilization (Fageria, 2014). A real picture of the response of rice K fertilization may occur due to the possibility of K being held in clay particles (Fageria,

2014). Potassium is held in clay particles and long-term K experiments are needed.

Similarly, several studies in the EAA have indicated that rice grown organic soils in the

EAA does not response to K fertilization (Jones et al., 1994). However, K is added routinely to sugarcane production in the EAA due to very low mineral content in the organic soils and very low native K levels. Potassium plays an important role in many physiological and biochemical processes in plants. It is also responsible for reducing diseases. Rice accumulates maximum amount of K; however, a major part of K is found in the straw (85%) and a small part (15%) transfers to the rice grain (Fageria, 2014).

Silicon showed a significant decrease after flooded rice cultivation from 91 mg kg1 pre flooded rice to 61.8 mg kg-1 after flooded rice cultivation and reflect high uptake of Si by the rice. There is a possibility of leaching of Si in drainage water also. Silicon amendments are routinely applied to rice as organic soils have very low content of Si.

Numerous field experiments under different soil and climatic conditions and with various plants clearly demonstrated the benefits of application of silicon fertilizer for crop productivity and crop quality including rice and sugarcane (Snyder et al., 2016). Other benefits include increased resistance of plant to diseases and insects. Calcium silicate, generally obtained as a byproduct of an industrial procedure (steel production, for example) is a widely used silicon fertilizers. Potassium silicate, though expensive, is

highly soluble and can be used in hydroponic culture (Snyder et al., 2016). Rice and

Sugarcane, both monocotyledons, are classified as high accumulators of Si and have a silicon content in the shoot that ranges from 1.0 % to 10 % dry weight. Because of the efficient silicon uptake system of the high accumulators, the amount of silicon uptake by the plant from the soil is substantial and may be twice the amount of N in rice (Tubaña and Heckman, 2015).

Micronutrients

We measured changes in four micronutrients (Fe, Mn, Cu, and Zn) due to flooded rice cultivation (Figure 2-3 & Table 2-3). Solubility of both Fe and Mn is impacted by redox, however, Cu and Zn solubility is not impacted by redox. In general, availability of all these micronutrients decrease with an increase in soil pH. Although we did not observe an increase or changes of soil pH pre- and post-flooded rice.

Iron concentrations significantly increased from 459.5 mg kg-1 to 537.2 mg kg-1 after flooded rice cultivation. The chemistry of flooded soils is dominated by Fe more than any other redox element. Fe3+ oxide compounds are reduced to Fe2+ and released into the soil solution. The major reason for this dominance is the large amount of soil Fe that can undergo reduction, which usually exceeds the total amount of other redox elements by a factor of 10 or more (Patrick and Reddy, 1978). This seems true for the organic soils in the EAA as Fe concentrations were at least 10x than that of the other micronutrients measured. Even with high concentrations of available Fe in the organic soils in the EAA, rice can show chlorosis before flooding in some locations, and chlorosis is alleviated after flooding (Snyder and Jones, 1988). However, seedling mortality occurs and therefore most EAA growers use Fe fertilizers with rice cultivation.

Manganese is another redox element where Mn4+oxide compounds get reduced to Mn2+. The Mn concentrations in the soils in our study did not increase with flooding; mean Mn concentration was at 20.20 mg kg-1 pre flooded rice and 21.85 mg kg-1 post flooded rice production (Table 2-4). Critical soil levels of Mn for rice varies but in general is between 4-8 mg kg-1 (Fageria, 2014) indicating sufficient levels of Mn in these soils to the rice crop. Manganese deficiency is reported in many rice growing regions of the world. The main causes are high pH soils, using high yielding cultivars that require high

Mn fertilizer rates, negative interactions with some macro- and micronutrients, and adsorption by organic matter. Some Histosols are naturally low in Mn (Fageria, 2014).

Manganese deficiency was determined to limit the growth of rice seedlings drill planted in several drained organic soils in the EAA having pH values ranging from 6.9 to 7.7

(Snyder et al., 1990). The application of approximately 15 kg Mn ha-1 as MnSO4 with the seed prevented the deficiency and provided near-maximum grain yields (Snyder et. al, 1990). Routine application of Mn to rice production is generally not practiced in the

EAA. However, Mn is added as part of a micronutrient mix to the sugarcane.

Rice is considered susceptible to Zn deficiency which is widely reported in lowland rice in most rice growing regions of the world (Fageria, 2014). Zinc deficiency in rice is related to low levels of Zn in highly weathered tropical soils, and the use of modern cultivars that require high Zn fertilizer rates. In addition, the availability of Zn to plants decreases with increases in soil pH and may be impacted by flooding if there is an increase in soil pH due to flooding. The pH of our soils was neutral before flooding and did not change much after flooding. Zn content was at 32.85 mg kg-1 in pre flooded rice and very similar content was observed after flooding at 34.62 mg kg-1. Overall, 1-5

mg kg-1 of soil is adequate for an upland and lowland rice production under most soil and climatic conditions (Fageria, 2014) which indicates there are adequate amounts of

Zn for rice plants in the soils sampled in this study.

Copper (Cu) is strongly held by organic matter in soils (Ellis and Foth, 1994) and most Cu deficiencies are found in organic soils. Indeed, Cu was identified as the nutrient needed to solve soil fertility issues in the Florida Everglades (Allison, 1950).

Copper availability and uptake by plants is also reduced with an increase in soil pH.

Complexation of Cu with organic matter occurs mainly at solution pH values above 6.5.

Copper content increased from 13.02 mg kg-1 pre flooded rice to 16.88 mg kg-1 post flooded rice production and the increase was significant (Table 2-3). Excess amounts of P, Zn, Fe, and Mn decrease Cu uptake in rice. In most rice producing soils, the critical level of copper varies from 0.5 to 3 mg kg-1 soil and depends on soil type and on the extracting solution used (Fageria, 2014). In an experiment in paddy soils in China, native Cu in soil was strongly absorbed by soil and most of the Cu was in the residual and organic matter fractions, but it transferred to the easily extracted fractions, such as, amorphous ferric oxides, carbonates, and exchangeable fractions after flooding (Liu et al., 2018).

Our results show increase in Cu solubility after flooding but not for Zn. Although the forms of Cu and Zn present in soils are not involved in oxidation-reduction reactions, their mobility may be affected by some of the consequences of soil submergence and flooding. The reduction of Fe and Mn and the production of organic complexing agents should increase the solubility of Cu and Zn. The increase in pH of acid soils however will lower their solubility. The net result of submergence is to increase the availability of

Cu and to depress that of Zn (Ponnamperuma, 1972), but Ponnamperuma (1984) illustrated a decrease in availability of Zn and Cu in soil solution after few weeks of submergence.

Objective 3: Determine impact of flooded rice production on nutrients and soil properties in shallow and deep soils.

We wanted to explore if shallow (<20”) and deep soils (>20”) showed differences in soil properties and nutrients due to flooded rice production. Results of the Wilcoxon sum rank (non-parametric t-test) are shown in Table 2-4 and box plots are presented in

Figures 2-4 to 2-8.

One common response in both shallow and deep soils is reduction in total C content which was significant in both categories of soils. As mentioned earlier, this was unexpected, and we hypothesized this is due to the production and accumulation of dissolved organic carbon (DOC) under anaerobic conditions which is then released with discharge water. More research is needed on this as maintaining the sustainability of the organic soils in the EAA is of prime concern to all farmers. Flooding soils and flooded rice production are recommended as sustainable management practices. In a modelling study, increasing water table depth or reducing the aerobic decomposition rate were found to help reduce soil subsidence (Rodriguez et al., 2020).

In shallow soils, pH, Mg, Cu and Mn all showed increases after flooded rice production. The pH of shallow soils increased from 6.9 to 7.31 and the increase was significant. Increases in pH after flooding are related to consumption of H ions in solution due to reduction of Fe and Mn in the soil (Inglett, 2015). Mn concentrations

increased significantly after flooding in the shallow soils from 26.37 to 33.42 mg Kg-1, however, there were no significant changes in Fe concentrations after flooding.

In addition, Mg concentrations increased after flooding, but Mg is not an element impacted by redox. The increase of extractable Mg (and Ca) due to submergence of flooding is normally linked to solubilization of CaCO3 and MgCO3 after the pH of calcareous soils decrease due to flooding. The pH of the shallow soils was at 7.31 so increased solubility of carbonates is not possible.

In deep soils, in addition to total C content, organic matter content, Pw, Si and K showed significant decreases after flooded rice production (due to uptake by the rice plant) while Fe concentrations increased (iron-reduction due to flooding). Similar to the decrease in total C content, the organic matter content decrease needs to be researched further.

A significant decrease in Pw, K, and Si was found in deep soils after flooded rice production but not in shallow soils. The decrease possibly reflects both plant uptake and leaching of these elements in rice drainage water. Silicon is added as a fertilizer for rice production but not P and K. Potassium decreased by 73.8 kg ha-1 in the deep soils and only 13.05 kg ha-1 in the shallow soils. Similarly, the decrease in Pw in deep soils was at 2.79 kg ha-1 while it decreased only 0.5 kg ha-1 in shallow soils. Another noticeable difference between deep and shallow soils is the significant increase of Fe after flooded rice production in the deep soils.

In a sugarcane lysimeter experiment trial with soil depths of 13 and 25 cm, water tables of constant and periodically flooded, periodic flooding increased plant uptake of manganese (Mn), silicon (Si), and boron (B) (Jennewein, et al., 2020). Soil depth

impacted sugarcane nutrient uptake with 25 cm of soil depth significantly affecting examined leaf nutrient concentrations except iron (Fe). Proximity to bedrock (the 13 cm soil depth) led to excessive calcium (Ca) uptake and low K and Fe DRIS (Diagnosis and

Recommendation Integrated System) values were below recommendations (Jennewein, et al., 2020).

Conclusions

This chapter focused on the impact of flooded rice production by sampling and analyzing data from 21 plots in the EAA that were planted in flooded rice summer 2018.

The plots were sampled twice before planting rice and after harvesting the crop. We also attempted to measure the impact of shallow soils vs deep soils on soil properties and nutrient chemistry. The designation was arbitrarily assigned as shallow soils <20 in and deep soil > 20 in of the O horizon above the limestone bedrock.

We observed a decrease in total C content in both shallow and deep soils, possibly reflecting the production and loss of dissolved organic carbon (DOC) with discharge water. Deep soils showed a decrease in Pw (P extracted with water), K, and

Si reflecting plant uptake, however the decrease of the same elements in shallow soils was not significant. Iron content increased in deep soils reflecting the reduction of Fe oxide compounds as the redox potential is reduced with flooded rice. However, Fe content did not increase in shallow soils after flooding.

A question to address in the future is the availability of nutrients like P, K, and Fe in shallow soils for rice production. Further research is needed to understand the chemistry of the shallow submerged organic soils in the area as soils are becoming shallower.

Table 2-1: Location of the 21 fields sampled in various farm regions located in the EAA, farm plot number, soil depth (we define shallow as <20 inches of soil and deep >20 inches, respectively), organic matter percentage and soil series. Soil series were defined by GPS (longitude and latitude) using the Web Soil Survey and Soil Data (Map is found in chapter 1). Sample Farm Region Farm Plot Soil Soil Soil Depth Organic Matter Soil Series Soil series – Location Type Depth (Inches) % (WSS) Depth measurements 1 Shelton 68-11-3 Muck Shallow 19(48cm) 81.7 Terra Ceia Dania 2 Shelton 68-11-5 Muck Shallow 17(43cm) 76.8 Terra Ceia Dania 3 Shelton 68-11-7 Muck Shallow 17.5(44cm) 78.9 Terra Ceia Dania 4 East Area CD18N Muck Deep 21(53cm) 76.5 Terra Ceia Lauderhill 5 East Area CD19N Muck Deep 20.5(52cm) 77.9 Terra Ceia Lauderhill 6 East Area CD20N Muck Deep 24.5(62cm) 78.9 Terra Ceia Lauderhill 7 Southern Ranch X6SW9 Muck Shallow 9.5(24cm) 71.6 Lauderhill Dania Lease 8 Southern Ranch X6SW10 Muck Shallow 11(28cm) 76.5 Lauderhill Dania Lease 9 Southern Ranch X6SW11 Muck Shallow 15.5(39cm) 79.0 Lauderhill Dania Lease 10 Manley (Blumberg) 64-E-11 Muck Shallow 10.5(27cm) 49.3 Pahokee Dania 11 Manley (Blumberg) 64-B-11 Muck Deep 37.5(95cm) 37.3 Pahokee Pahokee 12 Manley (Blumberg) 64-F-11 Muck Deep 55(140cm) 58.2 Pahokee Terra Ceia/Torry 13 West Organic Area 2ABE-W Muck Deep 23.5(57cm) 70.7 Terra Ceia Lauderhill 14 West Organic Area 3ABE-W Muck Deep 25(64cm) 73.0 Terra Ceia Lauderhill 15 West Organic Area 4ABE-W Muck Deep 25.5(65cm) 72.0 Terra Ceia Lauderhill 16 Zone I 28-CD-7S Muck Deep 55(140cm) 78.8 Terra Ceia Terra Ceia/Torry 17 Zone II 38-IM-14E Muck Deep 37.5(95cm) 73.0 Terra Ceia Pahokee 18 Veg 1 Roth Celery Roth Muck Deep 29.5(75cm) 78.9 Terra Ceia Lauderhill 19 Veg 2 Roth Lettuce Roth Muck Deep 23(59cm) 79.0 Terra Ceia Lauderhill 20 King Ranch 40920 Muck Shallow 13.5(34cm) 76.8 Terra Ceia Dania 21 King Ranch 41140 Muck Shallow 18(46cm) 74.6 Terra Ceia Dania

Table 2-2: Average values of soil properties, macronutrients, micronutrients, and beneficial element (Si) for shallow and deep muck soils located in the EAA before the cultivation of flooded rice. We define shallow soils as <20in. and deep soils as >20in. Pre-Flooding Nutrients Soil Depth p-value Shallow Deep

pH 6.94 7.13 0.231 Total C (%) 39.37 37.87 0.369 Total N (%) 2.48 2.46 0.982 OM (%) 74.67 71.91 0.240 Pm3 43.68 56.96 0.534

Pw(kg/ha) 5.98 5.15 0.128

Ca(kg/ha) 7988.02 7661.93 0.695 K(kg/ha) 83.69 124.76 0.134 Mg(kg/ha) 1137.23 819.58 0.050* Si(mg/kg) 74.55 102.87 0.259 Cu (mg/kg) 13.09 13.47 0.076

Fe(mg/kg) 386.02 504.18 0.002* Mn(mg/kg) 32.40 16.20 0.001* Zn(mg/kg) 36.02 32.50 0.016*

*Significant is p-value is <0.05.

Table 2-3: Statistical results using the Wilcoxon test for differences in soil properties and nutrients before and after the cultivation flooded rice in the EAA. Nutrients Pre-Flooded Rice Post-Flooded Rice Mean Difference Mean S.E. Mean S.E. Post-Pre p-value* pH 7.04 0.08 7.15 0.07 0.11 0.272 Total C (%) 37.8 1.12 35.9 1.24 -1.9 <0.001* Total N (%) 2.43 0.06 2.37 0.07 -0.06 0.2029 OM (%) 72.4 2.40 68.3 2.61 -4.1 0.014* Pm3 (kg/ha) 59.4 18.5 54.3 18.9 -5.1 0.563 Pw (kg/ha) 5.68 0.93 3.87 0.62 -1.81 0.015* Ca (kg/ha) 7963 744 10134 1024 2171 0.065 K (kg/ha) 109.8 17.20 62.0 6.65 -47.8 0.002* Mg (kg/ha) 960.8 81.9 1124.0 107.3 163.2 0.004* Si (mg/kg) 91.0 14.1 61.8 8.69 -29.2 <0.001* Cu (mg/kg) 13.02 2.51 16.88 2.83 3.86 <0.001* Fe (mg/kg) 459.5 19.6 537.2 30.3 77.7 0.001* Mn (mg/kg) 20.20 2.06 21.85 2.81 1.65 0.393 Zn (mg/kg) 32.85 3.52 34.62 3.72 1.77 0.128

Table 2-4: Differences in soil properties and nutrients as impacted by flooded rice cultivation and depth of soils in the organic soils in the EAA (<20 inches). Nutrients Shallow Soils (depth <20 inches) p-value Pre Post Difference (Pre and Post) pH 6.90 7.31 0.41 0.020* Total C (%) 38.18 36.44 -1.74 0.004* Total N (%) 2.43 2.34 -0.09 0.164 OM (%) 73.9 68.3 -5.6 0.164 Pm3(kg/ha) 52.1 61.4 9.28 0.476 Pw (kg/ha) 5.31 4.81 -0.50 0.575 Ca (kg/ha) 8061 11636 3575 0.203 K (kg/ha) 81.8 68.7 -13.05 0.301 Mg (kg/ha) 1047.7 1391.0 343.3 0.008* Si (mg/kg) 79.3 51.8 -27.50 0.074 Cu (mg/kg) 12.08 18.25 6.17 0.008* Fe (mg/kg) 412.1 409.8 -2.25 0.820 Mn(mg/kg) 26.37 33.42 7.05 0.012* Zn (mg/kg) 31.64 35.08 3.44 0.129

Table 2-5. Differences in soil properties and nutrients as impacted by flooded rice cultivation and depth of soils in the organic soils in the EAA (>20 inches). Nutrients Deep Soils (depth >20 inches) p-value Pre Post Difference Post - Pre pH 7.14 7.02 -0.12 0.301 Total C (%) 37.57 35.51 -2.06 <0.001* Total N (%) 2.42 2.39 -0.03 0.910 OM (%) 71.2 68.3 -2.9 0.027 Pm3(kg/ha) 64.84 49.03 -15.81 0.230 Pw (kg/ha) 5.95 3.16 -2.79 0.014 Ca (kg/ha) 7891 9008 1117 0.233 K (kg/ha) 130.8 57.0 -73.8 <0.001* Mg (kg/ha) 895.7 923.9 28.2 0.233 Si (mg/kg) 99.8 69.3 -30.5 <0.001* Cu (mg/kg) 13.73 15.85 2.12 0.077 Fe (mg/kg) 495.1 632.8 137.7 <0.001* Mn(mg/kg) 15.58 13.17 -2.41 0.064 Zn (mg/kg) 33.75 34.28 0.53 0.569

7.8 2.8 a a a 7.6 a 2.6 7.4 2.4 7.2 2.2 7.0 2.0

pH 6.8 1.8

Total N (%) N Total 6.6 1.6 6.4 1.4 6.2 1.2 Pre Post Pre Post

45 90 a b a 40 80 b

35 70

30 60 (%) OM

Total C (%) C Total 25 50

20 40

15 30 Pre Post Pre Post

Figure 2-1. Soil Properties pre- and post-flooded rice production (pH, Total C, Total Carbon, Total N [ Total Nitrogen, and OM, organic matter]).

22000 500 20000 18000 400 16000 a 14000 300 12000 200 a

K (kg/ha) K

Ca(kg/ha) a 10000 b 8000 100 6000 4000 0 Pre Post Pre Post

2200 25 2000 20 1800 1600 a b 15 1400 1200 10 a b 1000

Pw (kg/ha) Pw Mg (kg/ha) Mg 800 5 600 0 400 200 Pre Post Pre Post

500 400 350 400 300 300 250

200 200

Si(mg/kg) 150 a a a Pm3 (kg/ha) Pm3 100 b 100 0 50 0 Pre Post Pre Post Figure 2-2. Macronutrients and beneficial element pre- and post-flooded rice (Ca, K, Mg, Pw, Pm3, and Si).

80 1000

70 900

60 800

50 700 a b 40 600

Fe (mg/kg) Fe

Cu (mg/kg) Cu 30 a b 500 20 400

10 300

0 200 Pre Post Pre Post

50 90

80 40 a 70 a 30 60 50 a a 20 Zn (mg/kg) Zn 40

Mn (mg/kg) Mn 30 10 20

0 10 Pre Post Pre Post Figure 2-3. Micronutrients pre- and post-flooded rice (Cu, Fe, Mn, and Zn).

Shallow Soils Shallow Soils 7.8 42 a b 7.6 40 b 7.4 38 a 36 7.2

pH 34 7.0

Total C (%) C Total 32 6.8 30

6.6 28

6.4 26 Pre Post Pre Post

Shallow Soils Shallow Soils 2.8 85

a 80 a 2.6 a a 75

70 2.4 65

2.2 (%) OM Total N (%) N Total 60

55 2.0 50

1.8 45 Pre Post Pre Post

Figure 2-4. Soil Properties pre- and post-flooded rice in shallow soils: (pH, Total C, Total N and OM).

Figure 2-5. Soil Properties pre- and post-flooded rice in deep soils: (pH, Total C, Total N).

Shallow Soils Shallow Soils 22000 160 20000 a 140 18000

16000 120 14000 a 100 a 12000

K (kg/ha) K Ca(kg/ha) 10000 a 80 8000 60 6000

4000 40 Pre Post Pre Post

Shallow Soils Shallow Soils 2200 14

2000 b 12 1800 10 1600 a a 1400 8 a

1200 6 Pw (kg/ha) Pw Mg (kg/ha) Mg 1000 4 800 2 600

400 0 Pre Post Pre Post

Shallow Soils Shallow Soils 500 120

a 400 100 300 80

200 a 60

Si(mg/kg)

Pm3 (kg/ha) Pm3 100 a a 40 0

20 Pre Post Pre Post Figure 2-6. Macronutrients and beneficial element (Si) pre- and post-flooded rice in shallow soils: (Ca, K, Mg, Pw, Pm3, and Si).

Figure 2-7. Macronutrients and beneficial element (Si) pre- and post-flooded rice in deep soils (Ca, K, Mg, Pw, Pm3, and Si).

Shallow Soils Shallow Soils 80 600

550 60 500 a a 40 450

Cu (mg/kg) Cu b (mg/kg) Fe 400

20 a 350

0 300 Pre Post Pre Post

Shallow Soils Shallow Soils 50 60

45 b 55

40 a 50 35 45 a a 30 40

Zn (mg/kg) Zn 25 35 Mn (mg/kg) Mn 20 30

15 25

10 20 Pre Post Pre Post

Figure 2-8. Micronutrients pre- and post-flooded rice in shallow soils (Cu, Fe, Mn, and Zn).

Figure 2-9. Micronutrients pre- and post-flooded rice in deep soils (Cu, Fe, Mn, and Zn).

CHAPTER 3 IMPACT OF THE CULTIVATION OF FLOODED RICE ON SOIL MICROBIAL COMMUNITIES

Introduction of Chapter 3

The study of microbial communities and their role in carbon and nutrient cycling have expanded tremendously through application of next-generation DNA sequencing technology. Soil microorganisms drive many biogeochemical processes including recycling of organic matter and nutrients and are intimately connected plant growth. Soil microbes participate in complex interactions between abiotic and biotic soil components, soil management, and global climatic factors. In agricultural systems these interactions can sometimes dictate a crops health and the yield of crop production (e.g., through regulation of plant pathogens, mineralization of compost or organic matter). Soil management (e.g., though Best Management Practices, BMPs) can be an important factor to maintain or sustainably enhance soil fertility, which can influence soil microbial communities (i.e., flooded rice). Relatively little research has been conducted on microbial communities in organic soils in general and in the Everglades Agricultural

Area (EAA).

A significant problem in the EAA and other areas with organic soils is soil subsidence. Soils subside when they are drained continuously, primarily because of aerobic microbial activity (Morris et al., 2004). It has been shown that improved water management with increased water table can over time reduce the soil subsidence rates

(Wright and Snyder, 2009). For example, Stephan and Johnson, 1951, and Snyder et al., 1978, documented that the subsidence rate is closely aligned with water table depth, as organic matter decomposition is impaired by flooded conditions. It can be hypothesized that the restriction of aerobic breakdown of complex organic matter is the 69

critical factor to reduce the rate of soil subsidence. Under inundated conditions soil aeration and breakdown of complex organic matter by microbes is restricted by limited oxygen diffusion. Oxygen concentrations in the soil matrix drop rapidly due to microbial respiration and the soil becomes anoxic.

In the EAA farmers use flooded rice as a crop rotation following sugarcane and sometimes winter vegetables. BMPs that include flooding fields (e.g., flooded rice) could help stabilize the soil by stimulating anaerobic microbial communities and limiting aerobic degradation of complex organic matter. The diversity and composition of soil bacteria communities in general is thought to have a direct influence on a wide range of ecosystem processes (Fierer N. and Jackson R. B, 2006). There are between 107 and

109 bacterial and archaeal cells in each gram of soil. Insights into microbial community composition and the factors that determine it could therefore improve our understanding of biogeochemical processes, food web dynamics, biodegradation processes, and overall soil quality (Drenovsky, 2014). However, which parts of the microbial communities are beneficial or could help reduce the effects of soil borne plant pathogens, due to soil flooding and rice cultivation, are still unknown.

Under anoxic conditions prevailing during flooded rice cultivation, the microbial community can be expected to shift from predominantly aerobic microbes capable of braking down complex organic matter to predominantly facultative or strict anaerobic microbes (i.e., fermenting microbes, methanogenic archaea, and sulfate-reducing bacteria) (Drenovsky, 2014). We hypothesized that these shifts from aerobic microbial communities to predominantly anaerobic microbes should be associated with shifts in

phylogenetic composition of the overall microbial communities by comparing microbial communities before and after rice cultivation.

Microbial communities in flooded-rice paddy soils

Rice is staple crop and the world's most important agronomic plant.

Approximately 143 million ha rice are planted globally each year (Liesack et al., 2000).

About 75% of the rice is grown under inundated and hence anoxic conditions in the soil.

Rice cultivation is therefore considered a significant source of methane (CH4) emissions

(Liesack et al., 2000). Rice field soils and their microbial communities have been studied extensively in order to understand the processes leading to methane production, but also to gain general understanding about both structure and functional relationships between microbial groups and interactions of microorganisms with rice plants and anaerobic soil conditions (Liesack et al., 2000).

Flooded rice paddy soil can be considered as a compartmentalized system, with three compartments characterized by various physiochemical conditions: oxic surface soil, anoxic bulk soil, and rhizosphere soil plus rhizoplane (See Fig.3-1) (Liesack et al.,

2000). This compartmentalized system, defined microscale chemical gradients can be measured and give knowledge about the activity and spatial distribution of functional groups of microbes (Liesack et al., 2000).

Figure 3-1: Upper panel: Cross-section through a drained rice microcosm. The rice was cultivated for 90 days under flooded conditions in the greenhouse. The separation of the rice paddy soil into at least two distinct zones, i.e., the rhizosphere soil plus root system versus non-rooted, anoxic bulk soil, can clearly be discriminated. Lower panel: Schematic cross-section through the compartmentalized rice paddy soil. Distinct compartments: oxic–anoxic interfaces (red-colored=uppermost mm of the surface soil and the narrow regions around the root system) versus anoxic bulk soil (=reduced soil). Redox reactions characteristic of oxic and anoxic zones, respectively, are shown. (After Liesack et al., 2000).

Rice paddy soils harbor a large variety of microorganisms including many uncharacterized lineages. Several metabolic groups of microorganisms that are well whose ecological roles in rice paddy soils have been investigated in detail in the past will be introduced in the following section. Known metabolic groups of microorganisms present in rice paddy soils include oxygen respiring bacteria, nitrate reducers, iron reducers, sulfate reducers, acetogenic bacteria, fermenting bacteria and methanogenic

(CH4-producing) archaea (Liesack et al., 2000). Biogeochemical cycling like carbon,

nitrogen, sulfur, and iron cycle in rice paddy soil is carried out by the concerted action of these different functional groups of microorganisms (Liesack et al., 2000). After flooding of the rice fields oxygen is depleted fast in most regions of the soil through consumption of oxygen by aerobic bacteria. In the anoxic zone alternative electron acceptors are used by microorganisms in order of decreasing redox potential beginning with nitrate reduction, followed by manganese (Mn IV) and iron (Fe III)-reduction, and ultimately

2- sulfate (SO4 ) reduction and methanogenesis (Fig. 3-2) (Liesack et al., 2000).

Figure 3-2: Oxidation–reduction couples of various electron acceptors arranged from the strongest oxidants (positive reduction potentials) at the top to the strongest reductants (negative reduction potentials) at the bottom (after Liesack et al., 2000).

Denitrification

- Denitrification, the sequential reduction of NO3 via NO2 , NO, N2O to N2 is the most energetically favorable microbial respiration after aerobic respiration and the main biological process removing reactive nitrogen from the biosphere and returning it to the atmosphere as N2. However, with N2O as an intermediate in the pathway, it is also

associated with significant emissions of nitrous oxide (N2O). Denitrification is widespread in anoxic niches in soils and enhanced in waterlogged soils and wetlands

(Madigan et al. 2020). Similar to the direct or indirect reduction of manganese and iron, denitrification is widespread among many facultative anaerobic bacteria and archaea and not associated with specific microbial phyla. Recent studies have shown that the genes for denitrification are scattered among microbial lineages with many microbes containing only the partial pathway or individual enzymes (e.g., nitrate reductase, or nitrite reductase) (Kuypers et al., 2018). Detection of bacteria capable of these metabolisms by molecular techniques therefore requires the analysis of specific marker genes involved in these process (Bertagnolli et al., 2018).

In contrast to denitrifying microbes, the microbial capacity for sulfate reduction and methanogenesis is evolutionarily old and restricted to specific microbial groups

(Madigan et al., 2020). The capacity for sulfate reduction in temperate environments is found within specific clades of the bacterial phyla Deltaproteobacteria, Firmicutes, and

Nitrospira, whereas methanogenesis is restricted to the archaeal phylum Euryarchaea.

Following the depletion of inorganic electron acceptors nitrate, Mn(IV) and Fe(III) and further reduction of the redox potential microbial fermentations together with sulfate reduction or methanogenesis (reduction of bicarbonate to methane) will become the primary anaerobic microbial processes (Oremland 1988; Boone 1991; Crill et al., 1991).

Methanogenic habitats are found in many freshwater environments (sediments, wetlands, swamps, paddy fields, etc.), intestinal tracts of higher animals and insects, landfills, and anoxic soils (Oremland 1988; Boone 1991; Crill et al., 1991). In rice paddy soils the availability of sulfate for respiration is limited. Therefore, the fermentative

degradation of organic matter is accomplished by the mutualistic interaction between fermenting, acetogenic and methanogenic microbes based on interspecies hydrogen and formate transfer (Oude-Elferink et al., 1994).

Fermenting Bacteria

Fermenting bacteria such as Clostridia and Anaerolinea ferment polysaccharides and sugars into organic acids (e.g., lactic acid, acetic acid, butyric acid) and H2.

Acetogenic bacteria are also widespread in anoxic environments and are an essential link in the anaerobic mineralization of organic matter. Acetogenic bacteria are specialized in the oxidation of H2 released from fermentation reducing CO2 to acetate.

The acetate released can then be oxidized by sulfate-reducing bacteria or acetoclastic methanogens to complete the pathway to methane (Madigan, 2020).

The growth of acetotrophic sulfate-reducing bacteria is dependent on the acetate and sulfate concentration, whereas the growth of aceticlastic methanogenic bacteria is solely dependent on the concentration of acetate. At low sulfate concentrations the growth of the sulfate-reducing bacteria will be sulfate-limited, which in turn favors methanogens in the competition for acetate and H2. In the absence of sulfate, some sulfate-reducing bacteria can also grow by fermentation of e.g. pyruvate, lactate, ethanol, fumarate, malate, fructose, serine, choline, acetoin to acetate and hydrogen or acetogenesis (Widdel and Hansen (XX), making them the most flexible strict anaerobes.

However, under those conditions they often rely on syntrophic consumption of hydrogen and acetate by methanogens (Meyer et al., 2013).

Hypotheses and Objectives

The overall goal of this study was to investigate how microbial communities in

EAA soils respond to rice cultivation and whether this could have an influence on nutrient cycling and soil fertility. We hypothesized that the microbial communities would change in species composition between aerated EAA soils and cultivation of flooded rice. To test this hypothesis, we selected 21 farm plots within the EAA that were planted with flooded rice during the summer of 2018, and 7 additional control plots (4 flooded fallow plots and 3 dry fallow plots). Each of the plots were sampled prior and after flooding or fallow treatment and the microbial community structure was determined using 16S rRNA gene amplicon sequencing.

Specific objectives:

1. Determine how contrasting known metabolic groups of microbes respond to flooded rice cultivation. In this analysis we selected strictly anaerobic microbes (fermentative clostridia, sulfate-reducing bacteria, and methanogens), as well as nitrate-reducing methane oxidizers, and strictly oxygen dependent Thaumarchaeotal ammonia oxidizers.

2. Examine the overall structure of microbial communities to evaluate whether the rice cultivation would have a significant effect on overall microbial community structure.

For Objective 1 we focused on specific metabolic microbial groups that could be identified in 16S rRNA gene sequencing datasets and represent distinct physiological groups of microbes. As described above, fermenters, sulfate-reducers and methanogens represent important microbial groups in flooded rice paddy soils, whereas they represent a minor fraction of microbes in well aerated upland soils. We expected that these microbial groups would strongly benefit from flooded rice cultivation and possibly also from the flooded fallow treatment, whereas we did not expect significant changes in dry fallow soil treatments.

We further included two contrasting microbial groups that are often found in upland soils in different niches. Nitrate-reducing methane oxidizers, a group of denitrifying bacteria within the bacterial phylum Rokubacteria that is capable of oxidizing methane in the absence of oxygen represents a group of microbes that is often found under denitrifying conditions in soils and wetlands. We expected these organisms to become more significant during rice cultivation due to expected increase in methanogenesis and oxygen limitation of strictly aerobic methane oxidizers. Lastly, we included ammonia-oxidizing archaea within the archaeal phylum Thaumarchaeota.

Ammonia-oxidizing archaea represent the most common nitrifiers and one of the most frequent microbial groups in upland soils and is only known to grow in the presence of oxygen. Ammonia-oxidizing archaea could thus be expected to decline in abundance under anoxic conditions.

For Objective 2 we conducted a genus-level comparison of microbial communities prior and after flooded rice cultivation to detect which microbial taxa independent of specific metabolic function would increase or decrease during rice cultivation. We used a combination of principal coordinate analysis and differential abundance analysis to identify whether overall microbial communities changed significantly, and which specific taxa could be identified.

Methods

Soil Sampling and Analysis

We sampled organic soils (order: Histosols) from 28 farm plots located in various farm regions in the EAA between March 2018 and September 2018. Locations, depth, and soil series are given in Table 2-1 and Appendix Table 3-1. The soil series mainly varied in depth of the O horizon, percent of organic matter, and underlying parent 77

materials. Comparison of GPS coordinates taken at each sampling location and web soil survey (WSS) data indicated that the 28 farm plots included Lauderhill, Pahokee,

Terra Ceia soil. However, based on soil depth measurements, we assigned different soil series including the Dania series for some samples as they became shallower (Table 2-

1). We defined shallow soils in our study as having a depth of equal or less than 20 inches (51 cm) and deep soils as having a depth higher than 20 inches (>51 cm) with a total of 12 deep soils and 9 shallow soils. Soil samples were analyzed for pH, total N and C and organic matter content, macronutrients (P, K, Ca, Mg), micronutrients (Mn,

Cu, Fe, Zn), and Si content (see Chapter 2).

From each plot soils were sampled at two sites at opposite ends of the plot and labeled “A” and “B”. Soil samples were taken after the pre-crop had been harvested and before rice had been planted or prior to flooding (“pre”), and after the rice has been grown and field drained from flooding (“post”). Independent of soil depth the top ~ 15 cm were sampled and placed in a sterile plastic bag and immediately refrigerated on wet ice before returning to the EREC laboratory. In the laboratory, soil samples were homogenized thoroughly in the sterile plastic bag to obtain a uniform sample. Soil samples for molecular analyses were stored in the -80°C freezer prior to DNA extractions. The samples were homogenized well to ensure an accurately distributed sample.

DNA Extractions

DNA extractions were performed at the University of Florida Fort Lauderdale

Research and Education Center (FLREC). To prepare for DNA extraction, a mortar and pestle were wrapped in aluminum foil (evenly distributes heat) and autoclaved for 20 mins. About 10 grams of each soil sample were placed inside the mortar and ground 78

with the pestle under liquid nitrogen (LN2). DNA was isolated from approximately 0.5 g soil using the Qiagen Soil DNAeasy Powersoil Kit following the manufacturers protocol.

DNA concentrations of the purified DNA were measured using a Nanodrop Analyzer and DNA concentrations were normalized to 20 ng/µl and samples were aliquoted and inserted into a 96-well skirted PCR plate and shipped on dry-ice overnight to Argonne

National Laboratory in Chicago, Illinois for further analysis.

PCR products for 16S rRNA gene amplicon libraries were generated following

Earth Microbiome Project protocols (Thompson et al., 2018). A fragment of the 16S rRNA gene was amplified from each sample using primers 515f and 818r. Triplicate

PCR products were then pooled in equimolar amounts, purified using AMPure XP

Beads (Beckman Coulter, Brea, CA), quantified by Qubit DNA quantification kit

(Invitrogen, Carlsbad, CA), diluted, denatured, and then diluted to 6.75 pM final concentration with 10% PhiX spike. Sequencing was performed on an Illumina MiSeq instrument using 150 cycle MiSeq Reagent kit v3 (Illumina, San Diego, Ca).

The sequence data were examined with QIIME 1.9.1 program to draw conclusion about changes to carbon and nutrient microbial community structure due to flooded rice cultivation. QIIME is a bioinformatics program package utilized for performing microbiome analysis. After removing the sequencing barcodes and quality filtering, the samples were demultiplexed and sequences were grouped into operational taxonomic units (OTU). Taxonomic classification was assigned to each OTU using the Silva 132 dataset. We removed OTUs that only occurred as single sequences once in the whole dataset or that had no classification at least to the level of “Archaea” or “Bacteria”, i.e.

were “Unassigned”. Then we calculated the total number of remaining sequences in each sample and converted the abundances of each OTU into relative abundance (%).

We then investigated the relative abundance of specific bacterial and archaeal communities within the soil before and after the cultivation of flooded rice or fallow period. We focused on 5 specific groups that include methanogens, strictly anaerobic fermentative Clostridia, sulfate reducing bacteria, anaerobic nitrate-reducing methane oxidizers in the bacterial phylum Rokubacteria and ammonia-oxidizing

Thaumarchaeota.

Statistical analyses

Alpha and beta diversity metrics were calculated for genus-level taxonomic distributions using the package core_metrics_phylogenetic in Qiime 1.9.1. Principal coordinate analysis of weighted Unifrac distance matrix was then plotted in R version

3.6.3.

Results and Discussion

We analyzed microbial communities from 28 different farm plots in 7 different farm regions across the EAA before and after flooded rice cultivation to investigate impacts of flooded rice cultivation on soil microbial communities. This analysis included

21 plots with flooded rice cultivation and four control plots with flooded fallow and three plots with dry fallow treatment. The phylogenetic composition of the microbial communities was analyzed by 16S rRNA gene amplicon sequencing and bioinformatic analysis. We expected microbial communities to change during the cultivation of flooded.

Comparative analysis of selected metabolic groups of microbes

In our analysis we first focused on comparing and contrasting five distinct metabolic groups of microorganisms that can be tracked by 16SrRNA gene sequences and are examples of strictly anaerobic microbes, nitrate-reducing methane oxidizers, and strictly oxygen dependent microbes. The five metabolic groups of microbes observed include: clostridia, sulfate-reducing bacteria, methanogens, nitrate-reducing methane oxidizers in the Rokubacteria, and ammonia-oxidizing Thaumarchaeota. Next, we analyzed the overall structure of the microbial communities and evaluated whether the cultivation of flooded rice had an impact on the overall microbial communities within the 28 field plots.

Strictly-anaerobic fermenting Clostridia

The overall relative frequency of Clostridia in 16S rRNA gene amplicon sequence data ranged from 0.05-0.5% (Fig. 3-3A). In shallow and deep muck soils Clostridia ranged from in 0.05-0.5% (samples 1-3, 7-9,23-26 shallow; samples 4-6.16-22 deep), whereas in organic muck samples (13-15 deep) the relative abundance of Clostridia ranged from 0.15-0.35%, and shallow and deep sandy muck soils from 0.2-0.34%

(sample 10 shallow, 11-12 deep). After rice cultivation the overall abundance of

Clostridia did not change consistently among soils. It seems as though in deep muck the relative abundance of clostridia remained consistent pre and post flooded rice cultivation in seven out of ten soils and only increased in the remaining three soils.

Surprisingly, Clostridia abundance dropped in seven out of 12 shallow soils, and increased in five soils with doubling in three samples. Some fields were very shallow and might not have had consistent enough flooding for the Clostridium to thrive. Organic

muck samples 14 and 15 were very similar with almost the same amount clostridia taxa pre and post flooding. Sandy muck (10-12) were inconsistent.

As clostridia are characterized as strictly anaerobic inhabitants of anoxic bulk soils, their abundance could be expected correlate well with major environmental factors indicative of strict anoxia in flooded soils. We would have expected to see more significant increases of Clostridia abundance following flooded rice cultivation. It is possible that in many soils the flooding was not consistent or long enough to allow for a more consistent and significant increase of Clostridia over time. Alternatively, Clostridia might not have been successful under anoxic conditions in our soils due to competition with other fermenting or acetogenic microbes, for example due to the lack of appropriate substrates (e.g. xylan, pectin, and cellulose).

Figure 3-3: Relative abundance of Clostridia A), sulfate-reducing Deltaproteobacteria B), and methanogenic archaea C) prior and post flooded rice cultivation in EAA soils. Given are the relative percentages of 16S rRNA gene sequences of microbes falling into each of these metabolic groups.

Sulfate reducing bacteria (SRB)

Relative abundance of sulfate-reducing bacteria (SRB) within the

Deltaproteobacteria in shallow and deep muck soils ranged from in 0.3-1.2% (samples

1-3, 7-9,23-26 shallow; samples 4-6.16-22 deep) (Fig. 3-3 B). Relative abundance of

SRB ranged from 0.8-1.45% in deep organic muck (samples 13-15 deep) and from 0.4-

0.65% in shallow and deep sandy muck soils samples (sample 10 shallow, 11-12 deep)

(Fig:3-3 B). Significant increase of SRB were only seen in nine out of 28 samples and only in the sandy muck group all samples behaved identically and increased. Whereas in all other remaining 19 samples the number of SRB either stayed the same (only in first group decreased in more than 50% of the samples). In sandy muck, SRB were lowest on average pre rice and increased in all 3-field plots, whereas SRB increased consistently in any other group. Similar to Clostridia, in the first group shallow muck we observed several samples with decreased in SRB following rice cultivation. In most shallow and deep muck samples SRB stayed at the same level. Only in a few samples

SRB approximately doubled in relative frequency. In organic muck SRB increased in two out of three samples. All three of the sandy muck samples saw a significant increase in sulfate reducers.

Methanogens

Relative abundance of methanogens in shallow and deep muck soils ranged from in 0-0.001% (samples 1-3, 7-9,23-26 shallow; samples 4-6.16-22 deep) (Fig:3-3C).

In deep organic muck samples relative abundance ranged from 0.001-0.006% (13-15 deep) and in shallow and deep sandy muck soils samples 10-12(sample 10 shallow, 11-

12 deep) from 0-0.0015 % (Fig. 3-3C). Overall, the abundance of methanogens was 84

more than 2 orders of magnitude lower than of SRB. We expected the methanogen community to increase significantly due to soil flooding. However, only in organic muck was there a consistently detectable methanogen community pre and post flooded rice, whereas in most other samples methanogens were too low in abundance to. And only in organic muck and sandy muck the abundance of methanogens significantly increased following rice cultivation. In sandy muck showed a small amount of growth only in post flooded rice cultivation (samples 10-12).

We hypothesized that clostridia, sulfate reducers, and methanogens would be good indicator groups for this study because they are strict anaerobes at the low end of the redox spectrum and typically found in rice paddy soils. The abundance of all three groups changed much less than expected with only a few exceptions in deeper soils.

The fact that neither Clostridia nor SRB consistently increased following flooding and rice cultivation might suggest that the redox potential did not drop low enough for these groups to thrive. In some deeper soils perhaps, the redox potential did drop lower than in shallow soil fields. One possible reason for this could be the relatively high nitrate concentrations in EAA soils that may have been sufficient as electron acceptor for organic matter mineralization via denitrification.

Many sulfate-reducing bacteria can use H2 or acetate as a source of electrons, and they are more efficient in the uptake and metabolism of both substrates than methanogens engaging in CO2 reduction (Kristjansson et al., 1982, Achtnich et al.,

1995). Consequently, in most environments SRB outcompete methanogens and there is relatively little between the zone of methanogenesis and the zone of sulfate reduction in sediments (Lovley and Phillips 1986, Kuivila et al., 1989). In fact, sulfate inputs to

freshwater wetlands via acid deposition have been suggested to be sufficient to suppress CH4 flux (Dise and Verry 2001, Gauci et al., 2004, Gauci and Chapman 2006).

There was no significant increase in methanogens in shallow and deep muck soils. Besides a lack of sufficiently low redox potential, perhaps the moisture content could be another contributor to the lagging of methanogens throughout all the groups. In the sandy muck soils the moisture content was low pre-flooding and increased from an average of 21.5% pre-flooding to 38.8% post flooding, changing by about 18%, possibly raising the moisture level above the threshold level to form sufficient anoxic niches for methanogens to grow. In the three fields (13-15) that were organically managed the moisture content was 51.5% and did not change significantly pre and post rice cultivation. However, the not organically managed plots in the WOA area had significantly lower abundance of methanogens and the comparison of WOA rice plots and WOA fallow plots showed no difference in OM content or moisture content, suggesting that moisture alone was not a key factor and other unknown factors contributed to the increase of methanogens in the organically managed soils. Another possible reasons we did not see methanogens thrive could be due to the depth of the soil and the flooding period being too short to shift from primarily aerobic degradation to methanogenic degradation of organic matter. Once soils become saturated methanogenesis is not the first process in anaerobic OM mineralization (He et al.,

2010). Nitrate, ferric iron, and sulfate play vital roles in the mineralization process, especially during the beginning flooded stages of rice fields when nitrate, ferric iron, and sulfate have not been completely depleted (Yao et al., 1999). Only after these electron

acceptors have been depleted, methanogenesis would become the dominant terminal process in organic matter mineralization.

According to Yao et al., 1999, anaerobic soils which include rice paddies contribute between 60-80% of the global methane emission to our atmosphere. Most anaerobic microorganisms, including methanogenic bacteria, can survive aeration and desiccation and recover after the soils are flooded (Fetzer et al., 1993; Fukui & Takii

1990; Furusaka et al., 1991). Our results of the comparison of methanogen microbial community before and after flooded rice cultivation in shallow and deep soils suggest that methanogenesis was likely not a significant process for organic matter decomposition during flooded rice cultivation in the EAA. Methanogens were present at low numbers and only longer flooding periods and depletion of nitrate and other electron acceptors may have led to more significant increase of methanogenesis over time.

Figure 3-4: Relative abundance of methane-oxidizing Rokubacteria (A) and ammonia- oxidizing Thaumarchaeota (B) prior and post flooded rice cultivation in EAA soils. Given are the relative percentages of 16S rRNA gene sequences of microbes affiliated to each of these metabolic groups.

Denitrifying methane-oxidizing Rokubacteria.

The relative abundance of Rokubacteria in shallow and deep muck soils ranged from in 0.05-0.44% (samples 1-3, 7-9,23-26 shallow; samples 4-6.16-22 deep) (Fig. 3-4A). In deep organic muck samples (13-15 deep) their relative abundance ranged from 0.14-

0.3%, and in sandy muck soils (sample 10 shallow, 11-12 deep) from 0.05-0.2%.

Notably, there were no Rokubacteria in two out of three sandy muck soils pre-rice, and at least doubled post rice cultivation. These results suggest that under the flooded

conditions in sandy muck soils Rokubacteria were able to thrive. In contrast, no clear trend was seen in Rokubacteria abundance in the other soils with both increases and decreases in some plots within shallow and deep muck soils. Although Rokubacteria remain poorly characterized and significant unknown metabolic diversity may exist within this group, members of this group have been shown to carry out anaerobic nitrate-dependent methane oxidation and are often found under denitrifying conditions in soils and wetlands, suggesting that anaerobic methane oxidation was likely occurring within these soils. However, we expected these organisms to become more significant during rice cultivation due to oxygen limitation of strictly aerobic methane oxidizers. The variability of Rokubacteria abundance in our soils post-rice cultivation might suggest that other unknown factors must contribute to their ecological success.

Ammonia-oxidizing Thaumarchaeota

Among the five metabolic groups of microbes, Thaumarchaeota were the most abundant. Their relative abundance in shallow and deep muck soils ranged from in 1.2-

5.6% (samples 1-3, 7-9,23-26 shallow; samples 4-6.16-22 deep) (Fig. 3-4B). In deep organic muck (13-15 deep) and sandy muck soils (sample 10 shallow, 11-12 deep) their relative abundance ranged from 0.9-2.4% and 1.1-4.0 %, respectively. Indeed,

Thaumarchaeota showed the most consistent response to flooding and rice cultivation among all five groups, with between 20 and 70% drop in abundance in all but one shallow and deep muck soil plots cultivated with rice, while remaining of similar abundance or only slightly decreasing in the flooded fallow or fallow treatments. In sandy muck their relative abundance decreased consistently by more than 50%.

Thaumarchaeota are known to oxidize ammonia aerobically, hence contributing to the nitrate pool in EAA soils. Ammonia-oxidizing Thaumarchaeota are not known to be 89

capable to thrive under anoxic conditions. The consistent decrease in Thaumarchaeota abundance therefore strongly suggest that most of the soils experienced sufficient anoxia to inhibit ammonia oxidation and growth of Thaumarchaeota.

Conclusion

Modern molecular methods such as High-throughput 16Sr RNA gene amplicon sequencing allows to investigate microbial communities in great detail. In soil microbes of all three domains (Archaea, Bacteria, and Eukaryotes) are involved in soil microbial processes and their enzymatic activities and nutrient cycling are influenced by many environmental factors including temperature, moisture, pH, soil organic matter, and the abundance of microorganisms in the soil.

While strictly anaerobic microbes such as sulfate reducers and methanogens did not increase in abundance significantly, the pre- and post-treatment comparison of all microbial genera showed that strictly aerobic taxa declined and facultative and some strictly anaerobic taxa increased in abundance indicating that the flooded rice cultivation did alter the overall microbial community structure and that the soils were largely devoid of oxygen. However, sulfate reduction and methanogenesis were not significant as data imply that the redox potential did not drop below 200mV. These results suggest that production of flooded rice may not enhance methane production and might have the potential to reduce ammonia oxidation in the EAA organic soils.

Taken together, the decreasing abundance of strictly aerobic ammonia-oxidizing

Thaumarchaeota, and lack of increasing population sizes of strictly anaerobic microbial groups (Clostridia, SRB, and methanogens) suggest that the redox that the soils investigated experienced strict anoxia during rice cultivation. This suggests that the redox potential in the soils did not decrease below the level of denitrification or 90

manganese and iron reduction, not sufficient to allow growth of sulfate reducers and methanogens. Further research might be needed to determine which anaerobic microbial respirations were the dominant microbial processes. These results are in stark contrast to conventional rice paddy soils, where sulfate reduction and methanogenesis are the main respiratory processes during organic matter mineralization. Perhaps only after longer flooding methanogenesis would have become significant.

Comparison of overall microbial community structure: Whole microbial community structure of all 56 composite soil samples were compared using principal coordinate analysis based on weighted Unifrac distance matrix of a total of 2526 genus- level microbial taxa (Fig. 3-5). The ordination plot showed that most microbial communities changed in composition pre- and post-flooded rice cultivation. Most shallow and deep muck soils fell into one cluster and consistently moved along PC1 after rice cultivation or flooding. Interestingly, the organically-managed muck, and the sandy muck soils microbial communities changed more significantly than the other shallow and deep muck soils. Whereas “sandy muck” was characterized by lower organic matter contents than the other two soil types, there was no obvious difference between the organically-managed muck soils and the other muck soil in the area. And it is unclear which factors contributed to the large shifts in those soil communities.

Figure 3-5: Principal coordinate analysis of EAA soil microbial communities based on weighted Unifrac distance matrix of 16S rRNA gene sequences before (pre) and after (post) treatment. The explained variation of the first two principal components are given in %. Outer ring color denotes pre-and post-treatment and colors refer to soil types. Numbers refer to soil plots as follows: 1. Shelton 68-11-3, 2. Shelton 68-11-5, 3. Shelton 68-11-7, 4. East Area CD18N, 5. East Area CD19N, 6. East Area CD20N, 7. Southern Ranch Lease X6SW9, 8. Southern Ranch Lease X6SW10, 9. Southern Ranch Lease X6SW11, 10. Manley(Blumberg) 64-E-11, 11. Manley(Blumberg) 64-B-11, 12. Manley(Blumberg) 64-F-11, 13. West Organic Area 2ABE-W, 14. West Organic Area 3ABE-W, 15. West Organic Area 4ABE-W, 16. West Organic Area 26ABW, 17. West Organic Area 27ABW, 18. West Organic Area 28ABW, 19. Zone I 28-CD-7S, 20. Zone II 38-IM-14E, 21. Veg 1 Roth, 22. Veg 2 Roth, 23. King Ranch 55320 1372, 24. King Ranch 55340 1372, 25. King Ranch 52720 1252, 26. King Ranch 52740 1252, 27. King Ranch 40920, 28. King Ranch 41140.

We further did a pre- and post-treatment comparisons for genus-level microbial communities in each of the three groups “muck”, “sandy muck, and “organic muck”.

Figures 3-6 to 3-8 show post- versus pre-treatment comparison of log-transformed genus-level relative abundances for each of the three groups. This pre-post

comparison showed that primarily genera of predominantly aerobic microbial genera, e.g. Rhizobium sp., Massilia sp., Devosia sp., Lysobacter sp., Ca. Nitrosotenius sp., declined in relative abundance by at least 50% in all three groups (genera labeled in

Fig. 3-6 to 3-8). In contrast, genera that increased during treatment were almost exclusively genera known to be facultative or strict anaerobes or genera typically found in anoxic wetlands or freshwater sediments (e.g. Anerolinea sp., Ignavibacterium sp.,

Ellin6067 group, Geobacter sp., Aneromyxobacter sp.). Furthermore, detailed analysis of these genera may be required to identify trends of microbial metabolisms in these genera. Nonetheless, these results are further indication that flooded rice cultivation had a significant effect on many microbial taxa and shifted microbial community metabolism from primarily aerobic to primarily anaerobic organic matter degradation.

Figure. 3-6: Pre- and post-treatment comparison of genus-level microbial taxa in conventionally managed muck soils in the EAA. Plotted were log-transformed relative abundances of post-treatment versus pre-treatment microbial communities. Genera that increased or decreased in relative abundance by at least 50% are labeled. 93

Figure. 3-7: Pre- and post-treatment comparison of genus-level microbial taxa in conventionally managed sandy muck soils in the EAA. Plotted were log- transformed relative abundances of post-treatment versus pre-treatment microbial communities. Genera that increased or decreased in relative abundance by at least 50% are labeled.

Figure. 3-8: Pre- and post-treatment comparison of genus-level microbial taxa in organically managed muck soils in the EAA. Plotted were log-transformed relative abundances of post-treatment versus pre-treatment microbial communities. Genera that increased or decreased in relative abundance by at least 50% are labeled.

APPENDIX A FARM REGIONS AND MICROBIAL GROUPS

Table A-1: Sample number, Farm Region and Farm plot shown for the 28 soil samples analyzed in the EAA. Previous crop planted before flooded rice cultivation(or flooded fallow and fallow) shown as well as soil type, depth, and the sampling dates before the rice was planted and after the field was drain post flooding and before rice was harvested.

Sample Farm Region Farm Plot Company Pre-crop Post-crop Soil Type Soil Depth Pre-Rice Sampling Date (2018) Post-Rice Sampling Date (2018) 1 Shelton 68-11-3 Florida Crystals Sugarcane Rice Muck Shallow First week of March July 23rd 2 Shelton 68-11-5 Florida Crystals Sugarcane Rice Muck Shallow First week of March July 23rd 3 Shelton 68-11-7 Florida Crystals Sugarcane Rice Muck Shallow First week of March July 23rd 4 East Area CD18N Florida Crystals Sugarcane Rice Muck Deep Mid March July 23rd 5 East Area CD19N Florida Crystals Sugarcane Rice Muck Deep Mid March July 23rd 6 East Area CD20N Florida Crystals Sugarcane Rice Muck Deep Mid March July 23rd 7 Southern Ranch Lease X6SW9 Florida Crystals Sugarcane Rice Muck Shallow Mid March Mid August 8 Southern Ranch Lease X6SW10 Florida Crystals Sugarcane Rice Muck Shallow Mid March Mid August 9 Southern Ranch Lease X6SW11 Florida Crystals Sugarcane Rice Muck Shallow Mid March Mid August 10 Manley(Blumberg) 64-E-11 Florida Crystals Sugarcane Rice "Sandy" Muck Shallow Mid May August 15th 11 Manley(Blumberg) 64-B-11 Florida Crystals Sugarcane Rice "Sandy" Muck Deep Mid May August 15th 12 Manley(Blumberg) 64-F-11 Florida Crystals Sugarcane Rice "Sandy" Muck Deep Mid May August 15th 13 West Organic Area 2ABE-W Florida Crystals Sugarcane Rice "Organic" Muck Deep July 24th October 8th 14 West Organic Area 3ABE-W Florida Crystals Sugarcane Rice "Organic" Muck Deep July 24th October 8th 15 West Organic Area 4ABE-W Florida Crystals Sugarcane Rice "Organic" Muck Deep July 24th October 8th 16 West Organic Area 26ABW Florida Crystals Sugarcane Fallow Muck Deep July 24th October 8th 17 West Organic Area 27ABW Florida Crystals Sugarcane Fallow Muck Deep July 24th October 8th 18 West Organic Area 28ABW Florida Crystals Sugarcane Fallow Muck Deep July 24th October 8th 19 Zone I 28-CD-7S Florida Crystals Corn Rice Muck Deep March 8th August 29th 20 Zone II 38-IM-14E Florida Crystals Corn Rice Muck Deep March 8th August 29th 21 Veg 1 Roth Celery Roth Roth Farms Celery Rice Muck Deep June 5th September 25th 22 Veg 2 Roth Lettuce Roth Roth Farms Lettuce Rice Muck Deep June 5th September 25th 23 King Ranch 55320 1372 King Ranch Farms Sugarcane Fallow/Flooded Fallow Muck Shallow May 9th September 25th 24 King Ranch 55340 1372 King Ranch Farms Sugarcane Fallow/Flooded Fallow Muck Shallow May 9th September 25th 25 King Ranch 52720 1252 King Ranch Farms Corn Fallow/Flooded Fallow Muck Shallow May 9th September 25th 26 King Ranch 52740 1252 King Ranch Farms Corn Rice Muck Shallow May 9th September 25th 27 King Ranch 40920 King Ranch Farms Sugarcane Rice Muck Shallow May 9th August 23rd 28 King Ranch 41140 King Ranch Farms Sugarcane Rice Muck Shallow May 9th August 23rd

Table A-2: This table shows the 5 groups we observed in our statistics using a paired t- test and non-parametric testing. We used color coding to sort through our data. Methanogens, Clostridia, Sulfate-reducing bacteria, Methylomirabilis- related Rokubacteia, and Thaumarchaeotal ammonia oxidizers were the groups we found most relevant to flooded rice production. Group Characteristics Methanogens Strict anaerobic methanogens Clostridia Strict anaerobic fermentative microbes Strict anaerobic sulfate-respiring Sulfate-reducing bacteria microbes Methylomirabilis-related Anaerobic nitrate-reducing methane Rokubacteia oxidizers Thaumarchaeotal ammonia oxidizers Strictly dependent on oxygen

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BIOGRAPHICAL SKETCH

Rachelle Berger has been a student at the University of Florida for the past 5 years, achieving both her undergraduate degree in Environmental Management in in

Agriculture and Natural Resources and her Master’s in Science in Soil and Water

Sciences. She is a mother of two children, and also earned an Associate of Science in

Biotechnology and an Associate of Arts from Palm Beach State College. Rachelle started her undergraduate in 2015 at UF under Professor Curry in the Bachelor of

Sciences in Environmental Management in Agriculture and Natural Resources Major which inspired her to get a Master of Sciences in Soil and Water Sciences from the

University of Florida. Rachelle grew up in south Florida where she spent her summers at the beach, exploring nature trails, and camping. Rachelle always knew her passion was environmental science and decided to dedicate her life to improving agriculture sustainability and our natural resources. She plans to continue her studies for the third time at the University of Florida in the Agronomy Department for doctoral studies.

Rachelle is proud to be a Jewish American. All four of her grandparents were Holocaust survivors in Auschwitz, Poland. Rachelle’s family came to New York after the war in the late 1950s. Her maternal grandfather started his own jewelry business where she started working very young. As a child, she was taught to do the best you can in all endeavors, to work hard, and wake up each morning like it is your last. Her paternal grandfather started buying hotels with his 3 brothers and built a small fortune from scratch which has always inspired her in life. Rachelle’s mother is a registered nurse and would work night shifts in the hospital and study in the daytime. She grew up watching my family work very hard for what that have which is what has fueled my determination to become a doctoral student and contribute my life to my biggest 107

passion, environmentally sustainability. When Rachelle learned that soil, water, air, and sunlight are the fur components we could not live without, it became an easy choice to decide which path she wanted to study. With year 2050 approaching quickly, and the population continuing to rise, she is committed to improving agricultural practices.

Rachelle has two beautiful children (Ava and Isaac) and became a mother at the young age of 23. Despite any obstacles that have come her way, she has not once given up on my dreams, aspirations, and studies. Rachelle is proud to be a ”double” Florida

Gator at UF!

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