Sugarcane Filter Press Mud As a Substrate for Nursery Hardening of Tissue Culture Bananas

SUGARCANE FILTER PRESS MUD AS A SUBSTRATE FOR NURSERY HARDENING OF TISSUE CULTURE BANANAS

NICHOLAS ALEXANDER LARSEN

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2015

To my son, John Harper Larsen

ACKNOWLEDGMENTS

First, I would like to thank God for giving me the endurance and opportunity to complete my education. A path was cleared for me, and all the right people were placed along that path to ensure my success. I thank my wife, Kiley, for her encouragement and insistence that I finish my education. I thank the late John Harper for making the undertaking of a doctorate financially feasible. I thank the University of

Florida for their Employee Education Program that paid for 6 credit hours each semester. I thank Gregg Nuessly for allowing me to further my education while still working for him. I thank Ron Rice, Jim Shine and Chris LaBorde for providing me with samples of Filter Press Mud. Andy Fu was a godsend helping me to get all the nutrient analyses completed in a timely fashion. I thank Kimberly Moore for taking me as a doctoral student and allowing me the freedom to choose a project of my own. I thank the rest of my committee, Timothy Broschat, Samira Daroub, and Ann Wilkie for providing scientific guidance and encouraging me to investigate further. I thank my son

John, who inspired me to expedite my work.

I will lift up mine eyes unto the hills, from whence cometh my help. My help cometh from the Lord, which made heaven and earth. He will not suffer thy foot to be moved: he that keepeth thee will not slumber. Behold, he that keepeth Israel shall neither slumber nor sleep. The Lord is thy keeper: the Lord is thy shade upon thy right hand. The sun shall not smite thee by day, nor the moon by night. The Lord shall preserve thee from all evil: he shall preserve thy soul. The Lord shall preserve thy going out and thy coming in from this time forth, and even for evermore (Psalm 121,

KJV).

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 8

LIST OF FIGURES ...... 12

ABSTRACT ...... 14

CHAPTER

1 BACKGROUND ...... 16

The Horticulture Industry in South Florida...... 16 Water Use in South Florida Nurseries...... 16 Fertilizer Use in South Florida Nurseries ...... 17 Nursery Grower Needs ...... 17 Sugarcane Filter Press Mud: A Potential Solution ...... 18 Research Objectives ...... 18

2 LITERATURE REVIEW ...... 20

Nursery and Container Production of Plants ...... 20 Fertilization in the Nursery ...... 20 Container Nursery Best Management Practices ...... 21 The Ideal Substrate ...... 22 Peat Alternatives ...... 23 Sugarcane Filter Press Mud ...... 25 FPM Impacts on Soils ...... 26 FPM as a Substrate ...... 27 FPM Use in Horticultural Crop Production ...... 28 FPM Use in Landscapes ...... 29 FPM Use in Fruit Tree Nurseries ...... 30 FPM Use in Forest Tree Nurseries ...... 31 FPM Use in Propagation ...... 32 FPM use in Ornamental Nurseries ...... 32 Bananas ...... 33 Banana Propagation and Planting ...... 34 Banana Irrigation Requirements ...... 35

3 PHYSICAL AND CHEMICAL CHARACTERIZATION OF SUGARCANE FILTER PRESS MUD AS A SUBSTRATE FOR ACCLIMATING TISSUE CULTURE BANANAS ...... 40

Introduction ...... 40

Methods and Materials...... 41 FPM Source Nutrient Characterization ...... 42 FPM Age Nutrient Characterization ...... 43 FPM Total Nutrient and Heavy Metal Analysis ...... 44 Statistical Analysis ...... 44 Results ...... 44 General Observations About FPM ...... 44 Chemical Characterization ...... 45 Source Leaching Experiment ...... 46 Age Leaching Experiment ...... 47 Source Physical Characteristics ...... 47 Age Physical Characteristics ...... 47 Discussion ...... 48

4 IRRIGATION REQUIREMENT OF TISSUE CULTURE BANANA PLANTLETS GROWN IN A SUGARCANE FILTER PRESS MUD BASED SUBSTRATE ...... 58

Introduction ...... 58 Methods and Materials...... 59 Results and Discussion...... 63

5 FERTILIZATION OF TISSUE-CULTURE BANANA PLANTLETS GROWN IN SUGARCANE FILTER PRESS MUD BASED SUBSTRATES ...... 79

Introduction ...... 79 Methods and Materials...... 80 Results and Discussion...... 82

6 NUTRIENT LEACHING FROM TISSUE-CULTURE BANANA PLANTLETS GROWN IN SUGARCANE FILTER PRESS MUD BASED SUBSTRATES ...... 93

Introduction ...... 93 Methods and Materials...... 93 Results ...... 95 Discussion ...... 96

7 CONCLUSIONS ...... 109

APPENDIX

A IMPACT OF FERTILIZATION ON VINCA GROWN IN BINARY MIXTURES OF SUGARCANE FILTER PRESS MUD AND VERMICULITE ...... 111

Introduction ...... 111 Methods and Materials...... 112 Results ...... 114 Discussion ...... 115

B IMPACT OF SUGARCANE FILTER PRESS MUD ON GROWTH AND DEVELOPMENT OF MARIGOLD ...... 120

Introduction ...... 120 Methods and Materials...... 120 Results ...... 122 Discussion ...... 123

C ELECTRICAL CONDUCTIVITY AND NUTRIENT CONCENTRATIONS IN LEACHATE DURING SUGARCANE FILTER PRESS MUD CHARACTERIZATION ...... 129

D RESULTS OF STATISTICAL TESTS ...... 139

LIST OF REFERENCES ...... 148

BIOGRAPHICAL SKETCH ...... 157

LIST OF TABLES

Table page

2-1 Ideal chemical characteristics for soilless media amended with controlled- release fertilizer (Yeager, 2007). Nutrient values reported in mg/kg...... 36

2-2 Nutrient analyses of sugarcane filter press mud (FPM) reported in publications and research worldwide. Values are percent composition dry matter...... 37

2-3 Heavy metals reported in sugarcane filter press mud (FPM) as compared with established limits...... 39

3-1 Chemical nutrient analysis of 3 sources of sugarcane filter press mud (FPM) collected during 2012-2013 milling season...... 51

3-2 Chemical characteristics of different ages of FPM from sugar mill “B” ...... 52

3-3 Total elemental analysis of sugarcane filter press mud aged at least 12 months collected from sugar mill “B” between 2012 and 2014, as a percentage of dry matter...... 53

3-4 Heavy metals present in FPM from source “B” ...... 54

3-5 Total nitrate-nitrogen(NO3-N), ammonium-nitrogen(NH4-N), phosphate- phosphorus (PO4-P), and potassium (K) leached over 80 days from containers filled with sugarcane filter press mud from three different sugar mills in the Everglades Agricultural Area ...... 55

3-6 Total nitrate-nitrogen(NO3-N), ammonium-nitrogen(NH4-N), phosphate- phosphorus (PO4-P), and potassium (K) leached over 64 days from containers filled with different ages of sugarcane filter press mud from sugar mill “B” ...... 56

3-7 Bulk density, porosity, and air-filled porosity of sugarcane filter press mud (FPM) from 3 different sources and four ages of source “B” ...... 57

4-1 Physical characteristics of sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. FPM was 12 month aged sample from a sugar mill in the Everglades Agricultural Area...... 67

4-2 Daily evapotranspiration measured gravimetrically during 4 days from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction...... 68

4-3 Daily evapotranspiration measured gravimetrically during 4 days from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction...... 69

4-4 Total estimated evapotranspiration measured gravimetrically from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction...... 70

4-5. Plant heights of ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction...... 71

4-6. Stem diameter, dry weights and root-shoot ration of ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction...... 72

4-7 Leaf production of ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction...... 73

4-8 Leaf production of ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction...... 74

4-9 SPAD readings from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction...... 75

4-10 Porometer readings from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction...... 76

5-1 Analyses of the sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite, FPM was 12 month aged sample from a sugar mill in the Everglades Agricultural Area...... 86

5-2 Plant heights of ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container...... 87

5-3 Leaf counts Day 0-28 of Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite Plants were fertilized with 6, 12, or 18 grams of fertilizer per container...... 88

5-4 Leaf counts Day 39-64 of Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container...... 89

5-5 SPAD readings from youngest fully expanded leaf of Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container...... 90

5-6 Stem diameter, dry weights, and root to shoot ratio of ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container...... 92

6-1 Analyses of the sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite, FPM was 12 month aged sample from a sugar mill in the Everglades Agricultural Area...... 100

6-2 Contributions of nitrate-nitrogen, ammonium-nitrogen, phosphorus, and potassium from fertilizer and substrate in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.1 ...... 101

6-3 Estimated weekly leachate from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container...... 102

10 6-4 Nitrogen leached from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container...... 103

6-5 Phosphate-phosphorus and potassium leached from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container...... 104

A-1 pH and available nutrients in sugarcane filter press mud...... 117

A-2 Physical and chemical characteristics of the substrates composed of sugarcane filter press mud and vermiculite...... 117

A-3 Means of growth and development parameters for vinca grown in binary mixtures of sugarcane filter press mud and vermiculite...... 118

A-4 Results of statistical tests...... 119

B-1 Reported nutrient values in Filter Press Mud from various sources...... 125

B-2 Chemical Characteristics of FPM sources used in the experiment...... 126

B-3 Measurements of growth and development for marigold grown in different sources of sugarcane filter press mud...... 127

B-4 Days to wilting for marigold grown in different sources of sugarcane filter press mud ...... 128

D-1 Results of statistical tests performed for physical and chemical characterization of sugarcane filter press mud ...... 139

D-2 Results of statistical tests performed to determine irrigation frequency requirement of bananas produced in sugarcane filter press mud-based substrates and sphagnum peat based substrates ...... 142

D-3 Results of statistical tests performed to determine fertilization requirement of bananas produced in sugarcane filter press mud-based substrates and sphagnum peat based substrates ...... 144

D-4 Results of statistical tests performed to determine nutrient leaching from bananas produced in sugarcane filter press mud-based substrates and sphagnum peat based substrates ...... 146

LIST OF FIGURES

Figure page

4-1 Tensiometer readings from containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Containers were filled to container capacity and allowed to dry down over 25 days...... 77

4-2 Percentage of container capacity remaining from containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Containers were filled to container capacity and allowed to dry down over 25 days...... 78

5-1 Iron deficiency observed on ‘Williams’ bananas grown in sugarcane filter press mud based substrates...... 91

6-1 Nitrate concentrations in leachate from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container...... 105

6-2 Ammonium concentrations in leachate from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container...... 106

6-3 Phosphate concentrations in leachate from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container...... 107

6-4 Potassium concentrations in leachate from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container...... 108

C-1 Electrical conductivity of leachate over 80 days from containers filled with sugarcane filter press mud from three different sugar mills in the Everglades Agricultural Area...... 129

C-2 Nitrate concentrations in leachate over 80 days from containers filled with sugarcane filter press mud from three different sugar mills in the Everglades Agricultural Area...... 130

C-3 Ammonium concentrations in leachate leached over 80 days from containers filled with sugarcane filter press mud from three different sugar mills in the Everglades Agricultural Area...... 131

C-4 Phosphate concentrations of leachate over 80 days from containers filled with sugarcane filter press mud from three different sugar mills in the Everglades Agricultural Area...... 132

C-5 Potassium concentrations of leachate over 80 days from containers filled with sugarcane filter press mud from three different sugar mills in the Everglades Agricultural Area...... 133

C-6 Electrical conductivity of leachate over 64 days from containers filled with different ages of sugarcane filter press mud from sugar mill “B”...... 134

C-7 Nitrate concentrations in leachate over 64 days from containers filled with different ages of sugarcane filter press mud from sugar mill “B”...... 135

C-8 Ammonium concentrations in leachate over 64 days from containers filled with different ages of sugarcane filter press mud from sugar mill “B”...... 136

C-9 Phosphate concentrations in leachate over 64 days from containers filled with different ages of sugarcane filter press mud from sugar mill “B”...... 137

C-10 Potassium concentration in leachate over 64 days from containers filled with different ages of sugarcane filter press mud from sugar mill “B”...... 138

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

SUGARCANE FILTER PRESS MUD AS A SUBSTRATE FOR NURSERY HARDENING OF TISSUE CULTURE BANANAS

Nicholas Alexander Larsen

August 2015

Chair: Kimberly Anne Moore Major: Horticultural Sciences

Sugarcane filter press mud (FPM) is a waste product of the sugar milling industry. Around the world, it is used in nurseries as a component of substrates in container nurseries. It has been used to grow bananas Musa spp. in various countries with contradictory results. The objectives of this research is to determine the suitability of Florida-produced FPM for hardening tissue culture bananas, the optimum irrigation frequency for hardening tissue culture bananas in FPM, the optimum rate of controlled release fertilizer for hardening tissue culture bananas in FPM, and finally, the amount of nutrient leaching that can be expected to occur while hardening bananas in FPM.

Sugarcane filter press mud was characterized chemically and physically to determine its suitability as a substrate. Sugarcane filter press mud was then used as a substrate component for growing banana plantlets Musa acuminata ‘Williams’ (AAA) in a greenhouse. Experiments were designed to determine the optimum irrigation frequency and fertilization rate. For the irrigation experiment, plants were watered on one, two and four day intervals. For the fertilization experiment, a low, medium, and high rate of Osmocote Plus was incorporated at planting. Leachate from the fertilizer

experiment was collected to determine nutrient leaching. These results were compared to those obtained with sphagnum peat.

Physical characterization indicated that bulk density, porosity and air-filled porosity were within the acceptable range for substrates. Chemical characterization indicated that electrical conductivity and pH were initially high, as were nitrate and potassium levels. Sugarcane filter press grown plants evapotranspired less water, but also required daily irrigation. Plant growth increased with increasing fertilization with maximum growth occurring at the highest rate of fertilization. Leaching of nitrates was much higher in sugarcane filter press mud than in sphagnum peat, but nitrogen leaching from nitrates and ammonium was similar. Phosphorus leaching was much lower in sugarcane filter press mud.

Overall, FPM is a suitable substrate for hardening tissue culture bananas.

Growing tissue culture bananas in FPM is a sustainable choice for the grower as it makes use of a waste product, uses less water, and leaches smaller quantities of phosphorus.

CHAPTER 1 BACKGROUND

The Horticulture Industry in South Florida

Horticulture production is a multi-billion dollar industry in southeast Florida. In

2005, the industry generated over $4 billion in sales and $3.6 billion in output impact

(Hodges and Haydu, 2006). Southeast Florida is not only a major consumer of horticultural products and services ($3.1 billion), but also produces $976 million of products from its nurseries. The horticulture industry is a major employer in the region, and in 2005, employed 95,202 people including 18,239 and 29,543 in the nursery and landscape sectors, respectively (Hodges and Haydu, 2006). Container nurseries occupy around 59,000 acres statewide (Yeager et al., 2010), with much of that acreage in southeast Florida. Ornamental nurseries are considered heavy input operations using large amounts of water and fertilizer.

Water Use in South Florida Nurseries

Nurseries are considered major water users in southeast Florida, and most nurseries irrigate using highly inefficient overhead irrigation (Haman and Yeager, 1997).

Containerized plants need to be irrigated with enough water to replace losses due to evapotranspiration. Daily irrigation may range from 0mm-25mm depending on temperature, rainfall, and container size (Personal Communication, David Demaio,

Palm Beach Soil and Water Conservation District). Container BMPs require that no more than 15% of the water applied to a substrate is leached (Yeager, 2007).

Containerized plants are limited in their ability to hold water, so that heavy rainfall one day does not negate the need for irrigation the following day (Cabrera, 1999; Yeager et al., 2010). Recent droughts and irrigation restrictions have increased the use of more

efficient micro-irrigation strategies in those nurseries that have survived the 2008-2009 economic downturn. Micro-irrigation averages 80% application efficiency in a nursery as opposed to overhead irrigation, which averages an application efficiency of only 20%

(Haman et al., 2005).

Fertilizer Use in South Florida Nurseries

The Florida nursery industry purchased $81 million of fertilizer and lime in 2005

(Hodges and Haydu, 2006). Rates for fertilization depend on the type of plant and to a certain extent the type of fertilizer used. Per hectare application rates for nurseries tend to exceed those used for field crops and landscapes. The substrate generally has a low ability to hold and supply nutrients. A hectare will hold 250,000 trade sized 1 gallon plants. For a 3-month containerized ornamental crop that has low nitrogen needs the plant might need 225 kg/ha of nitrogen (N), for medium needs, 450 kg/ha and for high needs, 675 kg/ha. The high nitrogen rate for fertilizing St. Augustine grass lawns is 290 kg/ha/yr, and for sugarcane it is 200 kg/ha/year.

Nursery Grower Needs

Nursery growers and landscape professionals need an organic product that can serve as a substrate and nutrient source. The ideal product would be abundant, cheap, safe to handle, and have characteristics that would be beneficial to plant growth. Peat harvesting disrupts wetlands, and in the long term, transport of Canadian peat to Florida could become cost prohibitive due to increasing transportation costs (McNally, 2003).

Fertilizer demand has increased due to increasing row crop production worldwide. Some fertilizer components have doubled in price over the past 10 years

(USDA, 2012). Controlled release fertilizer (CRF) is more expensive than mineral salt

fertilizer, leading some researchers to start examining alternatives such as pasteurized poultry litter (Broschat, 2008).

Sugarcane Filter Press Mud: A Potential Solution

One possible solution comes from the sugarcane mills in south-central Florida around Lake Okeechobee. Sugarcane filter press mud (FPM) is a waste product of the sugar manufacturing industry that is composed of the solids flocculated out of the sugarcane juice by calcium phosphate. The flocculate is mixed with water and pumped out into settling ponds. FPM is removed from the mill facility and applied to agricultural fields, usually in close proximity to the sugarcane mill (Qureshi et al., 2001). FPM represents a liability to the mill. For each ton of cane processed 0.02-0.06 tons of FPM are produced, depending on sugarcane variety, harvesting practice, and milling practice. Based on this rate, Florida sugar mills produce between 285,000 and 855,000 tons of FPM, annually. The low range of the figure is enough material to fill 285 million trade-sized 1 gallon pots. The mills must pay to put the material in a landfill, or pay to deliver it to growers’ fields. Nutrient contents vary depending on the harvesting and milling practices (Gilbert et al., 2008). Application rates to agricultural fields are usually far in excess of nutrient needs. It is usually measured locally in terms of inches instead of a rate per hectare. In a Florida study, the rate used was 224 t/ha (Gilbert et al., 2008) in a sandy soil, while in Australia, Barry et al. (1998a) reported that 150 t/ha was the predominant application rate in spodosols and ultisols.

Research Objectives

FPM should be tested for adoption by the Florida nursery industry. FPM could replace some container substrate components. Worldwide, it has not had problems with heavy metals. Vast quantities are produced every year. FPM contains nutrients

that are required for plant growth. FPM has been widely tested around the world for a variety of crops. In other areas where sugarcane is grown, FPM constitutes a standard component of substrates. Only limited research on its potential for the Florida horticulture industry has been conducted. Florida produced FPM has not been adequately characterized physically and chemically for use in substrates. It can be assumed that FPM will change the amount of fertilization and irrigation required. It is unknown how much FPM source will impact plant growth. FPM is expected to be mixed with inorganic materials like vermiculite and perlite; therefore, the performance of plants in both lightweight and heavyweight mixtures requires investigation. Finally, environmental concerns require an investigation into the components of the leachate from FPM. Banana (Musa acuminata) was chosen as the test species because it is responsive to water deficits, nutrient deficiency, and has been used in other experiments involving FPM around the world. Tissue culture plants are produced for use throughout the tropics and sub-tropics. These plants must be hardened in a protective structure in order to prepare them for planting in an open-field situation. In order to investigate these issues the following experiments were performed:

1. Characterization and suitability assessment of sugarcane filter press mud as a substrate for growing banana (Musa acuminata).

2. Impact of irrigation frequency on banana (Musa acuminata), grown in sugarcane filter press mud.

3. Fertilization requirements of banana (Musa acuminata) grown in sugarcane filter press mud.

4. Nutrient leaching from bananas (Musa acuminata) grown in sugarcane filter press mud

CHAPTER 2 LITERATURE REVIEW

Nursery and Container Production of Plants

Substrates accounts for 3.4% ($100.8 million) of input purchases by nursery growers (Hodges and Haydu, 2006). Commercial grade media currently sells for up to

$88/m3 (Personal communication, Brenda Sellers, Winfield Solutions). For a trade-sized

1 gallon container substrate would account for $0.25 of the final production cost.

Originally, the substrate for container production would have been soil; however, sterile soilless substrates are the industry standard for the modern container nursery. These substrates will usually be a mixture of organic and inorganic components. The organic components are typically peat, pine bark, saw dust, and coconut coir. The inorganic components are typically sand, vermiculite, and perlite (Bilderback, 1982). The preceding components would be mixed in different ratios in order to give the plant an optimum space to grow. While pine bark and coir are renewable resources that will exist as long as there are pine trees and coconut palms, peats are a limited resource harvested from wetlands. Harvesting of sphagnum peat in Canada and Europe and reed/sedge peat in the southeast USA has caused some environmental concern

(McNally, 2003).

Fertilization in the Nursery

In the container nursery industry, there are several different fertilization practices.

The chosen method of fertilization will depend on plant species and ability to implement the practice. Fertilization practices include top-dressing, incorporation, layering, and fertigation. Container best management practices (BMPs) discourage fertigation due to the potential for off-target application. The use of controlled release fertilizers (CRFs) is

preferred but growth can vary. Yeager et al. (1989) reported that Ligustrum japonicum and azalea (Rhododendron sp.) shoot weight were higher when plastic-coated potassium nitrate was top-dressed. However, with Osmocote, the shoot weights for

Ligustrum were higher when incorporated, but there was no difference in azalea growth between top-dressed and incorporated. Broschat and Moore (2003) also compared top- dressing, incorporation, and layering of CRF and reported that the response was species dependent. For example, Areca Palm (Dypsis lutescens) grew better with CRF incorporation.

Application method can affect leaching as well. Yeager et al. (1989) found that leachates from incorporated fertilizer containers were higher than those that were top- dressed. Fertilization practices should take into consideration the length of the crop when applying CRF. Container BMPs encourage the use of organic substrate components that can contribute nutrients to the plant. Controlled release fertilizers labels recommend rates of incorporation ranging from 0.3 kg/m3 to 1.0 kg/m3. A producer can only layer or incorporate at planting so any supplemental fertilization would have to come through fertigation or top-dressing. The potential of blow-over must be considered when top-dressing fertilizer because it can move off-site when the container is toppled.

Container Nursery Best Management Practices

Container nursery best management practices (BMPs) in Florida are a voluntary program designed to improve environmental water quality, and decrease wasteful water-use (Yeager, 2007). The handbook encourages producers to limit water use and fertilizer use to the minimum required to produce a marketable plant. The recommendations are research-based. The program encourages growers to measure

the amount of water being used for irrigation, the amount of water actually being used by plants, and the amount of water being leached. In Palm Beach County, an employee of the Palm Beach Soil and Water Conservation District visits growers and measures water output, and uniformity by collecting irrigation water. The employee can measure water use gravimetrically and leaching by collecting leachate following irrigation. Some growers use an estimated evapotranspiration method to determine their irrigation needs

(Personal Communication, David Demaio, Palm Beach Soil and Water Conservation

District). Growers are encouraged to store substrates, soil, and fertilizer on impervious covered storage areas. The use of organic sources of nutrients is promoted. Blow-over mitigation is also discussed since lost fertilizer can potentially lead to eutrophication of waterways.

The Ideal Substrate

The ideal substrate would be free of pests, diseases and toxins, and provide water, nutrients, and air in the correct amounts as well as providing support for the plant. The ideal substrate for container plants would have a total porosity of 85%, 20%-

30% air-filled pore space, 20%-30% easily available water (-1 kPa to -5 kPa), and 4%-

10% water buffering content (-5 kPa to -10 kPa) (De Boodt and Verdonck, 1972).

These levels optimize the air and water available to plant roots. Cabrera (1999) reported slightly different amounts with porosity ranging from 70%-85%, 10%-20% air- filled pores, and greater than 30% easily available water. The bulk density of horticultural substrates tends to be much lower than field soils. Yeager (2007), reports a desirable range of 0.19 g/cm3 – 0.70 g/cm3 for horticultural substrate bulk density.

Increased bulk density negatively impacts aeration and water holding capacity

(Fernandes and Cora, 2004) as well as shipping costs. Cabrera (1999) reported that

the optimum particle size was 0.5 mm – 2.0 mm. The ideal pH and EC levels will depend on the plant being grown (Cavins et al., 2000). Yeager (2007) outlines ideal chemical characteristics of soilless media in his BMP manual for Florida container nurseries (Table 2-1).

Peat Alternatives

For many years, researchers have been investigating alternative substrates that make use of waste. Coconut coir and pine bark have been the most successful examples of a waste stream being turned into a substrate. Many of the other waste streams need composting prior to use. Fitzpatrick investigated the use of composted urban waste (1981), solid waste (1989), sewage sludge (Fitzpatrick and Carter, 1983), municipal solid waste, diapers, horse stable waste, yard trash and a cocompost of dewatered municipal sludge and yard trash (Fitzpatrick and Verkade, 1991). Beeson

(1996) examined the use of composted yard waste as a container substrate for azalea

(Rhododendron indicum) and pittosporum (Pittosporum tobira variegata), and found that growth in composted yard waste was similar or better than in a conventional substrate.

Bi et al. (2007) examined the use of pulp mill ash on the growth of Tagetes patula and

Lycopersicon esculentum and found that Tagetes patula in substrates with up to 50% pulp mill ash had similar growth to plants in commercial substrates. Lycopersicon esculentum was not as tolerant and only had similar growth in mixtures up to 30% pulp mill ash. Tripepi et al. (1996) examined pulp and paper sludge compost as a substitute for peat for the production of Syringa vulgaris, Acer tataricum, and Prunus x cistena.

Syringa vulgaris and Prunus x cistena plants were tallest in a media consisting of 25% compost. Acer tatricum exhibited a positive linear response to compost addition to the substrate. Boyer et al. (2007a) examined using WholeTree, where the entire hardwood

tree is ground up as a substrate. They found that when WholeTree was combined with peat, Lantana camara had similar growth to plants grown in commercial substrates. In another paper, Boyer et al. (2007b) examined clean chip residual (CCR) amended with composted poultry litter. They found that Lantana camara grown in CCR/poultry litter mixtures had similar or better growth than plants grown in pine bark and had greener leaves as indicated by SPAD readings. Bugbee (2002) examined the growth of containerized plants in biosolids compost and found that Begonia spp., dianthus

(Dianthus chinensis), Senecio cineraria, Nicotiana spp., petunia (Petunia x hybridida),

Agastache spp., Aster spp., Iberis spp., Coreopsis spp., Lavatera arborea, Thymus vulgaris, Forsythia spp., Juniperus spp., Syringea vulgaris, Spirea spp., and Weigela florida had a positive linear response to increasing levels of biosolids. Marigold

(Tagetes spp.), Hosta spp, Hylotelephium spectabile, and Andromeda polifolia showed no response. Wilson et al. (2006) tested the Florida-native shrubs Forestiera segregata var. pinetorum, Myricanthes fragrans, and Viburnum obovataum in yard waste/biosolids compost and found no difference from a peat-based media. Trochoulias et al. (1990) examined the use of bagasse, the ground up stalks of sugarcane, as a substrate.

African violets (Saintpaulia ionantha) grew more roots in bagasse, while Brachycome,

Schizocentron, Hibiscus, Pandorea, and Trachelospermum grew as well in composted bagasse as they did in peat-sand mixtures. Wang (1991) examined cotton waste compost effects on Radermachera sinica and Brassaia actinophylla and found that growth was greatest for both species in mixes with 25% compost. Moore (2005) provided an overview of the use of composts in potting mixes, and noted five types of responses to increasing amounts of compost in the substrate. Begonia (Begonia x

semperflorens-cultorum), impatiens (Impatiens wallerana), salvia (Salvia splendens), jasmine (Jasmine volubile), and viburnum (Viburnum suspensum) had no response.

Marigold (Tagetes erecta), deutzia (Deutzia gracilis), silver-leaf dogwood (Cornus alba), red-osier dogwood (Cornus sericea), ninebark (Physocarpus opulifolius), and oleander

(Nerium oleander) showed a plateau response where further increases beyond a certain amount did not increase growth. Impatiens, begonias, snapdragons (Antirrhinum majus), dogwood, ninebark, rhododendron (Rhododendron indicum), and pittosporum showed positive linear responses to increasing compost content. A bell-shaped response, likely due to increasing salt concentrations, was noted in salvia, dianthus, petunia, zinnia (Zinnia elegans), and marigold. Finally, impatiens, cat whiskers

(Orthosiphon stamineaus), angelonia (Angelonia angustifolia), golden shrimp plant

(Pachystachys lutea), golden globe (Lysimachia congestifloria), Mexican heather

(Cuphea hyssopifolia), Bolivian sunset (Gloxinia sylvatica), chrysanthemum

(Chrysanthemum x morifolium), snapdragon, geranium (Pelargonium x hortum), and queen palm (Syagrus romanzoffiana) had negative linear responses to increasing compost incorporation into the substrate. Impatiens had three different responses in three different types of compost, indicating that the source of compost is a predictor for plant performance in compost-containing substrates. Chong (2005) gives an overview of his 20 years of research into waste and composts in nursery substrates in his paper, and finds that despite higher pH and higher electrical conductivity (EC), most alternative substrates perform as well or better than commercially available substrates.

Sugarcane Filter Press Mud

Sugarcane Filter Press Mud (FPM) is a waste product of the sugarcane milling process. After the juice is extracted from the crop, lime is added to the juice to provide

reactive Ca+2, which can react with suspended solids and phosphorus (P) in the juice to flocculate impurities out of the juice. This flocculate settles out of the juice and is transported to a settling area where it dries out. The flocculate is the filter press mud and its components are soil, ash, plant residue, waxes and starches and sugars

(Doherty and Dye, 1999). FPM is normally returned to sugarcane fields, and this practice has been extensively researched. The product is not homogeneous, so it is expected that results would be variable depending on where the product originated, and is known to be affected by locality, cultivar of sugarcane, harvesting practice, and mill practices (Solaimalai et al., 2001). FPM can serve as a vital nutrient source, but it must be analyzed to determine the specific nutrient content, since it is not a standard product

(Table 2-2). FPM has been used to improve soils to grow vegetables, fruits, and ornamentals. FPM has also been used as a standard component of container media across Latin America.

FPM Impacts on Soils

In South Africa, Dee et al. (2002) report increased pH, organic carbon, and microbial activity with additions of 10-20 Mg/ha. They also report decreased Aluminum

(Al) availability. In Pakistan, Khan (2011) amended calcareous soils with 20 t/ha of

FPM and found decreased bulk density and pH and increased porosity, macronutrients, and micronutrients. In Solaimolai et al. (2001), the authors reviewed previous work documenting improvements in soil tilth, water holding capacity, infiltration, particle stability, organic matter, macronutrient and micronutrient levels and amelioration of saline, sodic conditions due to the elevated organic matter and cation exchange capacity binding the injurious ions.

Despite the improvement in soil characteristics, there are concerns with land applications of any organic waste product. This is why the USA and several other countries set limits on heavy metal content in biosolids and composts. It is also the reason that manure management plans that dictate manure loading rates to agricultural fields have had to be implemented around confined animal feeding operations. In

Australia, Barry et al. (1998a) concluded that the heavy metal content of FPM (Table2-

3) is not of concern even at application rates of 150 t/ha, and would be less than the heavy metal content provided by municipal biosolids. Kumar et al. (2011) found that the heavy metals iron (Fe), zinc (Zn), and manganese (Mn) were highly bio-available in

FPM, but concluded that FPM would not be a source of heavy metal pollution since the concentrations were not above regulatory limits.

FPM as a Substrate

Few journal articles have addressed the physical characteristics of FPM as a substrate for container use. Moreno-Álvarez (2002) reported that FPM in Cuba had 9 percent air-filled porosity, and only 19.8% of the water held was easily available.

Berrospe-Ochoa et al. (2012) measured a bulk density of 0.17 g/cm3 and a porosity of

91 percent for fresh FPM. Composted FPM had an increased bulk density of 0.36 g/cm3 and a decreased porosity of 81 percent. Gallego et al. (2008) measured a bulk density of 0.234 g/cm3 and a porosity of 86.86 percent. Air-filled porosity for this media was 31.69 percent. Only 11.97% of the water was easily available. Gallego et al.

(2008) also showed that as FPM decomposes it loses over 40% of its volume.

Electrical conductivity was reported to be 3.8 mS/cm for fresh FPM and 6.4 mS/cm for composted FPM. Abul Soad et al. (2012) reported EC values of 4.4 mS/cm in fresh

FPM and 4.7 mS/cm in composted FPM.

FPM Use in Horticultural Crop Production

Solaimalai et al. (2001) reported yield increases for Tomato (Lycopersicum esculentum) and Turmeric (Curcuma longa) in India, with an application rate of 20

Mg/ha. Sharath Pal et al. (2014) reported increases in yield following a 10 Mg/ha application of FPM, for ginger (Zingiber officianale) in the dry zone of Karnataka, India.

When FPM was combined with fertilizer, yield, plant height, number of leaves per clump, leaf size, number of tillers per clump, and leaf area increased. Smith and Hamill

(1996) reported, as standard practice, applying 125 Mg/ha of FPM prior to planting ginger in Queensland, Australia. Stofella and Graetz (1996) and Berrospe-Ochoa et al.

(2012) investigated the use of FPM as substrate for the production of tomato transplants. Both found no difference in percent germination between FPM and sphagnum peat commercial mixtures. However, Stofella and Graetz found that plants grown in FPM were shorter and had fewer roots while, Berrospe-Ochoa et al. found the opposite to be true. In Columbia a 75:25 mixture of FPM and ash produced the best tomato transplants when compared to several other ratios of FPM and ash and soil

(Rengifo et al., 1996). In Florida, application of 188 Mg/ha of FPM compost increased tomato plant height, shoot diameter and marketable yield (Stofella and Graetz, 2000).

Likewise, tomatoes grown in FPM amended soil in Columbia had much higher yields

(Bruzón and Abad, 1995). Vega-Ronquillo et al. (2006) used FPM at 20 Mg/ha as the control treatment as a substrate for growing organoponic and greenhouse cucumbers

(Cucumis sativus), and found that yield was drastically lower for FPM-grown cucumbers than for cucumbers grown in compost containing crop residue, grass, and cattle manure.

Solaimalai et al. (2001) reported yield increases for pomegranate (Punica granatum) and pineapple (Ananas comosus) amended with FPM. For pomegranate,

FPM applied at 75% of the gypsum requirement increased plant height and stem diameter. Pattison (2010) reported increased yields and decreased damage from parasitic nematodes in Australia when 40 Mg/ha FPM was used in banana (Musa spp). orchards. Armario-Aragón (2007) reported increased pseudostem circumference, foliar area, fruit number, yield per plant, and yield per hectare when a mixture of FPM, fertilizer and ash was applied to a banana orchard. The highest rate of FPM incorporation (14 kg per banana plant) resulted in a 6 Mg/ha increase in yield over chemical fertilization at recommended rates. However, plants fertilized FPM alone at

18kg per plant yielded less than those fertilized with chemical fertilization at recommended rates. García et al. (1998) also concluded that adding FPM increased banana yields in Cuba. In addition, they found that adding chemical fertilizer to the FPM further increased yields. Similar results were found with plantains (Musa spp.). In

Australia, FPM was used in avocado (Persea americana) orchards at the rate of 100

Mg/ha to act as a mulch, and provide 80% of the nitrogen (N) needs. FPM use in avocado orchards was also advised as a possible integrated pest management technique for managing Phytophthora cinnamomi since it encourages shoot growth and increases soil carbon (Pegg and Giblin, 2008).

FPM Use in Landscapes

Little has been written on FPM use as a soil amendment in landscaping. A company in Texas was marketing a co-compost of FPM, fly ash, clinker, and mulch that was being used in landscapes in the Rio Grande Valley of Texas. A personal visit with the owner of the business indicated that production had ceased earlier in 2014.

Landscape use in Florida is mostly anecdotal and tied to people involved in the sugar industry; no commercial use of FPM for landscapes in Florida exists.

FPM Use in Fruit Tree Nurseries

In Puerto Rico, Argueta et al. (1997) evaluated several substrates for a coffee

(Coffea Arabica) nursery and determined that a mix of soil, FPM, and river sand produced the largest plants. Aponte et al. (1998), in Puerto Rico, advise the use of

FPM in a 3:1 by volume mixture with alluvial soil for growing avocados in the nursery.

Morales-Payan (1998) reported that FPM is a common component of the propagation substrate for papaya (Carica papaya) in the Dominican Republic. In Brazil, Prucoli-

Posse et al. (2003) found that papaya grown in a 3:2 by volume mixture of bagasse and

FPM did not perform as well as standard potting mix in 53 cm3 container, but did do as well as in a 115 cm3 container. Stofella et al. (1996) found that citrus (Citrus spp.) rootstock seedling growth was improved by FPM.

Bananas may be the most well studied and evaluated fruit “tree” in a nursery setting with FPM. It may also be the plant with the greatest variability in results. In the acclimatization phase of tissue-culture bananas, Izquierdo et al. (2009) found that plants grown in a 3:1 by volume mixture of FPM and alluvial soil had 84% survival in that mixture, and were 5.86 cm tall after 60 days. Treating plants with an oligogalacturonide increased survival, pseudostem diameter, number of leaves, number of roots, and leaf proline content, indicating less stress to the plants. In Pakistan, Abul Soad et al. (2012) used fresh FPM and FPM compost as a substrate for hardening tissue-culture banana plants of the cultivar ‘Grand Naine’ and had no survival. This was possibly due to the high EC of the media. Silva et al. (1999) had very poor results when using FPM as substrate for hardening the cultivar ‘Prata’. The 100% FPM substrate consistently

produced the poorest plants. However, some improvement was seen when FPM was mixed with other components. In contrast, Vasane and Kothari (2006) found that bananas of the cultivar ‘Grand Naine’ performed well in mixtures of soil and FPM. In a comparison of 7 substrates, FPM grown plants had the greatest girth, leaf length, number of leaves, root mass, and chlorophyll. In another trial, Vasane and Kothari

(2008) found that mixtures of FPM and vermicompost produced larger and healthier banana plants than soil alone, or soil mixed with either FPM or vermicompost.

FPM Use in Forest Tree Nurseries

In a Cuban study on nursery growth of African mahogany (Khaya nyasica), it was determined that a mixture of 30% coffee processing residue (CPR) 10% FPM and 60% soil produced the tallest plants. However, in binary mixtures of soil and FPM or CPR, those with FPM produced taller plants (López et al., 2010). Fornes et al. (2002) investigated the use of FPM in mixtures with bagasse for the production of three species of Cedrela. Despite significant interaction terms, the authors ran their mean separations across all fertilizer and substrate combinations. For C. balanse and C. lilloi fertilized with 15-5-35 granular a 50-50 mixture of FPM and bagasse performed as well as plants grown in pine bark. For C. fissilis, the opposite was true. When the plants were fertilized with a nutritive solution, only C. lilloi grown in a 50-50 mixture of FPM and bagasse was able to achieve the same growth as plants grown in pine bark. Castillo et al. (2006) found that Eucalyptus (Eucalyptus grandis) grown in substrates with FPM as a principal component produced larger plants with greater foliar area and root mass.

These benefits were also apparent 16 months after planting in the forest.

FPM Use in Propagation

Soares de Freitas et al. (2009) used FPM as a component of propagation media for eucalyptus (Eucalyptus grandis x Eucalyptus urophylla) in Brazil. They found that a mixture of coconut coir and FPM was superior to a mixture of bagasse and FPM for rooting quality and plant survival. Do Nascimento et al. (2013) found that using FPM in mixtures with bagasse did not affect germination of Eucalyptus (Eucalyptus camaldulensis) but did affect the eventual growth of the plants as compared to a commercial standard media. This was attributed to the higher water holding capacity of the mixtures. Maldonado et al. (2005) used FPM in Venezuela for rooting Malphigia emarginata and found that those rooted in a mixture of FPM and river sand had longer roots than those in a mixture of coconut coir and river sand. The percent survival, percent rooted, and numbers of roots were also numerically higher, but the high variation prevented statistical separation.

FPM use in Ornamental Nurseries

In Cuba, FPM was mixed with soil and rice straw in variable amounts to determine the impact of micorrhizae on the growth of Begonia (Begonia spp.) The two highest rates of FPM incorporation with micorrhizae produced the largest begonias

(Morales-Alvero et al. 2011). In a study using anthuriums (Anthurium andreanum), a mixture of soilless media that included FPM produced plants that had the greatest height, fastest leaf emission rate, and longest peduncles (Corbera et al., 2008). In

Florida, Poole and Conover (1989) used a commercially available product containing

FPM mixed with bagasse and boiler ash, which had a high pH and EC. Gardenia jasminoides performed poorly in this substrate; however, Dieffenbachia spp. grew as well in this commercial substrate as it did in reed-sedge peat and better than it did in

pine bark. In Texas, plant height and stem diameter of Chrysanthemum morifolium was highest in plants grown in FPM. Plants grown in FPM also produced the most leaves but the difference was not significant (Soltenzad et al., 1982). In Mexico, the effect of growth regulators and substrate on the rooting of Kalenchoe blossfeldiana revealed that plants grown in FPM-peat moss mixtures had the most growth and nutrient contents

(Villanueva et al., 1998). Larsen and Moore (2014) used FPM as a substrate component in binary mixtures with vermiculite and found that vinca (Cataranthus roseus) grown in FPM were larger and more developed than those grown in a commercially available sphagnum peat-based substrate. Substrates composed of greater than 67% FPM, performed equally well, while the substrate composed of 50%

FPM was intermediate between the rest of the FPM treatments and the commercially available sphagnum peat-based substrate.

Bananas

Most commercially cultivated bananas are triploids of Musa acuminata. Musa balbisiana is also a contributor to many local varieties. Genomes are generally denoted after cultivar names indicating ploidy and the contribution of Musa acuminata (A) and

Musa balbisiana (B). The most popular cultivated varieties around the world are

Cavendish(AAA) (Stover and Simmonds, 1987).

The banana plant is a large monocotyledonous herb native to tropical Southeast

Asia. A corm emits leaves whose petioles form a 2-4 m long pseudostem. Eight to twelve months after planting, the plant will emit a flower consisting of a raceme of separate male and female flowers. The parthenocarpic, edible fruit is the ovary of the female flower. The plant has long fibrous shallow roots. Lateral offshoots called

suckers emerge from the corm, and a grouping of a mother plant and several suckers is called a mat.

Banana Propagation and Planting

The suckers represent the traditional propagation material used by growers since antiquity. Within the last 30 years, tissue-culture plantlets have become the starting point for modern plantations (Stover and Simmonds, 1987). Tissue culture plantlets offer the grower the ability to plant uniform, disease-free plants. In South Africa,

Robinson et al. (1993) found higher yields and greater stem diameters by using tissue culture plants. They did find out, however, that using 50 cm tall tissue-culture plantlets had a slightly negative impact on yield, and a slightly positive impact on months to first harvest. In Cameroon, Fonsah et al. (2007) found similar results of higher yields with tissue-culture plantlets. Galán Saúco and Robinson (2010) outlined establishment procedures for tissue culture plants in fields and in protected-culture. They state that the new plants can be considered established eight weeks after planting assuming the protocol is followed. They recommend hardened plants 20-50 cm tall, deep fertile soil free of pathogens, frequent micro-irrigation, and water with an EC less than 0.75 mS/cm. Plants should be planted so that the soil surface of the potting media is 10-15 cm below the surrounding soil surface in order to provide support. For one month plants should be irrigated twice a day for 15 minutes to decrease stress. For the second month 10 mm of irrigation twice per week is recommended. Fertilizer should be applied if necessary two weeks after planting and every two weeks thereafter for the first three months.

Banana Irrigation Requirements

Bananas have very large broad leaves with high leaf area indices and can have the potential of very high rates of evapotranspiration (ET). The potential for high ET coupled with a shallow root system can be a recipe for drought stress (Galán Saúco and

Robinson, 2010). FAO-ET for bananas shows a Kc of 0.5 for young plants and up to 1.2 for established plants (Allen et al., 1998). Most banana roots are found in the 0-300mm soil depth (Robinson and Alberts, 1989). Turner et al. (2007) found that bananas respond quickly to water deficit by shutting their stomata. Irrigation recommendations advise growers to irrigate with small amounts frequently instead of one high volume irrigation event (Lahav and Kalmar, 1981; Galán Saúco and Robinson, 2010).

Banana researchers have found that bananas have very high irrigation requirements. Field studies by Purseglove (1972) found that bananas require 100mm of irrigation or rainfall per month. Stover and Simmonds (1987) found that, in the tropics, bananas could consume 3-6 mm of water per day depending on climatic and plant factors.

Table 2-1. Ideal chemical characteristics for soilless media amended with controlled- release fertilizer (Yeager, 2007). Nutrient values reported in mg/kg. Analysis Desirable level pH 5.0-6.0 Electircal conductivity (EC) mS/cm 0.5-1.0 Nitrate-N (NO3-N) 15-25 Phosphorus (P) 5-10 Potassium (K) 10-20 Calcium (Ca) 20-40 Magnesium (Mg) 15-20 Manganese (Mn) 0.3 Iron (Fe) 0.5 Zinc (Zn) 0.2 Copper (Cu) 0.02 Boron (B) 0.05

Table 2-2. Nutrient analyses of sugarcane filter press mud (FPM) reported in publications and research worldwide. Values are percent composition dry matter. Nitrogen Phosphorus Potassium Calcium Magnesium Country (N) (P) (K) (Ca) (Mg) Barry et Australia 1.5 0.9 0.7 2.3 0.6 al.,1998b Dee et al., S. Africa 1.68 1.13 0.31 1.1 0.4 2002 Gilbert et Florida 1.05 0.31 0.15 7.8 0.50 al., 2008 Alexander, S. Africa 1.69 0.72 0.19 1.84 0.37 1971 López et Cuba 1.50 1.16 0.72 3.16 0.32 al., 2010 Stofella and Florida 2.42 1.81 0.10 7.62 0.43 Graetz, 2000 Moreno- Álvarez et Cuba 2.70 2.00 0.15 7.8 0.80 al., 2011 Bruzón and Abad, Columbia 2.2 2.8 0.4 3.1 0.4 1995 Corbera et Cuba 0.75 1.44 0.14 0.79 0.25 al., 2008 Villanueva et al., Mexico 1.64 0.47 0.15 0.34 0.18 1998 Berrospe- Ochoa et Mexico 2.4 0.04 0.06 0.11 0.24 al., 2012 Almeida- Júnior et Brazil 1.42 1.73 0.145 2.53 0.34 al., 2011 Ferreira et 2.91- Brazil 1.1-1.4 0.50-1.11 0.25-0.80 0.09-0.34 al., 1986 3.90 Cifuentes et al., Guatemala 1.83 1.61 0.63 7.80 0.60 2011

Table 2-2. Continued. Nitrogen Phosphorus Potassium Calcium Magnesium Country (N) (P) (K) (Ca) (Mg) Vega- Ronquillo Cuba 1.35-1.98 0.87-1.3 0.08-0.12 4.84-7.50 3.9-4.4 et al., 2006 Fidalsky and Dias- Brazil 2.38 0.29 0.19 0.14 0.10 Chaves, 2010 Pattison, Australia 1.4 0.39 0.5 6.3 0.23 2010 Arrieche and Mora, Venezuela 1.2 5.2 3.2 3.5 0.85 2005

Table 2-3. Heavy metals reported in sugarcane filter press mud (FPM) as compared with established limits. USEPA Biosolids Part 503 Ceiling Woods Barry et Stofella Concentrations End al. and Graetz Pattison, Element mg/kg or ppm QSAP1,2,3 1998a1 20001 20101 Arsenic (As) 75 - 2.8 - - Cadmium (Cd) 85 2.0 0.29 1.98 0.17 Chromium (Cr) - 100 35 - 74.4 Copper (Cu) 4300 100 34 245 76.1 Lead (Pb) 840 150 8 29.7 14.9 Mercury (Hg) 57 0.5 0.05 - - Nickel (Ni) 420 50 11 8.68 48.2 Zinc (Zn) 7500 400 136 313 143 1Value Reported in ppm 2Woods End Research Laboratory, 1998 3Quality seal of approval program

CHAPTER 3 PHYSICAL AND CHEMICAL CHARACTERIZATION OF SUGARCANE FILTER PRESS MUD AS A SUBSTRATE FOR ACCLIMATING TISSUE CULTURE BANANAS

Introduction

Worldwide production of tissue-culture banana (Musa spp.) plants is likely in the hundreds of millions of plants annually based on worldwide acreage and the planting/replanting requirements. Bananas are produced throughout the tropics and subtropics, and tissue culture has been shown to increase yields and crop uniformity.

Substrates for these millions of plants have been widely reported through the literature.

The ideal substrate for nursery plants has been thoroughly discussed by several researchers (DeBoodt and Verdonk, 1972; Poole et al., 1981, Cabrera, 1999; Yeager,

2007). However, only one paper outlines the ideal characteristics for growing tissue culture bananas in the nursery. Robinson and Galán-Saúco (2009b) recommend a substrate with a pH of 5.5-6.5, 10%-20% air-filled porosity (AFP) and a 40-50% water holding capacity. To meet this recommendation, they recommend a 70% peat moss and 30% vermiculite substrate.

Elevated salinity and/or nutrient status have been a proposed cause of poor growth in some substrates. Robinson and Galán-Saúco (2009b) reported foliage damage due to high salinity levels in coconut coir. In Brazil, Silva et al. (1999) found that plants growing in media with sugarcane filter press mud (FPM) had poor growth, which may have been caused by the elevated levels of phosphorus (P). In Pakistan,

Abul Soad (2012) also found poor growth in FPM, and in this case it was attributed to elevated electrical conductivity (EC). However in India, Vasane and Kothari (2006) found that FPM and soil mixtures were the best performing substrates tested. This finding was later confirmed in subsequent testing by Vasane et al. (2010). In Cuba,

Izquierdo et al. (2009) successfully used a 3:1 by volume mixture FPM and soil as their substrate for tissue-culture bananas.

FPM is known to be a variable material with characteristics being affected by locality, cultivar of sugarcane (Sacharrum spp.), harvesting practice, and mill practices

(Solaimalai et al., 2001). When Fitzpatrick et al. (1998) reviewed research into using composts as substrates they distinguished parent material, composting time, maturity, inert material, regulated elements and microbiological factors as being important to making successful compost-based substrate. The purpose of this research was to characterize and describe the chemical and physical attributes of Florida FPM in order to address its suitability as a substrate component for tissue culture bananas. The objectives of this research were to determine if the source of FPM affects the nutrients leached during an 80 day leaching experiment, to determine if the age of FPM affects the nutrients leached during a 64 day leaching experiment, and to determine if different sources and ages have bulk densities, porosities, and air-filled porosities that fit within the range of an ideal substrate.

Methods and Materials

From 2012 to 2014, samples of FPM were collected from 3 sugar mills in south

Florida. One mill provided a fresh sample and a pond-aged sample, while another mill provided several fresh samples, which could be compared with pile-aged samples a third source only provided a fresh sample. For anonymity, sources are identified as “A”,

“B” and “C.” Physical and chemical characterizations for the different sources were started shortly after reception. For different ages, characterizations were started after a certain age was reached. The physical properties of bulk density, porosity, and air-filled

porosity were measured using 16.5 cm x 16.5 cm containers due to their use as the container for later direct testing of bananas in an FPM based substrate. Porosity and air-filled porosity methods were based on those outlined by Bilderback (1982). Three

2200 mL samples per substrate were placed into containers with liners, which prevented drainage. These containers were saturated and weighed to calculate a saturated weight. The liners were then punctured, which allowed the containers to drain. A drained weight was recorded four hours after the liners were punctured. The substrate was then removed from the container and oven dried at 120C to determine dry weight. Bulk density was determined by dividing dry weight by the volume (2200 mL). Porosity was determined by calculating the difference between saturated weight and dry weight. Air-filled porosity was determined by measuring the difference between the saturated and drained weights and dividing by the total volume.

FPM Source Nutrient Characterization

FPM from the different sources was sent to the Everglades Soil Testing Lab for nutrient analysis for Phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and iron (Fe) (Table 3-1). Phosphorus was measured using a water extraction followed by measurement using the ascorbic acid-molybdenum blue method and a probe colorimeter (Murphy and Riley, 1962). Potassium, calcium (Ca), magnesium (Mg) were extracted with 0.5 N acetic acid (Morgan et al., 2009). Iron (Fe) was extracted with 6N hydrochloric acid. Potassium, Ca, Mg, and Fe was measured by atomic absorption spectrometry.

Nitrate-nitrogen (NO3-N), ammonium-nitrogen (NH4-N), phosphate-phosphorus

(PO4-P), and potassium (K) release characteristics were determined by packing the

different FPM materials into 5.1 cm x 30 cm cylindrical PVC containers, and collecting the daily leachate following 2.5 cm of irrigation with deionized water. Leachate was filtered as it leached to remove particulate matter. There were 3 containers per sample.

Every eight days, the volume of leachate was measured and a 20 mL subsample was taken and frozen until analysis could be performed. The experiment lasted 80 days.

Nitrate, ammonium, and potassium concentrations were determined using an Accumet

XL250 combination ion selective electrode (Fisher Scientific, Waltham, MA). Samples were prepared for the preceding ions by making a 2:5 dilution in a 120mL polyethylene bottle. Phosphate was determined by the ascorbic acid-molybdenum blue method method (Murphy and Riley, 1962) using a Spectronic 20D+ spectrophotometer (Thermo

Electron Corporation, Waltham, MA) measuring absorbance at 440 nm. The standard curve measured between 0 mg/L and 1 mg/L. Sample preparation involved dilution in a

5 mL vial to an appropriate concentration for interpolation on the standard curve. Mass leached per container was determined by summing the product of the nutrient concentration and leachate volume. Unfortunately, additional samples from source “A” and source “C” were not made available for more in-depth analysis.

FPM Age Nutrient Characterization

FPM from one sugar mill source “B” was collected and placed into 12-75L containers. Samples from these containers were collected at 3-month intervals in order to obtain characteristics of fresh, 3 month old, 6 month old, 9 month old, and 12 month old FPM. Samples of each age FPM were sent to Waters Agricultural Lab in Camilla,

GA for a saturated media extract (SME) characterization of nutrients available (Table 3-

2).

Treatment setup, leachate collection, and mass leached per container were the same as in the FPM source nutrient characterization. However, leachate was only collected for 64 days during this trial. Leachate was prepared for analysis by dilution in

5 mL vials so that concentration could be interpolated on the standard curve. Nitrate- nitrogen, NH4-N, and PO4-P analysis were performed by colorimetry with a Seal

Autoanalyzer 3 (Seal Analytical, Mequon, WI). Analysis for K was performed by atomic absorption spectrometry with a PerkinElmer AAnalyst 400 (PerkinElmer, Waltham, MA).

Both machines were located at the UF/IFAS Fort Lauderdale Research and Education

Center.

FPM Total Nutrient and Heavy Metal Analysis

Four samples of FPM from “B” collected between 2012 and 2014 were sent to

Waters Agricultural Lab in Camilla, GA for analysis of total macronutrients, micronutrients, and sodium (Na), aluminum (Al), cadmium (Cd), chromium (Cr), lead

(Pb), cobalt (Co), and nickel (Ni) (Table 3-3, Table 3-4).

Statistical Analysis

Statistical analysis was performed with JMP Pro 11.0 using ANOVA for tests on total amounts of nutrient leached. In tests involving several measurements, a least squares model with substrate, days after start and their interaction as effects. The results of statistical tests are reported in Table D-1.

Results

General Observations About FPM

When delivered fresh from the sugar mill, FPM has a distinct, unpleasant odor.

Soon after delivery the pile will develop a bright orange bloom caused by the fungus

Neurospora spp. As the pile ages it decreases in volume. In dry weather, the pile will

form a calcium-salt crust. Abundant cane fibers are visible in fresh material, but not in aged material. FPM forms aggregates that can make it difficult to mix with other components. Many fine particles are present in the material, but due to aggregation, hydraulic conductivity appears to be similar to sphagnum-peat based media. Saturation has not been a problem in tests in containers ranging in depth from 7.5 cm to 16.5 cm.

Chemical Characterization

The pH of the different sources of FPM were all around 7.0, ranging from 6.93 in the fresh sample of source “A” to 7.38 in the fresh source of “B” (Table 3-1). Water extractable P ranged from 37.50 mg/kg in the pond sample of source “A” to 266.18 mg/kg in the fresh source of “A”. Acetic acid extractable K ranged from 229.41 mg/kg in the fresh source “C” to 1519.85 mg/kg in fresh source “A”. Calcium (Ca) values ranged from 13,508.82 mg/kg in pile-aged source “B” to 1708.82 mg/kg in fresh source “C”.

Magnesium (Mg) values ranged from 2307.35 mg/kg in pile-aged source “B” to 155.88 mg/kg in fresh source “C”. Iron (Fe) values ranged from 9.61 mg/kg in pond-aged source “A” to 0.66 mg/kg in fresh source “C”.

For the aging experiment pH ranged from 7.1 in the fresh sample to 7.4 in the 9- month sample (Table 3-2). Organic matter ranged from 27.3% in the fresh sample to

36.3% in the 12-month sample. Saturated media extract (SME) NO3-N ranged from 9.1 mg/kg in the fresh sample to 535.2 in the 12-month sample. Ammonium-nitrogen (NH4-

N) showed the reverse trend the 28.7 mg/kg in the fresh sample and 9.1 mg/kg in the

12-month sample. Phosphorus ranged from 8.58 mg/kg in the fresh sample to 16.84 mg/kg in the 3-month sample. Potassium ranged from 110 mg/kg in the 6-month sample to 214 mg/kg in the 12-month sample. Calcium increased from 137 mg/kg in the fresh sample to 495 mg/kg in the 12-month sample. Magnesium ranged from 143

mg/kg in the 6-month sample to 230 mg/kg in the 12-month sample. Sulfur showed a marked decrease starting at 284 mg/kg in the fresh sample and then decreasing to 33 mg/kg in the 6-month sample followed by an increase to 60 mg/kg in the 12-month sample. Boron was fairly stable starting at 0.07 mg/kg and ending at 0.06 mg/kg. Zinc ranged from 0.03 mg/kg in the 6-month sample to 0.35 in the 12-month sample.

Manganese levels were between 3 mg/kg and 5 mg/kg until the 12-month sample, which showed a dip to 0.01. Iron levels started at 0.29 mg/kg and trended downward to

0.13 mg/kg in the 12-month sample. Copper levels started at 0.12 mg/kg and fell to

0.01 mg/kg in the 6-month sample where the copper level stabilized.

Across 4 different FPM source “B” deliveries, nutrient content was 1.4% N,

2.08% P, and 0.27% K. FPM was 3.85% Ca and 0.72% Mg. Micronutrient levels were generally low except for Fe, which was 0.48% (Table 3-3). Cadmium, Cr, Pb, Co and Ni means and ranges were all below levels of concern (Table 3-4).

Source Leaching Experiment

For the source experiment (Table 3-5), total NO3-N leached was highest in the fresh sample “A”, and it was lowest in the fresh sample of “B”. The range was 7.3 mg/container to 155.4 mg/container. Ammonium-nitrogen leaching was generally lower than NO3 leaching. The pile-aged source of “B” was an exception with 41.0 mg/container leached. The pond-aged source of “A” had no measured NH4-N in the leachate. Phosphate-phosphorus leaching ranged from 29.1 mg/container in the fresh source of “B”, to 273.9 mg/container in the pile-aged source of “B”. Potassium leaching ranged from 3.4 mg/container for the pond-aged source of “A” to 18.9 mg/container for the fresh source of “C”

Age Leaching Experiment

There were no significant differences in the amounts of nitrate, ammonium, phosphorus, and potassium leached across the different ages of FPM (Table 3-6).

Nitrate-nitrogen leached ranged from 444.2 mg/container in the 9-month aged sample to

295.4 mg/container in the 12-month aged sample. Ammonium-nitrogen leached ranged from 14.4 mg/container in the 9-month aged sample to 17.2 mg/container in the 3- month aged sample. Phosphate-phosphorus leached ranged from 5.1 mg/container in the 12-month aged sample to 10.6 mg/container in the 9-month aged sample.

Potassium leached ranged from 149.1 mg/container in the 9-month aged sample to

185.7 mg/container in the 6-month aged sample.

Source Physical Characteristics

Bulk densities of the different sources were all below 0.6 g/cm3 (Table 3-7). The lowest bulk densities were found in the fresh samples and ranged from 0.25 g/cm3 in “C” to 0.31 g/cm3 in “A”. Aged samples were higher with the pond source of “A” having a bulk density of 0.59 g/cm3 and the pile-aged sample of “B” having a bulk density of 0.51 g/cm3. Porosity of the samples did not differ much ranging from 63 percent in the fresh sample of “A” to 70 percent in the pile-aged sample of “B”. Air-filled porosity was higher in fresh samples ranging from 13 percent in “A” to 19 percent in “B”. Both aged samples had air-filled porosities below 10 percent with the pond aged sample of “A” having an air-filled porosity of 7 percent and the pile aged sample of “B” having an air- filled porosity of 9 percent.

Age Physical Characteristics

Bulk density ranged from 0.36 g/cm3 in the 3-month sample to 0.52 in the 9- month sample (Table 3-7). Porosity ranged from 67 percent in the 12-month sample to

70 percent in the 6-month sample. Air-filled porosity ranged from 14 percent in the 9- month sample to 22 percent in the 6-month sample.

Discussion

The source of FPM affected the nutrient leaching that occurred during the 80 day leaching study. The fresh sources of “A” and “C” had similar peak NO3-N concentrations, but “A” maintained higher concentrations for the remainder of the experiment (Figure C-2). Both sources of B had low NO3-N concentrations throughout the leaching study.

For NH4-N and PO4-P, pile-aged “B” stood out from the rest of the sources. The separation between the curve for NH4-N and PO4-P, the 50-fold increase in NH4-N leached, and the10-fold increase in PO4-P leached point to possible contamination

(Figure C-3, Figure C-4). The substrate test done on the material prior to use did not indicate differences in P that would have warranted the observed differences in leaching. If this data was removed, then source would have no effect on NH4-N and

PO4-P leaching.

For K, “A” and “C” had peak K concentrations at the same time (Figure C-5).

However, the peak for “C” had greater magnitude and total K leached was greatest in C.

The fresh sample of “B” peaked later in the study, and this fit well with the electrical conductivity data (Figure C-1).

There were no differences observed in leaching between the different ages.

Nitrate and K leaching were higher in the source study, while NH4-N and PO4-P leaching in these samples was lower than in the source study. While age may not be a significant predictor for nutrient release and leaching, high (>6 mS/cm) electrical conductivity was observed in the 3 month aged sample.

Physically, the samples of FPM generally meet or nearly meet the requirements for an ideal substrate. Bulk density was within the range reported by Yeager (2007) of

0.19 g/cm3 to 0.70 g/cm3. Porosity was on the lower than required by Cabrera (1999), and was too low for De Boodt and Verdonk (1972). Air-filled porosity was generally within the range reported by Cabrera (1999). Based on the aging experiment, use of

FPM should probably occur at least 6-9 months after delivery because bulk density almost doubled from 0.28 g/cm3 in fresh samples to 0.52 g/cm3 in 9-month samples.

This finding corroborates the reports of increasing bulk density after composting by

Berrospe-Ochoa et al. (2010).

Chemically, the pH was high and might limit the types of plants that can be produced. The EC had a tendency to spike above 3.0 mS/cm, which may be a problem for certain plants (Yeager, 2007). Nitrate, K, Ca, Mg, and Mn were all higher than the desirable values reported by Yeager (2007). However, the nutrient supplying power of

FPM is of great interest in that it can supply N, P, and K in plant relevant quantities.

Fertilizer rates could possibly decreased by using FPM.

In comparison to other sources of FPM throughout the world, fresh bulk density was roughly equivalent to that reported by Gallego et al. (2008), but higher than the reported bulk density by Berrospe-Ochoa et al. (2010). Both Gallego et al. (2008) and

Berrospe-Ochoa et al. (2010) reported porosity and air-filled porosity that were higher than the values observed in this study. Berrospe-Ochoa et al. (2010) reported that air- filled porosity increased with composting, which we did not observe in this study. One possible reason for the discrepancy could be differences in milling and harvesting practice. Physical properties of FPM are likely influenced by the amount of soil, trash,

and lime in the juice. Smaller amounts of soil and greater quantities of cane fiber may decrease bulk density and increase porosity.

In conclusion, FPM around the world has been successfully used as a substrate for growing plants in a nursery. FPM sourced from Florida sugarcane mills is likely to have similar success based on the physical and chemical properties. There are no heavy metal concerns. The product is variable from load to load in terms of nutrient content, so a user must be aware of the nutrient content before use. While age does impact the product physically, it has no impact on nutrient release and leaching.

Table 3-1. Chemical nutrient analysis of 3 sources of sugarcane filter press mud (FPM) collected during 2012-2013 milling season.1 Mill Storage pH Phosphorus Potassium Calcium Magnesium Iron “A” Fresh2 6.93 266.18 1519.85 3611.76 896.32 1.88 “B” Fresh2 7.38 100.00 1105.15 8750.00 2307.35 3.56 “C” Fresh2 7.15 108.09 229.41 1708.82 155.88 0.66 “A” Pond3 7.27 37.50 412.50 11037.50 1472.79 9.61 “B” Aged4 7.36 89.71 217.65 13508.82 1988.97 5.40 1Results reported as mg/kg 2Collected from sugarcane mill after being pumped out of the mill 3Collected from settling pond at the mill after unknown residence period 4Collected 12 months after fresh delivery from sugar mill, separate delivery from “B”-fresh

Table 3-2. Chemical characteristics of different ages of FPM from sugar mill “B”.1,2 Measurement Fresh 3 month 6 month 9 month 12 month pH 7.1 7.3 7.3 7.4 7.3 Organic 27.30 27.11 35.76 35.41 36.37 matter (%) Electrical 6.48 3.08 2.87 3.3 4.31 conductivity (mS/cm) Nitrate-N 9.1 286 276.2 356.7 535.2 Ammonia-N 28.7 14 14 14.7 9.1 Phosphorus 8.58 16.84 11.5 12.27 10.86 Potassium 132.7 136.2 109.7 144.2 213.9 Calcium 137.4 268.4 312.6 351.3 494.9 Magnesium 171.6 187.5 142.7 157.7 229.8 Sulfur 283.6 52.15 33.18 40.49 60.48 Boron 0.07 0.06 0.05 0.05 0.06 Zinc 0.11 0.21 0.03 0.16 0.35 Manganese 4.23 4.61 3.53 3.88 0.01 Iron 0.29 0.16 0.15 0.14 0.13 Copper 0.12 0.02 0.01 0.01 0.01 1Nutrient values reported in mg/kg 2Analysis performed by Waters Agricultural Laboratory, Camilla, GA

Table 3-3. Total elemental analysis of sugarcane filter press mud aged at least 12 months collected from sugar mill “B” between 2012 and 2014, as a percentage of dry matter.1 Element Mean (%) Range2 N 1.40 1.00 - 2.09 P 2.08 1.65 - 2.66 K 0.27 0.09 - 0.7 S 0.21 0.16 - 0.32 B 0.001 0.001 - 0.001 Zn 0.01 0.01 - 0.01 Mn 0.02 0.02 - 0.03 Fe 0.48 0.19 - 1.2 Cu 0.01 0.003 - 0.01 Ca 3.85 3.04 - 5.52 Mg 0.72 0.33 - 1.58 Na 0.02 0.01 - 0.04 Al 0.31 0.1 - 0.88 1Analysis performed by Waters Agricultural Laboratory, Camilla, GA 2Low and high values determined from collected samples

Table 3-4. Heavy metals present in FPM from source “B”.1,2 United States Source “B” Source “B” Environmental means range4 Element Protection Woods End Agency, QSAP3 Biosolids Part 503 Arsenic 75 - - - Cadmium 85 2.0 0.18 0.109-0.302 Chromium - 100 27.03 3.54-91.9 Copper 4300 100 52.5 30-100 Lead 840 150 2.21 1.01-3.02 Mercury 57 0.5 - - Nickel 420 50 9.85 0.285-36.89 Zinc 7500 400 100 100-100 1Analysis performed by Waters Agricultural Laboratory, Camilla, GA 2Values reported in mg/kg 3Quality seal of approval program (QSAP), Woods End Research Laboratory, 998 4Low and high values determined from collected samples

Table 3-5. Total nitrate-nitrogen(NO3-N), ammonium-nitrogen(NH4-N), phosphate-phosphorus (PO4-P), and potassium (K) leached over 80 days from containers filled with sugarcane filter press mud from three different sugar mills in the Everglades Agricultural Area.1,2 Substrate NO3-N NH4-N PO4-P K Mean SE Mean SE Mean SE Mean SE “A” – Fresh3 155.4 ± 3.5 A 0.1 ± 1.7 B 41.5 ±3.9 B 13.3 ± 0.3 B “A” – Pond4 52.0 ± 5.5 C 0.0 ± 0.0 B 33.1 ±2.4 B 3.4 ± 0.4 D “B” – Fresh3 7.3 ± 3.2 D 0.8 ± 0.2 B 29.1 ±2.0 B 11.4 ± 0.6 C “B” – Aged5 12.9 ± 0.8 D 41.0 ± 11.8 A 273.9 ±9.7 A 3.5 ± 0.4 D “C” – Fresh2 106.7 ± 10.5 B 13.0 ± 1.7 B 41.4 ±2.4 B 18.9 ± 0.2 A 1Results reported in mg/container 2Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05) 3Collected from sugarcane mill after being pumped out of the mill 4Collected from settling pond at the mill after unknown residence period 5Collected 12 months after fresh delivery from sugar mill, separate delivery from “B”-fresh

Table 3-6. Total nitrate-nitrogen(NO3-N), ammonium-nitrogen(NH4-N), phosphate-phosphorus (PO4-P), and potassium (K) leached over 64 days from containers filled with different ages of sugarcane filter press mud from sugar mill “B”.1,2 Substrate NO3-N NH4-N PO4-P K Mean SE Mean SE Mean SE Mean SE 3 Month 418.6 ± 45.8 17.2 ± 0.5 3.9 ± 0.8 172.0 ± 12.9 6 Month 370.0 ± 50.2 16.4 ± 0.7 2.7 ± 0.2 185.7 ± 9.8 9 Month 444.2 ± 56.4 14.4 ± 1.0 4.2 ± 1.1 149.1 ± 14.2 12 Month 295.4 ± 86.1 15.4 ± 1.3 2.0 ± 0.2 171.6 ± 25.0 1Results reported in mg/container 2Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

Table 3-7. Bulk density, porosity, and air-filled porosity of sugarcane filter press mud (FPM) from 3 different sources and four ages of source “B”. FPM Bulk Density1 Porosity2 Air-Filled Porosity2 “A” – Fresh2 0.31 63 14 “A” – Pond3 0.59 66 7 “B” – Fresh2 0.29 65 18 “B” – Aged4 0.51 70 9 “C” – Fresh2 0.25 65 19 3 month 0.36 67 18 6 month 0.44 70 22 9 month 0.52 69 14 12 month 0.49 67 17 1g/cm3 2Percent by volume 3Collected from sugarcane mill after being pumped out of the mill 4Collected from settling pond at the mill after unknown residence period 5Collected 12 months after fresh delivery from sugar mill

CHAPTER 4 IRRIGATION REQUIREMENT OF TISSUE CULTURE BANANA PLANTLETS GROWN IN A SUGARCANE FILTER PRESS MUD BASED SUBSTRATE

Introduction

Bananas (Musa spp.) are an important crop in Florida not only for their fruit production, but also as landscape plants. Current recommendations advise growers to start with tissue culture plantlets as they are free of pests and can produce a large uniform crop (Robinson et al. 1993; Fonsah et al. 2007; Galán-

Saúco and Robinson, 2010).

Irrigation of field grown bananas in plantations has been studied extensively. Shmueli (1953) found that bananas had very efficient regulation of their stomata and that there was a threshold at 66% total available moisture, where plant performance declined. Eckstein and Robinson (1996) recommended a three-day interval between irrigations for field grown plants. Turner and

Thomas (1998) found that five days after irrigation to container capacity, leaves quit emerging, stomata closed, and photosynthesis was 1/10 of the well-watered control, in 25 L pots.

Worldwide annual nursery production of tissue culture plantlets is likely in the hundreds of millions of plants per year based on worldwide acreage and requirements for planting/replanting. Despite this, there is only rudimentary data on proper irrigation of bananas during the nursery phase. Furthermore, there is a lack of information on the proper irrigation frequencies based on the type of substrate used. Robinson and Galán-Saúco (2009a) recommend daily watering for light substrates and less frequent watering for heavier substrates. Silva et al.

(1999) irrigated with 334 mL per 2.5 L pot twice weekly. However, Martins et al.

(2011) had twice a day 1.7 cm irrigation events for the first 30 days and once a day 1.7 cm irrigation events for days 31-105. Klock-Moore and Broschat (2001) found that areca palm (Dypsis lutescens) and Philodendron produced the greatest matter in a 60% biosolid compost when irrigated daily.

Sugarcane filter press mud (FPM) is a waste product of the sugar milling process that consists of soil, cane fiber, ash, and lime. Depending on the source of FPM, water holding capacity ranges from 49% to 61% by volume. The purpose of this research was to compare the growth of “Williams” bananas grown in FPM and sphagnum peat (SP) substrates under three irrigation frequencies.

The objectives of this research are to determine the effect of substrate on plant growth and water use, to determine the effect of irrigation frequency on plant growth and water use, and to determine if there is an interaction between substrate and irrigation frequency on plant growth and water use.

Methods and Materials

Tissue culture banana plantlets cv ‘Williams’ were purchased from

Sunscape Nursery in Apopka, FL. The plantlets were acclimated to greenhouse conditions under 50% shade and misting for two weeks. Plantlets were graded prior to planting to a uniform height of 4 cm and 6 leaves.

The substrate mixtures consisted of 90% and 55% FPM (12 month aged source from an Everglades Agricultural Area (EAA) sugar mill) or SP (Pro-Moss-

Fine, Premier Tech Horticulture, Quakertown, PA) by volume mixed with perlite and vermiculite. The perlite (Specialty Vermiculite, Pompano Beach, FL) and vermiculite (Specialty Vermiculite, Pompano Beach, FL) were mixed in equal parts to make up the complement of each mixture. The physical properties of

bulk density, porosity, and air-filled porosity were measured using 16.5 cm x 16.5 cm containers due to their use as the container for later direct testing of bananas in an FPM-based substrate. Porosity and air-filled porosity methods were based on those outlined by Bilderback (1982). Three 2200 mL samples per substrate were placed into containers with liners, which prevented drainage. These containers were saturated and weighed to calculate a saturated weight. The liners were then punctured, which allowed the containers to drain. A drained weight was recorded four hours after the liners were punctured. The substrate was then removed from the container and oven dried at 120C to determine dry weight. Bulk density was determined by dividing dry weight by the volume (2200 mL). Porosity was determined by calculating the difference between saturated weight and dry weight. Air-filled porosity was determined measuring the difference between the saturated and drained weights and dividing by the total volume. Water holding capacity was determined as grams of H20 retained in a drained pot per gram of dry substrate in the container. (Table 4-1). Media was prepared by mixing the components in their respective volumetric proportions in a concrete mixer. Osmocote Plus (Everris, Dublin, OH) 15N-2.6P-10K 3-4 month release was incorporated at the rate of 4.8 g fertilizer per liter of substrate. The experiment was conducted within a pad and fan cooled greenhouse at the

Everglades Research and Education Center in Belle Glade, FL.

There were 3 irrigation frequency treatments: daily, every other day, and every 4th day. Irrigation volumes were designed to provide 20% leaching.

Irrigation volumes were calculated every eight days by calculating the mass of

water required to bring each pot back to container capacity. Container capacity is the amount of water held in a container substrate after drainage. The average volume of water to bring each pot back to container capacity for each treatment was then multiplied by 1.2 to determine the following week’s irrigation amounts.

Irrigation amounts for week one were determined by calculating daily evaporative loss from an unplanted pot.

Leaf color was measured at planting, 24 days after planting (DAP), 40

DAP, and 64 DAP with a Minolta-502 SPAD Meter (Minolta, Ramsey, NJ).

Stomatal conductance was measured at planting, 24 DAP, 40 DAP, and 64 DAP with a Delta-T AT4 porometer (Delta-T, Cambridge, UK). Porometer readings were made between 10AM and 2PM on sunny days. SPAD readings and stomatal conductance measurements were made on the youngest fully expanded leaf approximately 1/3 of the leaf’s length basad of the leaf apex. Anthocyanin rich leaf blotches common to bananas were avoided. Leaf counts were done regularly to determine the leaf emission rate. Plant height was measured six times throughout the experiment by measuring from the plant base to the junction of the plant collar and the cigar leaf. After 64 days the experiment was terminated, the stem diameter and the number of functional leaves were measured. The plants were then removed from their pots, had the soil washed off their roots, and dried in a drying room at 65C until no further weight loss occurred. Dry weights of roots and shoots were then measured to determine dry matter accumulation and root/shoot ratio.

In order to determine differences in evaporative water use from the substrates, a 25-day dry down was conducted in unplanted pots. Three pots were prepared with 2200 mL each of the four substrates. Irrometer MLT tensiometers (Irrometer, Riverside, CA) were installed in the containers so that the tip was 10cm below the surface. The pots were saturated and allowed to drain free water before an initial tensiometer and mass readings were calculated.

Daily tensiometer and mass readings were taken for the first 4 days and then on days 6, 7, 10, 11, 15, 17, 22, and 25. At the end of the drying period, the soil was removed from the container in order to determine dry weight. Substrate dry weight was determined by oven drying at 120C until no further weight loss occurred. The substrate dry mass was used to determine the remaining container capacity by subtracting the substrate dry mass and container mass from the measured masses during the experiment. These numbers were converted to percentage of total container capacity by dividing by the mass of water in the container on day 1 after drainage.

The experiment was designed as a factorial with each media x irrigation treatment consisting of 6 replicates for a total of 72 experimental units. The experimental unit consisted of one banana plantlet in a 16.5 cm x 16.5 cm pot.

Statistical analysis was performed with JMP Pro 11.0 using a least squares model with substrate, irrigation frequency and their interaction being the effects in test involving only one measurement. In tests involving several measurements, testing date was added to the effects. The results of statistical tests are reported in Table D-2.

Results and Discussion

Water use in FPM treatments was lower than in SP treatments. Substrate and irrigation interval affected water use. There was also a significant substrate x irrigation interval interaction for water use. From 24 DAP until the end of the experiment, highest water use in FPM treatments occurred in the daily irrigated treatments (Table 4-2, Table 4-3). For SP55, the 2-day treatment had the greatest water use from 32 DAP until the end of the experiment (Table 4-2, Table

4-3). For SP90, the 4-day treatment had the greatest water use from 32 DAP until the end of the experiment (Table 4-2, Table 4-3). Total estimated evapotranspiration (Table 4-4) ranged from 8279 mL in SP55-2 day to 3193 mL in the FPM90-4 day, while water use in the FPM-4 day treatments was lower than in the FPM-1 day treatments, water use in SP-4 day treatments was equal to water use in SP-1 day treatments.

Plant height was responsive to substrate and irrigation, but differences in plant height were only measured starting 42 DAP (Table 4-5). Daily irrigation consistently produced the tallest plants in each treatment. However, in SP55 there were no differences in height between irrigation frequencies. This is likely due to greater root growth in this treatment, which would have allowed the plant to access water that other plants could not. There was a positive response to irrigation for the FPM treatments, but not for the SP treatments in terms of root growth. Silva et al. (1999) found that including FPM in substrates produced smaller plants than those produced in a commercial mixture when irrigated twice per week. This research confirms those findings at the 4-day interval, but disputes those findings with daily irrigation. The height to stem diameter ratio

does indicate some slight crowding since it was below 10:1 (Robinson and

Galán-Saúco, 2009a).

Leaf emission rates have been identified as the most sensitive indicator of soil water deficit (Turner et al., 2007)). In this study, differences that appeared were fairly small, and only appeared 39 DAP (Table 4-7, Table 4-8). At 64 DAP, the only treatment that showed any difference between daily irrigation and longer intervals was in FPM90, where the 1-day treatment separated from the 2-day and

4-day treatments. Some research indicates that drought stress induces leaf yellowing and early leaf senescence (Ke 1979). However, the current research found no differences in number of functional leaves 64 DAP. SPAD readings

(Table 4-9) consistently showed no differences between irrigation frequencies in the SP treatments. In the FPM treatments, which had lower SPAD readings than

SP, there was a trend toward lower SPAD readings with increasing intervals between irrigation events, which agreed with the findings of Ke (1979). The lower SPAD readings could also be indicative of a more intense iron deficiency due to precipitation of iron in dryer substrates.

Dry matter production (Table 4-6) and evapotranspiration (Table 4-10) relate well to porometer readings (Table 4-10) because they are indicative of stomatal aperture, which controls the rate of carbon assimilation and transpiration. Highest water use was found in the most massive plants in SP55-2 day, while the smallest plants were produced in the lowest water using treatment,

FPM90-4 day. Stomatal conductance has been shown to be an indicator of drought stress by several researchers (Shmueli, 1953; Ke, 1979; Turner and

Thomas, 1998; Turner et al., 2007). Drought stress, indicated by stomatal closure, was shown during the 24 DAP and 40 DAP measurement in all 4 day treatments except SP90. Surprisingly, no stress in any treatment was indicated

64 DAP. This could be a result of greater rooting that allowed the plants to access additional water. Under a 25 day unplanted dry down, FPM-based substrates had higher soil moisture and higher water potentials at the end of 25 days (Figure 4-1, Figure 4-2). This finding indicates that evaporation from the

SP-based substrates was higher than in the FPM-based substrates. Given the lack of difference between substrates in terms of stomatal conductance, the differences in water use are likely driven by differences in evaporation from the substrate surface.

Despite using less water, the plants grown in FPM seemed to be more sensitive to longer intervals between irrigation. Rather than being an indication of water in FPM being held at higher tension, as is suggested by Gallego et al.

(2008), the finding points to a possibility that less frequent irrigation caused a deleterious salt accumulation. Biernbaum (1992) noted that extensive leaching was required to maintain electrical conductivty levels in commercially available substrates. Levels of NO3 in an adjoining leaching study were frequently in excess of 100 mg/L and K levels were frequently in excess of 50 mg/L.

Decreasing root weights also point to salt accumulation as a possible reason for increased sensitivity to longer irrigation intervals. As moisture decreased, these concentrations would increase to potentially harmful levels. Interestingly, the proportion of FPM in the mixture did not visibly affect the quality of the plant

grown, so a grower would have some latitude in the proportion of perlite and vermiculite incorporated into the substrate.

In conclusion plant growth and water use are affected by substrate and irrigation frequency. There is also an interaction between substrate and irrigation frequency. Plants grown in FPM based substrates are more sensitive to decreased irrigation frequency. This is likely due to salt accumulation. Daily- irrigated FPM plants were equivalent in size and color to those grown in SP based substrates. These plants were able to reach marketable size and color while using ≈ 70% of the water used in the sphagnum peat based substrates.

Table 4-1. Physical characteristics of sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. FPM was 12 month aged sample from a sugar mill in the Everglades Agricultural Area. Water Bulk Air-Filled Holding Substrate Density1 Porosity2 Porosity Capacity3 FPM55 0.28 72 0.13 1.68 FPM90 0.35 69 0.1 2.22 SP55 0.15 83 0.25 1.65 SP90 0.19 79 0.15 2.04 1g/cm3 2Percent by volume 3 g H2O/g substrate

Table 4-2. Daily evapotranspiration measured gravimetrically during 4 days from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction.1,2 Substrate Irrigation Interval Day 8 Day 16 Day 24 Day 32 (Days) ET SE ET SE ET SE ET SE FPM55 1 40 ±7 46 ±8 120 ±14 A 118 ±13 CDE FPM55 2 51 ±9 48 ±8 88 ±12 CDE 96 ±13 DEF FPM55 4 44 ±3 48 ±3 66 ±5 EF 59 ±7 F FPM90 1 25 ±3 63 ±9 99 ±6 ABCD 124 ±8 CD FPM90 2 38 ±3 50 ±5 75 ±7 DE 85 ±12 EF FPM90 4 30 ±2 49 ±3 46 ±2 F 66 ±6 F SP55 1 43 ±10 83 ±20 118 ±21 AB 165 ±13 B SP55 2 56 ±10 77 ±13 106 ±3 ABC 218 ±21 A SP55 4 48 ±5 79 ±8 100 ±1 ABC 149 ±13 BC SP90 1 58 ±17 63 ±18 113 ±5 AB 139 ±21 BC SP90 2 56 ±10 63 ±11 108 ±7 ABC 132 ±19 BCD SP90 4 54 ±10 63 ±11 95 ±1 BCD 151 ±8 BC 1 ET reported in mL H20 per day 2Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

Table 4-3. Daily evapotranspiration measured gravimetrically during 4 days from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction.1,2 Substrate Irrigation Interval Day 40 Day 48 Day 56 Day 64 (Days) ET SE ET SE ET SE ET SE FPM55 1 142 ±17 AB 113 ±18 CDEF 168 ±16 AB 149 ±19 CD FPM55 2 119 ±9 BC 150 ±22 BCDE 152 ±23 BCD 159 ±24 BCD FPM55 4 89 ±8 C 82 ±9 EF 98 ±9 EF 99 ±10 E FPM90 1 153 ±31 AB 102 ±25 DEF 181 ±8 AB 150 ±21 CD FPM90 2 86 ±11 C 120 ±17 CDEF 116 ±13 DE 126 ±16 D FPM90 4 85 ±10 C 65 ±6 F 74 ±14 F 78 ±11 E SP55 1 158 ±21 AB 208 ±40 AB 158 ±17 BC 191 ±29 AB SP55 2 177 ±3 A 223 ±25 A 200 ±10 A 220 ±18 A SP55 4 166 ±3 A 163 ±8 ABCD 171 ±5 AB 176 ±7 B SP90 1 119 ±12 BC 179 ±19 ABC 128 ±12 CDE 162 ±16 BC SP90 2 154 ±11 AB 145 ±30 BCDE 149 ±18 BCD 156 ±26 BC SP90 4 166 ±7 A 211 ±41 AB 163 ±5 ABC 195 ±22 AB 1 ET reported in mL H20 per day 2Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

69 Table 4-4. Total estimated evapotranspiration measured gravimetrically from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction.1,2 Substrate Irrigation Interval Evapotranspiration (Days) FPM55 1 5829 ±316 CD FPM55 2 5430 ±579 DE FPM55 4 3676 ±293 F FPM90 1 5941 ±328 CD FPM90 2 4414 ±460 EF FPM90 4 3193 ±210 F SP55 1 7348 ±844 AB SP55 2 8279 ±307 A SP55 4 6847 ±189 BC SP90 1 6279 ±441 BCD SP90 2 6219 ±606 BCD SP90 4 7038 ±419 ABC 1 ET measured in mL H20 2Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

70 Table 4-5. Plant heights of ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction.1,2 Irrigatio Day 0 Day 14 Day 28 Day 42 Day 65 Substrat n Heigh S Heigh S Heigh S Heigh S Heigh S e Interval t t t t t E E E E E (Days) FPM55 1 10 ±0 24 ±2 35 ±2 46 ±2 A 50 ±1 A FPM55 2 10 ±0 23 ±2 31 ±1 42 ±2 AB 47 ±2 ABC CD FPM55 4 10 ±0 21 ±1 28 ±0 36 ±1 42 ±1 DEF E ABC FPM90 1 10 ±0 22 ±1 32 ±2 40 ±1 BC 47 ±2 D BC CDE FPM90 2 10 ±0 19 ±1 26 ±1 39 ±2 43 ±2 D F FPM90 4 10 ±0 19 ±1 23 ±1 34 ±1 E 39 ±2 F SP55 1 10 ±0 25 ±1 35 ±3 43 ±2 AB 49 ±2 AB SP55 2 10 ±0 26 ±1 35 ±2 42 ±1 AB 45 ±1 BCD ABC SP55 4 10 ±0 23 ±2 19 ±9 41 ±2 B 46 ±2 D ABC SP90 1 10 ±0 24 ±2 35 ±1 40 ±1 BC 47 ±2 D BCD SP90 2 10 ±0 23 ±2 33 ±1 43 ±2 AB 44 ±2 E SP90 4 10 ±0 23 ±1 30 ±0 35 ±1 DE 39 ±1 EF 1Measured in cm 2Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

71 Table 4-6. Stem diameter, dry weights and root-shoot ration of ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction.1 Substrate Irrigation Stem Diameter2 Shoot Dry Root Dry Weight3 Total Dry Root to Shoot Interval Weight3 Weight3 Ratio (Days) FPM55 1 42.2 ±2.2 AB 29.7 ±3.0 ABC 16.8 ±2.1 BCD 46.5 ±5.0 BC 0.6 ±0.0 CD FPM55 2 37.5 ±2.1 BCD 25.7 ±3.4 CD 12.4 ±1.9 CDEF 38.2 ±5.3 CDE 0.5 ±0.0 D FPM55 4 32.7 ±1.0 EF 20.4 ±1.6 DE 10.7 ±1.0 DEF 31.1 ±2.5 EF 0.5 ±0.0 D FPM90 1 43.0 ±2.1 A 32.5 ±1.3 AB 19.4 ±1.3 B 52.0 ±2.3 AB 0.6 ±0.0 BCD FPM90 2 34.2 ±2.0 DEF 22.0 ±2.4 DE 10.4 ±1.6 EF 32.5 ±4.0 DEF 0.5 ±0.0 D FPM90 4 30.2 ±1.1 F 16.4 ±1.7 E 7.6 ±0.7 F 24.0 ±2.4 F 0.5 ±0.0 D SP55 1 40.7 ±1.7 ABC 29.5 ±2.0 ABC 15.4 ±2.3 BCDE 44.9 ±4.2 BC 0.5 ±0.1 D SP55 2 42.2 ±0.7 AB 34.5 ±0.6 A 26.8 ±2.0 A 61.3 ±2.5 A 0.8 ±0.1 A SP55 4 39.0 ±1.2 ABC 28.2 ±1.4 BC 19.9 ±3.1 B 48.1 ±4.1 BC 0.7 ±0.1 ABC SP90 1 39.3 ±2.3 ABC 25.1 ±2.6 CD 19.2 ±3.8 B 44.2 ±6.3 BC 0.7 ±0.1 AB SP90 2 37.8 ±2.1 BCE 25.5 ±2.5 CD 17.8 ±3.0 BC 43.3 ±5.2 BCD 0.7 ±0.1 ABC SP90 4 37.2 ±0.6 CDE 26.0 ±1.0 CD 21.1 ±1.4 AB 47.1 ±2.2 BC 0.8 ±0.0 A 1Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05) 2Measured in mm 3Measured in g

72 Table 4-7. Leaf production of ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction. Irrigation Day 0 Day 12 Day 20 Day 28 Interval Substrate (Days) Leaves SE Leaves SE Leaves SE Leaves SE FPM55 1 6 ±0.0 8.2 ±0.2 9.2 ±0.2 10.8 ±0.2 FPM55 2 6 ±0.0 8 ±0.0 9 ±0.0 10 ±0.0 FPM55 4 6 ±0.0 8.7 ±0.4 9.7 ±0.4 10.7 ±0.4 FPM90 1 6 ±0.0 8.3 ±0.3 9.3 ±0.3 10.3 ±0.3 FPM90 2 6 ±0.0 7.8 ±0.5 8.8 ±0.5 9.8 ±0.5 FPM90 4 6 ±0.0 8.3 ±0.3 9.3 ±0.3 10 ±0.4 SP55 1 6 ±0.0 8.5 ±0.3 9.5 ±0.3 10.8 ±0.3 SP55 2 6 ±0.0 8.3 ±0.3 9.3 ±0.3 10.5 ±0.3 SP55 4 6 ±0.0 8.7 ±0.3 9.7 ±0.3 10.8 ±0.4 SP90 1 6 ±0.0 8.5 ±0.2 9.5 ±0.2 10.5 ±0.2 SP90 2 6 ±0.0 8.7 ±0.2 9.7 ±0.2 11.2 ±0.3 SP90 4 6 ±0.0 8.5 ±0.2 9.5 ±0.2 11 ±0.4

73 Table 4-8. Leaf production of ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction.1 Day 39 Day 47 Day 64 Irrigation Interval Substrate (Days) Leaves SE Leaves SE Leaves SE FPM55 1 11.8 ±0.2 ABC 12.8 ±0.2 ABCDE 14.2 ±0.3 AB FPM55 2 11.2 ±0.2 C 12.2 ±0.2 CDE 13.5 ±0.2 B FPM55 4 11.5 ±0.6 BC 12.5 ±0.4 BCDE 14.0 ±0.3 AB FPM90 1 12.0 ±0.4 ABC 12.8 ±0.3 ABCDE 14.3 ±0.2 A FPM90 2 11.2 ±0.7 C 12.0 ±0.6 DE 13.5 ±0.7 B FPM90 4 11.5 ±0.2 BC 11.8 ±0.4 E 13.5 ±0.2 B SP55 1 12.5 ±0.4 AB 13.3 ±0.3 AB 14.8 ±0.4 A SP55 2 12.2 ±0.3 ABC 12.8 ±0.4 ABCDE 14.5 ±0.3 A SP55 4 12.7 ±0.3 A 13.2 ±0.5 ABC 14.7 ±0.3 A SP90 1 12.3 ±0.2 AB 13.0 ±0.3 ABCD 14.2 ±0.3 AB SP90 2 12.8 ±0.2 A 13.7 ±0.3 A 14.8 ±0.2 A SP90 4 12.8 ±0.3 A 13.3 ±0.3 AB 14.7 ±0.3 A 1Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

74 Table 4-9. SPAD readings from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction.1 Substrate Irrigation Day 0 Day 24 Day 40 Day 64 Interval (Days) SPAD SPAD SPAD SPAD FPM55 1 41.8 ±3.5 37.1 ±2.7 ABCDE 42.2 ±2.8 D 44.5 ±2.3 BC FPM55 2 44.9 ±3.2 29.1 ±2.7 E 48.2 ±1.3 BC 42.4 ±1.8 CD FPM55 4 42.3 ±2.3 32.1 ±3.3 BCDE 43.7 ±1.2 CD 41.5 ±1.2 CD FPM90 1 47.2 ±1.8 31.7 ±3.3 CDE 44.4 ±1.2 CD 44.4 ±2.1 BC FPM90 2 39.9 ±3.8 39.8 ±3.4 ABC 38.8 ±2.7 D 38.0 ±2.1 D FPM90 4 45.2 ±2.3 30.7 ±2.3 DE 38.9 ±2.6 D 31.9 ±1.5 E SP55 1 39.9 ±3.8 40.1 ±3.3 AB 53.2 ±2.2 AB 48.6 ±1.0 AB SP55 2 45.2 ±2.3 42.0 ±4.0 A 53.3 ±2.1 AB 45.1 ±1.9 ABC SP55 4 45.3 ±2.9 39.9 ±3.9 ABC 54.3 ±2.3 A 47.7 ±1.4 AB SP90 1 39.6 ±3.6 38.9 ±2.2 ABCD 53.6 ±2.0 AB 46.7 ±1.6 ABC SP90 2 46.7 ±2.1 39.1 ±2.0 ABC 53.5 ±1.9 AB 50.4 ±3.2 A SP90 4 43.1 ±2.8 38.5 ±1.5 ABCD 54.2 ±1.9 A 45.6 ±1.4 ABC 1Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

75 Table 4-10. Porometer readings from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were irrigated every 1, 2, or 4 days to container capacity with a 20% leaching fraction.1 Substrate Irrigation Interval (Days) Day 0 Day 24 Day 40 Day 64 s/cm s/cm s/cm s/cm FPM55 1 0.6 ±0.1 0.1 ±0.0 c 1.0 ±0.1 c 0.4 ±0.2 FPM55 2 0.3 ±0.1 1.0 ±0.3 bc 1.3 ±0.1 c 0.4 ±0.1 FPM55 4 0.3 ±0.0 3.3 ±0.6 b 7.2 ±1.4 a 1.1 ±0.5 FPM90 1 0.5 ±0.1 0.3 ±0.1 c 1.4 ±0.3 c 0.4 ±0.2 FPM90 2 0.4 ±0.1 0.5 ±0.2 bc 1.3 ±0.3 c 1.6 ±1.0 FPM90 4 0.4 ±0.1 2.1 ±0.7 bc 9.0 ±3.9 a 0.7 ±0.3 SP55 1 0.5 ±0.1 0.2 ±0.1 c 1.2 ±0.3 c 0.2 ±0.1 SP55 2 0.3 ±0.1 1.4 ±0.4 bc 0.6 ±0.1 c 0.9 ±0.2 SP55 4 0.3 ±0.0 6.5 ±3.3 a 6.2 ±1.9 ab 0.6 ±0.4 SP90 1 0.5 ±0.1 0.3 ±0.1 c 1.4 ±0.3 c 0.5 ±0.2 SP90 2 0.3 ±0.1 0.3 ±0.1 c 0.7 ±0.1 c 0.8 ±0.5 SP90 4 0.4 ±0.1 1.8 ±0.8 bc 2.5 ±0.3 bc 0.2 ±0.1 1Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

76 Figure 4-1. Tensiometer readings from containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Containers were filled to container capacity and allowed to dry down over 25 days.

77 Figure 4-2. Percentage of container capacity remaining from containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Containers were filled to container capacity and allowed to dry down over 25 days.

78 CHAPTER 5 FERTILIZATION OF TISSUE-CULTURE BANANA PLANTLETS GROWN IN SUGARCANE FILTER PRESS MUD BASED SUBSTRATES

Introduction

Bananas, (Musa spp.), are an important crop in Florida not only for their fruit production, but also as landscape plants. According to the most recent

Census of Agriculture, Commercial banana farms currently occupy over 450 hectares in the state, primarily in Miami-Dade County (USDA, 2014). Current recommendations advise growers to start with tissue culture plantlets as they are free of pests and can produce a more uniform crop (Robinson et al., 1993;

Fonsah et al., 2007, Galán-Saúco and Robinson, 2010)

Worldwide annual nursery production of tissue culture plantlets is likely in the hundreds of millions of plants per year based on worldwide acreage and requirements for planting/replanting. Despite this, there is only rudimentary data on proper nursery fertilization of bananas during the nursery phase. For field planting, a fertilizer with a 1 part of nitrogen (N) per 2.5 parts of potassium (K) is recommended (Galán-Saúco and Robinson, 2010). Robinson and Galán-

Saúco (2009a) recommend 2-4 g fertilizer per liter substrate of a 2N-0.4P-1.7K fertilizer, although no published research could be found to support this recommendation over any other type of fertilization. Similarly, Broschat (2009) found that while field-grown palms (Arecaceae) required a fertilizer ratio of 2N-

0.4P-2.5K, he found that container grown palms required a 3N-0.4P-1.7K ratio.

This recommendation also does not account for the nutrient supplying capacity of the substrate itself. Furthermore, there is a lack of information on the proper fertilization rates based on the type of substrate used. Nomura et al. (2009)

79 appears to be the only paper that addresses the nutrient supplying power of the substrate, finding that in compost based substrates, no fertilizer was needed for bananas.

Sphagnum peat (SP) is widely available and is often used as a substrate component in nurseries in North America, while sugarcane filter press mud

(FPM) is a broadly used peat-alternative substrate component wherever sugar mills are nearby.

The purpose of this research was to determine the optimum rate of fertilization for ‘Williams’ bananas in SP-based and FPM-based substrates. The objectives of this research is to determine the effect of substrate on plant growth, the effect of fertilization on plant growth and to determine if there is an interaction between substrate and fertilization.

Methods and Materials

Tissue culture plantlets cv ‘Williams’ were purchased from Sunscape

Nursery in Apopka, FL. The plantlets were acclimated to greenhouse conditions under 50% shade and misting for two weeks. Plantlets were graded prior to planting to a uniform height of 4 cm and 6 leaves.

The substrate mixtures consisted of 90% and 55% FPM or SP by volume mixed with perlite and vermiculite. The nutrient analyses (Table 5-1) of these substrates were performed by saturated media extract at Waters Agricultural

Laboratory, Camilla, GA. Three 2200 mL samples per substrate were placed into containers with liners, which prevented drainage. These containers were saturated and weighed to calculate a saturated weight. The liners were then punctured, which allowed the containers to drain. A drained weight was recorded

80 four hours after the liners were punctured. The substrate was then removed from the container and oven dried at 120C to determine dry weight. Bulk density was determined by dividing dry weight by the 2200. Porosity was determined by calculating the difference between saturated weight and dry weight. Air-filled porosity was determined measuring the difference between the saturated and drained weights and dividing by the total volume. The perlite (Specialty

Vermiculite, Pompano Beach, FL) and vermiculite (Specialty Vermiculite,

Pompano Beach, FL) were mixed in equal parts to make up the complement of each mixture. Media was prepared by mixing the components in their respective volumetric proportions in a concrete mixer.

There were 3 fertilization treatments: Osmocote Plus 15N-2.6P-10K 3-4 month-release (Everris, Dublin, OH) incorporated at the rates of 2.4, 4.8, and 7.2 grams of fertilizer per liter of substrate. These rates correspond to 6, 12, and 18 grams of fertilizer per container.

Leaf color was measured at planting, 4 weeks after planting (WAP), 7

WAP and 10 WAP with a Minolta-502 SPAD Meter (Minolta, Ramsey, NJ).

These measurements were made on the youngest fully expanded approximately

1/3 of the leaf’s length basad of the leaf apex. Anthocyanin rich leaf blotches common to bananas were avoided. Plant height was measured six times throughout the experiment by measuring from the plant base to the junction of the plant collar and the cigar leaf. After plants had grown 64 days, the experiment was terminated. The plants were then removed from their pots, had the soil washed off their roots, and dried in a drying room at 65C until no further

81 weight loss occurred. Dry weights of roots and shoots were then measured to determine dry matter accumulation and root/shoot ratio.

The experiment was designed as a factorial with each media x fertilization treatment consisting of 6 replicates for a total of 72 experimental units. The experimental unit consisted of one banana plant in a 16.5 cm x 16.5 cm pot. The experiment was conducted within a pad and fan cooled greenhouse maintained between 20˚C and 30˚C at the Everglades Research and Education Center in

Belle Glade, FL. Statistical analysis was performed with JMP Pro 11.0 using a least squares model with substrate, fertilizer rate and their interaction being the effects in test involving only one measurement. In tests involving several measurements, testing date was added to the effects. The results of statistical tests are reported in table D-3.

Results and Discussion

Final plant height ranged from 41 cm in the SP90-6g treatment to a height of 54 cm in the SP55-12g and SP55-18g treatments (Table 5-2). 50 cm height is the reported upper boundary for field planting tissue culture banana plants

(Robinson and Galán-Saúco, 2009a; Robinson et al., 1993). Treatments with 12 g and 18 g of fertilizer per container were able to reach or exceed this maximum marketable height in 65 days. Not surprisingly, the largest, and most green plants were produced using the highest fertilizer rates. However, plants grown at the middle rate were usually statistically equivalent to those grown using the high fertilizer rate. When plant height was modeled over the entire experiment, substrate was a significant predictor for the model, while fertilizer rate was not.

At the final height measurement, fertilizer was clearly a factor as the plants

82 fertilized at the 6g rate separated from the 12g and 18g treatments (Table 5-2).

Lima et al. (2009) found that fertilization with 14N-5.6P-11.6K increased banana height in a commercial substrate and a compost containing substrate. Martins et al. (2009) in a test of different fertilizer sources and substrates found no interaction between fertilizer source and substrate, but did find that plants with

Osmocote 14N-5.6P-11.6K produced taller banana plants with larger stem diameters.

At the termination of the experiment, there were no differences between treatments in the number of functional leaves. For leaf counts, substrate was a significant factor, while fertilizer rate was not. However, at the final leaf count no significant differences were shown (Table 5-3; Table 5-4). All plants had approximately 10 leaves, which is the number advised for field planting.

SPAD readings were affected by both fertilizer rate and substrate. No significant differences were observed until 7 WAP (Table 5-5). At the final SPAD reading, the highest SPAD readings were observed in the SP treatments. All 6 g treatments were equivalent across substrates. FPM55 did not show a response to fertilizer for SPAD readings. Iron deficiency was observed in the FPM treatments (Figure 5-1). This was likely caused by the high pH of the substrate or possibly elevated container capacity (Lahav and Turner, 1989).

Substrate and fertilizer rate were significant predictors for stem diameter, shoot dry weight, root dry weight, and total dry weight. Final stem diameter

(Table 5-6) ranged from 36 mm in the SP90-6g treatment to 48 mm in the SP55-

18 g treatment. Stem diameter of plants grown in SP appears to be slightly more

83 responsive to fertilizer than plants grown in FPM; however, the trend falls just outside of the confidence limits. Also, none of the plants had the 10:1 height to stem diameter ratio reported by Robinson and Galán-Sáuco to be proper for field planting (2009a). This is likely due to some etiolation that occurred towards the end of the experiment as plants became crowded.

Shoot dry weight ranged from 19.6 g in the SP90-6g treatment to 34.2 g in the SP55-12g treatment. Root dry weight ranged from 11.0 g in the FPM55-6g treatment to 22.0 g in the SP55-6g treatment. Total dry weight ranged from 35.1 g in the FPM55-6g treatment to 54.5 g in the SP55-18g treatment. Dry weights tended to be higher with increasing rates of fertilizer, however in SP55 all fertilizer rates produced plants with equivalent total dry matter (Table 5-6). This is due primarily to increased root dry matter in the SP55-6g treatment, in comparison to the other 6g treatments. Root to shoot ratios ranged from 0.4 in

FPMM55-6g to 0.8 in SP55-6g. The highest root-shoot ratios were found in

SP55-6g and SP90-6g (Table 5-6). Root weight and root-shoot ratios were the only observations where there was a significant interaction effect. The root-shoot ratio response was contrasting between FPM and SP. The lowest root-shoot ratios were in the lowest rate of fertilizer in FPM, while the highest root-shoot ratios in SP occurred at the lowest rate of fertilizer. This observation is likely due to a lack of P. Broschat and Klock-Moore (2000) found that in areca palm

(Dypsis lutescens) root to shoot ratio was around 0.8 in when grown at 0mg P per week, while at 16mg P per week the ratio was around 0.2.

84 In conclusion, substrate and fertilization affect plant growth and color. The only interaction was in terms of root growth and root to shoot ratio. If available,

SP55 would be the preferred substrate to use, as plant growth measurements at the 12 g fertilizer rate were equivalent to those at the 18 g fertilizer rate. If FPM is readily available from a local sugar mill, the 18 g fertilizer rate would produce the largest plants. Caution must be used with the material though as there may be micronutrient deficiencies due to the high pH of the material. Results could be different if a micronutrient spray containing iron had been used in this study.

Also, the elevated nutrient concentrations of nutrients in FPM could necessitate a change in fertilizer to account for the initially high levels of NO3 and K. High levels of those nutrients can cause root damage. Ultimately, the costs of fertilizer and substrate would have to be considered.

85 Table 5-1. Analyses of the sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite, FPM was 12 month aged sample from a sugar mill in the Everglades Agricultural Area.1,2 Measurement FPM55 FPM90 SP55 SP90 pH 7.2 7.4 6.5 6.3 Organic 23.82 32.37 26.37 46.63 Matter3 Bulk Density4 0.28 0.35 0.15 0.19 Porosity 72 69 83 79 Air-Filled 13 10 25 15 Porosity5 Electrical 2.09 2.35 0.514 0.501 Conductivity6 Nitrate- 204.8 235.9 5.6 7.7 Nitrogen Ammonium- 8.75 12.25 9.8 13.3 Nitrogen Phosphorus 9.56 9.68 4.45 4.92 Potassium 44.73 77.78 18.12 19.39 Calcium 228.3 261.7 28.88 32.78 Magnesium 100.0 117.5 12.06 13.36 Sulfur 73.49 70.17 42.75 35.79 Boron 0.07 0.06 0.16 0.15 Zinc 0.55 0.62 0.17 0.09 Manganese 0.01 0.01 0.17 0.22 Iron 0.02 0.04 0.35 0.28 Copper 0.03 0.04 0.01 0.03 1Nutrient values reported as mg/kg 2Chemical analyses performed by Waters Agricultural Laboratory, Camilla, GA, Saturated media extract 3Percent 4g/cm3 5Percent by volume 6mS/cm

86 Table 5-2. Plant heights of ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.1,2 Day 0 Day 14 Day 28 Day 42 Day 65 Fertilizer Height Height Height Height Height Substrate Rate (cm) SE (cm) SE (cm) SE (cm) SE (cm) SE FPM55 6 10 ±0 24 ±1 ABCD 26 ±2 D 38 ±1 CDE 52 ±3 A FPM55 12 10 ±0 24 ±1 A 30 ±2 ABCD 40 ±2 BCDE 50 ±3 A FPM55 18 10 ±0 22 ±1 A 33 ±2 A 43 ±3 ABC 47 ±2 ABC FPM90 6 10 ±0 21 ±1 DEF 27 ±2 BCD 37 ±2 CDE 43 ±2 C FPM90 12 10 ±0 20 ±1 BCDE 28 ±1 CD 38 ±2 DE 50 ±3 A FPM90 18 10 ±0 20 ±1 CDEF 31 ±2 ABC 43 ±1 ABC 53 ±2 A SP55 6 10 ±0 23 ±1 AB 30 ±1 ABCD 38 ±2 CDE 43 ±2 C SP55 12 10 ±0 20 ±1 DEF 32 ±1 AB 46 ±2 A 54 ±2 A SP55 18 10 ±0 18 ±1 F 26 ±2 D 47 ±2 A 54 ±2 A SP90 6 10 ±0 19 ±0 EF 26 ±1 D 36 ±1 E 41 ±2 C SP90 12 10 ±0 20 ±0 BCDE 32 ±2 AB 41 ±1 BCD 49 ±2 AB SP90 18 10 ±0 22 ±1 ABC 32 ±1 AB 45 ±2 AB 53 ±3 A 1Measured in cm, from soil surface to junction of the top leaf collar and the cigar leaf. 2Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

87 Table 5-3. Leaf counts Day 0-28 of Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.1 Substrate Fertilizer Rate Day 0 Day 12 Day 20 Day 28 Leaves SE Leaves SE Leaves SE Leaves SE FPM55 6 6.0 ±0.0 8.2 ±0.5 BCDE 9.2 ±0.5 BCDE 10.5 ±0.4 ABC FPM55 12 6.0 ±0.0 7.7 ±0.3 E 8.7 ±0.3 E 9.8 ±0.3 C FPM55 18 6.0 ±0.0 8.3 ±0.3 ABCDE 9.3 ±0.3 ABCDE 10.5 ±0.3 ABC FPM90 6 6.0 ±0.0 8.2 ±0.4 BCDE 9.2 ±0.4 BCDE 10.3 ±0.4 BC FPM90 12 6.0 ±0.0 8.0 ±0.4 CDE 9.0 ±0.4 CDE 10.3 ±0.5 C FPM90 18 6.0 ±0.0 7.8 ±0.3 DE 8.8 ±0.3 DE 9.8 ±0.3 C SP55 6 6.0 ±0.0 9.0 ±0.4 AB 9.9 ±0.3 ABC 11.1 ±0.4 AB SP55 12 6.0 ±0.0 8.8 ±0.3 ABC 9.8 ±0.3 ABC 11.5 ±0.4 A SP55 18 6.0 ±0.0 9.2 ±0.2 A 10.2 ±0.2 A 11.4 ±0.4 AB SP90 6 6.0 ±0.0 8.7 ±0.2 ABCD 9.8 ±0.2 ABCD 11.2 ±0.4 AB SP90 12 6.0 ±0.0 8.3 ±0.2 ABCDE 9.3 ±0.2 ABCDE 11.0 ±0.4 AB SP90 18 6.0 ±0.0 9.0 ±0.3 AB 10.0 ±0.2 AB 11.1 ±0.3 AB 1Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

88 Table 5-4. Leaf counts Day 39-64 of Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.1 Substrate Fertilizer Day 39 Day 47 Day 64 Rate Leaves SE Leaves SE Leaves SE FPM55 6 11.8 ±0.3 ABCD 12.5 ±0.4 ABCD 14.0 ±0.4 FPM55 12 11.2 ±0.3 D 11.8 ±0.3 D 13.5 ±0.2 FPM55 18 12.0 ±0.3 BCD 12.5 ±0.3 ABCD 14.0 ±0.3 FPM90 6 11.8 ±0.5 BCD 12.0 ±0.4 CD 13.8 ±0.5 FPM90 12 11.7 ±0.6 CD 12.3 ±0.5 BCD 13.7 ±0.6 FPM90 18 11.2 ±0.4 D 12.0 ±0.4 CD 13.8 ±0.4 SP55 6 12.8 ±0.5 AB 13.0 ±0.5 ABC 14.7 ±0.6 SP55 12 12.5 ±0.4 ABC 13.5 ±0.4 A 14.8 ±0.6 SP55 18 13.0 ±0.3 A 13.5 ±0.3 A 14.8 ±0.4 SP90 6 12.5 ±0.2 ABC 13.0 ±0.3 ABC 14.5 ±0.2 SP90 12 12.3 ±0.2 ABC 13.0 ±0.4 ABC 14.3 ±0.2 SP90 18 12.3 ±0.3 ABC 13.3 ±0.3 AB 14.8 ±0.2 1Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

89 Table 5-5. SPAD readings from youngest fully expanded leaf of Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.1 Substrate Fertilizer Week 0 Week 4 Week 7 Week 10 Rate SPAD SPAD SPAD SPAD FPM55 6 43.1 ±2.8 33.5 ±2.9 48.3 ±1.5 DEF 42.4 ±0.9 CD FPM55 12 41.8 ±3.5 37.5 ±2.8 46.2 ±2.2 DEF 43.6 ±2.1 CD FPM55 18 46.7 ±2.1 40.8 ±3.1 48.7 ±3.0 CDE 43.3 ±1.8 CD FPM90 6 47.2 ±1.8 28.0 ±3.6 43.0 ±2.2 F 40.9 ±1.7 D FPM90 12 44.9 ±3.2 31.5 ±2.2 43.7 ±1.9 DEF 38.9 ±1.2 D FPM90 18 42.3 ±2.3 37.3 ±1.7 46.8 ±1.3 DEF 46.1 ±0.7 BC SP55 6 42.3 ±2.3 39.1 ±2.3 49.6 ±1.3 BCD 42.9 ±2.7 CD SP55 12 47.2 ±1.8 34.1 ±4.0 53.8 ±0.5 BC 49.0 ±1.5 AB SP55 18 39.9 ±3.8 40.8 ±3.9 50.5 ±1.9 BCD 49.6 ±1.2 AB SP90 6 45.2 ±2.3 36.6 ±3.5 50.6 ±2.4 BCD 42.9 ±2.7 CD SP90 12 45.3 ±2.9 38.9 ±3.8 54.6 ±2.4 AB 53.5 ±2.1 A SP90 18 39.6 ±3.6 38.2 ±3.6 59.6 ±1.1 A 53.6 ±1.9 A 1Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

90 Figure 5-1 Iron deficiency observed on ‘Williams’ bananas grown in sugarcane filter press mud based substrates.

91 Table 5-6. Stem diameter, dry weights, and root to shoot ratio of ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.1 Substrate Fertilization Stem Diameter2 Shoot Dry Root Dry Total Dry Root to Shoot Rate Weight3 Weight3 Weight3 Ratio FPM55 6 39.0 ±2.5 cd 24.1 ±2.3 de 11.0 ±1.8 c 35.1 ±4.0 c 0.4 ±0.0 f FPM55 12 41.8 ±2.9 c 27.1 ±2.5 cd 12.5 ±1.9 c 39.6 ±4.2 bc 0.5 ±0.0 f FPM55 18 42.8 ±0.8 bc 32.0 ±1.4 ab 19.6 ±2.4 ab 51.6 ±3.8 a 0.6 ±0.0 cde FPM90 6 39.7 ±2.2 cd 24.5 ±1.9 d 12.1 ±1.4 c 36.6 ±3.2 c 0.5 ±0.0 ef FPM90 12 39.2 ±2.0 cd 27.7 ±2.0 bcd 19.5 ±1.8 ab 47.2 ±3.7 ab 0.7 ±0.0 bc FPM90 18 42.3 ±1.4 c 31.7 ±1.5 abc 18.5 ±1.6 ab 50.2 ±3.0 a 0.6 ±0.0 cde SP55 6 40.2 ±1.3 cd 26.2 ±0.7 d 22.0 ±1.4 a 48.2 ±1.8 ab 0.8 ±0.0 a SP55 12 47.5 ±1.4 ab 34.2 ±1.9 a 18.9 ±1.6 ab 53.1 ±3.5 a 0.5 ±0.0 def SP55 18 47.8 ±1.2 a 33.5 ±1.0 a 21.0 ±1.5 a 54.5 ±2.0 a 0.6 ±0.0 cd SP90 6 35.5 ±1.1 d 19.6 ±1.0 e 15.9 ±1.7 bc 35.5 ±2.5 c 0.8 ±0.1 ab SP90 12 43.3 ±1.2 abc 27.8 ±1.3 bcd 17.8 ±1.9 ab 45.6 ±2.9 ab 0.6 ±0.1 cd SP90 18 47.5 ±1.4 ab 31.3 ±1.3 abc 19.7 ±1.4 ab 51.0 ±2.2 a 0.6 ±0.0 cd 1Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05) 2Measured in mm 3Measured in g

92 CHAPTER 6 NUTRIENT LEACHING FROM TISSUE-CULTURE BANANA PLANTLETS GROWN IN SUGARCANE FILTER PRESS MUD BASED SUBSTRATES

Introduction

Nutrient leaching from nursery containers represents both a monetary waste and an environmental liability. Leaching is essential to container production to prevent a build-up of salts and to ensure saturation of the media. Growers must try to limit both the volume of leachate and the nutrient concentration in the leachate.

Bananas (Musa spp.) are grown throughout the tropics and subtropics. Use of tissue culture banana plants is widely practiced and millions of plants are produced each year. These banana plants will spend 2-4 months in a container nursery.

Substrate choice, fertilization practices, and irrigation practices vary widely depending on the complexity and locale of the banana nursery. However, recommendations are outlined by Robinson and Galán Saúco (2009a).

This research seeks to quantify the amount of nitrate (NO3-N), ammonium (NH4-

N), phosphate-phosphorus (PO4-P), and potassium (K) leached from container-grown bananas fertilized with 3 different rates of fertilizer in 4 different substrates and irrigated daily. The objectives of this research will determine the effect of substrate and fertilizer rate on nutrient leaching, and will also determine if there is an interaction between substrate and fertilizer rate.

Methods and Materials

Tissue culture banana plantlets cv ‘Williams’ were purchased from Sunscape

Nursery in Apopka, FL. The plantlets were acclimated to greenhouse conditions under

50% shade and misting for two weeks. Plantlets were graded prior to planting to a uniform height of 4 cm and 6 leaves.

93 The substrate mixtures consisted of 90% and 55% FPM or SP by volume mixed with perlite and vermiculite. The perlite (Specialty Vermiculite, Pompano Beach, FL) and vermiculite (Specialty Vermiculite, Pompano Beach, FL) were mixed in equal parts to make up the complement of each mixture. Media was prepared by mixing the components in their respective volumetric proportions in a concrete mixer. The nutrient analyses (Table 6-1) of these substrates were performed by saturated media extract at

Waters Agricultural Laboratory, Camilla, GA. Contributions of NO3-N, NH4-N, P, and K by the substrate were determined by multiplying the concentrations reported by Waters

Agricultural Laboratory by the dry weight of 2500 mL of substrate.

There were 3 fertilization treatments: Osmocote Plus 15N-2.6P-10K 3-4 month- release (Everris, Dublin, OH) incorporated at the rates of 2.4, 4.8, and 7.2 grams of fertilizer per liter of substrate. These rates correspond to 6, 12, and 18 grams of fertilizer per container. Contributions of NO3-N, NH4-N, P, and K from substrate and fertilizer are reported in Table 6-2.

From 1-24 days after planting (DAP), 100mL of irrigation per pot was applied.

From 25-64 DAP, 200 mL of irrigation per pot was applied. At planting, and every 8 days thereafter a leachate sample was collected using the Pour-Thru method (Cavins et al., 2000). Daily leachate volume was estimated to be the difference between the irrigation amount and gravimetric evapotranspiration as measured in an adjoining irrigation experiment. Gravimetric evapotranspiration was calculated by measuring difference in mass of the plant, substrate and container before irrigation and after irrigation to container capacity followed by drainage.

94 Leachate was prepared for analysis by dilution in 5mL vials so that concentration could be interpolated on the standard curve. Analysis for NO3-N, NH4-N, and PO4-P were performed by colorimetry with a Seal Autoanalyzer 3. Analysis for K was performed by atomic absorption spectrometry with a PerkinElmer AAnalyst 400. Both machines were located at the UF/IFAS Fort Lauderdale Research and Education

Center.

The experiment was designed as a factorial with each media by fertilization treatment consisting of 3 replicates for a total of 36 experimental units. The experimental unit consisted of one banana plant in a 16.5 cm x 16.5 cm pot. The experiment was conducted within a pad and fan cooled greenhouse maintained between 20˚C and 30˚C at the Everglades Research and Education Center in Belle

Glade, FL. Statistical analysis was performed with JMP Pro 11.0 using a least squares model with substrate, fertilizer rate and their interaction being the effects. The results of statistical tests are reported in Table D-4.

Results

Total estimated leachate (Table 6-3) varied considerably from 2747 mL of H2O in the SP55 treatments to 4171 mL of H2O in the FPM 55 treatment. The SP55 treatment had one week where leaching would not have been expected to occur due to evapotranspiration that exceeded the 200 mL/day irrigation schedule.

Mass of NO3-N leached ranged from 992.34 mg/pot in FPM55-18g to 52.24 mg/pot in SP90-6g (Table 6-4). Nitrate leaching was higher in FPM treatments. The lowest rates of fertilization in FPM treatments leached more NO3-N than the SP treatments with the highest rates of fertilization.

95 Mass of NH4 leached ranged from 595.53 mg/pot in SP90-18g to 5.45 mg/pot in the FPM90-6g (Table 6-4). Ammonium leaching was higher in SP treatments, with only the lowest rates of fertilization in SP treatments having equivalent leaching to the FPM treatments.

Mass of N from NO3-N and NH4-N ranged from 1003.34 mg/pot in SP90-18g to

96.54 mg/pot in SP55-6g (Table 6-4). Increased rates of fertilizer were associated with increasing amounts of nitrogen leaching.

Mass of PO4-P leached ranged from 118.45 mg/pot in SP90-18g to 3.32 mg/pot in FPM90-18g (Table 6-5). Phosphorus leaching was higher in SP treatments. The 6g treatments of SP were statistically similar to the FPM treatments, but still had mean P-

PO4 leaching two times as high as the highest mean PO4-P leached in FPM treatments.

Mass of K leached ranged from 219.21 mg/pot in FPM55-18g to 18.75 mg/pot in

SP55-6g (Table 6-5). Potassium leaching in FPM treatments was higher than in SP treatments. Potassium leaching tended to increase with increasing fertilization.

Discussion

Nitrate-nitrogen leachate was elevated in the FPM treatments and some concentrations measured early in the crop would be of environmental concern (Figure

6-1). Curves for the two FPM-based substrates are similar except at the highest rate of fertilization. When comparing the 18g treatments of FPM55 and FPM90, it appears that

- this rate of fertilizer exceeds the capacity of the substrate to retain NO3 .

Concentrations in the FPM-18g treatments never were below the 10 mg/L threshold for drinking water. Beeson (1996) reported decreased NO3-N leachate when yardwaste compost consisted of 20-40 percent of the substrate in Pittosporum tobira variegata and

Rhododendron indicum when compared to a 3:1:1 pine bark, sledge peat and sand

96 substrate. This data suggest that the type of composted material may have a large impact on NO3-N leaching. This is not surprising, as the substrate was determined to have high levels of NO3-N prior to planting. In agreement with the work of Broschat

(1995) on Spathiphyllum and Dypsis lutescens, NO3-N concentrations did decrease as the experiment progressed.

Ammonium-nitrogen leachate concentrations were higher in the SP treatments

(Figure 6-2) than in the FPM treatments. Shober et al. (2010) found that sphagnum- based substrates had high ammonium loads, especially early in the growth phase of

Begonia x hybrida, Solenostemon scutellarioides, and Tagetes patula. There also appears to be a strong fertilizer rate signal as the concentrations of NH4-N increased with increasing rates of fertilizer. At 24 DAP peak NH4-N concentrations are reached and SP90 has greater amplitude than SP55.

The difference in leaching of N between FPM and SP based substrates seems to be in speciation. FPM primarily leached NO3-N, while SP primarily leaches NH4-N. A major component of FPM is the histosols of the Everglades Agricultural Area, which are flocculated out of the sugarcane juice solution with other contaminants. These histosols are highly humified reed-sedge peats. Shober et al. (2010) found that reed-sedge peats had higher leaching of NO3-N, while sphagnum peats had higher leaching of NH4-N. At the 12g fertilization rate N leaching was the same across substrates except for SP55-

12g. There seems to be a contrasting affect in terms of percentage of FPM or SP in terms of N leaching. FPM55 had numerically higher N leaching than FPM90 at each level of fertilization, while SP55 had numerically lower N leaching than SP90 at each level of fertilization. This is likely due to differences in cation exchange capacity (CEC)

97 and the charge of the primary species of nitrogen being leached. Vermiculite, which has an elevated CEC, was 22.5 percent of the media in the FPM55 and SP55

+ treatments. Vermiculite likely played a positive role in binding NH4 in SP treatments.

Lucas (1982) noted that muck soil, which is the primary constituent of FPM, has nearly eight times the CEC of sphagnum peat on a volume basis. This could explain why so little NH4-N was leached from the FPM treatments. The change in the shape of the

NO3-N release curves in response to increasing fertilization provides evidence of

- saturation of NO3 binding sites in FPM55 (Figure 6-1).

The findings of lower P leaching in FPM-based substrates is intriguing as FPM is roughly 2 percent P, and the FPM-based substrates has SME phosphorus readings twice as high as the SP-based substrates. Phosphate-phosphorus leaching was relatively flat throughout the experiment and was not impacted by fertilization in the

FPM treatments (Figure 6-5). This was likely due to precipitation and immobilization of the nutrient. The pH of the FPM-based substrates was higher and calcium (Ca) and magnesium (Mg) levels were 10-fold higher than in the SP-based substrates. The SP treatments did show decreasing levels of phosphorus as the experiment progressed, which seems to conflict with the finding of Broschat (1995) that P leaching increased over time. However, that experiment was seven months longer than the current study and involved several fertilization events. Phosphate-phosphorus leaching from SP55 was lower than in SP90 indicating that vermiculite might be binding some of the PO4-P.

Potassium leaching was generally not considered an environmental hazard, but did represent a loss of a resource. Since K levels in the FPM-based substrates were already higher at the beginning of the experiment, it was expected that FPM-based

98 substrates would leach more potassium than SP-based substrates (Figure 6-5). Also given the large quantities of divalent cations in FPM-based media, and its histosol provenance, potassium may have been unable to compete at cation-exchange sites, and stayed in soil solution available to be leached. The change in the shape of the K release curves in response to increasing fertilization provides evidence of saturation of

K binding sites in FPM55 (Figure 6-4). This change in shape follows the pattern see in the NO3-N release curves.

In conclusion, substrate, fertilization, and their interaction affect nutrient leaching.

The high levels of NO3-N leaching indicate that N fertilization practices when using

FPM-based media could be adjusted to match the amount of N added as fertilizer to the needs of the plant. Phosphorus leaching was much lower in FPM than in SP. This is important, as FPM would likely be used close to its source near the Everglades where P is a nutrient of concern. Adjusting the proportion of vermiculite and perlite incorporated into the substrate could be a nutrient management tool for managing N and P leaching.

Potassium leaching was higher in the FPM-based media; however, K is not an element of environmental concern. This finding may indicate that K fertilization practices could be altered. In a commercial nursery setting, it may be advisable to collect the early leachate from FPM-based media, treat it for pathogens, and then recycle the leachate by fertigation. The volume of leachate was higher for FPM-based substrates. Adopting an evapotranspiration based irrigation regime would decrease mass of nutrients of leached, and would benefit both the grower and the environment.

99 Table 6-1. Analyses of the sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite, FPM was 12 month aged sample from a sugar mill in the Everglades Agricultural Area.1,2 Measurement FPM55 FPM90 SP55 SP90 pH 7.2 7.4 6.5 6.3 Organic Matter3 23.82 32.37 26.37 46.63 Bulk Density4 0.28 0.35 0.15 0.19 Porosity5 72 69 83 79 Air-Filled5 13 10 25 15 Porosity Electrical 2.09 2.35 0.514 0.501 Conductivity6 Nitrate-Nitrogen 204.8 235.9 5.6 7.7

Ammonium- 8.75 12.25 9.8 13.3 Nitrogen Phosphorus 9.56 9.68 4.45 4.92 Potassium 44.73 77.78 18.12 19.39 Calcium 228.3 261.7 28.88 32.78 Magnesium 100.0 117.5 12.06 13.36 Sulfur 73.49 70.17 42.75 35.79 Boron 0.07 0.06 0.16 0.15 Zinc 0.55 0.62 0.17 0.09 Manganese 0.01 0.01 0.17 0.22 Iron 0.02 0.04 0.35 0.28 Copper 0.03 0.04 0.01 0.03 1Nutrient values reported as mg/kg 2Chemical analyses performed by Waters Agricultural Laboratory, Camilla, GA, Saturated media extract 3Percent dry weight 4g/cm3 5Percent by volume 6mS/cm

100 Table 6-2. Contributions of nitrate-nitrogen, ammonium-nitrogen, phosphorus, and potassium from fertilizer and substrate in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.1 Substrate Fertilizer Nitrate-nitrogen Ammonium-nitrogen Phosphorus Potassium rate Substrate Fertilizer Total Substrate Fertilizer Total Substrate Fertilizer Total Substrate Fertilizer Total FPM55 6 143 396 539 6 504 510 7 156 163 31 600 631 12 143 792 935 6 1008 1014 7 312 319 31 1200 1231 18 143 1188 1331 6 1188 1194 7 468 475 31 1800 1831 FPM90 6 206 396 602 11 504 515 8 156 164 68 600 668 12 206 792 998 11 1008 1019 8 312 320 68 1200 1268 18 206 1188 1394 11 1188 1199 8 468 476 68 1800 1868 SP55 6 2 396 398 4 504 508 2 156 158 7 600 607 12 2 792 794 4 1008 1012 2 312 314 7 1200 1207 18 2 1188 1190 4 1188 1192 2 468 470 7 1800 1807 SP90 6 4 396 400 6 504 510 2 156 158 9 600 609 12 4 792 796 6 1008 1014 2 312 314 9 1200 1209 18 4 1188 1192 6 1188 1194 2 468 470 9 1800 1809 1reported in mg/container

101 Table 6-3. Estimated weekly leachate from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.1 Day Day Day Day Day Day Day Day Total 1-8 9-16 17-24 25-32 33-40 41-48 48-56 56-64 Leachate FPM 55 547 480 432 640 656 464 696 256 4171 FPM 90 400 600 296 808 608 376 784 152 4024 SP 55 547 456 136 656 280 336 0 336 2747 SP 90 360 336 296 696 488 648 168 576 3568 1 Measured in mL H20 per pot.

102 Table 6-4. Nitrogen leached from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.1,2 Substrate Fertilizer Rate Nitrate-nitrogen SE Ammonium-nitrogen SE Nitrogen SE FPM55 6 539.07 17.37 CD 6.66 1.07 D 545.73 16.52 CD FPM55 12 604.76 37.06 BC 8.61 1.51 D 613.36 38.56 CD FPM55 18 992.34 30.78 A 11.00 1.44 D 1003.34 31.87 A FPM90 6 301.22 22.93 E 5.45 0.40 D 306.67 22.63 E FPM90 12 483.35 40.17 D 6.19 0.54 D 489.54 40.54 D FPM90 18 672.97 40.22 B 6.64 0.85 D 679.61 39.39 BC SP55 6 52.24 6.55 I 44.21 3.96 CD 96.45 3.42 F SP55 12 132.01 6.95 H 154.20 57.98 C 286.21 64.43 E SP55 18 206.44 20.26 FG 289.02 65.61 B 495.46 84.33 D SP90 6 50.87 10.38 I 47.93 6.19 CD 98.79 16.57 F SP90 12 142.51 9.78 GH 354.91 87.27 B 497.42 84.93 D SP90 18 226.53 22.31 F 595.53 66.34 A 822.06 88.44 B 1Measured in mg/pot 2Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

103 Table 6-5 Phosphate-phosphorus and potassium leached from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.1,2 Phosphate- Substrate Fertilizer Rate phosphorus SE Potassium SE FPM55 6 6.33 1.82 C 147.25 7.94 C FPM55 12 8.46 1.00 C 142.55 11.79 C FPM55 18 10.15 1.63 C 219.21 9.81 A FPM90 6 5.58 1.42 C 103.45 3.48 D FPM90 12 4.03 0.49 C 131.01 10.74 C FPM90 18 3.32 0.52 C 185.62 14.10 B SP55 6 19.42 1.40 C 18.75 1.97 F SP55 12 33.49 4.91 BC 57.36 3.01 E SP55 18 62.52 8.29 B 74.25 11.72 E SP90 6 25.63 4.38 C 19.62 2.64 F SP90 12 64.25 7.87 B 52.11 1.92 E SP90 18 118.45 34.82 A 73.51 14.18 E 1Measured in mg/pot 2Means in the same column followed by different letters are significantly different (Student’s t, JMP 11.0; α =0.05)

104 Figure 6-1. Nitrate concentrations in leachate from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.

105 Figure 6-2. Ammonium concentrations in leachate from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.

106 Figure 6-3. Phosphate concentrations in leachate from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.

107 Figure 6-4. Potassium concentrations in leachate from ‘Williams’ bananas grown in containers filled with sugarcane filter press mud (FPM) and sphagnum peat (SP) in 90% and 55% by volume mixtures with equal parts perlite and vermiculite. Plants were fertilized with 6, 12, or 18 grams of fertilizer per container.

108 CHAPTER 7 CONCLUSIONS

Sugarcane filter press mud (FPM) is an acceptable substrate for growing tissue culture banana plantlets during the acclimatization stage. The physical characteristics of the material provided adequate water holding, drainage, and aeration. The chemical characteristics were less than ideal based on pH, electrical conductivity, nitrate, and potassium levels, but those did not seem to have a deleterious impact on plant growth.

From the irrigation frequency experiment, it was shown that bananas grown in FPM- based substrates use less water than those grown in sphagnum peat-based substrates.

However, bananas grown in FPM-based substrates do require daily irrigation for best growth. In the fertilization experiment, bananas grown in FPM based substrates were shown to be responsive to increased amounts of fertilization, in spite of the high levels of nutrients in FPM. This could be a response to higher levels of nitrogen and potassium available later in the growth cycle, or a positive responsive to the additional iron from the fertilizer, since it was shown that bananas grown in FPM showed symptoms of iron deficiency. Leachate from bananas grown in FPM was high in nitrate-nitrogen (NO3-N) and potassium (K). Leaching of these nutrients can be a liability unless a method for recycling the leachate is employed. Nitrogen leaching was higher in FPM-based substrates than in SP-based substrates. Phosphorus leaching was much lower in FPM than it was in SP. Adjusting the proportion of perlite and vermiculite can affect the amount of nutrient leaching at the highest rate of fertilization tested.

This research can be synthesized to design an irrigation and fertilization scheme that takes advantage of the benefits of FPM and attempts to minimize the liabilities.

109 Given the high levels of NO3-N and K in the leachate, fertilization with these elements might be revised. Addition of foliar iron sprays would improve plant performance in

FPM, possibly changing the ranking of the various irrigation and fertilizer treatments. By taking advantage of the lower levels of evapotranspiration in FPM-based media, leachate volume might also be cut. By decreasing fertilizer and water use, the liability of enriched runoff is decreased, while sustainability is increased.

110 APPENDIX A IMPACT OF FERTILIZATION ON VINCA GROWN IN BINARY MIXTURES OF SUGARCANE FILTER PRESS MUD AND VERMICULITE

Introduction

FPM, and eventually dry out. During the off-season, the material is removed and applied to agricultural fields, usually in close proximity to the sugarcane mill (Qureshi et al., 2001). One analysis concluded that it would only be profitable to apply FPM to fields with sandy soils within 10 miles of the sugarcane mill and that the optimum rate for yields would be adding 20-25 cm of FPM to the soil (El-Hout, 2008). Currently, only one sugar mill is located in an area with sandy soils.

Fertilizer demand has increased due to increasing row crop production worldwide. Some fertilizer components have doubled in price over the past 10 years

(USDA, 2012). Controlled release fertilizer (CRF) is more expensive than mineral salt fertilizer leading some researchers to start examining alternatives such as pasteurized poultry litter (Broschat, 2008). FPM contains many nutrients needed for plant growth, especially nitrogen, phosphorus and calcium.

FPM has been evaluated for some horticultural uses, but has not been adopted as a substrate or amendment by nurserymen and landscape professionals in most of the United States. The exception is the Lower Rio Grande Valley of Texas where a commercial facility (Natural Soil Solutions, LLC, Santa Rosa, TX) exists to produce a

FPM based product for ornamental and landscape use. Their product, however, does

111 make use of ash, bagasse, and mulch. In Florida, Poole and Conover (1989) found that a FPM-based product with high pH and EC produced Gardenia jasminoides poorly; however, Dieffenbachia spp. grew as well in this commercial substrate as it did in reed- sedge peat and better than it did in pine bark. Soltenazad (1982) reported that plant height and stem diameter of Chrysanthemum morifolium was highest in plants grown in

FPM. Plants grown in FPM also produced the most leaves but the difference was not significant. Stofella et al. (1996) found that citrus rootstock seedling growth was improved by FPM. Stofella and Graetz (1996) and Berrospe-Ochoa et al. (2012) investigated the use of FPM as substrate for the production of tomato transplants. Both found no difference in percent germination between FPM and sphagnum peat commercial mixtures; however Stofella and Graetz found that plants grown in FPM were shorter and had fewer roots while Berrospe-Ochoa et al. found the opposite to be true.

In these studies, the impact of fertilization on growth of plants grown in FPM substrate was not addressed.

Vinca was chosen as a suitable test plant based on some preliminary knowledge and testing of the substrate. FPM is known to hold water, have high levels of phosphorus, and a pH near or above 7.0. The objective of this experiment was to determine if FPM was capable of supplying a significant portion of the nutrients required for plant growth and development.

Methods and Materials

FPM that was pile aged for over 12 months was mixed with fine vermiculite

(Therm-o-rock, Chandler, AZ) in 4 different volumetric ratios of FPM:Vermiculite : 9:1,

3:1, 2:1, and 1:1. Initial Pour-thru readings of the vermiculite indicated a pH of 9.5 and an EC of 0.1 mS/cm. Individual cells of a 306 tray were filled with 100% FPM, one of

112 the FPM:Vermiculite mixtures, or Fafard 2 (57% Sphagnum peat, 20% perlite, 23% vermiculite, Conrad Fafard, Inc, Agawam, MA). The trays were placed under irrigation one day before planting. Approximately 10mm of overhead irrigation was applied daily.

The irrigation water had a pH of 7.4 and an EC of 1.0 mS/cm. Cell liners (228) of

“Cooler Coconut” vinca were obtained from Knox Nursery, Winter Garden, FL and planted on 11 Apr 2013. 100 mL of 100ppm N KNO3 was applied to the fertilized treatment units weekly. Observations during growth were made on leaf color and flowering date. Plants were harvested 25 May 2013, and data was taken on plant height, number of flowers, number of branches and dry weight. Shoots were cut at the soil surface and dried at 48 C until there was no change in weight. Starting and ending pH and EC were measured using the Pour-thru technique (Cavins et al., 2000) using a

Hanna HI 98130 (Hanna Instruments Inc, Woonsocket, RI). Measurements were made on a bulk sample of the nine individual cells. Bulk density was measured by drying a 1L sample of the substrate at 120 C, and weighing it. Total porosity was measured by saturating that same sample and weighing it to determine the weight of the water added. A nutrient analysis of the filter press mud was made at the Everglades Soils

Testing Lab in Belle Glade, FL to determine available levels of P, K, Mg, Ca and Fe

(Table A-1). The experiment was designed as a factorial design with 6 substrates and 2 rates of fertilization. Statistical analysis was done with JMP 8.0 (SAS Institute, Cary,

NC) using a least-squares fit model. Means separations were accomplished with a student’s t-test when the model indicated statistical significance.

113 Results

There was little difference in initial or final pH among the treatments and all were within the acceptable range for plant growth (Table A-2). Initial EC appeared to increase as the amount of FPM increased from 50 to 75 but then decreased. Final EC measurements showed that fertilized treatments had increased EC for treatments with containing less than 75% FPM. However, the EC in the 90% FPM and 100%FPM treatments showed decreased EC with fertilization. There was little difference in bulk density or porosity among FPM treatments.

The fertilized treatments had visually larger plants by 30 April 2013, they remained visibly larger through the end of the experiment. Flowering began on 30 April

2013 and by 25 May 2013 all plants had flowered. Mean days to flowering in the unfertilized treatment ranged from 25.4 days in the 67% FPM treatment to 31.4 days in the Fafard 2 treatment (Table A-3). In the fertilized treatment, mean days to flowering ranged from 25.1 days in the 100% FPM treatment to 30.0 days in the Fafard 2 treatment. Fertilization did not have a significant impact on days to flowering (Table A-

4).

Mean height in the unfertilized treatments ranged from 13.9 cm in the Fafard 2 to

23.7cm in the 75% FPM (Table A-3). In the fertilized treatments, mean height ranged from 17.6 cm in the Fafard 2 to 26.6 in the 90% FPM. Mean number of flowers at harvest in the unfertilized treatments ranged from 1.4 in the Fafard 2 to 3.3 in the 100%

FPM (Table 3). In the fertilized treatments, numbers of flowers ranged from 2.3 in the

Fafard 2 to 5.0 in the 50% FPM. Mean number of branches in the unfertilized treatments ranged from 0.8 in the Fafard 2 to 10.1 in the 100% FPM (Table A-3). Mean number of branches in the fertilized treatments ranged from 4.0 in the Fafard 2 to 11.7

114 in the 75% FPM. Shoot dry weight in the unfertilized treatments ranged from 0.3 g in the Fafard 2 to 1.2 g in the 100% FPM. Shoot dry weight in the fertilized treatments ranged from 0.5 g in the Fafard 2 to 2.1 g in the 67% FPM.

For all variables tested, ANOVA indicated a significant response to treatment

(Table A-4). Substrate had a significant effect on all variables tested. Fertilizer had a significant effect on all variables except days to flowering. The interaction was significant for number of branches and shoot dry weight.

Discussion

Prior research has examined the nutrient supplying capacity of composts and bio-solids. Chaney et al. (1980) found that composted biosolids, when making up 33% of the soilless media, could supply the needed micronutrients, phosphorus and a portion of the Nitrogen required for growth of Tagetes erecta. FPM contained adequate plant- available iron to counteract the deleterious effects of high substrate pH and high irrigation water pH. Phosphorus was also not an apparent issue as plants grew very well without any supplemental phosphorus fertilization. The plants in unfertilized FPM were more developed than those grown in fertilized Fafard 2, indicating that the nutrient supplying power was greater than a once weekly fertilization of 100 ppm N application of KNO3. Thomas et al. (2012) indicated that vinca grew more in response to nitrate fertilization as opposed to ammonium fertilization. Mineralization of organic matter in the FPM would have released ammonium ions, so by adding nitrate fertilizer to the soil, the ratio of NO3/NH4 would increase. Moore (2004) evaluated petunias and impatiens at different rates and types of fertilization and compost incorporation and found that both plants were responsive to additions of fertilizer. However, if the fertilizer had a more rapid release then compost incorporation should not be over 30%, but at a slower

115 release rate up to 60% of the media could be comprised of compost. In the current study, biomass was shown to increase with increasing rates of FPM incorporation, however addition of fertilizer eliminated the differences between FPM incorporation rates. This would seem to indicate that there was perhaps a hidden hunger for either nitrogen or potassium that was eliminated with fertilization.

116 Table A-1. pH and available nutrients in sugarcane filter press mud.1 pH P K Ca Mg Fe 7.36 89.71 217.65 13508.82 1988.97 5.40 1Values reported in mg/kg

Table A-2. Physical and chemical characteristics of the substrates composed of sugarcane filter press mud and vermiculite. Treatment Initial pH1 Initial EC1,2 Bulk Density3 Total Final pH1 Final EC1,2

Porosity4 No Fertilizer Fertilized No Fertilizer Fertilized

FAFARD2 7 0.75 0.17 84 6.87 7.14 0.80 0.97

FPM50 7.4 1.41 0.36 78 7.00 7.15 1.20 1.87

FPM67 7.35 1.57 0.42 57 7.16 6.97 1.09 1.58

FPM75 7.44 1.51 0.41 57 7.21 7.08 0.97 1.29

FPM90 7.22 1.49 0.38 59 7.01 7.21 0.92 0.74

FPM100 7.47 0.57 0.35 57 7.14 6.91 0.84 0.73

1Detemined by pour-thru method bulking all samples within a treatment

2mS/cm

3g/cm3

4Percent by volume

117 Table A-3. Means of growth and development parameters for vinca grown in binary mixtures of sugarcane filter press mud and vermiculite.1 Substrate Fertilizer Flowering Date # Flowers Height # Branches Shoot Dry Weight

FAFARD2 No 31.4 A 1.4 E 13.9 F 0.8 F 0.3 DE FAFARD2 Yes 30.0 AB 2.3 CDE 17.6 E 4.0 E 0.5 E FPM100 No 29.7 AB 3.3 BC 23.6 CD 10.1 AB 1.2 B FPM100 Yes 25.1 C 4.3 AB 26.3 AB 11.1 A 2.0 A FPM50 No 28.4 ABC 1.9 DE 19.5 E 4.9 DE 0.7 CD FPM50 Yes 27.7 BC 5.0 A 24.2 BC 12.1 A 2.0 A FPM67 No 25.4 C 2.8 CD 22.0 D 6.8 CD 1.0 B FPM67 Yes 25.4 C 4.3 AB 25.4 ABC 11.3 A 2.1 A FPM75 No 27.8 ABC 3.0 CD 23.7 CD 8.2 BC 1.0 BC FPM75 Yes 27.7 BC 4.3 AB 26.1 AB 11.7 A 2.0 A FPM90 No 26.6 BC 2.8 CD 23.6 CD 6.9 CD 0.9 BC FPM90 Yes 25.6 C 4.2 AB 26.6 A 11.4 A 2.0 A 1 Means in the same column followed by different letters are significantly different (Student’s t, JMP 8.0)

118 Table A-4. Results of statistical tests.1 Response df F P Days to Flower ANOVA 11,96 2.4 0.01 Effect Tests Substrate 5 3.9 0.0031 Fertilizer 1 2.9 0.0898 Substrate x Fertilizer 5 0.8 0.5 Number of Flowers ANOVA 11,96 7.5 <0.0001 Effect Tests Substrate 5 6.0 <0.0001 Fertilizer 1 43.0 <0.0001 Substrate x Fertilizer 5 1.9 0.1 Height ANOVA 11,96 24.8 <0.0001 Effect Tests Substrate 5 43.0 <0.0001 Fertilizer 1 54.8 <0.0001 Substrate x Fertilizer 5 0.6 0.7 Number of Branches ANOVA 11,96 23.1 <0.0001 Effect Tests Substrate 5 30.7 <0.0001 Fertilizer 1 83.1 <0.0001 Substrate x Fertilizer 5 3.6 0.005 Shoot Dry Weight ANOVA 11,96 40.8 <0.0001 Effect Tests Substrate 5 38.3 <0.0001 Fertilizer 1 228.8 <0.0001 Substrate x Fertilizer 5 5.6 0.0001 1Fit Least Squares, JMP 8.0

119 APPENDIX B IMPACT OF SUGARCANE FILTER PRESS MUD ON GROWTH AND DEVELOPMENT OF MARIGOLD

Introduction

Filter Press Mud (FPM) is a waste product of the sugar manufacturing industry that is composed of the solids flocculated out of the sugarcane juice by calcium phosphate. These solids include ash, soil, waxes, sugars, and plant material. The flocculate is mixed with water and transported to open-air storage near the mill. Some mills will pile the FPM, while others will move it into settling ponds. During the off-season, the material is removed and applied to agricultural fields, usually in close proximity to the sugarcane mill (Qureshi et al., 2001).

Nutrient contents vary depending on the location of the mill and can vary widely depending on the harvesting and milling practices (Gilbert et al. 2008).

Soil type, cultivar, fertilization practices, flocculation practices, and weather can have impacts on FPM characteristics. Florida sugarcane is primarily grown on histosols underlain by limestone bedrock. Calcium phosphate is used to clarify the juice in Florida, while in some other locations calcium sulfate is used. Florida sugarcane is mostly burned while in other areas it is harvested green. Table B-

1 highlights the differences in nutrient content found in FPM throughout the world.

The purpose of this study is to characterize some of the different sources of sugarcane filter press mud available in Florida, and to determine their ability to produce a common landscape plant, marigold.

Methods and Materials

Individual cells of a 306 tray were filled with mixtures of the various sources of

120 FPM vermiculite, and perlite and Farfard 2 as a standard control treatment. The final mixture was 55% FPM, 25% vermiculite, and 20% perlite by volume. The trays were placed under irrigation one day before planting. Approximately 10mm of overhead irrigation was applied daily. The irrigation water had a pH of 7.4 and an EC of 1.0 mS/cm. Liners (228) of “Inca II Gold” marigold were produced on site in Belle Glade, FL and planted on 13 September 2013. Fertilization was accomplished by adding 100 mL of 100ppm N KNO3 weekly to each treatment unit. Observations during growth were made on leaf color and flowering date. At

45 days after transplanting, plants were removed from irrigation for the second phase of the trial. Irrigation was withheld from the plants in the greenhouse to determine days to wilting. Plants were observed at 8AM, 12:30PM, and 5:00PM to determine time of wilting. After wilting, shoots were cut at the soil surface, measured and dried at 60 C until there was no change in weight. Starting and ending pH and EC were measured using the Pour-thru technique (Cavins et al.,

2000) using a Hanna HI 98130 (Hanna Instruments Inc, Woonsocket, RI). EC and pH measurements were made on a bulk sample of the nine individual cells.

Bulk density was measured by drying a 1L sample of the substrate at 120 C, and weighing it. Total porosity was measured by saturating that same sample and weighing it to determine the weight of the water added. A nutrient analysis of the filter press mud was made at the Everglades Soils Testing Lab in Belle

Glade, FL to determine available levels of P, K, Mg, Ca and Fe (Table 2). The experiment was designed as a randomized complete block design with 6 treatments, 9 blocks, and 1 replicate per block. Statistical analysis was done

121 with JMP 8.0 (SAS Institute, Cary, NC) using a least-squares fit model. Means separations were accomplished with a student’s t-test when the model indicated statistical significance.

Results

Days to flowering ranged from 25.7 days in the “B”-aged sample to 28.9 days in the “C”-fresh sample. The “A”-pond and “C”-fresh grown marigolds did not differ in their days to flowering; however, only the “C”-fresh grown marigolds differed from the rest of the treatments (Table B-3).

Numbers of flowers at harvest ranged from a high of 3.1 flowers per plant in the “A”-fresh grown marigolds to a low of 1.8 flowers per plant in the Fafard 2 grown marigolds. “A”-fresh grown marigolds did not differ significantly from “B”- aged grown marigolds. All other FPM sources did not differ significantly from the

Fafard 2 grown marigolds in the number of flowers produced (Table B-3).

The shortest marigold plants measuring 38.3 cm were grown in “B”-fresh media, while the tallest marigolds, measuring 44.1 cm, were grown in “B”-aged media. “C”-fresh, “B”-fresh, and Fafard 2 grouped together to form the lowest growing plants, while “A”-fresh, “A”-pond, and “B”-aged grouped together to form the tallest growing plants. However, “A”-pond and Fafard 2 did not differ significantly(Table B-3).

Stem diameter was least in “C”-fresh at 3.7 mm, and greatest in “B”-aged at 4.9 mm. The stem diameter in Fafard 2 grown plants did not differ from those grown in “A”-fresh, “A”-pond, nor “B”-fresh. However, “A”-fresh, “B”-fresh and

“B”-aged did group together with the greatest stem diameters (Table B-3).

Dry weight varied from 2.7 g per plant for the “C”-fresh grown plants to

122

4.9g for the “B”-aged grown plants. Fafard 2, “A”-pond, and “C”-fresh grown plants produced the least dry matter. “A”-fresh and “B”-aged grown plants produced the greatest amount of dry matter. “B”-fresh plants did differed significantly from neither Fafard 2 plants nor the “A”-pond plants, but were larger than the smallest average plants (Table B-3).

Plants grown in Fafard-2 took the longest to wilt averaging 5.0 days after last irrigation. “C”-fres, “A”-fresh, “B”-fresh, and “B”-aged grown plants all grouped together wilting between 3.0 and 3.4 days after the final irrigation event.

“A”-pond grown plants separated from the rest of the FPM-based media grown plants lasting 4.0 days after the final irrigation. However, when days to wilting are weighted by the size of the plant, the results differ. When weighted by plant mass, Fafard-2, “A”-fresh, and “B” aged do not differ in days to wilting. “A”-pond,

“A”-fresh, “B”-fresh, and “B”-aged group together. “C”-fresh grown plants, however, did not remain turgid as long as the rest of the FPM-based media grown plants when taking the biomass produced into account (Table B-4).

Discussion

FPM source was an important factor in determining eventual growth of

Marigolds. Two of the sources “A”-fresh and “B”-aged seemed to consistently perform better than the rest of the plants. Neither of the sources seemed to share any physical or chemical characteristics. They did both outperform the standard Fafard-2 mixture. It is noteworthy, that the same substrates that produced the best quality plants also had the longest weighted days to wilting.

Source has been an important factor in other studies regarding the growth of Marigolds. Bugbee (2002) found no response in marigold to increasing

123 quantities of biosolids incorporation. Moore (2005) noted that depending on the compost type marigold might either have a plateau response or a bell curve- shaped response to increasing levels of compost incorporation. Shober et al.

(2010) found that marigold grown in peat/compost mixtures performed better when grown in sphagnum peat in comparison to reed-sedge peat.

In conclusion, further testing might be required to determine what makes certain sources of FPM better for nursery production than others. “B”-aged was also used in the study by Larsen and Moore (2013) to successfully produce vinca. While none of the FPM grown plants showed visible signs of nutrient deficiency, it is always possible that there was a hidden hunger.

124 Table B-1. Reported nutrient values in Filter Press Mud from various sources.1 Author Location Nitrogen Phosphorus Potassium Calcium Magnesium Barry et al., 1998a Australia 1.5 0.9 0.7 2.3 0.6 Dee et al., 2002 S. Africa 1.68 1.13 0.31 1.1 0.4 Gilbert et al., 2008 Florida 1.05 0.31 0.15 7.8 0.50 Alexander, 1971 S. Africa 1.69 0.72 0.19 1.84 0.37 López et al., 2010 Cuba 1.50 1.16 0.72 3.16 0.32 Stofella and Florida 2.42 1.81 0.10 7.62 0.43 Graetz, 2000 Moreno-Álvarez et Cuba 2.70 2.00 0.15 7.8 0.80 al., 2011 Bruzón and Abad, Columbia 2.2 2.8 0.4 3.1 0.4 1995 Corbera et al., Cuba 0.75 1.44 0.14 0.79 0.25 2008 Villanueva et al., Mexico 1.64 0.4 0.0015 0.0034 0.0018 1998 Berrospe-Ochoa et Mexico 2.4 0.04 0.00005 1.12 0.24 al., 2012 1 Results reported as percentage of dry matter

125 Table B-2. Chemical Characteristics of FPM sources used in the experiment.1 Mill Storage pH P K Ca Mg Fe

“A” Fresh 6.93 266.18 1519.85 3611.76 896.32 1.88

“C” Fresh 7.15 108.09 229.41 1708.82 155.88 0.66

“B” Fresh 7.38 100.00 1105.15 8750.00 2307.35 3.56

“A” Pond 7.27 37.50 412.50 11037.50 1472.79 9.61

“B” Aged 7.36 89.71 217.65 13508.82 1988.97 5.40

1 Results reported as mg/kg

126 Table B-3. Measurements of growth and development for marigold grown in different sources of sugarcane filter press mud. Substrate Days to Flowering Number of Flowers Height (cm) Stem Diameter (mm) Dry Weight (g)

Fafard 2 26.1 B 1.8 C 39.9 BC 4.5 BC 3.5 BC

“C” Fresh 28.9 A 2.1 BC 38.1 C 3.7 D 2.7 C

“A” Pond 27.0 AB 1.9 C 41.4 AB 4.3 C 3.4 BC

“A” Fresh 26.2 B 3.1 A 43.4 A 4.7 AB 4.8 A

“B” Fresh 26.6 B 2.2 BC 38.3 C 4.7 AB 3.9 B

“B” Aged 25.7 B 2.7 AB 44.1 A 4.9 A 4.9 A

Substrate P > F = 0.0414 P> F = 0.0094 P> F = 0.0003 P > F = <0.0001 P > F = <0.0001

Block P > F = 0.0815 P > F = 0.3549 P > F = 0.3975 P > F = 0.2052 P > F = 0.1402

127 Table B-4. Days to wilting for marigold grown in different sources of sugarcane filter press mud Substrate Weighted Days to Wilting (gram x days)

Fafard 2 17.4 A

“C” Pond 8.2 C

“A” Pond 13.5 B

“A” Fresh 15.5 AB

“B” Fresh 12.3 B

“B” Aged 14.6 AB

Substrate P > F = <0.0001

Block P > F = 0.2962

128 APPENDIX C ELECTRICAL CONDUCTIVITY AND NUTRIENT CONCENTRATIONS IN LEACHATE DURING SUGARCANE FILTER PRESS MUD CHARACTERIZATION

Figure C-1. Electrical conductivity of leachate over 80 days from containers filled with sugarcane filter press mud from three different sugar mills in the Everglades Agricultural Area.

129 Figure C-2. Nitrate concentrations in leachate over 80 days from containers filled with sugarcane filter press mud from three different sugar mills in the Everglades Agricultural Area.

130 Figure C-3. Ammonium concentrations in leachate leached over 80 days from containers filled with sugarcane filter press mud from three different sugar mills in the Everglades Agricultural Area.

131 Figure C-4. Phosphate concentrations of leachate over 80 days from containers filled with sugarcane filter press mud from three different sugar mills in the Everglades Agricultural Area.

132 Figure C-5. Potassium concentrations of leachate over 80 days from containers filled with sugarcane filter press mud from three different sugar mills in the Everglades Agricultural Area.

133 Figure C-6. Electrical conductivity of leachate over 64 days from containers filled with different ages of sugarcane filter press mud from sugar mill “B”.

134

Figure C-7. Nitrate concentrations in leachate over 64 days from containers filled with different ages of sugarcane filter press mud from sugar mill “B”.

135 Figure C-8. Ammonium concentrations in leachate over 64 days from containers filled with different ages of sugarcane filter press mud from sugar mill “B”.

136 Figure C-9. Phosphate concentrations in leachate over 64 days from containers filled with different ages of sugarcane filter press mud from sugar mill “B”.

137

Figure C-10. Potassium concentration in leachate over 64 days from containers filled with different ages of sugarcane filter press mud from sugar mill “B”.

138 APPENDIX D RESULTS OF STATISTICAL TESTS

Table D-1. Results of statistical tests performed for physical and chemical characterization of sugarcane filter press mud.

Response df F P

Source study Electrical conductivty ANOVA 9,140 15.9566 <0.0001

r2=0.51 Substrate 4 9.0000 <0.0001 Days after start (DAS) 1 76.4358 <0.0001

Substrate x DAS 4 7.7933 <0.0001

Nitrate-nitrogen ANOVA 9,140 18.8337 <0.0001

r2=0.55 Substrate 4 17.6692 <0.0001

DAS 1 58.8677 <0.0001

Substrate x DAS 4 9.9896 <0.0001

Ammonium-nitrogen ANOVA 9,140 8.9336 <0.0001

r2=0.36 Substrate 4 7.9775 <0.0001

DAS 1 13.9280 0.0003

Substrate x DAS 4 8.6412 <0.0001 Phosphate- phosphorus ANOVA 9,140 32.1222 <0.0001

r2=0.67 Substrate 4 68.9082 <0.0001

DAS 1 0.0014 0.9703

Substrate x DAS 4 3.3664 0.0115

139 Table D-1. Continued Response df F P

Potassium ANOVA 9,140 15.7238 <0.0001

r2=0.50 Substrate 4 10.0484 <0.0001

DAS 1 56.0484 <0.0001

Substrate x DAS 4 10.9819 <0.0001 Total nitrate- nitrogen 3,8 122.3794 <0.0001 Total ammonium- nitrogen 3,8 10.8843 0.0012 Total phosphate- phosphorus 3,8 451.7026 <0.0001

Total potassium 3,8 274.5526 <0.0001

Age study Electrical conductivty ANOVA 7,88 19.5165 <0.0001

r2=0.61 Substrate 3 1.8205 0.1493

DAS 1 123.5242 <0.0001

Substrate x DAS 3 2.5433 0.0613

Nitrate-nitrogen ANOVA 7,88 14.2313 <0.0001

r2=0.53 Substrate 3 0.5111 0.6757

DAS 1 97.1587 <0.0001

Substrate x DAS 3 0.3090 0.8188

Ammonium-nitrogen ANOVA 7,88 0.4210 0.8868

r2=0.03 Substrate 3 0.4312 0.7312

DAS 1 1.4242 0.2359

140 Table D-1. Continued. Response df F P

Substrate x DAS 3 0.0763 0.9726 Phosphate- phosphorus ANOVA 7,88 1.2883 0.2655

r2=0.09 Substrate 3 1.6861 0.1758

DAS 1 3.5811 0.0617

Substrate x DAS 3 0.1261 0.9444

Potassium ANOVA 7,88 17.8391 <0.0001

r2=0.59 Substrate 3 0.5195 0.6700

DAS 1 122.5873 <0.0001

Substrate x DAS 3 0.2426 0.8664 Total nitrate- nitrogen 3,8 1.1268 0.3944 Total ammonium- nitrogen 3,8 1.6235 0.2592 Total phosphate- phosphorus 3,8 2.1694 0.1696

Total potassium 3,8 0.8397 0.5093

141 Table D-2. Results of statistical tests performed to determine irrigation frequency requirement of bananas produced in sugarcane filter press mud-based substrates and sphagnum peat based substrates. Response df F P Stem diameter ANOVA 11,60 5.5281 <0.0001 Substrate 3 4.1944 0.0092 Fertilizer 2 14.8681 <0.0001 Fertilizer x 6 3.0817 0.0107 Substrate Functional leaves ANOVA 11,60 1.1917 0.3123 Substrate 3 1.1987 0.3180 Fertilizer 2 0.3009 0.7413 Fertilizer x 6 1.4852 0.1987 Substrate Shoot dry weight ANOVA 11,60 5.6859 <0.0001 Substrate 3 6.1848 0.0010 Fertilizer 2 9.4174 0.0003 Fertilizer x 6 4.1926 0.0014 Substrate Root dry weight ANOVA 11,60 6.0001 <0.0001 Substrate 3 10.6186 <0.0001 Fertilizer 2 1.8123 0.1721 Fertilizer x 6 5.0868 0.0003 Substrate Total dry weight ANOVA 11,60 6.0662 <0.0001 Substrate 3 8.4495 <0.0001 Fertilizer 2 5.4426 0.0067 Fertilizer x 6 5.0825 0.0003 Substrate Root to shoot ratio ANOVA 11,60 6.1737 <0.0001 Substrate 3 14.7436 <0.0001 Fertilizer 2 0.3370 0.7153 Fertilizer x 6 3.8342 0.0027 Substrate Estimated total ANOVA 11,60 10.3728 <0.0001 Evapotranspiration Substrate 3 25.9186 <0.0001 Fertilizer 2 6.8617 0.0021 Fertilizer x 6 3.7703 0.0030 Substrate SPAD reading ANOVA 23, 264 4.0370 <0.0001 r2=0.26 Days after 1 16.9009 <0.0001 Planting (DAP) Substrate 3 15.9068 <0.0001 Irrigation 2 0.9201 0.3997 DAP x Substrate 3 4.3955 0.0049 DAP x Irrigation 2 1.6587 0.1924 Substrate x 6 1.1356 0.3419 Fertilizer DAP x Substrate x 18 0.5119 0.7991 Irrigation Stomatal ANOVA 23, 264 2.2860 0.0010 Conductance r2=0.17 DAP 1 1.0485 0.3068

142 Table D-2. Continued. Response df F P Substrate 3 1.4102 0.2402 Irrigation 2 18.6533 <0.0001 DAP x Substrate 3 0.5325 0.6604 DAP x Irrigation 2 0.0863 0.9173 Substrate x 6 1.0975 0.3641 Irrigation DAP x Substrate x 6 0.2730 0.9493 Irrigation Leaf Production ANOVA 23, 480 204.0088 <0.0001 r2=0.91 DAP 1 4590.941 <0.0001 Substrate 3 39.0060 <0.0001 Irrigation 2 3.3746 0.0350 DAP x Substrate 3 4.8166 0.0026 DAP x Irrigation 2 0.8296 0.4368 Substrate x 6 3.1127 0.0053 Irrigation DAP x Substrate x 6 1.0162 0.4139 Irrigation Plant Height ANOVA 23, 361 161.4471 <0.0001 r2=0.91 DAP 1 3540.602 <0.0001 Substrate 3 11.2540 <0.0001 Irrigation 2 46.6114 <0.0001 DAP x Substrate 3 3.0679 0.0280 DAP x Irrigation 2 11.8467 <0.0001 Substrate x 6 1.0348 0.4022 Irrigation DAP x Substrate x 6 1.1757 0.3186 Irrigation Evapotranspiration ANOVA 23, 552 20.6153 <0.0001 r2=0.54 DAP 1 224.3189 <0.0001 Substrate 3 51.0097 <0.0001 Irrigation 2 13.0463 <0.0001 DAP x Substrate 3 2.6195 0.0505 DAP x Irrigation 2 1.2671 0.2828 Substrate x 6 7.8083 <0.0001 Irrigation DAP x Substrate x 6 2.2446 0.0383 Irrigation Tensiometer readings ANOVA 7,112 76.6069 <0.0001 r2=0.83 Days after start 1 391.6112 <0.0001 (DAS) Substrate 3 11.1033 <0.0001 DAS x Substrate 3 32.3848 <0.0001 Percentage of ANOVA 7,112 539.4618 <0.0001 container capacity r2=0.97 DAS 1 3284.737 <0.0001 Substrate 3 87.9618 <0.0001 DAS x Substrate 3 47.2141 <0.0001

143 Table D-3. Results of statistical tests performed to determine fertilization requirement of bananas produced in sugarcane filter press mud-based substrates and sphagnum peat based substrates. Response df F P Stem diameter ANOVA 11,60 5.0135 <0.0001 Substrate 3 4.3693 0.0075 Fertilizer 2 14.8798 <0.0001 Fertilizer x 6 2.0469 0.0732 substrate Functional leaves ANOVA 11,60 1.0628 0.4058 Substrate 3 1.0107 0.3943 Fertilizer 2 0.9213 0.4036 Fertilizer x 6 1.1360 0.3528 substrate Shoot dry weight ANOVA 11,60 7.0253 <0.0001 Substrate 3 5.0321 0.0035 Fertilizer 2 27.5916 <0.0001 Fertilizer x 6 1.1664 0.3363 substrate Root dry weight ANOVA 11,60 4.4613 <0.0001 Substrate 3 6.7776 0.0005 Fertilizer 2 6.6271 0.0025 Fertilizer x 6 2.5812 0.0272 substrate Total dry weight ANOVA 11,60 5.0928 <0.0001 Substrate 3 5.5770 0.0019 Fertilizer 2 16.9592 <0.0001 Fertilizer x 6 0.5043 0.5043 substrate Root to shoot ratio ANOVA 11,60 8.1363 <0.0001 Substrate 3 12.0769 <0.0001 Fertilizer 2 1.8082 0.1728 Fertilizer x 6 8.2753 <0.0001 substrate SPAD reading ANOVA 23, 264 3.8049 <0.0001 r2=0.25 Days after Planting 1 31.1279 <0.0001 (DAP) Substrate 3 7.5043 <0.0001 Fertilizer 2 3.8646 0.0222 DAP x substrate 9 23.3279 0.0202 DAP x fertilizer 6 2.2038 0.1124 Substrate x 6 0.6388 0.6992 fertilizer DAP x substrate x 18 1.3197 0.2485 fertilizer Leaf production ANOVA 23, 480 169.9160 <0.0001 r2=0.89 DAP 1 3791.748 <0.0001 Substrate 3 30.9979 <0.0001 Fertilizer 2 2.3637 0.0952 DAP x substrate 3 2.3778 0.0691 DAP x fertilizer 2 0.0560 0.9455 Substrate x 6 1.7388 0.1101 Fertilizer DAP x substrate x 6 0.1196 0.9940 fertilizer

144 Table D-3. Continued. Response df F P Plant height ANOVA 23, 336 194.9619 <0.0001 r2=0.93 DAP 1 263.0287 <0.0001 Substrate 3 3.0077 0.0304 Fertilizer 2 1.6995 0.1843 DAP x substrate 3 1.2192 0.3027 DAP x fertilizer 2 0.7164 0.4893 Substrate x 6 0.7012 0.6488 fertilizer DAP x substrate x 6 1.6571 0.1308 fertilizer

145 Table D-4. Results of statistical tests performed to determine nutrient leaching from bananas produced in sugarcane filter press mud-based substrates and sphagnum peat based substrates. Response df F P Nitrate- nitrogen ANOVA 23,300 44.6852 <0.0001 Days after planting r2=0.77 (DAP) 1 444.6091 <0.0001 Substrate 3 98.8902 <0.0001 Fertilizer rate 2 30.0412 <0.0001 Substrate x Fertilizer rate 6 1.4993 0.1780 DAP x substrate 3 64.3627 <0.0001 DAP x fertilizer rate 2 1.2695 0.2825 DAP x substrate x fertilizer rate 6 3.6293 0.0017 Ammonium- nitrogen ANOVA 23,300 8.7047 <0.0001 Days after planting r2=0.40 (DAP) 1 15.6235 <0.0001 Substrate 3 29.8101 <0.0001 Fertilizer rate 2 16.7678 <0.0001 Substrate x Fertilizer rate 6 6.4745 <0.0001 DAP x substrate 3 5.0200 0.0021 DAP x fertilizer rate 2 1.8567 0.1580 DAP x substrate x fertilizer rate 6 0.6663 0.6770 Phosphate- phosphorus ANOVA 23,300 13.8659 <0.0001 Days after planting r2=0.52 (DAP) 1 41.8363 <0.0001 Substrate 3 48.7853 <0.0001 Fertilizer rate 2 19.8843 <0.0001 Substrate x Fertilizer rate 6 7.6187 <0.0001 DAP x substrate 3 12.6871 <0.0001 DAP x fertilizer rate 2 1.2216 0.2962 DAP x substrate x fertilizer rate 6 0.7897 0.5786 Potassium ANOVA 23,300 27.1816 <0.0001 Days after planting r2=0.68 (DAP) 1 311.2243 <0.0001 Substrate 3 52.4995 <0.0001 Fertilizer rate 2 22.7916 <0.0001 Substrate x Fertilizer rate 6 0.8917 0.5012 DAP x substrate 3 28.5795 <0.0001 DAP x fertilizer rate 2 5.0128 0.0072 DAP x substrate x fertilizer rate 6 1.6260 0.1395

146 Table D-4. Continued. Response df F P Total nitrate- nitrogen ANOVA 11,24 135.0685 <0.0001 Substrate 3 381.0742 <0.0001 Fertilizer rate 2 135.2245 <0.0001 Substrate x fertilizer rate 6 12.0136 <0.0001 Total ammonium- nitrogen ANOVA 11,24 21.9562 <0.0001 Substrate 3 43.8869 <0.0001 Fertilizer 2 24.209 <0.0001 Substrate x fertilizer rate 6 10.24 <0.0001 Total nitrogen ANOVA 11,24 26.7542 <0.0001 Substrate 3 33.5379 <0.0001 Fertilizer rate 2 87.0673 <0.0001 Substrate x fertilizer rate 6 3.258 0.0174 Total phosphate- phosphorus ANOVA 11,24 10.6124 <0.0001 Substrate 3 23.6765 <0.0001 Fertilizer rate 2 10.3135 0.0006 Substrate x fertilizer rate 6 4.1799 0.0051 Total potassium ANOVA 11,24 49.8068 <0.0001 Substrate 3 140.9409 <0.0001 Fertilizer rate 2 54.237 <0.0001 Substrate x fertiliizer rate 6 2.763 0.0347

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156 BIOGRAPHICAL SKETCH

Nicholas Larsen was born in DeLand, Florida December 21st 1983. He graduated from the International Baccalaureate program at DeLand High School in

2002. He attended the University of Florida from 2002 to 2005 and earned a Bachelor of Arts degree in Spanish with a minor in plant science. He then worked for Sugar

Farms Co-Op in Loxahatchee, FL before beginning his Master of Science in Agronomy at Iowa State University in 2006. In 2007, he married his wife, Kiley, and in 2009, he completed his master’s degree and settled in Pahokee, FL. From 2006 to 2015, he has worked at the Everglades Research and Education Center in Belle Glade, FL starting as a biological scientist and was promoted to senior biological scientist in 2010. In 2012, he began his PhD program at the University of Florida, which was completed in 2015.

Beyond academics, Nicholas Larsen is heavily involved in his community. He has served as a board member for Leadership ‘Glades from 2008-2009. He was the treasurer of Pahokee Relay for Life in 2009. He has been a board member of the

Pahokee Chamber of Commerce since 2012. He was a supervisor and then treasurer for the Palm Beach County Soil and Water Conservation District until 2015. In 2010, he started serving the City of Pahokee first on the Nuisance Abatement Board, and then on the Zoning, Adjustment and Planning Board, which he currently chairs.

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