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Nutrient Supply and Uptake During Propagation of Petunia Cuttings

Permanent Link: http://ufdc.ufl.edu/UFE0024849/00001

Material Information

Title: Nutrient Supply and Uptake During Propagation of Petunia Cuttings
Physical Description: 1 online resource (138 p.)
Language: english
Creator: Santos, Kathryn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

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Abstract: 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 NUTRIENT SUPPLY AND UPTAKE DURING PROPAGATION OF PETUNIA CUTTINGS By Kathryn Marie Santos August 2009 Chair: Paul R. Fisher Major: Horticultural Sciences Nutrition strategies were found to be highly variable between grower operations surveyed across the country, emphasizing the need for further understanding of supply and uptake dynamics in propagation. The objectives of this research were to (1) survey water and fertilizer management strategies during propagation, (2) survey typical tissue nutrient levels in 44 herbaceous annual unrooted cuttings, (3) evaluate the effect timing of macronutrient supply on Petunia x hybrida Supertunia Royal Velvet cuttings, (4) evaluate the effect of stock plant nutritional status on fertilizer response in propagation, and (5) employ aeroponics design to quantify stem versus foliar uptake. Grower management of the timing and concentration of nutrients applied to vegetatively grown calibrachoa (Calibrachoa x hybrida) or petunia (Petunia x hybrida) ranged from 0.5 to 80 mg L-1 Nitrogen (N) in week 1, and from 64 to 158 mg L-1 N in week 4. Leached water volumes ranged from 4.5 to 46.1 L m-2 over 4 weeks and contained 0.29 to 1.81 g m-2 N, 0.11 to 0.45 g m-2 P, and 0.76 to 2.86 g m-2 K. Most of the nutrients and water were leached during the first seven days when the highest volume of water was typically applied to cuttings as a method to minimize transpiration until root emergence. Nutrient availability in the root zone at root emergence increased root length from 0.5 cm to 2.8 cm, however excessive application of water during the first week of propagation (greater than 1 container capacity) leached the preplant nutrient charge in the substrate and constant application of complete fertilizer at 100 mg N L-1 for 21 days reduced root dry weight. Tissue nutrient decline was observed prior to root emergence and when initial tissue nutrient concentrations were 4.9%N or lower, they quickly fell below recommended ranges prior to root emergence. Nutrient solutions applied in mist propagation rapidly affected tissue nutrient concentrations of cuttings with differences in tissue nutrient concentration apparent by 7 days after insertion into substrate, therefore foliar uptake of both macronutrient and micronutrients was effective in supplementing nutrient supply prior to root emergence and minimized the measured tissue nutrient decline during root initiation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kathryn Santos.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Fisher, Paul R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024849:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024849/00001

Material Information

Title: Nutrient Supply and Uptake During Propagation of Petunia Cuttings
Physical Description: 1 online resource (138 p.)
Language: english
Creator: Santos, Kathryn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: Horticultural Science -- Dissertations, Academic -- UF
Genre: Horticultural Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: 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 NUTRIENT SUPPLY AND UPTAKE DURING PROPAGATION OF PETUNIA CUTTINGS By Kathryn Marie Santos August 2009 Chair: Paul R. Fisher Major: Horticultural Sciences Nutrition strategies were found to be highly variable between grower operations surveyed across the country, emphasizing the need for further understanding of supply and uptake dynamics in propagation. The objectives of this research were to (1) survey water and fertilizer management strategies during propagation, (2) survey typical tissue nutrient levels in 44 herbaceous annual unrooted cuttings, (3) evaluate the effect timing of macronutrient supply on Petunia x hybrida Supertunia Royal Velvet cuttings, (4) evaluate the effect of stock plant nutritional status on fertilizer response in propagation, and (5) employ aeroponics design to quantify stem versus foliar uptake. Grower management of the timing and concentration of nutrients applied to vegetatively grown calibrachoa (Calibrachoa x hybrida) or petunia (Petunia x hybrida) ranged from 0.5 to 80 mg L-1 Nitrogen (N) in week 1, and from 64 to 158 mg L-1 N in week 4. Leached water volumes ranged from 4.5 to 46.1 L m-2 over 4 weeks and contained 0.29 to 1.81 g m-2 N, 0.11 to 0.45 g m-2 P, and 0.76 to 2.86 g m-2 K. Most of the nutrients and water were leached during the first seven days when the highest volume of water was typically applied to cuttings as a method to minimize transpiration until root emergence. Nutrient availability in the root zone at root emergence increased root length from 0.5 cm to 2.8 cm, however excessive application of water during the first week of propagation (greater than 1 container capacity) leached the preplant nutrient charge in the substrate and constant application of complete fertilizer at 100 mg N L-1 for 21 days reduced root dry weight. Tissue nutrient decline was observed prior to root emergence and when initial tissue nutrient concentrations were 4.9%N or lower, they quickly fell below recommended ranges prior to root emergence. Nutrient solutions applied in mist propagation rapidly affected tissue nutrient concentrations of cuttings with differences in tissue nutrient concentration apparent by 7 days after insertion into substrate, therefore foliar uptake of both macronutrient and micronutrients was effective in supplementing nutrient supply prior to root emergence and minimized the measured tissue nutrient decline during root initiation.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Kathryn Santos.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Fisher, Paul R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024849:00001


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1 NUTRIENT SUPPLY AND UPTAKE DURING PROPAG ATION OF PETUNIA CUTTINGS By KATHRYN MARIE SANTOS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DE GREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Kathryn Marie Santos

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3 To my parents for instilling in me the confidence that I can accomplish anything I put my mind to and for teaching me to pur sue all of my dr eams with integrity and purpose

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4 ACKNOWLEDGMENTS The following dissertation, while an individual work, benefited from the insights and direction of several people. I tha nk my advisor, Dr. Paul Fisher, for his confidence, patience, and guidance. As my mentor and friend he has taught me more than I could ever give him credit for here. He has shown me, by his example, what a good scientist (and person) should be. I would also like to thank Dr. Bill Argo, who was always there to answer a question, brainstorm, or trouble shoot a problem. His genuine nature helped keep me grounded and extensive technical knowledge challenged me to constantly think beyond the results to their application. Sincere thanks are given to my committee members, D rs. Eric Simonne, Thomas Yeager and Hannah Carter for their guidance and critical reviews of my dissertation. contributions were unique, teaching me the importance of utilizing diverse perspectives, all of which substantially improved the fi nished product. I am truly grateful for having a committee that never accepted less than my best. For their support of my research and help with data collection, I would like to thank the Young Plant Research Center Partners. Special recognition is given to the American Floral Endowment for financial support of the research. I would also like to thank Angelica Cretu, Ernesto Fonseca, Becky Hamilton, Jinsheng Huang, and Connie Johnson for their friendship, technical support, and unending patience when it came to washing roots. Finally I would like to thank my b oyfriend David, whose support extended beyond picking up and moving to FL, to tirelessly helping me with late night and weekend data collection. His support, encouragement and love carried me thro ugh this experience.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FI GURES ................................ ................................ ................................ ......................... 9 ABSTRACT ................................ ................................ ................................ ................................ ... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 12 Propagation Indust ry ................................ ................................ ................................ ............... 12 Physiology of Root Development ................................ ................................ ........................... 13 Environmental Factors Affecting Rooting ................................ ................................ .............. 13 Light ................................ ................................ ................................ ................................ 14 Moisture and Substrate ................................ ................................ ................................ .... 14 Carbohydrates ................................ ................................ ................................ .................. 16 Nutrition ................................ ................................ ................................ ................................ .. 16 Stock Plant Nutrition ................................ ................................ ................................ ....... 17 Foliar Fertilization ................................ ................................ ................................ ........... 18 Physiology of Foliar Uptake ................................ ................................ ............................ 21 2 A SURVEY OF WATER AND FERTILIZER MANAGEMENT DURING CUTTING PROPAGATION ................................ ................................ ................................ .................... 24 Introduction ................................ ................................ ................................ ............................. 24 Materials and Methods ................................ ................................ ................................ ........... 26 Quantify Leached Irrigation Water Volume. ................................ ................................ ... 27 Quantify Nutrient Levels in Tissue, Substrate, and Leachate. ................................ ........ 28 Compare Nutrient Use Efficiency At Each Location. ................................ ..................... 29 Resul ts and Discussion ................................ ................................ ................................ ........... 30 Quantify Leached Irrigation Water Volume. ................................ ................................ ... 30 Quantify Nutrient Levels in Nutrient Solution, Tissue, Substra te, and Leachate. .......... 31 Applied nutrients ................................ ................................ ................................ ...... 31 Tissue uptake ................................ ................................ ................................ ............ 31 Subst rate nutrients ................................ ................................ ................................ .... 32 Leachate nutrients ................................ ................................ ................................ .... 33 Compare Nutrient Use Efficiency at each Location. ................................ ....................... 33 Conclusion ................................ ................................ ................................ .............................. 34

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6 3 A SURVEY OF TISSUE NUTRIENT LEVELS IN VEGETATIVE CUTTINGS ............... 42 Introduction ................................ ................................ ................................ ............................. 42 Materials and Methods ................................ ................................ ................................ ........... 43 Results ................................ ................................ ................................ ................................ ..... 46 Nitrogen, Phosphorus, and Potassi um ................................ ................................ ............. 47 Calcium, Magnesium, and Sulfur ................................ ................................ .................... 48 Micronutrients ................................ ................................ ................................ ................. 48 Conclu sion ................................ ................................ ................................ .............................. 49 4 TIMING OF MACRONUTRIENT SUPPLY DURING CUTTING PROPAGATION OF PETUNIA ................................ ................................ ................................ .......................... 65 Introduction ................................ ................................ ................................ ............................. 65 Materials and Methods ................................ ................................ ................................ ........... 67 Results and Discussion ................................ ................................ ................................ ........... 70 Shoot and Root Growth ................................ ................................ ................................ ... 70 Nutrient Concentration ................................ ................................ ................................ .... 73 Conclusion ................................ ................................ ................................ .............................. 75 5 EFFECT OF PETUNIA STOCK PLANT NUTRITIONA L STATUS ON FERTILIZER RESPONSE DURING PROPAGATION ................................ ................................ ............... 85 Introduction ................................ ................................ ................................ ............................. 85 Materials and Methods ................................ ................................ ................................ ........... 90 Results and Discussion ................................ ................................ ................................ ........... 93 Conclusion ................................ ................................ ................................ .............................. 99 6 STEM VERSUS FOLIAR UPTAKE DURING PROPAGATION OF PETUNIA VEGETATIVE CUTTINGS ................................ ................................ ................................ 105 Introduction ................................ ................................ ................................ ........................... 105 Materials and Methods ................................ ................................ ................................ ......... 108 Aeroponics Design ................................ ................................ ................................ ........ 108 Results and Discussion ................................ ................................ ................................ .. 110 Nutrient concentration ................................ ................................ ............................ 110 Dry weight and root growth ................................ ................................ ................... 112 Conclusion ................................ ................................ ................................ ............................ 113 7 CONCLUSION ................................ ................................ ................................ ..................... 121 A Survey of Water and Fertilizer Management During Cutting Propagation ...................... 122 A Survey of Tissue Nutrient Levels in Vegetative Cuttings ................................ ................ 123 Timing of Macronutrient Supply During Cutting Propagation of Petunia ........................... 124 Effect of Petunia Stock Plant Nutritional Status on Fertilizer Response During Propagation ................................ ................................ ................................ ....................... 127

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7 Employing Aeroponics Design to Quantify Stem Versus Fo liar Uptake During Propagation o f Petunia Vegetative Cuttings ................................ ................................ ..... 129 LIST OF REFERENCES ................................ ................................ ................................ ............. 131 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 138

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8 LIST OF TABLES Table page 2 1 Cell count per tray, tray area, substrate components, crop, cultivar, start date, end date, mean day and night temperature are provided for each of the eight commercia l greenhouse locations surveyed. ................................ ................................ ......................... 35 2 2 Water leached by location over a 4 week crop cycle. ................................ ....................... 36 2 3 Nutrient solution applied over time at eight greenhouse locations. ................................ ... 37 2 4 Total nitrogen, phosphorus and potassium applied were t he sum of the tissue uptake, nutrients leached and fin al substrate nutrient content. ................................ ..................... 39 2 5 Nitrogen, phosphorus and potassium use efficiency for each location. ............................. 41 3 1 th and 90 th commended ranges for 14 species. ................................ ................................ ................................ .......................... 50 3 2 variation (standard deviation/mean), survey range (10 th and 90 th percentile s) for species in survey with a minimum of 10 samples from 2 or more locations. .................... 55 4 1 Fertilization treatments applied to Petunia x hybrida over a 21 day crop cycle.. ............. 78 5 1 Initial tissue nutrient concentration (% N, P, and K) in cuttings taken from plants treated with one of four stock treatments.. ................................ ................................ ....... 102 6 1 Tissue N, P and K concentration of Petunia x hybrida root development ................................ ................................ ................................ ........... 117 6 2 Tissue N, P and K uptake of of Petunia x hybrida development. ................................ ................................ ................................ .................... 118 6 3 Shoot dry weig ht of of Petunia x hybrida development. ................................ ................................ ................................ .................... 119 6 4 Root length and root number of of Petunia x hybrida ................................ ......... 120

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9 LIST OF FIGURES Figure page 4 1 Measured total dry weight (A), shoot dry weight (B), root dry weight (C), and shoot:root ratio (D) per cutting over time.. ................................ ................................ ........ 79 4 2 Nitrogen content in the combined shoot and root by treatme nt over time expressed as % of dry weight (A), mg per cutting (B), or nitrogen uptake per week (C). .................... 81 4 3 Nitrogen, P, and K in leachate, substrate and tissue over time for plants receiving c ontinuous Complete fertilizer. ................................ ................................ ......................... 83 5 1 Tissue nutrient concentration (% N, P and K) trends in petunia cuttings taken from stock plants grown under 4 different fertility treatments and propa ga ted under 3 mist treatments. ................................ ................................ ................................ ....................... 103 6 1 Aeroponics system design. ................................ ................................ ............................... 115 6 2 Aeroponics split plot design consisting of four whole pl ots (1 to 4). .............................. 116

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10 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 NUTRIENT SUPPLY AND UPTAKE DURING PROPAGATION OF PETUNIA CUTTINGS By Kathryn Marie Santos August 2009 Chair: Paul R. Fisher Major: Horticultural Science Nutrition strategies were found to be highly variable between grower operations surveyed across the country, emphasizing the need for further understanding of supply and uptake dynamics in propagation. The objectives of this research were to (1) survey water and fertilizer management strategies during propagation, (2) survey typical tissue nutrient levels in 44 herbaceous annual unrooted cuttings, (3) evaluate the effect timing of macronutrient supply on Petunia x hybrida cuttings, (4) evaluate the effect of stock plant nutritional status on fertilizer response in propagation, and (5) employ aeroponics design to quantify stem versus foliar uptake. Grower management of the timing and concentration of nutrients applied to vegetatively grown calibrachoa (Calibrachoa x hybrida ) or petunia (Petunia x hybrida ) ranged from 0.5 to 80 mgL -1 Nitrogen (N) in week 1, and from 64 to 158 mgL -1 N in week 4. Leached water volumes ranged from 4.5 to 46.1 Lm -2 over 4 weeks and contained 0.29 to 1.81 gm -2 N, 0.11 to 0.45 gm -2 P, and 0.76 to 2.86 gm -2 K. Most of the nutrients and water were leached during the first seven days when the highest volume of water was typically applied to cuttings as a method to minimize transpiration until root emergence. Nutrient availability in the root zone at root emergence increased root length from 0.5 cm to 2.8 cm, however excessive application of water during the first week of propagation (greater than 1 container capacity)

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11 leached the preplant nutrient charge in the substrate and constant application of complete fertilizer at 100 mg NL 1 for 21 days reduced root dry w eight Tissue nutrient decline was observed prior to root emergence and when initial tissue nutrient concentrations were 4.9%N or lower, they quickly fell below recommended ranges prior to root emergence. N utrient solutions applied in mist propagation r apidly affected tissue nutrient concentration s of cuttings with differences in tissue nutrient concentration apparent by 7 days after insertion into substrate therefore foliar uptake of both macronutrient and micronutrients was effective in supplementing nutrient supply prior to root emergence and minimized the measured tissue nutrient decline during root initiation

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12 CHAPTER 1 INTRODUCTION Propagation Industry Off shore production of unrooted cuttings of ornamental bedding plants is increasing, with th ree quarters of these imports coming from Costa Rica, Guatemala, and Mexico ( U.S. Department of Agriculture, 2007 ). Total sales increased from $23.7 million in 1997 to $61.2 million in 2006, and the total volume of units imported was near 900 million in 2 006, twice that imported in 2000 ( U.S. Department of Agriculture, 2007 ). Most horticultural production firms either propagate or buy seed cuttings, or tissue cultured propagules These propagules are planted into small cells called plugs or liners and placed under high humidity to germinate or produce roots, and subsequently grown to a saleable seedling plug or rooted liner This requires 4 6 weeks in the case of herbaceous cuttings Th e plug or liner is then transplanted into the field or landscape, or into a larger container for further growth before sale Th e total value of sales of propagative plant material for cut flowers, potted flowering plants, annual bedding and garden plants, herbaceous perennials, foliage, and cut cultivated greens for 200 5 was $439 million, 2 % above the previous year ( U.S. Department of Agriculture 2006). Annual bedding and garden plants accounted for 49 % of all propagative material, or $214 million. These propagation numbers probably underestimate the economic value of the seedling and cutting industry when including vegetable, woody ornamental, and fruit production. Due to minimal seed production, Supertunias are one of the many petunia types that are cultivars were the first in a multiflora (large flower number on large branched plants) petunia types from wild species and older cultivars such as P. axillaris (Griesbac k, 2006). Reintroduction of P. axillaris and

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13 multiflora genetics emphasized stress tolerance, plant vigor, trailing growth, floriferousness, and postproduction durability (Dole et al., 2002; Griesbach, 2006). New petunia cultivars with increased genetic va riability are developed annually (Gerats and Vandenbussche, 2005). P etunia continues to be important as a commercial ornamental in the horticulture industry and a s a plant model for dichotomous species (Gerats and Vandebussche, 2005). Physiology of Root Development Once severed from the stock plant, production of hormones such as ethylene, jasmonates, and auxins increase s and subsequently initiate adventitious root development (Blakesly et al, 1994; DeKlerk, 1999; Clark et al, 1999; Shibuya et al., 2004; Sorin, 2005; Schilmiller et al., 2005). The cutting begins to respond to the severance, first by callus and then by root formation, which are two in dependent processes (Dole and Gibson 2006). As soon as the cutting is removed from the stock plant the o uter cells form a protective layer of necrotic cells and suberin (hy drophobic substance) (Dole and Gibson 2006). The living cells beneath the protective layer begin to div ide and form callus (Dole and Gibson 2006). When the swollen callus area turns wh ite or tan, the epidermis ruptures and causes the callus to crack because of root differentiation (Dole and Gibson 2006). In petunia, callus is typically formed in 5 7 days, and roots develop in 9 14 days ( Dole and Gibson 2006). Root development can be divided into 4 stages, (1) cutting condition at insertion into substrate (2) callus formation, (3) root development, and (4) toning (Dole et al., 2006). Environmental Factors Affecting Rooting Callus formation and r oot development is affected by many factors including light, moisture, carbohydrate content, and concentration of nutrients including N.

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14 Light Vegetative cuttings require a minimum amount of photosynthetic light for callus and root development (Haissig, 1986; Veierskov, 1988). Light inten sities below the minimum light requirement can delay root growth and development leading to extended rooting and increased probability for poor rooting, increasing the mean daily light integral (DLI) from 3 6 molm 2 d 1 increased rooting in petunia by 10 days (Lopez and Runkle, 2008). Decreasing the p hotosynthetic daily light integral during propagation negatively affected rooting, dry weight and flowering in New Guinea Impatiens and Petunia (Lopez and Runkle, 2008). Conversely, excessive light can lead t o stomatal closure, reduced turgor, and lower osmostic potential which can inhibit root formation because of water stress and photoinhibition (Elias son and Brunes, 1980; Grange and Loach, 1985). Moisture and Substrate In a typical rooting environment for v egetative cuttings, water is initially supplied by either mist emitters or automated boom watering systems to minimize transpiration loss. The amount of water required is species dependent. For example artemi sia ( Artemisia spp.) gaura ( Gaura lindheimer i ) rosemary ( Rosemarinus officinalis ) or lavender ( Lavandula angustifolia ) cuttings rooting performance is reduced in high mist enviro nments (Dole and Gibson, 2006). Excessive water application can lead to increased use of water resources and associated production costs, reduce oxygen availability in the substrate, and thereby reduce rooting percentage (Geneve et al., 2004). Improved irrigation management may help reduce nutrient, pesticide, and trace element loads in irrigation runoff to surf ace waters as well as leaching of agricultural chemicals into groundwate r supplies (Schaible and Aillery 2003). High leaching volumes resulting from mist irrigation is intended to maintain cutting turgidity during root development and often water is supplied in ex cess of uptake and container

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15 capacity resulting in the potential for rapid leaching of fertilizer (Kerr and Hanan, 1985 ; Mudge et al., 1995, Biernbaum and Argo 1996; Santos et al., 2008 ). Propagators surveyed applied enough water to their crops to leach a s much as 46 Lm 2 in a 4 week crop cycle and six of eight operations leached over one container capacity (CC) during the same period (Santos et al., 2008). Mist application during propagation is significant because soilless substrates have a limited abil ity to retain nutrients, especially when total leaching rates are greater than 1 CC (Biernbaum et al, 1995; Kerr and Hanan, 1985). Container capacity can be defined as the total amount of water present in the container after the substrate i s saturated and then allowed to drain for one hour The loss of nutrients from leaching during stages 1 and 2 of root development, could render the substrate without a pre plant charge by stage 3 (root emergence) a critical stage for nutrient replenishment for the cutti ng. Therefore, commercial fertilizer application early in propagation before root formation may be required to recharge leached nutrients in the substrate. Water soluble fertilizer applications prior to root emergence may also facilitate foliar uptake of nutrients (Tukey, 1958) to reduce observed tissue nutrient declines during preliminary phases of propagation regardless (Svenson and Davies, 1995; Wilkerson and Gates, 2005) Proper water management during propagation should provide enough water to minimi ze transpiration from the cuttings until root formation, after which water supply should be reduced because it is divided between the substrate (from the roots) and overhead application (micro climate, humidity). Growers that use constant cycle mist timer s that do not account for changes in light and temperature, typically over apply water during periods of low light or temperature, when transpiration is slowed. A similar net nutrient supply can be achieved with either low fertilizer and leaching rates (re source efficient strategy) or high fertilizer and leaching rates (resource inefficient); and

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16 c ommercial horticultural practices vary widely (Yelanich and Biernbaum, 1993). The leaching fraction is defined as the volume of leachate divided by the volume of irrigation solution entering a container (Ku and Hershey 1997). Yelanich and Biernbaum (1993) described leaching fractions as high as 0.5 common in greenhouse potted plant production. The lower the leaching fraction with water soluble fertilizer, the gr eater the irrigation and nutrient use efficiency (defined as plant uptake divided by applied water volume (irrigation efficiency), or nutrient content (nutrient use efficiency)), and therefore the more resource efficient. Carbohydrates Carbohydrate levels can also affect callus and root development in cuttings. Increased root number and root length in Chrysanthemum cuttings at higher initial tissue N concentrations (5 6 %N) was correlated with higher sucrose:starch ratios in the leaves, and the rooting res ponse was attributed to increased carbohydrate partitioning towards export to the region of root development (Druege et al., 2000; Rapaka et al. 2005). A proportion ( 25 50%) of the carbohydrates produced in plant shoots are typically allocated to the roo ts (Marschner, 1995). Carbohydrate reserves in unrooted cuttings are highly dependent on photosynthesis during the stock plant phase (decline with decreasing photosynthetic photon flux density) and decline during cutting storage and shipment (Rapaka et al 2005). High nitrogen concentration (5 6 %N) was shown to increase root development in Chrysanthemum cuttings, independent of carbohydrate levels (Druege et al., 2000). However, increased N supply during propagation also has potential to increase the sh oot to root dry weight ratio, which is not horticulturally desirable for rooted cuttings (Levin et al., 1989; Olsthoorn et al., 1991). Nutrition Annual n itrogen fertilizer application rates as high as 3600 kg ha 1 N were estimated for chrysanthemum ( Dendra nthema x grandiflorum ) (Nelson, 1998) and poinsettia ( Euphorbia

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17 pulcherrima ) potted crops (Yelanich and Biernbaum, 1994). Much of the excess nitrogen applied in crops grown with high fertilizer concentrations and heavy leaching can be lost into the enviro nment, depositing as much as 100 mg of nitrate nitrogen ( NO 3 N ) (243 ml or of effluent with a NO 3 N concentration of 411.6 mgL) per irrigation from a 6 inch pot into the soi l profile (McAvoy et al. 1992). Soluble phosphate and micronutrients are also us ed more intensively per hectare in greenhouse production than in field crop production (Nels on, 1990). Stock Plant Nutrition Adequately fertilized stock plants provide cuttings with enough nutrient reserves to carry them through propagation. Nutrient r anges that were developed for finished plant production may underestimate the nutrient reserves required by unrooted cuttings, however ranges (recommended or survey) do not exist for unrooted cuttings. Growers need sufficiency ranges specifically develope d for vegetative cutting production, in order to adjust their irrigation and fertility programs accordingly. Stock plant nutrition has been shown to affect subsequent rooting success of the vegetative cuttings (Dole and Gibson, 2006). M ineral nutrients are required for root growth and development, specifically N, P, Ca, Mn, and Zn for root initiation and N, P, K, Ca, and B for root growth and development (Blazich, 1988). Nitrogen depletion restricts the growth of all plant organs, and any nutrient defic iency during cutting insertion into substrate has the potential to limit root and shoot growth ( Marschner, 1995; Dole and Gibson, 2006; Barker and Pilbeam, 2007 ). Rowe and Blazich (1999) found that loblolly pine, ( Pinus taeda ) cuttings taken from stocks plants grown with higher N developed more shoot growth during the 12 week propagation cycle, compared to cuttings taken from stock plants with low N. D eficiencies of P, Ca, and Zn in stock plants were shown to adversely affect rooting in vegetative strawf lower ( Bracteantha bracteata) cuttings (Gibson, 2003).

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18 Increased stock plant fertilizer concentrations in species other than petunia resulted in increased root growth up to an optimal level whereby when surpassed the additional fertilizer actually decrea sed cutting performance. Gibson (2003) found the greatest cutting yield, root number and shoot dry weight in cuttings taken from New Guinea impatiens ( Impatiens x hawkeri Bull.) and scaevola ( Scaevola aemula R. Br.) stock fertigated with 300 mgNL 1 compa red with 100 mg NL 1 applied at each irrigation through drip irrigation. Adventitious root number and root length of Chrysanthemum ( Dendranthema grandiflorum Ramat.) cuttings were positively correlated to the initial total tissue nitrogen concentration ( Druege et al., 2000 ). As initial N concentration increased from 2 to 7% N, root number in stored cuttings increased from approximately 5 to 15 and root length increased from 1 to 2.5 cm (Druege et al., 2000). Loblolly pine ( Pinus taeda L.) cuttings tak en from stock plants with higher mineral nutrient content (9 mg Ncutting 1 vs 3, 5, 6, 8 mg Ncutting 1 ) were also shown to maintain that higher concentration throughout propagation and showed more positive rooting (28 33%) compared to cuttings taken from stock grown under lower fertility regimes (17%) (Rowe and Blazich, 1999). However, fertilizer concentrations of 200 mg NL 1 applied 3 times a week to holly ( Ilex crenata Thunb.) and 4 0 mg NL 1 applied weekly to eastern red cedar ( Juniperus virginiana L .) during stock plant production decreased subsequent adventitious rooting compared with lower fertilizer concentrations (100 mg NL 1 and 20 NL 1 respectively) (Rein et al., 1991; Henry et al., 1992). Typical N fertilization recommendations for stock pr oduction of herbaceous ornamental crops are between 150 and 250 mg NL 1 applied at each irrigation (Dole and Gibson 2006). Foliar Fertilization Fertilization during propagation can alleviate deficiency symptoms and encourage rooting Foliar nutrient a pplications prior to root emergence potentially serve to (1) replenish pre plant nutrients leached from the substrate which are subsequently taken up by the cut stem

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19 through the transpiration stream or newly emergent roots or (2) supply nutrients for direc t foliar uptake. A ppropriate timing and concentration of nutrient supply in vegetative cutting propagation affects root development, uniform ity of plant growth uptake efficiency, nutrient runoff and transplant success. Fertigation recommendations for ve getative cuttings are currently correlated with root developmental stage. A n initial application of 50 75 mg NL 1 is recommended at visible callus development (stage 2) and subsequent applications of 100 mg NL 1 at root emergence (stage 3) (Dole and Gib son, 2006). N utrients can be supplied to vegetative cuttings through a combination of preplant nutrient charge in the substrate supplemental application of water soluble fertilizer and/or incorporation of controlled release fertilizers. Fertigation stra tegies during propagation of Petunia vary widely among commercial growers, resulting in a range of resource efficiency and suggesting a need to better understand nutrient uptake processes and fertilizer response, in order to develop best management practic es (Santos et al., 2008). Historically, fertilization through overhead emitters was not recommended for short term crops, due to the potential for clogged emitters and algae growth (Dole et al., 2006) until stage 3 (root development) with weekly applica tions of 100 mg NL 1 alternating between 15 0 15 and 20 10 20, and increased concentration of 150 200 mg NL 1 applied weekly during stage 4 (toning). However, a survey of commercial greenhouse cutting propagators of Petunia and Calibrachoa found that f ertigation began anywhere from stage 1 to 3 (Santos et al., 2008). Applied N concentrations ranged from 0.4 to 80.2 mg NL 1 during week 1 (stage 1 to 2), 0 to 194.8, 19.2 to 148.0, and 64.0 to 157.6 mg NL 1 during weeks 2, 3 and 4 respectively (Santos e t al., 2008). Supplemental application of nutrients to a range of herbaceous and woody cuttings through mist was recommended by Good and Tukey (1967). In particular, fertilization during root formation may benefit cuttings with suboptimal initial tissue nutrient concentrations.

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20 F oliar deficiency symptoms are likely to develop during propagation i f cuttings are deficient in Cu, Fe, Mg, Mn, N, or S (Gibson, 2003). Nutrients other than N have also been ascribed roles in root initiation, including P, Ca, M g, Mn, B and Zn (Anderson, 1986; Blazich 1988; Svenson and Davies, 1995). Due to low phloem mobility, Mn, Fe, Zn, Cu, B, and Mo deficiencies appear first in new growth (Marschner, 1995), emphasizing the importance of adequate micronutrient supply during s tock production to ensure sufficient concentrations in the plant apices when tip cuttings are taken. Manganese, B, Fe, Cu, Mo, and Zn were all shown to increase in concentration near the basal stem of poinsettia cuttings shortly after insertion into subst rate suggesting that concentrations of these elements may be important in root initiation or elongation (Svenson and Davies, 1995). Decrease in tissue nutrient concentration typically occurs during cutting propagation. T issue concentration s of both mac ronutrients and micronutrients changed in apical stem cuttings of poinsettia during the root initiation phase ( Euphorbia pulcherrima Willd. ex. Klotzch ) (Svenson and Davies, 1995). Specifically, N, P, and K concentrations (%) declined from day 1 to day 13 from 3.4 to 2.85, 0.35 to 0.32, and 1.86 to 1.70% respectively (Svenson and Davies, 1995). Nutrient shortages that arise during adventitious root development have been attributed to foliar leaching (mainly in hardwood cuttings), dilution, or low initial t issue nutrient concentrations (Good and Tukey, 1967). Nutrient loss in cuttings has been attributed to processes such as elimination of metabolites by special organs, for example sugars by nectaries, dissimilation or degeneration of plant tissue, or ion e xchange between the plant and foliar solution (Tukey, 1970). Increase in cutting dry weight prior to root development (stages 1 2) and without supplemental nutrient uptake can result in a dilution of the pre existing nutrient concentration in the cutting tissue and explain tissue nutrient decline (Blazich, 1988).

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21 Physiology of Foliar Uptake Nutrients applied to the foliage must overcome a number of barriers before entering the cytosol (Mengel, 2002). Foliar applied solutes must penetrate the cuticle, wh ereby entering the apoplast, and further penetration is dependant on the availability of water filled space which enables diffusion into the cytosol (Mengel, 2002). Nutrients can then be transported from cell to cell via the plasmodesmata, from where they can be loaded into the phloem and transported long distances (Mengel, 2002). Demand for a particular nutrient impacts foliar uptake efficiency, especially for macronutrients because the total quantity of uptake is low and more closely follows micronutrie nts than macronutrient demand (Mengel, 2002). Nitrogen foliar applications were shown to be the most practical macronutrient crop applications. Environmental, s tructural, and morph ological characteristics within a given plant species contribute to th e eff icacy of foliar nutrient applications. Physiological factors that affect the efficacy of foliar fertilization are the nutrient forms applied, the root temperature, root osmotic potential, leaf age, and current nutrient status in the tissue (Weinbaum, 1996 ; Mengel, 2002). Plant leaves are specialized organs primarily functioning in the production of organic compounds through photosynthesis and other related processes. F oliar applied compounds penetrate the leaf surface through the cuticle via cuticular cr acks and imperfections or through stomata, leaf hairs and other specialized epidermal cells (Tukey et al., 1961; Burkhardt and Eichert, 2001). In contrast to roots, the outer walls of the epidermal cells in all aerial plant organs are covered by a hydroph obic cuticle (Marschner, 1995) which has the potential to limit nutrient absorption The water phobic cuticle is a limiting barrier for two way transport of water and solutes in and out of the leaves (Marschner, 1995). Cuticular waxes embedded in the cut in/cutan matrix were found to be responsible for barrier properties and diffusion of non electrolytes. Neutral, non charged molecules have been found to penetrate the cuticle by diffusion and

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22 dissolution of cuticular waxes (Schonherr, 2001 ). However, the mechanism of cuticle penetration of water and ions is not fully understood but may occur due to the existence of aqueous pores (Schonherr and Schreiber, 2004). P lant cuticles are known to be poly electrolytes with isoelectric points arou nd 3.0 (Schonherr et. al, 1972 and Schonherr, 1977). Therefore, the ion exchange capacity of the cuticle will be altered by changed levels in pH. Foliar application solutions with pH values greater than 3 will then render the cuticle as negatively charged (Chamel and Vit ton 1996; Schonherr et al., 1977). A negatively charged cuticle means that positively charged ions will be attracted to the cuticle, whereas negatively charged ions will be repelled, and therefore less likely to be taken up. Mengel estimated that a catio n is 1000 times more likely to penetrate the cuticular membra ne compared to an anion (2002). During a typical petunia or calibrachoa production cycle (28 days) the water volume applied can exceed the container capacity of the substrate, causing leaching of preplant nutrient charge (Santos et. al., 2008). Transpiration was observed to increase by nearly 50% upon visible root emergence in poinsettia (Wilkerson and Gates, 2005). Water moves in plants along gradients of water potential typically generated b y transpirational water loss from leaves (Sheriff, 1984). Therefore, potential for nutrient uptake from the base of the stem (through the transpiration stream) should also increase at initial root emergence. Previous research on leaching in greenhouses (Yelanich and Biernbaum, 1994; Ku and Hershey 1997; Groves and Warren, 1998) has focused on potted plants, rather than propagation. We are unaware of research on leaching and fertilizer concentration in plug and liner trays where the water inputs relative to substrate volume may be much higher than in large containers. Based on the variability in production practices within the industry, management practices need to be

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23 evaluated and critical areas such as the amount of water and nutrients lost, along with nutritional strategies at different root developmental stages should be quantified in order to determine points of potential improvement.

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24 CHAPTER 2 A SURVEY OF WATER AND FERTILIZER MANAGEMENT DURING CUTTING PROPAGATION Introduction Most horticultural p roduction firms either propagate or buy seed cuttings, or tissue cultured propagules These propagules are planted into small cells called plugs or liners and placed under high humidity to germinate or produce roots, and are subsequently grown to a sale able seedling plug or rooted liner, which requires 4 6 weeks in the case of most herbaceous cuttings Th e plug or liner is then transplanted into the field or landscape, or into a larger container for further growth before sale Th e total value of sales o f propagative plant material for cut flowers, potted flowering plants, annual bedding and garden plants, herbaceous perennials, foliage, and cut cultivated gree ns for 2005 was $439 million, 2% above the previous year (U.S. Dept. Agr. 2006). Annual beddin g and garden plants accounted for 49% of all propagative material, or $214 million. These propagation numbers probably underestimate the economic value of the seedling and cutting industry when including vegetable, woody ornamental, and fruit production. Agriculture accounts for over 8 0 % of freshwater consumption in the U.S., and an increase in water use regulations necessitates improved irrigation strategies (Weibe and Gollehon, 2006). Greenhouse pro pagation involves considerable application of water for control of humidity, soil moisture, and as a means to apply water soluble fertilizer. In a typical rooting environment for vegetative cuttings, water is initially supplied by either mist emitters or automated boom watering systems to minimize transpirati on loss. The amount of water required is species dependent. For example artemi sia ( Artemisia spp.) gaura ( Gaura lindheimeri ) rosemary ( Rosemarinus officinalis ) or lavender ( Lavandula angustifolia ) cuttings rooting performance is reduced in high mist e nviro nments (Dole and Gibson, 2006). Excessive water

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25 application can lead to increased use of water resources and associated production costs, reduce oxygen availability in the substrate, and thereby reduce rooting percentage (Geneve et al., 2004). Impro ved irrigation management may help reduce nutrient, pesticide, and trace element loads in irrigation runoff to surf ace waters as well as leaching of agricultural chemicals into groundwate r supplies (Schaible and Aillery 2003). Annual n itrogen fertilizer application rates as high as 3600 kg N ha 1 were estimated for chrysanthemum ( Dendranthema x grandiflorum ) (Nelson, 1998) and poinsettia ( Euphorbia pulcherrima ) potted crops (Yelanich and Biernbaum, 1994). Much of the excess nitrogen applied in crops gro wn with high fertilizer concentrations and heavy leaching can be lost into the environment, depositing as much as 100 mg of nitrate nitrogen ( NO 3 N ) (243 ml or of effluent with a NO 3 N concentration of 411.6 mg N L 1 ) per irrigation from a 6 inch pot into the soi l profile (McAvoy et al. 1992). Soluble phosphate and micronutrients are also used more intensively per hectare in greenhouse production than in field crop production (Nels on, 1990). A similar net nutrient supply can be achieved with either low fertilizer and leaching rates (resource efficient strategy) or high fertilizer and leaching rates (resource inefficient); and c ommercial horticultural practices vary widely (Yelanich and Biernbaum, 1993). Fertilizers applied to both stock plants and duri ng propagation of cuttings impact successful rooting of vegetative cuttings in propagation (Blazich, 1988; Gibson, 2003; Lebude et al., 2004; Rowe and Blazich, 1999). Biernbaum et al. (1995) and Kerr and Hanan (1985) found that the majority of fertilizer salts were rapidly removed from container media following leaching of one container capacity (Biernbaum et al., 1995) or one soil volume (Kerr and Hanan, 1985). Container capacity (CC) can be defined as the total amount of water present in the container a fter the substrate i s saturated and then allowed to drain for 1 hour

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26 Previous research on leaching in greenhouses (Groves et al., 1998; Ku and Hershey 1997; Yelanich and Biernbaum, 1994) has focused on potted plants, rather than propagation. We are unaw are of research on leaching and fertilizer concentration in plug and liner trays where the water inputs relative to substrate volume may be much higher than in large containers. Based on the variability in growing practices within the industry, management practices need to be evaluated and critical areas such as the amount of water and nutrients lost should be quantified in order to determine points of potential improvement. Our objectives were to: (1) quantify levels of irrigation water leached during pro duction of liner trays in multiple commercial greenhouse operations, (2) quantify nutrient levels in substrate, tissue, and leachate of these commercial crops, and (3) compare nutrient use efficiency at each location. Materials and Methods Nutrient and ir rigation d ata were collected in 2006 from eight greenhouse locations in Michigan, Colorado, New Hampshire, and New Jersey, which represent a range in climatic conditions within the northern U.S. Two greenhouse locations were selected in each of four state s, each of which had at least 3 ha in plug and liner production Each greenhouse location represented an experimental unit. Although several locations had the capability to recirculate irrigation water, none were doing so with these crops because of dise ase susceptibility. The greenhouse businesses were leading propagators that had previously cooperated with the authors in ons ite trials and we were therefore confident about being able to collect reliable data. The timing for the experiments was determi ned based on the peak production season for U.S. propagation of annuals (January March). The experiment was run for one week at each location (conditions described in Table 2 1) and four crops of vegetatively grown liner trays were related that were eithe r 0 1, 1 2, 2 3, or 3 age group in one location. In six locations, calibrachoa was selected; and in two locations,

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27 petunia was selected. Species selection depended on the crops grown and ava ilable at each location, and both species had a similar 4 5 week crop time in a liner tray (Blackmore Company; Belleville, MI). The cultivar within the species was consistent within a given location, but varied between locations. Within each crop, there w ere five replicate measurements of each of the following variables, located randomly within the crop: volume, pH ( Nova Analytics Corporation Pinnacle Corning pH Meter model 430 ; Woburn, MA ) and electrical conductivity (EC) ( Thermo Scientific Orion Model 13 0 ; Waltham, MA ) of applied nutrient solution n=5, (using five in dividual irrigation collection funnels); volume, pH and EC of leached nutrient solution (using five leachate collection trays); substrate pH and substrate EC (using five liner propagation tra ys); and plant fresh and dry weights (combined root and shoot, using five groups of three plants each). Within each crop, there was a single replicate measurement of each of the following: substrate nutrient levels (combined from five trays), leachate nutr ient levels, n=1, (combined from five leachate samples), tissue nutrient levels (from 15 combined plants), and Data were analyzed as a split plot design with location as the main plot, crop age as the s ubplot, and each tray as a random block. There were no significant differences between species or cultivar therefore location was assigned as the main factor in the model. Proc Mixed and Proc GLM in SAS (SAS Institute Inc. version 9.1; Cary, NC) were use d for statistical analysis and Quantify L eached Irrigation W ater V olume. Growers recorded the number and schedule of irrigation events per day, NPK ratio, and concentration (parts per million) of fertilizer applied. The volume of applied irrigation solution

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28 plastic bottles topped with open funnels, surface area=24.4 cm and stood 17 cm a bove the bottle) in each of the four crops, or a total of 20 bottles (five bottles/crop four crops) per location. The irrigation collection funnels were left for one week, and growers were instructed to apply irrigation solution evenly across the crop s urface and funnels as per normal practices. The data for irrigation water volume applied were not shown due to too much variability in the measurements. The volume of solution leached from the propagation trays was measured by placing 20 the top of plug trays (in between the plug cells) were covered with water resistant tape to prevent irrigation water from running directly into the leachate collecting tray. A fter one week, the collection trays were removed from beneath the propagation trays and the leachate volumes were measured. Quantify Nutrient L evels in T issue, Substrate, and L eachate. From each of the five replicate samples per crop, 25 mL of applied ir rigation water (from the irrigation collection funnels) and leachate (from the leachate collection trays) were collected. The pH and electrical conductivity (EC) were recorded for each of those samples. The five samples from each crop were then combined into one sample per crop and sent to Quality Analytical Laboratories (Panama City, FL) for a complete nutrient analysis using inductively coupled plasma (ICP) atomic emission spectrophotometry (Thermo Jarrell Ash ICAP 61E; Franklin, MA) to measure P K C a Mg S Fe, Mn B, Cu Zn, molybdenum ( Mo ), aluminum (Al), sodium (Na) The leachate and substrate solution samples were analyzed for nitrate nitrogen (NO 3 N ) and ammonium nitrogen (NH 4 N ) using a Lachat QuikChem AE (Lachat Instruments; Loveland, CO). This instrument uses FIA (flow injection analysis) to colorimetrically determine NO 3 N and NH 4 N concentration. For tissue samples, N was

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29 measured as Total Kjeldahl N (TKN) where all the protein is converted to NH 4 using heat, a catalyst, sulfuric acid an d hydrogen peroxide. The sample was then run on a spectrophotometer using Nesslerization for N determination. A Hach (Loveland, CO) Digestahl apparatus was used for the conversion (digestion) and a Hach DR 4000 spectrophotometer (Hach; Loveland, CO) for the analysis. Plug squeeze tests were performed approximately one hour after irrigation with nutrient solution by pressing down firmly on the top of the substrate surface and collecting the solution from the hole at the bottom of the pressed plug cell, on each of the five replicate propagation trays per crop (Scoggins et al., 2002). The pH and EC was recorded individually for these samples. The plug squeeze samples were then combined into a single replicate per crop for complete nutrient analysis. Five gr oups of three plants were removed from random locations within trays in each crop. The plants were washed in four separate baths of deionized water to remove any substrate from the root mass and to clean the foliage. Fresh weight of each replicate sample of three combined plants was taken, placed in a forced air drying oven at 55 C, and weighed again after all liquids evaporated (3 d) to measure dry weight. The dry tissue samples were combined by crop for a complete nutrient analysis. The container capac ity (CC) was calculated for substrates at each location by measuring the total amount of water present in the container after the substrate has been saturated using subirrigation and allowed to drain for approximately 1 hour. Compare N utrient U se Efficienc y At Each L ocation. The resource use for each location was calculated using the following formulae: Tissue n utrient uptake= (2 1) [(Final DW z m 2 ) (Final Tissue % N, P or K)] [(Initial DWgm 2 ) (Initial Tissue % N, P or K)]

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30 z DW = dry weight (g) Fina l nutrient concentration in substrate = (2 2) (CC z m 2 ) (Final concentration (gL 1 ) N, P or K) z CC = container capacity (L/m 2 ) Total nutrient leached = (2 3) Total leachate volume (L) Total concentration (gL 1 ) N, P, or K % N Distribution = (2 4) [(A) / (Leachate N z + Tissue uptake N z + final substrate N z )] 100, where A= leachate N, tissue nutrient N, or final substrate N (Ku and Hershey, 1997). z N = grams (g) of N in leachate, tissue or substrate Results and Discussion Quantify L eached Irrigat ion Water V olume. The volume leached varied between locations ranging from 0.6 to 6.0 L per propagation tray, with corresponded to 44,900 to 460,769 Lha 1 over a 4 week crop period (Table 2 2). Six of the eight locations leached at least one CC over the 4 week crop cycle, and three locations leached 2.0 to 4.7 CC (Table 2 2). The greatest leaching also occurred during weeks 1 or 2, in six of the eight locations, and on average, significantly more water was leached during week 1 than in the subsequent 3 crop weeks (Table 2 3). The nature of vegetative propagation requires higher volumes of water to be applied to maintain humidity and plant turgidity until root formation. No previous research exists to provide a baseline to compare against, however betwe en these 8 operations we discovered less leaching at some indicating an excess at others. Given the rapid leaching of nutrients from soilless substrates following leaching of one CC (Kerr and Hanan, 1985; Biernbaum et al., 1995), a significant amount of pr e plant nutrients would have been leached early in the crop cycle before roots emerged and nutrients were taken up by the cuttings. These results indicate that irrigation volume was excessive at the beginning of the crop cycle when plant uptake would be r educed because of small leaf area and lack of plant roots.

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31 Quantify N utrient L evels in Nutrient Solution, T issue, S ubstrate, and L eachate. Applied n utrients Fertilizer strategy varied w idely between locations. Applied nutrient concentrations increased wit h crop age, as measured with both EC and N level of the applied solution (Table 2 3). On average, 24, 85, 93, and 101 mgL 1 N were applied during weeks 1 to 4, respectively, with a nutrient solution EC of 0.44, 0.88, 0.87, and 1.01 dSm 1 (Table 2 3). A veraged across the four weeks, the nutrient concentrations in mg.L 1 were 76 N, 4 P, 65 K, 61 Ca, 17 Mg, 21 S, 2 Fe, 0.4 Mn, 0.2 B, 0.3 Cu, 0.4 Zn, 0.05 Mo. Locations varied greatly in the applied N levels, particularly during the first 2 weeks (week 0 to 1: 0 to 80 mgL 1 N, week 1 to 2: 0 to 195 mgL 1 N, week 2 to 3: 19 to 148 mgL 1 N, and week 3 to 4: 64 to 158 mgL 1 N) (Table 2 3). The total applied N, P and K (sum of tissue uptake, nutrients leached and final substrate nutrient content) during the 4 week crop cycle ranged by a factor of approximately four between locations, from 1.4 to 8.3 gm 2 N, 0.3 to 1.2 gm 2 P, and 2.7 to 10.1 gm 2 K (Table 2 4); which represented 14 to 83 kgha 1 N, 3 to 12 kgha 1 P, and 27 to 101 kgha 1 K (Table 2 5). O n an annual basis, the applied N level (averaging 637 kgha 1 N) was lower than the rate reported for potted chrysanthemum and poinsettia ( Nelson, 1998 ; Yelanich and Biernbaum, 1994) Tissue u ptake Tissue uptake ranged from 0.9 to 4.5 gm 2 N, 0.2 to 0.6 g m 2 P and 1.5 to 4.6 gm 2 K (Table 2 4), varying by a factor of 3 4 between locations. This range was partly the result of differences in dry weight (0.072 to 0.177 g per cutting), but principally caused by differences in tissue N concentration between locations. The change in cutting dry weight (DW) was calculated by subtracting the final tissue DW from the tissue DW at day 0 (the date of cutting insertion into substrate ) (Table 2 4). Tissue percent N decreased from week 0 to week 4 (Table 2 4), but th ere was no significant change in P or K level. In an unpublished survey of tissue nutrient levels in

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32 visually healthy unrooted cuttings, we surveyed tissue from 291 petunia and 179 calibrachoa crops from 14 commercial locations. The mean 2 standard dev iations (SD) were 3.8% to 7.5% N, 0.3% to 0.9% P, and 3.4% to 6.6% K in petunia, or 3.4% to 6.3% N, 0.2% to 0.7% P, and 2.0% to 4.6% K in calibrachoa, P percent N in locations C, E, G, and H were below the mean 2 SD survey levels. Locations E, G, and H also had the three lowest total applied N. P levels were within the survey range, and final K level in location C was slightly below the mean 2 SD. There was no correlation between cha nge in DW and tissue percent of N, P, or K. Substrate n utrients The optimal EC range for greenhouse substrate using a SME is 0.75 to 2.0 mmho/cm for plugs ( Styer, 1997 ). According to Scoggins, the media squeeze (or press extraction method) averages 0.1 dSm 1 higher than the SME (saturated media extraction) method for petunia; therefore, a substrate EC lower than 0.85 or higher than 2.6 are beyond the acceptable limit when performing an media squeeze in a petunia crop (Scoggins, 2002; Styer, 1996). Subs trate EC at each location ranged from 0.2 3.2 dSm 1 during week 1; 0.4 3.2 dSm 1 during week 2; 0.3 1.4 dSm 1 during week 3; and 0.4 2.3 dS m 1 during week 4 (Table 2 3). On average, there was a significant decrease in substrate EC in weeks 1, 2, and 3 (2.0, 1.32 and 0.80 dS m 1 respectively) with a rise in week 4 (Table 2 3). Substrate EC during week 3 was below the acceptable limit (Styer, 1996). Seven of the eight locations showed a significant drop in substrate EC from week 1 to week 2 of propaga tion (Table 2 3). Most of the substrate pH values were in an acceptable range (5.6 to 6.4, Argo and Fisher (2002)) and averaged 5.3 to 6.5 (data not shown). The N, P and K content (g m 2 ) at the end of the four week crop cycle varied widely (between 0.14 to 2.92 g m 2 N, 0.02 to 0.41 g m 2 P, and 0.51 to 4.51 g m 2 K, Table 2 4). The lowest substrate N

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33 occurred in location E, which also had the lowest growth, N tissue uptake and leachate N (Table 2 4). Leachate n utrients The leachate EC during weeks 1, 2 3 and 4 averaged 2.77, 1.50, 0.89, and 1.40 dSm 1 respectively (Table 2 3). In contrast, applied nutrient solution EC increased over weeks 1 to 4, suggesting that the high leachate EC in week 1 resulted primarily from leaching of the pre plant nutrient charge. Based on the nutrient analysis results for the leachate samples, on average, locations leached 1.09 gm 2 N, 0.27 gm 2 P and 1.52 gm 2 K over the 4 week crop cycle (Table 2 4). However, leaching levels at individual locations were as high as 1 .81 gm 2 N, 0.45 gm 2 P, and 2.86 gm 2 K or as low as 0.29 gm 2 N, 0.11 gm 2 P and 0.76 gm 2 K (Table 2 4). Compare N utrient Use Efficiency at each L ocation. The fate of nutrients applied during vegetative cutting propagation can be divided into tiss ue, substrate, and leachate. Tissue uptake was calculated by change in percent nutrient DW from week 0 to week 4, and uptake efficiency was calculated from Eq. 4, where A = tissue uptake. More efficient growers would be defined as having a high tissue uptake and low level of nutrients leached on both a percentage and absolute basis. On average, locations had 50% N, 49% P, and 46% K nutrient uptake efficiencies (Table 2 5), with individual locations up to 77% N, 72% P, and 73% K uptake efficiency. In t erms of leachate, the average percent nutrients leached between locations ranged from 23% N, 34% P, and 28% K, with maximum leaching levels of 45% N, 45% P, and 55% K. Several factors should be considered when evaluating the efficiency of nutrient manageme nt within an individual location. For example, location E had one of the higher nutrient uptake efficiencies (Table 2 5), and low nutrient leaching on a percentage (Table 2 5) and absolute (Table 2. 4) basis, along with moderate leached water volume (Table 2 2).

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34 However, location E also had excessively low tissue percent N and low growth rate (Table 2 4) suggesting that inadequate N fertilizer was applied. Conclusion The variability in water and fertilizer use, and our observation that liners produced at a ll locations were saleable plant material, indicates that there is a broad range in practices and resource efficiencies that can be employed to produce a horticulturally acceptable product. That situation presents an educational opportunity (to improve ef ficiency) by minimizing leaching and optimizing uptake, and a challenge (to convince growers of a need for change when current practices are already producing acceptable crops). We attribute management differences to grower decisions and technology, rathe r than to differing geographic locations, because locations with similar greenhouse temperature and structures located only 10 km apart leached very different water levels. In follow up discussions with the grower businesses in this study, these leaching and fertilizer data were helpful as a training tool and baseline to review practices that could minimize leaching and more closely match water and nutrient supply with plant need. Examples of management practices operations with high leaching rates implem ented were to reduce the irrigation frequency during early propagation stages and/or to replace old mist nozzles with nozzles that supply smaller volumes of water. Further research should focus on optimizing strategies to supply necessary nutrients and wa ter during the root initiation and growth stages, and to measure the impact of grower training on improved resource efficiency.

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35 Table 2 1 Cell count per tray, tray area, substrate components, crop, cultivar, start date, end date, mean day and night tem perature are provided for each of the eight commercial greenhouse locations surveyed. Location Cells (no./ tray) Tray area (m 2 ) Substrate components Crop Cultivar Start date End date Mean day temp (C) z Mean night temp (C) A 84 0.13 Peat/Perlite Petuni a Supertunia Mini Strawberry Pink Vein 8 Feb. 15 Feb. 20 22 B 84 0.13 Peat/Perlite Calibrachoa Superbells Pink 6 Jan. 13 Jan. 21.1 20.4 C 105 0.15 Peat/Perlite/Polymer Calibrachoa Callie Rose Star 24 Feb. 2 Mar. 21.8 17.4 D 105 0.13 Peat/Perlite Calibra choa Minifamous Dark Violet 25 Jan. 1 Feb. 22.8 19.6 E 84 0.15 Peat/Perlite Petunia Petunia Surfinia Purple Veined 24 Jan. 31 Jan. 21.1 20.8 F 105 0.15 Peat/Perlite/Soil Calibrachoa Colorburst Violet 8 Feb. 15 Feb. 19.4 17 G 105 0.13 Peat/Polymer Calib rachoa Minifamous Caribbean Sunset 6 Jan. 13 Jan. 21.8 19.4 H 84 0.13 Peat/Perlite/Rockwool Calibrachoa Superbells Tequila Sunrise 23 Feb. 1 Mar. 22.3 18.8 z The day an d night temperature were recorded by Onset HOBO temperature data loggers (Onset; Bourne, MA).

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36 Table 2 2 Water leached by location (A H) over a 4 week crop cycle. Location Vol leached (Lm 2 ) z Vol leached (Lha 1 ) CC y CC Leached ( per 4 weeks) A 7.7 cd 77,215 9.8 0.8 B 46.1 a 460,769 9.8 4.7 C 14.2 b 141,686 10.5 1.3 D 4.5 d 44,900 11.8 0.4 E 8.7 bcd 87,333 8.6 1.0 F 13.7 b 136,667 10.6 1.3 G 14.7 bc 147,184 6.8 2.2 H 19.5 b 194,615 9.7 2.0 Avg 16.1 161,296 9.7 1.7 SD 12.1 121,487 1.4 1.3 z Leachate is quantified in terms of volume leached per tray, per hectare (ha), or container capacities (CC) leached per tray. stly Significant Difference (HSD) test, P y One container capacity (CC) was defined as the total amount of water present in the container after the substrate was saturated and then allowed to drain for one hour.

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37 Table 2 3. Nutrient solution applied over time at eight greenhouse locations. (Note: Mean P ) Location z Crop age (wee ks) Irrigation method y Applied N ( mg L 1 ) x Applied solution EC (dS m 1 ) w Substrate EC(dS m 1 ) Leachate EC (dS m 1 ) Leach vol (L) A 1 Boom 80.2 0.7 a 2.5 b 2.7 b 0.4 a 2 Boom & Hand 145.7 1.2 c 3.2 c 3.2 b 0.1 a 3 Boom & Hand 139.3 1.1 c 0.6 a 0.8 a 0. 1 a 4 Boom & Hand 109.8 0.9 b 0.4 a 0.3 a 0.4 a B 1 Boom 6.2 0.2 a 0.2 a 0.2 a 3.4 c 2 Boom 82.2 0.8 b 0.6 bc 0.5 ab 1.2 b 3 Hand 129.5 1.2 c 0.3 ab 0.5 ab 0.3 a 4 Hand 112.8 1.0 c 1.0 c 1.2 b 1.0 b C 1 Boom 67.8 0.9 a 1 .8 b 2.0 b 0.9 b 2 Boom 99.7 1.1 b 1.3 a 1.5 bc 0.1 a 3 Boom 82.2 0.9 a 0.9 a 1.1 ac 0.8 b 4 Boom 64.0 1.4 c 2.3 c 3.0 d 0.4 a D 1 Boom 0.5 0.3 a 1.8 b 3.4 c 0.4 b 2 Boom & Hand 194.8 1.7 c 0.9 a 1.5 ab 0.0 a 3 Boom & Hand 148.0 1.4 b 0.8 a 0.6 a 0.0 a 4 Boom & Hand 157.6 1.4 b 2.0 b 1.9 b 0.2 ab E 1 Boom 0.4 0.3 a 3.2 b 1.2 a 0.1 a 2 Boom 2.1 0.6 b 0.7 a 1.0 a 0.7 b 3 Boom & Hand 93.5 0.8 c 1.0 a 0.9 a 0.1 a 4 Boom & Hand 105.5 0.9 c 0.7 a 0.7 a 0.4 a F 1 Boom 5.0 0.3 a 3.1 c 4.8 c 0.3 b 2 Boom & Hand 0.0 0.2 a 2.5 b 2.9 b 0.0 a 3 Hand 52.4 0.5 c 1.4 a 1.7 a 1.2 c 4 Hand 72.7 0.7 b 2.1 b 2.0 a 0.5 b G 1 Mist 6.8 0.4 a 0.9 bc 1.5 b 1.4 c 2 Mist 11.7 0.5 a 0.4 a 0.4 a 0.4 b 3 Hand 19.2 0.4 a 0.7 ab 0.5 a 0.0 a 4 Hand 107.4 1.1 b 1.1 c 1.1 ab 0.1 ab H 1 Boom 23.3 0.5 a 2.6 b 6.4 b 0.2 a z Data were analyzed by location (denoted A to H) and crop age (1, 2, 3, an d 4 weeks after sticking of cuttings). y Irrigation method included boom, stationary mist, or hand watering, or a combination of more than one method during the same week. x The applied nitrogen (N) concentration (mgL 1 ) was measured on one sample for e ach crop and location, combined from 5 replicate irrigation collection funnels; 1 mg.L 1 = 1 ppm. w Electrical conductivity (EC) data were based on 5 replicate samples per crop age and location; 1 dS.m 1 = 1 mmho/cm.

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38 2 Boom & Hand 142.3 1.1 c 1.0 a 0.9 a 1.3 c 3 Hand 79.3 0.7 b 0.7 a 1.1 a 0.4 ab 4 Hand 78.3 0.7 b 0.9 a 1.1 a 0.7 b Stand ard Error 21.3 0.06 0.16 0.30 0.11 Avg 1 a 23.8 0.44 a 2.00 a 2.77 c 0.88 c Avg 2 ab 84.8 0.88 b 1.32 b 1.50 b 0.48 b Avg 3 b 92.9 0.87 b 0.80 c 0.89 a 0.36 a Avg 4 b 101.0 1.01 c 1.32 b 1.40 b 0.46 ab Avg All 75.6 0.8 1.4 1.6 0.5

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39 Table 2 4. Total nitrogen, phosphorus and potassium applied were t he sum of the tissue uptake, nutrients leached and fin al substrate nutrient content. (Note: Mean separation was calculated using P ) Location z Species Concn in tissue (%) y Change in DW (g/cutting) x Tissue uptake (gm 2 ) w Final substrate nutrient (gm 2 ) v Leached nutrient (gm 2 ) u Total applied (gm 2 ) Nitrogen Initial Final A Petunia 6.1 4.4 0.15 ab 4.0 b 0.38 0.78 b c 5.1 B Calibrachoa 3.9 3.8 0.07 e 1.7 ef 1.52 1.59 ab 4.8 C Petunia 4.8 3.6 0.10 cde 2.3 cd 2.26 0.90 ab c 5.4 D Calibrachoa 4.0 3.8 0.09 de 2.9 c 2.92 0.95 ab c 6.8 E Petunia 4.2 2.9 0.07 e 0.9 f 0.14 0.29 c 1.4 F Calibrachoa 3.0 3.6 0.18 a 4.5 a 2.08 1.69 a 8.3 G Calibrachoa 5.4 2.3 0.11 cd 1.3 f 1.57 0.73 bc 3.6 H Calibrachoa 4.6 3.2 0.12 bc 1.8 de 0.47 1.81 ab 4.0 Avg 4.5a 3.4b 0.11 2.4 1.42 1.09 4.9 Standard Error 0.12 0.12 0.01 0.1 0.16 Phosphorus A Petunia 0.7 0.5 0.5 b c 0.02 0.35 ab 0.8 B Calibrachoa 0.6 0.4 0.2 f 0.05 0.11 b 0.3 C Petunia 0.6 0.5 0.3 cd 0.41 0.45 a 1.2 D Calibrachoa 0.5 0.6 0.5 bc 0.05 0.15 b 0.7 E Petunia 0.5 0.6 0.2 ef 0.10 0.15 b 0.5 F Calibrachoa 0.4 0.5 0.6 a 0.17 0.23 ab 1.0 G Cal ibrachoa 0.5 0.4 0.3 de 0.17 0.36 ab 0.9 H Calibrachoa 0.7 0.5 0.3 cd e 0.18 0.38 ab 0.9 Avg 0.5a 0.5a 0.4 0.14 0.27 0.8 Standard Error 0.01 0.01 0.2 0.04 Potassium A Petunia 5.7 4.5 4.1 b 0.51 1.05 b 5.7 z Each letter (A H) represents an indiv idual location. y Initial and final percent Nitrogen (N), phosphorus (P), and potassium (K) in the tissue. x Change in dry weight (DW) for each species at each location. N, P, and K applied and distribution in the tissue, substrate and leachate at each lo cation. w Tissue uptake was c alculated by multiplying the dry weight of the vegetative cuttings at week 4 and week 0 by the difference. v Final n utrient concentration in the substrate was c alculated by multiplying the mgL 1 N, P and K in the soil solution by the container capacity for each tray and then converting to gm 2 u Nutrients leached were c alculated by multiplying the mgL 1 N, P and K by the total vo lume of leachate over 4 weeks.

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40 B Calibrachoa 3.6 3 .5 1.6 d 1.21 1.44 ab 4.3 C Petunia 4.3 3.3 2.1 c 2.43 1.49 ab 6.0 D Calibrachoa 4.0 4.7 3.8 b 4.51 1.86 ab 10.1 E Petunia 5.0 4.1 1.5 cd 0.42 0.76 b 2.7 F Calibrachoa 3.4 3.7 4.6 a 1.22 1.41 ab 7.2 G Calibrachoa 4.6 2.5 1.7 cd 1.90 1.32 b 4.9 H Calibrachoa 3.6 2.6 1.5 cd 0.85 2.86 a 5.2 Avg 4.3a 3.6a 2.6 1.63 1.52 5.8 Standard Error 0.12 0.12 0.2 0.27

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41 Table 2 5 Nitrogen, phosphorus and potassium use efficiency for each location. Location z Species Nutrient uptake effic iency (%) y Final substrate nutrient (%) x Nutrient leached (%) Total applied (kgha 1 ) Total leached (kgha 1 ) Nitrogen A Petunia 77 7 15 51 8 B Calibrachoa 36 31 33 48 16 C Petunia 42 41 17 54 9 D Calibrachoa 43 43 14 68 10 E Petunia 69 10 21 1 4 3 F Calibrachoa 55 25 20 83 17 G Calibrachoa 36 44 20 36 7 H Calibrachoa 44 12 45 40 18 Avg 50 27 23 49 11 Phosphorus A Petunia 55 3 42 8 3 B Calibrachoa 53 13 34 3 1 C Petunia 29 34 37 12 4 D Calibrachoa 72 7 21 7 1 E Petunia 49 20 32 5 2 F Calibrachoa 61 16 22 10 2 G Calibrachoa 38 20 42 9 4 H Calibrachoa 35 21 45 9 4 Avg 49 17 34 8 3 Potassium A Petunia 73 9 18 57 10 B Calibrachoa 38 28 34 43 14 C Petunia 35 40 25 60 15 D Calibrachoa 37 44 18 101 19 E P etunia 56 16 29 27 8 F Calibrachoa 64 17 20 72 14 G Calibrachoa 35 39 27 49 13 H Calibrachoa 29 16 55 52 29 Avg 46 26 28 58 16 z Each letter (A H) represents an individual location. y The % Nutrient Uptake (also termed uptake efficiency) was calculated by dividing the total mg/tray (Table 2.4) by the change in tissue dry weight in mg (Ta ble 2.4). x The Final % Nutrient in Substrate was calculated by dividing the total mg/tray (Table2.4) by the final substrate nutrient concentration (Table 2.4).

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42 CHAPTER 3 A SURVEY OF TISSUE NUTRIENT LEVELS IN VEGETATIVE CUTTINGS Introduction Off shore production of unrooted cuttings of ornamental bedding is increasing, with three quarters of these imports coming from Costa Rica, Guatemala, and Mexico (U.S. Dept. of Agr., 2007). Total sales increased from $23.7 million in 1997 to $61.2 million in 2006, and the total volume of units i mported was near 900 million in 2006, twice that imported in 2000 (U.S. Dept. of Agr., 2007). Stock plant nutrition has been shown to affect subsequent rooting success of the vegetative cuttings (Dole and Gibson, 2006). We found that tissue N, P, and K concentrations in petunia tissue declined during propagation even under constant fertigation from 6.6 %N to 4.7 %N (Ch. 3 in Santos (2009)). Unrooted cuttings with initially lower tissue nutrient concentrations are therefore at risk of falling below suffi ciency ranges during propagation. Tissue nutrient analysis is used to confirm visual or hidden deficiency symptoms, evaluate nutrient interactions, confirm uptake of applied nutrients, and establish recommended (sufficiency) or survey ranges. A recommende d range is determined by testing plant tissue that appears near or above toxicity or below deficiency symptoms (Mills and Jones, 1996). The goal is to develop a range that represents concentrations of a given nutrient that falls between deficiency and tox icity symptoms at which growth rate and plant quality are acceptable for horticultural production (Mills and Jones, 1996). During plant production the objectives should be for the plant tissue results of a given crop to fall in the median of the recommen ded range. In contrast, survey ranges describe observed nutrient levels, which may or may not be adequate for and Jones, 1996). The limitation is that the lowe r and upper limits of the survey range are not as

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43 clearly defined as com pared to the recommended range (Mills and Jones, 1996). Survey ranges were determined through evaluation of multiple tissue nutrient analyses of healthy looking cuttings over time, a process that required much less time and materials compared with the procedures involved in determining recommended ranges. Recommended and deficiency ranges were quantified by Gibson et al. (2007) for more than 25 popular bedding plants produced as seed or vegetative cuttings based on growth response in hydroponic studies. Adequately fertilized stock plants provide cuttings with enough nutrient reserves to carry them through propagation. Nutrient ranges that apply to finished plant production may undere stimate the nutrient reserves required by unrooted cuttings, however ranges (recommended or survey) do not exist for unrooted cuttings. If the recommended ranges determined for finished plant production could be applied to vegetative cutting production, g rowers could use those ranges to adjust their irrigation and fertility programs accordingly. The objectives of this survey were to (1) determine nutrient levels for commercially produced unrooted cuttings for which there are no recommended ranges and to (2 ) compare mean tissue nutrient levels from a survey of commercially produced unrooted cuttings to published recommended ranges. Materials and Methods Twenty two domestic and offshore stock plant facilities and propagation greenhouses provided tissue samp les from visually healthy stock plants or cuttings during 2004 08 to Quality Analytical Laboratories (Panama City, FL), Soil and Plant Laboratory (Orange, CA), and University of New Hampshire Plant Diagnostics Laboratory (Durham, NH). The tissue samples w ere taken from the terminal growing tip and included both the stem, 1 2 sets of expanded leaves and were representative of the cutting size, age and quality for each particular species. The tissue sampling method differed from normal tissue sampling proto cols as described by

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44 Mills and Jones (1996) because samples included the stem, recently mature leaves as well as immature leaves. Companies participating in this survey remained anonymous, but each company participating in this survey had several years of experience and demonstrated success in rooted liner and finished plant production. Stock plant fertilization programs varied from operation to operation, depending on substrate type, water quality, and plant species. All stock producers used water solub le fertilizers and N concentration rates ranged from between 150 250 mgL 1 as recommended by Dole and Gibson (2006). Samples came primarily from cutting suppliers, or were submitted immediately after cuttings were received in the U.S. by propagation gree nhouses. Participating companies were directed to follow the same sampling method. Samples were only to be taken from visually healthy and vigorous stock plants or cuttings, from a single cultivar per species, and the same cultivar was sampled up to three times per production season. Samples were taken from a minimum of 15 plants if a company sampled from their own stock, with 500 mg of sample taken from entire unrooted tip cuttings consistent with commercially produced cuttings. Each sample was air drie d to avoid Botrytis or other disease issues during shipping, bagged separately and labeled with the cultivar, species, location, and sample date. Samples were promptly shipped to the analytical labs within 24 hours of sampling. Total elemental content in oven dried, ground plant tissue samples, were determined by laboratory plant analysis (Jones, 2001). Nitrogen was measured as Total Kjeldahl Nitrogen (TKN) The Kjeldahl N determination method used wet digestion in the presence of an oxidizer, sulfuric a cid (H 2 SO 4 ), potassium sulfate (K 2 SO 4 ) to raise the boiling point of the acid, and a metal catalyst to transform the following forms of nitrogen (nucleic acids, NO 3 N, NH 4 N, and protein N) into ammonium (Mylavarapu and Kennelley, 2002). Quantification of ammonium

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45 was accomplished through colorimetry distillation, or ion specific electrode (Mylavarapu and Kennelley, 2002). Samples were prepared for analysis of the remaining nutrients by ashing, moistening with equal parts deionized water and hydrochloric acid (HCl), and then analysis of Ca, Mg, P, K, Na, S, Al, Mn, Cu, Fe, Zn, B, and Mo using Inductively Coupled Plasma (ICP) emission spectrometry (Mylavarapu and. Kennelley, 2002). Aluminum was included in the analysis because in nonaccumulating species, a t high concentrations, negatively affects growth at low substrate pH and in accumulating species, at low concentrations, can stimulate root growth (Marschner, 1995). Therefore the authors felt although Al was not defined as an essential element the typica l ranges found in plant tissue were worth recording and monitoring. Data were analyzed using Proc Means in SAS (version 9.1; SAS Institute, Cary, NC). Each sample submitted was identified as an experimental unit and all cultivars within a given genus wer e grouped, except for Impatiens and Pelargonium, which were separated on a species basis. Over 2004 to 2008, 4,863 tissue samples were collected from 22 companies for a total of 127 species. Forty four of those species were sampled a minimum of 10 times f rom a minimum of 2 locations. Ranges presented for each species in the survey represented the 10 th to the 90 th percentile of the overall sample nutrient ranges. These criterions were determined reasonable by the authors based on the sample size and sourc e. The high and low values within those ranges were eliminated through presentation of the 10 th to the 90 th percentile, although not a standard way of describing the data it was chosen because some of the data did not assume any particular frequency distr ibution. Means calculated in the tissue nutrient survey were compared to published ranges (Williams, 2004; Gibson, 2003; Pitchay, 2002; Mills and Jones, 1996; Erwin et al., 1992). These known ranges exist for fourteen bedding plant species or genera, whic h were compared to the

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46 mean nutrient levels for corresponding plants in the survey, including Argyranthemum, Angelonia, Sutera, Begonia, Brachyscome, Bracteantha Calibrachoa, Impatiens waller ana, Impatiens hawkeri, Nemesia, Osteospermum, Petunia, Salvia, and Vinca major Critical minimum ranges determined by research done at North Carolina State University were also included for comparison. The initial critical minimum values correlate to when visual deficiency symptoms were first apparent on the tissue, and the advanced values indicate when symptoms affected the growth and development of the plant tissue. Recommended and critical minimum ranges for macronutrients and micronutrients were determined with the exception of N, using a Perkin Elmer 3300 Induc tively Coupled Argon Plasma Emission Spectrophotometer, Norwalk, CT while total N was analyzed using Carlo Erba NA 1500 Nitrogen Analyzer (Pitchay, 2002). Results The survey data represent the typical nutrient levels in cuttings of each species and were di vided into two tables, depending on whether recommended ranges already existed for the genus or species (Table 3 1) or where there were no published recommended ranges (Table 3 2). Individual nutrient trends varied between plant species enough that groupi ng species based on nutrient survey range similarities was impossible. Survey tissue nutrient means and ranges were therefore best organized by plant species (Table 3 2). Data presented in Table 3 2 are the first ranges published specifically for unrooted cuttings of those species. Variability tended to be higher in the mean micronutrient values compared to the mean macronutrient values per species, as shown in the coefficients of variation (standard deviation/mean) for each species (Table 3 2). On avera ge the coefficients of variation by nutrient between species in Table 3. 2 were 17, 27, 22, 40, 37, and 50 % for N, P, K, Ca, Mg, and S respectively compared with 51, 66, 51, 66, 40, and 77 % for Fe, Mn, Zn, Cu, B, and Mo respectively. Similar trends were observed in Table 3 1, where coefficients of variation by nutrient between species were 17, 25, 20, 35, 36, and 47 % for

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47 N, P, K, Ca, Mg and S respectively compared with 62, 58, 51, 88, 42, and 83 % for Fe, Mn, Zn, Cu, B, and Mo (data not shown). Substrat e physical properties and pH varied between participating locations and could have influenced tissue nutrient concentrations, by affecting nutrient availability for uptake. Substrates ranged from peat based, to volcanic rock, to compost. In addition, wat er quality would affect the nutrients supplied to the stock plants at the different locations depending on the alkalinity and Ca, Mg, or Fe levels in the water. High coefficients of variation in Cu, Mn, Zn, or S may partly be a result of some plants that may have been treated with fungicides containing one or more of the above elements during the sampling period. Nitrogen, Phosphorus, and Potassium None of the plant species whose mean N, P, or K tissue nutrient levels were lower than the recommended range s fell below critical minimum levels (Table 3 1). No plant species showed higher mean tissue nitrogen levels than the recommended ranges, 50% ( Begonia, Impatiens wallerana, Nemesia, Impatiens hawkeri, Petunia, Salvia and Vinca major ) were within range and 50% ( Angelonia, Argyranthemum, Bacopa, Brachyscome, Bracteantha, Calibrachoa, and Osteospermum ) were lower than the recommended range. For phosphorus, 57% of plant species ( Bacopa, Begonia, Nemesia, Impatiens hawkeri, Osteospermum, Petunia, Salvia, and Vi nca major ) had mean P within range, with 7% (one genus, Calibrachoa ) showing higher P levels, and 36% ( Angelonia, Argyranthemum, Brachyscome, Bracteantha, and Impatiens wallerana ) with lower P levels. Half of the plant species ( Angelonia, Begonia, Calibrac hoa, Nemesia, Impatiens hawkeri, Petunia, and Salvia ) had mean K within the recommended range, 14% (Impatiens wallerana and Vinca major ) were higher than the recommended range, and 36% ( Argyranthemum, Bacopa, Brachyscome, Bracteantha, and Osteospermum ) wer e lower.

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48 Calcium, Magnesium, and Sulfur Mean tissue levels were above the recommended range in 14% ( Brachyscome and Osteospermum ) for Ca, 29% ( Argyranthemum, Brachyscome, Bracteantha, and Calibrachoa ) for Mg, and 36% ( Argyranthemum, Bacopa, Calibrachoa, Im patiens hawkeri, and Vinca major ) for S. 64% ( Angelonia, Argyranthemum, Bacopa, Begonia, Bracteantha, Calibrachoa, Petunia, Salvia, and Vinca major ) were lower than the recommended ranges for Ca, 21% ( Bacopa, Impatiens wallerana, and Vinca major ) for Mg, and 14% ( Impatiens wallerana and Salvia ) for S. None of the species whose Ca, Mg, or S tissue nutrient levels were lower than the recommended ranges fell below critical minimum levels. Micronutrients Mean Fe levels were higher than the recommended range in 43% of plant species ( Argyranthemum, Bacopa, Bracteantha, Calibrachoa, Impatiens wallerana, and Osteospermum ) compared to the recommended ranges, 29% ( Angelonia, Bacopa, Bracteantha, and Osteospermum ) for Mn, 36% ( Argyranthemum, Bacopa, Bracteantha, Cal ibrachoa, and Osteospermum ) for Zn, 43% ( Argyranthemum, Bacopa, Brachyscome, Bracteantha, Calibrachoa, and Osteospermum ) for Cu, and 29% ( Argyranthemum, Bracteantha, Calibrachoa, and Impatiens wallerana ) for B. 14% ( Angelonia and Vinca major ) were lower t han the recommended ranges for Fe, 29% ( Argyranthemum, Brachyscome, Impatiens wallerana, and Vinca major ) for Mn, 7% ( Brachyscome ) for Zn, 14% ( Angelonia and Impatiens wallerana ) for Cu, and 7% ( Brachyscome ) for B. Plant groups with Fe, Mn, Zn, Cu, and B tissue nutrient levels lower than the recommended ranges did not fall below critical minimum levels, and plant groups with Fe, Mn, Zn, Cu, and B higher than the recommended range did not reach toxicity levels.

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49 Conclusion Maintaining tissue nutrient levels within the recommended ranges for each species is a prerequisite (it is necessary but may not be sufficient) for rooting success and uniform performance in the propagation environment. The nutrient ranges presented in this survey represent the typical nut rient levels in cuttings of each species. These ranges can be used by growers as an additional resource when interpreting tissue analysis reports of their unrooted cuttings and making corrective nutrient management decisions should nutrient levels fall ou tside of the survey ranges. Overall, 48% of mean tissue nutrient measurements in Table 3 1 fell within the recommended range, 25% were higher, and 27% were lower. Tissue levels in unrooted cuttings were similar to ranges established for finished plants, in spite of the difference in sampling procedures. Species with nutrients that fell above or below the recommended ranges did not reach critical minimum deficiency or toxicity levels. We therefore conclude, based on the fact that 75% of the means calcul ated were either below or within the recommended ranges, that where those ranges exist for finished plants, they can be applied to stock plant production of vegetative cuttings. However, unlike the species in Table 3 1, recommended and critical minimum ra nges do not yet exist for each of the species in Table 3 2. The ranges presented in Table 3 2 should serve as a reference for typical tissue nutrient concentrations in cuttings of those species with the understanding that the determination of survey range s is based on healthy plant tissue compared with that of recommended ranges which incorporates more precision sampling of tissue with tissue nutrient deficiency symptoms, along with greenhouse controlled experiments that evaluate deficiency symptoms by nut rient in hydroponic solutions.

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50 Table 3 1 th and 90 th commended ranges for 14 species. z (Note: Superscripts for the Reco mmended Range and Critical Minimum columns indicate the source: 1) Gibson et al. (2007), 2) Mills and Jones (1996), 3) Pitchay (2002), 4) Williams (2004), 5) Gibson (2003), or Erwin et al. (1992). Ranges determined by were based on sufficiency (or recommended) data or source survey data from Mills and Jones (1996) Critical minimum values correlate to when initial and advanced deficiency symptoms were visible, as determined by North Carolina State University. ) Angelonia Angelonia angu stifolia Recommended Critical Minimum 4 Nutrient Mean y Std. Dev. Survey Range Range 1,4 Initial Advanced N (%) 4.05 0.55 3.43 4.66 4.63 to 5.06 2.82 1.70 P (%) 0.41 0.07 0.34 0.50 0.44 to 0.63 0.29 0.23 K (%) 2.88 0.50 2.36 3.34 2.82 to 3.47 2.03 1.49 Ca (%) 0.53 0.23 0.30 0.80 0.88 to 1.18 0.24 0.15 Mg (%) 0.28 0.09 0.19 0.39 0.24 to 0.30 0.16 0.13 S (%) 0.34 0.16 0.13 0.51 0.33 to 0.37 0.11 0.20 Fe ( mgL 1 ) 90.7 41.7 62.4 125.3 99.6 to 110.0 80.3 8.0 Mn ( mgL 1 ) 111.4 52.0 47.7 184.4 83.6 to 108.7 11.9 11.4 Zn ( mgL 1 ) 73.9 25.2 43.3 106.8 59.4 to 86.2 38.1 28.3 Cu ( mgL 1 ) 6.3 7.1 2.2 11.2 8.3 to 12.4 2.5 B ( mgL 1 ) 36.1 9.2 26.5 47.4 29.6 to 46.2 21.9 22.0 Mo ( mgL 1 ) 1.7 1.7 0.6 4.2 to Al ( mgL 1 ) 62.2 71.2 11.3 168.9 to Number of samples: 71 Number of locations: 5 Argyranthemum Argyranthemum frustescens 1,3 3 N (%) 5.39 0.76 4.50 6.40 6.53 to 7.28 2.92 1.48 P (%) 0.53 0.13 0.37 0.70 0.58 to 0.73 0.32 0.06 K (%) 4.72 0.93 3.30 6.04 6.49 to 7.05 0.62 0.42 Ca ( %) 1.30 0.28 0.99 1.67 1.78 to 1.79 0.92 0.24 Mg (%) 0.38 0.17 0.18 0.57 0.33 to 0.34 0.07 0.07 S (%) 0.90 0.41 0.45 1.44 0.27 to 0.30 0.18 0.06 Fe ( mgL 1 ) 117.9 66.1 64.0 201.4 56.0 to 66.0 36.9 23.0 Mn ( mgL 1 ) 188.8 97.8 82.3 296.8 234.0 to 236.0 1 0.3 4.9 Zn ( mgL 1 ) 44.3 37.7 20.2 79.5 21.5 to 30.9 11.5 7.9 Cu ( mgL 1 ) 15.1 23.3 4.6 25.0 5.3 to 7.8 1.9 1.2 B ( mgL 1 ) 60.6 28.5 31.1 87.9 47.5 to 58.8 11.7 8.0 Mo ( mgL 1 ) 5.24 4.94 1.30 12.40 to Al ( mgL 1 ) 49.48 37.09 16.29 109.71 to Number of samples: 117 Number of locations: 4 z Argyranthemum, Angelonia, Sutera, Begonia, Brachyscome, Bracteantha, Calibrachoa, Impatiens walleriana, Impatiens hawkeri, Nemesia, Osteospermum, Petunia, Salvia, and Vinca major. y Dashes indicate where reference values were not determined.

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51 Table 3 1. Continued Bacopa Sutera spp. Recommended Critical Minimum 3 Nutrient Mean Std. Dev. Survey Range Range 1,3 Initial Advanced N (%) 4.84 0.71 4.00 5.77 4.98 to 5.60 1.97 1.15 P ( %) 0.55 0.17 0.38 0.80 0.49 to 0.61 0.12 0.05 K (%) 3.76 0.77 2.86 4.71 5.07 to 5.11 0.57 0.40 Ca (%) 0.91 0.36 0.56 1.35 1.35 to 1.66 0.34 0.17 Mg (%) 0.36 0.11 0.23 0.50 0.40 to 0.41 0.08 0.11 S (%) 0.51 0.23 0.25 0.70 0.34 to 0.45 0.11 0.07 Fe ( mg L 1 ) 114.2 82.5 66.3 167.0 55.6 to 60.5 40.7 28.4 Mn ( mgL 1 ) 123.8 72.3 55.4 194.6 95.8 to 115.0 5.7 3.8 Zn ( mgL 1 ) 47.8 21.1 27.5 81.0 22.1 to 27.4 9.5 8.2 Cu ( mgL 1 ) 15.8 12.2 4.9 36.3 6.8 to 7.8 1.8 1.6 B ( mgL 1 ) 44.4 26.5 23.9 72.1 39.1 to 48.5 6.2 5.0 Mo ( mgL 1 ) 3.1 2.3 1.2 6.3 to Al ( mgL 1 ) 57.8 76.7 15.0 78.1 to Number of samples: 87 Number of locations: 6 Begonia Begonia hiemalis 2 3 N (%) 4.80 0.87 3.60 6.00 2.00 to 6.00 1.50 1.39 P (%) 0.44 0.11 0.30 0.60 0.29 to 0.75 0.10 0.06 K (%) 2.55 0.46 1.82 3.03 2.25 to 6.00 0.75 0.52 Ca (%) 0.99 0.25 0.70 1.30 1.00 to 3.10 0.55 0.43 Mg (%) 0.45 0.15 0.31 0.64 0.30 to 0.88 0.10 0.11 S (%) 0.61 0.21 0.22 0.90 0.22 to 0.70 0.13 0.06 Fe ( mgL 1 ) 171.5 131.1 72.9 337.7 50.0 to 200.0 62.6 Mn ( mgL 1 ) 77.4 36.3 27.4 127.4 45.0 to 200.0 9.4 8.2 Zn ( mgL 1 ) 55.2 43.6 33.1 85.5 25.0 to 100.0 21.8 Cu ( mgL 1 ) 10.9 7.0 5.8 16.8 7.0 to 33.0 1.7 1.7 B ( mgL 1 ) 47.1 16.8 32.8 69.5 20.0 to 75.0 9.1 6.7 Mo ( mgL 1 ) 14.5 11.2 1.5 26.6 to Al ( mgL 1 ) 94.1 67.2 27.6 189.8 to Number of samples: 49 Number of locations: 3 Brachyscome Brachyscome hybrid 1,4 4 N (%) 4.67 1.09 3.60 6.00 6.99 to 7.46 4.29 2.21 P (%) 0.56 0.14 0.39 0.70 0.58 to 0. 61 0.27 0.23 K (%) 3.77 0.65 2.90 4.75 4.77 to 4.95 2.32 1.27 Ca (%) 0.90 0.46 0.53 1.15 0.67 to 0.83 0.18 0.14 Mg (%) 0.29 0.14 0.15 0.43 0.23 to 0.26 0.13 0.11 S (%) 0.67 0.20 0.48 0.98 0.31 to 0.14 0.17 Fe ( mgL 1 ) 125.5 63.6 56.2 266.1 116.5 to 298.3 66.6 Mn ( mgL 1 ) 198.2 94.0 85.1 353.2 to Zn ( mgL 1 ) 54.9 28.0 24.0 78.5 to Cu ( mgL 1 ) 10.6 7.9 5.1 23.5 4.5 to 5.3 1.3 B ( mgL 1 ) 58.5 20.0 39.2 92.2 64.9 to 67.0 47.8 22.1 Mo ( mgL 1 ) 4.7 4.4 1.6 11.2 to Al ( mgL 1 ) 37.4 33.8 7.4 71.7 to Number of samples: 29 Number of locations: 2

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52 Table 3 1. Continued Bracteantha Bracteantha bracteata Recommended Critical Minimum 5 Nutrient Mean Std. Dev. Survey Range Range 1,5 Initial Advanced N (%) 4.91 0.83 3.83 5.90 5.51 to 6.33 3.19 1.76 P (%) 0.62 0.13 0.50 0.75 0.76 to 0.82 0.26 0.07 K (%) 4.92 0.97 3.60 6.31 6.04 to 6.74 1.08 0.77 Ca (%) 1.07 0.29 0.77 1.50 1.42 to 1.46 0.29 0.24 Mg (%) 0.37 0.13 0.20 0.52 0.21 to 0.29 0.12 0.06 S (%) 0.68 0.27 0.4 7 0.98 0.21 to 0.09 0.05 Fe ( mgL 1 ) 94.4 34.5 57.2 139.0 61.4 to 89.9 41.2 27.2 Mn ( mgL 1 ) 196.0 86.3 91.5 324.0 117.4 to 174.3 40.1 5.4 Zn ( mgL 1 ) 89.2 33.3 49.4 130.8 32.0 to 34.4 18.3 10.8 Cu ( mgL 1 ) 8.5 9.0 3.1 15.5 5.8 to 6.8 2.9 1.4 B ( mg L 1 ) 61.3 20.9 37.4 90.7 28.5 to 31.2 17.1 5.6 Mo ( mgL 1 ) 5.9 4.4 1.7 12.8 to Al ( mgL 1 ) 64.6 49.5 19.8 145.1 to Number of samples: 136 Number of locations: 5 Calibrachoa Calibrachoa hybrida 1,4 4 N (%) 4.86 0.74 4.06 5. 80 5.04 to 5.06 1.65 0.85 P (%) 0.46 0.14 0.30 0.63 0.36 to 0.42 0.16 0.05 K (%) 3.33 0.65 2.60 3.98 2.95 to 4.22 1.70 0.51 Ca (%) 0.90 0.41 0.48 1.37 1.48 to 1.84 0.37 0.11 Mg (%) 0.47 0.18 0.26 0.68 0.28 to 0.39 0.11 0.10 S (%) 0.67 0.38 0.23 1.13 0 .44 to 0.61 0.15 0.21 Fe ( mgL 1 ) 115.9 82.4 59.0 199.7 68.0 to 110.4 77.5 40.6 Mn ( mgL 1 ) 100.4 66.0 41.4 166.5 70.4 to 107.7 34.9 6.3 Zn ( mgL 1 ) 50.4 35.8 26.0 68.6 27.4 to 43.9 16.0 10.8 Cu ( mgL 1 ) 10.9 14.4 4.3 17.0 9.4 to 9.9 2.6 2.3 B ( mgL 1 ) 51.2 21.3 29.4 80.9 32.0 to 37.4 6.4 2.4 Mo ( mgL 1 ) 7.8 5.5 2.3 15.4 to Al ( mgL 1 ) 81.7 167.5 12.7 180.1 to Number of samples: 251 Number of locations: 14 Bedding plant impatiens Impatiens wallerana 2S 3 N (%) 4.49 0.79 3. 29 5.48 3.64 to 5.83 1.21 0.82 P (%) 0.64 0.19 0.42 0.86 0.77 to 0.92 0.08 0.07 K (%) 3.02 0.77 2.25 4.03 1.37 to 2.35 0.31 0.31 Ca (%) 2.13 0.43 1.50 2.61 1.75 to 2.40 0.34 Mg (%) 0.54 0.20 0.27 0.83 0.89 to 3.64 0.09 0.07 S (%) 0.59 0.45 0.29 1.18 0.83 to 0.87 0.21 0.10 Fe ( mgL 1 ) 196.4 130.6 89.7 308.6 107.0 to 130.0 55.4 43.8 Mn ( mgL 1 ) 175.0 109.7 83.1 321.0 329.0 to 419.0 10.4 7.6 Zn ( mgL 1 ) 62.3 29.7 33.0 93.8 57.0 to 67.0 26.1 23.4 Cu ( mgL 1 ) 12.2 7.4 5.8 19.0 20.0 to 37.0 2.5 2.7 B ( mgL 1 ) 38.5 19.9 21.3 69.3 23.0 to 25.0 14.1 10.0 Mo ( mgL 1 ) 13.6 7.7 4.4 25.9 to Al ( mgL 1 ) 196.0 276.2 26.0 583.2 to Number of samples: 52 Number of locations: 9

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53 Table 3 1. Continued Nemesia Nemesia fruticans, N. hybrid s Recommended Critical Minimum 3 Nutrient Mean Std. Dev. Survey Range Range 1,3 Initial Advanced N (%) 5.23 0.73 4.30 6.20 5.63 to 5.94 2.30 1.61 P (%) 0.57 0.10 0.46 0.71 0.51 to 0.59 0.10 0.04 K (%) 3.25 0.65 2.50 3.98 4.60 to 4.94 0.34 0.23 Ca (%) 1 .09 0.31 0.75 1.50 1.50 to 1.64 0.56 0.34 Mg (%) 0.45 0.15 0.21 0.64 0.33 to 0.08 0.06 S (%) 0.56 0.21 0.36 0.90 0.16 to 0.23 0.09 0.06 Fe ( mgL 1 ) 129.7 93.4 66.9 250.1 55.1 to 55.8 37.3 24.6 Mn ( mgL 1 ) 108.9 64.9 54.7 182.0 81.2 to 112.5 6.7 6.0 Zn ( mgL 1 ) 67.4 35.3 44.2 94.6 20.0 to 22.0 13.8 13.3 Cu ( mgL 1 ) 11.9 8.0 6.6 19.1 5.9 to 7.0 1.5 1.8 B ( mgL 1 ) 62.8 33.3 36.0 96.0 42.0 to 43.4 8.1 4.6 Mo ( mgL 1 ) 4.4 5.2 1.4 8.3 to Al ( mgL 1 ) 47.5 97.8 13.6 88.1 to Number of sam ples: 262 Number of locations: 7 New Guinea Impatiens Impatiens hawkeri 1,2,3,6 3 N (%) 4.25 0.85 3.15 5.36 2.00 to 4.50 1.95 1.09 P (%) 0.46 0.10 0.36 0.58 0.20 to 0.80 0.07 0.05 K (%) 2.35 0.43 1.86 2.87 1.50 to 4.50 0.53 0.50 Ca (%) 1.73 0.34 1.36 2.17 0.50 to 2.00 0.72 0.42 Mg (%) 0.36 0.14 0.22 0.56 0.30 to 0.80 0.07 0.07 S (%) 1.05 0.43 0.55 1.63 0.91 to 0.21 0.08 0.05 Fe ( mgL 1 ) 133.7 99.1 61.9 288.3 75.0 to 300.0 43.9 Mn ( mgL 1 ) 109.6 86.5 41.1 186.9 50.0 to 250.0 5.8 4.0 Zn ( mgL 1 ) 50.5 23.3 32.0 67.7 25.0 to 100.0 18.6 17.7 Cu ( mgL 1 ) 6.0 4.6 3.2 9.1 5.0 to 15.0 1.6 1.6 B ( mgL 1 ) 38.1 15.1 23.5 54.9 20.0 to 60.0 6.1 4.9 Mo ( mgL 1 ) 14.0 11.1 1.3 28.0 to Al ( mgL 1 ) 64.4 88.2 18.2 123.4 to Number of sa mples: 675 Number of locations: 9 Osteospermum Osteospermum hybrida 1,3 3 N (%) 5.25 1.05 3.77 6.53 5.70 to 6.50 2.50 1.50 P (%) 0.64 0.20 0.43 0.90 0.30 to 0.10 0.10 K (%) 3.78 0.76 2.97 4.67 4.50 to 5.00 0.70 0.50 Ca (%) 1.82 0.54 1.14 2.5 2 1.70 to 1.80 0.40 0.20 Mg (%) 0.68 0.25 0.39 1.02 0.50 to 0.10 0.10 S (%) 1.07 0.60 0.29 1.88 0.20 to 0.10 0.10 Fe ( mgL 1 ) 118.8 58.1 64.8 190.0 63.2 to 65.3 47.8 23.1 Mn ( mgL 1 ) 173.5 103.0 83.2 281.2 95.2 to 142.0 14.9 3.7 Zn ( mgL 1 ) 52.9 2 4.8 28.2 80.3 14.0 to 23.3 9.7 5.8 Cu ( mgL 1 ) 13.8 9.8 6.4 23.6 6.3 to 7.0 2.5 2.1 B ( mgL 1 ) 58.1 24.9 34.2 84.2 43.0 to 60.2 12.5 10.3 Mo ( mgL 1 ) 9.1 7.4 1.9 21.9 to Al ( mgL 1 ) 62.7 109.2 17.8 112.4 to Number of samples: 256 Numbe r of locations: 15

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54 Table 3 1. Continued Petunia Petunia hybrida Recommended Critical Minimum 3 Nutrient Mean Std. Dev. Survey Range Range 2S Initial Advanced N (%) 5.67 0.88 4.50 6.80 3.85 to 7.60 2.06 1.32 P (%) 0.59 0.15 0.40 0.75 0.47 to 0.93 0.07 0.05 K (%) 5.04 0.84 4.02 6.02 3.13 to 6.65 0.69 0.63 Ca (%) 1.16 0.46 0.69 1.74 1.20 to 2.81 0.32 0.38 Mg (%) 0.53 0.20 0.29 0.82 0.36 to 1.37 0.08 0.06 S (%) 0.79 0.33 0.38 1.19 0.33 to 0.80 0.11 0.06 Fe ( mgL 1 ) 119.3 104.6 62.6 211.0 84 .0 to 168.0 53.1 30.0 Mn ( mgL 1 ) 84.8 44.1 49.6 126.0 44.0 to 177.0 11.4 9.2 Zn ( mgL 1 ) 51.5 22.7 30.8 81.6 33.0 to 85.0 13.0 10.2 Cu ( mgL 1 ) 11.6 10.5 4.5 19.8 3.0 to 19.0 3.5 3.3 B ( mgL 1 ) 41.1 19.9 24.3 66.1 18.0 to 43.0 10.3 5.4 Mo ( mgL 1 ) 5. 5 4.5 1.9 10.7 to Al ( mgL 1 ) 95.9 139.2 16.2 248.3 to Number of samples: 373 Number of locations: 11 Salvia Salvia nemorosa, S. superba 2 3 N (%) 4.99 0.72 3.99 5.80 2.38 to 5.61 2.22 2.01 P (%) 0.34 0.12 0.21 0.50 0.30 to 1.24 0.07 0.07 K (%) 3.24 0.62 2.41 4.06 2.90 to 5.86 0.22 0.20 Ca (%) 0.99 0.49 0.64 1.30 1.00 to 2.50 0.66 0.46 Mg (%) 0.53 0.18 0.36 0.78 0.25 to 0.86 0.09 0.07 S (%) 0.58 0.25 0.37 0.84 0.73 to 0.10 0.13 Fe ( mgL 1 ) 125.1 67.5 60.1 225.8 60.0 to 300.0 52.0 Mn ( mgL 1 ) 83.5 62.1 36.5 185.4 30.0 to 284.0 5.1 4.2 Zn ( mgL 1 ) 48.7 19.0 28.1 73.3 25.0 to 115.0 12.0 11.3 Cu ( mgL 1 ) 10.2 7.2 4.9 17.3 7.0 to 35.0 1.8 1.8 B ( mgL 1 ) 50.3 19.8 30.4 71.0 25.0 to 75.0 9.3 8.8 Mo ( mgL 1 ) 4.8 3.9 1.2 12.6 to Al ( mgL 1 ) 94.8 81.3 26.8 236.0 to Number of samples: 56 Number of locations: 3 Vinca Vinca major 2 3 N (%) 4.93 0.86 3.56 5.84 2.72 to 6.28 2.66 1.04 P (%) 0.52 0.12 0.38 0.66 0.28 to 0.64 0.07 0.04 K (%) 4.19 1. 01 2.82 5.33 1.88 to 3.48 0.73 0.28 Ca (%) 0.67 0.35 0.40 0.90 0.93 to 1.13 0.32 0.28 Mg (%) 0.26 0.08 0.17 0.38 0.32 to 0.78 0.11 0.09 S (%) 0.97 0.59 0.27 1.87 0.22 to 0.50 0.09 0.07 Fe ( mgL 1 ) 63.1 38.7 32.5 106.9 72.0 to 277.0 29.0 16.4 Mn ( mgL 1 ) 51.6 33.3 29.0 89.2 135.0 to 302.0 9.3 5.2 Zn ( mgL 1 ) 49.3 17.9 30.1 70.0 30.0 to 51.0 12.3 6.4 Cu ( mgL 1 ) 9.1 7.3 3.0 16.8 6.0 to 16.0 1.4 1.0 B ( mgL 1 ) 40.8 12.9 26.6 62.6 21.0 to 49.0 10.5 7.9 Mo ( mgL 1 ) 9.2 7.2 1.4 20.6 to Al ( mgL 1 ) 52.4 60.6 11.9 130.9 to Number of samples: 74 Number of locations: 5

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55 Table 3 2. variation (standard deviation/mean), survey range (10 th and 90 th per centiles) for species in survey with a minimum of 10 samples from 2 or more locations. Nutrient Mean Std. Dev Coeff. Var. Survey Range Ajuga Ajuga reptans Samples: 43 Locations: 2 N (%) 4.50 0.58 13% 3.86 5.20 P (%) 0.49 0.12 25% 0.35 0.70 K ( %) 3.72 0.72 19% 2.60 4.61 Ca (%) 0.95 0.35 36% 0.59 1.40 Mg (%) 0.30 0.11 39% 0.18 0.46 S (%) 0.54 0.33 61% 0.24 0.93 Fe (mgL 1 ) 105.19 54.66 52% 61.01 172.00 Mn (mgL 1 ) 62.86 29.43 47% 38.12 83.23 Zn (mgL 1 ) 33.25 10.18 31% 24.78 50.33 Cu (mgL 1 ) 13.42 6.74 50% 7.13 22.57 B (mgL 1 ) 46.79 19.97 43% 29.17 66.51 Mo (mgL 1 ) 4.12 2.05 50% 2.05 6.93 Al (mgL 1 ) 115.31 236.60 205% 20.91 192.30 Bidens Bidens Samples: 20 Locations: 2 N (%) 5.57 0.69 12% 4.40 6.30 P (%) 0.64 0.08 13% 0.51 0.74 K (%) 3.71 0.59 16% 3.16 4.51 Ca (%) 0.72 0.14 19% 0.61 0.82 Mg (%) 0.44 0.10 24% 0.37 0.57 S (%) 0.63 0.12 18% 0.53 0.78 Fe (mgL 1 ) 162.53 149.96 92% 80.50 453.50 Mn (mgL 1 ) 91.99 73.22 80% 38.13 134.70 Zn (mgL 1 ) 56.06 20.95 37% 33.55 80. 38 Cu (mgL 1 ) 7.75 2.64 34% 4.83 10.38 B (mgL 1 ) 61.17 23.53 38% 39.20 91.14 Mo (mgL 1 ) 3.18 1.77 56% 1.71 4.22 Al (mgL 1 ) 32.91 34.30 104% 12.76 64.65 Ceratostigma Ceratostigma Samples: 10 Locations: 2 N (%) 3.38 0.66 20% 2.59 4.24 P (%) 0.24 0.06 26% 0.19 0.35 K (%) 1.54 0.47 31% 1.01 2.20 Ca (%) 0.68 0.59 86% 0.31 1.47 Mg (%) 0.44 0.24 55% 0.27 0.84 S (%) 0.46 0.16 34% 0.24 0.68 Fe (mgL 1 ) 68.90 34.93 51% 45.48 127.28 Mn (mgL 1 ) 50.43 51.03 101% 18.74 126.86 Zn (mgL 1 ) 51.31 27 .07 53% 27.50 91.16 Cu (mgL 1 ) 10.58 2.00 19% 7.65 12.97 B (mgL 1 ) 45.65 11.59 25% 28.34 58.37 Mo (mgL 1 ) 4.75 4.85 102% 1.22 13.45 Al (mgL 1 ) 65.86 81.28 123% 17.37 195.73

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56 Table 3 2. Continued Nutrient Mean Std. Dev Coeff. Var. Survey Range Co leus Solenostemon scutellariodes Samples: 56 Locations: 5 N (%) 4.72 0.59 13% 4.00 5.44 P (%) 0.78 0.22 28% 0.50 1.10 K (%) 4.40 1.17 27% 2.80 6.40 Ca (%) 1.51 0.35 23% 1.11 2.10 Mg (%) 0.59 0.19 32% 0.39 0.86 S (%) 0.45 0.28 61% 0.23 0.76 F e (mgL 1 ) 136.46 76.06 56% 70.71 252.00 Mn (mgL 1 ) 176.82 99.78 56% 69.33 338.20 Zn (mgL 1 ) 84.15 65.79 78% 40.70 134.60 Cu (mgL 1 ) 14.97 9.63 64% 7.12 28.89 B (mgL 1 ) 48.22 16.03 33% 28.18 72.00 Mo (mgL 1 ) 4.74 3.92 83% 1.42 8.19 Al (mgL 1 ) 9 3.16 79.55 85% 18.86 230.70 Dahlia Dahlia hybrids Samples: 30 Locations: 4 N (%) 5.07 1.04 21% 3.77 6.40 P (%) 0.78 0.20 25% 0.58 0.98 K (%) 4.34 0.74 17% 3.43 5.21 Ca (%) 1.12 0.34 31% 0.62 1.46 Mg (%) 0.50 0.13 27% 0.36 0.67 S (%) 0.43 0.20 46% 0.23 0.71 Fe (mgL 1 ) 135.99 64.44 47% 81.34 231.94 Mn (mgL 1 ) 123.92 75.78 61% 50.87 235.42 Zn (mgL 1 ) 70.32 27.18 39% 43.62 103.83 Cu (mgL 1 ) 16.13 8.01 50% 9.23 22.19 B (mgL 1 ) 69.08 32.60 47% 42.40 103.18 Mo (mgL 1 ) 2.76 2.79 101% 1 .12 7.31 Al (mgL 1 ) 85.84 79.55 93% 30.20 182.24 Diascia Diascia hybrids Samples: 40 Locations: 6 N (%) 5.48 0.75 14% 4.69 6.10 P (%) 0.61 0.13 21% 0.48 0.71 K (%) 3.60 0.73 20% 3.00 4.41 Ca (%) 1.06 0.29 28% 0.75 1.52 Mg (%) 0.40 0.17 4 3% 0.23 0.67 S (%) 0.51 0.27 54% 0.14 0.81 Fe (mgL 1 ) 131.23 52.76 40% 73.45 201.70 Mn (mgL 1 ) 99.16 84.34 85% 38.36 135.20 Zn (mgL 1 ) 46.68 14.91 32% 33.39 64.56 Cu (mgL 1 ) 9.90 9.42 95% 4.59 14.99 B (mgL 1 ) 42.46 15.27 36% 24.27 63.66 Mo (mg L 1 ) 2.84 1.87 66% 1.31 5.13 Al (mgL 1 ) 62.80 42.95 68% 20.37 121.67

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57 Table 3 2. Continued Nutrient Mean Std. Dev Coeff. Var. Survey Range Euphorbia Euphorbia Samples: 73 Locations: 3 N (%) 4.55 0.73 16% 3.60 5.60 P (%) 0.59 0.25 43% 0.35 0.8 6 K (%) 2.89 0.56 19% 2.23 3.62 Ca (%) 1.11 0.55 50% 0.60 1.70 Mg (%) 0.38 0.18 49% 0.21 0.57 S (%) 0.56 0.39 70% 0.26 0.91 Fe (mgL 1 ) 93.06 67.28 72% 50.67 138.90 Mn (mgL 1 ) 73.79 37.06 50% 38.91 131.70 Zn (mgL 1 ) 53.49 47.48 89% 28.69 66.91 Cu (mgL 1 ) 8.00 6.64 83% 3.92 11.02 B (mgL 1 ) 56.28 31.34 56% 24.14 106.10 Mo (mgL 1 ) 10.65 5.96 56% 3.71 16.71 Al (mgL 1 ) 53.48 54.40 102% 16.65 112.90 Fuchsia Fuchsia hybrids Samples: 64 Locations: 9 N (%) 4.34 0.66 15% 3.30 5.15 P (%) 0.50 0.12 24% 0.35 0.69 K (%) 2.70 0.54 20% 2.05 3.40 Ca (%) 1.22 0.33 27% 0.84 1.60 Mg (%) 0.42 0.14 34% 0.27 0.66 S (%) 0.32 0.17 53% 0.13 0.52 Fe (mgL 1 ) 200.74 122.82 61% 67.96 345.51 Mn (mgL 1 ) 97.10 79.21 82% 36.93 190.00 Zn (mgL 1 ) 52.65 19.51 37% 27.79 76.98 Cu (mgL 1 ) 20.90 22.99 110% 7.52 35.89 B (mgL 1 ) 49.69 17.68 36% 26.78 72.59 Mo (mgL 1 ) 7.38 6.30 85% 1.57 15.75 Al (mgL 1 ) 45.45 41.49 91% 11.75 106.20 Geranium (hybrid, non zonal) Pelargonium Samples: 30 Locations: 7 N (%) 3.55 0.79 22% 2.60 4.76 P (%) 0.52 0.10 19% 0.39 0.68 K (%) 2.96 0.42 14% 2.58 3.42 Ca (%) 1.11 0.24 22% 0.83 1.44 Mg (%) 0.25 0.05 20% 0.18 0.31 S (%) 0.15 0.04 25% 0.10 0.20 Fe (mgL 1 ) 104.56 44.37 42% 63.45 156.97 Mn (mgL 1 ) 190.17 89.3 5 47% 74.99 308.59 Zn (mgL 1 ) 39.28 13.20 34% 24.90 53.99 Cu (mgL 1 ) 9.17 4.04 44% 5.74 12.54 B (mgL 1 ) 40.98 13.52 33% 24.94 58.52 Mo (mgL 1 ) 3.36 1.58 47% 1.74 5.64 Al (mgL 1 ) 99.89 72.93 73% 30.62 226.96

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58 Table 3 2. Continued Nutrient Mean S td. Dev Coeff. Var. Survey Range Gerananium (ivy) Pelargonium peltatum Samples: 66 Locations: 7 N (%) 3.54 0.62 17% 2.97 4.09 P (%) 0.41 0.10 24% 0.30 0.57 K (%) 3.31 0.38 12% 2.80 3.76 Ca (%) 1.18 0.27 23% 0.90 1.55 Mg (%) 0.33 0.08 24% 0.24 0.38 S (%) 0.12 0.02 17% 0.10 0.15 Fe (mgL 1 ) 113.64 37.48 33% 71.32 166.96 Mn (mgL 1 ) 254.74 119.43 47% 103.55 444.19 Zn (mgL 1 ) 26.37 9.74 37% 19.56 35.89 Cu (mgL 1 ) 7.31 2.91 40% 4.07 12.49 B (mgL 1 ) 50.20 17.55 35% 25.23 76.16 Mo (mgL 1 ) 7 .98 5.42 68% 3.18 13.87 Al (mgL 1 ) 107.54 58.45 54% 33.83 194.31 Geranium (zonal) Pelargonium hortorum Samples: 235 Locations: 11 N (%) 3.55 0.81 23% 2.71 4.78 P (%) 0.41 0.11 28% 0.28 0.56 K (%) 2.76 0.57 21% 2.17 3.49 Ca (%) 1.15 0.30 26% 0.79 1.52 Mg (%) 0.32 0.09 27% 0.23 0.42 S (%) 0.15 0.07 43% 0.11 0.19 Fe (mgL 1 ) 151.57 70.68 47% 78.34 252.23 Mn (mgL 1 ) 302.64 152.58 50% 113.83 539.97 Zn (mgL 1 ) 55.89 25.06 45% 27.54 88.64 Cu (mgL 1 ) 10.10 8.14 81% 5.41 16.27 B (mgL 1 ) 40. 20 15.26 38% 25.31 56.24 Mo (mgL 1 ) 4.15 2.46 59% 2.12 6.63 Al (mgL 1 ) 167.96 107.17 64% 59.11 338.68 Helichrysum Helichrysum Samples: 14 Locations: 5 N (%) 3.65 0.84 23% 2.40 4.45 P (%) 0.35 0.13 36% 0.23 0.61 K (%) 3.29 1.10 33% 2.40 5.0 6 Ca (%) 0.84 0.30 36% 0.50 1.26 Mg (%) 0.26 0.16 60% 0.13 0.57 S (%) 0.37 0.22 60% 0.19 0.68 Fe (mgL 1 ) 175.07 106.89 61% 78.96 355.70 Mn (mgL 1 ) 168.15 152.40 91% 50.78 274.00 Zn (mgL 1 ) 69.49 45.31 65% 41.89 105.40 Cu (mgL 1 ) 9.36 6.34 68% 3. 82 19.42 B (mgL 1 ) 53.46 26.69 50% 25.00 91.01 Mo (mgL 1 ) 4.31 4.54 105% 0.73 10.00 Al (mgL 1 ) 180.85 279.54 155% 32.06 299.77

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59 Table 3 2. Continued Nutrient Mean Std. Dev Survey Range Heliotropium Heliotropium arborescens Samples: 84 Loc ations: 8 N (%) 4.50 0.71 16% 3.53 5.36 P (%) 0.71 0.18 25% 0.52 0.90 K (%) 3.10 0.74 24% 2.01 4.00 Ca (%) 2.50 0.71 28% 1.70 3.10 Mg (%) 0.68 0.25 37% 0.43 1.06 S (%) 0.69 0.28 41% 0.42 1.16 Fe (mgL 1 ) 155.57 91.28 59% 74.77 272.00 Mn (mgL 1 ) 13 8.68 81.18 59% 42.57 248.00 Zn (mgL 1 ) 78.65 32.01 41% 37.16 111.00 Cu (mgL 1 ) 22.05 16.93 77% 5.55 42.03 B (mgL 1 ) 66.60 18.60 28% 47.36 94.89 Mo (mgL 1 ) 4.13 3.82 92% 1.28 7.61 Al (mgL 1 ) 60.33 43.62 72% 21.88 122.40 Ipomea Ipomea batatas Samples: 61 Locations: 5 N (%) 5.11 0.78 15% 4.07 6.00 P (%) 0.72 0.20 28% 0.44 0.90 K (%) 4.17 0.50 12% 3.43 4.80 Ca (%) 1.10 0.38 35% 0.62 1.52 Mg (%) 0.41 0.15 36% 0.27 0.57 S (%) 0.67 0.31 46% 0.28 1.06 Fe (mgL 1 ) 103.33 31.23 30% 67.18 136.00 Mn (mgL 1 ) 118.87 66.17 56% 56.00 208.00 Zn (mgL 1 ) 55.08 19.17 35% 33.15 85.41 Cu (mgL 1 ) 16.20 19.20 119% 3.66 42.00 B (mgL 1 ) 51.99 16.80 32% 36.00 71.76 Mo (mgL 1 ) 5.20 3.82 73% 1.54 10.02 Al (mgL 1 ) 65.47 60.83 93% 15.52 151.00 Lam ium Lamium maculatum Samples: 19 Locations: 3 N (%) 4.78 0.98 21% 3.52 6.09 P (%) 0.49 0.14 28% 0.31 0.70 K (%) 3.65 0.84 23% 2.69 4.84 Ca (%) 0.98 0.33 34% 0.68 1.42 Mg (%) 0.58 0.21 37% 0.20 0.81 S (%) 0.43 0.15 35% 0.20 0.61 Fe (mgL 1 ) 76.89 36.10 47% 48.96 102.73 Mn (mgL 1 ) 103.31 82.90 80% 24.89 271.30 Zn (mgL 1 ) 41.15 22.04 54% 19.68 91.83 Cu (mgL 1 ) 7.81 3.41 44% 3.99 14.95 B (mgL 1 ) 47.30 18.02 38% 21.24 72.44 Mo (mgL 1 ) 7.22 5.29 73% 0.92 15.91 Al (mgL 1 ) 50.45 54.32 108% 19.53 88.82

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60 Table 3 2. Continued Nutrient Mean Std. Dev Coeff. Var. Survey Range Lantana Lantana camara and L. montevidensis Samples: 65 Locations: 11 N (%) 4.35 0.72 16% 3.60 5.20 P (%) 0.55 0.17 30% 0.39 0.72 K (%) 3.05 0.54 18% 2.60 3 .56 Ca (%) 1.18 0.26 22% 0.87 1.49 Mg (%) 0.49 0.14 28% 0.32 0.69 S (%) 0.41 0.34 81% 0.19 0.70 Fe (mgL 1 ) 109.50 53.14 49% 62.36 158.46 Mn (mgL 1 ) 126.50 80.73 64% 42.39 231.28 Zn (mgL 1 ) 55.29 17.39 31% 34.25 76.50 Cu (mgL 1 ) 10.75 5.55 52% 5. 44 20.89 B (mgL 1 ) 60.45 22.17 37% 36.83 88.58 Mo (mgL 1 ) 2.62 1.61 61% 1.12 5.49 Al (mgL 1 ) 70.21 90.86 129% 15.96 148.87 Lavandula Lavandula angustifolia, L. dentata, and L. stoechas Samples: 16 Locations: 7 N (%) 3.40 0.63 18% 2.54 4.10 P (%) 0.38 0.13 33% 0.20 0.50 K (%) 3.43 0.68 20% 2.40 4.40 Ca (%) 0.78 0.27 35% 0.49 1.10 Mg (%) 0.30 0.07 22% 0.23 0.41 S (%) 0.21 0.10 46% 0.11 0.31 Fe (mgL 1 ) 108.14 59.08 55% 46.58 222.55 Mn (mgL 1 ) 122.88 116.69 95% 30.20 196.23 Zn (mgL 1 ) 5 6.29 38.76 69% 23.13 129.63 Cu (mgL 1 ) 11.44 8.41 74% 5.16 17.88 B (mgL 1 ) 41.95 22.60 54% 19.60 83.19 Mo (mgL 1 ) 1.63 1.30 80% 0.65 3.48 Al (mgL 1 ) 110.09 96.64 88% 21.14 286.69 Leucanthemum Leucanthemum superbum Samples: 14 Locations: 5 N (%) 4.78 0.81 17% 4.10 6.10 P (%) 0.79 0.26 33% 0.41 1.20 K (%) 5.84 1.39 24% 4.34 7.90 Ca (%) 1.13 0.55 49% 0.50 1.80 Mg (%) 0.35 0.15 41% 0.18 0.60 S (%) 0.48 0.35 74% 0.13 1.05 Fe (mgL 1 ) 85.28 30.87 36% 64.07 128.17 Mn (mgL 1 ) 103.39 42.2 4 41% 48.27 159.90 Zn (mgL 1 ) 55.09 18.71 34% 34.44 81.89 Cu (mgL 1 ) 9.34 3.50 37% 5.11 12.47 B (mgL 1 ) 51.01 20.19 40% 31.49 87.88 Mo (mgL 1 ) 4.21 2.87 68% 1.39 7.72 Al (mgL 1 ) 55.38 39.29 71% 25.36 113.74

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61 Table 3 2. Continued Nutrient Mean S td. Dev Coeff. Var. Survey Range Lobelia Lobelia erinus Samples: 22 Locations: 5 N (%) 4.69 0.42 9% 4.34 5.21 P (%) 0.62 0.16 26% 0.41 0.78 K (%) 4.74 0.75 16% 3.96 5.62 Ca (%) 0.88 0.18 21% 0.67 1.10 Mg (%) 0.31 0.10 31% 0.22 0.47 S (%) 0. 30 0.06 21% 0.22 0.37 Fe (mgL 1 ) 170.11 99.30 58% 85.58 278.82 Mn (mgL 1 ) 105.44 60.55 57% 47.78 150.18 Zn (mgL 1 ) 75.23 48.54 65% 35.43 149.47 Cu (mgL 1 ) 8.53 6.97 82% 3.11 24.00 B (mgL 1 ) 33.56 19.11 57% 19.59 59.81 Mo (mgL 1 ) 3.54 4.38 124% 1.06 5.43 Al (mgL 1 ) 196.83 206.06 105% 52.54 504.80 Lysimachia Lysimachia congestiflora and L. nummularia Samples: 27 Locations: 3 N (%) 4.23 0.52 12% 3.70 5.10 P (%) 0.45 0.12 26% 0.29 0.60 K (%) 3.35 0.93 28% 2.49 4.60 Ca (%) 0.53 0.58 109% 0.28 0.79 Mg (%) 0.22 0.18 85% 0.11 0.32 S (%) 0.40 0.21 53% 0.22 0.76 Fe (mgL 1 ) 95.86 38.84 41% 65.88 172.90 Mn (mgL 1 ) 56.67 56.97 101% 13.86 115.60 Zn (mgL 1 ) 42.44 41.39 98% 18.67 74.40 Cu (mgL 1 ) 5.70 4.26 75% 1.70 12.08 B (mgL 1 ) 38.50 16.98 44% 19.92 70.24 Mo (mgL 1 ) 8.12 6.03 74% 1.65 16.22 Al (mgL 1 ) 56.90 44.70 79% 24.49 159.80 Mecardonia Mecardonia Samples: 10 Locations: 3 N (%) 3.77 0.60 16% 2.88 4.45 P (%) 0.42 0.10 25% 0.27 0.55 K (%) 2.78 0.72 26% 1.97 3.89 Ca (%) 0.94 1.07 113% 0.28 2.92 Mg (%) 0.37 0.22 60% 0.19 0.75 S (%) 0.85 0.36 42% 0.40 1.37 Fe (mgL 1 ) 93.80 66.71 71% 19.58 187.03 Mn (mgL 1 ) 80.06 36.37 45% 43.38 132.20 Zn (mgL 1 ) 75.77 63.16 83% 34.35 186.20 Cu (mgL 1 ) 16.90 18.01 107% 2.40 47. 87 B (mgL 1 ) 43.42 18.33 42% 14.51 66.20 Mo (mgL 1 ) 8.32 7.36 88% 2.00 21.17 Al (mgL 1 ) 69.32 64.88 94% 16.96 167.75

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62 Table 3 2. Continued Nutrient Mean Std. Dev Coeff. Var. Survey Range Penstemon Penstemon Samples: 17 Locations: 5 N (%) 3.82 0.75 20% 3.10 5.40 P (%) 0.51 0.09 17% 0.40 0.57 K (%) 2.72 0.69 25% 1.97 3.90 Ca (%) 0.75 0.24 32% 0.43 1.00 Mg (%) 0.36 0.09 26% 0.25 0.46 S (%) 0.50 0.54 108% 0.19 0.67 Fe (mgL 1 ) 94.33 67.45 71% 44.45 153.46 Mn (mgL 1 ) 70.19 47.79 68% 21 .54 155.70 Zn (mgL 1 ) 39.84 13.76 35% 27.88 67.14 Cu (mgL 1 ) 5.12 3.09 60% 2.04 10.95 B (mgL 1 ) 40.49 8.59 21% 26.95 54.00 Mo (mgL 1 ) 1.77 1.03 58% 0.70 3.60 Al (mgL 1 ) 33.61 23.51 70% 14.49 83.08 Perovskia Perovskia atriplicifolia Samples: 13 Locations: 3 N (%) 3.59 0.93 26% 2.20 4.57 P (%) 0.46 0.12 26% 0.27 0.54 K (%) 3.30 0.83 25% 2.05 4.14 Ca (%) 0.62 0.17 27% 0.44 0.79 Mg (%) 0.19 0.05 26% 0.14 0.25 S (%) 0.35 0.17 47% 0.17 0.62 Fe (mgL 1 ) 89.66 42.79 48% 43.60 136.89 Mn (mgL 1 ) 62.62 61.62 98% 21.85 85.96 Zn (mgL 1 ) 48.96 39.56 81% 27.07 75.87 Cu (mgL 1 ) 6.16 3.69 60% 2.92 12.66 B (mgL 1 ) 48.52 16.12 33% 31.85 71.61 Mo (mgL 1 ) 2.31 1.56 68% 0.92 4.12 Al (mgL 1 ) 116.80 218.59 187% 14.79 117.11 Phlox Phlox paniculata Samples: 108 Locations: 6 N (%) 4.98 0.90 18% 3.70 5.93 P (%) 0.63 0.16 25% 0.40 0.79 K (%) 3.58 0.66 18% 2.80 4.35 Ca (%) 1.09 0.45 41% 0.77 1.40 Mg (%) 0.35 0.14 40% 0.19 0.53 S (%) 0.87 0.46 52% 0.38 1.39 Fe (mgL 1 ) 171.77 120.6 0 70% 51.95 332.60 Mn (mgL 1 ) 101.24 81.40 80% 36.15 169.50 Zn (mgL 1 ) 73.80 52.21 71% 36.26 147.02 Cu (mgL 1 ) 8.04 3.24 40% 3.78 12.20 B (mgL 1 ) 63.56 33.72 53% 31.92 104.80 Mo (mgL 1 ) 3.77 3.18 85% 1.10 8.86 Al (mgL 1 ) 61.37 75.23 123% 13.92 143.20

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63 Table 3 2. Continued Nutrient Mean Std. Dev Coeff. Var. Survey Range Portulaca Portulaca oleracea Samples: 12 Locations: 2 N (%) 3.94 1.26 32% 1.68 5.35 P (%) 0.68 0.39 57% 0.24 1.14 K (%) 6.18 3.32 54% 2.64 11.50 Ca (%) 0.99 0.47 4 7% 0.54 1.58 Mg (%) 1.34 0.75 56% 0.27 2.35 S (%) 0.52 0.32 60% 0.18 1.15 Fe (mgL 1 ) 101.86 37.45 37% 74.50 160.90 Mn (mgL 1 ) 315.36 181.76 58% 107.50 558.70 Zn (mgL 1 ) 81.24 49.69 61% 26.94 155.00 Cu (mgL 1 ) 13.39 9.16 68% 2.55 24.00 B (mgL 1 ) 58.76 22.07 38% 43.36 73.94 Mo (mgL 1 ) 4.04 4.41 109% 1.30 14.39 Al (mgL 1 ) 77.16 89.55 116% 16.36 294.50 Scaevola Scaevola aemula Samples: 81 Locations: 6 N (%) 4.56 0.74 16% 3.68 5.58 P (%) 0.57 0.22 39% 0.36 0.87 K (%) 3.72 0.65 18% 3.05 4.62 Ca (%) 1.77 0.43 24% 1.30 2.20 Mg (%) 0.44 0.17 39% 0.23 0.68 S (%) 0.91 0.52 57% 0.30 1.58 Fe (mgL 1 ) 93.54 49.14 53% 61.99 126.71 Mn (mgL 1 ) 107.76 55.47 51% 57.80 194.81 Zn (mgL 1 ) 42.41 16.58 39% 25.58 65.48 Cu (mgL 1 ) 6.95 7.29 10 5% 2.28 13.32 B (mgL 1 ) 46.32 21.85 47% 31.64 61.32 Mo (mgL 1 ) 5.19 4.87 94% 1.42 10.08 Al (mgL 1 ) 60.85 62.37 103% 16.77 108.80 Sedum Sedum Samples: 12 Locations: 3 N (%) 4.94 0.62 13% 4.20 5.70 P (%) 0.51 0.10 19% 0.40 0.60 K (%) 4. 01 0.75 19% 3.30 5.10 Ca (%) 3.29 0.84 26% 2.40 4.12 Mg (%) 0.41 0.11 27% 0.30 0.52 S (%) 0.42 0.30 71% 0.19 0.80 Fe (mgL 1 ) 78.45 42.77 55% 43.83 106.90 Mn (mgL 1 ) 53.23 27.81 52% 15.51 85.28 Zn (mgL 1 ) 67.76 30.66 45% 38.71 95.13 Cu (mgL 1 ) 8. 98 3.60 40% 5.25 14.49 B (mgL 1 ) 44.80 22.07 49% 25.66 76.84 Mo (mgL 1 ) 7.97 6.50 82% 3.80 18.01 Al (mgL 1 ) 61.91 72.85 118% 16.86 108.48

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64 Table 3 2. Continued Nutrient Mean Std. Dev Coeff. Var. Survey Range Thunbergia Thunbergia Samples: 50 Locations: 2 N (%) 5.16 0.71 14% 4.30 6.10 P (%) 0.84 0.20 23% 0.61 1.06 K (%) 4.09 0.78 19% 3.11 4.91 Ca (%) 0.67 0.31 46% 0.33 1.06 Mg (%) 0.32 0.06 18% 0.26 0.37 S (%) 0.25 0.04 16% 0.21 0.29 Fe (mgL 1 ) 113.62 19.19 17% 91.73 134.72 Mn (mgL 1 ) 130.08 101.10 78% 64.89 270.95 Zn (mgL 1 ) 89.67 23.18 26% 60.76 111.82 Cu (mgL 1 ) 13.34 2.74 21% 9.53 16.73 B (mgL 1 ) 39.22 13.26 34% 27.52 62.34 Mo (mgL 1 ) 2.09 1.57 75% 0.99 3.01 Al (mgL 1 ) 37.51 26.05 69% 15.10 76.87 Torenia Torenia fournieri Samples: 86 Locations: 6 N (%) 3.87 0.65 17% 3.00 4.80 P (%) 0.53 0.15 28% 0.37 0.70 K (%) 2.66 0.89 33% 1.86 3.42 Ca (%) 0.45 0.34 75% 0.26 0.61 Mg (%) 0.38 0.16 44% 0.20 0.60 S (%) 0.37 0.17 45% 0.16 0.61 Fe (mgL 1 ) 130.74 53.16 4 1% 78.18 196.50 Mn (mgL 1 ) 129.45 74.42 57% 58.79 226.60 Zn (mgL 1 ) 54.48 24.70 45% 32.51 82.88 Cu (mgL 1 ) 12.46 11.15 89% 5.72 18.05 B (mgL 1 ) 63.52 22.74 36% 36.35 98.01 Mo (mgL 1 ) 4.01 2.29 57% 1.50 7.42 Al (mgL 1 ) 70.18 77.98 111% 18.03 126 .64 Verbena Verbena canadensis, V. X hybrida, V. rigida, V. tenera, and V. tenuisecta Samples: 249 Locations: 9 N (%) 5.04 0.90 18% 3.91 6.20 P (%) 0.56 0.13 23% 0.40 0.71 K (%) 3.10 0.67 22% 2.33 3.92 Ca (%) 1.39 0.49 35% 0.88 1.98 Mg (%) 0.51 0.17 34% 0.33 0.78 S (%) 0.73 0.36 49% 0.29 1.17 Fe (mgL 1 ) 96.08 40.83 42% 59.56 145.20 Mn (mgL 1 ) 90.23 46.05 51% 40.70 144.80 Zn (mgL 1 ) 48.59 21.09 43% 29.35 77.27 Cu (mgL 1 ) 9.42 7.42 79% 4.32 12.80 B (mgL 1 ) 54.54 24.19 44% 29.63 86.14 M o (mgL 1 ) 5.17 4.07 79% 1.72 10.51 Al (mgL 1 ) 59.03 57.81 98% 16.16 117.20

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65 CHAPTER 4 TIMING OF MACRONUTRIENT SUPPLY DURING CUTTING PROPAGATION OF PETUNIA Introduction A ppropriate timing and concentration of nutrient supply in vegetative cutting prop agation affects root development, uniform ity of plant growth uptake efficiency, nutrient runoff and transplant success. Root development can be divided into 4 stages, (1) cutting condition at insertion into substrate (2) callus formation, (3) root devel opm ent, and (4) toning (Dole and Gibson 2006). Fertigation recommendations for vegetative cuttings are currently correlated with root developmental stages, an initial application of 50 75 mg NL 1 is recommended at visible callus development (stage 2) an d subsequent applications of 100 mg NL 1 at root emergence (stage 3) (Dole and Gibson, 2006). N utrients can be supplied to vegetative cuttings through a combination of preplant nutrient charge in the substrate supplemental application of water soluble f ertilizer and/or incorporation of controlled release fertilizers. Historically, fertilization through overhead emitters was not recommended for short term crops, due to the potential for clogged emitte rs and algae growth (Dole and Gibson 2006). The conce ntration of fertilizer solutions applied during the propagation of vegetative petunia and calibrachoa cuttings used in commercial greenhouses rang ed from 0.5 to 80 and 64 to 158 mg L 1 N for 7 and 28 days after insertion into substrate respectively (Santos et al., 2008). Timing of fertigation varied from constant application of N for 28 days to application of N 14 days after insertion (Santos et al., 2008). The observed practices at these operations suggest that the current recommended fertilization strat egies may need to be refined with further consideration of fertilizer timing and nitrogen concentration. High leaching volumes resulting from mist irrigation is intended to maintain cutting turgidity during root development and often water is supplied in excess of uptake and container

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66 capacity resulting in the potential for rapid leaching of fertilizer (Kerr and Hanan, 1985 ; Mudge et al., 1995, Biernbaum and Argo 1996; Santos et al., 2008 ). Propagators surveyed leached as much as 46 Lm 2 in a 4 week cro p cycle and six of eight operations leached over one container capacity (CC) during the same period (Santos et al., 2008). Mist application during propagation is significant because soilless substrates have a limited ability to retain nutrients, especiall y when total leaching rates are greater than 1 CC (Biernbaum et al, 1995; Kerr and Hanan, 1985). The loss of nutrients from leaching during stages 1 and 2 of root development, could render the substrate without a pre plant charge by stage 3 (root emergenc e) a critical stage for nutrient replenishment for the cutting. Therefore, commercial fertilizer application early in propagation before root formation may be required to recharge leached nutrients in the substrate. Water soluble fertilizer applications prior to root emergence may also facilitate foliar uptake of nutrients (Tukey, 1958) to reduce observed tissue nutrient declines during preliminary phases of propagation regardless (Svenson and Davies, 1995; Wilkerson and Gates, 2005) Proper water manageme nt during propagation should provide enough water to minimize transpiration from the cuttings until root formation, then water supply should be reduced because it is divided between the substrate (from the roots) and overhead application (micro climate, hu midity). Growers that use constant cycle mist timers that do not account for changes in light and temperature, Matching timing of fertilizer supply to plant requirement has potential to increase uptake efficiency and reduce nutrient runoff. Macronutrie nts, in absolute terms, are required in higher quantities compared to micronutrients, therefore plant response to macronutrient supply is of particular interest when uptake capabilities are minimized, especially during phases 1 and 2 of rooting. The objec tive s of this research were to evaluate the efficacy of fertilizer applications

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67 applied directly through the mist during the first seven days of propagation and to quantify the effect of macronutrient supply on growth and nutrient uptake of petunia cutting s at (0 7), (7 14) and (14 21) days after insertion Materials and Methods T he experiment was conducted at the University of Florida, Environmental Horticulture Research Greenhouse Complex in Gainesville FL Greenhouse and irrigation management were co ntrolled by an environmental control system (Hortimax, Pijnacker, Netherlands). Unrooted cuttings of Petunia x hybrida Supertunia Royal Velvet were air freighted over two days from an off shore vegetative cutting supplier, InnovaPlant in Costa Rica Cuttings were immediately inserted into 21 x 50 cm, 102 count (19.6 mlcell 1 ) propagation trays filled with 70% peat/30% perlite (by volume) substrate containing 2.1 kgm 3 hydrated lime, and 0.14 Lm 3 wetting agent (AquaGro 2000 M, Aquatrols, New Jersey) Substrate container capacity was 2.3 L tray 1 The experimental unit was defined as an individual (21 x 50 cm 102 count) propagation tray. Mist was provided by bench risers fitte d with 1.2 L m in 1 JetRain nozzles (Dramm; Manitowac, WI) spaced 91.4 cm apart and alternated 30.5 cm and 40.6 cm above the crop until roots reached the side and bottom of each cell (14 days after insertion ). M ist frequency was adjusted according to light, temperature and root development stages. Average light levels were 216 .4 mmolm 2 s 1 per day with average temperatures of 23 C day and 19 C night. Prior to significant root growth (root length less than 3 cm) the mist frequency was every 17 to 28 minutes for 5 seconds triggered by an accumulated light threshold of 200 mmol m 2 s 1 of PAR light. After root length was greater than 3 cm, the light threshold was increased to 500 mmolm 2 s 1 resulting in a mist frequency of every 50 60 minutes for 5 seconds soluble fertilizer contai ning (in mg/L) 75 NO 3 N, 25 NH 4 N, 12 P, 83 K, 20 Ca, 10 Mg 1.4 S, 2 Fe, 1 Mn, 1 Zn, 0.5 Cu, 0.5 B, and 0.2 Mo

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68 was supplied over three weeks (27 Mar 2007 to 18 Apr 2007). Fertilizer salts used were ammonium nitrate, ammonium phosphate, calcium nitrate, m agnesium nitrate, potassium nitrate, boric acid, copper sulfate, iron EDDHA, manganese sulfate, sodium molybdate and zinc sulfate. For days 7, 14, and/or 21, as shown in Table 4 1, the alternative fertilizer treatment applied contained only the micronutri ent component of the Complete fertilizer and was considered the control Clear water was not used as the control because the objective was to evaluating the effect macronutrient fertilizer supply had on growth and tissue nutrient trends in petunia. After 14 days, cuttings were irrigated with the fertilizer solutions (based on need) twice a week by hand using a hose fitted with a 1000PL water breaker (Dramm; Manitowac, WI) for the remaining 7 days. Fertilizer solutions were pumped from separate 378.6 L st ock tanks beneath each bench. Irrigation volume applied was 4.0 and 2.3 L for 7 and 14 21 days after insertion respectively, resulting in total leaching of 1.1 container capacities over three weeks Average volume leached was 1.2 and 0.7 L experimental u nit 1 7 and 14 21 days after insertion respectively. The experiment was divided into four blocks each consisting of the eight treatments described in Table 4 1. At the start of the experiment each block contained 6 replicate trays per treatment combin ation. Two experimental units (trays) per treatment were collected from each block on each measurement date and destructively sampled n=8. Data were collected every 7 days for 21 days after sticking, equivalent to a typical petunia production cycle (Dole et al., 2006) Data were analyzed using Proc GLM in SAS (version 9.1; SAS Institute, Cary, NC) as a randomized complete block design, with each block consisting of a treatment and treatments with varying intervals of micronutrient only treatm ent s. M ean s were analyzed using

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69 Data collected from each experimental unit included SPAD chlorophyll index (Spectrum Technologies Minolta SPAD 502; Plainfield, Illinois), number of leaves per cutt ing, plant height, longest root length, rooting stage, dry weight and shoot and root tissue samples for nutrient analysis. R ooting stage for each treatment was determined using a rooting scale defined by Dole and Gibson (2006) whereby the rooting of veget ative cuttings was divided into four stages: stage 1, with the stuck cutting through to the development of a swollen base, stage 2, with callus development through to root initial protrusion, stage 3, with significant root and shoot growth, and stage 4, to ning stage prior to transplant ( Dole and Gibson, 2006 ). Tissue nitrogen concentration was measured as total Kjeldahl N where all the protein was converted to NH 4 using heat, a catalyst, sulfuric acid, and hydrogen peroxide. The sample was then run on a sp ectrophotometer (DR 4000; Hach, Loveland, CO) using Nesslerization forN determination. A Hach Digestahl apparatus was used for the conversion (digestion) and a spectrophotometer (DR 4000) for the analysis. Analysis of Ca, Mg, P, K, Na, Mn, Cu, Fe, Zn, B, and Mo was accomplished by dry ashing digestion followed by Inductively Coupled Plasma (ICP) emission spectrometry by Quality Analytical Laboratories (Panama City, FL.) using a Thermo Jarrell Ash ICAP 61E Samples were either the total cuttings 7 days a fter insertion in order to provide sufficient tissue for complete nutrient analysis, or separated roots and shoots 14 and 21 days after insertion Cutting nutrient content was calculated by multiplying cutting dry weight by the % N, P, or K respectively and nutrient uptake was calculated by subtracting g of N, P, or K on day 0 from the measurement date (g of N, P, or K on day 7, 14, or 21). Two additional experimental units per bench were randomly selected every 7 days (April 4, 7, and 18, 2007) for subst rate and leachate nutrient sampling. The plug press method was

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70 used to sample substrate by pressing down firmly on the top of the substrate surface and the solution was collected from the hole at the bottom of the pressed plug cell (Scoggins et al., 2002) Solution leached from the propagation trays was measured in collection trays following the protocol described by Santos et al., (2008). Solutions were submitted for nutrient analysis using inductively coupled plasma atomic emission spectrophotometry ( ICAP 61E; Thermo Jarrell Ash, Franklin, MA) to measure P, K, Ca, Mg, S, Fe, Mn, B, Cu, Zn, Mo, Al, and Na. The leachate and substrate solution samples were analyzed for NO 3 N and ammonium (NH 4 ) N using a Lachat QuikChem AE (Lachat Instruments, Loveland, C O). This instrument uses flow injection analysis to colorimetrically determine NO 3 N and NH 4 N concentration. Results and Discussion Shoot and Root G rowth There was a 100% survival in all treatments. There was a significant interaction between time and fe rtilizer treatment (main effect) with regards to shoot, root and combined root and shoot growth. Fertilization in the first 7 days of production, corresponding with root initiation and initial root emergence increased total dry weight 21 days after insert ion compared to fertilization with micronutrients only during the first 7 days. The combined root and shoot dry weights (Figure 4 1A) for treatments receiving less than complete fertilizer during days 0 to 21 did not differ from the Complete dry weight unt il 21 days after sticking, at which point treatments that received Complete fertilizer during the first 7 days (CMM, CMC, CCM, and CCC) were consistently higher in total dry weight than plants receiving micronutrients during those first 7 days (MMM, MMC, M CM, and MCC) (Figure 4 1A). Application of Complete fertilizer during the first seven days of production had a carryover effect that resulted in increased dry weight 21 days after insertion compared to plants that received micronutrients only 7 days after insertion

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71 Under constant Complete (C) fertilizer application, the combined shoot and root dry weight increased significantly by day 7 from 0.020 to 0.047 gcutting 1 Continued fertigation with the Complete fertilizer resulted in increased shoot dry wei ght compared with interrupted macronutrient supply ( Figure 4 1 B ). Shoot dry weight was greater in treatments that received Complete for 14 days (0.064 gcutting 1 ) compared to that of treatments that only received micronutrients during the first week foll owed by Complete the second (0.051 gcutting 1 ). The lowest shoot dry weights at 21 days after insertion were found in plants from treatments that received micronutrients for the first 14 days, followed by either micronutrients or Complete fertilizer in w eek 3 (0.069 or 0.065 gcutting 1 ) (MMM or MMC), respectively. Continued application of Complete fertilizer at 14 and 21 days after insertion (CC or CCC) resulted in reduced root dry weight compared to those plants with interrupted macronutrient supply (MM or CMM) (Figure 4 1C). Fertilization did not affect rooting stage (1, 2, 3, or 4) (data not shown) or longest root length between treatments. Initial root emergence was observed on day 4, and by day 7 primary roots averaged 2.6 cm in length. Root lengt h increased to 2.6, 12.2, and 17.9 cmcutting 1 by 7, 14, or 21 days after insertion respectively. Root dry weight at day 14 was lower in plants that received Complete fertilizer for 2 weeks (0.014 gcutting 1 ) (CC) compared to that of plants that receiv ed only micronutrients (0.019 gcutting 1 ) (MM). However, by 21 days there was a positive effect of early Complete fertilizer applications on root dry weight (CMM). Root dry weight was greater after 21 days for plants that received the Complete fertilize r in the first week, followed by interrupted supply (CMM) compared with plants that received only micronutrients during the first 7 days or Complete fertilizer for the entire 21 day period. Decrease in rooting with increased fertilizer rates was attribute d to increased shoot growth and decreased tissue maturation in holly (Rein et al., 1991).

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72 For commercial production of rooted cuttings, a low S:R ratio is desirable to reduce shipping costs, the need for chemical growth regulator application, and crop prod uction time required to produce a well rooted, compact plant. Increasing the duration of Complete fertilization increased the shoot:root ratio (S:R). The S:R ratio at day 14 (Figure 4 1D) was higher in plants that received constant Complete fertilizer (4 .5), (CC) compared to plants that received the other seven treatments (which averaged 3.2). The S:R ratio continued to be highest at 21 days in the CCC treatment (4.1), compared to an average of 2.4 for the other seven treatments. Constant Complete fer tilizer application increased shoot height and leaf number. Plant height averaged 1.7 cm to 2.1 cm/cutting on day 0 and day 7, regardless of fertilizer treatment. At 14 days, the cuttings that received the CC treatment were taller than (4.5 cm) the cutti ngs that received the MM treatment (3.5 cm). At 21 days, cuttings that received constant Complete fertilizer (CCC) were the tallest of all treatments (8.3 cm) and cuttings that received micronutrients for 21 days were the shortest (3.6 cm). Treatments ha d only minor effects on SPAD chlorophyll index 14 days after insertion SPAD were higher in plants that received Complete fertilizer for 7 days followed by micronutrients for 7 days (CM) (32.7) compared to plants that received micronutrients for 7 days fo llowed by Complete for 7 days (MC) (28.7). Constant Complete fertilizer (CC) resulted in increased leaf number by day 14. Treatments with the highest leaf number (8.1 leavescutting 1 ) received Complete fertilizer during the first 14 days, whereas treatme nts receiving only micronutrients had the lowest leaf counts (6.5 leavescutting 1 ). At 21 days, highest leaf counts were observed in plants receiving CCC or CCM treatments (10.7 or 10.4 leavescutting 1 ). The lowest leaf counts were observed in plants re ceiving the MMM (7.0 leavescutting 1 ) or MMC (7.2 leavescutting 1 ) treatments.

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73 Nutrient Concentration Tissue nutrient concentration decreased from day 0 to 7 in plants from all fertilizer treatments (Figure 4 2A). There was a significant interaction be tween time and fertilizer treatment (main effect) with regards to N, P and K tissue concentrations. During the first week of propagation using the Complete fer tilizer, tissue N concentration dropped from 6.6% on day 0 to 4. 3 % on day 2 1 (Figure 4 2A) Pe rcent P and K concentration s followed a similar trend to N, with decreasing nutrient concentration s over the first 14 days followed by stable to slightly increasing concentrations (data not shown) A greater decline in tissue nutrient concentration occurre d during the first week when micronutrients only were provided (Figure 4 2A). Cuttings that received micronutrients had 3.7% N, 0.3% P, and 2.7 %K (0.0016, 0.00010, and 0.00 11 g cutting 1 ) at day 7, compared to 4.8% N, 0.5% P, and 3.7% K (0.0020, 0.00020, and 0.0015 g cutting 1 ) with the Complete fertilizer The initial decrease in tissue nutrient concentrations could be attributed to dilution that resulted from an increase in total dry weight in conjunction with minimal uptake of nutrients (Blazich, 1988 ). Nutrients leached from the foliage, a phenonmenon observed by Good and Tukey (196 7), was not a factor in the initial tissue nutrient decline because a net loss of mg Ncutting 1 was not observed during the first 7 days of propagation (Figure 4 2B). Ti ssue nutrient decline regardless of total water volume applied was also observed in poinsettia (Svenson and Davies, 1995; Wilkerson and Gates, 2005). Application of Complete nutrient supply at any week in the crop cycle resulted in increased tissue N, P, o r K concentration at termination compared to micronutrients only (P and K data not shown). Tissue nutrient concentration s increased once Complete application resumed, however regardless of treatment, tissue N, P, and K concentration s did not return to ini tial percent nutrient concentration s suggesting that nutrient concentrations in the stock plant

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74 are very important to avoid nutrient deficiency symptoms in the unrooted cuttings (Rowe and Blazich, 1999). The goal throughout vegetative propagation is to m aintain tissue nutrient concentrations within the recommended ranges to avoid slowed root and shoot development caused by nutrient deficiency. Mills and Jones (1996) and Gibson et al. (2007) observed deficiency symptoms in petunia at 2.1%N, 0.07% P, and 0 .7% K whereas 1.8 % N, 0.2 %P, and 1.5 % K were observed for the MMM treatment at 21 days, indicating that N had dropped to deficient concentrations for that treatment. Mills and Jones (1996) reported minimum sufficiency concentrations of 3.9% N, 0.47% P, and 3.1% K for finished plants and with micronutrient fertilizer (MMM) only the tissue concentrations below these concentrations occurred on days 7, 14, and 21. Nitrogen content (dry weight x tissue concentration, Figure 4 2B ) and also content of P and K (data not shown) increased each week that Complete fertilizer was applied. N uptake was different between fertilizer by day 14 (Figure 4 2C). Weekly N uptake increased over time for plants that received Complete fertilizer. A small amount of N uptake d id occur in plants receiving continuous micronutrients only, presumably from the peat substrate. By week 3, N uptake in CCC treated plants was nearly 10X nutrient uptake of MMM cuttings receiving only micronutrients (MMM) days 0 21 (0.0028 versus 0.0003 9 gcutting 1 ) respectively. The fate of applied nutrients each week (leached, substrate accumulation, or plant uptake, Figure 4 3) showed increased uptake over time. The first week of propagation resulted in the least uptake, 20% uptake efficiency, ((pl ant uptake/nutrients applied)*100) which would be expected because root emergence did not occur until day 4 and uptake would only occur through foliage and the cut stem. In commercial propagation conditions, the highest amount of leaching often occurs dur ing the first 7 days of propagation (Santos et al., 2009) because of the large

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75 water volume applied to maintain cutting turgidity. Uptake efficiency increased to 70% and 80% in weeks 2 and 3, with the combination of a more developed root systems and reduc ed irrigation frequency. Conclusion The timing of macronutrient supply throughout a 21 day propagation cycle was found to affect the growth and development of petunia cuttings. Growth was dependent upon macronutrient supply, with decreasing shoot dry weig ht (Figure 4 1B) and slightly increasing root dry weight (Figure 4 1C) when micronutrients were applied. Continuous application of Complete fertilizer also resulted in more rapid development, as quantified by more leaves per plant, when compared to plants that received on micronutrients indicating that some macronutrient (probably P or K) had reached a minimum critical concentration to limit growth. A high compact heig ht. Therefore, under continuous Complete fertilizer, increased chemical or climate controlled growth regulation would be required to control the increased shoot growth, or a lower constant macronutrient concentration could be applied. In the case of petu rate of fertilizer used in this experiment may have been too high and a reduction in fertilizer rate to 75 mgNL 1 constant might control excessive shoot growth. The results from this experiment were based a particular plant species petunia, response to fertilization during propagation could vary between plant genera. During the first 7 days of propagation regardless of nutrient supply, tissue nutrient concentrations dropped. This response can be attributed to dilution because of g rowth that occurred in the first seven days in conjunction with minimal uptake. Nutrient decline was also observed in poinsettias after 7 days under mist and were uncorrelated to 3 different rates of water volume applied (Wilkerson and Gates, 2005) as wel l as in poinsettias that did not have any water

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76 applied to foliage 13 days after insertion (Svenson and Davies, 1995). The uniform decline in tissue nutrient concentrations during the first 7 days emphasizes the importance of high initial tissue nutrient concentrations in unrooted cuttings on subsequent plant health. If the tissue nutrient concentrations are low in the unrooted cutting, the fertilizer applications need to be started as soon as possible, or a reduction in growth and quality may occur as ti ssue nutrient concentrations drop below recommended ranges and nutrient deficiencies begin to limit growth. Complete fertilizer applications during this initial drop in tissue nutrient concentration were shown to sustain higher tissue nutrient concentrat ions compared to cuttings receiving micronutrients only. The positive response to Complete fertilizer applications early in propagation could be attributed to uptake via foliar, cut stem, or root initials. H igh humidity environments, such as propagation, enable nutrients to stay in solution longer and are more available for foliar uptake ( Dybing and Currier, 1960; Clor et al., 1962; Schonherr, 1972. Relative humidity and leaf water status have been shown to be key factors controlling foliar uptake (Bukov ac and Wittwer, 1959; Tukey and Marczynski, 1984; Schonherr, 2001). Overall, early nutrient supply had positive effects on growth and nutrient concentrations of petunia, but mist fertigation involves tradeoff in terms of increased potential for algae growt h, increased nutrient runoff if the fertigation solution is not recycled (as evidenced by the low uptake efficiency, Figure 4 3), and potential for phytotoxicity or minimal response in certain species. For example, we observed a positive response to mist fertigation for several species including Calibrachoa, Solenostemon, Phlox, Scaevola, Sutera and Bidens, but negative effects on quality of Helichrysum, Lavender, Poinsettia, Spathyphyllum, Anthurium, Guzmania, and ferns, and no response for osteospermum, vinca, woody hydrangea, viburnum, and fothergilla cuttings. Early mist fertigation would therefore be favored in combination with an irrigation

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77 system that includes capture and reuse of leachate in addition to water sanitation for algae control (for examp le, copper ionization or chlorination), for fast growing species where nutrient dilution may rapidly occur, in species that readily absorb nutrients through the foliage (lacking a thick cuticle, and not prone to salt damage), and where initial tissue nutri ent concentrations are low in the unrooted cuttings. Areas that warrant further investigation include the response to early application of macronutrients in plant species other than petunia, and understanding which sites (leaf, cut stem, or callus) are im portant pathways for nutrient uptake or loss before root emergence.

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78 Table 4 1 Fertilization treatments applied to Petunia x hybrida over a 21 day crop cycle. (Note: soluble fertilizer containin g (in mg/L) 75 NO 3 N, 25 NH 4 N, 12 P, 83 K, 20 Ca, 10 Mg, 1.4 S, 2 Fe, 1 only (1.4 S, 2 Fe, 1 Mn, 1 Zn, 0.5 Cu, 0.5 B, and 0.2 Mo) ) Treatment 7 Days 14 Days 21 Days Cod e 1 C C C CCC 2 C C M CCM 3 C M M CMM 4 C M C CMC 5 M M M MMM 6 M M C MMC 7 M C C MCC 8 M C M MCM

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79 Figure 4 1 Measured total dry weight (A), shoot dry weight (B), root dry weight (C), and shoot:root ratio (D) per cutting over time. (Note: Root dry weight was measured when root length was greater than 3 cm, corresponding to 14 days after insertion Root emergence began at day 4, regardless of treatment. Roots were not separated from shoots until 14 days after insertion White bars correspond to each data collection interval and treatment, calculated from the average of eight randomly selected trays cronutrient fertilizer treatment during a given week. Means were separated within each date using )

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80 Figure 4 1.

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81 Figure 4 2. N itrogen content in the combined shoot and root by treatment over time expressed as % of dry weight (A), mg per cutting (B), or nitrogen uptake per week (C). (Note: Nitrogen up take was calculated using the followi ng equation: [(day 7, 14, or 21 DW in g cutting 1 ) (day 7 14 21 tissue % N)] [(preceding week DW in g cutting 1 ) (preceding tissue % N)] where DW = dry weight. C fertilizer treatment and M represents the micronutrien t fertilizer treatment during a given week. Treatment labels are made up of three letters, the first letter corresponds to the fertilizer treatment week one, and so forth. The m ean separation used was )

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82 Figure 4 2.

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83 Figure 4 3 Nitrogen, P, and K in leachate, substrate and tissue over time for plants receiving continuous Complete fertilizer. (Note: Each bar represents the average of eight trays, divided by the number of cuttings per tray (102). Letters show mean comparisons across time within either tissue, substrate, or leac P 0.05 level.

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84 Figure 4 3.

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85 CHAPTER 5 EFFECT OF PETUNIA STOCK PLANT NUTRITIONAL STATUS ON FERTILIZER RESPONSE DURING PROPAGATION I ntroduction The value of imported annual and perennial unrooted cuttings was 61,232 million dollars in 2006 that represented approximately 878 units (U.S. Department of Agriculture, 2007). Unrooted cuttings are harvested from stock or donor plants, transp orted for 2 4 days to cutting propagation greenhouses in the United States, and inserted into a substrate in multi cell liner trays to develop roots. Dole and Gibson (2006) divided cutting propagation into four stages (1) cutting condition at insertion ( 2) callus development, (3) root development, and (4) toning the rooted cutting. P etunia is one genus that can be propagated by seed or cutting. Historically, P etunia was the first cultivated bedding plant and today continues to maintain its importance as a commercial ornamental in the horticulture industry and a plant model for dichotomous species (Gerats and Vandebussche, 2005). vegetatively breeding programs began redeveloping multiflora (large flower number on large branched plants) petunia types from wild species and older cultivars such as P. axillaris (Griesback, 2006). New petunia cultivars with increased genetic variability in terms of stress tolerance, plant vigor, trailing growth, flower size and number, and postproduction durability are developed annually ( Dole et al., 2002; Gerats and Vandenbussche, 2005; Griesbach, 2006 ) which could potentially result in variability in fertilizer response. Research with species other than petunia have found trends of either increasing root development with increasing stock plant fertilizer levels, or an optimum tissue nutrient range where excess fertilizer actually decreased cutting perfor mance. G ibson

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86 (2003) found 17% greater cutting yield, and increased root number and shoot dry weight in cuttings taken from New Guinea impatiens ( Impatiens x hawkeri Bull.) and scaevola ( Scaevola aemula R. Br.) stock fertigated with 300 mgNL 1 compared with 100 m g NL 1 applied at each irrigation through drip irrigation. Adventitious root number and root length of Chrysanthemum ( Dendranthema grandiflorum Ramat.) cuttings were positively correlated to the initial total tissue nitrogen concentration ( Druege et al ., 2000 ). As initial N concentration increased from 2 to 7% N, root number in stored cuttings increased from approximately 5 to 15 and root length increased from 1 to 2.5 cm (Druege et al., 2000). Loblolly pine ( Pinus taeda L.) cuttings taken from stock plants with higher mineral nutrient content (9 mg Ncutting 1 vs 3, 5, 6, 8 mg Ncutting 1 ) were also shown to maintain that higher concentration throughout propagation and showed more positive rooting (28 33%) compared to cuttings taken from stock grown u nder lower fertility regimes (17%) (Rowe and Blazich, 1999). However, fertilizer concentrations of 200 mg NL 1 applied 3 times a week in holly ( Ilex crenata Thunb.) and 4 0 mg NL 1 applied weekly to eastern red cedar ( Juniperus virginiana L.) during stoc k plant production resulted in decreased subsequent adventitious rooting compared with lower fertilizer concentrations (100 mg NL 1 and 20 NL 1 respectively) (Rein et al., 1991; Henry et al., 1992). Typical N fertilization recommendations for stock prod uction of herbaceous ornamental crops are between 150 and 250 mg NL 1 applied at each irrigation (Dole et al., 2006). Fertigation strategies for propagation of Petunia vary widely between commercial growers, resulting in a range of resource efficiencies suggesting a need to better understand nutrient uptake processes and fertilizer response, so that management

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87 practices can be developed (Santos et al., 2008). Fertigation of Petunia vegetative cuttings was not recomme nded by Dole and Gibson (2006) until stage 3 (root development) with weekly applications of 100 mg NL 1 alternating between 15 0 15 and 20 10 20, then increased concentration of 150 200 mg NL 1 applied weekly during stage 4 (toning). However, a survey of commercial greenhouse cutting pro pagators of Petunia and Calibrachoa found that fertigation began anywhere from stage 1 to 3 (Santos et al., 2008). Applied N concentrations ranged from 0.4 to 80.2 mg NL 1 during week 1 (stage 1 to 2), 0 to 194.8, 19.2 to 148.0, and 64.0 to 157.6 mg NL 1 during weeks 2, 3 and 4 respectively (Santos et al., 2008). Supplemental application of nutrients to a range of herbaceous and woody cuttings through mist was recommended by Good and Tukey (1967) and fertigation during stage 1 to 2 was shown to increase tissue N, P, and K concentrations in Petunia (Ch. 2 in Santos (2009)). In particular, fertilization during root formation may benefit cuttings with suboptimal initial tissue nutrient concentrations. Commercial stock plant production of unrooted Petunia cuttings can occasionally result in cuttings that have deficient macronutrient levels before propagation occurs. In a survey of tissue nutrient concentrations of 373 commercially produced crops of Petunia unrooted cuttings from 11 locations over 4 years, 4% of samples were below the published recommended minimum for the species of 3.85%N, 19% of samples were below the recommended P (0.47%), and 2% of samples were below the recommended K (3.13%) (Ch. 3 in Santos (2009)). Depletion in tissue nutrient concent ration typically occurs during cutting propagation. T issue concentration s of both macronutrients and micronutrients changed in apical stem cuttings of poinsettia during the root initiation phase ( Euphorbia pulcherrima

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88 Willd. ex. Klotzch ) (Svenson and Davi es, 1995). Specifically, N, P, and K concentrations (%) declined from day 1 to day 13 from 3.4 to 2.85, 0.35 to 0.32, and 1.86 to 1.70% respectively (Svenson and Davies, 1995). Nutrient shortages that arise during adventitious root development have been a ttributed to foliar leaching which has been attributed to processes such as elimination of metabolites by special organs, for example sugars by nectaries, dissimilation or degeneration of plant tissue, or ion exchange between the plant and foliar solution (Tukey, 1970). Additional factors could be dilution or low initial tissue nutrient concentrations (Good and Tukey, 1967). Increase in cutting dry weight prior to root development (stages 1 2) and without supplemental nutrient uptake can result in a dilu tion of the pre existing nutrient concentration in the cutting tissue and explain tissue nutrient decline (Blazich, 1988). Tissue nutrient decline was tings 7 days after insertion with 6.6% N to 4.7% N in cuttings t reated with a complete fertilizer at 100 mg N L 1 to 3.7% N in cuttings rec eiving micronutrients only (Ch.4 in Santos (2009)). Nitrogen uptake was not different from zero in cuttings treated with micronutrients only, however dry weight increased in these cutting from 20 to 43 mg/cutting. Therefore, the observed decline in %N with the micronutrients only fertilizer could be at tributed to dilution of N (Ch. 4 in Santos (2009)). Nitrogen P, and K uptake for cuttings that received a complete fertilizer trea tment was increased compared to initial concentration but was inadequate to maintain tissue N P and K at initial concentrations, although they were maintained at a higher concentration than cuttings that did not receive macronutrient fertilizer (Ch. 4 i n Santos (2009)).

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89 Root development is affected by many factors including light, moisture, carbohydrate content, and concentration of nutrients including N. Nitrogen depletion restricts the growth of all plant organs, and N deficiency during rooting has th e potential to limit root and shoot growth ( Marschner, 1995; Dole and Gibson, 2006; Barker and Pilbeam, 2007 ). Increased root number and root length in Chrysanthemum cuttings at higher initial tissue N concentrations (5 6 %N) was correlated with higher su crose:starch ratios in the leaves, and the rooting response was attributed to increased carbohydrate partitioning towards export to the region of root development (Druege et al., 2000; Rapaka et al. 2005). A significant percentage ( 25 50%) of the carbohy drates produced per day in plant shoots is typically allocated to the roots (Marschner, 1995). Carbohydrate reserves in unrooted cuttings are highly dependent on photosynthesis during the stock plant phase (decline with decreasing photosynthetic p hoton fl ux density) and decline during cutting storage and shipment (Rapaka et al. 2005). High nitrogen concentration (5 6 %N) was shown to increase root development in Chrysanthemum cuttings, independent of carbohydrate levels (Druege et al., 2000). However, inc reased N supply during propagation also has potential to increase the shoot to root dry weight ratio, which is not horticulturally desirable for rooted cuttings (Levin et al., 198 9; Olsthoorn et al., 1991; (Ch.4 in Santos, (2009)). Nutrients other than N h ave also been ascribed roles in root initiation, including P, Ca, Mg, Mn, B and Zn (Anderson, 1986; Blazich 1988; Svenson and Davies, 1995). Due to low phloem mobility, Mn, Fe, Zn, Cu, B, and Mo deficiencies appear first in new growth (Marschner, 1995), e mphasizing the importance of adequate micronutrient supply during stock production to ensure sufficient concentrations in the plant apices when tip

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90 cuttings are taken. Manganese, B, Fe, Cu, Mo, and Zn were all shown to result in increase d concentration ne ar the basal stem of poinsettia cuttings shortly after insertion in substrate suggesting that concentrations of these elements may be important in root initiation or elongation (Svenson and Davies, 1995). Our goal was to investigate the potential interact ion between initial tissue nutrient levels resulting from stock plant fertilization, and subsequent fertilizer response during cutting propagation. The specific objective was to evaluate the effect of initial tissue nutrient concentration on fertilizer re sponse during cutting propagation of Petunia x hybrida concentrations of a c omplete water soluble fertilizer and cuttings were subsequently grown with a complete, N + micronutrients, or mic ronutrients only nutrient solution. Materials and Methods sales (InnovaPlant, personal communicat ion), and this cultivar has been used in other nutritional studies by Santos (2009). The cuttings were inser ted on February 13, 2008 into 10.5 x 50 cm, 51 count propagation trays with 70% peat/30% perlite (by volume) substrate containing 2.1 kg m 3 hydrat ed lime, 0.14 L m 3 wetting agent (AquaGro 2000 M, Aquatrols, New Jersey) a nd no pre plant fertilizer. The plants were placed on benches fitted with an aluminum poly rail bench riser system with JetRain mist nozzles (Dramm; Manitowac, WI) spaced 91.4 cm ap art and were maintained as stock in a greenhouse at the University of Florida, FL, USA. Growing environment and mist frequency were controlled by a greenhouse environmental control system (Hortimax Gemlink Environmental Control System, Rancho Santa Margar ita, CA) and heating/ventilation

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91 set points were 20/22 C during the day and night, providing an average of 21.4C during the night and 24.6C during the day. The average daily light integral inside the greenhouse was 9.2 molm 2 d Mist frequency was con trolled during the day by light accumulation and set to 740 mol until root formation and then increased to 1000 mol, averaging every 25 60 minute frequencies respectively of 5 sec each Mist frequency at night was controlled by a timer, from every 35 minutes for 5 sec until root emergence to completely off at stage 3. The stock plants were fertigated through the mist with a water soluble fer tilizer containing (in mgL 1 ) 75 NO 3 N, 25 NH 4 N, 12P, 83 K, 20 Ca, 10 Mg 0.7S, 1Fe, 0.5Mn, 0.5Zn, 0.25Cu, 0.25B, a nd 0.1 Mo for seven days. After initial root emergence, the stock to produce cuttings with a range of initial tissue nutrient concentrations. Treatments were appl ied using deionized water at each irrigation for 21 days: (1) Control: 0.7S, 1Fe, 0.5Mn, 0.5Zn, 0.24Cu, 0.24B, and 0.1 Mo (2) Low: 37.5 NO 3 N, 12.5 NH 4 N, 6P, 41.5K, 10Ca, 5 Mg 0.7S, 1Fe, 0.5Mn, 0.5Zn, 0.24Cu, 0.24B, and 0.1 Mo (3) Moderate 75 NO 3 N, 25 NH 4 N, 12P, 83 K, 20 Ca, 10 Mg 0.7S, 1Fe, 0.5Mn, 0.5Zn, 0.25Cu, 0.25B, and 0.1 Mo and (4) High 150 NO 3 N, 50 NH 4 N, 24P, 166K, 40Ca, 20 Mg 0.7S, 1Fe, 0.5Mn, 0.5Zn, 0.24Cu, 0.24B, and 0.1 Mo Salts were ammonium nitrate, ammonium phosphate, calcium nitrate, magnesium nitrate, potassium nitrate, boric acid, copper sulfate, iron EDDHA, manganese sulfate, sodium molybdate and zinc sulfate. Sulfa te was supplied in each of the treatments through the micronutrient carriers and remained constant in all four treatments. In contrast to P, K, Ca, and Mg changed in proportion with the change in N. During this period, cuttings were sprayed with two

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92 applications of ethephon (2 chloroethyl) phosphonic acid at 500 mgL 1 to reduce flower bud formation. One cutting was harvested fr om each stock plant (1 cutting per cell = 51 cuttings per 51 cell count tray) grown under each of the four fertilizer treatments 28 days after insertion (March 19, 2008). Cuttings taken from each stock plant fertilizer treatment (6048 cuttings or 144 tray s with 42 cuttings per tray) and subsequently treated with three propagation fertilizer solutions (12 trays with 42 cuttings per tray) applied using deionized water for 17 days to evaluate overall response to (1) complete fertigation response to (2) N wit hout P, K, and Mg and a control with (3) micronutrients o nly : (1) NO 3 N, 25 NH 4 N, 12P, 83 K, 20 Ca, 10 Mg 0.7S, 1Fe, 0.5Mn, 0.5Zn, 0.24Cu, 0.24B, and 0.1 Mo NO 3 N, 25 NH 4 N, 20Ca 1Fe, 0.7S 0.5Mn, 0.5Zn, 0.24Cu, 0.24B, and 0.1 Mo (lacking P, K and Mg) and (3) Mo Calcium and sulfur were the nitrate nitrogen carrier and sulf ur was a carrier in the micronutrient formulations. M treatment group were phosphorus, potassium, and magnesium. The remaining cuttings 9 cuttings per stock treatment tray were combined and sent in for initi al tissue nutrient analysis. The experiment was split into two blocks with 6 replicates per stock and propagation fertilizer treatment combination per block. E ach block consis ted of 12 treatment combinations [ n=6. The experimental unit was defined as an individual (10.5 x 50 cm, 51 cell count)

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93 propagation tray containing 42 cuttings (due to even divisibility across 3 da ta collection periods, 14 cuttings per tray per stage). Four root development stages were recorded, with data measured when the majority of cuttings across treatments had reached each stage: (0) none on day 0, (1) initial visible root emergence on day 7, (2) roots to side and bottom of cell (cuttings removed from mist) on day 13, and (3) a thoroughly rooted cutting where lifting the shoot would remove the complete root ball and cell of substrate on day 17. Fourteen cuttings per replicate tray per block we re destructively sampled at each of those stages for tissue dry weight and tissue nutrient analyses. Three replicate trays per block per treatment combination were combined into one, for adequate tissue sam ple size for nutrient analysis ( 2 tissue samples per treatment combination per block per root development stage ) Tissue samples were analyzed for Total Kjeldahl Nitrogen (TKN) and other nutrients using inductively coupled plasma (ICP) emission spectrometry (Quality Analytical Laboratories, Panama City, FL.). Data were analyzed as a split plot design, with propagation fertilizer treatment as the main plot and stock fertilizer treatment as the sub plot, and statistical analysis utilized Proc Mixed in SAS (version 9.1; SAS Institute, Cary, NC). Results and Discussion Initial tissue nutrient concentrations at stage 0 differed between stock fertilizer treatments (Table 5 1), ranging from (in % N, P, and K respectively) 5.6, 0.75, and 5.1 in the High treated stock, 4.9, 0.50, and 4.3 in the Moderate treated st ock, 4.7, 0.39, and 3.7 in the Low treated stock, and 3.4, 0.24, and 2.5 in the Control treated stock. Initial N concentration s in the Moderate and Low stock treatment groups were not statistically different from each other (P<0.1882), but differed from N levels in High and Control treated plants. P and K levels were significantly different between cuttings from each

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94 stock plant fertilizer level. The initial nutrient levels of the High, Moderate and Low treated stock were within the ranges recommended by Gibson et al. (2007) of 3.9 to 7.6%N, 0.5 to 0.9%P, and 3.1 to 6.7% K, respectively. However, initial N, P and K concentrations in cuttings taken from Control treated stock were below recommended ranges of Gibson et al., (2007) Initial root emergence ( stage 1) was observed after 7 days and roots reached the side of most cells by 13 d ays after insertion (stage 2). C uttings were thoroughly rooted by 17 days (stage 3) after insertion This timing of root developmental stages was consistent with a schedul e described for pe tunia propagation by Dole and Gibson (2006). Data were not collected on timing of rooting stage of each replicate tray, however no obvious differences were noted between treatments. Rooting stage and stock treatment was affected total dr y weight (shoot plus root, P<0.0001 ; 0.0001 respectively ), and there was no interaction or effect of fertilizer treatments during propagation. Total dry weight across rooting stages was 11% greater in cuttings from the Low, Moderate and High treated stock compared with cuttings that had the lowest initial tissue nutrient levels (from Control treated stock). Total dry weight increased as rooted stage increased, from an initial dry weight at stage 0 (sticking) of 0.06 gcutting 1 to 0.08 gcutting 1 at stag e 1 (day 7), with a final dry weight of 0.10 gcutting 1 by the end of the experiment (day 17) (P<0.0001) Shoot dry weight was also affected by rooting stage and stock fertilizer treatment (P<0.0001 ; 0.0001 respectively ). Shoot dry weight was greatest fo r cuttings from the High treated stock fertilizer, 7% higher than any other treatment. Moderate and Low treated stock were not different from each other (P<0.7079), however they were 9%

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95 greater than shoot dry weight for cuttings harvested from the Control treated stock (P<0.0001). Shoot dry weight increased as ro oted stage increased from 1 to 2 from an initial dry weight at stage 0 (sticking) of 0.06 gcutting 1 to 0.08 gcutting 1 at stage 1 (day 7), to 0.09 gcutting 1 at stage 2 (day 13) (P<0.0001). S hoot dry weight was not different between stage 1 and stage 3 (P<0.8976). There was an interaction between rooting stage and stock fertilizer treatment effects (P<0.01) on root dry weight, but propagation treatment was not significant (P<0.0997). Stock tr eatment effects on root dry weight are therefore described by rooting stage, and only stages 2 and 3 were analyzed because root dry weight could not be measured at stage 0 (sticking) or 1 (initial root emergence). By the end of the experiment at stage 3, root dry weight in cuttings taken from the High fertilizer treated stock plants was 0.016 gcutting 1 which was lower than the Low and Moderate treated cuttings (0.021 gcutting 1 ). Control treated plants at stage 3 had an intermediate root dry weight of 0.018 gcutting 1 which were not differ ent from other treatments. At stage 2, root dry weight was greater in cuttings taken from Moderate and Low stock treatments (0.0106 gcutting 1 ) compared with High treated stock (0.0064 gcutting 1 ). Cuttings from High and Moderate stock treatments were not different from the Control (0.0086 gcutting ) at stage 2, but Low treated plants had 15% higher root dry weight than Control treated plants. At both stages 2 and 3, the High stock fertilizer therefore resulted in less rooting than Moderate and Low treated plants. A decline in rooting with increasing fertilizer rate applied to stock plants was also observed in holly ( Ilex crenata Thunb. and willow ( Salix sp.) (Garton et al., 1983; Rein et al., 1 991).

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96 Rooting stage and stock treatment affected the shoot/root (S:R) ratio (P<0.0002). The S:R decreased from rooting stage 2 to stage 3 (9.1 to 4.3, respectively) as roots increased in dry weight (P<0.05) The S:R was higher in cuttings taken from the High stock treatment compared with all other treatments (14.3 compared to 10.0) (P<0.05) In contrast to results in this study, where propagation fertilizer had no effect on root or shoot ound that increasing duration of fertilizer supply at 100 mg NL 1 during propagation resulted in decreased root growth and increased shoot growth by 21 days after sticking (Santos, 2009). Marschner attributed the increase in shoot/root dry weight ratio w ith increasing N supply to be a result of shoot growth being favored over root growth (Marschner, 1995). In this study, there was a carryover effect from increased fertilizer supply in the stock treatment that similarly favored shoot growth, and reduced r oot growth. There were interactions between rooting stage and stock propagation treatment, rooting stage and mist propagation treatment on tissue percent N, P, and K and stock propagation treatment and mist propagation treatme nt on tissue percent N (P<0.0 5 Figure 5 1). Plants that had lower initial tissue N, P, and K at stage 0 continued to have lower tissue N, P, and K at stages 1, 2 and 3 during propagation for the Micronutrients Only treatment, and for P and K with the Complete and N + Micronutrient T reatments (Figure 5 1). Loblolly pine cuttings taken from stock grown with five different fertilizer levels also showed carryover effects on tissue nutrient levels during sub sequent propagation (Rowe and Blazich 1999). At stage 1 (day 7), only the stock treatment affected tissue N (P<0.0001), whereas both stock and propagation treatment main effects influenced tissue P and K (P<0.0001). Tissue N was different for each stock treatment at

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97 stage 1, ranging from 2.6%N for cuttings from the Control treated s tock up to 4.9 %N from the High treated stock. Tissue P and K were different between cuttings taken from each stock treatment (P<0.0001), ranging from the highest concentrations in cuttings taken from the High treated stock (0.51%P and 3.3%K), to the lowe st concentrations in cuttings taken from the Control treated stock (0.18%P and 1.7%K). Tissue P and K were lower in the N + Micronutrients and Micronutrient Only propagation mist treatments At stage 2, stock treatment and propagation mist treatment continued to affect %N, P and K in the tissue (P<0.0001). Tissue N remained highest in cuttings taken from High treated stock (4.83%N), and there were no significant differences between cuttings from Moderate, Low, and Control treated stock (averaging 3.59%N). Propagation mist treatments that contained N fertilizer (the Complete and N+Micronutrients treatments) resulted in higher tissue N compared with cuttings under Micronutrients Only (4.1%N ve rsus 3.1%N, respectively, P<0.0001). Percent P and K remained highest in cuttings taken from High treated stock (0.42%P and 2.8%K), and P and K levels were significantly different between each of the stock treatments (P<0.0001 ; 0.0001 respectively ). Prop agation fertilizer treatments that contained N or Micronutrients Only (i.e., no P or K) led to a decrease in tissue P and K compared with cuttings receiving the T here was an interaction between stock and propag ation fertilizer treatment s for tissue N (P<0.02) at stage 3. The highest tissue N resulted from a combination of the High, Moderate, or Low stock treatment with the Complete propagation fertilizer, or the High and Moderate stock treatment with the N+Micr onutrients fertilizer. The lowest %N

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98 (2.3 % N) occurred in cuttings taken from the Control or Low stock tre atments grown with the M icronutrient Only propagation fertilizer (Figure 5 1C). Stock and propagation fertilizer main effects resulted in differen t tissue P and K percentages (P<0.0001 ; 0.0001 ). The highest %P occurred in cuttings taken from High treated stock compared to the other 3 treatments (0.45%P, P<0.0001), followed by the Moderate treated plants (0.33%P) and tissue P was not different betwe en the Low and Control treated cuttings (0.22%P, P<0.1958). All stock treatments resulted in significantly different tissue K levels at stage 3, ranging from 1.65%K for cuttings from the Control treated stock to 2.66%K from the High treated stock. Tissue %P and K were also highest in cuttings that received the Complete propagation mist treatment which did not statistically differ from each another (P<0.85). Both stock and propagation fertilizer treatments were important for maintaining tissue N, P, and K above recommended minimum levels for petunia (3.85%N, 0.47%P, and 3.13%K reported by Gibson et al., (2007)). Tissue N in cuttings from High treated stock plants did not d rop below 3.85%N regardless of propagation fertilizer. Tissue N dropped below 3.85%N with the other three stock treatments when no NPK was provided during propagation (the Micronutrients Only treatment). For cuttings from the Control treated stock, the C omplete Fertilizer was the only treatment that raised the mean tissue N to above 3.85%N. For tissue P and K, only the High or Moderate stock treatments in combination with the Complete fertilizer resulted in tissue levels at stage 3 equal to or above the recommended minimum tissue concentrations.

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99 The decline in tissue N observed in this study also occurred during root development of poinsettia cuttings that were propagated without additional nutrient supply and was attributed to dilution resulting from pla nt growth (Svenson and Davies, 1995). To determine if dilution explained the trend observed in results for the Micronutrients Only propagation treatment, total tissue N (%N times dry weight) was calculated in cuttings taken from each of the stock treatmen ts (difference in dry weight time %N from stage 0 to stage 1) and was not different from 0, (P<0.05). Given the measured increase in total dry weight from stage 0 to 1 (0.06 to 0.08 gcutting 1 ), the decline in %N for cuttings taken from each stock fertil izer treatment could therefore be explained as dilution. Focus on tissue nutrient decline during stages 0 1 only was because regardless of fertilizer treatment and initial tissue nutrient concentration tissue nutrient decline occurred during the first sev en days, however, after 7 days tissue nutrient decline was amended with subsequent fertilization. Conclusion Our results show that providing high fertilizer levels in petunia stock plant production increases the initial tissue nutrient content in harveste d unrooted cuttings, resulting in higher tissue nutrient concentration throughout the rooting cycle and increasing the likelihood that tissue nutrient concentrations do not fall to deficient levels. However, a moderate fertilizer level during stock produc tion may in fact lead to improved growth characteristics during rooting, whereby we observed reduced root growth, increased shoot growth, and a higher shoot to root ratio from cuttings that had the High stock fertilizer treatment compared with cuttings gro wn with Low or Moderate fertilizer. The specific tissue nutrient levels in unrooted cuttings that resulted from our fertilizer treatments would probably differ under commercial stock plant production,

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100 because in the experimental protocol, cuttings were ha rvested from 4 week old cutting s grown in liner trays whereas commercial stock plant facilities typically produce cuttings in larger containers from more mature plants. Cutting propagators do not always have control of the stock nutrition programs under wh ich the unrooted cuttings were produced. If cuttings are received with low initial tissue nutrient concentrations, application of a complete fertilizer through the mist will be effective in increasing tissue N concentration by stage 2 although this did no t affect plant growth rate. Because N, P, and K all declined during propagation with Micronutrients Only fertilizer, a complete fertilizer solution would be most effective at reducing potential nutrient deficiency during propagation. In previous research (Ch. 4 in Santos, 2009), application of a complete fertilizer at 100 mg NL 1 resulted in increased subsequent root growth, but continuous fertigation at this level over 3 weeks inc reased shoot growth and reduced root growth compared with interrupted macronutrient supply at weeks 2 or 3. Therefore, 100 mg NL 1 may be excessive as a continuous fertilizer rate over the entire propagation cycle under the irrigation, fertilizer, and cl imate conditions of that study. In interpreting this study and the results from Ch. 4 in Santos (2009), it is important to note that S and micronutrients were continuously supplied at a constant level in all propagation and stock fertilizer treatments. Thi s micronutrient fertilizer strategy is used by some commercial propagators, but not in cases where micronutrients are blended at a constant ratio with macronutrients. Dilution was the predominant factor that explained the drop in tissue N concentration dur ing the first week of propagation, similar to results in chrysanthemum,

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101 dianthus, and poinsettia herbaceous cuttings (Good and Tukey, 1967; Blazich, 1988; Svenson and Davies, 1995).

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102 Table 5 1. Initial tissue nutrient concentration (% N, P, and K) in cutt ings taken from plants treated with one of four stock treatments. Stock Treatment Initial Nutrient Concentration N P K High (200 mg NL 1 ) z 5.6 a y 0.75 a 5.1 a Moderate (100 mg NL 1 ) 4.9 b 0.50 b 4.3 b Low (50 mg NL 1 ) 4.7 b 0.39 c 3.7 c Contro l (0 mg NL 1 ) 3.4 c 0.24 d 2.5 d z The 200 mg N L 1 solution containe d 150NO 3 N, 50NH 4 N, 24P, 166K, 40Ca, 20Mg, 0.7S, 1Fe, 0.5Mn, 0.5Zn, 0.24Cu, 0.24B, and 0.1Mo Micronutrients and S were maintained at the same concentration in the 0, 50 and 100 mg N L 1 treatments, and N, P, K, Ca, and Mg were decreased proportionately with N y M ean s were separated using

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103 Figure 5 1 Tissue nutrient concentration (% N, P and K) trends in petunia cuttings taken from stock plants grown under 4 different fertility treatments and propagated under 3 mist treatments. (Note: Stock treatment 1 1 (open 1 (closed triangle) and micronutrients only (open triangle) are column. The lower recommended range limits for tissue nitrogen (3.85%), phosphorus (0.47%) and potassium (3.13 %) concentration in petunia were represented by the red line (Gibson et al., 2007). M ean s were separated within each measurement date using

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104 Figure 5 1. Stag e | Days 0 0 1 7 2 13 3 17

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105 CHAPTER 6 STEM VERSUS FOLIAR UPTAKE DURING PROPAGATION OF PETUNIA VEGETATIVE CUTTINGS Introduction A decline in tissue nutrient concentration was observed in petunia and poinsettia during the first phase of root development (Svens on and Davies, 1995) Nutrient applications during this phase have the potential to alleviate tissue nutrient decline and maintain nutrient levels within recommended ranges. Combined macronutrient and micronutrient mineral nutrient applications to petuni a apical stem cuttings during the first 7 days of propagation maintained tissue nutrient concentrations at higher concentrations (4.8 %N) compared to cuttings that received micronutrients only (3.7 %N) (Ch. 4 in Santos, (2009)). Tissue nutrient decline ha s been attributed to dilution and foliar leaching (Tukey, 1967; Blazich, 1988; Svenson and Davies, 1995). Foliar nutrient applications prior to root emergence potentially serve to (1) replenish pre plant nutrients leached from the substrate which are subs equently taken up by the cut stem through the transpiration stream or newly emergent roots or (2) supply nutrients for direct foliar uptake. Once severed from the stock plant, hormones such as ethylene, jasmonates, and auxins increase and subsequently pl ay diverse roles in initiating adventitious root development (Blakesly et al, 1994; DeKlerk, 1999; Clark et al, 1999; Shibuya et al., 2004; Sorin, 2005; Schilmiller et al., 2005 ; Ahkami et al., 2008 ). Physiologically, the plant begins to respond to the se verance, first by callus and then by root formation, which are two in dependent processes (Dole and Gibson 2006). As soon as the cutting is removed from the stock plant the outer cells form a protective layer of necrotic cells and suberin (hyd rophobic sub stance) (Dole and Gibson 2006). The living cells beneath the protective layer begin to div ide and form callus (Dole and Gibson 2006). When the

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106 swollen callus area turns white or tan, the epidermis ruptures and causes the callus to crack because of r oot differentiation (Dole and Gibson 2006). In petunia, callus is typically formed in 5 7 days, and roots d evelop in 9 14 days (Dole and Gibson 2006). Water mist applications during the first phase of propagation (prior to root emergence) are intended to maintain plant turgidity and minimize transpiration. During a typical petunia or calibrachoa production cycle (28 days) the water volume applied can exceed the container capacity of the substrate, causing leaching of preplant nutrient charge (Santos et. al., 2008). Transpiration was observed to increase by nearly 50% upon visible root emergence in poinsettia (Wilkerson and Gates, 2005). Water moves in plants along gradients of water potential typically generated by transpirational water loss from leaves (Sheriff, 1984). Therefore, potential for nutrient uptake from the base of the stem (through the transpiration stream) should also increase at initial root emergence. Environmental, s tructural, and morph ological characteristics within a given plant spe cies contribute to th e efficacy of foliar nutrient applications. Physiological factors that affect the efficacy of foliar fertilization are the nutrient forms applied, the root temperature, root osmotic potential, leaf age, and current nutrient status in the tissue (Weinbaum, 1996; Mengel, 2002). Plant leaves are specialized organs primarily functioning in the production of organic compounds through photosynthesis. F oliar applied compounds penetrate the leaf surface through the cuticle via cuticular crac ks and imperfections or through stomata, leaf hairs and other specialized epidermal cells (Tukey et al., 1961; Burkhardt and Eichert, 2001). In contrast to roots, the outer walls of the epidermal cells in all aerial plant organs are covered with a hydroph obic cuticle (Marschner, 1995) which has the potential to limit

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107 nutrient absorption The water phobic cuticle is a limiting barrier for two way transport of water and solutes in and out of the leaves (Marschner, 1995). Cuticular waxes embedded in the cut in/cutan matrix were found to be responsible for barrier properties and diffusion of non electrolytes. Neutral, non charged molecules have been found to penetrate the cuticle by diffusion and dissolution of cuticular waxes (Schonherr, 2001 ). However, the mechanism of cuticle penetration of water and ions is not fully understood but may occur due to the existence of aqueous pores (Schonherr and Schreiber, 2004). P lant cuticles are known to be poly electrolytes with isoelectric points around 3.0 (Schonherr et a l ; 1972, 1977). Therefore, the ion exchange capacity of the cuticle will be altered by changed levels in pH. Foliar application solutions with pH values greater than 3 will then render the cuticle as negatively charged (Chamel, 1996; Schonherr et al ., 1977). A negatively charged cuticle means that cat ions will be attracted to the cuticle, whereas an ions will be repelled, and therefore less likely to be taken up. Mengel estimated that a cation is 1000 times more likely to penetrate the cuticular memb ra ne compared to an anion (2002). In general, the micronutrient requirement can be better met by foliar application than macronutrients requirements because in absolute terms higher quantities of macronutrients are needed (Mengel, 2002). Given that our re search has found an increase in N, P and K when fertilizer was applied by root formation, our question was whether this uptake was occurring through some combination of basal and foliar uptake. Therefore we wanted to determine if applications were recharg ing nutrients in the substrate or if true foliar uptake occurred. The objective of this research was to quantify rooting response to fertility treatments and tissue nutrient concentration changes in response to basal or apical nutrient supply during

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108 three rooting phases in propagation of Petunia x hybrida Materials and Methods Aeroponics D esign An aeroponics system was developed with the capability to apply isolated nutrients solutions to the fo liar or basal portion of petunia vegetative cuttings simultaneously (Figure 6 1). The bottoms of ninety six 10.8 L plastic tubs (29.2 34.3 13.3 cm) were removed and opening was covered with a water resistant, flexible polystyrene foam sheet attached u sing contact adhesive to form a waterproof seal. Forty n ine equally spaced crosses (0.8cm x 0.8c m) were made in the foam. Petunia x hybrida Rica were inserted into eac h cross. The base of each cutting was approximately 0.5 to 0.8 mm below the foam sheet. The foliar portion of the cutting was above the foam sheet. A cross shaped, rather than a circular, hole was used because it held the plant in place while allowing f or the minor variability in stem diameter. One of two treatments (a complete fertilizer solution (in mgL 1 ) 56 NO 3 N, 19 NH 4 N, 13P, 88K, 39Ca, 28 Mg 20S, 11Na, 1.1Fe, 0.5Mn, 0.5Zn, 0.25Cu, 0.29B, 0.1 Mo and 0.01Al or clear water 3 NH 4 N, 0.2P, 3K, 19Ca, 18 Mg 19S, 11Na, 0.1Fe, 0.04B, 0.01 Mo and 0.01Al) were applied in a complete factorial arrangement to the apical or basal portion of each cutting. The experiment was run twice (1) December 3, 2008 and (2) January 30, 2009 and the design was a split plot de sign in a randomized complete block design with foliar fertilizer treatment as the whole plot and basal fertilizer treatment as the sub plot and (Figure 6 experiment due to unavailab

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109 13.3 cm), that contai ned 49 plants. Each experimental unit was placed above a JetRain mist nozzle (1.2 Lmin 1 ) (Dramm; Manitowac, WI). Each whole plot contained 24 units (12 units per split plot ) and there were a total of 4 whole plots 8 split plots for a total 96 units. Base treatments were randomized within each split plot, 6 ap plied complete fertilizer and 6 applied clea r water and overhead treatments were randomized between whole plots Plots were split in h alf by overhead mist treatment. Two overhead JetRain mist no zzles spaced 91.4 cm apart applied either the complete fertilizer or clear water treatment. Overhead and underneath mist intervals were set to 15 minutes for 5 seconds during the day and 35 minutes for 5 seconds at night for the duration of the experiment Growing environment and mist frequency were controlled by a greenhouse environmental control system (Hortimax Gemlink Environmental Control System, Rancho Santa Margarita, CA) and heating/ventilation set points were 20/22 C during the day and night, p roviding an average of 22.1 and 21.7 C during the night and 24.0 and 23.9 C during the day in experiments 1 and 2 respectively. The average daily light integral inside the greenhouse was 5.9 and 6.1 molm 2 d in experiments 1 and 2 respectively. Data w ere analyzed using Proc Mixed in SAS (version 9.1; SAS Institute, Cary, NC), as a split plot design with foliar fertilizer treatment as the main plot and base fertilizer treatment as the split plot. The experiments were analyzed separately because differe nt cultivars were used in each. Dry weight and tissue nutrient concentration were measured at three developmental stages, (1) prior to visible root emergence, (2) at initial

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110 visible root emergence, and (3) when average root length was greater than 1cm in the clear water above and clear water below treated plants. Plant tissue was washed in a 0.2% P free detergent solution, followed by 0.05N HCl, and deionized water prior to analysis as described by Jones, (2001). Data were analyzed by developmental stage which was treated as a categorical variable and means within each developmental stage were separated by least squared means analysis. Two experimental units per block per treatment combination (above x below) were destructively sampled (8 units per combin ation per block) at each developmental stage. Nutrient uptake was calculated by subtracting the product of the total dry weight (mg) and the tissue nutrient concentration (N, P or K) at stage 1, 2, or 3 by the product of the total dry weight (mg) and the tissue nutrient concentration (N, P or K) at stage 0. Root length, root number, and root dry weight were also measured on the final data collection date. Results a nd Discussion Callus formation (stage 1) was observed 3 and 4 days after placing cuttings in substrate initial root emergence (stage 2) was observed 6 and 7 days after insertion and average root lengths were greater than 1cm in the clear water above and clear water below treated plants (stage 3) 9 and 10 days after insertion in experiments 1 and 2 respectively. This timing of root developmental stages was consistent with a schedule described for petunia propagation by Dole et al. (2006). Nutrient c oncentration Tissue N, P and K concentrations declined as plant development increased from s tage 0 3 regardless of fertilizer treatment or location applied (Table 6 1). However, f oliar fertilization during the early stages of root development maintained higher tissue N, P and K concentrations in Petunia x hybrida upertunia

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111 e treated with clear water only (Table 6 1). F ertilizer treatments applied to the basal portion of the cuttings maintained higher tissue N (both experiments ) P (Exp t 2) and K (Exp t 2) concentra tions compared with cuttings treated with clear water (Table 6 1). Initial N, P and K tissue concentrations differed betwe en experiments 1 and 2 There was an interaction between stage and foliar treatment on percent N in Expt 1, percent K in Expt 2 an d between stage and basal treat ment on percent N in Expts 1 and 2 (data not shown) F ertilizer applied to the foliage resulted in increased percent N in stages 2 and 3 but not in stage 1 and percent K in plants that received foliar fertilizer at stage 2 w as equivalent to the percent K in plants that received clear water foliar applications at stage 1 Similarly, basal fertilizer applications maintained higher tissue N concentrations by stage 3 ( Expt 1) and by stage 2 ( Expt 2) compared with clear water app lications Foliar application of N, P and K during propagation maintained tissue nutrient concentration at higher levels prior to stage 2 (initial root emergence) with plants that received clear water only ( Expt 2), however overall a decline in concentra tion was measured from stage 1 to stage 3 T issue nutrient concentration decline prior to root formation was also observed in poinsettia cuttin gs that were grown without mist, therefore nutrient loss was not attributed to foliar leaching, but instead to d ilution (Svenson and Davies, 1995). Therefore, foliar fertilization with a complete fertilizer during propagation has the potential to maintain tissue nutrient levels and thereby reduce the likelihood of falling below recommended ranges.

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112 Measurable N, P and K uptake occurred during root development from the foliar and basal portions of the cuttings except for P uptake in Expt 2 (Table 6 2 ). There was an interaction between stage and foliar effects on N uptake in both Expts (P<0.05, 0.05) and K uptake in Expt 2 (P<0.05) (data not shown) Nitrogen uptake increased in cuttings when fertilizer was applied to the foliage during stages 2 and 3 but not stage 1, and no additional N uptake occurred in cuttings treated with foliar clear water at stages 1, 2, and 3. Potassium uptake occurred in cuttings when foliar fertilizer was applied compared with clear water treatments at stages 1, 2, and 3, no K additional uptake occurred in plants treated with clear water stages 1, 2 and 3. The lack of additional N and P u ptake in the clear water treatments is a result of the minimal amounts of those elements in the clear water treatment. In Expt 1, P and K uptake occurred by stage 3 and K uptake was higher in plants treated with foliar fertilizer (Table 6 2 ). Nitrogen up take was also higher in cuttings that were treated with fertilizer from the bottom (basal portion) compared with clear water (Table 6 2 ). A net loss of P was measured in experiment 2, and could be attributed to a combination of foliar leaching Dry weight and root g rowth Initial dry weight differed between the two experiments (P<0.05) (Table 6 3 ) Shoot dry weight increased across developmental stages (Table 6 3) Foliar fertilizer applications did not affect shoot dry weight, however total dry weight w as higher in plants that received fertilizer overhead in Expt 1, and root dry weight was higher in plants that received fertilize r from below in Expt 2 (Table 6 3). Basal fertilizer applications resulted in increased root length 17% from 1.15 cm to 7. 35 cm compared with plants treated with clear water in Expt 1 and 18% from 0.49 cm to 2.79 cm in Expt 2 (Table 6 4 ). Root number also increased with basal ferti lizer

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113 treatments (Table 6 4). Increase in root length and root number when complete fertilizer was applied directly to the root zone supports research reported by Marschner on localized nutrient supply (1995). Nitrogen and phosphorus were shown to be the most effective nutrients in eliciting enhanced root growth (Marschner, 1995). More specifically i n annual plant species rooting density rapidly increased in zones of higher nutrient concentrations, especially N (Marschner, 1995). Increased rooting with localized nutrient supply to the root zone observed in this study also emphasized the importance of the nutrient availability in the substrate at root emergence through the use of best management practices. Propagators that leached high volumes of water during propagation, a resource inefficient strategy, were shown to leach most, if not all of their p re plant charge prior to root emergence (Santos et al., 2008). Conclusion The results show that localized nutrient applications to either the foliage or the cut stem of petunia cuttings resulted in increased growth, uptake and nutrient concentration. More specifically foliar applications tended to increase total growth and tissue nutrient concentration, and basal applications in creased root growth (dry weight, length and number ). Foliar absorption and increased growth during and after propagation was also observed in Chrysanthemum cuttings (Tukey and Marczynski, 1984). Localized nutrient supply to the root zone increased root growth and length in accordance with research on root response to basal nutrient supply reported by Marschner (1995). In previ ous research preplant charge can be very easily leached from the substrate, emphasizing the importance of proper water management strategies to ensure nutrient availability at root emergence (Santos et al., 2008). Best management strategies to minimize wa ter use during propagation could include reducing the irrigation frequency during early

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114 propagation stages or replacing old mist nozzles with nozzles that supply smaller volumes of water (Santos et al., 2008). Foliar nutrient applications during propagat ion serve a similar role as those ascribed in agricultural practices under conditions when nutrient availability is low, topsoil is dry or root activity is reduced during reproductive stages (for a review Wojcik, 2004). During propagation foliar fertiliza tion supplemented uptake prior to significant root development and maintained higher tissue nutrient concentrations compared with cuttings that did not receive foliar nutrient supply. Differences were measured between the two experiments and these differe nces are attributed to both ti me and cultivar effects These results are specific to petunia and therefore foliar applications to other plant species should be trialed on a small group of plants before implementing foliar fertilization practices to an ent ire crop.

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115 Figure 6 1 Aeroponics system design. (Note: Separate nutrient solutions (75 mg L 1 of a complete fertilizer (F) or clear tap water (C)) were applied through mist nozzles to the foliar or basal portions of petunia unrooted cuttings placed in holes in a water resistant flexible polystyrene foam sheet in four p ossible combinations, F above F below; F above C below; C above F below; or C above C below )

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116 Figure 6 2 Aeroponics split plot design consisting of four whole plots (1 to 4). (Note: Plots were split by foliar nutrient solution treatment fertilizer (F dashed rectangles) or clear water (C white rectangles). Each sub plot was randomized with either fertilizer (F) or clear water (C) treatments applied to the basal portion of each cutting. Each square containing a letter represents a replicate container receiving either F or C. ) 1 2 3 4

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117 Table 6 1 Tissue N, P and K concentration of Petunia x hybrid a 2) cuttings during 3 stages of adventitious root development Tissue N(%) Tissue P(%) Tissue K(%) ANOVA Effect z Level Exp t 1 Exp t 2 Exp t 1 Exp t 2 Exp t 1 Exp t 2 Stage y 0 6.6 0 .19 5.49 0 .19 0.83 0 .02 0.78 0 .02 5.00 0 .13 5.10 0 .13 1 5.85 a 4.50a 0.70a 0.60a 4.04a 4.5a 2 4.75b 3.79b 0.55b 0.49b 3.13b 3.79b 3 4.21c 3.17c 0.56b 0.38c 3.14b 3.17c Foliar Clear 4.68a 3.30b 0.58a 0.47b 3.29a 3.3b Fertilizer 5.20a 4.34a 0.63a 0.51a 3.59a 4.34a Basal Clear 4.81b 3.69b 0.60a 0.48b 3.42a 3.6 9b Fertilizer 5.07a 3.95a 0.61a 0.50a 3.45a 3.95a Significance x Stage *** *** *** *** *** *** Foliar NS NS NS ** Stage*Foliar NS NS NS NS ** Basal *** ** NS ** NS ** Stage*Basal NS NS NS NS Foliar*Basal NS NS NS NS NS NS Stage*Foliar*Basal NS NS NS NS NS NS z Stage mean was (n=32 ) and foliar and basal means were (n=48) each. Within a given effect (Stage, Foliar, or Basal) least square means were compared using eparately. y Development stages were: (0) Condition at insertion, (1) callus formation, (2) initial root emergence, and (3) when root leng th in plants receiving clear water above and below averaged approximately 1cm. x Nonsignificant, or significant effe

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118 Table 6 2. Tissue N, P and K uptake of of Petunia x hybrid a cuttings during 3 stages of adventitious root development Uptak e N (mg/cutting) Uptake P (mg/cutting) Uptake K (mg/cutting) ANOVA Effect z Level Exp t 1 Exp t 2 Exp t 1 Exp t 2 Exp t 1 Exp t 2 Stage y 0 1 0.19b 0.34b 0.006b 0.009a 0.05b 0.09b 2 0.34a 0.50b 0.022b 0.010a 0.01b 0.09b 3 0.36a 0.89a 0.062a 0.003a 0.25a 0.25a Foliar Clear 0.17a 0.33b 0.021a 0.010a 0.05a 0.09b Fertilizer 0.43a 0.83a 0.040a 0.004a 0.21a 0.37a Basal Clear 0.24b 0.44b 0.027a 0.010a 0.12a 0.06b Fertilizer 0.36a 0.72a 0.033a 0.002a 0.14a 0.22a Significance x Stage *** *** NS ** Foliar NS NS NS NS Stage*Foliar ** NS NS NS ** Basal ** NS NS NS ** Stage*Basal NS NS NS NS NS NS Foliar*Basal NS NS NS NS NS NS Stage*Foliar*Basal NS NS NS NS NS NS z Within a given effect (Stage, Foliar, or Basal) least were analyzed separately. y Development stages were: (0) Condition at insertion, (1) callus formation, (2) initial root emergence, and (3) when root leng th in plants receiving clear water above and below averaged approximately 1cm. x Nonsignificant, or

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119 Table 6 3 Shoot, root and tota l dry weight of Petunia x hybrid a 2) cuttings during 3 stages of adventitious root development Shoot Dry Weight (mg) Root Dry Weight (mg) z Total Dry Weight (mg) ANOVA Effect y Level E xp t 1 Exp t 2 Exp t 1 Exp t 2 Exp t 1 Exp t 2 Stage 0 x 21.78 0 .8 24.80 0 .8 1 27.98c 29.94c 2 37.61b 36.53b 3 41.80a 46.58a 0.85.4 2.32.3 42.67.8 48.832 Foliar Clear 34.99a 37.32a 0.96a 2.60a 41.37a 47.29a Fertilizer 36.60a 38.04a 0.75a 2.03a 43.97a 50.37a Basal Clear 35.79a 37.09a 0.14b 1.35b 43.09a 47.85a Fertilizer 35.80a 38.27a 1.57a 3.28a 42.25a 49.81a Significance w Stage *** *** Foliar NS NS NS NS NS NS Stage*Foliar NS NS Basal NS NS ** NS NS Stage*Basal NS Foliar*Basal NS NS NS NS NS Stage*Foliar*Basal NS NS z Root dry weight was measured separate from shoot dry weight stage 3. y Stage mean was (n=32) and foliar and basal means were (n=48) and (n=16) for root and total dry weight. Within a given effect (Stage, Foliar, or Basal) least ately. x Stage 0 values were not included in the ANO VA because treatments occurred after Stage 0 data were collected. Mean standard error are presented for stage 0. w

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120 Table 6 4 Root length and root number of of Petunia x hybrid a during stage 3. Root Length (cm) Root Number ANOVA Effect z Level Exp t 1 Exp t 2 Exp t 1 Exp t 2 Foliar Clear 4.63a 1.68a 9.88a 19.06a Fertilizer 3.88a 1.60a 9.31a 18.30a Basal Clear 1.15b 0.49b 5.76b 17.69b Fertilizer 7.35a 2.79a 13.43a 19.68a Significa nce y Foliar NS NS NS NS Basal *** ** *** ** Foliar*Basal NS NS NS NS z Within a given ef fect (Stage, Foliar, or Basal) least square means were compared using Tukey adjustment at y identified by NS, *, **, and *** respectively.

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121 CHAPTER 7 CONCLUSION Nutrition strategies during vegetative propagation were measured to be highly variable in the propagation industry and revealed the need for fur ther understanding of the nutrient supply and uptake dynamics in vegetative propagation. A strategy should begin with an evaluation of initial tissue nutrient status in the cuttings. Tissue nutrient survey ranges were established for 44 vegetatively prop agated species to provide a baseline for growers to use when making decisions regarding subsequent fertilization strategies in propagation. For species where recommended ranges existed at the finished production stage, it was concluded that those ranges c ould also be applied to unrooted cuttings. The importance of starting with tissue nutrient levels within recommended ranges was emphasized in the results of cutting response during propagation with varied initial tissue nutrient levels whereby cuttings th at started with low tissue nutrient concentrations (4.9% or less) dropped below recommended ranges prior to significant root development. Measured growth and tissue nutrient decline were observed in multiple studies during the first seven days of propag ation regardless of subsequent fertilizer applied and was attributed to dilution. However, the decline was reduced when fertilizer was applied due to foliar nutrient uptake. Application of complete fertilizer during propagation increased overall growth a nd more specifically foliar applications during initial root emergence and elongation increased shoot growth and basal applications increased root elongation. Nutrient availability in the root zone at emergence is contingent on best management strategies. In a survey of leading propagators the nutrient charge was shown to be easily leached from the substrate in 75% of the operations, during a 4 week propagation cycle, with the most leaching occurring during the first seven days of propagation. Extended a pplications of complete fertilizer at 100 mg NL 1 did negatively

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122 affect overall root dry weight, therefore optimal constant fertilization strategies mig ht employ lower concentrations. Areas that warrant further investigation include the response to early application of macronutrients in plant species other than petunia, reduction of fertilizer concentrations to evaluate if root development effects are minimized. Also, application of the results from the basal fertilizer application in the aeroponics exper iments to a substrate based experiment to determine if localized nutrient supply in liner tray substrates promotes root elongation. Finally, evaluation of the impact of grower training on improved resource efficiency working towards optimal fertilization strategies during propagation. A Survey of Water and Fertilizer Management During Cutting Propagation W ater volume and nutrient content leached during propagation of herbaceous cuttings were quantified in commercial greenhouses Grower management of th e t iming and concentration of nutrients applied to vegetatively grown calibrachoa ( Calibrachoa x hybrida ) or petunia ( Petunia x hybrida ) liner trays varied between the 8 locations ranging from 0 .5 to 80 mgL 1 N itrogen (N) in week 1 and from 64 to 158 m g NL 1 in week 4. Over a 4 week crop period applied nutrients averaged 4.9 g Nm 2 0.8 g Pm 2 Phosphorus ( P ) and 5 .8 g Km 2 Potassium (K), and leached nutrients averaged 1.1 g Nm 2 0.3 g Pm 2 and 1.6 g Km 2 Leaching of nutrients and irrigatio n water was highly variable between locations Leached water volumes ranged from 4.5 to 46.1 Lm 2 over 4 weeks and contained 0.29 to 1.81 g Nm 2 0.11 to 0.45 g Pm 2 and 0.76 to 2.86 g Km 2 The broad range in current commercial fertigation practice s, including timing of nutrient supply, concentration of applied fertilizer, and leaching volume indicate considerable potential to improve efficiency of water and fertilization resources during propagation and reduce runoff.

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123 That situation presents an edu cational opportunity (to improve efficiency) by minimizing leaching and optimizing uptake, and a challenge (to convince growers of a need for change when current practices are already producing acceptable crops). Management differences were attributed to grower decisions and technology, rather than to differing geographic locations, because locations with similar greenhouse temperature and structures located only 10 km apart leached very different water levels. In follow up discussions with the grower bus inesses in this study, these leaching and fertilizer data were helpful as a training tool and baseline to review practices that could minimize leaching and more closely match water and nutrient supply with plant need. Examples of management practices oper ations with high leaching rates implemented were to reduce the irrigation frequency during early propagation stages and/or to replace old mist nozzles with nozzles that supply smaller volumes of water. A Survey of Tissue Nutrient Levels i n Vegetative Cut tings Nutrient ranges for finished plant production exist for many plant species however r anges (recommended or survey) did not exist for unrooted cuttings. A tissue nutrient survey conducted during 2004 08 on 44 plant genera commercially produced as unro oted cuttings compare d mean tissue nutrient levels from the selected plants to recommended ranges, and to provide d survey ranges for species for which sufficiency data were not available. Maintaining tissue nutrient levels within the recommended ranges fo r each species is a prerequisite (it is necessary but may not be sufficient) for rooting success and uniform performance in the propagation environment. Mean tissue levels in almost 50% of the unrooted cutting species surveyed were statistically similar to ranges established for finished plants. Species with nutrients that fell above or below the recommended ranges did not reach critical minimum deficiency or toxicity levels. Where ranges exist for finished plants, they can validly be applied to stock p lant production of vegetative cuttings. The nutrient ranges presented in the survey represent ed the

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124 typical nutrient levels in cuttings of each species. These ranges can be used by growers as an additional resource when interpreting tissue analysis repor ts of their unrooted cuttings and making corrective nutrient management decisions. Overall, 48% of mean tissue nutrient measurements fell within the recommended range, 25% were higher, and 27% were lower. Where those ranges exist for finished plants, they can be applied to stock plant production of vegetative cuttings. The survey ranges determined for species where no recommended ranges previously existed provide as a reference for typical tissue nutrient concentrations in cuttings of those species. Tim ing o f Macronutrient Supply During Cutting Propagation o f Petunia T iming of macronutrient applications on the early growth and development of Petunia x hybrida affected tissue nutrient concentration, uptake, and growth Growth was dependent upon macronutrient supply, with decreasing shoot dry weight and slightly increasing root dry weight when micronutrients were applied. Continuous application of Complete fertilizer also resulted in more rapid devel opment, as quantified by more leaves per plant, when compared to plants that received on micronutrients indicating that some macronutrient (probably P or K) had reached a minimum critical concentration to limit growth. With constant fertigation using the Complete fertilizer, plant dry weight (DW) doubled from day 0 (sticking of unrooted cuttings) to day 7 (.020 g to .047 g), root emergence was observed by day 4, and by day 7 the average length of primary roots was 2.6 cm. Tissue N concentrations decreased from an average of 6.6% on day 0 to a minimum of 3.9% on day 11, followed by an increase to 4.7% by day 28. Tissue P and K concentrations followed a similar trend to that of tissue N. During any week that the micronutrient fertilizer was substituted fo r the Complete fertilizer tissue N, P, and K concentrations decreased compared with plants receiving the Complete fertilizer. For example, plants receiving the micronutrient

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125 fertilizer between day 0 7 had 20% lower tissue N concentration at day 7 compared to those receiving the Complete fertilizer. Although both shoot DW and leaf count increased once macronutrient fertilization was resumed after day 7, final shoot DW and leaf count were lower than plants receiving Complete fertilizer from day 0 21. Time to first root emergence was not affected by fertigation. Shoot dry weight increased by 22% but root dry weight decreased by 34% when Complete fertilizer was applied from days 0 21 compared to the treatment with the highest root dry weight, resulting in a higher root to shoot ratio. Results emphasize the importance of early fertigation on petunia, a fast rooting species to maintain tissue nutrient levels within recommended ranges. A high shoot growth, and compact height. Therefore, under continuous Complete fertilizer, increased chemical or climate controlled growth regulation would be required to control the increased shoot growth, or a lower constant macronutrient concentration could b e applied. In the case of reduction in fertilizer rate to 75 mgNL 1 constant might control excessive shoot growth. The results from this experiment were b ased a particular plant species, petunia, response to fertilization during propagation could vary between plant genera. During the first 7 days of propagation regardless of nutrient supply, tissue nutrient concentrations dropped. This response can be attr ibuted to dilution because of growth that occurred in the first seven days in conjunction with minimal uptake. Nutrient decline was also observed in poinsettias after 7 days under mist and were uncorrelated to 3 different rates of water volume applied (Wi lkerson and Gates, 2005) as well as in poinsettias that did not have any water applied to foliage 13 days after insertion (Svenson and Davies, 1995). The uniform decline in

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126 tissue nutrient concentrations during the first 7 days emphasizes the importance o f high initial tissue nutrient concentrations in unrooted cuttings on subsequent plant health. If the tissue nutrient concentrations are low in the unrooted cutting, the fertilizer applications need to be started as soon as possible, or a reduction in gro wth and quality may occur as tissue nutrient concentrations drop below recommended ranges and nutrient deficiencies begin to limit growth. Complete fertilizer applications during this initial drop in tissue nutrient concentration were shown to sustain hi gher tissue nutrient concentrations compared to cuttings receiving micronutrients only. The positive response to Complete fertilizer applications early in propagation could be attributed to uptake via foliar, cut stem, or root initials. H igh humidity env ironments, such as propagation, enable nutrients to stay in solution longer and are more available for foliar uptake ( Dybing and Currier, 1960; Clor et al., 1962; Schonherr, 1972. Relative humidity and leaf water status have been shown to be key factors c ontrolling foliar uptake (Bukovac and Wittwer, 1959; Tukey and Marczynski, 1984; Schonherr, 2001). Overall, early nutrient supply had positive effects on growth and nutrient concentrations of petunia, but mist fertigation involves tradeoff in terms of incr eased potential for algae growth, increased nutrient runoff if the fertigation solution is not recycled (as evidenced by the low uptake efficiency), and potential for phytotoxicity or minimal response in certain species. For example, we observed a positiv e response to mist fertigation for several species including Calibrachoa, Solenostemon, Phlox, Scaevola, Sutera and Bidens but negative effects on quality of Helichrysum, Lavender, Poinsettia Spathyphyllum, Anthurium, Guzmania and ferns, and no response for osteospermum, vinca, woody hydrangea, viburnum, and fothergilla cuttings. Early mist fertigation would therefore be favored in combination with an irrigation system that includes capture and reuse of leachate in addition to water sanitation for algae control (for

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127 example, copper ionization or chlorination), for fast growing species where nutrient dilution may rapidly occur, in species that readily absorb nutrients through the foliage (lacking a thick cuticle, and not prone to salt damage), and where i nitial tissue nutrient concentrations are low in the unrooted cuttings. Effect o f Petunia Stock Plant Nutritional Status o n Fertilizer Response During Propagation Fertilization strategies during stock plant and cutting production are linked in terms of c utting nutrient levels and quality, and affect potential for nutrient runoff. The nutrient solutions applied in mist propagation rapidly affected tissue nutrient concentration s of cuttings in both experiments with differences apparent by 7 days after ins ertion A constant nutrient solution of 100 mg N L 1 complete fertilizer applied through the mist to Supertunia raised tissue N concentrations 17 days after insertion in plants taken from stock treated with 100 or 200 mg N L 1 comp ared to plants taken from 0 or mg N L 1 treated stock and received micronutrients only through the mist. Providing a complete fertilizer during propagation of petunia, beginning immediately after sticking of cuttings, reduced the risk of nutrient deficien cy. Particularly in situations where fertilizer is not applied early during propagation, stock plants should be managed to ensure unrooted cuttings have adequate nutrient reserves. Nutrient deficiency during propagation of vegetative cuttings negatively a ffects plant growth and health (Dole and Gibson, 2006; Gibson et al., 2007). Providing high fertilizer levels in petunia stock plant production increases the initial tissue nutrient content in harvested unrooted cuttings, resulting in higher tissue nutrie nt concentration throughout the rooting cycle and increasing the likelihood that tissue nutrient concentrations do not fall to deficient levels. However, a moderate fertilizer level during stock production may in fact lead to improved growth characteristi cs during rooting, whereby we observed reduced root growth, increased shoot growth, and a higher shoot to root ratio from cuttings that had the High stock fertilizer

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128 treatment compared with cuttings grown with Low or Moderate fertilizer. The specific tiss ue nutrient levels in unrooted cuttings that resulted from our fertilizer treatments would probably differ under commercial stock plant production, because in the experimental protocol, cuttings were harvested from 4 week old cutting grown in liner trays w hereas commercial stock plant facilities typically produce cuttings in larger containers from more mature plants. Cutting propagators do not always have control of the stock nutrition programs under which the unrooted cuttings were produced. If cuttings a re received with low initial tissue nutrient concentrations, application of a complete fertilizer through the mist was effective in increasing tissue nutrient concentration by stage 2 although this did not affect plant growth rate. Because N, P, and K all declined during propagation with a Micronutrients Only fertilizer, a complete fertilizer solution would be most effective at reducing potential nutrient deficiency during propagation. In research looking at fertilization timing, application of a complete fertilizer at 100 mg NL 1 propagation increased subsequent root growth, but continuous fertigation at this level over 3 weeks increased shoot growth and reduced root growth compared with inter rupted macronutrient supply during weeks 2 or 3. Therefore, 100 mg NL 1 may be excessive as a continuous fertilizer rate over the entire propagation cycle under the irrigation, fertilizer, and climate conditions of that study. In interpreting these studi es it is important to note that S and micronutrients were continuously supplied at a constant level in all propagation and stock fertilizer treatments. This micronutrient fertilizer strategy is utilized by some commercial propagators, but not in cases whe re micronutrients are blended at a constant ratio with macronutrients. Dilution was the predominant factor that explained the drop in tissue N concentration during the first week of

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129 propagation, similar to results in chrysanthemum, dianthus, and poinsetti a herbaceous cuttings (Good and Tukey, 1967; Blazich, 1988; Svenson and Davies, 1995). Employing Aeroponics Design t o Quantify Stem Versus Fo liar Uptake During Propagat ion o f Petunia Vegetative Cuttings Localized nutrient applications to either the foliag e or the cut stem of petunia cuttings increased growth, uptake and nutrient concentration. More specifically foliar applications tended to increase shoot growth and tissue nutrient concentration, and basal applications increased root growth (dry weight an d length). Foliar absorption and increased growth during propagation and after propagation was also observed in Chrysanthemum cuttings (Tukey and Marczynski, 1984). Localized nutrient supply to the root zone increased root growth and length in accordance with research on root response to basal nutrient supply reported by Marschner (1995). In previous research preplant charge can be very easily leached from the substrate, emphasizing the importance of proper water management strategies to ensure nutrient availability at root emergence (Santos et al., 2008). Best management strategies to minimize water use during propagation could include reducing the irrigation frequency during early propagation stages or replacing old mist nozzles with nozzles that suppl y smaller volumes of water (Santos et al., 2008). Foliar nutrient applications during propagation serve a similar role as those ascribed in agricultural practices under conditions when nutrient availability is low, topsoil is dry or root activity is redu ced during reproductive stages (for a review Wojcik, 2004). During propagation foliar fertilization supplemented uptake prior to significant root development and maintained higher tissue nutrient concentrations compared with cuttings that did not receive foliar nutrient supply. Differences were measured between the two experiments and these differences are attributed to both time and cultivar effects, however since both were main factors their individual

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130 effects on response cannot be isolated. These resu lts are specific to petunia and therefore foliar applications to other plant species should be trialed on a small group of plants before implementing foliar fertilization practices to an entire crop.

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1 31 LIST OF REFERENCES Ahkami, A.H., S. Lischewski, K.T. Haensch, S. Porfirova, J. Hofmann, H. Rolletschek, M. Melzer, P. Franken, B. Hause, U. Druege, and M.R. Hajirezaei. 2008. Molecular physiology of adventitious root formation in Petunia hybrida cuttings: inovolvement of wound response and prim ary metaboli sm. New Phytol 181:613 625. Anderson, A.S. 1986. Environmental influences on adventitious rooting in cuttings of non woody species, p. 223 253. In: M.B. Jackson (ed.). New root formation in plants and cuttings. Martinus Nijhoff Publishers, Boston MA Argo, W. R. and J.A. Biernbaum. 1996. Availability and persistence of macronutrients from lime and preplant nutrient c harge fertilizers in peat based root m edia. J. Amer. Soc. Hort. Sci. 121(3):453 460. Argo, W. R. and P. R. Fisher. 2002. Understanding pH management for container grown crops. 1st ed. Meister Publ., Willoughby, OH Barker, A. V. and D. J. Pilbeam. 2007. Handbook of plant nutrition. CRC Press, London, U.K.. Biernbaum, J.A., W.R. Argo, B. Weesies, A. Weesies, and K. Haack. 1995. Pers istence and replacement of preplant nutrient charge fertilizers from highly leached peat based root media. HortSci ence 30(4):763. Black, C. A. 1993. Soil fertility evaluation and c ontrol. Lewis Publishers, Boca Raton. Blakesley, D. 1994. Auxin metab olism and adventitious root initiation. In: Davis T D Haissig B E eds. Biology of adventitious root formation. New York, NY, USA: Plenum Press, 143 153. Blazich, F.A. 1988. Mineral nutrition and adventitious rooting, p. 61 69. In: T.D. Davis, B.E. Hassig, and N. Sankhla (eds.). Adventitious root formation in cuttings. Dioscorides Press, Portland OR Bukovac M. J. and S. H. Wittwer. 1957. Absorption and mobility of foliar applied nutrients. Plant Physiol. 32(5): 428 435. Burkhardt, J. and T. Eichert, 2001. Stomatal uptake as an important factor in foliar nutrition. p. 1046 1047. In: Plant Nutrition. W.J. Horst et al ed s. Kluwer Academic Publishers, Boston MA. Chamel, A. and N. Vitton. 1996 Sorption and diffusion of 14 C atrazine through i solated plant cuticles. Chemosphere 33(6): 995 1003. Clark, D.G., E.K. Gubr ium J.E. Barrett, T.A. Nell, H.J. Klee. 1999. Root formation in ethylene insen sitive plants. Plant Physiol 121:53 60.

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138 BIOGRAPHICAL SKETCH Kathryn Marie Santos was born in Methuen, M.A. to Mark J. Santos and Brenda C. Santos. She grew up with a younger brother, Mark and sister, Mary in Derry, NH within an hour from the White Mountains Boston, and the Atlant ic O cean. She graduated from Pinkerton Academy, Derry, N ew Hampshire in September 1999. Kathryn g raduated with a B.S. degree in plant b iology and a minor in English at the University of New Hampshire, Durham in May 2003. Massachuset ts as an Assistant Head Grower from June 2003 until November 2004. In November 2004 she began working as a technician for Dr. Paul Fisher at the University of New Hampshire and enrolled in graduate studies the following semester, January 2005. She tran s ferred into a doctoral program at the University of Florida, Gainesville in August of 2006. Currently, Kathryn is completing the requirements for Ph.D. degree in the environmental horticulture program under the direction of Dr. Paul Fisher.