Dra. Victoria Fernández - Foliar Fertilization

5/26/2018 Dra. Victoria Fernndez - Foliar Fertilization

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Foliar Fertilization

Scientifc Principles and Field Practices

V. Fernndez, T. Sotiropoulos and P. Brown

International Fertilizer Industry Association (IFA)Paris, France, 2013


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The designation employed and the presentation of material inthis information product do not imply the expression of any opi-nion whatsoever on the part of the International Fertilizer Industry

Association. This includes matters pertaining to the legal status of anycountry, territory, city or area or its authorities, or concerning the delimi-tation of its frontiers or boundaries.

International Fertilizer Industry Association

28, rue Marbeuf75008 ParisFranceTel: +33 1 53 93 05 00Fax: +33 1 53 93 05 45/ [email protected]: fertilizernews

Foliar Fertilization: Scientific Principles and Field PracticesV. Fernndez, T. Sotiropoulos and P. BrownFirst edition, IFA, Paris, France, March 2013Copyright 2013 IFA. All rights reservedISBN 979-10-92366-00-6

The publication can be downloaded from IFAs web site.

To obtain paper copies, contact IFA.

Printed in FranceCover photos: Apples (Bigstock), Wheat (123RF), Pistachios, Cantaloup (iStockphoto)Layout: Claudine Aholou-PutzGraphics: Hlne Ginet


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3

About the book 5

About the authors 5

Acknowledgements 7

List of abbreviations, acronyms, and symbols 7

1. Introduction and scope 101.1. A brief history of foliar fertilization 10

2. Mechanisms of penetration into the plant 12

2.1. Role of plant morphology and structure 152.1.1. Cuticles and their specialized epidermal structures 15

2.1.2. Effect of topography: micro- and nano-structure of the plant surface 18

2.2. Pathways and mechanisms of penetration 202.2.1. Cuticular permeability 20

2.3. Conclusions 25

3. Physico-chemical properties of spray solutions and their impacton penetration 27

3.1. Factors determining spray retention, leaf wetting, spreading and rate of

penetration 283.1.1. Concentration 28

3.1.2. Solubility 28

3.1.3. Molecular weight 29

3.1.4. Electric charge 293.1.5. Solution pH 30

3.1.6. Point of deliquescence 30

3.2. Environment 31

3.3. Formulations and adjuvants 323.3.1. Mineral compounds applied as foliar sprays 32

3.3.2. Formulation additives: adjuvants 33

3.4. Conclusions 40

4. Environmental, physiological and biological factors affecting plantresponse to foliar fertilization 42

4.1. Introduction 42

4.2.Leaf age, leaf surface, leaf ontogeny, leaf homogeneity and canopy development 44

Contents


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4 Foliar fertilization: scientific principles and field practices

4.3. Plant species and variety 49

4.4. Effect of the environment on efficacy of foliar-applied nutrients 534.4.1. Light 53

4.4.2. Temperature 554.4.3. Humidity 56

4.5. Summary of the effects of the environment on plant response to foliar

fertilization 59

4.6. Nutrient mobility and transport 60

4.7. Conclusions 70

5. Years of practice learning from the field 72

5.1. Spray application technology 72

5.2. Foliar formulations and application technology 745.3. Biological rationale for the use of foliar fertilizers 74

5.3.1. Role of crop phenology and the environment on plant response 75

5.3.2. Influence of the environment on the efficacy of foliar applicationsduring spring 76

5.3.3. Efficacy of foliar applications for flowering and grain set in field crops 81

5.3.4. Foliar fertilization during peaks of nutrient demand 83

5.3.5. Post-harvest and late season sprays 87

5.3.6. Foliar fertilization and crop quality 87

5.4. Impact of plant nutritional status on efficacy of foliar fertilizers 885.5. Source and formulation of nutrients for foliar spray 91

5.6. Toxicity 94

5.7. Conclusions 99

6. Regulatory and environmental considerations 101

6.1. Regulatory matters 101

6.2. Environmental and food quality considerations 102

6.3. Conclusions 104

7. Perspectives of foliar fertilization 106

7.1. Conclusions 108

8. References 112


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About the book

Foliar ertilization is a widely used crop nutrition strategy o increasing importanceworldwide. Used wisely, oliar ertilizers may be more environmentally riendly andtarget oriented than soil ertilization though plant responses to oliar sprays are variableand many o the principles o oliar ertilization remain poorly understood.

Te aim o the book is to provide up-to-date inormation and clarification onthe scientific basis o oliar ertilization and plant responses to it with reerence tothe underlying environmental, physiological and physico-chemical determinants.Inormation drawn rom research, field trials and observational studies, as well asdevelopments in ormulation and application techniques, are discussed.

About the authors

Victoria Fernndez

Forest Genetics and Ecophysiology Research Group, School of Forest Engineering, TechnicalUniversity of Madrid (Universidad Politcnica de Madrid), Madrid, Spain.

Victoria Fernndez holds a research tenure at the echnical University o Madrid,Spain. She gained a Bachelor o Science in Horticulture at University College, Dublin,Ireland and a PhD at Humboldt University o Berlin, Germany. For more than 12 years,Dr. Fernndez has been implementing applied and undamental research approachesto oliar ertilization as a means to improve the effectiveness o oliar sprays and haspublished various peer-reviewed articles in this regard. She is currently ocusing onanalyzing the physico-chemical properties o plant suraces rom an eco-physiologicaland agronomic viewpoint and also in relation to their interactions with oliar-applied

agro-chemicals.


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Thomas Sotiropoulos

Ministry of Rural Development and Food, Greek Agricultural Organization Demeter,

Pomology Institute, Naoussa, Greece.

Tomas Sotiropoulos received a Bachelor o Science in Agriculture at the AristotleUniversity o Tessaloniki, Greece in 1993, a MSc in Plant Breeding and PlantPhysiology in 1996 and a PhD rom the same University in 1999. Dr. Sotiropoulos iscurrently an Associate Researcher in the Greek Agricultural Organization Demeter,Pomology Institute, Naoussa. His main interests include applied and undamentalresearch dealing with ertilization as well as cultivar breeding and evaluation, mainlyon deciduous ruit trees. He has participated in several national and European researchprojects and published various peer-reviewed articles on the previous topics. He alsoserved as a part time Proessor in the School o Agriculture o the Aristotle Universityo Tessaloniki and the Alexander echnological Educational Institute o Tessaloniki.

Patrick Brown

Professor, Department of Plant Sciences, University of California, Davis, California, USA.

Patrick Brown received a Bachelor o Science (Hons) in agronomy and biochemistry atthe University o Adelaide, Australia in 1984 and a PhD in agronomy and internationalagricultural development rom Cornell University, USA in 1988. Dr. Brown is currentlyProessor o Plant Nutrition in the Department o Plant Sciences at the University oCaliornia, Davis. His research ocusses on the role o micronutrients in plant growthand development and encompasses research rom undamental biology to fieldapplication and extension. Dr. Brown is author o 150 scientific articles, books andbook chapters with significant contributions in the area o the physiology o boron,the role o nickel in plant biology and the mechanisms o elemental transport in plants.Current research ocuses on the optimization o nutrient use in orchard crops and the

development o decision support systems or growers. Dr. Brown has served as theDirector o International Programmes at the University o Caliornia, Davis and asPresident o the International Plant Nutrition Colloquium, as well as requently servingin an advisory role or governmental, industrial and grower organizations.


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Acknowledgements

Te authors wish to thank the many colleagues in academia and the ertilizer industrywho have responded to our requent questions and requests or inormation. Teauthors are especially grateul to the growers and consultants who have been criticalin our education and who ultimately demonstrate what works, what does not work andwhat makes no sense. We still have a lot to learn!

List of abbreviations, acronyms,and symbols

AP Adaptation to echnical Progress (as used in the book)B boronB(OH)

3or H

3BO

3 boric acid

Ca2+ calcium ionCaCl

2 calcium chloride

Ca(H2PO

4)

2 calcium phosphate

Ca(NO3)

2 calcium nitrate

Cu copperDAFB days afer ull bloomEC European CommissionEDDHSA ethylenediamine-di-(2-hydroxy-5-sulophenylacetate)EDDS ethylenediaminedisuccinateEDA ethylenediaminetetraacetateEU European UnionFe ironFeCl

3 iron chloride

Fe(NO3)3 iron nitrateHEDA N-2-hydroxyethyl-ethylenediaminetriacetateH

3PO

4 phosphoric acid

IDHA iminodisuccinic acid


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K potassiumkg ha-1 kg per hectareKCl potassium chloride also known as muriate o potash (MOP)

K2CO3 potassium carbonateKH2PO

4 monopotassium phosphate

K2HPO

4 dipotassium phosphate

KM potassium metalosateKNO

3 potassium nitrate

K2SO

4 potassium sulphate

KS potassium thiosulphatelbs acre-1 pounds per acreMg magnesiummg kg-1 milligram per kilogrammg L1 milligram per litreMgCl

2 magnesium chloride

Mg(NO3)

2 magnesium nitrate

MgSO4 magnesium sulphate

MKP monopotassium phosphatemM millimoleMn manganesemN m-1 miliNewton per meterMnSO

4

manganese sulphateMo molybdenumN nitrogenNa sodiumNa

2B

4O

7 borax

Na2B

8O

13 sodium-octoborate

NH4H

2PO

4 ammonium dihydrogen phosphate

(NH4)

5P

3O

10 ammonium tripolyphosphate

Ni nickelnm nanometer

P phosphorus32P phosphorus isotopePHP polyhydroxyphenylcarboxilatePO

43- phosphate

POD point o deliquescencePO(NH

2)

3 phosphoryl triamide

Q10 temperature coefficientRb rubidiumS sulphur

SEM Scanning Electron Microscopyg cm2- microgramme per square centimeterL microlitreM micromolar


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US United States (o America)UV ultra violetZn zinc

ZnSO4 zinc sulphate degreeC degree Celsius

List of terms

Uptake Te process o transport o oliar applied nutrients through thelea cuticular surace into cellular space where they can affectplant physiology and metabolism.

Adsorption Te adherence o oliar applied nutrients to the lea cuticularsurace. At any time a portion o adsorbed nutrients may notbe available or uptake into the cellular space where they canaffect plant physiology and metabolism.

Absorption Te term absorption is used here to include both the uptakeand adsorption o nutrients.


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1. Introduction and scope

Foliar fertilization is an important tool for the sustainable and productive managementof crops. However, current understanding of the factors that influence the ultimateefficacy of foliar applications remains incomplete. Tis book provides an integratedanalysis of the principles, both physico-chemical and biological, known to influencefoliar absorption and utilization by the plant, and reviews the available laboratoryand field experimental results to provide insights into the factors that ultimately

determine the efficacy of foliar applications. Advances in this field will require a soundunderstanding of the physical, chemical, biological and environmental principles thatgovern the absorption and utilization of foliar applied nutrients. Te aim of this bookis to describe in detail the state of knowledge on the mechanisms of uptake by plantorgans (leaves and fruits) of surface-applied nutrient solutions, and to describe theenvironmental and biological factors and interactions that are key to understandingthese processes. Empirical information gathered from foliar nutrient spray trials andfield practices will be merged with physical, chemical and biological principles toarrive at a greater understanding of this technology, its potential, its weaknesses and itsunknowns. Te authors will also strive to illustrate the challenges facing this technologyand the research and development required for its advancement. Te goal of this bookis to provide the reader with this understanding.

1.1. A brief history of foliar fertilization

Te ability of plant leaves to absorb water and nutrients was recognized approximatelythree centuries ago (Fernndez and Eichert, 2009). Te application of nutrient solutionsto the foliage of plants as an alternative means to fertilize crops such as grapevine

agriculture was noted in the early 19thcentury (Gris, 1843). Following this, researchefforts were applied to try and characterize the chemical and physical nature of the plantfoliar cuticle, the cellular physiology and structure of plant leaves as well as focusingon potential mechanisms of penetration by foliar sprays. With the advent of firstlyfluorescent and then radio-labelling techniques in the first half of the 20thcentury itbecame possible to develop more accurate methods to investigate the mechanisms ofleaf cuticular penetration and translocation within the plant following foliar applicationof nutrient solutions (Fernandez and Eichert, 2009; Fernandez et al., 2009; Kannan,2010).

Te role of stomata in the process of foliar uptake has been a matter of interest sincethe beginning of the 20thcentury. However in 1972 it was postulated that pure watermay not spontaneously infiltrate stomata unless a surface-active agent to lower surfacetension below 30 mN m-1is applied with the solution (Schnherr and Bukovac, 1972).


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1. Introduction and scope 11

As a consequence of this, most investigations were subsequently carried out on cuticularmembranes isolated from adaxial (upper) leaf surfaces of species in which enzymaticisolation procedures could be conducted, e.g. from poplar or pear leaves. Utilizing this

system it was found that cuticles are permeable to water and ions as well as to polarcompounds (Kerstiens, 2010). Furthermore the occurrence of two distinct penetrationpathways in the cuticle, one for hydrophilic and another for lipophilic substances, hasbeen suggested (Schnherr, 2006; Schreiber and Schnherr, 2009).

Te proposition that stomata could also contribute to the foliar penetration processwas re-assessed by Eichert and co-workers at the end of the 1990s and subsequentlyvalidated (Eichert and Burkhardt, 2001; Eichert and Goldbach, 2008; Eichert et al.,1998; Fernandez and Eichert, 2009). At present the quantitative significance of thispathway and the contribution of other surface structures such as lenticels to the uptake

of foliar applied solutions remain unclear.Since its first recorded use in the early 19thcentury (Gris, 1843), foliar fertilizationhas been the subject of considerable controlled environment and field research and hasbecome widely adopted as a standard practice for many crops. Te rationales for theuse of foliar fertilizers include: 1) when soil conditions limit availability of soil appliednutrients; 2) in conditions when high loss rates of soil applied nutrients may occur;3) when the stage of plant growth, the internal plant demand and the environmentconditions interact to limit delivery of nutrients to critical plant organs. In each of theseconditions, the decision to apply foliar fertilizers is determined by the magnitude of thefinancial risk associated with the failure to correct a deficiency of a nutrient and theperceived likelihood of the efficacy of the foliar fertilization.

Furthermore foliar fertilization is theoretically more environmentally friendly,immediate and target-oriented than soil fertilization since nutrients can be directlydelivered to plant tissues during critical stages of plant growth. However while theneed to correct a deficiency may be well defined, determining the efficacy of the foliarfertilization can be much more uncertain.


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2. Mechanisms of penetration into theplant

Te processes by which a nutrient solution applied to the oliage is ultimately utilizedby the plant include oliar adsorption, cuticular penetration, uptake and absorptioninto the metabolically active cellular compartments in the lea, then translocation andutilization o the absorbed nutrient by the plant. From a practical perspective it is ofendifficult to distinguish between these processes though many trials using the term oliaruptake ofen reer to an increase in tissue nutrient content without directly measuringthe relative biological benefit o the application to the plant as a whole. Tis conusionand imprecision greatly complicates the interpretation o both controlled environment/laboratory and field experimentation and has undoubtedly resulted in inconsistentplant response and general uncertainty in predicting the efficacy o oliar treatments.Tereore the challenges acing practitioners o oliar ertilization and or researchersattempting to understand the actors that determine the efficacy o oliar ertilizers aregreat.

Te aerial surace o the plant1 is characterized by a complex and diverse array ospecialized chemical and physical adaptations that serve to enhance plant toleranceto an extensive list o actors including unavorable irradiation, temperatures, vaporpressure deficits, wind, herbivory, physical damage, dust, rain, pollutants, anthropogenicchemicals, insects and pathogens. Aerial plant suraces and structures are also welladapted to control the passage o water vapor and gases, and to restrict the loss onutrients, metabolites and water rom the plant to the environment under unavourableconditions. Tese characteristics o aerial plant suraces that allow them to protect theplant rom environmental stress and to regulate water, gas and nutrient exchange alsoprovide the mechanisms affecting the uptake o oliar applied nutrients. Improvements

in the efficacy and reproducibility o oliar ertilization requires knowledge o thechemical and physical attributes o plant suraces and the processes o penetration intothe plant.

Aerial plant suraces are generally covered by a hydrophobic cuticle and very ofenpossess modified epidermal cells such as trichomes or stomata. Te outer surace othe cuticle is covered by waxes that may coner a hydrophobic character to the plantssurace. Te degree o hydrophobicity and polarity o the plant surace is determinedby the species, chemistry and topography which are also influenced by the epidermalcell structure at a microscopic level. Like leaves, ruits are also protected by a cuticle

1For simplicity we will use the term aerial plant suraces to mean the external suraceso all above ground plant organs including stems, leaves, trunks, ruits, reproductive andother above ground organs that can be targeted or oliar application.


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and may contain epidermal structures such as stomata2or trichomes3that influence thetranspiration pathway and contribute to its conductance o water (and nutrients) whichare critical actor or ruit growth and quality (Gibert et al., 2005; Morandi et al., 2010).

A transverse section o a typical angiosperm lea consists o a cuticle that covers theupper and lower epidermal cell layers enclosing the mesophyll as illustrated in Figure2.1 with a microscopic image shown in Figure 2.2.E. Leaves differ in their structurebetween species but generally consist o palisade parenchyma near the upper epidermisand spongy parenchyma (also reered to as spongy mesophyll) between the palisadelayer and the lower epidermis. Tere are large intercellular spaces among the mesophyllcells, especially in the spongy parenchyma (Epstein and Bloom, 2005). Te epidermisis a compact layer with sometimes two or more layers o cells (Figure 2.2.F) and theprincipal eatures, related to nutrient and water transport, which characterize theepidermis are the cuticle and the stomata.

2Stomata are pores surrounded by 2 guard cells that regulate their opening and closurewhich are present at high densities in leaves and are responsible or gaseous exchange andcontrolling water transpiration through the plant.3Epidermal cell hair or bristle-like outgrowth.

Figure 2.1. Typical structure of dicotyledonous leaf including vascular bundle in a leaf vein.(Reproduced with permission from Plant Physiology, 4thEdition, 2007, Sinauer Associates).


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Te surace topography and transversal structure o a peach lea and a ruit usingScanning Electron Microscopy (SEM) and optical microscopy afer tissue staining isshown in Figure 2.2. Both the peach ruit and lea surace stained with auramine O is

covered by a cuticle that emits a green-yellow fluorescence when observed under UVlight (Figure 2.2. C and D). Te lea has a cuticle protecting the abaxial (lower) andadaxial (upper) lea side and the trichomes on the peach ruit surace are also coveredby a cuticle. On the abaxial peach lea surace, stomata are present (approximately 220mm-2) while only a ew (approximately 3 mm-2) occur beneath the trichomes coveringthe peach ruit (Figure 2.2. A and B) (Fernandez et al., 2008a; Fernandez et al., 2011). A

Figure 2.2.Micrographs of a peach leaf versusa peach fruit. Surface topography of a leaf (A)and fruit (B) observed by Scanning Electron Microscopy (SEM) (x400). Transversal sections of apeach leaf and a peach fruit after tissue staining with auramine O (UV light observation; C andD) and toluidine blue (light transmission; E and F) (micrographs A and B by V. Fernndez; C andE by G. Lpez-Casado; D and F by E. Domnguez).


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layer o epidermal cells is observed beneath the abaxial and adaxial lea cuticle and ontop o the mesophyll cells (Figure 2.2. E). A multiserrate, disorganized epidermis withsingle-celled trichomes is ound above the parenchyma cells and underneath the peach

ruit surace (Figure 2.2. F).When present in deciduous plant species, and always in evergreens, the leavesrepresent the majority o the total surace o the aerial part and will capture most othe spray applied and will also interact with rain water, og or mist. While the primaryunction o the plant surace is to protect against dehydration, the permeability o plantsuraces to water and solutes may actually play a crucial eco-physiological role to absorbwater under water-limiting conditions (Fernandez and Eichert, 2009; Limm et al., 2009).

All aerial plant parts are covered by a hydrophobic cuticle that limits the bi-

directional exchange o water, solutes and gases between the plant and thesurrounding environment.

Epidermal structures such as stomata, trichomes or lenticels may occur on thesurace o different plant organs and play important physiological roles.

2.1. Role of plant morphology and structure

Te undamental requirement or an effective oliar nutrient spray is that the activeingredient penetrates the plant surace so it can become metabolically active in thetarget cells where the nutrient is required. A oliar applied chemical may cross the plantlea surace viathe cuticleper se, along cuticular cracks or imperections, or throughmodified epidermal structures such as stomata, trichomes or lenticels. Te cuticleproves an effective barrier against the loss o water and yet, at the same time, it provesan equally effective one against the uptake o oliar applied chemicals. Te presenceo cuticular cracks or the occurrence o modified epidermal structures can contributesignificantly to the rate o uptake o oliar nutrient sprays. Te structure and compositiono the plant lea surace will be briefly described as a basis or understanding their role

in the uptake and absorption o oliar applied nutrient sprays.

2.1.1. Cuticles and their specialized epidermal structuresTe cuticle covering aerial plant parts is an extra-cellular layer composed o a biopolymermatrix with waxes embedded into (intra-cuticular), or deposited onto (epi-cuticularwaxes), the surace (Heredia, 2003). On the inner side, a waxy substance called cutinis mixed with polysaccharide material rom the epidermal cell wall, which is chieflycomposed o cellulose, hemicellulose and pectin in a ratio similar to that ound inplant cell walls. Tereore the cuticle itsel can be considered as a cutinized cell wall,

which emphasizes the compositional and heterogeneous nature o this layer and itsphysiologically important interaction with the cell wall underneath (Dominguez et al.,2011).


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Te cuticle matrix is commonly made o the bio-polyester cutin orming anetwork o cross-esterified hydroxy C

16and/or C

18atty-acids (Kolattukudy, 1980). Te

composition o the biopolymer matrix may vary depending on the plant organ, species

and genotypes, stage o development and growing conditions (Heredia, 2003; Kerstiens,2010). While cutin is depolymerized and solubilized upon saponification, cuticles romsome species may contain an alternative non-saponifiable and non-extractable polymerknown as cutan, which yields a highly characteristic series o long chain n-alkenes andn-alkanes upon flash pyrolysis (Boom et al., 2005; Deshmukh et al., 2005; Villena etal., 1999). Recently, Boom et al.(2005) determined the presence o cutan in cuticles odrought-tolerant species such asAgave americana, Podocarpus sp. or Clusia roseaandsuggested that it might be a preserved biopolymer especially in xeromorphic (waterstoring) plants. Cutin is the only polymer present in cuticles o the ruits and leaves omany

Solanaceaeand

Citrusspecies (Jeffree, 2006) whereas in

Beta vulgariscutan is the

only polymer orming the lea cuticular matrix (Jeffree, 2006). Variable proportions ocutin and cutan have been determined in cuticular membranes extracted rom leaves osome plant species such asAgave americana(Villena et al., 1999) and in some ruit typessuch as sof-ruit berries, apples and peppers (Jarvinen et al., 2010; Johnson et al., 2007).

Te waxes present in the cuticle, either deposited onto, or embedded into, thecuticular matrix are mainly mixtures o long chain aliphatic molecules (mainly C

20-C

40

n-alcohols, n-aldehydes, very long-chain atty-acids and n-alkanes) and o aromatic(ring-chain) compounds (Samuels et al., 2008). Wax composition has been observedto vary between different plant species and organs, the stage o development and theprevailing environmental conditions (Koch et al., 2006; Kosma et al., 2009).

As well as the cutin and/or cutan matrix and the waxes, variable amounts andtypes o phenolics may be present in the cuticle either in ree orm embedded in thematrix or chemically bound to cutin or waxes by ester or ether bonds (Karabourniotisand Liakopoulos, 2005). Hydroxycinnamic acid derivatives (e.g. erulic, caffeic orp-coumaric acid), phenolic acids (e.g. vanillic acid) and flavonoids (e.g. naringenin)have been determined analytically in epicuticular wax and cuticle matrix extractsand observed by fluorescence microscopy (Karabourniotis and Liakopoulos, 2005;Liakopoulos et al., 2001). Besides the major role o phenols in protection against biotic

(microbes or herbivores) and abiotic (UV radiation, pollutants) stress actors, they arealso involved in the attraction o pollinators (Liakopoulos et al., 2001).

Many plant suraces are pubescent4to a greater or lesser degree as shown in Figure2.3. or soybean, maize and cherry lea adaxial suraces. According to Werker (2000),trichomes are defined as unicellular or multicellular appendages which originate romepidermal cells only, and which develop outwards rom the surace o various plantorgans. Scientific studies on these epidermal structures began in the 17 thcentury withemphasis being placed either on individual trichomes or on the collective properties othe trichome layer reerred to as the indumentum (Johnson, 1975). richomes can grow

on all plant parts and are chiefly classified as glandular or non-glandular. Whilenon-glandular trichomes are distinguished by their morphology, different kinds oglandular trichomes are defined by the secretory materials they excrete, accumulate or

4A surace covered by trichomes.


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absorb (Wagner et al., 2004; Werker, 2000). Non-glandular trichomes exhibit a majorvariability in size, morphology and unction and their presence is more prominentin plants thriving in dry habitats and usually on young plant organs (Fahn, 1986;Karabourniotis and Liakopoulos, 2005).

Stomata are modified epidermal cells that control lea gaseous exchange andtranspirational water losses. Tey are generally present on the abaxial lea side but insome plant species (known as amphistomatic), including maize and soybean, they alsooccur on the upper lea side (Eichert and Fernndez, 2011). Stomata also occur in theepidermis o many ruits such as peaches, nectarines, plums or cherries though at lowerdensities compared to the leaves. Stomatal density, morphology and unctionality mayvary between different plant species and organs (Figure 2.4) and can be affected by

Figure 2.4.Scanning electron micrographs of stomata present on the surface of: (A) peach fruit;(B) cherry fruit; (C) rose abaxial leaf surface; and (D) broccoli abaxial leaf surface (Micrographsby V. Fernndez, 2010).

Figure 2.3. Adaxial surface of: (A) soybean; (B) maize; and (C) cherry leaf (Micrographs by V.Fernndez, 2010).


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stress actors such as nutrient deficiencies (Fernandez et al., 2008a; Will et al., 2011), orthe prevailing environmental conditions such as light intensity and quality as illustratedby changes seen in plants growing in natural or artificial shade (Aranda et al., 2001;

Hunsche et al., 2010).Another example o epidermal structures that occur on plant suraces are lenticels(Figure 2.5). Lenticels are macroscopic structures that may occur in stems, pedicels orruit suraces (e.g. they are present on the skin o ruits such as apple, pear or mango)once the periderm (cork) has ormed. Teir evolutionary origin has been linked tostomata, epidermal cracks and trichomes (Du Plooy et al., 2006; Shaheen et al., 1981).

Figure 2.5.Scanning electron micrograph of a lenticel found on the surface of a Golden Deli-cious apple skin (Micrograph by V. Fernndez, 2010).

Te absorption o nutrient solutions by plant suraces may occur via: Te cuticle.

Cuticular cracks and imperections.

Stomata, trichomes, lenticels.

2.1.2. Effect of topography: micro- and nano-structure of the plantsurface

Te topography o the plant surace, as determined by the composition and structure othe epi-cuticular waxes in glabrous (trichome-ree) areas, or by the presence o trichomesor trichome layers in pubescent suraces, will determine its properties and interactionswith water, nutrient solutions, contaminants, micro-organisms, agrochemicals, etc.


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Plant suraces have different degrees o wettability when in contact with waterdroplets as shown in Figure 2.6 or the leaves and ruits o our different plant species.

In the last decade, the water and contaminant repellent properties o plant suraceswith rough topography have been described (Barthlott and Neinhuis, 1997; Wagner etal., 2003) and different types o epicuticular waxes have been classified or several plant

species (Barthlott et al., 1998; Koch and Ensikat, 2008).Te presence o a micro- and nano-relie structures associated with the suraces

over the epidermal cells, and the chemical properties o the waxes deposited onto thelea surace, may markedly increase its roughness and surace area and will ultimately

Plant organ and species Average contact anglewith pure H

2O ()

Drop image

Adaxial side of

Eucalyptus globulus leaf

140

Adaxial side ofFicus elasticaleaf

83

Calanda Peach (Prunuspersica L. Batsch)

130

Apple (Malus domesticaL.Borkh) fruit surface

84

Figure 2.6.Average contact angles with pure water drops of the adaxial Eucalyptus globulus(A) and Ficus elastica(B) leaves; and peach (C) and apple (D) fruit surfaces (V. Fernndez, 2011).


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determine the degree o polarity and hydrophobicity. Differences in surace polarityand hydrophobicity in relation to variable growing conditions, plant species, varietiesand organs can be expected and these will have an influence on the effectiveness o

oliar sprays. Fernndez et al.(2011) examined the properties o a peach variety whichis covered by a dense indumentum5as a model system or a pubescent plant suraces.Te peach skin investigated was ound to be very hydrophobic with contact angles orwater higher than 130. Properties such as the surace ree energy, polarity, and work oadhesion o the peach lea surace was determined by means o estimating the contactangle o three liquids - water, glycerol and di-iodomethane. Tis methodology hasproved a valuable tool or the characterization o plant suraces and should be urtherexplored and exploited or scientific and applied purposes (Figure 2.6).

2.2. Pathways and mechanisms of penetration

Te structure and chemistry o the plant surace will affect the bi-directional diffusiono substances between the plant, the lea surace and the surrounding environment andhence and thereore the rate o uptake o oliar ertilizers. In the ollowing sections, themost significant plant surace penetration pathways o chemical sprays will be described,with emphasis on the mechanisms o cuticular permeability and stomatal uptake.

2.2.1. Cuticular permeabilityTe cuticle consists o three layers (Figure 2.7), namely (rom the external to the internalsuraces o the plant organ), the epicuticular wax layer (EW), the cuticle proper (CP)and the cuticular layer (CL) (Jeffree, 2006).

Te EW layer is the outermost and most hydrophobic component o the cuticle. TeCP that lies beneath the epicuticular waxes contains mainly cutin and/or cutan and isby definition ree o polysaccharides (Jeffree, 2006). Te CL is located under the CP andconsists o cutin/cutan, pectin and hemicelluloses that increase the polarity o this layerdue to the presence o hydroxyl and carboxylic unctional groups. Te middle lamellaeand pectin layer (ML) is situated beneath the CL. Variable amounts o polysaccharide

fibrils and pectin lamellae may extend rom the cell wall (CW), binding the cuticle tothe underlying tissue (Jeffree, 2006).

A gradual increase in negative charge rom the epicuticular wax to the pectinlayer creates an electrochemical gradient that may increase the movement o cationsand water molecules (Franke, 1967). Te intra-cuticular waxes limit the exchange owater and solutes between the plant and the surrounding environment (Schreiber andSchnherr, 2009), while the epicuticular waxes influence the wettability (Holloway,1969; Koch and Ensikat, 2008), light reflectance (Lenk et al., 2007; Pndel et al., 2006)and surace properties o the plant organ.

Te lipophilic and hydrophobic nature o the structural components o the cuticlemake it an effective barrier against the diffusion o hydrophilic, polar compounds.However, lipophilic and a-polar compounds may penetrate the hydrophobic cuticular

5A covering o trichomes.


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membrane at high rates compared to polar electrolyte solutions which have not hadsurace-active agents added to them (Fernandez and Eichert, 2009). Indeed, severalstudies provide evidence or the penetration o polar solutes through intact astomatouscuticles by direct and indirect means (Heredia, 2003; Riederer and Schreiber, 2001;yree et al., 1992).

Experimental evidence has shown that plant cuticles are asymmetric membraneswith a gradient o fine structure and waxes rom the outer to the inner surace. Plantcuticles have a large inner sorption compartment consisting mainly o the biopolymer

matrix (cutin and/or cutan) and a comparably smaller (10% o total volume) outercompartment where waxes predominate (Schnherr and Riederer, 1988; yree et al.,1990).

Te current state o knowledge on the mechanisms o penetration o polar solutesand apolar lipophilic substances through the cuticle will be briefly discussed in theollowing paragraphs.

Te cuticle is an asymmetric membrane composed mainly o 3 layers:

Te epicuticular wax layer. Te cuticle proper, chiefly made o cutin/cutan and intracuticular waxes.

Te cuticular layer, containing cutin/cutan and polysaccharide material.

Figure 2.7.Schematic representation of the general structure of the plant cuticle covering twoadjacent epidermal cells (EC) separated from each other by the middle lamellae and pectinaceouslayer (ML) and the cell wall (CW). Epicuticular waxes (EW) are deposited onto the cuticle proper(CP) which is mainly composed of a biopolymer matrix and intra-cuticular waxes. The cuticularlayer (CL) chiefly contains cutin and/or cutan and polysaccharides of the CW.


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Permeability of lipophilic, apolar compoundsTe penetration o lipophilic6, apolar substances through the plant cuticle has beenproposed to ollow a dissolution-diffusion process (Riederer and Friedmann, 2006).

Tis model implies that the movement o a lipophilic, apolar molecule rom a solutiondeposited onto the plant surace into the cuticle precedes the diffusion o the moleculethrough the cuticle (Riederer and Friedmann, 2006). Te diffusion o a lipophilicmolecule has been proposed to be governed by partitioning and its penetration rate willbe proportional to the solubility and mobility o the compound in the cuticle (Riederer,1995; Schreiber, 2006). At a molecular level, both the dissolution and diffusion o amolecule in the cuticle can be viewed as passing into and between voids in the polymermatrix arising by molecular motion (Elshatshat et al., 2007).

aking into account Ficks first law, the diffusive flux (J; molm-2s-1) is related to theconcentration gradient with solutes moving rom regions o high to low concentrationwith a magnitude that is proportional to the concentration gradient (spatial derivative).According to the cuticular diffusion model, which has been thoroughly explained byRiederer and Friedmann (2006), the diffusive fluxJis proportional to the mass transercoefficient P(i.e. the permeance o the membrane; m s-1) multiplied by the concentrationdifference between the inner and the outer sides o the cuticle:

J= P* (Ci-C

o)

where: Ci

is the concentration (mol m-3) at the inner side o the cuticle and Co

is theconcentration in the outer side o the cuticle.

Under certain experimental conditions, the mobility o a molecule can be predictedby calculating the permeance which is a value specific to a given molecule and aparticular cuticular membrane (Riederer and Friedmann, 2006). Te permeance (P ms-1) is expressed as:

P = D * K* l-1

where: D(m2s-1) is the diffusion coefficient in the cuticle; Kthe partition coefficient

which is the ratio between the equilibrium molar concentrations in the cuticle andin the solution at the cuticle surace; and l(m) which is the path length o diffusionthrough the cuticle. Te diffusion path length may be tortuous and much larger than thecuticle thickness which is determined by the waxes embedded in the polymer matrix(Baur et al., 1999; Schnherr and Baur, 1994) and by the spatial disposition o cutinand/or cutan molecules (Fernandez and Eichert, 2009). Te diffusion coefficient D alsodepends on the temperature and fluid viscosity o the oliar nutrient solution and size othe chemical molecules it contains.

Methods to predict the mobility o lipophilic, apolar compounds through the cuticle

o a ew species that enable the enzymatic isolation o astomatous (adaxial) cuticleshave been developed in recent decades (Riederer and Friedmann, 2006; Schreiber, 2006;Schreiber and Schnherr, 2009).

6Compounds which are soluble in oils, ats, or organic solvents.


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Experimental evidence has shown that the cuticle is highly size selective (Buchholzet al., 1998) and that it may act as a molecular sieve. Te size o voids have been oundto ollow a log-normal distribution that may be in the same order o magnitude as some

agrochemicals which may be limiting their diffusion through the cuticle (Schreiber andSchnherr, 2009).

Permeability of hydrophilic, electrolytesTe permeability o cuticles to solutes has been investigated usingastomatous isolated cuticles using the same methodology used to assessthe penetration o apolar, lipophilic substances (Schreiber and Schnherr,2009). In the absence o surace-active agents solutions o ionic, hydrophilic7compounds have generally been ound to penetrate the cuticle at a lower rate comparedto lipophilic, apolar compounds. Tis finding is probably explained by the lipophilicnature o the cuticular constituents as well as the ease with which lipophilic compoundswill diffuse owing to their higher solubility in such media as compared to hydrophilics.However, some authors have suggested that the rate o penetration o electrolytesdetermined experimentally is too high to be explained by simple dis-solution anddiffusion in the cuticle and have proposed that hydrophilic solutes may penetratethrough the cuticle via a physically distinct pathway, along what have been calledpolar, aqueous or water-filled pores (Schnherr, 2006; Schreiber, 2005; Schreiber andSchnherr, 2009).

It has been hypothesized that such pores may arise rom the absorption o watermolecules onto polar moieties located in the cuticular layer (Schnherr, 2000; Schreiber,2005), such as unesterified carboxyl groups (Schnherr and Bukovac, 1972); ester andhydroxylic groups (Chamel et al., 1991) in the cutin network; and carboxylic groupso pectic cell wall material (Kerstiens, 2010; Schnherr and Huber, 1977). However,no conclusive experimental evidence has been ound so ar to support the presenceo such aqueous pores in cuticles as they are not visible or identifiable with currentmicroscope technologies (Fernandez and Eichert, 2009).

However the size o the aqueous pores o a ew plant species has been indirectlyderived rom permeability trials using astomatous, adaxial cuticles. Diameters o about

1 nm were calculated or de-waxed isolated citrus cuticles (Schnherr, 1976), andisolated ivy (Hedera helix) cuticles (Popp et al., 2005). Furthermore, pore diametersranging rom 4 to 5 nm have been calculated rom permeability trials carried out withintact coffee and poplar leaves (Eichert and Goldbach, 2008).

Lipophilic, apolar compounds have been proposed to penetrate cuticles by asolution-diffusion process.

Te mechanisms o penetration by hydrophilic, polar compounds are not ully

elucidated yet.

7Water miscible/soluble compounds such as mineral salts, chelates or complexes.


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Permeability of stomata and other plant surface structuresTe potential contribution o stomata to the penetration o lea-applied chemicals hasbeen a matter o controversy or many decades (Dybing and Currier, 1961; Schnherr

and Bukovac, 1978; urrell, 1947) and is still not ully understood (Fernandez andEichert, 2009). Early studies aimed at assessing the process o stomatal uptake suggestedthat it may occur via infiltration i.e. the mass flow o oliar-applied solutions into thelea interior through the open stomata (Dybing and Currier, 1961; urrell, 1947);Middleton and Sanderson, 1965). However, Schnherr and Bukovac (1972) showedthat the spontaneous infiltration o an open stoma by a oliar-applied aqueous solutioncould not occur in the absence o an external pressure or a surace-active agent thatcould lower the surace tension o the solution below a certain threshold (set to 30 mNm-1). Subsequently, many studies have provided evidence or increased uptake rates oplant suraces where stomata are present, especially when the prevailing experimentalconditions were avourable to the opening o the stomatal pores (Eichert and Burkhardt,2001; Fernandez and Eichert, 2009). Investigations carried out on leaves containingstomata only on the abaxial lea surace demonstrated higher oliar penetration ratesthrough the abaxial as compared to the adaxial side (Eichert and Goldbach, 2008;Kannan, 2010). Since this observation contradicts the premise o Schnherr andBukovac (1972) that the higher penetration rates associated with stomatal openingcould not be due to the mass flow through the stomatal pores unless the solutionssurace tension is below 30 mN m-1, several different hypotheses have been proposedto explain these subsequent observations. For instance, the higher penetration ratesin the presence o stomata have been attributed to the increased permeability o theperistomatal cuticle and the guard cells (Sargent and Blackman, 1962; Schlegel andSchnherr, 2002; Schlegel et al., 2005; Schnherr and Bukovac, 1978) but no conclusiveevidence supporting this has been orthcoming so ar (Fernandez and Eichert, 2009).

Te direct contribution o stomata to the process o penetration by oliar-appliedaqueous solutions in the absence o surace-active agents has been subsequently re-assessed (Eichert et al., 1998) in investigations on stomatal uptake perormed withwater-suspended hydrophilic particles (43 nm and 1 m diameter respectively) usingconocal laser scanning microscopy which demonstrated that the treatment solution

passed through the stomata by diffusing along the walls o the stomatal pores (Eichertand Goldbach, 2008). Tis process was reported to be slow and size selective sinceparticles with a diameter o 1 m were excluded while the 43 nm particles passed intothe pores.

Te mechanisms o solute movement into ruits has received only limitedinvestigation though several studies have estimated the permeability o apples to Casolutions either with intact ruits (Mason et al., 1974; Van Goor, 1973), ruit discs(Schlegel and Schnherr, 2002) or isolated cuticular membranes (Chamel, 1989; Glennand Poovaiah, 1985; Harker and Ferguson, 1988; Harker and Ferguson, 1991). Schlegel

and Schnherr (2002) reported a major contribution o stomata and trichomes to theuptake o surace-applied Ca-containing solutions during the early developmentalstages o ruits. However afer June drop the disappearance o stomata and trichomes


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and the sealing o the remaining scars by cutin and waxes may significantly reduce thepermeability o ruit suraces.

Tere have been a ew instances o the assessment o the contribution o trichomes or

lenticels on ruits to the process o uptake o surace-applied nutrient solutions. Harkerand Ferguson (1988) and others (Glenn and Poovaiah, 1985; Harker and Ferguson,1991) suggested that lenticels in mature apples were preerential sites or the uptake oCa solutions through the ruit surace though this possibility has not been assessed indetail so ar.

Stomata may play a major role in the absorption o nutrient solutions applied tothe oliage.

Te mechanisms o stomatal penetration by pure water are not yet ully elucidatedbut recent evidence points towards a process o diffusion along the stomatal porewalls.

Addition o certain suractants to the nutrient solution ormulation leads to theinfiltration o stomata (Chapter 3).

2.3. Conclusions

Te state-o-the-art concerning the process o uptake o solutions by plant suraces hasbeen described in Chapter 2. Plants are covered by a hydrophobic cuticle that controlsthe loss o water, solutes and gases to the environment though conversely it alsoprevents their unrestrained entry into the plant interior. Te structural and chemicaleatures o the plant surace render it difficult to wetting and thereore permeation by asurace-applied polar nutrient solution. In the light o the current state o knowledge,the ollowing certainties, uncertainties and opportunities or the application o oliarertilizers can be addressed.

Certainties

Plant surfaces are permeable to nutrient solutions.

Te ease by which a nutrient solution may penetrate into the plant interior willdepend on the characteristics o the plant surace, which may vary with organ,species, variety and growing conditions, and on the properties o the oliar sprayormulation applied.

Plant surfaces usually possess a hydrophobic coating provided by the epicuticularwaxes.

Te micro- and nano-relief associated with the structure of the epidermal cells,

and the epicuticular waxes deposited onto the surace, together with the chemicalcomposition o these waxes, will determine the polarity and hydrophobicity o eachparticular plant surace.


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Epidermal structures such as stomata and lenticels, which can be present on theleaves and ruits suraces, are permeable to surace-applied solutions and may play asignificant role in its uptake.

Apolar, lipophilic substances have been found to cross cuticles via a solution-diffusion process.

Uncertainties

Te mechanisms of cuticular penetration of polar, hydrophilic compounds (i.e. thoserelating to the uptake o aqueous oliar ertilizers) are currently not ully understood.

Te contribution of the stomatal pathway to the foliar uptake process should beurther elucidated as well as the role o other epidermal structures such as trichomesand lenticels.

Improvingthe eectiveness of foliar fertilizers will require a better understanding ofthe contact phenomena at the interace between the liquid (i.e. the oliar ertilizerormulation) and the solid (i.e. the plant surace).

Te eectiveness of foliar nutrient treatments will improve once the mechanisms ofoliar uptake are better understood.

Opportunities

Multiple scientic experiments and applied studies carried out in the last century

have shown that plant suraces are permeable to oliar nutrient ertilizers. Tis permeability presents the opportunity to supply nutrients to plant tissues and

organs, bye passing root uptake and translocation mechanisms which may limit thenutrient supply o the plant under certain growing conditions.

Foliar fertilization has great potential and should be further explored and exploitedin the uture.


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3.Physico-chemical properties of spray solutions and their impact on penetration 27

3. Physico-chemical properties of spraysolutions and their impact on penetration

Te absorption o oliar-applied nutrients by the plant surace involves a series ocomplex processes and events. Te main processes involved include ormulation othe nutrient solution; the atomization o the spray solution and transport o the spraydroplets to the plant surace; the wetting, spreading and retention o the solution by theplant surace; the ormation o a spray residue onto the surace; and the penetrationand distribution o the nutrient to a (metabolic) reaction site (Young, 1979). Te aboveevents are interrelated and overlap in that a change in one usually has an effect onthe others, and each process is affected by plant growth stage actors, environmentalconditions and application parameters (Bukovac, 1985).

Te properties o the spray ormulations are crucial in determining the perormanceo oliar ertilizers, especially since most o the conditions at the time o treatmentcannot be ully controlled. Foliar nutrient sprays are generally aqueous solutionscontaining mineral element compoundss as active ingredients. Te physico-chemicalcharacteristics o the specific nutrient compound in aqueous solution, such as itssolubility, pH, point o deliquescence (POD) and molecular weight will have a majorinfluence on the rate o absorption o the element by the lea. However, an array oadditives that may modiy the properties o the ertilizer solution are ofen included inthe ormulations with the aim o improving the perormance o nutrient sprays. Te rateo retention, wetting, spreading and rainastness o a nutrient oliar spray is governedby the physico-chemical properties o the ormulation which can contain chemicalcompounds with different characteristics that may interact with each other when theyare together in aqueous solution.

When an aqueous solution is applied to a lea, initially there is a high rate o

penetration which decreases with time resukting rom the drying o the appliedsolution (Sargent and Blackman, 1962). Tis drying is influenced by the prevailingenvironmental conditions and by the ormulation o the applied oliar spray solution.

In the ollowing sections, the principal physico-chemical properties o a ertilizerormulation that may affect and improve its perormance will be described in theoreticaland applied terms.

Water is the usual matrix o oliar nutrient sprays.

Plant suraces are hydrophobic to a greater or lesser degree and the contact area opure water drops can be limited depending on the characteristics o the surace.

Te prevailing environment will affect the physico-chemical properties andperormance o the ormulations on the lea suraces.


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3.1. Factors determining spray retention, leaf wetting,spreading and rate of penetration

Plant responses to oliar ertilizers may be affected by the properties o the spraysolution, which determine the success in achieving the absorption and translocation othe applied nutrients into plant organs. While the process o absorption o lea-appliedsolutions is complex and currently remains unclear (Chapter 2), the properties o theormulations are associated with strict chemical principles well as by the prevailingenvironmental conditions (e.g. relative humidity and ambient temperature) at the timeo treatment. An account o the principal physico-chemical actors in relation to theoliar application o nutrient solutions will be provided in the ollowing sections.

3.1.1. ConcentrationIn Chapter 2 it was shown that the current cuticular diffusion models are based onFicks first law and relate the diffusive flux to the concentration gradient between theouter and the inner parts o the plant surace. Te concentration o a nutrient presentin a oliar spray will always be significantly higher than the concentration ound withinthe plant organ. Tereore, a concentration gradient will be established when a nutrientsolution is applied onto the plant surace and this will potentially lead to the diffusiono the nutrient across the surace. Higher penetration rates in association with increasedconcentrations o several applied mineral elements have been reported in studiesperormed with isolated cuticles (Schnherr, 2001) and intact leaves (Zhang and Brown,1999a; Zhang and Brown, 1999b). However, the relationship between concentration othe applied solution and oliar penetration rates is currently not ully understood. Anegative correlation between increasing Fe-chelate concentrations and the penetrationrate through isolated cuticles and intact leaves, expressed as a percentage o the amountapplied, has been observed (Schlegel et al., 2006; Schnherr et al., 2005). A similarnegative correlation has been reported or oliar-applied K (Ferrandon and Chamel,1988) and other elements (ukey et al., 1961). It is hypothesized that the decrease inrelative penetration rates with higher K concentrations may be due to a progressivesaturation o the uptake sites (Chamel, 1988). As an alternative hypothesis, Fe-salts

and chelates may reduce the size o the hydrophilic pathway by inducing the partialdehydration o the pores in the cuticle (Schnherr et al., 2005; Weichert and Knoche,2006a; Weichert and Knoche, 2006b).

Te ideal concentration range o mineral nutrient solutions or oliar applicationshould be selected according to actors such as the kind o nutrient (e.g. macro- or micro-nutrient), plant species, plant age, nutritional status and weather conditions (Kannan,2010; Wittwer and eubner, 1959; Wojcik, 2004), and all o these will ultimately belimited by the need to avoid phyto-toxicity.

3.1.2. SolubilityBeore applying a oliar spray ormulation, it is crucial that the compounds it containsare appropriately dissolved or suspended. Foliar ertilizers are commonly dissolved orsuspended in water and contain as active ingredients chemical compounds as salts,


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chelates or complexes o mineral nutrients. Te solubility o a chemical compound in aspecific solvent (usually water) at a given temperature is a physical property which canbe altered through use o additives. Te highest limit o the solubility o a substance in

a solvent is reerred to as the saturation concentration where adding more solute doesnot increase solution concentration. Water solubility o the applied substance is a keyactor or oliar uptake, since absorption will occur only when the applied compoundis dissolved in a liquid phase on the plant surace that will subsequently diffuse into theplant organs.

3.1.3. Molecular weightTe size o the nutrient molecule in solution will affect the rate o penetration o aoliar ertilizer as a consequence o the mechanism o cuticular absorption. It has beensuggested that water and solutes cross the cuticle viaaqueous pores (Schnherr, 2006) orin an aqueous continuum (Beyer et al., 2005), and a ew studies have estimated the radiio such pores by indirect means. Te radiio cuticular aqueous pores has been estimatedat approximately 0.3 to 0.5 nm in leaves and 0.7 to 1.2 nm in ruits o some species(Beyer et al., 2005; Luque et al., 1995; Popp et al., 2005; Schnherr, 2006). However,larger pore radii between 2 and 2.4 nm have been calculated or the cuticle o coffeeand poplar leaves by Eichert and Goldbach (2008). Several experiments with differentsolutes and cuticular membranes have shown that the process o cuticular permeabilityis size-selective with high molecular weight (larger) compounds being discriminatedagainst low molecular weight molecules (Schreiber and Schnherr, 2009).

Recent evidence (Eichert and Goldbach, 2008) suggests that the oliar uptake pathwayis less size selective than would be predicted by the cuticular penetration route o entrywhich may indicate that there is a stomatal pathway (Chapter 2). However the processo stomatal uptake is also size-selective since particles with a diameter o 1 m did notenter the stomatal pore whereas particles o 43 nm diameter did penetrate into thestomata (Eichert and Goldbach, 2008).

3.1.4. Electric chargeSalts are electrolytes and will dissociate into ree ions when dissolved in water with the

final solution being electrically neutral. Anions and cations present in aqueous solutionwill be hydrated or solvated to different degrees depending upon their physico-chemicalcharacteristics. Te same phenomena will apply or nutrients supplied as chelates orcomplexes since with ew exceptions most o these compounds are not neutral and willthereore be ionized when dissolved in water. For example, many o the Fe-chelatesavailable on the market are negatively charged (Fernandez and Ebert, 2005). At apH > 3 plant cuticles are negatively charged (Schnherr and Huber, 1977) and cell wallshave charges corresponding to dissociated weak acids (Grignon and Sentenac, 1991).Consequently uncharged or electron-charged compounds and anions can penetrate the

lea and are translocated in the apoplast8 easier than positively-charged complexes orcations.

8Non-living, extracellular space surrounding the living cells (i.e. the symplast).


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However, when applying salts or chelates or complexes, the latter two being ormedby mixing metal salts with ligands accompanied with their own corresponding ions, theanions and cations present in solution can penetrate into the leaves. Te nature o the

anions and cations in the oliar applied solution will have physiological significance andmust be considered when designing a oliar spray ormulation.

3.1.5. Solution pHSince plant cuticles are poly-electrolytes, their ion exchange capacity will be altered withpH fluctuations (Chamel and Vitton, 1996). Cuticles were shown to have iso-electricpoints around pH 3 and when solution pH values are higher than this they will renderthe cuticle negatively charged and the cuticular carboxyl groups will then readily bindpositively charged cations (Schnherr and Bukovac, 1972; Schnherr and Huber, 1977).

While it is clear that the pH o the spray solution alters penetration there is noconsistency in plant response and it appears that the pH o the solution alone is notthat predictive o penetration and is influenced more significantly by the nutrient beingapplied and the plant species being treated. In most o the scientific reports on oliarertilization usually no reerence is made to the pH o the nutrient spray solution appliedto the oliage which is a critical oversight particularly in the case o pH unstable mineralelements such as Fe. Cook and Boynton (1952) recorded the greatest absorption o ureaby apple leaves in the pH range 5.4 to 6.6. Furthermore the highest uptake rates by citrusleaves afer oliar urea treatment were recorded when the pH o the solution was keptbetween 5.5 to 6.0 (El-Otmani et al., 2000). Working with Fe compounds, Fernandezet al.(2006) and Fernandez and Ebert (2005) observed that pH values around 5 wereoptimal or oliar uptake o Fe-containing solutions. Blanpied (1979) showed thatmaximum Ca absorption by apple leaves occurred when the solution pH ranged rom3.3 to 5.2. However, Lidster et al.(1977) reported the highest Ca absorption rates bysweet cherry (Prunus aviumL.) ruits when CaCl

2solution o pH 7 was applied. Reed

and ukey (1978) observed maximum P absorption by chrysanthemum leaves whenthe solution pH was between 3 to 6 or Na-phosphate and between 7 to 10 pH orK-phosphate.

Frequently oliar spray salts dissolved in pure water will alter spray solution pH

and some ormulations may have extreme pH values and hence will affect the uptakeprocess o by the oliage. For instance the majority o Fe(III)-salts are very acidic while1% CaCl

2or 8% K

2SO

4have pH values above 9.

3.1.6. Point of deliquescenceTe processes o hydration and dissolution o a salt are determined by its point odeliquescence (POD) which is a physical property associated with a compound at agiven temperature (Schnherr, 2001). Deliquescent salts are hygroscopic substances(i.e. capable o trapping water rom the surrounding environment) and will dissolve

once a critical relative humidity threshold has been attained. Te point o deliquescenceis defined as the relative humidity value at which the salt becomes a solute. Tereby, thelower the point o deliquescence o a salt is, the sooner it will dissolve upon exposureto ambient relative humidity (Fernandez and Eichert, 2009). When ambient relative


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humidity is higher than the point o deliquescence o the oliar applied compound,the substance will dissolve and will be available or absorption by the lea. Te effect orelative humidity on the solution or crystallization o salts has been assessed in studies

carried out with cuticular membranes and intact leaves and could be better exploredollowing the experimental practices used in aerosol research (Fernandez and Eichert,2009). Similarly, the physiological effects associated with the deposition o hygroscopicaerosol particles onto plant suraces are currently not ully understood, but it isconsidered that such particless may either act as lea desiccants or promote increaseduptake rates (Burkhardt, 2010).

3.2. Environment

Environmental actors such as relative humidity and temperature will play a role withregard to the perormance o a oliar sprays and the uptake o lea-applied solutions.Environment can also alter oliar spray efficacy through its influence on the biology othe plant - a process that will be discussed in Chapter 4.

Te most relevant environmental actors affecting the perormance o solutionswhen sprayed to the oliage will be described, considering that under field conditions,continuous interaction between such actors will result in different physiologicaland physico-chemical responses and effects. Te effect o the environment on oliaruptake-related phenomena will be discussed in more detail when describing thebiological actors affecting the efficacy o oliar ertilization in Chapter 4. Here the twoenvironmental actors that most directly affect the perormance o oliar nutrient spraysare temperature and relative humidity.

Relative humidity is a major actor influencing oliar uptake o nutrient sprays sinceit affects the permeability o the plant surace and the physico-chemical responses toapplied compounds. At high relative humidity permeability may be increased due tocuticular hydration and the delayed drying o the salts deposited onto the plant suraceollowing the application o a oliar spray. Salts with points o deliquescence above theprevailing relative humidity in the phyllosphere9will theoretically remain as solutes and

lea penetration will be prolonged.emperature will affect various physico-chemical parameters o the oliar spray

ormulation such as its surace tension, solubility, viscosity or point o deliquescence. Ingeneral, increasing temperature range (e.g. rom 0 to 40C) under any field conditionswill increase solubility o the active ingredients and adjuvants, but will decreaseviscosity, surace tension and the point o deliquescence. In addition, high temperatureswill speed the rate o evaporation rom the spray solutions deposited onto the oliagereducing the time until solution dryness occurs when lea penetration can no longeroccur.

Other environmental actors such as light intensity or precipitation may also affectthe perormance o oliar nutrient sprays. For instance, several Fe(III)-chelates areknown to be degraded by exposure to sun-light. On the other hand, the occurrence

9The aerial part of plants that can serve as a habitat for microorganisms.


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o precipitation shortly afer the application o a oliar spray may rapidly wash-off thetreatment. As a consequence, weather orecasts should be taken into consideration priorto oliar spray applications to avoid conditions that can reduce humidity or increase

drying speed such as high winds, heavy rain or extremes o temperature at the time ooliar application.

3.3. Formulations and adjuvants

Commercial oliar nutrient sprays are generally composed o at least two majorcomponents, namely: the active ingredient(s) and the inert material(s) or adjuvant(s).Adjuvants help to improve the spreading (wetting) and persistence (sticking) o theactive ingredient(s) or mineral element(s) on the lea surace as well as promote therate o uptake and bioactivity o the mineral element(s) applied. Limitations to theoliar uptake o applied mineral elements has led to the widespread use and continuoussearch or adjuvants that improve the perormance o spray treatments. In the ollowingparagraphs inormation on the active ingredients and adjuvants will be provided.

3.3.1. Mineral compounds applied as foliar spraysA preliminary distinction should be made concerning the application o either macro-or micro-nutrients, the latter being supplied at lower rates and concentrations andofen being unstable when applied as inorganic salts. An account o the most commonmineral element carriers according to recent articles is shown in ables 3.1 and 3.2. Teoliar ertilizer industry is characterized by a large number o proprietary products thatare requently derived rom common salts which can be occasionally mixed in novelratios and/or with addition o compounds that serve to complex, chelate or bind and/or adjuvants that can enhance efficiency o uptake.

Table 3.1.Macro-nutrient carriers normally used in foliar spray formulations.

Macronutrient Common element compounds References

N Urea, ammonium sulphate,ammonium nitrate

Zhang et al.(2009); Fageriaet al.(2009)

P H3PO4, KH2PO4, NH4H2PO4,Ca(H

2PO

4)

2, phosphites

Noack et al.(2011); Schreiner (2010); Hossainand Ryu (2009)

K K2SO

4, KCl, KNO

3, K

2CO

3, KH

2PO

4Lester et al.(2010), Restrepo-Daz et al.(2008)

Mg MgSO4, MgCl

2, Mg(NO

3)

2Dordas (2009a), Allen (1960)

S MgSO4

Orlovius (2001), Borowski and Michalek,(2010)

Ca CaCl2, Ca-propionate, Ca-acetate Val and Fernndez (2011); Wojcik et al.

(2010); Kraemer et al.(2009a,b).


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Table 3.2. Micro-nutrient carriers normally used in foliar spray formulations.

Micronutrient Common element compounds References

B Boric acid (B(OH)3

),

Borax (Na2

B4

O7

),Na-octoborate (Na

2B

8O

13), B-polyols

Will et al. (2011); Sarkar et al. (2007),Nyomora et al. (1999)

Fe FeSO4, Fe(III)-chelates, Fe-complexes

(lignosulphonates, glucoheptonates,etc.)

Rodrguez-Lucena et al. (2010a, 2000b);Fernndez et al. (2008b); Fernndez andEbert (2005); Moran (2004)

Mn MnSO4, Mn(II)-chelates Moosavi and Ronaghi (2010), Dordas

(2009a), Papadakis et al. (2007), Moran(2004)

Zn ZnSO4, Zn(II)-chelates, ZnO,

Zn-organic complexes

Amiri et al. (2008); Haslett et al. (2001),

Moran (2004); Zhang and Brown (1999).

Until the 1970s, the oliar micronutrient ertilizer market was dominated by productsbased on inorganic compounds particularly sulphates (Moran, 2004). During the1980s a wide variety o micronutrient chelates and complexes (e.g. synthetic chelatesusing EDA, glucoheptonates, polyols, amino-acids, or lignosulphonates, among manyother types) were offered as an alternative to the application o inorganic compounds.

Te recommended rates at which oliar ertilizers are used are highly variable and areusually based on the specific plant species being treated. As previously described thephysico-chemical properties o the active ingredients, e.g. molecular size, solubility orpoint o deliquescence, will influence the rate o uptake by oliage. In general, syntheticchelates are much larger and have higher points o deliquescence than the inorganicmineral salts commonly used as active ingredient carriers. While some materials arerecommended on the basis o rigorous controlled environment and extensive field trials,many requently utilize rates designed to merely ensure saety and satisy cost concerns.Optimal concentration rates or the many and varied oliar ertilizers available ordifferent crops are currently lacking and uture research efforts should ocus on trials toestablish clear concentration thresholds or oliar-applied nutrient solutions.

Foliar-applied nutrient solutions could be phytotoxic due to their high osmoticpotential and pH by affecting important physiological processes such as photosynthesisand/or stomatal opening (Bai et al., 2008; Elattal et al., 1984; Fageria et al., 2009; Kluge,1990; Swietlik et al., 1984; Weinbaum, 1988). Tese effects can be a critical actor orconsideration when spraying macro-nutrient ertilizers to the oliage.

3.3.2. Formulation additives: adjuvants

General information

As described in Chapter 2, plant surace topography may vary between plant speciesand varieties, organs and growing conditions. Te presence, chemistry and topographyo epicuticular waxes and epidermal structures such as trichomes may render thesurace difficult to wet. Under such circumstances, the proper wetting, spreading


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and penetration o oliar ertilizers may require the addition o co-ormulants suchas surace-active agents (adjuvants) that modiy the properties o the spray solution.Numerous oliar and cuticular uptake studies have shown the improved efficacy o

ormulations containing adjuvants that act by enhancing the wetting, spreading,retention, penetration and humectant properties o oliar sprays as compared to puremineral element solutions applied alone. Tereore the ormulation o mineral elementsolutions with adjuvants can have a significant effect on the uptake and bioactivityo the nutrients supplied to the oliage though this may also decrease or increase thephytotoxicity risk associated with the nutrient active ingredients applied. Tis impliesa fine-tuning o the nutrient active ingredients and the adjuvant compounds and theirrelative concentration which is necessary to develop a oliar nutrient ormulation thatprovides reproducible plant uptake responses without plant damage.

Adjuvants can be defined as any substance included in a ormulation or which isadded to the spray tank that modifies the nutrient active ingredient activity or the spraysolution characteristics (Hazen, 2000). Tey are generally classified as; (i) activatoradjuvants (e.g. surace active agents) which increase the activity, penetration, spreadingand retention o the active ingredient or; (ii) utility adjuvants (e.g. acidifiers) that modiythe properties o the solution without directly affecting the efficacy o the ormulation(Penner, 2000).

Although there are many commercially adjuvant co-ormulants on the market (able3.3) there is considerable conusion concerning the classification o such compoundsand their purported mode o action (Green and Foy, 2000).

Adjuvant names are usually related to the major properties they coner uponthe spray ormulations to which they are added. However the categorization anddistinction between activator and utility adjuvants is rather subjective and currentlylacks standardization. For instance, adjuvants described as penetrators, synergists oractivators may increase the rate o oliar uptake through different chemical or physicalmechanisms though the general principle o enhanced spray absorption is the same.Adjuvants described as buffering agents or neutralizers are generally chemicalsystems that adjust and stabilize spray solution pH; while other suractants may bereered to as detergents, wetting agents, or spreaders; but again or both types the

general principles are the same. Tere are several adjuvants types usually reered to asstickers that increase solution retention and rainastness and some o these may alsoprolong or retard the process o solution drying when included in oliar sprays.

Humectants are compounds with water-binding properties which can be eitherorganic, such as carboxy-methyl cellulose (Val and Fernandez, 2011), or inorganic,such as CaCl

2. Teir presence in the ormulation lowers the point o deliquescence

(POD) and prolongs the process o solution drying which is especially important toincrease the efficacy o oliar sprays in arid and semi-arid growing regions. Some typesso surace-active agents or utility adjuvants such as stickers or humectants can also

act to increase the rate o retention and rain astness o oliar applied ormulations(Blanco et al., 2010; Kraemer et al., 2009b; Schmitz-Eiberger et al., 2002) which can beparticularly important in regions o high rainall or where requent overhead irrigationis employed. ypical examples o stickers and humectants are latex and soy lecithin


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both o which can significantly improve the retention o oliar sprays on leaves andare requently included in commercial ormulations o many plant protection productsalthough there is an apparent lack o sound inormation concerning the effectiveness osuch adjuvants when used with oliar ertilisers.

Te reasons underlying this are that considerable research efforts have been made

in recent decades to develop adjuvants or oliar spray ormulations which enhancethe perormance o pesticides and herbicides while less attention has been paid todeveloping products specific or oliar nutrient sprays. Adjuvants are usually marketedseparately and may contain single compounds (e.g. surace-active agents alone) or aresold as mixtures o suractants, lecithin, synthetic latex, vegetable oils, tallow aminesor atty acid esters that coner a spectrum o the desired properties outlined previouslywhen included in a oliar-applied solution.

As a consequence since most commercial adjuvant products have been devisedor their application in combination with plant protection products to acilitate their

perormance when applied to the oliage, their suitability or combination with oliarnutrient sprays, which are normally hydrophilic solutes, cannot be a prioriassumed andshould thereore always be empirically tested. For oliar nutrient sprays it is critical thatthe treatments are not phytotoxic to leaves and plants since their value and marketability

Table 3.3. Example of adjuvants available on the market classified according to their purportedmode of action.

Adjuvant name on label Proposed mode of action

surfactant lowering surface tension

wetting agent equivalent to surfactant

detergent equivalent to surfactant

spreader equivalent to surfactant

sticker increasing solution retention; rainfastness

retention aid increasing solution retention; rainfastness

buffering agent pH buffering

neutraliser pH buffering

acidifier lowering pH

penetrator increasing the rate of foliar penetration (e.g. by solubilizingcuticular components)

synergist increasing the rate of foliar penetration

activator increasing the rate of foliar penetration

compatibility agent improving formulation compatibility

humectant retarding solution drying by lowering the formulations point ofdeliquescene (POD) on the leaf

drift retardant better spray targeting and deposition on foliage

bounce and shatter minimizer better spray targeting and deposition on foliage


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can be compromised by crop damage caused by such treatments. Unortunately it isnot currently possible to predict theoretically the perormance o any active ingredientwhether a herbicide, a pesticide or a mineral nutrient element in combination with a

particular adjuvant (Fernandez et al., 2008a; Liu, 2004).

SurfactantsSurace-active agents or suractants are the most widely-used type o adjuvant in oliarspray ormulations. One o the first examples o these compounds being added to oliarnutrient sprays was in the first hal o the 20thcentury with the use o the ionic suractantVatsol in combination with Fe compounds (Guest and Chapman, 1949).

One method used to assess the effect o a suractant is to measure the contact anglewith a paraffined microscope slide and the drop shape by the pending drop methodcomparing the surace tensions o pure water (A and B) with a 0.1% organosiliconsuractant solution (C and D) as shown in Figure 3.1.

Tese measurements were carried out at 25C and the contact angles (Figure 3.1 Aand C) or water and a 0.1% organosilicon suractant solutions were approximately 95and 45 respectively giving calculated surace tensions o approximately 72 and 22 mN

Figure 3.1.Contact angles (A and C) and pending drops used to calculate the surface tension(B and D) of distilled water (A and B) and a 0.1% organosilicon (C and D) distilled water solution(V. Fernndez, 2011).


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respectively. Tis experimental system demonstrates how the addition o a suractant toa pure water solution lowers its surace tension and increases dramatically the area ocontact between the liquid and the solid (in this case a paraffined surace) by lowering

the contact angle.Suractants are large molecules consisting o a non-polar, hydrophobic portionattached to a polar, hydrophilic group (Cross, 1998; adros, 1995). It is important thatthe ends o the hydrophobic and the hydrophilic parts o the suractant molecule are araway rom each other so that they can react independently o each other with suracesand solvent molecules (Cross, 1998). Te hydrophobic part o the suractant interactsweakly with water molecules while the polar or ionic head group interacts strongly withthese so rendering the suractant molecule water soluble.

Surace active agents are characterized by the abrupt change in their physicalproperties they undergo once a certain concentration has been reached. Tese changesin solubility, surace tension, equivalent conductivity or osmotic pressure are due tothe association o suractant ions or molecules in solution to orm larger units. Teseassociated units are called micelles and the concentration at which this association takesplace is known as the critical micelle concentration. Each particular suractant moleculehas a characteristic critical micelle concentration value or a given temperature andconcentration.

Te mechanisms o action o suractants when applied to the oliage are very complexand are only partially understood (Wang and Liu, 2007) although possible modes osuractant action have been suggested by Stock and Holloway (1993) and include:increasing the effective contact area o deposits; dissolving or disrupting epicuticularwaxes; solubilizing agrochemicals in deposits; preventing or delaying crystal ormationin deposits; retaining moisture in deposits; and promoting stomatal infiltration.However, it is now known that suractants can also alter the diffusion o substances viacuticular solubilization or hydration and that they can also affect the permeability o theplasma membrane. Tereore suractant composition and concentration are key actorsinfluencing the perormance o oliar sprays (Stock and Holloway, 1993).

Te hydrophilic portion o a suractant can be non-ionic, ionic or zwitterionic,accompanied by counter-ions in the last two cases. When present in a oliar spray

ormulation the polarity o the hydrophilic part o a suractant may determine actorssuch as the occurrence o interactions between the suractant and the active ingredientsor the contact properties between the spray solution and each particular plant surace.

Non-ionic surfactantsNon-ionic suractants are widely used in oliar sprays as they are theoretically lessprone to interact with other polar components o the ormulation. Te most commonhydrophilic polar group in non-ionic suractants is that based on ethylene oxide(adros, 1995) with the organosilicons, alkyl phenol ethoxylates, alkyl-polyglucosides,

atty alcohol ethoxylates, polyethoxylated atty acids, ethoxylated atty amines,alkanolamides or sorbitan esters belonging to this group o suractants.

An example o a non-ionic suractant molecule is shown in Figure 3.2.


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Figure 3.2.Molecular structure of the non-ionic surfactant, SilwetL-77.

According to Stock and Holloway (1993) the addition o non-ionic suractants withlow ethylene oxide contents, which are good spreaders with their low surace tensions,will avour the uptake o lipophilic pesticides; while conversely uptake o hydrophilicpesticides is improved by suractants with higher ethylene oxide units and thereorepoor spreading properties. However, conflicting evidence concerning the effect o highand low ethylene oxide containing suractants suggests that ethoxylated suractantsmay enhance the uptake o both hydrophilic and lipophilic compounds by differentmechanisms as yet not ully clarified (Haes et al., 2002; Kirkwood, 1993; Ramsey R. J.

L., 2005). For example, low ethylene oxide-content suractants that enhance uptake olipophilic compoundss were ound to alter the physical properties o cuticles and to bemore phytotoxic. By contrast, suractants with higher ethylene oxide contents appearto increase cuticular hydration and to be less phytotoxic (Coret and Chamel, 1993;Ramsey, 2005; Uhlig and Wissemeier, 2000). Suractants with either large hydrophobicgroups or long hydrophilic chains, or both, have been reported to be less phyto-toxicbecause o their lower water solubility and hence, slower rate o oliar uptake (Parr,1982). Studies perormed with Ca-containing compounds (CaCl

2and Ca-acetate) in

combination with ethoxylated rapeseed oil suractants with different ethylene oxide

contents (Kraemer et al., 2009a; Kraemer et al., 2009b; Schmitz-Eiberger et al., 2002)showed that they can affect the rate o cuticular permeability o Ca viathe distributiono the active ingredient in the droplet and the rain-astness o the ormulations.Organosilicon, non-ionic suractants, also known as super-spreaders, are a group o


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chemicals cont

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