Habtom PhD Thesis Final.pdf - Silicon in Agriculture

STUDIES ON THE USE OF BIOCONTROL AGENTS 

AND SOLUBLE SILICON AGAINST 

POWDERY MILDEW OF ZUCCHINI AND ZINNIA 

By 

Habtom Butsuamlak Tesfagiorgis 

B.Sc. (Hons) Eritrea, M.Sc. (Natal) 

Submitted in partial fulfilment of the 

requirements for the degree of 

Doctor of Philosophy 

In the 

Discipline of Plant Pathology 

School of Agricultural Sciences and Agribusiness 

Faculty of Science and Agriculture 

University of KwaZulu-Natal 

Pietermaritzburg 

Republic of South Africa 

December 2008

ABSTRACT 

Powdery mildew (PM) is an important foliar disease of many crops, occurring under both 

greenhouse and field conditions. The application of biological control and soluble silicon (Si) 

against PM has received increasing acceptance as a result of increased environmental and 

public concern over the use of fungicides for disease management, and because many key 

fungicides are no longer effective because of resistance problems. However, success with 

these control options depends on the development of effective antagonists and understanding 

how best to use Si in agriculture. 

Potential antagonists of PM were isolated from naturally infected leaves of different plants. A 

total of 2000 isolates were tested in a preliminary screening on detached leaves of zucchini. 

The best 30 isolates showing consistent results were further tested under greenhouse 

conditions for their efficacy against PM of zucchini. In a greenhouse trial, 23 isolates provided 

disease control to levels of 30 to 77%. Application of 29 isolates resulted in significant 

reductions in values of area under disease progress curve (AUDPC). The best five isolates 

were identified as Clonostachys rosea (Link) Schroers, Samuels, Seifert & Gams (syn. 

Gliocladium roseum) (Isolate EH), Trichothecium roseum (Pers.) Link (syn. Cephalothecium 

roseum) (Isolate H20) and Serratia marcescens (Bizio) (Isolates B15, Y15 and Y41). 

Three adjuvants (Break-Thru ® (BK), Partner ® (PR) and Tween-80 ® (T-80)) were compared 

for their ability to improve efficacy of spray application of silicon (Si) and biocontrol agents 

(BCAs) against PM. Both BK and PR improved the efficacy of Si significantly (P < 0.05). 

Microscopic studies showed that BK affected PM fungi directly and enhanced the deposition 

of BCAs on the pathogen. Break-Thru ® was only toxic to the pathogen mycelia when used at 

> 0.25 ml l -1 , but phytotoxic to zucchini plants when used at > 0.45ml l -1 . However, it did 

not affect the c.f.u. of bacterial BCAs. Use of BK at 0.2-0.4 ml l -1 can be recommended to 

assist spray application of Si (at 750 mg l -1 ) or BCAs for improved control of PM. 

i

The effect of concentration, frequency of application and runoff of Si sprays applied to the 

foliage was evaluated for control of PM of zucchini. Silicon (250-1000 mg l -1 ) + BK 

(0.25 ml l -1 ), was sprayed onto zucchini plants at frequencies of 1-3 wk -1 . Spraying Si 

reduced the severity of PM significantly (P < 0.05). Regardless of the concentration of Si, the 

best results were obtained when the frequency of the treatment was increased, and when spray 

drift or spray runoff were allowed to reach the rhizosphere of the plants. When Si was applied 

onto leaves, direct contact between the spray and the pathogen resulted in mycelial death. Part 

of the spray (i.e., drift and runoff) was absorbed by plant roots, and subsequently played an 

important role in the health of the plants. If affordable, soluble Si should be included in 

nutrient solutions of hydroponics or supplied with overhead irrigation schemes when PM 

susceptible crops are grown. 

Under greenhouse conditions, application of BCAs, with or without Si, reduced the severity 

and development of PM significantly (P < 0.001). Application of Si significantly reduced the 

severity and AUDPC values of PM (P < 0.05 for both parameters). Silicon alone reduced the 

final disease level and AUDPC values of PM by 23-32%, and improved the efficacy of most 

BCAs. In the course of the investigation, antagonistic fungi consistently provided superior 

performances to bacterial isolates, providing disease control levels of up to 90%. Higher 

overall disease levels reduced the efficacy of Si against PM, but did not affect the efficacy of 

BCAs. 

Under field conditions, Si alone reduced disease by 32-70%, Isolate B15 reduced disease by 

30-53% and Isolate B15 + Si reduced disease by 33-65%. Other BCAs applied alone or 

together with Si reduced the disease level by 9-68%. Most BCAs reduced AUDPC values of 

PM significantly. For most antagonists, better efficacy was obtained when Si was drenched 

into the rhizosphere of the plant. However, efficacy of some of the BCAs and Si were affected 

by environmental conditions in the field. Repeated trials and better understanding of how to 

use Si and the BCAs, in terms of their concentration and application frequency, and their 

interactions with the plant and the environment, are needed before they can be used for the 

commercial control of PM. 

ii

Elemental analysis was conducted to determine the impact of differing application levels of 

silicon (Si) in a form of potassium silicate (KSi) in solution in terms of Si accumulation and 

selected elements in different tissues of zucchini and zinnia and growth of these plants, and to 

study the effect of PM on the levels of selected elements in these two plant species. Plants 

were grown in re-circulating nutrient solutions supplied with Si at different concentrations and 

elemental composition in different parts were analysed using EDX and ICP-OES. Increased 

levels of Si in the solution increased the levels of Si in leaves and roots of both plants without 

affecting its distribution to other plant parts. In zucchini, the roots accumulated the highest 

levels of Si, substantially more than in the shoots. In contrast with zinnia, accumulation of Si 

was highest in the leaves. Accumulation of potassium (K) in shoots of both plants increased 

with increased levels of KSi in the nutrient solution. However, K levels in flower of zinnia, 

fruits of zucchini and roots of both plants remained unaffected. Increased level of Si reduced 

accumulation of calcium (Ca) in both plants. 

Adding Si into the nutrient solution at 50 mg l -1 resulted in increased growth of zucchini and 

increased uptake of P, Ca, and Mg by both plant species. However, application of higher 

levels of Si did not result in any further biomass increase in zucchini. Levels of Si in the 

nutrient solution had no effects on elemental composition and characteristics of the fruits of 

zucchini. In both plant species, the presence of PM on the leaves of plants resulted in these 

leaves accumulating higher levels of Si and Ca, but less P, than leaves of uninfected plants 

exposed to the same levels of soluble Si. The highest concentrations of Si were observed in 

leaf areas infected with PM, and around the bases of trichomes. For optimum disease control 

and maximum accumulation of different elements in these two plants, hydroponic applications 

of Si at 50-150 mg l -1 is recommended. 

Five selected biocontrol agents and potassium silicate, used as source of soluble Si, were 

tested under hydroponic conditions at various concentrations against PM of zinnia 

(Glovinomyces cichoracearum (DC) Gelyuta, V.P.). Application of BCAs resulted in 

reductions in final disease level and AUDPC values of PM by 38-68% and 30-65%, 

iii

espectively. Both severity and AUDPC values of PM were reduced by 87-95% when plants 

were supplied with Si (50-200 mg l -1 ). It is proposed that the provision of a continuous 

supply of Si and the ability of this plant species to accumulate high levels of Si in its leaves 

were the major reasons for the good response of zinnia to Si treatments against PM. Silicon 

played a protective role before infection and suppressed development of PM after infection. 

The combination of the best selected BCAs and Si can be used as an effective control option 

against PM of zinnia when grown in hydroponic system. 

iv

DECLARATION 

I, Habtom Butsuamlak Tesfagiorgis, declare that 

i. The research reported in this thesis, except where otherwise indicated, is my original work. 

ii. This thesis has not been submitted for any degree or examination at any other university. 

iii. This thesis does not contain other persons’ data, pictures, graphs or other information, 

unless specifically acknowledged as being sourced from other persons. 

iv. This thesis does not contain other persons’ writing, unless specifically acknowledged as 

being sourced from other researchers. Where other written sources have been quoted, then: 

a) their words have been re-written but the general information attributed to them has been 

referenced; 

b) where their exact words have been used, their writing has been placed inside quotation 

marks, and referenced. 

v. Where I have reproduced a publication of which I am an author, co-author or editor, I have 

indicated in detail which part of the publication was actually written by myself alone and 

have fully referenced such publications. 

vi. This thesis does not contain text, graphics or tables copied and pasted from the Internet, 

unless specifically acknowledged, and the source being detailed in the thesis and in the 

References sections. 

Signed:……………………………. 

H.B. Tesfagiorgis (Candidate) 

Signed:……………………………. 

Prof. M.D. Laing (Supervisor) 

v

ACKNOWLEDGEMENTS 

I wish to express my sincere appreciation to my supervisor, Prof. M.D. Laing, for his 

guidance, invaluable advice, encouragement and assistance in reviewing and editing this 

thesis. 

My thanks are extended to Dr M.J. Morris, for co-supervising the project and for his valuable 

insights and inputs. 

I also wish to thank my friends, Dr H.H. Kinfe, Dr M.G. Mengistu and Dr M.W. Bairu, for 

their support and encouragement at the crucial periods of this work. 

Further thanks go to: 

Proseed Pty Ltd, for providing the facilities to enable me to carry out the field investigation. 

The Department of Chemistry and the Electron Microscopy Unit, UKZN, for providing me 

their facilities to carry out analysis of plant samples. 

Dr C. Southway, for his technical advice and guidance during the elemental analysis. 

The staff and postgraduate students in the Disciplines of Microbiology and Plant Pathology for 

their assistance, whenever it was needed. 

Dr K.S. Yobo and Ms M.P. Mchunu for their assistance and encouragement in different parts 

of this study. 

I am also grateful to PQ Silicas SA, Plant Health Products (Pty) Ltd and National Research 

Foundation of SA for their funding of this research. 

Special thanks go to my parents, brothers and sisters for their love, encouragement and for 

always being there and believing in me. 

Finally, my sincere gratitude and thanks to God who makes all things possible. 

vi

DEDICATION 

To Bustuamlak Tesfagiorgis’s family for the support, 

understanding and spiritual encouragement 

during my studies. 

vii

TABLE OF CONTENTS 

ABSTRACT ................................................................................................................................. i 

DECLARATION ........................................................................................................................ v 

ACKNOWLEDGEMENTS ....................................................................................................... vi 

DEDICATION .......................................................................................................................... vii 

LIST OF FIGURES .................................................................................................................. xii 

LIST OF TABLES ................................................................................................................... xiv 

TABLE OF ACRONYMS ........................................................................................................ xv 

INTRODUCTION ...................................................................................................................... 1 

CHAPTER ONE ......................................................................................................................... 4 

Literature Review ....................................................................................................................... 4 

Application of biocontrol agents and soluble silicon for the control of powdery mildew ..... 4 

1.1 General introduction ......................................................................................... 4 

1.2 Powdery mildew ............................................................................................... 6 

1.2.1 Taxonomy of powdery mildew pathogens ........................................................ 6 

1.2.2 Epidemiology of powdery mildew .................................................................... 7 

1.2.3 Symptoms caused by powdery mildew ............................................................. 8 

1.2.4 Effects of powdery mildew in the plant ............................................................ 8 

1.2.5 Control strategies .............................................................................................. 9 

1.3 Biological control ........................................................................................... 13 

1.3.1 Characteristics of a successful biological control agent ................................. 16 

1.3.2 Mechanisms of action of biocontrol agents .................................................... 16 

1.3.3 Challenges and opportunities in the development of biocontrol against foliar 

diseases ........................................................................................................... 18 

1.3.4 Improvement of biocontrol efficacy ................................................................ 19 

1.4 Soluble silicon ................................................................................................. 20 

1.4.1 Effects of silicon on powdery mildew control ................................................ 20 

1.4.2 Effects of silicon on plant growth ................................................................... 21 

1.4.3 The role of silicon in amelioration of abiotic stresses ..................................... 21 

1.4.4 Possible mechanisms of action of silicon ........................................................ 21 

1.4.5 Methods of application of soluble silicon ....................................................... 22 

1.4.6 Challenges in using silicon.............................................................................. 23 

1.5 Opportunities for the use of biocontrol agents and silicon in an integrated 

disease management programme .................................................................... 23 

1.6 References ....................................................................................................... 26 

CHAPTER TWO ...................................................................................................................... 36 

Isolation and in vitro screening of potential biocontrol agents against powdery mildew .... 36 

Abstract ........................................................................................................... 36 

2.1 Introduction ..................................................................................................... 36 

2.2 Materials and methods .................................................................................... 38 

2.2.1 Collection of samples ...................................................................................... 38 

2.2.2 Isolation and culturing of potential biocontrol agents..................................... 38 

viii

2.2.3 Preparation of powdery mildew inoculum ...................................................... 39 

2.2.4 Screening of isolates on detached leaves ........................................................ 39 

2.2.5 Screening of isolates on whole plants ............................................................. 40 

2.2.6 Data analysis ................................................................................................... 41 

2.3 Results ............................................................................................................. 41 

2.3.1 Isolation and screening on detached leaves .................................................... 41 

2.3.2 Screening on whole plants .............................................................................. 44 

2.4 Discussion ....................................................................................................... 48 

2.5 References ....................................................................................................... 51 

CHAPTER THREE .................................................................................................................. 54 

Effects of adjuvants on the control of powdery mildew of zucchini when using foliar 

applications of soluble silicon and selected biocontrol agents ............................................. 54 

Abstract ........................................................................................................... 54 

3.1 Introduction ..................................................................................................... 55 


3.2.1 Effects of adjuvants and silicon on powdery mildew of zucchini .................. 56 

3.2.2 Scanning electron microscop studies on the effects of Break-Thru ® on 

Podosphaera xanthii and biocontrol agents.................................................... 57 

3.2.3 Biocompatibility test of Break-Thru ® ............................................................. 57 

3.2.4 Phytotoxicity test of Break-Thru ® on zucchini plants ..................................... 58 

3.3 Results ............................................................................................................. 58 

3.3.1 Effects of three adjuvants on powdery mildew control of with silicon .......... 58 

3.3.2 Visual and scanning electron microscopic observations on direct impact of 

Break-Thru ® on powdery mildew ................................................................... 60 

3.3.3 Biocompatibility of Break-Thru ® with biocontrol agents ............................... 63 

3.3.4 Phytotoxicity test of Break-Thru ® ................................................................... 66 

3.4 Discussion ....................................................................................................... 66 

3.5 References ....................................................................................................... 69 

CHAPTER FOUR ..................................................................................................................... 71 

Effects of concentration, frequency of application and runoff of foliar-applied silicon on 

powdery mildew of zucchini ................................................................................................. 71 

Abstract ........................................................................................................... 71 

4.1 Introduction ..................................................................................................... 72 


4.2.1 Preparation of plants ....................................................................................... 73 

4.2.2 Preparation of Podosphaera xanthii for inoculation ....................................... 73 

4.2.3 Application of soluble silicon ......................................................................... 73 

4.2.4 Disease assessment and statistical analysis ..................................................... 74 

4.3 Results ............................................................................................................. 74 

4.4 Discussion ....................................................................................................... 78 

4.5 References ....................................................................................................... 81 

ix

CHAPTER FIVE ...................................................................................................................... 84 

Studies on the effects of selected biocontrol agents and soluble silicon on the development 

of powdery mildew of zucchini, under greenhouse conditions ............................................ 84 

Abstract ........................................................................................................... 84 

5.1 Introduction ..................................................................................................... 85 


5.2.1 Preparion of plants and inoculation with Podosphaera xanthii ...................... 86 

5.2.2 Application of biocontrol agents and silicon .................................................. 86 

5.2.3 Disease assessment .......................................................................................... 87 

5.2.4 Data analysis ................................................................................................... 87 

5.3 Results ............................................................................................................. 88 

5.4 Discussion ....................................................................................................... 92 

5.5 References ....................................................................................................... 95 

CHAPTER SIX ......................................................................................................................... 99 

Use of selected biocontrol agents and silicon for the management of powdery mildew of 

zucchini under field conditions ............................................................................................ 99 

Abstract ........................................................................................................... 99 

6.1 Introduction ................................................................................................... 100 

6.2 Materials and methods .................................................................................. 101 

6.2.1 Trial site, land preparation and transplanting ................................................ 101 

6.2.2 Preparation of Podosphaera xanthii inoculum ............................................. 101 

6.2.3 Application of biocontrol agents and soluble silicon .................................... 102 

6.2.4 Disease assessment ........................................................................................ 102 

6.2.5 Statistical analysis ......................................................................................... 102 

6.3 Results ........................................................................................................... 103 

6.4 Discussion ..................................................................................................... 108 

6.5 References ..................................................................................................... 112 

CHAPTER SEVEN ................................................................................................................ 116 

Uptake and distribution of silicon in zucchini and zinnia, and its interaction with the uptake 

of other elements ................................................................................................................. 116 

Abstract ......................................................................................................... 116 

7.1 Introduction ................................................................................................... 117 


7.2.1 Preparation of plants ..................................................................................... 118 

7.2.2 Energy dispersive X-ray fluorescence (EDX) analysis ................................. 119 

7.2.3 Inductively coupled plasma-optical emission spectrometers (ICP-OES) 

analysis of plant tissues for elemental composisions .................................... 120 


7.3 Results ........................................................................................................... 122 

7.3.1 Observations from EDX-ESEM analysis and elemental mapping ............... 122 

7.3.2 Results of ICP-OES analysis ......................................................................... 126 

7.4 Discussion ..................................................................................................... 132 

7.4 References ..................................................................................................... 139 

x

CHAPTER EIGHT ................................................................................................................. 142 

Effects of selected biocontrol agents and silicon on powdery mildew of zinnia plants grown 

hydroponically .................................................................................................................... 142 

Abstract ......................................................................................................... 142 

8.1 Introduction ................................................................................................... 143 


8.2.1 Preparation of plants ..................................................................................... 144 

8.2.2 Design of the hydroponic system .................................................................. 144 

8.2.3 Preparation of biocontrol agents ................................................................... 145 

8.2.4 Preparation of powdery mildew inoculum and inoculation technique .......... 145 

8.2.5 Disease assessment ........................................................................................ 146 


8.3 Results ........................................................................................................... 147 

8.4 Discussion ..................................................................................................... 150 

8.5 References ..................................................................................................... 153 

CHAPTER NINE .................................................................................................................... 156 

General overview .................................................................................................................... 156 

9.1 References ....................................................................................................... 163 

xi

LIST OF FIGURES 

Figure 2.1 Plates showing preliminary screening of potential isolates using detached leaves inoculated with 

Podosphaera xanthii and treated with biocontrol agents ...............................…………….……………………...43 

Figure 2.2 Zucchini plants growing under greenhouse....………..…………………………………..……………43 

Figure 2.3 Efficacy of potential BCAs in controlling powdery mildew of zucchini after five weeks of treatment 

under greenshouse conditions…………………...………………………………………………………………...46 

Figure 2.4 Effects of selected isolates in development of powdery mildew of zucchini represented as area under 

disease progress curve (AUDPC) after five weeks of treatment under glasshouse conditions ..………………….47 

Figure 3.1 Relationships between adjuvants and Si applied at differing concentrations on severity (A) and 

AUDPC values (B) of powdery mildew of zucchini after five weeks of infection with Podosphaera xanthii …..61 

Figure 3.2 Effects of different adjuvants in controlling powdery mildew of zucchini in combination with foliarapplied 

Si at various concentrations. …………………………………………………………………...…………62 

Figure 3.3 Effects of different adjuvants in suppressing development of PM of zucchini with foliar-applied Si at 

various concentrations after five weeks of inoculation. AUDPC values are as a % of the untreated control 

AUDPC value……………………………………………………………………………………………………...62 

Figure 3.4 Plates of PDA showing the effects of Break-Thru ® on mycelia of Isolates EH (A) and H20 (B)…….63 

Figure 3.5 In vitro bioassay on the effects of Break-Thru ® mixed with growth medium on growth of BCAs. A: 

colony forming units of Isolates B15, Y15 and Y41 after two days of incubation at 30 o C. C.F.U. of Isolates B15 

and Y15 were diluted by 10 10 and Isolate Y41 by 10 9 ; B: mycelial growth of Isolates EH and H20 after two 

weeks of inoculation at 25 o C……….……………………………………………………………………………..64 

Figure 3.6 Environmental Scanning electron microscopic observations of the effects of Break-Thru® on 

Podosphaera xanthii and selected BCAs. A: Conidia and hyphae of the control; Plate 2: Collapsed and 

disintegrated hyphae and conidia of the fungus; C: Hyphae of the fungus sticking to each other; D: Conidia bond 

each other and BCAs deposited on top of the fungal structures; E: Enhanced deposition of BCAs on the surface 

of the pathogen; F: Trichothecium roseum (Isolate H20) establishing on the hyphae of the pathogen ………..…65 

Figure 4.1 Histograms showing the effects of spraying Si on the severity of powdery mildew of zucchini, when 

applied at various concentration and frequencies on plants grown under greenhouse conditions in open or covered 

pots in two different trials………………………………………………………………………… ……………...76 

Figure 4.2 Relationship between Si concentrations and frequency of sprays applied in open or covered pots on 

PM control of zucchini plants grown under glasshouse conditions in two different trials………………………..77 

xii

Figure 5.1 Percentage reduction in final disease levels of powdery mildew of greenhouse-grown zucchini by five 

biocontrol agents and soluble silicon compared to an Untreated Control treatment five weeks after inoculation 

with Podosphaera xanthii ………………………………………………………………………………………...90 

Figure 5.2 Percentage reduction of the area under disease progress curve (AUDPC) values of powdery mildew of 

greenhouse-grown zucchini by five biocontrol agents and soluble silicon compared to an Untreated Control 

treatment, five weeks after inoculation with Podosphaera xanthii ……………………………………………….91 

Figure 6.1 Efficacy of five biocontrol agents and silicon in reducing the severity of powdery mildew of zucchini 

after five weeks of inoculation with Podosphaera xanthii under field conditions …………..... ………. ……..106 

Figure 6.2 Efficacy of five biocontrol agents and silicon in reducing the AUDPC values of powdery mildew of 

zucchini after five weeks of inoculation with Podosphaera xanthii under field conditions .……………………107 

Figure 7.1 Energy dispersive X-ray (EDX) spectrums of silicon and other elements on leaves of zucchini and 

zinnia plants that received Si treatments of 0 (control), 100 mg l -1 and 100 mg l -1 plus Podosphaera xanthii and 

Glovinomyces cichoracearum, respectively……………………….. …… ……………………………………...123 

Figure 7.2 EDX mapping of silicon deposition on leaves of zucchini and zinnia plants that received Si treatments 

of 0, 100 mg l -1 and 100 mg l -1 plus Podosphaera xanthii and Glovinomyces cichoracearum……….……. ….124 

Figure 7.3 Scaning electron microscope and EDX mapping showing silicon deposition of Si at the base of 

trichomes of non-infected leaf of zucchini and in the leaf containing structures of the pathogen …………..…..125 

Figure 7.4 Effects of Si level in nutrient solution on the accumulation of Si in different parts of zucchini and 

zinnia plants after six weeks of growth in a nutrient recirculating system …..………………..………………...128 

Figure 7.5 Effects of concentrations of Si applied in nutrient solution on accumulations of K, P, Ca and Mg in 

different parts of zucchini after six weeks of growth in a nutrient re-circulating system……..………………....129 

Figure 7.6 Effects of concentration of Si applied in nutrient solution on accumulations of K, P, Ca and Mg in 

different parts of zinnia after six weeks of growth in a nutrient re-circulating system…………………………..130 

Figure 7.7 Effects of infection with powdery mildew fungi on the accumulation of Si, Ca and P in different parts 

of zucchini and zinnia plants grown in a nutrient re-circulating system for six weeks and supplied with Si at 

150 mg l -1 …………………………………………………………………………...……………………………131 

Figure 8.1 Horizontal mini troughs and their accessories used to supply nutrient solution to the plants in a recirculating 

system ……………….…………………………………………………………………………..…...145 

Figure 8.2 Percentage reductions in the disease severity and area under disease progress curve (AUDPC) of 

powdery mildew of zinnia after five weeks of treatment with five biocontrol agents soluble silicon, compared to a 

control inoculated with Golovinomyces cichoracearum…………………..…………..………………………149 

xiii

LIST OF TABLES 

Table 1.1 Lists of some fungicides that have been commonly used for specific reference to target powdery 

mildew fungi. .................................................................................................................................................. 11 

Table 1.2 Lists of powdery mildew species that have developed resistance to target fungicides .................. 11 

Table 1.3 Lists of biocontrol agents that have been tested against different species of powdery mildew of 

various crops. .................................................................................................................................................. 14 

Table 1.4 List of biocontrol products registered and commercially available (developed or being developed) 

against foliar pathogens. ................................................................................................................................. 15 

Table 1.5 Comparison of the benefits and limitations in terms of fungicides, silicon and biocontrol agents 

against plant diseases. ..................................................................................................................................... 25 

Table 2. 1 Sites where naturally infected leaves of host plants were collected .............................................. 42 

Table 2.2 Effect of selected biocontrol agents on final disease level (FDL) and AUDPC of powdery mildew 

of zucchini plans after 10 d and 5 wk of growth in a growth chamber and greenhouse, respectively. ........... 45 

Table 3.1 Effects of different adjuvants sprayed with soluble silicon (Si) at various concentrations on final 

disease level of powdery mildew of greenhouse grown zucchini after five weeks of infection with 

Podosphaera xanthii. ...................................................................................................................................... 59 

Table 3.2 Effects of different adjuvants and soluble silicon, sprayed at various concentrations, on AUDPC 

values of powdery mildew of zucchini grown under glasshouse conditions after 5 weeks of infection with 

Podosphaera xanthii ....................................................................................................................................... 60 

Table 4.1 Analysis of variance showing factorial interactions between concentration, frequency and runoff 

of foliar-applied silicon on severity of powdery mildew of zucchini. ............................................................ 78 

Table 5.1 Effects of selected biocontrol agents and soluble silicon (Si) on final disease level (FDL) and area 

under disease progress curve (AUDPC) of powdery mildew of greenhouse-grown zucchini five weeks after 

inoculation with Podosphaera xanthii. ........................................................................................................... 89 

Table 6.1 Effects of biocontrol agents and silicon on final disease level (FDL) and AUDPC values of 

powdery mildew of zucchini under field conditions .................................................................................... 105 

Table 8.1 Effects of selected biocontrol agents and silicon on the final disease level (FDL) and area under 

disease progress curve (AUDPC) of powdery mildew of zinnia, five weeks after inoculation with 

Golovinomyces cichoracearum .................................................................................................................... 148 

xiv

TABLE OF ACRONYMS 

ANOVA = Analysis of variance 

ARC-PPRI = Agricultural Research Council - Plant Protection Research Institute 

AUDPC = Area under disease progress curve 

BCA(s) = Biological control agent 

BK = Break-Thru ® 

DDW = De-ionized distilled water 

EDX = Energy dispersive X-ray fluorescence 

ESEM = Environmental scanning electron microscopy 

HDPE = High-density polyethylene () bottles 

ICP-OES = Inductively coupled plasma-optical emission spectrometers 

IPM = Integrated pest management 

ISR = Induced systemic resistance 

NA = Nutrient agar 

NB = Nutrient broth 

NDA = National Department of Agriculture 

PDA = Potato dextrose agar 

PM = Powdery mildew 

PR = Partner ® 650 

RCBD = Randomized Block Design 

SAR = Systemic acquired resistance 

T-80 = Tween-80 ® 

xv

INTRODUCTION 

In the Discipline of Plant Pathology, at the University of KwaZulu-Natal is a research team, 

called Biocontrol for Africa, which has been developing research momentum for the last 15 

years. As a result, promising progress is being made in identifying beneficial microorganisms 

for disease control and growth stimulation. The development and successful 

commercialization of Eco-T ® , a bio-fungicide product using a selected strain of Trichoderma 

harzianum Rifai. reflects the efforts being undertaken to achieve team goals, to deliver 

effective biocontrol products to farmers. The use of soluble silicon against biotic and abiotic 

stresses of plants has also shown promising results (Epstein, 1994, Bélanger et al., 1995). 

However, the relationships between Si and plants, Si and pathogens, and Si and the soil and 

other environmental factors, are not fully understood. In addition, Si is considered as safe and 

environmentally friendly. However, the option of using it integrated with application of BCAs 

for improved disease control, especially against PM, has not previously received much 

attention. 

As an alternative approach for sustainable disease management programme, the application of 

Si in agriculture is a fast growing area of interest, and central to our research group. Several 

projects are in progress to uncover some of the myths of Si in agriculture in order to obtain 

acceptable level of disease control and optimum plant growth, without compromising the 

quality of crop produce, or harming the environment. The research contained in this thesis 

reflects part of the ongoing research of the Biocontrol for Africa team. 

All the research was conducted at the University of KwaZulu-Natal, Pietermaritzburg, South 

Africa. The main emphasis of the research was to screen potential biocontrol agents and 

evaluate the efficacy of BCAs and silicon against PM of zucchini and zinnia caused by 

Podosphaera xanthii (Castagne) and Glovinomyces cichoracearum (DC) Gelyuta, V.P., 

respectively. 

1

Research activities included isolation of potential antagonists from various plant species that 

were naturally infected with powdery mildew (PM), and in vitro screening of these isolates 

against PM of zucchini. Applications of Si as drenches, sprays and in hydroponic nutrient 

solution were investigated in glasshouse conditions on the severity of PM of zucchini and 

zinnia. The efficacy of the best BCAs and Si applications, plus the combined effects of these 

two treatments against PM, was further evaluated under glasshouse and field conditions. 

Finally, the impact of Si levels in a nutrient solution on elemental uptake by zucchini and 

zinnia, and the relationship between PM-infection and elemental uptake by both plants was 

assessed using EDX and ICP-OES analysis. 

The objectives of this study were to: 

1. Review available literature on the use of biocontrol agents and silicon against PM plant 

diseases; 

2. Isolate potential biocontrol agents against PM, then evaluate their efficacy under 

greenhouse and field conditions; 

3. Study the effects of Si levels on PM control applied as drench, foliar treatment and in 

nutrient solution; 

4. Understand the mode of action of foliar-applied silicon, and to determine the effects of 

dosage, frequency and runoff of the spray on PM control; 

5. Improve the spray efficiency of Si and BCAs by testing various adjuvants; 

6. Evaluate the use of co-application of BCAs and silicon with Break-Thru ® for 

integrated disease management of PM; 

7. Evaluate the effects of Si levels in the nutrient solution on PM control, plant growth, 

quality of produce and nutrient uptake and accumulation by zucchini and zinnia; 

8. Study the relationship between PM-infection and elemental uptake by zucchini and 

zinnia; and 

9. Test the efficacy of the best BCAs plus Si on PM of zinnia grown hydroponically. 

The scope of this thesis is broad, containing nine chapters, each chapter covering specific 

aspect of the research conducted on the use of biocontrol agents and silicon Si against PM. 

Each of these chapters is presented as discrete paper, resulting in repetition of some references 

2

etween chapters. This is the standard format for MSc and PhD theses adopted by the 

University of KwaZulu-Natal. References have been formatted in the style of Crop Science. 

References 

Epstein, E. 1994. The anomaly of silicon in plant biology. Proceedings of the National Academy of 

Sciences of the United States of America, 91: 11-17. 

Bélanger, R.R., Bowen, P.A., Ehret, D.L., Menzies, J.G. 1995. Soluble silicon: Its role in crop and 

disease management of greenhouse crops. Plant Disease, 79: 329-336. 

3

CHAPTER ONE 

LITERATURE REVIEW 

APPLICATION OF BIOCONTROL AGENTS AND SOLUBLE SILICON FOR 

THE CONTROL OF POWDERY MILDEW 

1.1 GENERAL INTRODUCTION 

Production of enough palatable and safe food for the world's ever-increasing population 

presents a major challenge to the world’s farmers and agricultural experts. In the last few 

decades, agricultural production has increased as a result of an increase in cultivated land and 

the use of agrochemicals for crop production and protection. However, the deterioration in 

productivity of arable lands, coupled with climatic changes is a strong indicator that such 

environmentally unfriendly and unsustainable agricultural systems cannot solve the problem 

of food shortage in the long term. Food security and agricultural sustainability require both 

development of new and appropriate technologies and an understanding of the ecosystems in 

which they are to be implemented. The need is for a new type of agriculture, based on 

sustainable production without intense use of fertilizers and pesticides. This would lead to the 

development of different production strategies (Campbell, 1989). 

In the last 50 years attitudes toward the use of agrochemicals have changed, partly as a result 

of pressure from conservationists and consumers, and more recently from organic growers 

(Finch, 1992). Agricultural scientists are under pressure to find solutions for the needs 

presented by farmers who want to replace or reduce the use of agrochemicals for their organic 

production. Increased awareness of the impact of some agrochemicals to our health is also 

shifting consumers to demand products grown with a minimal use of chemicals. A demand for 

near zero residues in food crops is common in members of the Organizasion for Economic Cooperation 

and Develeopment (OECD). 

4

The use of biological control agents (BCAs) to control powdery mildew (PM) has been 

studied extensively. For many years, potential BCAs have been isolated from the phylloplane 

and tested for their ability to control PM. The most promising BCAs have been further 

developed and marketed as alternatives to the traditional chemical-based fungicides. Jutsum 

(1988) predicted that the global market value of biocontrol products should have a significant 

proportion of the market of the total expenditure on crop protection and public health remedies 

by 2000. However, the development in the use of BCAs has been slower than predicted, 

largely due to inconsistent results in different growing environments, the challenge of making 

biological control of foliar pathogens competitive with agrochemicals (Schippers, 1988) and 

excessively difficult registration procedures in OECD countries. For example, it currently 

takes 5-10 years to get a BCA registered in the EU (M.D. Laing, 2008, pers. comm.). To 

improve this situation, more fundamental knowledge is needed on the biotic and abiotic 

factors affecting the population dynamics, survival and antagonistic activity of BCAs in the 

phylloplane (Campbell, 1989). The successful development of BCAs is likely to depend on a 

thorough understanding of the biology and ecology of each pathogen and its antagonists, and 

their interactions with other phylloplane inhabitants. Other biotic and abiotic factors such as 

the concentration of the antagonist applied, inoculum density of the pathogen, host genotype 

and conduciveness of the environment to the disease also affect the survival and activities of 

BCAs (Landa et al., 2001). 

Silicon (Si), being the second most abundant element on the earth’s crust, is present in plants 

at 0.1-10% of their dry weight (Epstein, 1994, 1999). Research over the last 40-50 years has 

demonstrated that this element can benefit plants by ameliorating biotic and abiotic stresses. 

Significant control of PM, and a consequent increase in plant development and yield, has been 

demonstrated by a variety of plants as a result of the application of soluble Si, in both 

greenhouse and field trials (Bélanger et al., 1995; Bowen et al., 1992; Menzies et al., 1991b; 

Remus-Borel et al., 2005; Samuels et al., 1991a & 1994). Silicon is safe and environmentally 

friendly. However, since the level of disease control is related to the amount of Si absorbed by 

the plant, more research is needed to determine the optimum concentraion and delivery 

methods that can provide acceptable levels of PM control, without compromising the quality 

and quantity of crop products. 

5

1.2 POWDERY MILDEW 

Powdery mildews are widespread plant diseases that are conspicuous by their superficial white 

mycelia and powder-like conidia (Kiss and Szentivanyi, 2001; Yarwood, 1957). They are 

mostly host-specific, ranging from a single species to a family of plants, and are obligate 

parasites that cannot survive without their host plant. Most PM fungi infect only one family of 

plants. For instance, the species Podosphaera xanthii, (syn. Sphaerotheca fuliginea) that 

causes PM on the Cucurbitaceae, usually does not attack plants in any other family. There is, 

however, one report, where a PM of cucumber (P. xanthii) infected a bean plant (Phaseolus 

vulgaris) (Kiss and Szentivanyi, 2001). As a group, PM fungi infect many species of plants 

including cereals and grasses, vegetables, ornamentals, weeds, shrubs and trees. It is a 

common disease of vegetables under both field and greenhouse conditions in most areas 

worldwide. 

1.2.1 Taxonomy of powdery mildew pathogens 

Powdery mildew fungi belong to the ascomycete fungi, in the order of Erysiphales with only 

one family, the Erysiphaceae (Huckelhoven, 2005). They are further subdivided into five 

tribes (Erysipheae, Golovinomycetinae, Cystotheceae, Phyllactinieae, Blumerieae) and further 

sub-tribes, making more than 10 genera in total (Braun et al., 2002; McGrath, 1996). 

Classification of PM fungi is based on: the type of conidiophore, presence or absence of welldeveloped 

fibrisin bodies and mode of conidial germination (Sitterly, 1978). For instance, 

according to these criteria, this author classified major species of PM of Cucurbitaceae into 

three genera and six species. These include: E. cichoracearum (DC ex Mecat); E. polyphaga 

Hammarlund; Leveillula taurica (Lev) Arnaud; and S. fuliginea (Schlecht. ex Fr.) Poll. The 

genus Leveillula is considered as a synonym for Erysiphe. Among these, E. cichoracearum 

and S. fuliginea are the most common and important PM species on cucurbits. On the 

ornamental, zinnia, E. cichoracearum is the most common pathogen (Boyle and Wick, 1996; 

Kamp, 1985). Sphaerotheca fuliginea (syn. Podosphaera fusca) has been renamed recently as 

Podosphaera xanthii (Castagne) (Perez-Garcia et al., 2006; Shishkoff and McGrath, 2002), 

6

and E. cichoracearum renamed as Glovinomyces cichoracearum (DC) Gelyuta, V.P. (Keinath 

and DuBose, 2004; Kobori et al., 2004) 

1.2.2 Epidemiology of powdery mildew 

The PM fungi overwinter on plant debris as colonies of mycelium or in minute brown to black 

sexual reproductive structure (cleistothecia) (McGrath and Thomas, 1996; Yarwood, 1957 & 

1978). In the spring, the cleistothecia produce ascospores that can be transported onto 

susceptible host tissues by wind or rain splash (Iannotte, 2004). Once a spore (ascospore or 

conidium) lands on the host surface, it germinates within 2-3d and penetrates the host cell wall 

with its appressorium. Direct penetration of the host cell by PM fungi involves both enzymatic 

and mechanical power (Green et al., 2002 cited by Huckelhoven, 2005). Following penetration 

of the cell wall barrier, the fungus develops a haustorium (root-like structures) in the 

epidermal cells of the plant, from which it extracts nutrients through the plasma membrane of 

the host (Huckelhoven, 2005). 

Severity of PM depends on: the genotype, age and condition of the host plant and prevalent 

weather conditions of the growing season. The most favourable environmental conditions for 

PM are dry atmospheric and soil conditions, moderate temperatures, reduced light intensity, 

fertile soil and succulent plant growth (Yarwood, 1957). According to McGrath (1996) and 

Yarwood (1978), importance of PM in different regions varies, based on the above-mentioned 

conditions. Generally, incidence of PM increases as rainfall decreases. Unlike most other 

fungi, conidia of PM can germinate in the absence of water at relative humidity below 20% 

(McGrath and Thomas, 1996). This is because conidia of PM fungi have high water content 

with an extremely efficient water conservation system. In contrast, rain and availability of free 

moisture on the surface of the leave are unfavourable conditions for the development of the 

disease because they favour the survival and establishment of other microbes that are 

antagonists to the pathogen (McGrath, 1996). Increased relative humidity of the air is also 

reported to enhance germination of PM conidia (Sitterly, 1978). According to the same author, 

optimum temperature for germination of conidia is 23-31 o C, with a peak at 28 o C. The fungus 

dies in few hours if the temperature rises above 27 o C or drops below 1.1 o C. 

7

Powdery mildew is usually more severe under glasshouse than under field conditions. This is 

because glasshouses provide reduced air circulation and light intensities as well as higher 

temperature and continuous cropping, the combination that often results in severe PM 

epidemics (Howard et al., 1994). Incidence of PM increases as relative humidity (RH) rises to 

90 %, but it does not occur when leaf surfaces are wet (e.g., in a rain shower). This makes the 

disease common in crowded plantings, where air circulation is poor, and in damp, shaded 

areas. Howard et al. (1994) showed that young, succulent growth is usually more susceptible 

than older plant tissues. When the optimum environmental conditions are present, the disease 

spreads fast, resulting in production of large numbers of conidia for further cycles of infection 

(McGrath, 1996). 

1.2.3 Symptoms caused by powdery mildew 

The disease is noted with appearance of small, round, whitish, powder-like spots on leaf 

surfaces, petioles and stems (McGrath and Thomas, 1996). Symptoms appear first on crown 

leaves, on shaded lower leaves and on leaf undersurfaces. These white, powdery colonies 

grow in size and cover both sides of the leaf, petioles and young stems (Howard et al., 1994). 

Older leaves infected with P. xanthii turn a dirty-white with age; while those infected with 

G. cichoracearum remain white (Howard et al., 1994). Severely infected leaves become 

yellow, turn brown, and fall prematurely (Howard et al., 1994). Fruits rarely show visible 

symptoms of PM even in the presence of heavy infection. 

1.2.4 Effects of powdery mildew in the plant 

Although PM is rarely lethal, it is a major production problem, especially when the prevalent 

environmental conditions are ideal for its development (Choi et al., 2004). Severe infection of 

plants with PM can causes premature leaf senescence, resulting in reduced photosynthesis and 

transpiration efficiencies by the plant, leading to stunted and weakened plants (McGrath and 

Thomas, 1996). Loss of leaves and vigour in ornamental plants, as a consequence of severe 

PM infection, make the infected plants unsightly and of little commercial value as ornamentals 

(Gombert et al., 2001). The impact of PM on fruit is usually indirect because PM hardly ever 

infects fruit. However, it reduces the quality and quality of fruit by impairing the 

8

photosynthetic rate of the plant. This often results in sunburned or prematurely-ripen fruits that 

have a low market value (Bélanger et al., 1997). Fruits produced from severely infected plants 

are known to have a poor appearance, inferior storage quality and diminished flavor due to 

reduced levels of soluble solids (Keinath et al., 2000; McGrath and Thomas, 1996). In South 

Africa, annual economic loss of R7-8milions was estimated on cucurbits as a result of 

infection by PM (Haupt, 2007), and the value of fungicides used to control the disease on 

various PM-susceptible crops is estimated to be more than R40million per annum (M.D. 

Laing, 2004, pers. comm.). 

1.2.5 Control strategies 

(a) Cultural practices 

Any activity that alters the environmental conditions that favour germination of the conidia 

and survival of the PM fungi can reduce or prevent the disease. Howard et al. (1994) 

recommended that lowering plant populations in order to increase air circulation,reduce 

relative humidity and minimize shading. Avoiding excessive nitrogen fertilization can also 

reduce disease severity by avoiding succulent growth. In addition, removing infected leaves 

and destroying them, followed by cleaning the greenhouse thoroughly after each successive 

crop, can minimize spread of the disease and reduce survival of the fungus during winter and 

prevent a carry-over of PM from an infected crop to a new crop. Furthermore, since the fungus 

does not like high leaf wetness for its germination, spraying water onto infected leaves every 

2-3d can reduce its establishment. However, since leaf wetness can cause infection of the plant 

by other foliar pathogens, spraying should be commenced early in the morning in order to 

allow the leaf to be dry within 2-3hr (Howard et al., 1994). 

(b) Chemical control 

If cultural practices fail to prevent or control the disease, application of contact or systemic 

fungicides is the most widely used option (McGrath, 1996). Some of the commonly used 

fungicides against PM of various crops are listed in Table 1.1. Some of these fungicides 

provide effective control of PM when applied properly. McGrath and Shishkoff (1999) 

recommended that once the disease is detected, fungicides should be applied every 7-10d. 

9

Fungicide applications made to control PM should be effective against more than one disease 

since most crops suffer from several foliar diseases that are caused by more than one fungus 

(Keinath and Du Bose, 2004). 

Since PM develops on the upper and lower surfaces of the leaf, the spray must cover both 

sides for effective control (McGrath, 2001, McGrath et al., 1996). However, these authors 

believe that even with modern techniques of spray, it is difficult to directly deliver fungicide to 

the lower surface. Hence, the use of translaminar or systemic fungicides is the ultimate choice 

for fungicide spray programmes. Unfortunately, because of their single-site modes of action, 

these types of fungicides have been prone to the development of resistance by the pathogen 

(McGrath, 2005). 

In spite of some success in the use of fungicides for PM management, several researchers have 

reported on the development of resistance by PM fungi on multiple crops in different parts of 

the world (McGrath, 2001; McGrath et al., 1996). In addition, phytoxicity caused by 

fungicides may be a problem on some plants. For instance, during periods of intense solar 

radiation or high temperatures, application of chlorothalonil has been reported to injure mature 

watermelon fruit (Holmes et al., 2002). Similarly, Pasini et al. (1997) reported that repeated 

use of dodemorph resulted in the shortening of stems of roses and increased selection of 

resistant populations of PM. The rate at which populations of PM fungi became resistant to 

fungicides has been linked to repeated use of fungicides with one specific mode of action 

(Engels et al., 1996; O’Hara et al., 2000). Some of the PM species that have developed 

resistance to fungicides are presented in Table 1.2. 

10

Table 1.1 Lists of some fungicides that have been commonly used for specific reference to target powdery mildew fungi. 

Fungicide (active ingredient) Crop Target Pathogen Reference(s) 

azoxystrobin melon Podosphaera xanthii Romero et al., 2007 

azoxystrobin, benomyl, chlorothalonil, mancozeb, watermelon P. xanthii Keinath and DuBose, 2004 

myclobutanil, pyraclostrobin 

benomyl, bitertanol apple P. leucotrica NDA, 1999 

benomyl, bitertanol, bupirimate, carbendazim, fenarimol, tomato Oidium neolycopersici Jones et al., 2001 

pyrazophos, thiabendazole, triforine 

benomyl, fenarimol zucchini P. xanthii Bettiol, 1999 

benomyl, carbendazim, tebuconazole, triticonazole wheat Blumeria graminis f.sp. tritici NDA, 1999 

dodemorph rose Sphaerotheca pannosa var. Pasini et al., 1997 

rosae 

epoxiconazole barley B. graminis f.sp. hordei Barber et al., 2003 

fenarimol, polyoxin cucumber P. xanthii Choi et al., 2004. 

mancozeb, proquinazid, penconazole, grape Uncinula necator Oliva et al., 199; Pianella et al., 2006 

pyrazophos, triadimefon, tridemorph, triforine long melon P. xanthii Jiskani et al., 2000 

sulphur papaya Ovulariopsis papayae NDA, 1999 

triadimefon pumpkin P. xanthii McGrath, 1996 

NB: Sphaerotheca fuliginea and P. fusca are referred as P. xanthii (Perez-Garcia et al., 2006) 

Table 1.2 Lists of powdery mildew species that have developed resistance to target fungicides 

PM fungi Fungicides Host Reference(s) 

B. graminis f.sp. tritici fenpropimorph, propiconazole, quinoxyfen wheat Bernhard et al., 2002; Engels et al., 1996 

B. graminis f.sp. hordei fenpropimorph barley O’Hara et al., 2000 

P. xanthii azoxystrobin, benomyl, kresoxim-methyl, 

mycobutanil, propiconazole, triadimefon 

cucurbit Ishii et al., 2001; McGrath, 2001; McGrath and Shishkoff, 

2001; McGrath et al., 1996 

U. necator azoxystrobin, myclobutanil grape Northover and Homeyer, 2001; Wong and Wilcox, 2002 

11

(c) Other control options 

Pasini et al. (1997) showed that the use of several antifungal compounds such as salts, oils and 

plant extracts against PM of rose were as efficient as spraying the fungicide dodemorph. They 

also noted that spray applications of KH 2 P0 4 and NaHC0 3 at 0.5-1% offered good control of 

PM. Wine vinegar, JMS Stylet Oil ® , canola oil, synertrol and neem extract also provided 

satisfactory disease control. In addition, fatty acids formulated as potassium salts reduced the 

severity of the disease significantly. Partial control of PM was obtained from Milsana ® , a 

concentrated extract from leaves of Reynoutriu sachulinensis (F. Schmidt) Nakai (Pasini et al., 

1997). Bettiol et al. (1999) showed that foliar application of a mixture of cow’s milk and water 

was effective in preventing infection of caused by P. xanthii. Similarly, spraying milk-based 

products onto pumpkin reduced PM and other foliar symptoms by 50-70% and post harvest 

fruit rot by 40-50% compared to the chemical control (Ferrandino and Smith, 2007). Bélanger 

et al. (1997) have reviewed a range of plant extracts and biocompatible products that have 

been used against PM. 

(d) Integrated control 

Integrated disease management is the practice of using a combination of control measures to 

prevent and manage diseases in crops. Strategies that integrate BCAs and chemical fungicides 

can overcome the lower efficacy of antagonists, while reducing the residues of chemical 

fungicides on the final products and the environment. The idea of IPM is based on good 

agricultural practices needed to produce profitable and productive crops in a sustainable way. 

Once the symptom is observed or a potential for infection is identified, different techniques 

can be used as preventative or curative measures to minimize the risk of disease infection and 

spread. 

Combinations of different control strategies have provided promising disease control when 

tested against PM of various crops. For instance, application of Ampelomyces quisqualis Ces 

ex Schlect, Trichoderma harzianum Rifai and Bacillus subtilis (Ehrenberg) Cohn, with lower 

rates of fungicides provided the same level of control of PM of strawberry as fungicides 

applied at full rates (Pertot et al., 2008). Similarly, a mixture of BCAs (B. subtilis, Tilletiopsis 

minor Nyland and Lecanicillium lecanii (Zimm.) Viégas), mineral salts (KH 2 PO 4 ), an 

12

antitranspirant (kaolin) and an antioxidant (ascorbic acid) controlled PM of mango effectively 

(Nofal and Haggag, 2006). A combination of BCAs and chemicals can give better results than 

when they are applied separately. Combination of these approaches can control the pathogen 

in climatic conditions beyond the effective range of the bio-protectant, minimize 

environmental pollution and the likelihood of the pathogen developing resistance, while 

providing localized and persistent control (Tronsmo and Hjeljord, 1998). 

1.3 BIOLOGICAL CONTROL 

Biological control, as a crop protection strategy system, emerged as a response to the search 

for a safe, effective and environmentally friendly approach to replace or supplement the use of 

chemical pesticides. Biological control of plant diseases involves the use of antagonistic 

microorganisms to control a pathogen. One form of biological control occurs if the activity of 

a microorganism, e.g., a plant pathogen, is controlled by another member of the community 

(Campbell, 1989). 

Over the past three decades, research has repeatedly demonstrated that many microorganisms 

can act as natural antagonists to plant pathogens (Cook, 2000). Powdery mildew fungi are 

prime targets for biocontrol agents because of their superficial growth (Bélanger et al., 1997). 

This has attracted many researchers to conduct intensive investigations in order to find 

antagonists that can provide acceptable levels of disease control. The mostly studied 

microorganisms against PM of different species are listed in Table 1.3. Although consistency 

has been the major challenge in controlling PM with these antagonists, most of these BCAs 

have produced promising results in suppressing PM. The success and failure of biological 

control depends amongst other things, on the production and application of effective BCAs. 

Through research, some of the most successful BCAs have been processed into commercial 

products that are approved for the market. Table 1.4 is a summary of some of the promising 

biological products against foliar diseases that have been commercialized. However, it should 

be noted that there are many more which are not listed on Table 1.4. 

13

Table 1.3 Lists of biocontrol agents that have been tested against different species of powdery mildew of various crops. 

BCA Target Pathogen Crop Reference(s) 

Ampelomyces quisqualis E. cichoracearum cucumber Sundheim, 1982 

P. aphanis strawberry Pertot et al., 2008 

P. leucotricha apple Ozentivanyi and Kiss, 2003 

P. xanthii cucumber, melon, 

pumpkin, squash, winter 

squash 

P. xanthii cucumber Elad, 2000; Elad et al., 1998 

NB: Sphaerotheca fuliginea and P. fusca are referred as P. xanthii (Perez-Garcia et al., 2006); P. flocculosa as S. flocculosa and P. rugulosa as 

S. rugulosa (Eken, 2005) and Verticillium lecanii as Lecanicillium lecanii (Verhaar et al., 1998). 

14 

Abo-Foul et al., 1996; Dik et al., 1998; Elad et al., 1998; 

McGrath and Shishkoff, 1999; Romero et al., 2003; 

Shishkoff and McGrath, 2002; Sundheim, 1982 

S. pannosa var. rosae rose Pasini et al., 1997 

U. necator grape Falk et al., 1995 

Various spp. various crops Sztejnberg et al., 1989 

Acremonium alternatum P. xanthii melon Romero et al., 2003 

Bacillus sp. P. xanthii cucurbits Romero et al., 2004 

B. subtilis P. xanthii squash zucchini Bettiol, 1999 

P. aphanis strawberry Pertol et al., 2008 

Lecanicillium lecanii P. xanthii cucumber, melon Askary et al., 1998; Dik et al., 1998; Romero et al., 2003; 

Verhaar et al., 1996 

S. macularies f.sp. fragariae strawberry Miller et al., 2004 

Meira geulakonigii P. xanthii cucumber Sztejnberg et al., 2004 

Sporothrix flocculosa P. xanthii cucumber Dik et al., 1998 

S. pannosa var. rosae rose Bélanger et al., 1994 

Several spp. several plants Neveu et al., 2007 

Sporothrix rugulosa P. xanthii cucumber Verhaar et al., 1996, 1998 

Stephanoascus flocculosus P. xanthii cucumber Jarvis et al., 1989 

S. pannosa var. rosae rose Hajlaoui and Bélanger, 1991 

Stephanoascus rugulosus P. xanthii cucumber Jarvis et al., 1989 

S. pannosa var. rosae rose Hajlaoui and Bélanger, 1991 

Tilletiopsis albescens P. xanthii cucumber Knudsen and Skou, 1993 

E. graminis f.sp. hordei barley Knudsen and Skou, 1993 

T. minor P. xanthii cucumber Hijwegen, 1992 

T. washingtonensis S. pannosa var. rosae rose Hajlaoui and Bélanger, 1991; Sztejnberg et al., 1989 

Trichoderma harzianum S. mors-uvae gooseberry Picton and Hummer, 2003 

P. aphanis strawberry Pertot et al., 2008

Table 1.4 List of biocontrol products registered and commercially available (developed or being developed) against foliar pathogens. 

Product Name BCA (s) Target Pathogen Producer Reference(s) 

AQ10 Biofungicide Ampelomyces quisqualis Powdery mildews Ecogen Inc, USA Hofstein and Chapple, 1998; 

McGrath and Shishkoff, 1999; 

Shishkoff and McGrath, 2002 

Aspire Candida oleophila Botrytis and Penicillium spp. Ecogen Inc, USA Droby et al., 1998 

BINAB-T ® T. harzianum + T. 

polysporum 

Various fungal diseases Bio-Innovation EFTR 

AB, Sweden 

Moller et al., 2003; Mommaerts et 

al., 2008 

Eco-77 ® T. harzianum Botrytis cinerea, Eutypa lata, Plant Health Products, Plant-Health, 2008 

E. leptoplaca 

South Africa 

QST 713, 

AgraQuest, Inc., USA EPA, 2005; Marrone, 2002 

Serenade TM downy mildew, early blight, 

late blight, bacterial spot, 

walnut blight 

Sporodox ® P. flocculosa Powdery mildew Plant Products Co. Ltd, 

Canada 

Konstantinidou-Doltsinis et al., 

2007 

Trichodex T. harzianum Various fungal diseases Makteshim Chemical Dik et al., 1999; Elad et al., 1998; 

Works Ltd, Israel. Etebarian et al., 2000; Hofstein and 

Chapple, 1998 

Trichodowels T. harzianum + T. viride Various fungal disease Agrimm Technologies Monte, 2001 

Ltd, New Zealand 

TrichoFlow WP TM T. harzianum Various fungal diseases Agrimm Technologies Bal and Altintas, 2006; Fourie and 


Trichopel ® T. harzianum Wide range of fungal diseases Agrimm Technologies 


Halleen, 2006 

Bhuiyan et al., 2003 

Trichoseal T. viride Trunk and foliar pathogens Agrimm Technologies 


YieldPlus Cryptococcus albidus Botrytis and Penicillium spp. Anchor Yeast, South 

Africa 

http://www.tricho.com/trichotech 

Droby et al., 2002 

15

1.3.1 Characteristics of a successful biological control agent 

A BCA must firstly be safe to humans, animals and the environment. It should have the 

ability to grow and colonize the phylloplane fast, produce large number of spores and 

survive with a minimal requirement for nutrients and favourable environmental conditions 

(Blakeman and Fokkema, 1982; Chao et al., 1986). Ideally, a BCA should also have some 

means by which it can survive under unfavourable environmental conditions. For instance, 

Bacillus species produce spores which are resistant to UV light and desiccation and can be 

formulated easily (Raaijmakers et al., 2002). 

Active colonization of available substrates by BCAs can greatly reduce the success of 

pathogen inocula attempting to infect host plants. For instance, infection of host plants by 

PM usually occurs within 72h after the conidia of the pathogen land on a susceptible leaf 

(Huckelhoven, 2005). Therefore PM can only be controlled if there is active and rapid 

colonization of the phylloplane by the BCA after it is sprayed onto the leaves of the target 

crop. In addition, an ideal BCA should be competitive with agrochemicals in terms of cost 

and efficacy. 

1.3.2 Mechanisms of action of biocontrol agents 

During the development of beneficial microbes for the control of plant diseases, scientists 

recognized that an effective delivery system requires a thorough understanding of its 

biological relationship with its target (Bateman and Chapple, 2001). There are at least four 

mechanisms by which BCAs act against a target PM pathogen: competition for resources, 

antibiosis, mycoparasitism and induction of host resistance (Whipps, 1992). 

Competition for soluble substrates occurs when two (or more) organisms require the same 

resource (Tronsmo and Hjeljord, 1998), and the use by one reduces the amount available to 

the other (Campbell, 1989). In such cases, one organism uses most of the nutrients and 

grows, while the other has insufficient nutrients for its growth and dies. This is typical for a 

fungus or bacterium that grows very fast and overwhelms the target organism. However, 

the use of competition for nutrients on the phylloplane as a biocontrol strategy seems 

unlikely against PM because they can germinate without exogenous nutrients and cover 

16

elatively large areas of leaves within 1-2wk after infection (McGrath and Thomas, 1996). 

However, Elad et al. (1998) showed that competition is one of the mechanisms that is used 

by T. harzianum T39 during its establishment on the phylloplane. 

Antibiosis occurs when the production of toxic metabolites or antibiotics of one organism 

has a direct negative effect on another organism. In pure culture, antibiotic production is 

common by many potential BCAs (Tronsmo and Hjeljord, 1998). It appears to be important 

to the survival of microorganisms through elimination of microbial competition for food 

sources, which are usually very limited in the phylloplane. Several species of Bacillus 

(Romero et al., 2004 & 2007) and yeast (Hajlaoui and Bélanger, 1991 & 1993; Urquhart 

and Punja, 2002) are known to use antibiotic production as their primary mode of action in 

suppressing PM. Production of ß-1,3-exo and endo-glucanase, chitinase and antifungal 

compounds by these antagonists was reported to inhibit germ tube development of P. 

xanthii and plasmolyse its spores (Urquhart and Punja, 2002). Many Trichoderma spp. are 

known to produce antibiotics that are is effective against a wide range of pathogens (Elad et 

al., 1998). 

Another mechanism utilized by BCAs is mycoparasitism. This is a parasitism of PM fungus 

by the antagonist fungus. It involves direct contact between the fungi resulting in death of 

the host (PM pathogen), and nutrient absorption by the parasite (antagonist) (Whipps et al., 

1988). To break down the walls of their host, mycoparasites possess various enzymes such 

as: cellulases, chitinases, β-1,3-glucanases and proteases (Elad, 1996 & 2000; Hijwegen, 

1992; Patil et al., 2000; Urquhart and Punja, 2002). The interaction between mycoparasites 

and their target fungi occurs in four sequential, but overlapping phases: target location, 

recognition, contact and penetration (Whipps et al., 1988). In the first stage, a chemical 

stimulus from the pathogenic fungus attracts the parasite (the antagonist). The second step 

involves attack of the target pathogen by the mycoparasite with the help of enzymes. In the 

third step, the mycoparasitic antagonist attaches to the host (pathogenic fungi) either by 

coiling around or growing alongside it. In the final step, the mycoparasitic fungus degrades 

the pathogenic cell wall by producing various enzymes (Tronsmo and Hjeljord, 1998). 

17

Some researchers have shown that BCAs may act against the pathogen by triggering host 

defense mechanisms. In induced systemic resistance (ISR), the BCA is not directly 

involved with the pathogen; instead, it activates the defense mechanisms of the host (Wei et 

al., 1996). Once ISR is expressed, it activates multiple potential defense mechanisms 

including increased production of antifungal compounds by the host (Elad, 2000; Wei et al., 

1996). According to these authors, ISR has a wide range spectrum, protecting the plant 

from variety of pathogens. Trichoderma harzianum T39 (Elad, 2000; Elad et al., 1998) and 

some growth promoting bacteria such as B. pumulus Meyer and Gottheil, Pseudomonas 

putida (Trevisan) and Serratia marcescens (Bizio) (Wei et al., 1996) have been reported as 

BCAs that induced systemic resistance. 

During screening, it is important to consider the different mechanisms of action, because 

inhibition of the pathogen by an introduced antagonist may involve different interactions. In 

addition, since the control system involves BCAs, pathogen, host and the environmental 

conditions, it is important to incorporate these components during the screening. Screening 

techniques often focus on detecting one or two modes of action and therefore fail to detect 

BCAs using other modes of action. 

1.3.3 Challenges and opportunities in the development of biocontrol against foliar 

diseases 

Biological control has been less successful in the phyllosphere than the rhizosphere 

(Andrews, 1992). This is partly due to extreme fluctuations in environmental conditions in 

the aerial part of the plant compared to the soil parts. Blakeman and Fokkema (1982) noted 

that the survival and activities of both saprophytes and pathogens on leaves is dependent on 

the microclimatological conditions and chemical properties of the leaf surface. Because of 

their requirements for specific environmental conditions, BCAs are usually effective within 

a limited range of temperature and RH. For instance, most antagonists of PM are more 

efficient when RH is maintained above 80% (Bélanger et al., 1994 & 1997; Hijwegen, 

1992; Jarvis and Slingsby, 1977; Jarvis et al., 1989), which can be achieved by 

manipulating the greenhouse environment. However, this is not possible under field 

conditions. Hence, efficacy of most BCAs against PM and other foliar diseases has been 

inconsistent, especially under field conditions. In addition, BCAs rely on the availability of 

nutrients for their survival and production of antibiotics and siderophores. Unfortunately, 

18

the amount of nutrients on the phylloplane is often limited and may be leached as a result of 

irrigation, rain, dew and fog (Andrews, 1992). 

The rate of establishment and biocontrol activities of introduced antagonists is often slower 

than that of the pathogen. For instance, conidia of PM require 3-5d to germinate and infect 

the plant (Huckelhoven, 2005; McGrath and Thomas 1996). In contrast, except for a few 

antagonists such as A. quisqualis (Sundheim and Krekling, 1982), establishment of BCAs 

on the phylloplane may take 10-14d, by which time the disease is at a high level. Once the 

disease pressure is very high, efficacy of BCAs is limited (Elad et al., 1998; Pertot et al., 

2008). Moreover, some introduced BCAs need the presence of their host on the phylloplane 

in order to establish themselves. For example, T. minor was more effective when applied 2d 

after the plant was infected with PM (Hijwegen, 1986). 

In spite of these challenges, use of BCAs on the phylloplane does provide some 

opportunities. The method of application of BCAs is simple, and it is easy to see the effects 

of treatments because the disease is visible. Once applied and established, BCAs can move 

onto different parts of the foliage, and may provide long-term control without the risk of 

resistance. 

1.3.4 Improvement of biocontrol efficacy 

Since biological control is holistic in its approach, it is important to combine the 

manipulation of different aspects that are necessary to favour the antagonist and 

disadvantage the pathogen (Andrews, 1992). For instance, growth of introduced BCAs can 

be assisted by manipulation of the microclimate of greenhouses (Blakeman and Fokkema, 

1982). Adjusting the RH to the level required by the antagonist or providing free water on 

the leaf surface can improve the efficacy of biological control. However, continuous 

monitoring is needed because increased leaf wetness can result in infection of the plant by 

other pathogens. In addition, the chemical properties of the leaf surface can also be 

modified to suit the demand of the antagonist by applying the right chemical. Growing 

BCAs in suitable media, applying correct dosage of BCA inoculum, using appropriate 

formulations and storage and placing inoculum in favourable positions can facilitate active 

colonization of the phylloplane by the antagonist. Application of BCAs before the disease 

reaches a certain level, and use of selected adjuvants and oils can improve efficacy of 

19

iological control. Elad et al. (1996) noted that the efficacy of Tilletiopsis spp. was best 

when applied few days before or immediately after infection of the plant with PM. 

Furthermore, use of additives, such as sucrose, can increase the population of the antagonist 

on the phylloplane (Blakeman and Fokkema, 1982). However, growth of other pathogens 

must be managed carefully. 

1.4 SOLUBLE SILICON 

Silicon (Si) is the second most abundant element in the surface of the earth and can present 

in plants in amounts equivalent to those of macronutrients such as calcium, magnesium and 

phosphorus (Epstein, 1994). Although the level of Si in plant tissues ranges between 0.1- 

10%, sometimes exceeding that of nitrogen and potassium, its importance to agriculture has 

been underrated with the perception that most plants can grow in a nutrient solution without 

Si in their formulation (Epstein, 1999). However, when Si is readily available to plants, it 

plays a major role in their survival and growth. Some of the beneficial effects of Si to plants 

include increased growth and decreased susceptibility towards pathogens and insects, and 

amelioration of abiotic stresses (Epstein, 1994 & 1999). 

1.4.1 Effects of silicon on powdery mildew control 

The effects of Si on incidence and severity of PM on a range of crops has been studied by 

several researchers (Bélanger et al., 1995; Menzies et al., 1991b; Samuels et al., 1991a & 

1994). Bélanger et al. (1995) noted that adding Si into nutrient solutions reduced incidence 

of PM in cucumber and delayed its rate of development. According to Menzies et al. 

(1991a), the reduction in the severity of PM was due to inhibition of germination of PM 

conidia by Si. Similarly, treatment with Si reduced PM of barley (Wiese et al., 2005), grape 

(Bowen et al., 1992; Reynolds et al., 1996), muskmelon (Menzies et al., 1992), wheat 

(Bélanger et al., 2003; Remus-Borel et al., 2005) and zucchini (Menzies et al., 1992). 

Whilst the use of Si for disease management is safe and effective, in most reports complete 

control of the disease was not obtained, with results differing among cultivars (Liang et al., 

2005; Palmer et al., 2006), cropping seasons and temperature (Schuerger and Hammer, 

2003). 

20

1.4.2 Effects of silicon on plant growth 

Bélanger et al. (1997) noted that, in Europe, 60% of cucumber and 30% of rose growers 

were using soluble Si on a regular basis. Most of those users believed that they had 

benefited by reducing fungicide application and increasing yields. Even in the absence of 

disease, application of Si has been observed to increase yields of cucumber. However, it is 

not clear if this increase was as a result of disease control or the management of several 

biotic and abiotic stresses of the plants. 

1.4.3 The role of silicon in amelioration of abiotic stresses 

Silicon has been reported to ameliorate mineral toxicity effects in plants (Epstein, 1994; 

Marschner, 1995). For instance, Si can reduce or prevent toxicity effects caused by 

aluminum (Al), iron (Fe) and manganese (Mn) (Marschner, 1995; Tisdale et al., 1993). 

Equally, it can reduce the impact of nutrient deficiencies of phosphorus (P) and zinc (Zn) 

(Epstein, 1994; Marschner, 1995). In addition, it reduces stress of plants caused by soil 

salinity and excessive transpiration (Epstein, 1994; Marschner, 1995; Tisdale et al., 1993). 

Moreover, in sugar cane, Si protected leaves from ultraviolet radiation damage by filtering 

out harmful ultraviolet rays (Tisdale et al., 1993). 

1.4.4 Possible mechanisms of action of silicon 

Although consistent results have been reported on the positive effects of Si in disease 

management, the mechanisms by which Si reduces severity of diseases are the subject of 

ongoing debate (Ghanmi et al., 2004). While several researchers believe that the possible 

mechanism of action of Si is purely mechanical, others claim that it is entirely based on the 

catalysis of induction of systemically acquired resistance (SAR) by the host plants. 

Zeyen et al. (1992) associated reduction of PM to be a result of increased in silification of 

the epidermal cells, which would impede the penetration by the pathogen germ tubes. 

Supportive reports by Carver et al. (1998) and Winslow (1992) showed that increased 

resistance against fungal pathogens in cereal species was correlated with the accumulation 

of Si in these plants. However, further investigations revealed that silification takes place at 

the trichome bases on the epidermis (Bélanger et al., 1995, Samuels et al., 1994), and 

around the fungal hyphae and infection pegs in infected cells of the host plant (Bélanger et 

21

al., 1995; Datnoff et al., 1997). Microscopic studies by Menzies et al. (1991b) showed 

increased accumulation of phenolic compounds within cells of cucumber plants treated with 

Si. Similarly, Fawe et al. (1998) discovered that treatment with Si induced defense 

reactions in cucumber plants towards PM by promoting accumulation of antifungal 

flavonoids. Moreover, Bélanger et al. (2003) reported that treatment with Si induced 

defense mechanisms in wheat against B. graminis f.sp. tritici by promoting accumulation of 

electron-dense, phenolic compounds surrounding fungal haustoria within the epidermal 

cells of these plants. Using X-ray microanalysis and microscopic investigations, Ghanmi et 

al. (2004) found that treating Arabidopsis thaliana (L.) Heynh with Si induced the 

production of electron-dense, fungitoxic substance that accumulated within and around the 

collapsed haustoria of epidermal cells within the leaves of the disease-resistant plants. 

Furthermore, Si was shown to increase resistance against blast disease on rice caused by 

Magnaporthe grisea (T.T. Hebert) M.E. Barr. by catalysing the accelerated production of 

phenolic like compounds and antifungal momilactone phytoalexins (Rodrigues et al., 

2004). Most of these conclusions were made based on applying Si in the nutrient solution. 

However, foliar application of Si has also provided effective control of PM in several crops 

(Bowen et al., 1992; Guével et al., 2007; Liang et al., 2005; Menzies et al., 1992; Palmer et 

al., 2006; Reynolds et al., 1996). What is not clear is whether similar mechanisms of action 

are involved when Si is used as a foliar treatment as when it is applied to plant roots as a 

fertilizer. 

1.4.5 Methods of application of soluble silicon 

Usually Si has been applied to plants by adding soluble forms of the element into a nutrient 

solution of a hydroponics system. This system ensures a continuity of supply of Si to the 

plants while the pH and concentration of each element in the nutrient solution can be 

balanced according to the requirements of the plants. With drip irrigation, Si may be added 

into the irrigation stream at the required level, in order to supply plants through the 

irrigation system. Under field conditions, Si is normally applied as drench or through the 

irrigation system (i.e. sprinkler or other techniques). Silicon can also be applied to plants as 

a foliar spray. Foliar application is commonly used to control foliar diseases directly rather 

than through amelioration of abiotic stresses of the plant related to the soil. 

22

1.4.6 Challenges in using silicon 

Although many studies have been made on Si in agriculture in the last 50 years in order to 

understand the role of Si in agriculture, its exploitation has been limited. This is partly due 

to inconsistency of the results under different conditions on different species. Susceptibility 

of plants to stress, and weather conditions and the characteristics of the soils such as soil 

physical properties, soil organic matter and soil chemistry have direct effects on the role of 

Si to plants. Bélanger (2008) noted that beneficial effects of Si are noticeable only when the 

plant is under stress. Unlike curative fungicides that can eradicate the disease completely, 

Si is more effective when applied as a protective treatment (Liang et al., 2005; Samuels et 

al., 1991b), and it is less effective under situations of high disease pressure (Kanto et al., 

2007). Since Si is interactive with other elements, such as Ca, Al and Mn, its availability to 

plants is dependent on presence of other elements and the soil pH (Epstein, 1994 & 1999). 

A lack of knowledge on optimum concentrations and application frequencies is another 

reason for incomplete control of diseases. For instance, application of high concentrations 

of Si to cucumber may provide effective control of PM, but may result in reduced quality of 

the fruit (Bélanger et al., 1997). Many researchers believe that disease resistance is linked 

with the amount of Si accumulated by the plant (Epstein, 1994; Jansen, 2004). However the 

level of Si that should be applied in order to give optimum disease control without 

compromising growth and quality of the produce is an issue of continuous research 

(Bélanger et al., 1995). A further challenge is that uptake of Si varies not only between 

crops (Mitani and Ma, 2005), but may also vary between cultivars within a single crop 

(Ago et al., 2008). 

1.5 OPPORTUNITIES FOR THE USE OF BIOCONTROL AGENTS AND 

SILICON IN AN INTEGRATED DISEASE MANAGEMENT PROGRAMME 

Each control option has its unique strengths and weaknesses relative to the other. Individual 

disease management options often result in incomplete disease control. This raises a 

question whether the use of biocontrol agents and Si can supplement each other for better 

disease control in a sustainable way. 

23

An IPM programme may involves the combined use of chemical, biological, physical, 

biotechnical, genetic and agricultural techniques of disease control that offers the 

opportunity to growers to choose the most appropriate intervention once a threshold disease 

level is reached (Oliva et al., 1999). According to these researchers, unlike chemical 

control, where the main objective is to eradicate the disease, the aim of IPM is to keep the 

disease level below the tolerance threshold by intervening only when the severity of the 

pathogen exceeds a certain level. 

The challenge to control PM with BCAs and Si is the extremely rapid development of the 

disease. Therefore, there is a need to find a means by which the rate of disease progress can 

be reduced so that the use of BCAs and Si can be more effective. If this challenge remains 

unsolved, farmers will continue to depend on using fungicides as their only effective tool to 

control PM. In South Africa, the proposed approach of using biological control and Si for 

disease management and plant growth promotion is getting encouraging acceptance by the 

farmers. Most soils in Africa are acidic and deficient in plant-available Si due to leaching. 

Therefore, application of Si fertilizer to crops should benefit most crops in most situations. 

Use of Si, combined with BCAs, by commercial farmers could replace the intensive use of 

agrochemicals in Africa. 

24

Table 1.5 Comparison of the benefits and limitations in terms of fungicides, silicon and biocontrol 

agents against plant diseases. 

Criteria Fungicides Silicon BCAs 

Efficacy 

• Short term 3 2 1 

• Long term 1 2 3 

• Rapidity of effect 3 2 1 

• Effect under high disease pressure 3 1 1 

• Curative control 3 1 1 

• Preventative control 3 3 3 

Reliability 

• Environmental influences 3 2 1 

• Influence of plant and soil 2 1 1 

• Shelf life 2 3 1 

Reduction in risks 

• Risk of resistance 1 3 3 

• Side effects to humans and animals 1 3 3 

• Residues in food 1 3 3 

• Persistence in the environment 1 3 3 

Public acceptance 1 2 3 

Profitability to producer 2 3 2 

Cost effectiveness 1 2 3 

Long-term benefit for society 1 3 3 

1 (least), 2 (intermediate) and 3 (most) relative advantage of the control method. The table 

and criteria for comparison was adopted and modified from Yobo (2005). 

25

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35

CHAPTER TWO 

ISOLATION AND IN VITRO SCREENING OF POTENTIAL BIOCONTROL 

AGENTS AGAINST POWDERY MILDEW 

H.B. Tesfagiorgis a , M.D. Laing a and M.J. Morris b 

a Discipline of Plant Pathology, School of Agricultural Sciences and Agribusiness 

University of KwaZulu-Natal, Private Bag X01, Scottsville 3209, South Africa 

b Plant Health Products, P.O. Box 207, Nottingham Road, South Africa 

Abstract 

Powdery mildew (PM) is an important disease of many plants worldwide. Potential 

antagonists of powdery mildew were isolated from naturally infected leaves of different 

plants. A total of 2000 isolates of bacteria, fungi and yeasts were tested in a preliminary 

screening on detached leaves. The best 30 isolates showing consistent results were further 

tested under glasshouse conditions for their efficacy against PM of zucchini. In a 

glasshouse trial, disease control of 30-77% was provided by 23 isolates, and significant 

reductions in AUDPC values were recorded as a result of 29 isolates being applied. The 

best five isolates, identified as Gliocladium roseum (syn. Clonostachys rosea) (Isolate EH), 

Trichothecium roseum (syn. Cephalothecium roseum) (Isolate H20) and Serratia 

marcescens (i.e., Isolates B15, Y15 and Y41), were selected for further studies. 

2.1 INTRODUCTION 

Powdery mildew (PM) is one of the most important foliar diseases of many plants 

worldwide, occurring both under greenhouse and field conditions (Kiss et al., 2004). 

Application of systemic fungicides has been the principal control option (McGrath, 1996; 

2001). However, effective control of this disease with fungicides is becoming increasingly 

difficult due to concerns about health and environmental hazards, as well as the rapid 

development of fungicide resistance by these pathogenic fungi, rendering most fungicides 

ineffective against PM (McGrath, 2001). The possibility of using resistant cultivars towards 

this disease is also limited, especially in fruit and vegetable crops (Bélanger and 

36

Benyagoub, 1997). Therefore, there is great research interest in identifying effective 

antagonists against PM that can supplement or replace conventional agrochemical 

fungicides. 

Biocontrol of PM by various antagonists may involve competition for natural resources 

(Nofal and Haggag, 2006), antibiosis (Hajlaoui et al., 1992; 1994), mycoparasitism (Askary 

et al., 1997; Falk et al., 1995a; Kiss, 1998; Romero et al., 2003; Sundheim, 1982; 

Sztenjberg et al., 1989; 1995b; Verhaar et al., 1997) or induction of systemic resistance in 

plants against pathogens (Elad et al., 1998; Silva et al., 2004; Vogt and Buchenauer, 1997). 

The superficial growth of PM on leaves makes it an easy target for many antagonists that 

can be isolated and screened for their biocontrol efficacy. 

The success of biological control depends on the initial screening because subsequent 

results depend on identifying the best isolates during the initial process (Chiou and Wu, 

2003). Isolating potential antagonists from their natural hosts can make screening easier and 

more effective because they will be applied in the same environment, to which they should 

be well adapted. 

There are several techniques of preliminary screening isolates for biological control agents, 

with the dual culture method being preferred by many researchers (Landa et al., 1997; 

Morton and Stroube, 1955; Paulitz et al., 1992). This technique involves culturing the target 

pathogen and the potential antagonist(s) together on the same artificial medium. Its 

simplicity and rapid results, with some level of reliability, makes it a good primary 

screening method when the antagonists and the target pathogens can be grown 

saprophytically. However, obligate pathogens such as PM cannot be cultured on artificial 

media, limiting the application of this approach. Moreover, some of the modes of action 

cannot be observed by dual culture, resulting in the loss of some effective isolates if their 

modes of action are not detected using this technique. Therefore, an alternative approach is 

needed for obligate parasites that require the screening of isolates in a natural way. The 

objectives of this study were to isolate and screen various potential antagonists of PM from 

naturally infected leaves of susceptible plants. 

37

2.2 MATERIALS AND METHODS 

2.2.1 Collection of samples 

Samples of leaves of 16 plant species, naturally infected with PM, were collected from 8 

sites in the province of KwaZulu-Natal (Table 2.1). Two leaf samples (one young and one 

old infected leaf) were taken from each plant species that were growing at least 1km apart, 

or after an interval of 10 d, if taken from the same plant. 

2.2.2 Isolation and culturing of potential biocontrol agents 

(a) Fungi 

Mycelia of PM, and hence any associated fungi, were scratched from the plant with a 

sterilized scalpel, plated onto Potato Dextrose Agar (PDA) and incubated at 25 o C. Plates 

were monitored for 1 wk, while developing colonies of fungi were sub-cultured. Each 

isolate was incubated for 1-2 wk depending on its growth and sporulation rates. Mycelia 

and spores were harvested by pouring 5ml of sterilized distilled water onto the plates of 

PDA for 5min and scratching the mycelial mat with a sterilized scalpel. The mycelial 

suspension was transferred into sterile bottles, shaken vigorously and spores were filtered 

using four layers of cheesecloth. 

(b) Bacillus 

Samples of infected leaves were placed into conical flasks containing sterilized distilled 

water and shaken vigorously. After removing the leaf materials from the flask, the 

suspension was serially diluted, then heated to 80 o C for 15 min. Aliquots of 0.1 ml were 

plated on Tryptone Soy Agar (TSA) and incubated at 30 o C overnight. Individual bacterial 

colonies were transferred onto clean PDA plates and incubated for 24 h at 30 o C. For mass 

production, isolates were inoculated onto 100 ml flasks containing 50 ml of Tryptone Soya 

Broth (TSB) and incubated in a water bath operating at 30 o C and 200-250 revolutions per 

minute (rpm). After an incubation period of 72 h, cultures were centrifuged at 9000 rpm for 

15 min. The suspension was removed gently and the pellet was transferred into bottles 

containing sterilized distilled water. 

38

(c) Yeasts 

The same technique that was used for Bacillus isolates was adopted with slight 

modifications. Mycelial and spore suspensions of PM were prepared by putting infected 

leaves into conical flasks containing 100 ml of sterilized distilled water and shaken 

vigorously for few minutes. The suspensions were serially diluted in sterile distilled water 

and an aliquot of 0.1 ml of each dilution was plated onto a semi-selective media, prepared 

by adding Rose Bengal to PDA, with the objective of inhibiting bacterial growth. All 

colonies of yeast were re-streaked on Malt Extract Agar (MEA) to obtain pure cultures and 

incubated at 25 o C. After 48 h of incubation, isolates were transferred into 100 ml flasks 

containing Malt Extract Broth (MEB) and fermented in a water bath for 72 h at 25 o C with 

rotary agitation at 200-250 rpm. 

2.2.3 Preparation of powdery mildew inoculum 

Inoculum of PM was collected from naturally infected leaves of zucchini (Cucurbita pepo, 

F1-Hybrid Partenon) obtained from Starke Ayres 1 , and maintained by inoculating conidia 

onto disease-free plants and keeping them in a separate glasshouse. Conidia of PM were 

harvested using the method of Askary et al. (1998) and Dik et al. (1998). To ensure a high 

level of conidial viability and to maintain the same age of conidia, dead conidia were 

removed by shaking the source leaves 24 h before inoculation. Infected leaves were then 

immersed in sterilized distilled water and shaken to remove PM conidia. Conidia were 

counted using a haemocytometer and the concentration was adjusted to 10 3 conidia ml -1 . 

Inoculation was by spraying the conidial suspension onto leaves within two hours of 

counting. 

2.2.4 Screening of isolates on detached leaves 

Seeds of zucchini were planted in trays filled with a composted pine bark, and kept in a 

glasshouse (26-28 o C, 75-85% RH). After 2 wk, fully developed leaves were cut off with 

their petioles and transferred into pairs of 90 mm diameter Petri dishes, as described by 

Shishkoff and McGrath (2002). Each leaf petiole was inserted through a hole that connected 

the bottom of the top Petri dish to the lid of the bottom Petri dish that contained a nutrient 

solution (Figure 2.1). Petri dishes were transferred into growth chambers set at a 

1 Starke Ayres Seeds (Pty) Ltd., P. O. Box 304, Eppindust 7475, South Africa. 

39

temperature of 25 o C and 18 o C (day and night), with a photoperiod of 16 h and an RH of 65- 

80%. The lid of the top Petri dish was removed to avoid fluctuations of humidity that might 

have affected antagonistic activity of potential antagonists. Potential antagonists were 

applied by putting 10-15 droplets of the microbial suspension onto the leaves using 

sterilized Pasteur pipettes. In order to give the antagonists a competitive advantage, they 

were applied 2 d before inoculation of the leaves with PM, and reapplied 4 d after 

inoculation with Podosphaera xanthii. 

Ten days after inoculation, the percentage of leaf area covered with PM was recorded and 

transferred into a scale of 1-5 where: 1 = 0-20%, 2 = 20-40%, 3 = 40-60%, 4 = 60-80%, 5 = 

80-100% of leaf area covered by PM. This was done in order to separate the most effective 

ones from the least based on a range.To avoid any possibility of cross contamination 

between treatments, each isolate was tested in two replicates, placed 150-200 mm from 

other isolates. Isolates that showed superior performance against PM were selected for 

further screenings that continued until the number of isolates was reduced to the best 30. 

2.2.5 Screening of isolates on whole plants 

Seedlings of zucchini were raised in the same way as described above and kept in the 

glasshouse until they produced two fully developed leaves. Seedlings were then 

transplanted into pots (180mm diameter) containing a composted pine bark medium and 

transferred into another glasshouse (24-30 o C and 65-85% RH). The plants were supplied 

with a complete fertilizer [3:1:3 (38) from Ocean Agriculture 2 at 0.5g l -1 ] + [Ca(NO 3 ) 2 at 

0.5 g l -1 ] by means of drip irrigation. Plant distance was 400mm within rows and 1.5-2m 

between rows (Figure 2.1). Once seedlings were well established, they were inoculated with 

approximately 3-5 ml of a conidial suspension of P. xanthii (10 3 conidia ml -1 ) using a hand 

sprayer. Inoculation took place in the late afternoon to ensure sufficiently high relative 

humidity for germination of the conidia. 

All BCAs were applied using hand sprayers 2d before inoculation of P. xanthii and 

reapplied 4 d later, when symptoms started to appear, and continued every week for 4 wk. 

The concentrations of propagules of BCAs applied were 10 6 ml -1 for fungi and 10 8 ml -1 for 

bacteria and yeasts, respectively. All BCAs were applied in the evenings to ensure 

2 Ocean Agriculture (Pty) Ltd., P.O. Box 742, Muldersdrift, 1747, South Africa. 

40

sufficient relative humidity for establishment of the antagonists. The best five isolates that 

controlled PM consistently were selected for further evaluations and sent for identification 

using DNA sequence. The bacterial and fungal BCAs were identified by Inqaba 

Biotechnical Industries (Pty) Ltd 3 and Agricultural Research Council (ARC-PPRI) 4 , 

respectively. 

Disease assessments were made weekly by estimating the percentage of leaf area infected 

for each treatment, immediately before the next spray application of the BCA. Values of 

area under disease progress curve (AUDPC) were calculated from the disease severity 

values using an AUDPC program (Shaner and Finney, 1977). 

2.2.6 Data analysis 

The experiment was arranged in a randomized complete block design with each treatment 

having three replications. Data was analysed using GenStat ® Statistical Analysis Software 

(GenStat, 2006). Where the CV % was > 20%, the original data was transformed using a 

square root transformation and means of treatments were separated using Duncan's New 

Multiple Range Test. Effect of individual treatment (i.e. BCA) in controlling PM was 

assessed by calculating the percentages of final disease level and AUDPC value of the 

Untreated Control reduced by the biocontrol treatment. 

2.3 RESULTS 

2.3.1 Isolation and screening on detached leaves 

When PDA was used as a growth media, many types of microbes grew from the scrapings 

of PM mycelium that were plated onto it. However, only fungal colonies were picked up 

using a sterilized scalpel. Rate of germination of germ-tubes as well as shape, size and 

colour of mycelia of these fungi were variable when observed with the naked eye. 

Microscopic examinations showed that the isolates differed widely in their spore structures 

and hyphae. When Rose Bengal was added to the PDA, growth of bacterial species was 

inhibited and yeasts, together with some fungi, grew on the medium. However, there were a 

3 Inqaba Biotechnical Industries (Pty) Ltd., P.O. Box 14356, Hatfield 0028, South Africa. 

4 Agricultural Research Council (ARC-PPRI), P. Bag X134, Queenswood 0121, South Africa. 

41

few Gram negative bacteria that grew on PDA containing Rose Bengal. The isolation 

technique used for Bacillus species provided many isolates, mostly Gram positive bacteria. 

Only colonies that showed some morphological resemblance to typical Bacillus colonies 

were chosen as potential isolates. 

The isolation techniques were applied to leaves of 16 plant species infected with P. xanthii 

PM, collected from different sites. A total of 2,000 candidate microbes were selected for 

screening on detached leaves. Detached leaves started to show PM symptoms 3-4 d after 

inoculation and the disease could cover an entire leaf within 10 d because the 

environmental conditions were favourable for PM. Adventitious roots developed from the 

bottom of the petiole after 6-7 d. As long as the nutrient solution was available, the leaves 

could stay alive, in most cases, for more than one month. 

During the evaluation, some leaves died due to high levels of PM and were considered as 

100% diseased (i.e., 0% control). More than half of the potential BCA isolates reduced PM 

disease levels compared to the untreated control. From all the isolates tested, the best 100 

isolates were selected for a second phase of screening, from which 70% of them were 

discarded, before a third phase of selection was conducted, because the efficacy of some of 

these isolates was less consistent than others. Some isolates were observed to produce an 

inhibition zone around them, restricting the expansion of the PM colony, while other 

isolates inhibited the establishment of the disease. After a series of screening on the 

detached leaves, a total of 30 isolates (11 bacteria, 12 fungi and 7 yeasts) were selected that 

reduced PM disease levels by more than 60%. 

Table 2. 6 Sites where naturally infected leaves of host plants were collected 

Site 

Host plants 

Albert Falls weed 1 

Durban, KwaMashu 

cucumber, grape, pumpkin 

Pietermaritzburg, Agriculture Campus cabbage, cucumber, papaya, pepper, pumpkin, rose, tomato, 

weed 2, strawberry, zinnia, zucchini 

Pietermaritzburg, Cedara 

cucumber, pumpkin 

Pietermaritzburg, Scottsville 

grape, mulberry, pumpkin, weed 3, zucchini 

Pietermaritzburg, Ukulinga Farm beans, pepper, tomato, weed 4, zucchini 

Tala Valley 

pumpkin 

Tongaat 

pumpkin 

42

A 

B 

Figure 2.1 Plates showing preliminary screening of potential isolates using detached leaves 

inoculated with Podosphaera xanthii and treated with biocontrol agents (A). Note the formation of 

adventitious roots from the petioles, and the development of powdery mildew (B). 

Figure 2.2 Zucchini plants growing in greenhouse. 

43

2.3.2 Screening on whole plants 

BCA 

Control 

Under glasshouse condition, plants started to show symptoms of PM 4d after inoculation 

and the disease level could reach up to > 80% within 2 wk, if not treated. Application of 

BCAs had a significant effect on the severity of PM (P < 0.001). Compared to the treatment 

containing only water, 23 isolates significantly reduced the final disease level by 30-77%. 

Some isolates that showed promising results on detached leaves (e.g., Isolates CR, CK, 

CFC, CE, C, B14, E-77) were less efficient in controlling the disease when tested under 

glasshouse condition, although they caused significant control (Table 2.2). 

The AUDPC value of the Untreated Control, was significantly reduced (P < 0.001) by 29 

isolates (Figure 2.3). There was a direct relationship between final disease level and 

AUDPC values. Whenever the final disease was high, the corresponding value of AUDPC 

was high. 

The best five biocontrol isolates were identified as Clonostachys rosea (Link) Schroers, 

Samuels, Seifert & Gams (syn. Gliocladium roseum, Isolate EH), Trichothecium roseum 

(Pers.) Link (syn. Cephalothecium roseum, Isolate H20) and 3 isolates Serratia marcescens 

(Bizio) (i.e., Isolates B15, Y15 and Y41). 

44

Table 2.7 Effect of selected biocontrol agents on final disease level (FDL) and AUDPC of powdery 

mildew of zucchini plans after 10 d and 5 wk of growth in a growth chamber and greenhouse, 

respectively. 

Isolates (BCAs) 

Type Disease rating on 

F= fungus, B= bacterium, Y= yeast 

Values within column followed by a common letter were not significantly different according to Duncan's 

New Multiple Range Test at α = 0.05. 

detached leaves 

FDL 

Greenhouse assessment 

Values in brackets are means of data transformed using square root transformations. 

Ratings: 1 disease < 20%; 2 = < 40%, 3 = < 60%; 4 =

CFC 

CK 

B-14 

E-77 

C 

CR 

B-29 

DY 

B-21 

B-1272 

DS 

Y18 

Y38 

Y20 

B-52 

Y30 

Y27 

ML 

Y26 

C12 

BK 

Y31 

C218 

Y15 

Y9 

Y41 

EH 

H20 

B-15 

BCAs 

CE 

0 20 40 60 80 100 

Disease control (%) 

Figure 2.3 Efficacy of potential BCAs in controlling powdery mildew of zucchini after five weeks of 

treatment under greenhouse conditions. 

46

BCAs 

CK 

B-29 

E-77 

CFC 

C 

B-21 

CE 

DY 

CR 

DS 

B-14 

B-1272 

B-52 

Y18 

Y38 

Y20 

BK 

Y30 

ML 

Y27 

Y26 

C12 

B-15 

Y31 

C218 

EH 

Y41 

Y9 

Y15 

H20 

0 10 20 30 40 50 60 70 80 90 

Reduction in AUDPC (%) 

Figure 2.4 Effects of selected isolates in development of powdery mildew of zucchini 

represented as area under disease progress curve (AUDPC) after five weeks of treatment under 

greenhouse conditions. 

47

2.4 DISCUSSION 

The development of a proper isolation and in vitro screening protocol that provides rapid, 

repeatable and reliable results is an important initial step in screening efficient antagonists 

for biocontrol of plant diseases. This is because the success of all subsequent stages 

depends on the ability of the initial screening procedure to identify appropriate candidates 

(Whipps et al., 1988). Biological control of plant diseases typically involves three 

components: the biocontrol agent (BCA), the pathogen and the plant itself (Nevew et al., 

2007). If one of these components is missing, the screening process will be incomplete. 

In order to be an appropriate candidate, an isolate should consistently produce a promising 

result under various environmental conditions. It is a common challenge during the 

screening process that some potential candidates show good efficacy initially, but fail to 

perform at later stages, or when they are tested in different environments. 

During the screening process, BCAs were applied before and after inoculation of the 

pathogen. Application before infection was aimed at including competition as a possible 

mechanism of action in controlling the disease by allowing the antagonists to establish 

themselves before the disease. When spores or conidia of a pathogen require exogenous 

nutrients for germination and germ tube elongation, they are subjected to competition for 

these nutrients with the indigenous saprophytic microbial community (Brodie and 

Blackeman, 1975). However, Wilson (1997) determined that conidia of PM fungi do not 

require exogenous nutrients during germination, and host penetration occurs within a short 

period following germination, limiting the value of competition as biocontrol strategy. He 

concluded that suppressing the sporulation and dissemination of the pathogen using 

mycoparasites is the only viable approach to control PM fungi. However, other mechanisms 

of action such as antibiosis and induction of host resistance are important mechanisms for 

many antagonists. Hence, initial screening should minimize loss of potential antagonists by 

including every possible opportunity for all antagonists to display their potential biocontrol 

activity. 

In most cases, the performance of biocontrol isolates on agar has less predictive value than 

in vivo tests (Verhaar et al., 1998). This is because biocontrol activity of all antagonists is 

dependant on several factors that determine the survival of the introduced microbe. For 

48

their survival and establishment, BCAs may require the presence of their host on the 

phylloplane. For instance, growth of Pseudozyma flocculosa (Traquair, Shaw and Jarvis) 

Boekhout and Traquair is dependent on the presence of PM (Nevew et al., 2007). This may 

be related to availability of nutrients from the target pathogen. 

Many isolates of yeasts suppressed PM development. Often there was a clear zone around 

the antagonists. Yeasts are known to produce several inhibitory products (Hajilaoui et al., 

1994, Benyagoub et al., 1996a; Urquhart and Punja, 2002). For instance, several species of 

Tilletiopsis produce ß-1,3 exo- and endo-glucanase, chitinase and antifungal compounds 

(Urquhart and Punja, 2002). An active fraction of ester fatty acids produced by species of 

this genus was reported to inhibit germ tube development of P. xanthii and plasmolyse its 

spores (Urquhart and Punja, 2002). Pseudozyma flocculosa secretes modified long-chain 

fatty acids that caused cytoplasmic granulation and collapse of PM hyphae (Hajilaoui et al., 

1994, Benyagoub et al., 1996a). This might be the reason for the clear zone around many of 

the yeast isolates tested. 

The formation of an inhibition zone by bacterial isolates species is believed to be the result 

of antibiosis or competition, through which the antagonists control the disease. A report by 

Romero et al. (2004) indicated that these two mechanisms of action are common in 

bacteria. Further work by these researchers showed that production of lipopeptide 

antibiotics such as surfactin, fengycin, and iturin A or bacillomycin, and butanolic by 

Bacillis subtilis (Ehrenberg) Cohn showed that antibiosis is the main factor in PM 

suppression by Bacillus (Romero et al., 2007). Few candidates of fungal isolates showed 

hyperparasitism in addition to the modes of actions demonstrated by yeasts and bacterial 

isolates. 

The screening was performed using one species of plant, zucchini (F1 hybrid), infected 

with a single PM fungus (P. xanthii). The hybrid used for this experiment is very 

susceptible to PM and grows upward, making it an ideal choice for this research. Although 

all cucurbit plants are known to be susceptible to PM (McGrath and Thomas, 1996), most 

of them grow horizontally which requires large growing areas and it also increases the 

possibility of inter-plot interference between treatments. 

49

The approach adopted was based on the hypothesis that if an isolate can antagonize PM of 

this plant (i.e. zucchini), then it will probably be effective against other PM species of 

different plants. This approach has been used by several authors (Sztejnburg et al., 1989; 

Szentivanyi and Kiss, 2003). These groups of authors showed that Ampelomyces quisqualis 

Cesati ex Schlechtendahl isolated from a specific PM provided effective control against 

other PM species on different crops. 

The efficacy of some of the potential antagonists used was reduced when they were tested 

under greenhouse conditions. This could be due to greater fluctuations of the environmental 

conditions of the glasshouse compared to the growth chamber, where temperature, light 

intensity and relative humidity were all stable. However, these factors are more variable 

under glasshouse conditions subjected to fluctuating daily weather conditions. In spite of 

such fluctuations, some isolates gave consistent results, probably due to their adaptability to 

environmental fluctuations. 

Under both growing conditions, PM covered the whole leaf within 2wk after inoculation. 

This demonstrated the potential for the disease to destroy the whole plant if the 

environmental conditions were favourable for its development, and there were no effective 

control measures. 

The screening procedure followed in this experiment yielded potential antagonists that can 

be used under glasshouse conditions where PM is prevalent, with environmental conditions 

that favour both the antagonists and the pathogen. Paulitz and Bélanger (2001) described 

glasshouses as providing conducive environmental conditions under which BCAs can 

operate at their best. However, to be effective, the potential BCAs must be able to establish 

themselves soon after their application because the development of PM is so fast in such 

environments. The control efficacy of these potential isolates is promising in controlling the 

disease. However, the level of control provided by all BCAs tested was not complete. 

Therefore, there is a need to enhance their efficacy with a better understanding of these 

BCAs, the pathogen and the host plant. The efficacy of these antagonists can also be 

enhanced by co-application of compatible products or adjuvants. There is also a need to test 

these selected isolates under field conditions, where environmental conditions are not 

controlled, in order to develop biofungicides that can be used for integrated disease 

management purposes for field crops. 

50


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53

CHAPTER THREE 

EFFECTS OF ADJUVANTS ON THE CONTROL OF POWDERY MILDEW 

OF ZUCCHINI WHEN USING FOLIAR APPLICATIONS OF SOLUBLE 

SILICON AND SELECTED BIOCONTROL AGENTS 


a 

Discipline of Plant Pathology, School of Agricultural Sciences and Agribusiness 



Abstract 

Three adjuvants (Break-Thru® (BK), Partner® (PR) and Tween-80® (T-80)) were 

compared for their ability to improve the efficacy of soluble silicon (Si) applied as a foliar 

spray against powdery mildew of zucchini (PM). The best adjuvant was applied alone to 

evaluate its direct effect on the pathogen. Among the adjuvants tested, BK followed by 

PR, improved efficacy of Si significantly (P < 0.05). However, T-80 did not affect the 

efficacy of Si. Microscopic studies showed that BK inhibited germination of conidia, and 

caused collapse and disintegration of both conidia and mycelia of the pathogen. It also 

enhanced the deposition of BCAs on the pathogen. 

The compatibility of BK was tested with selected biocontrol agents (BCAs) and zucchini at 

various concentrations. Biocompatibility tests showed that mixing BK (0.2-1 ml l -1 ) to 

nutrient broth improved the c.f.u. of Isolates Y15 and Y41 while not affecting that of 

Isolate B15. However, mycelial growth of fungal Isolates EH and H20 was inhibited when 

BK was mixed into PDA agar at concentrations of more than 0.2 ml l -1 . Spraying BK at 

0.25 ml l -1 was compatible with zucchini plants, but showed phytotoxic effect at more 

than 0.5 ml l -1 . In the presence of BK, the efficacy of foliar spray of Si was improved by 

increasing the Si concentration to an optimum concentration of 750 mg l -1 . Break-Thru ® at 

concentraions of 0.2-0.4 ml l -1 enhanced the activity of Si sprays and three BCAs in 

controlling PM. 

54


Adjuvants are widely used with agrochemicals, with the objective of enhancing spray 

performance by improving the coverage, absorption and efficacy of the spray treatment 

(Gent et al., 2003). Amer et al. (1993) reported a 30% increase in control of powdery 

mildew (PM) of wheat (Erysiphe graminis f.sp. tritici Marchal) when adjuvants were 

mixed with fungicides. Several reports have indicated that adjuvants may have beneficial 

effects when applied with some BCAs (Bélanger et al., 1997; Phillip et al., 1990; Picton 

and Hummer, 2003). Enhanced survival as a result of homogenous distribution of BCAs on 

leaves was reported when Sigma Oil, Aqua Aid and Tween-80 were mixed with a 

suspension of BCAs for control of PM of cucumber (Dik et al., 1998). Efficacy of 

Sporothrix flocculosa Traquir, Shaw and Jarvis against PM of rose was also improved 

when surfactants were added into the spray mix (Bélanger et al., 1994). Moreover, the 

biocontrol activity of several yeasts against postharvest diseases of fruits and vegetables 

was improved by adding various adjuvants into the spray suspension (Lima et al., 2005) 

Foliar applications of silicon (Si) to control PM have been moderately successful on a few 

crops (Bowen et al., 1992; Guével et al., 2007; Liang et al., 2006; Menzies et al., 1992; 

Palmer et al., 2006; Reynolds et al., 1996). However, there is little information about the 

optimum concentration of Si that can provide acceptable levels of control, or on efforts to 

improve its efficacy. As with agrochemicals, the efficacy of foliar sprays of Si may be 

improved by adding adjuvants to enhance their coverage, and retention of Si on the 

phylloplane, and therefore reducing the concentrations of Si required for good control of 

PM. Similarly, adjuvants may enhance the even distribution of BCAs on leaves, and 

therefore improve the level of PM control that they can provide. 

Before any adjuvant may be recommended for large-scale usage, a thorough assessment on 

its potential risks is essential. Assessment of its direct impact on the plant, the BCA and the 

pathogen are needed. The use of adjuvants alone can limit development of PM by 

inhibiting germination of conidia and/or inhibiting its mycelial growth. For instance, 

mineral oil, Tween-80 and some surfactants were reported to have inhibitory effects on PM 

of cucumber (Dik et al., 1998), rose (Bélanger et al., 1994) and wheat (Ziv and 

Frederiksen, 1987). Therefore, applying such adjuvants with BCAs can inflate the 

perceived activity of the antagonists (Bélanger et al., 1997). 

55

The objectives of this study were to compare three adjuvants for their capacity to improve 

foliar-applied soluble Si; to study the direct effect of the best adjuvant on the pathogen; to 

test its compatibility with the plant and selected BCAs; and to determine the optimum 

concentration of Si that can give acceptable level of control, when combined with the best 

adjuvant. 


3.2.1 Effects of adjuvants and silicon on powdery mildew of zucchini 

Seedlings of zucchini were grown and inoculated with PM as described in Chapter 2. 

Three adjuvants were tested: Break-Thru ® (polyether-polymethylsiloxane-copolymer), 

Partner 650 ® (alkoxylated fatty alkylamine polymer/ethoxylated sorbitane ester) and 

Tween-80 ® (polyoxyethylene 20 sorbitan monooleate). These were obtained from 

Universal Crop Protection 1 , Degussa Africa 2 and Merck Chemicals 3 , respectively. 

As a source of soluble silicon (Si), potassium silicate (K 2 SiO 3 ) (a product K2550 containig 

20.5-20.9% SiO 2 and 8.0-8.15% K 2 O) was obtained from PQ Silicas SA 4 and 

concentrations of Si was calculated according to the product’s information. Break-Thru ® , 

Partner, and Tween-80 at a rate of 0.5, 0.5, and 0.2 ml l -1 , respectively, were mixed with 

Si (at 250, 500, 750 and 1000 mg l -1 ) and sprayed onto leaves of infected zucchini plants 

until runoff. Water was used as a control. Treatments were applied once a week in the late 

afternoon to avoid the hottest period of the day. Percentage of leaf area covered by PM was 

recorded weekly for five weeks and the area under disease progress curve (AUDPC) was 

calculated from the disease data using an AUDPC Program (Shaner et al., 1977). Analysis 

of variance (ANOVA) analysis was performed with GenStat ® Statistical Analysis Software 

(GenStat, 2006). Comparisons between means of treatments was made using Fisher’s 

protected least significant difference (FLSD) and effect of adjuvants on percentage 

reductions of final diseae level and AUDPC values were calculated by comparing the 

values of each treatment against that of the Untreated Control (only water). Polynomial 

1 Universal Crop Protection (Pty) Ltd., P.O. Box 801, Kempton Park 1620, South Africa. 

2 

Degussa Africa (Pty) Ltd., P.O. Box 261, Somerset West 7129, South Africa. 

3 

Merck Chemicals (Pty) Ltd., 259 Davidson Rd. Wadeville 1422, Gauteng, South Africa. 

4 PQ Silicas South Africa (Pty) Ltd, 169 Tedstone Rd, P.O. Box 14016 ,Wadeville 1422, Gauteng, South 

Africa. 

56

egression analysis was also performed to determine the effects of concentration of Si 

applied with different adjuvants at various rates on open or covered pots. The adjuvant that 

provided best result in suppressing PM with Si was selected for further investigations. 

3.2.2 Scanning electron microscop studies on the effects of Break-Thru ® on 

Podosphaera xanthii and biocontrol agents 

Two fungal (Clonostachys rosea (Link) Schroers, Samuels, Seifert & Gams, Isolate EH) 

and Trichothecium roseum (Pers.) Link, Isolate H20) and 3 isolates of Serratia marcescens 

(Bizio) (i.e., Isolates B15, Y15 and Y41) were prepared, as described in Chapter 2, and BK 

was added into the microbial suspension at a rate of 0.25 ml l -1 . All treatments were 

sprayed onto diseased plants that were growing in a greenhouse, with the environmental 

conditions set at 25 o C and 18 o C (day and night), a photoperiod of 16 h and an RH of 65- 

80%. Treatments were applied weekly during the late afternoon to ensure enough humidity 

and a favourable temperature for the establishment of the BCAs. After 5 wk, samples were 

taken from the infected leaves to investigate the direct impact of BK on the pathogen and 

BCAs using scanning electron microscopy. 

Samples of the infected leaves were cut into circles of approximately 10mm diameter and 

fixed overnight in 3% glutaraldehyde in cacodylate buffer (0.1 M; pH 7.0), and then 

dehydrated in a graded alcohol series. Specimens were critical point dried in a Hitachi 

HCP-2 using CO 2 as a transfusion fluid. Dried specimens were mounted on copper stubs 

using double-sided carbon tape. All stubs were then coated with gold-palladium in a 

Polaron E500 Sputter Coater and viewed with a Philips XL30 environmental scanning 

electron microscope (ESEM) operating at 12 KV. 

3.2.3 Biocompatibility test of Break-Thru ® 

Microbial media containing BK were prepared for fungal and bacterial BCAs. A solution 

of BK was prepared by mixing 10 ml l -1 of the adjuvant with 90ml of sterilized distilled 

water and filtered through a filter paper. Full strength Potato Dextrose Agar (PDA) was 

autoclaved for 15min at 121 o C and left at room temperature to cool down. When the 

temperature of the flask was approximately 40 o C, the solution of BK was added to the 

57

medium at concentrations of 0.02-1 ml l -1 . The PDA-BK medium was mixed thoroughly 

using a magnetic stirrer, poured to Petri dishes and kept in a laminar flow for 24 h. 

Approximately 3-5 mm-diameter agar plugs of Isolates EH and H20, taken from the 

leading edge of 2wk old cultures, were plated on the medium and incubated at 25 o C. After 

2 wk mycelial growth of Isolate EH was measured using a ruler. However, growth of 

Isolate H20 was patchy when BK was added to the agar and mycelial growth was 

measured by estimating the percentage of the plate covered by the fungus and converting 

the value into a diameter. Nutrient Broth (NB) was prepared in 100 ml conical flasks and 

autoclaved at 121 o C for 15 min. After the broth was cold, BK was added into the flasks at 

concentrations of 0.01-0.1 ml l -1 . The three isolates of S. marcescens (i.e., Isolates B15, 

Y15 and Y41) were inoculated into the broth using a sterile loop and fermented in a 

shaker, set at 150 rpm at 28 o C. After 48 h of incubation, cultures were serial-diluted in 

McCarthy bottles containing sterile distilled water and plated onto PDA. Finally, plates 

were incubated for 24 h at 30 o C and colony-forming units (c.f.u.) were counted. Data of 

each BCA was analysed as Randomized Block Design using GenStat ® Statistical Analysis 

Software separately (GenStat, 2006) and relationships between concentrations of BK and 

microbial growth were confirmed using polynomial regression analysis. 

3.2.4 Phytotoxicity test of Break-Thru ® on zucchini plants 

Break-Thru ® (0.25-2 ml l -1 ) was sprayed onto zucchini plants infected with PM once a 

week until runoff. After 3wk, visual assessments were made of the toxicity of BK to the 

plants. 

3.3 RESULTS 

3.3.1 Effects of three adjuvants on powdery mildew control of with silicon 

All adjuvants used in this study improved spray coverage and retention of Si on zucchini 

leaves. When leaves were sprayed with Si that contained any of these three adjuvants, they 

remained wet for longer periods than leaves sprayed with Si without an adjuvant. Adding 

BK and PR to Si sprays gave better results, reducing disease levels by an average of 18- 

20%. However, T-80 did not improve the efficacy of Si sprays significantly. Application of 

BK and PR alone significantly reduced disease severity levels by 20% and 18%, 

respectively. However, spraying T-80 alone exacerbated the disease level (Table 3.1). 

58

The concentration of Si in the spray solution had a significant impact on PM control 

(P < 0.001). Regardless of the adjuvant used, the efficacy of Si was improved as the spray 

concentration was increased, with 750 mg l -1 being the optimum dosage. The effect of 

adjuvants and Si applied at various concentrations on severity of PM of zucchini after 5 wk 

of infection and the level of disease control obtained by these treatments are presented in 

Table 3.1 and Figure 3.1, respectively. 

The effects of adjuvants on AUDPC values were highly significant (P < 0.001). Without 

Si, BK and PR reduced the AUDPC values significantly (Table 3.2). AUDPC values of Si 

plus BK or PR were also significantly low. Spraying Si containing BK and PR reduced the 

AUDPC value by 26 and 23%, respectively. Even without Si, these two adjuvants reduced 

AUDPC values by 20-25%. However, application of T-80 did not affect the AUDPC value 

significantly and the efficacy of Si was unaffected by the presence of T-80. When 

comparisons were made among concentrations, the lowest AUDPC values were obtained 

when Si was applied at 750 mg l -1 . The AUDPC values of PM as affected by three 

adjuvants, applied with Si at various concentrations, are presented in Figures 3.1 & 3.2. 

Table 8.1 Effects of different adjuvants sprayed with soluble silicon (Si) at various concentrations 

on final disease level of powdery mildew of greenhouse grown zucchini after five weeks of 

infection with Podosphaera xanthii. 

Si concentration (mg l -1 ) 

Adjuvants 

0 250 500 750 1000 

Break-Thru ® 73.3 b 66.7 ab 63.7 ab 60.0 a 61.7 ab 

Partner ® 75.0 b 65.0 ab 70.0 ab 58.3 a 60.0 a 

Tween-80 92.3 c 79.0 b 73.3 b 63.3 ab 75.0 b 

Water 91.7 c 78.3 b 76.7 b 76.7 b 75.0 b 

Effects 

P-Values 

Adjuvants < 0.001 

Concentration < 0.001 

Adjuvants*Concentration 0.704 

FLSD 

11.686 

CV (%) 

9.9 

Means followed by a common letter were not significantly different according to Fisher’s protected least 

significant difference (P < 0.05). 

59

Table 9.2 Effects of different adjuvants and soluble silicon, sprayed at various concentrations, on 

AUDPC values of powdery mildew of zucchini grown under glasshouse conditions after 5 weeks 

of infection with Podosphaera xanthii 

Si concentration (mg l -1 ) 

Adjuvants 

0 250 500 750 1000 

Break-Thru ® 1724 b 1392 ab 1416 ab 1031 a 1169 ab 

Partner ® 1636 b 1524 b 1456 b 1229 ab 1180 ab 

Tween-80 2164 c 1871 bc 1787 bc 1289 ab 1599 b 

Water 2168 c 1851 bc 1735 bc 1649 b 1712 b 

Effects 

P-Values 

Adjuvants < 0.001 

Concentration < 0.001 

Adjuvants*Concentration 0.955 

FLSD 420.9 

CV (%) 16.2 

Means followed by a common letter were not significantly different according to Fisher’s protected least 

significant difference (P

A 

× Break-Thru: y= 1.142x 2 - 9.851x + 82.06 

R² = 0.983 

●Partner: y = 0.476x 2 - 6.525x + 80.00 

R² = 0.715 

▲ Tween: y = 3.262x 2 - 24.60x + 114.5 

R² = 0.909 

■ Water: y= 1.785x 2 - 14.21x + 102.6 

R² = 0.900 

B 

× Break-Thru: = 37.92x 2 - 374.6x + 2053 

R² = 0.843 

●Partner: y = -2.357x 2 - 106.5x + 1750 

R² = 0.960 

▲ Tween: y = 56.57x 2 - 510.6x + 2651 

R² = 0.799 

■ Water: Water: y = 56.42x 2 - 449.9x + 255. 

R² = 0.991 

Figure 3.1 Relationships between adjuvants and Si applied at differing concentrations on 

severity (A) and AUDPC values (B) of powdery mildew of zucchini after five weeks of 

infection with Podosphaera xanthii. 

61

× Break-Thru: y = 9.308ln(x) + 20.48 

R² = 0.913 

● Partner: y = 10.09ln(x) + 18.53 

R² = 0.766 

▲Tween 80: y = 15.54ln(x) + 1.519 

R² = 0.726 

■ Water: y = 10.45ln(x) + 2.990 

R² = 0.817 

Figure 3.2 Effects of different adjuvants in controlling powdery mildew of zucchini in combination 

with foliar-applied Si at various concentrations. 

× Break-Thru: y = 17.55ln(x) + 21.09 

R² = 0.833 

● Partner: y = 13.35ln(x) + 22.41 

R² = 0.892 

▲Tween 80: y = 20.53ln(x) - 0.014 

R² = 0.758 

■ Water: y = 14.19ln(x) + 2.322 

R² = 0.899 

Figure 3.3 Effects of different adjuvants in suppressing development of PM of zucchini with foliarapplied 

Si at various concentrations after five weeks of inoculation. AUDPC values are as a % of 

the untreated control AUDPC value. 

62

3.3.3 Biocompatibility of Break-Thru ® with biocontrol agents 

Analysis of ANOVA on the effects of BK on colony forming units of Isolates B15, Y15 

and Y41 were not significant at P < 0.05. However, regression analysis showed that there 

was relationship between concentrations of BK in the medium and c.f.u. of Isolate B15, 

with increasing levels of BK inhibiting the c.f.u. of this isolate slightly. Adding BK at a 

concentration of 0.4 ml l -1 was optimum for growth of Isolates Y15 and Y41. The effect 

of BK on mycelial growth of Isolates EH and H20 was significant (P < 0.001) (Figure 3.4 

B). Growth of these two fungal BCAs was significantly inhibited when BK was added into 

the medium at a concentration of 0.4 ml l -1 . Mycelial growth of Isolates EH and H20 were 

reduced by 19 and 49%, respectively, when BK was mixed to PDA at 1 ml l -l (Figures 3.4 

and 3.5). Result of an in vitro bioassay on the effects of BK on growth of all BCAs is 

presented in Figure 3.5. 

Control BK at 1ml l -1 

A 

Control BK at 1ml l -1 

B 

Figure 3.4 Plates of PDA showing the effects of Break-Thru ® on mycelial growth of Isolates 

EH (A) and H20 (B). 

63

A 

× Y15: y = 1.551x 2 - 5.862x + 15.45 

R² = 0.355; P = 0.070 

■ B15: y = 1.037x 2 - 7.482x + 27.97 

R² = 0.757; P = 0.835 

▲ Y41: y = 0.845x + 2.24 

R² = 0.240; P = 0.291 

B 

× H20: y = 1.575x 2 - 21.07x + 112.8 

R² = 0.902; P < 0.001 

▲ EH: y = 0.216x 2 - 3.881x + 58.89 

R² = 0.800; P < 0.001 

Figure 3.5 In vitro bioassay on the effects of Break-Thru ® mixed with growth medium on growth of 

BCAs. A: colony forming units of Isolates B15, Y15 and Y41 after two days of incubation at 30 o C. 

C.F.U. of Isolates B15 and Y15 were diluted by 10 10 and Isolate Y41 by 10 9 ; B: mycelial growth of 

Isolates EH and H20 after two weeks of inoculation at 25 o C. 

64

A 

. 

B 

1 

C 

D 

D 

E 

F 

Figure 3.6 Environmental Scanning electron microscopic observations of the effects of Break- 

Thru® on Podosphaera xanthii and selected BCAs. A: Conidia and hyphae of the control; Plate 2: 

Collapsed and disintegrated hyphae and conidia of the fungus; C: Hyphae of the fungus sticking to 

each other; D: Conidia bond each other and BCAs deposited on top of the fungal structures; E: 

Enhanced deposition of BCAs on the surface of the pathogen; F: Trichothecium roseum (Isolate H20) 

establishing on the hyphae of the pathogen. 

65

3.3.4 Phytotoxicity test of Break-Thru ® 

Application of BK at 0.25 ml l -1 provided moderate disease control with no phytotoxic 

effects to the plant. When BK was applied at 0.5 ml l -1 , PM was reduced by more than 

60%. At 1 ml l -1 , BK provided complete control of PM. However, leaves of the plants 

showed symptoms of shrinking followed by burning when BK was applied at higher levels. 

Repeated application of BK, even at 0.5 ml l -1 , caused stunting of the plant, with young 

leaves being malformed and reduced in size. 


Selection of an effective adjuvant for an integrated disease management programme can 

reduce the impact of agrochemicals on the environment by reducing the quantity of active 

ingredient required to deliver disease control without compromising yield or quality 

(Kirkwood, 1993). When used properly, adjuvants are expected to have no toxic effect to 

the plants or the environment. However, recent studies have indicated that some adjuvants, 

primarily surfactants, can have direct impact on the BCAs and the target pest (Dr A. 

Charudattan, 2007 5 , personal communication). 

Although many available adjuvants provide application information on the label, each 

plant management project is unique and requires an assessment before the appropriate 

adjuvant can be selected and utilized. Adjuvants can increase the efficacy of foliar 

treatments by altering spray droplet sizes, distribution of sprays on the plant, viscosity 

(stickiness) of the sprays, their evaporation rate, the rate of uptake of agrochemicals by the 

target plant, and the solubility of agrochemicals in solution (Young, 2004). 

In this study, some of the above properties were shown by all adjuvants used. For instance, 

better coverage of zucchini leaves was achieved with the same amount of Si solution or 

BCA suspension when BK, PR and T-80 were added into the spray mixture. Prokop and 

Kejklícek (2002) noted that most water soluble adjuvants improve spray coverage by 

reducing the percentage of small droplets (i.e., < 75 µm) which are the major contributors 

to off-target drift. Longer retention of the spray on the surface of the leaves was assumed 

5 

Dr R. Charudattan, Department of Plant Pathology, University of Florida 

66

to be the result of increase in viscosity of the spray solution by these adjuvants. When any 

of these three adjuvants were mixed in the spray solution, they formed foam. Formation of 

foam is reported to control drifts, making the spray application more effective and 

economical (Young, 2004). 

At higher dosages (> 0.5 ml l -1 ), BK was shown to control PM effectively on its own, but 

it had phytotoxic effects on zucchini. Plants that were sprayed with BK at higher levels 

frequently showed stunted growth with malformed leaves. Young leaves were more 

sensitive to this adjuvant. Spraying BK at a concentration of 0.25 ml l -1 provided moderate 

disease control without affecting the plant negatively. Both BK and PR showed promising 

results in reducing PM of zucchini when they were sprayed alone or together with Si. 

Addition of BK and PR improved the efficacy of Si by improving distribution of the Si 

sprays, and by suppressing the pathogen directly. Foliar application of Si without adjuvants 

also resulted in significantly lower disease values, especially when used at higher 

concentrations. Regardless of the adjuvant used, the efficacy of Si was increased as 

concentration increased up to 750 mg l -1 . 

The addition of T-80 into spray mixtures was reported to have an inhibitory effect on PM 

fungi (Bélanger et al., 1994; Dik et al., 1998; Ziv and Frederiksen, 1987) and to have 

enhanced the efficacy of B. amyloliquefaciens Ribonuclease (Barnase) as a BCA by 

improving the distribution of the antagonist on the surface of the plant (Chiou and Wu, 

2003). Dik et al. (1998) also reported increased control of PM by A. quisqualis Ces ex 

Schlect when T-80 was mixed in the spray suspension. In this study, T-80 improved spray 

efficiency, but did not affect the disease directly. The size and shape of the PM colonies 

were not apparently affected when sprayed with T-80. Even when applied together with Si, 

the colonies of PM were large and fluffy, similar to colonies of the control treatment. In 

some cases, the disease severity and AUDPC value of treatments containing T-80 were 

even higher than treatments without any adjuvant. The reason for the failure of T-80 to 

have any impact on the disease is not known. Although each adjuvant was applied based 

on recommendations from previous studies and their registration labels, the concentration 

of T-80 was much less than that of both BK and PR. Therefore, the concentration of T-80 

67

used might not have been strong enough to affect the disease or the pathogen might have 

been resistant towards this adjuvant. 

Colonies of PM treated with BK were small, flat and dry. Further investigations with 

ESEM revealed that the disease suppression shown by BK resulted from the direct impact 

of the adjuvant on the pathogen. Conidia of PM were less in number and mycelial growth 

of the PM fungus was limited after treatment with BK. In addition, conidia and hyphae of 

the pathogen collapsed and disintegrated as a result of direct contact with BK. It is 

probable that this impact is related to the chemical properties of the adjuvant. The adhesive 

properties of BK also forced conidia and hyphae of the PM fungus to stick to each other, 

restricting spread of conidia and expansion of its colonies Moreover, mixing BK with 

suspensions of BCAs resulted in increased deposition of these antagonists on the pathogen, 

resulting in improved control of PM. When used at an appropriate rate, BK has the 

potential to improve the efficacy of BCA antagonists significantly. 

A major limitation to the effective use of BCAs and Si against PM has been due to the rate 

of disease development and spread. The direct impact of BK on the pathogen therefore 

gives a novel opportunity to enhance the management of PM with BCAs and Si. It is 

conceivable that BK would provide a short-term control function, followed by the 

combined effects of Si and a BCA to provide medium to long-term disease control. 

Growth of Isolate EH and Isolate H20 was inhibited when BK was added to PDA at 

concentration of 0.4 ml l -1 . In contrast, adding BK into NB at 0.2-1 ml l -1 had little effect 

on the c.f.u. of isolates of S. marcescens. Based on the biocompatibility and phyto-toxicity 

tests, it is recommended that for effective control of PM combined with BCAs or Si, BK 

must be used at a concentration range of 0.2-0.4 ml l -1 , depending on the sensitivity of the 

plant towards the adjuvant. During the course of disease control, repeated application may 

result in an accumulation of BK on the phylloplane, which may have adverse effect on the 

plant or the BCAs. To avoid such risks, further investigations on the impacts of BK 

deposition on the plant and antagonists is needed before the recommended rate is adopted 

for implementation. In addition, direct contact between the adjuvant and the BCA in target 

may affect the antagonistic properties of the BCAs. Therefore, more in vitro research is 

68

needed to determine effects of BK on the metabolic properties of the BCA isolates. 

Moreover, although most adjuvants are considered to have no pesticidal properties and are 

exempted from regulations of U.S. Environmental Protection Agency (Hock, 1998), 

investigation is needed on the hazards that BK may pose to consumers and beneficial 

microbial inhabitants of the phylloplane. 


Amer, M.A., Hoorne, D., Poppe, J. 1993. In-vivo evaluation of adjuvants for more effective control 

of celery leaf-spot (Septoria apiicola) and powdery mildew (Erysiphe graminis) of wheat 

with fungicides. Pesticide Science, 37: 113-120. 

Bélanger, R.R., Dik, A.J., Menzies, J.G. 1997. Powdery mildews: Recent advances toward 

integrated control. In: G.J Boland and L.D Kuykendall (Eds.). Plant-Microbe Interactions 

and Biological Control. Marcel Dekker, Inc, New York. 






Chiou, A.L., Wu, W.S. 2003. Formulation of Bacillus amyloliquefaciens B190 for control of lily 

grey mould (Botrytis elliptica). Journal of Phytopathology, 15: 13-18. 




GenStat, 2006. GenStat Statistical Analysis Software 9 th ed. Lawes Agricultural Trust, Oxford, UK. 

Gent, D.H., Schwartz, H.F., Nissen, S.J. 2003. Effect of commercial adjuvants on vegetable crop 

fungicide coverage, absorption and efficacy. Plant Disease, 87: 591-597. 

Guével, M.-H., Menzies J.G., Bélanger, R.R. 2007. Effect of root and foliar applications of soluble 


Pathology, 119: 429-436. 

Hock, W.K. 1998. Horticultural spray adjuvants. Agrochemical Fact Sheet, The Pennsylvania State 

University. http://pubs.cas.psu.edu/FreePubs/pdfs/uo202.pdf Captured on 15/08/2008. 



Pathology, 54: 678-685. 

69

Lima, G., Castoria, R., Spina, A.M., de Curis, F. 2005. Improvement of biocontrol activity of yeast 

against post harvest pathogens: recent experiences. Acta Horticulturae, 682: 2035-2040. 

Menzies, J., Bowen, P., Ehret, D., Glass, A.D.M. 1992. Soluble silicon sprays inhibit powdery 

mildew development on grape leaves. Journal of the American Society for Horticultural 

Science, 117: 902-905. 

Palmer, S., Scott, E., Stangoulis, J., Able, A.J. 2006. The effect of foliar-applied Ca and Si on the 

severity of powdery mildew in two strawberry cultivars. Acta Horticulturae, 708: 135-139. 

Philipp, W.-D., Beuther, E., Hermann, D., Klinkert, F., Oberwalder, C., Schmiditke, M., Straub, B. 

1990. Zur formulieurung des Mehltauhyperparasiten Ampelomyces quisqualis Ces, 

Zeitschrift fur Planzenkrankeheiten und Pflanzenschutz, 97: 120-132. 

Picton, D.D., Hummer, K.E. 2003. Oil application reduces white pine blister rust severity in black 

currants. HortTechnology, 13: 365-367. 

Prokop, M., Kejklícek, R. 2002. Effect of adjuvants on spray droplet size of water. Research in 

Agricultural Engineering, 48:144-148. 



70

CHAPTER FOUR 

EFFECTS OF CONCENTRATION, FREQUENCY OF APPLICATION AND 

RUNOFF OF FOLIAR-APPLIED SILICON ON POWDERY MILDEW OF 

ZUCCHINI 


a 




Abstract 

The effect of concentration, frequency of application and runoff of foliar-applied soluble 

silicon (Si) on the severity of powdery mildew (PM) (Podosphaera xanthii (syn. 

Sphaerotheca fuliginea) of zucchini (Cucurbita pepo L.) was evaluated. Soluble Si (250- 

1000 mg l -1 ), together with Break-Thru ® (0.25 ml l -1 ), was sprayed onto zucchini plants at 

frequencies of 1-3 wk -1 . The leaves of all plants were inoculated with a known 

concentration of conidia of P. xanthii 2 d after the sprays were applied. The effect of runoff 

was determined by covering some of the pots with polyethylene sheets, while others were 

left open. 

Spraying Si onto zucchini crops reduced the severity of PM significantly. The efficacy of 

Si was improved by increasing the spray frequency. However, concentration of Si did not 

have a major impact on the efficacy of Si. Regardless of the concentration of Si, the best 

results were obtained when the frequency of the treatment was increased, and when Si was 

allowed to reach the root zone of the plants. 

When Si was applied onto leaves, direct contact between the spray and the pathogen 

seemed to be the main mechanism of action involved in disease control, and part of the 

spray (i.e., the runoff) was absorbed by the plant roots, and subsequently played an 

important role in the health of the plants. If affordable, soluble Si should be included in 

nutrient solutions of hydroponic crops or supplied from overhead irrigation schemes. 

71


Powdery mildew (PM) of many crops causes significant losses in yield if not managed 

properly (Romero et al., 2003 & 2004). Soluble silicon (Si) has been investigated by many 

authors (Bélanger et al., 1995 & 1997; Epstein, 1994 & 1999; Liang et al., 2005) as an 

environmentally safe option for the management of this disease. These researchers have 

demonstrated that effective control of PM can be achieved by the application of soluble Si 

at appropriate dosages and frequencies. 

Foliar applications of Si have shown promising results in controlling PM of several crops 

(Bowen et al., 1992; Guével et al., 2007; Liang et al., 2005; Menzies et al., 1992; Palmer 

et al., 2006; Reynolds et al., 1996). These authors have showed that it is possible to replace 

or supplement the fungicides being used against PM with soluble Si sprays. The level of 

disease control that can be achieved using this approach is, however, limited due to a lack 

of information on the optimum concentration. Efficacy of Si can also be improved by 

increasing the frequency of application as this increases the deposition of the element on 

the phylloplane. 

Furthermore, when a foliar spray is used, there is always runoff which is intercepted by the 

soil. If the Si treatment is absorbed by the roots, as a quasi-essential element, it may play a 

role in the health of the plant. Therefore, to assess the direct effect of foliar application of 

Si on PM, it is important to differentiate the impact of runoff from the total effect of the 

treatment. Understanding the effect of such components can determine whether foliar 

applications of Si should be more or less effective than root drenches. 

The objectives of this study were to determine the effects of foliar applied Si on the 

severity of PM, determine the optimum concentration and application frequencies of Si on 

PM, and study the impact of runoff on the efficacy of foliar sprays of soluble Si. 

72


4.2.1 Preparation of plants 

Seeds of zucchini (Cucurbita pepo, F1-Hybrid Partenon) were planted in seedling trays 

and kept in a greenhouse at a temperature of 26-28 o C and relative humidity of 75-85%. 

Trays were irrigated with a complete fertilizer [Ocean Agriculture 1 3:1:3 (38) at 0.5 g l -1 ] 

+ [Ca(NO 3 ) 2 at 0.5 g l -1 ] for 2 wk using overhead irrigation. Once the germinated 

seedlings had fully developed a second leaf, they were transplanted into pots filled with 

composted pine bark and transferred to another glasshouse set at 26-28 o C, 75-85% RH. 

The plants were irrigated with the same nutrient solution using drip irrigation until the end 

of the experiment. 

4.2.2 Preparation of Podosphaera xanthii for inoculation 

Inoculum of PM was obtained from plants that were inoculated with previously collected 

samples of natural inoculum. Conidia of PM were harvested using the same technique, as 

described in Chapters 2-3, and counted using a haemocytometer. Finally, seedlings were 

inoculated by spraying 3-5 ml of conidial suspensions of PM (10 3 conidia ml -1 ) onto the 

leaves of each seedling using a hand sprayer within two hours of counting. Inoculation was 

done 2 d after Si was sprayed onto plants. 

4.2.3 Application of soluble silicon 

Soluble Si was sprayed onto zucchini plants in the form of KSi at four different 

concentrations (250, 500, 750 and 1000 mg l -1 ) until runoff. Treatments were applied at a 

frequency of 1-3 wk -1 . Impact of Si spray that is absorbed by the roots on disease level was 

assessed by controlling movement of Si onto the roots. The lower part of the plants and 

their pots were sealed with polyethylene sheets cut and taped into place to provide a 

waterproof barrier to stop any Si spray from reaching the root zone of the plants, in the 

form of runoff and drift,. The aerial parts of the plants were left open for spray. Other pots 

were left uncovered, allowing the drift and runoff of spray applications to reach to the 

rhizosphere and to be taken up by the roots. 

1 Ocean Agriculture (Pty) Ltd., P. O. Box 742, Muldersdrift, 1747, South Africa. 

73

For all treatments, Break-Thru ® (BK) was used as a wetter at concentration of 0.25 ml l -1 . 

Distilled water containing only BK was sprayed at the same frequencies as the control 

treatment. All the treatments were applied in the late afternoon in order to minimize 

dryness of the leaves due to heat. Disease levels, as percentage of leaf area infected, were 

evaluated 3 wk after inoculation. 

4.2.4 Disease assessment and statistical analysis 

The experiment was conducted twice, with each treatment having three replications. The 

treatments were arranged in a factorial randomized complete block design. Analysis of 

Variance (ANOVA) were performed using a factorial treatment structure. Where the CV % 

was > 20%, the original data was transformed using a square root transformation. 

Interactive effects of application concentration, frequency and runoff of Si on the severity 

of PM was analysed using GenStat ® Statistical Analysis Software (GenStat, 2006). 

Treatment means were separated using Fisher’s protected least significant difference 

(LSD). The percentage of disease reduction by each treatment was calculated by taking the 

final disease of the Untreated Contarol as a reference. Finally, polynomial regression 

analysis was performed on the percentage of disease reduced by different levels of Si 

applied on open or covered pots at 3 different frequencies. 

4.3 RESULTS 

In both experiments, application of Si reduced the disease severity significantly. The effect 

of concentration on the efficacy of soluble Si sprays was not significant (Table 4.1 and 

4.2). After spraying the plants with Si at various concentrations (250-1000 mg l -1 ) for 

3 wk, disease severity of the control treatment was reduced by 42-61% and 75-79% in the 

first and second trials, respectively. In both experiment, extra Si had little or no added 

effect on the efficacy of the spray. When the effect of Si at different concentrations on 

severity of PM was compared, there was no significant difference between the values. 

In both trials, spray frequency had a significant effect on the efficacy of Si in controlling 

PM. Using the same concentration of Si, efficacy of the treatment was increased initially 

by 30% and almost doubled in the subsequent experiment when the spray frequency was 

74

tripled. Even in the control treatment which contained only BK, the disease level was 

reduced when the treatment was sprayed at higher frequency (Figures 4.1 & 4.2). 

A comparison of PM severity between open and covered pots showed that drift and runoff 

of foliar-applied Si had a significant impact on disease control (Tables 4.1). In both trials, 

efficacy was improved significantly when Si was sprayed onto plants in open pots. An 

overall increase of 17% (Trial 2) and 18% (Trial 1) in disease reduction occurred on plants 

in open pots, where Si was allowed to reach the rhizosphere, compared to the sealed pots 

where the spray was restricted to the phylloplane (Figure 4.2). 

In both trials, the interaction of Si concentration and frequency was significant. Interactive 

effects of concentration and frequency on the efficacy of Si was significant in the second 

experiment but not in the first experiment. However, trends of these two experiments 

showed that the best results were obtained when Si was used at higher concentrations with 

higher frequencies (i.e., 3 times per week). For instance, when Si was applied 3 times a 

week at 1000 mg l -1 , then its efficacy was increased by more than two - three times 

(Figures 4.1 & 4.2). 

Interactive effects of Si concentration and application method were not significant on the 

efficacy of Si. However, improved efficacy was obtained when treatments with higher 

levels of Si were sprayed on plants in open pots. Interaction of frequency and runoff on the 

efficacy of Si was not significant. In both trials, efficacy of Si was improved when sprayed 

onto plants in open pots at higher frequencies. Increasing the frequency from 1 to 3 times 

per week increased the efficacy of the treatment by a mean of 35% in the first experiment 

and nearly 100% in the second experiment. Trends of the two trials also showed that the 

increase in efficacy was slightly higher when sprays were applied to plants in the open 

pots. 

Interaction of concentration, frequencies and application method was only significant on 

the second experiment. However, individual observations showed that the best results were 

obtained when Si was sprayed at higher frequencies to plants in open pots. Effects of Si at 

various concentrations sprayed onto plants in open and sealed pots, at different 

frequencies, in controlling PM are presented on Figures 4.1 & 4.2. 

75

Trial 1 

Trial 2 

Figure 4.1 Histograms showing the effects of spraying Si on the severity of powdery mildew of 

zucchini, when applied at various concentration and frequencies on plants grown under greenhouse 

conditions in open or covered pots in two different trials. 

76

Covered pots 

Open pots 

Trial 1 

Spray frequencies 

3 / wk (▲): y = 2.199x 2 - 8.252x + 61.77 

R² = 0.815 

2/wk (×): y = 0.844x 2 - 1.176x + 41.17 

R² = 0.972 

1 / wk (■): y = -2.488x 2 + 6.896x + 43.01 

R² = 0.707 


3 / wk (▲): y = 3.497x 2 - 14.22x + 75.78 

R² = 0.745 

2 / wk (×): y = 2.536x 2 - 12.34x + 65.82 

R² = 0.999 

1 / wk (■): y = -3.320x 2 + 16.88x + 34.82 

R² = 0.999 

Trial 2 


3 / wk (▲): y = 4.092x 2 - 16.25x + 89.46 

R² = 0.998 

2 / wk (×): y = 1.691x 2 - 7.780x + 87.88 

R² = 0.696 

1 / wk (■): y = -7.689x 2 + 30.20x + 48.92 

R² = 0.989 


3 / wk (▲): y = 3.508x 2 - 16.26x + 100.3 

R² = 0.945 

2 / wk (×): y = 2.867x 2 - 15.06x + 98.96 

R² = 0.718 

1 / wk (■): y = 0.823x 2 - 2.583x + 71.45 

R² = 0.235 

Figure 4.2 Relationship between Si concentrations and frequency of sprays applied in open or covered pots on PM control of zucchini 

plants grown under glasshouse conditions in two different trials. 

77

Table 10.1 Analysis of variance showing factorial interactions between concentration, 

frequency and runoff of foliar-applied silicon on severity of powdery mildew of zucchini. 

Source of variation 

Trial 1 Trial 2 

F-value P-value F-value P-value 

Concentration 1.03 0.389 0.84 0.481 

Frequency 32.56 < 0.001*** 25.89 < 0.001*** 

Runoff 21.86 < 0.001*** 5.19 0.027* 

Concentration*Frequency 2.26 0.053 3.54 0.006** 

Concentration * Runoff 0.33 0.805 1.84 0.153 

Frequency*Runoff 0.13 0.877 0.48 0.621 

Concentration * Frequency * Runoff 0.90 0.503 3.06 0.013* 

CV (%) 18.4 24.42 

Values with *= significant at P < 0.05; **= significant at P < 0.01; *** = significant at P < 0.001 


In this study, spraying soluble Si onto the leaves of zucchini reduced the severity of PM 

significantly. This confirmed the findings of previous research on the use of Si sprays for 

the control of PM of cucumber (Menzies et al., 1992; Liang et al., 2005), grape (Bowen et 

al., 1992; Reynolds et al., 1996), muskmelon (Menzies et al., 1992), strawberry (Kanto et 

al., 2004; Palmer et al., 2006), wheat (Guével et al., 2007) and zucchini (Menzies et al., 

1992). 

In most cases, foliar applications of Si provides less disease control compared to when the 

element is fed to the plant in a nutrient solution or as a soil amendment (Guével et al., 

2007; Liang et al., 2005). However, in some plants foliar application provides better 

efficacy than supplying the element to the roots. For instance, Bowen et al. (1992) found 

that spraying Si at 1000mg l -1 onto grape leaves reduced the severity of PM significantly; 

whereas treating the plant with a Si-amended solution did not. This was considered to be 

because foliar application allows Si to be applied to non-hydroponic crops or crops that are 

unable to transport Si from their root to shoots through their vascular system, (Bélanger et 

al., 1995). 

78

When Si is applied as a foliar treatment, its mode of action in reducing severity of PM may 

be different from when it is fed through the roots (Wang and Galletta, 1998). Spraying Si 

on cucumber can result in the formation of a coating (film) on the leaves (Menzies et al., 

1992), which would act as a physical barrier, preventing the penetration of fungal hyphae 

into the host (Bowen et al., 1992). Guével et al. (2007) showed that foliar application of Si 

has a direct effect on PM over and above any effects mediated by the plant, as results from 

root amendments which may lead to induced resistance. A direct action by Si spays on PM 

was confirmed by our results when Si was sprayed onto plants where the element was 

restricted from reaching the roots of the plants. When KSi is used against PM, the active 

ingredient of the spray appears to be Si (Menzies et al., 1992). 

Results of this study showed that in the range of 250-1000 mg l -1 , the level of Si in the 

spray solution had little impact on the efficacy of Si in PM control. Severity of PM was 

significantly reduced by all treatments, indicating that application of Si at lower 

concentrations could provide the optimum disease control while minimizing the cost of 

control. Observations by Menzies et al. (1992) on the use of Si against PM of different 

hosts showed that the efficacy of a foliar spray of Si at 1000 mg l -1 was equivalent to a 

solution of 100 mg l -1 applied as a soil amendment. 

In this study, the efficacy of Si was improved by increasing the spray frequency. When the 

application frequency was increased from 1 to 3 times per week, efficacy of Si was 

doubled. This agrees with a report of Reynolds et al. (1996), who increased the efficacy of 

Si sprays against PM of grape (Uncinula necator (Schwein.) Burrill) by increasing spray 

frequency. Increasing application frequencies could increase the total amount of Si 

deposited on the surface of the leaf, resulting in better efficacy. Spraying Si at a 

concentration of 250 mg l -1 with a frequency of 3 times per week could be expected to 

result in a lower amount of Si being deposited on the leaf than if it was applied once a 

week at a concentration of 1000 mg l -1 . However, this study showed that spraying Si at 

250 mg l -1 x 3 x wk -1 was more effective than spraying 1000 mg l -1 x 1 x wk -1 . Even for 

the adjacent treatment, which did not contain Si, disease severity was reduced by 

increasing the frequency of spray treatment. Therefore, when spray frequency is increased, 

there must be other factors that are involved in improving the efficacy of Si other than the 

total amount of the element deposited on the phylloplane. The wetting agent (BK) and 

increased leaf wetness could have their own impact on the pathogen. Break-Thru ® was 

79

shown to have a direct effect on PM restricting expansion of its colonies and collapsing 

conidia and hyphae of the pathogen (Chapter 3). Although extended periods of leaf 

wetness can favour infection of zucchini by several foliar pathogens, it has a negative 

effect on the development of PM by inhibiting germination of the conidia (Bushnell and 

Rowell, 1967; Quinn and Powell, 1982; Sakurai and Hirata, 1959). Most importantly, 

increasing the frequency of applications could improve the continuity of Si supply to plants 

roots through runoff. Even though the major factors remain unknown, this study 

demonstrated that better disease control could be achieved by spraying Si at lower 

concentrations with increased spray frequencies instead of applying higher concentrations 

of Si at lower frequencies. 

When the same concentration of Si was sprayed at the same frequencies per week, the 

severity of PM recorded from plants grown in uncovered pots was less than that of plants 

grown in covered pots. This was because, in open pots, part of the spray solution was 

intercepted by the soil as a result of drift and runoff. Once the Si solution was in the 

rhizosphere of the plant, it could be absorbed by the roots and translocated to different 

parts of the plant. Reviews by Epstein (1994, 1995 & 2001) showed that adding Si into the 

nutrient solution or adding it to the soil benefits plants by providing protection against 

pathogens through physical barriers and triggering plant resistance against pathogens, and 

ameliorating other biotic and abiotic stresses of the plant. However, when KSi is sprayed 

onto the leaf, it may be deposited on the surface of the leaf without penetrating into the 

plant (Buck et al., 2008). Therefore, the possible mechanisms of protection it provides 

might have been through direct contact with the pathogen and alteration of the chemical 

properties of the leaf such as pH, which could lead to changes in osmotic properties of the 

leaf surface (Liang et al., 2005). Using scanning electron micrographs, Bowen et al. (1992) 

observed an active accumulation of Si around the appressoria of the PM fungi when Si was 

sprayed onto leaves of cucumber plants. However, Reynolds et al. (1996) suggested that 

exogenously applied silicates act to augment the activity of their endogenous counterparts, 

resulting in the accumulation of the element around the pathogen. 

80

In most research conducted on the management of PM using foliar-applied Si, the effects 

of the drift or runoff of soluble Si on the disease has not been considered. However, as 

seen in this study, such drift and runoff can have a significant impact on the health of the 

plant and give a confounded conclusion because different modes of action can be involved, 

once the element is absorbed by the plant via its roots. For instance, foliar application of Si 

did not reduce severity of PM of strawberry (Palmer et al., 2006), but enhanced growth of 

the plant by increasing its chlorophyll content (Wang and Galletta, 1998). Therefore, such 

metabolic changes of the plant might be related to the absorption of Si through the roots as 

a result of drift and runoff of foliar-applied Si. 

In this study, the plants were irrigated using drip irrigation, avoiding any wash-off of Si 

from leaves to the soil. However, due to size of the growing area and other technical 

limitations, most growers prefer overhead irrigation than drip irrigation. In such cases, Si 

can be mixed with the nutrient solution and supplied to the plant as part of the irrigation. 

Using that technique could improve the efficacy and minimize/avoid the costs of labour 

that would be involved in spraying of the element three times a week to control the disease. 

This technique may improve the efficacy of the treatment by increasing the spray coverage, 

and ensuring a better contact between the spray and the pathogen. If proven economical, 

the foliar use of Si at appropriate application intervals and concentrations could replace 

fungicides for the management of PM on zucchini, a welcome development for organic 

farmers. 


Bélanger, R.R, Bowen, P.A., Ehret, D.L., Menzies, J.G. 1995. Soluble silicon: its role in crop and 


Bélanger, R.R., Dik, A.J., Menzies, J.G. 1997. Powdery mildew: recent advances toward integrated 

control. In: G.S. Boland and L.D. Kuykendall (Eds.). Plant-microbe interactions and biological 

control. Marcel Dekker, New York, USA. Pp. 89-109. 




Buck, G.B., Korndorfer, G.H., Nolla, A., Coelho, L. 2008. Potassium silicate as foliar spray and 

rice blast control. Journal of Plant Nutrition, 31: 23-237. 

81

Bushnell, W.R, Rowell, J.B. 1967. Fluorochemical liquid as a carrier for spores of Erysiphe 

graminis and Puccinia graminis. Plant Disease Report, 51: 447-448. 



Epstein, E. 1999. Silicon. Annual Review of Plant Physiology and Plant Molecular Biology, 

50: 641-64. 

Epstein, E. 2001. Silicon in plants: Facts vs. concepts. Silicon in Agriculture. In: L.E. Datnoff, 

G.H. Snyder, and G.H. Korndörfer (Eds.). Elsevier Science BV, Amsterdam, Pp. 1-15. 

Genstat. 2006. GenStat Statistical Analysis Software. 8 th ed. Lawes Agricultural Trust, Oxford, 

UK. 

Guével, M.H., Menzies J.G., Bélanger, R.R. 2007. Effect of root and foliar applications of soluble 


Pathology, 119: 429-436. 

Kanto, T., Miyoshi, A., Ogawa, T., Maekawa, K., Aino, M. 2004. Suppressive effect of potassium 

silicate on powdery mildew of strawberry in hydroponics. Journal of General Plant 

Pathology, 70: 207-211. 



Pathology, 54: 678-685. 

Menzies, J., Bowen, P., Ehret, D., Glass, A.D. M. 1992. Foliar applications of potassium silicate 

reduce severity of powdery mildew on cucumber, muskmelon, and zucchini squash. 

Journal of the American Society for Horticultural Science, 117: 902-905. 

Palmer, S., Scott, E., Stangoulis, J., Able, A.A. 2006. The effect of foliar-applied Ca and Si on the 

severity of powdery mildew of strawberry cultivars. 2006. Acta Horticulturae, No. 708: 

135-139. 

Quinn, JA, Powell, CC Jr. 1982. Effects of temperature, light, and relative humidity on powdery 

mildew of begonia. Phytopathology, 72: 480-484. 

Reynolds, A.G., Veto, L.J., Sholberg, P.L., Wardle, D.A., Haag, P. 1996. Use of potassium silicate 

for the control of powdery mildew [Uncinula necator (Schwein) Burrill] in Vitis vinifera L. 

cultivar Bacchus. American Journal of Enology and Viticulture, 47: 42-428. 

Romero, D., Perez-Garcia, A. Rivera, M.E., Cazorla, F.M., de Vicente, A., 2004. Isolation and 



Romero, D., Rivera, M.E., Cazorla, F.M., de Vicente, A., Perez-Garcia, A. 2003. Effects of 


Mycological Researches, 107: 64-67. 

82

Sakurai, H., Hirata, K. 1959. Some observations on the relation between the penetration hypha and 

haustorium of the barley powdery mildew and the host cell. V. Influence of water spray on 

the pathogen and the host tissue. Annals of the Phytopathological Society of Japan, 4: 239- 

245. 

Wang, S.Y. and Galletta, G.J. 1998. Foliar application of potassium silicate induces metabolic 

changes in strawberry plants. Journal of Plant Nutrition, 21: 157-167. 

83

CHAPTER FIVE 

STUDIES ON THE EFFECTS OF SELECTED BIOCONTROL AGENTS 

AND SOLUBLE SILICON ON THE DEVELOPMENT OF POWDERY 

MILDEW OF ZUCCHINI, UNDER GREENHOUSE CONDITIONS 


a 



b Plant Health Products, P. O. Box 207, Nottingham Road, South Africa 

Abstract 

Duplicate trials were conducted under greenhouse conditions to evaluate the effects of five 

potential biocontrol agents and soluble silicon (Si) for the control of powdery mildew of 

zucchini caused by Podosphaera xanthii. Biocontrol agents were sprayed onto leaves with 

a wetter (Break-Thru ® ), and Si was drenched onto zucchini roots (250ml of K 2 SiO 3 at 

100mg l -1 , applied weekly). All five BCAs provided significant control of PM, whether Si 

was drenched to the roots or not. The effects of Si applied alone on disease severity and 

AUDPC were significant, reducing them by 23% and 32%, respectively. Application of Si 

improved the efficacy of most BCAs significantly. Of the five BCAs, the fungi provided 

better control of PM than the bacterial isolates, reducing disease levels by up to 90%. 

Higher disease pressure reduced the efficacy of Si against PM but, did not affect the 

performance of BCAs. 

84


Powdery mildews (PM) are prime targets for biocontrol agents (BCAs) because they are 

superficial pathogens, and are therefore accessible to external agencies. As a result, several 

biocontrol studies have been conducted to control PM under greenhouse conditions (Dik et 

al., 1998; Elad et al., 1998; Hijwegen, 1992; Verhaar et al., 1993, 1996). Controlled 

environmental variables of greenhouses provide optimum environmental conditions for the 

establishment and survival of antagonists. Most antagonists of PM require high humidity, 

making greenhouses an ideal environment for implementation of biocontrol. 

Ampelomyces quisqualis Ces. (Pertot et al., 2008; Sundheim, 1982), Bacillus subtilis 

(Ehrenberg) Cohn (Keinath and DuBose, 2004), Lecanicillium lecanii (Zimm.) Zare & W. 

Gams (syn. Verticillium lecanii (Zimm.) Viegas) (Askary et al., 1997; Miller et al., 2004; 

Verhaar et al., 1993; 1996), Sporothrix flocculosa Traquir, Shaw & Jarvis (syn. 

Pseudozyma flocculosa) (Bélanger et al., 1994; Jarvis et al., 1989; Hajlaoui and Bélanger, 

1991), Tilletiopsis spp. (Urquhart et al., 1994; Hijwegen, 1992), and Trichoderma 

harzianum Rifai (Elad, 2000; Elad et al., 1998; Pertot et al., 2008) have all shown 

moderate to good disease control of PM when tested in greenhouses. Some of these 

antagonists have been commercialized. For instance, A. quisqualis strain AQ10 was 

marketed as AQBiofungicide (Elad et al., 1998), B. subtilis Strain QST 713 as Serenade TM 

(Ngugi et al., 2005) and T. harzianum strain T39 as TRIXODEX TM (Elad et al., 1998). 

Low humidity levels have been the major factor inhibiting the use of BCAs against PM on 

field crops. As a result, biocontrol of this disease has been limited to greenhouse 

conditions, where humidity is not a limiting factor. To overcome this limit under field 

conditions, scientists have tried amending the BCAs with adjuvants or oils (Bélanger et al., 

1994; Philipp et al., 1990). 

The efficacy of BCAs depends on the climatic conditions of the crop. Powdery mildew can 

thrive under dry conditions, whereas most BCAs need relative humidity above 70% 

(Hajlaoui and Bélanger, 1991). Furthermore, the rate of development of PM may influence 

the level of control of PM by BCAs, especially in the case of hyperparasites. This means 

that the efficacy of BCAs may differ from season to season, from cultivar to cultivar, and 

may be influenced by other control measures such as fungicides. 

85

Use of soluble silicon (Si) in PM management has been studied under controlled 

conditions with some degree of success (Bélanger et al., 1995; Menzies et al., 1991b; 

Samuels et al., 1991a & 1994). Both BCAs and Si have drawn considerable attention 

because they are considered as environmentally friendly. However, the level of disease 

control obtained from each control options is often incomplete, raising the possibility of 

using them together in order to supplement each other. 

The objective of this study was to compare the efficacy of five selected BCAs against PM 

of zucchini (caused by Podosphaera xanthii (Castagne)), to determine the efficacy of Si in 

controlling PM and improving efficacy of BCAs by combining them with this element. 


5.2.1 Preparion of plants and inoculation with Podosphaera xanthii 

Seedlings of zucchini (Cucurbita pepo L., F1-Hybrid Partenon), were raised in a 

greenhouse operating at a temperature of 26-28 o C and relative humidity (RH) of 75-85%. 

After producing two fully developed leaves, they were transplanted into pots (180 mm in 

diameter) containing composted pine bark and transferred into another greenhouse (24- 

30 o C and 65-85% RH) permanently. The plants were irrigated with complete fertilizer 

[Ocean Agriculture 1 3:1:3(38) at 0.5 g l -1 ] + [Ca(NO 3 ) 2 at 0.5 g l -1 ] by means of drip 

irrigation. Pots were kept at a distance of 40 cm within rows and 150-200 cm between 

rows. After 3 d of transplanting, seedlings were inoculated by spraying 3-5 ml of conidial 

suspensions of P. xanthii (10 3 conidia ml -1 ) onto the leaves of each seedling using a hand 

sprayer. First, the source plants that were growing in a separated greenhouse were shaken 

24 h before spores harvested for inoculation. This was done to ensure that all conidia in the 

suspension were fresh and of the same age. Once the conidia had been counted, inoculation 

commenced immediately, within 2 h during late afternoon to ensure a sufficiently high 

temperature and relative humidity for infection. 

5.2.2 Application of biocontrol agents and silicon 

Clonostachys rosea (Link) Schroers, Samuels, Seifert & Gams (Isolate EH), Trichothecium 

roseum (Pers.) Link (Isolate H20) and 3 isolates of Serratia marcescens (Bizio) (i.e., 


86

Isolates B15, Y15 and Y41) were used as biocontrol agents against PM. All BCAs were 

produced according to their requirements as described in Chapter 2 and applied until runoff 

using a hand sprayer 2d before inoculation of the seedlings with PM and repeated after 4d 

when PM symptom started to appear and then continued weekly for 5 wk. The 

concentration of propagules per ml of each BCA was 10 6 for Isolates EH and H20 and 10 8 

for isolates of S. marcescens. As a wetter, Break-Thru ® (at 0.25 ml l -1 ) was added to each 

microbial suspension and mixed thoroughly. All BCAs were applied in the evenings to 

improve establishment of the antagonists by providing sufficient relative humidity. Si, in 

the form of KSi, was drenched onto the roots of each plant 3 d before inoculation and 

weekly thereafter (250 ml at 100 mg l -1 ). 

5.2.3 Disease assessment 

Assessment of the percentage of leaf area infected was recorded for each treatment weekly 

before the next application. Ratings on percentage of the leaf covered by PM was recorded 

for 3 leaves every week to represent disease severity and averaged to give the percentage 

of disease. The development of disease was very high, especially for the Untreated 

Control. This created a problem because the rated leaves soon developed 100% disease. 

To deal with this problem, ratings of the severity level of PM of the control plants were 

made on new leaves that were not fully covered by the PM colonies. Then the leaves that 

corresponded to the leaves rated for the Untreated Control plants were assessed to get a 

severity rating for the treatments. Finally, AUDPC was calculated from PM levels using an 

AUDPC Program (Shaner and Finney, 1977). 

5.2.4 Data analysis 

Two trials were conducted, with each treatment having three replications, arranged in a 

randomized complete block design. Analysis of variance and contrast analysis between 

treatments that contained Si and without Si were performed using GenStat ® Statistical 

Analysis Software (GenStat, 2006). Comparison between means of treatments was 

performed using Fisher’s Protected LSD and efficacy of each treatment on percentage 

reductions of FDL and AUDPC values were calculated by comparing the values of each 

treatment with that of the Untreated Control. 

87

5.3 RESULTS 

One week after inoculation, the mean leaf area infected of 3% was recorded on the first 

three leaves of the Untreated Control. The level of disease increased exponentially and at 

the end of the second week, these leaves were 100% diseased, and the newly emerging 

leaves were infected. In the second trial, the mean disease level after one week was 18% 

and subsequently followed a similar pattern. At the end of the month, the level of leaf area 

infected of of plants from the Untreated Control was 61% and 89% for the first and second 

trials, respectively. In both trials, the effects of treatments on the severity and AUDPC 

values were highly significant (F < 0.001). 

In the first week of the first trial, the severity levels of PM recorded for all treatments were 

similar, ranging from 0-5%. However, severity levels of PM started to differ after the 

second week of treatments. In the second trial, the severity levels of PM were obvious after 

only one week of infection, with the antagonistic fungi Isolates EH and H20, with and 

without Si, reducing disease severity to a level of < 3% versus a level of 18% recorded for 

the Untreated Control. The levels of PM of plants treated by bacterial BCAs alone, or with 

Si, were within the range of 2-7%; while plants treated with Si alone developed an initial 

infection level of 10%. In both trials, all BCAs reduced PM level significantly (P < 0.001). 

The ranking order of treatments and the percentage of reduction obtained by all treatments 

are presented in Table 5.1 and Figures 5.1 and 5.2, respectively. 

Application of Si had a significant effect on the severity of PM (P < 0.05), and the contrast 

in severity levels of PM between plants treated with Si and those that were not treated with 

Si was highly significant (P < 0.05). Furthermore, most BCAs provided improved control 

of PM when they were applied to plants that were being treated with Si. 

The effects of treatments on AUDPC were significant (P < 0.001) for both trials. All BCAs 

reduced PM development, as reflected by significantly lower AUDPC values. Similarly, 

application of Si had a significant impact on AUDPC values of the second trial, but not in 

the first trial. In both trials, Si alone significantly reduced the AUDPC values. Integrated 

applications of Si and BCAs resulted in significantly lower AUDPC values, with most 

BCAs treatments showing an improved efficacy, compared to their respective values 

without Si (Table 5.1). Overall, application of Si provided a mean reduction of 13 % in 

disease severity and AUDPC values. 

88

Table 11.1 Effects of selected biocontrol agents and soluble silicon (Si) on final disease level (FDL) and area under disease progress curve 

(AUDPC) of powdery mildew of greenhouse-grown zucchini five weeks after inoculation with Podosphaera xanthii. 

Treatments 

FDL AUDPC FDL AUDPC 

-Si +Si -Si +Si -Si +Si -Si +Si 

Control 61.3 c 41.7 b 688.8 c 470.2 b 89.3 e 68.7 d 965.0 e 758.2 d 

B15 43.3 b 37.7 b 448.2 bc 396.7 b 39.0 c 35.0 bc 379.2 c 221.8 b 

EH 32.0 ab 30.0 ab 361.7 ab 331.3 ab 11.0 a 6.3 a 145.8 ab 126.0 a 

H20 36.0 b 30.0 ab 374.5 ab 309.2 ab 5.3 a 9.0 a 71.2 a 128.3 a 

Y15 25.3 a 34.0 ab 249.7 a 361.7 ab 28.0 b 26.3 b 275.3 b 271.8 b 

Y41 36.0 b 30.3 ab 399.0 b 337.2 ab 29.0 b 31.3 bc 255.5 b 292.8 bc 

Average 39.0 33.9 420.3 367.7 33.6 29.4 348.7 299.8 

Effects F-values F-values F-values F-values 

- Si = treatments without Si, + Si= treatments with Si 


BCAs < 0.001 < 0.001 < 0.001 < 0 .001 

Si 0.020 0.052 0.045 0.009 

BCAs * Si 0.020 0.041 0.022 < 0.001 

FLSD 10.25 130.14 9.95 87.08 

CV (%) 16.6 19.5 18.6 15.9 

Means within column followed by a common letter were not significantly different according to Fisher’s protected least significant difference (P


BCAs 

PM control (%) 

Figure 5.1 Percentage reduction in final disease levels of powdery mildew of greenhouse-grown zucchini by five biocontrol agents 

and soluble silicon compared to an Untreated Control treatment five weeks after inoculation with Podosphaera xanthii. 

90


BCAs 


Figure 5.2 Percentage reduction of the Area under disease progress curve (AUDPC) values of powdery mildew of greenhouse-grown 

zucchini by five biocontrol agents and soluble silicon compared to an Untreated Control treatment, five weeks after inoculation with 

Podosphaera xanthii. 

91


Infection by PM and consequently, development of PM, was very rapid in both trials, 

partly due to the environmental conditions of the greenhouse that favoured PM. Powdery 

mildew progress on the Untreated Control plants demonstrated the ideal conductions of the 

greenhouse to the development of PM. Powdery mildew is a very common disease of crops 

that are grown under such conditions. This is because its high RH, high temperature and 

restricted air circulation provide an ideal environment for germination of conidia of PM 

fungi and their establishment (Howard et al., 1994). Once infection starts in such 

environment, the disease spreads throughout the greenhouse at a fast rate and causes a 

massive impact on the yield and quality of the fruit, if not managed properly (Dik et al., 

1998). 

Restrictions on the use of fungicides have intensified the use of biological control as an 

alternative approach in controlling PM. As a result, some promising results have been 

reported by several researchers (Dik et al., 1998; Elad et al., 1998; Hijwegen, 1992; Miller 

et al., 2004; Verhaar et al., 1993 & 1996). This is because PM grows superficially on the 

leaf and its exposure to the external environment makes it vulnerable to attack by many 

microbes (Bélanger et al., 1998). However, due to the dependency of antagonists on 

specific environmental conditions for establishment and survival, most of them operate 

more effectively within a limited range of temperature and RH (Bélanger et al., 1998; 

Jarvis and Slingsby, 1977; Jarvis et al., 1989). When BCAs are tested against PM, the most 

limiting factor has been their requirements for a high RH. In this regard, greenhouses are 

ideal sites for exploitation of biological control because they provide the best 

environmental conditions for the development and activity of BCAs. This was 

demonstrated in our trials where the severity levels of PM were very high in the Untreated 

Control. 

In both trials, all BCAs reduced disease severity by suppressing the infection rate and 

development of PM. After 4wk of treatments with these BCAs, promising levels of disease 

reduction (40-90%) were obtained. Reductions in the levels of PM can increase the amount 

and quality of yields by zucchini. We did not find a publication on the impact of PM on 

yields of zucchini. However, in cucumber, a level of infection as little as 5% for 60d will 

translate into a yield loss of 6% (Bélanger et al., 1998) and severe infection can reduce the 

92

yield by more than 50% (Sundheim, 1982). Therefore, the ability of our isolates to 

suppress PM level by 40-90% shows their potential to be used against PM under 

greenhouse conditions, giving the growers an alternative option to managing PM 

effectively. 

Most of our isolates controlled PM better when they were applied to plants treated with Si. 

Drenching the pots of zucchini weekly with 250 ml of a Si solution at a concentration of 

100 mg l -1 reduced PM by an average of 12-14% over all treatments. Such additive effects 

were likely due to a combination of mechanisms that are involved by the mixed treatment 

in controlling PM as opposed to the modes of action provided by Si or individual 

antagonist, applied alone. Although the mechanism by which Si exerts its protective effects 

against plant diseases is the subject of debate and controversy (Ghanmi et al., 2004), 

prevention of penetration of PM fungi as a result of physical barrier (Bowen et al., 1992; 

Samuels et al, 1991a & b), priming the resistance of the host (Cherif et al, 1994; Fawe et 

al., 1998; Menzies, et al., 1991b) and ameliorating the biotic and abiotic stresses of the 

plant (Epstein, 1994, 1999, 2001) are currently considered to be the main modes of action 

of the element. In case of biological control, the known mechanisms of action employed by 

different antagonists in suppressing PM include: antibiosis (Dik et al., 1998; Hajlaoui et 

al., 1992 & 1994; Urquart et al., 1994 ), competition (Nofal and Haggag, 2006), 

mycoparasitism (Askary et al., 1997; Falk et al., 1995a & b; Kiss 1998; Romero et al., 

2003; Sundheim, 1982; Sztenjberg et al., 1989; Verhaar et al., 1997) and inducing host 

resistance (Elad et al., 1998; Silva et al., 2004; Vogt and Buchenauer, 1997). Therefore, 

when some of these mechanisms of action are used in controlling PM, better disease 

control could be obtained. Sometimes, co-application of BCAs together or with Si might 

not give an improved efficacy against the target disease, PM. However, it might still 

benefit the plant because one of the treatments in the combination could provide protection 

against non-target pathogens. For instance, co-application of AQ10 and Trichodex did 

not improve efficacy of the application in controlling PM of cucumber but gave better 

control of grey mold (Elad et al., 1998). 

The level of control obtained by Si alone, or in combination with the BCAs, was 

promising. However, there is still a possibility that such level of control can be improved 

by manipulating the environment of the greenhouse further. For instance, setting the 

operating temperature at 20-25 o C can give better results (Schuerger and Hammer, 2003). 

93

According to these authors, suppression of PM of cucumber by Si was independent of light 

intensity, but was significantly inhibited when the temperature was higher than 24 o C. 

Co-application of BCAs with other control options has also given an improved success in 

controlling PM of different crops in similar environment (Dik et al., 1998; Elad et al., 

1998; Sundheim, 1982). This is because when some BCAs are used alone, they cannot 

offer protection for the entire season so that they can be viable alternatives to chemicals 

(Pertot et al., 2008). This is common especially when mycoparasites are used as BCAs, 

which need a certain level of disease since they can only attack established infections (e.g., 

A. quisqualis) (Fokkema, 1993). In contrast, epidemics of PM develop very fast and can 

overtake the mycoparasite. For this reason, repeated applications of BCAs may increase 

the likelihood of their controlling PM. However, if the mode of action of the BCA is 

antibiosis, the antagonist does not have to be in direct contact with the pathogenic fungi 

because the molecules will diffuse over the leaf surface (Dik et al., 1998). In this study the 

use of Break-Thru ® has improved efficacy of BCAs by enhancing their deposition on the 

leaf and the pathogen and directly affecting the PM fungi. 

In most reports where Si produced promising results, the research has been conducted 

under controlled environments by growing plants in hydroponics (Kanto et al., 2004; 

Schuerger and Hammer, 2003) or recycling-nutrient solutions (Adatia and Besford, 1986). 

In both systems, there is continuous supply of Si to the plant, giving an improved efficacy 

compared to the technique used in this study. In our case, Si was supplied to the 

rhizosphere once a week, from which some of the solution was drained out of the pots due 

to gravity, or leached as a result of irrigation, leaving little opportunity for the plant to 

utilize the supply. This means that the amount of Si that could be absorbed by the plant 

was lower than when the element is supplied continuously to the roots. When Si is used to 

provide control of diseases, the availability of Si over time is more important than its 

amount. This is because if the supply is interrupted, even for one day, the plant may be 

infected by the pathogen (Samuels et al., 1991b). Once infection occurs, protective 

measures have little impact on PM. Samuels et al. (1991b) have described the type 

protection offered by Si against diseases as “non-systemic resistance”. Therefore, we 

believe that improved efficacy can be obtained by increasing the frequency of supply of Si 

to plant roots. Alternatively, reducing the loss of Si by reducing the drainage can play a 

significant role in improving the availability of Si to the plant. 

94

The overall disease levels were high in Trial 2, which made most BCAs perform better. 

However, Si performed worse under the high disease pressure of Trial 2. In Trial 2, the 

disease severity level increased by 31%, reflecting a 28% reduction in the efficacy of Si. 

This was assumed to be a direct response to the increase in disease pressure compared to 

the first trial. High levels of infection at the start of Trial 2 might have played a role in 

reducing the efficacy of the Si treatment by increasing the development of PM, which 

ultimately resulted in a high final disease level. Kanto et al. (2007) demonstrated that the 

impact of Si applications is reduced when the plants are already infected with PM. 

Therefore, for best control of PM, Si must be applied prior to infection, by relying on the 

previous history of the growing environment or disease forecasts. If Si fails to provide 

protection against infection by the pathogen or if the pressure of PM is too high, the use of 

other control options, including fungicides, may be needed as emergency measures so that 

Si can function at a manageable level of PM. In spite of the reduced efficacy of Si in 

Trial 2, the fact that both control options provided promising results gives hope that they 

can provide economic control under natural levels of infection, where PM levels are 

usually much lower than the levels tested here. 


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98

CHAPTER SIX 

USE OF SELECTED BIOCONTROL AGENTS AND SILICON FOR THE 

MANAGEMENT OF POWDERY MILDEW OF ZUCCHINI 

UNDER FIELD CONDITIONS 


a 



b Plant Health Products, P. O .Box 207, Nottingham Road, South Africa 

Abstract 

Two field trials were conducted at the Ukulinga Research Farm to evaluate the efficacy of 

five selected biocontrol agents and soluble silicon (Si) in controlling powdery mildew of 

zucchini caused by Podosphaera xanthii. Biocontrol agents were applied as foliar sprays at a 

concentration of 10 8 propagules ml -1 . One litre of Si at 100 mg l -1 was drenched weekly 

into the rhizosphere of treated plants. Although statistically not significant, disease 

reductions of 32-70% by Si alone, 30-53% by Isolate B15, and 33-65% by Isolate B15 + Si 

were achieved. Other BCAs applied alone or together with Si also reduced disease levels 

by 9-68%. Plants treated with most of the BCAs showed significantly lower AUDPC 

values. For most antagonists, better efficacy was obtained when Si was drenched weekly 

into the rhizosphere of the plants. Efficacy of some of the BCAs and Si were affected by 

the environmental conditions of the field. A low temperature with a high humidity 

enhanced the performances of Isolate B15 plus Si whereas these conditions suppressed the 

fungal BCAs. However, at a high temperature and a low relative humidity, the efficacy of 

the fungal antagonists was superior, and the rest of the treatments provided reduced disease 

control. Although promising results were achieved by all treatments, repeated trials and 

better understanding of the use of Si and the BCAs in their dosage and application 

frequency, as well as interactions with the host plant and the environment, are needed 

before they can be implemented on a commercial scale for sustainable control of the 

powdery mildew. 

99


The development of fungicide resistant fungal mutants combined with increased concerns 

over health and environmental hazards caused by fungicides have intensified the interest in 

identifying biocompatible products that can supplement or replace conventional fungicides, 

especially to control diseases such as powdery mildew (PM) (Shishkoff and McGrath, 

2002). 

Effective control of PM with several biocontrol agents has been achieved under 

greenhouse conditions (Bélanger et al., 1997; Elad et al., 1996 & 1998; Falk et al., 1995; 

Jarvis and Slingsby, 1977; Verhaar et al., 1996). This has resulted in the development of 

some promising biocontrol products on the market. However, efficacy of many of these 

products has been inconsistent when tested under field conditions, partly due to the 

variability of the many factors that govern the establishment of BCAs (Bélanger et al., 

1994; Schuerger and Hammer, 2003). Unlike greenhouse conditions, where humidity, 

temperature and other growth factors are relatively stable, field conditions are variable and 

unpredictable. Humidity, temperature, light intensity and rainfall are some of the main 

factors that determine the success of a BCA. These factors have a direct impact on disease 

development, and the survival and antagonistic activities of BCAs on the phylloplane. 

Adding silicon (Si) into nutrient solutions has reduced PM of cucurbits grown in 

hydroponics (Bélanger et al., 1995; Menzies et al., 1991a & b; Samuels et al., 1991a; 

Schuerger and Hammer, 2003). As a foliar spray, Si has also been reported to effectively 

control PM on cucumber, muskmelon and zucchini (Menzies et al., 1992), and on grape 

(Bowen et al., 1992). 

However, under both greenhouse and field conditions, the level of control achieved with 

BCAs and Si is often incomplete. This is as a result of the slow establishment of BCAs as 

opposed to the fast development of the disease. One of the limitations of Si has been the 

failure to provide curative protection once the plant is infected by the pathogen (Kanto et 

al., 2007). Co-application of BCAs and Si may complement each other to give a better 

disease control. 

100

To date, we have found no publications in which Si was directly supplied to the roots of 

zucchini plants growing under field conditions. Objectives of this study were to evaluate 

the efficacy of five selected isolates and Si against PM of zucchini caused by Podosphaera 

xanthii (Castagne) under field conditions, and to study the possibility of using these two 

control options in the development of an integrated field management strategy against this 

disease. 


6.2.1 Trial site, land preparation and transplanting 

The field trials were conducted at Ukulinga Research Farm located at E 29 o 40E and 30 o 24’ 

S, 715 m above sea level, in the Southern Tall Grassveld of South Africa (Morris, 2002) on 

heavy, deep soil of Bonheimer clays (Dr R. Melis 1 , 2002, pers. comm.). 

The field was thoroughly plowed twice with a tractor and all the existing weeds were 

removed by hand. To moisten the field and enhance survival of the seedlings, irrigation 

was commenced one day before transplantation. Seedlings of zucchini, raised as described 

in previous sections, were transplanted into the field once they had a fully developed 

second leaf. Planting distance was 1.25m between plants and 1.5m between rows. During 

the experimental periods, moisture level of the field was monitored daily. The field was 

irrigated with overhead irrigation and kept free of weeds with herbicides, as needed. 

6.2.2 Preparation of Podosphaera xanthii inoculum 

Collection, storage and preparation of P. xanthii were the same as described in the previous 

Chapters except that inoculations were performed by spraying the conidia onto the 

zucchini crop with a CP3 knapsack sprayer instead of a hand sprayer. 

1 Dr. R. Melis, Pro-Seed C.C., P.O. Box 101477, Scottsville 3209, South Africa . 

101

6.2.3 Application of biocontrol agents and soluble silicon 

Microbial suspensions of three Serratia marcescens (Bizio) isolates (i.e., Isolates B15, Y15 

and Y41) and two fungi (Clonostachys rosea (Link) Schroers, Samuels, Seifert & Gams 

(syn. Gliocladium roseum) (Isolate EH) and Trichothecium roseum (Pers.) Link (syn. 

Cephalothecium roseum) (Isolate H20) ) were prepared as described in the previous 

sections and were applied onto plant leaves at a concentration of 10 8 propagules ml -1 , 

together with a wetter (Break-Thru ® , 0.25 ml l -1 ) 3 d before inoculation and continued 

weekly 4 d after inoculation. All BCAs were applied using a CP3 Knapsack sprayer until 

runoff. 

One litre of Si at 100 mg l -1 , in the form of KSi, was drenched onto the roots of each plant 

3 d before inoculation and weekly thereafter. Where Si was not used as a treatment, the 

same volume of clean water was drenched onto the rhizosphere. All treatments were made 

during the late afternoon or early evening to provide favourable conditions for the 

establishment of BCAs and to increase the availability of Si to the plant roots by reducing 

evaporation. 


The first disease rating was performed 10 d after inoculation with conidia of P. xanthii and 

continued weekly for 4 wk. Percentage of the area of leaves covered by PM was recorded 

weekly as the disease severity and area under disease progress curve (AUDPC) was 

calculated from the disease percentage during the course of the trial using an AUDPC 

Program (Shaner and Finney, 1977). 

6.2.5 Statistical analysis 

Two trials were conducted, with each treatment having three replications, arranged in a 

randomized complete blocks design. Each replication was represented by a plot of 12 

plants. To reduce inter-plot interference, data was collected from the central row of each 

plot. Data was analyzed using Genstat (R) Statistical Analysis Software (Genstat, 2006). 

Comparison between treatments with Si and without Si was performed using contrast 

analysis. Where the CV (%) was > 20%, data was transformed using a square root 

102

transformation. Means of treatments were compared using Fisher’s Protected Least 

Significant Difference (FLSD) and efficacy of each treatment on percentage reductions of 

FDL and AUDPC values were calculated by comparing the values of each treatment with 

that of the Untreated Control. 

6.3 RESULTS 

Symptoms of PM started to appear one week after inoculation and developed slowly. In all 

plots, the disease was mainly concentrated on the older leaves, which senesced with 

increasing PM severity. Late-emerging leaves were susceptible to infection, but severity of 

the disease was relatively low on them. To assess levels of infection, disease progress 

within the same period was analysed for each plot. 

The impact of the selected BCAs and Si on the severity levels of PM after 5 wk of 

inoculation are presented in Table 6.1. Some of the treatments significantly reduced the 

severity of the disease in the first trial (P < 0.05), but not in the second trial (Table 6.1). In 

contrast, the effects of treatments on disease progress, represented by AUDPC values, were 

not significant for the first trial. However, they were significant for the second trial 

conducted during January - March, 2007. In both experiments, the effects of Si on 

individual treatments were not significant, as shown by contrast analysis. However, most 

BCAs provided improved control when applied to plants treated with Si (Figures 6.1 & 

6.2). 

In the first trial (March - May, 2006), Si alone and Isolate B15 with/without Si controlled 

the disease significantly. Disease reduction of 73, 53 and 65% was obtained by treating the 

plant with Si, Isolate B15 and Isolate B15 + Si, respectively. The levels of disease after 

treatment by the rest of the individual treatments were not statistically lower than that of 

the control. However, they reduced the disease severity by 9 - 51%. In this trial, the 

bacterial isolates showed better antagonistic activity than the two fungal isolates (Figure 

6.1). Similarly, significant reductions in AUDPC values of 67 and 60% was recorded when 

plants were treated with Si and B15 + Si, respectively. In contrast, application of Isolate 

EH + Si resulted in an AUDPC value similar to that of the Untreated Control. In most 

cases, treatments that showed high level of disease had high AUDPC values (Table 6.1). 

103

However, if the disease levels recorded throughout the trial remained almost constant, it 

resulted in low or high AUDPC values depending on the initial disease level. 

In the second trial, which was conducted between January - March, 2007, the effects of 

treatments on the severity of PM were not significant at P < 0.05. However, Isolates H20 

and EH, without Si, reduced disease severity by 68 and 61%, respectively. Although not 

significant, application of the rest of BCAs, with or without Si, reduced PM by 22-41% 

and Si alone reduced disease severity by 32% (Figure 6.1). The effect of treatments on the 

development of disease, as represented by AUDPC, was significant at P < 0.05 when the 

original data was used, but none were significant when transformed data was analysed. In 

spite of these statistical differences, all BCAs, with or without Si, reduced the AUDPC 

values by 44-77%, in parallel with reductions in disease severity. 

In both trials, the effect of Si on individual treatments was not significant when contrasts 

were made between treatments that contained Si against those without Si. In spite of 

difference in disease levels between treatments, there were no obvious differences in terms 

of time to flowering and fruit setting among plots. 

104

Table 12.1 Effects of biocontrol agents and silicon on final disease level (FDL) and AUDPC values 

of powdery mildew of zucchini under field conditions 

Treatment Silicon Trial 1 Trial 2 

FDL AUDPC FDL AUDPC 

Water - 25.7 (4.91) b 233.3 (14.87) b 29.0(5.34) b 532.3(22.99) b 

Water + 7.0 (2.59) a 77 (8.59) a 19.7(4.16) ab 306.0(16.26) b 

B15 - 12.0 (3.45) ab 126.0 (11.17) ab 20.3(4.27) ab 266.3(16.13) ab 

B15 + 9.0 (2.98) a 92.2 (9.5)ª 19.3(4.29) ab 229.0(14.86) ab 

EH - 17.0 (4.1) b 129.5 (11.37) ab 11.3(3.35) a 168.0(12.71) ab 

EH + 23.3 (4.79) b 190.2 (13.68) b 20.0(4.47) ab 266.7(16.31) ab 

H20 - 20.7 (4.51 b 165.7 (12.75) ab 9.3(2.90) a 127.7(11.16) a 

H20 + 16.0 (3.97) ab 141.2 (11.67) ab 17.0(4.08) ab 201.0(14.02) ab 

Y15 - 16.0 (3.9) ab 136.5 (11.37) ab 22.0(4.67) ab 217.3(14.73) ab 

Y15 + 16.3 (4.03) b 151.7 (12.12) ab 22.7(4.72) ab 304.0(17.35) b 

Y41 - 14.3 (3.75) ab 143.5 (11.89) ab 20.0(4.28) ab 220.7(14.57) ab 

Y41 + 12.7 (3.55) ab 124.8 (11.14) ab 20.0(4.4) ab 283.7(16.64) ab 

Effects P-value P-value P-value P-value 

Treatment 0.030 0.200 0.587 0.068 

-Si vs. +Si 0.084 0.168 0.576 0.655 

F.LS.D. 1.266 3.992 1.98 5.891 

CV (%) 19.4 20.3 27.6 22.2 

- = no Si, += Si present 

Means within column followed by a common letter were not significantly different according to Fisher’s 

protected least significant difference (P < 0.05). 

Values in brackets are means of data transformed using square root transformations 

105


BCAs 

PM control (%) 

Figure 6.1 Efficacy of five biocontrol agents and silicon in reducing the severity of powdery mildew of zucchini under 

field conditions five weeks after inoculation with Podosphaera xanthii in two trials. 

106


BCAs 


Figure 6.2 Efficacy of five biocontrol agents and silicon in reducing the AUDPC values of powdery mildew of zucchini after five 

weeks of inoculation with Podosphaera xanthii under field conditions. 

107


The BCAs tested and a Si drench demonstrated the potential to reduce the final disease 

levels of PM of zucchini effectively. Isolate B15 alone, or together with Si, provided 

significant disease control. Compared to the rest of the isolates tested, Isolate B15 showed 

superior biocontrol activity, although its efficacy was lower in the second trial. However, 

the efficacies of most treatments were reduced in the second trial, with the exceptions of 

Isolates EH and H20 without Si. Five weeks after inoculation with P. xanthii, the epidemic 

of the disease, as represented by AUDPC values, was significantly reduced by all 

treatments. 

These two trials showed that, in spite of their potential to control the disease, inconsistency 

was observed with some of the isolates tested. For example, Isolate B15 was the best in the 

first trial, but performed poorly in the second trial. In contrast, Isolates EH and H20 were 

inferior at the first trial and were the best in the second trial. Interestingly, the performance 

of Isolates Y15 and Y41 were relatively stable in both trials. For most treatments, the 

disease level of the second trial was slightly lower than the first one. Cook and Baker 

(1983) suggested that effective biocontrol activity can only be obtained within a specific 

level of disease. According to these authors, if the disease level caused by a target 

pathogen is minimal compared to the Uninfected Control, then the efficiencies of various 

treatments in controlling the disease appear to be negligible, masking the efficacy of some 

genuinely effective isolates. In contrast, if the disease level is higher than certain level, 

especially for fast developing diseases such as PM, then the contribution of each control 

measure becomes minimal, and comparison between treatments usually results in nonsignificant 

differences. 

Significant control of diseases and subsequent increases in yield has been obtained in a 

number of field studies of biological control. The major problem, however, is the failure to 

repeat these results consistently in different soils or in different years in naturally infected 

fields and to make biological control of plant diseases competitive with chemical control 

(Schippers, 1988). Unlike greenhouse trials, where most environmental conditions are 

controlled, field trials results are affected by several environmental factors operating 

independently or in combination with each other. Reports of previous studies on biocontrol 

of several diseases indicated that fluctuations in environmental conditions in the field have 

been the main reason for the failure or inconsistent performance of biocontrol agents 

108

(Paulitz and Bélanger, 2001). Because of their requirements for specific environmental 

conditions, BCAs are usually effective within a limited range of temperature and RH 

(Blakeman and Fokkema, 1982). For instance, most antagonists of PM operate more 

efficiently when RH is maintained above 80% (Bélanger et al., 1997; Jarvis and Slingsby, 

1977), which can be achieved by manipulating the greenhouse environment, but this is not 

possible under field conditions. The requirement of many BCAs for a high RH has been 

confirmed by Jarvis et al. (1989) and Bélanger et al. (1994), who showed that the efficacy 

of Stephanoascus flocculosus Traquair , L.A. Shaw and Jarvis, Sporothrix flocculosa 

Traquair, Shaw & Jarvis and S. rugulosus Traquair, Shaw & Jarvis against PM was 

reduced when RH levels dropped below 60%. Hijwegen (1992) also showed that survival 

and antagonistic activities of Tilletiopsis minor Nyland against P. xanthii was reduced 

significantly when RH levels were reduced below 70-75%. Relative humidity is often the 

primary limiting factor under field conditions, when it drops below a minimum 

requirement of the BCAs. 

Climatic data secured for the two periods of the trials showed that during the first trial, the 

mean temperature was < 15 o C and the mean RH was > 70%. Although the prevalent RH 

was ideal for germination of PM spores and infection of the plant by the pathogen, 

development of the disease was retarded because of the low temperature, which was less 

than optimum for the fungus (Cheah and Falloon, 2001). Most BCAs prefer warmer 

environmental conditions for their establishment and best biocontrol activities. In spite of 

that, the performances of the bacterial isolates were favoured by high RH of the field, 

which was within the range of their requirement for their survival and biocontrol activities. 

In the second trial, where day time temperatures in the field were high (i.e. 27-29 o C) and 

RH was lower than < 54%, the efficacy of antagonistic fungi was better than that of 

bacterial isolates. This might have been related to the sensitivity/tolerance of these BCAs 

towards RH. Based on the results of these two trials, it was concluded that the fungal 

BCAs were relatively tolerant to low RH but were sensitive to low temperature; 

specifically, Isolate B15 was more sensitive to a low RH; and Isolates Y15 and Y41 were 

less sensitive to fluctuations in temperature and RH. 

Although the contrast analysis between treatments containing Si against treatments without 

Si showed no significant effect, application of Si reduced the disease severity consistently 

and improved the efficacy of most of the BCAs, especially in the first trial. Reduced 

109

performances of Si in the second trial might have been related to the high temperatures in 

the field. Schuerger and Hammer (2003) reported similar results, where the efficacy of Si 

in controlling PM of glasshouse-grown cucumber crop was reduced at higher temperatures 

(24-32 o C). According to these researchers, the optimum temperature for best control of the 

disease using Si was in the range of 20 o C, which supports the results of our first trial. 

Despite its potential, this study showed that the potential role of Si in controlling PM of 

zucchini under field condition may be limited, especially when compared to previous 

reports that were conducted under greenhouse conditions (Bélanger et al., 1995; Guevel et 

al., 2007; Menzies et al., 1991a & b; Schuerger and Hammer, 2003). Some of the 

following reasons might have contributed for this shortcoming. 

For effective control or disease prevention, Si must be supplied to the plant continuously. 

Interruption of the supply, even for one day, can result in infection of the plant by the 

pathogen. Samuels et al. (1991b) reported that enhanced resistance of cucumber against 

PM lasted only 24 h after Si was removed from the nutrient solution. Firstly, in this field 

study, one litre of Si was applied to the rhizosphere at a frequency of once per week. Most 

of the water content of the solution can be lost soon to evaporation, leaving dry Si in the 

rhizosphere. In this form, Si cannot be absorbed by plant roots because the element is not 

in the form of a solution. Secondly, some of the applied Si may have become permanently 

unavailable to the plant roots if it bounds to Al 3+ or Mn 2+ ions in the soil (M.D. Laing, 

2008, pers. comm.). Therefore, if the applied Si is not being taken up by the plant due to 

unavailability, the plant could be infected easily by the pathogen after 1-2 d of treatment. 

Once the symptoms appear on the susceptible plant, applying Si as curative measure has 

little impact because the disease can cover the whole plant within few days. Kanto et al. 

(2006) and Liang et al. (2005) noted that Si is more effective when used preventively than 

curatively. Therefore, increasing the application frequency or switching to using slow 

release formulations of Si could be used to improve the availability of Si to plants over an 

extended period of time. 

In greenhouse trials, Kanto et al. (2006) showed that the efficacy of Si in controlling PM of 

strawberry was affected by the susceptibility of the plants to the disease. These researchers 

showed that the less susceptible cultivar responded better to Si treatment than more 

susceptible cultivar. 

110

In both trials, there was little variation in terms of time of flowering and production of fruit 

among the treatments. From the lack of correlation between the treatments, and time of 

flowering and fruit setting, it would appear that the linkage between PM infection and the 

development of zucchini under field conditions is weak. Severe infection of PM causes 

indirect effects on the quality and yield of fruit by reducing the photosynthetic area of the 

plant through premature senescence of leaves (Cheah and Falloon, 2001; Keinath et al., 

2000). Fruit set is indirectly affected as a result of the reduced photosynthetic activities of 

the plant. Therefore, one explanation for our observation would be that the cultivar used in 

these studies may be highly susceptible, but has a relatively high level of tolerance to the 

disease, continuing to yield well despite high disease levels. 

The efficacy of the tested antagonistic fungi was generally superior in the second trial 

compared to the first one. In the first trial conducted from March to May 2006, the relative 

humidity was in the range of 70-75%, with a temperature of 4-12 o C. In the second trial, the 

mean temperature and RH during the growing period of January to February 2007 was 27- 

29 o C and 54-59%, respectively. From this information, it was concluded that Isolates B15, 

Y15 and Y41 have the potential to perform well at lower temperatures, provided that there 

are high RH levels. 

Some of the tested isolates provide some degree of control, especially when they were used 

together with Si. This is an encouraging result because it provides the growers with an 

option to include this combination as an alternative in PM control. Farmers who do not 

want to use conventional fungicides can use this option. However, since there were 

inconsistent performances by some of the BCAs tested, there is a need for repeated 

investigation under various environmental conditions. Repeated application of Si may give 

a better control as compared to the frequency used in this study. Hence, further research on 

the dosage/frequency of Si applications is needed before its use is adopted widely as a 

control strategy against this disease. In addition, the cultivar used in this study was very 

susceptible to the disease and the potential of the treatments might have been hindered due 

to the extremely fast development of the disease. It is possible that the efficacy of BCAs 

and Si treatments would have been improved if the cultivar used was less susceptible to the 

disease. In studies conducted by Bélanger et al. (1994) and Dik et al. (1998), it was 

observed that the degree of control provided by BCAs was better when resistant cultivars 

were used compared to susceptible ones. Similarly, the efficacy of sprays of inorganic salts 

111

such as potassium bicarbonate was affected by disease pressure (McGrath and Shishkoff, 

1999; Muza and Travis, 1995). Other reports also showed that efficacies of Ampelomyces 

quisqualis Ces ex Schlect, Trichoderma harzianum Rifai and Bacillus subtilis (Ehrenberg) 

Cohn were dependant on environmental conditions and disease pressure (Elad et al., 1998; 

Pertot et al., 2008). 

Moreover, co-application of some of the antagonists tested in this study might have 

provided enhanced efficacy in controlling the disease. In a number of studies, synergistic 

effects have been observed by combining antagonists. For instance, the use of 

Pseudomonas fluorescens Migula and a mixture of Trichoderma spp. provided effective 

control of PM and downy mildew (Abd-El-Moity et al., 2003). They also reported that 

when B. subtilis was added to that combination and applied at an early age (i.e. 4wk old), 

the crop gave its highest yields. The additive effects were probably due to a combination of 

multiple mechanisms that affect the pathogens, as opposed to the fewer control 

mechanisms provided by a single antagonist. 

There is also a need to understand more about the epidemiology of the disease, the 

resistance level of the plant and to identify the optimal environmental requirements of the 

BCAs in order to improve their efficacy by manipulating some of these variables. 

Moreover, since disease suppression by an introduced beneficial organism depends on the 

amount of inoculum applied onto the phylloplane, it may be necessary to alter the 

application protocol using higher rates, shorter spray intervals or applying chemical 

fungicides when the disease pressure is too high. 


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CHAPTER SEVEN 

UPTAKE AND DISTRIBUTION OF SILICON IN ZUCCHINI AND ZINNIA, 

AND ITS INTERACTION WITH THE UPTAKE OF OTHER ELEMENTS 

H.B. Tesfagiorgis a and M.D. Laing a 

a 



Abstract 

Elemental analysis was conducted to determine the impact of differing application levels of 

silicon (Si) in nutrient solutions on: (1) the uptake and distribution of Si into different 

organs (tissues) of zucchini and zinnia; (2) its impact on the uptake and accumulation of 

other elements; (3) the effect of powdery mildew on the levels of selected elements on 

these two plant species; and (4) the effects of Si uptake on the growth of zucchini and 

zinnia plants. Plants were grown in re-circulating nutrient solutions, supplied with Si at 

different concentrations. Samples were taken from different organs of each plant and 

analysed using energy dispersive X-ray fluorescence scanning electron microscopy (EDX) 

and inductively coupled plasma-optical emission spectrometers (ICP-OES). Increased 

levels of Si in the solution increased accumulation of Si in leaves and roots of both plants 

without affecting its distribution in other parts. In zucchini, roots accumulated higher levels 

of Si g dw -1 than leaves. With zinnia, accumulation of Si g dw- 1 was highest in leaves. 

Accumulation of K in shoots of both plants increased with increased levels of KSi in the 

nutrient solution. However, K levels in flower of zinnia, fruits of zucchini and roots of both 

plants remained unaffected. Increased level of Si reduced accumulation of Ca in both 

plants. 

Adding Si into the nutrient solution at a lower level (i.e., 50 mg l -1 ) increased the growth 

of zucchini plants and resulted in a maximal uptake of P, Ca, and Mg in both plants. 

However, application of higher levels of Si did not provide further growth enhancement. 

Levels of Si in the nutrient solution had no effects on elemental composition and 

characteristics of the fruits of zucchini. However, when Si was applied at > 50 mg l -1 , then 

116

the P level of the fruit was reduced by 50%. In both plants, infected leaves accumulated 

higher levels of Si and Ca, but less P, than leaves of uninfected plants exposed to the same 

levels of soluble Si. The highest concentrations of Si were observed in leaf areas infected 

with PM, and around the bases of trichomes of both plants. For optimum disease control 

and maximum accumulation of different elements in these two plants, application of Si at 

50-150 mg l -1 is recommended. 


Silicon (Si) is the second most abundant element in the earth’s crust. It has been considered 

as not essential to the growth of plants (Epstein, 1994). This is because of a perception that 

many plants can grow normally in its absence (Epstein, 1999) and symptoms for its 

deficiency and toxicity are not apparent (Ma and Yamaji, 2006). Even today, some 

scientists do not consider Si as an essential element for plant growth, although its beneficial 

roles on plant growth and resistance to biotic and abiotic stresses have received 

considerable attention (Datnoff et al., 1997; Epstein, 1994, 1999 & 2001; Ma, 2004; Ma 

and Yamaji, 2006; Meyer and Keeping, 2005). However, these researchers have shown that 

when crops with a high Si demand are repeatedly grown in soils with low levels of plantavailable 

Si, then symptoms of Si deficiency are being manifested by low productivity, and 

susceptibility of the crops to biotic and abiotic stresses. 

The mechanisms of uptake, translocation and accumulation of Si on different plants have 

been investigated by several researchers (Liang et al., 2005; Ma and Yamaji, 2006; Rains 

et al., 2006; Raven, 2001; Tamai and Ma, 2003). In some plants, the uptake of Si can be 

equal or even greater than Ca, Mg, S and P (Epstein, 1994 & 1999; Ma and Yamaji, 2006), 

ranging 0.1-10 % (Tamai and Ma, 2003), with the cell walls of epidermal layers being the 

main site of Si deposition (Adatia and Besford, 1986). The level and mechanisms of uptake 

varies among plants species (Adatia and Besford, 1986; Ma and Yamaji, 2006). Beneficial 

effects of the element on growth and yield of several crops have been linearly related to the 

level of Si supplied to the plants (Bélanger et al., 1997; Ma, 2004; Ma and Takahashi, 

2002). However, an excessive supply of Si to cucumber (Bélanger et al., 1997; Samuels et 

al., 1993) and strawberry plants (Lieten et al., 2002) may result in poor fruit quality. 

117

Therefore, there is a need to determine the optimum level of Si supply in order to obtain 

maximum benefits without compromising the quantity and quality of yield, or the cost of 

applying Si to plants. With the increased application of Si to soil-less media in order to 

enhance plant growth and disease protection, careful investigation of the uptake and 

distribution of this element in different part of the plants is needed. 

Increased accumulation of Si in trichomes, around pathogen sites of infection and at 

different parts of the cell has been reported on powdery mildew (PM) infected plants 

(Cherif et al., 1992a; Menzies et al., 1991; Samuels et al., 1991a, 1991b & 1993). The 

presence of Si may result in different levels of activation, or speed of activation, of defence 

reactions in infected and uninfected plants (Cherif et al., 1992b). Where a plant is infected 

by P. xanthii, its uptake of Si increases, and then resistance to the disease is enhanced 

(Cherif et al., 1994; Rodrigues et al., 2005). To date, there is little information available 

that maps out the distribution of Si to different parts of different plant species. 

Understanding the level of uptake and accumulation of Si into different organs of plants, 

and its effects on the total elemental uptake of plants can lead to the better use of this 

element in crop production. The objectives of this research were (1) to assess uptake and 

distribution of Si to different parts of zucchini and zinnia, in relation to Si supply and its 

impact on the uptake and accumulation of other elements by these plants; (2) to determine 

the relationship between infection of these plants by P. xanthii and accumulation of Si and 

selected elements by each plant; and (3) to study the effects of Si supply on characteristics 

of fruits of zucchini and flowers of zinnia and accumulation of other elements in these 

organs. 



Seeds of zucchini (Cucurbita pepo, F1-Hybrid Partenon) and zinnia (Zinnia elegans cv. 

Jakobrekop Sunbow), obtained from Starke Ayres 1 and McDonald Seeds 2 , respectively, 

were used for this study. Seeds of both plants were planted in Speedling® trays containing 

1 Starke Ayres, P. O. Box 304, Eppindust 7475, South Africa. 

2 McDonald Seeds (Pty) Ltd, P. O. Box 238, Pietermaritzburg, 3200, South Africa. 

118

composted pine bark. These were transferred into a tunnel operating at 26-28 o C and 75- 

85%. Trays were irrigated with a balanced fertilizer [3:1:3 (38) from Ocean Agriculture 3 , 

at 0.5 g l -1 ] + [Ca(NO 3 ) 2 at 0.5 g l -1 ] until seedlings were transplanted into pots filled with 

composted pine bark. Transplanting was commenced after seedlings of zucchini developed 

their first leaf, and when zinnias reached a height of 50-70 mm. Finally, pots were 

transferred into a nutrient re-circulating system that is in a greenhouse (24-30 o C and RH of 

60-70%) and supplied with a complete hydroponics nutrient solution. Soluble Si was added 

in the form of KSi , at 11 different concentrations (0, 50, 100, 150, 200, 250, 300, 400, 500, 

750 and 1000 mg l -1 ). To see the effect of infection with P. xanthii on elemental uptake by 

these two plant species, both plants were inoculated with the pathogen and grown on 

separate benches while being supplied with Si (150 mg l -1 ). Inoculations were made by 

dusting conidia of the pathogen from infected leaves onto healthy zinnia leaves, or 

spraying a conidial suspension onto zucchini leaves using a hand sprayer. The levels of Si 

used were based on previous results for both plants. Plants of zucchini and zinnia were 

kept in the system for 6 wk, by which time all the plants had produced well-developed 

fruits and flowers. 

7.2.2 Energy dispersive X-ray fluorescence (EDX) analysis 

Leaf samples were taken from different positions of each plant and washed thoroughly 

with distilled water to remove superficial dust, cut into pieces of approximately 10 mm 

diameter, and fixed overnight in 3% glutaraldehyde in cacodylate buffer (0.1 M; pH 7). 

Samples were then dehydrated in a graded alcohol series. Specimens were critical point 

dried in a Hitachi HCP-2 using carbon dioxide as the transfusion fluid. Dried specimens 

were mounted onto copper stubs using double-sided carbon tape. All stubs were then 

coated with gold-palladium in a Polaron E500 Sputter Coater and the accumulation of Si 

and other elements in the samples was assayed using an environmental scanning electron 

microscope with an energy dispersive X-ray analysis system (ESEM-EDX). Analysis was 

performed on leaves of uninfected plants and areas of infected leaves that were covered or 

uncovered with colonies of the pathogen. 


119

Quantitative calculations were made using the fundamental parameter method of the EDX 

program as used by Marguí et al. (2005). Elements that were considered for analysis and 

quantification included: carbon (C), nitrogen (N), calcium (Ca), magnesium (Mg), 

potassium (K), oxygen (O), phosphorus (P) and silicon (Si). 

To further study the effects of infection and presence of the pathogen on Si accumulation 

in leaves, elemental mapping was performed on (1) entirely disease-free leaves, (2) 

disease-free areas of infected plants, and (3) infected areas of infected plants covered by 

PM colony. Elemental mapping was also performed to examine the distribution of Si to 

different parts of the leaf. 

7.2.3 Inductively coupled plasma-optical emission spectrometers (ICP-OES) 

analysis of plant tissues for elemental composisions 

(a) Preparation of samples 

Plants of zinnia and zucchini were removed from their growing medium and washed 

thoroughly using tap water and finally rinsed in distilled water, to remove superficial dust. 

The plant materials were cut into four parts: leaves, roots, flower/fruits, and 

stem + petioles. Samples were then oven-dried at 70 o C for 72 h, ground into particles less 

than 1.0 mm in diameter, using a blender and then thoroughly homogenized. A sample of 

0.5 g of each plant material was put into a crucible and kept overnight in a furnace set at 

650 o C. Ashes of the samples were processed using microwave digestion. 

(b) Microwave digestion 

All sample digestions were carried with the CEM Microwave Digester (CEM MARS5 

Microwave) using tarred 100 ml Teflon ® PFA digestion vessels. The vessels were 

thoroughly washed using acid and rinsed with distilled water to free them from any 

particulate matter and then dried. Ash was transferred into the vessels and 5 ml of 65% 

HNO 3 and 0.1 ml HF were added to each sample and the vessels were thereafter sealed 

with their lids. Thirteen vessels, including a reagent blank vessel, were arranged in a 

scrubber and digestion was performed using the following procedures. The temperature 

was ramped to 165 o C within 10 min with the application of 1200W power, followed by a 

120

dwell time of 20 min at 165 o C. The temperature and pressure limits were set to 175 o C and 

15.2 bar (220 psi), respectively. Following completion of the digestion program, the 

vessels were allowed to remain in the microwave cavity until the internal temperature 

cooled to < 65 o C. Then the scrubber was removed from the microwave, placed in a fume 

exhaust system and then the vessels were vented slowly to release the residual pressure. 

Roots and leaves of zinnia and zucchini produced a small quantity of precipitate at the end 

of the cycle, so the procedure was repeated after adding 0.9 ml HF to the digest. Once the 

samples were fully digested, they were diluted to 100ml with de-ionized distilled water 

(DDW) to make a dilution factor of 100 (v/v) and transferred into high-density 

polyethylene (HDPE) bottles. After every cycle, the vessels were washed with tap water 

and rinsed with DDW. 

(c) Preparation of standards 

A 100 ml reagent blank was prepared by mixing DDW with 5 ml of HNO 3 and 0.1 or 

1 ml of HF, depending on the volume of HF used for the digest. Two sets of five standard 

solutions (i.e., 2, 15, 50, 100 and 200 mg l -1 ) were prepared to avoid formation of Si 

precipitates, with the first set containing only Si and the second set including Ca, K, Mg 

and P. 

(d) ICP-OES system 

All measurements were made using an Inductively Coupled Plasma-Optical Emission 

Spectrometer (ICP-OES) (Varian Model 720-ES). To avoid instrument damage and 

contamination from the free HF of the solution, a V-groove nebulizer, a Sturman-Masters 

Spray Chamber, and a Radial Torch were used instead of the glassy materials of the 

machine. The system components used were recommended by the supplier as being 

suitable for solutions containing up to 30% HF. Samples were taken automatically at a rate 

of 1 ml min-1 using an SPS3 AutoSampler. The plasma forward power was 1000W with 

plasma and auxiliary gas flow rates of 15 and 1.5 l min-1, and a pump rate of 15 rpm was 

used to aspire the sample solutions. The ICP system was calibrated using ICP Expert II 

software, with each calibration curve being constructed linearly through zero after 

subtraction of the reagent blank, as used by Feng et al. (1999). 

121


Data collected from the ICP-OES was analysed using polynomial regression to study the 

relationships of Si in the nutrient solution with elemental compositions of different tissues 

of zucchini and zinnia plants. 

7.3 RESULTS 

7.3.1 Observations from EDX-ESEM analysis and elemental mapping 

Results of the EDX analysis and elemental mapping showed that leaves of both plants 

accumulated high levels of Si. Even in the control, where Si was not added into the nutrient 

solution, leaves of both plants accumulated some levels of Si (Figures 7.1 & 7.2). As the 

concentration of Si in the nutrient solution was increased, the level of Si in the leaves of 

both plants increased. In some cases, the level of Si was even higher than that of C and O. 

In both plants, infected leaves accumulated more Si than the uninfected leaves, with Si 

mainly concentrated around the infected areas. Further investigations of these areas 

showed that Si was accumulated in the plant and not in the hyphae of the PM fungus 

(Figure 7.3B). Regardless of Si concentration in the nutrient solution, Si was always highly 

concentrated at the base of trichomes (Figure 7.3A). 

122

0 mg l -1 

150 mg l -1 150 mg l -1 + PM 

150 mg l -1 + PM 

Zucchini 

0 mg l -1 150 mg l -1 

Zinnia 

Figure 7.1 Energy dispersive X-ray (EDX) spectrums of silicon and other elements on leaves of zucchini and zinnia plants that 

received Si treatments of 0 (control), 100 mg l -1 and 100 mg l -1 plus Podosphaera xanthii and Glovinomyces cichoracearum, respectively. 

123

0 mg l -1 150 mg l -1 150 mg l -1 + PM 



Figure 7.2 EDX mapping of silicon deposition on leaves of zucchini and zinnia plants that received Si treatments of 0 

(control), 100 mg l -1 and 100 mg l -1 plus Podosphaera xanthii and Glovinomyces cichoracearum, respectively. 

124

A 

B 

Figure 7.3 Scaning electron microscope and EDX mapping showing silicon deposition of Si at the 

base of trichomes of non-infected leaf of zucchini (A) and in the leaf containing structures of the 

pathogen (B). 

125

7.3.2 Results of ICP-OES analysis 

When samples were completely digested through a microwave treatment, the digest 

became colourless. An amount of 5 ml HNO3 and 0.1 ml HF were sufficient to yield full 

digests of samples of flowers of zinnia and fruits of zucchini and the stem and petioles of 

these two plants. However, leaf and root samples required an extra 0.9 ml of HF for a 

complete digestion. 

As with the EDX analysis, both plants accumulated considerable amounts of Si in their 

tissue, even if the element was not added to the nutrient solution. The source of this 

element in the control was probably from the composted pine bark that was used as the 

growing medium, plus the Si in the irrigation water. Accumulation of Si by both plants 

was directly related to the level of the element in the nutrient solution. With zucchini, 

adding as little Si as 50 mg l-1 increased growth of the plant. However, extra applications 

of Si did not result in further increases in plant growth. Adding Si into the nutrient solution 

at 50 mg l-1 doubled the total amount of Si accumulated in different tissues of the plant, 

with leaves accumulating more than six times as much. As the levels of Si in the nutrient 

solution was increased up to 400 mg l-1, the level of Si accumulated by leaves was 

increased by 8-9 times, reaching its maximum level of 68.8 mg g-1 dry weight (dw). 

However, the extra accumulation of Si in the leaves obtained by supplying Si at 150- 

400 mg l-1 was negligible, and extra applications had little impact. Similarly, 

accumulation of Si in roots was almost doubled when the concentration of Si in the 

solution was kept between 50-200 mg l-1. However, application of Si at higher rates (i.e., 

> 200 mg l-1) had little impact on the level of Si accumulated by roots. In addition, the 

level of Si accumulated in stems and petioles of zucchini remained constant when Si was 

supplied at 0-200 mg l-1. However, at 250-500 mg l-1, that amount increased by 35-65%. 

The amount of Si in the fruit of zucchini was not affected by nutrient concentrations of Si 

(Figure 7.4). 

The effect of Si levels in the nutrient solutions on the accumulation of Si by zinnia was 

similar. As the level of Si in the nutrient solution increased, leaves of zinnia accumulated 

as high as 92.6 mg g-1 dw, which was more than double the value obtained for the control 

treatment. Optimum concentration of Si in the nutrient solution was considered to be 

126

250 mg l-1 because increasing the Si level further had a negligible impact on 

accumulation of the element. Roots of this plant also accumulated high amount of Si (24.2 

mg g-1 dw) as the level of Si in the solution was set at 150 mg l-1. Flowers of zinnia 

doubled their Si content when the plant was supplied with 50 mg l-1. However, 

subsequent increases in Si supply did not result in increased Si levels in zinnia flowers. 

Changing Si level in the nutrient solution did not affect the Si content of the stems and 

petioles of zinnia (Figure 7.4). 

Both plant species responded positively to the levels of KSi in the solution by increasing 

the total amount of K obtained in their tissues. With zucchini, adding Si at 100 mg l-1 to 

the solution increased the amount of K found in the leaves by 44%. However, applying 

more Si did not increase accumulation of K further. In contrast, leaves of zinnia 

accumulated higher levels of K when the plant was supplied with higher levels of Si. In 

addition, the K content of the stems and petioles of both plants was increased as the 

concentration of the Si supply was increased. When KSi was added to the nutrient solution 

at the maximum concentration (i.e., 1000 mg l-1), the amount of K accumulated by stem 

and petioles of both plants almost doubled. In contrast, the levels of K in the roots and 

fruits of zucchini, and flowers of zinnia were not affected by Si levels of nutrient solutions 

(Figures 7.5 & 7.6). 

The levels of Mg in leaves of both plants were increased in both plants as a result of 

increasing Si level in the nutrient solution. At a Si level of 50 mg l -1 , the level of Ca in 

different parts of both plants was increased. However, extra levels of Si in the solution did 

not increase their Ca contents. Instead, reduced accumulation of Ca was observed, 

especially in roots, flowers and fruits. Level of P in fruits of zucchini and flowers of zinnia 

were increased slightly with increased levels of Si in the nutrient solution. In addition, the 

Mg content in leaves of both plants increased as the level of Si of the nutrient solutions 

increased. Measurement of levels of Ca, Mg and P in both plants showed that the highest 

levels of these elements were obtained when Si was added to the nutrient solution at 

50 mg l -1 (Figures 7.5 & 7.6). 

127

Si in plant tissue 

(mg g -1 dw) 

A 


● Leaves: y = -0.898x 2 + 6.866x + 6.275 

R² = 0.58 

■ Roots: y = -0.192x2 + 2.596x + 12.70 

R² = 0.215 

▲ Fruits: y = -0.014x2 + 0.138x + 4.958 

R² = 0.225 

× Stem+ petioles: y = -0.056x2 + 0.794x + 2.334 

R² = 0.437 

Si in plant tissue 

(mg g -1 dw) 

B 


● Leaves: y = -0.532x 2 + 10.476x + 28.94 

R² = 0.728 

■ Roots: y = -0.354x 2 + 4.726x + 24.52 

R² = 0.290 

▲ Flowers: y = -0.036x 2 + 0.678x + 9.780 

R² = 0.384 

× Stem+ petioles: y = 0.046x 2 - 0.936x + 11.662 

R² = 0.676 

Figure 7.4 Effects of Si level in nutrient solution on the accumulation of Si in different parts of 

zucchini (A) and zinnia (B) plants after six weeks of growth in a nutrient recirculating system. 

128

K in plant tissue 

(mg g -1 dw) 


× Stem + petioles: y = 0.044x 2 - 0.082x + 4.496 

R² = 0.713 

● Leaves: y = -0.016x 2 + 0.308x + 3.96 

R² = 0.168 

■ Roots: y = 0.024x 2 - 0.214x + 2.456 

R² = 0.169 

▲ Fruits: y = 0.014x 2 - 0.224x + 3.438 

R² = 0.075 

Mg in plant tissue 

(mg g -1 dw) 


● Leaves: y = 0.002x 2 + 0.154x + 5.95 

R² = 0.204 

■ Roots: y = 0.002x 2 - 0.10x + 2.288 

R² = 0.358 

▲ Fruits: y = -0.012x 2 + 0.106x + 2.486 

R² = 0.033 

× Stem + petioles: y = -0.022x 2 + 0.062x + 4.402 

R² = 0.643 

Ca in plant tissue 

(mg g -1 dw) 


● Leaves: y = -0.200x 2 + 2.844x + 20.90 

R² = 0.188 

■ Roots: y = -0.002x 2 - 0.182x + 7.934 

R² = 0.268 

▲Fruits: y = -0.030x 2 + 0.318x + 1.378 

R² = 0.080 


R² = 0.505 

P in plant tissue 

(mg g -1 dw) 


● Leaves: y = -0.128x 2 + 1.78x + 3.778 

R² = 0.108 

■ Roots: y = 0.070x 2 - 0.766x + 9.362 

R² = 0.14 

▲ Fruits: y = 0.018x 2 - 0.328x + 7.392 

R² = 0.144 

× Stem + petioles: y = 0.002x 2 + 0.174x + 5.868 

R² = 0.161 

Figure 7.5 Effects of concentrations of Si applied in nutrient solution on accumulations of K, P, Ca, and Mg in different parts of zucchini after six weeks of 

growth in a nutrient re-circulating system. 

129

K in plant tissue 

(mg g -1 dw) 


● Leaves: y = 0.01x 2 - 0.038x + 5.264 

R² = 0.435 

■ Roots: y = -0.002x 2 + 0.088x + 2.316 

R² = 0.209 

▲ Flowers: y = -0.002x 2 + 0.158x + 1.852 

R² = 0.585 

× Stem + petioles: y = 0.004x 2 + 0.156x + 4.108 

R² = 0.426 

P in plant tissue 

(mg g -1 dw) 


▲ Flowers: y = 0.088x 2 - 1.352x + 11.554 

R² = 0.41 

● Leaves: y = 0.028x 2 - 0.352x + 6.276 

R² = 0.302 


R² = 0.112 

■ Roots: y = -0.002x 2 - 0.034x + 4.016 

R² = 0.093 

Ca in plant tissue 

(mg g -1 dw) 


● Leaves: y = -0.056x 2 + 0.112x +10.926 

R² = 0.840 

■ Roots: y = -0.026x 2 + 0.254x + 4.144 

R² = 0.121 


R² = 0.444 

▲ Flowers: y = -0.086x 2 + 0.794x + 3.34 

R² = 0.585 

Mg in plant tissue 

(mg g -1 dw) 


▲ Flowers: y = -0.064x 2 + 0.994x + 2.574 

R² = 0.577 

● Leaves: y = -0.014x 2 + 0.002x + 6.764 

R² = 0.578 


R² = 0.201 

■ Roots: y = -0.006x 2 + 0.092x + 0.402 

R² = 0.101 

Figure 7.6 Effects of concentration of Si applied in nutrient solution on accumulations of K, P, Ca and Mg in different parts of zinnia after six weeks of growth 

in a nutrient re-circulating system. 

130



Figure 7.7 Effects of infection with powdery mildew fungi on the accumulation of Si, Ca and P in 

different parts of zucchini and zinnia plants grown in a nutrient re-circulating system for six weeks 

and supplied with Si at 150 mg l -1 . 

131

Effects of powdery mildew on accumulation of selected elements 

Infected plants of zucchini accumulated higher levels of Si in their leaves and roots than 

disease-free plants. However, the amount of Si in other parts of infected plants was almost 

unaffected. The total amount of K found in different parts of infected zucchini plants was not 

significantly affected as a result of infection with the pathogen. The total amount of P 

accumulated by all parts of infected zucchini plants declined by 30%, with leaves of infected 

zucchini plants accumulating 56% less than uninfected zucchini plants. In addition, the P level 

in stems and petioles of infected zucchini plants was reduced by 29%. However, the level of 

Ca accumulated in different parts of infected zucchini plants was increased by 81%. Infection 

doubled the amount of Ca in leaves and increased that in roots by 26%. However, fruit of 

infected zucchini plants accumulated less Ca. Infection resulted in increased levels of Mg in 

leaves (+45%), while reducing that of roots and fruits (-62% and -34%, respectively) (Figure 

7.7). 

Plants of zinnia infected with G. cichoracearum accumulated higher levels of Si in their roots 

and leaves than similar tissues of disease-free plants. However, other parts of infected and 

non-infected plants accumulated similar levels of Si. Similarly, infected plants showed 

increased levels of Ca and Mg in their different parts than the uninfected plants. Infection with 

zinnia with G. cichoracearum did not have a major impact on the total amount of K and P 

accumulated by this plant, although the level of P in leaves was reduced slightly. The effect of 

infection on accumulation of different elements by zucchini and zinnia plants are presented on 

Figures 7.7. 


To our knowledge, this is the first investigation to determine the uptake and distribution of Si, 

K, Ca, P and Mg to different parts of zinnia and zucchini plants. The analytical techniques 

employed in this study provided unbiased and reliable information on the concentration of 

different elements in different tissues of both plants. EDX analysis was faster and required 

lesser chemicals in comparison to the ICP-OES. However, ICP-OES has a higher precision 

and lower detection limit than EDX (Einhäuser, 1997). This is because the sample size taken 

by EDX is often small and superficial. For instance, if the reading is taken from the surface of 

132

a leaf, the value may vary depending on the presence of the pathogen on the leaf. In some 

cases, where the leaf was covered by the pathogen, the Si values were much higher than that 

of a disease-free leaf. In addition, the data obtained from EDX was relative (i.e. the graph 

represents a percentage of each element from the total in consideration). Moreover, depending 

on the age of the plant, distribution of Si on both sides of the leaf can differ (Hodson and 

Sangster, 1988), with older tissues accumulating more Si than the young tissues (Ma and 

Yamaji, 2006). According to these authors, the increased accumulation of Si in specific tissues 

is related to immobility of Si in older tissues. Therefore, it is not possible to make firm 

conclusions on the exact amount of a specific element based on EDX assessment alone. 

Despite these limitations, the EDX can give useful information about a sample’s composition 

and its elemental distribution (Einhäuser, 1997). Furthermore, it provides an opportunity to 

observe the effect of Si treatments on the morphology of the pathogen and the plant, as well 

identifying the distribution of the element in specific plant tissues. The main advantage of ICP 

over EDX was that it provided accurate results by taking representative samples, while 

minimizing the level of sampling error. Hence, only the results of the ICP analyses were 

considered for interpretation. 

Although the mechanism of uptake and distribution of Si in different plants remains poorly 

understood, Epstein (1999) suggests that Si is absorbed by plant roots in the form of 

monosilicic acid [Si(OH) 4 ] and translocated to different parts of the plant through the xylem 

(Hodson & Sangster, 1989), making for uneven distribution within shoots (Ma and Takahashi, 

2002; Ma and Yamaji, 2006). In monocots, once silicic acid is translocated from roots to 

shoots, it becomes more concentrated as a result of loss of water through transpiration and 

polymerizes to form silica gel (SiO 2 .nH 2 O). In most monocots, polymerization starts once the 

concentration of Si(OH) 4 in the xylem exceeds 2 mM, although higher concentrations are 

needed in some plants such as rice and wheat (Ma and Yamaji, 2006). If Si is deposited in a 

polymerized form, it is not available for redistribution or for physiological activity (Ma and 

Yamaji, 2006). However, these studies were conducted in monocots, which routinely deposit 

Si as phytoliths in their leaves. In contrast, most dicots do not deposit phytoliths (some 

cucurbits are the exceptions), and it is therefore unlikely that the same process applies to 

dicots. 

133

Leaves have been the main organ selected for analysis when determining uptake and 

accumulation of elements by plants (Adatia and Besford, 1986; Ranganathan et al., 2006; 

Samuels et al., 1991b). However, such observations may lead to biased and inconclusive 

results because elements are distributed to different organs of plants at different levels. Even 

when samples are being taken only from leaves, their orientation can affect the outcome. 

Adatia and Besford (1986) discovered that leaves of cucumber positioned at the bottom of 

shoots had a higher Si content than the top leaves. Such differences might be related to 

differences in the age of the leaves, or to their proximity to the source of Si (Ma and Yamaji, 

2006). In young leaves of wheat, Si is mainly concentrated in the abaxial (lower) epidermal 

cells, whereas in old leaves both abaxial and adaxial (upper) epidermal cells have the same 

levels of Si (Hodson, and Sangster, 1988). Moreover, Epstein (1994) noted that distribution of 

this Si in different parts of the roots and reproductive parts is often variable. In this study such 

variables were avoided by taking representative samples of each organ from the entire plant 

and blending it thoroughly after drying. 

Accumulation of Si by some plant species may reach levels, or higher, than recognized plant 

macronutrients such as P, K, Mg, Ca and S (Epstein, 1994 & 1999). Our investigation showed 

that there was accumulation of Si in both plants. Even in the control, where no Si was added 

into the nutrient solution, the levels of Si in the roots and leaves of zinnia and zucchini plants 

were higher than that of Ca, P, K and Mg. We believe that these plants obtained the element 

from the composted pine bark and water, which were used in the growing system. This is in 

agreement with the observation that Si is ubiquitous in nature (Epstein, 1994 & 1999). 

Regardless of the level of Si in the nutrient solution, the daily uptake of the element by both 

plants was related to their water consumption. However, the fact that both plants reached 

saturation at a certain level of Si in the solution demonstrates that they absorb the element 

actively rather than passively. The difference in accumulation of Si by different species of 

plants has been attributed to differences in the ability of the roots to take up this element (Ma 

and Takahashi, 2002). The uptake, translocation and polymerization of Si in rice is affected by 

the transpiration rate and other metabolic activities of the plant (Ma and Yamaji, 2006; Rains 

et al., 2006). However, Ma and Yamaji (2006) have identified a Si transporter gene in rice 

that is believed to be responsible for increased accumulation of Si in this plant. 

134

In zucchini and zinnia plants, Si was mainly accumulated in roots and leaves. In contrast, the 

remaining parts of these two plant species accumulated relatively little Si. High levels of Si in 

the root were assumed to be as a result of a slow rate of translocation of the element from the 

roots to shoots, or that the amount of Si required by other organs of the plant had reached its 

saturation stage. A high level of Si in leaves might be because of high transpiration rate of this 

organ (Raven, 2001), coupled with other metabolic activities that take place in this part of the 

plant (Liang et al., 2005; Rains et al., 2006). 

Although both plants accumulated more Si as the levels of Si in the nutrient solution were 

increased, zucchini attained its optimal level of Si with a lower level of Si in the nutrient 

solution than zinnia. Supplying Si at levels as low as 50 mg l -1 increased the growth of 

zucchini because the uptake of most of the elements studied was maximal at that level of Si. 

Adding Si at > 50 mg l -1 did not result in increased growth because the levels of most of the 

other macronutrient elements in the plant remained almost constant. With cucumber, 

application of Si increased the weight of leaves of the plant without affecting their size (Adatia 

and Besford, 1986). According to these authors, leaves of Si- fed plants were stronger than the 

control because they had higher chlorophyll contents. Surprisingly, the effect of Si on the 

growth of zinnia was not noticeable. It is possible that the difference could not be detected 

easily because the plant does not produce a big crown. Accumulation of Si and K in the petiole 

and stems of Si supplemented plants was not different compared to the control. In addition, 

there was no apparent difference in the morphology of the zinnia flowers as a response to the 

increased supply of Si in the nutrient solutions. Plant size also appeared similar. However, 

this may have been because the beneficial effects of Si treatment of plants are usually 

expressed when the plants are subjected to various stress conditions (Epstein, 1994). However, 

in this study, plants were not stressed because they were raised under optimum growth 

conditions and kept free of diseases, except PM. 

In addition to protection against biotic and abiotic stresses, Si has also been shown to 

strengthen the plant by thickening its stem and stiffening the leaves (Takahashi, 1995). This 

role can be of great importance especially for zinnia and other ornamental plants, and 

vegetables, where their ornamental value or their fruit quality are affected by the ability of the 

135

plant to stand erect and support its organs properly. Application of Si can also improve the 

yield of zucchini, as well as increasing the quantity, as has been mentioned for other crops 

(Datnoff et al., 1997; Ma, 2004; Ma and Takahashi, 2002; Samuels et al., 1993). 

Although the mode of action by which Si exerts its protective effects against diseases and 

pests are complex and remain controversial (Fawe et al., 1998; Epstein, 1994; Ghanmi et al., 

2004), the relationship between the accumulation of Si in specific areas of the plant and 

consequent disease protection remains of great importance. Therefore, accumulation of a high 

level of Si in leaves of both plants may protect these plants against PM and other foliar 

diseases. 

Adding Si into the nutrient solution did not affect the elemental composition of fruits of 

zucchini. With the exception of P, accumulation of all other elements (i.e. Ca, Mg, K and Si) 

was slightly increased by supplying the plant with low levels of Si (i.e. 50 mg l -1 ). As the 

concentration of Si in the nutrient solution was raised beyond 50 mg l -1 , the levels of Ca and P 

in zucchini fruit were slightly lower than that of the control, while the level of Mg remained 

unaffected and levels of both K and Si increased slightly. Even increased in K and Si levels in 

response to extra Si application were negligible. 

Time of flowering and fruit settings and the size of the fruit were not affected by Si supply in 

the solution. In addition, visual estimations were that the colour and texture of Si-treated and 

untreated zucchini fruit were the same, suggesting that treatment with Si did not have major 

impact on the characteristics of the zucchini fruit. Contrary to our findings, a study by 

Bélanger et al. (1997) reported that application of Si at > 100 mg l -1 increased the cucumber 

yield, but at the same time, it hardened the fruit of cucumber, resulting in poor fruit quality 

which limited farmers to using Si at low levels. Samuels et al. (1993) found that cucumber 

plants produced unusual, dull appearing fruits, when they were grown in hydroponics supplied 

with Si. Therefore, the response of cucurbits to Si treatment may vary among species or even 

the cultivar level (Ago et al., 2008). Hence, no generalization can be made on each plant 

species and cultivar without proper testing. 

136

Elemental analysis of the whole plant showed that application of 100-150 mg l -1 Si was 

optimal for zinnia. Application of Si at 150 mg l -1 provided effective control of PM and 

resulted in accumulation of high levels of Si in all parts of the plant. Moreover, concentrations 

of Ca, P, K, and Mg in all parts of the plant were little affected at that level. For zucchini, 

supplying Si at 200 mg l -1 resulted in the highest level of Si accumulation in roots, although 

leaves could accumulate more with an extra supply of Si. However, the differences in 

accumulated Si as a result of applications of 50-400 mg l -1 were small, indicating that 

translocation of Si from roots to leaves was not increased significantly as a result of increasing 

the supply of Si. Since our main objective was to control PM, the impact of lower levels Si on 

growth and composition of zucchini and zinnia were not investigated. We believe that when 

the risk of infection by PM is minimal, the use of Si at lower levels (i.e., < 50 mg l -1 ) can 

increase the growth of both plants because accumulations of most of the elements studied 

seemed to be optimal. However, when the risk of PM is high, Si should be used at higher rates 

(100-150 mg l -1 ). This level could be altered, based on the need to balance the uptake and 

distribution of other elements, which ultimately determines the health of the plant. For 

example, K was part of the solution (i.e. KSi) and its level in the plant was increased by 

increasing the concentration of potassium silicate applied. However, this can result in reduced 

uptake of Ca, Mn, Fe and other elements by the plant (Ma and Takahashi, 1993). For instance, 

uptake of Ca by rice plants was reduced when Si was added into the nutrient solution (Ma and 

Takahashi, 1993). With cucumber, reduced uptake of Ca as a response to Si supply resulted in 

increased growth of the plant (Marschner et al. (1990). However, adding Ca to the solution did 

not affect uptake and distribution of Si in rice (Ma and Takahashi, 1990). 

Previous research on the effects of Si on uptake and accumulation of P in various plants 

provided contradictory results (Ma and Takashi, 1990; Islam and Saha, 1969). This was 

because Si is possibly involved in the metabolic or physiological changes in the plants by 

promoting or suppressing uptake and transportation of selected elements, depending on the 

stress conditions (Liang, 1999). Islam and Saha (1969) discovered that the application of Si 

resulted in increased levels of P, Ca and Mg in rice. Interestingly, Ma and Takashi (1990) 

found that the concentration of Si in the shoots of cucumber was slightly reduced with 

increased level of P in the same organ, although uptake of Si was not significantly affected by 

the presence of P in the nutrient solution. They concluded that Si could increase availability of 

137

P when Si is deficient or reduce uptake of P when levels of Si are high, reflecting that Si plays 

a major role in balancing P uptake. Within a small range, adding low levels of Si to the 

nutrient solution improved accumulation of P in most parts of both plants studied here. 

Infections of zinnia and zucchini with PM resulted in increased accumulation of Si and Ca in 

their leaves. Similar observations have been reported for various plant species (Samuels et al., 

1991b; Koga, 1994: Cherif et al., 1992a). Even in the same leaf, the concentration of Si 

around infected areas was higher than disease-free areas. This agrees with previous 

investigation by Menzies et al. (1991) and Samuels et al. (1991b) who demonstrated that 

infection of the plant with the pathogen results in increased accumulation of Si in the leaves. 

These authors observed high levels of Si at the infection sites and around the hyphae of the 

pathogen. Our observations using EDX mapping gave extra information, showing 

conclusively that Si was accumulated in the leaf and not in the pathogen. The conclusion was 

based on the observation that when the concentration of Si in the pathogen and the leaf tissue 

(infected site) were compared using contrast mapping, Si levels were low in the pathogen, but 

were at high levels in leaf tissue adjacent to PM hyphae. Even though the total uptake of K 

and Mg by both plants was not affected by infection, their accumulation on leaves was 

increased by infection. This indicates that they may also have some roles in disease control. 

Regardless of infection, the highest concentration of Si was observed at the base of trichomes, 

confirming observations of other researchers (Iwasaki and Matsumura, 1999; Samuels et al., 

1991a, 1991b & 1993). 

Leaves of zucchini and zinnia infected their respective pathogens accumulated more Ca and 

less P than leaves of uninfected plants. Similar observations were reported for other crops 

infected with different pathogens (Goodenough and Maw, 2008; Kalamera and Heath, 1998). 

Reduced uptake of P by infected plants may have been affected by the increased uptake of Ca 

by the infected plant. Zhang et al. (2006) showed that the uptake of P was reduced when 

plants were supplied with calcium silicate (CaSiO). It has been noted that under stress 

conditions, Si enhances absorption of Ca by the plant at the expenses of Fe and Mn (Islam and 

Saha, 1969; Liang, 1999). However, whether the increase in Ca content of infected leaves is 

related to the expression of the resistance to PM or not is still not clear. 

138


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141

CHAPTER EIGHT 

EFFECTS OF SELECTED BIOCONTROL AGENTS AND SILICON ON 

POWDERY MILDEW OF ZINNIA PLANTS GROWN HYDROPONICALLY 


a 



b Plant Health Products, P. O. Box 207, Nottingham Road, South Africa 

Abstract 

Five selected biocontrol agents, and soluble silicon (Si) at various concentrations, were tested 

for their ability to control powdery mildew of hydroponically grown zinnia plants. Biocontrol 

treatments were applied before infection and continued weekly, while Si was supplied to 

plants continuously. Both BCAs and Si reduced the severity and AUDPC values of PM 

significantly. Application of BCAs resulted in reductions in severity and AUDPC values of 

PM by 38-68% and 30-65%, respectively. Both severity and AUDPC values of PM were 

reduced by 87-95% when plants were supplied with soluble Si (50-200 mg l -1 ). It is proposed 

that the provision of a continuous supply of Si and the ability of zinnia to accumulate high 

levels of Si in its leaves were the major reasons for the good response of zinnia plants to Si 

treatments against PM. Silicon played a protective role before infection and suppressed 

development of PM after infection. The combination of the tested BCAs and Si can be used as 

effective control options against PM of zinnia grown hydroponically. 

142


Zinnia (Zinnia elegans L) is a popular ornamental flower grown in many parts of the world. 

Production of attractive flowers, fast growth, a short production cycle and minimal labor 

requirements makes zinnia a commercially important crop as a cut flower (Pinto et al., 2005), 

as well as a popular bedding plant. However, its susceptibility to several diseases often 

reduces its value as an ornamental plant (Linderman and Ewart, 1990). Powdery mildew (PM), 

caused by Golovinomyces cichoracearum (DC.) VP Heluta is one of the common diseases of 

zinnia, resulting in plant losses and decreased ornamental values by weakening plant growth, 

resulting in poor flowering (Boyle and Wick, 1996; Kamp, 1985). 

The use of fungicides to control PM of annual bedding plants is limited by their toxicity to the 

flower crop (Kamp, 1985) and other non-target organisms. Furthermore, development of 

fungicide resistance by several species of PM fungi as a response to intensive use of 

fungicides has also been a major challenge (McGrath, 1996; McGrath, 2001; McGrath and 

Shishkoff, 2001; O'Hara et al., 2000; Wong and Wilcox, 2002). As an alternative approach to 

the control of PM of zinnia, Kamp (1958) successfully used a polymer-based anti-transpirant. 

Similarly, applications of biocontrol agents (BCAs) and soluble silicon (Si) to PM susceptible 

crops have produced promising results in suppressing the disease caused by different PM 

species, under controlled environmental conditions (Dik et al., 1998; Guével et al., 2007; 

Liang et al., 2005; Menzies and Bélanger, 1996; Verhaar et al., 1996, 1997). However, in spite 

of the challenges that PM poses to growers of zinnia, no alternative control measures have 

been developed to date to supplement or replace fungicides. 

Based on prior research, the objectives of this study were to test the efficacy of five selected 

biocontrol agents, and Si applied at various concentrations, for control of PM and to assess 

whether a continuous supply of Si could improve its efficacy. Zinnia was selected as an 

ornamental test plant for this study under hydroponics condition because of its relatively small 

size and its susceptibility to PM. 

143



Seeds of Zinnia (Zinnia elegans L., cv. Jakobrekop Sunbow), obtained from McDonald 

Seeds 1 , were planted in Speedling ® trays containing composted pine bark and transferred into 

a greenhouse tunnel operating at 26-28 o C and an RH of 75-85%. Trays were irrigated with a 

balanced fertilizer [3:1:3 (38) from Ocean Agriculture 2 at 0.5 g l -1 ] + [Ca(NO 3 ) 2 at 0.5 g l -1 ] 

for 3 wk. Seedlings were transplanted into pots (150 mm diameter) once they had produced 2- 

3 well developed leaves and attained a height of approximately 50-70 mm. Thereafter, pots 

were transferred into a greenhouse (mean temperature, 28 o C and RH of 60-70%). All pots 

were placed into horizontal mini troughs with the nutrient solution being supplied in a recirculating 

system (Figure 8.1). 

8.2.2 Design of the hydroponic system 

Horizontal mini troughs, constructed from 1mm thick black plastic, were placed on a table. 

The troughs were arranged in alternating directions to provide enough space for the reservoirs 

(8l) to fit next to each other. Each reservoir contained a Project Powerhead submersible pump 

(100w) fitted to supply lines which carried the nutrient solution to the top end of the trough 

from where it flowed through the trough and back to the reservoir (Neumann, 2003). The 

pumps ran continuously and the level of nutrient solution in each reservoir was maintained by 

adding the nutrient solution as needed. Where Si was used as a treatment, its concentration in 

the nutrient solution was set at 50, 100, 150 and 200 mg l -1 by adding KSi . Finally, the 

seedlings of zinnia, in pots, were placed into the troughs with each row containing four or five 

pots for the first and second trials, respectively. 

1 McDonald Seeds (Pty) Ltd, P. O. Box 238, Pietermaritzburg, 3200, South Africa 


144

Figure 8.1. Horizontal mini troughs and their accessories used to supply nutrient solution to the plants 

in a re-circulating system. 

8.2.3 Preparation of biocontrol agents 

Microbial suspensions of three Serratia marcescens (Bizio) isolates (i.e., Isolates B15, Y15 

and Y41) and two fungi (Clonostachys rosea (Link) Schroers, Samuels, Seifert & Gams (syn. 

Gliocladium roseum) (Isolate EH) and Trichothecium roseum (Pers.) Link (syn. 

Cephalothecium roseum) (Isolate H20)) were prepared as described in Chapter 3. Biocontrol 

agents were applied to plants once a week at a concentrations of 10 8 and 10 6 spores ml -1 for 

bacterial and fungal BCAs, respectively. Treatments with BCAs were applied 2 d before 

plants were inoculated with PM, and repeated 4 d after the first symptoms of PM developed 

and continued weekly. To enhance survival of BCAs on the phylloplane, spraying was 

commenced during the late afternoon using hand sprayers. 

8.2.4 Preparation of powdery mildew inoculum and inoculation technique 

Seedling of zinnia were prepared as described in the above and put into ice cream containers 

(2l) and kept in an open environment to allow natural infection by placing them in batches, 

placed at different positions in the Controlled Environmental Facility (CEF) of the university. 

Plants were watered manually by drenching them with a complete nutrient solution. After the 

first symptoms of PM, infected plants were kept in a separate glasshouse to enhance 

development of the disease and to avoid contamination between experiments. The inoculum 

145

was maintained by infecting fresh plants through direct contact of infected and uninfected 

leaves. 

Three inoculation techniques were tested: (1) conidia of PM were sprayed onto both sides of 

zinnia leaves until run-off, using a hydraulic hand sprayer; (2) dusting of dry conidia collected 

from infected leaves onto disease-free plants; and (3) physical contact between infected and 

disease free leaves. However, the spray technique of inoculation did not result in infection of 

zinnia. Therefore, the last two methods were combined as the standard inoculation technique 

for zinnia. Infected leaves of the same age were collected from the source plants, and one 

infected leaf was used to infect one plant by dusting the conidia using a brush and by gently 

rubbing the infected leaf against 3 leaves of the test plant. Inoculation was performed during 

the late afternoon to enhance establishment of the disease. 


Percentage of leaf area covered by PM was assessed weekly before spraying of BCAs for 

5 wk, and AUDPC was calculated using an AUDPC Program (Shaner and Finney, 1977). The 

final disease level recorded at the end of the experiment was recorded as the final disease 

level. 


The experiment was conducted twice, with each treatment having four replications in the first 

trial, and five replications in the second trial. The arrangement was determined by access to 

space in the hydroponics system. Because of their homoginuity, the results of these two trials 

were mixed together and ANOVA analysis was performed using GenStat (R) Statistical 

Analysis Software (GenStat, 2006). To reduce the coefficient of variation, data was 

transformed a using square root transformation. Means of treatments were compared using 

Fisher’s Protected LSD at P < 0.05. Percentage reduction in disease severity and AUDPC 

values were calculated by comparing each treatment against the control. 

146

8.3 RESULTS 

Symptoms of PM started to appear on the leaves one week after inoculation and developed 

slowly. After 5 wk, disease level of the control plants reached > 45% in the first trial and 31% 

in the second trial. 

In both trials, the BCAs and Si had significant effects on the severity of PM and its AUDPC 

values. In the first trial, all BCAs reduced the severity of PM significantly, with reductions of 

35-84%. Isolates EH and H20 reduced the severity of PM by 83% and 60%, respectively. 

Similarly, treatments with the Isolates B15, Y15 and Y41 caused significant reductions in 

disease severity of 35-76%. Among the three bacterial isolates tested against the disease, 

Isolate Y41 performed best. In the second trial, only Isolates EH, H20 and Y41 reduced the 

severity of PM significantly. Treatments with these three isolates provided disease reductions 

of 41-47%. Although statistically not significant, Isolate B15 and Isolate Y41 also reduced the 

final disease level by 30% and 34%, respectively. In both trials, application of BCAs reduced 

the AUDPC values significantly (P < 0.001). Among the BCAs tested, Isolate EH gave the 

best results in suppressing the development of PM (Figure 8.2). 

Adding Si at concentrations of 50-200 mg l -1 into the nutrient solution reduced the severity 

and AUDPC values of PM significantly. After one month of infection, the final disease level 

recorded for plants treated with Si was reduced by 86-100% and 75-90%, in the first and 

second experiments, respectively. AUDPC values were also significantly reduced when plants 

were treated with Si (Figure 8.2). The performance of Si was better in the first trial, providing 

disease control as much as 100% when Si was added into the nutrient solution at 150 mg l -1 . 

147

Table 8.13 Effects of selected biocontrol agents and silicon on the final disease level (FDL) and area 

under disease progress curve (AUDPC) of powdery mildew of zinnia, five weeks after inoculation with 

Golovinomyces cichoracearum 

Treatments FDL AUDPC 

Control 36.9 (5.92) e 358.2 (18.02) e 

BCAs 

Silicon 

B15 22.8 (4.45) d 220.5 (13.86) cde 

EH 12.1 (3.00) bc 126.4 (9.74) bc 

H20 17.8 (4.07) cd 182.0 (13.06) cd 

Y15 22.6 (4.53) de 250.8 (15.18) de 

Y41 15.7 (3.47) cd 145.8 (11.00) cd 

50 mg l -1 4.3 (1.92) ab 43.2 (6.04) ab 

100 mg l -1 2.3 (1.29) a 21.4 (3.87) a 

150 mg l -1 4.1 (1.39) a 31.9 (3.93) a 

200 mg l -1 4.7 (1.82) ab 47.4 (5.69) ab 

Effects P-values P-values 

F < 0.001 < 0.001 

CV% 46.8 46.6 

MSE 2.222 21.885 

FLSD 1.398 4.389 

Means within column followed by a common letter were not significantly different according to Fisher’s 

protected least significant difference (P < 0.05). 

Values in brackets are means of data transformed using a square root transformation. 

148

A 

Si level 

(mg l -1 ) 

Si 

BCA 

Isolates 

BCAs 

B 

Si level 

(mg l -1 ) 

Si 

BCA 

Isolates 

BCAs 

Figure 8.2 Percentage reductions in the disease severity (A) and Area under disease progress curve 

(AUDPC) (B) of powdery mildew of zinnia after five weeks of treatment with five biocontrol agents 

soluble silicon, compared to a control inoculated with Golovinomyces cichoracearum. 

149


Unlike other crops, where inoculation PM fungi can be performed by spraying on a conidial 

suspension of the pathogen in water, inoculation of zinnia with conidial spray onto leaves was 

not effective. It has been reported that high leaf wetness has detrimental effects on the 

germination of conidia of PM fungi (Bushnell and Rowell, 1967; Quinn and Powell, 1982; 

Sakurai and Hirata, 1959; Sivapalan, 1993). In this study, even though the time between 

harvest of conidia and spraying was minimized to less than 30 min and a high concentration of 

conidia was used, the inoculation method did not produce consistent infection, indicating that 

this pathogen was sensitive to water. Therefore, dusting conidia by shaking the infected leaf 

over the test plant, and physical contact between infected and disease-free leaves were used as 

standard technique of inoculation. Although these techniques lacked information on the exact 

amount of inoculum used to cause a certain level of infection, both techniques consistently 

produced a level of infection that developed to > 45% within one month after inoculation. 

From the hydroponic trials, it was demonstrated that all the tested BCAs and Si reduced the 

level of PM significantly. In both trials, the level of control obtained from Si treatments was 

better than that of BCAs. If zinnia plants absorb Si before infection, then they become highly 

resistant to infection by G. cichoracearum. Richmond and Sussman (2003) noted that plants 

treated with Si before infection displayed an accelerated activation of SAR resistance of the 

plant, thereby inducing fungal cell death. In contrast, although some BCAs which can 

antagonize the pathogen within one week after application (Sundheim and Krekling, 1982), 

most antagonists require an average of 10-14 d to establish themselves on the phylloplane and 

to start their antagonistic activities (Verhaar et al., 1997). Therefore, by the time the BCAs 

started their activities, the pathogen could have already covered a large part of the phylloplane, 

making biocontrol less effective. In previous chapters, fast development of the disease versus 

slow establishment of the BCAs has been shown to be one of the challenges in controlling PM 

with BCAs. To overcome a similar problem, Verhaar et al. (1997) suggested that BCAs 

should be used as preventatively (i.e. applied approximately 1 wk before mildew inoculation) 

or as an early curative treatment (within 2 d after PM inoculation). 

150

Both BCAs and Si gave some control of PM. Most isolates tested in this study showed 

promising performance by reducing the severity of PM and suppressing its progress. Among 

the BCA isolates tested, Isolate EH performed best, providing disease control that was 

comparable to that provided by Si. In spite of fluctuations in disease levels between the two 

experiments, the relative efficacies of all isolates were almost constant and were less than Si 

treatments. 

Several researchers have reported that treatments with Si produced promising results in 

controlling PM of different crops (Bélanger et al., 1995; Guével et al., 2007; Menzies et al., 

1991). However, most of these researchers indicated that the level of control was usually 

incomplete. In contrast to these reports, our results demonstrated the possibility of obtaining 

complete control of PM when zinnia plants were grown in hydroponics supplied with Si. 

Unlike hydroponics, where a plant is supplied with a nutrient solution continuously, in most 

glasshouses and field trials, Si is usually supplied to plants as a spray or drench, at limited 

frequencies per week. Samuels et al. (1991) observed a rapid decline in Si-induced resistance 

against PM when cucumber plants growing with a Si containing solution were transferred into 

a Si-free solution. However, the same plant species supplied with Si continuously at a 

concentration of 100 mg l -1 or higher, showed a reduction in PM severity by as much as 98% 

(Menzies et al., 1991). Similarly, as much as 100% control was obtained when zinnia plants 

were continuously supplied with Si (50-150 mg l -1 ), leading to a conclusion that for maximum 

disease protection, a continuous supply of Si is needed. This, according to Heine et al. (2006), 

is because Si-enhanced resistance of plants against diseases is linked to the maintenance of a 

high Si status in the plant. Another reason for the inconsistency in efficacy of Si is the effect 

of the soil. Soil pH and nutritional balance can determine the availability of Si to the plant. For 

instance, soils that have large amounts of Fe, Al, and Mn can bind Si and make it unavailable 

to the plant (M.D. Laing, 2008, pers. comm.). 

In this study, complete control of PM was obtained where plants did not show any symptom of 

the disease one week after artificial inoculation with the pathogen. Once symptoms of PM 

were observed on the leaves, it was not possible to cure the plant completely regardless of the 

concentration of Si added into the nutrient solution, indicating that Si was more effective as 

151

protective treatment. Although Si did not provide a complete curative treatment, it kept PM 

levels low, probably by inducing resistance of the plants against the disease (Bélanger et al., 

2003; Fawe et al., 1998; Liang et al., 2005). 

The response of zinnia to Si treatment for the management of PM was promising. To date, 

there is no information on the uptake and accumulation of Si by zinnia and subsequent disease 

suppression. In Chapter 7, however, we showed that leaves of zinnia could accumulate Si to 

levels of 10% of dry matter. Although the mode of action by which Si provides its protective 

role against plant pathogens remains a controversial issue (Fauteux et al., 2005; Ghanmi et al., 

2004; Rodrigues et al., 2004 and 2005), reports by Ma and Yamaji (2006) indicated that there 

is a positive relationship between the amount of Si accumulated in the plant and the level of 

disease resistance. Therefore, the high level of PM control observed on zinnia by Si treatment 

could be the result of high uptake and accumulation of the element by this plant species. 

The experiments were conducted as a preliminary testing of both control options and they 

were not used together as part of an integrated approach in managing the disease because there 

was a lack of space in the glasshouse. However, given the additive effects of BCAs and Si 

shown in our previous research, the promising results obtained in this study, suggest that coapplication 

of these two components should provide better control of PM, leading to increased 

growth and quality of zinnia plants. 

152


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155

CHAPTER NINE 

GENERAL OVERVIEW 

The impact of powdery mildew (PM) on crop production and the control strategies that have 

been used against this disease has been widely covered in the literature (Bélanger et al., 1997; 

McGrath and Thomas, 1996; Yarwood, 1957 & 1978). Increasingly, reports in development of 

fungicide resistance by a number of PM species, coupled with public concerns for 

environmental and health hazards, have made the use of fungicides less popular. To meet the 

need for safe and environmentally friendly control measures, biocontrol agents and soluble 

silicon have emerged as viable alternatives that can be used individually, or combined with 

other control strategies for integrated disease management (Bélanger et al., 1997; McGrath, 

2001; McGrath et al., 1996). 

The use of biological control against PM of several species has been well documented 

(Bélanger et al., 1997; Jarvis and Slingsby, 1977; Sztejnberg et al., 1989; Verhaar et al., 1996 

& 1998). This has resulted in development of some promising biocontrol products on the 

market. However, their use has been limited due to their inconsistency under variable 

environmental conditions. To improve the efficacy of biocontrol agents (BCAs), which has 

been associated with their specific requirements for specific environmental conditions, various 

techniques have been used. To a certain extent, the lack of appropriate screening protocols, 

combined with insufficient environmental experiments during evaluation, and a lack of 

knowledge of the modes of action of the specific BCA, have all contributed to this problem. 

Similarly, promising progress has been made in the use of soluble silicon (Si) against PM and 

other diseases (Bélanger et al., 1997; Epstein, 1994 & 1999; Ma and Takahashi, 2002). In 

some plants, increased growth and yields have been reported as a result of enhanced resistance 

against biotic and abiotic stresses, especially when Si was applied to soils that were deficient 

in Si (Datnoff et al., 2001). The ability of Si to benefit plants without affecting the 

environment negatively has made it an “agronomically essential element” (Ma and Takahashi, 

2002) or quasi-essential (Epstein, 1999). In spite of this, due to the incomplete and 

156

inconsistent control it provides, the use of Si against PM and other pathogens has been limited. 

The main reasons for the low efficacy of Si are a lack of knowledge on the optimum 

application conditions (i.e., concentration, frequency and methods of application), mode of 

action in controlling the disease and the relationship between Si and the plant, and other biotic 

and abiotic factors that are involved in the production system. 

The research presented in this thesis focused on the isolation and in vitro screening of 

antagonists, using a series of in vitro and glasshouses experiments, for the evaluation and 

improvement of the efficacy of BCAs and Si against PM under different environmental 

conditions. It was established that: 

• At the preliminary stages, 29 BCA isolates reduced severity of PM by 30-77%. Under 

glasshouses and field conditions, five isolates (i.e., Gliocladium roseum (Isolate EH). 

Trichothecium roseum (Isolate H20) and 3 isolates of Serratia marcescens (Isolates 

B15, Y15 and Y41)) reduced the severity of PM by 50-90%. 

• Break-Thru ® (BK) at lower concentrations (i.e., 0.25 ml l -1 ) improved the efficacy of 

spray applications of BCAs and Si by enhancing their deposition on the leaves and the 

pathogen, and by directly affecting the pathogen. Break-Thru ® was compatible with 

the zucchini and BCA isolates when used at < 0.40 ml l -1 . However, at higher rates, it 

was toxic to zucchini and to the BCAs, especially Isolates EH and H20. 

• When Si was applied as a foliar treatment, it inhibited PM through direct contact with 

the pathogen. Efficacy of Si spray was improved by increasing the spray frequency. 

Although no Si was absorbed directly through the leaf, part of the foliar treatment was 

absorbed by the plant roots as runoff and drift, and played a role in enhancing host 

resistance of the plant against PM. 

• The efficacy of Si was better when added into the nutrient solution than when it was 

drenched onto roots of plants or sprayed on as a foliar treatment. In all these 

application methods, increased concentrations provided better disease control. 

Drenching 250 ml (under greenhouse conditions) or 1 l (under field conditions) of Si 

at 100 mg l -1 per week provided significant control of PM of zucchini. Spraying 

750 mg l -1 of Si also gave significant reduction in the severity of PM. When added into 

157

the nutrient solution, 50-150 mg l -1 of Si reduced the severity of PM of zinnia by 85- 

100%. 

• A combination of BCAs + Si + BK provided better control of PM under both field and 

glasshouse conditions, and can be used for integrated management of PM. 

• Adding KSi into the nutrient solution increased the levels of Si and K in zucchini and 

zinnia. It also enhanced the growth of zucchini without affecting morphological 

characteristics of the fruits. However, it did not cause obvious changes to the growth of 

zinnia plants and their flowers. 

• Infections of zucchini and zinnia plants with PM resulted in increased uptake of Si and 

Ca by these plants, while reducing their P uptake. However, it did not affect the uptake 

of K and Mg in these plants. 

The development of a proper isolation and in vitro screening protocol that provides rapid, 

repeatable and reliable results is an important initial step in screening effective antagonists for 

biocontrol of PM. Since the performance of biocontrol isolates on agar has less predictive 

value than in vivo tests (Verhaar et al., 1998), the screening protocol adopted in this thesis 

involved three components: the biocontrol agent (BCA), the pathogen and the plant itself. If 

one of these three components were to be missing, then the screening procedure would have 

been incomplete. Isolates that showed consistent results against PM of zucchini (P. xanthii) 

under different growth conditions also controlled PM of zinnia caused by G. cichoracearum, 

confirming a previous hypothesis that if a BCA isolate can antagonize PM of one species of 

plant, then it can often be used against PM species of different plants (Sztejnburg et al., 1989; 

Szentivanyi and Kiss, 2003). The process of screening and rating systems followed in this 

study can serve in developing a more comprehensive protocol, especially where isolates are to 

be screened against obligate pathogens, and their antifungal activity is not noticeable during in 

vitro assays. 

Clonostachys rosea has been reported as mycoparasite of many fungal species (Kwasna et al., 

1999) and could be used as BCA against PM (Sutton and Peng, 1993) and other foliar (Cota et 

al., 2008; Morandi et al., 2008; Yu and Sutton, 1997) and root diseases (Tarantino et al., 

2007). Serratia marcescens has been identified as a growth promoting rhizobacterium that 

158

produces several antibiotics and can induce resistance of plants against several diseases 

(Battaglino et al., 1991; Jeun, 2004; Kobayashi et al., 1995; Maji et al., 2003; Ordentlich et 

al., 1988; Roberts et al., 2005; Strobel et al., 1999; Sujay et al., 2003; Wei et al., 1996). 

Similarly, a report by Iida et al (1996) identified Trichothecium roseum as producing 

antifungal antibiotics and inducing host resistances in plants. In other research, it has provided 

promising results when tested against PM and other diseases (Hijwegen and Buchenauer, 

1984; Huang et al., 2000; Vanneste et al., 2002). 

All the adjuvants tested (i.e., Break-Thru ® , Partner ® and Tween-80 ® ) improved spray 

efficiency of BCAs and Si by enhancing coverage of the spray and reducing the total volume. 

Both Break-Thru ® and Partner ® suppressed PM directly. However, Break-Thru ® was chosen 

for further studies because it was more compatible with our BCAs and the plant. Electron 

microscopy investigations showed that Break-Thru ® increased the deposition of BCAs on the 

pathogen and on the leaves. It also inhibited germination of conidia, and caused the collapse 

and disintegration of conidia and mycelia of the pathogen. The fact that it was biocompatible 

at lower dosages provides the opportunity to be used in the spray mix in controlling PM with 

spray applications. 

Si was more effective against PM when supplied to plants as a nutrient solution in a 

hydroponics system than when applied as drench or foliar spray. For best PM control, plants 

need an uninterrupted supply of Si, which was manageable under hydroponics. Regular 

drenching of Si onto the roots of plants was also effective in reducing the severity of PM of 

zucchini under both glasshouse and field conditions. When Si is added to the soil as a 

fertilizer, it usually improves the resistance of plants against PM, and other biotic and abiotic 

stresses of the plant, resulting in increased growth and yield of the plants. These benefits are 

noticeable when the plants are grown under sub-optimal conditions (Datnoff et al., 2001). 

However, if the aim is to control PM, then Si should be applied before infection because once 

the plant is infected, curative treatment with Si is less effective (Liang et al., 2005). In 

addition, continuity of supply of Si to plants is more important than the total volume or level 

of Si in the solution. Therefore, to improve the continuity of plant-available Si in the fields, 

the development of slow release formulations of Si, or increasing the application frequencies 

159

are recommended. As a foliar treatment, increasing the concentration and frequency of Si 

applications provided the best control of PM. This was partly due to increasing availability of 

Si to plant roots via drift and runoff. Spraying Si three times per week at lower levels of Si 

(i.e., 250mg l -1 ) provided the best results while reducing the total amount of Si that gave 

equivalent PM control when applied once per week. When KSi is used as a foliar treatment 

against PM, Si has been identified as the active ingredient of the solution (Menzies et al., 

1992). Direct contact between Si and the pathogen, or the changes in osmotic characteristics 

of the leaf created by Si deposition, are one of the mechanisms of disease control (Liang et al., 

2005). In addition, increased deposition of Si on the leaf surface can prevent penetration of 

fungal hyphae into the host (Bowen et al., 1992). However, there is no evidence that shows 

penetration of the leaf surface by Si directly. Therefore, any priming of resistance of the plant 

by foliar applications of Si can only be the result of Si that is absorbed by plant roots after the 

element is intercepted by the plant roots as a result of runoff or drift. 

Although the use of BCAs and Si against PM has been studied intensively, there are not many 

studies that incorporated both control options as part of an integrated control package. 

Combinations of BCAs and Si were more effective, and should result in a better PM control 

than when these two control measures are used separately. As expected, better control was 

obtained under both glasshouse and field conditions, when BCAs were applied with Si. The 

additive effects were probably due to the combination of different modes of action that affect 

the pathogen, as opposed to the fewer control mechanisms provided by the BCA or Si. Unlike 

most fungicides, Si has a broad spectrum of activities that affect economically important 

diseases (Datnoff et al., 2001). Therefore, the use of BCAs and Si for disease control should 

be developed and practiced in an integrated disease management programmes. 

The main challenge in the use of BCAs and Si against PM has been the speed of PM 

development. In this study, the use of Break-Thru ® at lower concentrations improved the 

performances of BCAs and Si spray by reducing the rate of PM development and contributed 

substantially towards an integrated programme under which BCAs and Si can actually control 

this disease. It might be possible to use combination of BCAs + Si + Break-Thru ® against 

other foliar diseases of economic crops. Treatment with Si resulted in less infection of the 

160

plant and the presence of Break-Thru ® weakened the pathogen, increasing the vulnerability of 

the pathogen to attacks by the antagonists. If the combination fails, the addition of 1-2 sprays 

of effective, systemic fungicides may provide better control, leading to an integrated disease 

management programme that would minimize the frequency of fungicide application, reduce 

the risk of fungicide resistance, and reduce environmental pollution, while providing a high 

level of PM control. 

In using Si, a lack of knowledge on optimum concentrations for optimal disease control and 

plant growth, without compromising quality has been the main challenge. Epstein (1994) and 

Feng (2004) have noted that disease resistance is linked with the amount of Si accumulated by 

the plant, which is species and cultivar dependant, and is linked to the level of physiologically 

available Si. Although high levels of Si provide effective PM control, at high concentrations, 

Si can reduce the quality of fruits of some crops (Bélanger et al., 1995 & 1997; Lieten et al., 

2002). In this study, the use of Si at 100-150 mg l -1 as a soil drench, or in nutrient solutions, 

provided effective control of PM on zucchini and zinnia. Increasing the level of Si in the 

nutrient solution resulted in an increased accumulation of Si in roots and leaves of both plant 

species, without affecting its distribution to other parts of these plants. The fact that growth of 

zucchini and the uptake of most other elements in both plants were optimal when Si was added 

into the nutrient solution at 50 mg l -1 showed that, where the likelihood of infection by the PM 

fungi is low, then application of Si at lower levels (50 mg l -1 ) can improve growth of the 

plant, while minimizing the cost of Si applications. However, since the experiment was 

conducted under optimal condition, these results need to be confirmed on different species of 

plants growing under different growth conditions. 

Electron microscopic observations on increased accumulation of Si in the base of trichomes 

and around the leaf areas infected by PM confirmed previous reports by Menzies et al. (1991) 

and Samuels et al. (1991a & b). The role of Si in infected plant was to priming defense 

mechanisms of the plant (Cherif et al., 1992). Leaves of zucchini and zinnia infected by their 

respective pathogens accumulated more calcium and less phosphorus than leaves of uninfected 

plants. Similar observations were reported on other crops infected with different pathogens 

(Goodenough and Maw, 2008; Kalamera and Heath, 1998). Cell wall strengthening and 

thickness are some of the functions of Ca (White and Broadley, 2003). However, whether the 

161

increase in Ca content of infected leaves is related to the expression of the resistance to 

infection or not is still not clear. 

The way forward 

• This study presents some promising isolates that have the potential to be used against PM 

and other foliar diseases. Some of them may also be used as growth promoters and in 

priming the resistance status of the plant (Jeun et al., 2004; Kobayashi et al., 1995; Iida et 

al., 1996). However, before these BCAs are developed further for commercialization, a 

series of studies is needed to assess their efficacy against other pathogens under various 

conditions. More investigations on the formulation, shelf life and cost-benefit analysis of 

these isolates are needed before they are released as bio-products. Toxicological studies 

will also be essential. 

• Most of the trials on Si were conducted under controlled growth conditions, where the 

plants were almost free of stresses except for the infection with PM. We believe that effects 

of Si would have been more visible if the plants were grown under more stressful 

conditions. 

• Most available information on uptake, transportation and accumulation of Si in plants are 

based on studies conducted in monocots. It is not known whether the same principle applies 

to dicots or not. Since the form of Si inside the plant is more important than total, more 

investigations is recommend on dicots in order to determine the mechanisms of uptake and 

levels of different forms of Si in different plants species. 

• In our study, the level of Si application did not cause obvious effects on the quality of the 

fruits of zucchini. However, since this effect may differ from species to species, it is 

important to assess this effect on other crops and determine the impacts of the fluctuation of 

selected nutrients in the fruit quality as influenced by Si application. 

• It is only recently that Si has been studied as an option for disease management. It is 

concluded that much research is still needed to fully understand the impact of this element 

on plant metabolic activities. Even in areas where much research has been conducted, e.g., 

disease control mechanisms, there is still much to be learned. However, since the effects of 

162

Si on plants seemed more complex than it was initially imagined, inter-disciplinary 

research is needed in order to exploit the full potential of Si for control of abiotic and biotic 

stress, and to prpmote plant growth. 


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