i

THE CONTROL OF RED SPIDER MITES ON TOMATOES USING NEEM AND SYRINGA EXTRACTS

by

NEPHIOUS JAMES KAMALENJE MWANDILA

Submitted in accordance with the requirements for the degree of

DOCTOR OF PHILOSOPHY

in the subject

ENVIRONMENTAL MANAGEMENT

at the

UNIVERSITY OF SOUTH AFRICA

SUPERVISOR: PROF J. OLIVIER

Co - SUPERVISORS: DR. D. VISSER PROF D.C. MUNTHALI

NOVEMBER 2009

ii

Student No: 3660-961-7 Declaration I declare that THE CONTROL OF RED SPIDER MITES ON TOMATOES USING NEEM AND SYRINGA EXTRACTS is my own work and that all the sources that I have used or quoted have been indicated and acknowledged by means of complete references. ------------------------------ ------------------- SIGNATURE DATE (MR N.J.K MWANDILA)

iii

Abstract

The efficacy of Neem (Azadirachta indica A. Juss) and Syringa (Melia azedarach L.)

against red spider mites (RSM) life phases (adult, nymphs and eggs) was assessed at

different concentrations (0.1%, 1%, 10%, 20%, 50%, 75%, 100%) and at exposure time

of 24, 48 and 72 hours using tomato leaf dip assays on water agar in plastic Petri dishes.

Tomato plants were grown in the greenhouse as a source of leaves and for the greenhouse

trial. A Greenhouse trial was carried out to simulate field conditions. Neem seeds were

sourced from Botswana, India, and Zambia. Syringa seeds were sourced from Botswana

and South Africa. Laboratory and Greenhouse trials were carried out at the Agriculture

Research Council, in the Vegetable and Ornamental Institute laboratories and green

houses in Pretoria South Africa.

Data was analysed by using the GenStat statistical program. Overall results of both

Neem and Syringa assays indicated that all levels of concentrations and time of exposure

had significant effects on mortalities of adult RSM and compared significantly with

commercial acaricides (Abamectin-plus, Hunter and Selecron). Both Neem and Syringa

caused significant mortalities at low concentration of 0.1% as early as 24 hours of

exposure. Both Neem and Syringa assays had significant mortalities on RSM nymphs as

early as 24 hours and with longer periods of exposure. Both Neem and Syringa had

significant effects on the hatching of RSM eggs at 48 hours and 72 hours of exposure. In

general, effects occurred in a dose (concentration) dependent manner. Based on the

findings and evidence in the literature, Neem and Syringa extracts could be useful as

botanical acaricides in the control of red spider mites (RSM) on tomatoes.

iv

Key terms:

Neem (Azadirachta indica); Syringa (Melia azedarach); Azadirachtin compound;

Azadirachtin standard; High Performance Liquid Chromatography (HPLC); Samples;

homogenous; Analysis of Variance; Randomized Designs; Abamectin-plus; Hunter;

Selecron; liquid–cooling agar; red spider mites; life cycle; heterogeneous; antifungal;

antibacterial; antifeedant; larva; protonymph; deutonymph.; Globalisation; food

production; food consumption; cereals; vegetables; pests; pesticides; limonoids.

v

Dedication

This thesis is dedicated to:

• the memory of my mother Elinala and my father James Kamalenje Mwandila--

two individuals who made a lasting impact on my life by bringing me up and

affording me an opportunity to attend school.

• my late son Daniel who kept encouraging me till he met his death by a robber’s

bullet.

• my eldest son Harvesto Malombo and his siblings- Elizabeth, Tiyezye, Bangala,

Newton, Esnart, Kasimba and Khumbo

vi

Acknowledgements

I am grateful to members of my family for their unstinting support throughout the

duration of this study. Special thanks are due to my children Harvest-Malombo, Daniel

(late), Elizabeth, Tiyezye, Esnart, Bangala, Newton, Kasimba, Khumbo and my nephew

Conerlius who always kept me smiling and feeling lighthearted in those anxious moments

of thesis-writing when I was very sick. I also wish to thank, most profoundly, my

promoter, Professor Jana Olivier, for her encouragement, her intelligence, her care, and

above all, her nurturing approach to the whole process of supervision. For me, working

with her was always a source of much immense joy even when I was seriously sick and

quitting seemed to be the only option especially after losing my son Daniel.

I want to thank

• Dr. Diedrich Visser my co-promoter, for his support and advice throughout the

time when I carried out the Laboratory bioassays at the Vegetable and Ornamental

Plant Institute (VOPI) and his valuable comments during the process of

supervision.

• Professor David Munthali my co-promoter, for his advice while in Botswana.

• The staff at the Agricultural Research Council’s (ARC) Vegetable and

Ornamental Plant Institute, Roodeplaat in particular Dr. Nolwazi Mkize and Sakki

Sambo for their support and help in purchasing several useful materials that were

used in the bioassays during my research experiments.

• Nanga Irrigation Research Station - Zambia for harvesting and providing me with

Neem seeds.

vii

• Mrs. Jeyasseeli Michael for sourcing me Neem seeds from India.

• My heart goes to all the VOPI staff for welcoming me as part of their team.

• Dr. Gerhard Prinsloo for providing me with much advice on research activities,

especially in the handling of High Performance Liquid Chromatography (HPLC)

• Subject Librarian, Mrs. Leanne Tracy Brown for her efficiency, integrity and

charm.

• Marie Smith, Elise Robbertse and Poloko Chepete for helping with statistics.

• Charnie Creamer ARC - Plant Protection Research Institute, for the identification

of the mites used in the bioassays

• Dr. Fetson Kalua of UNISA for making my life easier while in South Africa by

providing accommodation and valuable advice and encouragement.

• G.N. Mthombeni for financial assistance during the time I desperately needed to

pay my fees at UNISA.

• Letsholo Bongalo for helping me with statistical graphs during my write up.

• Not forgetting other friends and individuals too many to mention who stood by

me when getting the thesis done.

Finally, I wish to acknowledge the Department of Agriculture and Environmental

Sciences and the Agricultural Research Council (ARC) for funding part of this project.

Yebo/ Thank you.

viii

TABLE OF CONTENTS

Declaration ii

Abstract iii

Dedication iv

Acknowledgements v

Table of contents vii

Abbreviations viii

Appendices iv

Chapter 1 General Introduction 1 1.1 Background to the study 1

1.1.1 Overview on food production 1

1.1.1.1 Food production and consumption patterns 2

1.1.1.2 World cereal production 2

1.1.1.3 World vegetable production 7

1.1.1.4 Vegetable production in the SADC region 11

1.1.1.5 Vegetable production in Botswana 12

1.1.1.5.1 Tomato production in Botswana 13

1.1.2 Factors affecting food production 14

1.1.2.1 Crop pests 14

1.1.2.2 Types of pests 15

1.1.2.3 Pesticides and their problems 16

1.1.2.4 Possible solutions: Botanical pesticides 18

ix

1.2 Summary and problem statement 20

1.3 Aim 22

1.3.2 Objectives of the study 22

1.4 Chapter layout 22

Chapter 2 Literature Review 23

2.1 Introduction 23

2.2 Red spider mites (RSM) 24

2.2.1 Taxonomy 24

2.2.2 Life cycle 25

2.2.3 Damage caused by red spider mites 30

2.3 Control measures 31

2.3.1 Natural control measures 31

2.3.2 Chemical control measures 32

2.3.3 Biological control measures 34

2.4 Botanicals as an option 35

2.4.1 Introduction 35

2.4.1 Neem (Azadirachta indica A. Juss) as a botanical pesticide. 36

2.4.2.1 Origin and distribution of Neem (Azadirachta indica A. Juss) 38

2.4.2.2 Uses as pesticides 38

2.4.2.3 Chemical composition 39

2.4.3 Syringa (Melia azedarach L) as a botanical pesticide 41

2.4.3.1 Origin and distribution of Syringa (Melia azedarach L.) 44

x

2.4.3.2 Uses as pesticides 44

2.4.3.3 Chemical composition 45

2.2.3.4 Summary 46

Chapter 3 Research Design and Methodology 47

3.1 Introduction 47

3.2 Study area 48

3.3. Materials and Methods 49

3.3.1 Collection of seeds 49

3.3.1.1 Neem seed collection 49

3.3.1.2 Syringa seed collection 49

3.3. 2 Determination of azadrachtin content of seeds 50

3.3.2.1 Preparation of standards 50

3.3.2.2 Extraction of the active constituents from Neem and Syringa seeds 50

3.3.3 Experimental plants 51

3.3.4 Data collection 52

3.3.4.1 Laboratory bioassays 52

3.3.4.2 Testing the effect of Neem and Syringa extracts on adult RSM 52

3.3.4.3 Testing the effect of Neem and Syringa extracts on nymph

red spider mites 54

3.3.4.4 Testing the effect of neem and syringa extracts on red spider mite eggs 55

3.3.5 Greenhouse trial 56

xi

Chapter 4 Comparison of azadirachtin composition in Neem and Syringa from different parts and regions of the world 59

4.1 Introduction 59

4.2. Materials and Methods 60

4.2.1 Extraction of the active constituents from Neem and Syringa seeds 60

4.2.2 Determination of Azadirachtin content in Neem and Syringa 60

4.3 Results and discussions 62

4.3.1 Peaks for azadirachtin standard at three concentrations 62

4.3.2 HPLC for Neem and Syringa 62

4.3.3 Neem Samples 63

4.3.4 Syringa samples 64

4.3.5 Comparison of Neem and Syringa 65

Chapter 5 Results of Neem and Syringa extract treatments on red spider mites 67 5.1 Introduction 67

5.2 Experimental design 67

5.2.1 Effect of Neem and Syringa extracts on adult mites 68

5.2.1.1 Neem : adult mites 68

5.2.1.2. Syringa : adult mites 72

5.2.1.3 Syringa leaves : adult mites 74

5.3 The effect of Neem and Syringa on nymphs 76

5.3.1 Neem: nymphs mites 77

5.3.2 Syringa: nymph mites 79

xii

5.4 Effect of Neem and Syringa extracts on eggs 81

5.4.1 Neem: mite eggs 81

5.4.2 Syringa: mite eggs 83

5.5. The greenhouse trial 86

Chapter 6 Overview summary, conclusions and recommendations 88 6.1 Introduction 88

6.1.1 High Performance Liquid Chromatography (HPLC) 88

6.1.2. Neem and Syringa seed extracts 89

6.1.2.1 NSE results with adult RSM 89

6.1.2.2 NSE results with nymphs 90

6.1.2.3. NSE results with eggs 90

6.1.3 Syringa seed extracts (SSE) and crushed Syringa leaves: Results 91

6.1.3.1 Adult red spider mites: SSE and crushed Syringa leaves results 91

6.1.3.2 Nymphs: Syringa seed extracts 92

6.1.3.3 Eggs: Syringa seed extracts 92

6.1.3.4 Greenhouse trials 94

6.2. Summary 94

6.3 Conclusions 95

6.4 Recommendations 96

Appendices 1, 2 and 3 97 References 105

xiii

Abbreviations and short forms used for primary texts

Red Spider Mites – RSM

Neem Seed Extracts – NSE

Syringa Seed Extracts – SSE

Integrated Pest Management – IPM

Neem Seed Kernel Extracts – NSKE

Neem Oil – NO

Northern American Free Trade Agreement – NAFTA

Tetranychus – T

Safety and Quality Assurance – SQA

Commercial farmers, providing food for the population and beyond – Globalisation

Dusting powder – DP

Emulsifiable concentrate – EC

Gas – GA

Granule – GR

Suspension concentrate – SC

Soluble concentrate – SL

Water soluble powder – SP

Wettable powder – WP

xiv

List of Tables

Table 1 Percentage world cereal crop production in 2000 20

Table 2 World cereal production in 2003 21

Table 3 Production and yield of vegetables in Africa and globally as compared to those of other food commodities (1990) 24

Table 4 Vegetable production by SADC countries (in 1000 tonnes 27 and as percentage of world vegetable production)

Table 5 A few vegetable chemical pesticides used in South Africa and 47

the world

Table 6 Mean percentage mortalities of adult red spider mites 80 (untransformed means) that died using Neem seed extracts at 24, 48 & 72 hours Table 7 Mean percentage mortalities of red spider mite adults 83

(untransformed means) for Syringa Seed Extracts (SSE) at 24, 48 & 72 hours.

Table 8 Mean percentage mortalities of red spider mite adults 87 (untransformed means) feeding on tomato leaves treated with Syringa leaf extracts at 24, 48 & 72 hours. Table 9 Mean percentage mortalities of red spider mite nymphs 89 (untransformed means) feeding on tomato leaves treated with Neem seed extracts at 24, 48 & 72 hours Table 10 Mean percentage mortalities of red spider mite nymphs 90 (untransformed means) feeding on tomato leaves treated

with Syringa seed extracts at 24, 48 & 72 hours Table 11 Mean percentage red spider mite eggs that hatched after 48 91 & 72 hours exposure to Neem seed extracts Table 12 Mean percentage red spider mite eggs that hatched after 48 98 & 72 hours exposure to Syringa seed extracts Table 13 Mean percentage mortalities of red spider mite adults 99 (untransformed means) feeding on tomato leaves treated with Neem and Syringa seed extracts at 24, 48 & 72 hours in the greenhouse trial

xv

APPENDICES

APPENDIX 1

1 A Dead adult mites on Neem seed extracts

1 B Dead adult mites on Syringa seed extracts

1 C Dead adult mites on Syringa leaf extracts

1 D Dead mite nymph on Neem seed extracts

1 E Dead mite nymph on Syringa seed extracts

1 F Number of hatched eggs on Neem seed extracts

1 G Number of hatched eggs on Syringa seed extracts

1 H The greenhouse trial

APPENDIX 2

Calculation of percentage efficacy of Neem and Syringa seed extracts

APPENDIX 3

A protocol for the control of red spider mites using Neem and Syringa

extracts

1

CHAPTER 1: GENERAL INTRODUCTION

1.1 Background to the study

1.1.1 Introduction

This chapter presents the background of the study which includes an overview of global

food production and consumption, the problem statement, the aim and objectives of the

study and the significance of study.

1.1.1.1 Overview on food production

Low agriculture productivity and declining production efficiencies pose a threat to global

food production. According to FAO (2006a), in 2006, world food production rose by less

than 1%. As a consequence, per capita food production was estimated to have fallen by

about 0.2%, representing the first decline since 1993 (WTO, 2201). The food production

levels in SADC are in no way different from the crisis in the rest of the World (SADC,

2002). Declining agricultural output is part of a wider pattern whereby governments have

continuously failed to recognize the role of small farmers in increasing agricultural

production. In SADC, over 80% of the population is engaged in subsistence farming. Yet

the thrust of governments and donors to improve agricultural output has largely been

toward the more powerful and politically organized modern commercial farming sector,

leading in general to low levels of food production.

In Botswana, as a result of some good rains received during the 2007/08 growing season,

cereal production increased by 26%, from 29,000 tonnes in 2007 to 37,000 tonnes in 2008.

2

Maize production alone increased from 1,000 tonnes in 2007 to about 8,000 tonnes in

2008, while the combined production of sorghum/millet increased only slightly, from

28,100 tonnes to 28,500 tonnes. The overall food supply/demand assessment indicated a

revised cereal deficit of about 253,000 tonnes. By the end of July 2008, the country had

already imported 119,000 tonnes of cereals or 41% of its planned imports for the

marketing year. Thus Botswana food production is very low and the country meets its

food requirements from imports. This chapter will include a discussion on world cereal

and vegetable production and their patterns.

1.1.1.2 Food production and consumption patterns

Food is vital for survival. Food production changed gradually from subsistence

agriculture to the development of commercial farming to provide food for the local

populations and beyond. With trade extending over borders, the consumer base expanded

from one region to another (Duncan, 1997). However, subsistence or barter farming is

still practiced in poorer communities and in developing countries. Cereals together with

vegetables are the most consumed crops in many parts of the world (United Nations,

1993). Trends in food production can thus be represented by patterns of cereal production

and consumption.

1.1.1.3 World cereal production

Since the 1950s, the growth of world cereal production has exceeded that of World

population growth.

3

World output of cereals, the main food source for the majority of consumers, increased by

2.7% per year while the population grew by about 1.9% per year (Duncan, 1997).

This increased production has led to an increase in per capita calorie consumption in the

world, especially in developing countries, where the increase was by about 27% (United

Nations 1993; Duncan, 1997). The globalisation of food production implies that a set of

pronounced extended linkages exists between the sites of production and consumption

(Goodman, 1999) (Figure 1.1 & 1.2). Oosterveer (2007) has observed that the transition

towards globalising food production increased the choices for food consumers in the

world. Consumers are now demanding greater variety. For example, consumers who a

decade ago consumed most of their food cereals such as rice or maize, now demand meat,

fruits and vegetables (Pamplona-Roger, 2004).

s

Figure 1.1 Linkages showing the sites of production and consumption. Adap

Decreasing Distance

Places of production

Places of consumption

ted from Oosterveer (2007)

4

Figure 1.2. World Map showing the movement of cereal grains moving from its country of production to country of consumption as shown by the direction of arrows (FAO, 2006a).

Due to transportation and advanced communication systems, the production of food can

now take place at a considerable distance from its eventual consumption (Bonnano, 1994;

Motarjemi et al., 2001).

Tables 1.1 & 1.2 show the world total cereal crop production (in tonnes) for selected

countries and their percentage of the global cereal production for the year 2000 and 2003

respectively. Only countries contributing significantly to at least more than 1% to world

cereal crop totals are taken into account.

5

___________________________________________________________________ Table 1.1. Percentage world cereal crop production in 2000 ___________________________________________________________________

Country Production in 1000 metric Tonnes

Share in World (Percentage)

China 420 308 20.16 USA 334 614 16.05 India 238 012 11.42 Russia 67 190 3.22 France 63 426 3.04 Indonesia 60 484 2.90 Brazil 50 148 2.41 Canada 49 502 2.37 Germany 46 473 2.33 Bangladesh 37 960 1.82 Australia 36 232 1.74 Vietnam 33 984 1.64 Thailand 30 132 1.45 Nigeria 21 288 1.02 Total selected countries Cereal Production

1 489 753

71. 67

Source: FAO Statistical year book (2005)

Despite these impressive figures and the global character of food trade in general, only a

very limited number of countries dominate the international trade in food products. This

state of affairs brings about price distortions because trade is monopolised by the few

countries which are able to produce both for the local market and for world trade.

McMichael (2000) and Einarsson (2000) claim that most (roughly 90%) of the world’s

food consumption occurs in the country where it is produced. The production of cereals

has been declining since 2000, and continues to do so. According to the FAO (2006a),

cereal production growth slowed down since 1990. In 2003 the world cereal production

declined even further (Table 1.2). Table 1.2 shows the decline in the selected world

countries total cereal production as compared to that of 2000 (Table 1.1).

6

_______________________________________________________________________ Table 1.2. World cereal production in 2003 _______________________________________________________________________

Country

Production in (1000Metric tonnes)

Share in World (in percentage)

China 376 123 18.03 USA 348 897 16.73 India 233 406 11.19 Brazil 67 453 3.23 Russia 65 562 3.14 Indonesia 63 024 3.02 France 54 940 2.63 Canada 50 174 2.41 Australia 41 652 2.00 Bangladesh 40 876 1.96 Germany 39 426 1.89 Vietnam 37 705 1.81 Thailand 31 420 1.51 Nigeria 22 616 1.08

Total selected countries Cereal Production

1 473 274

70.63

Source: FAO Statistical year book (2005)

When one compares the years 2000 and 2003 (Table 1.1 and Table 1.2) in terms of cereal

production, a decline in total world cereal production is noticeable (in percentage terms).

What could have caused this decline in world cereal production? A number of factors are

plausible and some of these include:

1. Drought

Inadequate rainfall, especially where countries experience early-season dry spells, result in

delayed planting (SADC, 2002). This effectively shortens the duration of the growing

season. As a result of this, yields are greatly reduced. In some cases rain does not come at

all, resulting in a scenario of total drought.

7

2. Bio-fuels

The other reason is the new trend where crops are produced for bio-fuels rather than for

food (WRR, 2007). In an article entitled “The world’s choice: food or bio-fuels” which

appeared in The Sunday Times of March 9 2008 page 6, Beddington (2008) pointed out

that the world today is concentrating on the production of crops for bio-fuels rather than

for food supply, thereby contributing to the decline in cereal production. In the 2006

annual assessment of the global agriculture, it was noted that increased use of grains for

biofuels would affect food production and that food prices would be kept higher than

average. Among many cereals, maize is noted to be the main cereal used for the

production of ethanol, one of the byproducts of bio-fuels (FAO, 2006b).

1.1.1.3 World vegetable Production

Vegetables contribute about 40% of the world food trade (Okigbo, 1990). Vegetables are

generally herbaceous (non-woody) plants that are cultivated in farms as well as backyard

gardens for home use. Usually all the botanical parts of these plants (leaves, buds, flowers,

fruits, stalks, roots or tubers), can be consumed fresh, steamed or boiled separately or in

combination with other foodstuffs (Okigbo, 1990; Pamplona-Roger, 2004). The growing

of vegetables plays a major role in providing food for people, creating employment, and

acting as a source of income in many parts of the world including the Southern African

Development Community (SADC) region (Bandeke, 1996). Vegetables are the main source of

micronutrients which are essential in preventing malnutrition, and are also becoming increasingly important

with respect to preventive medicine, as a source of fibre, for their special proteins and oils, and other

nutritive qualities (McDonald & Low, (1990).

8

Pamplona-Roger (2004) has commented that vegetables should no longer be considered a mere

side dish to the “main course”; quite the contrary. Vegetables, together with grains and fruits,

should be principal elements of a truly healthy and nutritious diet. The World Health

Organisation is also advocating an increase in the consumption of fruits and vegetables (WHO,

1999). According to FAO (1989) (cited in Okigbo 1990), vegetables constitute the fourth

largest agricultural commodity group produced worldwide, and the fifth largest in the African

region (Table 1.3). Vegetables have been grown in such climatic diversity. Consequently,

plant species adapted to specific climate and soil conditions have evolved and a wide array of

annual and perennial crops are used as vegetables (Shanmugasundaram, 1990). Okigbo (1990)

defines vegetable as inclusive of separated roots, tubers and pulses (Table 1.3). Thus vegetable

production in Africa and globally is now greater than cereals (Okigbo, 1990).

9

_____________________________________________________________________________ Table 1.3 Production and yield of vegetables in Africa and globally as compared to those of other

food commodities (1990)

AFRICA WORLD

Food commodity

Production (million t)

Yield (t/ha)

% production of all commodities

Production (million t)

Yield (t/ha)

% production of all commodities

Cereals 89.0 1.2 25.9 1743.0 2.5 39.5

Roots and

tubers

98.0 7.0 28.5 571.0 12.3 12.9

Pulses 6.9 0.61 2.0 55.0 0.80 1.2

Vegetables 30.8 - 9.0 423.4 - 9.6

Fruits 41.6 - 12.1 332.3 - 7.5

Sugar 77.1 53.1 22.4 1282.9 47.5 29.1

Nuts 0.3 - <0.01 4.1 - 0.1

Source: Okigbo (1990).

Global production and trade in fruit and vegetables have been growing at a faster rate than in

any other agriculture commodities over the past 40 years and their share in world agriculture

trade have increased. More recently, (2000 to 2004) the value of trade in fruit and vegetables

increased by over 40%, from $52 billion to $74 billion at an annual growth rate of 9.35% (FAO,

2006b). The variety of commodities on offer is also increasing as is the frequency at which

these new varieties are being traded. For example, the inclusion of cassava, yams and sweet

potatoes, beans and peas (pulses) as vegetables (Okigbo 1990; FAO, 2006b) to the list of

vegetables has enriched the vegetable commodities (Figure.1.3).

10

World Vegetable Production in millions of tons per year (2006)

13.4 6.813.3 5.827.9

9.129.4

37.2

69.3

131.7

157.7

296.6

0

50

100

150

200

250

300

350

Potato

Cassava

Sweet Potato

Tomato

Cabbage

Yam

Onion

Carrot

Cucumber

Pepper

Pumpkin

Eggplant

Fig.1.3. Selected World Vegetable produced in millions of tonnes (2004).

Source: ENCYCLOPEDIA of Foods.

Most of the trade in fruits and vegetables occurs within three geographic regions namely, the

European Union (EU), the North American Free Trade Agreement (NAFTA) countries and East

Asia (China and Japan). However, this trend where trade was concentrated within the

mentioned countries has changed over the past few years, with greater imports of fruits and

vegetables coming from SADC countries (Table 1.4) and other developing countries in the

southern hemisphere.

11

1.1.1.4. Vegetable production in the SADC region

Until recently, vegetable production was an ignored and little-known industry in the SADC

region (Mnzava, 1990). For a long time, the production of vegetables was restricted to areas

with favourable climate, and invariably where the major consumers had established themselves.

This has now changed. Critically important for Africa and SADC countries is the fact that the

produce is harvested when the crop is off-season in countries in the Northern hemisphere

(Mnzava, 1990). Within Africa and the SADC region, however, only a few countries

contribute to the world’s vegetable production and trade. According to FAO (2006b) Production

Yearbook, South Africa is the highest contributor to the world vegetable production and trade in

the SADC region, while most of the SADC countries produce only for their domestic

consumption (Table 1.4).

12

Table 1.4. Vegetable Production by SADC Countries (in 1000 tonnes and as percentage of world vegetable production). _______________________________________________________________________________ Country 1981 1991 2001 2003 2004 Prdn % Prdn % Prdn % Prdn % Prdn % Angola 661 0.1 663 0.08 669 0.06 721 0.05 721 0.05 Botswana 26 0.00 28 0.00 27 0.00 27 0.00 27 0.00 DRCongo 3 094 0.49 3 833 0.47 2 867 0.24 2 962 0.22 2 893 0.21 Lesotho 36 0.01 42 0.01 32 0 31 0 31 0 Madagascar 1 002 0.16 1 118 0.14 1 231 0.1 1 234 0.09 1 234 0.09 Malawi 588 0.09 732 0.09 778 0.06 918 0.07 1188 0.09 Mozambique 513 0.08 558 0.07 461 0.04 451 0.03 451 0.03 Namibia 14 0 19 0 33 0 41 0 41 0 Seychelles 3 0 4 0 4 0 4 0 4 0 South Africa 4 662 0.74 5 801 0.71 7 141 0.59 7 897 0.59 7769 0.56 Swaziland 133 0.02 153 0.02 113 0.01 122 0.01 122 0 Tanzania 2 227 0.35 2 505 0.31 2 482 0.21 2 522 0.19 2528 0.18 Zambia 285 0.05 378 0.05 366 0.03 369 0.03 369 0.03 Zimbabwe 244 0.04 325 0.04 373 0.03 378 0.03 378 0.03 Total 13 488 2.13 16 159 1.99 16 607 1.37 17 686 1.31 17 765 1.3

Source: FAO production Yearbook 2006b.

According to FAO Production Yearbook (2006b), Namibia, the Seychelles and Botswana

do not contribute significantly to the world vegetable trade (Table 1.4).

1.1.1.5 Vegetable production in Botswana

In Botswana, most of the vegetables are produced by small scale farmers, and these

vegetables are consumed locally. It is estimated that this production (by small scale

farmers) accounts for only between 20% to 30% of the national demand (Bok et al.,

2006). Records show that 70% of Botswana vegetable requirements are met from imports

from South Africa (Bandeke, 1996; Mosie, 2004).

13

The most widely grown vegetable crops in Botswana are cabbages, potatoes, tomatoes,

onion, rape, spinach, kale (choumolier) and green mealies. Out of these vegetable crops,

spinach and tomatoes top the list (Bandeke, 1996; Bok et al., 2006). For purpose of this

study, a brief review of tomatoes will be given.

1.1.1.5.1 Tomato production in Botswana

Tomato (Lycoperiscon esculentum L.) is a vegetable crop that is grown worldwide. Its

selection and preference as a crop is due to its nutritional value and economic importance.

Records reveal that tomato is the second most important vegetable crop next to potato

(Solanum tuberosum L.) (Pamplona-Roger, 2004), (Figure.1.3). According to FAO

(2005), 125 million tonnes of tomatoes were produced in the world in 2005. The largest

producers of tomatoes (in tonnes) were: China, accounting for about one-fourth of the

global output; the United States is second, with Turkey third. In South Africa, tomatoes

are among the most important and highly valued horticultural products (Louw, 2005).

Louw (2005), has observed that in 2004 tomato production in South Africa was worth

R1.6 billion (an equivalent of US $246 million) per year.

In Botswana, (one of the SADC member countries), most of its tomatoes are produced by

local farmers and a few commercial farmers in the ‘Tuli block’ along the Limpopo River,

bordering with South Africa (Bok et al., 2006). Poor performance in the production of

tomatoes and other vegetables in Botswana is attributed to a number of factors, including

unreliable and inadequate rainfall as well as pests. Pests are the most important factors or

determinants in the production of vegetable crops (Bandeke, 1996; Molefi, 1996).

14

1.1.2 Factors affecting food production

There are a number of factors that affect food production. However, pests are among the

most contributing factors that affect food production. Pests affect harvests in all cereals

and other food plants. However, pest problems are mostly experienced in Africa due to the

high importation costs (and therefore unavailability) of pesticides (FAO, 2006b).

1.1.2.1 Crop pests

In nature, pest densities tend to fluctuate and the environment plays a major role in this

trend. Changes in environmental conditions lead to changes in the pest population levels

that attack and affect yields of cultivated crops and in particular tomatoes (Molefi, 1996;

Bok et al., 2006). Crop losses due to pests have a great impact on the decline of food

production. There have been major pest infestations leading to total crop failure. Crop

production losses to pests are estimated to exceed 35% annually (Henneberry et al., 1991).

The damage caused by pests to crops increase with the increase in pest population. Pest

damage to crops causes loss in crop yields and affects the quality of the produce which

results in loss of revenue to the farmer (Kasozi et al., 1999). In many cases, the pest

attacks the final product such as the leaves or fruits and this drastically reduces the market

value of the crop. For example, buyers are reluctant to buy spinach, cabbage or other leafy

vegetables with holes in them. Tomatoes which have larvae in them or are covered with

red spider mites are equally unacceptable.

15

1.1.2.2 Types of pests

Pests are defined as any insect, rodent, nematode, fungus, mite, weed or any other form of

terrestrial, aquatic plant, or animal life, or virus, bacteria or other micro-organism that

damage or kill crops or reduce the value of the crops before or after harvest (Kasozi et al.,

1999; Biswas, et al., 2004). The major pests of vegetables are lepidoterous caterpillars,

e.g. Agrotis species and Plutella xylostella which feed on the leaves of cabbage and other

brassicas, and Helicoverpa armigera which bores into tomato fruits. There are a number

of other pests that cause damage to tomatoes and reduce yields. These include pests like

tomato semi-looper (Chrysodeixis acuta), and nematodes (Meloidogyne species) (Kasozi

et al., 1999). However, one of the most common pests of tomato is the red spider mite

(Wikipedia, 2007). Bok et al., (2006) reported that various species of red spider mites

attack the tomato crop in Botswana reducing the yield to very low levels. This may be one

of the reasons why Botswana is not included among the world and SADC tomato

producing countries (FAO, 2005). Red spider mites (Tetranychus species) are a

polyphagous, parenchyma cell feeding pest on over 200 host plant species and have a

serious economic impact on many crops, especially tomatoes (Spencer, 1990; Flaherty &

Wilson, 1999; Van den Boom et al., 2003). These phytophagus mites attack mainly the

mature and old leaves of the tomato plant by sucking cell sap and damaging the

chlorophyll-producing organs, thus reducing photosynthesis, causing a great deal of yield

loss (Biswas et al., 2004). One of the methods of limiting damage to these crops is by

applying chemical pesticides.

16

1.1.2.3 Pesticides and their problems

Although the use of insecticides in the production of these crops has become unavoidable,

chemical insecticides have their own problems and may have severe environmental

consequences. They also appear to follow a pattern of initially being very successful,

resulting in high yields. After a number of years the target insect develops some degree of

tolerance. A series of events then occurs: more frequent application of pesticides and

higher dosages are needed to obtain effective control; insect population often increase

rapidly after treatments and the pest population gradually becomes increasingly tolerant to

the pesticide and its efficacy decreases (Ellis & Mellor, 1995). As a result, another

pesticide is substituted and the cycle is repeated. Resistance to more than one pesticide is

then usually the end result. It is estimated that only 1% of the applied insecticide actually

reaches the target (Daka, 2003). A large proportion of the insecticides end up in the

environment where they may affect non-target species. Insecticides may also have

adverse effects on wildlife and may pollute soil and water (Fig. 1.4). Other disadvantages

include the presence of pesticide residues in foods and animal feed which is causing health

concerns among consumers (Dent, 1991; Ellis & Mellor, 1995; Daka, 2003). Figure 1.4

illustrates the numerous ways in which pesticides can contaminate the environment via

drainage water, dust and aerial drift.

17

Figure 1.4. Different pathways through which insecticides may reach the environment

Source: Adapted from Daka (2003).

As the world population increases, so does the demand for food. This leads to the use of

more pesticides in order to eradicate pests on ever increasing areas of food production.

However, this draws a substantial amount of foreign currency resources for the

importation of insecticides (Bok et al., 2006). Most farmers, especially the resource poor

farmers, do not have the knowledge to use pesticides correctly. The reality is that pesticide

abuse leads to fatalities. The World Health Organisation attributes about 20,000 deaths

and more than a million illnesses each year to pesticides being mishandled or used in

excess (USOIA, 1992). In addition, chemical pesticides are usually very expensive and

beyond the ability or reach of resource poor farmers.

18

It is clear that some solution must be found to assist such farmers in fighting the effects of

crop pests. One way to rectify this would be to select crop varieties that are naturally

resistant to pests and that do not need pesticides. The other way would be to identify

botanical pesticides that are not harmful to animals or humans (Rembold, 1993).

1.1.2.3 Possible solutions: Botanical pesticides

Most farmers in sub-Saharan Africa are resource poor in terms of access to natural

resources, credit, information and external inputs (van Huis & Meerman, 1997). These

farmers rely on low-input traditional farming and cultural control techniques. These

farming and control techniques that contribute either directly or indirectly to pest

management, include sanitation, seed selection, rotation, weeding, multiple cropping,

tillage, fire, flooding and natural pesticides (van Huis & Meerman, 1997). One option,

therefore, is to use locally available pesticides, which can be obtained and applied by local

farmers themselves. It is also important that the pesticides are not harmful to humans or

animals. Thus botanical pesticides which are mostly found within easy reach and in most

cases do not interfere with parasitoid foraging (Charleston, 2004) can be a good

alternative for the resource poor farmers. It is documented that several substances of plant

origin have been tried in the control of insect pests. For example, secondary metabolites

present in Amoora ruhituka (Meliaceae), Annona reticulata and Annona squamosa

(Annonaceae), act as insect feeding deterrents and growth regulators. Anti ovipositional

properties of extracts from custard apple oil were found to reduce the egg–laying of the

female pulse beetle (Völlinger, 1995; Charleston, 2004).

19

Plants form the basis for many medicines and have been used for centuries to protect

humans and animals. Plants synthesize secondary plant compounds which can partly be

considered as weapons to defend themselves against pests and diseases that have

competed with them since time immemorial (Schmutterer, 1995). Since the manufacture

of chemical pesticides require chemicals and laboratory facilities, the use of plant

materials may offer a solution. Extracts from plants contain numerous compounds in

comparison to synthetic pesticides and therefore delay the build up in resistance (Rice,

1993; Völlinger, 1995; Charleston, 2004). Research showed that the seed kernel extracts

of Neem (Azadirachtin indica) have anti-feedant effects (feeding inhibition) and growth

inhibition properties and cause abnormal development in many insects (Hedge, 1996; Juan

& Sans, 2000). Melia azedarach (L.) (also known as the Syringa tree) has anti-feedant

properties (Ascher et al., 1995; Singh et al., 1998; Nathan, et al., 2006). According to

Schmutterer (1995), and Charleston (2004), triterpenoids and tetranortriterpenoids are the

main active ingredients found in these two plants. Azadirachtin, a tetraterpenoid, is found

in the Neem tree, while two other tetraterpenoids, meliacin and meliacarpin besides

azadirachtin, are found in the Syringa fruits. The growth inhibition and anti-feedant effects

of these two tetraterpenoids from Syringa compare favourably with that of azadirachtin

(Lee et al., 1991; Juan & Sans, 2000). The advantage of using botanicals is that they are

easily available; Syringa for example grows easily in many parts of the SADC region. In

South Africa it is even considered a weed (Ascher et al., 1995; Charleston, 2004).

20

1.1.2 Summary and problem statement

In 1998, the world population increased at a historically high annual average rate of 1.8%

(since 1950) (Gretchen et al., 1998). Cereal production more than kept pace (accounting

for more than 50% of the energy intake of the world’s poor at that time) (Duncan, 1997;

Gretchen, et al., 1998). Cereal production in the world has declined to an alarming level,

resulting in unrest and furious debates in the media on these shortages of food. Recently,

the United Nations warned that 82 countries, including China, face food emergencies, as

stock piles of wheat drop to the lowest level since 1980, resulting in food prices rising to a

record high (Gretchen et al., 1998; McMichael, 2000). The prospect of food shortages

over the next 20 years is so acute that urgent attention is required to increase food

production. However, growing enough food is getting more difficult because of:

1. climate change, which leads to shortage of water in many regions.

2. the new policy of changing from growing crops for food to that of bio–fuels.

3. pests, which reduce the yield of many crops.

The first two factors are important in helping to bring about an understanding of the

problem of food shortage, and deserve merit. However, it is the third factor for which

intervention strategies can be implemented, some aspects of which are dealt with in this

study.

21

In summary, the importance of the need to increase food production and the impact of

pests, especially red spider mites, in tomato crop production needs attention. The

application of insecticides as a solution has revealed numerous problems.

Botswana farmers are mostly resource poor who cannot afford the expensive and intricate

usage of conventional pesticides and most of the food that they produce is consumed

locally. One of the main vegetable food crops produced in Botswana and in the world is

tomato. However, this crop is heavily attacked by red spider mites (Bok et al., 2006). It is

important therefore that a solution be sought. A solution could be the development of a

botanical pesticide against red spider mites that can be produced cost-effectively by the

small scale farmers themselves. The question is, can effective pesticides be developed

against red spider mites on tomatoes for use by resource-poor farmers in Botswana?

Research has shown that Neem (Azadirachta indica A. Juss) and Syringa (Melia

azedarach L) are effective insecticides. These plants are readily available to farmers in

Botswana as they are found in large parts of the country. Little or no research has been

done to determine whether Neem and Syringa are effective acaricides.

Research Questions:

• Neem and Syringa are effective insecticides but are they effective as acaricides?

• Can Neem and Syringa extracts be used to control red spider mites in tomatoes?

22

1.3 Aim:

To explore the potential of Neem (Azadirachta indica) and Syringa (Melia azedarach)

extracts to control red spider mites (Tetranychus spp.) on tomatoes.

1.3.1 Objectives of the study:

The objectives of the study were to:

• investigate the possible geographical variations in the chemical composition of

Neem and Syringa

• to determine whether Neem and Syringa extracts are as effective against red spider

mites on tomato plants as the conventional acaricides.

• to establish the optimum concentrations of Neem and Syringa extracts (which does

not cause phytotoxicity) for red spider mite control.

1.4 Chapter layout

Chapter 1 is a general introduction of the study. Chapter 2 provides an overview of the

literature related to red spider mites as well as Neem and Syringa as botanical pesticides

and Chapter 3 deals with the Research Design and the Methodology used. Chapter 4 gives

a comparison of azadirachtin composition in Neem and Syringa samples. Chapter 5 deals

with the analysis and discussion of the results, and Chapter 6 gives an overview, summary,

conclusions and recommendations. This is followed by a section on appendix then

References.

23

CHAPTER 2: Literature Review

2.1. Introduction Cultivated tomatoes, Lycoperiscon esculentum L., have a variety of pests, the most serious

being the red spider mite. This has resulted in dependence on intensive use of pesticides,

especially when the crop is grown in open fields (Engindeniz, 2005). The overwhelming

nature of red spider mites attack often prompts desperate farmers to apply any available

pesticide in a bid to bring the infestation under control (Luchen & Mingochi, 1994). Such

indiscriminate application of chemical measures has limited effect on red spider mites and

often leads to loss of the crop (Messiaen, 1992). However, the introduction of pest

management strategies in the form of Integrated Pest Management (IPM) has helped in

controlling red spider mites and other tomato pests such as African bollworm

(Helicoverpa armigera) (Reganold, et al., 1990). The idea of Integrated Pest Management

took root in the 1960s in response to the pesticides dilemma. The principle behind IPM

was to use a variety of insect controls instead of relying solely on chemical insecticides.

These methods may include the use of cultural practices, natural enemies, and selective

pesticides (Reganold et al., 1990; Bohmont, 1997). Cultural practices are simple

techniques such as vacuuming out insects, the introduction of certain plants to ward off

pests that attack a particular crop or dislodging insects with strong jets of water. However,

a successful IPM program depends on a thorough understanding of pest populations, the

associated ecosystems, and the available management tactics. IPM is based on proper pest

identification, periodic scouting, and the application of pest management practices during

the precise stage of the crop’s development where no control actions would result in

significant economic losses (Bues et al., 2003).

24

The following section gives an overview of the characteristics of the red spider mites.

2.2. Red spider mites (RSM)

2.2.1. Taxonomy

Red spider mites belong to the class Arachnida and genus Tetranychus. Both the class and

genus include at least three well known species which are Tetranychus cinnabarinus

(Boisduval), also called the carmine mite; Tetranychus urticae Koch, also called the two

spotted red spider mite (Bok et al., 2006); and the tobacco spider mite, Tetranychus evansi

Baker & Prichard (Visser, 2005). Differentiating these three red spider mite species is not

easy. Wang (1987) tried to differentiate between T. urticae Koch and T. cinnabarinus

Boisduval by using morphological characters. This proved difficult because they are both

polymorphic and there was a significant variation in morphology among populations

found on different host plants and in different geographic locations (Wang, 1987). Meyer

(1987), considered T. cinnabarinus and T. urticae to be one and the same organism. This

was subsequently accepted by specialists of the Tetranychidae (Ehara, 1993; Baker &

Tuttle, 1994; Bolland et al., 1998). However, Kuang & Cheng (1990), using

morphological, biological and molecular data, showed that there were in fact marked

differences between T. urticae and T. cinnabarinus in that T. urticae females have 10 setae

on tibia I whereas T. cinnabarinus has 10-13 setae (an addition of up to three solenidia) on

tibia I. Zhi-qiang & Jacobson (2000), in their study on greenhouse tomato plants in the

UK, confirmed that the colour of the mite cannot be reliably used to separate T.urticae and

T. cinnabarinus.

25

The lack of clarity in terms of classification has now resulted in the green form of these

complex species being referred to as T.urticae while the red form is called T. cinnabarinus

(Baker & Tuttle, 1994; Bok et al., 2006). In this study, no differentiation is made between

the different mite species. This is because normally more than one type of mite occur

within a single infestation and the eventual goal of the research is to develop an effective

acaricide against all red spider mites.

2.2.2 Life cycle

The spider mite’s life cycle starts with a small, round egg (Figure 2.1). There are three

active immature stages (larva, protonymph and deutonymph), each separated by a resting

stage before a final moult to the adult (Klubertanz et al., 1991). The life cycle of spider

mites is temperature-regulated and occurs rapidly at warmer temperatures (Mau &

Kessing, 1992). Both T. cinnabarinus and T. urticae complete their life cycle from egg to

adult in about a week or two when temperatures are favourable (Mau & Kessing, 1992;

Bolland & Valla, 2000). Spherical shiny eggs are laid singly by the adult on the underside

of the leaf surface or are attached to the silken web span (Figure 2.2.).

Larvae

Protonymph Deutonymph

Adult

Eggs

26

Figure 2.1. Red spider mite life cycle. Adapted from Wikipedia, the free encyclopedia (Online) Accessed 6th August 2007

Figure 2.2. A silken web span produced by adult mites on tomato leaves.

The stages in the life cycle of the red spider mite are shown in Figure 2.1. It takes three

days for the eggs to hatch and the resultant larvae are six legged and pinkish in colour.

After a resting phase, the larva moults into a protonymph, which is eight-legged. The

protonymph feeds before going into another resting stage. It then changes into the

deutonymph before moulting into the adult (Knapp et al., 2003). Adult female mites are

0.5 mm long while the males are slightly smaller and wedge-shaped with a black spot on

either side of its colourless body (Figure 2.3a, 2.3b and Figure 2.4a). Figure 2.4b shows

the five stages of the two-spotted red spider mites, namely the eggs, larvae, protonymphs,

deutonymphs and adult.

27

(a) (b)

Figure 2.3. (a) Adult Female red spider mite. Fig. 2.3.(b) Adult male red spider mite

Source: Meyer (1987)

Figure 2.4 (a) Adult red spider mites.

Source: http:// www.bio-bee.com (on line) Accessed 26th March 2009

28

Figure 2.4 (b) The four stages of a red spider mite life cycle (egg, larva, nymph and adult)

Source: http://www.bio-bee.com (on line) Accessed 26th March 2009

The adult females may live up to 24 days and may lay up to 200 eggs (Meyer, 1987).

Thus, at a temperature of between 21 to 31 degrees Celsius in October, 10 spider mites

are capable of multiplying so fast as to reach 1000 by November and 100,000 by

December (Collyer, 1998). Biswas et al. (2004) suggested that the alarming rate of

reproduction by T. cinnabarinus (Boisd.) and T.urticae (Koch) was due to an increase in

the fecundity of these mites during high temperatures. Tetranychus evansi Baker &

Prichard are the third most common spider mite species. This species has only recently

been identified in southern Africa. It is commonly known as the tobacco spider mite and

originates from South America (Visser, 2005). Adult females of T. evansi are 0.5 mm

long, oval, orange red with a distinct dark blotch on each side of the body.

29

Males are smaller and straw to orange coloured (Bolland and Valla, 2000; Castagnoli et

al., 2006) (Figure 2.4c).

Fig 2.4c. A female tobacco spider mite (Tetranychus evansi) with a smaller male on top.

Wikipedia, the free encyclopedia (Online) Accessed 6th July 2008

Tetranychus evansi is currently the most important dry season pest of tomatoes in southern

Africa (Knapp et al., 2003). It is known to occur in South Africa, Namibia, Malawi,

Mozambique, Zambia, Zimbabwe, Kenya, Democratic Republic of Congo, Somalia,

Morocco, and Tunisia (Knapp et al., 2003).

30

2.2.3 Damage caused by red spider mites

Red spider mites (Tetranychus spp.) attack nearly 100 cultivated crops, including maize,

tobacco, cotton, beans, eggplant, pepper, tomatoes, cucurbits and many other vegetables

(Mau & Kessing, 1992).

They are also pests of papaya, passion fruit and are a common pest of many flowers such

as carnation, chrysanthemum, cymbidium, gladiolus, marigold and roses (Guo et al., 1998;

Tadmor et al., 1999; Bolland & Valla, 2000; Batta, 2003; Knapp et al., 2003). Red spider

mites are often found in pockets on the undersides of leaves near the midribs and veins.

Adult and nymphs of the red spider mites suck sap especially from the mature and older

leaves. This causes the upper surface to become stippled with little dots. These dots on the

upper surfaces usually indicate the presence of feeding punctures on the underside of the

leaf (Goff, 1986; Lu & Wang, 2005). Continued feeding may result in the collapse of

mesophyll cells.

The leaves eventually become bleached and discoloured. Leaf drop can occur following

heavy infestations (Figure 2.5) due to an increase in the mite population especially under

hot, dry conditions (Knapp et al., 2003). This drastically reduces the crop yield (Hill,

1983; Visser, 2005; Bok et al., 2006).

31

Figure. 2.5 A tomato plant with heavily infested leaves in a greenhouse.

2.3. Control measures

In the past the red spider mite was thought to be an insect rather than a mite (Luchen &

Mingochi, 1994). This led to the use of insecticides which, paradoxically, resulted in an

increase in mite infestation (Dagli & Tunc, 2001). With the proper classification of these

pests as mites, a variety of other control measures were developed such as natural control,

biological control, chemical miticides and the use of botanical extracts (Greathead et al.,

1990).

2.3.1 Natural control measures

The red spider mite can also be controlled by using natural methods such as spraying with

water and using pest resistant varieties.

32

Since all Tetranychus species infestations happen during hot and dry weather, rain helps to

reduce spider mite numbers, especially during moulting, by washing them off leaves of the

infested plants (Brandenburg & Kennedy, 1982; Rosenheim & Corbett, 2003). A number

of researchers have reported on crop varieties which are resistant to the red spider mite

(Knapp et al., 2003). For example, various cucurbits such as melon and water melon are

resistant to the carmine spider mite (Mansour et al., 1987; Mansour & Bar-Zur, 1990;

Scully et al., 1991; Mansour et al., 1994).

2.3.2 Chemical control measures

About 50 chemicals are registered in South Africa and the world for the control of red

spider mites. A few of the common miticides include; Dicofol, Abamectin, Profenofos and

Chlorphenapyr Table 2.1 (Ho, 2000; Chapman & Martin, 2003).

33

_______________________________________________________________________________ Table 2.1. A few chemical pesticides used in vegetables in South Africa and the world Trade Name Active Ingredient Formulation

Acathrin fenpropathrin EC ACE acephate SP

Abamectin-plus abamectin EC Agriphos alminium phosphide GE (tablet)

Blue death gamma-BHC DP, EC Carbaryl dust carbaryl DP Carbofuran carbofuran GR Cymite chlorphenapyr SC

Deltamethrin deltamethrin EC Demeton demeton-S-methyl EC Diazinon 275 EC diazinon EC Dursban chlorpyrifos EC Endosulfan endosulfan EC, SC, WP Fenthion fenthion EC Furadan carbofuran GR (soil application) Kelthane dicofol WP Methaphos methamidophos SL

Mevinphos mevinphos EC, SL Omite propargite EC, WP Parathion parathion EC Phorate phorate GR Rogor dimethoate EC Selecron profenofos EC Talstar bifethrine SC

Source: Safety and quality assurance (2007)

Unfortunately, control of red spider mites with both contact and systemic pesticides has

become increasingly difficult. This is because the mite has become resistant to a number of

acaricides.

34

In China, red spider mites first developed resistance to parathion, demeton and malathion,

then fenpropathrin, and thus became resistant to a number of organophosphates (Bohmont,

1997; Lin et al., 2005). According to Guo et al. (1998), red spider mites have now

developed a resistance to at least 25 pesticides.

Experiments with various pesticides have shown that Kelthane MF (dicofol), at a dose of

0.5 kg per 500 litres, was very effective, especially against mite eggs (Chapman & Martin,

2003). Even though the use of pesticides has been growing at an alarming rate, users are

becoming more aware of their side-effects and are seeking alternative ways of controlling

or preventing red spider mite infestations. Currently natural organic pesticides have been

found to be very effective (Ngugi et al., 1990). Most of these natural organic products are

homemade pest control substances extracted from botanicals.

2.3.3 Biological control measures

While it is becoming difficult to control the red spider mites with conventional acaricides

such as chlorphenapyr and dicofol, there are a few predators known to control the red

spider mites (Hurd & Eisenberg, 1990; Wise, 1993; Memmott et al., 2000; Chase, 2000;

Polis et al., 2000; Rosenheim, 2001; Shurin et al., 2002; Schmitz et. al., 2004). It has

been found that Stethorus spp. (Coccinellidae) and Oligota spp. (Staphylinidae) are the

main predators of T. cinnabarinus (Boisduval) (Rosenheim & Corbett, 2003). Rosenheim

et al., (2004) reported that the lady bird beetle Stethorus siphonulus suppressed the spider

mite T. cinnabarinus. The predacious mite, Phytoseiulus persimilis Athias - Henriot

(Phytoseiidae: Acarina) has also been utilised successfully to control T. urticae.

35

In Zambia, however, this predatious mite did not successfully control the red spider mites

when the infestation was very high (Mingochi et al., 1994). While advocating the use of

predatious mites it should be noted that alkaloids in tomatoes are harmful to some

predators which may hamper biological control.

It has been found that mites can also be controlled by pathogens. Brandenburg &

Kennedy, (1982) has documented that mite infestations were reduced by the fungus,

Neozygites floridana, especially during periods with high relative humidity. These

conditions favour the quick multiplication of the fungus. Wekesa (2005), reported that the

pathogen Metarhium anisopliae successfully controlled the tobacco spider mite T. evansi.

Batta (2003), found that this fungus also controlled the nymphs and adult stage of red

spider mites on eggplant.

2.4. Botanicals as an option

2.4.1 Introduction

Nature has provided mankind with a rich repository of plants which can produce a huge

variety of usable compounds. It is estimated that there are about two to five million

different plant species in the world today (Schmutterer, 1995; Charleston, 2004).

However, only 10 percent of these have been examined chemically, indicating a

potentially vast resource which still remains untapped (Dhaliwal et al., 2004).

36

Extracts prepared from plants (botanical pesticides) have a number of properties including

antifeedant effects, insect growth regulation, as well as having other negative effects on

nematodes and other agricultural pests. They also have antifungal and antibacterial

properties (Prakash & Rao, 1997; Boeke et al., 2001; Charleston, 2004). Most studies on

botanical pesticides have centred on plants from the mahogany family, Meliaceae, and in

particular members from the genera Azadirachta and Melia appear to be effective against

insect pests (Schmutterer, 1995; Charleston, 2004).

2.4.2. Neem (Azadirachta indica A. Juss) as a botanical pesticide

Neem (Fig. 2.6) is a fast growing tree which may reach a height of 25 m, a girth of

2.5 m, with a crown of 10 m across (USNRC, 1992). It is evergreen but under severe

drought it may shed some or all of its leaves.

Figure 2.6. A Neem tree branch with leaves

37

Neem can be established and propagated from cuttings, stumps, tissue cultures or by seed.

Seed propagations in nurseries and transplanting seedlings in fields is the most commonly

accepted method to produce plantation stands (Puri, 1999). Neem trees begin fruiting at 3

to 5 years, but do not become fully reproductive until they are ten years old. From this

time on, the tree yields an average of about 21 kilograms of fruit per year (USNRC, 1992).

It usually bears its flowers in September and October and fruit maturity occurs in March

and April in the Southern Hemisphere (Rembold, 1993). However, it sometimes fruits as

early as November and December (Saxena, 2004). The fruit is about 2 cm long and when

ripe, has a yellow fleshly pericarp, a white hard shell and a brown, oil-rich seed kernel

(Figure 2.7) (Kraus, 2002; Saxena, 2004).

Fig 2.7. Neem tree branches with seeds

Wikipedia, the free encyclopedia (Online) Accessed 6th July 2008

38

2.4.3.1. Origin and distribution of Neem (Azadirachta indica A. Juss)

The Neem tree is native to India, Bangladesh, Myanmar and Pakistan, and can grow in

most arid, subtropical and tropical areas of the world (Schmutterer, 1990; Ascher, 1993;

Copping, 2001). Neem is susceptible to damage at temperatures of 0°C and below. Its

distribution is thus limited to the temperate and tropical regions of the world (Ascher,

1993; Koul & Wahab, 2004). At present, Neem is widely distributed in the arid tropical

and sub-tropical countries of Asia, the Americas, Australia and South Pacific Islands and

has been planted in many parts of Asia: Bangladesh, Cambodia, India, Indonesia, Iran,

Malaysia, Myanmar, Nepal, Pakistan, Sri Lanka, Thailand and Vietnam. It has also been

introduced into Saudi Arabia, and the northern parts of Yemen and China (USNRC, 1992;

Hedge, 1996; Rembold, 1996). To date, Neem trees are found growing in the African

countries Somalia, Kenya, Tanzania, Malawi and Mozambique (GTZ Report, 2000), and

have recently been introduced into Zambia and Botswana where they are grown at

research stations and on road sides, providing shade, based on this researcher’s

observation.

2.4.2.2. Uses as pesticide

Although research on the use of Neem started in the early 1920s in India (Ruckin, 1992),

there was little global attention given to the species until 1959, when a German

entomologist noticed that Neem trees in the Sudan resisted an attack by the migratory

locusts (Schistocerca sp.) (Ruckin, 1992). Thereafter, a number of scientists started

studying the plant (Koul, 1996). The major interest in Neem has been its recognition as a

source of valuable plant allelochemicals, which have insecticidal, insect repellent,

antifeedant and growth regulatory properties (Mordue and Blackwell, 1993; Koul, 1996).

39

Extracts from various parts of the Neem plant are used for pest control. The dried leaves

and powdered seeds are applied to crop plants to prevent insects from feeding and laying

eggs (Schmutterer & Ascher, 1987). Extracts of Neem twigs, stem bark and root bark

have also been studied and a number of compounds have been isolated for use as

insecticides (Koul et al., 1990; Koul, 1996; Luo-Xiao et al., 2000).

Singh et al. (1998) reported that the seed is the most important part of Neem, as most

biologically active materials are concentrated in this part of the tree. Studies carried out

by Murgan & Ancy (1992) revealed that Neem seed kernel extract (NSKE) and Neem oil

(NO) affect the American bollworm (Helicoverpa armigera) by interfering with the

efficiency of feeding and causing a significant decline in its protein and lipid

concentration. The advantage of Neem extracts compared to most of the commonly

available insecticides includes low costs, environmentally friendly properties, and non-

toxicity to man (Mordue, & Blackwell, 1993; Sharma & Ansiri, 1993). Neem extracts are

also said to be of low toxicity to beneficial insects like bees, butterflies and natural

predators of pests. This is an important characteristic as most insecticides used in the

control of insect pests are detrimental to predators and beneficial insects (Mansour et al.,

1987; Riechert & Lockley, 1984; Luo-Xiao et al., 2000).

2.4.2.3. Chemical composition

Neem has a bitter taste, the bitterness being due to the presence of an array of complex

compounds called “limonoids”. The limonoids in Neem belong to nine basic structure

groups.

40

One of these compounds is azadirachtin from Neem seed kernel extract, a

tetranortriterpenoid. It exhibits insect growth regulatory effects on the immature stages of

insects by preventing insects from moulting (Broughton et al., 1986; Mordue &

Blackwell, 1993; Thacker, 2002). It is known to be chemically similar to ecdysonlids, the

hormone responsible for triggering moulting (Weinzierl & Henn, 1991).

Other compounds include azadirone (from seed oil), amoorastaitin (from fresh leaves),

vepinin (from seed oil), vilasinin (from green leaves), gedunin (from seed oil and bark),

nimbin (from leaves and seed), nimbolin (from kernels), and salannin (from fresh leaves

and seed) (Schmutterer, 1995). This cocktail of compounds significantly reduces the

chances of tolerance or resistance developing in any of the affected organisms.

Azadirachtin is said to be the most bioactive of all the compounds found in the Neem tree.

Although such assertions may be due to the fact that azadirachtin has been investigated

more thoroughly than other Neem compounds (Quarles, 1994; Thacker, 2002), research

conducted on a number of limonoids (Azadirachtin, Salannin, Gedunin, 17-

Hydroxyazadiradione and Deacetylnimbin) by Nathan et al., (2006), found azadirachtin to

be most potent for the control of the rice leaf folder Cnaphalocrosis medinalis (Nathan et

al., 2004).

The tetranortriterpenoid, azadirachtin (Fig. 2.8), has also received much attention as a

pesticide because it is relatively abundant in Neem kernels and has shown biological

activity on a wide range of insects.

41

It is reported that azadirachtin is actually a mixture of seven isomeric compounds labeled

as Azadirachtin-A to Azadirachtin-G with Azadirachtin-A being present in the highest

quantity and Azadirachtin-E regarded as the most effective insect growth regulator

(Verkerk & Wright, 1993). It has also been reported that azadirachtin interacts with the

corpus cardiacum, thereby blocking the activity of the moulting hormone. Thus the

compound acts as an insect growth regulator, suppressing fecundity, moulting, pupation

and adult formation (Ascher, 1993; Schmutterer, 1995).

Fig 2.8. The structure of azadirachtin a tetraterpenoid

Source: Siddiqui et al. (1993)

2.4.3 Syringa (Melia azedarach l) as a botanical pesticide

Syringa (Fig.2.9 and 2.10), also commonly known as “Chinaberry” or “Bead Tree”, is a

deciduous tree belonging to the mahogany family Meliaceae. The roots are suckering,

forming thickets of shrubby plants. Syringa may flower and fruit from shrub size onwards.

42

The adult tree has a rounded crown, and measures between 7 and 12 m in height. The

leaves grow up to 50 cm long, are alternate, long-petioled, 2 or 3 times compound (odd-

pinnate); the leaflets are dark green above and lighter green below, with serrate margins

(Ascher et al., 1995). (Figure 2.9, 2.10 and 2.11). The Syringa tree produces flowers

during the months of September to October, and fruits mature around March and April in

the Southern Hemisphere. The flowers are small and fragrant, with five pale purple or

lilac petals, growing in clusters. The fruit is a drupe, marble-sized, light yellow at

maturity, hanging on the tree all winter, and gradually becoming wrinkled (Figure 2.11).

Fruit berries are believed to be poisonous to humans and some other mammals (Russell et

al. 1997; Visser, 2004), but seeds are commonly dispersed by a variety of songbirds,

which relish the drupes and sometimes “gorge themselves to the point of temporary

intoxication” (Langeland & Burks, 2005).

Fig 2.9. Typical purple flowers of the Syringa tree.

43

Fig 2.10. Green Syringa seeds during the growing season.

Fig 2.11. A Syringa tree

44

2.4.3.1. Origin and distribution of Syringa (Melia azedarach L.)

The Syringa tree (Fig. 2.11) originates from north-western India. It is however, found in

nearly all warm climatic regions (Palacios et al., 1993). It also grows in Croatia, Southern

China, Northern Argentina, Northern Italy, Southern France and Australia. It is also

widely distributed in dry regions of the Southern and Western United States (Schmutterer,

1995). In South Africa, Zambia, Zimbabwe, and Botswana, the Syringa was planted as a

drought-resistant ornamental and shade tree (Ascher et al., 1995). The tree leaf litter has a

potential soil amendment activity that can increase mineralisable nitrogen and increase the

soil pH in acidic soils (Noble et al., 1996), but is now considered as an invasive species in

South Africa (Ascher et al., 1995). The tree occurs primarily in disturbed areas such as

road right-of-ways and fence rows, but has also invaded flood plains and marshes (Ascher

et al., 1995; Chung Huang et al., 1996).

2.4.3.2. Uses as pesticide

Syringa, just like Neem, is one of the promising plants with pest control properties from

an entomological perspective (Schmutterer, 1990, 1995). Aqueous extracts of Syringa

seeds have been used to control some insect pests in cotton (Gupta & Sharma, 1997). Lee

et al. (1987) found that methanolic extracts of Syringa fruits possess insecticidal potency

that is comparable or equivalent to that of Neem seed extracts or to that of azadirachtin

(Abou-Fakhr Hammad et al., 2001).

45

2.4.3.3. Chemical compounds

Syringa is known to have repellent antifeedant properties (Saxena, 1987; Abou-Fakhr

Hammad et al., 2001) and other pesticide properties against insect pests (Abou-Fakhr

Hammad et al., 2000). Many limonoids (tetranortriterpenoids) have been isolated from

Syringa (M. azedarach L.) (Chung Huang et al., 1996) which are chemically related but

are not azadirachtin (Langeland & Burks, 2005). Thus they are able to act as insect growth

regulators, suppressing fecundity, moulting, pupation and adult formation, similar to

azadirachtin (Ascher et al., 1995). Although the fruits are the poisonous part of the tree

(Visser, 2004), they have been used for the treatment of a variety of diseases such as

dermatitis and the treatment of viral infections such as herpes (Mendez et al., 2002). A

number of potent pharmaceutical limonoids and triterpenoids have also been isolated from

fruits and the bark (Lee et al., 1991).

46

2.4.3.4 Summary

This chapter describes the problems that farmers face in the control of red spider mites.

Even though Integrated Pest Management (IPM) seemed to show some degree of success

in controlling red spider mites, certain studies indicated that caution needs to be taken by

ensuring that pest populations and the associated ecosystem is known before practicing

IPM as noted by Bues et al. (2003).

The three most commonly encountered red spider mite species on tomatoes in southern

Africa are:

• Tetranychus urticae Koch

• Tetranychus cinnabarinus Boisduval

• Tetranychus evansi Baker & Prichard

This chapter describes various methods of controlling red spider mites, in particular the

effectiveness of the botanical pesticides extracted from Neem and Syringa.

47

CHAPTER 3: Research Design and Methodology

3.1 Introduction

This Chapter outlines the research design and methodology used to determine whether

extracts of Neem and Syringa could be used in controlling red spider mites on tomatoes.

Red spider mites are economically important pest with worldwide distribution, inflicting

damage to a number of field and horticultural crops including cotton, tomato, tobacco,

maize and ornamentals such as roses (Lin et al., 2005). Resistance to most conventional

insecticides, including organophosphates, carbamates and pyrethroids, has been reported

in many countries due to misuses of these chemicals in tomato and cotton fields

(Luchen & Mingochi, 1994; Prischmann et al., 2005).

Even though insecticides and their residues often have direct effects on spider mites,

including decreased fecundity, they do not appear to affect spider mites’ life span

(Cross & Berrie, 1994; Blumel & Hausdorf, 2002). Mites are a classic example of an

induced pest that exhibits population outbreaks when pesticides intended to reduce

primary pest densities also kill natural enemies such as predatious mites (Acari:

Phytoseiidae), Stethorus spp. (Coleoptera: Coccinellidae), and generalist macro predators

in the orders Hemiptera, Neuroptera, and Thysanoptera (Flaherty & Wilson, 1999). Such

population outbreaks also occurred with the application of acaricides such as abamectin

and methrine (Meng et al., 2000; Zhao et al., 2001).

48

In this chapter the following methods will be discussed:

• collection of seeds

• determination of azadirachtin composition in collected seeds by using a standard

azadirachtin

• extraction of the active constituents from Neem (Azadirachta indica) and Syringa

(Melia azedarach) seeds

• determination of the efficacy of the Neem and Syringa extracts using red spider

mite adults, nymphs and eggs

• greenhouse trials to determine the efficacy of Neem and Syringa in comparison

with the conventional acaricides

3.2 Study area

The experiments were carried out at the Vegetable and Ornamental Plant Institute

(VOPI), one of the Institutes of the Agricultural Research Council (ARC) in South

Africa, located at Roodeplaat (25º 36´ S, 28º 36´E), north east of Pretoria. The studies

were conducted between August to December 2007, and in April 2008. The average

temperature in the laboratory and the greenhouse was 22 ± 40C and the average

relative humidity was 54 ± 2%. These two variables were monitored constantly because

they are known to affect the activity and therefore feeding of the mites (Meyer, 1987;

Collyer, 1998).

49

3.3. Materials and Methods

3.3.1 Collection of seeds

3.3.1.1 Neem seed collection

Dried out Neem seeds were collected from three countries, namely Botswana, India and

Zambia. These three countries were selected on the basis of easy availability of the Neem

seeds. In India, Mrs. Jayeseeli helped in the collecting and transporting of the seeds to

Botswana, the seeds having been collected from Chennai on the south Coast of India, (800

17´ N, 130 04´E). In Zambia, personnel at the Nanga Irrigation Research Station collected

seeds. From there, the seeds were transported to Botswana and the study area. In

Botswana, Neem seeds were collected from Serowe village, 120 kilometres north of the

Tropic of Capricorn. The seeds were pre-dried to make transportation easier and also to

reduce the chances of fungal infestation. In Zambia, 10 kilograms of the seeds were

collected from Nanga Irrigation Research Station in Mazabuka district, 100 kilometres

from the capital city Lusaka. Nanga is located at 150 46´ N, 270 55´ E. The seeds were

picked from the ground and dried before transportation.

3.3.1.2 Syringa seed collection

The Syringa seeds were collected from two countries, namely Botswana and South Africa.

In Botswana the seeds were collected from Tonota College of Education campus (210 26´

S, 270.28´ E) which is 220 kilometres north of the Tropic of Capricorn. In South Africa,

they were collected from the ARC – Roodeplaat Vegetable and Ornamental Plant Institute

(VOPI), north-east of Pretoria. In all the instances, 10 kg of dried seeds were collected in

April by shaking the trees and collecting the fallen seeds from the ground.

50

3.3. 2 Determination of azadirachtin content of seeds

Determination of azadirachtin composition in samples from the different countries was

done in order to establish whether geographical location affected the azadirachtin content

in the genotypes and to select the source of Neem and Syringa materials to be used in the

experiments.

3.3.2.1 Preparation of standards

To determine the amount of azadirachtin in the samples of Neem and Syringa from

different countries, a standard comprising of 99% pure azadirachtin obtained from a

solvent mixture of 80 ml methanol, 20 mg sodium phosphate and a buffer (of pH 2.6) was

run through the analytical reversed-phase High Performance Liquid Chromatography

(HPLC). This was performed on a 5 µm particle size column using different (standard)

crystal concentrations of azadirachtin: 10 mg/ml; 5 mg/ml and 2.5 mg/ml, at a run time of

30 minutes and a rate of 1 ml/min. The full details of the results are given in Chapter 4.

3.3.2.2 Extraction of the active constituents from Neem and Syringa seeds

A 500 g sub-sample of each of the dried Syringa and Neem seeds were selected for the

experiments. These were crushed to fine powder using a rotary blender (Fig. 4.1). The

seeds were later dried for 24 hours at room temperature. Extraction was carried out

according to Warthen et al. (1984). The powder was weighed and 1000 ml of 96%

methanol was added and shaken for three hours using a magnetic stirring vibrating shaker

in a beaker. The mixture was left in the shaker overnight, followed by filtration using

Whatman filter paper No 40.

51

The filtrate was poured into a round bottom flask and concentrated to 500 ml for three

hours on a rotary vacuum evaporator at 40oC. The preparation of the stock solution was

done using the water method as described by Copping (2001). For example, assuming

100 ml of the concentrated extracts were used for the preparation of the stock solutions, to

prepare a 1%, 10%, 20% or 30% Neem and Syringa stock solution, 99 ml ; 90 ml ; 80

ml and 70 ml distilled water was added, respectively.

3.3.3 Experimental plants

Tomato plants (Fig. 3.1) were grown in a glasshouse at the Roodeplaat Research Station.

Tomato seeds of the variety Money Maker were sown in pots containing a vermiculite

growth medium (pot sizes were 7 cm diameter and a height of 17 cm). When plants

reached a height of three to four centimetres, nine seedlings were transplanted into

separate individual pots.

Fig 3.1. The experimental tomato plants in the greenhouse.

52

3.3.4 Data collection

Data was collected from bioassays in the laboratory and the greenhouse experiments.

3.3.4.1 Laboratory bioassays

The efficacy of Neem and Syringa extracts was tested on adults, nymphs and eggs of red

spider mites using the leaf dip method on water agar in Petri dishes (Levent et al., 2005).

Autoclave sterilized water agar (15 g/litre) was added to Petri dishes. The water agar was

prepared one day before the experiment day, by dissolving 15 g of agar powder in one liter

of water and sterilizing it for 15 minutes at 1210C.

3.3.4.2 Testing the effect of Neem and Syringa extracts on adult red spider mites

Different concentrations (0.1%, 1%, 10%, 20%, 50%, 75% and 100%) of Neem

(Azadirachta indica) and Syringa (Melia azedarach) were used in the treatments. Three

commercial acaricides were compared with the two botanicals and were applied according

to specifications on the labels; Abamectin-plus - 0.6 ml/liter, Hunter – 0.4 ml/liter and

Selecron – 3 ml/liter. Control treatments with distilled water were included. The

experiment was carried out in a completely randomized design with 12 treatments and

three replicates. The treatments were:

• 7 botanicals

• 3 acaricides

• 2 controls

Each treatment consisted of three plastic Petri dishes filled with water agar.

53

Tomato leaves were picked from plants in the greenhouse and taken to the laboratory for

use in the bioassays using the leaf–dip method (Levent et al., 2005). For each treatment

(Fig. 3.2), nine tomato leaves were immersed into one of the concentrations of Neem,

Syringa or the 3 acaricides (Abamectin-plus, Hunter, and Selecron) for thirty seconds. The

control leaves were immersed in distilled water only for thirty seconds and allowed to dry

for thirty minutes on a filter paper under room temperature. Three treated tomato leaf discs

were placed onto 3 ml water agar within each Petri dish (Fig. 3.3). Four adult mites were

transferred onto each of the three leaves using a pencil brush. A total of twelve adult mites

were transferred onto each leaf in each Petri dish and left to feed on the tomato leaves in

the Petri dish.

Fig. 3.2 Treated tomato leaves left to dry on towel paper.

54

Figure 3.3 Treated tomato leaves placed onto 3 ml water agar within Petri dishes

The counting of dead/live mites was carried out at 24 hours, 48 hours and 72 hours after

placing the mites on the leaves. Temperature and relative humidity sensors were installed

in the laboratory to monitor these two variables during the experimental period. This

experiment was repeated once in an effort to verify the data.

3.3.4.3 Testing the effect of Neem and Syringa extracts on nymph red spider mites

Red spider mite nymphs were subjected to concentrations of Neem and Syringa extracts

and 3 conventional acaricides. The method used was the same as that used for the adult red

spider mites. Four nymphs were transferred on each of the three leaves using a pencil

brush.

55

A total of 12 nymphs were transferred on the tomato leaves in each water agar Petri dish

and left to feed on the leaves in the Petri dish. The counting of the dead/live nymphs was

carried out at 24, 48 and 72 hours after the placing of the mites.

3.3.4.4 Testing the effect of Neem and Syringa extracts on red spider mite eggs

Red spider mite eggs were subjected to concentrations of Neem and Syringa extracts and 3

conventional acaricides. The method used was the same as that used for the adult red

spider mites. Six mite eggs were transferred on to each of the three leaves using a

pencil brush. A total of 18 mite eggs were transferred on the tomato leaves into each

water agar Petri dish. The counting of the hatched mite eggs was carried out at 24, 48, and

72 hours after placing the eggs.

The treatments were as follows:

Treat. 1 = Control 1: Untreated

Treat. 2 = Control 2: Tomato leaf discs dipped in distilled water

Treat. 3 = Neem or Syringa conc. at 0.1%

Treat. 4 = Neem or Syringa conc. at 1.0%

Treat. 5 = Neem or Syringa conc. at 10%

Treat. 6 = Neem or Syringa conc. at 20%

Treat. 7 = Neem or Syringa conc. at 50%

Treat. 8 = Neem or Syringa conc. at 75%

Treat. 9 = Neem or Syringa conc. at 100%

Treat 10 = Abamectin-plus - 0.6 ml/liter

Treat 11= Hunter – 0.4 ml/liter and

Treat 12 = Selecron – 3 ml/liter

56

3.3.5 Greenhouse trials

The greenhouse experiment was carried out in an effort to simulate field conditions. The

findings in the efficacy of Neem and Syringa on adult, nymphs and eggs bioassays were

used to select the Neem and Syringa extract concentration to be used in the greenhouse

trials.

Tomato seedlings of the variety Money Maker were transplanted into 16 pots of the same

size as in the laboratory experiment. The pots were arranged in four rows. The rows were

70 cm apart and the pots 45 cm from each other in each row. Each row comprised of four

pots. When the plants reached 15 centimeters in height, 5 adult mites (3 to 5 days old)

were transferred to the lower surface of the plant’s upper leaves.

Ten days after inoculation with the mites, and before inoculation with the extracts, the

tomato leaves were examined to determine the presence and population density of the red

spider mites, using the direct examination method recommended by Steinkraus et al.

(1999) where:

none - no spider mites present. light - one to 10 spider mites present under the leaf with little leaf damage (russeting, speckling). medium - 11 to 50 spider mites present per leaf (leaves speckled, mottled yellow or red). heavy - more than 50 spider mites present per leaf on most plants (many leaves reddish – brown in colour).

57

Three leaves from each experimental row were collected randomly to check on the

number of red spider mites’ presence and population. Thereafter, on the same day, the

Neem seed extracts (NSE) and Syringa seed extracts (SSE) at 50% and the conventional

acaricides were applied to the leaves of the potted tomato plants using a hand held

spraying bottle. Each treatment was replicated four times.

Five leaves from each experimental plot were collected randomly. Records of live and

dead mites were taken after 24, 48 and 72 hours respectively. An ANOVA was

performed to reveal whether there was a significant difference between the Neem or

Syringa extracts and the two acaricides Abamectin-plus and Hunter. (Selecron was left

out because it did not perform well compared to Abamectin-plus and Hunter in the

Bioassays results. The treatments were as follows;

Treat. 1 = No application (Control)

Treat. 2 = Distilled water (Control)

Treat. 3 = 50% of NSE

Treat 4 = 50% of SSE

Treat 5 = Abamectin-plus - 0.6 ml/liter

Treat 6 = Hunter – 0.4 ml/liter

58

The followings information was recorded:

• the date of transplanting seedlings

• leaf damage symptoms (ranked) before and after treatment

• age of the tomato plants at pest inoculation period

• the number of leaves per plant before and after treatment

• the number of mites on the sampled leaves before and after treatment

• temperature and relative humidity records throughout the experimental period

The data obtained from the trial were analysed by using Analysis of Variance (ANOVA).

Treatment averages were separated using the GenStat statistical programme.

59

CHAPTER 4: Comparison of azadirachtin composition in Neem and Syringa from

different parts and regions of the world.

4.1 Introduction

Nature has provided a rich repository of plants which are a source of a number of

compounds that are used in industry, medicine, and agriculture. Chemical examination of

such natural products for crop plant protection against pests over the past sixty years has

been quite fruitful. Plants such as Neem, and recently Syringa, which belongs to the same

family, have been among those investigated for such chemicals (Subrahmanyam & Rao,

1993). Neem (Azadirachta indica A Juss) and Syringa (Melia azedarach L.) have been

credited as trees with a potential for use in pest control (Koul & Wahab, 2004). At

present, these trees are widely distributed mainly in the arid tropical and subtropical

countries (Rembord, 1996). Previous research has established that there are differences in

yields of the main extract azadirachtin in Neem (Dhaliwal et al., 2004). For example

Kumar et al. (2000) evaluated the insecticidal property of Neem seed kernel extracts from

38 Neem trees, sampled from six locations in India, by means of laboratory bioassays.

These revealed that there are significant differences between trees originating from

different ecotypes. However, information on the environmental effects on the chemical

composition of Syringa is inadequate or non-existent. This chapter deals with the study

aimed at evaluating the concentration of azadirachtin in Neem and Syringa seeds obtained

from different regions (Botswana, India and Zambia for Neem) and (Botswana and South

Africa for Syringa).

60

4.2. Materials and methods

4.2.1 Extraction of the active constituents from Neem and Syringa seeds

Dried seeds of Neem and Syringa were weighed, and from each plant material, a 500 g

Sample was collected as described in section 3.3.2.2.

4.2.2 Determination of Azadirachtin content in Neem and Syringa

The standards as described in section 3.3.2.1 were passed through the analytical reversed-

phase HPLC, performed on (5µm particle size) column at a run time of 20 minutes and a

rate of 1ml/min. Three trials with different concentrations (10 mg/ml; 5 mg/ml; 2.5

mg/ml) were used to establish and ascertain the purity of the azadirachtin standard (Figure

4.2. and Figure 4.3). The peaks appeared between 3.5 and 3.8 minutes.

Figure 4.1. The rotary vacuum evaporator used in the extraction of active ingredients.

61

Figure 4.2. The high performance liquid chromatography (HPLC) used to determine the

azadirachtin compound in the samples.

Azadirachtin: 10 mg/ml

2.5 3.0 3.5 4.0 4.5 min

0

50

100

mAUExtract-190nm,4nm (1.00)

Azadirachtin: 5 mg/ml

2.5 3.0 3.5 4.0 4.5 min

0.0000

24.5439

-10.4076

mAUExtract-190nm,4nm (1.00)

62

Azadirachtin: 2. 5 mg/ml

2.5 3.0 3.5 4.0 4.5 min

0

10

mAUExtract-190nm,4nm (1.00)

Figure 4.3. Peaks of azadrachtin at the three concentrations from the high performance

liquid chromatography (HPLC)

4.3 Results and discussion

4.3.1 Peaks for azadirachtin standard at three concentrations

The 99% Azadirachtin was divided into three portions of 10 mg/ml’ 5 mg/ml and 2.5

mg/ml and run through the analytical reversed-phase HPLC using a sodium phosphate

buffer at a pH of 2.6 and a running rate of 20 minutes. Sample concentrates were run at 30

minutes. The difference was made to avoid overlapping of some compounds. The results

on all three divisions showed the peaks in all the three appearing at between 3.5 and 3.8

minutes. This proved that the standard was pure and could be used to determine

azadirachtin presence in the Neem and Syringa samples.

4.3.2 HPLC for Neem and Syringa

Dried acqueous extracts were prepared as explained in section 3.3.2.2 after which the

samples were removed for high-performance liquid chromatography (HPLC).

Concentrates from the samples (Neem & Syringa) were run through the analytical

reversed-phase HPLC, performed on a 5 µm particle size column with 99% azadirachtin

as the standard at a run time of 30 minutes and a rate of 1 ml/min.

63

The retention time of peaks detected at 3.5 – 3.8 min were measured for Neem and

Syringa extracts from different geographical regions. See Figures 4.4a, 4.4b and 4.4c for

Neem and Figures 4.5a and 4.5b for Syringa.

4.3.3 Neem samples

Neem samples peaked between 3.5 and 3.8 minutes as determined by the standard. The

concentration for Azadirachtin in the Botswana sample was 30 mAU while the samples

from India were 35 mAU and Zambia 44 mAU, as shown by the peaks (Fig. 4.4a, Fig.

4.4b, and Fig 4.4c).

2.5 3.0 3.5 4.0 4.5 min

0

25

50mAUExtract-190nm,4nm (1.00)

Figure 4.4a Peaks at 3.5 to 3.8 minutes showing concentration of azadirachtin in the Botswana’s

Neem sample

2.5 3.0 3.5 4.0 4.5 min

0

25

50mAUExtract-190nm,4nm (1.00)

Figure 4.4b Peaks at 3.5 to 3.8 minutes showing concentration of azadirachtin in the Indian

Neem sample

64

2.5 3.0 3.5 4.0 4.5 min

0

25

mAUExtract-190nm,4nm (1.00)

Figure 4.4.c Peaks at 3.5 to 3.8 minutes showing concentration of azadirachtin in the Zambian

Neem sample

The results in the Neem samples showed that there are differences in the concentration of

the chemical azadirachtin in Neem grown in different locations. This is clearly shown by

the peak figures between India and Zambia Neem samples and the Botswana sample. This

revealed that there are differences between trees from different ecotypes as was previously

observed by Kumar et. al. (2000).

4.3.4 Syringa samples

The concentration of azadirachtin in Botswana Syringa sample was 1 mAU. The South

Africa Syringa Sample was 2 mAU (Fig. 4.5a and Fig. 4.5b).

2.5 3.0 3.5 4.0 4.5 min

0

5

10mAUExtract-190nm,4nm (1.00)

Figure 4.5.a Peaks at 3.5 to 3.8 minutes showing azadirachtin concentration in the Botswana

Syringa sample

65

3.0 3.5 4.0 4.5 min

0

5

10mAUExtract-190nm,4nm (1.00)

Figure 4.5b. Peaks at 3.5 to 3.8 minutes showing azadirachtin concentration in the South Africa

Syringa sample

Although the amount of azadirachtin differed in Syringa obtained from the two locations,

the differences were not significant. Even though the level of azadirachtin concentration

was low in the Syringa tree, Syringa extracts have previously been reported to be quite

effective on a number of insect pests and has also been reported as containing a variety of

compounds which together with azadirachtin show insecticidal, anti-feedant, growth

regulating and development-modifying properties (Schmutterer, 1990; Mordue &

Blackwell, 1993; Carpinella et al., 2003; Nathan et al., 2005). Ascher et al. 1995) reported

that seed extracts of Syringa (M. azedarach) elicited a variety of effects in insects such as

growth retardation, reduced fecundity and moulting disorders.

4.3.5 Comparison of Neem and Syringa

The results show that there was a higher level of azadirachtin in the Neem samples than in

the Syringa samples. This indicates that it may not be azadirachtin which is responsible for

the efficacy of Syringa extracts in the control of insect pests. The study revealed that while

the geographical origin of Neem has an influence on its azadirachtin composition, Syringa

origin does not affect its composition.

66

More studies are needed though, to identify the different insecticidal chemical compounds

that are found in Syringa. The results revealed clearly that it is probably not azadirachtin

that is the most potent insecticidal compound in Syringa.

67

CHAPTER 5: Results of Neem and Syringa extracts treatments on red spider mites

5.1 Introduction

Bioassay investigations were carried out using different concentrations (0.1%, 1%, 10%,

20%, 50%, 75% and 100%) of Neem (Azadirachta indica) and Syringa (Melia azedarach)

to determine whether there was any significant differences in effectiveness between Neem

and Syringa against red spider mites at different growth stages at incubation periods of 24,

48 and 72 hours. The effectiveness of the Neem and Syringa extracts were also compared

against the conventional acaricides: Abamectin-plus (abamectin), Hunter (chlorphenapyr)

and Selecron (profenofos). In addition, a greenhouse trial was carried out to simulate field

conditions.

5.2 Experimental design

A completely randomized design was used for the experiments. An analysis of variance

(ANOVA) was used to test for differences between the 12 treatments (2 controls; 7

concentrations and 3 conventional acaricides). Differences in treatment means were

identified using Fisher’s protected t-test least significant difference (LSD) at the 1% level

of significance (Snedecor & Cochran, 1980). Data were analysed using the GenStat (2003)

statistical program.

The results of the analyses are provided in Tables 5.1 to 5.8 together with Figures 5.1 to

5.8 which present the results in graphical form.

68

Tables 5.1 to 5.3 and their corresponding graphs present the information about the death

rates of adult mites using Neem, Syringa and Syringa leaf extracts while Tables 5.4 and

5.5 depict the information on the nymphs mortality. Tables 5.6 and 5.7 provide data on

eggs, while Table 5.8 provides data on the greenhouse experiment. Data for Figures 1 to 8

is shown in the Appendices (Appendix A1 to A8).

5.2.1 Effect of Neem and Syringa extracts on adult mites

5.2.1.1 Neem: Adult mites

The results in Table 5.1 indicate that distilled water and the control with 0% Neem seed

extract (NSE) had no effect on the adult mites for the given periods of 24, 48 and 72

hours. Specifically, the effects of these two treatments were statistically negligible (0% of

dead adult mites) for the 72 hours of exposure. At 24 hours the 0.1%, of the NSE had the

same effect as the two controls. At 48 hours NSE (ranging from 0.1% to 75%), were

statistically similar (represented by the small letter “d”). However, these NSE effects were

statistically different (LSD > 21.09 at α = 0.01) and more effective than the two controls.

69

________________________________________________________________________ Table 5.1. Mean percentage mortalities of red spider mite adults (untransformed means) feeding on tomato leaves treated with Neem seed extracts at 24, 48 & 72 hours. __________________________________________________________________________

Treatment % mortality at 24 hours

% Mortality at 48 hours

% Mortality at 72 hours

Control Azad-0 0.0 (0.0) e 0.0 (0.0) e 0.0 (0.0) d Distilled water 0.0 (0.0) e 0.0 (0.0) e 0.0 (0.0) d NSE – 0.1 0.0 (0.0) e 31.54 (27.78)d 38.35 (30.56)d NSE – 1 33.2 (30.6) d 46.6 (52.8) d 54.8 (66.7) c NSE – 10 35.0 (33.3) d 49.9 (58.3) d 58.5 (72.2) c NSE – 20 36.8 (36.1) d 51.4 (61.1) d 58.6 (72.2) c NSE – 50 38.6 (38.9) d 56.5 (69.4) cd 90.0 (100.0) a NSE – 75 41.8 (44.4) cd 62.0 (77.8) bcd 90.0 (100.0) a NSE – 100 59.8 (72.2) bc 78.2 (88.9) ab 90.0 (100.0) a Abamectin - 0.6 ml/liter 90.0 (100.0) a 90.0 (100.0) a 90.0 (100.0) a Hunter - 0.4 ml/liter 90.0 (100.0) a 90.0 (100.0) a 90.0 (100.0) a Selecron – 3 ml/liter 66.5 (77.8) b 73.9 (88.9) abc 90.0 (100.0) a SEM 5.33 4.92 3.02 F probability <0.001 <0.001 <0.001 LSD (1%) 21.09 19.46 11.93

CV% 21.1 15.6 8.0 Note: SEM is the standard error of the mean. Values in parenthesis: Percentage mortalities

LSD is the t-test least significant difference at the 1% level. Means within columns followed by the same lower case letter did not differ significantly at the 1% level. CV% is the coefficient of variation of each experiment.

In addition, the Neem seed extracts at 100% was similar to that of Neem seed extract

concentrations of 75% at 72 hours but superior to the treatment using lower concentrations

(small letter c) at 24 and 72 hour exposure times. The second column in Table 5.1 also

indicates that the effects of the two conventional acaricides, Abamectin-plus 0.6 mℓ/ℓ and

Hunter 0.4 mℓ/ℓ, had the same effects at 24 hours on adult mites but were statistically

different from the other treatments for the same time period.

70

The third conventional acaricide, Selecron, had a significantly similar effect as the Neem

seed extracts at 100% after 24and 48 hours and both showed a weaker effect in

comparison to Abamectin-plus and Hunter. The picture emerging after 48 hours of

treatment (third column, Table 5.1) is that various concentration levels of the Neem seed

extracts (up to 75%) had the same effect “d” on the treatment of adult mites. Something

worth noting, however, is that although the treatment with these concentrations produced

the same effects “d”, the percentage mean values of dead adult mites for concentrations of

50% and 75% were above 55% and were numerically higher than those of 0.1%, 1%, 10%

and 20%. They also had a significantly similar effect “c” as the conventional acaricide

Selecron at 3 mℓ/ℓ.

Further, at 48 hours of treatment, there was no significant difference between the

treatments with the Neem seed extracts at 100% and the three conventional acaricides

(Abamectin-plus, Hunter, Selecron (shown by letter “a”). The performance of 50% Neem

seed extracts at 72 hours was as good as any of the higher concentrations (75% and 100%)

as well as the three conventional acaricides. On the whole, there is more variability

(heterogeneity) among the treatments used at 24 hours as indicated by the coefficient of

variation (CV). Variability decreased as the duration of the treatment continued: 24hrs

(21.1); 48hrs (15.6); 72hrs (8.0). It was, however, interesting to note that at 48 hours

exposure, treatment with 0.1% Neem seed extracts had similar effects as concentrations

50% and 75% (“d”). Figure 5.1 shows the values of the dead spider mites in percentage

form, including their standard deviations (error bars) of the set of readings for each

experiment at the three time periods (24 hours, 48 hours and 78 hours).

71

The data used for graphs in this chapter are given in parenthesis from Table 5.1 to Table

5. 8, and also in Appendices 1A to Appendix 1H, together with their standard errors. The

line graph indicates that for each chemical used in the treatment, the number of dead mites

increased with the increase in the number of hours of treatment. That is, the trend lines

were in the expected order with 24 hours at the bottom and 72 hours at the top. The

treatments with Abamectin-plus and Hunter indicated that all adult mites were killed

within the first 24 hours of treatment. The same effect was achieved by exposing the adult

mites to NSE 50%, 75%, 100% and Selecron for 72 hours.

Figure 5.1: The percentage mortalities of adult mites on tomato leaves treated with different concentrations of Neem seed extracts.

72

5.2.1.2. Syringa seeds: Adult Mites Table 5.2 shows the treatments using the Syringa (Melia azedarach) seed extracts (SSE).

The results in Table 5.2 have a similar pattern to those in Table 5.1. The effects of the

treatments on adult mites using distilled water and 0% Melia azedarach concentration are

statistically negligible at all time periods (24 hours, 48 hours and 72 hours).

________________________________________________________________________ Table 5.2. Mean percentage mortalities of red spider mite adults (untransformed means) feeding on tomato leaves treated with Syringa seed extracts at 24, 48 & 72 hours ________________________________________________________________________

Note: SEM is the standard error of the mean. Values in parenthesis: Percentage mortalities

LSD is the t-test least significant difference at the 1% level. Means within columns followed by the same lower case letter did not differ significantly at the 1% level. CV% is the coefficient of variation of each experiment.

Treatment

Perc. mortality at 24 hours

Perc. mortality at 48 hours

Perc. mortality at 72 hours

Control M.azedar - 0 (0.0) e (0.0) e (0.0) e Distilled water (0.0) e (0.0) e (0.0) e M.azedarach – 0.1 31.4 (27.8) d 46.6 (52.8) d 48.3 (55.6) c M.azedarach – 1 31.5 (27.8) d 50.0(58.3) cd 76.4 (91.7) b M.azedarach – 10 46.6 (52.8) cd 60.0(75.0)bcd 84.4 (97.2) ab M.azedarach – 20 49.9 (58.3) cd 70.2 (83.3) abc 90.0 (100.0) a M.azedarach – 50 49.9 (58.3) cd 73.9(88.9) ab 90.0 (100.0) a M.azedarach – 75 62.0 (77.8) bc 74.4(88.9) ab 90.0(100.0) a M.azedarach – 100 84.4 (97.2) a 90.0(100.0) a 90.0 (100.0) a Abamectin - 0.6ml/liter 90.0 (100.0) a 90.0(100.0) a 90.0 (100.0) a Hunter – 0.4ml/liter 78.2 (88.9) ab 90.0(100.0) a 90.0 (100.0) a Selecron – 3ml/liter 41.8 (44.4) d 90.0(100.0) a 90.0 (100.0) a SEM 4.87 5.40 2.89 F probability <0.001 <0.001 <0.001 LSD (1%) 19.28 21.34 11.44 CV% 17.9 15.2 7.2

73

At 24 hours, the percentage of dead adult mites increased with increases in the

concentration of the SSE. Statistically, there was no difference in the percentage of dead

adult mites when using treatments of 0.1%, 1%, 10%, 20% and 50% or Selecron

(identified by letter “d”). SSE at 100% performed the same as Abamectin-plus and Hunter

(letter “a”) and better than the lower concentrations of SSE.

Worthy of note is that the treatment with Hunter has a significantly similar effect (though

numerically higher) as the treatment with Syringa seed extracts at 75%. At 48 hours, the

treatment with 20% SSE had similar effects as any of the higher concentrations including

the three conventional acaricides (Abamectin-plus, Hunter and Selecron).

The effect of 100% Syringa extracts on adult mites is statistically the same as the effects

of Abamectin-plus and Hunter at all periods (24 hours, 48 hours and 72 hours) while for

Neem, the effects were statistically equivalent from 48 hours onwards. The graph

depicting the effect of Syringa extracts on adult mites is presented in Figure 5.2. Once

again the order of the trend lines for the three periods (24 hours, 48 hours and 72 hours)

was as expected with lower effects at 24 hour exposure. When comparing the effects of

treatments with different concentration levels of Neem with those of Syringa, the Syringa

treatment seemed to perform better than that of Neem in several ways. At 48 hours, the

Syringa treatments at low concentrations (20%) worked equally well as any of the higher

concentrations (including the conventional acaricides). With Neem, that same effect was

achieved only when using a concentration of 50% and at a longer period of 72 hours of

exposure. In addition, the treatments with Syringa extracts produced more homogenous

results (CV) than those with Neem which had higher values of the coefficient of variation.

74

Figure 5.2: The percentage mortalities of adult mites on tomato leaves treated with different

concentrations of Syringa seed extracts 5.2.1.3 Syringa leaves: Adult mites This leaf dip bioassay was done to establish if the Syringa leaves could also be used to

control mites (Table 5.3), since earlier research had indicated that Syringa leaf extracts

were effective in the control of the potato tuber moth, Phthorimaea operculella (Zeller)

(Visser, 2005).

75

_________________________________________________________________________ Table 5.3. Mean percentage mortalities of red spider mite adults (untransformed means) feeding on tomato leaves treated with Syringa leaf extracts at 24, 48 & 72 hours _________________________________________________________________________

Treatment

% mortality at 24 hours

% mortality at 48 hours

% mortality at 72 hours

Control M.azedar - 0 0.0 (0.0) f 0.0 (0.0) e 0.0 (0.0) e Distilled water 0.0 (0.0) f 0.0 (0.0) e 0.0 (0.0) e M.azedarach – 0.1 38.4 (38.9) e 48.3 (55.6)d 54.8 (66.7)cd M.azedarach – 1 45.0 (50.0) de 49.8 (58.3)d 60.2 (75.0)cd M.azedarach – 10 46.6 (52.8) cde 53.1 (63.9)cd 63.9 (80.6)cd M.azedarach – 20 51.5 (61.1) bcd 53.3(63.9) cd 63.9 (80.6) cd M.azedarach – 50 53.1 (63.9) bcd 60.2(75.0) bc 72.0 (86.1) bc M.azedarach – 75 58.6 (72.2) bc 66.4 (83.3) b 90.0 (100.0) a M.azedarach – 100 84.4 (97.2) a 90.0(100.0) a 90.0 (100.0) a Abamectin- 0.6ml/liter 90.0 (100.0) a 90.0(100.0) a 90.0 (100.0) a Hunter – 0.4ml/liter 90.0 (100.0) a 90.0(100.0) a 90.0 (100.0) a Selecron – 3ml/liter 60.2 (75.0) b 63.9 (80.6) b 84.4 (97.2) ab SEM 3.19 2.40 3.44 F probability <0.001 <0.001 <0.001 LSD (1%) 12.63 9.49 13.62 CV% 10.7 7.5 9.4

Note: SEM is the standard error of the mean. Values in parenthesis: Percentage mortalities

LSD is the t-test least significant difference at the 1% level. Means within columns followed by the same lower case letter did not differ significantly at the 1% level. CV% is the coefficient of variation of each experiment.

Distilled water and the control (M. Azedarach-0) had no effect on the lives of adult mites

for the three different time periods of exposure to treatment. At 24 hours, the mean

percentage dead mites increased steadily with increases in crushed Syringa leaf

concentration levels. At 48 hours, crushed Syringa leaf at 50% and 75% had statistically

the same effect as the conventional acaricide, Selecron. The 100% crushed Syringa leaf

extracts performed as well as Abamectin-plus and Hunter for all time periods (24 hours,

48 hours and 72 hours). The visual representation is given in Figure 5.3.

76

Figure 5.3: The percentage mortalities of adult mites on tomato leaves treated with different

concentrations of Syringa leaf extracts

When comparing the graphs representing dead adult mites using the extracts of Neem,

Syringa seed extracts and Syringa leaves, Syringa shows more stable results and in the

expected direction, e.g. higher mortalities with higher dosages. However, the Syringa

seed extracts were found to have superior acaricide properties to Syringa leaf extracts

which were more effective than Neem seed extracts.

5.3 The effect of Neem and Syringa on nymphs

Leaf dip bioassays were carried out to determine the effect of Neem and Syringa extracts on mite nymphs.

77

5.3.1 Neem: Mite nymphs

__________________________________________________________________ Table 5.4. Mean percentage mortality of red spider mite nymphs (untransformed means) feeding on tomato leaves treated with Neem seed extracts at 24, 48 & 72 hours _________________________________________________________________

Treatment

% mortality at 24 hours

% mortality at 48 hours

% mortality at 72 hours

Control Azadr – 0 0.0 (0.0) e 0.0 (0.0) d 0.0 (0.0) c Distilled water 0.0 (0.0) e 0.0 (0.0) d 0.0 (0.0) c NSE – 0.1 48.2 (55.6) d 58.5 (72.2) c 76.4 (91.7) b NSE – 1 49.8 (58.3) d 70.8 (88.9) b 84.4(97.2) ab NSE – 10 54.8(66.7) cd 70.8 (88.9) b 90.0(100.0) a NSE – 20 56.6(69.4) cd 84.4 (97.2) a 90.0(100.0) a NSE – 50 66.4(83.3) bc 90.0(100.0) a 90.0(100.0) a NSE – 75 76.4 (91.7) ab 90.0(100.0) a 90.0 (10.0) a NSE – 100 84.4 (97.2) a 90.0(100.0) a 90.0 (100.0) a Abamectin - 0.6 ml/liter 90.0 (100.0) a 90.0(100.0) a 90.0 (100.0) a Hunter – 0.4 ml/liter 90.0 (100.0) a 90.0(100.0) a 90.0 (100.) a Selecron – 3 ml/liter 41.3 (44.4) d 58.5 (72.2) c 90.0 (100.0) a SEM 4.18 2.43 2.62 F probability <0.001 <0.001 <0.001 LSD (1%) 16.51 9.62 10.35 CV% 13.2 6.4 6.2

Note: SEM is the standard error of the mean. Values in parenthesis: Percentage mortalities

LSD is the t-test least significant difference at the 1% level. Means within columns followed by the same lower case letter did not differ significantly at the 1% level. CV% is the coefficient of variation of each experiment.

Table 5.4 revealed that the effect of distilled water was statistically similar to that of the

control for the three periods. At 24 hours, the treatment with Selecron had the same effect

as the treatment with 0.1%, 1%, 10% and 20% NSE, while treatments with 75% and 100%

NSE were statistically similar to that with the conventional acaricides Abamectin-plus and

Hunter.

78

At 48 hours, the average percent of dead nymphs, when treated with Selecron, was

statistically similar as when treated with 0.1% to 10% NSE and these treatment effects

were statistically inferior to the treatment with 20% NSE which had significantly similar

effects as the higher concentrations of 50%, 75%, and 100% NSE and the two

conventional acaricides (Abamectin-plus and Hunter). The data in Table 5.4 is also

portrayed graphically in Figure 5.4.

The trend lines for 24 hours, 48 hours and 72 hours were in the expected order with the 24

hour trend line at the bottom and 72 hours at the top. This indicated that more nymphs

died at longer periods of exposure and with increase in the levels of percentage Neem seed

extract (NSE) concentrations. The trend lines for 48 hours and 72 hours showed an

overlap at concentration levels of 50%, 75%, 100% NSE, and the two acaricides

(Abamectin-plus and Hunter). The same average number of nymphs died at those

concentration levels. Specifically, all the nymphs were dead at the above concentrations

(50%, 75% and 100%) NSE, at periods 48 hours and 72 hours. The bars (standard

deviations) showed a variation of dead nymphs at 24 hours, indicating that the number of

dead nymphs varied in each of the repeated experiments for the same period and

concentration level.

79

Figure 5.4: The percentage mortalities of mite nymphs on tomato leaves treated with different concentrations of Neem Seed extracts

5.3.2 Syringa: Nymph mites

__________________________________________________________________________

Table 5.5 Mean percentage mortalities of red spider mite nymphs (untransformed means) feeding on tomato leaves treated with Syringa seed extracts at 24, 48 & 72 hours

________________________________________________________________________

Treatment

% mortality at 24 hours

% mortality at 48 Hours

% mortality at 72 Hours

Control M.azeda - 0 0.00 (0.00)e 0.00 (0.00)e 0.00 (0.00)b Dist.Water 0.00 (0.00)e 0.00 (0.00)e 0.00 (0.00)b M.azedarach – 0.1 43.35 (47.22)d 54.84 (66.67)d 71.97 (86.67)a M.azedarach – 1 48.20 (55.56)d 56.49 (69.44)d 73.94 (88.11)a M.azedarach – 10 52.60 (60.33)d 60.21 (75.00)cd 76.38 (91.67)a M.azedarach – 20 60.21 (75.00)c 63.94 (80.56)bc 82.38 (94.83)a M.azedarach – 50 68.34 (86.11)bc 90.00 (100.00)a 90.00 (100.00)a M.azedarach – 75 70.78 (88.89)b 90.00 (100.00)a 90.00 (100.00)a M.azedarach – 100 84.41 (97.22)a 90.00 (100.00)a 90.00 (100.00)a Abamectin 90.00 (100.00)a 90.00 (100.00)a 90.00 (100.00)a Hunter 90.00 (100.00)a 90.00 (100.00)a 90.00 (100.00)a Selecron 61.97 (77.78)bc 68.34 (86.11)b 84.41 (97.22a SEM 2.60 1.63 4.85 F probability <0.001 <0.001 <0.001 LSD (1%) 10.27 6.46 19.17 CV% 8.1 4.5 12.1

80

Note: SEM is the standard error of the mean. Values in parenthesis: Percentage mortalities

LSD is the t-test least significant difference at the 1% level. Means within columns followed by the same lower case letter did not differ significantly at the 1% level. CV% is the coefficient of variation of each experiment.

Treatments with distilled water gave the same results as the control where nothing was

applied at all three periods. At 24 hours, the treatment with 100% was significantly similar

to treatments with Abamectin-plus and Hunter and performed better than the other

treatments. At 48 hours of exposure, 50% of the Syringa extract was as effective as all the

above concentrations and the conventional acaricides Abamectin-plus and Hunter but was

superior to Selecron. At 72 hours of exposure, even 0.1% of the Syringa extract was as

effective as the conventional acaricides.

Figure 5.5 shows the effect of the Syringa seed extracts on nymphs at three periods of 24

hours, 48 hours and 72 hours. The trend lines indicate that more nymphs died with

increasing concentrations and with increasing periods of exposure. The treatment with

SSE (50%), had significantly similar effect as treatments with higher concentrations of

SSE (75% and 100%) and as Abamectin-plus and Hunter at 48 and 72 hours of exposure.

Although not significant, Selecron seemed to be less effective compared to SSE (50% to

100%) and the other two acaricides.

The effect of the Syringa seed extracts had a similar trend as that of Neem seed extracts. In

particular, more nymphs died with longer periods of exposure to the treatments and with

increases in concentration levels.

81

The standard deviations were smaller (shorter bars) compared to those with Neem as

treatments. When replicating the experiments for each treatment, the number of dead

nymphs was almost the same, making the results more reliable.

Figure 5.5: The percentage mortalities of mite nymphs on tomato leaves treated with

different concentrations of Syringa Seed extracts

5.4 Effect of Neem and Syringa seed extracts on eggs

A leaf dip bioassay was carried out to determine the effect of Neem and Syringa seed

extracts on mite eggs.

5.4.1 Neem: Mite eggs Table 5.6 shows the percentage of eggs (N = 18) that hatched after exposure to different

concentrations of NSE.

82

_______________________________________________________________________ Table 5.6. Mean percentage red spider mite eggs that hatched after 48 & 72 hours exposure to Neem seed extracts. Eighteen eggs were used for each test. The means were untransformed but an angular transformation was used to normalise percentages

____________________________________________________________________

Treatment

% egg hatch at 48 hours

% egg hatch at 72 hours

Control -NSE- 0 39.53 (40.74) a 61.11 (68.52) a Distilled water 32.18 (29.36)ab 45.09 (50.00) a NSE – 0.1 31.34 (27.78) ab 43.94 (48.15) ab NSE – 1 30.51 (25.93) ab 39.38 (40.74) ab NSE – 10 27.62 (22.22) ab 36.69 (37.04) ab NSE – 20 23.90 (16.67)abc 29.80 (25.93) abc NSE – 50 21.95 (16.67)abc 28.85 (24.07) abc NSE – 75 6.49 (3.70) bc 15.87 (11.11) bc NSE -100 0.00 (0.00)c 0.00 (0.00)c Abamectin -0.6 ml/liter 13.92 (9.26) bc 13.92 (9.26) bc Hunter – 0.4 ml/liter 11.75 (11.11) bc 29.28 (27.78) abc Selecron – 3 ml/liter 39.38 (40.74) a 45.16 (50.00) ab SEM 6.50 9.17 F probability 0.003 0.007 LSD (1%) 25.70 36.29 CV% 48.5 49.0

Note: SEM is the standard error of the mean. Values in parenthesis: Percentage hatched eggs

LSD is the t-test least significant difference at the 1% level. Means within columns followed by the same lower case letter did not differ significantly at the 1% level. CV% is the coefficient of variation of each experiment.

The column for the 24 hour period was omitted from the table because no mortalities were

observed at this exposure period. Although each of the chemicals had a deterring effect

on the hatching of eggs, the only concentration that ensured that no eggs hatched was

100% NSE. At 48 hours, the only treatments that performed statistically better than

distilled water were 75%, 100% NSE, Abamectin-plus and Hunter. The effect of the

treatments on the hatching of eggs is much clearer when depicted in graphical form as

shown in Figure 5.6.

83

Figure 5.6: Percentage hatched eggs of mites on tomato leaves treated with different concentrations of Neem seed extracts

Mite eggs normally hatch two to three days after being laid (Bolland & Valla, 2000;

Knapp et al., 2003). In this study however, no eggs hatched at 24 hours with any of the

treatments, except Selecron. On the whole, the average number of hatched eggs decreased

with increases in NSE concentrations from 10% upwards; and treatments with 100% NSE

produced the best results of all the treatments.

5.4.2 Syringa: Mite eggs Table 5.6 shows the percentage of eggs (N = 18) that hatched after exposure to different

concentrations of SSE.

84

________________________________________________________________________________ Table 5.7 Mean percentage red spider mites eggs that hatched after 48 & 72 hours exposure to Syringa seed extracts. 18 eggs were used for each test. The means were untransformed but an. Angular transformation was used to normalise percentages. __________________________________________________________________

Treatment

% Eggs hatched at 48 hours

% Eggs hatched at 72 hours

Control – M.azedar - 0 31.34 (27.78) a 71.52 (83.33) a Distilled water 26.28 (20.37) ab 62.62 (77.78) ab M.azedarach – 0.1 24.09 (16.67) ab 38.34 (38.89) abc M.azedarach – 1 19.07 (11.11)abc 31.66 (29.63) bc M.azedarach – 10 4.54 (1.85) bc 13.63 (5.56) c M.azedarach – 20 4.54 (1.85) bc 11.75 (11.11) c M.azedarach – 50 4.54 (1.85) bc 11.03 (5.56) c M.azedarach – 75 4.54 (1.85) bc 8.03 (5.56) c M.azedarach – 100 4.54 (1.85) c 0.00 (0.00) c Abamectin - 0.6 ml/liter 10.60 (9.26) abc 12.86 (12.96) c Hunter – 0.4 ml/liter 4.54 (1.85) bc 11.03 (5.56) c Selecron – 3 ml/liter 19.54 (18.52) abc 21.49 (20.37) c SEM 6.00 8.91 F probability 0.006 <0.001 LSD (1%) 23.74 35.23 CV% 83.7 62.0

Note: SEM is the standard error of the mean. Values in parenthesis: Percentage hatched eggs

LSD is the t-test least significant difference at the 1% level. Means within columns followed by the same lower case letter did not differ significantly at the 1% level. CV% is the coefficient of variation of each experiment.

Table 5.7 shows the results of the treatments using Syringa (Melia azedarach) seed

extracts. The column for the 24 hour period was omitted from the table because no

mortalities were observed in the eggs at this exposure period. At 48 hours 0%, 0.1% and

1% SSE had no significant effects (letter “a”) on the hatching of eggs similar to the

treatments with distilled water.

85

At 48 hours of exposure distilled water had the same effect on hatched eggs as that of the

SSE treatments of 0.1%, 1%, 20%, 50%, 75% and the three acaricides (letter “b”).

However, at 72 hours, distilled water was only similar to that of 0.1% and 1% SSE. The

treatment at 0.1% had the same effect on hatched eggs after 72 hours of exposure as those

of higher concentrations and the three acaricides (Figure 5.7 and Appendix A). The trend

lines indicate that for each treatment the average number of hatched eggs increased with

increased hours of exposure to the treatment - more eggs hatched at 72 hours exposure

than at 48 hours. It is, however, difficult to compare between the two exposure periods (48

hours and 72 hours) since mite eggs were not at the same stage of development. The

highest percentage of hatched eggs is observed under the treatment with distilled water at

72 hours of exposure, and the lowest is observed under treatment with SSE 100% at the

same time period (72 hrs).

Figure 5.7: Percentage hatched eggs of mites on tomato leaves treated with different concentrations of Syringa seed extracts

86

5.5. The greenhouse trial The greenhouse trial simulated field conditions. Table 5.8 shows the results of the

greenhouse trials with the Neem and Syringa extracts and the two acaricides. Fifty percent

concentrations were used for the Neem and Syringa extracts because this concentration

showed stable results which compared effectively with the conventional acaricides.

Selecron was omitted because it did not perform well in the bioassay results. In the

greenhouse trials, red spider mites were exposed to the two botanicals and the acaricides

for 24 hours, 48 hours and 72 hours.

_______________________________________________________________________________________ Table 5.8. Mean percentage mortalities of red spider mite adults (untransformed means) feeding on tomato leaves treated with Neem and Syringa seed extracts at 24, 48 & 72 hours in the greenhouse _______________________________________________________________

Note: SEM is the standard error of the mean. Values in parenthesis: Percentage mortalities

LSD is the t-test least significant difference at the 1% level. Means within columns followed by the same lower case letter did not differ significantly at the 1% level. CV% is the coefficient of variation of each experiment.

Treatment % mortality at 24 hours

% mortality at 48 hours

% mortality at 72 hours

Control 16.91 (9.12)e 19.21 (10.26)c 18.26 (10.98)d Water 28.27 (22.59)c 30.31 (25.76)d 34.52 (33.08)c Neem 50% 41.06 (43.22)bc 42.60 (45.84)c 47.20 (53.77) bc Syringa 50% 44.22 (48.64)abc 50.03 (58.73)bc 57.37 (70.59)abc Abamectin 54.70 (66.18)ab 57.88 (78.17)ab 64.36 (82.69)a Hunter 57.02 (69.97)a 62.38 (71.49)a 66.00 (80.66)a SEM 3.86 2.52 2.87 F probability <0.001 <0.001 <0.001 LSD (1%) 15.70 10.28 11.67 CV% 18.5 11.6 12.2

87

As a whole, it was observed that at 50%, Syringa performed numerically better than Neem

and had significantly similar effects as Abamectin-plus and Hunter at 24 hours and 72

hours (letter “a”).

Although the effect of Neem (50%) and Syringa (50%) on adult mites was significantly

similar “c” at all three time periods, Syringa 50% had an added advantage in that its

average effect also fell in the same statistical category as Abamectin-plus and Hunter

(letter “a”). The greenhouse experiment confirmed to some extent what the bioassays had

revealed. The order of the average number of dead mites was as expected with lower

average numbers at 24 hours, higher at 48 hours and highest at 72 hours.

Figure 5.8. The percentage mortalities of adult mites on tomato leaves treated with 50% concentration of Neem and Syringa seed extracts

88

CHAPTER 6. Discussion and comparison of efficacy of Neem and Syringa on red spider mite infestations

6.1 Introduction

This chapter gives an overview of the aim of this study which was to explore the

effectiveness and eventual use of the Neem and Syringa extracts in the control of red

spider mites on tomatoes. It also discusses the HPLC techniques and results, the Bioassay

results and the Greenhouse treatments of the Neem and Syringa seed extracts (NSE and

SSE) on the red spider mite life stages (eggs, nymphs and adult) using tomato leaves as

substrate.

6.1.1 High Performance liquid chromatography (HPLC)

An HPLC was carried out to determine the level of azadirachtin compound in Neem and

Syringa which was showed to be responsible for the anti-feedant and growth reduction on

a number of insect pests (Kumar & Sannaveerappanar, 2004).

The results in the Neem samples showed that azadirachtin composition levels varied

according to Neem plants grown in different locations. This is clearly shown in Chapter 4

from the figures of the Neem samples obtained from India, Zambia and Botswana. The

results revealed that there were significant differences between trees from different

ecotypes which support the findings of Kumar et al. (2000). The results of the Syringa

samples revealed that there is a negligible content of azadirachtin in both samples as

shown in Chapter 4.

89

This shows that with Syringa, geographical differences will have no impact on the amount

of azadirachtin while with Neem, geographical differences tend to influence the amount of

azadirachtin. This is an important point because it means that Syringa trees from any

geographical location can be used in extracting compounds to be used in the control of

pests assuming that acaricide tests were found to be positive.

6.1.2. Neem and Syringa seed extracts

Laboratory bioassays were conducted to evaluate the efficacy of Neem and Syringa seed

and Syringa leaf extracts against red spider mites (RSM) on tomatoes. All treatments were

compared with the synthetic acaricides Abamectin-plus, Hunter and Selecron, which are

commonly used in the country (South Africa / Botswana) for control of red spider mites.

6.1.2.1 Neem seed extracts results

Laboratory bioassays were carried out on adult mites, nymph mites and mite eggs to

determine the effectiveness of Neem seed extracts on them.

6.1.2.1.1. NSE results with adult red spider mites Laboratory bioassays with adult red spider mites showed that the effect of 100% NSE was

similar to that of 75%. Efficacy increased with time after application. It was observed that

at 100% NSE, tomato plant leaves showed phytotoxicity with dark discoloration,

especially on the younger leaves. This level of phytotoxicity could be harmful to the plant

by reducing photosynthesis. It would therefore not be advisable to use a 100% NSE for the

control of red spider mites.

90

It is also interesting to note that at 72 hours of mite exposure to Neem concentrations, 50%

NSE application was able to control red spider mites just as effectively as the two

conventional acaricides, Abamectin-plus at 0.6 mls/l and Hunter at 0.4 mls/l.

6.1.2.3 NSE results with nymphs

The bioassay results using nymphs revealed that NSE was more effective when applied to

red spider mite nymphs (in relation to SSE). The results revealed that all nymphs died at

concentrations of 50%, 75% and 100%, at periods 48 hours and 72 hours. This indicates

that Neem is more effective against nymphs than against adult mites. This is important

since both adult and nymphs of the red spider mites feed by sucking sap from mature

leaves as reported by Lu and Wang (2005). In view of the seriousness of the red spider

mites infestation on tomatoes (Knapp et al., 2003), controlling mites at the nymph stage

would yield best results and prevent them from further multiplication.

6.1.2.4. NSE results with eggs

At 24 hours no eggs hatched with any of the treatments except when using the

conventional acaricide Selecron. At 48 hours the average number of hatched eggs

decreased with increases in NSE concentrations from a concentration of 10% onwards;

and treatments with 75% and 100% NSE produced the best results. It is, however,

unfortunate that a 100% NSE treatment cannot be used due to phytotoxicity to the tomato

leaves.

91

6.1.3 Syringa seed extracts (SSE) and crushed Syringa leaves results

Bioassays were carried out on adult mites, nymph mites and mite eggs to determine the

effectiveness of Syringa seed extracts on them.

6.1.3.1 SSE and crushed Syringa leaves: adult red spider mites results

The bioassay results for Syringa applications were different from those for Neem. At 48

hours, 20% SSE was able to control red spider mites as were all of the three conventional

acaricides (Abamectin-plus, Hunter and Selecron), while NSE was only able to compare

favourably with the conventional acaricides at 50% concentration after 72 hours of

exposure. It was also observed that at 72 hours, a 10% Syringa extract treatment was as

effective as the commercial acaricides. The effect of 100% Syringa extracts on adult mites

was statistically the same as the effects of Abamectin-plus and Hunter at all periods (24

hours, 48 hours and 72 hours).

However, just as with Neem, 100% SSE had the same level of phytotoxicity to the tomato

plant leaves. It is evident that the level of azadirachtin in SSE is lower than that of NSE.

This seems to suggest that NSE would perform better than SSE. However, this was not the

case. One reason could be the presence of other compounds such as meliacin and

meliacarpin in SSE (Juan & Sans, 2000). These, together with azadirachtin are able to kill

red spider mite adults and nymphs. The results with crushed Syringa leaves showed some

similarity with SSE. After 24 hours of exposure, 20% crushed Syringa leaf extracts were

able to control red spider mites and compared well with 50% and 75% crushed Syringa

leaf extracts as well as conventional acaricides.

92

This showed clearly that SSE and Syringa leaves could be used in the control of red spider

mites as an alternative to conventional acaricides and may even be more effective than

Neem.

6.1.3.2 Nymphs: Syringa seed extracts results

The bioassay results revealed that after 24 hours of exposure, 20% SSE was able to kill the

same number of nymphs as 50% SSE. 50% SSE killed a similar number of nymphs as did

the conventional acaricides (Abamectin-plus and Hunter). The results also revealed that all

nymphs were killed at a concentration of 50% SSE.

6.1.3.3 Eggs: Syringa seed extracts results

The results showed that 20% SSE was able to prevent 88% of mite eggs from hatching

after 48 hours of exposure. It was also noticed that some eggs do not hatch at 10% SSE. It

is, however, clear that SSE are effective in preventing mite eggs from hatching. This

indicated that using Syringa, red spider mites could be controlled as early as at the egg

stage of their development. Earlier reports stated that Dicofol at 0.5 kg per 500L was able

to stop or reduce mite eggs from hatching with great success (Chapman & Martin, 2003).

However, these conventional acaricides have side effects (Ngugi et al., 1990). This study

has revealed that SSE is more effective than Syringa leaf extracts and NSE. It was

observed that Syringa leaf extracts were more effective than NSE. It is clear however that

the superiority of SSE is probably not due to azadirachtin but rather due to other chemical

compounds present in Syringa.

93

6.1.4 Greenhouse trials

Greenhouse trials were conducted to simulate field conditions and to compare them with

the laboratory bioassays. Fifty percent of both the Neem and Syringa extracts was used in

the treatments because this concentration showed stable results in the bioassays which

compared well with the conventional acaricides. For synthetic acaricides, Selecron was

left out as it did not compare effectively with the botanicals or with other acaricides.

94

6.2. Summary

This study has revealed that botanicals could be used to reduce the number of red spider

mite eggs hatching, with similar effects as conventional acaricides. It has been reported

that pests can develop resistance to conventional pesticides, resulting in higher dosages

being used (Lin et al., 2005). However, botanicals have the advantage in that they contain

a cocktail of compounds which may significantly reduce the chances of tolerance or

resistance build-up by mites (Thacker, 2002). These results suggested that botanicals can

be used to effectively control red spider mites and can be a good substitute for

conventional acaricides. The fact that they were able to prevent mite eggs from hatching

makes them more useful and important as preventive acaricides. The study has also

revealed that botanical extracts such as azadirachtin in Neem and Syringa can be used as

alternatives to these conventional acaricides with great success.

It is clear from the literature that the long-term use of synthetic chemicals poses potential

environmental risks by killing beneficial insects and destroying the ecological balance

between pests and their natural enemies (Levent et al., 2005). Some botanical extracts, on

the other hand, are also said to be of low toxicity to insects such as bees, butterflies and

natural predators of pests (Mordue & Blackwell, 1993). This study has also revealed that

the azadirachtin in Syringa is very low and did not differ significantly between the two

sources of origin as shown in Chapter 4. Thus Syringa extracts could be taken from any

Syringa tree regardless of origin. This is important especially for the subsistence farmer

who will be able to produce their own acaricides from backyard trees to control red spider

mites (Bues et al., 2003; Bok et al., 2006).

95

6.3 Conclusions

From the literature, it has been shown that red spider mites are the most prevalent pests on

tomatoes in Botswana (Bok et al., 2006). The ability of the Neem and Syringa extracts to

prevent red spider mite eggs from hatching and to control red spider mites at the nymph

stage makes these botanicals effective acaricides in the prevention and control of this

major pest.

Red spider mites can thus be effectively controlled on tomatoes by spraying Neem and

Syringa seed extracts as acaricides. The advantage of using Neem and Syringa is that no

special equipment is required to manufacture the seed extracts. Syringa is easy to acquire

as it grows easily in many types of soil and different environments. Neem is also easy to

acquire as it is grown in many parts of the tropical regions. Considering the red spider

mite’s short life cycle, which may be as short as a week, especially during hot and dry

seasons, a weekly application is recommended. Since red spider mites are commonly

found on the underside of the leaf surfaces, thorough applications should be done - all

parts of the plant, as well as the underside of leaves should be treated.

Botswana and other southern African countries could save millions of dollars on

expensive acaricides. The introduction of easily home-made Neem and Syringa extracts as

organic agricultural chemicals by using locally available facilities would be a tremendous

help to many tomato growers. Neem and Syringa as a botanical source of acaricides could

play a significant role in reducing the indiscriminate use of synthetic acaricides or

insecticides which are potentially dangerous to man and the environment.

96

The adverse effects on non-target species, the development of insecticide resistance and

consequent pest resurgence, pesticide residues in both soils and crops are all potential

concerns that may be eliminated when using botanical pesticides.

The introduction and success of botanical pesticides to Botswana resource-poor farmers

will improve food production and bring in food export opportunities. This study therefore

was aimed at exploring the potential of Neem (Azadirachta indica A. Juss) and Syringa

(Melia azedarach L.) extracts as alternatives to conventional acaricides in the control of

red spider mites, (Tetranychus species).

6.4 Recommendations

• Further studies are needed to determine the efficacy of Neem leaf and the Syringa

bark extracts against red spider mites (RSM).

• It would be worthwhile to test the efficacy of the Neem or Syringa seed extracts

against important fungal diseases.

• The effects of Neem and Syringa extracts on natural enemies should be

investigated.

• It would be interesting to isolate and determine the acaricidal effects of other

chemical compounds in Syringa.

• It is important that farmers are informed about the problems of RSM and how to

control them. It is therefore recommended that a protocol for control, including the

ecology of the pest, be compiled in the future.

97

7. APPENDICES

Appendix 1 A

Table 5.1: The percentage mortality of adult mites on tomato leaves treated with different concentrations of Neem seed extracts .

% St Dev

Neem

24 hours

48 hours

72 hours

24 hours

48 hours

72 hours

Control 0 0 0 0.00 0.00 0.00 Water 100ml 0 0 0 0.00 0.00 0.00 Azadrachtin0.1% 0 27.78 30.56 0.00 16.67 9.62 Azadrachtin 1% 30.56 58.33 66.67 12.73 14.43 8.33 Azadrachtin 10% 33.33 61.11 72.22 14.43 4.81 9.62 Azadrachtin 20% 38.89 52.78 72.22 4.81 12.73 12.73 Azadrachtin 50% 36.11 69.44 100 9.62 4.81 0.00 Azadrachtin 75% 44.44 77.78 100 12.73 4.81 0.00 Azadrachtin100% 72.22 88.89 100 26.79 19.24 0.00 Abamectin 100 100 100 0.00 0.00 0.00 Hunter 100 100 100 0.00 0.00 0.00 Selecron 77.78 88.89 100 19.25 9.62 0.00

Appendix 1B Table 5.2: The percentage mortality of adult mites on tomato leaves treated with different concentrations of Syringa seed extracts

% St Dev

Syringa 24 hours

48 hours

72 hours

24 hours

48 hours 72 hours

Control 0 0 0 0.00 0.00 0.00 Dist. Water 0 0 0 0.00 0.00 0.00 M.azedarach0.1% 27.78 52.78 55.56 12.73 17.35 12.73 M.azedarach 1% 27.78 58.33 91.67 9.62 22.05 8.33 M.azedarach 10% 52.78 75 97.22 12.73 8.33 4.81 M.azedarach 20% 58.33 83.33 100 14.43 16.67 0.00 M.azedarach 50% 58.33 88.89 100 14.43 9.62 0.00 M.azedarach 75% 77.78 88.89 100 4.81 12.73 0.00 M.azedarach100% 97.22 100 100 4.81 0.00 0.00 Abamectin 100 100 100 0.00 0.00 0.00 Hunter 88.89 100 100 19.25 0.00 0.00 Selecron 44.44 100 100 9.62 0.00 0.00

98

Appendix 1C Table 5.3: The percentage mortality of adult mites on tomato leaves treated with different concentrations of Syringa leaf extracts

% St Dev Syringa leaves

24 hours

48 hours

72 hours

24 hours

48 hours

72 hours

Control 0 0 0 0.00 0.00 0.00 Water 100ml 0 0 0 0.00 0.00 0.00 M.azedarach0.1% 38.89 55.56 66.7 17.35 4.81 4.81 M.azedarach 1% 50 58.33 75 8.33 8.33 8.33 M.azedarach 10% 52.78 63.9 80.6 4.81 12.73 8.33 M.azedarach 20% 61.11 63.89 80.56 9.62 12.73 4.81 M.azedarach 50% 63.89 75 86.11 4.81 8.33 12.73 M.azedarach 75% 72.22 83.33 100 12.73 8.33 0.00 M.azedarach100% 97.22 100 100 4.81 0.00 0.00 Abamectin 100 100 100 0.00 0.00 0.00 Hunter 100 100 100 0.00 0.00 0.00 Selecron 75 80.56 97.22 8.33 4.81 4.81

Appendix 1D

Table 5.4: The percentage mortality of mite nymphs on tomato leaves treated with different concentrations of Neem seed extracts

.

% St Dev

Neem

24 hours

48 hours

72 hours

24 hours

48 hours

72 hours

Control 0 0 0 0.00 0.00 0.00 Water 100ml 0 0 0 0.00 0.00 0.00 NSE 0.1% 55.56 83.33 91.67 4.81 16.67 8.33 NSE 1% 58.33 88.89 97.22 8.33 4.81 4.81 NSE 10% 66.67 88.89 100 8.33 4.81 0.00 NSE 20% 69.44 97.22 100 9.62 4.81 0.00 NSE 50% 83.33 100 100 8.33 0.00 0.00 NSE 75% 91.67 100 100 8.33 0.00 0.00 NSE 100% 97.22 100 100 4.81 0.00 0.00 Abamectin 100 100 100 0.00 0.00 0.00 Hunter 100 100 100 0.00 0.00 0.00 Selecron 44.44 72.22 100 25.46 9.62 0.00

99

Appendix 1E Table 5.5 The percentage mortality of mite nymphs on tomato leaves treated with different

concentrations of Syringa seed extracts

% St Dev

Syringa

24 hours

48 hours

72 hours

24 hours

48 hours

72 hours

Control 0 0 0 0.00 0.00 0.00 Water 100ml 0 0 0 0.00 0.00 0.00 M.azedarach0.1% 47.22 66.67 86.11 8.33 8.33 12.73 M.azedarach 1% 55.56 69.44 88.89 4.81 4.81 8.33 M.azedarach 10% 60.33 75 91.67 8.1 8.33 9.62 M.azedarach 20% 75 80.56 94.83 8.33 4.81 12.73 M.azedarach 50% 86.11 100 100 4.81 0.00 0.00 M.azedarach 75% 88.89 100 100 4.81 0.00 0.00 M.azedarach100% 97.22 100 100 4.81 0.00 0.00 Abamectin 100 100 100 0.00 0.00 0.00 Hunter 100 100 100 0.00 0.00 0.00 Selecron 77.78 86.11 97.22 4.81 4.81 4.81

Appendix 1F Table 5.6 Percentage hatched eggs of mites on tomato leaves treated with different

concentrations of Neem seed extracts

% St Dev

Syringa

24 hours

48 hours

72 hours

24 hours

48 hours

72 hours

Control 0 40.74 68.25 0 13.98 30..60 Water 100ml 0 29.63 50 0 16.92 27..96 M.azedarach 0.1% 0 27.78 48.15 0 14.70 27..96 M.azedarach 1% 0 25.93 40.74 0 6.42 16..97 M.azedarach 10% 0 22.22 37.04 0 11.11 35.28 M.azedarach 20% 0 16.67 25.93 0 5.56 16.97 M.azedarach 50% 0 16.67 24.07 0 5.56 11.56 M.azedarach 75% 0 3.70 11.11 0 2.85 6.42 M.azedarach100% 0 0 0 0 0.00 0.00 Abamectin 0 9.26 9.26 0 4.50 4.50 Hunter 0 11.11 27.78 0 6.42 14.70 Selecron 22.22 40.74 50 0.00 23.13 25.46

100

Appendix 1G Table 5.7 Percentage hatched eggs of mites on tomato leaves treated with different

concentrations of Syringa seed extracts

St Dev Syringa

48 hours

72 hours

48 hours

72 hours

Control 27.78 83.33 14.70 24.22 Water 100ml 20.37 77.78 11.56 14.70 M.azedarach0.1% 16.67 38.89 5.56 19.25 M.azedarach 1% 11.11 29.63 5.56 27.40 M.azedarach 10% 1.85 5.56 3.21 0.00 M.azedarach 20% 1.85 11.11 3.21 5.56 M.azedarach 50% 1.85 5.56 3.21 0.00 M.azedarach 75% 1.85 5.56 3.21 0.00 M.azedarach100% 1.85 0.00 3.21 0.00 Abamectin 9.25 12.96 4.80 5.85 Hunter 1.85 5.56 3.21 0.00 Selecron 18.51 20.37 9.26 14.70

APPENDIX 2

Calculation of % efficacy

In view of pre-treatment differences, corrected percentage efficacy can be calculated

according to the following modification of Abbott’s formula as described by Henderson &

Tilton (1955):

%Efficacy = [1 - (Ta/Ca x Cb/Tb)] x 100,

Where Tb is infestation in treated plot prior to application;

Ta is infestation in treated plot after application;

Cb is infestation in control plot prior to application;

Ca is infestation in control plot after application.

If calculated per experimental unit, these percentages can be analysed by ANOVA.

101

APPENDIX 3

A protocol for the control of red spider mites using Neem and Syringa extracts.

PROGRAMME FOR THE CONTROL OF RED SPIDER MITES USING NEEM AND

SYRINGA EXTRACTS

102

Background

Red spider mites (Tetranychus spp.) attack nearly 100 types of cultivated crops like maize,

tobacco, cotton, beans, eggplant, pepper, tomatoes, cucurbits and many other vegetables

(Mau et al., 1992). They are also pests of papaya, passion fruit as well as being a common

pest of many flowers such as carnation, chrysanthemum, cymbidium, gladiolus, marigold

and roses (Guo, 1998; Tadmor et al., 1999; Bolland and Vella, 2000; Batta, 2003; Knapp

et al., 2003).

Red spider mites are often found in pockets on the undersides of leaves near the midribs

and veins. Adult and nymphs of the red spider mite suck sap especially from mature

leaves. This causes the upper surface to become stippled with little dots that are signs of

the feeding puncture (Goff 1986; Lu & Wang, 2005). Continued feeding may result in the

collapse of mesophyll cells. The leaves eventually become bleached and discoloured.

Leaf drop can occur following heavy infestations due to an increase in the mite population

especially under hot, dry or alkaline conditions (Knapp et al., 2003). This drastically

reduces the crop yield (Hill 1983; Visser, 2005; Bok et al., 2006). Hot dry weather is

favourable for mite infestations whereas very high relative humidity and rain tend to kill

red spider mites during moulting, by washing them off leaves.

Field determination of spider mite infestations

Spider mite infestations can be determined by direct examination of suspected infested

leaves by using a hand lens or by shaking symptomatic leaves onto a sheet of white paper.

103

Using the following criteria (Steinkraus et al., 1999), mite populations can be classified as

follows:

• none - no spider mites

• light - 1 to 10 spider mites found on occasional plants

• medium - 11 to 50 spider mites per leaf

• heavy - more than 50 spider mites per leaf on most plants. Many leaves appear reddish–brown in colour.

Management of red spider mites

It should be known that natural enemies of mites are present in and around fields and can

keep mite populations low. Many insecticides used to control red spider mites severely

reduce the number of beneficial insects or other mites that keep red spider mites

population in check. Moreover, the insecticides are very expensive and not always readily

available. Botanical acaricides may be a solution to this challenge.

Before applying any chemical, including botanical acaricides, ensure that weeds are

properly taken care of - the field should be weed-free.

Preparation of extracts

To prepare 5 litres of spray, take two kilograms of Syringa seeds, crush them using a

mortar and pistil and add 5 litres of water. Boil the mixture for one hour and leave it to

cool overnight. The next day filter it through a fine cloth. Neem seed extracts can be

prepared using the same method.

104

Application of the Neem and Syringa seed extracts

Since red spider mites take about a week to complete a life cycle, it is recommended that

botanical acaricides should be applied at least once a week, using a Knapsack sprayer.

105

8 REFERENCES

Abou-Fakhr Hammad, E.M., Nemer, N.M. & Kawar, N. S. (2000). Efficacy of Chinaberry tree (Meliaceae) aqueous extracts and certain insecticides against the pea leafminer (Diptera: Agromyzidae). Journal of Agriculture Science 134, 413-420. Abou-Fakhr Hammad, E.M., Zournajian H. & Talhouk S. (2001). Efficacy of extracts of Melia azedarach L. callus, leaves and fruits against adults of the sweet potato whitefly Bemisia tabaci (Hom., Aleyrodidae). Journal of Applied Entomology 125, 483-488. Ascher, K.R.S. (1993). Non-conventional insecticide effects of pesticides available from the Neem tree, Azadrachta indica. Archtectural. Insect Biochemistry & Physiology 22, 433-449. Ascher, K. R. S., Schmutterer, H., Zebitz, C.P.W. & Naqvi, S. N. H. (1995). The Persian lilac or Chinaberry tree: Melia azedarach L. In: H. Schmutterer (Ed). The neem tree: Source of unique natural products for integrated pest management, medicine, industry and other purposes (pp. 605-642), VCH, Weinheim, Germany. Baker, E. D. & Tuttle, D. M. (1994). A Guide to Spider Mites. (Tetranychidae) from the United States pp 347. Westbloomfield: Indra Pulishing House, Michigan. Bandeke, T. J. (1996). “Food and nutrition situation in Botswana: An overview” in J. Kiamba, & M. Mhango (Eds). Proceedings of the Home Economics Research Capacity Building Workshop. (pp. 36-46). Botswana, University of Botswana.

Batta, Y.A. (2003). Symptomatology of tobacco white fly and red spider mite infection with the entomopathogenic fungus Metarhizium anisopliae (Melsch) Sorokin. Agriculture Science, 30(3), 294–303. Beddington, J. (2008). The world’s choice: food or biofuels, South Africa. Sunday Times, March 9 p. 6. Biswas, G.C., Islam, I., Haque, M.M., Saha, R.K., Hoque, K.M.F, Islam, M.S. & Haque, M. E. (2004). Some biological aspects of carmine spider mite, Tetranychus cinnabarinus Boisd.(Acari: Tetranychidae) infesting eggplant from Rajshadhi. Journal of Biological Sciences 4 (5), 588–591.

106

Blumel, S. & Hausdorf, H. (2002). Results of the 8th and 9th IOBC joint pesticides testing programme: In: H. Vogt, & U. Heimbach (Eds.), Proceedings of the Meeting at San Michele All’Adige, Persistence Test with Phytoseiulus persimilis vol. 25. Athias Henriot (Acari: Phytoseiidae). Bulletin- OILB-SROP (pp 43-51). Trento, (Italy).

Bohmont, B. L. (1997). The Standard Pesticide: Users Guide. Prentice Hall Upper Saddle River, New Jersey. Bok, I., Madisa M., Machacha, D, Moamongwe M. & More, K. (2006). Manual for Vegetable Production in Botswana. Ministry of Agriculture, Botswana. Boeke, S. J., Van Loon J. J. A, Van Huis A., Kossou & Dicke, M. (2001). The use of plant material to protect stored leguminous seeds against seed beetles: a review, Wageningen University papers 2001-3, The Netherlands. Bolland, H. R. & Valla, F. (2000). First record of the spider mite Tetranychus evansi (Acari: Tetranychidae) from Portugal. Entomologische Berichten 60 (9), 180. Bolland, H. R., Guitierrez, J. & Flechtmann, C. H. W. (1998). World Catalogue of Spider Mites Family (Acari: Tetranychidae). (pp 392). Brill, Leiden, Boston, Köln. Bonnano, A (Ed.) (1994). From Columbus to ConAgra. The Globalization of Agriculture and Food. Lawrence: University Press of Kansas. Brandenburg, R.L. & Kennedy, G.G. 1982. Relationship of Neozyites floridana (Entomophthorales: Entomophthoraceae) to two spotted spider mite (Acari: Tetranychidae) population in field corn. Journal of Economic Entomology 75, 691-694. Broughton, H. B., Ley, S.V., Slawin, A. M. Z., Williams, D. J. & Morgan, E. D. (1986). X-ray Crystallographic Structure of Azadirachtin and Reassignment of the Structure of the Limonoids. Eschborn: Martinsried Unit. Bues, R., Dadomo, M., Lyannaz, J.P., Di Lucca, G., Macua Gonzalez, J.I., Prieto Losada, H., & Dumas, Y. (2003). Evaluation of the environmental impact of pesticides applied in processing tomato. Acta Horticulture 613, 255-258.

107

Carpinella, M.C., Defago, M.T., Valladares, G. & Palacios, S.M. (2003). Antifeedant and insecticide properties of limonoids from Melia azedarach (Meliaceae) with potential use for pest management. Agric. Food Chemistry 15, 369-674. Castagnoli M., Nannelli R., & Simon S. (2006). Tetranychus evansi (Baker & Pritchard) (Acari: Tetrannychidae), a new pest for Italy Informatore. Fitopatologico 5, 50-52. Chapman R.B. & Martin, N. A., (2003). Spider mite resistance strategy. New Zealand Plant Protection Society. Charleston. D.S. (2004). Integrating Biological Control and Botanical Pesticides for management of Plutella xylostella. PhD thesis Wageningen University. Chase, J.M. (2000). Are there real differences among aquatic and terrestrial food webs? Trends in Ecology and Evolution 15, 408–412. Chung-Huang, R.., Tadera, K.., Yagi, F., Minami,Y., Okamura, H., Iwagawa, T. & Nakatani, M., (1996). Limonoids from Melia azedarach. Phytochem 43 (3), 581-583. Collyer E. (1998). The Horticulture and Food Research Institute of New Zealand, Kluwer Academic Publishers. Copping, L.G. (Eds) (2001). The Bio-pesticides Manual. Second Edition. British Crop Protection Counsel. Cross, J.V. & Berrie. A.M. (1994). Effects of repeated foliar sprays of insecticides or fungicides on organophosphate-resistant strains of the orchard predatory mite Typhlodromus pyri on apple. Crop Protection Journal 13, (39-44). Dagli, F & Tunc, I. (2001). Dicofol resistance and stability of resistance in populations of Tetranychus cinnabarinus. Pest Management Science 57 (7), 609-614 Daka, P.S. (2003). A study of Deltamethrin in sediment from the Okavango delta - Botswana. Unpublished Masters Dissertation. University of Botswana.

Dent, D. (1991). Insect Pest Management. Cab International, London.

108

Dhaliwal, G. S., Arora, R. & Koul, O. (2004). Neem Research: Present Status and Future Outlook in Neem: Today and in the New Millennium O. Koul and S. Wahab (Eds), (pp. 65-96) the Netherlands, Kluwer Academic Publishers. Duncan, R. (1997). World food markets into the 21st century: commodity risk management policies. The Australian Journal of Agricultural and Resource Economics 4(3), 429-43. Ehara, S. (Ed) (1993). Plant Mites of Japan in colours. (pp 298). Tokyo, Zenkoku Noson Kyoiku, Kyokai. Einarsson, P. (2000). Agricultural Trade Policy; as if food security and ecological sustainability mattered. Review and Analysis of Alternative Proposals for the Renegotiation of the WTO Agreement on Agriculture, Stockholm: Forum Syd. Sweden.

Ellis, C. & Mellor, G.T. Jr. (1995). Living in the Environment. Belmont: Thomson Learning, Inc.

Encyclopedia of Foods and their Healing Power, (2004). A Guide to Food Science and Diet Therapy, (p103). Education and Health Library. Engindeniz, S. (2005). Economic analysis of pesticide use on processing tomato growing: A case study for Turkey. Crop Protection (4), 234-241 ELSEVIER. FAO. (1989). FAO Production Yearbook. Rome: Food and Agriculture Organisation of the United Nations. FAO (2005) Statistical Service: FAO Rome (Online) www.http://apps.fao.org. Accessed 03/4/2008. FAO. (2006a) FAO Statistical Year book, Rome: Food and Agriculture Organisation of the United Nations.

FAO. (2006b), FAO Production year book, Rome: Food and Agriculture Organisation of the United Nations.

109

Flaherty, D. L., & Wilson, W.T. (1999). Biological control of insects and mites on grapes. In: T.S Bellows & T.W. Fisher, (Eds.), Handbook of Biological Control (pp 853-869). Academic Press, NewYork, USA.

GenStat® for Windows® (2003). Introduction– (Ed) R.W Payne, (7th Edition) Published VSN International.

Goff, L. (1986). Spider mites (Acari: Tetranychidae) in the Hawaiian Islands. International Journal of Acarology 12 (1), 43 –49 Goodman, D. (1999). Agro-food studies in the “age of ecology”: Nature, corporeality, bio-politics. Sociologia Ruralis 39(1), 17-38. Greathead D. J. Waag, P & Doi, A.C (1990). When and how to introduce exotic control agents. In D. J. Girling (Ed). UNDP/FAO Manual for Biological control. C.A.B. International Institute of Biological Control, (pp 1-3), Berkshire, U.K. Gretchen, D., Dasgupta, P., Bolin, B., Crosson, P & Waterlow, J.C. (1998). Food production population growth and the environmental security. Science 281, 1291-1292. GTZ (Deutsche Gesellschaft fur Technische Zusammenarbeit Report (2000). Status Report on Global Neem usage, GTZ, Eschborn, Germany. Gupta, G. P. & Sharma, K. (1997). Neem-based pest management strategy in cotton system. Pesticide Research Journal 9, 190-197. Guo, F.Y., Zhang, Z., & Zhao, Z.M. (1998). Pesticide resistance of Tetranychus cinnabarinus (Acari: Tetranychidae) in China: a review. Systematic and Applied Acarology, 3, 3-7. Hedge, N.G. (1996). Improving the productivity of Neem trees, In Neem and Environment, Vol.1,. R.P. Singh, M.S. Chari, A.K. Raheja & W. Kraus Eds, (pp.69-79) Oxford & IBH Publishing Co. Pvt. Ltd., New Delhi,. Henneberry, T.J., Glass, E. H., Gilbert, R. G., King, E. G Jr., Miller, R. W & Whitten, C.J. (1991). Integrated Pest Management, A sustainable technology. In: D.T Smith (Ed), Agriculture and the Environment (pp140 -150) USDA.

110

Henderson, C.F. & Tilton, E.T. (1955): Test with acaricides against the Brown wheat mite. J. Econ. Entomology 48: 157-161. Hill, D.S. (1983). Tetranychus cinnabarinus (Boisd.). In: Agricultural Insect Pests of the Tropics and their Control, 2nd Edition (pp501–502). Cambridge UK, Cambridge University Press. Ho, C.C., (2000). Spider mite problems and control in Taiwan. Experimental and Applied Acarology 24 (5-6), 453–462 Hurd, L.E. & Eisenberg R.M (1990). Arthropod community responses to manipulation of a bitrophic predator guild. Ecology 71, 2107–2114 Juan, A. & Sans, A. (2000). Antifeedant activity of fruit and seed extracts of Melia azedarach and Azadirachta indica on larvae of Sesamia nonagrioides. Phytoparasitica 28, 311-319.

Kasozi, J., Mahonde, L., McLeod G & Simela, T. (1999). New Trends in Agriculture, (pp.107-120). Botswana. Macmillan Publishing Co. Klubertanz, T.H., Pedigo, L.P. & Carlson, R.E. (1991). Impact of fungal epizootics on the biology and management of the two spotted spider mite Tetranychus urticae in soyabean. Environ. Entomology. 20, 731-735. Knapp, M., Saunyama, I.G.M, Sarr, I & DE- Moraes, G.J. (2003). Tetranychus evansi in Africa – Status, Distribution, Damage and Control Options. International Centre of Insect Physiology and Ecology (ICIPE), Nairobi, Kenya. Koul, O, (1996), Mode of Azadrachtin action, in Neem, eds. N.S.. Randhawa and B.S. Palmer (pp160-170). New Age International Publishers Ltd., New Delhi, Koul, O. & Wahab, S. (2004). Neem: Today and in the New Millennium Kluwer (pp 1-19). Academic Publishers. NetherLands. Koul, O., Isman, M. B. & Ketkar, C. M., (1990). Properties and uses of Neem, Azadrachta indica. Canadian Journal of Botany, 68, 1-11.

111

Kraus, W. (2002). Biologically active ingredients, in The Neem Tree, 2nd Edition,.H. Schmutterer Ed, The Neem Foundation (pp.39-111), Mumbai, India. Kuang, H. & Cheng, L. (1990). Studies on differentiation between two sibling species Tetranychus cinnabarinus and T. urticae. Acta Entomologia Sinica 33, 109-115. Kumar, G.V.S & Sannaveerappanavar, V.T. (2004). Evaluation of bio-efficacy of insecticide and seed oil combinations against Plutella xylostella (L) on cabbage. Journal of Agricultural Sciences 38(3), 302-305. Kumar, A.R.V, Jayappa, J & Chandrashekara, K. (2000). Relative insecticidal value: An index for identifying Neem trees with high insecticidal yield. Current Science 79, 1474-1478.

Langeland, K.A & Burks K.C. (2005). Identification and Biology of Non-Native Plants in Florida's Natural Areas, 96-97. Lee, S.M.; Klocke, J.A. & Balandrin, M.F., (1987). The structure of L-cinnamolymelianolone, new insecticidal tetranortriterpenoids, from Melia azedarach L (Meliaceae) Tetrahedron Ltt. 28, 3543-3546 Lee, S. M.; Klocke, J. A., Barnby, M. A. & Balandrin, M.F., (1991). Insecticidal constituents of Azadrachta indica and Melia azedarach (Meliaceae) In: P.A. Hedin (Ed), Natural occurring pest Bioregulators 449 (pp 293-304), Washington DC. Levent, E., Enanc, O. & Erdal, N. Y. (2005). Effect of a Commercial Neem Insecticide (Neem Azal T/S) on Early and Late development Stages of the Beet Army Worm, Spodoptera exiqua (Hubner) (Lepodoptera: Noctuidae) Lin, H.E., Zhimo, Z., XiaoFang, C., Deng, X-P, & Wang J-.J. (2005). Resistance selection of Tetranychus cinnabarinus to fenpropathrin and genetic analysis. Agriculture Science in China, 4 (11) 851 – 856. Louw, A. (2005). Regoverning Markets. World Food and Agribusiness Symposium, Chicago, Illinois.

112

Lu, C.T. & Wang, C.L. (2005). An investigation of spider mites on papaya and re-evaluation of some acaricides. Plant Protection Bulletin Taipei, 47 (3) 273 – 279. Luchen, S.W. & Mingochi, D.S., (1994). Red spider mites in vegetables: Can we combat them? In: Productive farming pp24: Government printer Zambia. Luo-Xiao, Dong, Wu-Shao, Hua, Ma-Yun, Bao, Wu-Da, Gang, Luo-X.D., Wu, X.H. , Ma, Y.B. & Wu, D.G. (2000). A new triterpenoids from Azadrachta indica. Fitoterapia 71, 66–672. MacDonald, I. & Low, J. (1990). Fruit and vegetables p86. London: Evans Brothers.

McMichael, P. (2000), ‘The power of food’. Agriculture and Human Values 17, 21-33. Mansour, F., & Bar-Zur, A. (1990). Resistance of maize inbred lines to the carmine spider mite Tetranychus cinnabarinus (Boisduval) (Acari: Tetranychidae) Maydica 37, 343–345 Mansour, F., Karachi, Z. , & Omari, N. (1987). Resistance of melon to the carmine spider mite Tetranychus cinnabarinus (Boisduval) (Acari: Tetranychidae) Bulletin of Entomological Research 77, 603-607 Mansour, F., Shain, Z. , Karachi, Z., & Gerzon, M. (1994). Resistance of selected melon lines to the carmine spider mite Tetranychus cinnabarinus (Boisduval) (Acari: Tetranychidae). Field and laboratory experiments. Bulletin of Entomological Research, 84, 26 –267 Mau, R.F.L. & Kessing M. (1992). Tetranychus cinnabarinus (Boisduval) Department of Entomology Leaflet, Honolulu, Hawaii. Memmott, J., Martinez, N.D. & Cohen, J.E. (2000). Predators, parasitoids and pathogens: species richness, trophic generality and body size in natural food web. Journal of Animal Ecology 69, 1-15 Mendez, M.C., Elfas, F., Aragao, M., Gimeno, E. J. & Riet-Correa, F. (2002). Intoxication of cattle by the fruits of Melia azedarach. Vet. Hum. Toxicology 44, 145-148. Meng, H., Wang, J., Jiang, X. & Yi, M., (2000). Studies on the resistance of Panonychus citiri to several acaricides. Pesticides Journal 39, 26-28.

113

Messiaen, C.M., (1992). The tropical vegetable garden (pp333). Macmillan Press in association with CTA. Netherlands. Meyer, (M.K.P. (1987). African Tetranychidae (Acari: Prostigmata) – with reference to the world genera. Republic of South Africa, Department of Agriculture and Water Supply. Entomology Memoir 69, 1- 175. Mingochi, D.S., Luchen, S.W. & Kembo, J. (1994). Biological control of vegetable pests in Zambia. In: Integrating biological control and host plant resistance, pp.107-111. Mnzava, N.A. (1990), The vegetable Industry: A SADC Regional Perspective. In: R.T. Opena and M.L. Kyomo (Eds), Proceedings of a workshop held at Arusha –Tanzania Vegetable Research and Development in SADC Countries (pp.63-70)AVRDC/SACCAR.

Molefi, R.K.K. (1996). A medical history of Botswana from 1885-1966 Gaborone. Government Printer, Gaborone, Botswana. Mordue, A.J. & Blackwell, A. (1993). Azadrachtin: an update. Journal of Insect

Physiology 39, 903- 924.

Mosie, E. (2004). The Nutritional quality of feeding programs in Botswana Junior Secondary schools and the contribution of family and consumer sciences to meal quality. PhD thesis, Texas Tech University, USA.

Motarjemi, Y., van Schothorst, M. & Kaferstein, F. (2001), Future challenges in global harmonization of food safety legislation. Food Control 12(6), 339-46. Murugan, K. & Ancy George Sr. (1992). Feeding and nutritional influence on the growth and reproduction of Dorphinis nerii Linn. (Lepidoptera: Sphingidae). J. Insect Physiol. 38, 961–968. Nathan S.S., Chung, P.G. & Murugan, K. (2004). Effect of botanicals and bacterial toxin on gut enzyme of Cnaphalocrosis medinalis. Phytoparasitica 32, 433-443. Nathan S. S., Kalaivani, K., Chung, P.G. & Murugan, K., (2006). Effect of neem limonoids on lactate dehydrogenase (LDH) of the rice leaffolder, Cnaphalocrocis medinalis (Guenée). Chemosphere 62 1388-1393.

114

Nathan, S.S., Chung, P.G. & Murugan, K. (2005). Effect of biopesticides applied separately or together on nutritional indices of the rice leaffolder Cnaphalocrosis medinalis (Guenee) (Lepidoptera: Pyralidae) Phytoparasitica 33, 187-195.

Nathan, S.S., Kalaivani, K., Scoon K. & Murugan, K. (2006).The toxicity and behavioural effects of Neem limonoids on Cnaphalocrocis medinalis (Guenee), Chemosphere 62(8), (1381-1387). Ngugi, D.N., Karau, P.K. & Nguyo, W. (1990). East African Agriculture. London: Macmillan. Noble, A.D., Zenneck, I., & Randal, P.J., (1996). Leaf litter ash alkalinity and neutralization soil acidity. Plant and Soil 179 (2), 293-302. Okigbo, B.N. (1990). Vegetables in Tropical Africa. In: R.T. Opena & M.L. Kyomo (Eds.), Proceedings of a workshop held at Arusha – Tanzania. Vegetable Research and Development in SADC Countries, (pp29-54) AVRDC/SACCAR. Oosterveer, P. (2007). Global Governance of Food Production and Consumption Issues and Challenges. Chetenham (pp 3-63): Edward Elgar Publishing Limited, Glensanda House, UK. Palacios, S. Vallandares, G. & Ferreyadra, D. (1993) Preliminary results in the searching of an insecticide from Melia azedarach extracts. In: H.G. Kleeberg (Ed), Proceedings of 2nd workshop practice-oriented results on use and production of neem – ingredients and pheromones (pp 99-105). Trifolio-M GmbH, Druck and Graphic. Pamplona-Roger, G.D. (2004). Encyclopaedia of Foods and their healing power.: A guide to food science and diet therapy p103. Education and healing library Editorial Safeliz. Polis, G.A., Sears, A. L. W. Huxel, G.R. , Strong, D. R. , & Maron, J. (2000). When is a trophic cascade a trophic cascade? Trends in Ecology and Evolution 15, 473–475 Prakash, A. & Rao, J. (1997). Botanical pesticides in Agriculture. CRC Lewis Publishers, Boca Raton.

115

Prischmann, D.A., James, D.G. & Snyder, W.E. (2005). Impact of management intensity on mites (Acari: Tetranychidae, Phytoseiidae) in central Washington wine grapes. International Journal Acarol 31, 10-15. Puri, H.S. (1999). Neem the divine tree Azadirachta indica. Harwood Academic Publishers, Amsterdam. Quarles, W. (1994). Neem tree pesticides products & ornamental plants. The IPM Practitioner 16 (10), 1-13. Rembold, H. (1993). Neem and its general development for pest control. Proceedings of the world Neem Conference (pp 8-9), India. Rembold, H. (1996). Neem and its natural development for pest control. In Neem and Environment, Vol.1, (Ed). R.P. Singh, M.S. Chari, A.K. Raheja and W.Kraus, Oxford & IBH Publishing Co. Pvt. Ltd., New Delhi, pp.1-10.

Reganold, J.P., Papendick, R.I. & James F.P. (1990). Sustainable Agriculture. Scientific American, pp. 112-120.

Riechert, S.E. & Lockley, T. (1984). Spiders as biological control agents. A. Rev. Ent. 29,

288–320.

Rice, M. (1993). Built in resistance prevention (BIRP): a valuable property of Azadirachtin, pp.13-14. In: World Neem Conference, Bangalore, India. Rosenheim, J.A. (2001) Source–sink dynamics for generalist insect predator in a habitat with strong higher–order predation. Ecol. Monogr. 71, 93 –116. Rosenheim, J.A., & Corbett, A. (2003). Omnivory and the indeterminacy of predator function: can knowledge of foraging behaviour help? Ecology 84, 2538–2548. Rosenheim, J.A, Tobias E., Rachel E., Goeriz & Ramert B. (2004). Linking a predator’s foraging behaviour with its effects on herbivore population suppression. Ecology 85 (12), 3362–3372.

116

Ruckin, F.R. (1992) (Ed). Neem: a tree for solving global problems. National Academy Press .Washington, D.C. Russell, A. B., Hardin, J.W. & Larry, G. (1997). Melia azedarach. In: Poisonous Plants of North Carolina. SADC. (2002). SADC Food Security Ministerial Briefing (pp1-9). Safety & Quality Assurance (2007). A guide for the control of plant pests. 40, 22-39. National Department of Agriculture. Republic of South Africa. Saxena, R.C. (1987). Antifeedants and tropical pest management. Insect Science. Appl. 8, 731-736. Saxena, R.C. (2004). Neem for ecological pest and vector management in Africa: Outlook for the new Millennium. Schmitz, O., Krivan, J. & Ovadia O. (2004). Trophic cascades: the primacy of trait – mediated indirect interactions. Ecology Letters 7, 153–163. Schmutterer, H. (1990). Properties and potential of natural pesticides from the Neem tree, Azadrachta indica. Ann. Rev. Entomol. 35, 271-297. Schmutterer, H. (1995). The Neem Tree Azadirachta indica A. Juss., and other Meliaceceous plants: Source of Unique Natural Products for Integrated Pest Management, Medicine, Industry and Other Purposes. VCH, Weinheim. Schmutterer, H. & Ascher K.R.S. Eds. (1987). Natural pesticides from the Neem tree and other tropical plants. Proceedings of the 3rd Int. Neem Conf. Nairobi, GTZ Schriftenreih, Eschborn. Scully, T.B., East, D.A., Eidelson, V.J & Cox, L.E. (1991). Resistance to the two spotted spider mite in musk melon. Proceedings of the Florida State Horticulture Society. 104, 276–278.

117

Sharma, V.P. & Ansiri M.A. (1993). Protection from mosquito by burning Neem oil in

kerosene. Journal of Medicine Entomology p24.

Shanmugasundaram, S. (1990). Vegetable research in southern Asia. In S. Shanmugasundaram (ed). Proceedings of a workshop held at Islamabad – Pakistan. Asian Vegetable Research and Development Center AVRDC (pp29-49). Shurin, J.B., Borer, E.T Seabloom, E. W. , Anderson, K. , Blanchette, A., Broitman, B., Cooper, S.D. & Halpern, B.S., (2002). A cross–ecosystem comparison of strength of trophic cascades. Ecology Letters 5, 785 – 791. Siddiqui, B.S., Ghiasuddin, S.F. & Siddiqui, S. (1993). Chemistry of Neem (Azadirachta indica), a sustainable source of natural pesticides. World Neem Conference, India. Singh, R.P., Singh, S. & Wahab, S. (1998). Biodiversity and importance of botanical pesticides, In: Ecological Agriculture and Sustainable Development. Vol. 2, (Eds). G.S. Dhaliwal, N.S. Randhawa, R. Arora and A.K. Dhawan, Indian Ecological Society and Centre for Research in Rural and Industrial Development, (pp 128 – 145), Chandigarh. Snedecor, GW & Cochran, W.G. (1980). Statistical methods (7th ed.). Ames: Iowa State University Press. Spencer, K.A. (1990). Host specialization in the world Agromyzidae (Diptera). Pp. 444. London: Kluwer Academic Publishers. Steinkraus, D., Zawislak, J., Lorenz, G., Layton, B. & Leonard, R. (1999). Spider mites on cotton in the Midsouth. University of Arkansas. Subrahmanyam, B. & Rao, P.J. (1993). Biological effects of certain oils and terpene fractions of plant origin on insects: World Neem Conference Vol. 116, (pp 1020 -1029). India. Tadmor, Y., Lewinsohn, E., Abo-Moch, F., Bar-Zur, A. & Mansour, F. (1999). Antibiosis of maize inbred lines to the Carmine spider mite Tetranychus cinnabarinus (Boisd.) (Acari: Tetranychidae) Phytoparastica 27, 35-41.

118

Thacker, J.R.M. (2002). An Introduction to Arthropod Pest Control. Cambridge University Press. United Nations. (1993) World population prospects: The 1992 Revision, United Nations New York. United States Office of International Affairs (USOIA). (2006). Neem: A tree for solving global problems (pp1-10). The National Academic Press. 500 Fifth Str. NW. Washington, D.C. 20001. US NRC (National Research Council (1992). NEEM: A Tree for Solving Global Problems, National Academy Press, Washington, DC. Van den Boom, C.E.M, Van Beek, T. A. & Dicke, M. (2003). Differences among plant species in acceptance by the spider mite Tetranychus urticae Koch. Journal of Applied Entomology 127,177-183. Van Huis & Meerman, (1997). Can we make IPM work for resource-poor farmers in Sub-Sahara Africa International Journal of Pest Management, 43(4): 313-320. Verkerk, R.H.J. & Wright, D.J. (1993). Biological activity of Neem seed kernel extracts and synthetic azadirachtin against larvae of Plutella xylostella L. Pesticide Science 37, 83-91. Visser, D. (2004). The potato tuber moth, Phthorimaea operculella (Zeller), in South Africa: potential control measures in non-refrigerated store environments. Ph.D. Thesis, University of Pretoria, Pretoria, South Africa. Visser, D. (2005). Guide to potato pests and their natural enemies in South Africa. Agricultural Research Council, South Africa. Völlinger, M. (1995). Studies of the probability development of resistance of Plutella xylostella to Neem products. In: H. Schmutterer (Ed). The Neem Tree: Source of unique Natural products for Integrated Pest Management, Medicine, Industry and other purposes pp. 477-483. Weinhein: VCH Verlagsgesellschaft.

119

Wang, F.H. (1987) Acariforms: Tetranychoideae. Economic Insect Fauna of China 23, 1-150. Warthen Jr., J.D., Stokes, J.B., Jacobson, M. & Kozempel, M.P. (1984). Estimation of azadirachtin content in Neem extracts and formulations. Journal of Liquid Chromatography 7 (591-598). Weinzierl, R. & Henn, T. (1991). Alternatives in insect management: biological and bio rational approaches. North Central Regional Extension, Publication 401. Wekesa, V.W. (2005). Pathogenicity of Beauveria bassiana and Metarhizium anisopliae to the tobacco spider mite Tetranychus evansi. Experimental and Applied Acarology 36, 1-2. http:// www. bio-bee.com/ (on line) Accessed 26th March 2009 www.http://en.wikipedia.org the free encyclopedia (Online) Accessed 6th August 2007 www.http://en.wikipedia.org the free encyclopedia (Online) Accessed 6th July 2008 Wikipedia, the free encyclopaedia (Online) www.http://en.wikipedia.org/wik/tomato#production trends Accessed 02/11/2007. Wise, D.H. (1993). Spiders in ecological webs. Cambridge University Press, Cambridge. World Health Organization(WHO) (1999). School feeding handbook. Rome. WRR. (2007) World and Regional Review: A long term Perspective Part II. pp 119 -135. WTO (World Trade Organisation) (2001). International Trade Statistics, Geneva: WTO, URL. Zhao, W., Wang, K., & Jiang. X. (2001). The monitoring of resistance of Tetranychus urticae Koch to several insecticides. Chinese Journal of Pesticide Science 3, 86-88.

120

Zhi-qiang Z. & Jacobson, R. (2000). Using adult female morphological characters for differentiation Tetranychus urticae complex (Acari: Tetranychidae) from greenhouse tomato in UK. Systematic and Applied Acarology 5, 69-76.