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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.
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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).
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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.
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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.
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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
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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
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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
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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.
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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
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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.
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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.
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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.
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8 REFERENCES
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