Baseline Study of Coconut Scale, Aspidiotus Destructor Signoret (Hemiptera: Diaspididae) and Its Management on Mango (Mangifera Indica L.)

BASELINE STUDY OF COCONUT SCALE, ASPIDIOTUS DESTRUCTOR SIGNORET (HEMIPTERA: DIASPIDIDAE) AND ITS MANAGEMENT ON MANGO (MANGIFERA INDICA L.)

SALAHUDDIN

A dissertation submitted for partial fulfillment of the requirement for the degree of

DOCTOR OF PHILOSOPHY

ENTOMOLOGY

DEPARTMENT OF ENTOMOLOGY FACULTY OF AGRICULTURE GOMAL UNIVERSITY DERA ISMAIL KHAN

2016

DEDICATED TO MY PARENTS

ACKNOWLEDGEMENT All glory be to Allah, the beneficial, the merciful. I would like to thank Almighty Allah who enabled me to complete this painstaking study of PhD well in time. I would like to express the deepest appreciation to my supervisor Dr. Habib ur Rahman, co- supervisors Dr. Inamullah Khan and Dr. Muhammad Daud Khan for their substantial guidance, continuous inspiration, invaluable assistance and suggestions from initial planning of experiments to the preparation of manuscripts in bringing this dissertation to its present form. I would like to thank my IRSIP program supervisor Dr. Steven P. Arthurs at Mid- Florida Research and Education Center, IFAS, University of Florida, USA for his cooperation to conduct research in his laboratory and proof reading and correction of my PhD dissertation. His deep knowledge and experience helped me to learn advance skills in Entomology and improve my scientific writing. I am grateful to my other supervisory committee members Professor Dr. Muhammad Ayaz Khan, Agha Shah Hussain and Dr. Muhammad Mamoon ur Rashid for their excellent guidance and suggestions on how to improve my work. Dr. Gillian W. Watson, Senior Insect Biosystematist, California Department of Food and Agriculture, Plant Pest Diagnostic Center, Sacramento California, U.S.A., is gratefully acknowledged for identification of Aspidiotus destructor. I would like to acknowledge the Higher Education commission (HEC), Islamabad, Pakistan for providing financial assistance for the duration of my PhD research through Indigenous 5000 PhD fellowship program and Department of Agriculture Extension, Government of Khyber Pakhtunkhwa for granting three years study leave. I am thankful to The Nuclear Institute for Food and Agriculture (NIFA), Peshawar, Kohat University of Science and Technology, Kohat and Mid-Florida Research and Education center (MREC), Apopka, University of Florida, USA for allowing me to work in their laboratories. I am thankfull to Arid Zone Research Institute (AZRI), Ratta Kulachi, D. I. Khan for providing metereological data during 2011-12 and 2012-13. I thank Imran Khan (AHO) and his staff to allow me to work in Fruit Nursery Farm and provided technical and labor help in growing and handling mango nursery plants. I am grateful for the technical help of Dr. Hafiz Mumtaz Khan, Dr. Farmanullah, Dr. Kalimullah, Tariq Nawaz, Waqas Yousafzai and Muhammad Suleman Shah. I would like to deeply appreciate Mishkatullah, Muhammad Nisar, Ejaz Malook, Saeed Hassan, Syed Fayaz Ali Shah, Luis Aristizábal, Robert Leckel, Nastaran Tofangsazi, Runquian Mao and Yingfang Xiao for supporting me as a lab assistant and as a friend. I iv

would like to thank all of them for making Peshawar, Kohat and Florida a home away from home. I appreciate the love and support that I received from friends Shah Namir Khan, Muhammad Gul, Imran Khan, H. Attaullah, Muhammad Riaz, H. Nabi Gul, Adnan khan, M. Tariq Khan, Sher Zaman, Waliullah, and all others whose names are much important but not written here. I have no words at command of acknowledging my late parents who taught me values, hard work and truthfulness in life, without whom I would not be the person as today. My love and pray for them is eternal. Their sincere prayers empowered me to achieve success in every sphere of life. May their soul rest in peace. Ameen. I appreciate the determination of my wife and kids, Rimsha Gul, Hammad Ahmad Khan, Iffat Gul and Fanila Gul who endured my long absence from home. I will never forget the moment when I would go to Peshawar for research work, my son would sit angry in the corner of home that “why Dad is going to Peshawar”. Their love provided me inspiration and was my driving force. I owe them everytime and wish I could show them how much I love them. I would like to thank my brothers Dr. Razaman Bittan, Dr. Ibrahim Gul Bittani, Advocate Noor zaman Bittani, my sister, my nephews Humaira, Sawaira, Afaq, Jawad and Hurrain for their long distance support. Salahuddin Bittani

List of Contents Chapter Contents Page No. ACKNOWLEDGEMENT iv LIST OF CONTENTS vi LIST OF TABLES xiii LIST OF FIGURES xv LIST OF ABBREVIATIONS xvii ABSTRACT xix 1. GENERAL INTRODUCTION AND REVIEW OF LITERATURE 1 1.1. Economic impact of mango 1 1.2. Pests of mango 1 1.3. Overview of scale insects 2 1.4. Armored scales 3 1.5. Aspidiotus destructor 3 1.5.1. Distribution 4 1.5.2. Economic importance 5 1.5.3. Use of common names of A. destructor 5 1.5.4. Seasonal population dynamics of A. destructor 7 1.5.5. Natural enemies of A. destructor 7 1.5.6. Reproduction in A. destructor 8 1.5.6.1. Common feature of A. destructor 8 1.5.6.2. Morphological description 9 1.5.7. Physiological, biochemical and molecular changes in mango 10 leaves under scale insects infestation 1.5.7.1. Ions analysis 11 1.5.7.2. Physiological changes in mango plant under insect 12 infestation 1.5.7.3. Biochemical changes in mango plant under insect 13 infestation 1.5.7.4. Molecular changes in mango plant under insect infestation 15 1.5.8. Development of generic doses of irradiation for the growth inhibition of A. destructor 16

1.5.9. Physiochemical analysis and sensory evaluation of mango fruits 18 1.5.10. Efficacy of different Insecticides against A. destructor 21 1.6. Aims 23 2. GENERAL MATERIALS AND METHODS 24 2.1. Study area at a glance 24 2.2. Raising mango nursery plants and release of A. destructor crawlers on test plants 24 2.3. Collection and Identification of scale insects on mango plants 24 2.4. Natural efficiency of parasitoids on A. destructor: 25 2.5. Biology of A. destructor on mango plants under laboratory and greenhouse conditions 25 2.6. Scale-induced, physiological, biochemical and molecular changes in mango leaves 25 2.6.1. Physiological changes in mango leaves under A. destructor infestation 26 2.6.2. Biochemical changes in mango leaves under A. destructor infestation 26 2.6.3. Molecular changes in mango leaves under A. destructor infestation 26 2.7. Effect of irradiation on pest mortality and development inhibition 26 2.8. Effect of irradiation on physiochemical analysis and sensory quality of mango fruit 27 2.9. Evaluation of different insecticides against A. destructor on mango nursery plants 27 2.10. Evaluation of different insecticides against A. destructor on mature mango trees 28 2.11. Experimental design / statistical analysis 28 3. INCIDENCE OF ASPIDIOTUS DESTRUCTOR IN RELATION TO ABIOTIC FACTORS AND NATURAL EFFICIENCY OF ITS 29 PARASITOIDS 3.1. Introduction 29 3.2. Materials and Methods 30

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3.2.1. Population dynamics of A. destructor on four mango c cultivars at five locations 30 3.2.2. Natural efficiency of parasitoids on A. destructor 30 3.3. Results 31 3.3.1. Population dynamics of A. destructor on four mango 31 cultivars 3.3.2. Population dynamics of A. destructor at five locations on 33 mango plants 3.3.3. Relation of A. destructor population fluctuation with abiotic 37 factors 3.3.4. Natural efficiency of A. melinus and Anagyrus sp. on A. 38 destructor 40 3.4. Discussion and conclusion 4. BIOLOGY OF A. DESTRUCTOR ON MANGO PLANTS (MANGIFERA SP.) UNDER LABORATORY AND GREENHOUSE 43 CONDITIONS 4.1. Introduction 43 4.2. Materials and Methods 44 4.2.1. Laboratory study 44 4.2.2. Greenhouse study 45 4.3. Results 45 4.3.1. Laboratory study 45 4.3.1.1. Aspidiotus destructor developmental stages and morphometry 45 4.3.1.2. Development of male Aspidiotus destructor 48 4.3.1.3. Development of female Aspidiotus destructor 48 4.3.2. Greenhouse study 49 4.4. Discussion and conclusion 51

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5. PHYSIOLOGICAL AND BIOCHEMICAL CHANGES IN MANGO LEAVE UNDER A. DESTRUCTOR STRESS 54 5.1. Introduction 54 5.2. Materials and Methods 56 5.2.1. Raising nursery of test plants 56 5.2.2. Determination of leaf biomass, leaf area and photosynthetic pigments 56 5.2.3. Cell viability and cell injury 57 5.2.3.1. Cell viability 57 5.2.3.2. Membrane leakage 57 5.2.4. Determination of various ions (Na+, K+, Ca++) 58 5.2.5. Determination of stress markers 58 5.2.6. Determination of total soluble proteins (TSP) and antioxidants 59 5.2.7. Proline accumulation in mango leaves under scale insects infestation 59 5.3. RESULTS 60 5.3.1.Physiological changes in mango leaves under scale 60 infestation 5.3.2. Changes in photosynthetic pigments 61 5.3.3. Changes in cell viability under scale insect infestation 62 5.3.3.1. TTC method 62 5.3.3.2. Electrolyte leakage under scale insect infestation 63 5.3.4. Scale-induced ionic changes in mango leaves 63 5.3.5. Effect of scale insect infestation on total soluble protein (TSP) contents and proline accumulation 64 5.3.6. Changes in stress markers under scale insects infestation 65 5.3.7. Changes in the antioxidant enzymes of mango leaves under 66 scale insects infestation 5.4. Discussion and conclusion 68

6. MOLECULAR CHANGES IN MANGO LEAVES UNDER SCALE INSECT INFESTATION 74 6.1. Introduction 74 6.2. Materials and Methods 74 6.2.1. DNA damage analysis 74 6.2.1.1. DNA isolation 74 6.2.1.2. RAPD primers 75 6.2.1.3. Amplification condition 75 6.2.1.4. Gel electrophoresis 75 6.2.1.5. RAPD data analysis and scoring 76 6.3. Results 76 6.4. Discussion and conclusion 81 7. EFFECT OF CO-60 GAMMA IRRADIATION ON THE MORTALITY AND GROWTH INHIBITION OF A. DESTRUCTOR 83 7.1. Introduction 83 7.2. Materials and Methods 84 7.2.1. Preparing test plants 84 7.2.2. Radiation treatments 84 7.2.3. Data analysis 85 7.3. Results 85 7.4. Discussion and Conclusion 90

8. PHYSIOCHEMICAL ANALYSIS AND SENSORY EVALUATION OF MANGO FRUIT 92 8.1. Introduction 92 8.2. Materials and Methods 93 8.2.1. Weight loss 93 8.2.2. Total soluble solids (TSS) 93 8.2.3. Titrable acidity 93 8.2.4. Ascorbic acid (vitamin C) 94 8.2.5. pH 94 8.2.6. Sensory analysis of irradiated and unirradiated mango fruits 94 8.2.7. Statistical analysis 95 8.3. Results 95 8.3.1. Percent weight loss 95 8.3.2. Firmness 96 8.3.3. Total soluble solids (TSS) 99 8.3.4. Titrable acidity 99 8.3.5. Ascorbic acid (vitamin c) 100 8.3.6. pH 101 8.3.7. Sensory analysis of irradiated and unirradiated mango fruits 101 8.4. Discussion and conclusion 104 9. EFFICACY OF DIFFERENT INSECTICIDES AGAINST VARIOUS DEVELOPMENTAL STAGES OF A. DESTRUCTOR 107 9.1. Introduction 107 9.2. Materials and Methods 108 9.2.1. Evaluation of insecticides on mango nursery plants 108 9.2.2. Evaluation of insecticides on mature mango trees 109 9.3. Results 109 9.3.1. Efficacy of insecticides against various stages of A. destructor on mango nursery plants 109 9.3.2. Efficacy of insecticides against A. destructor on mature mango plants 112 9.4. Discussion and conclusion 115

10. GENERAL DISCUSSION AND CONCLUSION 117 10.1. Summary 117 10.2. Conclusion 120 10.3. Future work 121 REFERENCES 122 FIGURES WITH CAPTIONS 159 APPENDIX 165

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List of Tables Table Title Page No. 3.1. Relationship of A. destructor population with abiotic factors during 2011– 38 12 and 2012–13. 4.1. Morphometry (length and width) and developmental rate (days) of male 47 and female A. destructor developmental stages on mango nursery plants in laboratory at temperature 30 °C and RH 64 %. 4.2. Developmental rate (days) of male and female A. destructor on different 50 cultivars of mango nursery plants in greenhouse at temperature 25°C and RH 60 %. 5.1. Effect of A. destructor on leaf area and leaf biomass in mango leaves 61 5.2. Effect of A. destructor on chlorophyll a and b and carotenoid contents in 62 mango leaves 5.3. Effect of A. destructor on cell viability and electrolyte leakage in mango 63 leaves 5.4. Effect of A. destructor on ions analysis in mango plant leaves 64 5.5. Effect of A. destructor on total soluble protein (TSP) contents and proline 65 accumulation in mango leaves 5.6. Effect of A. destructor on malandialdihyde (MDA) contents and hydrogen 66

peroxide (H2O2) in mango leaves 5.7. Effect of A. destructor on peroxidase (POD) and superoxide dismutase 67 (SOD) in mango leaves 5.8. Effect of A. destructor on ascorbate peroxidase (APX) and catalase 68 (CAT) activities in mango leaves 6.1. RAPD primers with their sequences and GC %. 76 6.2. Changes of total bands in control and of polymorphic bands and varied 79 bands in mango leaves under mango scale insects stress in three mango cultivars. 6.3. Molecular size (base pair, bp) of appearance and disappearance of bands 80 for all primers in mango leaves under mango scale insects stress. 7.1. Effect of gamma irradiation on maturation development of exposed 1st 86 instar nymph of A. destructor on mango leaves

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7.2. Effect of gamma irradiation on maturation of 2nd instar A. destructor 87 7.3. Effect of gamma irradiation on mature female of A. destructor 87 7.4. Effect of gamma irradiation on eggs hatch and mature female with eggs 88 of A. destructor 7.5. Growth inhibition (%) of A. destructor at different development stages 89 after 60 days of irradiation 7.6. Large-scale validation tests irradiating A. destructor females with eggs 89 and without eggs 8.1. Regression coefficient for the effect of different doses (0–250 Gy) of 98 Gamma Irradiation on the physiochemical analysis of mango fruit at various storage duration. 8.2. Changes in titrable acidity of mango fruits after exposing to different 100 doses of gamma irradiation compared with control. 8.3. Changes in vitamin c contents of mango fruits after exposing to different 100 doses of gamma irradiation compared with control. 8.4. Changes in pH of mango fruits after exposing to different doses of 101 gamma irradiation compared with control. 9.1. Formulations and recommended rate of different Insecticides evaluated as 108 spray treatments on mango nursery plants and mature trees. 9.2. Efficacy (% mortality) of different insecticides against A. destructor life 111 stages on mango nursery plants during 2012 9.3. Efficacy (% mortality) of different insecticides against A. destructor life 113 stages on mango nursery plants during 2013 9.4. Efficacy (% mortality) of different insecticides against A. destructor on 114 mature mango trees during 2012 and 2013

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List of Figures Figure Title Page No. 3.1 Population dynamics (±SEM) of A. destructor on different mango 32 varieties during 2011–12. 3.2. Population dynamics (±SEM) of A. destructor on different mango 33 varieties during 2012–13. 3.3. Population dynamics (±SEM) of A. destructor at different locations on 34 mango plants during 2011–12. 3.4. Population dynamics (±SEM) of coconut scale, A. destructor at various 34 locations on mango during 2012–13. 3.5. Overall Population dynamics of A. destructor during 2011–12 and 2012– 35 13. 3.6. Population trends in A. destructor infestation during 2011–12 (a) and 37 2012–13 (b). 3.7. Per leaf populations (±SEM) of A. destructor and percent parasitization 39 (±SEM) of Aphytis melinus and Anagyrus sp. on mango plants during 2011–12. 3.8. Per leaf populations (±SEM) of scale insect, A. destructor and 39 parasitization (±SEM) of Aphytis melinus and Anagyrus sp. on mango plants in 2012–13. 6.1. RAPD profile of genomic DNA in the leaves of mango plant exposed to 78 0, 10, 20 and 30 days scale insect infestation using primer OPA 10. a. Langra, b. Kala Chounsa, c. Anwar Ratoul. Marker (100–1000 bp). 6.2. RAPD profile of genomic DNA in the leaves of mango plant exposed to 78 0, 10, 20 and 30 days scale insect infestation using primer OPC 01. a. Langra, b. Kala Chounsa, c. Anwar Ratoul. Marker (100–3000 bp). 6.3. RAPD profile of genomic DNA in the leaves of mango plant exposed to 78 0, 10, 20 and 30 days scale insect infestation using primer RPl 3. a. Langra, b. Kala Chounsa, c. Anwar Ratoul. Marker (100–3000bp). 8.1. Changes in % weight loss (±SEM) of mango fruits after exposing to 96 different gamma irradiation doses compared with control. 8.2. Regression lines of gamma irradiation doses at different time intervals in 97

respect to: (A) % weight loss (B) Firmness (C) TSS (D) Acidity (E) Vitamin c (F) pH in mango fruit 8.3. Firmness changes (±SEM) in mango fruits after exposing to different 97 gamma irradiation doses compared with control. 8.4. Total soluble solids changes (±SEM) in mango fruits after exposing to 99 different gamma irradiation doses compared with control. 8.5. External appearance scoring of mango fruits after exposing to different 102 gamma irradiation doses compared with control. 8.6. Odor scoring of mango fruits after exposing to different gamma 102 irradiation doses compared with control. 8.7. Taste scoring of mango fruits after exposing to different gamma 103 irradiation doses compared with control. 8.8. Texture scoring of mango fruits after exposing to different gamma 103 irradiation doses compared with control. 8.9. Overall acceptability scoring of mango fruits after exposing to different 104 gamma irradiation doses compared with control.

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List of abbreviations µm Microgram µmol Micromole ABI Applied Biosystem Inc., USA. AFLP Amplified fragment length polymorphism ANOVA Analysis of variance APX Ascorbate peroxidase bp Base pair CAT Catalase CI Chloroform Isomylalcohal DIKhan Dera Ismail Khan DNA Deoxyribonucleic acid EB Ethidium bromide EC Emulsifiable concentrate EDTA Ethylenediamine tetra acetic acid FAO Food and Agriculture organization FW Fresh weight GC Guanine cytosine gm Gram Gy Grays

H2O2 Hydrogen per oxide HCl Hydrochloric acid IGR Insect growth regulator IPM Integrated pest management JH Juvenile hormone Kb Kilobyte KP Khyber Pakhtunkhwa L Liter MDA Malondialdehyde Mg Magnesium mg Milligram Mgcl2 Megnesium chloride ml Milliliter xvii

mM Millimole Nacl Sodium chloride NIFA Nuclear Institute for Food and Agriculture O– Super oxide OH– Hydroxyl ion PARAS Pakistan Radiation services PCR Polymerase chain reaction POD Peroxidase PVP Polyvinyl-Pyrrolidine QTL Quantitative trait loci RAPD Random amplified Polymorphic DNA RCBD Randomized Complete Block Design REFL Restriction fragment length polymorphism RH Relative humidity ROS Reactive oxygen species SB Stylet bundle SDS Sodium Dodecyl sulfate SL Soluble liquid SOD Superoxide dismutase SSR simple sequence repeats TSP Total soluble proteins TTC Triphenyltetrazolium chloride USDA U.S. Department of Agriculture v/v Volume by volume VNTR Variable number tandem repeats VOCs Volatile organic compounds W/W Weight by weight % WG Wettable Granules – O 2 Superoxide radical OH– Hydroxyl radical ROOH Hydroperoxides ROO. Alkylperoxy RO Alkoxy radicl xviii

ABSTRACT Aspidiotus destructor Signoret (Hemiptera: Diaspididae) is a very serious insect pest in both tropical and subtropical regions. The pest causes chlorosis, defoliation and dieback on wide range of crops. In Pakistan, both early and late season mango cultivars are frequently infested by A. destructor from July to December each year. Despite heavy losses to mango crops, little information about the seasonal population fluctuation, general biology and management are known about this pest in Pakistan. This study was undertaken to (1) establish baseline information regarding population dynamics, efficiency of native parasitoids of A. destructor and biology; (2) physiological, biochemical and molecular changes in mango plant leaves under A. destructor infestation; and (3) use of gamma irradiation and insecticides for the control of various stages of A. destructor. In the first objective, I determined that the populations of A. destructor built up in July with peak in October to November and lowest from December to June and having a significant correlation with wind velocity and rainfall. Parasitism by Aphytis melinus DeBach (Hymenoptera: Aphelinidae) and an Anagyrus sp. (Hymenoptera: Encyrtidae) on A. destructor reached to peaks in November (13.6 and 16.3 %) and December (14.2 and 14.9 %) during 2011–12 and 2012–13, respectively. In developmental biology study, I determined that males had four immature stages and an adult stage and females had two immature stages with pre-oviposition and oviposition stages; and total developmental span for male and female was 27 and 39.5 days, respectively. Physiological parameters such as leaf area, leaf biomass, chlorophyll a, b and carotene contents as well as total soluble protein (TSP) decreased while proline accumulation increased with increasing A. destructor infestation period. Stress markers like malandialdihyde (MDA) and hydrogen peroxide (H2O2) as well as antioxidants enzymes such as superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT) were determined as per their established protocols. Increasing trend in MDA contents indicated lipid peroxidation while up regulation of antioxidants enzymes revealed a well- defined defense response to scale insects infestation in the three mango cultivars. DNA (Deoxyribonucliec acid) damage study was conducted by random amplified polymorphic DNA (RAPD) technique using different markers. This technique confirmed that RAPD is a very effective tool in investigating genotoxicity caused by A. destructor in mango leaves. Co- 60 gamma irradiation was used to determine mortality of various stages of A. destructor at Nuclear Institute for Food and Agriculture (NIFA), Peshawar. I found egg stage as the most susceptible and 2nd instar nymphs as the most tolerant stage to gamma irradiation. In large- xix

scale validation tests, a dose of 228 Gy provided quarantine security for all stages of A. destructor on exported commodities. Physiochemical analysis and sensory quality of irradiated and un-irradiated mango fruits were investigated. I determined that irradiation had no negative impact on mango fruit, but rather imcrease it’s shelf life as irradiated fruits retained its nutritional quality and were acceptable to consumer for long time on the basis of sensory quality scoring than un-irradiated fruits. I evaluated eight insecticides against A. destructor, in which highest percent mortalities was recorded with petroleum oil, imidacloprid and pyriproxyfen as compared to mineral oil, thiamethoxam, bifenthrin and profenofos against all life stage of the scale insect. Overall results of my dissertation suggest that management strategy plan for A. destructor may be designed in June-July with focus on crawler stage and insecticides application may be done in July or early August when there is least parasitoid ctivities in the field. I recommend irradiation dose of 228 Gy for 100 % mortality of A. destructor in mango fruits for export purpose. For future study predators, viruses and other parasitiods associated with A. destructor can be explored with quantification of irradiation stress to mango plant.

Chapter 1.

General introduction and review of literature

1.1. Economic impact of mango

Mango (Mangifera indica L.) (Anacardiaceae) has been called the ‘king of all fruits’ and is grown in more than 100 countries worldwide (Galan Sauco, 2002; Tharanathan et al., 2006; Singh and Singh, 2011). Mango cultivation in South Asia has occurred for at least 4,000 years, which is why this region is considered as the home of mango (Salunkhe and Desai, 1984). Mango is a rich source of carbohydrates, vitamin A and C, iron, potassium, calcium and protein (Baloch, 1994).

The total production of mango among the top 100 mango producing countries in tropical and subtropical areas exceeds 34.3 million tons per annum (Galan Sauco, 2010). Overall, 80 % of mango is produced and 90 % is consumed by nine countries including India, China, Indonesia, Mexico, Thailand, Pakistan, Brazil, The Philippines and Nigeria. Asia is the largest producer region of mango with 26 million tones, Africa 3.4 million, North and Central America 2.9 million and South America 2.0 million tones (Galan Sauco, 2010). Mango is mainly exported by India (9.7 % of world production), Mexica (3.2 %) and Peru (10.3 %). The largest importing regions include European communities (34 %), USA (20 %) Asia (27 %) and Arabian Penunsula (14 %) (Gerbaud, 2009).

Mango is ranked as the second most important fruit after citrus, based on area in production, and third most valuable cash crop after cotton and rice in Pakistan (Anonymous, 2011). Pakistan is the 5th major producer (FAO, 2013) and 3rd major exporter (Shahnawaz et al., 2012) of mango worldwide. During 2010–11, mango crop occupied 171.9 thousand hectares and its total production was 1885.9 thousand tones/annum in Pakistan. The share of Khyber Pakhtunkhwa was 0.3, Punjab 111.9, Sindh 59.2 and Baluchistan was 0.5 thousand hectares. In production, Punjab produced 1503.2, Sindh 381.3, Khyber Pakhtunkhwa 0.3 and Baluchistan 1.1 thousand tones per annum (Anonymous, 2010–11).

1.2. Pests of mango

Mango is attacked by at least 86 different species of insect pests in Pakistan (Qureshi and Mohyuddin, 1982) which include the fruit flies (Dacus zonatus Sunsers and D. darsalis

Hendel), scale insects (Aspidiotus destructor Signoret, Lindingaspis ferrisi McKenzie, Aulacaspis tubercularis Newstead, Parlatoria crypta McKenzie, Coccus hesperidum L., C. mangiferae L. and Pulvinaria psiddi Maskell, mealybug (Drosicha stebbingi Green) and mango hoppers (Idioscopus niveosparsus Lethierry, I. nagpurensis Pruthi and Amritodus atkinsoni Leth.) (Mohyuddin and Mahmood, 1993). Of these, 27 scale insects species recorded from Pakistan (Mohyuddin, 1981), in which Aspidiotus destructor Signoret and Lindingaspis ferrisi belonging to family Diaspididae are the most serious pest of mango. Beside mango, A. destructor also attacks Cocos nucifer L. in Pakistan. Every year, heavy infestation has been observed on mango in both coastal and sub-coastal areas of Pakistan from August to December (Ahmad and Ghani, 1972). In Pakistan, A. destructor was first reported on mango plants from Punjab Province by Rehman and Ansari (1941) and Sindh Province by Cavin in 1956 (UK CAB International, 1966). A short account of the damage caused by A. destructor to the mango and its distribution in Pakistan was provided by Ansari (1942).

1.3. Overview of scale insects

Scales (Hemiptera: Coccoidea) are diverse group of insects consisting of eight thousand species in 30 families (Gullan and Cook, 2007). The superfamily Coccoidea includes family Diaspididae with 2369 species (32 %), Pseudococcidae 2048 (28 %), Coccidae 1129 (15 %) and 1179 species (16 %) are in all other families (Ben Dov et al., 2003). Kondo and Kawai (1995) reported 28 species in 22 genera and 5 families in Colombia and were the 2nd most important pests of mango after fruit flies. Among them, armored scale insects constitute 50 % of the total reported species. Many species of scale insects are extraordinarily invasive due to the adaptation of both the individual and the population to varying environmental conditions (Ben-Dov, 1994). However, the impact of infestation depends on the species, the host, environmental factors and natural enemies (Moiler, 1996). Scale insects have a wide host range including mango (Germain et al., 2010), tomato (Culik and Gullan, 2005), and cotton (Wu and Zhang, 2009; Nagrare et al., 2009). Scale insects reproduce both sexually and parthenogenetically with some species have sexual diamorphism (Ghabbour, 1995, Ghabbour and Mohammad, 1998 and Moharum, 2006). Life cycle and biological activities of scale insects vary in their natural environment (Vennila et al., 2010).

1.4. Armored scales

Armored scales insects (Diaspididae) constitute an important group of agriculture pests (Rosen and DeBach, 1978). These insects infest a variety of crops and cause symptoms including leaf curl, defoliation and in case of severe infestation, fruits become malformed, dwarfed and substandard in quality (Ahmad and Ghani, 1972; Gupta and Singh, 1988; Moffit, 1999; Miller and Miller, 2003). Plants are damaged by direct feeding as well as injecting toxins by the insects to the leaves causing stunted growth of plants, defoliation, shedding of flowers and may kill the plant (Plant Protection Daklak, 2004). In 2004–05, coffee plantations in Daklak (Vietnam) were severely affected by scale insects damaging 2,873 out of 27,418 hectares area.

Armored scales have a circular, sub circular, elongate, thread like or a characteristic oyster shell shape (Ghabbour and Mohammad, 1998). Two pairs of thoracic spiracles are present in the females, but have no abdominal spiracles. The wax is secreted by the wax glands of the pygidium and the exuviae are added to the armor during molting. Body is not attached to the scale cover between molts. The crawlers of armored scale insects are either born alive (viviparous) or hatched from eggs (oviparous). Unlike other scale insects, the armored scales do not produce honeydew (Beardsley and Gonzalez, 1975).

Armored scale insects reproduce both sexually and asexually, while some species are oviparous, ovoviviparous and viviparous (Koteja, 1990a). Sometime females lay eggs that produce only male scales. It is, therefore, assumed that fertilized reproduction may also occur which is influenced by food availability (Taylor, 1935). Boratynski (1953) illustrated dimorphism in the second instar of seven armored scale insects species and provided valuable information pertaining to the morphological difference between the two sexes at immature stages. Ghabbour (1995) and Ghabbour and Mohammad (1998) reported sexual dimorphism in the second instar of Aonidiella orientalis Newstead (Diaspididae). Females have three immature feeding instars while in male, the two feeding instars are followed by pre-pupal, pupal and adults stage (Koteja, 1990b).

1.5. Aspidiotus destructor

Among armored scales, Aspidiotus destructor Signoret is a polyphagous and cosmopolitan pest of over 75 genera in over 60 plant families (Davidson and Miller, 1990; Ben-Dov 2014).

It commonly infests coconut and banana but attacks on wide range of fruit trees and ornamental plants including various species of palms, avocado, mango, guava, citrus, papaya, rubber plant, breadfruit, ginger, bird of paradise, sugarcane, mountain apple, plumeria, tea, brassica, capsicum, grape and tomato (Ferris, 1938; Williams and Watson, 1988; Davidson and Miller, 1990; Chua and Wood, 1990; Howard et al., 2001; Mariau, 2001; CPC, 2007). Dekle (1965) recorded 140 hosts of A. destructor from Florida, USA. It is considered as the most damaging pest for coconut palm in Philippines where it attacks both foliage and fruits but it is much more destructive for nursery plants than mature palm trees (Nafus and Schreiner, 1989; Figure 1.1).

1.5.1. Distribution

A. destructor was first described by Signoret in 1869 from coconut palm in South Asia (Signoret, 1869). The pest appears to be native to South Asia; but has been spread all over the world mainly on infested coconut and banana (UK CAB International, 1966; Figure 1.2). A. destructor reached many Caribbean islands by 1902 but its pest status was not noted until 1950 (Goberdhab, 1962). In the 20th century, the pest was regularly reported from many tropical areas of the eastern and western hemisphere in 1962 (Chazeau, 1981), San Andresen Caribbean in 1974 (Mosquera, 1977) and Hawai’ian island in 1968 (Tenoria, 1969). It also remained a pest in the northern hemisphere in temperate areas like China, Russia and Georgia under glass or greenhouses (Dzhashi, 1989; Tang and Qin, 1991; Danzig and Pellizzari, 1998). The pest has been reported on banana worldwide including Mozambique (Pais, 1973), Costa Rica (Quezada et al., 1973), and the Philippines (Tabibullah and Gabriel, 1975). The pest was present on many Pacific Islands but it was not known until 1968 when discovered on coconut palms at Kailua, Oahu (Tenorio, 1969). It has also been recorded from China, Southeast Asia, India, Pakistan, Russia, Brazil, Central America and Caribbean, the Pacific Islands, Africa and North America (UK CAB International 1966; Bennett and Alam, 1985; Burger and Ulenberg, 1990; Tao 1999, Claps et al. 2001). In the United States, A. destructor has been recorded from Florida, Georgia, Pennsylvania, Connecticut and Hawaii (Nakahara, 1982; Heu, 2002). It is also found in American Samoa, Australia, Fiji, Northern Mariana Islands, Solomon Islands (Williams and Watson, 1988; CPC, 2007). A. destructor was first documented from North America during the 1930s. It was a minor pest of ornamental plants in Florida but was under control of natural enemies introduced from Puerto Rico (Clausen, 1956). 4

1.5.2. Economic importance

A. destructor mainly infests the under side of leaves but also frond stalks, flower, fruits and younger plants; however, trees more than 4 years old or in well-drained soil are less infested (Tabibullah and Gabriel, 1973; Watson, 2005; Figure 1.3). A. destructor sucks cell sap from leaves, stems and fruits, causing yellowing, tissue distortion and dieback. Yellowing of the leaves are caused by the sap sucked by the scale and the toxic saliva injected to the leaves (Waterhouse and Norris, 1987). The infested leaves may prematurely die, vigor of plant reduced, fronds drop off and the crown may dies within short period of time (Chua and Wood, 1990; Figure 1.4). In an environment, where natural enemies are scarce, complete defoliation has been observed due to the infestation of A. destructor (Debach, 1974).

A. destructor has been kept under control by natural enemies in most part of the world but still there are outbreaks recorded in recent years. Copra (coconut) production was reduced to one third (from 1400 to 500 tones/year) in Principe Island, Sao (West coast of Africa) in 1955 after three years of the invasion of A. destructor before the introduction of biological control strategies (Simmonds, 1960; Rosen, 1990a). A. destructor damaged 300,000 out of 400,000 coconut plants over three years in North Celebes (Indonesia) and reduced the coconut production up to one third in 1955 within three years of its invasion (Reyne, 1948; Simmonds, 1960). The coconut industry was destroyed by A. destructor in Mauritius in 10 years and the coconut plants nearly disappeared (Rosen and DeBach, 1978). In the Ivory Coast, heavy attack of A. destructor reduced the yield of coconut up to 25 % in 2 to 3 years (Mariau and Julia, 1977).

A. destructor is of great concern due to its relatively short life cycle of ~ 35 days and 9–10 overlapping generations per year. The pest is mainly dispersed by other creatures like birds, insects, bats and some time by human through the transport of nursery plants and goods like coconut baskets made of plant materials (Taylor, 1935). It is transported to distant areas by animals or commercial transport as well as by suckers of banana (Dharmaraju and Laird, 1984).

1.5.3. Use of common names of A. destructor

Since reported by Signoret in 1869, A. destructor has received various names including bourbon scale and transparent scale. According to the Entomological Society of America

(ESA), its current official name is coconut scale. Other scientific names which are no more in use are Aspidiotus cocotis Newstead, 1893, Aspidiotus lataniae Green, 1896, Aspidiotus simillimus translucens Fernald, Aspidiotus translucens Cockerell and Robinson, 1915, Aspidiotus transparens Green, 1890, Aspidiotus vastatrix Leroy, Aspidiotus watanabei Takagi, 1969 and Temnaspidiotus destructor (Signoret) Borchsenius. Its international common names are. Its local common names are Schildlaus and Kokospalmen (Germany) and Cocciniglia del cocco (Italy). Its taxonomic tree is given below:

Domain: Eukaryota

Kingdom: Metazoa

Phylum: Arthropoda

Subphylum: Uniramia

Class: Insecta

Order: Hemiptera

Suborder: Sternorrhyncha

Superfamily: Coccoidea

Family: Diaspididae

Genus: Aspidiotus

Species: destructor

Source: http://www.cabi.org/isc/datasheet/7415 (February, 2015).

Other species confused with A. destructor include Aspidiotus cryptomeriae Kuwana and Aspidiotus excisus (Williams and Watson, 1988). Aspidiotus cryptomeriae Kuwana has shorter marginal setae of the median lobes by the outer corners while Aspidiotus excisus has marginal prepygidial macroducts on either side of abdomen, dorsal setae on the outer corner of median lobs and short dorsal pygidial macroduct. Reyne (1948) reported subspecies of A. destructor as Aspidiotus destructor rigidus from Sulawesi (Indonesia) which had thicker prosomal cuticle than A. destructor.

1.5.4. Seasonal population dynamics of A. destructor

Monitoring of coconut scales is mostly based on direct counts of the total number of scale insects per leaf and the proportion of plant parts infested (Nestle et al., 1995; Berlinger et al., 1999; Shaw et al., 2000). Pheromone trapping and yellow sticky traps are used to estimate the pest population densities (Gieselmann et al., 1979; Jactel et al., 1996). However, a detailed survey of the pest on mango plants and screening of mango resistant/susceptible cultivars in relation to abiotic factors have not been undertaken. The pest has 9 generations in tropical areas while 3 generations in Hunan (China) (Tabibullah and Gebriel, 1975; Tang and Qin, 1991; Zhou et al., 1993). Very little information is available about the peak and dormant season of A. destructor on mango in Pakistan.

1.5.5. Natural enemies of A. destructor

Reliance on natural enemies for the management of A. destructor and implementation of biological control measures has a long history in many countries of the world (Moutia and Mamet, 1946; Reyne, 1948; Simmonds, 1960; Cochereau, 1965; Gaprindashvili, 1975; Wu and Tao, 1976; Lai and Funasaki, 1983; Kinawy, 1991). The distribution of predators and parasitoids depends upon the climatic condition and the density of hosts (Rosen and DeBach, 1978; Hely et al., 1982; Foster et al., 1995). In general, parasitoids are more effective than predators as well as have economic edge over chemical control (DeBach et al., 1953; Hely et al., 1982; Sorribas et al., 2010).

Coccinellid predators like Pseudoscymnus anomalus Chapin, Rhyzobius satelles Blackburn, Cryptognatha nodices Marshall, C. gemellata Mulsant, Chilocorus nigritus F. and C. malasiae Crotch have been recorded to be very effective against A. destructor in Fiji, Mauritius and Indonesia (Taylor, 1935; Beardsley, 1970; Waterhouse and Norris, 1987). C. nodiceps is a voracious predator having high preference for coconut scale while P. anomalus is a specific predator of A. destructor that requires 14 days to develop to adulthood with high reproductive capacity and long life at adult stage (Waterhouse and Norris, 1987). Other predators that reduced A. destructor population in different regions include Telsimia nitida Chapin and Lindorus lophanthae Blaisdell and predaceous thrips, Aleurdothrips fasciapennis Franklin (Beardsley, 1970; Waterhouse and Norris, 1987). I observed many predators feeding on A. destructor but predator study was not covered by my PhD research proposal. 7

Aphytis spp. are the most common external parasitoids of A. destructor and play a major role in controlling the scale insect population in different areas (Taylor and Paine, 1935; Beardsley, 1970). A. destructor remained unchecked in Fiji, New Hebrides (Vanuatu) and Mauritius until parasitoids was introduced. DeBach (1974) discovered that A. melinus is the most effective parasitoid of A. destructor in Fiji. Hosts of A. melinus include coconut scale, red scale, yellow scale, oleander scale, walnut scale, San Jose scale and cactus scale (Rosen and Debach, 1979, 1978). Australia established first commercial insectary of Biological Service Inc. in 1971 for mass rearing of A. melinus (Furness et al., 1983; George, 1984).

Aphytis melinus DeBach was described by Paul DeBach in 1959 from the specimens collected from red scale in Pakistan in 1956 (DeBach, 1959; Figure 1.5). Ahmad and Ghani, (1972) recovered A. melinus from A. destructor and studied its life cycle but its natural efficiency in the field has not been documented in Pakistan.

1.5.6. Reproduction in A. destructor

In general, diaspidid scale insects are neotenic i.e. reproduction of female at larval stage (Nur, 1990). Some diaspidid females start reproduction while they are still at larviform stage and continue uptill oviposition stage. Normally there is 1:1 (male to female) ratio in diaspidids but sex ratio is influenced by environmental condition or the structure of population (Nur, 1990a). Sometimes females produce female colonies in young age and male colonies in old age. In one study, 133 species were examined in which 17 % were parthenogenetic or having some parthenogenetic population (Brown, 1965). Some species of armored scale insects lay 50–150 eggs/female but some may produce as few as 10 eggs and as many as 600 eggs per female at a rate of 1–10 eggs/day under the scale cover (Jalaluddin et al., 1992). One male can fertilize many females in sexual fertilization (Nur, 1990a).

1.5.6.1. Common features of A. destructor

A. destructor has a soft body inside a hard, distinct and separable cover (Metcalf, 1962). The cover is made of thread like wax secreted from body wall as discarded old skin which is cast during molts. The following description was provided by Williams and Watson (1988). The common features of A. destructor include lack of paraphyses or intersegmental scleroses (Figure 1.6). The pest has three pairs of lobes with no indication of a fourth pair, plates are long, flat and fringed (two between median lobes, two between median and second lobes,

three between second and third lobes, and a variable number beyond third lobe) and characteristic sclerotization on dorsum of pygidium.

1.5.6.2. Morphological description

Eggs: Adult female of A. destructor deposit eggs under scale cover in concentric circles around the body of the female in batches of 3 to 4 (Taylor and Paine, 1935; Ahmad and Ghani, 1972; Waterhouse and Norris, 1987; Figure 4.1 c). Freshly laid eggs are white and elongate becoming pale yellow. Fecundity of A. destructor depends upon the climatic conditions (Jalaluddin et al., 1992; Zhou et al., 1993).

Crawler: Newly hatched crawlers are free–moving and can be recognized by the presence of legs, antennae and a pair of bristles at the tip of abdomen (Figure 4.1 d). Crawlers are light green to yellowish brown, translucent and oblong (Waterhouse and Norris, 1987

Second instar female: Up to the second molt there is no physical differences between the sexes, both are pale yellow. Differences between the sexes start in the middle of 2nd larval stage (Dekle, 1965). Females remain pale yellow, circular and somewhat transparent in the second instar, which lasts for 8–10 days. Females nymphs are 0.6–1.1 mm long (Williams and Watson, 1988).

Immature male: Development of male characteristics also starts in the middle of the 2nd instar which lasts for 5–8 days (Williams and Watson, 1988). Male covers become reddish brown and more elliptical in shape and then transform into the pre-pupal, pupal and adult stages (Figure 4.1 g). The male pre-pupal and pupal stages are spent under the scale produced by the second instar.

Adult male: Small two-winged and reddish gnat-like insects having eyes, antennae, three pairs of legs and long appendages arise from the pupae by pushing out the scale cover. Adult males do not feed and are short lived (Dekle, 1965; Figure 4.1 h).

Adult female: After 2nd instar, females shift to pre–oviposition stage having orange yellow body under the milky white, semitransparent, circular or broadly oval cover, 1.5–2.0 mm in diameter (Figure 4.1 i). Gravid females can be easily recognized with naked eyes by their closely packed colonies (Dekle, 1965). The cover is flat and translucent with a sub central pale exuviae. Bodies of slide–mounted specimens are 0.7–1.2 mm in length with a pyriform

and membranous body. Sexual reproduction is influenced by food availability, but typically the sex ratio is 1:1 (male: female); although all male and all female colonies have been observed (Ahmad and Ghani, 1972; Taylor, 1935).

Developmental biology.

A. destructor development depends upon climatic condition: at a mean temperature of 30°C and RH 65 %, total life cycle from egg to the beginning of oviposition of a female was 34–35 days and male was 30–35 days on seedling coconut in Fiji with 8–10 generations per year (Taylor, 1935). Threshold temperature for egg laying and female development of A. destructor was 10.5°C and 12.3°C in China while survival temperature for eggs was 12.33°C ± 1.47°C reported in a research conducted in Shangi (Tang and Qin, 1991). ). Crawlers search to find a suitable site on leaves for settlement at which stage all the appendages are lost (Dekle, 1965; Beardsley and Gonzalez 1975; Miller and Davidson, 2005; Fasulo and Brooks, 2010). After settling, females remain sessile throughout development; adult males undergo pseudo-pupation, develop a pair of wings and can disperse by flying to find mates (Ghauri, 1962). Early crawler’s mortality in A. destructor is very high during rainfall as the scales are dislodged from leaf surface (Watson, 2005). Crawlers are primarily the stage responsible for dispersal of A. destructor within and between host trees. However, comprehensive biology with description of length and width and developmental duration at various temperatures and RH on different mango cultivars has not been studied in detail.

1.5.7. Physiological, biochemical and molecular changes in mango leaves under scale insects infestation Due to their long association, plants and insects have developed a variety of beneficial and harmful interactions with each other (Stotz et al., 1999). To avoid damage by herbivorous insects, plants have developed chemical defenses against feeding by insects (Harborne, 1989). These defenses are arranged in complex regulatory networks in time and space, which are further modulated by interactions with other signaling pathways. These integrated responses ultimately lead to a characteristic gene expression pattern in the production of phytochemicals that are directed against the invader. Damages caused by insects induce signal responses in plants, which may be due to the saliva secretion of the herbivores or up– regulation of phytoalexins by the plant (Agrawal, 1998; Ryan and Moura, 2002; Stratmann 2003; Zavala et al., 2004). Phytoalexins are active against insects and pathogens that infest

plants which cause local as well as systemic induction of volatile emissions (Paré and Tumlinson, 1997; Dicke et al., 1999; Thaler et al., 2002). Volatiles are produced by several plants, as a form of direct and indirect defense against insect infestation (Karban and Baldwin, 1997; Tholl, 2006).

1.5.7.1. Ions analysis The sodium ion (Na+) is considered harmful to the cell activities for most plants when it is present in the cytosol at a concentration higher than the adequate level (1–10 mM) (Munns, 2006). It interferes with the natural balance of K+, which activates more than 50 enzymes in cytosol. At higher concentration, Na+ can displace calcium (Ca2+) from the plasma membrane that produces a change in cell membrane permeability which can be reflected by a leakage of K+ from the cells (Cramer et al., 1989). Na+ ions induce toxic damages to the plant cells in both osmotic and ionic effects, causing growth retardants and low productivity prior to death (Chinnusamy et al., 2005). Garcia et al. (2007) reported that increased levels of Na+ disturbed the relationships of Na+/Ca2+, Na+/K+, Na+/Mg2+, but decreased Ca2+, Mg2+ and K+ contents, causing imbalance and nutritional stress which consequently progressed to plant stress. Excess levels of Na+ and Cl– inhibit the uptake of K+ that lead to the appearance of symptoms like those in K+ deficiency. The deficiency of potassium (K+) in plants initially leads to chlorosis and then necrosis. K+ is vital for osmoregulation and protein synthesis, which maintains cell turgor and stimulates photosynthesis. Both K+ and Ca2+ are necessary to maintain the integrity and functioning of plasma membranes. Carden et al. (2003) investigated that maintenance of adequate level of K+ in plant tissue under salinity stress might be due to selective K+ uptake and cellular K+ /Na+ compartmentation and distribution in the shoots. Ca2+ is a signaling molecule that plays an important role in mediating mechanisms which are involved in recognition and response to various stresses in plants. Under stress conditions, plants either accumulate or release intracellular cytosolic Ca2+ that acts as a signal and regulates different physiological processes for the adjustment of these stresses (Kader and Lindberg, 2010). Ca2+ restricts the entry of Na+ into the plant cells (Hussain et al., 2010). In Jaya cultivar of rice, Ca2+ application (4 mM Ca2+) has been reported to improve the survival percentage and limited the cellular levels of Na+ (Anil et al., 2005).

1.5.7.2. Physiological changes in mango plant under insect infestation Herbivore insects feeding on plants affect photosynthesis, which induces physiological and biochemical changes in the plants as well as reduce the leaf gas exchange (Gomez et al., 2004). A phloem feeding insect like scales remove carbohydrates from leaves when combined with reduced photoassimilates under shade condition, resulting in reduced plant growth (Risebrow and Dixon, 1987). Normally this situation is compensated by the plants in the form of increasing photosynthetic activities (Thomson et al., 2003). This compensation is regulated by chlorophyll contents, which is an important component of plant–insect interaction in the leaf tissues (Ni et al., 2002). Despite negative effects on leaf physiology, herboviry improves photosynthesis in plant by reducing constraints in environmental conditions and penetratiing light in to defoliated canopy (Anten and Ackerly, 2001). It also increases the availability of water, hormones and nutrients to the plants (Mc Naughton, 1983). Leaf, root, stem and leaf dry weights, nitrogen concentration and chlorophyll contents were reduced in a single leaf of subtropical tree specie, Guaiacum sanctum L. in USA upon feeding by scale insect, Toumeyella sp (Schaffer and Mason, 1990). Retuerto et al. (2004), in Spain, examined the ability of a plant, Ilex aquifolium L. (European Holly) to recover from the scale insects-induced stress condition by increasing photosynthesis. Chlorophyll fluorescene, monitor photosynthetic performance in algae and plants (Baker, 2008), parameter was used for the plant growing under natural light and temperature. Photosynthesis in scale infested leaves, uninfested leaves of infested trees and uninfested leaves of uninfested trees was determined. In all the trees, photosynthesis was significantly increased by scale insects infestation due to light and temperature treatments. They concluded that an increase in the feeding rate of scale insects, followed by high temperature and light, increases the demand of carbon, which results in the greater compensatory photosynthesis. Nabil (2013) stated that mango shield scale, Kilifia acuminata Signoret (Hemiptera: Coccidae) caused considerable damage to the chlorophyll contents of mango leaves in Egypt. Positive and highly significant correlation between percent chlorophyll loss and the number of attacking scale insects was found during the course of study. Golan et al. (2014) studied the response of two host plants (5 lemon and 5 fern plants) to the infestation of scale insect, Coccus hesperidum L. (Sterrnorhyncha: Coccoiddea: Coccidae) after 6 months in Poland. Plants were divided into five density series according to the infestation. In all treated plants, chlorophyll and carotenoid contents were greatly decreased. Highest decrease was observed in smallest abundance of scale insects on lemon leaves (first 12

class density) than in fern leaves (second class density). Maximum reactin of the chlorophyll fluorescence (a technique used to evaluate physiological response of plant to herbivory or impact of feeding of insects on the plant light reaction) was observed in density class 3 and 4 in the leaves of fern and density class 4 and 5 in lemon leaves. Oveall reaction of the chlorophyll fluorescene depends upon the properties of host plant and the abundance of scale insects feeding on the host. Juarez-Hernandez et al. (2014) histologically analyzed the penetration of stylet bundle (SB) of white mango scale, Aulacaspis tubercularis Newstead (Hemiptera: Diaspididae) inserted into mango plant tissues to determine the cellular path, damage of leaf cell and sucking sap of phloem. The length of SB was 3 times (3 mm) the total length (1 mm) of a female scale body. The path of the SB was intracellular, passing through mesophyll and pierced xylem cells and phloem tissues. The attacked site was of thick red masses having phenolic compounds due to sucking of sap by scale inscts. However, little is known about the effects of scale insect on the photosynthetic pigments of plants. 1.5.7.3. Biochemical changes in plant under insect infestation Plants have developed enzymatic and non-enzymatic antioxidant defense system against almost all kind of stress conditions. (Foyer et al. 1994; Sanita et al. 1999; Clijsters et al., 1999; Mittler, 2002; Scandalios, 2002; Razinger et al., 2006). The enzymatic antioxidant system is one of the important protective mechanisms, which works simultaneously with different enzymes including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX). SODs are present in different cell compartments and – catalyze the disproportionation of two O2 radicals to H2O2 and O2 (Salin, 1987). H2O2 is removed by CAT, which is located in peroxisomes and mitochondria, and scavenge photo respiratory and respiratory H2O2 (Willekens et al., 1997). POD plays a role in lignin biosynthesis, Indole Acetic Acid (IAA) degradation, and changing H2O2 to water using a wide range of electron donors like guaiacol which is why it is called guaiacol peroxidases (Ádám et al., 1995). Ascorbate peroxidase is mostly present in chloroplasts and cytosol, and is the key enzyme of the ascorbate cycle. It detoxifies peroxides by converting ascorbic acid to dehydroascorbate (Mittler and Zilinskas, 1994; Zhang and Kirkhan, 1996). One of the characteristic differences in the reaction mechanisms among these enzymes is: CAT without co-substrates can disproportionate H2O2, and POD and APX require co-substrates to detoxify hydrogen peroxide.

Under biotic and abiotic stress, plants generate reactive oxygen species (ROS) from free – radicals (O 2) which are produced during photosynthesis (Mittler, 2002; Appel and Hort, 2004; Mahajan and Tuteja, 2005). These ROS are the main cause of cellular death in the plant under stress condition. Highly reactive and toxic reactive oxygen species are hydrogen per – – oxide (H2O2), superoxide radical (O 2), hydroxyl radical (OH ), hydroperoxides (ROOH), alkylperoxy (ROO.) and alkoxy radicl (RO). They cause damage to proteins, lipids, carbohydrates and DNA; ultimately causing plant death. Major cause of low yield in the world is due to ROS accumulation in the plant cell (Tuteja, 2007; Khan and Singh, 2008; Tuteja and Hirts, 2010). ROS have the potential to interact with many cellular components, causing significant damage to membranes and other cellular structures, and consequently cause growth inhibition (Verma and Mishra, 2005; Gao et al., 2008). Some of the ROS are highly toxic and need to be detoxified by cellular responses, if the plant is to survive and grow (Gratäo et al., 2005). ROS scavenging depends on the detoxification mechanism, which may occur as a result of sequential and simultaneous action of a number of antioxidant enzymes, including CAT, POD, SOD and APX. Plants with high constitutive and induced antioxidant levels have better resistance to damage (Parida and Das, 2005). In the cell, proteins, DNA, carbohydrates and lipids are the main vulnerable biomolecules to free radicals. The action of ROS on cell membrane causes peroxidation of polyunsaturated fatty acids (PUFA) in membrane lipids. Lipid–based signaling and their relationship to the defense of plant against mechanical stress, pathogens and herbivores are highly acknowledged and is still a field of increasing interest. In biotic and abiotic stress reaction, oxylipins group of fatty acid derivatives play a vital role as a source for signaling compounds. Although infections by pathogens, herbivore attacks and heavy metal treatment are quite different forms of stress. However plants show a common response like an induced synthesis and accumulation of secondary compounds (Farmer et al., 2003). Mithofer et al. (2004) proposed a mechanism of biotic and abiotic stress in higher plants in which reactive oxygen species were linked with lipid per oxidation processes; as a result similar and highly reactive signaling compounds are formed. They observed that for both biotic and heavy metal stress, enzymatic and non–enzymatic reactions were different however; ROS was involved in the oxidation of unsaturated fatty acids which produced oxylipins. Oxylipins are highly variable class of lipid–derived compounds in plants as well as represent new endogenous signals involved in biotic and abiotic induced stress responses. 14

Howe and Jander (2008) stated that upon insect feeding, plant responded by producing toxin as well as defensive proteins that acted on the physiological processes of insects. Plants under insects attack also emit volatiles that attract predators and hence increase plant resistance indirectly. In the course of this process, an insect’s oral secretion and transduction of signal from the injured cells are first recognized. In the second phase, these initial cues are transmitted by different signaling pathways like calcium ion fluxes, phosphorylation cascades, and in particular the jasmonate pathways. These signaling pathways play a key role in the resistance of plant against various insect.

1.5.7.4. Molecular changes in plant under infestation of insect Plants are exposed to different types of biotic stresses including infection of fungi, insects . . pests and bacteria which produce ROS like O2 , H2O2 and OH (Mahajan and Tuteja, 2005). ROS damage cellular molecules including DNA in a plant cell. DNA damage is both structural and chemical, resulting in mutation in the organism which has deleterious and sometime advantageous (rare) effect on the affected orgamism (Friedberg et al., 1995; Tuteja and Tuteja, 2001). The damage of DNA consist of pyrimidine dimers, base deletion, strand breaks, cross link and modification of base which lead the cell to genotoxic stress or genomic instability (Britt, 1996; Tuteja et al., 2001; Tuteja and Tuteja, 2001). In many organisms, DNA damage response pathways such as cell-cycle arrest, direct activation of network repair and apoptosis are activated in response to DNA damage (Zhou and Elledge, 2000). It is a kind of signal transduction pathway which consists of sensor, effector and transducers (Tuteja et al., 2001). For the measurement of DNA damage, single cell gel electrophoresis method, based on the movement of damaged DNA in an agarose gel in an electric field, is used (Singh et al., 1988; Tice, 2000). Nature of the DNA is visualized by ethidium bromide, fluorescene dyes or silver stain. Usually, damaged DNA, having strand breaks, moves to anode farther in the agarose gel as compared to intact DNA which creates a shape like celestial comet. In the recent years, many sensitive and selective DNA assay methods have been developed in the field of genotoxicology and advances in molecular biology. A polymerase chain reaction (PCR) based technique called random amplified Polymorphic DNA, developed by Welsh and McClelland, (1990) and Williams et al. (1990) amplify random fragments of DNA with a single primer of neoclitide sequence at low condition for annealing. This technique is used for classification of species, phylogeny and genetic mapping as well as for various types of DNA damage and mutational events in plants, bacteria, vertebrate and invertebrate (Savva,

1998; Atienzar et al., 2000). With the help of RAPD, variations in the intensity and gain and loss of DNA band is detected following exposure to toxicant which is the indication of DNA damage (Atienzar and Jha, 2006). Liu et al. (2005) used DNA (RAPD) profile for the determination of reduction in total soluble protiens and root growth inhibition in barley seedlings (Hordeum vulgare L.) under cadmium (Cd) stressed plants in China. Variation in the intensity of bands like loss of normal bands, appearance of new bands was taken as criteria for comparison with normal seedling of barley. RAPD DNA polymorphism analysis can be used as investigation tool in environmental toxicology and as a beneficial biomarker assay for the determination of genotoxicity in the Cd stressed plants. Cenkci et al. (2009) used RAPD to determine the DNA damage in the leaves and roots of seedling of bean (Phaseolus vulgaris L.) under stress of toxic chemicals like B, Hg, Cr and Zn at various concentrations in Turkey. Changes in DNA bands, as detected through RAPD in the root and leave of bean seedling, were very usefull tools to evaluate the genotoxicity of toxic chemicals. Kakec et al. (2010) studied the genotoxic effect of various concentrations of boron on wheat (Triticum aestivum L.) and bean (Phaseolus vulgaris L.) plants through DNA damage assay using RAPD analysis in Turkey. Variation in RAPD results at different concentration of boron revealed that it is a very effective biomarker for the investigation of genotoxicity caused by varios factors in the plants. 1.5.8. Development of generic doses of irradiation for the growth inhibition of A. destructor

Fruit flies and scale insects disrupt trade among different countries. These insects need to be treated for disinfestation that is not harmful to the user, worker and commodity (Couey, 1983). Use of ionizing energy (gamma irradiation) below 1 kilo Gray (Gy) has promising applications against different species of pests such as fruit flies, naval orange worm, mango seed weevil, potato tuber moth, scale insects and codling moth (Burditt, 1982; Tilton and Burditt, 1983; Moy, 1985). Ionizing radiation is used to prevent the development or reproduction of pest for phytosanitary purpose to ship the commodities out of regulated areas (Hallman, 2011).

Following its discovery in the late 19th century, it was found that ionizing radiation at low doses sterilize organisms with no major ill effects on the commodities and consumers and 16

thus serves as alternative to methyl bromide for the disinfestation of insect pests (Hunter, 1912; Follett and Armstrong, 2004; Follett, 2006). Irradiation has the advantages over other treatments like hot water, vacuum treatment and methyl bromide as it can be easily applied in packing, safe for shelf life and is also a pesticide free treatment (Watters, 1968; Hallman, 2011). Its disadvantages are that many organic food industries do not accept irradiated food; lack of availability of technology as well as certification and inspection problems.

USA Department of Agriculture-Animal and Plant Health Inspection Services (APHIS) published landmark rules for generic radiation doses in 2006 (Follet, 2009). In these rules, they approved radiation doses of 150 Gy for tephrid fruit fly and 400 Gy for other insects except pupae and adult of Lepidoptera for all horticultural commodities to facilitate safe trade among different countries. Irradiation prevents the reproduction of the pest rather than acute mortality as irradiated insects still remain alive after irradiation but stop feeding and do not reproduce (Tilton, 1974).

First study on irradiation as a phytosanitary measure was conducted in Taiwan in the late 1920s against fruit flies and in Hawaii after 25 years as quarantine treatment (Koidsumi, 1930; Balock et al., 1956). Irradiation was commercially used for the first time as insects control method in 1929 against Lasioderma serricorne F. (Diehl, 1995). Commercial use of irradiation in mango was started when mango was irradiated and shipped from Puerto Rico to Florida, USA (Hallman, 2001a). In 1989 first phytosanitary treatment of 150 Gy was published against fruit flies in Hawaiian papayas (Glosser, 1989). In 1992, first commercial facility of irradiation as a phytosanitary treatment was built in Mulberry, Florida to replace the treatment of ethylene dibromide fumigation for grapefruit (Heather and Hallman, 2008). Irradiation doses of 250 Gy were first used in Australian mangoes as phytosanitary treatments for international shipment from Australia to New Zealand in 2004. Pakistan has expressed interest in the use of irradiation technology for disinfection of mango fruits for export expansion (Arshad, 2010). An agreement was signed between Ministry of food, Agriculture and Livestock, Pakistan and USDA in 2007 for the approval of irradiation as a phytosanitary measure for the export of mango fruit. The quarantine act of Pakistan was also reviewed recently and updated to fulfil the requirement of sanitary and phytosanitary measures (SPS) agreements of agriculture. In 2010, United States of America through U.S. Department of Agriculture (USDA) approved phytosanitary treatments of mango fruit of Pakistan with gamma irradiations (USDA, 2010b). Some researchers are of the opinion that irradiation 17

prolong shelf life of mango, if picked at mature green stage (Mumtaz et al., 1969; Akhter et al., 2005). Most of the studies showed that there is no significant loss found in the phytonutrients at a dose that is optimal for quarantine security (Thomas, 2001; Akhtar et al., 2008).

A. destructor is a quarantine pest and is mainly transported through human agency to various parts of the world (Burger and Ulenberg, 1990; Dharmaraju and Laird, 1984). The damage caused by A. destructor downgrade the market value which might cause the rejection of fruits exported to new market (Beardsley, 1970). Highly rigid quarantine regulation procedures including gamma irradiation are recommended to stop the entry of the pest to new area (Follet, 2006). Detailed study on the growth inhibition of A. destructor at various doses of gamma irradiation on mango plants has not been conducted so far.

1.5.9. Physiochemical analysis and sensory evaluation of mango fruit

Acceptance of fruits by consumers is mostly depending upon its color of epidermis (González–Aguilar et al., 2001; Crisosto et al., 2003); while sweetness and flavor arouses emotional feeling of man (Bayarri et al., 2001). Sensory analysis of pulps and products are important tool for the correlation of physicochemical properties with that of human perception. Market value of the fruit is determined by its appearance and profit is increased if quality is better. Good looking fruits get higher prices than poor looking fruits (Nakamura, 1977; Kitagawa et al., 1980).

In fruits and vegetables, there are almost 30 to 50 % postharvest losses occur due to poor production and postharvest management as well as lack of processing and marketing facilities (Expert Consultation, 2010). Mango fruits deteriorate very rapidly due to various biotic and a biotic stresses which causes huge postharvest losses, especially in less developed countries (Kader, 2002). Due to poor information about proper postharvest treatments and storing situations, mango fruits are not only lose its quality but also meet a significant postharvest damage (Salunkhe and Desai, 1984). Studies have been undertaken to reduce the ripening rate of fruit by chemical and physical methods like controlled atmosphere, carbonate and bicarbonate solution and low and high temperature (Chaplin et al., 1991; Fallik et al., 2000; Larrigaudiere et al., 2002). In this situation, income of farmers, consumer prices and quality of the product are negatively affected due to immediate marketing of the product without necessary processing and packaging. According to FAO (1981) report, handling technologies, 18

marketing and transportation are not present in less developed countries. Efforts have been made to increase mango production but to get maximum benefits from production will be a dream unless decrease their postharvest damages which are 30–40 per cent.

Gamma irradiations are used as a quarantine treatment for the agriculture commodities for 70 years with enough research has been done in the last 50 years (Koidsumi, 1930). However, practical application was much delayed due to technical and scientific reasons (Hallman, 2001a). The phasing out of methyl bromide (MB) particularly increased the interest in this technology for being recognized as an acceptable alternative method to chemical fumigation (Burditt, 1994). Food irradiation has been studied extensively and found that irradiation does not cause any harm to the food or become radioactive (Urbain, 1986). The joint expert committee of food and agriculture organization (FAO), international atomic energy commission (IAEA) and world health organization (WHO) concluded, in a meeting held in 1980 on the wholesome of food, that irradiation of food up to an overall average dose of 10 kGy has no toxicological hazards or add any nutritional or microbial issues in the food (FAO/IAEA/WHO Joint Expert Committee, 1981). Radiation of food with ionizing irradiation reduces the long term storage losses in the fruits and vegetables (Benkeblia, 2000). In the recent years, studies on tangerines, Citrus reticulata and clementines, Citrus clementina showed promising results of fruits irradiation (Mahrouz et al., 2002). Irradiation also reduces losses in the food by destroying insects, bacteria and mold that cause food contamination (Urbain, 1986; Ahmad, 1998). Low doses of irradiation up to 1–2 k Gy are considered suitable to increase the shelf life and storage life of fruits and vegetables for commercial use (Mahmood, 1972).

Gamma irradiation is a very effective technique for preservation of food items (Antonio et al., 2012). It is used an alternative method for post–harvest processing of citrus to qualify for the international trade law for phytosanitary. Its purpose is to prevent the invasive pests from propagation in new area (FAO, 2009). Irradiation doses of gamma rays have been developed as post–harvest disinfection method for many fruits and vegetables and showed great promise as irradiations have no detrimental effect on fruits but sterilize the insects (Kader, 1986; Follet, 2007). McGuire (2000) stated that gamma irradiation of dose up to 0.1–0.3 kGy had no deleterious effect on fruit quality of Logan. Ahmad (1987) studied that irradiation dose of 0.5 kGy had no ill effect on external appearance of Feutrels early mandarin. Hallman and Martinez (2001) also reported that irradiation dose of 0.5 kGy were safe for the organoleptic 19

quality of fruit. Hammad et al. (1996) observed that irradiation doses of 1 and 2 kGy had no objectionable effect in term of quality degradation on strawberry.

Khalil et al. (2009) found less decrease in weight loss, less increase in TSS and high values of acidity and ascorbic acid in blood red orange irradiated at 0.25 and 0.5 Gy and stored up to 42 days as compared to unirradiated fruits. Chouksey et al. (2013) treated custard apple, Annona squamosal with 0, 0.25, 0.50, 1.0, 1.25, 1.50, 1.75 kGy gamma irradiation and the fruits were then treated with 50 and 100 ppm benzyl adenine and stored at 25±5 °C and RH 90±2 % for 12 days in India. Irradiation dose of 1.5 kGy with benzyl adenine of 50 ppm increased the shelf life of fruit for 6 days as well as maintained good sensory qualities like texture, color, flavor and nutritional value at room temperature as compared to control. Yadav et al. (2013) studied the effect of Co– 60 gamma irradiation and storage temperature on the quality of mango fruit var. Kesar in India. Irradiation doses of 0.00 kGy, 0.20 kGy, 0.40 kGy and 0.06 kGy were applied from radio isotope and were stored at 27±2oC and RH 60–70%,

9°C and RH 90%, 12°C and RH 90% and control (12°C, O2 2%, CO2 3% and RH 90%). Fruits irradiated at 0.40 kGy and stored at temperature of 9°C with RH 90 % showed maximum weight loss and ripening reduction while minimum physiological weight loss and maximum marketability was observed in the fruits irradiated at 0.40 kGy with 12°C and RH 90 % at 41.43 days shelf life along with highest score of pulp color, skin color, texture, taste and overall acceptability.

Yadav et al. (2014) concluded that irradiation significantly reduced the physiological weight loss and ripening while increased the marketability of mango fruit of cv. Alphonso irradiated at 0.40 kGy and stored at 9°C. Highest scores of sensory qualities of fruits including texture, pulp color and overall acceptability were recorded at irradiation dose of 0.40 kGy and storage at 12°C.

Zhang, (2014) tested the effect of gamma irradiation doses from 0.2 to 0.6 kGy on the nutrients composition of citrus fruit cv. Shatang mandarin in China at a temperature of 4°C. Shelf life of fruit increased at dose range of 0.2 to 0.4 kGy while unirradiated fruits decayed after 15 days. However, shelf life of fruits irradiated at high doses of 0.5 and 0.6 kGy extended to 45 days in refrigerated storage. Total soluble solids (TSS) contents, ascorbic acid, total sugars and titerable acidity remained same in irradiated and unirradiated fruits at 15 days

storage period. Results indicated that irradiation was a useful and safe method for quarantine treatment of different commodities.

1.5.10. Efficacy of different insecticides against A. destructor

Various insecticides have been registered for the control of scale insects in ornamental and fruit crops. Usually crawler stages of scale insects are more susceptible to insecticides than mature stages. The efficacy of contact insecticides decreases after the scale insects develop their protective waxy covers. Oil at the rate of 1.5 % v/v is effective against overwintering stages while organophosphorus and pyrethriod insecticides at the rate of 0.15 % v/v are effective against crawlers (Aleksidze et al., 1995). Organophosphorus insecticides when mixed with mineral oil (Volck oil) gave better results against Latania scale Hemiberlesia latania Signoret as compared to oil alone (El–Kifl et al., 1980; Nada et al., 1990). Oil is used successfully in citrus against scale insects at 2 % v/v concentration; especially nymphal stage is the most sensitive stage to oils. Among the scale insects species, purple scale, Lepidosaphes beckii Newman is very sensitive to insecticides, followed by red scale Aonidiella aurantii Maskell while olive scale Parlatoria oleae Colvee show resistant to oils (Ibrahim, 1990).

Organophosphate insecticides, mineral and horticultural oils have been used against A. destructor since 1920 to 1970 (Hely et al., 1982; Beattie, 2005). Similarly, Diamethoate was successfully used against scale insects on large area of coconut plants in southern Oman (Kinawy, 1991). Insect growth regulator (IGRs) and systemic insecticides are effective against A. destructor, if they are applied at immature stages. Chemicals like dimethoate, diazinon, malathion, formothion and nicotine are effective against A. destructor as that are used against sucking insects like mealybug (Copland and Ibrahim, 1985; Jalaluddin and Mohanasundaram, 1989; Kinawy, 1991). A. destructor is also controlled through the combination of cultural practices like pruning of infested leaves and branches and chemical using diazinon as a general chemical against homopteran (Cochereau, 1969; Beardsley, 1970). In Hawaii, diazinon was the only pesticide registered for the control of banana pest including A. destructor (Morse and Brawner, 1986).

Chemical control is generally very costly due to the height of plants. In addition many armored scale insect developed resistant against insecticides (Tauili’ili and Vargo, 1993; Smith et al., 1997). Similarly, broad spectrum insecticides disrupt the activities of natural 21

enemies as parasitoids in particular are more susceptible to the insecticides than pests (Croft and Brown, 1975; Tomlin, 1994). Integrated pest management (IPM) tactics based on cultural, biological and chemical control are essential against coconut scale as using chemical alone may kill the natural enemies and result in the resurgence of the pest (Copland and Ibrahim, 1985; Mohyuddin and Mahmood, 1993). In many crops, IPM tactics have been developed and successfully applied in different localities for the control of A. destructor (Nafus and Schreiner, 1989; Pinese and Piper, 1994). A. destructor was suppressed on bananas by using insecticidal oils, buprofezin (IGR) and natural enemies (Chou, 2003). Four applications of buprofezin were used per season on bananas with cultural practices like pruning of scale infested leaves and branches for complete control of A. destructor. Fish-oil- rosin soap is used in India on coconut nursery plants against A. destructor which is comparatively safe and effective (Jalaluddin and Mohanasundaram, 1989). However, use of insecticides on the basis of monitoring data of pest and natural efficiency of parasitoids has not undertaken.

Imidacloprid has systemic and contact activity against a wide range of sucking insects and comparatively less toxic to animals and beneficial insects than Organophosphate and Pyrethroid (Mullins, 1993). It has systemic mode of action and is effective against those insects that have developed resistant against other insecticides (Elbert et al., 1990; Nauen et al., 1996). Another second–generation neonicotinoid insecticide, in the subclass thianicotinyl, is thiamethoxam which has good activity against many sucking pests including scale insects (Horowitz et al., 1998; Maienfisch et al., 1999). Thiamethoxam has both contact and oral toxicity (Lawson et al., 1999). This group does not kill the pest immediately. The insects first stop feeding and ultimately die of starvation (Koenig et al., 1999). Pyriproxyfen is an insect growth regulator (IGR) of juvenile hormone (JH) mimics which disturb the hormonal balance and also causes chitin deposition in the juvenile insect’s stage (Ishaaya and Horowitz, 1992). It causes deformation and death during the molting process of insects and pupation. Pyriproxyfen causes suppression of metamorphosis, embryogenesis as well as adult formation. Pyriproxyfen and fenoxycarb stop hatching of egg in pear psylla, Casopsylla pyricola Foerster (Higbee et al., 1995), egg hatch and adult formation in B. tabaci (Ishaaya and Horowitz, 1992) and Haematobia irritans L. (Bull and Meola, 1993). Pyriproxyfen and methoprene had an obvious lethal effect on the egg hatching of Ziposcelis entomophila Enderlein (Ding et al., 2002).

1.6. Aims

Keeping in mind the afore-mentioned problems and their solutions, the present studies were designed with the following objectives:

1. Monitor seasonal population dynamic of A. destructor and parasitoids activity in the field

2. Compare mango varieties for susceptibility to A. destructor

3. Developmental biology of A. destructor

4. Examine physiological, biochemical and molecular changes in mango plant leaves under A. destructor infestation

5. Determine minimum generic doses of Co–60 gamma irradiation for the growth inhibition/mortality of various stages of A. destructor

6. Determine the effect of irradiation on the physiochemical analysis and sensory evaluation of mango fruits

7. Evaluate different insecticides for the control of A. destructor on mango nursery plants and mature mango trees under field conditions.

Chapter 2.

General Materials and Methods

2.1. Study area (Dera Ismail Khan) at a glance

Dera Ismail Khan (D. I. Khan) is the extreme southern District of the province Khyber Pakhtunkhwa (KP), Pakistan (Figure 2.1). It lies at 31° 49' /North, Latitude and 70° 55' /East, Longitude. The climate is arid to semi–arid, hot and dry in summer with average annual rainfall of 180 to 200 mm. The elevation is 121–210 meters above sea level. The mean maximum and minimum temperature in summer and winter are 45 and 8°C, and mean RH ranges from 51 % in June to 78 % in October, respectively (Appendix 2.1and 2.2).

2.2. Raising mango nursery plants and release of A. destructor crawlers on test plants

Nursery of mango plants was raised by sowing mango seeds in Fruit Nursery Farm (FNF), Department of Agriculture (Extension) D.I.Khan in 2010, which were T-bud grafted after one year with three mango cultivars i.e. Langra, Kala Chounsa and Anwar Ratoul while Desi mango was kept without grafting. These plants were kept under close supervision as well as similar cultural practices including watering, hoeing and application of farm yard manure (FYM) were applied. Crawlers of scale insects emerged in the FNF in the first week of July at the termination of dry and hot season and increased humidity conditions due to the beginning of monsoon rains. The plants were exposed to 100 scale crawlers by placing the pots under mature mango trees that were infested with the insect at the FNF. The upper portion of the nursery plants were wrapped with scale-infested leaves so that newly hatched crawlers could attach to the nursery plants. The plants were exposed to the infested leaves with scale insects for 24 hours and thereafter removed to obtain crawlers of uniform age (Figure 4.1 b).

2.3. Collection and identification of scale insects of mango plants

Surveys were conducted in five mango orchards including Karamat Abad at Bannu road (15km North), Fruit Nursery Farm, Department of Agriculture Extension (5 km North West), Qureshi Morr at Multan road (10 km South), bank of River Indus (10 km East) and Panyala (60 km East North) from the study area in the extreme southern part of KP (Chapter 3). Four mango cultivars i.e. Langra, Kala Chounsa, Anwar Ratoul and Sindhry were tagged for data recording in the selected orchards. Number of adult scales and crawlers were counted

fortnightly between July 2011 and June 2013. Meteorological data was received from Arid Zone Research Institute, Pakistan Agriculture Research Council, Ratta Kulachi, D. I. Khan. Scales collected from each site were sent to Dr. Gillian W. Watson, Senior Insect Biosystematist, California Department of Food and Agriculture, Plant Pest Diagnostic Center, Sacramento California, U.S.A. and were identified as Aspidiotus destructor Signoret (Hemiptera: Diaspididae).

2.4. Natural efficiency of parasitoids on A. destructor

Two parasitoids Aphytis melinus (Hymenoptera: Aphelinidae) and Anagyrus sp. (Hymenoptera: Encyrtidae) were recorded from A. destructor collected from mango plants in the 5 selected orchards from July 2011 to June 2013. Aphytis melinus (Hymenoptera: Aphelinidae) was reported by DeBach (1959) from Pakistan and confirmed by Dr. Inamullah Khan, Principle Scientist, Nuclear Institute for Food and Agriculture (NIFA), Tarnab, Peshawar. Anagyrus sp. (Hymenoptera: Encyrtidea) was identified to genera level by Prof. Dr. Inayatullah, Department of Entomology, University of Agriculture, Peshawar, Pakistan.

2.5. Biology of A. destructor on mango plants under laboratory and greenhouse conditions

Mango nursery plants, manually infested with A. destructor, were obtained from FNF, D. I. Khan, transferred into 19 (height) × 18 (width) cm plastic pots and shifted to Nuclear Institute for Food and Agriculture (NIFA), Peshawar at a distance of 350 km North of D. I. Khan (Figure 2.2). The scale insects were reared on mango nursery plants in laboratory and greenhouse in NIFA for pest biology (Chapter 4).

2.6. Scale-induced, physiological, biochemical and molecular changes in mango leaves

Studies on physiological, biochemical and molecular changes in mango leaves under A. destructor infestation were conducted in department of biotechnology and genetic engineering, Kohat University of Science and Technology, Kohat (Chapter 5, 6). Thirty six mango nursery plants (12 plants from each variety i.e. Langra, Kala Chounsa and Anwar Ratoul) of uniform age (two years) and size (60–70 cm) were obtained in pots from mango orchard of FNF, D. I. Khan. 100 scale crawlers were released on these plants according to the procedure given in 2.2. The infested leaves were picked after 0, 10, 20 and 30 days and were snapped frozen in liquid nitrogen for further study.

2.6.1. Physiological changes in mango leaves under A. destructor infestation

Photosynthetic pigment, chlorophyll a and b, and carotene contents of the plant were determined by using spectrophotometer (Dr/4000 Spectrophotometer, HACH, USA) (Lichtentaler and Wellburn, 1983).

The chlorophylls “a” (I), chlorophyll “b” (II) and total carotenes (III) in methanol were calculated by the formula:

Ca = 15.65 a666 – 7.340 a653………………... (I)

Cb = 27.05 a653 – 11.21 a666…………….….. (II)

Cx+c = 1000 a470 – 2.860 ca – 129.2 cb / 245… (III)

Cell viability of mango leaves was determined using TTC (Triphenyltetrazolium chloride) reduction method (Jiang, 1999) and cell injury was determined by membrane leakage method (Lu et al., 2008). The ionic contents of the plant were determined by using flame absorption spectrophotometer (Awan and Salim, 1997).

2.6.2. Biochemical changes in mango leaves under A. destructor infestation

Reactive Oxygen species (ROS), scavenging enzymes like POD (Peroxidase), SOD (Super oxide dismutase), APX (Ascorbate peroxidase), GPX (Glutathione peroxidase) and other biochemical assays like CAT (Catalase), MDA (Melondealdehyde), H2O2 (Hydrogen per oxide), were found by ultra violet UV–2450 spectrophotometer according to established protocols as per detail given in relevant chapters. Free proline contents were quantified by the procedure of Bates et al. (1973).

2.6.3. Molecular changes in mango leaves under A. destructor infestation

In order to study scale–induced molecular changes in mango leaves, DNA damage analysis was performed using Random Amplified Polymorphic DNA (RAPD) markers using the method of Doyle and Doyle (1987).

2.7. Effect of irradiation on pest mortality and development inhibition

Effects of gamma radiation on mortality and growth inhibition of A. destructor were determined by exposing the scales infested mango plants to gamma radiation doses of 0, 100,

150, 200, 250 and 300 Gy in a Cobalt–60 gamma irradiator source (Azotop, Hungary) having a dose rate of 8.5 kGy/h measured with a Frickie dosimeter at Pakistan Radiation Services (PARAS), Lahore (Chapter 7).

2.8. Effect of irradiation on physiochemical analysis and sensory quality of mango fruit

Physiochemical analysis and sensory quality of mango fruit cv. Langra were determined at irradiation doses of 0–250 Gy in food science and technology section in NIFA and stored at 29.3±1.4 °C and RH 42±2.6 % (Chapter 8). Physiochemical analysis of fruits was done using following formulas.

Weight loss = (Fresh weight – Weight at storage interval) × 100 Fresh weight Acidity (%) = 0.01 × 0.064 × Titre × 100 × 100 10 × 20 Where 0.01 is dye normality of NaOH solution and 0.064 is constant factor.

Ascorbic acid = 0.1 × titre × 100 × 100 10 × 20 Where 0.1 is dye factor or Normality of NaOH solution

Titre is ml of dye used in sample–ml of dye used in blank

Organoleptic characteristics were judged by a panel of 20 semi-trained judges through 9 points hedonic scale with number 9 was considered as the best one (Larmond, 1977).

2.9. Evaluation of different insecticides against A. destructor on mango nursery plants

Efficacy of different insecticide against A. destructor was evaluated at FNF during the year 2012 and 2013. Winhort (861g/L, 86.1 % v/v Petroleum oil), Foliol winter oil (84 % w/w mineral oils), Imidacloprid 20 % SL (imidacloprid 200w/v, gm/L), Priority (pyriproxyfen 10.8 EC), Actara 25 WG (thiamethoxam), Seven star 10 % EC (bifenthrin) 10% w/v (10.77 % w/w) Denadem (profenofos) and Curacron 500 EC (profenofos) were applied at their recommended rate (Chapter 9). The experiments were designed in Completely Randomized Design (CRD) with 9 treatments including control and 3 replications. Spray was done by locally made hand held atomizer sprayer. Scales were recorded on the upper as well as lower

surface of three leaves taken randomly (top, middle and lower) per plant. Pre-spray data was taken to compare the efficacy of insecticides on the basis of percent scales mortality.

2.10. Evaluation of different insecticides against A. destructor on mature mango trees

Efficacy of insecticide for the control of A. destructor was evaluated at FNF during the year 2012 and 2013 on mature mango trees of age 15 years or more. Insecticides treatments and experimental design were kept the same as in 2.9. Spray was done by a motorized sprayer, BJ Power sprayer GX 120 Honda 4.0, Honda Motor Co. Ltd, Japan. Scales were recorded on the upper and lower surface of four infested leaves per tree (North, South, West and East). Pre spray data were taken to compare the efficacy of insecticides on the basis of percent scales mortality.

2.11. Experimental Design / Statistical analysis:

The data were analyzed using analysis of variance (ANOVA) with statistix 9 version software. Least significant differences (LSD) and standard errors (SE) were used to further assess the difference between means (Gomez and Gomez, 1984). The ANOVA was applied for each treatment followed by the Tukey HSD and P≤ 0.05 was used as criterion for significance. Microsoft Excel spreadsheet was also used for regression analysis and graphs. Percentage mortality and growth inhibition of A. destructor were subjected to probit analysis using PoloPlus (LeOra computer software) to estimate the dosage response of various stages of scale insects to irradiation. Detail experimental procedure for each experiment is given in the relevant chapter.

Chapter 3.

Incidence of A. destructor in relation to abiotic factors and natural efficiency of its parasitoids

3.1. Introduction

A. destructor is a very serious pest of mango and coconut in Pakistan (Ahmad and Ghan, 1972). The pest causes severe damage to mango from August-December every year at various locations in both coastal and plain areas. The pest completed life cycle in 35–44 days on Cucurbita maxima Dichesne in Pakistan with 3–4 generations per year. In China, A. destructor has three generations per year on Actinidia chinensis Planch (Kivi fruit), as a host, where fertilized females overwinter on the stem (Zhou et al., 1993). In Japan, it had one generation in a year on tea plants (Murakami, 1970) while in most tropical areas, A. destructor continuous to reproduce throughout the year (Goberdhab, 1962).

Aphytis melinus DeBach (Hymenoptera: Aphelinidae) is an important parasitoid of A. destructor (Ahamad and Ghani, 1972). In Pakistan, 37 % parasitism by A. melinus was observed on A. destructor infesting mango plants. The parasitoid lays 25–49 eggs per female in 7–16 days on A. destructor within 24 hours after emergence. It parasitizes the host at both low and high densities and has been recorded from all parts of Pakistan. A. melinus is also an important parasitoid of other scale insects including Aonidiella auranti Mast., A. citrina Coq. and A. orientalis Newst. in Pakistan and India (DeBach, 1959; Ahmad and Ghani, 1972). The parasitoid was introduced into California and it substantially controlled an outbreak of A. aurantii (DeBach, 1959).

Biology of A. destructor has been extensively studied on guava (Chiu, 1986) but forecasting of its population and seasonal monitoring has not developed, which is required for preventing and predicting the outbreaks of the pest. Usually timing and efficacy of insecticides against scale insects are based on direct count or plant parts or plants infested (Berlinger et al., 1999; Shaw et al., 2000). Similarly, pheromone traps and yellow sticky traps are also used for population density estimation (Jactel et al., 1996).

The present study was designed to monitor population fluctuation of A. destructor on four mango cultivars at five locations in D.I.Khan in relation to abiotic factors over 2 years

(between 2011 and 2013). Natural efficiency of two parasitoids, Aphytis melinus and Anagyrus sp. on A. destructor was also recorded during the two study years.

3.2. Material and methods

3.2.1. Population dynamics of A. destructor on four mango cultivars and at five locations

A survey was conducted in five mango orchards including Karamat Abad, FNF, Qureshi Morr, Bank of River Indus and Panyala in D. I. Khan. A total of 12 mango trees (3 trees from each of fourmango cultivars i.e. Langra, Kala Chounsa, Sindhry and Anwar Ratoul) were tagged for data recording in each orchard. The selected orchards received normal field practices like hoeing, weeding and irrigation. No chemical was applied throughout the study period. Number of adult scales and crawlers were counted fortnightly between July 2011 and June 2013. Weather conditions were regularly recorded. For monitoring, 20 randomly selected leaves from each tree (5 leaves from each side) in the range of 2–4 m height were cut and examined under binocular microscope in the laboratory. Number of adult and immature stages was counted fortnightly from July 2011 to June 2013. Scales were considered alive if they had pale bodies or released yellow body fluid when pricked while those having a dark brown or desiccated body were assessed as dead.

3.2.2. Natural efficiency of parasitoids on A. destructor

Two parasitoids A. melinus and Anagyrus sp. were recorded from A. destructor collected from mango plants. Infested leaves/twigs were collected from the same five locations as above in the study area in transparent polythene bags separately from July 2011 to June 2013. Infested plant materials were transferred to plastic pots (15 cm diameter and 20 cm height) to observe parasitoid emergence. The pots were covered with muslin cloth for proper aeration and held with rubber bands. Three leaves were kept in each plastic pot and replicated 5 times. The pots were observed daily and the emerged parasitoids were collected with aspirator, counted and then the percentage parasitism was calculated as follows:

Parasitoids emerged from scales Percent parasitism = × 100 Total scales

Statistical analysis

Monitoring data of A. destructor and its parasitoids were presented by bar graphs with standard errors using MS Excel spread sheet. The population dynamics data were correlated with abiotic factors using regression analysis and the trends in population were plotted against weather data throughout the year.

3.3. Results

3.3.1. Population dynamics of A. destructor on four mango cultivars

A. destructor infestation started in July 2011 with the emergence of new crawlers on all the four tested mango cultivars having highest infestation on Langra (15.2 scales/leaf) and lowest infestation on Sindhry cultivar (4.7 scales/leaf; Figure 3.1). The infestation gradually increased and reached a maximum of 130.0 scales/leaf on Langra, followed by Kala Chounsa (49.0scales/leaf) in November 2011 while minimum A. destructor infestation was observed on Anwar Ratoul (27.6 scales/leaf) and Sindhry (18.0 scales/leaf) in October, the same year. The scale population declined from December onward and reached a lowest infestation in June 2012 on all the four cultivars. Among the four mango cultivars, minimum infestation was recorded in Sindhry (0.3 scale/leaf), followed by Kala Chounsa (0.5 scale/leaf), Anwar Ratoul (0.7 scale/leaf) and Langra (1.58 scale/leaf) in the month of June 2012. Overall populations of A. destructor were in aggregation in the form of colonies. One branch or twig may be fully infested with scales but other will be totally free of infestation on the same tree. High values of the SE in the data indicated the variation in population due to the scattered nature of living in the A. destructor.

Figure 3.1. Populations dynamic (±SEM) of A. destructor on different mango varieties during 2011–12.

During the second study year (2012–13), infestation of A. destructor also started in July 2012 with maximum infestation of 9.0 scales/leaf on Langra cultivar and 0.1 scale/leaf on Sindhry cultivar (Figure 3.2). A. destructor population increased gradually the following months and reached a highest infestation on Langra cultivar (99.5 scales/leaf) in October 2012. Sindhry was the least affected cultivar by A. destructor having the lowest infestation of 11.6 scales/leaf in October 2012 as compared to Kala Chounsa (42.1 scale/leaf) and Anwar Ratoul (18.8 scale/leaf). The scale infestation declined from December onward and minimum infestation was noticed from January to June 2013 on all the tested mango cultivars.

Figure 3.2. Populations dynamic (±SEM) of A. destructor on different mango varieties during 2012–13.

3.3.2. Population dynamics of A. destructor at five locations on mango plants

A. destructor infestation started in July 2011 at the five selected locations including Karamat abad, FNF, Qureshi Morr, Bank of River Indus and Panyala during the two study years (Figure 3.3). Maximum scale insects count (12.1 scales/leaf) was observed at Karamat abad in July 2011, followed by FNF (9.4 scales/leaf), Qureshi Morr (9.4 scales/leaf), Bank of river Indus (8.3 scales/leaf) while lowest scale count was recorded in Panyala (5.1 scales/leaf). The scale insects infestation gradually increased and reached to its peak in October–November at all locations. Highest per leaf scale infestation in 2011 was recorded at Karamat abad (88.0 scales/leaf) in November, followed by Fruit Nursery Farm (73.3 scales/leaf) in October, Bank of river Indus (65.8 scales/leaf) in November, Qureshi Morr (42.5 scales/leaf) in November and Panayal (28.5 scales/leaf) in October. From November onward, there was a sharp decline in population dynamics with lowest population was recorded in May–June 2012.

Figure 3.3. Populations dynamic (±SEM) of A. destructor at different locations on mango plants during 2011–12.

During the second year (2012–13), the scale insects infestation started in July 2012 with releasing of new crawlers (Figure 3.4). Maximum infestation of A. destructor in 2012 was recorded at Karamat Abad (5.6 scales/leaf) in October, followed by Bank of River Indus (4.6 scale/leaf) in November, Qureshi Morr (2.7 scale/leaf) in November, FNF (2.7 scale/leaf) in October and Panyala (0.1 scale/leaf) in October. The infestation declined from November onward and minimum infestation was recorded from December 2013 to June 2013 at all locations.

Figure 3.4. Populations dynamic (±SEM) of A. destructor at different locations on mango plant during 2012–13.

Overall data showed that during 2011–12, scale insects infestation started with releasing newly hatched crawlers from July (8.9 scales/leaf) and continued for six consecutive months (Figure 3.5). Maximum scale insect infestation was recorded in October and November (51.0 and 54.0 scales/leaf), consecutively during 2011–12. From December onward, the scale infestation dropped suddenly with declining temperature and lowest scale insect infestation was noted in February (1.3 scales/leaf). There is slight increase in scale insects population during March and April but the generation did not complete as hot and dry weather started in May and June where scale insects infestation declined to the minimum in June (0.8 scales/leaf). During the 2nd year (2012–13), the scale insects population reached to its peak in October and November (43.5 and 38.5 scales/leaf). The population decreased gradually and the lowest insect infestation was recorded in March (1.4 scales/leaf). A slight increase in population of scale insects was observed in April but soon the number decreased to the smallest value in May (1.0 scales/leaf).

Figure 3.5. Overall population dynamics of A. destructor during 2011–12 and 2012–13.

For more precise presentation of the population fluctuation of scale insects, the year has divided into static trend, increasing trend and decreasing trend (Figure 3.6. a and b).

During the two years, the scale insects showed static trend from January to June (0–270 day number) in population as mostly the insects were present in hibernation on the leaf as mature females. From July to November (180–270 day number), there was active period and the

scale insects rapidly released crawlers which completed generation in shortest time during hot and humid season. The scale insects population started declining from mid–November to January (270–360 day number).

Figure 3.6. Population trends in A. destructor infestation during 2011–12 (a) and 2012–13 (b).

3.3.3. Relation of A. destructor population fluctuation with abiotic factors

Relation of A. destructor population fluctuations with major weather parameters was observed during 2011–12 and 2012–13. Coefficient of correlation and other statistics about

scale insects and weather factors like maximum, minimum and mean temperatures, % RH observed at 8.00 am and 2.00 pm, wind speed (km/hour), rainfall (mm), and mean relative humidity are given in table 3.1. (Meteorological data is attached in Appendix 2.1 and 2.2).

During 2011–12 and 2012–13, per leaf and per month scale insects populations were affected by wind and rainfall and similar trend was observed in the population data (Table 3.1). The variation in scale insects population and its relation to wind and rainfall was significant and positive during 2011–12 and 2012–13. The scale populations were non-significantly correlated with maximum temperature, minimum temperature, mean temperature, RH at 8.00 am, 2.00 pm.

Table 3.1. Relationship of A. destructor population with abiotic factors during 2011–12 and 2012–13.

Weather Regression coefficients Parameter Year factors a b r2 Probability wind velocity 3.1±0.24 0.03±0.01 0.71 0.01 Population 2011-12 dynamics of A. Rainfall 2.5±0.43 0.05±0.02 0.68 0.01 destructor with wind velocity 3.3±0.25 –0.04±0.01 0.72 0.01 2012-13 abiotic factors Rainfall 1.5±0.57 0.13±0.03 0.83 0.001 3.3.4. Natural efficiency of A. melinus and Anagyrus sp. on A. destructor

A. melinus and Anagyrus sp. emerged from A. destructor collected from fields in July and August 2011, respectively. The emergence of the parasitoids gradually increased and reached a peak in November for A. melinus and in December for Anagyrus sp. during 2011–12 and 2012–13 (Figure 3.7 and 3.8). Highest percent parasitism of 13.6 and 16.3 % of A. melinus was recorded in November while 14.2 and 14.9 % parasitism of Anagyrus sp. was observed in December during the two study years, consecutively. The percent parasitism of A. melinus and Anagyrus sp. dropped from January onward with the declining A. destructor population and no parasitoid was observed from February to June during 2011–12 and 2012–13.

Figure 3.7. Per leaf populations (±SEM) of A. destructor and percent parasitism (±SEM) of Aphytis melinus and Anagyrus sp. on mango plants during 2011–12.

Figure 3.8. per leaf populations (±SEM) of A. destructor and percent parasitism (±SEM) of Aphytis melinus and Anagyrus sp. on mango plants during 2012–13.

3.4. Discussion and conclusion

The data showed seasonal variation in A. destructor populations on mango trees. New crawlers of A. destructor emerged in July when temperature decreased due to monsoon rainfalls and RH increased. The populations reached a maximum in October–November and then declined to a minimum from January to June due to less rainfall and high temperature in the summer months (May and June). Wright and Diez (2005) showed that A. destructor populations build up in July–August on banana in Hawaii, reaching a peak in October– November with multiple generations, and then decline to the lowest scale count in May–June due to dry weather and high temperature. Findings of our study are compatible with those of Chiu (1986) who observed fluctuations in the populations of A. destructor with seasonal variation pattern in Hawaii on guava plants. Maximum A. destructor populations on guava plants were recorded in September to January and comparatively lower populations in the summer months due to natural mortality caused by high temperature of 28–30 °C. Wright and Diaz (2005) showed a positive relationship between rainfall and A. destructor population expansion in Hawaii as both colony expansion and dispersal of A. destructor to non-infested plants are the main factors for population increase. Nabil et al. (2012) showed peak of scale insects, Aulacaspis tubercularis Newstead on mango plants in November in Egypt during two study years. Ascher et al. (1995) reported that A.tubercularis had its population peaks in August in Kaapmuiden where as in Nelspruit, the population reached to its peak in November. Chou (2003) and Nestel et al. (1995) reported aggregation form of living is Diaspididae and found that below 10 % of infestation on leaves had very little chance of fruit infestation.

During monitoring, it was observed that A. destructor infestation was more prevalent on Langra cultivar than Kala Chounsa and Anwar Ratoul while Sindhry was the least affected cultivar by scale insects. It is possible that Sindhry has unfavorable characteristics (physical or chemical) for this scale insect. The semi erect branching system of Sindhry provides less shade and more aeration whereas A. destructor prefers the dense canopy structure provided by Langra. These findings are in agreement with Bakrey (2009) who reported that mango variety, Alphonso showed resistant to scale insect infestation in Egypt due to its unfavorable characteristics including thicker epidermis and sclerenchyma, deeper vascular bundle, condense pericyclic fibers and lignin as well as it has fewer numbers of resins (food sources) which act as physically difficult and energetically cost for insect penetration of mouth parts. 40

Radwan (2007) chemically analyzed the leaves of mango and found that Hendi variety was susceptible to scale insects due to high quantities of total carbohydrate as compared to Alphonso which had not only low carbohydrates but also had toxic secondary metabolites against scale insects.

A. melinus and Anagyrus sp. started emergence from A. destructor in late August and September with peak percent parasitism in October and November. However; no parasitoid emerged from February to July during 2011–12 and 2012–13 due to nonavailability of host as well as unfavorable environmental condition. With the increasing A. destructor infestation, the percent parasitism of the two parasitoids also increased. From November onward, the infestation of A. destructor declined while percent parasitism of the parasitoids reached a maximum in November which indicated that besides weather factors, the parasitoids also played major role in the control of the pest. These findings are in agreement with that of Quednau (1964) who reported that in South Africa, A. melinus effectively controlled A. aurantii, Chrysomphalus dictyospermi Morgan and C. ficus Ashm. Nabil et al. (2012) observed mean percent parasitism of 2.71 and 1.93 % of Aphytis sp. on Aulacaspis tubercularis Newstead during the two study years in Egypt. Kamel et al. (2003) observed 0.8 and 14.6 % parasitism of 18 species of genus Aphytis on eleven armored scale insects species (Diaspidids) from Egypt. In Australia, 70 % of citrus growers control red scale and yellow scale with the two parsitoids, Aphytis linganensis and Comperiella bifasciata which reduce the scale infestation below 0.05 scales per fruit with 68–81 % reduction in cost of chemical control (Smith, 1978; Papacek and Smith, 1992). Noyes (2012) reported that Anagyrus sp. play major role in the control of scale insects. The parasitoid has been recorded with different parasitism levels from many scale insects species including Coccus hesperidum L. (Coccidae), Ferrisia virgata Cockerell, Nipaecoccus viridis Newstead, Phenacoccus hirsutus Granara de Willink, Planococcus citri Risso and Formicococcus robustus Ezzat and McConnell. It has generally observed that closely packed plants and the plants having high amount of under growth parts are less infested by A. destructor as these plants harbor natural enemies (Simmonds, 1921).

Results of the current study showed that peak of A. destructor population was noted from September– November and lowest count was recorded in May-June during both the two study years. With the start of rainfall in July, the scale number increased with increase in RH and temperature. Dabek (1998) investigated that main cause of outbreak of A. destructor in 41

Nanumaga Island, Tuvalu in March, 1994 on coconut was speculated as the prevailing dry weather (mean annual rainfall 2610 mm) in Nanumaga as compared to other islands like Funafuti where mean annual rainfall was 3465 mm. Similarly, Wright and Diez (2005) observed that increase in number of A. destructor was positively correlated with rainfall for both Cavendish and Brazillian banana cultivars in Hawaii. It is possible that mature females terminated hibernation and released more crawlers during humid season (Mariau, 2001) and the wind played role in dispersion of crawlers to new plants to expand its colony (Mau and Kessing, 1992). Willard (1974) reported that number of crawlers of A. destructor in the air current had significant and positive correlation with wind speed and temperature but negative correlation with relative humidity. Crawlers can establish colonies about 80 km away from the original place. It was inferred from my study that life cycle was affected by the temperature and RH followed by long day light from July to November in my study area. After November the temperature decreased and RH also remained optimum but still the scale population dropped to the minimum because day light was very short. During May-June though the temperature and day light were high yet the scale population did not flared up because the RH was low which was very essential factor for population flare up of scale insects. It is predicted from the study that potential severity of scale population must be on the basis of varying degrees of the three mentioned meteorological factors.

During the previous study, it was noted that A. destructor changed its life cycle at varying meteorological condition. There were many over lapping generations lasted from July to December in the environmental condition of my study area. Similarly, some varieties were more infested with A. destructor than the others which indicated that life cycle was influenced by the nature of mango plant. It was necessary to get detail information about the biology of A. destructor in different temperatures and RH and on different mango varieties. In the next chapter biology of A. destructor will be studied at various temperature and different mango cultivars in detail.

Chapter 4.

Biology of A. destructor on mango plants (Mangifera sp.) under laboratory and greenhouse conditions

4.1. Introduction

A. destructor is an important pest of many tropical crops, especially coconut throughout the world (Taylor, 1935). The pest is found in Africa, Asia, the Pacific region and in some parts of Australia (C. I. E., 1960). It has a wide host range including mango, citrus, sour sop, custard apple, umbrella trees, mulberry and lillypilly. A. destructor lays eggs under the scale which hatch into free moving crawlers (Taylor, 1935; Waterhouse and Norris, 1987). The crawler stage is the dispersal stage as they have legs and can move up to one meter, but are often carried longer distances by animals, other insects or the wind to infest other plants (Greathead, 1990). In females, the main function of first and second instar is feeding and third instar is reproduction (Koteja, 1990), while in males, the first two instars feed, followed by non–feeding pre–pupa, pupa and adult stages. Adult females are sessile while adult males are provided with single pair of wings for locomotion and antennae to locate pheromones (Moreno, 1972) released by females for mating (Gieselmann, 1990).

At a mean temperature of 30°C, total life cycle of male and female A. destructor is 34–35 and 30–34 days, respectively with 8–10 generation per year in tropical areas while at 29°C and RH 55 % on Cucurbita maxima, as a host, the total life cycle is 33 and 27 days (Taylor, 1935; Ahmad and Ghani, 1972). In general, male to female sex ratio is 1:1; however, in some cases all males have been observed in A. destructor colonies. Taylor (1935) had recorded whole female colonies of coconut scales in Fiji. In one study, life cycle of coconut scale was reported to last for 32 days for females and 27 days for males in greenhouse in Philippines (Tabibullah and Gabriel, 1975).

The present study was aimed to investigate biological characteristics, such as developmental duration, life cycle, fecundity and morphology of coconut scale on Desi mango cultivar under laboratory conditions as well as on different mango cultivars in the greenhouse. The study provides useful information for effective identification, monitoring and population management of the pest.

4.2. Materials and methods

4.2.1. Laboratory study

Twenty four mango plants (Desi mango) of uniform age (two years) and size (60–70 cm) were obtained from the Fruit Nursery Farm, D. I. Khan. The plants were individually transferred into 19 (height) × 18 (width) cm plastic pots in February and March, 2013. These plants were kept under close supervision and similar cultural practices including hoeing, weeding and irrigation were applied to all the plants. Holes were made in the sides and bottom of the pots for aeration. After the plants acclimatized in the pots in 30 to 40 days, they were marked separately for A. destructor infestation. Crawlers of scale insects emerged in the FNF in the first week of July at the termination of dry and hot season and increased humidity conditions due to the beginning of monsoon rains.

The plants were exposed to scale crawlers by placing the pots under mature mango trees that were infested with the insect at the FNF. The upper portion of the nursery plants were wrapped with scale-infested leaves so that newly hatched crawlers could attach to the test plants (Fig. 4.1 a, b). The plants were exposed to the infested leaves with scale insects for 24 hours and thereafter removed to obtain crawlers of uniform age.

Following initial infestation, biology of coconut scale was studied in the laboratory on mango nursery plants in pots at 27.9 to 32.1°C (mean temperature 30°C) and 61.9 to 66.2 % RH (mean 64 %). All life stages were marked separately to observe change in size, color and shape. For these observations, newly laid eggs were examined daily under a binocular microscope. Time of egg laying and number of eggs laid were counted under scale cover. Egg dimensions were measured by an ocular and stage micrometer. Newly hatched crawlers were observed daily with a head lens to determine molting. Morphometry (length and width of various instars) was performed using the ocular and stage micrometer.

Data concerning oviposition period, total number of eggs laid, male and female longevity, and sex ratio were recorded throughout the study period. For fecundity, gravid females were observed under the microscope and total number of eggs laid per female was counted. In the same manner, all life stages of males and their width at thorax, length from head to abdomen, antenna and extended wing positions were measured.

4.2.2. Greenhouse study

A second study was conducted in a greenhouse from July to December, 2013 under a temperature range of 23.2°C to 26.8°C (mean 25°C) and RH range of 55.9 and 64.2 % (mean 60 % RH). In this study, biology of the scale insect was studied on Desi, Langra, Kala Chounsa and Anwar Ratoul mango cultivars. Three nursery plants were selected from each cultivar. Temperature and RH data were obtained from the nearby meteorological station at Arid Zone Research Institute, Pakistan Agriculture Research Council, Ratta Kulachi, Dera Ismail Khan, KPK, Pakistan. The procedure for infestation of nursery plants with scale insects was the same as described above for the laboratory study.

4.3. Results

4.3.1. Laboratory study

4.3.1.1. Aspidiotus destructor developmental stages and morphometry a. Eggs: Mature females laid eggs in concentric circles in 3–4 batches with 10–17 eggs per batch which were easily observed under scale cover (Figure 4.1.c). Newly laid eggs were whitish, smooth and elongate. Mean length and width of freshly laid eggs were 0.22 mm and 0.09 mm, respectively (Table 4.1). The eggs hatched after 4–6 days in the laboratory at 30°C and 64 % RH. b. Crawlers: Crawlers emerged from underneath the scale cover and moved on the leaf for 24–48 hours in search of a suitable place for fixation. At this stage, the body of the crawler was light green, cylindrical and somewhat oblong (Figure 4.1 d). Mean length and width were 0.23 mm and 0.11 mm, respectively (Table 4.1). They had most characteristics of a typical larva including legs, antennae and segmented body. More than 98 % of the crawlers settled on the lower surface of mango leaf, the remainder settled on petioles and stems and upper surfaces of the nursery plants. c. White cap stage: Crawlers feeding through their rostrum started to secrete a waxy substance over their body which gave them white color (Figure 4.1 e). The ‘white cap’ stage began after 24 hours of settlement and could be observed by naked eye. Mean length and width were 0.23 mm and 0.21 mm, respectively (Table 4.1). White cap stage continued for 4– 6 days in the laboratory at 30°C and 64 % RH.

d. Nipple stage: At nipple stage, the external surface of the scales changed from white grey to pale green and concentric ridges appeared on their body. Total time taken by the insect from emerging crawlers to nipple stage was 9–14 days. At the termination of nipple stage, 1st instar stage was completed and the molting process started. Molting process continued for 4– 5 days before 2nd instar started. At the molting stage, color changed to light green and the body became oblong with one end pointed and pygidium developed.

Table 4.1. Morphometry (length and width) and developmental rate (days) of male and female A. destructor developmental stages on mango nursery plants in laboratory at temperature 30°C and RH 64 %.

Length (mm) Width (mm) Developmental rate (days) Scale Stages Range Mean ± SE Range Mean ± SE Range (days) Mean ±SE Egg 0.2–0.3 0.2±0.006 0.1–0.2 0.1±0.002 4–6 5.0 ± 0.15 Crawlers 0.2–0.3 0.2±0.006 0.1–0.2 0.1±0.002 1–2 – White Cap. 0.2–0.3 0.2±0.006 0.2–0.3 0.2±0.006 4–6 5.0±0.15 Nipple Stage. 0.3–0.4 0.3±0.004 0.3–0.4 0.3±0.002 3–5 4.0±0.16 2nd Instar 0.4–0.5 0.4±0.004 0.3–0.4 0.3±0.004 4–6 5.0±0.15 Pre–oviposition 0.6–0.8 0.7±0.016 0.5–0.7 0.6±0.012 7–9 8.0±0.16 Oviposition 0.6–0.8 0.7±0.014 0.5–0.8 0.7±0.012 10–12 11±0.17 Total life cycle – – – – 33–46 39.5 ♂ Pupae 0.3–0.4 0.3±0.004 0.1–0.2 0.1±0.002 3–5 4.0±0.17 ♂ Adult – – – – 2–3 2.5±0.10 Width at thorax 0.2–0.3 0.2±0.006 – – – – Head to abdomen 0.5–0.6 0.5±0.004 – – – – Antenna 0.5–0.6 0.5±0.006 – – – – Extended wings 1.2–1.5 1.4±0.040 – – – – Total life cycle – – – – 21–33 27 SE = Standard error. RH = Relative Humidity, Data based on 25 individuals.

e. Second instar: Second instar of the scale insect was marked with re–insertion of rostrum into the leaf surface for feeding purpose and remained for 4–6 days (Table 4.1). The main diagnostic character of this stage was the presence of orange molt ring (Figure 4.1 f). Size of the scale insects at 2nd instar was 0.4–0.5 mm with an average of 0.4 mm in length and 0.3– 0.4 mm with an average of 0.3 mm in width

4.3.1.2. Development of male Aspidiotus destructor

After second instar, the body of males elongated and the color changed to brown/orange with two black spots of eyes appearing at one end. Males transformed to pre-pupa which had a very short life span; pre-pupa changed to pupa and then adult. At the pupal stage, eyes could be clearly identified, genitalia were well developed and the body tapered (v-shaped) towards the posterior end (Figure 4. 1 g). Pupal stage remained for 3–5 days with an average of 4.0 day. From pre-pupal stage onward, orange pigmentation reduced. After 4–5 days, adult males emerged from pupae having slender body (Figure 4.1 h), orange brown head, two well- developed black eyes, one pair of long membranous wing, three pairs of jointed legs and filiform antennae. Width of adult male at thorax, Head to abdomen, Antenna and length at extended wing position were 0.2–0.3 mm, 0.5–0.6 mm, 0.5–0.6 and 1.2–1.5 mm, respectively. Total adult life stage of males lasted for 2–3 days and the entire life cycle (eggs to adult) was completed in 21–33 days (Table 4.1).

4.3.1.3. Development of female Aspidiotus destructor

Sexual variation in A. destructor started after the second molt, when male developed into pre- pupa and pupa, while female transformed to mature adult (pre-oviposition and oviposition stages). The third instar was characterized by pale green body, circular with internal ridges and concentric pale rings with ridges in the body (Figure 4.1 i). After 1 to 2 days, female body and its cover separated. At this stage, mating with males occurred. Fully mature females were apterous (without wings), apodeous (without legs), soft bodied, light green in color, and tapered anteriorly. Mean length and width of mature female were 0.66 mm and 0.61 mm, respectively (Table 4.1). Pre-oviposition stage remained for 7–9 days at 30 °C and 64 % RH. At oviposition stage, female increased in size and started egg laying.

Reproduction: Oviposition stage lasted for 10–12 days at 30°C and 64 % RH conditions (Table 4.1). Females laid 28–65 (mean 47) eggs in 3–4 batches in lines near the posterior end

under the scale cover. The recorded sex ratio on nursery plants was 1:2.1 (male: female); amongst a total of 2710 post 2nd instar nymphs, 1840 were female and 870 were male. Total life cycle (from eggs to gravid female) lasted for 33–46 days at 30°C and 64 % RH.

4.3.2. Greenhouse study

Developmental durations of different stages of A. destructor on Desi and Langra cultivars were similar, whereas on Kala Chounsa and Anwar Ratoul, the life span of the insect was prolonged by 2 to 3 days (Table 4.2). In greenhouse, egg, white cap, 2nd instar, 3rd instar and oviposition stages remained for 5–7 days, 5–8 days, 6–8 days, 8–11 days and 11–13 days, respectively at 25 °C and 60 % RH on Desi mango cultivar. Total duration of life span of A. destructor from egg to adult stage on Desi, Langra, Kala Chounsa and Anwar Ratoul was 32– 46 days, 32–46 days, 36–47 days and 37–50 days for males, and 43–58 days, 44–59 days, 47–60 days and 47–60 days, for females.

Table 4.2. Developmental rate (days) of male and female scale insects on different cultivars of mango nursery plants in greenhouse at temperature 25 °C and RH 60 %. Cultivars Scale Stage Desi mango Langra Kala Chounsa Anwar Ratoul Range (days) Mean ± SE Range (days) Mean ± SE Range (days) Mean ± SE Range (days) Mean ± SE Egg 5–7 6.0±0.17 5–7 6.0±0.17 5–7 6.0±0.17 5–7 6.0±0.17 Crawlers 1–2 – 1–2 – 1–2 – 1–2 – White Cap. 5–8 6.2±0.24 5–8 7.0±0.17 7–9 5.9±0.17 7–9 5.9±0.17 Nipple Stage. 7–9 8.0±0.17 7–9 8.0±0.17 8–9 8.5±0.10 8–9 8.5±0.10 2nd Instar 6–8 7.0±0.17 7–9 5.9±0.17 7–9 5.9±0.17 7–9 5.9±0.17 Pre–Pupae 2–3 2.5±0.10 2–3 2.5±0.10 2–3 2.5±0.10 2–3 2.9±0.10 Pupae 4–6 5.0±0.15 4–5 4.0±0.16 4–7 5.7±0.22 4–7 5.7±0.22 Adult 2–3 2.5±0.10 2–3 2.5±0.10 2–3 2.5±0.10 2–3 2.5±0.10 Total life cycle of male 32–46 39 32–46 39 36–47 41.5 37–50 43.5 3rd Instar female 8–11 9.3±0.23 8–11 9.3±0.23 8–11 9.3±0.23 8–11 9.3±0.23 Oviposition 11–13 12.0±0.17 11–13 12.0±0.17 11–13 12.0±0.17 11–13 12.0±0.17 Total life cycle of female 43–58 50.5 44–59 51.5 47–60 53.5 47–60 53.5 SE = Standard error. RH = Relative Humidity, Data based on 25 individuals

4.4. Discussion and conclusion A. destructor is a serious pest of mango in Pakistan having 3–4 overlapping generations on the host plants from July to December each year (Salahuddin et al., 2015). Heavy infestations were recorded at several localities in coastal and subcoastal areas of Punjab and Sindh provinces of Pakistan (Ahmad and Ghani, 1972). Other researchers also found that the A. destructor has many overlapping generations in tropical regions while in Japan, China and India it has 1, 3 and 6 generations per year, respectively (Murakami, 1970; Tabibullah and Gabriel, 1975; Aisagbonhi and Agwu, 1985; Gupta and Singh, 1988; Mau and Kessing, 1992; Tang and Qin, 1991; Zhou et al., 1993). The pest developed and survived under moderately hot (20–35°C) and humid (50–70 % RH) climatic conditions in our study area. Developmental variations in males and female life stages at two temperatures of 30 °C and 25 °C and relative humidities of 64 % and 60 % were observed during the course of our study. Findings of this study are compatible with those of Chiu (1986) who showed increase/decrease in population of A. destructor with seasonal variation in Hawaii on guava plants. Wright and Diaz, (2005) reported a positive relationship between rainfall and A. destructor population expansion on banana in Hawaii. Elwan (2005) and Trencheva et al. (2004) observed that two species of scale insects, Lepidosaphes pallidula Williams and Pseudaulacaspis pentagona Targioni Tozzetti had four and three generations per year on mango (Mangifera indica) in Egypt. Waterhouse and Norris (1987) and Taylor (1935) had reported 8–10 generations of A. destructor per year in tropical regions depending upon climatic conditions. Stathas et al. (2007) had shown that one generation of Parlatoria ziziphi Lucas lasted for 62 days in southern Greece at 25 °C 65% RH and 16 h light/ day. Zhou et al. (1993) reported that in China, total life cycle from egg to adult stage for male and female was 38 and 44 days, respectively. Watson and El-Serwy (2007) recorded shortest generation of Acanthomytilus Sacchari Hall (Hemiptera: Daispididae) as 27 days at 29 °C and 62.2 % RH.

Results of the present study revealed that after settlement on leaf surface, female A. destructor remained sessile throughout her life but its male in the last stage had well- developed wings and were able to fly. Females passed through three instars while males had two feeding instars with pre–pual, pupal and adult stages. The results of this study agree with those of Dekle (1965) who reported that all the appendages of crawlers in coconut scales were lost once they settled on leaves. Similar results were reported by Tabibullah and Gabriel (1973), Koteja (1990b) and Gieselmann (1990) who studied biology of A. destructor. Foster

et al. (1995) studied biology of Aonidiella aurantii Mask. in detail and reported similar results.

During development, color, shape, length and width of A. destructor changed as the insect developed to the next stage. Overall, male and female ranged from 0.2 mm to 1.16 mm and 0.2 mm to 0.8 mm, respectively. These observations agree with those of Williams and Watson (1988) who reported that slide–mounted adult female A. destructor were 0.7–1.2 mm in length with pyriform and membranous body with egg, larval and pre-oviposition stages were 5, 17 and 25 days, respectively.

A. destructor laid 65–110 (average 90) eggs in Fiji at a mean temperature of 30 ˚C however; in China on Actinidia, as a host, the female laid 32–43 eggs (Taylor, 1935; Zhou et al., 1993). Watson and El-Serwy (2007) discovered the egg laying range of 39–48 eggs per female in Acanthomytilus sacchari Hall under scale cover. In the present study, relatively high fecundity (28–65 eggs) with long oviposition period (10–12) in A. destructor was noted on mango plants. Crawlers emerged from early laid eggs while the pest still continued eggs laying. Continuous reproduction enables the pest to have multiple overlapping generations during its peak season from July to December. Similarly, length and width of 1st instar, 2nd instar, adult male and adult female changed along with developmental stages. These results are in line with that of Williams and Watson (1988) who studied that slide mounted adult female was of 0.7–1.2 mm in length with membranous body

In the present investigation the crawlers that emerged from July to October were mostly females with male to female sex ratio of 1:2.1; however, from October onwards male crawlers were dominant. Nur (1990) also reported 1:1 male to female ratio during July to August in diaspidid scale insects but late in the season, number of males increased to as many as twice the number of females. Bonafonte (1981) discovered that fecundity of Chrysomphalus aunidum L. was influenced by temperature and humidity. Quayle (1911a) had reported that male Aonidiella aurantii Maskell armored scales were more common in spring in southern California, USA, compared to autumn, summer and winter seasons; sex ratio of the insect was 1: 0.6 (male: female) from January to July, and 1:1 from mid-summer to mid-winter. Sex ratio in coconut scale was much influenced by temperature and RH variations.

Developmental duration of A. destructor life stages did not generally differ amongst different mango varieties in the present study. These results are in agreement with those of Ahmad and Ghani (1972) who found that duration of different life stages of A. destructor on squash, Cucurbita maxima was not significantly different from coconut palms, studied by Taylor (1935). Tabibullah and Gabriel, (1975) also studied the biology of A. destructor in the field and greenhouse in the Philippines on three coconut cultivars: Laguna, Coco nino and Tambuliid, and reported no significant differences in the developmental duration of A. destructor on the three coconut cultivars.

From results of the present study, it can be concluded that A. destructor has a rather high reproductive capacity on mango plant as compared to Actinidia in China and has many overlapping generations from July to December in Pakistan each year. The results of the study of biology at various temperatures and RH would be helpful in predicting population explosions of the pest insect for timely adopting of population management strategies.

Diaspidids scale insects cause severe damage to mango plants due to sucking of cell sap from leaves, shoots, twigs and fruits (Elwan, 1990). As a result, both photosynthesis and respiration affect badly and ultimately, yield is dropped considerably. The infested leaves become curl, yellow and at last drop from trees (Moffit, 1999). In some cases, complete defoliation occurs in mango nursery plants and mature trees.

In the next chapter, physiological and biochemical changes occurred in mango plant leaves under scale insects infestation have been studied in detail.

Chapter 5.

Physiological and biochemical changes in mango leaves under A. destructor infestation.

5.1. Introduction.

Aspidiotus destructor is a polyphagous pest of both tropical and subtropical plants that suck cell sap from leaves, stems and fruits, causing yellowing, tissue distortion and dieback. Yellowing of the leaves is due to sucking of large amount of sap sucked by the scale and injecting toxic saliva into the leaves of host (Waterhouse and Norris, 1987). In an environment, where natural enemies are scarce, complete defoliation has been observed due to scale insects (Debach, 1974). Both insects and plants have adopted tactics to escape each other’s defense systems which has resulted in the development of a sophisticated protection system in plants that has the ability to recognize the signals from damaged cells and activates the plant immune response against the herbivores (Howe and Jander, 2008; Hare, 2011). On the other hand, insects have the ability to trace their hosts even when the host plants are concealed in group of other plants. Plant volatiles such as terpenoids, aromatic compounds and green leaf volatiles play a pivotal role in the host location process (Bruce et al., 2005). Numerous stages are involved from detecting insect feeding to the production of indirect plant responses. These include disruption of leaf tissues, release of elicitors, signaling cascades and the activation of transcription factors eventually leading to the plant cell response (Maffei et al., 2007). In an insect induced-stress condition, rate of photosynthesis is increased in the damaged tissues causing nutritional difficiency in the plant as more nutrients are required for the plant to help in repair of the damaged tissues (Nowak and Caldwell, 1984; Golawska et al., 2010). Studies show that response of plant to insect infestation has different effect on photosynthetic pigments (chlorophyll a and b). Stress condition may some time be helpful to explore the mechanism of resistance as well as photosynthetic pigments can be used as markers for the identification of chlorosis caused by herbivores (Haung et al., 2014). Different techniques like chlorophyll a fluorescene have been developed to assess the physiological response of plants to herbivory (Calatayud and Le Ru, 2006). These techniques help to evaluate the impact of insect infestation on non–photochemical energy dissipation, photochemistry and photosynthetic performance of plant under insect stress (Nabity et al., 2009). 54

In stress condition, plant generates excess reactive oxygen species (ROS), which cause changes in plant physiology (Muhajan and Tuteja, 2005). Basically, ROS are reduced forms of atmospheric oxygen (O2), which are produced by the oxidation of oxygen to form a singlet 1 – oxygen ( O2), or transfer an electron to form superoxide radical (O2 ), and form hydrogen peroxide (H2O2) if two electrons are transferred to oxygen. Similarly, if three electrons are transferred to oxygen, they produce hydroxyl radical (OH–). These reduced forms of oxygen are highly reactive and oxidize different cellular components causing oxidative damage to the plant cells (Mittler, 2002). ROS are produced in different parts of cell like the chloroplast, mitochondria, plasma membrane, endoplasmic reticulum, apoplastic space and cell wall (Blokhina et al., 2013). ROS are produced in normal metabolism in plant like photosynthesis and respiration but when plant tissues are exposed to stress condition, accessive amount of ROS are formed in the plant cells, which have detrimental effects on biological activities of cells (Bolwell et al., 2002). ROS has also beneficial effects on plant cell due to their importance in signaling pathways to necessitate fine regulation of their level in the cell by ROS– scavenging enzymes like superoxide dismutase (SOD), peroxidase (POD), glutathione peroxidase (GPX), Ascorbate peroxidase (APX), catalase (CAT), glutathione peroxidase (GR) (Shigeoka et al., .– 2002). Highly reactive oxygen species are superoxide radical (O2 ), hydrogen peroxide 1 . – (H2O2), singlet oxygen ( O2), perhydroxy radical (HO2 ), hydroxyl radical (OH ), and alkoxy radicals (RO.). They damage proteins, lipids, carbohydrates and DNA, and ultimately cause plant death (Khan and Singh, 2008). Under biotic or abiotic stress conditions, the balance between production and scavenging of ROS (antioxidants) is disturbed, which results in increased level of intercellular ROS (Demiral and Turkan, 2005). The degree of damage by ROS depends on the balance between the production of ROS and its removal by the antioxidant defense systems.

Salivary glands of insect inject enzymes like H2O2 through saliva to the plant at the time of feeding. H2O2 generated glucose oxidase contribute to the increase in the concentrations of reactive oxygen species at the site of herbivore attack (Felton and Eichenseer, 1999). Although production of ROS in the plant as a result of herbivore feeding is yet to be explored but wounds by insect feeding induce ROS in the damaged tissues (Gatehouse, 2002; Kessler and Baldwin, 2002). In the present study, physiological and biochemical changes in mango plant leaves under A. destructor infestation have been investigated.

5.2. Material and Methods

5.2.1. Raising nursery of test plants

Nursery plants of three mango cultivars i.e. Langra, Kala Chounsa and Anwar Raotul of uniform age (2 years) and size (60–70 cm) were obtained from FNF, D. I. Khan. The plants were infested with 100 scale insects (crawlers) on each leaf to establish cohorts of even age scale insects according to the procedure given in Chapter 2 (2.2). The plants were checked daily untill the settlement of scale insects on leaves. Number of insects was counted and the extra scales were pricked by needles to stop their development. Infested leaves were collected after 0, 10, 20 and 30 days from each variety. The leaves were thoroughly washed with double distilled water and immediately put in ice and moved to the laboratory for further experimentations.

5.2.2. Determination of leaf biomass, leaf area and photosynthetic pigments Leaf biomass, leaf area and photosynthetic pigments were determined taking three leaves (upper, lower and middle) from each plant and replicated three times with one nursery plant per replication.

For fresh weight, three scale infested leaves (lower, middle and upper) from each nursery plant were picked, washed with distilled water, dried and weighed separately on electrical balance and then average weight of the three leaves was calculated. For dry biomasses, three leaves per replication were put in an incubator at 70ºC for 48 hours. Dry weight was measured on electric balance.

Leaf length and width of three scale infested leaves (lower, middle and upper) from each nursery plant were recorded and then leaf area was calculated according to Hunt et al. (1987).

Formula for leaf area

Leaf area = Leaf length × leaf width × 0.62

Where 0.62 is correction factor for mango leaf calculated manually.

Photosynthetic pigments (chlorophyll a, b and total carotenoids) of scale infested fresh mango leaves were determined by using the method of Lichtenthaler and Welburn (1985). Fresh mango leaf sample of 25 mg were taken in 15 mL falcon tube with equal volume (25

ml) of magnesium oxide. Then, 5 ml methanol was added to it and the mixture was homogenized on orbital shaker for 2 hours. After homogenization, turbid pigment extract (methanol extract) was transferred to 5 mL graduated centrifuge tube. Centrifugation was done for 5 minutes at 4000 revolution per minute (rpm) at room temperature. The supernatant was transferred with pipette to a 1–cm path length cuvette and took an absorbance reading against a solvent blank in a UV-VIS spectrophotometer at three wavelengths as per given formulas:

666 nm (chlorophyll a maximum using 100 % acetone)

653 nm (chlorophyll b maximum using 100 % acetone)

470 nm (carotenoids)

5.2.3. Cell viability and cell injury.

5.2.3.1. Cell viability

Cell viability was determined by measuring dehydrogenase activity using the triphenyltetrazolium chloride (TTC) reduction technique (Knievel, 1973). Fresh mango leaves samples of 0.2 g were weighed and incubated in 0.6 % (v/v) TTC in phosphate buffer (pH 7.8) at 30oC for 24 h. Leaves were thoroughly washed with distilled water and extracted with 95 % ethanol at 60°C for 4 h. The absorbance was recorded at 490 nm to estimate the amount of viable leaf tissues. Leaf viability was expressed as the absorbance per gram of fresh weight and the data were replicated three time.

5.2.3.2. Membrane leakage

Membrane injury in scale infested leaves was determined and was indicated by membrane leakage measurements (Lu et al., 2008). 20 pieces of 1 cm of leaves were taken and placed in 20 mL of sterile distilled water in a test tube. The samples were incubated at 10°C for 24 hours and then kept at room temperature for 10–15 minutes. Electrical conductivity “EC–1” was measured using conductivity meter (BMC EC meter–400 L). The samples were autoclaved at 121oC for 10 minutes and then kept at room temperature for 10–15 minutes. Electrical conductivity ''EC–II'' was examined again with the help of conductivity meter. Membrane leakage was determined by the following formula:

Membrane leakage = EC-1 × 100 EC-2 5.2.4. Determination of various ions (Na+, K+, Ca++)

Scale-infested mango leaves were placed in an incubator at 74°C for 24 hours for drying. 25 mg of dried and well–grounded samples were taken in a beaker which was added with concentrated sulphuric acid (H2SO4) and H2O2, warmed and stirred smoothly by hand till the formation of black slurry. The liquid was allowed to evaporate with contineous heating until a drop is remained in the beaker. 25 ml distilled water was added to the extract and the samples were analyzed for determination of various ions using flame photometer by the method of Awan and Salim (1997).

For each ion analysis, 100 ppm, 150 ppm, 200 ppm and 250 ppm standard solution were prepared. After preliminary standardization of the flame photometer with standard solutions, the concentrations of Na+, K+ and Ca+2 were determined in all the plant samples.

5.2.5. Determination of stress markers

To determine lipid peroxidation and reactive oxygen species, 0.5 g fresh leaf materials of mango nursery plant infested with 100 mango scale insects for different time periods were taken. Malondialdehyde (MDA) was determined by the method of Zhou and Leul (1998). 0.5 g of mango leaf sample was taken and homogenized in 10 mL of 0.25 % TBA (thiobarbitaric acid) which was dissolved in 10 % TCA (trichloroacetic acid). The samples were heated at 95 °C for 30 minutes and cooled in ice. The samples were again centrifuged at 5000 rpm for 10 minutes. The absorbance was recorded at 600 nm and 532 nm, where nonspecific absorbance of 600 nm was subtracted from 532 nm. MDA contents were expressed as µmolg–1 fresh weight (FW) using extinction coefficient of 155 mMcm–1.

Concentration of H2O2 was determined according to Velikova et al. (2000). 0.5 g crushed mango leaves were taken in an ice cold conditions which was added with 5.0 mL of TCA (0.1 %, w/v). The homogenate was centrifuged for 20 minutes at 14,000 rpm. For the preparation of 4 mL reaction solution, 1 mL supernatant, 1 mL phosphate buffer (pH 7.8) and 2 mL of potassium iodide (1 M KI) were used. The reaction mixture was transferred with pipette to a cuvette and took absorbance reading in a UV-VIS spectrophotometer at 390 nm. The H2O2 content was determined by using an extinction coefficient of 0.28 μMcm–1 and expressed as µmol g–1 FW.

5.2.6. Determination of total soluble proteins (TSP) and antioxidants

Fully crushed scale infested mango leaves sample of 0.5 g was taken and homogenized in 8 mL of 50 mM potassium phosphate buffer (pH 7.8) containing 2 mM ethylenediamine–tetra acetic acid (EDTA) and 1 % (w/v) polyvinyl-polypyrrolidin (PVP) under ice cold condition. The crude extract was centrifuged for 15 min at 14,000 rpm at a temperature of 4°C. The supernatant was taken and used for the determination of total soluble proteins and different enzymatic and non–enzymatic antioxidants. TSP content was determined by the method of Bradford (1976) and bovine serum albumin was used as a standard. The activity of superoxide dismutase (SOD) (EC1.15.1.1) was determined using the protocol of Zhang et al. (2008) following the inhibition of photochemical reduction due to nitro blue tetrazolium (NBT). The reaction solution consists of 50 mM potassium phosphate buffer (pH

7.8), 13 mM methionine, 75 mM NBT, 2 mM riboflavin, 0.1 mM EDTA-Na2 and 100 µL of enzyme extract in a 3–ml volume). For the measurement of one unit of SOD activity, reading was taken to know the amount of enzyme required to cause 50% inhibition of the NBT reduction at an absorbance of 560 nm. For the measurement of ascorbate peroxidase (APX) (EC 1.11.1.11) activity, a reaction mixture was prepared which includes 3 mL of 50 mM potassium phosphate buffer (pH 7.8),

0.1 mM EDTA-Na2, 0.3 mM ascorbic acid, 0.06 mM H2O2 and 100 µL enzyme extract. The solution was added with H2O2 and after 30 seconds, absorption was taken at 290 nm (Nakano and Asada, 1981). Catalase (CAT) (EC1.11.1.6) activity was determined using the method of –1 Aebi (1984). H2O2 (extinction coefficient 39.4 mM cm ) was used for 1 minute at A240 in 3 mL reaction solution which includes 50 mM potassium phosphate buffer (pH 7.8), 2 mM

EDTA-Na2, 10 mM H2O2 and 100 µL enzyme extract. The activity of peroxidase (POD) (EC1.11.1.7) was determined by the method of Zhou and Leul (1999) using guaiacol as the substrate in a total volume of 3 mL. The reactant solution contained 50 mM potassium phosphate buffer (pH 7.8), 1% guaiacol, 0.4% H2O2 and 100 mL enzyme extract. Changes in absorbance by the addition of guaiacol were measured at 470 nm per g FW per min at 25±2°C. 5.2.9. Proline accumulation in mango leaves under scale insects infestation. Proline quantification in mango leaves under mango scale insect infestation was done by using the method of Bates et al. (1973). 100 mg fresh plant leaves were crushed in liquid nitrogen and completely homogenized in 5 mL of 3% (w/v) salfosalisylic acid. The residues

were centrifuged at 4000 rpm for 30 minutes. Supernatant of the homogenized tissues (~1 mL) were put in 1 mL acid ninhydrin (1.25 g ninhydrin warmed in 30 mL of glacial acetic acid with 20 mL of 6 M phosphoric acid with continues stirring at room temperature) for reaction. The mixture was put in oven at 100°C for 1 hour and color phase containing toluene was placed in room temperature (25°C) for 20 minutes. The reaction mixture was vigorously mixed with 2 mL toluene. The optical density was measured at 520 nm by using spectrophotometer (Dr. /4000 spectrophotometer, HACH, USA) while toluene was used as blank. Concentration of proline on fresh weight basis was determined using D-proline standard curve (µmol proline/g fw). The data were analyzed using the one way analysis of variance (ANOVA). The ANOVA was applied for each treatment with three replications followed by the Tukey HSD at P≤ 0.05 level of significance. 5.3. Results

5.3.1. Physiological changes in mango leaves under scale infestation

Leaf area, leaf fresh and dry weight of scale infested mango leaves decreased with increasing stress duration (10, 20 and 30 days). In the three varieties, leaf area decreased significantly (P ≤ 0.05) after 20 and 30 days exposure to scale insects as compared to threir respective controls (Table 5.1). However, maximum percent decrease in leaf area was recorded in Kala Chounsa (76.8 %), followed by Anwar Ratoul (52.3 %) and Langra (42.7 %). Mango plant exposed to scale insects for 10 days showed no significant decline (P ≤ 0.05) in leaf fresh weight in Langra and Anwar Ratoul cultivars whereas significant reduction was noted at 20 and 30 days post scale infestation period. In Kala Chounsa, evident decrease (47.8 %) was observed in leaf fresh weight after 10 days exposure to scale insects. Kala Chounsa (74.8 %) and Anwar Ratoul (66.7 %) showed maximum decrease in leaf fresh weight after 30 days exposure to scale insect infestation as compared to Langra (57.3 %).

Table 5.1 Effect of A. destructor on leaf area and leaf biomass in mango leaves Leaf area (cm2) Leaf fresh weight (gm) Leaf dry weight (gm)

Days Kala Anwar Kala Anwar Kala Anwar Langra Langra Langra Chounsa Ratoul Chounsa Ratoul Chounsa Ratoul

0 47.8 a 50.3 a 35.4 a 1.4 a 1.6 a 1.1 a 0.3 a 0.3 a 0.4 a

31.0 10 38.4 b 35.4 b 1.2 b 0.8 b 1.0 a 0.2 b 0.3 a 0.3 b ab

20 31.7 bc 19.0 c 25.0 b 0.7 c 0.5 c 0.6 b 0.2 c 0.2 b 0.2 c

30 27.4 c 11.7 d 16.9 c 0.6 c 0.5 c 0.4 c 0.1 c 0.1 b 0.1 d

LSD 7.39 3.01 8.04 0.17 0.15 0.12 0.03 0.05 0.06 (0.05)

Different letters within columns indicate significant differences (P ≤/0.05) among treatments (0, 10, 20, 30 days).

Significant (P ≤ 0.05) decrease in leaf dry weight was observed after 20 days (0.15 cm and 0.16 cm) and 30 days (0.13 cm and 0.12 cm) exposure to scale insects in Langra and Kala Chounsa varieties, respectively as compared to control (0.30 cm and 0.31 cm). In Anwar Ratoul significant decline (P ≤ 0.05) in leaf dry weight was noted at 10 days (0.28 cm) and 20 days (0.17 cm) infestation by scale insects compared to control (0.44 cm). Overall, maximum percent reduction in leaf dry weight was recorded in Anwar Ratoul (70.45 %), followed by Kala Chounsa (61.3 %) and Langra (56.7 %).

5.3.2. Changes in photosynthetic pigments

The mean data of all the three components of photosynthesis (chlorophyll a and b and Carotenoids) were significantly reduced in Langra, Kala Chounsa and Anwar Ratoul (Table 5.2). Maximum chlorophyll a content was recorded in control in all the three cultivars, which was decreased along with increasing scale insect infestation duration. Maximum reduction in chlorophyll a contents was observed in Anwar Ratoul (55.5 %), followed by Langra (53.4 %) and Kala Chounsa (53.3 %) after 30 days post scale insects stress span.

Table 5.2 Effect of A. destructor on chlorophyll a and b and carotenoids contents in mango leaves

Chlorophyll a mg/gmFW Chlorophyll b mg/gmFW Total carotenoids µg/gFW

Days Kala Anwar Kala Anwar Kala Anwar Langra Langra Langra Chounsa Ratoul Chounsa Ratoul Chounsa Ratoul

0 20.3 a 20.6 a 21.4 a 26.1 a 38.0 a 36.5 a 2618.6 a 2721.4 a 2594.2 a

19.9 10 19.1 a 13.8 b 23.8 a 34.8 a 36.1 a 2183.9 b 2576.9 a 2438.0 b ab

20 17.7 b 12.7 b 10.8 c 16.9 b 19.5 b 25.8 b 1317.3 c 1721.1 b 1592.1 c

30 9.5 c 9.6 c 9.5 c 15.8 b 16.1 b 18.5 c 998.0 d 1070.7 c 1089.7 d

LSD 2.53 1.99 1.59 4.45 3.46 3.95 160.03 206.3 141.47 (0.05)

Different letters within columns indicate significant differences (P ≤/0.05) among treatments (0, 10, 20, 30 days).

Highest chlorophyll b contents were observed in control in Kala Chounsa (38.0 mg/gm FW), followed by Anwar Ratoul (36.5 mg/gm FW) and Langra (26.1 mg/gm FW). In the three tested varieties, chlorophyll b contents gradually decreased as the scale infestation period increased. Maximum reduction (57.51 %) was found in Kala Chounsa at 30 days scale insects infestation period, followed by Anwar ratoul (49.3 %) and Langra (39.3 %).

In varietal comparison, maximum reduction in carotene contents were observed in Langra (61.89 %), followed by Kala Chounsa (60.66 %) while least reduction in carotene contents was recorded in Anwar Ratoul (57.99 %) after 30 days exposure to scale insects infestation.

5.3.3. Changes in cell viability under scale insect infestation

5.3.3.1. TTC (Triphenyltetrazolium chloride) method

Due to sucking sap from leaves, the cell viability in mango leaves under mango scale insects stress reduced along with increasing infestation period. Maximum cell viability was recorded in the leaves of control plant in all the three varieties of mango i.e. Langra (9.6 Pb), Kala Chounsa (10.0 Pb) and Anwar Ratoul (8.7 Pb), which were declined in Langra (9.0 Pb), Kala Chounsa (7.4 Pb) and Anwar Ratoul (7.8 Pb) after 10 days post infestation interval as compared to control (Table 5.3). However significant decrease in cell viability was observed

after 30 days scale insects infestation span in all the three selected mango varieties. Among the three varieties, maximum decrease in cell vaibility was observed in Langra (85.9%), followed by Kala Chounsa (83.8%) and Anwar Ratoul (62.4 %) at 30 days post treatment of scale insects.

5.3.3.2. Electrolyte leakage under scale insect infestation

Cell injury increased significantly (P ≤ 0.05) in all the treated varieties with increasing insects stress on mango leaves as compared to control (Table 5.3). However, non–significant effect was observed during 10–30 days exposure to scale insects in the three mango cultivars while cell injury at control was significantly different from other treatments in Langra. In Kala Chounsa and Anwar Ratoul, significant difference was found at 30 days exposure to scale insects infestation as compared to control.

Table 5.3 Effect of A. destructor on cell viability and electrolyte leakage in mango leaves Cell viability (Pb) Electrolyte leakage (Pb)

Days Kala Anwar Kala Anwar Langra Langra Chounsa Ratoul Chounsa Ratoul

0 9.6 a 10.0 a 8.7 a 48.2 b 58.3 b 56.3 b

10 9.0 a 7.8 b 7.4 a 77.4 a 63.5 ab 65.4 ab

20 4.3 b 4.1 c 4.8 b 80.2 a 75.2 a 67.8 ab

30 1.4 c 1.6 d 3.3 b 83.4 a 77.6 a 71.6 a

LSD 2.29 1.46 2.21 13.21 16.62 14.14 (0.05)

Different letters within columns indicate significant differences (P ≤/0.05) among treatments (0, 10, 20, 30 days).

5.3.4 Scale-induced ionic changes in mango leaves

Both Na+ and Ca+ ions contents gradually increased with increasing scale insect infestation period while K+ ion decreased in all the three mango cultivars (Table 5.4). Maximum Na+ ions concentration was recorded at 30 days exposure of mango leaves to scale insect infestation in all varieties. As a whole highest increase in Na+ content (84.1 %) was recorded in Langra which was statistically at par (P ≤ 0.05) with Kala Chounsa and Anwar Ratoul. Similarly, K+ contents were decreased with increasing scale insect stress. In the three mango 63

varieties, 56–60% decrease in K+ contents was recorded in Anwar Ratoul at 30 days post scale infestation periods as compared to control. Overall effect of scale infestation on three mango varieties in respect to K+ contents was observed in Anwar Ratoul (59.5 %), which was statistically at par with Langra (57.8 %) and Kala chounsa (56.4 %). Mango scale insect infestation caused gradual increase in the level of Ca2+ content. Highest increase in Ca2+ contents were noted in Langra (89.2 %) while Kala Chounsa (74.4 %) and Anwar Ratoul (51.9 %) showed comparatively less increase in C+2 contents but were statistaically at par with Langra. Table 5.4 Effect of A. destructor infestation on ions analysis in mango plant leaves

Sodium (Na+) Potassium (K+) Calcium (Ca+2)

Days Kala Anwar Kala Anwar Kala Anwar Langra Langra Langra Chounsa Ratoul Chounsa Ratoul Chounsa Ratoul

0 60.7 b 58.7 c 58.3 b 51.3 a 52.0 a 68.3 a 92.2 d 94.9 d 102.0 c

10 97.0 a 72.7 b 59.7 b 37.3 b 39.7 b 59.0 b 116.8 c 117.4 c 118.8 b

20 102.3 a 74.3 b 84.3 a 28.0 c 32.3 c 39.3 c 140.6 b 146.3 b 127.3 b

30 111.7 a 91.3 a 95.0 a 21.7 d 22.7 d 27.7 d 174.5 a 165.5 a 154.9 a

LSD 36.29 12.88 12.03 6.43 5.02 7.74 12.35 9.98 9.25 (0.05)

Different letters within columns indicate significant differences (P ≤/0.05) among treatments (0, 10, 20, 30 days).

5.3.5. Effect of scale insect infestation on total soluble protin (TSP) contents and proline accumulation in mango leaves

The recorded data illustrate that significant (P ≤ 0.05) decrease in TSP contents was found in all varieties along with increasing infestation duration (Table 5.5). Maximum TSP was found in control of all the three varieties while lowest TSP contents were present in plant exposed to scale insect for 30 days. Among the three mango varieties maximum decrease in TSP was observed in Kala Chounsa after 30 days (80.3 %) exposure to scale insects, followed by Lngra (71.6 %) and Anwar Ratoul (30.9 %). Data pertaining to proline accumulation showed that as the infestation period of scale insects increased, the proline accumulation increased significantly as compared to control (table 5.5). In all the three mango varieties, minimum proline contents were recorded in control i.e. 64

langra (4.57µmol/gfw), Kala Chaunsa (8.06 µmol/gfw) and Anwar Ratoul (9.66 µmol/gfw). The proline contents increased significantly (P ≤ 0.05) in all the three varieties with varying degrees. Maximum increase was observed in Langra (61 %), followed by Kala Chounsa (56.48 %) while Anwar Ratoul (50.46 %) showed lowest increase in proline accumulation after 30 days exposure to scale insects infestation. Table 5.5 Effect of A. destructor on total soluble proteins (TSP) contents and proline accumulation in mango leaves Total soluble proteins (mg/ml) Proline accumulation (µmol/gfw)

Days Kala Anwar Langra Kala Chounsa Anwar Ratoul Langra Chounsa Ratoul

0 3.4 a 3.5 a 3.0 a 4.6 c 8.1 b 9.7 b

10 2.2 b 2.9 a 2.5 b 9.2 b 10.66 b 13.1 ab

20 1.3 c 1.3 b 1.3 c 10.5 ab 15.8 a 16.1 ab

30 1.0 c 0.7 b 2.1 c 11.9 a 18.5 a 19.6 a

LSD 0.63 0.67 0.45 2.0 3.1 7.1 (0.05)

Different letters within columns indicate significant differences (P ≤/0.05) among treatments (0, 10, 20, 30 days).

5.3.6. Changes in stress markers under scale insect infestion

Malondialdehyde (MDA) contents are the indication of lipid peroxidation in the cell. In stress condition MDA contents increase along with the increase in stress duration. In the present study, considerable increase in the MDA was noticed in the mango plant leaves infested with scale insects (Table 5.6.). The most obvious increase in MDA contents was observed in Langra at 20 days (119.7 %), followed by Kala Chounsa (112.6 %) post scale infestation while minimum increase (72.8 %) in MDA contents was observed in Anwar Ratoul. In all the three varieties, MDA contents decreased at 30 days scale insects infestation time after continuous increase up to 20 days as compared to controls.

Highly significant (P ≤ 0.05) increase in H2O2 concentration was observed in mango leaves under mango scale insect stress at various time periods (Table 5.6). Overall response of the three selected mango cultivars in term of H2O2 was significantly different (P ≤ 0.05) from each other under mango scale insects infestation. Highest H2O2 concentration was recorded in

Anwar Ratoul (498.7 µM/g FW), followed by Kala chounsa (469.1 µM/g FW) while Langra showed the least (422.6 µM/g FW) H2O2 activity after 30 days exposure to scale insect stress.

Table 5.6 Effect of A. destructor on malandialdihyde (MDA) contents and hydrogen peroxide

(H2O2) in mango leaves

Malandialdihyde (MDA) Contents Hudrogen peroxide (H2O2) contents (µM/g FW) (µM/g FW) Days Kala Anwar Anwar Langra Langra Kala Chounsa Chounsa Ratoul Ratoul

0 13.4 b 15.4 c 18.9 c 236.3 d 132.5 d 266.0 d

10 25.6 ab 22.8 b 26.5 b 288.3 c 178.3 c 301.4 c

20 29.4 a 32.7 a 32.7 a 334.1 b 373.6 b 423.7 a

30 26.8 a 21.7 b 30.2 ab 422.6 a 469.1 a 498.7 a

LSD 12.67 1.56 5.51 43.34 26.77 21.08 (0.05)

Different letters within columns indicate significant differences (P ≤/0.05) among treatments (0, 10, 20, 30 days).

5.3.7 Changes in the antioxidant enzymes of mango leaves under scale insect infestion

Data showed that peroxidase (POD) activity was significantly (P ≤ 0.05) induced by scale insects feeding in all the three varieties (Table 5.7). Among the three mango cultivars Langra showed maximum increase (52.3 %) in POD activity followed by Kala Chounsa (36.8 %) at 30 days post scale stress period while Anwar Ratoul was less (20.8 %) influenced by scale insects in respect to POD activity. Similarly, significant increase (P ≤ 0.05) in POD activity was observed in mango leaves at 10 days and 30 days exposure to scale insects as compared to control. Activities of superoxide dismutase (SOD) increased with increasing stress duration (10, 20 and 30 days) in all the three varieties (Table 5.7). Maximum increase in SOD activity was recorded in Anwar Ratoul (157.1 (µ/gFW)), followed by Kala Chounsa (156.1 (µ/gFW)) and Langra (119.5 (µ/gFW)) after 30 days exposure to scale insect stress which were statistically non–significant to each other but were significantly different from control.

Table 5.7 Effect of A. destructor on peroxidase (POD) and superoxide dismutase in mango leaves POD (µM/gfw) SOD (µ/gFW)

Days Kala Anwar Kala Anwar Langra Langra Chounsa Ratoul Chounsa Ratoul

0 2.62 c 2.88 c 3.37 b 100 b 100 c 100 c

10 2.93 bc 3.11bc 3.68 ab 59.8 c 54.9 d 95.5 c

20 3.28 b 3.60 ab 3.87 a 85.2 b 144.5 b 118.9 b

30 3.99 a 3.94 a 4.07 a 119.5 a 156.1 a 157.1 a

LSD (0.05) 0.44 0.67 0.46 15.69 5.31 13.05

Different letters within columns indicate significant differences (P ≤/0.05) among treatments (0, 10, 20, 30 days).

In the present study the ascorbate peroxidase (APX) activity was determined which were greatly varied in all the three mango varieties (Table 5.8). There were significant differences (P ≤ 0.05) found in the three selected varieties of mango exposed to scale insect stress. Maximum mean value was observed in Langra (66.7 mM/g FW) which was statistically at par with Kala Chounsa (64.9 mM/g FW) and Anwar Ratoul (62.14 mM/g FW) at 30 days exposure to scale insecs infestation. APX activities increased up to 42.4 % in Langra at 30 days post scale stress period followed by Anwar Ratoul (30.0 %) while lowest increase was recorded in Kala Chounsa (19.3 %).

The specific activity of catalase (CAT) in the leaves of mango plant under mango scale insect stress is given in Table 5.3.8.In all the three cultivars, highly significant differences (P ≤ 0.05) in CAT activity were found under the scale insect infestation. Cultivar Langra showed maximum CAT activity (0.43 mM/g FW) at 20 days post scale insects treatment which was at par with Kala Chounsa (0.33 mM/g FW) and Anwar Ratoul (0.36 mM/g FW). Interestingly, the CAT activity decreased at 30 days exposure to scale insects in all the three varieties which was also statistically at par with each other.

Table 5.8 Effect of A. destructor on ascorbate peroxidase and catalase activities in mango leaves

APX (mM/g FW) CAT (mM/g FW)

Days Kala Anwar Kala Langra Langra Anwar Ratoul Chounsa Ratoul Chounsa

0 46.9 d 54.4 b 47.8 c 0.31 b 0.18 c 0.19 b

10 53.2c 57.5 b 55.4 b 0.36 b 0.25 ab 0.33 a

20 60.6 b 64.8 a 60.1 ab 0.43 a 0.33 a 0.36 a

30 66.7 a 64.9 a 62.1 a 0.33 b 0.27 a 0.35 a

LSD 1.96 2.08 4.74 0.06 0.0.08 0.08 (0.05)

Different letters within columns indicate significant differences (P ≤/0.05) among treatments (0, 10, 20, 30 days).

5.4. Discussion and conclusion

Scale insects feed on different parts of their host plants like leaves, twigs, branches and roots (William and Granara de Willink, 1992). The pests suck cell sap from leaves and twigs due to which both photosynthesis and respiration are negatively affected, and ultimately leaf area and leaf biomass are reduced on a wide range of crops. Our results indicated that with increasing scale insect infestation, leaf area and biomass decreased as compared to control at various levels of pest infestation. Kala Chounsa was the most affected variety as 76.8 % and 74.8 % decrease in leaf area and leaf fresh weight was noted, respectively after 30 days infestation by scale insects as compared to control. Maximum reduction (70.4 %) in dry weight was observed in Anwar Ratoul at 30 days scale infestation. Carson and Richard (1999) found that insect herbivory (xylem–tapping spittle bug, Philaenus spumarius) greatly reduced total plant biomass of Solidago altissima in New York, USA. These results are supported by the findings of Whittaker (1970) as well as Polis and Strong (1996).

Chlorophyll a, b, carotenoids and total soluble proteins contents in mango leaves under scale insect infestation decreased. Among the three varieties, maximum decrease in chlorophyll a contents was recorded in Anwar Ratoul (55.5 %). Langra and Kala Chounsa varieties were more affected by scale insect in respect to photosynthetic pigments and TSP, while Anwar Ratoul showed stability against insect stress. It is possible that Anwar Ratoul has unfavorable 68

characteristics (physical or chemical) for this scale insect. The semi erect branching system of Anwar Ratoul provides less shade and more aeration whereas A. destructor prefers the dense canopy structure provided by Kala chounsa and Langra. Bakrey (2009) reported that mango variety, Alphonso showed resistant to scale insect infestation in Egypt due to its unfavorable characteristics including thicker epidermis and sclerenchyma, deeper vascular bundle, condense pericyclic fibers and lignin as well as fewer numbers of resins (food sources) which act as physically difficult and energetically cost for insect penetration of mouth parts. In contrast, Langra and Kala Chounsa had thick leaves, heavily branched and round spreading system, which provided an ideal condition to scale insects for long association with mango plant causing slow but steady loss in chlorophyll and carotenoid contents. Scale insects, which are passed through feeding stage are called instar and molt when dormant stage. After settlement on leaf surface, crawlers started feeding for few days and then detached its rostrum (feeding tube) and stopped feeding (Foster et al., 1995). At this stage, leaves got some relief from scale feeding, but after 5–6 days, it re–entered its rostrum and again started feeding. Therefore, there were increasing and decreasing trends in chlorophyll and carotenoid contents in the mango leaf under scale insects stress. These findings are in agreement with Nabil (2013) who stated that mango shield scale Kilifia acuminata Signoret infested leaves have less chlorophyll a and b contents as compared to control. Fanizza et al. (1991) also reported chlorophyll contents decline under different stresses. Leaves of plants under stress produce less chlorophyll contents. Rafi et al. (1996) noted reduced chlorophyll level in cereal attacked by Diuraphis noxia (Mordvilko) (Homoptera: Aphididae). Heng-moss et al. (2003) found that under feeding by D. noxia, total chlorophyll and carotenoids concentration differed between Betta wheat isolines. Similarly, Burd and Elliott (1996) found significant decrease in chlorophyll contents infested by D. noxia in susceptible wheat and barley varieties. Significant loss in chlorophyll a and b was also found by Golawska et al. (2010) in four fabaceae species infested by pea aphid Acyrthosiphon pisum (Homoptera: Aphididae).

In the present study, cell viability greatly decreased in the mango plant leaves under scale insect infestation where as cell injury increased. It might be due to the fact that insects suck cell sap from leaves, injure cell membrane, which disturb the physiological processes of cell. TTC (Triphenyltetrazolium chloride) method is widely used as a general procedure to determine vitality in plant tissues (Coria et al., 1998; Schrazi and Muir, 1998; Sulpice et al., 1998). Triphenyltetrazolium chloride remains colorless until reduced to formazan, which is a

bright red pigment and is easily extracted and photometrically measured (Steponkus & Lanphear, 1967). In plant tissue, it is reduced by an enzyme called dehydrogenase, associated with mitochondrial function. With increasing scale insect stress, the Na+ and C++ level increased in the entire three mango varieties while K+ level decreased continuously. Weimberg (1987) stated that if Na+ increased inside the cell, it causes inhibition of K+ uptake in to the cell and an imbalance in Na+/ K+ ratio is created in the cell which is subsidized by flow of Ca++ from the cytosol to the perisome. Cicek and Cakirlar (2002) found that imbalance in Na+/ K+ ratio disturbs function of different enzymes and regulation of biological processes.

In stress physiology studies, MDA is used as suitable biomarker to assess the oxidative damage (Ohkawa et al., 1979). In our study, considerable increase in MDA concentration was observed in mango plant exposed to mango scale insect stress as compared to the control. It indicated that mango plants subjected to scale insect feeding induced oxidative stress at varying degrees in respect to infestation duration. Langra variety showed maximum MDA value exposed to scale insect stress as compared to Kala Chounsa and Anwar Ratoul. Form these results, it is inferred that scale insects infestation by their sucking cell sap and injecting toxin into the cell caused oxidative damage to the cells. Thus H2O2 levels in the plant cell increased as indicated by lipid peroxidation. Berglund and Ohlsson (1995) concluded that defense system of plant is induced by H2O2 as indicated by MDA which in turn enhance the production of secondary metabolites.

Rise in lipid peroxidation and H2O2 concentrations in mango leaves revealed that the scale insect caused oxidative damage to mango plant. That’s why, antioxidant enzymes activities increased which reflect varying degrees of stability or tolerance of mango plants to scale insects infestation. The enzymatic response to scale insects attack varied with changing time course. Here in this study, as the insects feeding continued, the plant tried to sustain the cellular sap loss throughout the study period (one generation of scale insects) with highest values at 30 days exposure to scale insects infestation. These results are augmented by Zhang et al. (2008) who reported that POD and SOD activities, and MDA concentration reached to its peak values at 6, 24, 12, and 48 hours during white fly, Bemesia tabaci infestation in China. However, POD activity was higher than other enzymes in response to B. tabaci stress which not only play role in stiffening of cell walls but are also actively involved in removal of H2O2. 70

In response to oxidative stress, proline contents consistently increased as defense mechanism in all the three mango varieties i.e. Langra (61 %), Kala Chounsa (56.48 %) and Anwar Ratoul (50.66 %). Hare and Cress (1997) reported that accumulation of proline contents in leaves is a well-established response to herbivory and drought as 40 % of the total free amino acids is made of proline in response to the cell sap sucked by plant hopper from leaves of Spartina alterniflora (Bacheller and Romeo, 1992). Free proline is also accumulated in Petunia hybrida plants where the increased proline served as an osmoprotectant that increased the drought resistance of the transformed plants (Yamada et al., 2005). It is also observed that though proline has its significant role in plant protection against different kinds of stress, yet it has also been shown to act as a feeding stimulant and to be an efficient way to store energy for insects (Behmer and Joern, 1994; Meyer et al., 2006; Gade and Auerswald, 2002). In our study, the POD activity was also strongly induced by scale insects along with increasing stress duration in the three mango varieties. It might be due to the disturbance of internal cellular components of cells by continuous sucking sap from the mango leaves. Because POD activity is very strongly correlated with the diverse physiological activities of plants i.e. lignifcatin, suberizatin, somatic embryogenesis and auxin metabolism (Zimmerlin et al., 1994). Lignificatin induced by enhanced POD activity causes increase in the toughness of leaves, which ultimately deter the sucking of herbivores feeding (Cipollini, 1997). Activation of POD produces phenoxy and other oxidative free radicals which deter herbivore feeding directly or generate toxins, which decrease digestibility of plant for insects (Felton et al., 1989). It is important to exhibit increase in POD activity, in different plants, in response to damage by insects feeding (Cipollini, 1997; Tscharntke et al., 2001; Allison and Schultz, 2004). Our results are in agreement with Viswanathan et al. (2005) who observed that plants under tortoise beetle feeding induce POD activity in leaves. Early-season POD activity was also differentially induced (up to 85 %) by Tortoise beetle and flea beetle herbivory. In field studies, it has been noted that specificity of plant-herbivore interaction can lead to complex, plant–mediated relation within herbivore communities (Van Zandt and Agrawal, 2004b, Viswanathan et al., 2005). In short, scale insects infestation significantly increased the enzymatic activities which in turn lead the plant to biosynthesis of phenylpropanoid and other secondary metabolites which demonstrated a very high resistance to insects attack. However, antioxidant enzymes and MDA showed different activity levels at different time after

infestation. Due to this factor, these enzymes might have different molecular bases to express corresponding genes for regulation of programmed defense mechanism. In mango leaves, the enzymatic system activated to varied levels in all the treatments of scale insects stress. The activity of enzymes was time dependent and almost all tested enzymes increased and then followed by a short period of decline. It means that the plant showed differential responses of antioxidative enzymes to scale insects in mango leaves. In the present study, with increasing H2O2 level in the leaves, the APX, SOD and CAT, which are main enzymes for scavenging of H2O2, increased due to toxic species exported to cytosol from organelles having an adverse effect on the plant function due to the formation of hydroxyl radicals which is formed by the reaction of H2O2 with superoxide radicals. These findings are in agreement with Dixit et al. (2001) who found that APX and CAT collectively work for the removal of H2O2. Similarly, increasing trend of SOD in mango leaves under – scale insect infestation revealed that superoxide radicals (O2 ) are formed in the cells, which are to be removed by SOD through the process of dismutation to O2 and H2O2, and then H2O2 – is sequentially reduced to H2O and O2 by CAT (Kashiwagi et al., 1997). As O 2 play role as precursor of more cytotoxic or highly reactive oxygen derivatives such as peroxynitrite or OH– (Halliwell and Gutteridge, 1999), SOD levels in leaves are an important protective – mechanism against cellular oxidative damage (O 2). Our results coincide with the observation of Bafana et al. (2011) and Celino et al. (2011) who concluded that SOD is key constituent of cellular defense against oxidative stress, and is among the most powerful antioxidants known in nature. Apel and Hirt (2004) recorded that when Lima bean is attacked by insects, SOD is one of the early enzyme activated for the reduction of superoxide anion which is produced either by Mehler reaction and photorespiration or by the reduction of molecular oxygen in mitochondria and oxidase reaction. Yang and Poovaiah (2002) and Maffei et al. (2006) observed that SOD, CAT and APX activity was found when Spodoptera littoralis started feeding on leaves of Lima bean. But among these, SOD activity is almost 7–37 fold higher than CAT while APX activity was lowest in Lima bean attacked by herbivores.

The effect of herbivores on CAT activity in leaves showed differential results. According to Liu et al. (2005), CAT activity subsidized in resistant rice while enhanced in susceptible varieties attacked by plant leaf hopper. Chen et al. (2004) observed increased CAT activity in pear leaves infested by Eriophyes pyri. It is very difficult to exactly compare different studies

due to variations in levels of induced responses along with differences in plant ontogeny, growing conditions and experimental methods.

In conclusion, it was confirmed that sucking sap from mango leaves by scale insects decreased leaf area and biomass, disturbed photosynthetic pigments, total soluble proteins and caused ionic imbalance. It was also inferred that scale insects infestation interrupted with the redox balance which lead to oxidative stress. It showed that damages caused by sucking insects were a chief potential factor for leaf chlorosis and creating oxidative stress products. These factors were collectively responsible for deteriorating photosynthetic activities which ultimately affect the health of attacked plant. To reduce this stress, antioxidant enzymes offerred a well organized antioxidant defense mechanism that provides protection to the plant. During this study, the antioxidant enzymes like APX, SOD and CAT activities provided an effective defense mechanism against oxidative destruction created by the buildup of ROS

Chapter 6.

Molecular changes in mango leaves under scale insects infestation

6.1. Introduction

Recent advancements in molecular biology led the discovery of different selective and sensitive analysis for DNA in eco–genotoxicology (Theodorakis et al., 2001, 2006; Castano and Becerril, 2004). Various DNA based techniques like RFLP (Restriction fragment length polymorphism), QTL (Quantitative trait loci), RAPD (Random amplified Polymorphic DNA), AFLP (Amplified fragment length polymorphism), SSR (Simple sequence repeats) and VNTR (Variable number tandem repeats) are used to asses the changes occurred at the DNA level. Random amplified polymorphic DNA (RAPD) is one of the useful techniques, which can be used to analyze genotoxicity and differences in DNA fingerprinting of untreated and treated individuals to genotoxic agents (Martins et al., 2005; Enan, 2006; Liu et al., 2007). RAPD PCR is a widely used technique to detecting changes in intensity as well as gain and loss of DNA bands as exposed to toxicant which is an indicator for DNA variation. This technique is developed by Williams et al. (1990) and Welsh and McClelland (1990). This technique is widely used for classification of species, genetic mapping, phylogeny as well as the novel biomarker assay for the discovery of DNA damage and mutational events in plants, invertebrates and vertebrates animals (Savva, 1996; Atienzar et al., 2000). The fragments of RAPD are detected after agarose gel electrophoresis and ethidium bromide (EB), which is stained to visualize bands shift, missing bands and appearance of new bands.

Objective of the present study is to evaluate the changes occurred in DNA of mango seedling induced by scale insects using RAPD technique.

6.2. Materials and methods.

6.2.1. DNA Damage Analysis

6.2.1.1. DNA Isolation

The total genomic DNA of mango seedling leaves under different infestation levels (0, 10, 20 and 30 days) of A. destructor, as per procedure given in Chapter 2 (2.6), was extracted using the method of Doyle and Doyle (1987). 0.05g fresh leaves were collected in 1.5 ml eppendorf tubes, crushed in liquid nitrogen in morter and pestle and mixed with 800 μL extraction 74

buffer comprising of (1% SDS, 500 mM NaCl, 100 mM Tris HCL (pH 8.5), 50 mM EDTA). The samples were taken in Eppendorf tubes and added with 20 μL ß mercaptoethanol and placed at 65 °C in an incubator for about one hour with regular shaking after every 15 minutes. The mixtures were then vortexed for one minute and 700 μL chloroform isomylalcohal (CI) was added and mixed properly with gentle shaking. The samples were centrifuged at 12000 rpm for 10 minutes at 25 °C. Supernatant was taken in a separate Eppendorf tube and 0.6 of chilled isopropanol added and incubated at –20 °C for 24 hours. The mixture was again centrifuged at 12000 rpm at 4 °C for 10 minutes. The pellet was washed with 70 % ethanol and air dried at room temperature and then dissolved in TE buffer and kept at –20 °C for PCR analysis.

6.2.1.2 RAPD primers

Total 8 primers were used (Operon Technologies, Alameda, CA, USA) including OPA –07, OPA–10, OPA–18, OPC–01, OPC–13, OPC–20, OPF–07 AND RPl–3. The sequence and GC % are given in table 6.1.

6.2.1.3 Amplification conditions

The extracted DNA was quantified spectrophotometrically and by electrophoresis on 0.8 % agarose gels. The amplification of DNA was performed according to the methodology of William et al. (1990) in an ABI (Applied Biosystem Inc, USA) thermal cycler. For PCR, 25

μL reaction mixture, 10 X buffer, 1.5 mM Mgcl2, 0.2 mM dNTPs, IU Taq DNA polemerase, 0.2 picomole primers, 50 mg of template DNA and PCR water were mixed. First denaturation process of DNA started at 94 °C for 5 minutes and then thermal cycling was performed with denaturation at 94 °C for 45 seconds. Then annealing at 37 °C for 1 minute and extension at 72 °C for 1.5 minutes and final extension at 72 °C for 10 minutes was done.

6.2.1.4 Gel electrophoresis

The products of amplified RAPD were run on 1.5 % agarose gel by electrophoresis at 125 V in 1 X TBE buffer for one hour which was stained with ethedium bromide and the gels were scanned using gel documentation system (Alpha Innotech, Alpha Imager EP, and USA). The PCR results were repeated twice and clear reproducing bands were selected for band scoring.

6.2.1.5 RAPD data analysis and scoring

The fragments patterns of DNA were evaluated by similarity index. Reproducible bands were scored manually as ‘1’ or ‘0’ for presence or absence of the bands. The data were used for similarity based analysis using the software program NTSYS (2.20). RAPD analyses were analyzed using the Nei genetic similarity index Nei and Li (1979).

Table 6.1 RAPD primers with their sequences and GC %.

S.No. Primers Sequence (5’__3’) GC % 1 OPA 10 GTGATCGCAG 60

2 OPC 01 TTCGAGCCAG 60

3 RPl 3 ACGCGACCTG 70

4 OPA 07 GAAACGGGTG 60

5 OPA 18 AGGTGACCGT 60

6 OPC 13 AAGCCTCGTC 60

7 OPC 20 ACTTCGCCAC 60

8 OPF 07 CCGATATCCC 60

6.3 Results

To verify the toxic effect of sucking of scale insects on mango leaves, the analysis of RAPD was used to amplify the fragments of DNA, which was extracted from 0, 10, 20 and 30 days infested leaves of mango. RAPD profile in mango leaves exposed to scale insects showed a variation in the appearance and/or disappearance of bands and increase and decrease in band intensities. Eight primers of 60–70 % GC were used for screening the genome of leaves under scale insects infestation. Only three primers gave specific, reproducible and stable results. Total 57 numbers of bands were observed in the three primers and three mango cultivars ranging from 150 to1200 base pair (bp). There were imminent differences between the bands shown in treated and untreated seedlings of mango leaves exposed to scale insects on the basis of appearance and disappearance in number and size of polymorphic bands of DNA fragments. Results of changes in RAPD profile of mango leaves are summarized in Table 6.2 and Table 6.3 with number of bands, appearance and disappearance of bands and bands intensity; and also in Figures 6.1 to 6.3. Various polymorphic bands were recorded at

different levels of scale insects infestation for different primers. For primer OPA–10 in Langra variety, one new band of 200 bp appeared at 10 days and two bands of 180 and 400 bp appeared at 20 days while one normal band of 290 bp disappeared at 20 days infestation by scale insects as compared to control. For OPC–01, 1, 3 and 1 normal bands ranging from 300 to 1200 bp disappeared at 10, 20 and 30 days. Similarly, for RPl–3, two normal bands of 150 and 500 bp disappeared at 10 days, two new bands of 200 and 1500 bp appeared at 20 days and one new band of 800 bp appeared and three bands of 150, 250 and 500 bp disappeared at 30 days post scale insects infestation. In Kala Chounsa, one new band of 500 bp appeared at 10 days for OPA–10 while two bands of 600 and 700 bp appeared at 10 and that of 200 and 300 bp appeared at 20 days, and one band of 800 bp appeared at 30 days for OPC–01. For RPl–3, one new band of 600 bp appeared and 800 bp disappeared at 10. Similarly, two bands of 100 and 200 bp appeared at 20 days while one normal band of 800 bp disappeared at 10, 20 and 30 days.

In Anwar Ratoul for OPA–19 only one new band of 400 bp appeared at 30 days, for OPC– 01, 2, 2 and 3 new bands (200–1000 bp) appeared at 10, 20 and 30 days consecutively while for Rpl–3 two new bands of 100 and 200 bp appeared in leaves exposed to 20 days scale insects.

In langra, appearance of new bands was less as compared to disappearance of normal bands but the bands intensity increased with increasing infestation period. Maximum band intensity was observed at 20 and 30 days in OPA–10 and RPl–3. In Kala Chounsa and Anwar Ratoul, new RAPD bands were high than disappearance of normal bands. Percent polymorphism in Langra was 41.7, 66.7 and 33.3 %; in Kala Chounsa was 55.6, 44.4 and 22.2 % while in Anwar Ratoul was 40, 80 and 80 % at 10, 20 and 30 days respectively using different primers. In almost all treatments, polymorphism was due to the gain and loss of RAPD bands in respect to control. The loss and gain and increase and decrease in intensity of bands demonstrated at 10 and 20 days but stability in bands were observed at 30 days.

Figure.6.1. RAPD profile of genomic DNA in the leaves of mango plant exposed to 0, 10, 20 and 30 days scale insect infestation using primer OPA 10. a. Langra, b. Kala Chounsa, c. Anwar Ratoul. Marker (100–1000 bp).

Figure.6.2. RAPD profile of genomic DNA in the leaves of mango plant exposed to 0, 10, 20 and 30 days scale insect infestation using primer OPC 01. a. Langra, b. Kala Chounsa, c. Anwar Ratoul Marker (100–3000 bp).

Figure.6.3. RAPD profile of genomic DNA in the leaves of mango plant exposed to 0, 10, 20 and 30 days scale insect infestation using primer RPl 3. a. Langra, b. Kala Chounsa, c. Anwar Ratoul. Marker (100–3000bp).

Table 6.2. Changes of total bands in control and of polymorphic bands and varied bands in mango leaves under mango scale insects infestation in three mango cultivars. Langra

0 days 10 days 20 days 30 days

Name of primer a B C d a b c d a b c d

OPA–10 2 1 0 1 0 2 1 0 2 0 0 0 2

OPC–01 5 0 1 2 0 0 3 2 0 0 1 2 1

RPl–3 5 0 2 3 0 2 0 0 2 1 2 2 2

Total bands 12 1 4 6 0 4 4 2 4 1 3 4 5

a+b 5 8 4

Polymorphism% 41.7 66.7 33.3

a+b+c+d 11 14 13

Kala Chounsa

OPA–10 2 1 0 0 0 0 0 0 0 0 0 2 0

OPC–01 3 2 0 0 1 2 0 0 1 1 0 0 1

RPl–3 4 1 1 1 2 1 1 0 2 0 1 1 1

Total bands 9 4 1 1 3 3 1 0 3 1 1 3 2

A+b 5 4 2

Polymorphism% 55.6 44.4 22.2

A+b+c+d 9 7 7

Anwar Ratoul

OPA–10 2 0 0 2 0 0 0 2 0 1 0 0 2

OPC–01 2 2 0 1 2 2 0 1 2 3 0 0 3

RPl–3 1 0 0 0 0 2 0 1 1 0 0 0 0

Total bands 5 2 0 3 2 4 0 4 3 4 0 0 5

a+b 2 4 4

Polymorphism% 40 80 80

a+b+c+d 7 11 9 a, b, c, d, a+b and a+b+c+d indicate appearance of new bands, disappearance of normal bands, decrease in bands intensities, increase in bands intensities, polymorphic bands and varied band, respectively.

Table 6.3. Molecular size (base pair, bp) of appearance and disappearance of bands for all primers in mango leaves under mango scale insects infestation. Langra

Name of 0 days 10 days 20 days 30 days primer

OPA–10 290, 300 200, 290, 300 180, 300, 400 290, 300

+ 200 + 180,400 + 0

- 0 - 290 - 0

OPC–01 300, 400, 600, 900, 1200 300, 400, 900, 1200 300, 400 400, 900, 1200

+ 0 + 0 + 0

- 600 - 600,900,1200 - 300,600,900

RPl–3 150, 250, 300, 500, 900 250, 300, 900 200, 250,500, 900,1500 250, 300, 800, 900

+ 0 + 200,1500 + 800

- 150, 500 - 150,300, - 150,250,500

Kala Chounsa

OPA–10 600,700 500,600,700 600, 700 600,700

+ 500 + 0 + 0

- 0 - 0 - 0

OPC–01 900,1000,1100 600,700,900,1100 200,300,900,1000,1100 800,900,1000,1100

+ 600, 700 + 200, 300, + 800

- 1000 - 0 - 0

RPl–3 250,500,800,900 250,500,600,900 100,200,250,500,900 250,500,900

+ 600 + 100, 200 + 0

- 800 - 0 - 800

Anwar Ratoul

OPA–10 900, 1000 900, 1000 900, 1000 400, 900, 1000

+ 0 + 0 + 400

- 0 - 0 - 0

OPC–01 1100, 1200 200,300, 1100,1200 200,300,1100,1200 200, 300, 1000,1100, 1200

+ 200,300 + 200,300 + 200,300, 1000

- 0 - 0 - 0

RPl–3 500 500 100, 200, 500 500

+ 0 + 100, 200 + 0

- 0 - 0 - 0 ‘+’ and ‘-’ indicate appearance of new bands and disappearance of normal bands, respectively.

6.4. Discussion and conclusion

It is widely accepted that the disappearance of normal bands represents DNA damage due to the breakage of single and double strand, modification of bases, oxidized bases, DNA protein cross link, point mutation as well as complex rearrangement of chromosomes which are induced by genotoxins (Atienzar et al., 2002; Wolf et al., 2004). It is reported that when DNA adduct is encountered by Taq polymerase, the possible outcomes are block, bypass and also the dissociation of enzymes complex. As a result, the normal bands are lost. Mutation causes appearance of new bands while in case of DNA damage the normal bands disappeared (Atienzar and Jha, 2006). The appearance of extra bands in RAPD profiles may be due to change in oligonucleotide priming sites by mutation, large deletions or homologous recombination i.e. juxtaposing the two separate sequences which match the sequence of primer used (Atienzar et al., 1999). Atienzar et al. (2000) further reported that new bands appeared only due to mutation when they occur in sufficient number of cells at same locus (10 % mutation needed for new PCR products in agarose gel).

In this study, RAPD was used to measure the DNA damage in mango leaves under scale insects infestation. In our results, the number of lost bands was more than that of appearing extra bands. In the same way, number of disappeared and appeared bands was higher in Langra than Kala Chounsa and Anwar Ratoul. RAPD analysis demonstrated that Langra was more vulnerable to scale insects than other two varieties. The results explained low genotoxicity in Kala Chounsa and Anwar Ratoul. Furthermore, disappearance of normal bands was increased with increasing infestation of scale insects. This suggested that scale insects had time specific effect on mango leaves. As Kala Chounsa and Anwar Ratoul were comparatively resistant to scale insects than Langra, they sustained the scale insects infestation through antioxidant enzymes defense mechanism that might protected the DNA damage in the leaves. These findings suggested that different mango cultivars showed different genotoxic effects under scale insects infestation as well as provided the level of resistant and susceptibility in mango varieties. In the light of above discussion, it is concluded that RAPD analysis is a very sensitive method for detection of DNA damage which can be used as a tool to investigate genotoxicity caused by scale insects in mango leaves. It has been previously studied that any change in the fingerprint of DNA from single base to complicated chromosomal rearrangement, noted in stressed organism, show the reflection of alteration in DNA, which is very efficiently presented by DNA fingerprinting biomarker assay in the field 81

of ecotoxicology (Atienzar et al., 2002; Savva, 1998). Here also, the change in DNA fingerprinting through appearance of new bands and disappearance of normal bands verified that scale insect caused considerable change in the molecular set up of the cell.

Chapter 7.

Effect of Co–60 gamma irradiation on the mortality and growth inhibition of A. destructor

7.1. Introduction

Scale insects are very minute insects, usually hidden under the scale cover or concealed under the bark of host plants or stick to the fruits, which are easily transferred to new areas in international export through commodities (Miller et al., 2002). Many of these insects are quarantine pests and fruits infested with scale insects are often rejected at the port of entry. Scale insect may arrive from any country, therefore regional expertise and diagnostic tools for scale insects must be available through state and federal inspection services (Hardy, 2013). In the USA, invasive scale insects are introduced from other areas and cause billions of dollars damage to the crops (Miller and Miller, 2003; Kondo et al., 2008). At the entry point of California, even a single live insect species may cause regulatory action against the whole lot of fruit (Miller and Chang, 1998).

Recently irradiation has been adopted as safe measure for disinfestations of the quarantine pests and has gained much importance in the export of agriculture fresh commodities (Heather and Hallman, 2008). According to general rules of U.S. Department of Agriculture (USDA) and Animal and Plant Health Inspection Service (APHIS), a generic dose of 400 Gy has been approved for the control of all types of insects except pupae and adults of Lepidoptera (Federal Register, 2006). In Hawaii, fruit flies on exported crops are controlled at an irradiation dose of 150 Gy (Follett and Armstrong, 2004), while it is suggested that green scale can be controlled with an irradiation dose of 250 Gy (Hara et al., 2002). Hallman (2011) investigated whether mangos from Pakistan could be exported to the United States when irradiated at dose of 400 Gy for the control of six species of scale insects representing quarantine pests in Pakistan.

Irradiation has merits over other post-harvest treatments like cold and hot water treatment, pressure washing and air tight treatments (Hallman, 2011). Heat and cold treatments require a balance between killing the pest and negative effects on commodity quality, whereas radiation does not typically affect quality of commodities at doses that kill the pest (Sommers and Fan, 2008; Hallman and Hellmich, 2009; Follet and Weinert, 2012). Historically for its

approval, the USDA required the quarantine treatments of irradiation for fruit flies to achieve 99.99 % mortality with not less than 93,613 test insects (Follet and Neven, 2006; Couey and Chew, 1986). More recently the USDA relaxed its testing criteria for insects that have no economic or environmental impacts (Follet and Neven, 2006). Japan, Australia and New Zealand permit irradiation for quarantine treatment with 99.99 % mortality at 95 % confidence level when treating not less than 30,000 insects (Couey and Chew, 1986; Follet and Neven, 2006). Japan requires 30,000 insects in three to four trials and New Zealand accept irradiation treatment of 10,000 individuals having no test individual survivor.

Little data is available on the disinfestations of scale insects through irradiation in Pakistan. Currently, the Government of Pakistan has adopted radiation dose of 400 Gy for disinfestations of fruit flies and all other insects including armored scales. Specific studies on development of irradiation doses that would inhibit the development of scales, reduce the risk of damage to fresh commodities as well as reduce cost and time of treatment are required (Follet, 2006a; Hallman, 2012). The current studies aim to determine the minimum effective irradiation dose to reliably inhibit development and emergence of various stages of A. destructor.

7.2. Materials and Methods

7.2.1. Preparing test plants

Mango nursery plants were manually infested with scale insect according to the procedure given in Chapter 2 (2.2). When crawlers moved and settled on the test plants, they were removed from field and placed in a greenhouse. Batches of 6–7 plants per dose in all 35 test plants were infested in the same way at 4–7 day intervals. Scales on the prior infested plants developed to the next stage while fresh infested plants were added into the lot. Plants infestation continued until 6–7 infested plants with particular stage of scale were obtained. Leaves on the abaxial side were marked on plants for specific stage of the scale.

7.2.2. Radiation treatments

Following infestation, all infested plants were moved to the commercial irradiator at Pakistan Radiation Services (PARAS), Lahore where irradiation studies were conducted using a product overlap source irradiator Cobalt 60Co MDS Nordion, Canada (47, 000 Ci). A dose mapping of the area within the irradiation chamber, where samples were placed, was

conducted to verify the accuracy and range of the doses applied. The absorbed dose was measured using Fricke dosimeters at upper, lower and lateral positions with dose uniformity ratio (DUR) was consistently < 1.04 Gy during all treatments. Response of irradiation was examined on 1st, 2nd instar nymphs, mature females and females with eggs in a series of irradiation doses between 100–300 Gy. In large scale validation test, more than 90,000 scale insects of different stages were exposed to various doses of gamma irradiation for the most tolerant and susceptible stages to irradiations. Individual plants with specific stage of scales served as replicates and every treatment included 3–4 replicates depending on the number of scales on the plants. Irradiated plants were shifted to the Nuclear Institute for Food and Agriculture and kept in the greenhouse at 30±2 °C, and RH 60±5 %. Scale insects on plants were examined twice a week to see their development to the next stage while eggs under the translucent body of scales were counted using magnifying lens. Recording of data from treated plants was discontinued when scales dried off and stopped to progress to the next stage. Untreated controls from each stage were held under identical conditions and examined similarly.

Data Analysis

Data on the effect of gamma irradiation on mortality/survival were subjected to analysis of variance (ANOVA) after testing for equal variances and normality. Means separations were done using a Tukey’s HSD test. All statistical analyses were done using Statistix 8.1 (Analytical Software, Tallahassee, FL). In addition, percentage mortality and adult inhibition data were subjected to probit analysis using PoloPlus (LeOra computer software) to estimate the dosage response of exposed eggs and nymphs. 7.3. Results

Response of first instar (white cap) scale insects to various doses of irradiation is given in table 7.1. At a dose of 100 Gy (DUR < 1.04), total 1105 1st instar nymphs were irradiated in which 7.87 % scales matured to gravid female and laid eggs while scales irradiated at doses of 150 Gy and above did not survive to further stages and died within 6–8 days after arrested development. In control treatments only 7.2 % natural mortality was recorded at 1st instar stage.

Table 7.1. Effect of gamma irradiation on maturation development of exposed1st instar nymphs of A. destructor on mango leaves.

Target dose F1 1st instar F1 2nd instar Total gravid Gravid female* (%) (Gy) nymphs nymphs females

0 4158 3733 2425 58.3 a

100 1105 1007 87 7.87 b

150 2857 0.0 0.0 0.00 c

200 5107 0.0 0.0 0.00 c

250 2301 0.0 0.0 0.00 c

300 1865 0.0 0.0 0.00 c

* Approximately 41.7 % of scales were males. No mortality was recorded in control treatments. Different letters within column 6 indicate significant differences using Tukey’s HSD test at (P ≤ 0.05) among treatments (0, 100, 150, 200, 250 and 300 Gy).

Response of 2nd instar nymphs irradiated at various doses of gamma irradiation is given in table 7.2. Scale insects were comparatively tolerant at this stage and reached gravid stage except those irradiated at 250 Gy. At a dose of 100 Gy, 68 % of scales developed to gravid females. At a dose of 150 and 200 Gy, 25 % and 22 % of scales developed to gravid female, respectively. All insects stopped growth and desiccated after few days when irradiated at 250 Gy and above. Mortality in control treatments was only 1.1 %.

Table 7.2. Effect of gamma irradiation on maturation of 2nd instar A. destructor

Target dose F1 2nd Instar F1 mature Total Gravid Gravid female (Gy) nymphs female females (%)

0 1568 1551 1314 83.8 a

100 1770 1325 1208 68.2 b

150 1402 385 344 24.54 c

200 1359 332 302 22.22 c

250 1426 9.47 0.0 0.0 d

300 1020 0.0 0.0 0.0 d

Different letters within column 5 indicate significant differences using Tukey’s HSD test at

(P ≤ 0.05) among treatments (0, 100, 150, 200 and 250 Gy).

Mature female scales irradiated at various doses of gamma irradiation showed highest tolerance to irradiation (Table 7.3). At a dose of 100 Gy, 54 % of scales developed to gravid females while at doses of 150 Gy and 200 Gy, 44 % and 34 % of scales developed to gravid female, respectively. No mortality in control treatments was recorded at this stage.

Table 7.3. Effect of gamma irradiation on mature female of A. destructor

Target dose F1 3rd stage Total Gravid Gravid female Number of eggs under (Gy) females females (%) female

0 2116 1778 84.02 a 47.6 ± 05

100 3076 1661 54.01 b 33.5 ± 04

150 2992 1324 44.24 bc 7.0 ± 11

200 2456 840 34.2 c 1.0 ± 01

250 2670 0.00 0.0 d 0.0

300 2007 0.00 0.0 d 0.0

Different letters within column 4 indicate significant differences using Tukey’s HSD test at (P ≤ 0.05) among treatments (0, 100, 150, 200 and 250 Gy). At a dose of 100 Gy, 311 gravid females with approximately 14611 eggs at a rate of 47 eggs/female were irradiated where 0.6 % crawlers reached to gravid stage. At an irradiation

doses of 150 Gy and above, not a single crawler released from the eggs which indicated that eggs stage was the most susceptible stage to irradiation. In control 355 gravid females with total of 16685 eggs @ 47eggs/female released crawlers, in which 5666, 5258 and 5200 scales shifted to 1st, 2nd instar and mature stages while 3034 (58 %) females developed to gravid stage.

Table 7.4. Effect of gamma irradiation on eggs hatch and mature female with eggs of A. destructor

Target Total Gravid No. Parent No. of eggs F1 1st instar F1 mature dose gravid female females under female nymphs female (GY) female (%)*

0 355 16685 5666 5200 3034 58 a

100 311 14617 1105 1007 87 0.6 b

150 385 18095 0.0 0.0 0.0 0.0 c

200 272 12784 0.0 0.0 0.0 0.0 c

250 289 13583 0.0 0.0 0.0 0.0 c

300 221 10387 0.0 0.0 0.0 0.0 c

* Approximate 42% of scales were females, Different letters within columns indicate significant differences using Tukey’s HSD test at (P≤ 0.05) among treatments (0, 100, 150, 200 and 250 Gy). Total number of eggs per female 47 (Salahuddin et al., 2015).

Probit analysis for large scale validation test of most tolerant and susceptible stage to expected dose of 156.9 Gy for eggs and 178.9 Gy for 1st instar nymph is given in table 7.5

Second instar nymphs were comparatively more tolerant to gamma irradiation and a dose of 217 Gy was recorded for 100 % inhibition of this stage. However, the expected mortality of scale insects at 200 Gy and 250 Gy were not significantly different from each other. In large scale conformity test, 220 Gy dose prevented complete development of scales to the next stages.

Table 7.5. Growth inhibition (%) of A. destructor at different development stages after 60 days of irradiation

Percent Inhibition Predicted dose Development for 100 % Dose (Gy) Slope ± SD stage inhibition (FL) 0 100 150 200 250 300 ± 95%

156.9 Egg stage 18.1 85.8 99.0 a 100 100 100 12.3 ± 0.6 (151.1–164.3)

1st instar 178.9 17.8 87.8 98.3 a 100 100 100 7.3 ± 0.6 nymphs (161.2–204.7)

2nd instar 217.7 19.4 27.6 81.6 96.4 100 100 11.9 ± 0.8 nymphs (206.9–233.4)

Mature 215.1 16.9 28.7 63.5 96.3 100 100 19.8 ± 0.95 females (208.3–223.7)

Data were subjected to probit analysis using PoloPlus (LeOra computer software) to estimate the dosage response of exposed eggs and nymphs. Table 7.6. Large–scale validation tests irradiating of A. destructor females with eggs and without eggs

Number of Number of Number of F1 F1 Target dose Number of Measured adult scales generation generation (Gy) plants dose irradiated white caps mature adults

Control 12 – 43,274 53,727 31,939

200 10 190–202 40,531 201 3.0

220 12 210–224 51,101 0.0 0.0 All the treatments exposed to various doses of irradiation affected the survival of eggs, nymphs and mature stages of A. destructor. The inhibition rate increased with the increasing doses of gamma irradiations. Similarly, tolerance to irradiation in scale insects also increased with the progressive stages of scales. Irradiation doses from 100 to 200 Gy did not cause 100 % inhibition in all life stages of scale insects. However, scales irradiated at higher doses (250

Gy and above) dried off and dislodged from leaves within 20–25 days. In large scale conformity test, 220 Gy dose was sufficient to prevent scale development with the addition of DUR value of 1.04 Gy.

7.4. Discussion and conclusion

In our study the scale insects were irradiated at their various stages of development including 1st instar (white cap), 2nd instar, mature female scales and female with eggs. Eggs stage was found very susceptible while mature scales were the most resistant stages to gamma irradiation. Similar increase in the resistance of various stages of scale insects to gamma irradiation has been reported by other researchers (Hallman, et al. 2010; Mansour and Al- Attar, 2012; The et al., 2012).

In the large scale validation tests approximately 40,532 scales were exposed to 200 Gy and 51,101 scales to 220 Gy (total 91,633 scales) with all stages of scale insects. Irradiation dose of 220 Gy prevented complete development of all scales to the next stages. In the present experiments the dose uniformity ratio during all treatments was consistently < 1.04, meaning that some plants received 1.04 times the target dose (dose of 220–228 Gy) during irradiation treatment. To meet the current requirement for a minimum absorbed dose, 228 Gy would be the more secure dose to stop reproduction of A. destructor. In order to achieve probit 9 mortality, a benchmark for the efficacy of phytosanitary treatments with 99.9968329 % mortality of a pest (IPPC, 2010) at the 95% confidence level, a minimum of 93,613 insects need to be tested with no survivors (Heather and Hallman, 2008). In the current studies I tested 91,633 insects in the large scale validation tests and about 50,000 scales of various stages during our initial studies. Thus adding together, the number exceeds the proposed limit of 93,631 insects. Based on this I propose a dose of 228 Gy as the level that would provide quarantine security. These results are in agreement with the probit 9 formula approved by different countries like Japan, Australia and New Zealand for scale insects (Couey and Chew, 1986). USDA approves the quarantine treatments of gamma irradiation for fruit flies with 99.99 % efficacy which is considered as minimum generic dose for quarantine security (Follet and Neven, 2006; Follet, 2006b).

It is concluded that irradiation of gamma rays provide excellent quarantine security against A. destructor in exporting commodities. Egg stage of A. destructor was the most susceptible while 2nd instar nymph was the most resistant stage to gamma irradiation. After large scale 90

validation test, an irradiation dose of 228 Gy was found safe for all the life stages of A. destructor for quarantine security.

Chapter 8.

Physiochemical analysis and sensory evaluation of mango fruit under gamma irradiation

8.1. Introduction A. destructoris a very destructive pest of both tropical and sub-tropical crops. The pest has a wide host range (Tenorio, 1969) and has been reported from many economically important crops. The pest is quarantined at the entry point of California and even a single live insect may cause regulatory action which may lead to the rejection of full carriage or whole lot of fruit from export (Miller and Chang, 1998).

U.S. Department of Agriculture (USDA) and Animal and Plant Health Inspection Service (APHIS) has been approved a generic irradiation dose of 400 Gy for the control of all types of insects except pupae and adults of Lepidoptera (Federal Register, 2006). However, scale insets are controlled with an irradiation dose of 200–250 Gy (Hara et al., 2002, Khan et al., 2014; Follet, 2006).

Gamma irradiation is a very effective method of postharvest fruit processing as compared to other techniques including cold and hot water immersion, methyl bromide fumigation and heated air (Antanio et al., 2012; Hallman, 2011). Irradiation has no detrimental effect on the quality of fruits in term of color, nutritional value and flavor. Irradiation has been widely investigated as post-harvest disinfection methods for different fruits and vegetables at doses that sterilize the insects but have no ill effects on fruit quality (Kader, 1986; Follet, 2007).

Gamma irradiation has been used in citrus as an alternative technology to fulfill the requirements of phytosanitary laws of trade in preventing the exotic pests (FAO, 2009). A mandarin cultivar (Clemenules) has also been reported to be very tolerant to application of X- ray irradiation for the control of Medfly (Alonso, 2007). Similarly, grapefruit (Rio Red) did not show any ill effect on appearance, soluble solids, titratable acidity and organoleptic quality on exposing to 0.5 kGy irradiation doses (Hallman and Martinez, 2001). Acidity and ascorbic acid values were reported to be higher in irradiated citrus fruits than un-irradiated along with decrease in weight loss and increase in TSS (total soluble solids) (Khalil, 2009). Here I assessed the effect of Co–60 gamma irradiation for controlling A. destructor on mango fruit quality for physiochemical analysis and storage.

8.2. Materials and methods

Mango fruit of Cultivar Langra of uniform size was harvested at 3/4th maturity stage early in the morning at temperature of 27 °C from Fruit Nursery Farm, D. I. Khan. Fruits were thoroughly washed with clean tap water and dried using blower at room temperature. Clean and dry fruits were irradiated at doses of 100, 150, 200 and 250 Gy in a Co60 gamma irradiator source (Izotop, Hungary) at NIFA, Peshawar having a dose rate of 8.5 kGy/hour using Fricke dosimeter calibration. Fruits in control were kept unirradiated throughout the study period. The fruits were stored at room temperature of 25.3±1.4 °C and RH 42±2.6 %. Physiological weight loss (%), firmness, TSS, vitamin C content, Acidity and pH were determined at 2 days intervals over 8 days. Organoleptic characteristics such as appearance, taste, odor, texture, firmness, flavor and consumer acceptability were judged by a panel of 20 volunteers udges through 9 points hedonic scale with number 9 was considered as the best (Larmond, 1977). Hand refractometer (Kernco Instruments Co. Texas) was used for the measurement of total soluble solids (°Brix). Acidity was determined by neutralization reaction as described in Association of Official Analytical Chemists (A.O.A.C., 2006).

8.2.1. Weight loss Three mango fruits of equal size in each irradiation treatment were separated, washed and dried. Percent weight loss in irradiated mango fruits and control was determined by the following formula:

Weight loss = (Fresh weight – Weight at storage interval) × 100 Fresh weight 8.2.2. Total soluble solids (TSS)

Total soluble solids in irradiated mango juice and control were determined by the method of Awan (2001). Fine paste of mango fruits was prepared using common grinder (juicer) (Deuron, Model No. GL–107, Power 350 W). A drop of well mixed juice was put on refractometer (Brix, Atago, Japan) for TSS reading through eye piece mirror graduated inside.

8.2.3. Titrable acidity

The titrable acidity of mango juice irradiated at different doses of gamma irradiation was determined by the method of Awan (2001). A fine paste of mango juice was filtered through number 4 whatman paper. 20 ml juice from the paste was dissolved in 80 ml distilled water in

a 250 ml beaker. Three drops of phenolphthalein were put in the solution and titrated against 0.01 N NaOH till the change of color. Reading was taken and put in the following formula for acidity determination:

Acidity (%) = 0.01 × 0.064 × Titre × 100 × 100 10 × 20 Where 0.01 is dye normality of NaOH solution and 0.064 is constant factor.

8.2.4. Ascorbic acid (Vitamin C)

The ascorbic acid of mango juice irradiated at different doses of gamma irradiation was determined by the method of Awan (2001). A fine paste of mango juice was filtered through number 4 Whatman paper. 20 ml juice from the fine paste was dissolved in 80 ml Oxalic acid in a 250 ml beaker. Three drops of phenolphthalein were put in the solution and then titrated against dye till the change of color. Reading was taken and put in the following formula for ascorbic acid determination.

Ascorbic acid = 0.01 × Titre × 100 × 100 10 × 20 Where 0.01 is dye factor or Normality of NaOH solution

Titre is ml of dye used in sample – ml of dye used in blank

8.2.5. pH

The pH of mango juice extracted from irradiated mango fruits and control was determined by the method of Awan (2001).

The pH meter was Standardized at two standards of pH 4.0 and 7.0 buffers. 5 gm pure mango juice was dissolved in 50 ml of dH2O (w/v) in 50 ml beaker and electrode of pH meter was put into the solution for pH reading. The electrode was washed with distilled water from wash bottle, dried using piece of filter paper and continued with the next reading.

8.2.6. Sensory analysis of irradiated and unirradiated mango fruits.

Sensory evaluation of mango fruit for color, texture, flavor and odor was done by 20 volunteers (10 males and 10 females). The volunteers belonged to agriculture research, soil conservation, food technology, water management and agriculture extension departments. 94

The fruits were evaluated after every two days for 8 days storage period at room temperature. Each person was given a proforma of 9 points for evaluation with number 9 as the best. Tasteless biscuits were given for eating before sensory evaluation of mango fruit.

8.2.7. Statistical analysis

Statistical data analysis was carried out by one way analysis of variance (ANOVA) in randomized complete block design (RCBD) with three replications and the means were separated by Tukey’s HSD test using Statistix 8 analytical software (version 8.1) at 1 % significance level. All the results were expressed in graphs and tables in mean values with standard errors. Correlation among the fruit quality deterioration and gamma irradiation doses was also done with regression analysis using MS Excel spread sheet.

8.3. Results

8.3.1. Percent weight loss

Figure 8.1 showed the effect of different doses of gamma irradiation on the percent weight loss in mango fruit at various storage intervals. Weight loss was significantly (P≤ 0.01) affected by both gamma irradiation doses and storage intervals. Among the different treatments, weight loss was significantly (P≤ 0.01) less (9.53 %) at 250 Gy after 8 days while maximum in control (20.68 %) at the same storage level. In the same way, fruits irradiated at low doses also showed stability in weight loss as compared to unirradiated fruits. After 8 days the experiment was terminated due to unacceptable condition of fruits in control on the basis of sensory evaluation.

Figure 8.1. Changes in % weight loss (±SEM) of mango fruits after exposing to different gamma irradiation doses compared with control.

Negative correlation between wieght loss and irradiation doses and possitive correlation between weight loss and storage intervals (Table 8.1, Figure 8.2 A) were observed during the course of study.

8.3.2. Firmness

Firmness of fruit decrease with increasing storage interval. There was significant (P≤ 0.01) stability observed in the firmness of fruits irradiated at various doses as compared to control from 2 to 8 days (Figure 8.3). Firmness decreased up to 74.51 % in fruits irradiated at 250 Gy while that of control was 87.25 % after 8 days storage time. There was strong negative correlation between firmness loss and irradiation doses at different storage interval as indicated from r2 values while possitive correlation between firmness loss and storage intervals was observes (Table 8.1, Figure 8.2 B).

Figure 8.2. Regression lines of gamma irradiation doses at different time intervals in respect to: (A) % weight loss (B) Firmness (C) TSS (D) Acidity (E) Vitamin c (F) pH in mango fruit

Figure 8.3. Firmness changes (±SEM) in mango fruits after exposing to different gamma irradiation doses compared with control. 97

Table 8.1. Regression coefficient for the effect of different doses (0–250 Gy) of Gamma Irradiation on the physiochemical analysis of mango fruit at various storage duration.

Parameter Days a b r2 Probability 2 3.8 (±0.37) –0.01 (±0.002) 0.89 P≤ 0.05 4 9.1 (±0.13) –0.02 (±0.001) 0.99 P≤ 0.00 Weight loss (%) 6 15.4 (±1.16) –0.04 (±0.007) 0.90 P≤ 0.01 8 20.2 (±0.58) –0.05 (±0.004) 0.98 P≤ 0.00 2 3.5 (±0.06) 0.001 (±0.0004) 0.96 P≤ 0.01 4 2.1 (±0.08) 0.006 (±0.001) 0.98 P≤ 0.00 Firmness 6 1.4 (±0.05) 0.005 (±0.0003) 0.99 P≤ 0.01 8 0.6 (±0.09) 0.002 (±0.001) 0.86 P≤ 0.05 2 17.0 (±0.05) –0.005 (±0.0003) 0.99 P≤ 0.00 4 19.8 (±0.31) –0.008 (±0.002) 0.85 P≤ 0.05 TSS (Brixo) 6 21.8 (±0.53) –0.013 (±0.003) 0.85 P≤ 0.05 8 24.2 (±0.50) –0.019 (±0.003) 0.93 P≤ 0.01 2 1.4 (±0.01) 0.001 (±0.0001) 0.98 P≤ 0.01 4 1.1 (±0.01) 0.002 (±0.0001) 0.99 P≤ 0.00 Acidity 6 0.8 (±0.08) 0.003 (±0.001) 0.89 P≤ 0.05 8 0.7 (±0.04) 0.003 (±0.0002) 0.98 P≤ 0.00 2 21.3 (±0.19) 0.015 (±0.001) 0.98 P≤ 0.00 4 15.8 (±1.04) 0.030 (±0.006) 0.88 P≤ 0.05 Vitamin C 6 11.6 (±0.21) 0.034 (±0.001) 0.99 P≤ 0.00 8 7.3 (±0.37) 0.041 (±0.002) 0.99 P≤ 0.00 2 4.3 (±0.06) 0.003 (±0.0004) 0.97 P≤ 0.01 4 3.4 (±0.13) 0.005 (±0.001) 0.93 P≤ 0.01 pH 6 3.0 (±0.16) 0.006 (±0.001) 0.93 P≤ 0.01 8 2.9 (±0.15) 0.006 (±0.001) 0.92 P≤ 0.01

8.3.3. Total Soluble Solids (TSS)

Figure 8. 4 depicts that minimum TSS was recorded in fruit irradiated at 250 Gy (37.93 %) as compared to control (70.14 %) after eight days storage. At every storage interval, higher TSS was recorded in control and lower in irradiated fruits but all the treatments were non- significant to each other. Irradiation doses and TSS contents were also negatively correlated with each other while TSS contents were possitively correlated with storage durations (Table 8.1, Figure 8.2 C).

Figure 8.4. Total soluble solids changes (±SEM) in mango fruits after exposing to different gamma irradiation doses compared with control.

8.3.4. Titrable acidity (g/L)

During storage, acidity of mango fruit decreased in both irradiated and unirradiated fruits. However, effect of irradiation on acidity was significant (P≤ 0.01) as more decrease in acidity was recorded in control as compared to rest of the treatments at all storage level (Table 8.2). At 250 Gy, acidity was more stable (21.08 % decrease) as compared to control (63.05 %). There was significant and strong possitive correlation between % acidity and irradiation doses at different storage intervals as indicated by the r2 values which were 0.976 (P≤ 0.005), 0.993 (P≤ 0.000), 0.892 (P≤ 0.05) and 0.979 (P≤ 0.005) at 2, 4, 6 and 8 days, consecutively (Table 8.1). Negative correlation was observed in % acidity and storage intervals (Figure 8.2 D ).

Table 8.2. Changes in titrable acidity (g/L) of mango fruits after exposing to different doses of gamma irradiation compared with control.

Storage duration (days) Irradiation doses (Gy) 2 4 6 8 Control 1.4 ± 0.05 b 1.1 ± 0.05 b 0.7 ± 0.02 b 0.6 ± 0.03 b 100 1.5 ± 0.02 ab 1.3 ± 0.08 ab 1.2 ± 0.11 a 1.0 ± 0.08 ab 150 1.5 ± 0.04 ab 1.3 ± 0.10 ab 1.2 ± 0.09 a 1.1 ± 0.09 a 200 1.5 ± 0.02 ab 1.4 ± 0.07 ab 1.2 ± 0.08 a 1.2 ± 0.12 a 250 1.6 ± 0.05 a 1.5 ± 0.08 a 1.4 ± 0.06 a 1.2 ± 0.10 a LSD values for 2 (0.22), 4 (0.36), 6 (0.36) and 8 days (0.42). Means followed by the same letters in a column are not significantly different using Tukey’s HSD test at (P ≤ 0.01). 8.3.5. Ascorbic acid (vitamin C)

The data regarding vitmin C contents of mango fruits as effected by irradiation doses are shown in table 8.3. The data showed that during storage vitmin C contents of mango fruit decreased in both irradiated and unirradiated fruits. However, in control significantly (P≤ 0.01) higher loss of vitamin C was recorded than irradiated fruits. With increase in doses of irradiation, the ascorbic acid contents decreased. Highest stability in vitamin C contents was observed in fruits irradiated at 250 Gy (36.36 %) as compared to control (74.55 %). There was significant and strong possitive correlation between ascorbic acid contents and irradiation doses at different storage intervals as indicated by the r2 values (Table 8.1) while negative correlation was noted between vitamin c contents and storage peroids (Figure 8.2 E).

Table 8.3. Changes in vitamin c (mg) contents of mango fruits after exposing to different doses of gamma irradiation compared with control.

Storage duration (days) Irradiation doses (Gy) 2 4 6 8 Control 21.5 ± 0.29 c 16.8 ± 0.17 d 11.5 ± 0.29 d 7.0 ± 0.29 d 100 22.7 ± 0.17 bc 17.5 ± 0.29 d 15.0 ± 0.29 c 12.0 ± 0.29 c 150 23.3 ± 0.67 abc 19.3 ± 0.17 c 17.0 ± 0.29 b 13.5 ± 0.29 c 200 24.5 ± 0.29 ab 21.8 ± 0.17 b 18.5 ± 0.29 ab 15.2 ± 0.44 b 250 25.2 ± 0.44 a 24.2 ± 0.17 a 19.8 ± 0.44 a 17.5 ± 0.29 a LSD values for 2 (1.90), 4 (0.92), 6 (1.51) and 8 days (1.51). Different letters in a column are significantly different using Tukey’s HSD test at (P ≤ 0.01).

100

8.3.6. pH [-log(mol/L)]

Table 8.4 revealed that during storage pH of mango fruit decreased in both irradiated and unirradiated fruits. However, irradiated fruits showed stability in pH change as compared to unirradiated fruits. pH of fruits irradiated at 250 Gy decreased upto 23.75 % as compared to control (46.43 %) after 8 days storage duration. There was significant and strong correlation between pH and irradiation doses at different storage intervals as indicated by the r2 values which were 0.966 (P≤ 0.005), 0.929 (P≤ 0.05), 0.928 (P≤ 0.05) and 0.921 (P≤ 0.01) at 2, 4, 6 and 8 days consecutively while pH value declined with storage duration which was negatively correlated with time peroids (Table 8.1, Figure 8.2 F).

Table 8.4. Changes in pH [-log(mol/L)] of mango fruits after exposing to different doses of gamma irradiation compared with control.

Storage duration (days) Irradiation doses (Gy) 2 4 6 8 Control 4.3 ± 0.03 c 3.3 ± 0.12 c 3.1 ± 0.06 b 3.0 ± 0.06 c 100 4.6 ± 0.06 b 4.1 ± 0.03 b 3.4 ± 0.12 b 3.2 ± 0.09 c 150 4.7 ± 0.04 b 4.3 ± 0.06 ab 4.1 ± 0.03 a 3.7 ± 0.12 b 200 4.8 ± 0.04 b 4.5 ± 0.09 a 4.4 ± 0.07 a 4.1 ± 0.06 a 250 5.1 ± 0.06 a 4.5 ± 0.09 a 4.4 ± 0.14 a 4.3 ± 0.09 a LSD values for 2 (0.23), 4 (0.39), 6 (0.43) and 8 days (0.39). Different letters in a column are significantly different using Tukey’s HSD test at (P ≤ 0.01). 8.3.7. Sensory quality or organoleptic characteristics

Data pertaining to organoleptic characteristics or sensory evaluation like external appearance, odor, taste, texture and over all acceptability by the consumer are given in figures 8.5–8.9. There was gradual decrease in all the five parameters along with storage duration. Maximum baseline test score of 8.5 was recorded in fresh fruits (before irradiation) which were decreased to the minimum level after 8 day storage duration (after irradiation) in all five parameters at temperature of 29.3±1.4 oC and RH of 42±2.6 %. However, at day 8, the external appearance, odor, taste, texture and overall acceptability of fruits irradiated at 250 Gy was still in acceptable range and look more fresh and attractive than other treatments while unirradiated fruits in control were least attractive in my consumer tests. After 8 days, percent decrease in scores of appearance, odor, taste, texture and overall acceptability of fruit in control and 250 Gy were: 55.29 % (control) and 35.29 % (250 Gy), 49.41 % (control) and 31.76 % (250 Gy), 55.88 % (control) and 26.47 % (250 Gy), 55.88% (control) and 44.12 %

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(250 Gy) and 55.88 % (control) and 35.29 % (250 Gy), respectively. Irradiation at 250 Gy resulted in very high scores for odor and taste as compared to other treatments. Overall irradiation was found to maintain the sensory quality of fruit for a period of 8 days at the above mentioned temperature and RH.

Figure 8.5. External appearance scoring of mango fruits after exposing to different gamma irradiation doses compared with control.

Figure 8.6. Odor scoring of mango fruits after exposing to different gamma irradiation doses compared with control.

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Figure 8.7. Taste scoring of mango fruits after exposing to different gamma irradiation doses compared with control.

Figure 8.8. Texture scoring of mango fruits after exposing to different gamma irradiation doses compared with control.

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Figure 8.9. Overall acceptability scoring of mango fruits after exposing to different gamma irradiation doses compared with control.

8.4. Discussion and Conclusion

Water continuously expires from the surface of fruit after harvesting which causes weight loss due to opening of surface pores for evaporation of moisture contents (Farooqi et al., 1975). This loss of moisture contents decreases visual appearance, salable weight and physiological dysfunctions. Irradiation doses up to 1.5 kGy keep rate of respiration in mango fruits (Tommy Atkins) at normal level (Moreno et al., 2006). Electron beam irradiation of mangoes at 1.0 kGy is the recommended treatment to maintain the overall fruit quality attributes. Abbasi (2009) reported that irradiated Crab chitosan (200 kGy) were superior in quality and extended shelf-life of mango fruit as well as maintained their eating quality upto 4 weeks storage period as copmarred to control. In the present study weight loss was noticed in both irradiated and unirradiated fruits. However, weight loss was comparatively less in irradiated fruits than unirradiated mango fruits. Similarly, firmness was retained in irradiated fruit for longer time than unirradiated mango fruits. D'innocenzo and Lajolo (2001) also stated that the firmness of irradiated fruits was retained at least 2 days longer than in normal fruits and also had a slower rate of softening. The findings of this research for weight loss of mango fruit are in agreement with that of (Reddy and Raju, 1988) who reported an average of 3.96 % weight loss in mango variety Alphonso stored at ambient temperature for five days as compared to the weight loss 104

of 3.9 and 3.7 % occurred in Tommy Atkins and Palmer mango varieties when stored at 20°C for five days. Some other researchers also reported same values for weight loss in mango fruit (Mahayothee et al., 2002; Krishnamurthy, 1988). Arthur and Wiendl (2000) also reported that weight loss in star–fruit irradiated at 0.5 kGy was less than unirradiated fruit. During mango fruit ripening, the texture of the fruit changed which is attributed to the degradation of pectic compounds by the pectic enzymes. The activity of this enzyme increase as the fruit ripens (Tridjaja and Mahendra, 2000). Thus firmness in mango fruit has vital importance as consumer first look at the soundness and firmness of fruit during purchase. In this study though the firmness decreased regularly with storage duration yet the decrease was slower in fruit irradiated with Gamma irradiation than controls. Jacobi et al. (1998) reported that a mango variety Kensington show softness at 4 day of storage while after 11 day the fruit become fully soft. Saks et al. (1999) also reported that Tommy Atkins mango fruit showed less firmness stored at 20 °C than 13°C. Similar results were also reported by Mahayothee et al. (2002) who observed that Thai cultivars of mango when harvested at mature green stage showed decrease in firmness at 3 and 4 day when stored at 23°C.

Increase in total soluble solids (TSS) in fruits is due to the breakdown of very complex carbohydrates (Nawaz, 1971). That is why, a general tendency in fruit that TSS increases as the fruit ripens. The increase or decrease in TSS is an indication of metabolic activity. As water contents were high in fresh fruit therefore they have low TSS. With increasing storage duration the TSS values increased due to evaporation of moisture. As irradiation inhibit the enzymatic activities of fruit and break the DNA strands, water loss is controlled and hence comparatively less decrease was observed in the TSS contents of irradiated fruits. My study also revealed that less increasein TSS contents was noted in the fruits treated with gamma irradiation than untreated fruits but all of them were non–significant at each storage levels. McGuire (2000) also stated that soluble solids were low in irradiated fruits than unirradiated fruits. However O’Mahony et al. (1985) found no significant differences between irradiated and unirradiated fruits in respect to soluble solids.

Acidity, ascorbic acid and pH are the important characteristics of mango fruit. Acidity is due to the presence of citric, malic, oxalic, succinic and benzoic acids. After harvesting the loss of acidity is due to the loss of moisture and conversion of sugar as ripening advance. As organic acids are the intermediate metabolites of citric acid cycles which utilized during the process of respiration. Therefore, acidity increased along with increase in storage period (Min and Or, 105

1977; Sheikh et al., 1979). Similarly, vitamin C is the most complex vitamin to be protected from degradation after harvesting due to rapid degradation by increased enzymatic activities. In the same way, pH of fruit decrease along with increasing storage duration as a result of microbial degradation of nutrients which produce acids and alcohols (Khan et al., 2012). In the present study acidity, vitamin C and pH contents were more stable in irradiated fruits than control. These findings are in agreement with that of Domarco et al. (1999) who observed that titrable acidity and pH of a grape variety italica significantly increased with increasing doses of irradiation. Abdellaousi et al. (1995) and Mahrouz et al. (2002) also reported that vitamin C contents were higher in clementine irradiated with gamma–irradiation than control.

It is concluded that mango fruits regularly decline in visual quality and taste in storage. This decrease in taste and visual quality is attributed by the decrease in acidity and vitamin C contents. In contrast weight loss and TSS increased during storage. Therefore to maintain the quality of fruit, acidity, vitamin C, weight loss and TSS may be retained in the fruits. Irradiation is an effective technique of post–harvest to check the changes in physiochemical analysis and sensory quality in mango fruits. Irradiation of mango fruit at dose of 250 Gy very efficiently control all the stages of coconut scale as well as it does not affect the quality of fruit but rather improve it. Because irradiation stops the enzymatic activities of fruit, as a result fruit retains its quality and freshness for longer time.

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Chapter 9.

Efficacy of different insecticides against various developmental stages of A. destructor

9.1. Introduction

A. destructor is a serious economic pest of mango in many countries of the world where they feed on different parts of their host such as leaves, twigs, branches and roots (Chua and Wood, 1990; Suomi, 1996; Nabity et al., 2009). The pest causes severe damage to mango, and kills the plant by sucking sap, injecting toxins and transmitting viruses. In heavily infested trees the yield is dropped mainly due to reduction of photosynthesis and respiration. A. destructor is also a very serious pest of coconut in the South Pacific (Dabek, 1998).

Insecticides like imidacloprid and thiamethoxam are effective against sucking pests including scale insects (Nauen et al., 1996; Maienfisch et al., 1999). Due to their systemic mode of action these insecticides are mostly distributed in the plant system uniformly. Acephate 75 WP (O,S-Dimethyl acetylphosphoramidothioate) @ 1.5 g/l, methyl parathion @ 2ml/l and imidacloprid @ 0.3 ml/l are very effective against A. destructor (Kumari et al., 2014). Similarly, pyriproxyfen has strong suppression against California red scale, Aonidiella aurantii Maskel and other coccids like Ceroplastes floridensis Comstock (Peleg, 1988). Pyriproxyfen is used against red scale in South Africa on citrus plants (Hattingh and Tate, 1995), hemipteran pests like sweet potato whitefly, Bimisia tabaci Gennadius and also against aphids (Devine et al., 1999). Mineral oil alone as well as when mixed with fenitrothion (Organophosphate) and pyriproxyfen are effective against scale insect while pyriproxyfen is more effective during summer time than spring; and chemicals with summer oil are effective in spring than winter (Nada et al., 1990; Mohamed, 2002). Excessive use of chemical insecticides against scale insects often negatively disrupts their parasitoids and predators and may cause acute and chronic health and environmental hazards and development of resistance in insects (Beardsley, 1970; Malr, 1997). Integrated pest management program on the basis of monitoring scale insects populations with natural enemies have also been developed and successfully applied in many regions (Pinese and Piper, 1994; Tauili’ili and Vargo, 1993). Cultural practices in the form of pruning A.

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destructor infested leaves from the plant are also useful for the management of coconut scale (Chou, 2003).

The aim of the present work was to investigate efficacy of different insecticides against various developmental stages of A. destructor on mango nursery plants and mature trees.

9.2. Materials and methods

9.2.1. Evaluation of insecticides on mango nursery plants

Mango nursery plants were obtained in July and August 2012 from FNF, D. I. Khan. The plants were manually infested with newly hatched crawlers according to the procedure given in Chapter 4 (4.2.1). Infested nursery plants with crawlers of uniform age were kept for 10, 15 and 20 day to get 1st instar, 2nd instar and mature stage of scale insects. Nursery plants infested with particular stages were sprayed using a hand held atomizer sprayer with recommended rates (Table 9.1). Experiments were arranged in a completely randomized block design with nine treatments including the control and with three replicates.

Table 9.1. Formulations and recommended rate of different Insecticides evaluated as spray treatments on mango nursery plants and mature trees.

Rate per 100 L of Trade names Common names Formulations water 861 gm/L (86.1 % Winhort Petroleum oil 2 L v/v)

Foliol Mineral oil 84 gm/L (84 % w/w) 2 L

Imidacloprid Imidacloprid 200 w/v 250 ml

Priority Pyriproxyfen 10.8 EC 50 ml

Actara Thiamethoxam 25 WG 10 gm

Seven Star Bifenthrin 10 % EC 250 ml

Denadem Profenofos 500 EC 400 ml

Curacron Profenofos 500 EC 300 ml

Control – – –

Data were taken by counting the number of scale insects on three tagged leaves (upper, middle and lower) on each individual nursery plant. Efficacy of insecticides was assessed 108

from the number of living and dead scales on treated leaves compared to control. Data were recorded before spraying and at day 1, day 7, day 14 and day 21 post spray application. Scales were considered dead if they had desiccated bodies or had their scale cover removed.

Percent mortality of scale insects was calculated by the following formula:

(Pre-spray data – Post-spray data) Percent mortality = × 100 Pre-spray data

9.2.2. Evaluation of insecticides on mature mango trees

The second experiment was conducted at the same location on 15 year old fruit bearing mango trees cultivar Langra in FNF during October 2012. A motorized sprayer (BJ Power sprayer GX 120 Honda 4.0, Honda Motor Co. Ltd, Japan) was used to spray the mature trees. Three replicate trees were sprayed with same insecticides as given in table 9.1. Twelve leaves (3 leave from each side in the height range of 2–4 m) were cut from each trees to observe alive and dead scale insects. Data were recorded before spraying and at day 1, day 7, day 14 and day 21 post spray application. Percentage mortality of scale insects was calculated as before.

Data were subjected to statistical analysis with one way ANOVA using Statistix 8 (version 8.1, Analytical software) Software. Means were separated by Tukey HSD test at P ≤ 0.05.

9.3. Results

9.3.1. Efficacy of insecticides against various stages of A. destructor on mango nursery plants

Petroleum oil (97.2 %), imidacloprid (97.4 %) and pyriproxyfen (98.2 %) showed the highest percent mortality against 1st instar A. destructor at day 21 post treatment which were significantly (P ≤ 0.05) superior to mineral oils (71.4 %), thiamethoxam (85.1 %), bifenthrin (57.4 %) and profenofos (66.0 %) (Table 9.2). Imidacloprid and pyriproxyfen were significantly (P ≤ 0.05) more effective than the other insecticides against 2nd instars and mature scale insects. In thiamethoxam and profenofos treated plants, the percent mortality of A. destructor increased with increasing time period while bifenthrin showed maximum

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mortality within the first 14 days against all the stages of scale insects. In control plants very little natural mortality of scale insects was observed throughout the study period.

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Table 9.2. Efficacy (% mortality) of different insecticides against A. destructor life stages on mango nursery plants during 2012.

Rate/100 liter 1st instar 2nd instar Mature scale Treatments of water 1 d 7 d 14 d 21 d 1 d 7 d 14 d 21 d 1 d 7 d 14 d 21 d Winhort 2 L 22.2 a 42.5 b 84.7 a 97.2 a 23.3 c 40.1 c 52.2 b 94.3 a 16.7 b 54.9 b 81.5 a 94.2 a

Foliol 2 L 12.7 b 24.6 c 56.5 bc 71.4 c 17.6 d 30.5 de 36.9 d 87.3 b 11.2 cd 38.4 c 61.8 b 83.9 b

Imidacloprid 50 ml 23.0 a 52.0 a 83.1 a 97.4 a 40.6 a 52.2 ab 66.5 a 99.2 a 28.3 a 65.8 a 83.0 a 97.9 a

Priority 250 ml 15.1 b 49.5 a 90.5 a 98.2 a 34.0 b 54.1 a 68.2 a 98.7 a 12.8 c 67.6 a 79.1 a 99.4 a

Actara 10 gm 14.0 b 27.9 c 54.3 bc 85.1 b 11.6 e 37.3 cd 53.7 b 77.0 c 5.7e 44.5 c 56.8 bc 82.5 b

Seven Star 250 ml 10.8 b 54.0 a 57.1 b 57.4 d 17.6 d 55.7 a 59.1 c 59.1 d 8.6 de 53.9 b 58.5 bc 58.5 d

Denadem 400 ml 13.9 b 30.6 c 48.3 cd 61.9 cd 19.1 d 42.8 bc 53.8 b 73.5 c 9.6 cd 17.5 d 54.7 cd 67.6 c

Curacron 300 ml 13.6 b 28.5 c 41.6 d 65.6 cd 12.2 e 24.4 e 36.7d 73.4 c 11.7 cd 15.9 d 49.4 d 71.3 c Control – 2.7 c 2.7 d 2.7 e 2.7 e 4.1 f 4.1 f 4.1 e 4.1 e 1.3 f 1.3 e 1.3 1.3 LSD values 6.0 6.5 9.0 10.1 4.2 9.4 6.4 6.2 3.9 7.6 5.5 5.8 – SEM± – 1.7 1.9 2.6 10.1 1.2 2.7 1.8 1.8 1.1 2.2 1.6 1.7 – CV % 14.7 6.6 5.5 5.0 7.3 8.6 4.8 3.0 11.5 6.7 3.3 2.8 d = days, SEM = standard error of mean

Different letters within columns indicate significant differences using Tukey’s HSD test at (P≤ 0.05) among treatments 111

During 2013, all the insecticides were superior over the control. Among the different insecticides, petroleum oil and pyriproxyfen showed maximum % mortality of all the stages of A. destructor at day 14 and day 21 (Table 9.3). Imidacloprid was effective from day 1 with more than 30 % mortality of A. destructor and its effect continued up to day 21 at which stage more than 90 percent mortality of all the stages of A. destructor was achieved. Percent mortality of different stages of A. destructor, treated with thiamethoxam and profenofos, consistently increased over time; however bifenthrin showed quick result against all the stages of A. destructor within first 14 days; after that no increase in percent mortality of scale insects was noted.

9.3.2. Efficacy of insecticides against A. destructor on mature mango plants

During the two study years, maximum percent mortality of A. destructor was recorded in plants treated with petroleum oil (75.3 and 76.0 %), imidacloprid (78.8 and 79.6 %) and pyriproxyfen (77.2 and 78.7 %), consecutively at day 21 (Table 9.4). Mineral oil, bifenthrin, thiamethoxam and profenofos were also effective against A. destructor but the percent mortality was comparatively less than petroleum oil, imidacloprid and pyriproxyfen. Subsequent increase in percent mortality of A. destructor was noted with thiamethoxam and profenofos while bifenthrin caused maximum mortality during the first 14 days; after that no increase in percent mortality of A. destructor was observed.

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Table 9.3. Efficacy (% mortality) of different insecticides against A. destructor life stages on mango nursery plants during 2013

Rate/100 1st instar 2nd instar Mature scale Treatments liter of 1 d 7 d 14 d 21 d 1 d 7 d 14 d 21 d 1 d 7 d 14 d 21 water Winhort 2 L 29.9 a 52.7 b 84.3 a 96.0 a 24.3 c 56.1 b 79.9 a 97.1 a 18.8 b 52.9 cd 88.3 a 97.9 a

Foliol 2 L 19.9 b 41.3 c 68.5 bc 80.0 bc 16.2 e 31.6 d 63.3 b 84.9 b 15.2 bcd 41.5 e 70.8 be 84.8 b

Imidacloprid 50 ml 32.3 a 59.9 a 84.3 a 97.8 a 43.4 a 66.5 a 86.4 a 99.4 a 29.1 a 76.3 a 88.0 a 97.5 a

Priority 250 ml 19.0 b 49.2 b 86.8 a 97.6 a 34.0 b 54.1 b 81.0 a 99.0 a 14.6 bcd 66.9 b 82.1 a 97.0 a

Actara 10 gm 13.8 b 30.5 d 56.8 c 90.8 ab 20.9 cd 42.9 c 55.2 b 79.6 c 9.3 e 46.9 de 59.4 cd 83.2 b

Seven Star 250 ml 12.8 b 64.6 a 71.0 b 72.0 c 17.4 de 47.6 c 58.9 b 67.6 d 14.1 cd 56.7 c 62.8 c 65.4 c

Denadem 400 ml 15.8 b 33.2 d 58.6 c 71.7 c 31.7 b 42.8 c 66.7 b 77.6 c 11.5 de 19.4 f 56.6 cd 69.9 c

Curacron 300 ml 16.3 b 30.5 d 39.9 d 73.5 c 15.0 e 36.7 d 57.3 b 76.5 c 17.2 b 22.9 f 55.3 d 68.9 c Control – 4.0 c 4.0 e 4.0 e 4.0 d 1.7 f 2.1 e 2.1 c 2.1 e 1.1 f 1.1 g 1.1 e 1.1 d LSD values 8.7 7.1 11.8 14.9 4.2 5.7 11.8 4.6 4.6 6.5 7.2 11.5 – SEM± – 2.5 2.0 3.4 4.2 1.2 1.6 3.4 1.3 1.3 1.9 2.1 3.3 – CV % 16.7 6.1 6.7 6.9 6.4 4.7 6.7 2.1 11.0 5.3 4.0 5.4 d = days, SEM = standard error of mean. Different letters within columns showed significant differences using Tukey’s HSD test at (P≤ 0.05) among treatments 113

Table 9.4. Efficacy (% mortality) of different insecticides against A. destructor on mature mango trees during 2012 and 2013

Rate/100 liter of 2012 2013 Treatments water 1 d 7 d 14 d 21 1 d 7 d 14 d 21 d Winhort 2 L 24.3 a 52.7 b 71.8 ab 75.3 a 26.3 ab 54.0 b 72.6 ab 76.0 a

Foliol 2 L 15.2 b 26.5 d 52.7 de 60.4 c 19.1 bc 29.9 d 54.9 cd 62.2 cd

Imidacloprid 50 ml 26.8 a 59.5 a 76.3 a 78.8 a 29.7 a 61.2 a 77.2 a 79.6 a

Priority 250 ml 16.1 b 49.2 b 68.4 bc 77.2 a 21.5 b 52.5 b 70.4 ab 78.7 a

Actara 10 gm 10.3 c 27.2 d 46.9 ef 59.1 c 12.7 c 29.2 d 48.3 d 60.2 d

Seven Star 250 ml 11.7 c 62.3 a 65.7 c 65.7 b 14.2 c 63.3 a 66.6 b 66.6 b

Denadem 400 ml 11.0 c 35.3 c 53.8 d 63.6 bc 14.0 c 37.5 c 55.4 c 64.8 bc

Curacron 300 ml 11.2 c 25.7 d 44.0 f 53.9 d 22.5 ab 35.1 cd 51.1 cd 59.9 d Control – 1.2 d 1.3 e 1.3 g 1.3 e 1.66 q 1.79 q 1.79 q 1.79 q LSD values 3.3 4.8 6.0 5.1 7.2 6.4 7.0 3.8 SEM± 0.9 1.4 1.7 1.4 2.1 1.8 2.0 1.1 CV % 8.2 4.4 3.9 3.0 14.1 5.5 4.4 2.2 d = days, SEM = standard error of mean

Different letters in a column indicate significant differences using Tukey’s HSD test at (P≤/0.05) among treatments 114

9.4. Discussion and conclusion

Results of the present study revealed that petroleum oil, imidacloprid and pyriproxyfen were very effective against A. destructor while thiamethoxam, profenofos and curacron were comparatively less effective during 2012 and 2013. Bifenthrin caused an early rapid mortality on scale insects but later on the spray effect subsidized. Our results agree with Abo–Shanab (2005) and El-Deeb (2004) who investigated that insect growth regulators (IGRs) and oil mixtures efficiently control scale insects Lepidosaphes beckii (Newman). Aleksidze et al. (1995) found that mineral oil at the rate of 1.5 % v/v was effective against overwintering stages of scales while organophosphorus and pyrethriods at the rate of 0.15 % (v/v) were effective against crawlers. Handa et al. (1999) found 85.3 % effectiveness of fenvalerate against scale insects. Thiamethoxam application at the point of drip irrigation was very effective against scale insects and mealybug in South Africa on mango plants (Legadec et al., 2005). These findings are in agreement with that of Asfoor (1997) who investigated that cidial, sumi oil, diazinon, fenitrothion, misrona oil and fenvalerate were very effective against the nymphal stages of Parlatoria oleae Colvee on pear, plum and apple trees while ovipositing females and non-ovipositing females showed resistant to the tested products. Mangoud (2008) found that misrona oil and buprofezin were most effective against Parlatoria ziziphi Lucas for long time. The results of present study also match with the findings of Hix et al. (1999), Twaite at al. (1999), Salyani et al. (1999) and Lainga et al. (1999). As A. destructor has aggregation nature of living, therefore spraying of insecticides on infested patches on leaves, twigs and fruits are preffered instead of covering whole plant. Similarly, only those plants which have heavy infestation based on monitoring data be treated with insecticides. Chou (2003) and Nestel et al. (1995) also reported that aggregation nature of living is typical in all Diaspidids. Chou (2003) stated that below 10 % of infestation on leaves has very little chance of fruit infestation in A. destructor. For control strategies of A. destructor in D. I. khan, early instars should be focused during July-August when crawlers, which can be easily controlled, are released. Also, there are fewer natural enemies present in the filed during these months. Agriculture scientists have to look for the alternative control measures including encouragement of parasitoid, oils, IGR and systemic insecticides as they represent the latest management tactics for commercial insect control. These insecticides have fewer harmful effects on non–target organisms; their

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specificity gives them superiority over conventional insecticides and has more or less harmony with integrated pest management (IPM) program. These insecticides have a good margin of safety to invertebrates, birds, fish and other wild life fauna as well as safe for man and their domestic mammalians. According to management decision plan based on population dynamic study of A. destructor, 1st spray must be applied in early August (before parasitoids emergence started), 2nd spray in October and 3rd spray in February after pruning of mango trees to get the orchard free of scale insect for next season. In this study, integrated approaches for the management of scale insects is suggested involving prevention of new infestation, monitoring, mechanical removal of infested leaves and twigs and encouraging natural enemies as effective tool for management tactics. In an extreme cases, need base application of oils, IGR and systemic insecticides are recommended as a last option for the control of A. destructor.

A. destructor is a very serious pest of mango with different infestation levels from July to December in D. I. Khan. Heavy parasitism of A. melinus and Anagyrus sp. playvery effective role in the control of the pest however; to keep the pest below economic threshold level, use of insecticides is necessary. The present study suggests that in case of moderate infestation, one spray either of petroleum oil, pyriproxifen or imidacloprid in early August, before parasitoids emergence, and second spray in November–December control A. destructor on mango plant. However; in severe infestation, three sprays of the above mentioned insecticides in early August, late September and November provide good control of A. destructor. Cultural practices such as cutting infested leaves and twigs also give excellent results if the scale infestation is in patches together with selective spray for management of coconut scales (Chou, 2003).

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CHAPTER 10.

GENERAL DISCUSSION AND CONCLUSION

10.1. Summary

Coconut scale, Aspidiotus destructor Signoret (Hemiptera: Diaspididae) is one of the most important pests of coconut, banana, mango and citrus, in Africa, Asia, the Pacific Region and Australia. The immature stage feeds on leaves, twigs, branches and roots of its host and damages its host by sucking sap and injecting toxins. In Pakistan, A. destructor infests both early and late varieties of mango. This challenging pest gained more importance due to high fecundity, easy dispersal by wind and birds, many overlapping generations in a season, and its scale cover which provides protection against insecticides. This is why the detail study of the pest was necessary to use it for management strategies.Since previous work about A. destructor in Pakistan are mainly focused on rearing the pest inlaboratory, reported infestation in different areas with damages caused to mango plant and parasitoids (Ahmad and Ghani, 1972; Mohyuddin and Mahmood, 1993), the present study was undertaken with the aim to generate new information about biology, population fluctuation and management of A. destructor. In Chapter 1, I reviewed the scattered literature about the population dynamics and natural efficiency of parasitoids, developmental biology, and physiological, biochemical and molecular changes to plants under scale infestation. As A. destructor disrupt in trade of fruits among different countries, in the present study generic doses of irradiation for growth inhibition/mortality of A. destructor was developed for exporting the fruits. To know the effect of irradiation on quality of fruit, physiochemical analysis and sensory evaluation of mango fruits were undertaken. At the end efficacy of different insecticides for the control of A. destructor was evaluated.

In chapter 3, I evaluated the seasonal population dynamics of A. destructor and its major parasitoids on mango plants in D. I. Khan. Monitoring population fluctuations helps in predicting where and when infestations may occur, and control measures may be needed. The correlation of pest infestation with weather factors may help growers to be vigilant for the possible flare up of population in the start of season. I recorded very high infestation of A. destructor on mango plants from July to December with 3–4 overlapping generations per year having maximum infestation in October and November. The populations of the pest were significantly correlated with wind velocity and rainfall which helps in dispersal and 117

establishing colonies on new plants. These results will help growers and nurserymen to locate new colonies in the orchard and apply management strategies such as prune the infested leaves or twigs (Chou, 2003) or application of insecticides on infested patches before flare up of the pest.

In chapter 3, my results showed that A. melinus and Anagyrus sp. were the most abundant parasitoids of A. destructor during the peak infestation of the pest which indicated that the parasitoids play a significant role in the control of scale insects. Thus use of insecticides is recommended at that time when there are least activities of natural enemies in the field.

Predicting the pest population occurrence in relation to weather condition helps the growers to spotlight the time and incidence of infestation. Thus developmental biology of A. destructor was studied on mango cultivar Desi in the laboratory and greenhouse at two different temperatures and RH to develop precise model of the pest distribution and seasonal biology in the study area (Chapter 4). The pest was more prevalent in the months of high RH and temperature (July to November) compared with months of high temperature with low RH (April to June) or high RH with low temperature (December to March). This study will assists the mango growers to develop a management decision plant on the basis of monitoring and meteorological data as well as prediction about the flare up of the pest population for the control of scale insects.

Physiological, biochemical and molecular changes in mango leaves of three cultivars were investigated under A. destructor infestation (Chapter 5, 6). Physiological activities of mango leaves decreased up to 50 % in scale infested leaves which indicated that chlorosis and dieback caused by the scale is due to large amount of cell sap sucked by the pest while the cellular injury was indicated by the ions analysis. In scale infested leaves, reduction in cell viability and increasing trend in electrolyte leakage with high amount of MDA and H2O2 indicated lipid peroxidation in the leaves. High value of POD, SOD, APX and CAT in the infested leaves specified the activation of antioxidant defense system against scale insects stress. The findings of this study encouraged the use of RAPD in investigation of genotoxicity in mango leaves under scale insects stress.

A. destructor is a quarantine pest of many fruits of tropical regions and the presence of even a single live female scale may cause the rejection of whole consignments. I tested gamma irradiation doses of 100–300 Gy against 1st, and 2nd instar nymphs and mature females with 118

and without eggs as a potential phytosanitary treatment (Chapter 7). The egg stage was found as the most susceptible and 2nd instar nymphs as the most tolerant stage. In large-scale validation tests a dose of 228 Gy provided quarantine security for mango scale on exported commodities. This is the first study in Pakistan to develop generic doses of gamma irradiation for growth inhibition of the pest and identify the tolerant and susceptible stages of A. destructor in mango fruits for export purpose. The effect of irradiation on mango fruit quality was evaluated in chapter 8. In physiochemical analysis and sensory quality of mango fruits of variety Langra, irradiated at 100–250 Gy, I confirmed that irradiations have no adverse effect on the overall quality of mango fruits but rather improved the organoleptic characteristics as irradiated fruits had improved nutrient stability and retaining quality for longer time than unirradiated fruits. This result has a promising implication against quarantine insects as well as extends shelf life of commodities by delaying the senescence of fruits and vegetables. Chemical insecticides can be expensive and harmful to natural enemies while biological control is time consuming and slow process. Usually growers do not wait to establish parasitoids and predators in the field. Thus I evaluated different insecticides with special attention to the activity of natural enemies including parasitoids (Chapter 9). On the basis of population dynamics data and percent parasitism of parasitoids, it is suggested that use of insecticides in July and early August will have two benefits 1) scales will be in crawler’s stage which is more susceptible stage to insecticides; 2) parasitoids have not yet emerged up to the mid–August which allows safe use of pesticides for natural enemies of the pest. Similarly, one dormant spray in February after pruning the plants to kill estray scales provide good control for the next season. It is also possible to combine two insecticides for broad spectrum control of A. destructor subject to their interaction or rotated the newer insecticides like neonicotinoid and oils with older ones like pyrethroids and organophosphates to discourage resistant development in the target pest.

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10.2. Conclusions

The findings of this study can be summarized as follows:

1. Populations of A. destructor build up in July with peak in October and November. I screened mango cultivars and found that Langra was the most susceptible variety while Sindhry was less susceptible to scale insects infestation. Two parasitoids, A. melinus and Anagyrus sp. were the most important natural enemies of A. destructor in the study area. Male A. destructor passed through two feeding instar with pre–pupal, pupal and adult stages while females had two immature instars with pre-oviposition and oviposition stages. Total life span of male and females A. destructor was 27 and 39.5 days, respectively.

2. Leaf area, leaf biomass, photosynthetic pigments, soluble proteins and cell viability decreased with increasing scale insects infestation duration while electrolyte leakage increased.

3. An increasing trend in MDA and H2O2 indicated stressful condition in the cell under scale insects infestation while POD, SOD, APX and CAT activity showed antioxidant defense system activated by the leaves to the scale infestation.

4. Changes occurred in RAPD DNA profile of mango leaves indicated by the appearance of new bands and disappearance of existing bands in the PCR products of mango leaves encouraged the use of RAPD as an excellent tool to find out scale insects stress.

5. The egg stage was the most susceptible while 2nd instar was the most tolerant stage of A. destructor to irradiation while 228 Gy was recommended for complete mortality of A. destructor in exporting commodities. 6. Physiochemical analysis showed stability of nutrients in irradiated mango fruits than unirradiated fruits. 7. Petroleum oil, imidacloprid and pyriproxyfen proved to be the best insecticides for the control of A. destructor on both mango nursery plants and mature trees.

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10.3. Future work

The present study addressed the population dynamics, biology and managements of A. destructor on mango plants and possible solutions for both farmers in the field and exporters of mango fruits. However, efficiency of predators in the field was not covered by this study which play very important role in the management of A. destructor. Another parasitoid Pakencyrcus pakistanensis Ahmad (Hymenoptera: Encyrtidae) was not recovered which is expected to be in the study area but need more precise survey and collecting more samples from more locations to confirm its presence and % parasitism. Since Gamma irradiation doses affect the physiology of plant (Figure 7.1) further study is needed to quantify the effect of irradiation on photosynthetic pigments, antioxidant enzymes and DNA damage. Study is also needed for possible combination of oils, IGRs and insecticides as well as to develop a best management IPM program which includes mechanical and cultural practices with effective use of predators, parasitoids and insecticides to reduce the cost of applying insecticides.

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References

Abbasi, N. A., Z. Iqbal, M. Maqbool and I. A. Hafiz. 2009. Postharvest quality of mango (Mangifera indica L.) fruit as affected by chitosan coating. Pak. J. of Bot. 41 (1): 343- 357. Abdellaousi, S., M. Lacroix, M. Jobin, M. Boubekri, M. Gagnon. 1995. Effect of gamma irradiation combined with hot water treatment on the physiological properties, vitamin c content and organoleptic quality of Clementines. Sciences-des-Aliments. 15: 217– 235.

Abo-Shanab, A. S. H. 2005. Efficacy of some IGRs/Insecticides, KZ oil and binary mixtures on mortality and enzyme activity of Egyptian mealybug Icerya aegyptiaca Douglas attacked guava trees in Alexandria Governorate. J. of Pest Cont. and Environ. Sci. 13: 73–85. Ádám, A. L., C. S. Bestwick, B. Barna and J. W. Mansfield. 1995. Enzymes regulating the accumulation of active oxygen species during the hypersensitive reaction of bean to Pseudomonas syringae pv. Phaseolicola. Planta. 197: 240–249. Aebi, H. 1984. Catalase in vitro. Methods Enzymol. 105: 121–126. Agrawal, A. A. 1998. Induced responses to herbivory and increased plant performance. Science. 279: 1201–1202. Ahmad, R. and M. A. Ghani. 1972. Studies on Aspidiotus destructor Sign. (Hem.: Diaspididae) and its parasites, Aphytis melinus Debach (Hymenoptera: Aphelinidae) and Pakencyyrtus pakistanensis Ahmad (Hymenoptera: Encyrtidae) in Pakistan. Commonwealth Institute of Biological control, Technical Bulliton. 15: 51–57.

Ahmad, S. 1987. Effect of Irradiation on the Keeping Quality of Feutrell’s Early. MSc Thesis. Department of Food Scie. Tech., NWFP Agricultural University, Peshawar, Pakistan Aisagbonhi, C. I. and S. I. Agwu. 1985. The life cycle of Aspidiotus destructor Signoret (Homoptera: Diaspididae) on coconut seedlings. J. of the Nigerian Inst. for Oil Palm Res. 7 (1): 120–129 Akhter, A. B. M., S. A. Khan, H. Akter, M. S. Islam and M. A. Howlader. 2008. Evaluation of gamma irradiation as a quarantine treatment against the oriental fruit fly, Bactrocera dorsalis Hendel. J. of Asiat. Soc. Bangladesh. Sci. 34: 157–168.

122

Akhter, H., S. A. Khan, K. Seheli, M. A. Wadud, A. A. Taslim, A. Yasmin and S. Islam. 2005. Irradiation, quarantine, shelf life extension and nutritional quality of fresh fruits and vegetables. Proceedings of first national seminar on use of irradiation quarantine treatment of fresh fruits and vegetables. Bangladesh Atomic Energy Commission, Dhaka. pp. 25–32. Aleksidze, G., K. R. Aseher and D. Y. Ben. 1995. Armored scale insects (Diaspididae) pests of fruit orchards and their control in the Republic of Georgia. Israel J. of Entomol. 29: 187–190. Allison, S. D. and J. C. Schultz. 2004. Differential activity of peroxidase isozymes in response to wounding of gypsy moth and plant hormones in northern red oak (Quercus rubra L.). J. of Chem. Ecol. 30 (7): 1363–1379. Alonso, M., L. Palou and M. A. Del Rio. 2007. Effect of X–ray irradiation on fruit quality of clementine mandarin cv. ‘Clemenules’, Radiat. Phys. Chem. 76 (10): 1631–1635. Anil, V. S., P. Krishnamurthy, S. Kuruvilla, K. Sucharitha, G. Thomas and M. K. Mathew. 2005. Regulation of the uptake and distribution of Na+ in shoots of rice (Oryza sativa) variety Pokkali: Role of Ca2+ in salt tolerance response. Physiol. Plant. 124: 451–464. Anonymous. 2010–11. Agriculture Statistics of Pakistan. Pakistan Bureau of Statistics, Ministry of Food and Agric. Econ. Wing, Islamabad, Pakistan 44th Ed. 90 P.

Anonymous. 2011. Fruit, vegetable and condiment. Ministry of Food, Agriculture and Livestock, Islamabad. Ansari, A. R. 1942. Short notes and exhibits. Occurrence of Bourbon scale (Aspidiotus destructor Sign.) in the Punjab. Indian J. of Entomol. 4 (2): 233 p.

Anten, N. P. R. and D. D. Ackerly. 2001. Canopy–level photosynthetic compensation after defoliation in a tropical understorey palm. Functional Ecology. 15: 252–262. Antonio, A. L., M. Carocho and A. Bento. 2012. Effects of gamma radiation on the biological, physico–chemical, nutritional and antioxidant parameters of chestnuts–a review. Food Chem. Toxicol. 50 (9): 3234–3242. AOAC. 2006. Official Methods of Analysis. Association of Official Analytical Chemists International. 18th Ed. Horwitz, W. (ed.). AOAC Press, Arlington, VA, USA. Apel, K. and H. Hirt. 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55: 373–399.

123

Arthur, V. and F. M. Wiendl. 2000. Gamma irradiation of star fruit to increase shelf life. Revista de Agri, Piracicaba. 75: 425–429. Ascher, K. R. S., Y. Ben-Dov, T. I. Labuschagne, H. Van hamburg and I. J. Froneman. 1995. Population dynamics of the mango scale, Aulocaspis tubercularis Newstead (Coccoidea: Daispididae), in South Africa. Israel J. of Entomol. 29:207–217. Asfoor, M. A. 1997. Seasonal abundance and control of the plum scale insect, Parlatoria oleae Clove on some deciduous trees. Ph. D. Thesis, Fac. of Agric. Zagazig University. pp. 398. Atienzar, F. A. and N. A. Jha. 2006. The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review. Mutat. Res. 613: 76–102. Atienzar, F. A., B. Cordi and A. J. Evenden. 1999. Qualitative assessment of genotoxicity using random amplified polymorphic DNA: comparison of genomic template stability with key fitness parameters in Daphnia magna exposed to benzo[a]pyrene. Environ. Toxicol. Chem. 18: 2275–2282. Atienzar, F. A., P. Venier and A. N. Jha et al. 2002a. Evaluation of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations. Mutat. Res. 521: 151–163. Atienzar, F.A., B. Cordi, M. E. Donkin, A. J. Evenden, A. N. Jha and M. H. Depledge. 2000. Comparison of ultraviolet-induced genotoxicity detected by random amplified polymorphic DNA with chlorophyll fluorescence and growth in a marine macroalgae, Palmaria palmate. Aquatic Toxicol. 50: 1–12. Atienzar, F.A., P. Venier, A. N. Jha and M. H. Depledge, 2002. Evaluation of the random amplified polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations. Mutat. Res. 521: 151–163. Awan, A. J. and S. Rehman. 2001. Food Analysis Manual. 3rd edn. Published by Unitech communication. Faisalabad, Pakistan. Awan, J. A. and U. R. Salim. 1997. Food analysis manual. Veterinary Agric. Publication. 5: 2–7.

Bacheller, J. D. and J. T. Romeo. 1992. Biotic and abiotic stress effects on nitrogen chemistry in the salt marsh cordgrass Spartina alterniflora (Poaceae). Chemoecology. 3:74– 80

124

Bafana, A., S. Dutt, A. Kumar, S. Kumar and P. S. Ahuja. 2011. The basic and applied aspects of superoxide dismutase. J. of Mol. Catal. B: Enzym. 68: 129–138. Baker, N. R. 2008. Chlorophyll fluorescence: A probe of photosynthesis In Vivo. Annual Review of Plant biology. 59: 89-113. Bakery, M. M. S. 2009. Studies on some scale insects and mealybugs infesting mango trees in Qena Governorate, Egypt. M.Sc. thesis, Fac. Agric. Minia University.

Baloch, A. F. 1994. Mango. In: Horticulture. National Book Foundation, Islamabad, Pakistan.

Balock, J. W., L. D. Christenson and G. O. Burr. 1956. Effects of gamma rays from cobalt 60 on immature stages of the oriental fruit fly, Dacus dorsalis Hendel and possible application to commodity treatment problems. Proc. 31st Annual Meeting Hawaii Acad. Sci., Honolulu, Hawaii. pp. 1–18. Bates, L. S., R. P. Waldren and I. D. Teare. 1973. Rapid determination of free proline for water studies. Plant Soil. 39: 205–208.

Beardsley, J. W and R. H. Gonzalez. 1975. The biology and ecology of armored scales. Annual Review of Entomology. 20: 47–73 Beardsley, J. W. 1970. Aspidiotus detructor Signoret, an armored scale pest new to the Hawaiian Islands. Proc. Hawaiian Entomol. Soci. 20: 505–508.

Beattie, G. A. C. 2005. Using petroleum-based spray oils in citrus. AGFACT, Sydney: NSW Department of Primary Industries.

Behmer, S. T. and A. Joern. 1994. The influence of proline on diet selection-sex-specific feeding preferences by the grasshoppers Ageneotettix deorum and Phoetaliotes nebrascensis (Orthoptera: Acrididae). Oecologia. 98: 76–82. Ben-Dov, Y. 1994. A systematic catalogue of the mealybugs of the world. Intercept Limited, Andover, UK pp. 686.

Ben-Dov, Y. 2014. ScaleNet, Aspidiotus destructor [WWW document]. URL http://www.sel.barc.usda.gov/catalogs/diaspidi/aspidiotusdestructor.html [accessed on 3 February 2016].

125

Ben-Dov, Y., D. R. Miller and G. A. C. Gibson. 2003. ScaleNet (web page) http://www.sel.barc.usda.gov/SCALENET/economic.htm. Benkeblia, N. 2000. Food irradiation of agricultural products in Algebra. Present situation and future developments (a short communication). Special Issue 11. 3rd International Conference on Physics of Agro. and Goods Products, Dabrowica–Lublin, Poland, 26– 28 May, 1998. Intenational Agrophysics, 14: 259–261.

Bennett, F. D. and M. M. Alam. 1985. An annotated check-list of the insects and allied terrestrial arthropods of Barbados. Bridgetown, Barbados, Caribbean Agric. Res. and Dev. Inst. 81 pp.

Berlinger, M. J., L. Segre, H. Podoler and R. A. J. Taylor. 1999. Distribution and abundance of the oleander scale (Homoptera: Diaspididae) on jojoba. J. of Econ. Entomol. 92 (50): 1113–1119.

Blokhina, O., E. Virolainen and K. V. Fagerstedt. 2003. Antioxidants, oxidative and oxygen deprivation stress: a review. Annals of Bot. 91: 179–194. Bolwell, G. P., I. V. Bindschedler, K. A. Blee, V. S. Butt, D. R. Davies, S. L. Gardner, C. Gerrish and F. Minibayeva. 2002. The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. J. of Exp. Boil. 53: 1367–1376. Bonafonte, P. 1981. Influence of environmental factors on armored scale insects, their biology, natural enemies and control. World Crop Pests, Elsevier, Amsterdam, the Netherlands. 4: 319–330.

Boratynski, K. L. 1953. Sexual dimorphism in the second instar of some Diaspididae (Homoptera: Coccoidea). Trans. R. Entomol. Soc. London. 4: 451–479.

Bradford, N. M. 1976. Rapid and sensitive method for quantification of microgram quantities of protein utilizing principle of protein- dye binding. Analyt. Biochem. 72: 248–254. Britt, A. B. 1996. DNA damage and repair in plants. Annual Review of Plant Physiol. Plant Mol. Biol. 47: 75–100. Brown, S. W. 1965. Chromosomal survey of armored and palm scale insects (Coccoidea: Diaspididae and Phoenicococcidae). Hilgardia. 36: 189–294.

126

Bruce, T. J. A., L. J. Wadhams and C. M. Woodcock. 2005. Insect host location: a volatile situation. Trends Plant Sci. 10: 269–274. Bull, D. L. and R. W. Meola. 1993. Effect and fate of the insect growth regulator pyriproxyfen after application to the horn fly (Diptera: Muscidae). J. of Econ. Entomol. 86: 1754–1760.

Burd, J. D. and N. C. Elliott. 1996. Changes in chlorophyll a fluorescence induction kinetics in cereals infested with Russian white aphis (Homoptera: Aphididae). J. of Econ. Entomol. 89: 1332–1337. Burditt, A. K. 1994. Irradiation In: Quarantine treatments for pests of food plants, J. L. Sharp and G. J. Hallman (ed.). pp. 101–117. Westview Press, Boulder, Colorado. Burditt, A. K. Jr. 1982. Food irradiation as a quarantine treatment of fruits. Food Technol. 36 (11): 51. Burger, H. C. and S. A. Ulenberg. 1990. Quarantine problems and procedures. In: D. Rosen (ed.). Armored scale insects, their biology, natural enemies and control. World Crop Pests. Elsevier, Amsterdam, the Netherlands. 4 (B): 313–327.

Calatayud, P. A. and B. Le Ru. 2006. Cassava-mealybug interaction. Institut de Recherche´ Pour le Development, Paris. Carden, D. E., D. J. Walker, T. J. Flower and A. J. Miller. 2003. Single-Cell measurement of the contribution of cytosolic Na+ and K+ to salt tolerance. J. of Plant Physiology. 131 (2): 676–683. Carson, W. P. and B. R. Richard. 1999. Top-down effects of insect herbivores during early succession: influence on biomass and plant dominance. Oecologia. 121: 260–272. Castano, A. and C. Becerril. 2004. In vitro assessment of DNA damage after short and long term exposure to benzo (a) pyrene using RAPD and the RTG–2 fish cell line. Mutat. Res. 552: 141–151. Celino, F. T., S. Yamaguchi, C. Miura, T. Ohta, Y. Tozawa, T. Iwai and T. Miura. 2011. Tolerance of spermatogonia to oxidative stress is due to high levels of Zn and Cu/Zn superoxide dismutase. PLoS One. 6: 916–938.

Cenkci, S., M. Yildiz, I. H. Cigerci, M. Konuk and A. bozdag. 2009. Toxic chemicals- induced genotoxicity detected by random amplified polymorphic DNA (RAPD) in bean (Phaseolus vulgaris L.) seedling. Chemosphere. 76: 900–906.

127

Chaplin, G. R., S. P. Cole, P. A. Landrigan, P. F. Lam and D. Gram. 1991. Chilling injury and storage of mango (Mangifera indica L.) fruit held under low temperatures. Acta Hortic. 29: 1461–1471.

Chazeau, J. 1981. The biological control of the transparent coconut scale Temnaspidiotus destructor Signoret in the New Hebrides (Homoptera, Diaspididae). Cahiers Orstom, Serie Biologie. 44: 11–22.

Chen, Y. W., C. H. Dou, X. H. Zhang and X. R. Li. 2004. Physiological variation of damaged leaves of pears by Eriophyes pyri Pagenstecher. J. of Lanzhou Univ. 40 (1): 68–71. Chinnusamy, V., K. Schumaker and J. K. Zhu. 2005. Molecular genetic perspectives on cross-talk and specificity in abiotic stresses signaling in plants. J. of Exp. Bot. 55: 225–236. Chiu, C.H. 1986. Impact of natural enemies and climate on population dynamics of coconut scale (Aspidiotus destructor Signoret). PhD dissertation, University of Hawaii at Manoa. 148 pp.

Chou, M. Y. 2003. Occurrence and control of Aspidiotus destructor Signoret in bananas. Unpublished M.Sc. thesis, University of Hawaii at Manao. Chua, T. H. and B. J. Wood. 1990. Other tropical fruit trees and shrubs. In: D. Rosen (ed.) Armored scale insects, their biology, natural enemies and control. World Crop Pests. Elsevier, Amsterdam, the Netherlands. 4: 543–552.

Cicek, N. and H. Cakirlar. 2002. The effect of salinity on some physiological parameters in two maize cultivars. Bulg. J. of Plant Physiol. 28: 66–74. Cipollini, D. F. 1997. Wind–induced mechanical stimulation increases pest resistance in common bean. Oecologia. 111: 84–90.

Claps, L. E., V. R. S. Wolff and R. H. González. 2001. Catálogo de las Diaspididae (Hemiptera: Coccoidea) exóticas de la Argentina, Brasil y Chile. Revista de la Socieded Entomológica Argentina. 60: 9–34.

Clausen, C. P. 1956. Biological control of insect pest in the continental United States. U. S. Dept. Agric. Tech. Bull. 1139: 151 pp.

128

Clijsters, H., A. Cuypers and J. Vangronsveld. 1999. Physiological responses to heavy metals in higher plants, Defense against oxidative stress. Verlag der Zeitschrift fu¨r Naturforschung. 54: 730–734. Cochereau, P. 1965. Control biologique d’ Aspidiotus destructor sign. Dans I’ile vate, Nouvelles–Hebrides, au moyen de Lindorus lophanthae Blasid. Compte Rendu de I’Academic Agriculture, France. 51 (5): 318–321. Cochereau, P. 1969. Biological control of Aspidiotus destructor Signoret (Homoptera: Diaspididae) on the island of Vate (New Hebrides) by means of Rhizobius puchellus Montrouzier (Coleopteran: Coccinellidae). Cahiers Orstom. 8: 57–100.

Copland, M. J. W. and A. G. Ibrahim. 1985. Chapter 2.10. Biology of glasshouse scale insects and their parasitoids. pp. 87–90. In: Biological Pest Control. The Glasshouse Experience. Eds. Hussey, N.W. and N. Scopes. Cornell University Press; Ithaca New York.

Coria, N. A., J. I. Sarquis, I. Penalosa and M. Urzua. 1998. Heat-induced damage in potato (Solanum tuberosum) tubers: membrane stability, tissue viability, and accumulation of glycoalkaloids. J. of Agric. Food Chemistry. 46: 4524–4528. Couey, H. M. 1983. Development of quarantine systems for host fruits of the medfly. Hort. Science. 18:45. Couey, H. M. and V. Chew. 1986. Confidence limits and sample size in quarantine research. J. of Econ. Entomol. 79: 887–890. CPC. 2007. Crop Protection Compendium. Aspidiotus destructor; distribution. Cramer, G. R., E. Epstein and A. Läuchli. 1989. Na+ and Ca++ interactions in barley seedlings: relationship to ion transport and growth. Plant Cell Environ. 12: 551–558. Crisosto, C. H., G. M. Crisosto and P. Metheney. 2003. Consumer acceptance of ‘Brooks’ and ‘Bing’ cherries are mainly dependent on fruit SSC and visual skin color. Postharvest Biol. Technol. 28:159–167. Croft, B. A. and W. A. Brown. 1975. Response of arthropod natural enemies to insecticides. Annual Review of Entomology. 20: 285–335. Culik, M. P. and P. J. Gullan. 2005. A new pest of tomato and other records of mealybugs (Hemiptera: Pseudococcidae) from Espirito Santo, Brazil. Zootaxa. 964: 1–8.

129

Dabek, A. J. 1998. Final report: Entomology and plant protection project, Tuvalu (August 1996–August, 1998). Commonwealth Fund for Technical Cooperation (CFTC) Pp.

Danzig, E. M. and G. Pellizzari. 1998. Diaspididae. In: Kozer, F. (ed.) Catalogue of Palaearctic Coccoidea. Hungarian Academy of Sciences. Budapest, Hungary: Akaprint Nyomdaipari Kft. 172–370.

Davidson, J. A. and D. R. Miller. 1990. Ornamental plants. In Rosen, D (ed.) Armored scale insects, their biology, natural enemies and control. Elsevier, Amsterdam, the Netherlands. 4 (B): 603–632. DeBach, P. 1959. New species and strains of Aphytis (Hymenoptera: Eulophidae) parasite on the California red scale, Aonidiella aurantii Mask., in the Orient. Annals of the Entomol. Soc. of America. 52 (4): 354–362.

DeBach, P. 1974. A. destructor in Fiji. In biological control by natural enemies. P. DeBach, ed. Cambridge, Cambridge Univ. Press. 133–135.

DeBach, P., C. A. Fleschner and E. J. Dietrick. 1953. Natural control of the California red scale in untreated citrus orchards in Southern California. Proceedings of the Seventh Pacific Science Congress of the Pacific Science Association, Auckland and Christchurch, New Zealand, 2 February to 4 March 1949. pp. 236–249.

Dekle, G. W. 1965. Florida Armored Scale Insects. Florida Department of Agriculture, Gainseville. pp. 265.

Demiral, T. and I. Turkan. 2005. Comparative lipid peroxidation, antioxidant defense system and proline contents in roots of two rice cultivars differing in salt tolerance. Env. and Exp. Botany. 53: 247–257. Devine, G., I. Ishaaya, A. R. Horowitz and I. Denholm. 1999. The response of pyriproxyfen- resistant and susceptible Bemisia tabaci Genn. (Homoptera: Aleyrodidae) to pyriproxyfen and fenoxycarb alone and in combination with piperonyl butoxide. Pesticide Sci. 55: 404–411. Dharmaraju, E. and M. Laird. 1984. Transport and the spread of crop pests in tropical Polynesia. In: M. Laird (ed.), Commerce and the spread of pests and disease vectors. Prager, Eastbourne, UK. 257–272.

130

Dicke, M., R. Gols, D. Ludeking and M. A. Posthumus. 1999. Jasmonic acid and herbivory differentially induces carnivore-attracting volatiles in lima bean plants. J. of Chemical Ecology. 25:1907–1922. Diehl, J. F. 1995. Safety of Irradiated Foods. Marcel Dekker, New York. Ding, W., E. Shaaya, J. J. Wang, Z. M. Zhao and F. Goa. 2002. Acute lethal effect of two insect growth regulators on Liposcelis entomophila (Psocoptera: Liposcellididae). Zool. Res. 23: 173–176.

D'innocenzo, M and F. M. Lajolo. 2001. Effect of gamma irradiation on softening changes and enzyme activities during ripening of papaya fruit. J. of Food Biochemistry. 25 (5):425-438. Dixit, V., V. Pandey and R. Shyam. 2001. Differential antioxidative response to cadmium in roots and leaves of pea (Pisum sativum l. cv. Azad). J. of Exp. Botany. 52: 1101– 1109. Domarco, R. E., M. H. F. Spoto, L. Blumer and J. M. M. Walder. 1999. Synergy of ionizing and of heating on the shelf life of the “Italia” grape. Scientia Agricola. 46: 981–986. Doyle, J. J. and J. L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19: 11–15.

Dzhashi, V. S. 1989. Results of study of the destructive shield bug. Subtropicheskie kul’tury. 3: 134–143.

Elbert, A., H. Overbeck and K. T. Iwaya. 1990. In: Imidacloprid, a novel systemic nitromethylene analogue insecticide for the crop protection. Brighton Crop Protection Conference-Pests and Disease, Brighton, England. Surrey: British Crop Protection Council. El-Deeb, M. F. 2004. Field toxicity and biochemical assessment of IGRs, Kz oil and their mixtures on the soft scale insect Icerya seychellurum Westwood (Homoptera: Margarodidae) attacking guava trees. J. of Adv. Agric. Res. (Fac.Agric. Saba Basha). 9: 389–400. El-Kifl, A. H., H. S. Salama and M. K. Hamdy. 1980. Chemical control of scale insects infesting fig trees in Egypt. J. Fac. of Agric., Al-Azhar Univ., Egypt. Pp. 609–615.

131

Elwan, E. A. 1990. Ecological and biological studies on certain insect pests of Coccoidea (Homoptera) infesting mango trees. Ph.D. Thesis, Fac. of Agric., Al-Azhar Univ., Egypt, 175 pp.

Elwan, E. A. H. 2005. Population dynamics of Maskell scale, Insulaspis pallidula Grees (Homoptera: Diaspididae), on mango trees in Egypt. Egyptian J. of Agri. Res. 83 (3): 1199–1212. Enan, M. R. 2006. Application of random amplified polymorphic DNA (RAPD) to detect the genotoxic effect of heavy metals. Biotechnol. Appl. Biochem. 43: 147– 154. Expert Consultation Meeting On Postharvest and Value Addition of Horticultural Produce. 2010. GFAR/APARI Date: 29, November – 2, December 2010 http://www.apcoab.org/uploads/ExCon_PHTHort.pdf FAO (1979). Preliminary report on the Review Agriculture Towards 2000. Fallik, E., Y. Aharoni, A. Copel, V. Rodov, S. Tuvia-Alkalai, B. Horev, O. Yekutieli, A. Wiseblum and R. Regev. 2000. Reduction of postharvest losses of Galia melon by a short hot-water rinse. Plant Pathol. 49: 333–338. Fanizza, G., I. Ricciardi and C. Bagnulo. 1991. Leaf greenness measurements to evaluate water stressed genotypes in vitis vififere. Euphytica. 55: 27–31. FAO. 1981. Food loss prevention in perishable crops. FAO, Agricultural Services Bulletin No. 43. FAO. 2013. Food and Agriculture Organization of the United Nations, FAOSTAT- Production: Countries by Commodity, Crop. http://faostat3.fao.org/home/index.html#DOWNLOAD (17th February, 2015). Farmer, E. E., E. Almeras and V. Krishnamurthy. 2003. Curr. Opin. Plant Biol. 6: 372–378. Farooqi, W. A., M. Ahmad, A. M. Hussain and Qureshi, M. J. 1975. Effects of thiabendazol and kininig materials on kinnow mandrins during storage. The Nucleus. 12: 25–29.

Fasulo, T. R. and R. F. Brooks. 2010. Scale pest of Florida citrus. EDIS. (19 May 2013.) Federal Register. 2006. Treatments for fruits and vegetables. Rules and regulations 71: 4451– 4464. Felton, G. W., K. Donato, R. J. Del Vecchio and S. S. Duffey. 1989. Activation of plant foliar oxidases by insect feeding reduces nutritive quality of foliage for noctuid herbivores. J. of Chem. Ecol. 15: 2667– 2694.

132

Ferris, G. F. 1938. Atlas of the scale insects of North America. Series 2. Stanford University Press, Palo Alto, California. Follet, P. A. 2006. Irradiation as a phytosanitary treatment for white peach scale (Homoptera: Diaspididae). J. of Econ. Entomol. 99 (6): 1974–1978.

Follet, P. A. 2009. Generic radiation quarantine treatments: the next steps. J. of Econ. Entomol. 101: 710–715.

Follet, P. A. and J. W. Armstrong. 2004. Revised irradiation doses to control melon fly, Mediterranean fruit fly, and Oriental fruit fly (Diptera: Tephritidae) and a generic dose for tephritid fruit flies. J. of Econ. Entomol. 97: 1254–1262.

Follet, P.A. 2006a. Irradiation as a Phytosanitary treeatments for Aspidiotus destructor (Homoptera: Diaspididae), commodity treatment and quarantine Entomolgy. J. of Econ. Entomol. 99 (4): 1138–1142. Follett, P. A. 2006b. Irradiation as a Phytosanitary Treatment for White Peach Scale (Homoptera: Diaspididae). J. of Econ. Entomol. 99: 1974–1978. Follett, P. A. 2007. Postharvest phytosanitary radiation treatments: less–than–probit 9, generic dose, and high dose applications, in: Area–Wide Controlof Insect Pests, pp. 425–433. Follett, P. A. and E. D. Weinert. 2012. Phytosanitary irradiation of fresh tropical commodities in Hawaii: Generic treatments, commercial adoption, and current issues. Radiat. Phys. Chem. 81: 1064–1067. Follett, P. and L. Neven. 2006. Current trends in quarantine entomology. Annual Review of Entomology. 51: 359. Food and Agricultural Organization (FAO). 2009. Phytosanitary Treatments for Regulated Pests. ISPM# 28, Food and Agricultural Organization, Rome, Italy. Food and Agriculture Organization. 2010. FAOSTAT database collections, agricultural data, Food and Agriculture Organization of the United Nations. Available from http://faostat.fao.org/faostat/collections?subset=agriculture. Foster, L. D., F. L. Robret and E. G. Elizabeth. 1995. Life stages of California red scale and its parasitoids. Univ. of California Division of Agric. and Natural Resources Publication # 21529.

133

Foyer, C. H., M. Lelandais and K. J. Kunert. 1994. Photooxidative stress in plants. Physiol. Plant. 92: 696–717. Friedberg, E. C., G. C. Walker, W. Siede. 1995. DNA repair and mutagenesis. ASM Press, Washington, DC. Furness, G. O., G. A. Buchanan, R. S. George and N. L. Richardson. 1983. A history of the biological and integrated control of red scale, Aonidiella aurantii on citrus in the Lower Murray Valley of Australia. Entomophaga. 28: 199–212.

Gade, G. and L. Auerswald. 2002. Beetles’ choice-proline for energy output: control by AKHs. Comp. Biochem. Physiol. 132:117–129.

Galán Saúco, V. 2002. Published. Mango production and world market: Current situation and future prospects, pp. 107–116. In, VII International Mango Symposium 645. Galán Saúco, V. 2010. Worldwide mango production and market. Current situation and future prospects. In: international mango symposium, 9, 2010. Sanya, Proceeding. Sanya: International Soc. for Horticultural Science. Gao, L. L., J. P. Klingler, J. P. Anderson, O. R. Edwards and K. B. Singh. 2008. Characterization of pea aphid resistance in Medicago truncatula. Plant Physiol. 146: 996–1009.

Gaprindashvili, N. K. 1975. Biological control of the main pests on tea plantations in the Georgian SSR. VIII International Plant protection conference, Moscow. 29–33.

Garcia, A. B., J. D. A. Engler, S. Iyer, T. Gerats, M. Van Montagu and A. B. Caplan. 2007. Effects of osmoprotectants upon NaCl stress in rice. Plant Physiology. 115:159–169.

Gatehouse, J. A. 2002. New Phytol. 156: 145–169.

George, R. 1984. Commercial production of parasites for control of red scale, Aonidiella aurantii Mask. in southern Australia. In: Bailey P, Swincer, D. (eds), Proceedings of the Fourth Australian Applied Entomol. Res. Conference, Adelaide, South Australia, 24–28 September 1984. pp. 110–113.

Gerbaud, P. 2009. Close-up mango. Fruit Crop, Paris. 164: pp. 7–39. Germain, J. F., J. F. Vayssieres and G. Matile-Ferrero. 2010. Preliminary inventory of scale insects on mango trees in Benin. Entomologia Hellenica. 19:124–131. 134

Ghabbour, M. W. 1995. Descriptions of the first instar of the Egyptian species in the Genus Aonidiella (Homoptera: Coccoidea: Diaspididae). Egypt. J. of Agric. Res. 73 (2): 379–387. Ghabbour, M. W. and Z. K. Mohammad. 1998. Descriptions of the second immature instar and adult male Aonidiella orientalis Newstead (Homoptera: Diaspididae). Bull. Entomol. Soc. Egypt. 76 (87): 87–97. Ghauri, M. 1962. The morphology and taxonomy of male scale insects (Hemiptera: Coccoidea). British Museum (Natural History). Adlard and Son, Dorking, UK. 221 pp.

Gieselmann, M. J. 1990. Pheromones and mating behavior. In: D. Rosen, (ed.) Armored scales insects, their biology, natural enemies and control. World Crop Pests. Elsevier, Amsterdam, the Netherlands. 4 (A): 221–224.

Glosser, M. 1989. Use of irradiation as a quarantine treatment for fresh fruits of papaya from Hawaii. Fed. Reg. 54: 387–393. Goberdhab, L. C.1962. Scale insects of the coconut palm with special reference to Aspidiotus destructor. J. of the Agric. Soc. of Trinidad and Tobago. 62: 49–70.

Golan, K., K. Rubinowska, K. Kmiec, I. Kot, E. Gorska-Drabik, B. Lagowska and W. Michalek. 2014. Impact of scale insect infestation on the content of photosynthetic pigments and chlorophyll fluorescene in two host plant species. Arthropod–Plant interaction. 9: 55–65.

Golawska, S., R. Krzyzanowski and I. Lukasik. 2010. Relationship between aphid infestation and chlorophyll contents in fabaceae species. Acta Biol. Cracoviensia. 52: 76–80.

Gomez, K. A. and A. A. Gomez. 1984. Statistical Procedures for Agriculture Research, John Wiley and sons, New York, USA, 1984.

Gomez, K. S., D. M. Oosterhuis, S. N. Rajguru, D. R. Johnson. 2004. Molecular biology and physiology. Foliar antioxidant enzyme responses in cotton after aphid herbivory. J. Cotton Sci. 8: 99–104.

135

González-Aguilar, G. A., J. G. Buta, C. Y. Wang. 2001. Methyl jasmonate reduces chilling injury symptoms and enhances color development of ‘Kent’ mangoes. J. of Sci. Food Agric. 81: 1244–1249. Gratao, P. L., A. Polle, P. J. Lea and R. A. Azevedo. 2005. Making the life of heavy metal stressed plants a little easier. Funct. Plant Biol. 32: 481–494. Greathead, D. J. 1990. Crawler behaviour and dispersal. In: D. Rosen (ed), Armored Scales, their biology, natural enemies and control. Vol. 4A. World Crop Pests. Elsevier, Amsterdam, the Netherlands. 4 (A): pp. 305–308.

Gullan, P. J. and L. G. Cook. 2007. Phylogeny and higher classification of the scale insects (Hemiptera: Sternorrhyncha: Coccoidea). Zootaxa. 1168: 413– 425. Gupta, B. P. and Y. P. Singh. 1988. Mango scale insects occurrence in western Uttar Pradesh and their control. Progressive Horticulture. 20 (3–4): 357–361. Halliwell, B. and J. M. C. Gutteridge. 1999. Free radicals in biology and medicine, 3rd edn, Oxford University Press. Hallman, G. J. 2001a. Irradiation as a quarantine treatment. In: Food Irradiation: Principles and Applications. R. A. Molins, ed., Wiley Interscience, New York. pp. 113–130. Hallman, G. J. 2011. Phytosanitary applications of irradiation, Compr. Review Food Sci. and Food Saf. 10 (2): 143–151. Hallman, G. J. 2011. Phytosanitary Applications of Irradiation. Compr. Review Food Sci. and Food Saf. 10: 143–151. Hallman, G. J. 2012. Generic phytosanitary irradiation treatments. Radiat. Phys. Chem. 81: 861–866. Hallman, G. J. and L. R. Martinez. 2001. Ionizing irradiation quarantine treatment against Mexican fruit fly (Diptera: Tephritidae) in citrus fruits. Post–harvest Biol. Technol. 23 (1): 71–77. Hallman, G. J. and R. L. Hellmich. 2009. Ionizing radiation as a phytosanitary treatment against european corn borer (Lepidoptera: Crambidae) in ambient, low oxygen, and cold conditions. J. of Econ. Entomol. 102: 64–68. Hallman, G. J., N. M. Levang–Brilz, J. L. Zettler and I. C. Winborne. 2010. Factors affecting ionizing radiation phytosanitary treatments, and implications for research and generic treatments. J. of Econ. Entomol. 103: 1950–1963.

136

Hammad, A. A. I., T. M. E. Mongy, M. R. A. Shady and S. M. Taha. 1996. Microbial changes in strawberries treated with gamma irradiation to improve their quality. Egyptian J. of Food Science. 23: 117–132. Handa, S. and K. K. Dahiya. 1999. Chemical management of mango scales. Agric. Sci. Degest, (Karnal). 19 (2): 112–114. Hara, A. H., Yalemar, J. A., Jang, E. B. and Moy, J. H. 2002. Irradiation as a possible quarantine treatment for green scale Coccus viridis Green (Homoptera: Coccidae). Postharvest Biol. Technol. 25: 349–358. Harborne, J. B. 1989. Methods in plant biochemistry, Vol. 1 plant phenolics, Dey, P. M. and Harborne, J. B. (Eds.), Academics press, London, 1.

Hardy, N. B. 2013. The status and future of scale insects (Coccoidea) systematics. Systematic Entomology. 38 (3): 453–458.

Hare, P. D. and W. A. Cress. 1997. Metabolic implications of stress–induced proline accumulation in plants. Plant Growth Regul. 21: 79–102. Hattingh, V. and B. Tata. 1995. Effect of field-weathered residues of insects growth regulators on some coccinellidae (Colleoptera) of economic importance as biological agents. Bull. Entomol. Res. 85: 489–493. Haunt, T. I., D. A. Reed, T. M. Perring and T. M. Palumbo. 2014. Feeding damage by Bagrada hilaris (Hemiptera: Pentatomidae) and impact on growth and chlorophyll contents of Brassicaceous plant species. Arthropod-Plant Interact. Heather, N. H. and G. J. Hallman. 2008. Pest management and phytosanitary trade barriers. CAB International, Oxford shire, UK. pp. 132–152. Hely, P. C., G. Pasfield and J. G. Gellatley. 1982. Insect pests of fruit and vegetable in NSW (New South Wales). Inkata Press, Melbourne. Heng-Moss, T. M., X. Ni, T. Macedo, J. P. Markwell, F. P. Baxendale, S. S. Quisenberry and V. Tolmay. 2003. Comparison of chlorophyll and carotenoid concentration among Russian wheat aphid (Homoptera: Aphididae) infested wheat isolines. Plant resistance. 96: 475–481.

Heu, R. A. (ed.). 2002. Distribution and host records of agricultural pests and other organisms in Hawaii. State of Hawaii Department of Agriculture, USA. 70 pp.

137

Higbee, B. S., D. R. Horton and J. L. Krysan. 1995. Reduction of egg hatch in pear psylla (Homoptera: Psyllidae) after contact by adults with insect growth regulators. J. of Econ. Entomol. 88: 1420–1424.

Hix, R., C. D. Pleass, D. E. Deyton and C. E. Sams. 1999. Management of San Jose scale on apple with soybean-oil dormant sprays. Hort. Science. 34 (1): 106–108.

Horowitz, A. R., Z. Mendelson, P. G. Weintraub and I. Ishaaya. 1998. Comparative toxicity of foliar systemic applications of two chloronicotinyl insecticides, acetamiprid and imidacloprid, against the cotton whitefly, Bemisia tabaci. Bull. Entomol. Res. 88: 437–442. Howard, F. W., D. Moore, R. M. Giblin-Davis and R. Abad. 2001. Insects on Palms. CAB Publishing, Wallingford, UK. 400 pp. Howe. G. A. and G. Jander. 2008. Plant immunity to insect herbivores. Annual Review of Plant Biol. 59: 41–66. Hunter, W. D. 1912. Results of experiments to determine the effect of Roentgen rays upon insects. J. of Econ. Entomol. 5: 188–193. Hussain, S. S., H. Raza and M. A. Kayani. 2010. Transgenic plants as vital approach for enhanced drought tolerance: Potentials and perspective. Arch. Agron. Soil Sci. in Press. Ibrahim, F. A. 1990. Morphological effects of mineral oils used to control scale insects on citrus trees. M. Sc. Thesis, Fac. of Agric., Cairo Univ., Egypt. 127 pp. International Plant Protection Convention (IPPC). 2010. Draft appendix to ISPM 15:2009. Submission of new treatments for inclusion in ISPM 15. Food and Agriculture Organization of the United Nations, Rome, Italy Ishaaya, I. and A. R. Horowitz. 1992. Novel phenoxy juvenile hormone analog (pyriproxyfen) suppresses embryogenesis and adult emergence of sweet potato whitefly (Homoptera: Aleyrodidae). J. of Econ. Entomol. 85: 2113–2117.

Jacobi, K. K., E. A. Macrae and S. E. Hetherington. 1998. Early detection of abnormal skin ripening characteristics of ‘Kensington’ mango (Mangifera indica Linn). Sci. Hortic. 72: 215–225.

Jactal, H., N. Perthuisot, P. Menassieu, G. Raise and C. Burban. 1996. A sampling design for within–tree larval population of maritime pine scale, Matsucoccus feytaudi Duc. 138

(Homoptera: Margarodidae), and their relationship between larval population estimates and male catch in pheromone traps. Canada Entomol. 128: 1143–1156.

Jalaluddin, M. and M. Mohanasundaram. 1989. Control of the coconut scale Aspidiotus destructor Sign. In the nursery. Entomol. 14: 3–4.

Jalaluddin, S. M., M. Mohanasundaram and P. C. Sundarababu. 1992. Influence of weather on fecundity potential of Aspidiotus destructor Signoret. Indian Coconut Journal. 22 (12): 5–7. Jiang, D. A. 1999. Experimental guide for plant physiology, Chendu Science and Technological University Publisher, Chendu China. pp. 39–42. (In Chinese)

Juárez-Hernández, P., J. Valdez-Carrasco, G. Valdovinos-Ponce, J. A. Mora-Aguilera, G. Otero-Colina, D. Téliz-Ortiz, E. Hernández-Castro, I. Ramírez-Ramírez and V. A. González-Hernández. 2014. Leaf penetration pattern of Aulacaspis tubercularis (Hemiptera: Diaspididae) stylet in mango. Florida Entomologist. 97 (1): 100–107. Kader, A. A. 1986. Potential applications of ionizing radiation in postharvest handling of fresh fruits and vegetables. Food Technol. 40 (6): 117–121. Kader, A. A. 2002. Postharvest Technology of Horticultural Crops. University of California Agriculture and Natural Resources Communications Services, USA, 535 pp. Kader, M. D. A. and S. Lindberg. 2010. Cytosolic calcium and pH signaling in plants under salinity stress. Plant Signaling and Behavior. 5 (3): 233–238. Kamel, A., S. Abd-Rabou, N. Hilmy, S. Allam and M. Moustafa. 2003. Seasonal abundance of certain Aphytis species (Hymenoptera: Aphelinidae) from Egypt. Egypt. J. of Agric. Res. 81 (3): 1009–1023.

Karban, R. and I. T. Baldwin. 1997. “Mechanisms of induced responses.” in Induced responses to herbivory., ed. R. Karban and I. T. Baldwin, University of Chicago Press, Chicago. pp 58–90. Kashiwagi, A., K. Kashiwagi, M. Takase, H. Hanada and M. Nakamura. 1997. Comparison of catalase in diploid and haploid Rana rugosa using heat and chemical inactivation techniques. Comp. Biochem. Physiol. Part B. 118: 499–503. Kekec, G., M. S. Sakcali and I. Uzonur. 2010. Assessment of genotoxic effects of boron on Wheat (Triticum aestivum L.) and bean (Phaseolus vulgaris L.) by using RAPD analysis. Bull. Environ. Contam. Toxicol., 84: 759–764. 139

Kessler, A. and I. T. Baldwin. 2002. Annu. Rev. Plant Biol. 53: 299–328. Khalil, S. A., S. Hussain and M. Khan. 2009. Effects of gamma irradiation on quality of Pakistani blood red oranges (Citrus sinensis L. Osbeck), Int. J. of Food Sci. Technol. 44 (5): 927–931. Khan, I., M. Zahid, K. Alamzeb and F. Mahmood. 2014. Effect of gamma irradiation on the mortality and growth inhibition of citrus scale Aonidiella aurantii Mask (Hemiptera: Diaspdidiae). Fla. Entomol. Khan, N. A. and S. Singh. 2008. A biotic stress and Plant responses, IK International, New Dehli.

Khan, R. U., S. R. Afridi, M. Ilyas, H. Abid, M. Sohail and S. A. Khan. 2012. Effect of different chemical preservatives on the storage stability of mango–sea buckthorn blended juice. Pak. J. of Biochem. Mol. Biol. 45 (1): 6–10. Kinawy, M. M. 1991. Biological control of the coconut scale insect, Aspidiotus destructor Sign. (Homoptera: Diaspididae) in the southern region of Oman (Dhofar). Tropical Pest Management. 37 (4): 387–389. Kitagawa, H., T. Kubo and T. Kusaragi. 1980. Retail trade in Florida grape fruit in Japan. Proceedings of the Florida State Horticultural Soc. 93: 327–331. Knievel, D. 1973. Procedures for estimating ratio of live or dry matter in root samples. Crop Sci. 13:124–126. Koenig, J., D. Lawson, N. Ngo, B. Minton, C. Ishida, K. Lovelace and S. Moore. 1999. Field trial results with pymetrozine (fulfill) and thiamethoxam (Centric/Actara) for the control of cotton aphid (Aphis gossypii). Proceeding, San Antonio, Taxas., Memphis, Tenn. National Cotton Council of America. Koidsumi, K. 1930. Quarantine studies on the lethal action of X-rays upon certain insects (Japan). J. of Soc. Tropical Agric. 2: 243–263. Kondo, T. and S. Kawai. 1995 Scale insects (Homoptera: Coccoidea) on mango in Colombia. Japan J. of Tropical Agriculture. 39: 57−58. Extra Issue 1. Tokyo University of Agriculture. Tokyo, Japan Paper No.29.

Kondo, T., P. J. Gullan and D. J. Williams. 2008. Coccidology. The study of scale insects (Hemiptera: Sternorrhymcha: Coccoidea). Corpoica. 9 (2).

140

Koteja, J. 1990. Paleontology. In: Armored scale insects: Their biology, natural enemies and control. D. Rosen, (ed.) Amsterdam., Elsvier. 4 (A): 149–163.

Koteja, J. 1990b. Life history. In: D. Rosen (ed.), Armored scale insects, their biology, natural enemies and control. World Crop Pests. Elsevier, Amsterdam, the Netherlands. 4 (A): 243–254.

Koteja, J. 1990a. Embryonic development; ovipary and vivipary. In: D. Rosen (ed.), Armored scale insects, their biology, natural enemies and control. World Crop Pests. Elsevier, Amsterdam, the Netherlands. 4 (A): 233–242.

Krishnamurthy, S. 1988. Effects of tal-prolonged on shelf-life and quality attributes of mango. Acta Hort. 213: 675–678. Kumari, D. A., V. Anitha, B. K. M. Lakshmi. 2014. Evaluation of insecticides for the management of scale insects in mango (Mangifera indica). Internat. J. of Plant Protec. 7 (1): 64–66.

Lagadec, M. D. L. and C. Ueckemann. 2002. Laboratory screening of chemicals for the control of mango scales. SAMGA-Mango Navorsings joernaal. pp. 93–97.

Lai, P. Y. and G. Y. Funasaki. 1983. List of beneficial organisms purposely introduced for biological control in Hawaii 1890 to 1982. Honolulu, Hawaii Dept. Agri., Division of Plant Industry, Plant Pest Control Branch. Lainga, W. G., G. A. C. Beattie, A. W. Meats, R. Spooner-Hart and L. Jiang. 1999. Efficacy of horticultural mineral oil for the control of purple scale and white louse scale in orange orchards. Spray oils Beyon 2000. Sustainable Pest and Disease Management Conference Sydney, October, 1999.

Larmond, E. 1977. Method of sensory evaluation of food. Canada deoartment of Agriculture, Publication no. 1284, Ottawa, Canada. Pp. 36–37. Larrigaudiere, C., J. Pons, R. Torres and J. Usall. 2002. Storage performance of clementines treated with hot water, sodium carbonate and sodium bicarbonate dips. J. of Hort. Sci. Biotechnol. 77: 314–319. Lawson, D., D. Dunbar, S. White and N. Ngo. 1999. Actara 25 WG: control of cotton pest with a new neonicotinoid insecticide, thiamethoxam. In: proceedings. Lever, R. J. A. W. 1969. Pest of the coconut palm. FAO Agricultural Studies, Series No: 77. 141

Lichtenthaler, H. K. and A. R. Wellburn. 1983. Determination of total carotenoids and chlorophylls a and b of leaves in different solvents. Biological Soc. Transactions. 11: 591–592.

Liu, W., P. J. Li, X. M. Qi, Q. X. Zhou, L. Zheng, T. H. Sun and Y. S. Yang. 2005. DNA changes in barley (Hordeum vulgare) seedlings induced by cadmium pollution RAPD analysis. Chemosphere. 61: 158–167. Liu, W., Y. S. Yang, Q. Zhou, L. Xie, P. Li and T. Sun. 2007. Impact assessment of cadmium contamination on rice (Oryza sativa L.) seedlings at molecular and population levels using multiple biomarkers. Chemosphere. 67: 1155–1163. Liu, Y. Q., L. Jiang, L. H. Sun, C. M. Wang, H. Q. Zhai and J. M. Wan. 2005. Changes in some defensive enzyme activity induced by the piercing–sucking of brown plant hopper in rice. J. of Plant Physiol. Mol. Biol. 31 (6): 643–650. Lu, S., Z. Wang, Y. Niu, Z. Guo and B. Huang. 2008. Antioxidant responses of radiation induced dwarf mutants of Bermuda grass to drought stress. J. of American Soc. Horticult. Sci. 133: 360–366. Maffei, M. E., A. Mitho’fer, G. I. Arimura, H. Uchtenhagen, S. Bossi, C. M. Bertea, L. S. Cucuzza, M. Novero, V. Volpe, S. Quadro and W. Boland. 2006. Effects of feeding Spodoptera littoralis on Lima bean leaves. III. Membrane depolarization and involvement of hydrogen peroxide. Plant Physiol. 140: 1022–1035.

Maffei, M. E., A. Mithöfer and W. Boland. 2007. Insects feeding on plants: Rapid signals and responses preceding the induction of phytochemical release. Phytochemistry. 68: 2946–2959.

Mahajan, S. and N. Tuteja. 2005. Cold, salinity and drought stresses: an overview. Arch. Biochem. Biophys. 444:139–158. Mahayothee, B., M. Leitenberger, S. Neidhart, W. Muhlbauer and R. Carke. 2002. Non- destructive determination of fruit maturity of Thai mango cultivars by near infrared spectroscopy. In International Symposium Sustaining Food Security and Managing Natural Resources in Southeast Asia – Challenges for the 21st Century, (ed.) Proceedings of the International Symparium, Chiang Mai, Thailand, January 8–11: 1995.

142

Mahmood, T. 1972. Use of Co– 60 gamma irradiation against some postharvest decay pathogens of citrus fruit in Turkey. Pakistan J. of Sci. and Industrial Res., 15: 268– 271. Mahrouz, M., M. Lacroix, G. D’Aprano, H. Oufedjikh, C. Boubekri and M. Gagnon. 2002. Effect of gamma-irradiation combined with washing and waxing treatment on physicochemical properties, vitamin C, and organoleptic quality of citrus Clementina Hort. ex. Tanaka. J. of Agric. and Food Chemistry. 50: 7271–7276.

Maienfisch, P., L. Gsell and A. Rindlisbacher. 1999. Synthesis and insecticidal activity of CGA 293’343 a novel broad spectrum insecticide. Pesticide Sci. 55 (3): 351–355.

Malr, P. 1997. Agricultural pest control program. Annual book of Ministry of Agric. and Land Reclamation, Egypt. pp. 5–40. Mangoud, A. A. H. 2008. Insecticidal effect of some chemical and natural control agents against the black parlatoria scale, Parlatoria ziziphi Lucas and associated parasitoids on citrus trees. Egypt. J. of Agric. Res. 86 (6): 2157–2167. Mansour, M. and J. Al-Attar. 2012. Effects of gamma irradiation on the grape vine moth, Lobesia botrana eggs. Radiat. Phys. Chem. 81: 1776–1780. Mariau, D. 2001. The fauna of oil palm and coconut. Insect and mite pests and their natural enemies. Translated from the French by Peter Biggins. Centre de coopération internationaleen recherche agronomique pour le développement (CIRAD). Pp 89–90. Mariau, D. and J. F. Julia. 1977. Nouvelle recherches sur la cochenille du cocotier Aspidiotus destructor Signoret. Oléagineux. 32 (5): 217–224. Martins, N., I. Lopes, A. Brehm and R. Ribeiro. 2005. Cytochrome B gene partial sequence and RAPD analysis of two Daphnia longispina lineages differing in their resistance to copper. Bull. Environ. Contam. Toxicol. 74: 755–760. Mau, R. F. L. and J. L. M. Kessing. 1992. Aspidiotus destructor Signoret. Crop knowledge master, University of Hawaii.

Mau, R. F. L. and J. L. M. Kessing. 1992. Crop knowledge master. Cocus Longulus Douglas. http://www.extento.hawaii.edu/kbase/crop/type/d_neobre.htm.

Mcguire, R.G. 2000. The response of Longan fruits to cold and gamma irradiation. J. of Hort. and Biotech. 73: 687–690.

143

McNaughton, S. J. 1983. Compensatory plant growth as a response to herbivory. Oikos. 40: 329–336. Metcalf, C. L. 1962. Scale insects in: destructive and useful insects, their habits and control. McGraw-Hill Book Company, New York, San Francisco, Toronto, London. pp. 866– 869. Meyer, S. T., F. Roces and R. Wirth. 2006. Selecting the drought stressed: effects of plant stress on intraspecific and within–plant herbivory patterns of the leaf–cutting ant Atta colombica. Funct. Ecol. 20: 973–981. Miller, C. E. and L. W. Chang. 1998. Inspection requirements for green bananas from Hawaii. Risk Analysis Systems, Policy and Program Development, Animal and Plant Health Inspection Service, U.S. Dep. Agric., Riverdale, MD. Miller, C. E. and L. W. Chang. 1998. Inspection requirements for green bananas from Hawaii. Risk Analysis Systems, Policy and Program Development, Animal and Plant Health Inspection Service, U.S. Dep. Agric., Riverdale, MD. Miller, D. R. and J. A. Davidson. 2005. Armored scale insect pests of trees and shrubs. Cornell Univ. Press, Ithaca, USA. 442 pp.

Miller, D. R., G. I. Miller and G. W. Watson. 2002. Invasive species of mealybugs (Hemipteran: Pseudococcidae) and their threat to U. S. agriculture. Proceeding of the Entomol. Soc. of Washington. 104 (4): 825–836.

Miller, G. L. and D. R. Miller. 2003. Invasive soft scales (Hemiptera: Coccidae) and their threat to U. S. Agriculture. Proceeding of the Entomol. Soc. of Washington. 105: 832–846.

Min, B.Y. and S. L. Or. 1977. Studies on the CA storage of sweet persimmon in polyethylene film pack. Korean J. 9: 96. Mithofer. A., S. Birgit and B. Wilhelm. 2004. Biotic and heavy metal stress response in plants: evidence for common signals. Federation of European Biochemical societies (FEBS) Letters. 566: 1–5.

Mittler, R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science. 7: 405–410.

144

Mittler, R. and B. A. Zilinskas. 1994. Regulation of pea cytosolic ascorbate peroxidase and other antioxidant enzymes during the progression of drought stress and following recovery from drought. Plant J. 5: 397–405. Moffit, L. J. 1999. Economic risk to United States agriculture of pink hibiscus. European and Mediterranean Plant Protection Organization, 2005. http.//www. eppo.org/ Quarantine/ insects/Ds_Maconellicoccus_hirsutus. Mohamed, A. A. 2002. Integrated control of scale insects on certain fruit trees. Ph. D. Thesis, Fac. of Agric., Al-Azhar University, pp. 173. Mohamed, A. A. 2010. Integrated control of scale insects on certain fruit trees. Ph. D. Thesis, Fac. of Agric., Al-Azhar University. 173 pp. Moharum, F. A. 2006. Ecological and morphological studies of white peach scale Pseudaulacaspis pentagona Targioni–Tozzetti and its natural enemies. Ph.D Thesis, Fac. of Agric., Moshtohor Benha University. 192 pp.

Mohyuddin, A. I. 1981. A review of biological control of insect and weed pests in Pakistan. Proc. 2nd Pakistan congr. Zool. Tandojam, 26–27, December, 1981: 31–79.

Mohyuddin, A. I. and R. Mahmood. 1993. Integrated control of mango pests in Pakistan. Acta Horticulture. 341: 467–483.

Moiler, H. 1996. Lessons for invasion theory from social insects. Biol. control. 78:125–42

Moreno, D. S. 1972. Location of the site of production of the sex pheromone in the yellow scale and the California red scale. Annals of the Entomol. Soc. of America. 65: 1283– 1286.

Moreno, M., M. Elena Castell-Perez*, C. Gomes, P. F. Da Silva and R. G. Moreira. 2006. Effects of Electron Beam Irradiation on Physical, Textural, and Microstructural Properties of “Tommy Atkins” Mangoes (Mangifera indica L.). J. of Food Science. 71 (2): 80-86.

Morse, J. G. and O. L. Browner. 1986. Toxicity of pesticides to Scirtothrips citri (Thysanoptera: Thripidae) and implications to resistance management. J. of Econ. Entomol. 79: 565–570.

145

Mosquera, F. 1977. Estudio preliminary sobre las escamas protegidas (Homoptera: Diaspididae) en cocotero en las Isla de San Andres y su Posible control. Revista Colombiana de Entomologia. 3: 7–16.

Moutia, L. A. and R. Mamet. 1946. Review of twenty–five years of economic entomology in Mauritius. Bulletin of Entomol. Res. 36: 439–472. Moy, J. H. 1985. “Radiation disinfestation of food and agriculture products.” Univ. of Hawaii, Honolulu.

Mullins, J. W. 1993. Imidacloprid. A new nitroguanidine insecticide. ACS symposium series–American Chemical Soc. 524: 183–198.

Mumtaz, A., W. A. Farooqi and M. Amir. 1969. Preservation of mangoes (Magnifera indica L.) by gamma radiation. Food Irrad. 9: 8–13. Munns, R. 2006. Genes and salt tolerance: bringing them together. New Phytol. 167:645– 663. Murakami, Y. 1970. A review of biology and ecology of diaspine scales in Japan (Homoptera: Diaspididae). Mushi. 43 (Pt. 7): 65–114. Nabil, H. A. 2013. Relationship between Kilifia acuminate Signoret and chlorophyll percentage loss on mango leaves. J. of Entomology. 10 (2): 110–114. Nabil, H. A., A. A. Shahein, K. A. Hammad and A. S. Hassan. 2012. Ecological studies of Aulacaspis tubercularis (Hemiptera: Diaspididae) and its natural enemies infesting mango trees in Sharkia Governarate, Egypt. Egypt. Acad. J. Biology. Sci. 5 (3): 9–17.

Nabity, P. D., J. A. Zavala and E. H. DeLucia. 2009. Indirect suppression of photosynthesis on individual leaves by arthropod herbivory. Annals of Bot. 103: 655–663. Nada, S., S. Abd–Rabou and G. Hussein. 1990. Scale insects infesting mango trees in Egypt (Homopetra: Coccidea). Proc. Issis VI, Part Krakow II: 133 – 134. Nafus, D. and P. Schreiner. 1989. Biological control activities in the Mariana islands Pacific Ocean from 1911 to 1988. Micronesica. 22 (1): 65–106. Nagrare, V. S., S. Kranthi, V. K. Biradar, N. N. Zade, V. Sangode, G. Kakde, R. M. Shukla, D. Shivare, B. M. Khadi and K. R. Kranthi. 2009. Wide spread infestation of the

146

exotic mealybug species, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), on cotton in India. Bull Entomol. Res. 99: 537–541

Nakahara, S. 1982. Checklist of the armored scales (Homoptera: Diapididae) of the Conterminous United States. Washington, USA: USDA, Animal and Plant Health Inspection Service, Plant Protection and Quarantine. 110 pp.

Nakamura, A. 1977. Japanese relation and the transaction of its marketing. Proceedings of the International Soc. of Citriculture. 2: 347–350. Nauen, R., J. Strobel, K. Tietjen, Y. Otsu, C. Erdelen and A. Elbert. 1996. Aphicidal activity of imidacloprid against a tobacco feeding strain of Myzus persicae (Homoptera: Aphididae) from Japan closely related to Myzus nicotianae and highly resistant to carbamates and organophosphates. Bull. Entomol. Res. 68 (2): 165–171. Nawaz, R. 1971. Effect of heat on the quality of pectin in mango juice. M.Sc (Hons) Thesis, Univ. of Agric. Faisalabad, Pakistan. Nei, M. and W. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleasis. Proc. Natl, Acad. Sci., 76: 5269–5273. RAPD markers. Ecotoxicology. 13: 303–309. Nestle, D., H. Cohen, N. Saphir, M. Klein and Z. Mendel. 1995. Spatial distribution of scale insects: comparative study using Taylor’s power law. Environ. Entomol. 24: 506– 512. Nowak, R. S. and M. M. Caldwell. 1984. A test of compensatory photosynthesis in the field: implications for herbivory tolerance. Oecologia. 61: 11–318. Noyes, J. S. 2012. Universal chalcidoidea database. In: http://www.nhm.ac.uk/chalcidoids; accessed: October, 2012. Nur, U. 1990. Chromosomes sex–ratios and sex determination in armored scale insects, their biology, natural enemies and control. World crop pests. Elsevier, Amsterdam, The Netherlands. 4 (A): 179–190. Nur, U. 1990a. Parthenogenesis. In: D. Rosen (ed.), armored scale insects, their biology, natural enemies and control. World Crop Pests. Elsevier, Amsterdam, the Netherlands. 4 (A): 191–197.

O’Mahony, M., S. Y. Wong and N. Odbert. 1985. Sensory evaluation of Navel oranges treated with low doses of gamma irradiation. J. of Food Science. 50: 639–649.

147

Ohkawa, H., N. K. Ohishi and K. Yagi. 1979. Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Annals of Biochem. 95:351. Ohlsson, K. and T. A. B. Berglund. 1995. Defensive and secondary metabolism in plant tissue cultures, with special reference to nicotinamide, glutathione and oxidative stress. Plant Cell Tiss. Org. Cult. 43:137–145. Pais, M. H. 1973. Insects on banana in Mozambique. Agronomia Mocambicana. 7: 131–138. Papacek, D. and D. Smith. 1992. Integrated pest management of citrus in Queensland, Australia. Recent developments and current status. Proceedings of the Seventh International Soc. of Citriculture Congress. 8–13 March 1992, Acireale, Italy. 3: 973– 977.

Paré, P. W. and J. H. Tumlinson. 1999. Plant volatiles as a defense against insect herbivores. Plant Physiology. 121:325–331. Parida, A. K. and A. B. Das. 2005. Salt tolerance and salinity effects on plants: a review. Ecotoxicol. Environ. Saf. 60: 324. Peleg, B. 1988. Effect of a new phenoxy juvenile hormone analog on California res scale (Homoptera: Diaspididae), Florida wax scale (Homoptera: coccidae) and the ectoparasite Aphytis holoxanthus DeBache (Hymenoptera: Aphelinida). J. of Econ. Entomol. 81 (1): 88–92. Pines, B. and R. Piper. 1994. Bananas insect and mite management. Queensland Department of Primary Industries. Plant Protection Sub-department of Daklak. 2004. Annual report, Daklak, Vietnam. 20 pp. Polis, G. A. and D. R. Strong. 1996. Food web complexity and community dynamics. Am Nat. 147:813–846.

Quayle, H. J. 1911a. The red or orange scale. Bulletin 222. Berkeley, California: University of California Publications. Quednau, F. W. 1964. A contribution on the genus Aphytis Howard in South Arica (Hymenoptera: Aphelinidae). J. of Entomol. Soc. S. Africa. 27 (1): 115 p. Quezada, J. R., P. DeBach and D. Rosen. 1973. Biological and taxonomic studies of Signiphora borinquensis, new species (Hymenoptera: Signiphoridae), a primary parasite of Diaspine scales. Hilgardia. 41: 543–603. Qureshi, S. A. and S. Mohyuddin. 1982. Insect pests of mango recorded from Pakistan. Proc. Ent. Soc. Karachi. 11/12: 19–22.

148

Radwan, D. E. M. 2007. Salicylic acid alleviates growth inhibition and oxidative stress caused by zucchini yellow mosaic virus infection in Cucurbita pepo leaves. Physiol. Molecular Plant Pathol. 69 (4–6): 172–181. Rafi, M. M., R. S. Zemetra and S. S. Quisenberry. 1996. Interaction between wheat aphid (Homoptera: Aphididae) and resistant and susceptible benotypes of wheat. J. of Econ. Entomol. 89 : 239–246. Razinger, J., M. Dermastia, L. Drinovec, D. Drobne, A. Zrimec and K. J. Dolenc. 2006. Antioxidative responses of duckweed (Lemna minor L.) to short term copper exposure. Environ. Sci. and Pollution Res. Inter. 14: 194–201. Reddy, L. S. and K. R. T. Raju. 1988. Effects of prepackaging and postharvest treatments on the storage behaviour of mango fruits cv. Alphonso. Acta Hort. 231: 670–674. Rehman, K. A. and A. R. Ansari. 1941. Scale insects of the Punjab and North West Frontier Province usually mistaken for San Jose scale (with descriptions of two new species). Indian J. of Agric. Sci. 11 (5): 816–830.

Retuerto, R., B. Fernandez-Lema, R. Roiloa and J. R. Obeso. 2004. Increased photosynthetic performance in holly trees infested by scale insect. Functional Ecology. 18: 664–669. Reyne, A. 1947. Notes on the biology of Comperiella unifasciata Ishii and its host Aspidiotus destructor rigidue nov. subsp. Tijdschr. Ent. 88: 294–302.

Reyne, A., 1948. Studies on a serious outbreak of Aspidiotus destructor rigidus in the coconut palms of Sangi (North Celebes). Tihdschrift voor Entomologie. 89: 83– 120.

Risebrow, A and A. F. G. Dixon. 1987. Nutritional ecology of phloem–feeding insectsin: Slansky, F. and Rodriguez, J. F. (eds) nutritional ecology of insects, mites, spiders, and related invertibrates. John Wiley and Sons, New York. Pp. 422–448. Rong, Z. H. Y. and H. W. Yin. 2004. A method for genotoxicity detection using random amplified polymorphism DNA with Danio rerio. Ecotoxicol. Environ. Safety. 58: 96– 103. Rosen, D. (ed.). 1990a. Armored scale insects, their biology, natural enemies and control. World Crop Pests. Elsevier, Amsterdam, the Netherlands. 4: 688 pp.

149

Rosen, D. and P. DeBach. 1978. Introduced parasites and predators of arthropod pests and weeds: a world review. In: Agriculture handbook. C. P. Clausen, ed. Washington, D. c., Agric. Res. Service, US dept. of Agriculture. 480:78–128.

Rosen, D. and P. DeBach. 1979. Species of Aphytis of the World (Hymenoptera: Aphelinidae). The Hague: Dr. W Junk Publishers.

Ryan, C. A. and D. S. Moura. 2002. Systemic wound signaling in plants: a new perception. Proc. Natl. Acad. Sci. U.SA. 99: 6519– 6520. Saks, Y., P. J. Hofman and G. F. Meiburg. 1999. Potential for improvement of mango skin color during storage. Acta Hort. 485: 325–329. Salunkhe, D. K. and B. B. Desai. 1984. Postharvest biotechnology of fruit, vol.1and 2, CRC Press, Boca Raton, Florda. USA. P. 85.

Salyani, M., W. Timmer, C. W. McCoy and R. C. Littell. 1999. Petroleum spray oils in Florida citrus applications. Spray oils Beyond 2000. Sustainable Pest and Disease Management Conference Sydney, October. 1999.

Sanita, L., D. Toppi and R. Gabbrielli. 1999. Response to cadmium in higher plants. Environ. Exp. Bot. 41: 105–130.

Savva, D. 1996. DNA fingerprinting as a biomarker assay in ecotoxicology. Toxicol. Ecotoxicol. News Rev. 3: 110–114.

Savva, D. 1998. Use of DNA fingerprinting to detect genotoxic effects. Ecotoxicol. Environ. Safety. 41: 103–106.

Schaffer, B. and L. J. Mason. 1990. Effect of scale insect herbivory and shading on net gas exchange and growth of a subtropical tree species (Guaiacum sanctum L.). Oecologia. 84: 467–473. Schrazi, A. M. and P. S. Muir. 1998. In vitro effect of formaldehyde on Douglas fir pollen. Plant, Cell and Environment. 21: 341–346. Shahnawz, M., S. A. Sheikh and S. G. Khaskheli. 2012. Effect of storage on the physicochemical characteristics of the mango (Mangifera indica L.) Variety, Langra. African J. of Biotechnology. 11 (41): 9825–9828.

150

Shaw, P. W., S. J. Bradley and T. S. Walker. 2000. Efficacy and timing of insecticides for the control of San Jose scale on apple. N. Z. J. of Plant Prot. 53: 13–17.

Sheikh, L. A., S. S. Ali, A. F. M. Etheshamudin and M. Y. Haque. 1979. Preservation of mangoes with fungicidal wax emulsion. Food Sci. Technol. 2: 79. Shigeoka, S., T. Ishikawa, M. Tamoi, Y. Miyagawa, T. Takeda, Y. Yabuta and K. Yoshimura. 2002. Regulation and function of ascorbate peroxidase isoenzymes. J. of Exp. Bot. 53: 1305–1319.

Signoret, V. 1869. Essai sur les cochenilles ou Gallinsectes (Homopteres: Coccides). Annals de la Societe Entomologique de France. 9: 109–138.

Simmonds, F.J. 1960. Biological control of coconut scale (Aspidiotus destructor) in Principe, Portuguese West Africa. Bull Ent. Res. 51: 223–237.

Simmonds, H. W. 1921. The transparent Aspidiotus destructor and its enemies in Southern Pacific. Fiji Department of Agric. Bulletin. 14: 4pp. Singh, N. P., M. T. McCoy, R. R. Tice and E. L. Schneider. 1998. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175: 184–191. Singh, Z. and S. P. Singh. 2011. Mango, Wiley Online Library. Sinnathamby, S. V. 1980. Developments in the control of coconut scale, Aspidiotus destructor Sign. in Sri Lanka. Ceylon Coconut Quarterly. 28: 81–88.

Smith, D. 1978. Biological control of scale insects on citrus in southeastern Queensland, control of red scale Aonidiella aurantii Maskell. J. of the Australian Entomol. Soc. 17: 367–372.

Smith, D., G. A. C. Beattie and R. Broadley. 1997. Citrus pests and their natural enemies: Integrated pest management in Australia. Brisbane: Queensland department of primary industries. Sommers, C. H. and X. Fan. 2008. Food Irradiation Research and Technology, Wiley.

Sorribas, J., R. Rodríguez and F. Garcia–Marí. 2010. Parasitoid competitive displacement and coexistence in citrus agro–ecosystems: Linking species distribution with climate. Ecological Applications. 20: 1101–1113. 151

Stathas, G. J., P. A. Eliopopoulos and G. Japoshvili. 2007. A study on the biology of the diaspidid scale Parlatoria zzizphi Lucas (Hemiptera: Coccoidea: Diaspididae) in Greece. Proceeding of the XI International Symposium on Scale Insect Studies. 24–27 September, 2007. Oeiras, (Portugal), ISA Press, Lisbon, 322 p.

Steponkus, P. L. and F. O. Lanphear. 1967. Refinement of the triphenyl tetrazolium chloride method of determining cold injury. Plant Physiology. 42: 1423–1426. Stotz, H. U., J. Kroymann and T. Mitchell–Olds. 1999. Plant–insect interactions. Current opinion in Plant Biology. 2: 268–272. Stratmann, W. J. 2003. Long distance run in the wound response jasmonic acid is pulling ahead. Trends in Plant Science. 8 (6): 247–250. Sulpice, R., Y. Gibon, A. Bouchereau and F. Larher. 1998. Exogenously supplied glycine betaine in spinach and rapeseed leaf discs: compatibility or non–compatibility. Plant, Cell and Environ. 21: 1285–1292. Suomi, D. A. 1996. Scale insects on ornamentals. Cooperative Extension, College of Agriculture and Home Economics, Washington State University. Tabibullah, M. and B. F. Gabriel. 1975. Biological study of Aspidiotus destructor Signoret in different coconut varieties and other host plants. Philippine Entomol. 2 (6): 409–426. Tang, S. J. and H. Z. Qin. 1991. Study on Temnaspidiotus destructor Signoret. J. of Shanghai Agric. College. 9 (3): 190–196.

Tao, C. 1999. List of Coccoidea (Homoptera) of China. Taichung, Taiwan: Taiwan Agric. Res. Inst., Wufeng. 1–176.

Tauili’ili, P., A. M. Vargo. 1993. History of biological control in American Samoa, Micronesica: 4: 57–60.

Taylor, T. H. C. 1935. The campaign against Aspidiotus destructor, Signoret in Fiji. Bull. Entomol. Res. 26: 1–102.

Taylor, T. H. C. and R. W. Paine. 1935. The campaign against Aspidiotus destructor in Fiji. Bulletin of Entomol. Res. 26: 1–102. Tenorio, J. A. 1969. Notes and exhibitions. Proceeding of the Hawaiian Entomol. Soc. 20: 279.

152

Tenorio, J. A. 1969. Notes and exhibitions. Proceeding of the Hawaiian Entomol. Soc. 20: 279. Thaler, J. S., M. A. Farag, P. W. Pare and M. Dicke. 2002. Jasmonate–deficient plants have reduced direct and indirect defenses against herbivores. Ecology Letters. 5: 764–774. Tharanathan, R., H. Yashoda and T. Prabha. 2006. Mango (Mangifera indica L.),“The king of fruits”. An overview. Food Rev. Int. 22: 95–123. The, D. T., N. T. Khan, V. T. K. Lang C. C. Van, T. T. T. An and N. H. H. Thi. 2012. Effects of gamma irradiation on different stages of mealybug Dysmicoccus neobrevipes (Hemiptera: Pseudococcidae). Radiat. Phys. Chem. 81: 97–100.

Theodorakis, C. W., J. W. Bickham, T. Lamb, P. A. Medica and T. B. Lyne. 2001. Integration of genotoxicity and population genetic analyses in kangaroo rats (Dipodimys merriami) exposed to radionuclide contamination at the Nevada test site, USA. Environ. Toxicol. Chem. 20: 317–326. Theodorakis, C. W., K. L. Lee, S. M. Adams and C. B. Law. 2006. Evidence of altered gene flow, mutation rate, and genetic diversity in redbreast sunfish from a pulp– millcontaminated river. Environ. Sci. Technol. 40: 377. Tholl, D. 2006. Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Current Opinions in Plant Biology. 9: 297–304. Thomas, P. 2001. Irradiation of fruits and vegetables. In: Food Irradiation: Principles and Applications. RA Molins, ed., Wiley Interscience, New York. pp. 213–238. Thomson, V. P., S. A. Cunningham, M. C. Ball and A. B. Nicotra. 2003. Compensation for herbivory by Cucumis sativus through increased photosynthetic capacity and efficiency. Oecologia. 134: 167–175. Tice, R. R., E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, J. C. Ryu and Y. F. Sasaki. 2000. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35: 206–221. Tilton, E. W. 1974. Achievements and limitations of ionizing radiation for stored–product insect control. In: Proceedings of the 1st International Working Conference on Stored Product Entomology, Savannah, Georgia, pp. 354–361.

153

Tilton, E. W. and A. K. Jr. Burditt. 1983. Insect’s disinfestation of grain and fruit. In preservation of food by ionizing radiation,” ed. E. S. Josephson and M. S. Peterson, 3: 215 p. CRC Press, Boca Raton, FL. Tomlin, C. 1994. The pesticide Manual: incorporating the agrochemicals handbook. Tenth Edition. Cambridge, UK, Crop protection Publications. The Royal Soc. of Chemistry. Trencheva, K., S. Gounari, G. Trenchev, E. Kapaxidi and G. Karetsos. 2004. Scale insects on oak trees (Quercus spp) in Balgaria and Greece. Proceeding of the 11th International symposium on Scale Insect Studies. pp 137–141. Tridjaja, N. O. and M. S. Mahendra. 2000. Maturity indices and harvesting practice of ‘Arumanis’ mango related to the target market. In Quality Assurance in Agricultural Produce, (G.I. Johnson, L.V. To, N.D. Duc and M.C. Webb, eds.) pp 129–133. Tscharntke, T., S. Thiessen, R. Dolch and W. Boland. 2001. Herbivory–induced resistance, and interplant signal transfer in Alnus glutinosa. Biochem Syst. Ecol. 29: 1025–1047.

Tuteja, N. 2007. Mechanism of high salinity tolerance in plant, Meth. Enzymol: Osmosens. Osmosignal. 428: 419–438.

Tuteja, N. and R. Tuteja. 2001. Unravelling DNA repair in human: molecular mechanisms and consequences of repair defect, Crit. Rev. Biochem. Mol. Biol. 36: 261– 290.

Tuteja, N. and H. Hirt. 2010. Cold, salt and drought stress in: Plant biology: From genomics towards system biology. Wiley–Blackwell, Weinheim, Germany. pp 137–159.

Tuteja, N., M. B. Singh, M. K. Misra, P. L. Bhalla and R. Tuteja. 2001. Molecular mechanisms of DNA damage and repair: progress in plants, Crit. Rev. Biochem. Mol. Biol. 36: 337–397. Tuteja, N., M. B. Singh, M. K. Misra, P. L. Bhalla and R. Tuteja. 2001. Molecular mechanisms of DNA damage and repair: progress in plants, Crit. Rev. Biochem. Mol. Biol. 36:337–397. Twaite, W. G., M. A. Eslick and H. Nicol. 1999. Evaluation of petroleum–derived oils for integrated pest and disease management in apple orchard. Spray oils Beyon 2000. Sustainable Pest and Disease Management Conference, Sydney, October, 1999.

154

UK CAB International, 1966. Aspidiotus destructor. Distribution maps of plant pests. Wallingford, UK: CAB International, Map 218. http://www.cabi.org/isc/datasheet/7415 (17th February 2015) Urbain, W. M. 1986. Radiation chemistry of food components and of foods. In: Food Irradiation (edited by B.S. Schweigert). Pp. 37–82. Orlando, FL: Food Irradiation, Academic Press. USDA (US Department of Agriculture) (2010a). Mangoes from India. 2010. CFR. 319: 56– 46.

USDA. 2010b. Citrus Greening. Regulatory Updates. Plant Health. http://www.aphis.usda.gov/plant_health/plant_pest_info/citrus_greening/regs.shtml (2 3 June 2010).

Uzonur, I., M. F. Abasiyanik, B. Bostanci, M. Eyidemir, N. Ocba, K. C. Yani and M. Petek. 2004. Re-exploring planaria as a model organism for genotoxicity monitoring by an ‘improved random amplified polymorphic DNA’ approach. FEB. 13:1420–1426. Van Zandt, P. A. and A. A. Agrawal. 2004b. Specificity of induced plant responses to specialist herbivores of the common milkweed Asclepias syriaca . Oikos. 104: 401– 409.

Velikova, V., I. Yordanov and A. Edreva. 2000. Oxidative stress and some antioxidant system in acid rain–treated bean plants. Plant Sci. 151: 59–66.

Vennila, S., A. J. Deshmukh, D. Pinjarkar, M. Agarwal, V. V. Ramamurthy, S. Joshi, K. R. Kranthi and O. M. Bambawale. 2010. Biology of the mealybug, Phenacoccus solenopsis on cotton in the laboratory. J. of Insect Science. 10: 115

Verma, S. and S. N. Mishra. 2005. Putrescine alleviation of growth in salt stressed Brassica juncea by inducing antioxidative defence system. J. of Plant Physiol. 162: 669–677.

Viswanathan, D. V. 2005. Specificity in induced plant responses shapes patterns of herbivore occurrence on Solanum dulcamara. Ecology. 86: 886–896.

Waterhouse, D. F. and K. R. Norris. 1987. Aspidiotus destructor Signoret in biological control Pacific prospects. Inkata Press, Melbourne. Chapter 8: pp. 62–71.

155

Watson, G. W. 2005. Aspidiotus destructor. Diaspididae of the world. Arthropods of economic importance. World Biodiversity database. Watson, G. W. and S. A. EL-Serwy. 2007. Biology and ecology of scale insects. Proceeding of the XI International Symposium on Scale Insect Studies, 24–27 September, 2007. Oeiras, Portugal, ISA Press, Lisbon, 322 p.

Watson, G. W. Diaspididae. Arthropods of economic importance. World biodiversity database. http://wbp.etibioinformatics.nl/bis/ (04/10/2015) Watters, F. L. 1968. An appraisal of gamma irradiation for insect control in cereal foods. Manitoba Entomol. 2: 37–45 Weimberg, R. 1987. Solute adjustments in leaves of two species of wheat at two different stages of growth in response to salinity. Physiol. Plant. 70: 381–388. Welsh, J. and M. McClelland. 1990. Genomic fingerprinting using arbitrarily primed PCR and a matrix of pairwise combinations of primers. Nucleic Acid Res. 18: 7213–7218. Whittaker, J. B. 1970. Cercopid spittle as a microhabitat. Oikos. 21: 59–64.

Willard, J. R. 1974. Survival of crawlers of California red scale, Aonidiella aurantii Mask. (Homoptera: Diaspididae). Australian J. of Zoology. 21: 567–573. Willekens, H., S. Chamnongpol, M. Davey, M. Schraud-Ner, C. Langebartels, M. Van

Montagu, D. Inze and W. C. Van. 1997. Catalase is a sink for H2O2 and is

indispensable for stress defense in C3 plants. EMBO J. 16: 4806–4816. William, D. J. and M. C. Granara de willink. 1992. Mealy bug of central and south America C.A.B. International, walling ford. 635pp.

Williams, D. J. and G. W. Watson. 1988. The scale insects of the tropical South Pacific region. Part 1. The armored scales (Diaspididae). CAB International, Wallingford, UK. 290 pp.

Williams, J. G. K., A. R. Kubelik, K. J. Livak, J. A. Rofalski and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nuc Acids Res. 18: 6531–6535.

Williams, J., A. R. Kubelik, K. J. Livak. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res. 18: 6531–6535. Wolf, H. D., R. Blust and T. Backeljau. 2004. The use of RAPD in ecotoxicology. Mutat. Res. 566: 249–262. 156

Wright, M. G. and J. M. Diez. 2005. Coconut scale Aspidiotus destructor (Hemiptera: Diaspididae) seasonal occurrence, dispersion and sampling on banana in Hawaii. International J. of Tropical Insect Science. 25 (2): 80–85 pp.

Wu, K. C. and C. C. C. Tao. 1976. Natural enemies of the transparent scale and control of the leaf bud beetle attacking coconut palm in Taiwan. J. of Agric. Res. of China. 25: 141– 155.

Wu, S. A. and R. Z. Zhang. 2009. A new invasive pest, Phenacoccus solenopsis threatening seriously to cotton production. Chinese Bull Entomol. 46 (1): 159–162. Yadav, M. K., N. L. Patel, A. D. Choudhry and P. K. Modi. 2014. Optimization of Irradiation and Storage Temperature for Maintaining Physiological Changes, Marketable Fruits, and Sensory Acceptance of Alphonso Mango (Mangifera indica). Agric. Res. 3 (4): 360–369. Yadav, M. K., N. L. Patel, B. R. Parmar and D. Nayak. 2013. Evaluation of physiological and organoleptic properties of mango cv. Kesar as influenced by ionizing radiation and storage temperature. SAARC J. of Agri. 11 (2): 69–80. Yamada, M., H. Morishita, K. Urano, N. Shiozaki, K. Yamaguchi-Shinozaki, K. Shinozaki and Y. Yoshiba. 2005. Effects of free proline accumulation in petunias under drought stress. J. of Exp. Bot. 56: 1975–1981. Yang, T. and B. W. Poovaiah. 2002. Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin. Proc. Natl. Acad. Sci. USA. 99: 4097–4102. Zahradník, J. 1990. Conifers. In: D. Rosen, (ed.), Armored scale insects, their biology, natural enemies and control. World Crop Pests. Elsevier, Amsterdam, the Netherlands. 4 (B): 633–644. Zavala, J. A., A. G. Patankar, K. Gase and I. T. Baldwin. 2004. Constitutive and inducible trypsin proteinase inhibitor production incurs large fitness costs in Nicotiana attenuata. Proc. Natl. Acad. Sci. U.S.A. 101:1607–1612. Zhang, J. and M. B. Kirkham.1996. Enzymatic responses of the ascorbate–glutathione cycle to drought in sorghum and sunflower plants. Plant Sci. 113: 139–147. Zhang, K., Y. Deng, H. Fu and Q. Weng. 2014. Effect of Co–60 gamma irradiation and refrigerated storage on the quality of Shatang mandarin. Food sci. and Human Wellness. 3: 9–15.

157

Zhang, S. Z., B. Z. Hua and F. Zhang. 2008. Induction of the activities of antioxidative enzymes and the levels of malondialdehyde in cucumber seedling as a consequence of Bemisia tabaci (Hemiptera: Aleyrodidae) infestation. Arthropod-Plant Interaction. 2: 209–213. Zhou, B. B. and S. J. Elledge. 2000. The DNA damage response: putting check points in perspective. Nature. 408: 433–439. Zhou, C. A., J. J. Zou and J. C. Peng. 1993. Bionomics of coconut scale–a main pest insect on Actinidia and its control. Entomol. Knowledge. 30 (1): 18–20.

Zhou, W. J and M. Leul. 1998. Uniconazole-induced alleviation of freezing injury in relation to changes in hormonal balance, enzyme activities and lipid peroxidation in winter rape. J. of Plant Growth Regul. 26: 41–47.

Zhou, W. J. and M. leul. 1999. Uniconazole–induced tolerance of rape plants to heat stress in relation to changes in hormonal levels, enzyme activities and lipid peroxidation. J. of Plant Growth regul. 27: 99–104.

Zimmerlin, A., P. Wojtaszek and G. P. Bolwell. 1994. Synthesis of dehydrogenation polymers of ferulic acid with high specificity by a purified cellwall peroxidase from French bean (Phaseolus vulgaris L.). Biochem. J. 299: 747–753.

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Figures with captions

Figure 1.1. A. destructor infested mango tree.

Figure 1.2. Distribution map of Aspidiotus destructor. UK CAB International 1966, Bennett and Alam, 1985; Tao, 1999; Claps et al.,2001).

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Figure 1.3. Immature stage of A. destrcutor on lower surface of mango leaf.

Figure 1.4. A. destructor infested mango twig.

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Figure 1.5. Aphytis melinus, pupa (left) and adult (right).

Figure 1.6. Body of an armored scale inside the scale cover. Photo: Matt Bertone.

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Figure 2.1. Map of Pakistan showing my study area (D. I. Khan)

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Figure 2.2.Manually infested mango nursery plant in pot with symptoms of A. destructor

Figure 4.1. Life cycle of A. destructor. (a) A. destructor infested mango plant (b) Transferring crawlers from infested mango plants to nursery plants (c) A. destructor eggs under scale cover (d) Crawler (e) White cap stage (f) Second instar (g) Pupa (h) Adult male (i) Adult female. 163

Figure 7.1. Mortality of A. destructor caused by gamma irradiation and its effect on mango leaves

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Appendix 2.1. Agro-meteorological data during 2011–12 recorded at Arid Zone Research Institute, Ratta Kulach, D. I. Khan, KP, Pakistan.

Wind Temperature °C Relative Humidity (%) Rainfall Months speed Maxi Mini Average 8:00 am 2:00 pm Average Km/day mm July, 2011 38 26 32 82 62 72 82 35 August, 2011 37 26 31.5 83 61 72 72 50 September, 2011 36 24 30 84 61 72.5 51 17 October, 2011 33 17 25 78 47 62.5 37 4 November, 2011 28 12 20 79 54 66.5 34 – December, 2011 21 3 12 70 51 60.5 44 – January, 2012 19 2 10.5 72 47 59.5 40 – February, 2012 19 4 11.5 69 47 56 90 4.5 March, 2012 27 12 19.5 76 61 68.5 74 1 April, 2012 32 18 25 83 67 75 79 40.5 May, 2012 38 23 30.5 81 62 71.5 65 2.5 June, 2012 40 26 33 83 64 73.5 85 3 Appendix 2.2. Agro-meteorological data during 2012–13 recorded at Arid Zone Research Institute, Ratta Kulach, D. I. Khan, KP, Pakistan.

Wind Temperature °C Relative Humidity (%) Rainfall Months speed Maxi Mini Average 8:00 am 2:00 pm Average Km/day mm July, 2012 37 27 32 83 62 72.5 90 49 August, 2012 35 26 30.5 80 61 70.5 77 36 September, 2012 33 23 28 82 69 75.5 50 75 October, 2012 32 16 24 70 34 52 47 – November, 2012 27 11 19 82 50 66 36 – December, 2012 22 6 14 82 46 64 58 13.5 January, 2013 19 4 11.5 79 57 68 47 – February, 2013 21 8 14.5 84 60 72 54 100 March, 2013 26 14 20 79 48 63.5 56 51 April, 2013 32 17 24.5 65 35 50 67 10 May, 2013 40 23 31.5 37 18 27.5 8 – June, 2013 41 26 33.5 54 39 46.5 4 80

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