Faculty of Agriculture Sciences and Technology, Bahauddin Zakariya University, Multan-Pakistan 2015

Seasonal Abundance and Management of Mango Inflorescence Midges in South Punjab Pakistan

HAFIZ MAHMOOD UR REHMAN M.Sc (Hons.) Agricultural Entomology

A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN AGRICULTURAL ENTOMOLOGY

Faculty of Agriculture Sciences and Technology, Bahauddin Zakariya University, Multan-Pakistan 2015

Statement and declaration

The work submitted in this thesis under the title “Seasonal abundance and management of mango inflorescence midges in South Punjab Pakistan” is in fulfillment of the requirement for the degree of Doctor of Philosophy.

I declare here that this work is the result of my own investigations and has not already been accepted in substance for any degree, nor it is currently submitted for any other degree.

All authors, work referred to in this thesis has been fully acknowledged.

______

Hafiz Mahmood Ur Rehman

(Candidate)

I certify that the above statement is correct.

______

Dr. Muhammad Razaq

Supervisor/Associate Professor/Chairman

Table of Contents

Chapter Description Page Abstract 1 1 General introduction and objectives 5 References 14 2 Phenology, population dynamics and within tree distribution 21 of Dasineura amaramanjarae Grover, 1965 (Diptera: Cecidomyiidae) in Punjab, Pakistan 2.1 Introduction 22 2.2 Materials and methods 25 2.3 Results 28 2.4 Discussion 37 References 40 3 Occurrence, Monitoring Techniques and Management of 44 Dasineura amaramanjarae Grover (Diptera: Cecidomyiidae) in Punjab Pakistan 3.1 Introduction 45 3.2 Materials and methods 50 3.3 Results 54 3.4 Discussion 61 References 64 4 Preference of Dasineura amaramanjarae Grover and 70 Procontarinia mangiferae Felt (Diptera: Cecidomyiidae) for different commercial cultivars of mango and their patterns of distribution 4.1 Introduction 71 4.2 Materials and methods 74 4.3 Results 76 4.4 Discussion 83 References 86 5 Damage patterns, biology, monitoring and management of 91 Procontarinia mangiferae Felt in Pakistan 5.1 Introduction 92 5.2 Materials and methods 96 5.3 Results 100 5.4 Discussion 113 References 118 6 Phenology, distribution, biology and population trends of 124 Procontarinia matteiana Kieffer and Cecconi (Diptera: Cecidomyiidae) in Punjab, Pakistan 6.1 Introduction 125 6.2 Materials and methods 126 6.3 Results 130 6.4 Discussion 137 References 139 Conclusions and recommendation for future research 141 Annexures 146

List of Tables

Chapter Description Page 3 Table I Treatments evaluated for the managing D. amaramanjarae 53 at Rahim Yar Khan during 2011 and 2012 4 Table I Mean numbers of larvae and adults of D. amaramanjarae 77 in 2011 and 2012 on different mango varieties at Rahim Yar Khan

4 Table II Mean numbers of larvae and adults of P. mangiferae in 78 2011 and 2012 on different mango varieties at Rahim Yar Khan

4 Table III Mean numbers of larvae of D. amaramanjarae and P. 80 mangiferae in 2011 and 2012 on various locations of mango orchard at Rahim Yar Khan

4 Table IV Mean numbers of larvae of D. amaramanjarae and P. 81 mangiferae in 2011 and 2012 on South, North, East and West Quadrants of mango tree at Rahim Yar Khan.

4 Table V Mean numbers of larvae of D. amaramanjarae and P. 82 mangiferae in 2011 and 2012 on lower and upper parts of mango tree canopy at Rahim Yar Khan

List Figures

Chapter Description Page 2 Fig. 1 Seasonal activity of D. amaramanjarae at Rahim Yar Khan 31 2 Fig. 2 Mean length (cm) and numbers of flower buds of an 32 inflorescence at Rahim Yar Khan : (Left) in 2010 (Right) in 2011 2 Fig. 3 Co-efficient of correlation between mean populations of D. 33 amaramanjarae and weather factors at Rahim Yar Khan in 2008-11 2 Fig. 4 Mean weekly populations of larvae of D. amaramanjarae/sheet at 34 Rahim Yar Khan in the months of February, March and April from 2008 to 2011 2 Fig. 5 Mean weekly weather factors at Rahim Yar Khan in the months of 34 February, March and April from 2008 to 2011 2 Fig.6 Percent mean of larvae of D. amaramanjare collected in 36 funnels hanged on mango trees at different vertical and horizontal strata at Rahim Yar Khan in 2009 and 2010 3 Fig. 1 Percent mean (Mean ± SE) infestation of D. amaramanjarae 55 on trees in commercial orchards and farmer fields Rahim Yar Khan, Bahawalpur and Multan in 2010(left) and 2011(right) 3 Fig. 2 Comparison of different color traps (Mean±SE) for monitoring 57 of D. amaramanjarae at Rahim Yar khan in 2011 3 Fig. 3 Comparison of different color traps(Mean±SE) for monitoring 57 of D. amaramanjarae at Rahim Yar khan in 2012 3 Fig. 4 Monitoring of D. amaramanjarae with traps of different colors 58 at Rahim Yar khan in 2011 3 Fig. 5 Monitoring of D. amaramanjarae with traps of different colors 58 at Rahim Yar khan in 2012 3 Fig. 6 Means of D. amaramanjarae populations in different 59 treatments (Mean±SE) at Rahim Yar Khan in 2011. (1. Sp. Ne. So= Spray of neem on soil. 2. Sp. Ne. Ca= Spray of neem on canopy, 3. Sp. Bi. Ca= Spray of bifenthrin on canopy) 3 Fig. 7 Means of D. amaramanjarae populations in different 60 treatments (Mean±SE) at Rahim Yar Khan in 2012. (1. Sp. Ne. So= Spray of neem on soil. 2. Sp. Ne. Ca= Spray of neem on canopy, 3. Sp. Bi. Ca= Spray of bifenthrin on canopy) 5 Fig. 1 Infestation of P. mangiferae : Exit holes (Left) and gallery 101 (Right) on mango inflorescence axillaries 5 Fig. 2 Infestation of P. mangiferae : Mango axillary bent at right 102 angle (Left) and damaged inflorescence (Right) 5 Fig. 3 Infestation of P. mangiferae : Damaged small sized mango 102 fruits 5 Fig. 4 Percent infestation of P. mangiferae on trees in commercial 104 orchards and farmer fields at Rahim Yar Khan, Bahawalpur and Multan in 2010 and 2011 5 Fig. 5 Mean weekly populations of P. magiferae at Rahim Yar Khan in the 105 months of February March and April from 2009 to 2011 5 Fig. 6 Weather factors at Rahim Yar Khan in the months of February March 106 and April from 2009 to 2011 Fig. 7 Montoring of P. mangiferae and its parasitoid at Rahim Yar 106 Khan in 2009 and 2010 5 Fig 8 Monitoring of P. mangiferae with different color traps at 107 Rahim Yar khan in 2011 5 Fig. 9 Monitoring of P. mangiferae with different color traps at 107 Rahim Yar khan in 2012 5 Fig . 10 Comparison of different color traps(Mean±SE) for 108 monitoring of P. mangiferae at Rahim Yar khan in 2011 5 Fig. 11Comparison of different color traps(Mean±SE) for monitoring 109 of P. mangiferae at Rahim Yar khan in 2012 5 Fig. 12 Means (Mean±SE) of P. mangiferae populations in different 110 treatments at Rahim Yar Khan in 2011 5 Fig. 13 Means (Mean±SE) of P. mangiferae populations in different 111 treatments at Rahim Yar Khan in 2012 6 Fig. 1 Steps involved in studying biology and mass rearing of gall 129 midge and parasitoids, S. temporale and C. pulcherrimus 6 Fig. 2 P. matteianna damaged curled mango leaves 133 6 Fig. 3 Male (left) and female (right) of P. matteiana 134 6 Fig. 4 Parasitoids of P. matteiana; S. temporale and C. pulcherrimus 135 6 Fig. 5 Numbers of S. temporale and C. pulcherrimus and their host P. 136 matteiana adults reared from 50 mango leaves in 2008-2010 at Rahim Yar Khan

Acknowledgements

I am highly indebted to Almighty Allah (The Most Beneficial and Merciful) who blessed me to conduct this piece of research work presented in this dissertation. I offer my humblest thanks from the deepest sense of heart to the Great Prophet of Allah, Muhammad (Peace Be Upon Him) the highest of mankind who enlighten and turned the lives of mankind into peace and good practices. The success initiates with the role of parents to culture ethics while completes with the guidance of a teacher. I feel fortunate to be associated with my affectionate supervisor Dr. Muhammad Razaq, Assistant Professor, Department Entomology, Faculty of Agriculture Sciences and Technology, Bahauddin Zakaryia University Multan (BZU) for providing me with facilities, guidance and advice. This lead to the final submission of this thesis after completing required objectives of research work. Thanks are also due to Agriculture Linkage Programme (ALP) for the financial assistance in conducting this piece of research in Pakistan. I am also grateful to Dr. Muhammad Ashraf Poswal, Global Director CABI for his frequent encouragement in successful completion this work. The consistent support of Mr. Riaz Mahmood, Project Manager, CABI South and West Asia is also highly acknowledged. I also say thanks to Mr. Peter Kolesik, Australia and Nami Uechi, Japan for their guidance during the course of work. I am also thankful for support, encouragement and patience of my parents, brothers and sisters during this study. Thank goes to my wife and children for their patience and love.

Hafiz Mahmood Ur Rehman

ABSTRACT

Mango, Mangifera indica L. (Anacardiaceae) is one of the most favorite fruit crops in the world. It ranks second in the fruit industry of Pakistan. Mango gall midges (Diptera:

Cecidomyiidae) damage many parts of the plant including the bark, shoots, leaves, inflorescence buds, axillaries, flowers, newly formed fruits and twigs in the world. The severely infested leaves and inflorescences fall on the ground. Moreover, these are source of inoculums of anthracnose.

They were reported first time in the last decade from mango growing areas of the Pakistan and ranked number one pests of mango among others during a survey in 2006. There was no published literature regarding identification and management.

Research was initiated encompassing the survey for incidence, identification, damage patterns, seasonal abundance, distribution in tree and field, varietal preference and monitoring/sampling for the developing IPM strategy for gall midges in mango growing areas of

Punjab, Multan, Bahawalpur and Rahim Yar Khan in 2008 to 2012. Three species of mango gall midges, Dasineura amaramanjarae Grover, 1965, Procontarinia mangiferae (Felt), and

Procontarinia matteiana Kieffer & Cecconi were recorded from Punjab. Their identification was confirmed from experts at Natural History Museum U. K. Voucher specimens have been deposited at CABI – Central and West Asia, Rawalpindi (Pakistan).

Dasineura amaramanjarae was active from February to April with a population peak in

March. The flower buds were the only parts for oviposition and the larvae moved to soil for pupation after feeding inside the buds. No stage of the pest was observed from mango trees from

May to January when flowers were not available. Studies on within tree distribution through trapping of the larvae with funnel rings indicated that D. amaramanjarae was not uniformly distributed vertically and horizontally in mango tree canopy. Highest numbers of D.

amaramanjarae larvae were found at the height 1.5m from ground and southern side of tree canopy.

Higher infestation of D. amaramanjarae was noted on mango trees in commercial orchards as compared to the trees scattered in small patches in farmer fields grown for personal consumption. Yellow/green sticky traps were more effective than blue and colorless traps for monitoring of adults. During two consecutive growing seasons the adults of the pest were observed from February to April at Rahim Yar Khan. The use of bifenthrin and neem seed kernel extract (NSKE) with integration of racking of soil under the mango tree was effective for the control of the pest.

Procontarinia mangiferae was recorded from January/February to April on inflorescence buds, axillaries and small mustard sized fruits. Female laid eggs on inflorescence tissues and larvae, after feeding, dropped to the soil under the mango tree to diapause after pupation. Mango trees in commercial orchards were damaged more by P. mangiferae than isolated trees in farmer fields. The adults of P. mangiferae were recorded on all colour traps from January to May with higher numbers on yellow/green followed by blue and colorless traps. A hymenopteran parasitoid was reared from mango inflorescence and its phenology was well synchronized with

P. mangiferae. Besides synthetic insecticide bifenthrin (Talstar 10 EC), the application of neem seed kernel extract (NSKE) on tree canopy integrated with soil raking was effective in managing the pest population.

Both the gall midge species, D. amaramanjarae and P. mangiferae were recorded on all mango cultivars, i. e., Chaunsa, Fajri, Dusehri, Surkha, Sindhri and Anwar Ratul with maximum damage on Surkha/ Dusehri. Research on their distribution patterns in different parts of mango

orchard, i. e., central, southern, northern, eastern and western sides proved that this species was found on all parts with maximum numbers on central/southern side. They were the most abundant on lower parts of mango tree canopy.

Initially it was hypothesized that gall midges only damaged the inflorescence of mango in

Pakistan but the infestation of P. matteiana was also observed on vegetative parts of mango tree during the survey. It formed solitary or grouped galls on the upper and lower surfaces of the leaves. Females oviposited on newly developed leaves, larval and pupal stages were completed in galls and adults emerged from galls through small holes. It was active from February/March to

November with two peaks of its population, first in March/April and second in

September/October. It underwent diapause as pupae in galls from December to February. The phenology of two parasitoids, Closterocerus pulcherrimus (Kerrich) and Synopeas temporale

Austin reared from galls of P. matteiana was well synchronized with their host. Their population was low in the year 2008. However with conservation measures it increased during 2009-10 in experimental orchard.

This research is an initial attempt in understanding the behavior of mango gall midges for developing long term management strategies on the large scale against the pest. Further research is needed on the biology/life history, determination of losses and economic threshold level of gall midges for designing guidelines for application of insecticides. Arthropod fauna should be further explored for parasitoids of the gall midges. Moreover, rearing techniques of discovered parasitoids should be developed for their possibility of augmentation. Biology of the parasitoids is also required for developing conservation techniques. Effectiveness of insecticides being currently applied should be screened regularly in the laboratory and field for occurrence of resistance in the gall midges. Currently no botanical insecticides like neem products are

registered for their use in the country. Such products should be evaluated for sustainable control of the pest as well as to improve the quality of fruits to avoid export problems. Finally, as with most of the developing countries application equipments being employed for pest control should be confirmed for minimum standards of FAO along with their efficacy.

Chapter 1

General introduction and objectives

1. GENERAL INTRODUCTION AND OBJECTIVES

Agriculture, the major part in the economy of Pakistan, is the main source of employment for 66 % of the population and a principal driving force for development and poverty reduction.

This sector provides 21.4% of GDP and 45% employment to labor force in the country

(Anonymous, 2013). Fruits and vegetables is a fast growing area of agriculture in Pakistan (Ali,

2008). They are a rich source of vitamins, minerals, dietary fiber and potassium (Kaiser et al.,

2003). Their regular use prevents from chronic disorders like heart diseases, stroke, cancer (Liu,

2003), cardiovascular diseases (Bazzano et al., 2002) and type-2 diabetes (Carter et al., 2010).

Mango, Mangifera indica L. (Anacardiaceae) is one of the most favorite fruit crop in the world. It provides minerals, amino acids, vitamins, fatty acids, carbohydrates, proteins and organic acids (Litz, 2009). It is also a valuable ornamental and shady tree which protects soil against erosion (D’ Almeida, 1995). Mango cultivation started four thousand years ago in its original home, South Asia (Salunkhe and Desai, 1984) and now has been spread in more than 100 countries of the world (Sauco, 1997). In Pakistan, it ranks second in the fruit industry (Asif et al.,

2011). Pakistan exports 60-70 thousand tons of mangos mostly to the countries of Middle East,

Europe and Far East (PHDEB, 2005). In 2013 the export of Pakistani mango to the international market was $20.5 million, five times more as compared to the previous year 2012 (Klasra, 2013).

It was grown on an area of 156.6 thousand hectares in Pakistan with the production of

1753.9 thousand tons in 2005-06 (Anonymous, 2007). Punjab and Sindh provinces cover 52.66 and 45.68% of total mango growing areas in the country. The major mango growing areas in

Punjab are Multan, Bahawalpur, Muzzaffar Garh and Rahim Yar Khan. Mango growers are facing many issues which have created a wide difference between potential yield (21.6 tons/ hectare) and average yield (9.96 tons/ hectare) of mango in Pakistan. This difference is attributed

to multiple factors like insect pests, diseases, weather hazards, defective marking system and poor pre and post harvest practices (Lodhi et al., 2006; Khan et al., 2008). Two hundred insect pests of mango have been recorded in the world (Veeresh, 1989). Among these, gall midges, caterpillars, leafhoppers, thrips and mites are the most important pests damaging mango inflorescences (Peña et al., 1998).

The gall midges (Diptera: Cecidomyiidae) are the minute and delicate flies with moniliform antennae, long legs and reduced wing venation. The name of family Cecidomyiidae is derived from the Latin word cecidium meaning galls due to the ability of their larvae to induce galls or abnormal outgrowths on different organs of plant. They are found in various ecosystems like forests, fields and meadows. The most damaging larval stage contains three to four or even five instars in gall midges. The mature larvae are elongate, cylindrical and orange, white, yellow or red in color. Larval development in gall midges may take less than two weeks in some species and more than two years in others (Ananthakrishnan, 1986).

Gall midges cause damage to mango by feeding in inflorescence and fruit tissues, making galls on leaves. In late 1990s more than 20 species of gall midges were known worldwide from various parts of mango plant including bark, shoots, leaves, pre- and post flowering shoot buds, inflorescence buds, axillaries, flowers, newly formed fruit and twigs (Srivastava, 1998). The leaves seriously infested by gall midges fell to the ground much earlier than usual. Shoots of heavily infested mango trees have almost no inflorescences resulting in low yields of mango trees (Uechi et al., 2002; Kolisek et al., 2009).

Leaves damaged by gall midges provide inoculums to anthracnose. Anthracnose is a group of fungal diseases (caused by Colletotrichum or Gloeosporium) including leaf spot, blossom blight, wither-tip or die-back (Bailey and Jeger, 1992). This disease is of major

economic importance in commercially mango growing areas of India, Philippines, Indonesia,

Peru, Hawaii and Portugal (Singh, 2000). Anthracnose occurs on all parts of mango trees but is the most common on flowers and flower stalks. Young and mature fruits are also infested. If early infection of fruit takes place they fall off (Litz, 2009). Losses due to anthracnose have been estimated to be 2-39% in India (Prakash and Srivastava, 1996).

Fungi causing this disease grow on wounds formed by feeding of gall midges and other insects (Daquioseand Quimio, 1979). Leaves of mango trees infested with gall midges provide a reservoir to anthracnose fungus (Colletotrichum gloeosporioides (Penzig) Penzig and Saccardo) which can reduce the photosynthetic capacity (Harris and Schreiner, 1992; Uechi et al., 2002;

Kolisek et al., 2009). As there is a synchronization of abundance of populations of mango gall midges with conidia of causal fungi during flush growth and flowering of mango trees, therefore higher populations of gall midges may help in epidemics of the disease (Singh, 2000).

Mango gall midges were first described by (Felt) in 1911 from West Indies but have now been recorded from China, Hawaii, India, Indonesia, Japan, Kenya, Mauritius, Oman,

Philippines, Reunion, South Africa, Taiwan, United Arab Emirates and many other parts of the world (Ahmed et al., 2005). They are still being spread rapidly in many areas of the world. For example, a mango leaf gall midge, Procontarinia mangicola (Shi) damages fresh mango leaves and produces circular blister galls, causing the leaves to crinkle. It has been recorded on eight islands in Okinawa Prefecture, Japan (Uechi et al., 2002). Similarly, another mango gall midge,

Procontarinia pustulata Kolisek has been found in many regions of Australia (Kolisek et al.,

2009).

In India, the occurrence of gall midges with mango dates back to ancient times. Some distinct galls observed on fossil leaves of the ancestor of mango, Eomangiferophyllum

damalgiriensis from the upper paleocene aged flora of north-eastern India are similar with the galls of mango midge Amradiplosis echinogalliperda Mani (Srivastava et al., 2000). Now, 17 species of gall midges infesting leaves, flowers and stem of the mango are known from different mango growing areas of India (Raman et al., 2009).

Dasineura amaramanjarae Grover and Procontarinia mangiferae (Felt) feed on mango inflorescence. D. amaramanjarae lays eggs between petals and sepals and larvae feed on style of gynaecieum and filament of stamen. In India, higher infestation is characterized by brownish red petals and dried up sepals (Prashad, 1970). P. mangiferae feeds on pre- flowering shoot buds, inflorescence buds, axis of inflorescence panicles, post-flowering shoot buds and newly formed fruits. Females lay eggs between leaves and buds, larvae feed inside the inflorescence tissues and at final instar come out with swift jump to move into soil for pupation. Their damage can be recognized by the black spots of varying size on inflorescences bent at right angle. Moreover, the infested fruits ultimately become deformed, pale, stop growing and subsequently drop down (Prashad, 1971). In Pakistan, mango gall midges are a new pest. Their damage has been recorded in last decade from all mango growing areas of Punjab including Multan, Lodhran, Rahim Yar Khan and Faisalabad

(Ahmed et al., 2005). They were ranked number one pest during a survey in 2006

(Anonymous, 2006). However, these were not recognized taxonomically. There was no published literature on identification, biology and ecology of mango gall midges from the country to develop pest management strategies.

The inception and growth of IPM as a discipline in agriculture is a relatively recent phenomenon that traces back to the advent of synthetic organic pesticides and the harmful effects they began to exert on agriculture during the late 1940s and 1950s (Castle and Naranjo, 2009).

Overuse, misuse and improper use of pesticides endangered the health of farm workers and consumers of agricultural products worldwide (Goodell, 1984). IPM emerged as a science-based approach to minimize the risks associated with the use of pesticides (Nagrajan, 1990; NRC,

1992). As defined, ―It is a decision support system for the selection and use of pest control tactics, singly or harmoniously coordinated into a management strategy, based on cost/benefit analyses that take into account the interests of and impacts on producers, society and the environment. It is considered the central paradigm of insect pest management and is often characterized as a comprehensive use of multiple control tactics to reduce pest status while minimizing economic and environmental costs (Kogan, 1998).‖

IPM is organized into different layers (avoidance, sampling and effective chemical use) identifying the major pest control components and their interactions. Avoidance techniques provide basis to manage pests by delaying them from reaching economic threshold levels. These include pest biology and ecology, cultural control, host plant resistance and biological control if taken area wide (Naranjo, 2010). First of all identification which involves determining the correct name is a key in publishing the literature about the pest. It aids in research and development of pest management programs and act as reference to deliver such a program to practitioners (Pedigo and Rice, 2009). The information about the phenology and timing of infestation is useful in developing management strategies against insect pests (Hanhn and Isaacs,

2012). In South Africa, phenology models have been used to predict optimum periods for insecticide applications against deciduous fruit pests resulting in reduced pesticide applications.

Similarly, in Peru, different potato IPM strategies based on the knowledge of biology and pest behavior, seasonal occurrence and spatial distribution have been used to avoid economic damage both in the field and storage (Maredia et al., 2003). In India D. amaramanjarae has been

effectively controlled by determining the peak period of abundance to apply insecticides in mango production system (Grover, 1985).

Cultural practices do not provide high levels of pest control therefore did not receive full attention in the time when all the reliance was placed on chemical control. Presently often scheme is to use several methods in combination. Each method achieves a certain level of control in this way, so that desired level is achieved with minimal ecological disruption. The bulk of the soil insect populations (particularly pupating stages from Lepidoptera, Coleoptera and Diptera) are in top 10 cm. Tillage will bring these insects to the surface to be exposed to sunlight desiccation and natural enemies (Hill, 2008). In India, the cultural practices like cleaning, manuring, irrigating and hoeing the soil under the mango trees had been used for managing mango gall midges (Prasad, 1966; Grover and Prasad, 1966; Grover, 1985).

Like cultural practices, biological control rarely gave desired level of pest control alone in majority of attempts in the past. At global level arthropod natural enemies have been attempted to manage 602 species. However the rate of complete control was 16% and partial success (integrated with other measures) was 58% (Pedigo and Rice, 2009). In Africa, conservation of two parasitoids, Platygaster diplosisae and Aprostocetus procerae not only suppressed the population of African rice gall midge (AfRGM), Orseolia oryzivora (Diptera:

Cecidomyiidae), but also provided African farmers with low-cost non-chemical control of

AfRGM (Nwilenea et al., 2008). Successful examples of integration of biological control with other methods include management of insect pests of citrus from Australia. Integrating biological control with other tactics is considered crucial to reduce insecticide inputs (Zalucki et al., 2009).

Plant resistance based upon push pull strategy mostly integrated with chemical control makes the whole IPM package more sustainable both environmentally and economically (Cook et al., 2006;

Birch et al., 2011). Presently more than 500 insect resistant cultivars of rice, maize, wheat cotton, sorghum and alfalfa are worldwide providing control of more than 50 key pests (Atwal and Dhaliwal, 2015).

Besides avoidance, the other layers of pest management are sampling and the effective use of chemicals. Sampling is a fundamental requisite for implementing any type of prescriptive control through adherence to thresholds and also for developing basic foundational knowledge of pest dynamics (Castle and Naranjo, 2009). In IPM pesticides are considered as a last resort but their application is inevitable due to unavailability of effective alternatives in most of the pest management systems (Zalucki et al., 2009).

Neem-based insecticides with low in mammalian toxicity and ecological hazards are another IPM option (Schmutterer, 1990). They have potential for management of variety of insect pests belonging to insect orders Orthoptera, Isoptera, Homoptera, Diptera, Coleoptera,

Lepidoptera and phytophagous mites. Pest management programmes currently being practiced incorporate neem pesticides in more than 70 countries of the world (Dhawan et al., 2013). In

Nigeria, the use of neem seed extract suppressed significantly the population of African rice gall midge (AfRGM), O. oryzivora (Ogah and Ogbodo, 2012).

As mentioned earlier there was no published research for managing gall midges from

Pakistan. Therefore, present research on different aspects of mango gall midges to develop IPM with following the objectives was initiated. The results of this research will provide foundational data to manage mango gall midges

1. Survey of mango growing areas for the incidence of mango inflorescence gall midges

2. Identification of mango inflorescence gall midges and their natural enemies

3. Seasonal abundance of mango inflorescence gall midges

4. Distribution of mango inflorescence gall midges in trees and fields

5. Development of monitoring/sampling techniques

6. Varietal preference of mango inflorescence gall midges

7. Evaluation of management options for mango inflorescence gall midges

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Chapter 2

Phenology, population dynamics and within tree distribution of Dasineura amaramanjarae Grover, 1965 (Diptera: Cecidomyiidae) in Punjab, Pakistan

This chapter has been published as;

REHMAN, H. M., MAHMOOD, R. AND RAZAQ, M., 2013. Phenology, population dynamics and within tree distribution of Dasineura amaramanjarae Grover, 1965 (Diptera: Cecidomyiidae) in Punjab, Pakistan.Pakistan J. Zool., 45: 1563-1572 (Impact Factor 0.33)

2.1 INTRODUCTION

Mango, Mangifera indica L. is grown in lowland tropical and sub-tropical areas throughout the world. It is one of the foreign exchange earning crops that ranks second in the fruit industry of Pakistan (Asif et al., 2011). The production of mango is undermined by more than 250 insect pests in the world (Peña and Mohyuddin, 1997; Peña et al., 1998). Among these, about 20 species of gall midges are known worldwide associated with various parts of mango plant including bark, shoots, leaves, pre and post flowering shoot buds, inflorescence buds, axillaries, flowers, newly formed fruit and twigs (Srivastava, 1998).

In neighboring country of Pakistan, India, Dasineura amaramanjarae Grover, 1965 is one of the midge pests of mango which feeds on flower buds. Female lays eggs near the stamens, larvae feed inside the flower buds and full grown larvae drop to the soil for pupation. In the case of severe infestation, the reduction in yield can reach 100% (Grover and Prasad, 1966; Prasad,

1966). It has been reported as a pest in mango growing areas of India like Saharanpur, Dehradun,

Delhi, Aligarh, Allahabad and Varanasi. The adults emerge from the soil and immediately pair at the start of the flowering season. A single female lays 40-50 eggs which are hatched in 30-36 hours, depending upon the temperature and humidity. There are four larval instars and the second and third instars cause the most of the damage. Pupation and diapause take place in soil under mango tree (Prasad and Grover, 1966).

Farmers apply insecticides and cultural practices for the control of D. amaramanjarae.

Wetting the soil at the time of flowering under the mango tree delays the emergence of adults from the soil and slows down the build-up of the pest population, enabling the inflorescence buds to escape the damage of gall midges. Literature reports the effectiveness of insecticides in the

past available at that time in India where it was reported as pest in 1980s. For example, the treatment of aldrin on the soil and spray of 0.02% phosphamidon + 0.03% diazinon, 0.25% demeton-S-methyl + 0.03% malathion, phosphamidon and demeton-S-methyl on mango trees at the peak of population proved to be effective in controlling D. amaramanjarae (Grover, 1985).

Pesticide sprays which are the most common control measure used to suppress insect pests of mango have been reported to be toxic to some hymenopteran parasitoids (Saifullah et al.,

2007; Prabhaker et al., 2007). Moreover, the intensive use of pesticides has led to problems such as insecticide resistance, loss of natural enemies, secondary pest outbreaks and environmental contamination (Zadocks, 1993; Dent, 1995). Estimating population abundance or density is necessary to understand the population dynamics of a species for making decisions in developing successful pest management programs (Krebs, 1978; Ekbom and Xu, 1990; Dent, 1997).

The problem of mango gall midges is recent in the Pakistan. Anecdotal reports of damage due to this pest were among the mango growers since last decade. However, it was not clear whether the damage was due to mango gall midges because of unawareness of identification of the pest. We initiated study and confirmed the presence of the complex of the gall midges in mango orchards. Among the different species of gall midge complex, we identified and explored population trends and natural enemies of Procontarinia matteiana Kieffer and Cecconi, a gall midge forming galls on mango leaves from the complex of mango gall midges (Rehman et al.,

2013). Two other species recorded were D. amaramanjarae and Procontarnia mangiferae.

Further studies were extended to collect baseline data for developing pest management by rearing D. amaramanjarae for identification with following specific objectives;

 To determine incidence and abundance of D. amaramanjara in the mango orchards

 To compare the population of D. amaramanjarae at horizontal (south, north, east and

west) and vertical (1.5, 3 and 6m) strata of mango tree

Nothing has been published about this pest species earlier than this study from Pakistan.

Integrated pest management (IPM) encompasses multiple control tactics to reduce pest status while minimizing economic and environmental costs. The model of IPM is organized into three steps i.e. avoidance tactics that delay or prevent the pest to achieve economic status, sampling and selective chemical application provide effective control (Naranjo, 2011). Outcome of the research will provide information about designing of cultural control practices (avoidance of the pest), the timing of the insecticide application and parts of the mango trees to be sprayed. In

India, based upon the phenology of the pest the optimum time to apply the insecticides on mango trees at a peak of population and 13 days later effectively controlled D. amaramanjarae (Grover,

1985). Only due to the timing of insecticide application 50% chemicals can be reduced (Metcalf and Luckmann, 1994). Moreover, the data on spatiotemporal distribution of the pest will be the foundation for future research to develop forecasting system, evaluation of pesticide alternatives and selective chemicals for fine tuning of pest management strategies (Castle and Naranjo,

2009).

2.2 MATERIALS AND METHODS

2.2.1 Identification

During the first year of the research larvae of the gall midge complex were collected from different parts of mango tree like flowers and axillaries. However, we separately reared the larvae collected from above said parts in the laboratory developed by CABI-Central and West

Asia at Rahim Yar Khan. The adults emerging from the pupae were preserved in 70-75% ethanol for their identification (Gagné, 1994).

2.2.2 Phenology and population dynamics of D. amaramanjarae

Phenology and population dynamics of D. amaramanjarae were investigated in a mango orchard at Taranda Saway Khan, Rahim Yar Khan. For the collection of larvae of D. amaramanjarae, plastic sheet method previously employed was used with some modifications

(Rehman, unpublished). Three mango trees of varity Fajri having a uniform flowering were selected and 12 plastic sheets (2x2m) were spread and fixed on ground under the canopies of these trees. Four sheets were spread under a mango tree and this individual tree was considered a replicate. Mature larvae of D. amaramanjarae falling on plastic sheets were counted daily and pooled to weekly basis. The experiment was continued throughout the study period starting from

January 2008 to April 201l. However damaged plastic sheets were changed when required.

In order to determine the association/synchronization of appearance of inflorescence and its growth to the population development of D. amaramanjarae, the numbers of flowers present on inflorescence were counted and length of inflorescence (cm) was measured with scale at weekly intervals from February to April of 2010 and 2011. For this purpose, five mango trees were selected and measurements were taken from four inflorescences of each tree. Data on

weather factors ie., temperature, relative humidity and rainfall were obtained from regional centre of Pakistan Meteorological Department at Rahim Yar Khan.

2.2.3 Vertical and horizontal distribution of D. amaramanjarae on mango tree canopy

To study the distribution of midges at different vertical and horizontal strata of the mango trees, funnel rings of 1 meter diameter with plastic jars fixed below were suspended on each side of the four strata/all four sides of the tree i.e. east, west, north and south (variety Chounsa) at 1.5,

3 and 6 meters above from ground. It was replicated on three trees. There were 12 traps total per tree. Number of larvae of D. amaramanjarae trapped in these funnel rings were counted weekly.

This study was started at Rahim Yar Khan from July 2008 and continued to June 2010.

2.2.4 Statistical analysis

A simple linear correlation was worked out between the mean weekly population of D. amaramanjarae and weekly mean, maximum and minimum temperatures, relative humidity and rainfall in January, February and March of 2008 to 2011 for Rahim Yar Khan location only. The significance of association of the weather factors and numbers of pest was determined from absolute co-efficient of correlation (r-value) by the method of Gomez and Gomez (1984). The numbers of gall midges of four sides and three heights were converted to percentage for each side and height, respectively. Sides and heights where funnel rings were tied were considered as treatments. As there were four rings attached on each horizontal side (South, North, East and

West) and three rings on vertical side (heights of 1.5, 3 and 6m) on each tree and there were total three trees for the experiment. Each tree was considered as a replicate. A one way analysis of variance (ANOVA) was carried out to determine the significance of treatments. Differences among the numbers of D. amaramanjarae among the treatments were calculated by LSD test at

5% level of significance. All the statistical analysis was carried out with software Statistix 8.1.

2.3 RESULTS

2.3.1 Occurrence of D. amaramanjarae

D. amaramanajarae was first time recorded from Pakistan. It was found only in flowers of mango from all the areas surveyed in Punjab including Rahim Yar Khan, Bahawalpur Multan and Lahore. The specimens were identified by the experts from Natural History Museum (NHM)

London, UK.

2.3.2. Phenology and population dynamics of D. amaramanjarae

In the four years (2008-11) of this study, D. amaramanjarae was observed in the months of February, March and April. Only fully developed larvae of this species drop to the soil (see introduction). They dropped from mango flowers to soil under mango tree for 63, 70, 63 and 35 days (Mean 57.75±7.76) in 2008, 2009, 2010 and 2011, respectively. The highest numbers of larvae were found in March (88%) followed by April (10%) and February (2%). During research period of all years, peaks of population of D. amaramanjarae were observed in March.

Maximum numbers were recorded in second week of March during 2008-09 and in third and fourth week in 2010 and 2011, respectively.

Studies on population dynamics in 2008 indicated that the larvae of D. amaramanjarae were first observed on 3rd week of February (mean temp. 22.01°C and RH 53.42%). Their numbers increased steadily and reached at peak in 3rd week of March (mean temp. 29.535°C and

RH 60.33%). After that, numbers of larvae decreased suddenly and reached at minimum level in the 3rd week of April. They were not found on mango trees from May 2008 and onwards. In next year (2009), small numbers of larvae started falling from flowers onto the ground in the 2nd week of February (mean temp. 18.87°C and RH 75.01%). Highest numbers were recorded in 2nd week

of March (mean temp. 25.57°C and RH 52.86%). They gradually decreased to zero in 4th week of April (mean temp. 33.86°C and RH 56.2%). Damage was not observed after April 09 till the

3rd week of February 2010 when population of D. amaramanjarae again emerged (mean temp.

19.32°C and RH 72.85%) and reached at peak in 3rd week of March (mean temp. 30.31°C and

RH 56.85%) then reduced to zero in 4th week of April (mean temp. 32.48°C and RH 51.5%). In

2011, a delay was observed in falling of larvae from flowers to ground due to frequent rainfall from 3rd week of February to 2nd March. Its emergence commenced during second week of

March (mean temp. 27.28°C and RH 73.43%). The peak period of pest was recorded in last week of March (mean temp. 28.5°C and RH 59.2%). Thereafter, their numbers decreased gradually and were not observed after second week of April 11(Fig. 4-5).

D. amaramanjarae was noted only when flowers were available and it was not found from

May to January on mango tree even on remaining parts of malformed inflorescences present on mango trees (Figs. 1, 2). A simple linear correlation between the mean weekly population of D. amaramanjarae and weekly mean, maximum and minimum temperatures, relative humidity and rainfall in January, February and March of 2008 to 2011 for Rahim Yar Khan location revealed non significant association of the weather factors and numbers of larvae (Fig. 3).

2.3.3 Vertical and horizontal distribution of D. amaramanjarae on mango tree canopy

The larvae were collected in funnels from February to first fortnight of April and they were not found in the remainder of the year between April to January during 2009-10. Though they were found all over tree canopy and their numbers varied on different aspects of canopy. In

2009, vertical distribution of the pest on tree canopy was statistically significant (F = 7.84; df =

2, 35; P < 0.001). About 38.69% population of D. amaramanjarae was found at the height of

1.5m of tree canopy from ground level while 34.86 and 26.44% was recorded at 3 and 6m, respectively. Pair wise comparison of population at various heights with least significant difference test (LSD at α = 0.05) indicated that the mean numbers of larvae at 1.5 and 3m were similar but higher than at 6 m.

Population of D. amaramanjare was not equally found (F = 7.31; df = 3, 35; P < 0.001) on the four horizontal sides (South, North, East and West) of tree. More numbers of larvae were found on the southern side (31.24%) followed by east, west and north sides with 23.42, 22.95 and 22.37 %, respectively. LSD test (α=0.05) showed that they were significantly higher at the southern side as compared to other three sides and there was no statistically significant difference among other three horizontal strata.

Fig. 1 Seasonal activity of D. amaramanjarae at Rahim Yar Khan

Fig. 2 Mean length (cm) and numbers of flower buds of an inflorescence with standard error at Rahim Yar Khan: (Left) in 2010 (Right) in 2011

Fig. 3 Co-efficient of correlation between mean populations of D. amaramanjarae (n=12) and weather factors at Rahim Yar Khan in 2008-11

Fig. 4 Mean weekly populations of larvae of D. amaramanjarae/sheet at Rahim Yar Khan in the months of February, March and April from 2008 to 2011

Fig. 5 Mean weekly weather factors at Rahim Yar Khan in the months of February, March and April from 2008 to 2011

In 2010, analysis of variance of numbers of D. amaramanjarae larvae among the different vertical strata (1.5, 3 and 6m) of mango tree showed that its distribution was significantly different (F = 8.11; df = 2, 35; P < 0.001). Highest numbers of larvae were recorded in the funnels at 1.5m (44.41%) followed by 3m (41.93%) and 6m 13.65%). LSD (α=0.05) indicated no significant difference among the populations at 1.5 and 3m though slightly more larvae were caught at lower areas. However population at 6m was significantly lower than other two strata.

Population differed considerably among the four horizontal strata of the tree (F = 4.51; df = 3,

35; P < 0.01). Funnel rings at the southern part of tree received higher numbers of the larvae

(42.15%) as compared to east (20.47%), north (18.95%) and west (18.42%). The aggregation of the pest on southern side was statistically significant from other three strata. However, there was no substantial variation in among east, north and west sides (Fig. 6).

a a a

b b b b b b b b

Fig. 6 Percent mean of larvae of D. amaramanjare collected in funnels hanged on mango trees at different vertical and horizontal strata at Rahim Yar Khan in 2009 and 2010

Note: Bars topped with different letters are significantly different for the same year (LSD Test, α = 0.05)

2.4 DISCUSSION

The larvae of D. amaramanjarae dropped on to the soil under the mango tree for pupation.

The pest was noted from February to April when flowers were available and was not found from

May to January on of mango trees. During inactive period (May to January) they were not recorded on malformed inflorescences on mango trees and flowers of other trees in/surrounding areas of experimental orchard. In India, larvae of D. amaramanjarae damaged mango trees by feeding on buds and flowers. Although emergence of D. amaramanjarae varied in different years yet it was synchronized with flowering on mango (Prasad, 1966; Grover, 1986). Larvae of

D. amaramanjarae pupate/hibernated in soil under mango trees (Prasad and Grover, 1966).

In present findings, D. amaramanjarae followed aggregated pattern of distribution.

Numbers of the larvae were significantly higher on the southern side of the trees than all other horizontal sides and significantly less at 6m height of tree canopy as compared to 3m and 1.5m.

In India, a mango gall midge, P. matteiana followed an aggregated distribution (Verghese and

Rao, 1988). Another mango gall midge species, Erosomyia indica (now Procontarinia mangiferae) showed a random pattern of distribution in a mango orchard. However, infestations in east lower, south lower and south upper were significantly correlated to the total infestation

(Verghese et al., 1988). In contrast to random distribution of closely related gall midge species

(E. indica) in India, D. amaramanjarae aggregated on southern quadrant of mango trees. There might be several reasons for difference in distribution. Preference for utilization of different parts of the tree for feeding and shelter reduces the competition among the closely related species.

Therefore both the species might have different preference. Moreover, the difference in congregation may be due to the effect of weather of both the regions. As depicted in the research

(Verghese et al., 1988) that infestation of E. indica on east lower, south lower and south upper

was significantly correlated the total infestation. But D. amaramanjarae was significantly greater on lower parts and southern side of mango tree canopy. This difference might be due to severity of winter and wind speed. In Punjab wind blows from north and the movement of the sun in winter and spring months is with its lower arc on southern side (Gosal, 2004; Lea, 2010).

Therefore, this side may provide some cushion from cool wind and favor congregation of gall midges due to sunlight. It is recommended to keep the honeybee hives southern east sides of walls to protect bees activities from ill effect of wind as they blow from the north (Hashmi,

1994). It has been also noted that gall midges are attracted to light (Kashyap, 1986), aside from other factor southern side also provides more light as compared to other sides due to sun movement. These reasons seem plausible for greater populations of midges on southern side.

Moreover, it is suggested that quantitative effect of above mentioned variables in combination with development of reproductive stages of the trees may be explored in the future.

Variation in flushing and blooming of mango trees which changes the distribution of inflorescences, a feeding and ovipostion site for pests on a mango tree and might be the species preference. In a study at Faisalabad Pakistan, it was observed that April flushes (bunches of newly emerged leaves) were more important for blooming, while April based May, June and July flushes showed more vegetative growth with less blooming percentage. The bloomed panicles on

April and April based May, June and July flushes were 31.50, 0.25, 27.99 and 10 %, respectively

(Anwar et al., 2006). Mature flushes with high starch contents would bloom readily (Chacko,

1984). The variations in growth patterns of flushes have an impact on blooming of mango tree which may induce the changes in distribution patterns of various mango gall midges species.

In conclusions, midge was found in all surveyed areas of Rahim Yar Khan, Bahawalpur and Multan Phenology showed that activity of D. amaramanjarae mostly started in February and

reached a peak in March. It was not found on mango trees from May to January. Present studies also revealed that the association between population of midge and weather parameters was not significant. Its distribution was not uniform on vertical and horizontal strata of mango tree. It was more abundant on 1.5m and on south side of mango tree.

This piece of research is an initiative in understanding eco-biology of D. amaramanjarae for developing its eco-friendly management tactics in Pakistan. The pest causes damage to flower buds from February to April with a peak in March. Its attack is significantly higher on lower and southern sides of tree canopy, therefore, insecticides can be applied at or near peak on mango trees canopy. We recognize that there is lot to do on its biology for the development of comprehensive phenogram showing the occurrence and duration of all life stages of the pest. As the pest remains active for three months, there is need to determine the losses by applying insecticides at varying intervals. More over the economic threshold level should be determined to avoid economic and environmental costs due to over use of insecticides. Role of cultural practices like hoeing and irrigating the orchards and natural enemies also demands evaluation for reducing midge population. These components are essential for developing any IPM program as recognized since late 1950s (Metcalf and Luckmann, 1994).

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regarding plant protection, harvesting and post-harvest technologies among the mango

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Lucknow, India. pp. 175–299.

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60.

Chapter 3

Occurrence, monitoring techniques and management of Dasineura amaramanjarae Grover (Diptera: Cecidomyiidae) in Punjab Pakistan

This chapter has been published as;

REHMAN, H. M., MAHMOOD, R. AND RAZAQ, M., 2014. Occurrence, monitoring techniques and management of Dasineura amaramanjarae Grover (Diptera: Cecidomyiidae) in Punjab, Pakistan. Pakistan J. Zool., 46: 45-52. (Impact Factor 0.33)

3.1 INTRODUCTION

Mango, Mangifera indica L. (Anacardiaceae) has been in cultivation in tropics and subtropics for several thousand years (Mukherjee, 1953, 1972; Purseglove, 1972). It is grown over 90 countries in the world and ranks fifth among major fruit crops. Asia accounts for approximately 77.0% of global mango production whereas the USA and Africa account for approximately 13.0% and 9.0%, respectively. Pakistan is also one of leading mango-producing and exporting countries. During 2003–2005, it contributed 4.5% and 6.9% to world production and export trade, respectively. This country is the major supplier of mango to the West Asian market (Litz, 2009).

In Pakistan, the potential yield per hectare of mango is 21.6 tons as compared to its average yield of 9.96 tons. This shows a wide difference that is attributed to multifarious factors like insect pests, diseases, weather hazards, defective marketing system and poor post and pre harvest practices (Srivastava, 1998; Lodhi et al., 2006).

About 250 insect pests of mango have been recorded in the world. Among these, gall midges, caterpillars, leafhoppers, thrips, and mites are the most important pests attacking mango

(Peña and Mohyuddin, 1997; Peña et al., 1998). Gall midges have recently been emerged as a pest of mangoes in Pakistan. They have been recorded since last decade in all mango growing areas of Pakistan particularly in Multan, Bahawlpur, Lodhran, Rahim Yar Khan, Lahore and

Faisalabad (Ahmed et al., 2005; Rehman et al., 2013). Researchers emphasized that this group of pest was needed to determine its status in the Punjab province.

Previous researchers from India reported that Dasineura amaramanjarae Grover (Diptera:

Cecidomyiidae) was recorded as a significant gall midge pest of mango which causes damage due to direct feeding of its larvae on flower buds. In the case of severe infestation, the reduction

in yield can reach 100%. Usually, 3-4 larvae feed in a single bud but their number can increase up to 6-8 or even 10-12 where several females have oviposited (Prasad, 1966).

The female of D. amaramanjarae has been reported to lay eggs near the stamens. A single female can lay 40-50 eggs, up to six eggs per bud. They are hatched in 30-36 hours, depending upon the temperature and humidity. There are four larval instars and the most of the damage is caused by the second and third instars. The larvae at final stage drop to the soil for pupation or diapause (Grover and Prasad, 1966).

In Pakistan, flowering on mango starts in February and fruits become mature in June/July depending upon type of cultivars and mango growing area (Iqbal et al., 2012). Recent research reports that the appearance of the adults of D. amaramanjarae has a close synchronization with initiation of flowering on mango trees in Punjab, Pakistan. They emerged from the soil after completion of pupation/diapause and cause damage from February to April i.e. during the availability of flowers. These have not been recorded on the mango orchards from May to

January when mango flowers are not available (Rehman et al., 2013 a).

Insect pests respond differently to various colors in their environment. For example, pea leaf miner, Liriomyza huidobrensis (Blanchard) are attracted to yellow and green sticky traps more than white, blue, red and violet traps. Many important crop pests such as plant hoppers, leafhoppers, aphids, whiteflies, thrips, and leaf miner flies are attracted to yellowish traps (Vaishampayan et al., 1975; Esker et al., 2004; Mainali and Lim, 2010). In contrast, the onion fly, Delia antique (Meigen) prefers white more than yellow color (Vernon and Bartel,

1985). The western flower thrips, Frankliniella occidentalis (Pergande) are attracted to blue sticky traps (Gillespie and Vernon, 1990).

The differential attractiveness of insect pests to color has been manipulated for their surveillance, monitoring and management to protect valued resources. In Japan, incandescent lights, mercury lamps, and black lights have been employed for forecasting pest outbreaks.

Yellow fluorescent lamps are used in the orchard at night to prevent the damage of fruit-piercing moths such as Eudocima tyrannus Guene´e and Oraesia emarginata Fabricius. (Nomura, 1967;

Nomura et al., 1965). Blue fluorescent light traps have been deployed widely to control rice stem borer, Chilo suppressalis Walker, and Tryporyza incertulas Walker moths in paddy fields across

Japan (Ishikura, 1950). For the detection of gall midges, sticky traps of different colors, light trap, and plastic sheet method have been employed in world (Prasad, 1968; Kashyap, 1986;

Sharma and Franzmann, 2001; Plažanin et al., 2012).

Insecticides are powerful tool in insect pest management as they are highly effective, rapid in curative action, adaptable to most situations, flexible in meeting changing agronomical and ecological conditions and relatively economical. However, much use of insecticides have been ecologically unsound and can induce pest resistance, outbreaks of secondary pests, adverse effect on non target organisms, objectionable pesticide residues and direct hazards to user (Metcalf and

Luckmann, 1994). Despite of their disadvantages insecticides are still major component of IPM systems (Zalucki et at., 2009).

Neem-based insecticides are alternate pest management option as they are extremely low in mammalian toxicity and most forms are nonirritating to skin and mucous membranes. Their effects on insects include repellence, feeding deterrence, oviposition deterrence, reduced growth and development, and interference with reproduction. The female of some lepidopterous insects like cabbage webworm, Crocidolomia binotalis Zeller, Afro-Asian cotton bollworm,

Helicoverpa armigera (Hüb.) and fall army worm, Spodoptera frugiperda (J.E. Smith) did not

lay eggs on the plant parts treated with neem products. Similarly dipterous insects like Lucilia sericata (Meigen) was also deterred from egg laying on neem treated surface (Schmutterer,

1990). It also has a deterrent effect on feeding behavior of Schistocerca gregaria (Forskal) nymphs on barley seedlings at low doses (2 ppm) (Nasiruddin and Mordue (Luntz), 1993).

Similarly African cotton leafworm, Spodoptera littoralis (Boisd.) fall armyworm, S. frugiperda , tobacco budworm, Heliothis virescens (F.) and old world bollworm, H. armigera were prevented from feeding at concentrations of 0.1 - 10 ppm dependent upon species

(Blaney et al., 1990; Simmonds et al., 1990; Mordue (Luntz) et al., 1998).

Tillage which is an effective element of integrated pest management interrupts insect life- cycle stages in the soil or in crop residues. Soil-inhabiting pests such as rootworms, white grubs, wireworms and the overwintering larvae and pupae of Lepidoptera and Coleoptera may be exposed to desiccation or bird predation by ploughing. The pests that feed on stubble after harvest may starve if the ground is tilled (Speight et al., 1999). For example, the ploughing of stubble may result in a 90% reduction of hibernating larvae of European corn borer, Ostrinia nubilalis (Hübner) (Horn, 1988). Shallow spring and autumn tillage may provide up to 25% and

90% sawfly control, respectively (Steffey et al., 1992). There is scarcity of literature on the detection methods and the pest status and management of D. amaramanjarae from mango growing areas of Pakistan. The present research aimed at to develop monitoring techniques and management strategies of D. amaramanjarae. The specific objectives were;

 To determine occurrence and abundance of D. amaramanjarae in commercial mango

orchards and a few mango trees intercropped with wheat and cotton for family

consumptions

 To compare the relative effectiveness of different sticky traps (yellow, green, blue,

and colorless) for developing simple monitoring techniques

 To evaluate synthetic insecticides, neem seed kernel extract (NSKE) and soil hoeing

under mango trees to develop management techniques for D. amaramanjarae

3.2 MATERIALS AND METHODS

3.2 .1 Severity of infestation of D. amaramanjarae

Severity of D. amaramanjarae infestation at major mango growing areas was studied in commercial mango orchards having no intercrops planted in a geometry and keeping uniform distance from tree to tree at three locations i.e. Taranda Saway Khan, Rahim Yar Khan (District

Rahim Yar Khan: 28.3°N, 65.23°E), Regional Agricultural Research Institute, Bahawalpur

(District Bahawalpur: 29.59° N, 73.19° E), and Bosan Road, Multan (District Multan: 31.32° N,

71.4°E) during 2010 and 2011. These orchards consisted of different mango varieties like

Chounsa, Tota Pari, Langra, Sindhri, Anwar Ratool, Dosehri, Surrkha, Sarooli, Late Chounsa,

Lahotia, and Dasi. Mango trees were 8-40 years old. Annually, they were given 5-14 irrigations and 1-2 ploughing and manuring.

In March during 2010 and 2011, four trees having consistent inflorescences were selected randomly at each of location and 15 inflorescences were taken at 1- 3m from each of four mango trees. Inflorescences where larva was found were considered infested. Small farmers also plant mango trees usually intercropped with wheat and cotton for producing fruits at small scale.

Infestation of D. amaramanjarae was also recorded from four trees at three locations with similar methodologies mentioned earlier at same locations. Percent infestation was calculated from each locality with following formula.

Total Inflorescences-Healthy Inflorescences Percent infestation = x100 Total Inflorescences

In both seasons 2010 and 2011, percent mean of adults with standard error (Mean+SE) on

each locality was calculated. A comparison in infestation of D. amaramanjarae on mango trees among the commercial orchards at three localities, Rahim Yar Khan, Bahawalpur, and Multan and orchard types, commercial orchards and the orchards grown by small farmers was made by using two way analysis of variance (ANOVA).

3.2.2 Relative effectiveness of different traps for monitoring of D. amaramanjarae

Three sticky traps yellow, green, and blue measuring 10x10cm were tested for their efficacies to monitor the adults of D. amaramanjarae from January to April (for 120 days) in

2011 and 2012. For the preparing traps, plastic sheet was attached on color paper and sticky material (crystalline grease) was pasted on plastic sheet (annexure 3). We selected four mango trees and six traps of each color were hanged per tree in experimental orchard at Rahim Yar

Khan. Each tree was considered as a replication. Six colorless sticky traps also tied as control on each four trees. Traps were hanged on the tree for 24 hour in a week and brought to laboratory in plastic bags. Adults of D. amaramanjarae on the traps were counted using magnifying lens.

Mean numbers of adults with standard error (Mean+SE) on each trap was calculated and the efficacy of four color traps was determined by making comparison with ANOVA. LSD test

(95%) was used for separating the difference among various traps.

3.2.3 Management of D. amaramanjarae

During two mango inflorescence seasons in 2011 and 2012, trials on management of D. amaramanjarae were conducted at Rahim Yar Khan in randomized complete block design

(RCBD) Water solution of neem seed kernel extract (NSKE) was prepared by grinding and mixing it (@10 g/100 ml) in distilled water for 3-4 h in a beaker. It was then filtered through a muslin cloth which was squeezed into the beaker. The suspension thus obtained was taken as

10.0% solution (Singh and Singh, 1998). We selected five mango trees (Chounsa variety) for each treatment and each tree was considered as replicate. There were thus total 30 trees for the experiment. These treatments are listed in Table 1. During each year 2011 and 2012, hoeing

(7cm deep) and spraying of NSKE was done from December to April (for 151 days) at fortnightly intervals while bifenthrin was sprayed by farmer once in March. A plastic sheet

(1x1m) was spread under the canopy of each replicate. Numbers of the larvae of D. amaramanjarae dropped on plastic sheets spread under all trees were counted daily from

February to April (for 89 days) in 2011 and 2012. Mean numbers of the larvae per sheet (a tree) was calculated for each treatment and further analyzed with ANOVA. Differences among the treatments were separated by LSD test (95%).

Table I. Treatments evaluated for the managing D. amaramanjarae at Rahim Yar Khan during 2011 and 2012

Sr. No Treatments

T1 Spray of neem seed kernel extract (NSKE) (10.0% sol.) on the soil under mango tree

T2 Spray of neem seed kernel extract (NSKE) (10.0% sol.) on canopy of mango tree

T3 Spray of neem seed kernel extract (NSKE) on mango tree canopy+ racking (hoeing) of soil under the mango tree canopy

T4 Racking (hoeing) of soil under the mango tree canopy

T5 Talstar, 10 EC (Bifenthrin, an insecticide of pyrethroid group from FMC United group Lahore) sprayed by farmer with tractor mounted boom sprayer @ 125ml/250 Liters of water for one hectare of commercial formulation

T6 Control

3. 3 RESULTS

3.3.1 Severity of infestation of D. amaramanjarae

D. amaramanjarae was recorded from all the areas surveyed in Punjab including Rahim

Yar Khan, Bahawalpur, and Multan. It had earlier been reported from mango trees in India in

1960s and we reported it recently from Pakistan (Rehman et al., 2013a). Severity of infestation was determined in the terms of infestation on inflorescences of mango trees in commercial orchards and farmer fields at three localities. In 2010, the population of the pest among three localities i.e. Rahim Yar Khan, Bahawalpur and Multan was similar (F = 1.71; df = 2, 23; P <

0.21). However, it was significantly different between two types of the mango orchards (F = 923; df = 1, 23; P < 0.001). Mango trees planted in patches at small farmer fields received significantly lower infestation of D. amaramanjarae than commercial orchards. Interaction of mango region and orchard type was not significant (F = 1.35; df = 2, 23; P < 0.28). In following year, 2011, infestation did not vary significantly on all regions (F = 1.7; df = 2, 23; P < 0.22).

Mango trees in commercial orchards were damaged more by D. amaramanjarae than isolated trees at farmer fields (F = 181; df = 1, 23; P < 0.001). The interaction between regions and orchard types was not significant (F = 1.4; df = 2, 23; P < 0.25) (Fig. 1).

3.3.2 Relative effectiveness of different traps for monitoring of D. amaramanjarae

During 2011-12, the adults of D. amaramanjarae were caught on all traps at Rahim Yar

Khan. The pest remained active at the site from February to April as observed in weekly captures on different color traps. Maximum numbers were observed in March followed by February and

April. Adults caught on yellow trap were higher followed by green, blue, and colorless for the both years (percentages calculated from Fig. 2 and 4). Figure 3 and 5 present actual numbers of

adults captured in different color traps however for comparison percentages calculated from the

seasonal means for each color trap are described. In 2011, population of D. amaramanjarae was

first observed in third week of February on all traps and continued up to first/second week of

April. There was a significant difference among the mean numbers of adults on these four kinds

traps (F = 5.05; df = 3, 15; P < 0.02). Numbers of adults on colorless trap was significantly lesser

than all other three traps. However, no significant difference was observed in D. amaramanjarae

population on the yellow, green, and blue traps in this season. All treatments were more effective

than control colorless trap.

a a a a a a

b b b b b b

Fig.adults 1 Percent were mean caught (Mean on ± SE) all infestation traps from of secondD. amaramanjarae week of February on trees in tocommercial the third orchards week ofand April. farmer fields Rahim Yar Khan, Bahawalpur and Multan in 2010(left) and 2011(right) Maximum numbers were recorded in March followed by April and February. There was a Note: Bars topped with different letters are significantly different (LSD Test, α = 0.05)

In next year 2012, there was a significant difference in attraction of D. amaramanjarae towards

the four traps (F = 6.48; df = 3, 15; P < 0.01). Yellow and green received significantly higher

numbers of adults than control. In both the years, catches of adults of D. amaramanjarae were

not consistent on all sampling dates in each trap (Figs. 2-5).

3.3.3 Management of D. amaramanjarae

During 2011-12, neem seed kernel extract (NSKE), cultural practices and insecticide were applied on the canopy and the soil beneath the mango trees for the management of D. amaramanjarae at Rahim Yar Khan. Mean numbers of larvae plastic sheet-1 were the relative estimate of damage by D. amaramanjarae on all treatments. In 2011, analysis of variance

(ANOVA) detected a significant difference in the mean numbers of the larvae among the various treatments used for the management of the pest (F= 45.84; df, 5, 29; P<0.001). Pair wise comparison with least significant difference test (LSD at α =0.05) showed that control treatment recorded highest numbers of D. amaramanjarae larvae. Bifenthrin and NSKE spray on tree canopy with raking of soil had significantly better control of the pest than other treatments.

Dunnett's Multiple Comparisons with control (α=0.05) also indicated that numbers of larvae in all treatments were significantly lower than control (Fig. 6). In 2012, the difference among the various treatments was also significant (F=65.74; df=5, 29; P < 0.001). LSD (α=0.05) showed that mean larval population in all treatments were significantly lower than control. There was no significant difference in infestation between the treatment of bifenthrin and NSKE spray on tree canopy along with racking where least numbers of larvae were recorded (Fig. 6 and 7).

a a

Fig. 2 Comparison of different color traps (Mean±SE) for monitoring of D. amaramanjarae at

Rahim Yar khan in 2011

Note: Bars topped with different letters are significantly different (LSD Test, α = 0.05)

Fig. 3 Comparison of different color traps (Mean±SE) for monitoring of D. amaramanjarae at Rahim Yar khan in 2012

Note: Bars topped with different letters are significantly different (LSD Test, α = 0.05)

Fig. 4 Monitoring of D. amaramanjarae with traps of different colors at Rahim Yar Khan in 2011

Fig . 5 Monitoring of D. amaramanjarae with traps of different colors at Rahim Yar khan in 2012

b b bc c c

Fig. 6 Means of D. amaramanjarae populations in different treatments (Mean±SE) at Rahim

Yar Khan in 2011. (1. Sp. Ne. So= Spray of neem on soil. 2. Sp. Ne. Ca= Spray of neem on canopy, 3. Sp. Bi. Ca= Spray of bifenthrin on canopy)

Note: Bars topped with different letters are significantly different (LSD Test, α = 0.05)

b bc c cd d

Fig. 7 Means of D. amaramanjarae populations in different treatments (Mean±SE) at Rahim Yar Khan in 2012. (1. Sp. Ne. So= Spray of neem on soil. 2. Sp. Ne. Ca= Spray of neem on canopy, 3. Sp. Bi. Ca= Spray of bifenthrin on canopy)

Note: Bars topped with different letters are significantly different (LSD Test, α = 0.05)

3. 4 DISCUSSION

Studies on severity of infestation at different localities and planting pattern at Rahim Yar

Khan, Bahawalpur, and Multan indicated that mango trees grown by small farmer in their fields received significantly lower infestation of D. amaramanjarae than commercial orchards on all sites during two years 2010 and 2011. The lower infestation may be due to the reason that small farmers grow mango trees usually intercropped with wheat and cotton and use more numbers of cultural practices (annually 10 ploughings and 5 hoeings) for seed bed preparation and weeding of these crops (Khalil and Amanullah, 2004). Another reason might be the availability of food source for midges which is obviously more abundant in commercial orchards. Farmers use only

1-2 hoeings in commercial orchards in a year. D. amaramanjarae pupates and hibernates in the soil under the mango tree and higher numbers of cultural practices may interrupt life cycle of the pest by breaking down its pupation and hibernating places under mango tree (Prasad, 1966;

Grover and Prasad, 1966).

Numbers of adults on all sampling dates were not consistent. It was noted that there was no significant difference among color traps in 2011. Higher numbers (seasonal long mean) of D. amaramanjarae adult were caught on yellow, green and blue traps than colorless. In 2012, yellow and green received significantly higher numbers of adults than control. The phytophagous insects preferred yellow color more than darker colors such as blue and black (Meyerdirk et al.,

1979). Among the gall midges, sorghum gall midge, Stenodiplosis sorghicola females were attracted to yellow followed by green, red, and blue traps. They responded more quickly to yellow, followed by red, green, blue traps (Sharma and Franzmann, 2001). The use of yellow sticky traps has been documented for the monitoring of blueberry gall midge Dasineura oxycoccana Johnson (Plažanin et al., 2012).

The results illustrated that mean larval population on all treatments for management was significantly lower than control. In both the years, bifenthrin and NSKE spray on tree canopy with raking of soil had significantly better control of the pest than other treatments. Bifenthrin has already been reported to be used for controlling insect pests of mango in Pakistan (Saifullah et al., 2007). However, it has been reported to be toxic to some hymenopterans parasitoids

(Prabhaker et al., 2007). The neem based insecticides are considered as economical for controlling insect pests and safe for human beings and beneficial insects due to lesser residual toxicity (Caboni et al., 2006; Hasan et al., 1996). They act as systemic and as contact poisons and their effects are antifeedant, toxicological, repellent, sterility inducing or insect growth inhibiting. Furthermore, they are environmentally safe and have the potential to be adopted on commercial scale, together with other control measures in order to devise a low cost management strategy (Gahukar, 2000).

In conclusions, D. amaramanjarae was found active from February to April, therefore it is recommended to start monitoring of this pest from February or with the initiation of flowering.

We recommend detailed assessment of plant development scale (BBCH) of mango during active period of D. amaramanjarae. The use of NSKE with integration of racking of soil under the mango tree was as effective as bifenthrin for the control of the pest. Initially, the use of NSKE with integration of racking of soil under the mango tree on a small scale (on five trees) proved very effective in controlling the pest population. It is also more eco-friendly than synthetic pyrethroid group and contact-gastrointestinal track insecticides. In the future, there is need to evaluate the impact of these techniques on large scale for sustainable control of the pest. It is also recommended to explore the natural enemies fauna of D. amaramanjarae in order to improve the integrated pest management. Moreover, losses should be determined due to D. amaramanjarae

and timing of application of pest management practices need to be explored along with development of economic threshold or action threshold levels.

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Chapter 4

Preference of Dasineura amaramanjarae Grover and Procontarinia mangiferae (Felt) (Diptera: Cecidomyiidae) for different commercial cultivars of mango and their patterns of distribution

4. 1 INTRODUCTION

In natural ecosystems, plants and insects are continuously interacting in different ways.

Insects damage the plants through feeding, oviposition and seeking shelter. On the other hand, plants develop different defense mechanisms to reduce insect damage including chemical and physical barriers such as the induction of defensive proteins (Haruta et al., 2001), volatiles that attract predators of the insect herbivores (Birkett et al., 2000), secondary metabolites (Baldwin,

2001; Kliebenstein et al., 2001) and trichome density (Fordyce and Agrawal, 2001).

Different heritable characters (defense mechanisms) in host plants induce differential susceptibility to infestation of insect herbivores. This host plant resistance provides basis for pest management strategies. It is a simple and convenient method for insect pest control without any additional cost (Dent, 2000). The benefits from the resistance can be complete or partial. The economic advantage of using pest-resistant cultivars sometimes can reach at 120-fold greater return on investment (Lugenbill, 1969; Smith and Quisenberry, 1994). The Hessian-fly,

Mayetiola destructor (Say) resistant wheat provided a 9:1 return on investment of research in

Morocco (Koul et al., 2004).

In addition, resistant cultivars also reduce insecticide residues, protect ground water from contamination and improve the health, food and safety of people. This is usually because of the lower quantities of insecticides needed to manage insect pests in integration with the host plant resistance (Pedigo and Rice, 2009).

There is scarcity of research on host plant resistance in mango and its integration with other pest management options. However, some studies report the screening of mango cultivars for comparative the resistance to insect pests. For example, mango has a resistance for Noorda sp, Idioscopus sp. (Bagle and Prasad, 1984; Cunningham, 1989) and Sternochetum mangiferae

(Hansen, 1993) and different degrees of susceptibility towards A. oblique (Carvalho et al.,

1996). In Philippines, a wild mango, Mangifera altissima has been proved to be resistant to hoppers, tip borers and seed borers (Angeles, 1991). In South Africa and India various mango cultivars showed differing levels of susceptibility to mango gall fly, Procontarinia matteiana Kieffer and Cecconi (Githure et al., 1998). Similarly, a differential susceptibility of mango cultivars against leaf gall midge, Procontarinia mangicola has also been reported in

Pakistan (Muhammed et al., 2013). It was noted that susceptibilities of the mango cultivars to P. matteiana were associated with the certain terpenes emitted by mango flush (Augustyn et al.,

2010). Most of this research, however, needs to be assessed further.

Insect pest species are distributed in a habitat with a characteristic pattern according to their predisposed genetic behavior and the conditions of environment. Understanding the pattern of this spatial distribution provides the information about the structure of population which affects precision of the estimation population parameters in sampling. Moreover, the distribution of pests also governs the distribution of their natural enemies like predators and parasitoids as they usually follow similar patterns to their host (Kumari, 2003).

Mango industry in Pakistan is facing a number of problems including diseases and insect pests (Mahmood and Gill, 2002; Ishaq et al., 2004). About 250 insect pests of mango have been recorded in the world (Peña & Mohyuddin, 1997). Gall midges, thrips, leafhoppers, caterpillars, and mites are the major pests damaging mango (Peña et al., 1998). Mango gall midges are cosmopolitan in distribution on mango trees. They have been recorded feeding on inflorescence and fruit tissues, making galls on leaves of mango and their injury supports for the spread of anthracnose (Uechi et al., 2002; Askari and Bagheri, 2005; Kolisek et al., 2009).

Studies were initiated on mango gall midges in Pakistan recently. Dasineura amaramanjarae Grover and Procontarinia mangiferae (Felt) (Erosomiya mangiferae Felt) are among the gall midges complex damaging mango in Pakistan. They have been recorded from all mango areas surveyed in Punjab Pakistan. D. amaramanjarae feeds on flower buds. Female lays eggs near the stamens, larvae feed inside the flower buds and full grown larvae drop to the soil for pupation. In the case of severe infestation, total fruits may be destroyed (Grover and Prasad,

1966; Prasad, 1966; Rehman et al., 2013). P. mangiferae feeds on inflorescences and fruits

(Gagné, 2010; Rehman unpublished).

Present research was designed to determine varietal preference of newly discovered inflorescences midges on commercially grown varieties of mango and their distribution pattern on various parts of mango tree and orchard in Punjab, Pakistan. The specific objectives of present study were;

 To record populations of D. amaramanjarae and P. mangiferae on commercially grown

varieties of mango i.e., Chaunsa, Dusehri, Fajri, Sindhri, Anwar Ratul and Surkha

 To know distribution patterns of both gall midge species in different parts of mango

orchard with respect to the direction i.e., southern, northern, eastern, western and central

 To determine within tree distribution of these gall midges encompassing southern,

northern, eastern and western sides of upper and lower canopies of mango tree

4.2 MATERIALS AND MATHODS

4.2.1 Varietal preference

All experiments were conducted in a mango orchard at Rahim Yar Khan (28.3°N,

65.23°E) in 2011 and 2012. Adults and larvae of D. amaramanjarae and P. mangiferae were recorded on six commercially grown cultivars of mango i.e., Chaunsa, Dusehri, Fajri, Sindhri,

Anwar Ratul and Surkha. The experiment was arranged in randomized complete block design

(RCBD). Four trees of similar size and age (18-20 years) of each cultivar were selected and each tree was considered as a replicate. To record the numbers of adults, a yellow sticky band (10x10 cm) was tightened on inflorescences at 2 m height from the ground on each tree for two days in a week during flowering. Mango trees remain in flowering stage in this region from February to

April (Rehman et al., 2013). Numbers of adult of both gall midge species were noted from traps with magnifying glass (10X).

To record the larvae, four inflorescences (one from each tree) were covered separately with a plastic bag (45cm long) having few pin holes for ventilation in the morning hours. The bags remained for 24 hours in each week during flowering period. The larvae were recorded from the same trees selected for recording adults. These plastic bags were removed and numbers of larvae of each species were counted. Mean numbers of adults (from traps) and larvae (from plastic bags) of both species captured on each cultivar were calculated and analyzed for significance of difference in cultivars with analyses of variance (ANOVA) using Software

Satatistix 8 (Analytical Software 2003). The difference among the mean numbers of larvae and adults was calculated with LSD at 5% level of significance (Gomez and Gomez 1984).

4. 2. 2 Distribution patterns

To determine the distribution patterns within orchard, eight trees were selected from each of five regions i.e., central, southern, northern, eastern and western sides of mango orchard (total trees 40) of an area of 5.26 ha. An inflorescence from each selected tree of the region (i.e. eight from each region and total 40) was covered with a plastic bag for 24 hours for recording gall midges at weekly intervals. Mature larvae escaped from the inflorescence into plastic bags were collected and brought to laboratory then number of larvae of both gall midge species were separated and counted. Mean numbers of larvae were calculated and analyses of variance

(ANOVA) were employed for comparing population of both species on various parts of orchard and mango tree.

To determine the distribution patterns within tree, five mango trees were selected randomly in orchard. Four inflorescences from the southern, northern, eastern and western sides of lower canopy (1-3m) from each of five selected trees (total 20) were covered with plastic bag for 24 hours. Similarly four inflorescences per tree (total 20) from upper canopy (3-6m) from four directions as mentioned earlier were also covered. Gall midge larvae trapped in plastic bags were separated into both species and their numbers were counted.

4. 3. RESULTS

4. 3.1 Varietal preference

Analysis of variance (ANOVA) detected no significance difference among the mean numbers of adults of D. amaramanjarae trapped on six varieties in both the years (F2011= 1.34; df=5, 25; P < 0.3 and F2012= 1.35; df=5, 25; P < 0.29). However, there was a significant difference among the larvae of D. amaramanjarae on different varieties (F=5.19;df=5, 25; P <

0.001) in 2011. More numbers of larvae were recorded on Surkha and Dusehri as compared to

Chaunsa, Anwar Ratul and Sindhri but the number of larvae in these two varieties didn’t differ significantly from Fajri in 2011. In the next season (2012), a significant difference among the mean numbers of larvae as also observed among the varieties (F=8.79;df=5, 25; P < 0.001). The population on Surkha was significantly higher than all other varieties (LSD at α = 0.05).

However, gall midge density of Dusehri didn’t differ significantly from Chuansa in 2012 (Table

I).

Numbers of adults of P. mangiferae on six varieties were similar in 2011 and 2012

(F2011= 1.56; df=5, 25; P < 0.22 and F2012= 1.32; df=5, 25; P < 0.30). Similarly in 2011 larvae were also similar in numbers on all tested varieties (F=2.22; df=5, 25; P < 0.10). However, in

2012 there was a significant difference in the numbers of P. mangiferae larvae among different varieties (F=13.8; df=5, 25; P < 0.001). Surkah had statistically higher infestations than all other varieties. Numbers of P. mangiferae adults and larvae were higher than that of D. amaramanjarae on most of the varieties (Table II).

4. 3.2 Distribution patterns

Analysis of variance (ANOVA) detected a significant difference in numbers of larvae of

D. amaramanjarae among various locations of orchard in 2011(F=6.21;df=4, 39; P < 0.001).

Significantly higher numbers of larvae were recorded in trees located on Southern and central part of orchard. These parts received statistically more numbers of larvae than north and west sides in 2012 (F=3.11;df=4, 39; P < 0.03).

Table. I Mean numbers of larvae and adults of D. amaramanjarae in 2011 and 2012 on different mango varieties at Rahim Yar Khan

Mango

Cultivars 2011 2012 2011 2012

Adults Larvae

Chaunsa 13.5±2.8 6.7±1.1 1.5±0.6b 1.2±0.6bc

Dusehri 17.0±3.0 5.5±1.3 4.5±0.6a 2.5±0.3b

Fajri 9.2±1.3 3.5±0.2 3.2±1.4ab 0.2±0.1c

Sindhri 8.5±1.7 4.0±2.2 0.7±0.5b 1.5b±0.6c

Anwar Ratul 11.5±5.6 6.0±1.2 1.0±0.4b 0.2±0.2c

Surkha 17.0±2.6 7.0±0.9 5.5±0.8a 4.0±0.4a

LSD N S* N S 2.6 1.4

*Non-significant

Table II. Mean numbers of larvae and adults of P. mangiferae in 2011 and 2012 on different mango varieties at Rahim Yar Khan

Mango

Cultivars 2011 2012 2011 2012

Adults Larvae

Chaunsa 179.75±27.4 303.5±16.2 0.5±0.5 15.7±1.7bc

Dusehri 214.0±26.9 350.7±15.0 2.2±0.7 13.0±0.8bc

Fajri 185.0±14.5 334.7±14.0 2.7±1.7 12.5±1.9bc

Sindhri 186.25±19.6 320.2±30.1 0.2±0.2 12.0±1.7c

Anwar Ratul 188.75±37.1 357.0±11.9 0.7±0.4 18.2±2.0b

Surkha 291.0±57.2 357.0±14.5 3.7±1.0 32.2± 4.0a

LSD N S* N S N S 6.2

*Non-significant

In 2011 mean numbers of larvae of P. mangiferae on various orchard sites were not significantly different (F = 1.5; df = 4, 39; P < 0.22). The difference in mean numbers of larvae among various sites was significant in 2012 (F = 9.65; df = 4, 39; P < 0.001). They were significantly higher in Southern sites than other parts of the orchard (Table III).

Larvae of D. amaramanjarae were different in all four quadrants of mango tree and higher on Southern side in 2011 (F = 6.41; df = 3, 39; P < 0.001). In 2012, the difference between mean numbers of larvae was not significant (F = 0.45; df = 3, 39; P < 0.72). There was a significant difference in mean numbers of larvae of P. mangiferae among four quadrants in

2011(F = 5.81; df = 3, 39; P < 0.001). Southern parts received higher numbers of the larvae than other parts. Infestation was similar on all strata in 2012 (F = 2.8; df = 3, 39; P < 0.06) (Table

IV).

Numbers of larvae of D. amaramanjarae were higher in lower half of the trees in

2011and (F2011 = 14.7; df = 1, 39; P < 0.001) in 2012 (F2012 = 9.2; df = 1, 39; P < 0.001).

Similarly, a significant difference was observed in mean numbers of larvae of P. mangiferae between upper and lower parts of mango tree (F2011 = 4.2; df = 1, 39; P < 0.04) (F2012 = 8.44; df =

1, 39; P < 0.001) for both the years. More larvae were recorded on lower canopy than upper parts of the tree (Table V).

Table. III Mean numbers of larvae of D. amaramanjarae and P. mangiferae in 2011 and

2012 on various locations of mango orchard at Rahim Yar Khan

Orientations of

mango orchard 2011 2012 2011 2012

D. amaramanjarae P. mangiferae

South 7.8±1.6a 8.7±1.7a 13.6±5.7 86.7±16.8a

North 2.2±0.9b 3.2±2.1c 2.5±1.4 27.7±8.1bc

East 2.7±0.3b 4.0±0.7bc 6.5±3.7 21.6±3.7c

West 3.2±1.2b 3.2±1.2c 8.6±2.8 24.5±2.7bc

Centre 7.5±1.2a 7.6±1.1ab 5.7±1.4 47.5±6.7b

LSD 3.1 4.2 N S* 25.3

*Non-significant

Table. IV Mean numbers of larvae of D. amaramanjarae and P. mangiferae in 2011 and

2012 on South, North, East and West quadrants of mango tree at Rahim Yar Khan.

Orientations of D. amaramanjarae P. mangiferae

mango tree 2011 2012 2011 2012

Mean Mean Mean Mean

South 60.0±5.8a 3.7±1.6 73.7±7.5a 21.4±4.8

North 30.4±4.0b 1.9±0.7 45.5±5.4b 10.5±2.4

East 41.0±4.8b 2.5±0.8 52.9±5.5b 12.3±3.3

West 41.8±3.6b 3.2±1.1 47.1±5.8b 9.2±2.3

LSD 13.9 N S* 15.5 N S

*Non-significant

Table. V Mean numbers of larvae of D. amaramanjarae and P. mangiferae in 2011 and 2012 on lower and upper parts of mango tree canopy at Rahim Yar Khan.

Orientations of

mango tree 2011 2012 2011 2012

D. amaramanjarae P. mangiferae

Lower 52.8±3.3a 4.4±0.9a 61.14.0a 18.1±2.4a

Upper 33.7±3.4b 1.2±0.4b 48.55.3b 8.6±2.2b

LSD 10.1 2.1 12.4 6.6

4.4 DISCUSSION

From the research it was noted that there was no significant difference among the mean numbers of adults of D. amaramanjarae and P. mangiferae trapped on six varieties in both the years. However, the larvae of D. amaramanjarae were more abundant on cultivar Surkha,

Dusehri and Fajri in 2011 and on Surkha in 2012. Though, the varietal differences in mango induce differential susceptibility towards insect pests in different parts of the world, there is scarcity of consistent research regarding the resistance in mango cultivars to gall midges. Most of the published research reports only susceptibility/resistance based upon the occurrence and abundance of their population or their damage. In South Africa and India, P. matteiana caused different levels of damage to various mango cultivars (Githure et al., 1998). Similarly, a differential susceptibility of mango cultivars against leaf gall midge, P. mangicola has also been reported in Pakistan (Muhammed et al., 2013).

Variations in the density of both gall midges were observed in cultivars like Chaunsa,

Anwar Ratul and Sindhri during two seasons. Chaunsa is the most popular commercial cultivar due to its taste (personal observations). Anwar Ratul and Sindhri are second to commercial preference of growers. The variation towards density of gall midges on cultivars might be contributed due to several reasons like nutritional level, types of the nutrients, weather factors etc. These factors have been proved to be responsible for changing resistance level in host to insects since early studies on plant resistance (Zafar et al., 2010; Khan et al., 2013) and recently in some studies it was reported that change in the biological and chemical properties of soils had negative effect on the pest damage (Altieri & Nicholls, 2003). Susceptibilities of the mango cultivars to P. matteiana were associated with the certain terpenes emitted by mango flush

(Augustyn et al., 2010). Interactive effect of chemical substances associated with attraction of

D. amaramanjarae and P. mangiferae in mango cultivars and populations of these gall midges need to be explored for the possible application plant resistance component in IPM.

The infestation of both inflorescence gall midges was more on lower (1-3m) region of tree canopy than upper parts (3-6m). The populations of D. amaramanjarae and P. mangiferae were higher on the southern sides of mango tree in 2011. In another study, we used funnel ring method for sampling the larvae to determine within tree distribution of D. amaramanjarae in the same region in 2009 and 2010 (Rehman et al., 2013). But funnel ring method was labor intensive as larvae of mango leaf gall midges were also captured in the funnel rings. This method took more time to separate the larvae of leaf gall midge species. However, both methods proved that the density of D. amaramanjarae was higher on lower and southern sides of the trees.

Studies of distribution patterns in a mango orchard showed that it was in the most abundant on southern sides and central parts. Density of P. mangiferae was higher on the southern side but changed on other sides in two seasons. Mango gall midges follow different patterns of distribution. In India, P. matteiana shows an aggregated pattern of distribution while Erosomyia indica (now synonymized with P. mangiferae) tends to follows a random pattern. Its distribution was not significantly different on various horizontal and vertical sections of mango tree

(Verghese et al., 1988; Verghese and Rao, 1988).

Several reasons seem plausible for greater abundance of midges on southern side of trees and orchard with respect to environment of Punjab. Direction of the sun in winter and spring months with its lower arc on southern side provides more light to this side (Lea, 2010). As reported in the past light attracts gall midges (Kashyap, 1986) and also influences their distribution in trees (Lebel et al., 2008; Khan, 2010). So there will be greater intensity of light on southern sides subsequently attracting more midges. Similarly, cool winds blow from north

during the activity period of gall midges (Gosal, 2004) and southern side of orchard and trees may provide cushion from coolness (also discussed in chapter 2). The difference in distribution patterns of P. mangiferae and D. amaramanjarae from other gall midge species may also be attributed to unequal distribution of inflorescences on mango tree canopy as a result of seasonal variation in flushing and blooming behavior of mango (Issarakraisila et al., 1991). However, these reasons for greater populations of midges on south and variations on other quadrants shall stand provisional unless confirmed by quantitative measurements of weather factors, presence of reproductive parts in trees and their interaction with abundance of midges. The degree of variability in the capacity of mango cultivars to recover/tolerate after damage by midges should also be determined for incorporation of tolerance mechanism of resistance in IPM.

It is concluded that densities of D. amaramanjarae and P. mangiferae were more on cultivar Surkha/Dusehri, southern side of mango orchard, lower parts and southern quadrant of mango tree canopy. From the pest management point of view, popular commercial cultivars

(Chaunsa, Anwar Ratul and Sindhri) are not the most preferred for gall midges. A caution is needed while applying insecticides that these reach to lower part of canopy as well as the

Southern sides of the trees. Further research is needed to determine the relationship between numbers of gall midges and yield loss on commercial cultivars. This will also help in applying insecticides too. Moreover, as a long term strategy, yield loss relationship should also be studied in germplasm particularly rootstock for developing resistant cultivars.

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Chapter 5

Biology, damage patterns, monitoring and management of newly recorded mango gall midge, Procontarinia mangiferae (Felt) in Pakistan

5. 1 INTRODUCTION

The gall midges (Diptera: Cecidomyiidae) are small delicate flies with different range of feeding behavior being phytophagous, predaceous, parasitic or saprophagous. The phytophagous midges feed on or within plants as cecidogenous or gall inducing species (Barnes, 1948; Hill,

1987). They are the pests of mango, Mangifera indica L. in Brazil, China, Guadeloupe, Hawaii,

India, Iran, Japan, Kenya, Mauritius, Oman, Philippines, Reunion, South Africa, Taiwan, United

Arab Emirates and many other parts of the world (De Villiers, 1998; Gagné 2004; Ahmed, et al.,

2005). Gall midges destroy inflorescence and fruit tissues of mango trees by making galls

(Askari and Bagheri, 2005; Kolisek et al., 2009). Damaged parts assist epidemics of anthracnose,

Colletotrichum gloeosporioides Penzig and Saccardo, 1884 (Uechi et al., 2002). Losses due to anthracnose have been estimated to be 2-39% in India (Prakash and Srivastava, 1996).

Gall midges were not reported as the pest of mango from Pakistan till recent. They were ranked number one pest in a survey during 2006 in Pakistan. There was scarcity of knowledge for the management of this group of the pest (Ahmed et al., 2005; Anonymous, 2006). Even the species were not reported from the mangoes. Studies were initiated on identification, damage patterns, biology and ecology to develop management strategies for gall midges associated with mangoes in Pakistan. In the recent research Procontarinia matteiana Kieffer & Cecconi,

Dasinura amaramanjarae Grover (Rehman et al., 2013 and 2014) and Procontarinia mangiferae

(Felt) (see section 5.2.2) have been recorded among the mango gall midges complex from

Pakistan.

Phenology, population trends, biology, distribution, damage patterns and management of

P. matteiana and D. amaramanjarae in Punjab Pakistan has been reported. P. matteiana damaged mangoes by forming solitary or grouped galls on the upper and lower surfaces of the

leaves. It was active from February/March to November with two peaks of its population, first in

March/April and second in September/October. The phenology of two parasitoids, Closterocerus pulcherrimus (Kerrich) and Synopeas temporale Austin reared from galls of P. matteiana was well synchronized with their host (Rehman et al., 2013).

Dasinura amaramanjarae was active from January/February to April with peak in

March. Female laid eggs on inflorescence tissues and larvae after feeding dropped to the soil under the mango tree for pupation/diapuse (Rehman et al., 2013a). Application of bifenthrin

(Talstar 10EC) and seed kernel extracts (NSKE) with racking of soil under the tree copy were proved the best in controlling D. amaramanjarae (Rehman et al., 2014).

Procontarinia mangiferae was recorded from all the areas surveyed in Punjab including

Rahim Yar Khan, Bahawalpur and Multan. This pest has spread from north-eastern Indian to

Guadeloupe, Brazil, West Indies, Kenya, South Africa, Java, Indonesia Thailand, Mauritius,

Reunion Island, Iran and Brazil (De Villiers, 1998; CABI, 2004). Other synonyms of the species are Erosomyia mangiferae (Felt) 1911, Mangodiplosis mangiferae Tavares 1918, Rhabdophaga mangiferae Mani 1938 and Erosomyia indica Grover 1965 (Gagne´ and Medina, 2004;

Amouroux et al., 2013).

Famers relay on the use of pesticide sprays to control mango insect pests in Pakistan

(Saifullah et al., 2007). However, the intensive use of pesticides may result in the problems such as insecticide resistance, loss of natural enemies, secondary pest outbreaks and environmental contamination (Zadocks, 1993; Dent, 1995). It is important to focus on the optimal combination and integration of monitoring tools and management tactics for the early detection of pests and precision spray applications to reduce the treatment area and quantity of pesticides. Earlier

detection could also allow the grower to use non-chemical measures like biological control, application of plant extracts, beneficial organisms and culturing measures more efficiently

(Zijlstra et al., 2011).

Integrated pest management system which involves optimal combination and integration pest management strategies includes obtaining the information about the pest, making decisions based on this information and taking action against a pest (Ruesink and Onstad, 1994).

Monitoring is the foundation of IPM as it provides the information about distribution of pest, efficacy of control measures and the variables required to develop forecasting system for pest outbreaks (Conway, 1984). Trapping is used in monitoring of the pest population and reduction of pesticide by providing information for accurate timing of sprays (Vale, 1982; Grieshbach,

2011). In India, the application of pesticides on mango trees and soil at the peak of population have been proved very effective in controlling P. mangiferae and other mango inflorescence gall midges (Grover, 1985).

Botanicals and cultural practices are another integral part of integrated pest management as they are more environment friendly. Botanicals reduce the resistance of the pest, application of pesticides and thus the cost of pest management (Regnault-Roger, 1997; Khorram et al.,

2011). Neem products have a potential value in pest management due to their selectivity towards phytophagous pests and minimal toxicity to beneficial insects (Naumann and Isman 1996;

Bhanukiran and Panwar, 2000). In Nigeria, the use of neem seed extract suppressed significantly the population of African rice gall midge (AfRGM), Orseolia oryzivora (Ogah and Ogbodo,

2012). In India, the cultural practices like cleaning, manuring, irrigating and hoeing the soil under the mango trees had been used for controlling mango gall midges (Prasad, 1966; Grover and Prasad, 1966; Grover, 1985). Present research aimed at;

 To survey for collection and presence of P. mangiferae.

 To compare the severity of infestation in commercial mango orchards with the mango

trees usually intercropped with wheat and cotton for producing fruits at small scale.

 To determine the seasonal abundance of P. mangiferae

 To evaluate the relative effectiveness of different color traps (yellow, green, blue, and

colorless) for developing monitoring techniques of P. mangiferae adults

 To compare various control methods for managing the pest

5. 2. MATERIALS AND METHODS

5. 2.1 Surveys at different regions for presence of P. mangiferae

Surveys were conducted for the presence of P. mangiferae mango trees in various regions of Punjab. Mango orchards were selected at Tranda Sway Khan (District Rahim Yar Khan, three commercial orchards), Regional Agricultural Research Institute (District Bahawalpur, two commercial orchards) and near Bahaudin Zakaria University at Bosan Road, (District Multan, three commercial orchards) in 2008 to 2010. Ten inflorescences were examined at least once a month from 10-12 mango trees in each orchard at all locations except for District Rahim Yar

Khan where inflorescences were weekly sampled during February to April of each year (Rehman et al., 2013a).

5.2.2 Damage patterns

Larvae of the gall midge complex were collected from different parts of mango trees like leaves, flowers, axillaries and small sized fruits and reared separately in the laboratory at Rahim

Yar Khan. The larvae reared from axillaries and small sized fruits were of P. mangiferae. Adults were preserved in 70-75% ethanol and deposited as voucher specimens at CABI – Central and

West Asia, Rawalpindi. The identification was confirmed from experts at Natural History

Museum U. K.

Severity of P. mangiferae infestation was determined in commercial mango orchards having no intercrops planted in a geometry and keeping uniform distance from tree to tree at three locations (mentioned above) during 2010 and 2011. In March, four trees were selected randomly at each of location and 15 inflorescences were selected from each of four mango trees.

The inflorescences with the symptoms of damage (black spots with holes on axillaries and small

fruits) were considered infested. Small farmers also plant mango trees usually intercropped with wheat and cotton for producing fruits at small scale. Infestation of midges was also recorded from five trees at three locations with similar methodologies mentioned earlier at same locations

(Rehman et al., 2014).

For both the seasons (2010 and 2011) percent infestation of inflorescences was calculated and converted to means with standard error (Mean+SE) for all localities. A two way analysis of variance (ANOVA) was done for making comparison in infestation on mango trees at three localities, Rahim Yar Khan, Bahawalpur, and Multan and orchard types, i.e., commercial orchards and the mango tree of small farmers.

5.2.3 Monitoring the larvae of P. mangiferae

Monitoring of larval population of P. mangiferae was carried in a mango orchard at

RahimYar Khan during from initiation of mango to maturity of the fruits (February to April) in each of three years 2009-11. For this purpose five trees of commercially important variety of mango, i.e. Chaunsa were selected two twigs carrying inflorescences from each tree were cut at about 1.5 m height above ground weekly and bought to laboratory. These were kept in small jars with their lower ends dipped in water as described earlier. The small jars were then placed in large plastic jars covered with muslin cloth tightened with rubber bands. These infested inflorescences were examined for the numbers of the larvae of P. mangiferae and its parasitoids.

Literature reports that P. mangiferae damages mango in almost whole the year in some countries

(Amouroux et al., 2013). Therefore monitoring of P. mangiferae was continued from May to

January after fruits had been matured. For this purpose 50 mango infested leaves were examined

at weekly intervals for the larvae of P. mangiferae using the methodology mentioned for inflorescences (Rehman et al., 2013).

5.2.4 Monitoring of adults of P. mangiferae and relative efficacies of different color traps

The efficacies of three color sticky traps (10x10cm) yellow, green, and blue were tested for monitoring the adults of P. mangiferae from January to April (for 120 days) in 2011 and

2012. We selected four mango trees and six traps of each color were hanged on inflorescence of each tree in experimental orchard at Rahim Yar Khan (See 3.2.2 of chapter 3 and annexure 3 for details). Each tree was considered as a replicate. Six colorless sticky traps also tied as control on each four trees. Traps were hung on the tree for 24 hours in a week and brought to laboratory.

Adults of P. mangiferae on the traps were counted using magnifying glass. Mean numbers of adults per trap was calculated and the efficacy of four color traps was determined by making comparison of four traps with ANOVA. LSD test was employed for separating the difference among various traps (Rehman et al., 2014).

5.2.5 Management of P. mangiferae

Trials on management of P. mangiferae were conducted at Rahim Yar Khan in

Randomized Complete Block Design (RCBD) in 2011 and 2012. Six treatments applied were;

(T1) Spray of neem seed kernel extract (NSKE) (10% sol.) on the soil under mango tree, (T2) spray of neem seed kernel extract (NSKE) (10% sol.) on canopy of mango tree, (T3) spray of neem seed kernel extract (NSKE) on mango tree canopy with racking (hoeing) of soil under the mango tree canopy, (T4) racking (hoeing) of soil under the mango tree canopy, (T5) Talstar, 10

EC (Bifenthrin, an insecticide of pyrethroid group from FMC United group Lahore) sprayed with tractor mounted boom sprayer @ 125ml/250 Liters of water for one hectare of commercial

formulation and (T6) control Water solution of neem seed kernel extract (NSKE) was prepared by grinding and mixing it (@10 g/100 ml) in distilled water for 3-4 h in a beaker. It was then filtered through a muslin cloth which was squeezed into the beaker. The suspension thus obtained was taken as 10% solution (Singh and Sing, 1998).

We selected five mango trees (Chounsa variety) for each treatment and each tree was considered as replicate. There were thus total 30 trees for the experiment. During each year 2011 and 2012, hoeing and spraying of neem seed kernel extract (NSKE) was done from December to

April (for 151 days) at fortnightly intervals while bifenthrin was sprayed once in March. A plastic sheet (1x1m) was spread under the canopy of each replicate. Numbers of the larvae of P. mangiferae dropped on plastic sheets spread under all trees were counted daily from February to

April (for 89 days) in 2011 and 2012. Mean numbers of the larvae per sheet (a tree) were calculated for each treatment of a season and further analyzed for ANOVA. Differences among the treatments were separated by LSD test (Rehman et al., 2014).

RESULTS

5.3.1 Surveys at different regions for presence of P. mangiferae

Procontarinia mangiferae was recorded in all mango areas of Punjab surveyed in Rahim

Yar Khan, Bahawalpur and Multan.

5.3.2 Damage patterns

Infestation of P. mangiferae was observed on inflorescence buds, small leaves, branches

(axillaries) and small sized fruits. Its larvae caused damage by inducing galls on inflorescence buds and leaves, making tunnels in the branches (axillaries) and feeding on small sized fruits. In the case of severe damage, branches of inflorescences bent at right angle (Fig. 1-3). The infested leaves, inflorescences and fruits usually dried and dropped from the mango tree. At their final stage, larvae moved to the soil for pupation.

The damage of P. mangiferae at different regions of Southern Punjab was recorded in the terms of presences of larvae, black spots with small holes, tunnels inside the inflorescence

(axillaries) and small damaged fruits. In both the years, P. mangiferae was recorded on all mango trees in commercial orchards and farmer fields at Rahim Yar Khan, Bahawalpur and

Multan. In 2010, its damage was similar (F = 1.22; df = 2, 23; P < 0.32) in orchards among three localities Rahim Yar Kahn, Bahawalpur and Multan. The difference in damage between commercial orchard and the trees at small farmer fields was statistically significant (F = 4890; df

= 1, 23; P < 0.001). Commercial orchards received higher population of P. mangiferae than trees at farmer field. The interaction between regions and farm types (Region x Farm type) was not significant (F = 2.88; df = 2, 23; P < 0.08). In next season 2011, There was no significant difference among the damages at three locations (F = 1.4; df = 2, 23; P < 0.34). Greater

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infestations of P. mangiferae were in commercial orchards as compared to the scattered trees intercropped with wheat and cotton (F = 274; df = 1, 23; P < 0.001). Localities and orchard types

(commercial vs small) did not affect the infestation of P. mangiferae (F = 3.04; df = 2, 23; P <

0.07) (Fig. 4).

Fig. 1 Infestation of P. mangiferae: Exit holes (Left) and gallery (Right) on mango inflorescence axillaries.

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Fig. 2 Infestation of P. mangiferae : Mango axillary bent at right angle (Left) and damaged inflorescence (Right).

Fig. 3 Infestation of P. mangiferae : Damaged small sized mango fruits

5.3.4 Monitoring the larvae of P. mangiferae

Studies on monitoring of P mangiferae larvae were conducted at Rahim Yar Khan from

February to April from 2009 to 2011. In 2009 they first appeared in second week of February

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and reached at peak in the second week of the March. They were reduced to zero in third week of

April and were not recorded onward on mango inflorescence. In the next season 2010, they appeared in third week of February with peak in second week of the March.. Population decreased onward and reached to zero in first week of the April. Larvae were not observed onward till next flowering season (May 2010 to January 2011). In 2011, larvae of the pest were recorded for the first time in last week of the February. Maximum numbers were observed in third week of the March which reduced to zero in the third week of April (Fig. 5- 6).

A hymenopteran parasitoid was recorded from P. mangiferae in inflorescence axillaries and small mustard sized fruit at Rahim Yar Khan. Species could not be identified as it culture declined due to unknown reasons. Its populations fluctuated along with its host P. mangiferae. It was present from February to April with peak of population in March (Fig. 7).

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a a a a a a

b b b b b b

Fig. 4 Percent infestation of P. mangiferae on trees in commercial orchards and farmer fields at Rahim Yar Khan, Bahawalpur and Multan in 2010 and 2011

5.3.3 Monitoring of adults of P. mangiferae and relative efficacies of different color traps

Monitoring of P. mangiferae adult was carried out in a mango orchard at Rahim Yar

Khan from January to April in 2011 and 2012. Adults of P. mangiferae were recorded on all traps in 2011. A small numbers of the adults were collected in the third week of the January which swiftly increased in February and reached to minimum in the last week the April. More than two peaks were observed during whole period of activity. Maximum numbers were recorded on yellow trap followed by green, blue and control (for season long means on all traps).

The difference among the numbers of the adults on traps was statistically significant (F = 6.67; df = 3, 15; P < 0.01). Numbers of adults on colorless trap was significantly lower than all traps.

There was no significant different among color traps. In the coming season 2012, the population of P. mangiferae started appearing in January, increased in February and reached at lowest level at the end of April. Numbers of the adults collected from four traps were significantly different

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(F = 93.32; df = 3, 15; P < 0.001) with maximum numbers on yellow (32.46%) followed by green (25.88%), blue (25.71%) and control traps (15.94%). Numbers of adults on yellow trap were significantly higher than all traps. It was noted that overall numbers of adults captured on yellow trap were higher than traps. The catches of adults on all traps were not consistent during different sampling dates (Fig. 8-11).

Fig. 5 Mean weekly populations of P. magiferae at Rahim Yar Khan in the months of February March and April from 2009 to 2011

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Fig. 6 Weather factors at Rahim Yar Khan in the months of February March and April from 2009 to 2011

Fig. 7 Monitoring of P. mangiferae and its parasitoid at Rahim Yar Khan in 2009 and 2010

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2011

Fig . 8 Monitoring of P. mangiferae with different color traps at Rahim Yar khan in 2011

2012

Fig . 9 Monitoring of P. mangiferae with different color traps at Rahim Yar khan in 2012

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2011 a

a a

Fig. 10 Comparison of different color traps (Mean±SE) for monitoring of P. mangiferae at Rahim Yar khan in 2011

Note: Bars topped with different letters are significantly different (LSD Test, α = 0.05)

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a 2012

b b

Fig. 11 Comparison of different color traps(Mean±SE) for monitoring of P. mangiferae at Rahim Yar khan in 2012

Note: Bars topped with different letters are significantly different (LSD Test, α = 0.05)

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5.3.5 Management of P. mangiferae

Experiments were conducted to evaluate management options and farmer practices in mango orchard at Rahim Yar Khan in mango flowering season during 2011 and 2012. The efficacy of different management practices was estimated through the mean numbers of the larvae of P. mangiferae per plastic sheet spread under the mango trees. In 2011, the numbers of the larvae collected from in all treatments were significantly different (F = 228; df = 5, 29; P <

0.001). Bifenthrin, with minimum numbers of the larvae was the most effective in suppressing the population of P. mangiferae followed by neem seed kernel extract (NSKE) spray on tree canopy with racking of soil, neem seed kernel extract (NSKE) spray on tree canopy, racking of soil, Spray of neem seed kernel extract (NSKE) on the soil under mango tree and control. The larvae in control were significantly higher than all treatments.

In 2012, a significant difference was observed among all the treatments at Rahim Yar

Khan (F = 212.78; df = 5, 29; P < 0.001). The second best treatment was neem seed kernel extract (NSKE) spray on tree canopy with racking of soil where the numbers of larvae were also significantly lesser than control. The numbers of the larvae collected in best treatment

(bifenthrin) were not significantly lower than this treatment. The remaining three treatments as racking of soil, neem seed kernel extract (NSKE) spray on tree canopy and spray of neem seed kernel extract (NSKE) on the soil under mango tree were intermediate as compared to others

(Fig. 12 and 13).

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b c c d e

Fig. 12 Means (Mean±SE) of P. mangiferae populations in different treatments at Rahim Yar Khan in 2011

(1. Soil= Spray of neem on soil. 2. Canopy= Spray of neem on canopy, 3. Bifenthrin = Spray of bifenthrin on canopy, 4. Racking= Racking of soil), 5. Canopy+ra= Spray of neem on canopy+racking of soil) Note: Bars topped with different letters are significantly different (LSD Test, α = 0.05)

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b c d e e

Fig. 13 Means (Mean±SE) of P. mangiferae e populations in different treatments at Rahim Yar Khan in 2012

Note: Bars topped with different letters are significantly different (LSD Test, α = 0.05)

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5. 4. DISCUSSION

Present research is the first report of distribution, damage patterns, intensity of infestation, biology, monitoring and management of P. mangiferae in mango growing areas of

Punjab Pakistan. It was recorded from all mango growing areas surveyed including Rahim Yar

Khan, Bahawalpur and Multan. Larvae damaged inflorescence buds, leaves, branches (axillaries) and small sized fruits. The infestation of this species was identified through black spots with circular exit holes and tunnels formed by the larvae inside the inflorescence axillaries usually bent at right angle. Damage on small fruits was identified by the signs of larval feeding, small holes. Such fruits become dried.

Similar damage pattern of P. mangiferae has been reported in other mango growing areas of the world. At Hormozgan, Iran, females laid eggs on inflorescence tissues, larvae after hatching started feeding and making small tunnels in branches. After feeding had been completed, they entered the soil for pupation (Pezhman and Askari, 2004; Askari and Bagheri,

2005). In India, three generations of P. mangiferae were recorded; first usually induced blister like galls on fleshy leaves surrounding the flower buds, second fed on inflorescence tissues causing it to bend at right angle and ultimately dry up and the third infested the small sized fruits making them completely hollowed (Prasad, 1966).

Populations of P. mangiferae were significantly higher on the mango trees in commercial orchards than farmer fields at all locations at Rahim Yar Khan, Bahawalpur and Multan. Several factors can be responsible for reduction of population in farmer fields including crop rotation, less availability of food sources, more cultural practices (annually 10 cultivations and 5 hoeings) for seed bed preparation and weeding in wheat and cotton (Khalil and Amanullah, 2002). Crop

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rotation is an effective tool in the management of insect pests particularly for those pests which have narrow host range (Dent, 2000). In India hoeing of soil under the mango tree canopy reduced gall midges populations (Prasad, 1966; Grover and Prasad, 1966).

P. mangiferae continued breeding from January to April with more than one generation.

A small numbers of adults appeared in January which swiftly increased in February and March and reduced to minimum at the end of April. They laid eggs on mango inflorescences and larvae after feeding moved to the soil for pupation. They were first observed in February, reached at peak in March and decreased to zero in April. It was observed that larvae dropped from mango organs to ground for pupation from February to April. No larva was recorded from May to

January. Present findings are similar to the earlier findings of Prasad, (1971) and Grover (1986) who reported that female of P. mangiferae laid eggs on inflorescence, larvae after hatching penetrated in to the tissue of mango organs, dropped to the ground after feeding at final instar, got buried in the soil and adults after emergence caused out breaks on mango flowering in India.

The whole population underwent diapause after flowering season (Prasad, 1971). The adults first appeared in January, reached at peak in March and reduced to zero in April after the activity of 85 days. There were three peaks in the population of adults of P. mangiferae (Grover, 1986).

The climatic conditions of our country are close to those of India with some variations. However, in Reunion Island, its larvae were collected throughout the year; from inflorescences in flowering and from leaves in vegetative growth period. Population of P. mangiferae was more during flowering season from June to October while it was less during vegetative growth from

November to May. The permanency of the population may be attributed to sporadic production of new inflorescence and leave throughout the year and ability of P. mangiferae to feed on different organs of mango under diverse eco-cultural conditions (Amouroux et al, 2012).

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Yellow traps were effective in monitoring of adults of P. mangiferae followed by green, blue and control traps for season long populations but not for all sampling dates. Monitoring is the foundation of IPM as it provides the information about distribution of pest, efficacy of control measures and the variables required to develop forecasting system for pest outbreaks

(Conway, 1984). Trapping is used in monitoring of the pest population and reduction of pesticide by providing information for accurate timing of sprays (Vale, 1982; Grieshbach, 2011). Yellow color traps have been used for the monitoring of sorghum gall midge, Stenodiplosis sorghicola, blueberry gall midge, Dasineura oxycoccana Johnson and other phytophagous insects. They were more effective than green, red, black and blue color traps (Meyerdirk et al., 1979; Sharma and Franzmann, 2001; Plažanin et al., 2012).

It was noted that the weekly captures of P. mangiferae in the most attractive color traps were not consistent. They varied on different sampling dates in both the seasons. Temporal variation towards color preference has been reported for different insects. For example, the adults of blunt-nosed leaf hopper, Limotettix vaccinii (Van Duzee) (Hemiptera: Cicadellidae) on cranberry were more attracted to red traps earlier in the season and then to yellow traps later in the season. Similarly, the attraction to color of pirate bugs and honey bees was also influenced by time of year (Rodriguez-Saona et al., 2012). The variation in color preference by P. mangiferae may be attributed to the changes in background color of traps due to the occurrence of various phenological events in mango trees like flushing, flowering and fruit setting etc. Temporal variations due to trap background changes have been reported earlier by Saxena and Saxena,

(1975) for certain leafhoppers. Moreover in addition to background, blowing of winds may have passive effect on the small sized gall midges (only 2–3 mm in length) for adherence to different

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color traps. However, this assumption may be tested with P. mangiferae by recording variations in wind speed and attraction of gall midges on traps.

A hymenopterans parasitoid was reared from the inflorescences infested with P. mangiferae. In two years, its phenology was well synchronized with its host at Rahim Yar Khan.

In literature, it has been noted that the conservation of local and well synchronized parasitoids are good option for suppressing the pest population. In Pakistan, the population of a mango gall midge, P. matteiana was suppressed from 81.35% to 27.25% by conserving its parasitoids, C. pulcherrimus and S. temporale (Rehman et al., 2013). In Africa, conservation of two parasitoids,

Platygaster diplosisae and Aprostocetus procerae not only suppressed the population of African rice gall midge (AfRGM), O. oryzivora, but also provided African farmers with low-cost non- chemical control of AfRGM (Nwilenea et al., 2008).

Mango trees sprayed with neem seed kernel extracts (NSKE) received significantly lesser numbers of the larvae of P. mangiferae than untreated control. The neem seed kernel extract

(NSKE) spray on tree canopy with integration of racking of soil was the most effective among the other neem treatments. Numbers of larvae collected in this treatment were not significantly higher than the best treatment bifenthrin. In Pakistan, this insecticide is usually recommended for controlling insect pests of mango (Saifullah et al., 2007). Literature reports that bifenthrin is toxic to some hymenopterans parasitoids (Prabhaker et al., 2007). The neem based insecticides due to their lesser residual toxicities are safer for human beings and beneficial insects thus more economical for insect pest management (Caboni et al., 2006; Hasan et al., 1996; Gahukar, 2000).

These are environmentally safe and due to different mode of action like systemic, contact, antifeedant, toxicological, repellent, sterility inducing or insect growth inhibiting can be adopted on commercial scale. Neem extracts with integration of other control measures can provide a low

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cost management tool (Gahukar, 2000). In Nigeria, the use of neem seed extract suppressed significantly the population of AfRGM as compared to untreated check (Ogah and Ogbodo,

The adults of P. mangiferae were recorded on all color traps from January to May with maximum adults on yellow trap. However, more research is required on refining of monitoring techniques on weekly intervals with color traps. It was also noted that the larvae of P. mangiferae found on mango from February to May, dropping on soil under the tree canopy for pupation/diapause. A local hymenopteran parasitoid was also recorded during this period. Its phenology was well synchronized with its host, P. mangiferae. The application of neem seed kernel extract (NSKE) with integration of cultural practices was best eco-friendly management option for long term and sustainable control of the pest. On the basis of these findings, it is recommended that monitoring and management should be initiated from January. More research is needed in future for the development threshold level of the pest with these traps, identification and conservation of the parasitoid and application of neem seed kernel extract (NSKE) on large scale.

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Chapter 6

Phenology, distribution, biology and population trends of Procontarinia matteiana Kieffer and Cecconi (Diptera: Cecidomyiidae) in Punjab, Pakistan

This paper has been published as;

REHMAN, H. M., MAHMOOD, R. AND RAZAQ, M., 2013. Phenology, distribution, biology and population trends of Procontarinia matteiana Kieffer and Cecconi (Diptera: Cecidomyiidae) in Punjab, Pakistan. Pakistan J. Zool., 45: 941-47. (Impact Factor 0.33)

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6.1 INTRODUCTION

Mango (Mangifera indica L.) is infested with 250 species of plant-feeding arthropods throughout the world. About 26 of these produce galls on various organs of mango tree (Peña and Mohyuddin, 1997; Raman et al., 2009; Gagne and Medina, 2004). Most of the mango gall inducing species belong to genus Procontarinia (Cecidomyiidae: Diptera) (Boucek, 1986).

Procontarinia matteiana Kieffer & Cecconi is a common gall midge on mango in India,

Guadeloupe, Brazil, West Indies, Kenya, South Africa, Java, Indonesia and Iran (De Villiers,

1998, Askari & Radjabi, 2003). In India, it attacks mango throughout the year prominently during vegetative and fruit maturity period (September and April) of the crop (Kaushik et al,

2012). P. matteiana was reported an economic pest during 1980s in Indian Gujarat as it damaged

25.80 to 47.70% leaves of 3 varieties (Alphonso, Kesar and Rajapuri) of mango in 17 places

(Jhala et al., 1987). This pest can cause serious damage in the absence of natural enemies (Austin, 1984). In Oman, P. matteiana caused considerable damage to mango plantations which necessitated a search for natural enemies to control it (Austin, 1984).

The objectives of this study were;

To collect and rear gall midge for the identification and biology

To survey different mango growing areas for the presences/absences of P. matteiana

To study the phenology and population trends of the gall midge and its natural enemies

To evaluate the efficacy of bio-control for management of the pest at Rahim Yar Khan

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6.2 MATERIALS AND METHODS

6.2.1 Survey sites

Mango orchards were surveyed for gall midges and their natural enemies in 2007-09 at different localities in Punjab, Tranda Sway Khan (Rahim Yar Khan: 28.3°N, 65.23°E), Regional

Agricultural Research Institute (Bahawalpur: 29.59° N, 73.19° E), orchard near Bahaudin

Zakaria University at Bosan Road, (Multan: 31.32° N, 71.4°E) and Thokar Niaz Beg (Lahore:

32.2° N, 74.2° E).

6.2.2 Biology, phenology and population development of P. matteiana and its parasitoids

The mango orchard where regular observations of population development of P. matteiana and its parasitoids were conducted is situated at Tranda Sway Khan, a small town near Rahim

Yar Khan. It consisted of about 302 mango trees of different varieties including Chounsa (222),

Sindhri (20), Fajri (15), Dosehri (21), Anwar Ratol (4), Late Chounsa (1), Sarooli (6), Surrakh

Sarooli (4), Tota Pari (2), Langra (2), Lahotia (2) and Desi (3). Most of the trees were about 30-

40 years old while a few were 5-15 years.

The studies on the phenology and population trends of P. matteiana and its parasitoids were conducted on commercially important variety of mango, i.e. Chaunsa. For this purpose 50 mango leaves, 10 each from five twigs from top of the branch at about 1.5 m height above ground were taken at fortnightly intervals. The twigs carrying these leaves were kept in jars with their lower ends dipped in water. Leaves infested with the gall midges were examined for numbers of P. matteiana and its parasitoids.

To study the biology of P. matteiana, mango seedlings with newly developed leaves were

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placed in jars in the laboratory together with field collected infested leaves for 7 days so that the adults emerging from them could lay eggs on the new leaves. The leaves having signs of oviposition were tagged and the seedlings were shifted to field cages (Fig. 1A-C). Tagged leaves were examined daily for stage of development of P. matteiana (egg to adult).

6.2.3 Management

6.2.3.1 Mass rearing of parasitoids

Locally available parasitoids of P. matteiana were reared in the laboratory and field and released in the experimental orchard. Their effect on the population of P. matteiana was calculated in terms of degree of parasitism.

To rear parasitoids in the laboratory, mango twigs infested with P. matteiana were dipped in small jars filled with water. The small jars were placed in large plastic jars covered with muslin cloth to collect the parasitoids emerging from P. matteiana galls. Adult parasitoids emerging from galls were collected with aspirator in glass vials. The mouths of glass vials were covered with muslin cloth secured with rubber band for ventilation. These vials were then transported to the experimental orchards for releasing on weekly basis.

In the field the culture of P. matteiana and its parasitoids was maintained in cloth tunnels

(3X2X2 m) secured by bamboo sticks in the orchard. More than 7000 mango seeds were grown in the field on small beds after the mango picking season closed (August, 2008). When seedlings reached a stage of 3-5 leaves, they were taken out with earthen balls by digging the soil. Before digging, nursery beds were irrigated for optimum softening of the soil. These seedlings were then shifted to small plastic bags and placed under mango trees for few days for their establishment in the plastic bags in February – March, 2009. Some mango seedlings were also infested with P.

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matteiana. Established mango seedlings were finally transferred to above described cloth tunnels

(@500/tunnel) (Fig. 1D, E). Some mango seedlings with galls on the leaves, infested dry twigs and fallen leaves with galls of P. matteiana were also placed in the tunnel to rear gall midges adults from them. During the experiment, dead plants even if infested leaves were present were removed and replaced with live plants. The adults of P. matteiana emerging from galls of infested seedlings, twigs and fallen leaves oviposited on newly developed leaves of seedlings kept in the tunnels. The parasitoids emerging from infested plants also oviposited on galls of P. matteiana. Parasitoids emerging from the twigs collected from infested trees that were reared in the in the laboratory were also released into tunnels to enhance oviposition rate on the galls of P. matteiana. The mango seedlings treated in this way had mostly parasitized galls. They were placed under preselected mango trees (700 seedlings/tree) in an experimental orchard on first appearance of midges in 2009-10 (Fig. 1F, G). These trees had been kept free from insecticides since 2008.

Data on number of adults of P. matteiana and its parasitoids/50 infested leaves was recorded at fortnightly intervals from preselected trees before and after the release of parasitoids.

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A B C

D E F

Fig. 1 Steps involved in studying biology and mass rearing of gall midge and parasitoids, S. temporale and C. pulcherrimus. Mango seedlings with newly emerged leaves and old leaves with galls of midge in jars (A), leaves with signs of oviposition of P. matteiana (B), seedlings shifted to field cages (C), and then to the cloth tunnels (D,E), mango seedling carrying P. matteiana galls mostly parasitized (F), seedlings with parasitized galls Mango seedlings under mango trees in experimental orchard for release of parasitoids (G). More details in materials and methods section

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6.3 RESULTS

6.3.1 Procontarinia matteiana Kieffer & Cecconi

This species is for the first time reported from Pakistan. The specimens were identified by experts from Natural History Museum (NHM), London, UK. It was recorded from all areas surveyed in Punjab. Gall midges caused solitary or grouped galls on the upper and lower surfaces of the leaves. Thickness of solitary galls ranged from 3-3.25 mm and diameter 4-4.4 mm. In case of severe attack of this species, leaves became curled and ultimately dried (Fig. 2).

6.3.2 Phenology

This species is multivoltine. All stages, egg, larva, pupa and adult were recorded from

March to November. Only the larval and pupal stages were detected in the winter months

(December onward to February) in galls.

6.3.3 Biology

On hatching larvae entered leaves and started forming galls. At the beginning of gall development it was light green, increased in size and gradually became hard and concave at oviposition site. In July 08 at Rahim Yar Khan they completed development from egg to adult in

40 to 45 (mean 42.5+ 3.6) days. The abdomen of males is brown and females is light green (Fig.

3).

6.3.4 Population trends

Observations on population trends ran from April 08 at Rahim Yar Khan. In 2008 it was most abundant in April (Fig. 5) and from then onwards its population remained low. The population again started increasing in July, reached its peak in second week of September;

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numbers decreased in October and November and it was not recorded in winter months

December 08 and in January and February 09. Though the population was very low compared with 2008, its maximum numbers were recorded in March. The numbers decreased in April and remained very low afterward and could not increase its population up to September when maximum numbers were observed. In 2010 its numbers started increasing in February and reached at peak in March. In 2009-2010 the population of P. matteiana remained low because of high numbers of the two parasitoids Closterocerus pulcherrimus and Synopeas temporale.

6.3.5 Parasitoids

6.3.5. 1 Synopeas temporale Austin

This is known from India on P. matteiana. For Pakistan it is a new record. The specimens were identified by experts from Natural History Museum (NHM), London, UK. This parasitoid

(Fig 4) was reared from the galls of P. matteiana.

6.3.5.1.1 Biology, phenology and population trends: These parasitoids completed egg to adult development within host gall. After completing development adults emerged from the galls. Its phenology is well synchronized with its host P. matteiana. The three years (2008-10) study indicates that its population was high in March/April and September/October (Fig. 5). Its breeding rate slowed down in winter months (November – February) when it was reared in small numbers.

6.3.5.2 Closterocerus pulcherrimus (Kerrich)

Its known host is P. matteiana and is distributed in India, Pakistan, Sri Lanka, UAE, and

Afro tropical region. The specimens were identified by experts from Natural History Museum

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(NHM), London, UK. This parasitoid was reared from the galls of P. matteiana. (Fig. 4)

6.3.5.2.1 Biology, Phenology and population trends:

This parasitoid completed its development within the host gall and after completion of development only adults emerged from the galls. This parasitoid was reared from galls of the host P. matteiana from middle of March to November (Fig 5). It was not reared in winter months

(December – February). Though this parasitoid was reared together with S. temporale its numbers remained lower than S. temporale.

6.3.6 Management

The impact of parasitoids was determined in terms of parasitism in experimental orchard at

Rahim Yar Khan where regular observations on population trends of P. matteiana and its parasitoids were being followed since inception of the studies. For this purpose, more than 7000 mango seedlings with gall midges, most having been parasitized were placed under the marked mango trees in 2009-2010 kept free from pesticides since 2008.

The population of P. matteiana on the trees where these mango seedlings were placed decreased about three fold compared with the population of these gall midges in the year 2008.

Parasitism success of C. pulcherrimus and S. temporale increased and the population of host P. matteiana decreased. In 2008, before the application of parasitoids the population of P. matteiana was 81.35% which decreased to 27.25% in 2009-10 after regular releases of parasitoids. Similarly the populations of C. pulcherrimus and S. temporale were 3.72 and 14.91

%, respectively in 2008. Their numbers increased to 17.69 and 55.05% in 2009-10 after the implementation of biological control (Fig. 5).

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Curling of leaves by P. matteianna Galls of P. matteianna

Fig. 2 P. matteianna damaged curled mango leaves

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Fig. 3 Male (left) and female (right) of P. matteiana

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Fig. 4 Parasitoids of P. matteiana; S. temporale and C. pulcherrimus

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Fig. 5 Numbers (Mean±SE) of S. temporale and C. pulcherrimus and their host P. matteiana adults/leaf in 2008-2010 at Rahim Yar Khan

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6.4 DISCUSSION

During the present study at Rahim Yar Khan, annually two generations were observed in the population of P. matteiana; first in March/April and second in September/October. This result coincides with the observation of Botha and Kotzé (1987). They found two generations of

P. matteiana in South-Africa; first appeared in February-March and the other in October-

November. This result is also similar to observations of Kaushik et al, (2012) who observed two generations in India; first in April and second in September. However, our observation differs from Askari and Radjabi (2003). They reported three overlapping generations in Syahoo and four in Minab Iran. In the same way, Gupta (1952) also reported three generations per year in India.

The result of this research indicates that P. metteiana completes its life cycle within galls.

The same observations have also been reported by Botha and Kotzé (1987) and Askari and

Bagheri (2005). Findings of this present study show that this pest completes its life cycle in

42.5±3.6 days. This corresponds the results of Askari and Radjabi (2003) who demonstrated that this pest completed its development in 46.594± 0.933 days in Iran.

Present work also reveals that the phenologies of two parasitoids S. temporale and C. pulcherrimus are well synchoronized with their host P. matteiana. Some initial attempts of their mass rearing showed positive impacts in reducing the population of P. matteiana from 81.35 to

27.25%. This finding coincides with observations of Annecke and Moran (1982). They reported that parasitoids attacked the mango gall midges so heavily that only parasitoids emerged from galls. This creates the incorrect impression that they are not parasitoids but gall inducing wasps.

This phenomenon is probably not happening in the regions where the mango gall midges have been introduced accidentally and became pests of economic significance in the absence of

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natural enemies.

In conclusion, P. matteiana has been recorded from all mango growing areas surveyed in

Punjab, Pakistan. It forms solitary or grouped galls on the upper and lower surfaces of the leaves.

It is multivoltine and remains active almost throughout the year with highest populations in April and September. It completes larval and pupal stages in plant tissues, adults emerge from the galls through small holes and females oviposit on young leaves. It completes its whole life cycle in

42.5±3.6 days. However, there is need for more detailed research work on its biology.

Two parasitoids S. temporale and C. pulcherrimus are new records from Pakistan. Both parasitoids attack the galls of P. matteiana and their phenologies are well synchronized with their host. Since they were well adapted to the local environment, they were selected for mass rearing. Preliminary efforts on mass rearing and introduction of these parasitoids indicated very positive impact on controlling the population of P. matteiana. However, much needs to be done to improve and encourage the use of these mass rearing techniques for application on a large scale to achieve long term control of pest. We also recommend more research work is needed on biology of these parasitoids.

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REFERENCES

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Africa. Butterworths, Durban/Pretoria, vol. 14, pp. 383.

ASKARI, M. AND BAGHERI, A., 2005. Biology and comparative morphology of two

cecidomyiid flies, Procontarinia mattiana and Erosomyia mangifera (Diptera:

Cecidomyiidae) in Hormozgan Province. J. entomol. Soc. Iran., 25: 27-42.

ASKARI, M. AND RADJABI, G., 2003. Study on the biology and population fluctuations of

mango midge gall Procontarinia mattiana (Diptera: Cecidomyiidae) in Hormozgan

province. Appl. Ent. Phytopath., 70 : 121-35.

AUSTIN, A. D., 1984. New species of Platygastridae (Hymenoptera) from India which parasitise

pests of mango, particularly Procontarinia. Bull. entomol. Res., 74: 549-557.

BOTHA, W., AND KOTZE, J. M., 1987. Life cycle of the mango gall fly, Procontarinia

matteiana. South African Mango Growers' Association. Year book.

BOUCEK, Z., 1986. Taxonomic study of Chalcidoid wasps (Hymenoptera) associated with

midges (Diptera: Cecidomyiidae) on mango trees. Bull. entomol. Res., 76:393-407.

DE VILLIERS, E. A. (Ed.), 1998. The cultivation of mangoes. Nelspruit, South Africa: Institute

for Tropical and Subtropical Crops., pp. 216.

GAGNE, R.J. AND MEDINA, C.D. 2004. A new species of Procontarinia (Diptera:

Cecidomyiidae), an important new pest of mango in the Philippines. Proc. entomol. Soc.

Washington, 106 : 19-25.

GUPTA, R. L., 1952. Prolonged larval period and delayed emergence of adults in Procontarinia

matteiana Kieff. & Cecc. (Itonididae: Diptera). Curr. Sci., 21: 139.

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JHALA, R.C., PATEL, Z.P. AND SHAH, A.H., 1987. Studies on the relative occurrence of leaf-

gall midge (Procontarinia matteiana Kieffer and Cecconi) on different varieties of mango

in south Gujarat, India. Trop. Pest Manage., 33: 277-279.

KAUSHIK, D. K, BARAIHA, U., THAKUR, B. S. AND PARGANIHA, O. P., 2012. Pest

complex and their succession on mango (Mangifera indica) in Chhattisgarh, India. Plant

Arch., 12: 303-306.

PEÑA, J. E. AND MOHYUDDIN, A. I., 1997. Insect pests. In: The mango — botany,

production and uses (ed. R.E. Litz). CAB International, Oxfordshire, UK, pp. 327-362.

RAMAN, A., BURCKHARDT, D. AND HARRIS, K. M., 2009. Biology and adaptive radiation

in the gall-inducing Cecidomyiidae (Insecta: Diptera) and Calophyidae (Insecta:

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Conclusions and recommendations for future research

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CONCLUSIONS AND RECOMMENDATIONS FOR FUTURE RESEARCH

The study was set out to describe seasonal abundance and management of newly recorded mango blossom gall midges (Diptera: Cecidomyiidae) in Southern Punjab Pakistan.

This pest damages many parts of the mango tree including the bark, shoots, leaves, inflorescence buds, axillaries, flowers, newly formed fruits and twigs in the world. In Pakistan, the damage of mango gall midges has been recorded in last decade from all mango growing areas of Punjab including Multan, Lodhran, Rahim Yar Khan and Faisalabad. They were ranked number one pest during a survey in 2006. There was no literature on its identification, biology, ecology and management strategies from the country. The aim of this research was to gather baseline data regarding incidence, identification, damage patterns, seasonal abundance, distribution in tree and field, varietal preference and monitoring/sampling for the developing IPM strategy for gall midges in mango production systems of Southern Punjab.

Two species of gall midges, Procontarinia mangiferae (Felt) and Dasineura amaramanjarae Grover were recorded from inflorescence of mango trees in Pakistan. P. mangiferae damaged inflorescence buds, making holes and tunnels in the axillaries that ultimately bent them at right angle. D. amaramanjarae damaged flowers by feeding inside at the base of stamen and carpel. The adults of P. mangiferae and D. amaramanjarae appeared in

January/February, with peak in the March and reduced to zero in April. Their females laid eggs on inflorescences and larvae after feeding dropped to the soil under the mango tree for pupation/diapuse. Larvae were not found on the ground from May to January. Their damage was significantly higher on the trees in commercial orchards as compared to the trees in farmer fields usually intercropped with wheat and cotton grown for domestic purpose.

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Yellow/green sticky traps were effective in attracting the adults of both gall midges as compared to blue and colorless traps. Both the species were recorded from whole canopy of trees with greater numbers on southern sides and in lower parts of tree canopy. These species preferred the mango variety Surkha/Dusehri more than Chaunsa, Sindhri and Anwar Ratul.

Application of bifenthrin (Talstar 10EC) and neem seed kernel extracts (NSKE) with racking of soil under the tree copy were effective in reducing their populations.

Since both mango inflorescence gall midges species were found active from February to

April, therefore it is recommended to start monitoring of this pest with the initiation of flowering. Their life cycles should be interrupted by racking of soil under mango trees canopy because the larvae pupated in soil from February to April. Lower part of canopy and southern side of the trees must be properly covered with insecticides during application. Similarly, greater attention is required for monitoring and control of the pest in the commercial orchards as they were more vulnerable to pest damage than the trees in farmer fields usually intercropped with wheat and cotton grown for domestic purpose. Bifenthrin and neem seed kernel extracts (NSKE) with integration of cultural control were effective against P. mangiferae and D. amaramanjarae.

The neem based insecticides are more economical for insect pest management due to their lesser residual toxicities for human beings and beneficial insects. Neem extracts with integration of other control measures can provide a low cost management tool.

Another species of midges recorded was Procontarinia matteiana. This species was present on vegetative parts of mango trees. It formed solitary or grouped galls on the upper and lower surfaces of the leaves. Females laid eggs on newly developed leaves, larval and pupal stages were completed in galls and adults emerged from galls through small holes. It was active from February/March to November with two peaks of its population, first in March/April and

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second in September/October. Two parasitoids Closterocerus pulcherrimus (Kerrich) and

Synopeas temporale Austin were recorded from galls of P. matteiana. Their abundance was well synchronized with their host populations. For managing P. matteiana it is recommended to enhance the populations of biological control agents (two parasitoids) by rearing and releasing them when the populations of midges appear in March/April. However, proper time of release is needed to be determined.

As mentioned earlier present research was first attempt towards the gall midge complex as insect pests of mango in Pakistan with available limited resources. The outcome of this research is basic in nature to develop IPM. However, future research is required to formulate/fine tune guidelines for the famers to manage gall midge complex. First of all quantification of losses due to different species of midges in mango trees at different growth stages is required.

Determination of action threshold will tell proper timing of insecticide applications.

Sophisticated modeling will be required for population development of midges and growth of mango trees (based upon BBCH scale) in relation to weather factors and other variables like biological control agents. Development of simple techniques for rearing of natural enemies and their release will be needed to avoid development of midges. Moreover, other parasitoids of gall midge species should be explored from Pakistan as well as from regions of their origin for possibilities of their introduction and augmentation. As, attraction towards color traps for P. mangiferae and D. amaramanjarae was inconsistent in different weeks of the season therefore, further research is needed to fine tune monitoring techniques.

Plant metabolites (terpenes) responsible for attraction of midges should be quantified in mango germplasm for possibilities of grafting to develop resistant cultivars. The degree of variability in the capacity of mango cultivars to recover/tolerate after damage by midges should

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also be determined for incorporation of tolerance mechanism of resistance in IPM. Education of the farmers for implementation of research is next step for area wide pest management.

Implementation phase usually requires more funds than research development phase.

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ANNEXURE 1: SURVEY AND EXPERIMENTAL SITES AT DIFFERENT

MANGO GROWING AREAS OF PUNJAB PAKISTAN

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ANNEXURE 2: FIELD IDENTIFICATION OF MANGO GALL MIDGES SPECIES

1. Dasineura amaramanjarae Grover

Larvae of Dasineura amaramanjarae Grover damage mango by direct feeding inside the buds, at the base of stamen and carpel of flowers. They become pinkish red at final stage and move to the soil to pupate. Adults are of red color

Life stages of D. amaramanjarae: A) Larvae feeding inside the flowers B) Larvae at final stage C) Pupa D) Adults.

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2. Procontarinia mangiferae (Felt)

Larvae of Procontarinia mangiferae (Felt) feed on inflorescence buds, small leaves, make tunnels in branches (axillaries) and small sized fruits. In the case of severe damage, branches of inflorescences bent at right angle. The infested inflorescences and fruits usually dry and drop from the mango tree

Damages of P. mangiferae : A) Damaged inflorescences B) Damaged small fruits

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3. Procontarinia matteiana Kieffer and Cecconi

Procontarinia matteiana Kieffer & Cecconi forms solitary or grouped galls on the upper and lower surfaces of the leaves. In case of severe attack, leaves become curled, ultimately dry and fall

Male Female

Single and grouped galls of P. matteiana A) On lower side B) Upper

side of mango leaf C) Male gall midge D) Female gall midge

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ANNEXURE 3: TRAPS FOR MONITORING

Traps for monitoring: A) Yellow B) colorless Traps

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