Department of Entomology, Faculty of Agriculture, University of Agriculture, Faisalabad, Pakistan

ENTOMOPATHOGENS AS POTENTIAL BIOCONTROL AGENTS AGAINST RED PALM WEEVIL, RHYNCHOPHORUS FERRUGINEUS (OLIVIER)

MUHAMMAD YASIN M.Sc. (Hons.) Entomology

A thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSPHY (Ph.D.) In ENTOMOLOGY

DEPARTMENT OF ENTOMOLOGY, FACULTY OF AGRICULTURE, UNIVERSITY OF AGRICULTURE, FAISALABAD, PAKISTAN

2016

DECLARATION

I hereby declare that that the contents of the thesis “Entomopathogens as Potential Biocontrol Agents Against Red Palm Weevil, Rhynchophorus ferrugineus (Olivier)” are product of my own research and no part has been copied from published sources (except references, standard mathematical or genetic models/equations/formulas/protocols etc.). I further declare that this work has not been submitted for the award of any other degree/diploma. The University may take action if the information provided found inaccurate at any stage (In case of any default, the scholar will be proceeded against as per HEC plagiarism policy).

Muhammad Yasin

The Controller of Examinations, University of Agriculture, Faisalabad.

We, the Supervisory Committee , certify that the contents and

form of thesis submitted by Muhammad Yasin, Registration No. 2005-

ag-1886 have been found satisfactory and recommend that it be

processed for evaluation, by the External Examiner (s) for the award of

degree.

Supervisory Committee

Chairman ______Dr. Waqas Wakil

Member ______Prof. Dr. Muhammad Jalal Arifl

Member ______Prof. Dr. Shahbaz Talib Sahil

This thesis is dedicated with Love and Respect to My Parents

ACKNOWLEDGEMENTS First of all I would like to bow my head before “ALMIGHTY ALLAH” the Omnipotent, the Omnipresent, the Merciful, the Beneficial who presented me in a Muslim community and also bestowed and blessed me with such a lucid intelligence as I could endeavor my services toward this manuscript. Countless salutations are upon the HOLY PROPHET MUHAMMAD (May Peace Be Upon Him), the fountains of knowledge, who has guided his “Ummah” to seek knowledge for cradle to grave. The work presented in this manuscript was accomplished under the sympathetic attitude, animate directions, observant pursuit, scholarly criticism, cheering perspective and enlightened supervision of Dr. Waqas Wakil, Assistant Professor, Department of Entomology, University of Agriculture, Faisalabad. His thoughtful guidance helped me in all the time of research, writing of dissertation/publications etc. and his rigorous critique improved my overall understanding of the subject. I am grateful to his ever inspiring guidance, keen interest, scholarly comments and constructive suggestions throughout my PhD studies.

I wish to acknowledge my deep sense of profound gratitude to the worthy member of my research supervisory panel Dr. Muhammad Jalal Arif and Dr. Shahbaz Talib Sahi, for their constructive criticism, illuminating and inspiring guidance and continuous encouragement throughout course of my study. I really have no words to express my sincere thankful feelings and emotions for all my Teachers, seniors and friends especially Dr. M. Usman Ghazanfar, Dr. M. Khalid Bashir, Dr. M. Ejaz Ashraf, Dr. Mirza Abdul Qayyum, Dr. Kashif Ali, Dr. M. Tahir and Dr. M. Irfan Akram for their cooperation, well wishes and moral support from time to time during the course of study. I am also thankful to my junior fellows specially Muhammmad Farooq, Muhammad Asif, Muhammad Ismail, Muhammad Usman, Muhammad Shoaib Qazi, Muhammad Ahmad Rana, Shumaila Rasoool, Sehrish Gulzar, Zeshna Khaliq, Muhammad Tufail, Mehwish Ayaz, Kanza Syed and Ayesha Faraz for their support towards my PhD studies. Words are lacking to express my humble obligation to my affectionate grandparents, Father, Mother, Brothers, Sisters, uncles, aunts, cousins and specially my lovely wife and my children Rehmin Fatima and Muhammad Abdullah for patience in absence of their Dad, and all family members for their love, good wishes, inspirations and unceasing prayers for me, without which the present destination would have been mere a dream.

I would like to express my deepest gratitude to the Higher Education Commission, Islamabad (Pakistan) for the scholarship under PhD Indigenous Fellowship Program is greatly acknowledged. I would like to say special thanks to Dr. Richard Stouthamer and Paul Rugman-Jones UCR, USA for accepting me towards IRSIP as an international student. Last but least I will pay my special thanks to Ms. Aima Bashir who always prayed for my success, may God bless her. Muhammad Yasin

CONTENTS

Chapter 1. Introduction……………………………………………………………………. 01

Chapter 2. Literature Reviewed 2.1 Invasive Red Palm Weevil (RPW)……………………………… 07 2.2 Taxonomic position ……………………………………………... 07 2.3 Classification…………………………………………………….. 07 2.4 Spatial distribution………………………………………………. 07 2.5 Control measures………………………………………………… 08 2.5.1 Microbial control………………………………………………… 08 2.5.2 History of microbial control……………………………………... 08 2.5.3 Entomopathogenic Fungi (EPFs)………………………………... 10 2.5.3.1 History…………………………………………………………… 10 2.5.3.2 Geographical distribution and occurrence……………………….. 11 2.5.3.3 Classification…………………………………………………….. 11 2.5.3.4 Host range……………………………………………………….. 11 2.5.3.5 Mode of infection………………………………………………... 12 2.5.3.6 Enzymes and toxins of EPFs…………………………………….. 12 2.5.3.7 Chitinases………………………………………………………... 12 2.5.3.8 Proteases and peptidases………………………………………… 13 2.5.3.9 Lipases…………………………………………………………… 13 2.5.3.10 Toxins……………………………………………………………. 13 2.5.3.11 Destruxins………………………………………………………... 13 2.5.3.12 Oosporein………………………………………………………... 14 2.5.3.13 Beauvericin and beauveriolide…………………………………... 14 2.5.3.14 Bassianolide……………………………………………………... 14 2.5.3.15 Beauveriolide……………………………………………………. 14 2.5.3.16 Host range……………………………………………………….. 14 2.5.3.17 Effect of abiotic factors………………………………………….. 15 2.5.3.17.1 Temperature……………………………………………………... 15 2.5.3.17.2 Relative humidity………………………………………………... 15 2.5.3.18 Effect of EPFs on non-target organisms………………………… 15 2.5.3.19 Integration of EPFs with other control measures……………….. 16 2.5.3.20 EPF against RPW………………………………………………... 17 2.5.3.21 Natural incidence of EPFs on RPW……………………………... 17 2.5.3.22 Susceptibility of RPW to EPFs infections under lab condition…. 18 2.5.3.23 Field and Semi-field assessment of fungi for RPW management.. 19 2.5.4 Endophytic fungi………………………………………………… 20 2.5.5 Future prospects of entomopathogenic fungi...... 20 2.5.6 Entomopathogenic Nematodes (EPNs)...... 21 2.5.6.1 Natural incidence………………………………………………… 21 2.5.6.2 Susceptibility of RPW to EPNs infections under lab conditions... 22 2.5.6.3 Field and semi-field assessment of EPNs for RPW management.. 22 2.5.6.4 Interactions between EPNs and pesticides...... 23 2.5.7 Entomopathogenic Bacteria...... 24

2.5.7.1 History...... 24 2.5.7.2 Classification…………………………………………………….. 24 2.5.7.3 Life cycle………………………………………………………… 25 2.5.7.4 Ecology…………………………………………………………... 25 2.5.7.5 Mechanism of action…………………………………………….. 26 2.5.7.6 Commercial formulations………………………………………... 26 2.5.7.7 Methods of applications of Bt products…………………………. 27 2.5.7.8 Superiority of Bt products over synthetic insecticides…………... 27 2.5.7.9 Concerns to use of Bt……………………………………………. 27 2.5.7.10 Interaction of Bt products and other toxins……………………… 28 2.5.7.11 Effect of Bacillus thuringiensis on non-target invertebrates…….. 28 2.5.7.12 Mode of infection………………………………………………... 29 2.5.7.13 Important entomopathogenic bacteria…………………………… 29 2.5.7.13.1 Bacillus thuringiensis……………………………………………. 29 2.5.7.13.1 Paenibacillus popilliae…………………………………………... 30 2.5.7.13.2 Brevibacillus laterosporus………………………………………. 30 2.5.7.13.3 Bacillus subtilis………………………………………………….. 30 2.5.7.13.4 Bacillus sphaericus……………………………………………… 30 2.5.7.13.5 Wolbachia………………………………………………………... 31 2.5.7.14 Host range of B. thuringiensis…………………………………… 31 2.5.7.15 Natural incidence………………………………………………… 32 2.5.7.16 Susceptibility of RPW to EPB under lab conditions….………… 32 2.5.7.17 Field and Semi-field assessment of EPB for RPW management.. 32 2.5.8 Microbial control agents as a component of RPW IPM…………. 33 2.5.9 Ecological engineering and agricultural practices to conserve microbial control agents…………………………………………. 33 2.5.10 Biotechnological approaches to enhance virulence of microbial control agents……………………………………………………. 34 2.6 References……………………………………………………….. 35

Chapter 3 Genetic variation among populations of Red Palm Weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) from the Punjab and Khyber Pakhtunkhwa provinces of Pakistan Abstract………………………………………………………….. 61 3.1 Introduction ……………………………………………………... 62 3.2 Materials and methods…………………………………………... 63 3.2.1 Specimen collections…………………………………………….. 63 3.2.2 DNA extraction and amplification………………………………. 63 3.2.3 Cleaning and sequencing………………………………………… 64 3.2.4 Genetic analysis………………………………………………….. 64 3.3 Results…………………………………………………………… 64 3.4 Discussion……………………………………………………….. 65 3.5 References……………………………………………………….. 68

Chapter 4 Resistance to commonly used insecticides and phosphine (PH3) against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) in Punjab and Khyber Pakhtunkhwa, Pakistan Abstract………………………………………………………….. 74 4.1 Introduction……………………………………………………… 75 4.2 Materials and methods…………………………………………... 76 4.2.1 RPW collection and rearing……………………………………... 76 4.2.2 Test chemicals…………………………………………………… 76 4.2.3 Generation of phosphine gas…………………………………...... 76 4.2.4 Bioassay…………………………………………………………. 77 4.3 Statistical analysis……………………………………………….. 77 4.4 Results…………………………………………………………… 78 4.4.1 Imidacloprid……………………………………………………... 78 4.4.2 Spinosad…………………………………………………………. 78 4.4.3 Lambda cyhalothrin……………………………………………… 78 4.4.4 Chlorpyrifos…………………………………………………...... 78 4.4.5 Profenophos……………………………………………………… 78 4.4.6 Deltamethrin……………………………………………………... 78 4.4.7 Cypermethrin…………………………………………………….. 79 4.4.8 Phosphine………………………………………………………... 79 4.5 Discussion……………………………………………………….. 79 4.6 References……………………………………………………….. 81

Insecticidal potential of Beauveria bassiana and Metarhizium Chapter 5 anisopliae isolates against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) Abstract………………………………………………………….. 89 5.1 Introduction……………………………………………………… 90 5.2 Materials and methods…………………………………………... 91 5.2.1 RPW collection and rearing……………………………………... 91 5.2.2 Culture collection………………………………………………... 91 5.2.3 Isolation from RPW cadavers…………………………………… 92 5.2.4 Screening assay …………………………………………………. 92 5.2.5 Virulence assay………………………………………………….. 92 5.2.6 Statistical analysis……………………………………………….. 93 5.3 Results…………………………………………………………… 93 5.3.1 Screening assay …..……………………………………………… 93 5.3.2 Virulence assay………………………………………………….. 93 5.4 Discussion……………………………………………………….. 94 5.5 References……………………………………………………….. 97

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Chapter 6 Combined effectiveness of endophytically colonized Beauveria bassiana and Bacillus thuringiensis against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) Abstract………………………………………………………….. 111 6.1 Introduction ……………………………………………………... 112 6.2 Materials and methods…………………………………………... 113 6.2.1 RPW collection and rearing…………………………………….. 113 6.2.2 Preparation of fungi……………………………………………… 113 6.2.3 Preparation of Bacillus thuringiensis spore-crystal mixtures…… 114 6.2.4 Screening of fugal isolates……………………………………… 114 6.2.5 Bioassay procedure……………………………………………… 114 6.2.6 Bioassay on development of R. ferrugineus……………………... 115 6.2.7 Bioassay on larval development ………………………………… 115 6.2.8 Statistical analysis……………………………………………….. 115 6.3 Results …………………………………………………………... 115 6.3.1 Fungal colonization of date palm petioles……………………….. 115 6.3.2 Toxicity of microbial agents…………………………………….. 116 6.3.3. Development of R. ferrugineus………………………………….. 116 6.3.4 Effect on larval development……………………………………. 117 6.4 Discussion ……………………………………………………….. 117 6.5 References……………………………………………………….. 120

Chapter 7 Integrated effect of entomopathogenic fungi and entomopathogenic nematodes against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) Abstract………………………………………………………….. 134 7.1 Introduction……………………………………………………… 135 7.2 Materials and methods…………………………………………... 136 7.2.1 RPW collection and rearing……………………………………... 136 7.2.2 Nematode………………………………………………………... 136 7.2.3 Preparation of fungi……………………………………………… 136 7.2.4 Treatment with entomopathogenic fungi………………………... 136 7.2.5 Treatment with H. bacteriophora ………………………………. 137 7.2.6 Treatment with entomopathogenic fungi and nematode ………... 137 7.2.7 Effects of entomopathogens on R. ferrugineus development…… 137 7.2.8 Effects of entomopathogens on larval development…………….. 138 7.2.9 Statistical analysis……………………………………………….. 138 7.3 Results………………………………………………………….... 138 7.3.1 Entomopathogenic fungi and nematode interaction……………... 138 7.3.2 Development of R. ferrugineus …………………………………. 139 7.3.3 Effect on larval development ………………………………….... 139 7.4 Discussion……………………………………………………….. 139 7.5 References……………………………………………………….. 142

Chapter 8 Combined toxicity of Beauveria bassiana, Bacillus thuringiensis and entomopathogenic nematodes against red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) Abstract………………………………………………………….. 155 8.1 Introduction……………………………………………………… 156 8.2 Materials and methods…………………………………………... 157 8.2.1 RPW collection………………………………………………….. 157 8.2.2 Preparation of B. thuringiensis spore-crystal mixtures………….. 157 8.2.3 Entomopathogenic nematode……………………………………. 157 8.2.4 Preparation of fungi……………………………………………… 158 8.2.5 Treatment with B. bassiana……………………………………… 158 8.2.6 Treatment with B. thuringiensis…………………………………. 158 8.2.7 Treatment with H. bacteriophora……………………………….. 158 8.2.8 Treatment with B. bassiana, Bt-k and H. bacteriophora………... 159 8.2.9 Sporulation and Nematode production………………………….. 159 8.2.10 Statistical analysis……………………………………………….. 159 8.3 Results…………………………………………………………… 159 8.3.1 Mortality of larvae and adult…………………………………….. 159 8.3.2 Mycosis and sporulation…………………………………………. 160 8.3.3 Insects affected by EPNs and EPNs production…………………. 160 8.4 Discussion……………………………………………………….. 160 8.5 References……………………………………………………….. 162 Summary………………………………………………………… 172

List of Tables

No Title Page No. Table 3.1 Sampling information for RPW populations collected from date palm Phoenix dactylifera in Punjab and KPK provinces of Pakistan……………... 70 Table 3.2 Genetic characterization of five RPW populations from the Punjab and KPK provinces of Pakistan based on a 528 bp section of the mitochondrial COI gene. For population abbreviations, see Table 1……………………….. 70 Table 3.3 Variation in a 528 bp segment of the cytochrome oxidase subunit I (COI) region of mitochondrial DNA (mtDNA) of Rhynchophorus ferrugineus. Average number of pairwise nucleotide differences (k) within (diagonal element) and between (below diagonal) populations in the Punjab and KPK provinces of Pakistan. For population abbreviations………………………... 71 Table 4.1 Geographical characteristics of the localities from where R. ferrugineus populations were collected in Punjab and Khyber Pakhtunkhwa, Pakistan…. 84 Table 4.2 Resistance to commonly used insecticides and phosphine against susceptible strains and field-collected populations of R. ferrugineus……….. 85 Table 5.1 Characterization of B. bassiana and M. anisopliae isolates obtained from soils and insect cadavers…………………………………………………….. 103 Table 5.2 Factorial analysis of screening and mycosis of R. ferrugineus exposed to B. bassiana and M. anisopliae isolates…………………………………………. 104 Table 5.3 Percentage pathogenicity (%±SE) of 19 isolates of B. bassiana and M. anisopliae isolates against R. ferrugineus larvae after 12 days of incubation. 104 Table 5.4 Percentage pathogenicity (%±SE) of 19 isolates of B. bassiana and M. anisopliae isolates against R. ferrugineus adult after 12 days of incubation... 105 Table 5.5 Factorial analysis for virulence of B. bassiana and M. anisopliae isolates against larvae and adult of R. ferrugineus…………………………………… 106 Table 5.6 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 7 days of exposure treated with B. bassiana and M. anisopliae isolates…………….… 106 Table 5.7 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 14 days of exposure treated with B. bassiana and M. anisopliae isolates……………. 107 Table 5.8 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 21 days of exposure treated with B. bassiana and M. anisopliae……………………. 107 Table 5.9 LC50 and LC90 values of B. bassiana and M. anisopliae isolates tested against larvae and adult of R. ferrugineus ………………………………….. 108 Table 5.10 LT50 and LT90 values of B. bassiana and M. anisopliae isolates tested against larvae of R. ferrugineus……………………………………………… 109 Table 5.11 LT50 and LT90 values of B. bassiana and M. anisopliae isolates tested against adult of R. ferrugineus………………………………………………. 110 Table 6.1 Percentage of petiole fragments colonized by entomopathogenic (E) and other (O) fungi in live palm petioles experiments…………………………… 124 Table 6.2 Factorial analysis of mortality, pupation, adult emergence and egg eclosion of R. ferrugineus exposed to endophytically colonized B. bassiana and B. thuringiensis…………………………………………………………………. 125 Table 6.3 Mean mortality (%±SE) of 2nd, 4th and 6th instar larvae of R. ferrugineus

treated with endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05) …………………………………... 126 Table 6.4 Pupation, adult emergence and egg eclosion (%±SE) of 2nd, 4th and 6th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)…………… 127 Table 6.5 Growth parameters e.g. larval duration (days), larval weight (grams) pre- pupal duration (days), pre-pupal weight (grams), pupal duration (days), pupal weight (grams), adult longevity (days) and adult weight (grams) (%±SE) of 2nd instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 4 cm away from inoculation site) and Bt-k (Bt1: 10 µg; Bt2: 15 µg; Bt3: 20 µg ml-1) alone and in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05) …………………………………………………………………….. 128 Table 6.6 Analysis of Co-variance for 10th instar larvae of R. ferrugineus regarding weight gain, frass production and diet consumption when treated with endophytic B. bassiana (Bb: 4 cm away from inoculation site) and Bt-k (Bt: 10 µg ml-1). Initial weight of larvae and diet consumption were taken as covariate……………………………………………………………………... 129 Table 7.1 Mean mortality (%±SE) of 2nd instar larvae of R. ferrugineus treated with B. bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae were used each @ 1×106 spore ml-1 and H. Bacteriophora was applied @ 100 IJs ml-1……………………………………………………….. 146 Table 7. 2 Mean mortality (%±SE) of 4th instar larvae of R. ferrugineus treated with B. bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae were used each @ 1×106 spore ml-1 and H. Bacteriophora was applied @ 100 IJs ml-1……………………………………………………….. 147 Table 7.3 Mean mortality (%±SE) of 6th instar larvae of R. ferrugineus treated with B. bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae were used each @ 1×106 spore ml-1 and H. Bacteriophora was applied @ 100 IJs ml-1……………………………………………………….. 148 Table 7.4 Factorial analysis of pupation, adult emergence and egg eclosion of R. ferrugineus exposed to B. bassiana, M. anisopliae and H. Bacteriophora….. 148 Table 7.5 Pupation, adult emergence and egg eclosion (%±SE) of 2nd, 4th and 6th instar R. ferrugineus larvae treated with B. bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae were used each @ 1×106 spore ml-1 and H. Bacteriophora was applied @ 100 IJs ml-1. Mean sharing the same letters are not significantly different. Means sharing the same letters within columns are not significantly different………………………. 149 Table 7.6 Effect of B. bassiana, M. anisopliae and H. Bacteriophora on the development of R. ferrugineus. B. bassiana and M. anisopliae were used each @ 1×104 spore ml-1 and H. Bacteriophora was applied @ 50 IJs ml-1. Mean sharing the same letters are not significantly different……………….. 150

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Table 7.7 Analysis of co-variance for 2nd, 4th and 6th instar larvae of R. ferrugineus regarding weight gain and frass production at a given level of diet consumption when treated with B. bassiana and H. Bacteriophora alone and in combination. Initial weight of larvae and diet consumption were taken as covariate……………………………………………………………. 151 Table 8.1 ANOVA parameters for the main effects and associated interactions for mortality levels of R. ferrugineus larvae and adults…………………………. 166 Table 8.2 Mean mortality (%±SE) of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination after 7 days of exposure (means followed by the same letter within each treatment and insect populations not significantly different; HSD test P≤0.05) …………………………………... 166 Table 8.3 Mean mortality (%±SE) of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination after 14 days of exposure (means followed by the same letter within each treatment and insect populations not significantly different; HSD test P≤0.05) …………………………………... 167 Table 8.4 Mean mortality (%±SE) of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination after 21 days of exposure (means followed by the same letter within each treatment and insect populations not significantly different; HSD test P≤0.05)…………………………………… 167

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List of Figures

No. Title Page No. Figure 3.1 Map of collection sites in Punjab and KPK provinces of Pakistan………….. 71 Figure 3.2 Distribution of mitochondrial haplotypes across five populations of RPW from the Punjab and KPK provinces of Pakistan…………………………… 72 Figure 3.3 Relationships between four Pakistani COI haplotypes and 48 others are occurring around the world. Haplotype network constructed from 539 COI sequences (each 528 bp long) generated by the present study and three earlier studies (see text). Each haplotype is represented by an oval or for that with the highest outgroup probability, a rectangle. Size of each haplotype is indicative of the number of specimens sharing that haplotype; also given inside each haplotype. H1-43 is numbered according to El Mergawy et al. (2011) and Rugman-Jones et al. (2013); H44-50 corresponds to additional haplotypes from Wang et al. (2015); and H51-52 are new to this study………………………………………………………… 73 Figure 4.1 Map of collection sites in Punjab and Khyber Pakhtunkhwa provinces of Pakistan (1. Bahawalpur 2. Rahim Yar Khan 3. Vehari 4. Dera Ghazi Kahn 5. Muzaffargarh 6. Layyah 7: Dera Ismail Khan)…………………………. 87 Figure 4.2 Resistance ratio (RR) of chemical insecticides and phosphine against susceptible strains and field-collected populations of R. ferrugineus populations of R. ferrugineus from various localities in Punjab and Khyber Pakhtunkhwa, Pakistan……………………………………………………… 88 Figure 6.1 Mean mycosis (%±SE) in cadavers of R. ferrugineus treated with endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)…………………………………………………. 130 Figure 6.2 Sporulation (conidia ml-1) on R. ferrugineus cadavers treated with endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)…………………………………………………. 130 Figure 6.3 Diet consumption (grams) in 10th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt- k (Bt: 10 µg ml-1) …………………………………………………………… 131 Figure 6.4 Frass production (grams) in 10th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-k (Bt: 10 µg ml-1) ………………………………………………………………….. 132 Figure 6.5 Weight gain (grams) in 10th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-k (Bt: 10 µg ml-1) …………………………………………………………………. 133 Figure 7.1 Diet consumption in last instar larvae of R. ferrugineus when treated with B. bassiana and H. bacteriophora…………………………………………... 152 Figure 7.2 Frass production in last instar larvae of R. ferrugineus when treated with B.

bassiana and H. bacteriophora……………………………………………… 153 Figure 7.3 Weight gain in last instar larvae of R. ferrugineus when treated with B. bassiana and H. bacteriophora……………………………………………… 154 Figure 8.1a Mean mycosis (%±SE) in larvae of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)……………………………………………………………………… 168 Figure 8.1b Mean mycosis (%±SE) in adults of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)…………………………..………………………………………… 168 Figure 8.2a Sporulation (conidia ml-1) in larvae of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)……………………………………………………………………… 169 Figure 8.2b Sporulation (conidia ml-1) in adult of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)………………………………………………………………………. 169 Figure 8.3a R. ferrugineus larvae affected by H. bacteriophora (%±SE) from different populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05) …………………………………………………………………. 170 Figure 8.3b R. ferrugineus adult affected by H. bacteriophora (%±SE) from different populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05) …………………...... 170 Figure 8.4a Nematode production (IJs ml-1) in larvae of R. ferrugineus affected by H. bacteriophora from different populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)…………………………… 171 Figure 8.4b Nematode production (IJs ml-1) in adult of R. ferrugineus affected by H.

x bacteriophora from different populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)…………………………… 171

Abstract

The red palm weevil red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) is one of the voracious pest among invasive insect pests. Pakistani populations of R. ferrugineus distributed among different areas have been found genetically diverse. Four different haplotypes were recorded across Punjab and Khyber Pakhtunkhwa (KPK) provinces. Haplotype H1, H5, H51 were found from all collection sites while H52 was rare haplotype that was only present in populations of Layyah and Dera Ghazi Khan only. Study indicated the native range of R. ferrugineus instead of invaded from other parts of the world, since the weevil was recorded from Pakistan in 1913 and may have been present before that. The populations of R. ferrugineus have gained resistance to commonly used chemical insecticides and phosphine due to the excessive and unwise use of these chemical insecticides. Resistance against seven different populations of R. ferrugineus was determined from very low to low and moderate to high level of resistance against commonly used insecticides. Phosphine, cypermethrin and deltamethrin exhibited high resistance against almost all the populations. To overcome the insecticide resistance entomopathogens were evaluated against R. ferrugineus. Nineteen different isolates of Beauveria bassiana s.l. and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales) were screened which exhibited varying level of susceptibility towards larvae and adult. Three isolates of B. bassiana (WG-41, WG-42 and WG-43) and two isolates of M. anisopliae (WG-44 and WG-45) exhibited highest larval and adult mortality after 12 days of application and the same isolates were tested for their virulence at different exposure intervals against larvae and adult which caused almost 100% mortality for certain isolates. B. bassiana are capable of colonizing endophytically in live date palm petioles even after 30 days of inoculation and can significantly reduce the weevil population when exposed to the endophytically colonized date palm petiole pieces. Moreover Bacillus thuringiensis var. kurstaki (Bt-k) is also an effective agent that can cause detrimental effects on R. ferrugineus survival alone and in combination with endophytically colonized date palm. Both agents exerted influence on developmental parameters such as larval duration, larval weight, pre-pula duration, pre-pupal weight, pupal duration, pupal weight, adult longevity (male and female) and adult weight (male and female) etc. Moreover, diet consumption, frass production and weight gain was affected by the treatments applied. Entomopathogenic fungi in integration with Heterorhabditis bacteriophora Poinar applied either simultaneously or in sequential manners exert detrimental effects on growth and development of R. ferrugineus larvae. Combined application of three agents i.e. B. bassiana, Bt-k and H. bacteriophora also suppress the larvae and adult population collected from 4 different areas of Punjab and KPK, Pakistan under laboratory conditions. Hence we can use microbial entomopathogens against this voracious pest which are safer to human beings and compatible to environment.

CHAPTER 1 Introduction Date palm is a tropical fruit crop which belongs to the family Arecaceae. It is a main source of dietary fiber and provides livelihood to a number of peoples in the old and new world. The tree is named as “tree of life” in the Bible due to its long life (100 year), productivity and elevated nutritional value (UN, 2003). It is one of the oldest cultivated plants on the earth (Lee, 1963; Riad, 2006). Date fruits have key importance in the Muslim culture and among the few fruits repeatedly mentioned in the Holy Quran, the tree has been mentioned as a humble tree as it does not affect the growth and development of any other plant (Goes straight and does not pour the shade on other plants or inhibit their growth). The date palm cultivation may have been practiced 7000 year ago (Popenoe, 1924), but the domestication of date palm is assumed to be started in Mesopotamia by 3000 B.C. (Nixon, 1951). Excavation of godowns from Mohenjo Daro indicated the presence of date seeds which depict the date palm cultivation in Sindh province of Pakistan since 5000 year back (Marshal, 1931). Some school of thought believes that Alexander the Great brought the date palm to the Indian subcontinent (Nixon, 1951; Pasha et al., 1972). But some scientists believe that the date palm prevailed in subcontinent before the time of Alexander as Greek army use to eat harvested date from the gardens of Kech valley (Balochistan) when they travel through the Makran coasts during 4th century BC (Qasim and Naqvi, 2012). Latter on the mass spread of date seeds came to existence by the arrival of Mohammed Bin Qasim in Sindh as early as 712 AD, when he came for the preach of Islam. During camping the Arab soldiers (threw) discarded date seeds at that site which caused spread of date palm cultivation in Sindh valley (Ahmad and Tahir, 2005; Dhillon et al., 2005; Jatoi et al., 2010). In Indian subcontinent off-shoots of highly potent verities of date palm (Dayri, Halawy, Khadrawy, Zahidi and Sayer) were imported from Basra (Iraq) during the colonial period (1910-1912) by the British Indian Government and were planted in Muzaffargarh and Multan (Punjab, Pakistan) (Milne, 1918). Pakistan is ranked 6th in date production in the world which produced 0.6 million tones dates in 2013 cultivated over 93,088 ha (Al-Khayri et al., 2015), largest presence of the dates in the world. The major importer countries include India, UK, USA, Canada, Malaysia, Germany, Indonesia and Denmark (Faostat, 2013). In Pakistan the major cultivars are Begum Jangi (Baluchistan), Aseel (Sindh) and Dhakki of (Dera Ismail Khan). The major date growing areas in Pakistan are Kech (the administrative center is Turbat), Panjgur, Sukkur and Khairpur, Jhang, Dera Ismail Khan, Dera Ghazi Khan, Multan, Muzaffargarh and Bahawalpur. Date palm is attacked by a numerous insect pests and diseases (Al-Doghairi, 2004). Among these insects Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) caused 10-20% loss in production to different varieties of dates in Pakistan (Baloach et al., 1992). The pest is cryptic in nature and has been found devastating 29 different palm species belonging to 18 genera and 3 families (Malumphy and Moran, 2009; Hussain et al., 2013). It is a voracious feeder and most prolific. A single female may give birth to about ﬁve million weevils in four generations, within 14 months (Nirula, 1956) which is a very high reproduction rate (Rahalkar et al., 1972; Avand-Faghih, 1996; Esteban-Duran et al., 1998; Cabello, 2006). Male RPW produces aggregation pheromones which attract weevils to damaged plants (Gunawardena and Bandarage, 1995). Larvae are damaging stages that remain confined within tree trunk, exploiting the stem vascular system and bore into the trunk (heart of the host) causing death of the palm (Ju et al., 2011; Hussain et al., 2013). Usually 3-4 generations comprised of different stages of the insect may be seen inside an infested palm (Rahalkar et al.,

1972) but in Egypt 21 generations have been reported in a single year by Salama et al. (2002). This high rate of multiplication may be attributed to continuous egg laying throughout the year, with some periods being more intense than others. During her life span a single RPW female can lay 58-760 eggs (Avand-Faghih 1996; Abraham et al. 2002; Kaakeh, 2005; Faleiro, 2006, Prabhu and Patil, 2009) which incubate for 1-6 days, before hatching into whitish yellow larvae (grubs), which live for 25-105 days depending on the weather conditions (Wattanapongsiri, 1966; Avand-Faghih, 1996; Abraham et al., 2002). The neonate larvae chew plant fibers and penetrate the interior leaving behind the chewed-up frass that has a typical fermented odor. The completely developed grubs pupate in a cocoon fabricated from chewed ﬁbers and the pupal period lasts for 11-45 days. The life cycle of the pest may vary from just 45-139 days reported from Philippines and Spain respectively (Esteban-Duran et al., 1998; Murphy and Briscoe, 1999). Adult weevils can interbreed and live within the same host until they are required to colonize a new palm. If the plant remains untreated the palm can die within 6-8 months (Kurian and Mathen, 1971; Avand-Faghih, 1996; Rajamanickam et al., 1995). By now the beetle is distributed over more than 50% of the date palm cultivated areas worldwide causing wide-spread damage. The ancient records of the beetle date back to the 1750s when Rhumph first found this species on sago palm Sagu Campas, in Ceylon in 1750-1755. He also first described the larvae, cocoon and dorsal and ventral views of the adult as Cossus sagurios. This name was not valid according to the ICZN article 3, 11 (a), and 86. In 1776, Sulzer identified and described the weevil from India as Curculios hemipterus Linnaeus, 1758. The weevil was later described as Curculios ferrugineus by Olivier in 1790 and this name is currently used. When Herbst erected the genus Rhynchophorus, he pointed out that R. ferrugineus (Olivier) was commonly mistaken for C. hemipterus Linn. He also pointed out the difference between two species. In 1797 Thunberg described a male specimen from India as Cordyle sexmaculatus. It was placed in synonymy with R. ferrugineus by Csiki in 1936. Chevrolate 1882 described the male and female specimens from Assam as “R. indostanus” and a male from Ceylon as “R. signaticillis” based on the shapes and number of spots on the pronotum (Wattanapongsiri, 1966). As described earlier the beetle is native to the Indian sub-continent that was identified by Olivier in 1790. It was observed first time from India in 1891, but devastation to coconut palms was not reported until 1906 (Lefroy, 1906). Until 1917, RPW was considered the only pest of coconut palm but latter on it was found attacking date palms in Punjab, India (Mohan, 1917; Buxton, 1920). During the same period (1918) RPW also inflicted harmful effects to date palm in Mesopotamia, but damage authentication was not confirmed by collecting any insect specimen. Furthermore, RPW is generally considered to be invasive in Pakistan, although it was first formerly reported in what are now the Multan, Muzaffargarh and Dera Ghazi Khan Districts of the Pakistani province of Punjab, and the neighboring Indian state of Punjab, almost a century ago (Mohan, 1917; Milne, 1918). The native range of RPW is thought to be restricted to Southeast Asia and Melanesia, stretching: through the countries bordering the Bay of Bengal from Sri-Lanka to the Malayan peninsula and Singapore; through Thailand, Cambodia and Vietnam; across the South China Sea to Taiwan and the Philippines; and down through the Sunda Islands (Java, Sumatra and Borneo) (Wattanapongsiri, 1966). Latter on worldwide distribution of the pest was recorded from Japan in 1975 (Matsuura, 1993). Since the mid-1980s, weevils advanced westward rapidly from Southern Asia and Melanesia (Gomez and Ferry, 1999) and the Kingdom of United Arab Emirates (UAE), Kingdom of Saudi Arabia (KSA) and Oman in 1985 (El-Ezaby, 1997), south of Spain in 1994

(Barranco et al., 1996), Savaran region of Iran in 1996 (Avand-Faghih, 1996), Palestine, Israel and Jordan 1999 (Kehat, 1999) Sharquiya region of Egypt in 1992 (Cox, 1993) in China the weevil was detected in 2007 (Li et al., 2009) and recently been reported in Cyprus, Morocco, Italy, France, Turkey, Greece, Portugal, Aruba and Syria (Zhang et al., 2008). The weevil has invaded every country of Southern, South Eastern and Western Asia (EPPO 2005, 2008) and lastly in Australia (Li et al., 2009) and California USA (NAPPO, 2010). It was added to the EPPO Alert list in 1999, since 2006 has been included on the A2 list of pests recommended for regulation (no. 339) (Melifronidou-Pantelidou, 2009; Nardi et al., 2011). This pest has spread to different climatic regions including Mediterranean, Monsoon, coastal, Arid and Semi-Arid (Avand-Faghih, 1996; Faleiro, 2006; El-Mergawy et al., 2011; Hussain et al., 2014). According to Rahalkar et al. (1972), the environment does not have a marked influence on the growth and development of the weevil. However, Ramachandran (1991, 1998) revealed variations in morphology and habit of RPW samples collected from different parts of India and suggested that fecundity and sex ratio may influence F1 and F2 progeny. DNA finger prints of three morphologically different forms of RPW collected from Egyptian date plantations indicated major genetic variations in the three forms (Salama and Saker, 2002). Agro-climatic conditions of the region, morphology of the date palm and modern farming systems have provided an environment conducive to the rapid establishment of RPW in the Middle East (Abraham et al., 1998). Different control tactics have been employed against RPW within an IPM strategy. The main component used against RPW is phyto-sanitation, mechanical control and deployment of chemical insecticides (Nirula, 1956; Abraham, 1971; Butani, 1975; Faleiro, 2006; Ajlan et al., 2000), use of plant extracts (Nassar and Abdullah, 2001), and pheromone trapping (Hagley, 1965; Hallet et al., 1993; Oehlschlager et al., 1993). Chemical insecticides are efficient in RPW control but they are short-lived and need to be applied periodically with possible negative consequences for human health and the induction of resistance in the insect (Abraham et al., 1998; Ferry and Gomez, 2002; Faleiro, 2006; Llácer et al., 2012a). Moreover, the unwise use of chemical insecticides has led the resistance against this pest (Abraham et al., 1998); To combat this problem some control measures should be initiated that are environmentally friendly and compatible with human health. Bio-control agents are an alternate method to potentially replace many chemical pesticides now used against RPW (Gauglar and Kaya, 1990). Entomopathogenic fungi (EPFs), entomopathogenic bacteria and entomopathogenic nematodes (EPNs) have been found very effective against a vast array of insect orders. Microorganisms have been successfully used to control a number of insect pests of economic importance (Francesca et al., 2015). Among them EPFs are an important microbial control agent and their effectiveness has been studied by a number of scientists (Murphy and Briscoe, 1999; Faleiro, 2006), particularly Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Clavicipitaceae) and, to a lesser extent, Metarhizium anisopliae (Metschnikoff) Sokorin (Ascomycota: Clavicipitaceae) (Deadman et al., 2001; Ghazavi and Avand-Faghih, 2002). Microbiological treatments with B. bassiana and M. anisopliae offer an alternative and bio-rational pest management strategy (Inglis et al., 2001) and this is the alternate method where potential replacement of many chemical pesticides may occur against RPW (Gauglar and Kaya, 1990; Merghem, 2011; Deadman et al., 2001;Gindin et al., 2006; El-Sufty et al., 2007; 2009; 2011;Sewify et al., 2009; Torta et al., 2009; Vitale et al., 2009; Dembilio et al., 2010; Francardi et al., 2012).

EPFs cause natural epizootics in insect pests through contact to the host body following penetrating, germination and proliferation into the host body and ultimately killing the host (Zimmermann, 2007). Infection is caused by direct application or by horizontal transmission from one developmental stage to the other (subsequent developmental stages) (Lacey et al., 1999; Quesada-Moraga et al., 2004). Similarly, mechanical transmission within populations has also been recorded for M. anisopliae, B. bassiana and Isaria fumosorosea (Lacey et al., 1999; Quesada-Moraga et al., 2004, 2008). These peculiar characteristics enable EPFs to combat concealed insect pests. Same is the case with RPW, whose most stages live into the tree trunk, enabling the pest to direct contact with the treatments applied except the adult stages which can be infected on emergence. Being the main pathogens of Lepidopteran pests EPFs can actively participate in the control of coleopteran pests as well due to its active mode of infestation on the outer surface of host cuticle (Hajek and St Leger, 1994; Lacey et al., 1999). Some researchers believe that EPFs can be effective as bio-control agents against RPW (Lacey et al., 1999; Dembilio et al., 2010; Francardi et al., 2013; Ricaño et al., 2013). The recent identification of strains of M. anisopliae and B. bassiana with high virulence against the RPW has increased the possibility of a more efficient microbiological control of the Curculionid. Entomopathogenic bacteria also play a significant role in managing insect pest populations which include the members of genus Bacillus (Salama et al., 2004). A number of species from this genus are successfully deployed against a variety of insect pests including the member of order Coleoptera; the species are stage specific. Bacillus thuringiensis (Bt), B. lentimorbus, B. sphaericus and B. popilliae synthesize insecticidal proteins (Bulla et al., 1975). B. thuringiensis is a spore-forming gram positive bacterium is the selectively toxic products of Bt; products that are harmless to mammals and acceptable to environment (Entwistle et al., 1993). B. thuringiensis produces protein crystal when larvae ingested this crystal protein through his food, crystal protein dissolved in the alkaline environment of larval midgut. Actual toxic fragment (protein) is produced when the dissolved crystal protein is proteolytically processed. This proteoltically processed protein adheres to the intima membrane of midgut columnar cells. The spores are produced on the epithelial cell membranes by membrane bound proteins. Finally, as a result of spore formation the cells of the larvae die (Bauer, 1995; Aronson et al., 1986; Gill et al., 1992). Manachini et al. (2009) evaluated a Bt based commercial product registered against coleopteran pests for the control of RPW and found the pathogenicity with small increase in concentrations than the recommended dose for Coleoptera. Experiments showed that the total number of circulating hemocytes (mainly the plasmatocytes) gradually decreased after 19 hours when RPW larvae fed with Bt spores. In this experiment for the first time a very high number of Bt vegetative forms were recorded in the hemolymph of RPW larvae after exposure to the Bt commercial product. As far as the mode of action is concerned, the Cry toxins produced after the ingestion of Bt spores in alkaline media that lyse larval midgut epithelial cells (Bravo et al., 2007). Cell contents together with other components promote spore germination which leads to the severe septicemia and ultimately cause the insect death. It has been proposed that during vegetative growth, Bt release some kind of new insecticidal proteins (Soberón, 2005; Salamitou et al., 2000; De Maagd et al., 2001; Bravo et al., 2007). The ﬁndings that bacteria, as a vegetative form, are in RPW hemolymph suggests that Bt is able to bypass the various above described steps to reach the hemolymph and affect the defense system. THC were dramatically reduced, especially the plasmatocytes. Many parasites must avoid hemocyte-mediated immune responses to growth in

4 host larvae, and many species achieve this by suppressing one or more components of the host immune defense system, e.g. alteration of THC, inhibition of hemocyte spreading, apoptosis in circulating hemocytes (Adamo, 2005; Eleftherianos et al., 2008; Ericsson et al., 2009). The interaction between entomopathogenic bacteria and hemocytes is little studied in insects and the available literature is mainly on Lepidoptera. However similar results to our ﬁndings have been found in insect response to other entomopathogenic bacteria, for example Bt-k in Trichoplusia ni (Ericsson et al., 2009) and Photorhabdus in ﬁfth-stage larvae of Manduca sexta (Eleftherianos et al., 2008). Differences in antibacterial responses have been attributed to bacterial species and virulence levels (Dettloff et al., 2001; Giannoulis et al., 2007), however some quite important gaps in understanding general mode of action of Bt still exist (Then, 2009). Indeed, there are several contradictions among the different models (Then, 2009). Thus, question of how bacteria act in RPW larvae is still open. EPNs are obligate parasites in the families Steinernematidae and Heterorhabditidae. They kill insects with the aid of mutualistic bacteria, which are carried in their intestine (Xenorhabdus spp. and Photorhabdus spp. are associated with Steinernema spp. and Heterorhabditis spp., respectively) (Poinar, 1990). The nematodes complete 2-3 generations within the host, after which free-living Infective Juveniles (IJs) emerge to seek new hosts (Poinar, 1990). The pathogenicity of EPNs to Helicoverpa sp. has been demonstrated previously (Bong, 1986). Furthermore, they have been found effective against a variety of insect pests including foliage feeders and they have been effective mainly against soil-inhabiting pests (Kaya, 1990). Several formulations have been developed to improve the activity of nematodes on plant and in stored products. In coleopteran pests larvae of several weevil species (Coleoptera: Curculionidae) such as the black vine weevil, Otiorhynchus sulcatus (F.), and the Diaprepes root weevil, Diaprepes abbreviatus (L.) (Shapiro-Ilan et al., 2002) are susceptible to EPNs. One approach to controlling H. armigera with EPNs would be to target the larvae when they drop to the ground or after burrowing into the soil for pupate. Currently efforts are focused on developing integrated control strategies against RPW by combining more than one control agent e.g. integrated use of EPFs, EPNs and entomopathogenic bacteria. Several researchers have demonstrated successful control of multivoltine coleopterans by combining microbial agents. In view of the extent of damage to the date palms attributed to RPW, it was considered worthwhile to exploit bio-rational approaches. The present study was carried out for evaluating the efficacy of different microbial control agents with the aim to meet the following objectives;

 To check the genetic variation among populations of Red Palm Weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) from the Punjab and Khyber Pakhtunkhwa provinces of Pakistan

 To check the resistance to commonly used insecticides and phosphine (PH3) against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) in Punjab and Khyber Pakhtunkhwa, Pakistan

 To check the Insecticidal potential of Beauveria bassiana and Metarhizium anisopliae isolates against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

 To check the combined effectiveness of endophytically colonized Beauveria bassiana and Bacillus thuringiensis against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

 To check the integrated effect of entomopathogenic fungi and entomopathogenic nematode against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

 To evaluate the combined toxicity of Beauveria bassiana, Bacillus thuringiensis and Heterorhabditis bacteriophora against red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

CHAPTER 2

2.1 Invasive Red Palm Weevil (RPW) Some invasive insect pests are of great importance due to their habit of severely damaging the agricultural products, and imposing severe threats to ecology and causing serious economic losses (Kenis et al., 2009; Simberloff et al., 2013). Resultantly, these annoying pests cause direct losses of worth thousand million dollars annually and money involved in management efforts to reduce populations below economic thresh hold level (Pimentel et al., 2005; Kovacs et al., 2010; Van Driesche et al., 2010; Simberloff et al., 2013). RPW is an important invasive pest that has invaded more than 50% of the date palm growing areas of the world. This is attributed to a higher fecundity than most species (Faleiro, 2006), capability to live and interbreed in the same tree even for several years (Avand-Faghih, 1996; Rajamanickam et al., 1995), adult flight capacity (Wattanapongsiri, 1966) and pest tolerance to a wide range of climatic conditions due to its protected habit within palm trees.

2.2 Taxonomic position A key to revision of this species and related genera was previously provided by Wattanapongsiri (1966). RPW was classified under order Coleoptera, the family Curculionidae and the subfamily Rhynchophorinae (Wattanapongsiri, 1966; EPPO, 2007). Synonymously it is also called Asian Palm Weevil, Indian Palm Weevil or Pakistani Weevil. This genus has 10 other species, three of them identified from New World, two African and five tropical Asian countries. Among these R. bilineatus, R. quadrangulus, R. palmarum, R. bilineatus, R. lobatus, R. distinctus R. ritcheri, R. vulneratus are severe pest of palms (Booth et al., 1990; Hallet et al., 2004).

2.3 Classification Kingdom: Animalia (Animals) Phylum: Arthropoda (Arthropods) Subphylum: Hexapoda (Hexapods) Class: Insecta (Insects) Order: Coleoptera (Beetles) Suborder: Polyphaga (Water, Rove, Scarab, Leaf and Snout Beetles) Superfamily: Curculionoidea (Snout and Bark Beetles) Family: Curculionoidea (Snout and Bark Beetles) Subfamily: Dryophthorinae Tribe: Rhynchophorini Genus: Rhynchophorus Species: ferrugineus (Red Palm Weevil)

2.4 Spatial Distribution The aboriginal home of RPW is considered to be the Southern Asia and Melanesia, it is cryptic in nature and has been reported to attack more than 29 different palm species belonging to 18 genera and 3 families (Malumphy and Moran, 2009; Hussain et al., 2013), including date palms and coconut palms, as well as the Mediterranean fan palms, the native Cretan date palms (Kontodimas et al., 2006; Dembilio et al., 2011) and Canary Islands date palms (Dembilio et al., 2009). Currently, this pest has spread to many areas of the world; its range now includes much of

Asia, regions of Oceania, Southern Europe, Middle-East, North Africa, the Caribbean, and in October 2010, five specimens belonging to the genus Rhynchophorus sp., were found in southern California (EPPO, 2008; 2009; 2011).

2.5 Control Measures Different control practices have been deployed to combat RPW among date palm growing areas of the world. Treatments revolve around the use of conventional chemical insecticides, sterile insect techniques and use of semio-chemicals (Paoli et al., 2014) and biocontrol agents (Wattanapongsiri, 1966; Murphy and Briscoe, 1999; Faleiro, 2006a, b). Integration of RPW associated microbial control agents with other control practices such as biocontrol agents with chemical insecticides and attract-and-kill techniques.

2.5.1 Microbial control Microbial pest control relies on use of microbes such as EPFs, EPNs, entomopathogenic bacteria and viruses. Very few researchers have systematically studied the effect of entomopathogens on RPW (Murphy and Briscoe, 1999; Faleiro, 2006). Conversely, entomopathogenic microorganisms, in particular mitosporic ascomycetes, have been reported to naturally regulate RPW populations (Dembilio et al., 2010b). The deployment of microbial control agents in pest control is an important step towards mitigating reliance on conventional chemical insecticides. Microorganism exhibit high degrees of host specificity that accounts for their distinguish ability to search their host. The use of entomopathogenic microbes has a number of advantages such as safer to environment and non-target organisms, cheaper and self- perpetuation. Environment friendly control practices against RPW are getting serious attention in many parts of the world. Concentrations are focused on use of entomopathogens such as EPFs, EPNs, entomopathogenic bacteria and their integration with the chemical insecticides and plant extracts.

2.5.2 History of microbial control Insects and microorganisms have ancient relationships well described by insects conserved in amber 15 to 20 million years ago, as the collection of several insect cadavers dressed with entomopathogens like neucleopolyhedrovirus (NPV), EPNs, trypanosomes is reported (Poinar and Poinar, 2005). It has been an old profession; however, its roots can be traced back to the time of Aristotle (2700 BC), who observed the diseased silk worm with whitish growth on the dead larvae of silk worm during 335 BC. It was not until the work of Agostino Bassi (1773-1856) an Italian lawyer and scientist reported the fungus, B. bassiana on the larvae as a whitish sooty growth. This led to the germ theory of disease and named “Calcinaccio” disease because the dead larvae exhibit whitish calcium powder like coverings (Steinhaus, 1956, 1975). Agostino Bassi observed that the causal agent of the disease as “vegetable parasite” a fungus now called B. bassiana that may be transferred through inoculation, contact or by the ingestion of the leaves by the caterpillar. This was the first research of Bassi which confirmed that microorganisms could cause disease and also it was the important contribution towards disproving the idea of spontaneous generation. Calcinaccio disease was found plaguing the silk industry first in Italy (1805) and then in France (1841). Bassi conducted scientific studies on Calcinaccio disease in 1807. After long and comprehensive observations, in 1835 Bassi

8 confirmed that this disease causing entity is a living organism which produced whitish growth on the dead larvae. He was honored for rescuing the precious and economically important silk industry by suggesting separation of the rows of caterpillars feeding on the mulberry leaves, disinfection process, destroying dead cadavers and keeping the rearing room clean and infection free. His findings were translated and distributed throughout the Europe and greatly helped Louis Pasteur (1822-1895) to study the cause and potential cure of the disease in Europe (Porter, 1973). In the same year, a famous Italian naturalist Giuseppe Gabriel Balsamo-Crivelli studied and named the fungus, Botrytis bassiana in the honor of Bassi (Steinhaus, 1949; Müller-Kögler, 1965; Rehner, 2005). The species B. bassiana came into existence when in 1911 Beauverie studied the fungus again and Vuillemin created the new genus Beauveria in honor of Beauverie in 1912, since then the species B. bassiana became the type. In 1865, French silk industry was badly devastated and Louis Pasteur was asked to identify the disease. He was not reluctant to accept the offer, although he was not fully aware of silk worms, he was persuaded by his teacher and friend Senator Jean-Baptist Dumas to move and consult the famous entomologist Jean Henri Fabre (1823-1915) in Alés Village in the south of France. After several years he came to the conclusion that two silk worm diseases "pébrine" and "flacherie" (thought to be caused by bacterium) are responsible for the decline of silk industry. He proposed that pébrine is characterized by tiny black spots on the surface of dead larvae of silk worm caused by the microorganism Nosema bombycis, previously described by Nägeli (1857). For the potential elimination of the disease, he proposed that careful handling, segregating of healthy and diseased larvae, and well maintained sanitation conditions may be helpful in disease prevention (Debré, 1998). During the study it is found that disease can be transmitted by contaminated food, contact with the infected caterpillar and even from mother to the offspring. This is the first study demonstrating the vertical transmission of the disease (Pasteur, 1874). He published his findings in two series to make people aware of silk worm disease and its prevention (Pasteur, 1874). This work laid the foundation for advances in sericulture in Japan dealing with the molecular and biochemical biology of the silk worm. The scientists like, Agostino Bassi, Louis Pasteur and Elie Metchnikoff, the19th century pioneers also proposed that these micro-organisms can be a good solution for controlling economically important insect pests (Steinhaus, 1956, 1975). The first half and end of the 19th century was the period of most development in invertebrate pathology, it was not until the discovery of B. thuringiensis (Bt) Berliner, that practical and massive use of entomopathogens started (Lacey and Goettel, 1995). In 1879, Metchnikoff discovered the diseased larvae of wheat cockchafer and later on Cleonus punctiventris near Odessa (Ukraine). He named this fungus the green muscardine fungus. The genus Metarhizium was first established by Sorokin (1883). For this fungus, he first proposed the name Entomophthora anisopliae, and later renamed as Isaria destructor. The history of the description, discovery the scientific research and on the use of fungus in biological control is described in detail by Steinhaus (1949) and Müller-Kögler (1965). In the start of 20th century B. thuringiensis was recovered first time from infected silk worm larvae by a Japanese bacteriologist (Ishiwata, 1901) and subsequently in 1911 German biologist Berliner re- discovered the disease. He isolated the bacterium from infected larvae of Mediterranean flour moth (Berliner, 1915) so named it B. thuringiensis. Because of high and knock down mortality effects with small amount of B. thuringiensis preparations, the agronomist get aware about the insecticidal properties of this bacterium. The first B. thuringiensis based commercial formulation

“Sporéine” was developed in France in 1938, but the 1st well documented record of commercial procedure for producing Bt-based product dates from 1959 by the “Bactospéine” under the 1st French patent as a bio-pesticide formulation. Since after, a vast array of microorganisms like fungi, bacteria, viruses and protozoans has been identified as potential biocontrol agent against insect pests (Riba and Silvy, 1989). So far, even though more than 100 species of entomopathogenic bacteria have been identified, only a few Bacillus species have met with commercial success, B. thuringiensis in particular (Starnes et al., 1993). Today a vast array of entomopathogens are deployed against insect pests of agriculture importance such as fruits, cereals, ornamentals, stored commodities, insect pests of households and insect vector of medical and veterinary importance (Tanada and Kaya,1993; Lacey and Kaya, 2007). Microbial control agents used against insect pests includes EPFs, EPNs, viruses, protozoa and bacteria. Keeping in mind the harmful effects of chemical insecticides and their impact on the environment and human health, insecticides based on entomopathogens exert only a small fraction of hazards on environment and human health as compared to the conventional insecticides. The share of bio-pesticide in crop protection market is about 600 million US$ which accounts for only 2% of the total pesticides, with about 90% of all bio-pesticide sales involving products based on B. thuringiensis. The comparison of microbial pesticides with chemical pesticides is usually exclusively cost effectiveness. These microbial insecticides particularly offer unique advantages when there are environmental and human safety concerns along with the increasing need of enriched biodiversity in an ecosystem and increased activity of natural enemies (Shahid et al., 2012). Furthermore, ease of application, production on artificial medium and long term storage are further distinct features of these bio-insecticides over other insect control tactics.

2.5.3 Entomopathogenic Fungi (EPFs) 2.5.3.1 History The history and research on mycopathogens invading insect pests is ancient. Before the invention of microscopes fungi could be seen with naked eye and this observation helped to establish invertebrate pathology as a modern study. Fungi are categorized in a number of taxa that exhibit greater diversity in properties, requirements and found in all arthropod habitats. As a result, great attention was diverted to possible use of fungi as microbial control of insect pests. The fungi are heterotrophic, eukaryotic, absorptive individuals which may develop in different patterns like diffuse, branched, or tubular body that can reproduce sexually as well as asexually (Kendrick, 2000). The primitive studies regarding entomopathogenic fungi were conducted during start of 18th century with an aim to develop control strategies for managing muscardine diseases of silk worm (Steinhaus, 1975). Bassi (1835 as cited by Steinhaus, 1975) proposed the germ theory using silkworm and invading fungus, later this was named Beauveria bassiana in the honor of Bassi. His studies on silkworm disease assisted him to introduce the fungal biocontrol agents for the control of insect vector that elicit disease in human beings. The silkworm diseases provided gross root foundations for the control of insect pests by employing entomopathogens. Nevertheless, the major efforts were attempted in deploying EPFs for the control of insect pests carried out during 1950’s when chemical insecticides were invented. There are many fungal based products commercially available worldwide now-a-days (Shah and Goettel, 1999; Copping, 2001). EPFs have a long primordial historic recognition; their illustrated descriptions can be seen centuries back, infection of B. bassiana and Cordyceps sp. to silk worm described in ancient

Japanese paintings infections of insects date from the 19th century (Samson et al., 1988). As a vocation, invertebrate pathology is an organized discipline. Historic stories can be drawn from the solution of silk worm and honey bee diseases prevention from entomopathogens (Steinhaus, 1956, 1975). Very first reports of managing insect pests in insect pathology with entomopathogenic fungi were proposed by the legend pioneers like Louis Pasteur, Elie Metchnikoff and Agostino Bassi (Steinhaus, 1975). Currently numerous entomopathogens are deploying for managing insect pests in lawn and turf, orchards, glasshouse, ornamentals, row crops, forestry, range lands, stored products, pest and insect vectors of medical and veterinary importance (Tanada and Kaya, 1993; Lacey and Kaya, 2007).

2.5.3.2 Geographical distribution and occurrence Soil is vital source for a number of EPFs especially species belonging to Ascomycota thus they serve to regulate the insect populations in soil (Keller and Zimmermann, 1989; Hajek, 1997) as most arthropods spent some of their life stages into the soil. The knowledge about the indigenous isolates of EPFs, their diversity, distribution and composition is key factor to conserve these indigenous fungal species for the natural control of the insect pest populations within the agro-ecosystem. The detailed studies have been conducted on the 14 occurrences? of soil dwelling entomopathogenic fungi in different countries, the data from around the world suggest them to be ubiquitous inhabitants of the soil (Chandler et al., 1997). The effect of different factors like climatic conditions, geographical distribution, habitat type, and soil properties, soil pH (Foth, 1984; Ali-Shtayeh et al., 2002; Padmavathi et al., 2003), soil organic matter (Milner, 1989; Mietkiewski et al., 1997), soil type (Storey and Gardner, 1988; Rath et al., 1992; Inglis et al., 2001; Derakhshan, 2008), soil moisture contents (Ali-Shtayeh et al., 2002) on fungal incidence and dispersal has been studied several times (Vänninen et al., 1989; Chandler et al., 1997; Meyling and Eilenberg, 2006; Zimmerman, 2007). The worldwide distribution of EPFs in insects from different habitats is also reported by many authors (MacLeod, 1954; Evans, 1982; Wraight et al., 1993; Aung et al., 2008; Thakur and Sandhu, 2010). The naturally occurring EPFs can be obtained by collecting insects from the field then incubating them under laboratory conditions and checking for outgrowth of fungi (Meyling and Eilenberg, 2007). EPFs from eight genera (Entomphthora, Batkoa, Conidiobolus, Pandora, Erynia, Neozygites, Zoospora and Tarichium) have been isolated from aphids. The most common entomopathogenic fungi, B. bassiana, P. fumosoroseus, and P. farinosus were recently isolated from some new insect hosts such as beetles of Agrilus species and hairy caterpillar of Lymantria species from Central India (Thakur and Sandhu, 2010).

2.5.3.3 Classification Among different fungal divisions, EPFs belongs to Ascomycota, Zygomycota and Deuteromycota (Samson et al., 1988), Oomycota and Chytridiomycota (classifies within fungi previously). Many of the genera of EPFs currently under research belong either to the class Hyphomycetes in the Deuteromycota or to the class Entomophthorales in the Zygomycota.

2.5.3.4 Host range Fungal infections to the most insect orders with all life stages have been observed, while infection to the immatures of holometabolous insects have been reported more commonly (Tanada and Kaya, 1993). The host range may differ significantly among different species of EPFs and even among different strains of the same single species. For obligate pathogens,

11 specifically restricted to a narrow host range and complicated life cycles associated to their insect host like Strongwellsea castrans (Phycomycetes: Entomophthoraceae), restricted to flies like anthomyiid (Eilenberg and Michelsen, 1999) and Entomophthorales, Massospora sp. are restricted to a single genus belonging to cicadas (Soper, 1974). In contrast, Deuteromycetes, particularly B. bassiana, have wide host range including numerous genera of insects (McCoy et al., 1988). It must be kept under consideration that description of host range to some extent mainly relies on laboratory studies which do not reflect the true picture in nature. Some factors like insect host, fungal biology and ecology may be responsible for reducing infection in insect host. It is important to mention that fungi are capable of infecting several other arthropods, insects and the species which are not pests of cultivated crops (Gibellula spp. predator of spiders and, Erynia and Cordyceps sp. infects ants).

2.5.3.5 Mode of infection The fungal infection of insect hosts is a complex process, involving chemical and physical procedures starting from spore attachment to host death. Following steps are undertaken during infection process: (1) spore attachment to the host cuticle, (2) germination of fungal spore, (3) diffusion into the host cuticle, (4) overcoming the immune defense mechanism, (5) formation and proliferation of hyphal bodies into the hemocoel, (6) saprophytic outgrowth from the dead host, production and dissemination of new conidia. For the successful attachment, mainly hydrophobicity of the spore and cuticular surface play significant role. Furthermore, the germination and infection is influenced by a number of factors e.g., humidity, optimal temperature, susceptible host stage and cuticular lipids, such as aldehydes, ketones, wax, short- chain fatty acids, alcohols and esters which may exhibit antimicrobial activity. Generally, fungal spores breach through the non-sclerotised parts of the cuticle such as joints, between segments or the mouthparts. The conidial germination starts after 10 h of attachment and may complete by 20 h at 20-25 oC. Before infection process, germ tube produce appressorium or penetration pegs which is accompanied by mechanical and chemical processes by the production of several enzymes (Ortiz-Urquiza and Keyhani, 2013).

2.5.3.6 Enzymes and toxins of EPFs Along with different degrading enzymes (such as lipase, protease, chitinase) which account for the virulence of different entomopathogenic fungi (Joshi et al., 1995; Fan et al., 2007), certain secondary metabolites of these fungi also possess insecticidal activities and contribute to the pathogenesis of the fungal strains (Mollier et al., 1994). Some metabolites may also act as the defensive tool by protecting the fungi from certain hostile factors such as competitive micro-organism (Dowd, 1992; Bandani et al., 2000). The type of toxins produced may also be helpful in defining the mode of action of the entomopathogenic fungi (Vey et al., 1993). The toxins produced by different entomopathogenic fungi, their role in fungal efficacy and safety concerns about the utilization of these compounds have been discussed by several researchers (Roberts, 1981; Strasser et al., 2000; Vey et al., 2001).

2.5.3.7 Chitinases The chitin is a major constituent of the insect cuticle, therefore, endo and exo chitanses are important enzymes for the breakdown of N-acetylglucosamine polymer of insect cuticle into monomers and a key factor determining the fungal virulence (Khachatourians, 1991).

Endochitinases, N-acetyl-β-D-glucosaminidases and chitinolytic enzymes from M. anisopliae and M. flavovirid and B. bassiana were presented in broth culture nourished with insect cuticles.

2.5.3.8 Proteases and peptidases Chitin and protein are the main constituents of insect cuticle; hence proteases and peptidases of EPFs is considered key component in degradation of insect cuticle, saprophytic growth, initiation of prophenol oxidase in insect hemolymph, furthermore they are also responsible for virulence in EPFs. Chymotrypsin (CHY1) of 374 amino acids, with pI of 5.07 and MW38279 were investigated from M. anisopliae by Screen and St Leger (2000). Some genes of overlapping response with a unique expiration pattern were observed when encountered with the cuticle of Blaberus giganteus, Popilla japonica and Lymantria dispar and using cDNA counted gene expression responses to the cuticles of number of host insects and constructed microarrays from expressed sequence tags, clone of 837 genes (Freimoser et al., 2005).

2.5.3.9 Lipases The epicuticle of the insects is chiefly composed of non-polar lipids which play an important role in chemical signaling between insect host and EPFs (Blomquist and Vogt, 2003), and keeps cuticular outer surface dry which aids to avoid the penetration of chemicals and insecticides (Blomquist et al., 1987; Juárez, 1994). They are chemically stable with high molecular mass, mainly due to the presence of specific physicochemical characteristics, like number of carbons, length of the chain and the kind and position of double bond and the functional groups. The long chain HC, free fatty acids, fatty alcohols and wax esters are ample components of the insect epicuticle. It also contains fats, waxy layers and lipoproteins which act as a barrier to the action of lipoxygenases and lipases of eontomopathogenic fungus. Among these compounds some have anti-fungal activities (Khachatourians, 1996) while some other possess saturated fatty acids which can inhibit the fungal growth.

2.5.3.10 Toxins The biochemical properties and structure of some major fungal metabolites have been investigated in detail (Vey et al., 2001), but very few studies have been conducted regarding the metabolite production under field conditions (Bandani et al., 2000; Strasser et al., 2000). One major problem to fungal toxins is that one type of fungi can produce variety of bioactive metabolites and risk assessment to these entire compounds would be enormous. Furthermore, fate of their toxins is little known in the environment, which would be the key question for their registration.

2.5.3.11 Destruxins Destruxins are moderately dissimilar compounds which exist in the form of isomers. Basically destruxins contain 5 amino acids and α-hydroxy acid which may be found in many different forms. Till now 28 different but structurally similar destruxins have been isolated from different EPFs and most of these are discovered from M. anisopliae isolates (Vey et al., 2001). Insects exhibit varying susceptibility levels to destruxins and Lepidopterans have been reported as the most susceptible amongst the all studied insect orders (Samuels et al., 1988; Kershaw et al., 1999). The toxicosis symptoms also vary among insect pests the most peculiar symptom are an immediate tetanus; which at low concentrations develops for up to three minutes period, while, brief or no paralysis is depicted at high dose rates (Abalis, 1981; Samuels et al., 1988).

2.5.3.12 Oosporein Oosporein is produced mainly from the soil inhibiting fungi like Beauveria spp. which contain red colored di-benzoquinone (Eyal et al., 1994). It reacts with amino acids and proteins through redox reaction by altering the SH-groups and results malfunctioning in enzymes (Wilson, 1971). Like bassianin and tenellin, oosporein also inhibit the activity of erythrocyte membrane ATPase which is directly proportional to the dose rate of oosporein. Up to 50% activity can be ceased at 200g/ml. All these pigments greatly influenced Ca2+-ATPases compared to the activity of Na+/K+-ATPase. Antibiotic effect of oosporein against gram-positive bacteria has also been observed with no or little effect on gram negative bacteria (Taniguchi et al., 1984; Wainwright et al., 1986).

2.5.3.13 Beauvericin and beauveriolide Beauvericin is also an important toxin isolated from Beauveria, Paecilomyces sp., the plant pathogenic fungi Polyporus fumosoroseus and Fusarium sp. (Gupta et al., 1991; Plattner and Nelson, 1994). Gupta et al. (1995) described two different forms of these toxicants Beauvericin A and B forms K+ and Na+ complexes, which increase the membranes permeability (Ovchinnikov et al., 1971). It also exhibits antibiotic activity against a number of bacteria like, Mycobacterium phlei, Escherichia coli, Sarcinea lutea, Bacillus subtilis, Staphylococcus aureus and Streptococcus faecalis (Ovchinnikov et al., 1971).

2.5.3.14 Bassianolide Another toxin cyclo-octadepsipeptide also called bassianolide is secreted by B. bassiana (Suzuki et al., 1977). Bassianolide is also an ionophore which exhibits different reactions with different hosts (Kanaoka et al., 1978). Very little knowledge about the toxic nature of bassianolide against plants and animals, the synergistic interaction with the structurally associated myco-toxin moniliformin may be possible.

2.5.3.15 Beauveriolide Beauveriolide are isolated from Beauveria spp. which is structurally similar to bassianolide and beauvericin (Namatame et al., 1999). The toxic effect of beauveriolid towards plants and animals is still unknown except beauveriolide I (Mochizuki et al., 1993). Overall these cyclodepsipeptides may still have an unreported health hazard effects is common. Except the above mentioned metabolites these B. bassiana also produce bassianin, tenellin and two non- peptide toxins isolated from Beauveria spp. which aid in inhibiting the erythrocyte membrane ATPases (Jeffs and Khachatourians, 1997).

2.5.3.16 Host range EPFs are the diverse group of insect pathogens that contains a large number of genera and species, the exact figure is unidentified but according to some estimation there are approximately 700 species in 100 genera of entomopathogenic fungi, however, Onofre et al. (2001) described it as 90 genera. These insect pathogens have broader host range and have been found naturally occurring in various populations of insect pests. The more extensive work regarding the natural host identification in order to recognize the biological activity of the entomopathogenic fungi is done on B. bassiana and M. anisopliae. The host range for B. bassiana is stated as 700 species including beneficial insects too (Goettel et al., 1990).

2.5.3.17 Effect of abiotic factors 2.5.3.17.1 Temperature Among different abiotic factors affecting the fungal propagation and survival (Roberts and Campbell, 1977; Fuxa, 1995); temperature is very important in determining the germination, growth rate and viability of the fungal conidia not only in the host but in the environment as well. Furthermore; the efficacy of fungal biopesticides has been highly influenced by the environmental temperatures (Inglis et al., 1996; Klass et al., 2007). The awareness about the fungal growth in association with the prevailing temperature is the preliminary step in selection of any fungal strain (Fargues et al., 1992). Ferron (1978) described that optimum values for different entomopathogenic fungi are between 20-30 ºC, which has been verified also by certain other researchers as well such as Ekesi et al. (1999), Dimbi et al. (2004) and Kiewnick (2006). The ability to tolerate different temperature profiles not only varies between the strains but the thermal tolerance between the isolates is also significant (Parker et al., 2003; Dimbi et al., 2004). Bugeme et al. (2008) observed the variable responses of B. bassiana and M. anisopliae isolates and preeminent germination was seen at 25 and 30 ºC whereas 30 ºC was best for the radial growth of the colony. The strains of M. anisopliae had better adoptability to tolerate high temperatures for germination (Inglis et al., 1997; Milner, 1997). The temperature is also considered as a factor which influences the virulence of different fungal isolates (Tefera and Pringle, 2003). As far as the stored grain insect pest management is concerned, mostly the virulence of different entomopathogenic fungi is evaluated under the mutual effect of temperature and relative humidity (Sheeba et al., 2001; Batta, 2004; Athanassiou et al., 2008). The geographical location of the isolates also account for their thermal tolerance as studied by Vidal et al. (1997) who found that isolates of I. fumosorosea (P. fumosoroseus) collected from Europe depicted growth at temperatures between 8-30 ºC (optimum growth rates 20-25 ºC), the isolates from southern United States and West Asia tolerated 8-35 ºC (optimum growth rates 25-28 ºC) whereas Indian isolates showed optimum growth at relatively high temperature range i.e. 32-35 ºC.

2.5.3.17.2 Relative humidity Humidity is also a key abiotic factor which greatly influences the efficacy and viability of entomopathogenic fungi. The role of relative humidity (r.h.) is more significant in spore germination and post mortal sporulation of the entomopathogens (Inglis et al., 2001). The viability of the conidia over a period of time is the important parameter which is mainly influenced by the interaction of relative humidity with different temperature levels (Zimmermann, 2007). The prolonged stability of fungal conidia is reported mostly under cool and dry conditions (Hedgecock et al., 1995; Hong et al., 1997). High relative humidity is the prerequisite for the mycosis on dead cadavers (Fernandes et al., 1989) as optimum sporulation on dead locusts (Schistocera gregaria) at >96% r.h. was observed by Arthurs and Thomas (2001). After the treatment of different stored grain beetle’s eggs with B. bassiana among different relative humidity levels, 92% r.h. reduced eggs hatchability up to 83 and 87% in R. dominica and T. castaneum, respectively (Lord, 2009). Some studies have also revealed that entomopathogenic fungi can germinate and infect the host even at low relative humidity levels (Inglis et al., 2001).

2.5.3.18 Effect of EPFs on non-target organisms EPFs are developed as commercial formulations (biopesticides) to combat the insect pests of agriculture and veterinary importance (Goettel et al., 1990; Kooyman et al., 1997;

Thungrabeab and Tongma, 2007; Reddy et al., 2008; Mahmoud, 2009). Different laboratory trials had also been conducted to test the biological activity of formulated conidia against various insect pests both under laboratory and field conditions (Ibrahim et al., 1999; Inglis et al., 2002; Batta, 2003; Ugine et al., 2005). The effect on non-targeted organisms is one of the basic principles for the evaluation of biopesticides. The broad spectrum activity of fungal entomopathogens (Zimmermann, 2007) is the key point for their successful adaptation as biological control agents but at the same time it might have some effects on non-target or beneficial insects. Peveling and Demba (1997) recommended the mycopesticide as economically sound and ecologically safe control measure of desert locust in date palm as M. flavoviride was safe to the natural enemy (Pharoscymnus anchorago F.) of scale insects of date palm. Cottrell and Shapri-Ilan (2003) also found that exotic Asian lady beetle (Harmonia axyridis) was less susceptible to GHA strain of B. bassiana. On the other hand the negative effect of EPFs has also been reported by several authors. The pathogenicity of eight EPFs isolated from natural populations of Coccinellids was revealed against Coccinella septempunctata (Kubilay et al., 2008). Some previous studies indicated the pathogenicity of EPFs to C. septempunctata (Manjula and Padmavathamma, 1996; Haseeb and Murad, 1997; Cagan and Uhlik, 1999) and other coccinellids as well (James and Lighthart, 1994; Pell and Vandenberg, 2002; Ashouri et al., 2003).

2.5.3.19 Integration of EPF with other control measures The publication “Silent Spring” by Rachel Carson in 1962, not only realized the side effects of chemical insecticides to human health (Purwar and Sachan, 2006) and environment but also lead a way to search for eco-friendly control strategies for the insect pest management. The continuous search for biologically safe and ecologically sound control measures identified some natural agents as potential candidates for insect control. The microbial control of insect pests using viruses, bacteria, EPNs and EPFs (Bhattacharya et al., 2003; Sabbour and Sahab, 2005) has become an important element of integrated pest management (Inglis et al., 2001). Among these microbial agents, EPFs are known as promising alternative to conventional insecticides which can effectively be used against a large number of pest species (McCoy et al., 1988; Zimmermann, 1993; Kaur and Padmaja, 2008). The potential role of EPFs as microbial control agents has been reviewed by various researchers (Evan, 1989; Ferron et al., 1991; Tanada and Kaya, 1993; Boucias and Pendland, 1998; Inglis et al., 2001). One of the best strategies is the combining EPFs with low lethal doses of chemical insecticides (Anderson et al., 1989). As pesticides may have a variety of effects on EPFs (Alves and Leucona, 1998), therefore, care should be taken while selecting the chemical substance that it should enhance the efficacy without any adverse impact on the fungal strain (Inglis et al., 2001). The first report of carbofuran being effectively combined with B. bassiana against Ostrinia nubilalis (European corn borer) came from Lewis et al. (1996). In another study from two carbamate insecticides (carbosulfan and carbofuran), carbosulfan inhibited the growth of B. bassiana and B. brongniartii but overall effect revealed the possibility of using these insecticides in IPM of Melolontha melolontha L. (Bednarek et al., 2004). Among organophosphates, chlopyriphos 20 EC was less toxic while triazophos 40 EC and profenophos 50 EC were moderately toxic to B. bassiana (Amutha et al., 2010). In addition to the chemical stressors various EPFs have also been evaluated in combination with botanicals (phytoproducts); the most prominent of them is neem and neem based insecticides. Akbar et al. (2005) evaluated the effectiveness of B. bassiana for T. castaneum in integrated manner with plant essential oils and

16 organosilicone carriers. Azadirachtin was combined with P. fumosoroseus against Bemisia argentifolii (James, 2003). The interaction and compatibility of different insect growth regulators (IGRs) and herbicides with EPFs is in vague (Inglis et al., 2001). The fungus Lecanicillium muscarium when simultaneously applied with buprofezin a higher mortality of B. tabaci 2nd instar larvae was seen (Cuthbertson et al., 2010). Triflumuron, a benzoylphenyl urea (BPU) which is a chitin synthesis inhibiter acted as “general stressor” and made the Lepidopteran larvae more susceptible to fungal infection by M. anisopline (Hassan and Charnley, 1989). Along with other agrochemicals; the herbicides are also considered as potential inhibitor to entomopathogenic fungi (Inglis et al., 2001). While studying the effects of four commonly used herbicides on vegetative growth and sporulation of six EPFs under laboratory conditions. Poprawski and Majchrowicz (1995) found totally impaired fungal growth at all tested temperatures. The EPFs have also been integrated with some other microbial pathogens as an alternative use of chemical substances (Zimmermann, 1993). The interaction of B. bassiana and B. thuringiensis var. israelensis for the control of Musca domestica in poultry houses was studied in field trials (Mwamburi et al., 2009). In the same scenario the B. thuringiensis has been incorporated with EPFs against a number of insect pests under different agro ecosystems (Kryukov et al., 2009; Lawo et al., 2008; Wraight and Ramos, 2005; Lacey et al., 2001; Brousseau et al., 1998; Molina et al., 2007).

2.5.3.20 EPF against RPW EPFs are commonly found in the nature and cause epizootics in insect populations, thus play a significant role in regulating insect population. Mostly, Entomophthorales and Hyphomycetes attack on terrestrial insects. EPFs from various strains of B. bassiana and M. anisopliae have been found in association with RPW. EPFs are among the most relevant biological agents suggested to control RPW (Faleiro, 2006). Some of these EPF strains were tested against RPW, M. anisopliae being more effective than the latter one (Gindin et al., 2006). However, no strain was originally isolated from RPW in this study. A number of studies were carried out to investigate the effectiveness of EPFs against RPW, the investigations led to the detection of more active isolates against RPW individuals in laboratory and field trials (Gazavi and Avand-Faghih, 2002; Shawir and Al-Jabr, 2010; Shaju et al., 2003; El-Sufty et al., 2007; Tarasco et al., 2007; El-Sufty et al., 2009; Sewify et al., 2009; Dembilio et al., 2010). Usually, EPFs infect their host through contact action which makes them superior than the other entomopathogens (Butt and Goettel, 2000). In RPW infection mostly occur via direct contact to the inoculum, transmission from diseased to the healthy ones (horizontal transmission) and transmission from the subsequent developmental stages (vertical transmission) via new generation of spores (Lacey et al., 1999; Quesada-Moraga et al., 2004). Thus, EPFs must be put forward as potential bio-control agents in IPM to control RPW current outbreaks (Faleiro, 2006; Murphy and Briscoe, 1999).

2.5.3.21 Natural incidence of EPFs on RPW In the beginning, intentions were focused on isolation of fungal strains from RPW; different strains of B. bassiana and M. anisopliae were recovered from pupae and adults of RPW (Ghazavi and Avand-Faghih, 2002). Very first natural infection of M. anisopliae was recorded from R. bilineatus as a result of accidental infection when treatments were applied against Scapanes australis (Bedford, 1974) with M. anisopliae spore based commercial formulation (Prior and Arur, 1985). Latter on different researchers found naturally infected RPW specimens

17 with B. bassiana and M. anisopliae (Salama et al., 2001; Shaiju-Simon and Gokulapalan, 2003; Salama et al., 2004; Gindin et al., 2006; Güerri-Agulló et al., 2007, unpublished; El-Sufty et al., 2007; Güerri-Agulló et al., 2008; Merghem, 2011). In 2007, RPW fungal infected pupae were reported from date palm garden in Spain (Dembilio et al., 2010b). Colonies of B. bassiana, Aspergillus sp., Metarhizium sp., Fusarium sp., Trichothecium sp. and Penicillium sp. were also recovered from different developmental stages of RPW in Italy (Torta et al., 2009; Tarasco et al., 2008). A B. bassiana isolate (B-SA3) isolated from Al-Qatif province (Saudi Arabia) from dead RPW by Hegazy et al. (2007) which latter on was used against RPW in laboratory study. Lo-Verde et al. (2014) isolated B. bassiana from RPW adults collected from Villagrazia and Cinisi (Palermo Province, Sicily). Recently, M. pingshaense recovered from RPW in Vietnam which kill the adults in a very short time (Cito et al., 2014). Thanks to an efficient enzymes and toxin production.

2.5.3.22 Susceptibility of RPW to EPFs infections under laboratory conditions Laboratory studies found that M. anisopliae strains caused 80-100% mortality in RPW larvae and adult. Adults withstand for 4-5 week against spore suspension, while dried formulation took 2-3 weeks to kill 100% RPW adults. Moreover, M. anisopliae caused 100% larval mortality within 6 and 7 days, but this took longer time to B. bassiana for getting the same level of mortality (Gindin et al., 2006). Similar findings were recorded in Egypt (Merghem, 2011) and Italy (Francardi et al., 2012, 2013) against RPW larvae and adults. Research studies suggested that effectiveness of EPFs against RPW was about 85% under controlled and semi- natural conditions (El-Sufty et al., 2009; Dembilio et al., 2010). Significantly higher mortality was recorded for the bio-control of RPW by Vitale et al. (2009) when treated with the commercial formulations of using a commercial product of B. bassiana and M. anisopliae alone and an integrated manners, whereas sole application of B. bassiana recovered from dead RPW cadavers did not gave promising results against adults. This might be attributed to the fact that polar extracts of adult may inhibit adhesion and germination of pores. In contrast efficient results were recorded by deploying B. bassiana as a bio-control agent recovered from naturally infected RPW (Sewifi et al., 2009; Dembilio et al., 2010; Güerri- Agulló et al., 2010). Thus, we cannot deny its importance in bio-controls of RPW. Dembilio et al. (2010b) performed laboratory experiments to check the vulnerability of RPWs to B. bassiana. The strain was infective to eggs, larvae and adult stages. They further reported adult lifespan was reduced from 1/2 to 1/10 and adults of either sex transmitted 55 and 60% disease to the healthy adults during courtship. B. bassiana not only induced mortality but also significantly affect the fecundity (approximately 62.6%) and egg hatchability (32.8%). Likewise, larvae obtained from infected female eggs exhibited 30-35% more mortality than the healthy ones, overall 78% less progeny were recorded as compared to the check treatment (Dembilio et al., 2010a). These finding are in accordance with the findings of Torta et al. (2009) and El-Sufty et al. (2009) who reported significantly higher mortality of RPW small larvae and adults to indigenous strains of B. bassiana. They further revealed that susceptibility was more evident in young larvae than old ones that might be due to the scarcity of antimicrobial cuticular compounds in younger larvae (Mazza et al., 2011a). Most recently Hussain et al. (2014) reported varied susceptibility level of different larval instars to four different isolates of B. bassiana. Other scientists also revealed the same results with EPFs against different developmental stages of RWP (Ghazavi and Avand-Faghih 2002; Shaiju-Simon and Gokulapalan, 2003; Gindin et al., 2006; Dembilio et al., 2010; Cito et

18 al., 2014). Lo-Verde et al. (2014) evaluated B. bassiana against egg, larvae and adult, the strain was isolated from infected adults and were found quite effective against all the tested stages. For the very first time 3 isolates of another different EPF Isaria fumosorosea showed promising results against RPW (Sabbour and Abdel-Raheem, 2014).

2.5.3.23 Field and Semi-field assessment of fungi for RPW management The efficacy of EPFs depends to a great extent on formulation. Sewify et al. (2009) reported successful reduction in RPW incidence in Egypt with indigenous strain of B. bassiana isolated from a RPW cadaver under field conditions. In field trials El-Sufty et al. (2009) reported 13-47% mortality in adult RPW population using a strain of B. bassiana isolated in UAE. Later on this strain was effectively deployed in auto-dissemination traps in date palm groves (El-Sufty et al., 2011). Field studies were conducted using two formulations of local strains of B. bassiana at UAE. The oil-based formulation of EPF exhibited 13.7-19.2% adult mortality, while dust formulations imparted only 8.9% mortality (El-Sufty et al., 2007). However, Abdel-Samad et al. (2011) observed little effect of oil based commercial formulation of B. bassiana on RPW, hence not recommended for formulation and field application, since it was quite expensive as compared to the other formulations. Moreover, polar extracts from adults were found to inhibit the spore germination of B. bassiana commercial formulation (Mazza et al., 2011a). Besse et al. (2011) reported high pathogenic potential of an indigenous strain of B. bassiana against RPW, hence recommended as promising agent for bio-control. A preventive and curative treatment with solid formulation of highly virulent strain of B. bassiana with high persistence was applied against RPW under semi filed conditions on 5 year old P. canariensis palms. The efficacies were >85.7%, confirming the pathogenic potential of this strain as a biocontrol agent against RPW (Güerri-Agulló et al., 2011). However, the few field studies carried out so far lacked adequate experimental designs, used fewer replication, had high infection rates, etc. (Güerri-Agulló et al., 2011). Lecanicillium (Verticillium) lecanii significantly affected the mortality of various larval instars and adults and the egg hatching percentages of adult females. Moreover, yield losses in date production decreased from 56 and 60% to 22 and 22% in El-Esraa (Nobarya) and El-Kassaseen (Ismailia) respectively (Sabbour and Solieman, 2014). Solid state formulations of two B. bassiana isolates deployed against RPW under field condition exhibited 100% mortality even after 30 days post application and the efficacy persisted for 3 months (Ricaño et al., 2013). Sabbour and Abdel-Raheem (2014) applied Iseria fumosorosea in date palm plantations and reported significant reductions in date palm weight loss. Results revealed that palm weight significantly increased in El-Kassaseen compared to El-Esraa to 5341±40.30 kg Feddan-1 as compared to 1981±80.54 kg Feddan-1 in the control during season 2012. During 2012 season yield losses were 59 and 62% in El-Esraa and El-Kassaseen which decreased to 28 and 27% in these respective regions; the same results obtained during 2013 season. These results called for expanded open field trials with I. fumosorosea strains to explore their bio-control potential. Most recently, Jalinas et al. (2015), using acoustic recording methods, found greatly reduced feeding activity after infesting palm trees with B. bassiana treated larvae. Retarded larval movement and feeding noises suggested that B. bassiana infection weakened RPW larvae, which reduced detectable feeding activity. The main difficulty in the implementation of acoustical detection methods is accessibility of these bio-control agents to the pest insects. Given the concealed nature of RPW, a systemic distribution of the agent, while highly desirable, is difficult to achieve practically. Consequently, adults are the targeted stage for fungal delivery because

19 this is the only free living stage and future research should be focused at attracting-and-infecting RPW adults could be most effective in managing this pest (Dembilio et al., 2010b).

2.5.4 Endophytic fungi Fungal endophytes may be beneficial in preventing disease by induction of host defense mechanisms (Sivasithamparam, 1998) or by directly affecting plant pests (Arnold et al., 2003). Reports on endophytic colonization of EPFs in date palms have been published which may be useful in the future (Gómez-Vidal et al., 2006). They inoculated B. bassiana, Lecanicillium dimorphum and L. c.f. psalliotae within young and adult date palms petioles and exhibited the fungal survival even after 30 days of inoculation; fungi were detected inside the parenchyma and sparsely within vascular tissue using microscopy techniques without any detrimental effect on date palm. Arab and El-Deeb (2012) applied endophytic fungi on date palm seedlings after 6 months the date palm pulp was offered to the larvae in laboratory which inflicted 80.3% mortality after 14 days. Ben Chobba et al. (2013) provided the report on 13 different fungal isolates from date palms from roots and leaves in Tunisia, although these might be affiliated to some fungal diseases in date palms but provided the way to the researchers towards endophytic colonization of EPFs. Nevertheless, the deployment of EPF strains with endophytic behavior is not well understood for systemic protection of palms against RPW. Field trials with B. bassiana strains to explore their bio-control potential are urgently needed.

2.5.5 Future prospects of entomopathogenic fungi The successful utilization of EPFs is well recognized in biocontrol programs but the inconsistent performance of these control agents is attributed by a variety of factors and the major one is their great dependency on the environmental conditions. Different fungal strains have limiting temperature ranges for germination, infection and post mortal sporulation. Same is the case for humidity conditions as many strains require high humidity for spore germination and sporulation (therefore oil formulations have been developed). The other restricting factors for the broad spectrum application of the mycoinsecticides include; the limited production of toxins (from the view point of registration authorities, the production of toxins is a hurdle for registration and practical use) by the fungi, the slow rate of activity of the fungal conidia, the higher doses of the conidia required for the effective control which ultimately yields inconsistent results compared to the chemical insecticides (Gressel, 2001), and the pathogenicity of various fungal strains to non-target organisms. Therefore, different approaches have been proposed to tackle these limitations especially with regard of decreasing application rates and increasing virulence of the fungal pathogens. The most significant among them is the integrated use of EPFs with other biological control agents. The second and the most promising approach is the implementation of biotechnology which has great potential to play a vital role in the EPFs development process from the identification of virulent strains to final formulation (Glare, 2003). Moreover, the high production rates of commercialized fungal formulations can be compensated if the equivalent control is attained at lower concentrations. The transgenic and genomic recombinant approaches yielding the reduced median lethal concentration (LC50) of the pathogens and shorter survival time of the target species (St. Leger et al., 1996) not only tend to improve the infection rate but also reduce the cost of the applied formulation. The herbicide and fungicide resistant genes have also been induced to various fungal strains (Bernier et al., 1989; Fang et al., 2004) which

20 ultimately make use of the fungal pathogens in combination with certain herbicides and fungicides.

2.5.6 Entomopathogenic Nematodes (EPNs) Interest in the use of EPNs as bio-control agents against a variety of pests has increased in last two decades (Dolinski and Lacey, 2007; Lacey and Shapiro-Ilan, 2008). Researchers are expanding the pathogenic potential of EPNs against a variety of plant nematodes, harmful insects, soil-borne plant pathogens and mollusks (Grewal et al., 2005). So far more than 30 families of nematodes, associated with insects have been reported, but because of the bio-control potential concentrations are focused on seven families of nematode including Sphaerularidae, Rhabditidae, Allantonematidae, Mermithidae, Heterorhabditidae, Steinernematidae and Neotylenchidae. For the biological control of RPW, Steinernematidae and Heterorhabditidae, received the most attention. These nematodes carry species-speciﬁc pathogenic bacteria, Photorhabdus by Heterorhabditidae and Xenorhabdus by Steinernematidae, which are released into the insect hemocoel when infective juveniles (IJ), penetrate into the insect host body. The third infective juvenile (IJ) stage of these EPNs actively searches for a suitable host, invades it, and releases symbiotic bacteria into the insect hemocoel. This process kills the invaded insect via bacterial septicemia and/or toxemia (Kaya and Gaugler, 1993).

2.5.6.1 Natural incidence Few species of EPNs have been recorded as naturally infecting RPW. The efforts to infect RPW with EPNs started with the recovery of parasitic species, Praecocilenchus rhaphidophorus Poinar, from Rhynchophorus bilineatus in Papua New Guinea and New Britain (Poinar, 1969) and Praecocilenchus ferruginophorus, isolated from infected RPW in India (Rao and Reddy, 1980). P. ferruginophorus was recovered from hemocoel and hemocoel of adults, and fat tissue, trachea and intestine of RPW larvae. Usually, the nematodes are released out of the body from infected insect during oviposition or may also be released with the feces via intestine. As a consequence of being released from the body the ovaries of infected weevils are harmed due to the production of eggs (Triggiani and Cravedi, 2011). The practical importance of nematode fauna associated with the Rhynchophorus palmarum lead researchers to study the Bursaphelenchus cocophilus (Cobb) Baujard a causative agent of red ring disease in palms in neo tropics (Giblin-Davis, 1993). Morover, Bursaphelenchus gerberae, Caenorhabditis angaria and Mononchoides sp. have also been reported from R. palmarum (Gerber and Giblin-Davis, 1990; Giblin-Davis et al., 2006; Kanzaki et al., 2008; Sudhaus et al., 2011). But none beyond Rhynchophorus spp. has been effectively surveyed. Other non-pathogenic nematodes inflicted no harmful effects on RPW are known from three Rhynchophorus spp.: Acrostichus rhynchophori (named Diplogasteritus in older publications; Kanzaki et al., 2009) and Teratorhabditis palmarum were isolated from R. palmarum and R. cruentatus respectively (Gerber and Giblin-Davis, 1990), while Teratorhabditis synpapillata Sudhaus was recovered from RPW in India and Japan (Kanzaki et al., 2008). Salama and Abd-Elgawad (2001) isolated Heterorhabditis spp. from ﬁve sites in Egypt. However, only two out of the ﬁve isolated nematode strains survived for 24 hours exposure in RPW infested palm tissue, nematode had a low viability of only 14-19%. The retarded growth of nematodes is thought to be due to the generation of acetic acid, ethyl acetate and ethyl alcohol from the infested palm tissue that limit the use of EPNs especially Heterorhabditis indica (Monzer and El-Rahman, 2003) in addition to the concealed nature of

RPW (Abraham et al., 2002). On the other hand, El-Bishry et al. (2000) reported that the host finding ability of juveniles decreased when the palm tissues were washed and sterilized. Anti-desiccants such as Leaf Shield and Liqua-Gel were reported to improve the efficacy of EPNs isolated from date plantations in Saudi Arabia (Hanounik et al., 2000). Steinernema sp. was isolated from naturally infected field collected adult of RPW inform the eastern province in Saudi Arabia (Saleh et al., 2011). Recently, Mononchoides sp., Teratorhabditis sp. and Koerneria sp. were found infecting pupae and adults of RPW in southern Italy, but their species identification, clarification on their biological parameters and type of association between RPW and these nematode species are still in progress (Oreste et al., 2013).

2.5.6.2 Susceptibility of RPW to EPNs infections under laboratory conditions Laboratory studies revealed that both larvae and adults of RPW were infected by Steinernema riobrave, S. carpocapsae and Heterorhabditis sp. (Abbas and Hononik, 1999). Similar results were reported by Salama and Abd-Elgawad (2001) when tested five strains of Heterorhabditis, were more virulent to RPW than other tested entomophilic nematode species (Salama and Abd-Elgawad, 2001). Laboratory studies showed that RPW larvae were suitable host for H. indicus (Banu et al., 1998). Similarly, Elawad et al. (2007) reported high mortality of H. indicus against RPW in UAE under laboratory conditions. Laboratory studies performed in Italy H. bacteriophora Poinar was reported to be the most effective against RPW larvae and adults (Triggiani and Tarasco, 2011). Additionally, exposure of RPW larvae to genetically modified strains of Heterorhabditis and Steinernema exhibited 95-100% and 50% mortality under and laboratory and field conditions respectively (Hanounik, 1998). Similar findings were reported from Turkey in which H. bacteriophora inflicted 69% and 80% larval and pupal mortality respectively in RPW (Atakan et al., 2009). This might be attributed to the fact that S. carpocaspsae was not encapsulated by RPW hemocytes, thus it is necessary to discover the phenomenon which contributed to the lack of reproduction in larvae and adult of RPW (Manachini et al., 2013). Shahina et al. (2009) evaluated seven Pakistani strains of EPNs against eggs, first, third, sixth and final larval instar, and adults of RPW under laboratory conditions. Significant differences were observed in the mortality of various life stages of the weevil, while the highest egg mortality was found from S. siamkayai and H. bacteriophora (95±2.1 and 97±2.2% at 150 infective juveniles (IJs ml-1). Recently, Atwa and Hegazi, 2014 evaluated 12 EPNs, that all were infective against first instars of RPW larvae. Some species were selective for a specific host stage while others were effective against all stages.

2.5.6.3 Field and semi-field assessment of EPNs for RPW management For EPNs applications in the field a preliminary agarose assay is preferred. This is a simple and rapid laboratory test for measuring chemo-attraction of nematodes to host diffusates and host recognition as a predictive screening tool for ﬁeld testing of new Heterorhabditis isolates (Monzer, 2004). Earlier field studies with EPNs such as Steinernematid and Heterorhabditid in date palms did not exhibit efficacious results due to the environmental constraints and RPW ecology (Hanounik, 1998; Abbas et al., 2001). Similarly, Koppenhöfer and Fuzy (2004) reported gradually decreased susceptibility of RPW larvae to S. carpocapsae IJs. However, later studies in the date palms from Middle East reported adult and larval mortality from the same nematode spp. isolated from respective hosts. The soil treatment of these nematodes around palms with 8×106 IJs palm-1 led to 33-87% adult mortality, while spraying the

22 palm trunk with the same nematode suspension resulted in only 8-13% adult mortality (Abbas et al., 2000). Similar results were recorded by Santhi et al. (2015) who evaluated S. carpocapsae and H. bacteriophora against the RPW under simulated natural conditions and reported that pupae in cocoons and adults exhibited a high susceptibility to S. carpocapsae. These findings are important when considering optimal use of EPNs for the control of RPW under natural conditions. Studies in curative and preventive assays of S. carpocapsae with chitosan by spraying the product at a dose of 3.6×106 IJs + 36 ml chitosan palm-1 in about 2 l of water on trunk and the bases of the fronds of each palm until the run off with the help of a manually operated backpack compact sprayer. The treatment showed efficacies of around 80% in curative assays and 98% in preventive assays in various palms. Accordingly, Llácer et al. (2009) and Dembilio et al. (2010a) performed experiments on P. canariensis and P. theophrasti respectively by using S. carpocapsae with chitosan, efficacies ranged from 83.8-99.7% and palm survival significantly increased as compared to the check treatments. From these experiments it was proved that chitosan as adjuvant can be effectively used with EPNs, particularly S. carpocapsae, which extend their period and protect them from environmental conditions (Llácer et al., 2009; Dembilio et al., 2010a). Apart from weather factors, other organisms associated with RPW can interfere with the efficiency of EPNs. The RPW associated organisms can interfere with the effectiveness of entomopathogens such as RPW predatory mites Centrouropoda almerodai Wisniewski and Hirschmann can reduce the efficacy of the nematode S. carpocapsae (Morton and Garcia-del-Pino, 2011; Mazza et al., 2011b). In Egypt, encouraging results were also recorded with another Steinernema sp. recovered from pupae and adults of RPW; this strain along with two other indigenous strains of the same genus inflicted considerable mortality against larvae and adults of RPW both under laboratory and field conditions (Shamseldean and Atwa, 2004). Shamseldean (2002) performed field studies on date palm trees with Egyptian isolates H. indicus (strain EGBB) H. bacteriophora (strain EKB20) and Steinernema sp. (strain EBNUE). He reported no symptoms of old or new infestation in treated palm trees at all treatments. The Saudi Arabian strain of H. indica induced 60 and 46% larval and adult mortalities, respectively, when the nematode was applied at the base of tree into the soil (Saleh and Alheji, 2003). Similar finding were observed from the field trials in France but raised an important point regarding defining optimal application standards (Chapin and André, 2010; Pérez et al., 2010). Because the efficacies in the field study of Dembilio et al. (2010a) were not significantly different when S. carpocapsae was applied singly or integrated manner with imidacloprid. Two successive application of Steinernema sp. by trunk injection resulted in significant reduction in RPW population after 3 weeks. Efficacies ranged from 48-88% in the curative assay and significant increase in palm survival was recorded as compared to the control treatment (Atwa and Hegazi, 2014). Recently, 42 billion worms were imported from Germany to Israel to combat voraciously feeding RPW spp. in Israel (Anonymous, 2015). The use of EPNs should be considered when designing integrated management strategies against RPW.

2.5.6.4 Interactions between EPNs and pesticides Sole or the integrated application S. carpocapsae and imidacloprid under natural conditions were not significantly different from each other with RPW mortalities ranging from 73-95% and significant increase in plant growth (Dembilio et al., 2010a). Similar results were

23 also observed by Tapia et al. (2011) and suggested application of S. carpocapsae and imidacloprid after every 60 days as preventive measure during field studies in Southern Spain. In the light of above findings the results suggested that the combined effect of S. carpocapsae in chitosan formulation and imidacloprid greatly enhanced the efficacy against RPW under field conditions and significantly reduced the reproductive potential of the RPW (Tapia et al., 2011).

2.5.7 Entomopathogenic Bacteria 2.5.7.1 History Existence of bacteria is as old as the history of life on earth. Evidence of bacterial fossils dates back to the Devonian period (416-359.2 million years ago) and considerable signs depict their presence from Precambrian time, about 3.5 billion years ago. The fossils found in north- west Australia's Pilbara region are thought to be nearly 3.5 billion years old and considered the oldest ones on earth planet. In Proterozoic Eon (about 1.5 billion years ago), when the activity of cyanobacteria resulted in oxygen production, bacteria became widespread (Anonymous, 2013). The gradual evolution of the bacteria made them able to survive under a wide range of environmental conditions with several descendent forms. As a result of this, today an uncountable and immeasurable diversity in morphology, physiology and taxonomy of bacteria prevails. Bacteria have been found living very close to every living organism including human beings. Both beneficial and harmful forms of bacteria have been thriving in various climates like soil, water, air and hot water springs etc. Confirmatory evidence of using entomopathogens for the control of insect pests are not known in ancient times, however, human interest in exploiting microbes particularly bacteria th rose to its extreme after the discovery and the commercial availability of microscope in late 19 th and early 20 century. Scientific efforts for the survival of the famous Japanese silk industry against sudden death of caterpillars proved fruitful resulting in the discovery of a spore forming bacterium, Bacillus sotto, by Sigetane Ishiwata (1868-1941) (Aizawa, 2001). This discovery lead to the world’s first ever demonstration of toxins when many other scientists including Aoki and Chigasaki (1915) and Mitani and Watari (1916) found enhanced lethal actions of bacterial cultures on silk worms when they were applied in alkaline solution (Aizawa, 2001). Doors of discoveries were opened for man and a German scientist Ernst Berliner in 1909 isolated a bacterium named by him as B. thuringiensis that killed the flour moth, Ephestia kuhniella Guenée (Lepidoptera: Pyralidae). Within the Prokaryotes, bacteria are the microorganisms that lack a nuclear membrane which separates genetic material from cytoplasmic contents and other membrane bounded organelles. Bacteria surround us all around and thus, can be isolated from any environment and hence their enriched flora can be given the name of metabolic strategy which they use to earn energy such as phototrophs (gain energy from sunlight), lytotrophs (obtain energy form inorganic material) and organotrophs (receive energy from organic material). The variation in their size is from one to few microns, and depending upon the morphologies, they can be grouped as cocci (spherical), bacilli (rod shaped) and spirochetes (spiral shaped). Propagation in bacteria is carried out through binary fission, a mode of asexual reproductions in which daughter cells are produced from mother cell as clonal copies (Jurat-Fuentes and Jackson, 2012).

2.5.7.2 Classification Entomopathogenic bacteria mostly belong to the families Bacillaceae, Enterobacteriaceae, Pseudomonadaceae, Micrococaceae and Streptococcaceae (Tanada and

Kaya, 1993). Although many bacteria are beneficial and essential but members from families Eubacteriales, Bacillus and Serratia have been registered against insect pests (Tanada and Kaya, 1993). For the successful control of RPW, bacterium has been exclusively isolated from different developmental stages of RPW and deployed under laboratory conditions and field conditions. The recognized factor for classifying bacteria involves the sequence of 16S ribosomal RNA. Two important groups of bacteria are Eubacteria (true bacteria) and Archaea containing bacteria having similar features of DNA replication, transcription and translation as exhibited by eukaryotes. Three major divisions within Eubacteria are primarily based on the presence or structure of cell wall: Gracilicutes (gram negative typed cell wall bacteria), Firmicutes (gram positive typed cell wall bacteria) and Tenericutes (Eubacteria which are devoid of cell wall). Most recent classification within Eubacteria mostly relies on the use of polyphasic taxonomy that includes analysis of nucleotide sequence of RNA (16S rDNA), DNA-DNA hybridization, genotypic, phenotypic and phylogenetic aspects (Brenner et al., 2005). Entomopathogenic bacteria; greatly concerned with entomological studies are grouped in Eubacteria. The cell wall of bacteria greatly serves the purpose to classify, support the molecules and organelles. In gram-positive bacteria, the cell wall is formed of cross-linked peptidoglycan while on the other hand, cell wall in gram-negative bacteria is formed of rather complex thin layer of peptidoglycan and lipoproteins and an outer polysaccharide membrane. Gram-negative bacteria are distinguished from gram-positive bacteria by lacking the ability to retain crystal violet dyes. Gram-positive are endospore forming, rod and cocci shaped bacteria often undergoing sporulation. Gram-negative bacteria on the other hand appear to be in rod or cocciform. They are much more diverse in their distribution and hence isolation can be successful from diseased and dead insect specimens (Jurat-Fuentes and Jackson, 2012).

2.5.7.3 Life cycle Life cycle of B. thuringiensis can be divided into different phases for convenience in understanding; Phase-I (vegetative growth); Phase-II (transition to sporulation); Phase-III (sporulation); and Phase-IV (spore maturation and cell lysis) (Berbert-Molina et al., 2008). Specific insecticidal (Cry) proteins lying deposited in crystals within mother cells starts to form with the onset of sporulation (Pérez-García et al., 2010). There are some evidences of the production of the insecticidal proteins within culture medium during vegetative growth (Singh et al., 2010; Abdelkefi-Mesrati et al., 2011). Distinctive characteristic insecticidal properties to Bt are conferred by another additional virulence factor phospholipase C, proteases and hemolysins (George and Crickmore, 2012) which are under control of pleiotropic regulator plc R. Removal of plc R gene results in drastic reduction in the virulence of Bt in orally infected insects (Salamitou et al., 2000). Sporulation leads to the production of two types of insecticidal proteins (cry toxins and cyt-toxins) within crystalline bodies. A single Bt strain is naturally provided with one or more toxins packaged into a single or multiple crystals (de Maagd et al., 2001). The Cry toxin is named for its production within crystals whereas Cyt-toxin got the mnemonic Cyt due to their in vitro cytolytic activity (Crickmore et al., 1998). The Cry toxins acquired the mnemonic Cry from the fact that they are found in the crystal while the Cyt-toxins acquired the mnemonic Cyt because of their in vitro cytolytic activity (Crickmore et al., 1998).

2.5.7.4 Ecology Earlier, B. thuringiensis was thought to have confined to soil only, but advanced isolation techniques in ‘Insect Pathology’ discovers its various sources of origin (Chaufaux et al., 1997).

Now this school of thought has expired and Bt has been isolated from dust, stored grain and silos materials (Iriarte et al., 1998). The probability of isolation of Bt from a site varies with its climate, geographical and environmental conditions. In much of the findings till today reveal most common shapes of crystals as bipyramidal and spherical. More often, Bt is regarded as soil inhabiting, but it lacks the capacity to multiply in soil or water that offers healthy environment for other bacteria to compete (Furlaneto et al., 2000). Reproduction in regular practice is carried within host insects. Most of the commercial Bt formulations are isolation from infected insects, so it would be obvious to say that soil acts as a reservoir for bacterium instead of multiplication site.

2.5.7.5 Mechanism of action The infection cycle start with the ingestion of Bt spores by insects making its way to alkaline environment of midgut (pH >9.5). The exposure to higher pH of the gut solublizes the inactive proteins that on the other hand remain insoluble. This activation results in the release of crystal proteins that produces δ-endotoxins. Insecticidal activities of δ-endotoxins get magnifies as a result of proteolytic activation and activated toxin readily get bound to specific receptors present at apical brush border of the midgut microvillae in target insects (Hofmann et al., 1988). The toxic action of proteins is because of N-terminal half consisting of seven anti-parallels α- helices. Loss of integrity of insect’s gut is the outcome of Bt activity that ultimately leads to death of insect due to starvation and septicemia (Kumar et al., 2013).

2.5.7.6 Commercial formulations A rapid development of interest in bio-pesticides has led to the commercial preparation of several Bt products. Now a day, wide variety of commercial products is accessible to farmers infecting a wide range of host insects. While developing commercial products, Bt strains used against lepidopterous insects belong to subspecies; thuringiensis, kurstaki, morrisoni and aizawai. For Dipterous insects, Bt strains include subspecies israelensis while coleopterans pests infecting products include subspecies tenebrionis. The total bio-pesticides used in agriculture globally, Bt products contribute the share of about 80% (Whalon and Wingerd, 2003). A series of complex changes are involved in Preparation of Bt products require standard fermentation batch process including vegetative phase, a sporulation phase release of spores and sporangia in final phase. Fermented solids at this phase are concentrated and mixed with inert material for packing as finished product (JuratFuentes and Jackson, 2012). Recently, over 400 of Bt originated commercial products are marketed over the world in different names registered against pests in different formulations (solid and liquid). These products contain in them various insecticidal proteins and viable spores, yet some products are also available with inactivated spores (Ahmedani et al., 2008). Several valuable products have been prepared from B. thuringiensis var. israelensis (Gnatrol, Aquabee, Bactimos, LarvX, Teknar AND Mosquito Attack etc.), B. thuringiensis var. kurstaki (Bioworm, Bactur, Dipel, Topside, Caterpillar Killer, Javelin, Futura, Thuricide, Worthy Attack and Tribactur), B. thuringiensis var. tenebrionis (M-One, Foil, Novardo, M-Track and Trident), B. sphaericus (Vectolex WDG and Vectolex CG etc.), B. thuringiensis var. aizawai (Certan) B. lentimorbus and B. popilliae (Japidemic, Milky Spore Disease, Doom and Grub Attack etc.).

2.5.7.7 Methods of applications of Bt products B. thuringiensis is undoubtedly regarded as biologically active pesticide effective against important insect pests well suited to IPM strategies. Based on the ecological aspects of target pest and infection cycle of active insecticidal proteins, Bt products in solid form as well as liquid form are applied. Bt products in solid form are either dust or granules (spread over the area of infection) while in liquid form (foliar sprays), Bt products are sprayed directly (Ali et al., 2010) to point of infection. A more persistent and biotechnologically advanced way of supplying toxins to target insect is to genetically express toxin-encoding genes within transgenic plants (Walter et al., 2010; Chen et al., 2011). This practice leaves no chances of escape of insect from active Bt components as toxin remained concealed along with the preferred diet of insects. Moreover, the presence of toxin-encoding genes leaves no harm to non-target fauna, no obvious changes in the physiology of host plant and no chemical change in the products and byproducts from host plant.

2.5.7.8 Superiority of Bt products over synthetic insecticides Environmental concern of pesticides has ever remained the hot issue. The hazards posed with the use of insecticides predominantly broad spectrum nature, quick onset of resistance in insects (Ahmad et al., 2008), environmental persistence, effect to non-target individuals, and phenomenon of bio-magnification have declared pesticides an evil for mankind. Another peculiar reason for the considering bio-control agents as key weapon against notorious insect pests is the selective (Stevens et al., 2011) and environmental friendly nature (Chen et al., 2011). Several laboratory and field studies have declared Bt toxins as a necessary component of insect control strategies and a widely preferred tool over the synthetic chemistries. Bt toxins have specific insecticidal impacts on insect pests of order coleopteran (Sharma et al., 2010), Diptera (Roh et al., 2010), Hymenoptera (Sharma et al., 2008), Lepidoptera (Baig et al., 2010) and non-insect hosts like nematodes (Hu et al., 2010). Although no such reports of harm to non-target individual has been reported, yet some studies provide insight into reduction in reproduction capacity in bumblebee (Bombus terrestris) workers after using commercial Bt aizawai strain (Mommaerts et al., 2010). Beside of this, Bt products still rule over bio-pesticide market and remains hub of biocontrol policies launched against insect pests.

2.5.7.9 Concerns to use of Bt Bt has several advantages over chemical insecticides: it is host specific and highly toxic to the target insects highly toxic to insects and yet highly specific. Bt toxins are safer to environment, animals, human beings and vast array of non-target pests. Therefore, Bt can be considered an ideal component for IPM programs (Nester et al., 2002). Besides these advantages, Bt formulations have some reservations to be considered (McGaughey and Whalon, 1992). Most of the microbial based products need repeated application for effective control of insect pests and sometimes officious only against immature stages immature stages feeding externally. This seems to be a concern limiting its worth for internal plant feeding insects. This can be overcome by incorporation of Bt gene (s) in transgenic plants (Krattiger, 1997). Some recent studies are reporting the development of resistance and cross resistance developed after the continuous use of Bt toxins is one of the emerging concern about the future of biological control. Even the laboratory investigations are confirming the incidence of resistance development in some insects (Pereira et al., 2010) and field populations (Sayyed et al. 2004). The term ‘cross resistance’ is used by some researchers (Gong et al., 2010; Xu et al., 2010) for the previously resistant insects (for a specific toxin) showing resistance to other

27 toxin/toxins they have not been exposed yet. Different routes and mechanism of resistance have been discovered, the most important explanation includes the reduced binding of toxins to receptors in midgut lining of insects, reduction in solubilisation of protoxin, precipitation by proteases, toxin degradation and or alteration in proteolytic processing of protoxins (Bruce et al., 2007). Several mode of resistance has been verified about the development of resistance, the ‘Mode 1’ being the most accepted that hypothesizes that reduction is the outcome of reduction in binding of Cry1A toxin to specific receptors (Heckel et al., 2007). The alteration of protease profile in the midgut of Cry1Ac resistant American boll worm is due to proteolytic processing of Cry1Ac ultimately producing 95 and 68 kDa toxins normally producing active 65 kDa toxins by midgut protease in vulnerable insects (Rajagopal et al., 2009). Apart from resistance, another constraint in use of Bt is the narrow host range and limited efficacy. Bt toxins are very limited in regard of host infection (Shu et al., 2009) and in most of Bt strains, infections remain limited to a single specie only a few Bt strains exhibit activity span against two or more insect orders (Zhong et al. 2000).

2.5.7.10 Interaction of Bt products and other toxins Narrow spectrum of activity in case of Bt is one of the constraint making biopesticides a second choice after synthetic insecticides. Most of Bt isolates show rather poor control over insects but their pathogenicity can be magnified by using in synchrony with some other suitable toxin. For instance, Cyt1Aa is a weak toxin to mosquitoes but synergistic action is found when combined with toxins like Cry4Ba and Cry11Aa (Fernandez-Luna et al., 2010). Combining Bt insecticidal toxins in combination with other proteins not only boosts their pathogenic effect but also helps to lower the resistance developed by insect pests. Proteins like cadherin fragments have been found to be successfully synergizing the efficacy of several insecticidal toxins (Peng et al., 2010). The simultaneous use of Cry1Ac and Cry2Ab results in powerful synergistic interaction against H. armigera (Ibargutxi et al., 2008). Mixing of spores and crystal proteins from same strain also yields synergistic insecticidal action (Johnson et al., 1998). A dose dependent interaction of Bt was also recorded against H. armigera in Pakistan from soil isolated M. anisopliae which integrated synergistically as well as antagonistically at lower and higher doses (Wakil et al., 2013). Kalantari et al. (2013) interpreted synergistic effect of Bt and HaSNPV by combining a suitable dose among several tested doses against H. armigera. Future research will open the horizon of success in integrating Bt with several agents to boost up its efficacy against a wide host range.

2.5.7.11 Effect of Bacillus thuringiensis on non-target invertebrates From the last few decades, Bt pesticides are being studied to control crop, forest and aquatic insect pests. Most of Cry toxins are specific to insects belonging to one of the insect orders either Lepidoptera, coleopteran and Diptera. Cry2 is an exception to this fact as it exhibits insecticidal activity against several families of Diptera and Lepidoptera (Schnepf et al., 1998). Many of the Bt formulations containing purified Cry toxins registered against Lepidopteran orders show no harm to non-lepidopterous insects (MacIntosh et al., 1990; Sims, 1997). Conversely, there is an exception as non-target Lepidoptera are not necessarily secure from Bt treated plants especially in forests (Sample et al., 1996; Herms et al., 1997). The drifting of aerially applied Bt subsp. kurstaki (Bt-k) to control gypsy moth was also found to be lethal to non-target Lepidoptera 3000 m away from treated site as demonstrated by Whaley et al. (1998). However no or negligible effect was found for aquatic habitats in Bt treated sites when

Kreutzweiser et al. (1992) demonstrated high concentrations of Bt-k on drift and mortality of Ephemeroptera, Plecoptera, and Prichoptera. Predators that preyed upon Bt treated hosts were not found susceptible except the Chrysoperla carnea. So in this regard, it would rather be justified statement to declare Bt toxin rather safe, specific in action and compatible to non-target individuals.

2.5.7.12 Mode of infection The ingestion of B. thuringiensis compounds by insects follows the route of midgut to expose it to alkaline environment of gut (pH >9.5). Here the higher pH of the gut solublizes the inactive, otherwise insoluble proteins resulting in the release of crystal proteins that produces δ- endotoxins. This proteolytic activation of δ-endotoxins offers an extraordinary insecticidal activity to insects and this activated toxin readily gets bound to specific receptors present at apical brush border of the midgut microvillae in target insects (Hofmann et al., 1988). The toxic action of proteins is attributed to N-terminal half consisting of seven anti-parallels α-helices. These α-helices offers potential gradient by penetrating the membrane and forming an ion channel in apical brush border membrane allowing rapid flux of ions. Loss of integrity of insect’s gut is the outcome of B. thuringiensis activity causes starvation and septicemia which leads to the death of insects (Kumar et al., 2013). The penetration of α-helices in the apical brush border membrane forms an ion channel (Knowles and Dow, 1993). As a result, rapid flux of ions takes place because of toxin-induced pores formation (Wolfersberger, 1989). Consequently the gut integrity gets lost that resultant starvation and/or septicemia leads to insect death. A wide array of B. thuringiensis products formulated for commercial uses have an extended spectrum of action effective to secure food crops, forest trees, stored grains and ornamentals (Meadows, 1993). Contrary to hazards associated with chemical pesticides, B. thuringiensis formulation offers a wide range of benefits. Although it is highly virulent to target insects, yet it is harmless to non-target insects due to its specificity. In spite of decades of use in field, B. thuringiensis toxins are still reported as non-hazardous to animals, human beings and other non-target pests. All these characteristics render it highly suited to include IPM programs (Nester et al., 2002). Besides these benefits, B. thuringiensis formulations have some associated limitations (McGaughey and Whalon, 1992). One of the limitations is its effectiveness against specific stage of insect especially immature stage. For this reason, an effective control of targeted insect requires repeated application. B. thuringiensis products perform better against insect exposed pests than insects concealed within plant parts or some other structures. But the expression of B. thuringiensis gene (s) using transgenic cultivars (Krattiger, 1997) may be able to address such concerns.

2.5.7.13 Important entomopathogenic bacteria 2.5.7.13.1 Bacillus thuringiensis Bacillus thuringiensis (Bt) holds a prominent position among commercial chemical compounds important for agricultural insect pests. It is a naturally occurring spore forming, gram-positive bacterium. It has been found as a source and reservoir of several important insecticidal proteins like δ-endotoxins, vegetative insecticidal proteins (vip) and cytolytic proteins etc. Among these proteins, δ-endotoxins have a vital role in protecting number of important crops from various insect pests. B. thuringiensis based insecticides have proved their worth as a bio-pesticide to protect food crops, cash crops, ornamentals, forest trees and stored commodities (Meadows, 1993). For convenience, life cycle of B. thuringiensis can be divided

29 into different phases; Phase-I: vegetative growth; Phase-II: transition to sporulation; Phase-III: sporulation; and Phase-IV: spore maturation and cell lysis (Berbert-Molina et al., 2008). More than 150 genes of exhibiting insecticidal nature have been identified from Bt δ-endotoxins family of proteins (Crickmore et al., 1998). These crystalline (cry) proteins remain inactive until the exposure to alkaline contents (pH >9.5) of insect mid gut, solubilize them (Milne and Kaplan, 1993) and ultimately liberating δ-endotoxins proteins.

2.5.7.13.1 Paenibacillus popilliae Paenibacillus popilliae previously known as B. popilliae is a gram-positive spore- forming bacterium which was initially isolated from infected Japanese beetle (Popillia japonica) (Coleoptera: Scarabaeidae) larvae in the late 1930s and then named after the name of its first host. The spore forming capability of bacterium protects it from heat, cold, drying and other harsh environmental regimes. P. popilliae plays a major role in biologically managing scarabs, particularly Japanese beetle (Petterson et al., 1999). B. popilliae has been reported from at least 29 scarabs, mostly from Melolonthinae and Rutelinae. P. popilliae causes milky spore disease in P. japonica and it is the first pathogen registered as insect biological control in USA.

2.5.7.13.2 Brevibacillus laterosporus Brevibacillus laterosporus is a gram-positive, rod-shaped, endospore-forming bacterium and is considered an important entomopathogenic and antimicrobial agent. It is morphologically distinguished by producing characteristic canoe-shaped parasporal body (CSPB) firmly attached at one end of the spore imparting it lateral position in the sporangium. Ubiquitous existence of this bacterium has enabled its isolation from various reservoirs particularly soils, insect bodies, fresh and sea water, leaf surfaces, compost, milk, honey, factory effluents, animal hide, wool and many other materials (Ruiu, 2013). It was discovered by White (1912) during 20th century associated with honey bees determined during an investigation on European foulbrood.

2.5.7.13.3 Bacillus subtilis German botanist Ferdinand Cohn in 1877, while working on hay Bacillus, discovered two new forms of Bacillus strain named Bacillus subtilis; one of them was heat sensitive (without endospore) while other was heat tolerant (endospore). A significant genomic diversity in the bacterium has been publicized using genomics analysis based on microarray-based techniques. It is competent for growth in many environmental conditions and is often considered as soil dweller. Most common sources of its isolation are air, soil, water and decomposing plants. However in most of the cases, it is not found naturally in biologically active but occurs in spore forms. Bacillus subtilis is scientifically fabulous for its ability to produce a number of antibiotics especially bacitracin and iturin. It regulates the development of adult mosquitoes by inhibiting their growth (Ramathilaga et al., 2012).

2.5.7.13.4 Bacillus sphaericus Bacillus sphaericus is a naturally occurring spore-forming gram positive bacterium that exhibits strong insecticidal properties. It possesses efficient larvicidal properties against mosquito by producing delta-endotoxins via sporulation that binds strongly to receptors in midgut epithelial lining of mosquito larvae. The bacterium has narrow spectrum and quite specific activity that sometimes decreases its demand for use in field. Enhanced time of lethal action against some mosquito species and recycling of toxin within dead mosquito sometimes

30 works as limiting factors for its use. One of the advantages exhibits over B. thuringiensis var. israelensis is its longer persistence that provides long lasting control (Filha et al., 2008).

2.5.7.13.5 Wolbachia Wolbachia are α-protobacteria, the members of the order Rickettsiales; a varied group of intracellular bacteria that comprises species exhibiting parasitic, mutualistic and commensal associations with their hosts. With its pathogenic nature extended to arthropods and filarial nematodes, it is regarded as the most common endosymbiotic bacterial species on the globe. The only member contained with genus Wolbachia in family Anaplasmataceae and order Rickettsiales is Wolbachia pipientis; the rest of the species; W. melophagi and W. persica have been recently declared as unrelated (Dumler et al., 2001). An insight into the intracellular life study of the bacterium ensures its obligate nature of infection to hosts and it has been found successfully infesting about 66% of the insect species (Hilgenboecker et al., 2008). Wolbachia being intracellular bacterium are vertically transmitted through the egg. Wolbachia sometimes manipulate the reproduction of host insects by cytoplasmic incompatibility. One of the vital reasons behind the successful propagation of Wolbachia in arthropods is its inherent ability to take control of the host’s reproductive cycle by providing nutrients and protecting host from other pathogens (Hosokawa et al., 2010). The genera closely related to Wolbachia; Anaplasma, Ehrlichia and Neorickettsia during their life stages include an invertebrate ‘vector’ and mammalian ‘host’ and in some cases invertebrate associations in some species have also been found. But contrary to unlike members, Wolbachia does not necessarily affect vertebrates. One of the important reasons behind increased interest for Wolbachia is their immense diversity, interesting phenomena shown while infecting their hosts such as reproductive manipulation, and their possible exploitations for pest and disease vector control (Bourtzis, 2008).

2.5.7.14 Host range of B. thuringiensis Different commercial products of B. thuringiensis for use in crops, forests and aquatic system do not necessarily contain β-exotoxin, but most of the B. thuringiensis products registered against insect pests contain Cry toxins (also known as δ-endotoxins). Normally, a single Cry protein works perfectly against a single order and sometimes against several families within an order. The Cry2 is an exception to this fact as it exhibits insecticidal nature against several families of Diptera and Lepidoptera (Schnepf et al., 1998). Most of the commercial B. thuringiensis products or purified Cry toxins formulated for lepidopterous insects are non- hazardous to a vast variety of non-target organisms (Sims, 1997). However, non-target Lepidopterans are mostly at risk in B. thuringiensis treated plants particularly in forests (Herms et al., 1997). For instance, the aerial spraying of B. thuringiensis subsp. kurstaki (Bt-k) to control gypsy moth was found to be lethal to non-target Lepidoptera 3000 m away from treated site (Whaley et al., 1998). However, no or a negligible effect was found for aquatic habitats in Bt treated sites when Kreutzweiser et al. (1992) demonstrated high concentrations of Bt-k on drift and mortality of Ephemeroptera, Plecoptera, and Trichoptera. Predators that preyed upon B. thuringiensis treated hosts were not found susceptible except the Chrysoperla carnea. So in this regards, it would rather be justified statement to declare B. thuringiensis toxin rather safe, specific in action and compatible to non-target individuals.

2.5.7.15 Natural incidence Effect of different bacterial spp. on RPW has been reported by many scientists. Dangar and Banerjee (1993) discovered some bacteria species belonging to Serratia sp., Bacillus sp. and the coryneform group from adult and larval stages of RPW in India, while Bacillus sphaericus Meyer and Neide and B. thuringiensis Berliner were isolated from larvae and adult of RPW in Egypt (Alfazairy et al., 2003; Alfazariy, 2004). Later on (Banerjee and Dangar, 1995) isolated Pseudomonas aeruginosa from larvae infected with this agent from Kerala, India. In Egypt, Salama et al. (2004) recovered three potential spore-forming bacilli from RPW larvae. The three bacteria belonged to the genus Bacillus and were identiﬁed as variants of B. laterosporus Laubach (strain 27), B. sphaericus (strain 73) and B. megaterium de Bary (strain 15). Under in vitro conditions, B. sphaericus caused 40% and 60% mortality in 2nd instar larvae of RPW when use different isolates of Bt. B. sphaericus considered being the most active culture which produces crystalline endotoxins and spherical endospores which are responsible for disease production in RPW. In Italy (Sicily), B. sphaericus B. thuringiensis and B. megaterium were recovered from RPW cadavers, but these isolates exhibited weaker pathogenic effect against eggs of RPW (Francesca et al., 2008), although lacking antimicrobial compounds (Mazza et al., 2011a). Most recently Francesca et al. (2015) isolated distinct strains of seven species from RPW beetle cadavers (B. cereus, B. amyloliquefaciens, B. pumilus, B. licheniformis, B. subtilis, B. megaterium and Lysinibacillus sphaericus) from Sicily.

2.5.7.16 Susceptibility of RPW to entomopathogenic bacteria under laboratory conditions Regarding the pathogenicity of Bt strains against different developmental stages of RPW very few studies has been conducted so for. In laboratory study application of P. aeruginosa suspension, either by inoculation by forced feeding, injecting and wading RPW larvae in the suspension. Complete mortality occurred eight days post inoculation in case of forced feeding and wading, while injection took 6 days, moreover, younger larvae were more vulnerable than the older ones (Banerjee and Dangar, 1995). This might be attributed to the fact that younger larvae probably lacking antimicrobial cuticular compounds (Mazza et al., 2011a). Alfazariy (2004) revealed successful control of RPW in laboratory conditions by infecting with B. thuringiensis var kurstaki (Bt-k). Albite this, other scientists revealed different susceptibility of RPW to the same bacterium (Bauce et al., 2002; Sivasupramaniam et al., 2007; Birda and Akhursta, 2007; Manachini et al., 2008a, b; Manachini et al., 2009). Evidences suggested that feeding cessation and midgut damage were observed amongst surviving larvae. Manachini et al. (2009) integrated commercially available B. thuringiensis into RPW larvae diet and revealed moderate pathogenicity against RPW larvae. Similar results were also recorded by Dembilio and Jacas (2013). Under laboratory conditions this bacterium can be effective against RPW larvae when ingested but their commercial application does not give satisfactory control (Manachini et al., 2009). However, retarded larval growth and its effect on hemocytes was primarily described, exhibiting that the bacterium is capable of growing in the hemolymph when uptake by the larvae (Manachini et al., 2011).

2.5.7.17 Field and Semi-field assessment of bacteria for RPW management Sequentially, several investigators tested certain commercial products based on Bt against RPW and reported the difficulty of using such products as a good control agent against the insect due to different reasons (Dembilio and Jacas, 2013). So for no solid evidence regarding susceptibility of RPW to entomopathogenic bacteria and confirmation of successful control

32 under field or semi-field conditions has been reported. The reduced susceptibility might be due to the host defense mechanism. Manachini et al. (2011) conducted preliminary study by deploying B. thuringiensis and Saccharomyces cerevisiae against the cellular immune response of RPW and exhibited that Bt is a stress factor for RPW. Future experiments to control RPW by deploying microorganisms must be designed to explore the molecular mechanism of disease resistance among RPWs and interaction between them and RPW immune system. Most recently Francesca et al. (2015) tested nine distinct stains of bacterium which significantly reduced the egg hatching, while B. licheniformis exhibited significant insecticidal activity against RPW larvae. Dangar (1997) evaluated the bio-control potential of free living unidentified yeast recovered from RPW haemolymph. The calculated lethal dose was 8,000,000 yeasts insect-1, while lethal time was recorded 4 days. Latter on Salama et al. (2004) isolated yeast from infected pupae of RPW in Egypt which caused 20-35% mortality in 2nd instar larvae of RPW after 7 days of application.

2.5.8 Microbial control agents as a component of RPW IPM As components of RPW IPM, entomopathogens can provide significant and selective control. An integrated approach is needed which can provide maximum effectiveness when combined with other control practices (Edwards, 1989). In the near future efforts are being made to study the synergistic interaction between entomopathogens and other pest control tactics (integration with soft chemical pesticides, semiochemicals, resistant plants, other natural enemies, remote sensing and chemigation etc.). These efforts will enhance the efficacy and sustainability of entomopathogens. Different formulations of EPNs have been employed in order to enhance the bio-control potential of EPN against RPW. A commercial formulation (Biorend-R® Palmeras) S. carpocapsae has been found effective both for preventive and curative measure under semi-filed conditions (Llácer et al., 2009). EPNs exhibit varying degrees of dispersal to find and invade the insect host in their habitat, and this affects the fitness traits of parasitism and infectivity (Koppenhöfer and Fuzy, 2008). Nematodes disperse by following long-distance cues to search for hosts. The IJs use distinctive foraging patterns to discover potential prey; either vigorously hunting for insect hosts (cruisers), or standing on their tails in an upright position over the surface and waiting for the host insect to pass by (ambushers) (Lewis et al., 2006). These foraging and dispersal strategies have a significant impact on effective traits of EPNs and on determining their relationship with the host, an important aspect in predicting EPN efficacy. Moreover, EPFs particularly B. bassiana and M. anisopliae have been reported to be effective against RPW but promising results under field conditions are not recorded except with solid formulation of B. bassiana which exhibited high RPW pathogenicity and persistence for preventive and curative treatments. The integration of microbial control agents with suitable control measures can be effectively used to combat RPW both under laboratory and field conditions.

2.5.9 Ecological engineering and agricultural practices to conserve microbial control agents Ecological Engineering (EE) in IPM of pest control is a comprehensive strategy integrating modern technologies with traditional cultural techniques to promote entomopathogens as a cornerstone for sustainable agricultural productivity (Gurr et al., 2004). It is argued that these ecological based approaches for managing insect populations of agricultural

33 importance can be safer and more sustainable than sole dependence on conventional chemical insecticides, hence the need to examine the concept and practice of ecological engineering as a component of modern agricultural activity. Ecological based pest control strategies are more appealing to the researchers, conservation bio-control in particular that increase the number and activity of natural enemies in the natural conditions by manipulation of habitat (Gurr et al., 2004). The conservation of antagonistic organisms in date palm gardens mostly targets indigenous strains with knowledge of local natural microbe communities instead of inundative applications of microbial antagonists. The idea behind conservation is to increase the number of entomopathogens and protect refuges in the orchards which resultantly will encourage entomopathogens to reduce pest infestation. Unluckily, in date palm pest control intention are mostly focused to inundative applications rather than conservation. The enhanced efficacy of EPNs can be achieved by providing supplementary food sources such as organic amendments in the soil enhance the efficacy and persistence of EPNs. Contrarily, some amendments with manures and plants containing allelopathic compounds can exert also hazardous effects to EPNs. In case of endophytic colonization of entomopathogens plant genotype can interfere with rhizosphere colonization and antagonist’s metabolites production, as well as the expression of induced resistance by plants. Ultimately, indicators will need to be identified, such as the presence of particular antagonists, which can guide decisions on where it is practical to use conservation biological control. Combination of entomopathogens with conservation practices can be helpful in improving the effectiveness and persistence of entomopathogens. Future research must be focused on the greater use of bioassays that accounts for the RPW suppression as effectiveness of a particular entomopathogen against RPW is not significantly affected by their abundance.

2.5.10 Biotechnological approaches to enhance virulence of microbial control agents Biotechnology provides magnificent opportunities to enhance the virulence of microbial control agents through incorporation of gene of interest exhibiting excellent control against insect pests e.g. the transformation of M. anisopliae by Aspergillus nidulans. To understand the virulence mechanism efforts are being made, emphasizing the cuticuler area where mostly penetrations occur and possessing a key enzyme, an endoprotease (St. Leger et al., 1986). In the future, insect killing speed of EPFs may be enhanced by inserting delta-endotoxin genes from B. thuringiensis into fungi which certainly will achieve improved strains. Except from the Bt delta- endotoxin there are several other proteins of insecticidal properties such as alpha-endotoxin, Vegetative Insecticidal Proteins (VIP) and numerous secondary metabolites that are prone genetic modification a (Attathom, 2002). The introduction of gene coding for proteinaceous insect toxins (scorpion toxin, mite toxin, trypsin inhibitor), hormones (eclosion hormone, diuretic hormone) or metabolic enzymes (juvenile hormone esterase) into nucleopolyhedroviruses genome are some approaches to increase speed of kill, enhanced virulence and extend host specificity of the virus (Attathom, 2002). Future, attention should be focused on formulation development and targeting RPW populations. Advantages of this approach include reduced risk for development of resistance and greater safety to the environment, and lack of effect on non-target and beneficial organisms. We believe that biotechnology and genetic engineering will come up with the effective use of insect antagonists as an integral part of integrated pest management program worldwide.

2.6 References Abbas, M.S.T. and S.P. Hanonik, 1999. Pathogenicity of entomopathogenic nematodes to red palm weevil, Rynchophorus ferrugineus. Inter. J. Nematol., 9: 84-86. Abbas, M.S.T., S.B. Hanounik, S.A. Mousa and M.I. Mansour, 2001. Pathogenicity of Steinernema abbasi and Heterorhabditis indicus isolated from adult Rhynchophorus ferrugineus (Coleoptera). Inter. J. Nematol., 11: 69-72. Abbas, M.S.T., S.B. Hanounik, S.A. Mousa and S.H. Albagham, 2000. Soil application of entomopathogenic nematodes as a new approach for controlling Rhynchophorus ferrugineus on date palm. Inter. J. Nematol., 10: 215-218. Abdelkefi Mesrati, L., H. Boukedi, M. Dammak-Karray, T. Sellami-Boudawara, S. Jaoua and S. Tounsi, 2011. Study of the Bacillus thuringiensis Vip3Aa16 histopathological effects and determination of its putative binding proteins in the midgut of Spodoptera littoralis. J. Inver. Pathol., 106(2): 250-254. Abdel-Samad, S.S.M., B.A. Mahmoud and M.S.T. Abbas, 2011. Evaluation of the fungus, Beauveria bassiana (Bals.) Vuill as a bio-control agent against the red palm weevil, Rhynchophorus ferrugineus (Oliv.) (Coleoptera: Curculionidae). Egyp. J. Biol. Pest Control, 21: 125-129. Abraham, V.A., 1971. Prevention of red palm weevil entry into coconut palms through wounds. Mysore J. Agric. Sci., 5:121-122. Abraham, V.A., J.R. Faleiro, C.P.R. Nair and S.S. Nair, 2002. Present management technologies for red palm weevil Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) in palms and future thrusts. Pest Manag. Hort. Ecosy., 8: 69-82. Abraham, V.A., M.A. Al-Shuaibi, J.R. Faleiro, R.A. Abozuhairah and P.S.P.V. Vidyasagar, 1998. An integrated approach for the management of red palm weevil Rhynchophorus ferrugineus Oliv. A key pest of date palm in the Middle East. Sultan Qaboos Uni. J. Sci. Res., 3: 77-83. Adamo, A.S., 2005. Parasitic suppression of feeding in the tobacco hornworm, Manduca sexta: parallels with feeding depression after an immune challenge. Arch. Insect Biochem. Physiol., 60: 185-197. Ahmad, M., A.H. Sayyed, M.A. Saleem and M. Ahmad, 2008. Evidence for field evolved resistance to newer insecticides in Spodoptera litura (Lepidoptera: Noctuidae) from Pakistan. Crop Prot., 27: 1367-1372. Ahmad, S. and A. Tahir, 2005. Dates culture. In: Ahmad, S. (Ed.). Date palm. Horticulture Foundation of Pakistan, Islamabad, and Pakistan Science Foundation, Islamabad. Pp. 4145. Ahmedani, M.S., M.I. Haque, S.N. Afzal, U. Iqbal and S. Naz, 2008. Scope of commercial formulations of Bacillus thuringiensis Berliner as an alternative to Methyl bromide against Tribolium castaneum adults. Pak. J. Bot., 40: 2149-2156. Aizawa, S.I., 2001. Bacterial flagella and type III secretion systems. FEMS Microbiol. Lett., 202: 157-164. Ajlan, A.M., M. Shawir, M.A. Abo-El-Saad Rezk and K.S. Abdulsalam, 2000. Laboratory evaluation of certain organophosphorus insecticides against the red palm weevil (Olivier). Sci. J. King Faisal Uni., 1: 15-26. Akbar, W., J.C. Lord, J.R. Nechlos and T.M. Loughin, 2005. Efficacy of Beauveria bassiana for red flour beetle when applied with plant essential oils or mineral oil and organosilicone carriers. J. Econ. Entomol., 98: 683-688.

Al-Doghairi, M.A., 2004. Effect of eight acaricides against the date dust mite, Oligonychus afrasiaticus (McGregor) (Acari: 13 Tetranychidae). Pak. J. Biol. Sci., 7(7): 1168-1171. Alfazairy, A.A., 2004. Notes on the survival capacity of two naturally occurring entomopathogens of the red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). Egyp. J. Biol. Pest Control, 14: 423. Alfazairy, A.A., R. Hendi, A.M. El-Minshawy and H.H. Karam, 2003. Entomopathogenic agents isolated from Coleopteran insect pests in Egypt. Egyp. J. Biol. Pest Control, 13: 125. Ali, S., Z. Huang and S.X. Ren, 2010. Production of cuticle degrading enzymes by Isaria fumosorosea and their evaluation as a biocontrol agent against diamondback moth. J. Pest Sci., 83: 361-370. Ali-Shtayeh, M.S., B.M. Abdel-Basit, B.M. Marai and R.M. Jamous, 2002. Distribution, occurrence and characterization of entomopathogenic fungi in agricultural soil in the Palestinian area. Mycopathol., 156: 235-244. Alves, S.B. and R.E. Leucona, 1998. Epizootiologia aplicada ao controle microbiano de insetos. In: Alves, S.B. (Ed.). Controle microbiano de insetos. Piracicaba: FEALQ. Pp. 97-170. Amutha, M., J.G. Banu, T. Surulivelu and N. Gopalakrishnan, 2010. Effect of commonly used insecticides on the growth of white Muscardine fungus, Beauveria bassiana under laboratory conditions. J. Biopesti., 3(1): 143-146. Anderson, T.E., A.E. Hajek, D.W. Roberts, K. Preisler and J.L. Robertson, 1989. Colorado potato beetle (Coleoptera: Chrysomelidae): effects of combinations of Beauveria bassiana with insecticides. J. Econ. Entomol., 82: 83-89. Anonymous, 2013. Problems of agriculture in Pakistan. Available at: www.zaraimedia.com. Accessed on May 06, 2013. Anonymous, 2015. 42 billion worms airlifted from Germany to combat palm weevil crisis. Avaialable at: http://www.jpost.com/Israel-News/42-billion-worms-airlifted-from- Germany-to-combat-palm-weevil-crisis-391817. Assessed on 10 Feb 2016. Aoki, K. and Y. Chigasaki, 1915. Uber des toxin von sog. Sotto-bacillen Mitt. Med. Fak. Kais. Univ. Tokyo, 14: 59-80. Arab, Y.A., and H.M. El-Deeb, 2012. The Use of endophyte Beauveria Bassiana for bio- protection of date palm seedlings against red palm weevil and Rhizoctonia Root-Rot Disease. Sci. J. King Faisal Uni., 13(2): 91-101. Arnold, A.E., L.C. Mejía, D. Kyllo, E.I. Rojas, Z. Maynard, N. Robbins and E.A. Herre, 2003. Fungal endophytes limit pathogen damage in a tropical tree. Proc. Nat. Acad. Sci. USA., 100: 15649-15654. Aronson, A.I., W. Beckman and P. Dunn, 1986. Bacillus thuringiensis and related insect pathogens. Microbiol. Rev., 50: 1-24. Arthurs, S. and M.B. Thomas, 2001. Behavioral changes in Schistocerca gregaria following infection with a fungal pathogen: implications for susceptibility to predation. Ecol . Entomol., 26: 227-234. Ashouri, A., N. Arzanian and H. Askary, 2003. Interactions of Verticillium lecanii (Zimm.) Viegas and Adonia variegata (Col.: Coccinellidae), pathogen and predator of aphids. Colloque international tomate sous abri, protection integree agriculture biologique, Avignon, France. Pp. 158-162. Atakan, E., H. Elekçioğlu, U. Gözel and O. Yüksel, 2009. First report of Heterorhabditis bacteriophora (Poinar, 1975) (Nematoda: Heterorhabditidae) isaloted from

Rhynchophorus ferrugineus (Oliver, 1790) (Coleoptera: Curculionidae) in Turkey. Bull. OEPP/EPPO., 39: 155-160. Athanassiou, C.G., N.G. Kavallieratos, B.J. Vayias, J.B. Tsakiri, N.H. Mikeli, C.M. Meletsis and Z. Tomanovic, 2008. Persistence and efficacy of Metarhizium anisopliae (Metschnikoff) Sorokin (Deuteromycotina: Hyphomycetes) and diatomaceous earth against Sitophilus oryzae (L.) (Coleoptera: Curculinoidae) and Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) on wheat and maize. Crop Prot., 27(10): 1303-1311. Attathom, T., 2002. Biotechnology for insect pest control. In: Proc. Sat. Forum, "Sustainable Agricultural System in Asia," Nagoya. Pp. 73-84. Atwa, A.A. and E.M. Hegazi, 2014. Comparative susceptibilities of different life stages of the red palm weevil (Coleoptera: Curculionidae) treated by entomopathogenic nematodes. J. Econ. Entomol., 107(4): 1339-1347. Aung, O.M., K. Soytong and K.D. Hyde, 2008. Diversity of entomopathogenic fungi in rainforests of Chiang Mai Province, Thailand. Fungal Diver., 30: 15-22. Avand-Faghih, A., 1996. The biology of red palm weevil, Rhynchophorus ferrugineus Oliv. (Coleoptera: Curculionidae) in Saravan region (Sistan and Balouchistan Province, Iran). Appl. Entomol. Phytopathol., 63: 16-18. Azam, K.M. and S.A. Razvi, 2001. Control of red palm weevil, Rhynchophorus ferrugineus Oliver using prophylactic spraying of date palms and trunk injection. In: Proceedings of the 2nd international conference on date palms, Al-Ain, UAE, March 2001. Pp. 216-222. Baig, D.N., D.A. Bukhari and A.R. Shakoori, 2010. Cry genes profiling and the toxicity of isolates of Bacillus thuringiensis from soil samples against American bollworm, Helicoverpa armigera. J. Appl. Microbiol., 109(6): 1967-1978. Baloach, H.B., M.A. Rustamani, R.D. Khuro, M.A. Talpur and T. Hussain, 1992. Incidence and abundance of date palm weevil in different cultivars of date palm. Proc. of 12th Cong. Zool. Pak., 12: 445-447. Bandani, A.R., B.P.S. Khamba, J. Faul, R. Newton, M. Deadman and T.M. Butt, 2000. Production of efrapeptins by Tolypocladium species and evaluation of their insecticidal and antimicrobial properties. Mycol. Res., 104: 537-544. Banerjee A. and T.K. Dangar, 1995. Pseudomonas aeruginosa, a facultative pathogen of red palm weevil, Rhynchophorus ferrugineus. World J. Microb. Biot., 11: 618-620. Banu, J.G., V.K. Sosamma and P.K. Koshy, 1998. Natural occurrence of an entomopathogenic nematode, Heterorhabditis indicus from Kerala, India. Nematology: Challenges and Opportunities in 21st Century. Proceedings of the 3rd International Symposium of Afro- Asian Society of Nematologists (TISAASN), Sugarcane Breeding Institute (ICAR), Coimbatore, India. Pp. 274-280. Barranco, P., J. de la Peña and T. Cabello, 1996. El picudo rojo de las palmeras, Rhynchophorus ferrugineus (Olivier), nueva plaga en Europa. Phytoma España, 67:36-40. Batta, Y.A., 2003. Production and testing of novel formulations of the entomopathogenic fungus Metarhizium anisopliae (Metschinkoff) Sorokin (Deuteromycotina: Hyphomycetes). Crop Prot., 22: 415-422. Batta, Y.A., 2004. Control of rice weevil (Sitophilus oryzae L. Coleoptera: Curculinoidae) with various formulations of Metarhizium anisopliae. Crop Prot., 23: 103-108. Bauce E., Y. Bidon and R. Berthiaume, 2002. Effects of food nutritive quality and Bacillus thuringiensis on feeding behaviour, food utilization and larval growth of spruce budworm

Choristoneura fumiferana (Clem.) when exposed as fourth-and sixth-instar larvae. Agri. Forest Entomol., 4: 57-70. Bauer, L.S., 1995. Resistance: a threat to the insecticidal crystal proteins of Bacillus thuringiensis. Florida Entomol., 78(3): 414-442. Bedford, G.O., 1974. Parasitism of the palm weevil Rhynchophorus bilineatus (Montrouzier) (Coleoptera: Curculionidae) by Praecocilenchus rhaphidophorus (Poinar) (Nematoda: Aphelenchoidea) in New Britain. J. Austr. Entomol. Soc., 13: 155-156. Bednarek, A., E. Popowska-nowak, E. Pezowicz and M. Kamionek, 2004. Integrated methods in pest control: effect of insecticides on entomopathogenic fungi (Beauveria bassiana (Bals.) Vuill., B. Brongniartii (Sacc.) and nematodes (Heterorhabditis megidis Poinar, Jackson, Klein, Steinernema feltiae Filipjev, S. glaseri Steiner). Polish J. Ecol., 52(2): 223-228. Ben-Chobba, I., A. Elleuch, I. Ayadi, L. Khannous, A. Namsi, F. Cerqueira, N. Drira, N. Gharsallah and T. Vallaeys, 2013. Fungal diversity in adult date palm (Phoenix dactylifera L.) revealed by culture-dependent and culture-independent approaches. J. Zhejiang Uni. Sci., 14(12): 1084-1099. Berbert-Molina, M.A., A.M.R. Prata, L.G. Pessanha and M.M. Silveira, 2008. Kinetics of Bacillus thuringiensis var. israelensis growth on high glucose concentrations. J. Indust. Microbiol. Biotech., 35(11): 1397-1404. Berliner, E., 1915. Ueber die Schlaffsucht der Mehlmottenraupe (Ephestia kuhniella Zell) und ihren Erreger Bacillus thuringiensis n. sp. Zeitschrift für Angewandte Entomologie, 2: 29-56. Bernier, L., R.M. Cooper, A.K. Charnley and J.M. Clarkson, 1989. Transformation of the fungus Metarhizium anisopliae to benomyl resistance. FESM Microbiol. Lett., 60(3): 261-265. Besse, S., L. Crabos and K. Panchaud, 2011. Efficacite de 2 souches de Beauveria bassiana sur le charançon rounge du palmier, Rhynchophorus ferrugineus. In: AFPPNeuvième Conférence Internationale sur les ravageurs en agriculture, Montpellier, France. Pp. 404- 409. Bhattacharya, A.K., P. Mondal, V.V. Ramamurthy and R.P. Srivastava, 2003. Beuveria bassiana. In: Srivastava, R.P. (Ed.). A potential bioagent for innovative integrated pest management programme. Biopesticides and Bioagents in Integrated Pest Management of Agricultural Crops. International Books Distributing Co., Lucknow. Pp. 381-491. Birda, L.J. and J.R. Akhursta, 2007. Variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) in Australia to two Bacillus thuringiensis toxins. J. Inver. Pathol., 94: 84-94. Blomquist, G. and R. Vogt, 2003. Insect pheromone biochemistry and molecular biology: the biosynthesis and detection of pheromones and plant volatiles. Boston (MA): Academic Press. Bong, C.F., 1986. Field control of Heliolhis zeo (Boddie) (Lepidoptera: Noctuidae) using a parasitic Nematode. Ins. Sci. Appl., 7:23-25. Booth, R.G., M.L. Cox and R.B. Madge, 1990. IIE Guides to insects of importance to Man. 3 New Guinea records of economically important beetles (Coleoptera). CABI Publishing, Walllingford, UK. Pp. 384. Boucias, D.G., J.C. Pendland and J.P. Latge, 1988. Non-specific factors involved in attachment of entomopathogenic deuteromycetes to host insect cuticle. Appl. Environ. Microbiol., 54: 1795-1805.

Bourtzis, K., 2008. Wolbachia based technologies for insect pest population control. Adv. Exp. Med. Biol., 627: 104-113. Bravo, A., S.S. Gill and M. Soberon, 2007. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, 49: 423-435. Brenner, C., R. Deplus, C. Didelot, A. Loriot, E. Vire, C. De-Smet, A. Gutierrez, D. Danovi, D. Bernard, T. Boon, P. Giuseppe Pelicci, B. Amati, T. Kouzarides, Y. de Launoit, L. Di Croce and F. Fuks, 2005. Myc represses transcription through recruitment of DNA methyltransferase corepressor. EMBO. J., 24: 336-346. Brousseau, C., G. Charpentier and S. Belloncik, 1998. Effects of Bacillus thuringiensis and Destruxins (Metarhizium anisopliae Mycotoxins) combinations on Spruce Budworm (Lepidoptera: Tortricidae). J. Inver. Pathol., 72(3): 262-268. Bruce, M.J., R. Gatsi, N. Crickmore and A.H. Sayyed, 2007. Mechanisms of resistance to Bacillus thuringiensis in the Diamondback Moth. Biopesti. Inter., 3(1): 1-12. Bugeme, D.M., N.K. Maniania, M. Knapp and H.I. Boga, 2008. Effect of temperature on virulence of Beauveria bassiana and Metarhizium anisopliae isolates to Tetranychus evansi. Experi. Appl. Acarol., 46: 275-285. Bulla, L.A. Jr, R.A. Rhodes and G.S. Julian, 1975. Bacteria as insect pathogens. Annu. Rev. Microbiol., 29: 163-190. Bunerjee, A. and T.K. Dangar, 1995. Pseudomonas aeruginosa, a facultative pathogen of red palm weevil, Rhynchophorus ferrugineus. World J. Microbiol. Biotechnol. 11: 618-620. Butani, D.K., 1975. Insect pests of fruit crops and their control, sapota-11. Pesticide Res. J., 9: 40-42. Butt, T.M. and M.S. Goettel, 2000. Bioassays of entomogenous fungi In: Navon, A. and K.R.S. Ascher (Eds.). Bioassays of Entomopathogenic Microbes and Nematodes, CABI. Pp. 141-195. Buxton, P.A., 1920. Insect pests of dates and the date palm in Mesopotamia and elsewhere. Bull. Entomol. Res., 11: 287-303. Cabello, T.P., 2006. Biology and population dynamics of red palm weevil in Spain. In Proceedings of the 1st International Workshop on Red Palm Weevil, 28-29, November 2005, IVIA, Valencia, Spain (in press). Cagan, L. and V. Uhlik, 1999. Pathogenicity of Beauveria bassiana strains isolated from Ostrinia nubilalis Hbn. (Lepidoptera: Pyralidae) to original host larvae and to ladybirds (Coleoptera: Coccinellidae). Plant Prot. Sci., 35: 108-112. Chandler, D., D. Hay and A.P. Reid, 1997. Sampling and occurrence of entomopathogenic fungi and nematodes in UK soils. Appl. Soil Ecol., 5: 133-141. Chapin, E. and N. André, 2010. Nouveau moyen de lutte biologique contre le papillon palmivore. Phytoma La Défense des Végétaux, 635: 27-30. Chaufaux, J., M. Marchal, N. Gilois, I. Jehanno and C. Buisson, 1997. Recherche de souches naturelles du Bacillus thuringiensis dans differents biotopes, átravers le monde. Canad. J. Microbiol., 43: 337-343. Chen, M., A. Shelton and G.Y. Ye, 2011. Insect-resistant genetically modified rice in China: from research to commercialization. Ann. Rev. Entomol., 56: 81-101. Cito, A., G. Mazza, A. Strangi, C. Benvenuti, G.P. Barzanti, E. Dreassi, T. Turchetti, V. Francardi and P.F. Roversi, 2014. Characterization and comparison of Metarhizium strains isolated from Rhynchophorus ferrugineus. FEMS Microbiol. Lett., 355: 108-115.

Cottrell, T.E. and D.I. Shapiro-Ilan, 2003. Susceptibility of a native and an exotic lady beetle (Coleoptera: Coccinellidae) to Beauveria bassiana. J. Inver. Pathol., 84: 137-144. Cox, M.L., 1993. Red palm weevil, Rhynchophorus ferrugineus in Egypt. FAO Plant Prot. Bull., 41: 30-31. Crickmore, N., D.R. Zeigler, J. Feitelson, E. Schnepf, J.V. Rie, D. Lereclus, J. Baum and D.H. Dean, 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Molecul. Biol. Rev., 62(3): 807-813. Dangar T. K. and A. Banerjee, 1993. Infection of red palm weevil by microbial pathogens. In: Nair, M.K., H.H. Khan, P. Gopalasundaram and E.V.V. Bhaskara Rao (Eds.). Advances in coconut research and development. Oxford IBM Publishing Co., New Delhi. Pp. 531- 533. Dangar, T.K., 1997. Infection of red palm weevil, Rhynchophorus ferrugineus, by a yeast. J. Plant. Crops, 25: 193-196. Deadman, M.L., K.M. Azam, S.A. Ravzi and W. Kaakeh, 2001. Preliminary investigation into the biological control of the red palm weevil using Beauveria bassiana. Proceedings of the Second International Conference on Date Palm, Al-Ain, UAE. March 25-27. Pp. 225- 232. Debré, P., 1998. Louis Pasteur. E. Forster, translator. Johns Hopkins University Press, Baltimore, Maryland, USA. De-Maagd, R.A., A. Bravo and N. Crickmore, 2001. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet., 17: 193-199. Dembilio, Ó., E. Llácer, D.E. Martinez, M.M. Altube and J.A. Jacas, 2010a. Field efficacy of imidacloprid and Steinernema carpocapsae in a chitosan formulation against the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) in Phoenix canariensis. Pest Manag. Sci., 66: 365-370. Dembilio, Ó. and J.A. Jacas, 2013. Biological control of Rhynchophorus ferrugineus. AFPP palm pest mediterranean conference nice-16, 17 and 18 January, 2013. Dembilio, Ó., E. Quesada-Moraga, C. Santiago-Alvarez and J.A. Jacas, 2010b. Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the red palm weevil, Rhynchophorus ferrugineus. J. Inver. Pathol., 104(3): 214-221. Dembilio, Ó., J.A. Jacas and E. Llacer, 2009. Are the new palms Washintonia filifera and Chamaerops humilis suitable hosts for the red palm weevil, Rhynchophorus ferrugineus (Col. Curculionidae). J. Appl. Entomol., 133(7): 565-567. Derakhshan, A., 2008. Natural occurrence and distribution of soil borne entomopathogenic fungi in Shahrood Region, Northeast of Iran. International Meeting on Soil Fertility Land Management and Agroclimatology. Turkey. Pp. 873-877. Dettloff, M., B. Kaiser and A. Wiesner, 2001. Localization of injected apolipophorin III in-vivo new insights into the immune activation process directed by this protein. J. Insect Physiol., 47: 789-797. Dhillon, B.S., R.K. Tyaagi and S. Saxena, 2005. Plant genetic resources: horticultural crops. Narosa Publishing House, New Delhi. Pp. 174-176. Dimbi, S., N.K. Maniania, S.A. Lux and J.M. Mueke, 2004. Effect of constant temperatures on germination, radial growth and virulence of Metarhizium anisopliae to three species of African tephritid fruit flies. Biocontrol, 49: 83-94.

Dolinski, C. and L.A. Lacey, 2007. Microbial control of arthropod pests of tropical tree fruit. Neotropi. Entomol., 36: 161-179. Dowd, P.F., 1992. Insect interactions with mycotoxin-producing fungi and their hosts. In: Bhatnagar, D. E.B. Lillehoj and D.K. Arora (Eds.). Handbook of Applied Mycology. Volume 5. Mycotoxins in Ecological Systems. Marcel Dekker, New York. Pp. 137-155. Dumler, J.S., A.F. Barbet, C.P. Bekker, G.A. Dasch, G.H. Palmer, S.C. Ray, Y. Rikihisia and F.R. Rurangirwa, 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ’HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. Inter. J. Syst. Evol. Microbiol., 51: 2145-2165. Edwards, S., 1989. Real Exchange Rates, Devaluation, and Adjustment, Cambridge, Massachusetts: MIT Press. Eilenberg, J. and V. Michelsen, 1999. Natural host range and prevalence of the genus Strongwellsea (Zygomycota: Entomophthorales) in Denmark. J. Inver. Pathol., 73: 189- 198. Ekesi, S., N.K. Maniania and K. Ampong-Nyarko, 1999. Effect of temperature on germination, radial growth and virulence of Metarhizium anisopliae and Beauveria bassiana on Megalurothrips sjostedti. Biocontrol Sci. Technol., 9: 177-185. Elawad, S.A., S.A. Mousa, A.S. Shahbad, S.A. Alawaash and A.M.A. Alamari, 2007. Efficacy of entomopathogenic nematodes against red palm weevil in UAE. Acta Horti., 736: 415- 420. El-Bishry, M.H., Y. El-Sebay and M.H. Al-Elimi, 2000. Impact of the environment in date palm infested with Rhynchophorus ferrugineus on five entomopathogenic nematodes (Rhabditida). Inter. J. Nematol., 10(1): 75-80. Eleftherianos, I., H. Baldwin, R.H. French-Constant, S.E. Reynolds, 2008. Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus. J. Insect Physiol., 54: 309-318. El-Ezaby, F.A, 1997. A biological in-vitro study on the red Indian date palm weevil. Arab J. Plant Prot., 15(2): 84-87. El-Mergawy, R.A.A.M. and A.M. Al-Ajlan, 2011. Red palm weevil, Rhynchophorus ferrugineus (Olivier): economic importance, biology, biogeography, and integrated pest management. J. Agri. Sci. Technol., 1: 1-23. El-Sufty, R., S. Al-Bgham, S. Al-Awash, A. Shahdad and A. Al-Bathra, 2011. A trap for auto dissemination of the entomopathogenic fungus Beauveria bassiana by red palm weevil adults in date palm plantations. Egyp. J. Biol. Pest Control, 21: 271-276. El-Sufty, R., S.A. Al-Awash, A.M. Al-Amiri, A.S. Shahdad, A.H. Al-Bathra and S.A. Musa, 2007. Biological control of red palm weevil, Rhynchophorus ferrugineus (Col.: Curculionidae) by the entomopathogenic fungus Beauveria bassiana in United Arab Emirates. Proceeding of the 3rd International Conference on Date Palm. Acta Horti., 736: 399-404. El-Sufty, R., S.A. Al-Awash, S. Al-Bgham, A.S. Shahdad and A.H. Al-Bathra, 2009. Pathogenicity of the fungus Beauveria bassiana (Bals.) Vuill to the Red Palm Weevil,

Rhynchophorus ferrugineus (Oliv.) (Col.: Curculionidae) under laboratory and field conditions. Egyp. J. Biol. Pest Control, 19: 81-85. Entwistle, P.F., J.S. Cory, M. Bailey and S. Higgs, 1993. Bacillus thuringiensis, an environmental bio-pesticide: theory and practice. New York, NY: Wiley. EPPO (European and Mediterranean Plant Protection Organization), 2008. Data sheets on quarantine pests. Rhynchophorus ferrugineus. EPPO Bull., 38: 55-59. EPPO (European and Mediterranean Plant Protection Organization), 2009. EPPO Reporting Service. 2009/002 - First record of Rhynchophorus ferrugineus in Curacao, Netherlands Antilles, January 26, 2009. EPPO (European and Mediterranean Plant Protection Organization), 2010. EPPO Reporting Service. 2010/176 - First record of Rhynchophorus ferrugineus in the USA, November 1, 2010. EPPO (European and Mediterranean Plant Protection Organization), 2008. Data sheets on quarantine pests. Rhynchophorus ferrugineus. EPPO Bull., 38: 55-59. Ericsson, J.D., A.F. Janmaat, C. Lowenberger and J.H. Myers, 2009. Is decreased generalized immunity a cost of Bt resistance in cabbage loopers Trichoplusia ni? J. Inver. Pathol., 100: 61-67. Esteban-Duran J., J.L. Yela, F. Beitia Crespo and A. Jimenez Alvarez, 1998. Biology of red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae: Rhynchophorinae), in the laboratory and field, life cycle, biological characteristics in its zone of introduction in Spain, biological method of detection and possible control. Boletin de Sanidad Vegetal Plagas, 24: 737-748. Esteban-Duran, J., J.L. Yela, F. Beitia-Crespo and A. Jimenez-Alvarez, 1998. Biologia del Curculionido ferruginoso de las palmeras Rhynchophorus ferrugineus (Olivier) en laboratorio y campo: Ciclo en cautividad, peculiaridades biologicas en su zona de introduccion en Espana y metodos biologicos de deteccion y posible control (Coleoptera: Curculionidae: Rhynchophorinae). Boletín de Sanidad Vegetal - Plagas, 24: 737-748. Evans, H.C., 1989. Mycopathogens of insects of epigeal and aerial habitats. In: Wilding, N.N.M., P.M. Collins and J.F.W. Hammond (Eds.). Insect-Fungus interactions. Pp. 205-238. Eyal, M.D., A. Mabud, K.L. Fishbein, J.F. Walter, L.S. Osbourne and Z. Landa, 1994. Assessment of Beauveria bassiana Nov. EO-1 Strain, Which Produces a Red Pigment for Microbial Control. Appl. Biochem. Biochem., 44: 65-80. Faghih, A.A., 1996. The biology of red palm weevil, Rhynchophorus ferrugineus Oliver (Coleopter, Curculionidae) in Savaran region (Sistan province, Iran). Appl. Entomol. Phytopathol., 63: 16-18. Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm during the last one hundred years. Inter. J. Tropi. Insect Sci., 26: 135-154. Fan, Y., W.S. Fang, S. Guo, X. Pei, Y. Zhang, Y. Xiao, D. Li, K. Jin, M.J. Bidochka and Y. Pei, 2007. Increased insect virulence in Beauveria bassiana strains over expressing an engineered chitinase. Appl. Environ. Microbiol., 73: 295-302. Fang, W., Y. Zhang, X. Yang, X. Zheng, H. Duan, Y. Li, and Y. Peia, 2004. Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker. J. Inver. Pathol., 85: 18-24. FAO Statistics Division, 2013. Production, harvested area, import and export of dates in Pakistan in 2011. Available at: http://faostat.fao.org.

Fargues, J., N.K. Maniania, J.C. Delmas and N. Smits, 1992. Influence de la température sur la croissance in vitro d’hyphomycètes entomopathogènes. Agronomie, 12: 557-564. Fernandes, P.M., B.P. Magalhaes, S.B. Alves and D.W. Roberts, 1989. Effect of physical and biological factors on the conidiogenisis and survival of Beauveria bassiana (Bals.) Vuill. inside cadavers of Cerotoma arcuata (Olivier, 1791). (Coleoptera: Chrysomelidae). Anais da Sociedade Entomologia do Brasil, 18: 147-156. Fernandez-Luna, M.T., H. Lanz-Mendoza, S.S. Gill, A. Bravo, M. Soberon and J. MirandaRios, 2010. An alpha-amylase is a novel receptor for Bacillus thuringiensis ssp. israelensis Cry4Ba and Cry11Aa toxins in the malaria vector mosquito Anopheles albimanus (Diptera: Culicidae). Environ. Microbiol., 12: 746-757. Ferron, P., 1978. Biological control of insect pests by entomopathogenic fungi. Ann. Rev. Entomol., 23: 409-423. Ferron, P., J. Fargues and G. Riba, 1991. Fungi as microbial insecticides against pests. In: Arora, D.K., L. Ajello and K.G. Mukerji (Eds.). Handbook of Applied Mycology, Vol. 2: Humans, Animals and Insects. Marcel Dekker, Inc, New York. Pp. 665-706. Ferry, M. and S. Gomez, 2002. The red palm weevil in the Mediterranean area. Palms, 46(4): 172-178. Foth, H.D., 1984. Fundamentals of Soil Science. John Wiley & Sons, London. Francardi, V., C. Benventi, G.P. Barzanti and P.F. Rovers, 2013. Auto contamination trap with entomopathogenic fungi: A possible strategy in the control of Rhynchophorus ferrugineus (olivier) (Coleoptera Curculionidae). REDIA, XCVI: 57-67. Francardi, V., C. Benvenuti, P.F. Roversi, P. Rumine and G. Barzanti, 2012. Entomopathogenicity of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorokin isolated from different sources in the control of Rhynchophorus ferrugineus (Olivier) (Coleoptera Curculionidae). Redia, 95: 49-55. Francesca, N., A. Alfonzo, G.L. Verde, L. Settanni, M. Sinacori, P. Lucido and G. Moschetti, 2015. Biological activity of Bacillus spp. evaluated on eggs and larvae of red palm weevil Rhynchophorus ferrugineus. Ann. Microbiol., 65: 477-485. Francesca, N., C.G. Caldarella and G. Moschetti, 2008. Indagini preliminari su bacilli sporigeni associati ad adulti di Punteruolo rosso e loro possibili impieghi in lotta biologica, In: La ricerca scientifica sul punteruolo rosso e gli altri fitofagi delle palme in Sicilia, Regione Siciliana-Assessorato Agricoltura e Foreste Dipartimento Interventi Infrastrutturali, Servizi allo Sviluppo. Pp. 69-72. Freimoser, F.M., G. Hu and R.J. St Leger, 2005. Variation in gene expression patterns as the insect pathogen Metarhizium anisopliae adapts to different host cuticles or nutrient deprivation in vitro. Microbiol., 151: 361-371. Furlaneto, L., H.O. Saridakis and O.M.N. Arantes, 2000. Survival and conjugal transfer between Bacillus thuringiensis strains in aquatic environment. Braz. J. Microbiol., 31: 233-238. Fuxa, J.R., 1995. Ecological factors critical to the exploitation of entomopathogens in pest control. In: Franklin R. Hall and J.W. Barry (Eds.), Biorational Pest Control Agents Formulation and Delivery, Vol. 595. American Chemical Society. Gauglar, R. and H.K. Kaya, 1990. Entomopathogenic nematodes in biological control. Boca Raton, Florida, USA: CRC press. Pp. 365. George, Z. and N. Crickmore, 2012. Bacillus thuringiensis Applications in Agriculture. In: Estibaliz S.E. (Ed.). Bacillus thuringiensis Biotechnology. Springer Science + Business Media B.V. Springer Netherlands.

Gerber, K. and R.M. Giblin-Davis, 1990. Association of the red ring nematode and other nematode species with the palm weevil, Rhynchophorus palmarum. J. Nematol., 22: 143- 149. Ghazavi, M., A. Avand-Faghih, 2002. Isolation of two entomopathogenic fungi on red palm weevil, Rhynchophorus ferrugineus (Olivier) (Col., Curculionidae) in Iran. Appl. Entomol. Phytopathol., 9: 44-45. Giannoulis, P., C.L. Brooks, G.B. Dunphy, C.A. Mandato, D.F. Niven and R.J. Zakarian, 2007. Interaction of the bacteria Xenorhabdus nematophila (Enterobactericeae) and Bacillus subtilis (Bacillaceae) with the hemocytes of larval Malacosoma disstria (Insecta: Lepidoptera: Lasiocampidae). J. Inver. Pathol., 94: 20-30. Giblin-Davis, R.M., 1993. Interactions of nematodes with insects, In: W. Khan (Ed.). Nematode Interactions. Chapman & Hall, London. Pp. 302-344. Giblin-Davis, R.M., N. Kanzaki, W. Ye, B.J. Center and W.K. Thomas, 2006. Morphology and systematics of Bursaphelenchus gerberae n. sp. (Nematoda: Parasitaphelenchidae), a rare associate of the palm weevil, Rhynchophorus palmarum in Trinidad. Zootaxa, 1189: 39- 53. Gill, S.S., E.A. Cowles and P.V. Pietrantonio, 1992. The mode of action of Bacillus thuringiensis δ-endotoxins. Ann. Rev. Entomol., 37: 615-636. Gindin, G., S. Levski, I. Glazer and V. Soroker, 2006. Evaluation of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana against the red palm weevil Rhynchophorus ferrugineus. Phytoparasitica, 34: 370-379. Glare, T.R., 2003. Biotechnology potential of entomopathogenic fungi. In: Arora, D.K., D. Bridge and D. Bhatnagar (Eds.). Fungal biotechnology in agricultural, food and environmental applications. CRC Press. Goettel, M.S., T.J. Poprawski, J.D. Vandenberg, Z. Li and D.W. Roberts, 1990. Safety to non- target invertebrate of fungal bicontrol agents. In: Laird, M., L.A. Lacey and E.W. Davidson (Eds.). Safty of Microbial insecticides. Boca Raton, CA: CRS Press. Pp. 209- 232. Gómez-Vidal, S. and M. Ferry, 1999. Attempts at biological control of date palm pests recently found in Spain. In: M. Canard and V. Beyssatarnaouty (Eds.). Proceedings of the First Regional Symposium for Applied Biological Control in Mediterranean Countries. Cairo, 25-29 October 1998. Imprimerie Sacco, Toulouse, France. Pp. 121-125. Gómez-Vidal, S., L.V. Lopez-Llorca, H.B. Jansson and J. Salinas, 2006. Endophytic colonization of date palm (Phoenix dactylifera L.) leaves by entomopathogenic fungi. Micron, 37: 624-632. Gong, Y.J., C.L. Wang, Y.H. Yang, S.W. Wu and Y.D. Wu, 2010. Characterization of resistance to Bacillus thuringiensis toxin Cry1Ac in Plutella xylostella from China. J. Inver. Pathol., 104(2): 90-96. Gressel, J., 2001. Potential failsafe mechanisms against the spread and introgression of transgenic hyper virulent biocontrol fungi. Trend. Biotech., 19: 149-154. Grewal, P.S., R.U. Ehlers and D.I. Shapiro-Ilan, 2005. Nematodes as Biocontrol Agents, CABI, New York, Wallingford. Pp. 513. Güerri-Agulló, B., L. Asensio., P. Barranco, S. Gómez-Vidal and L.V. Lopez-Llorca, 2008. Use of Beauveria bassiana as a tool for biological control of Rhynchophorus ferrugineus, In: 41st Annual Meeting of Society for Invertebrate pathology and 9th international Conference on Bacillus thuringiensis, Warwick, UK, 3-7 August, 2008. Pp. 125.

Güerri-Agulló, B., R. López-Follana, L. Asensio, P. Barranco and L.V. Lopez-Llorca, 2011. Use of a solid formulation of Beauveria bassiana for biocontrol of the red palm weevil (Rhynchophorus ferrugineus) (Coleoptera: Dryophthoridae) under field conditions in SE Spain. Florida Entomol., 94: 737-747. Güerri-Agulló, B., S. Gómez-Vidal, L. Asensio, P. Barranco and L.V. Lopez-Llorca, 2010. Infection of the red palm weevil (Rhynchophorus ferrugineus) by the entomopathogenic fungus Beauveria bassiana: a SEM study. Micros. Res. Tech., 73: 714-725. Gunawardena, N.E. and U.K. Bandarage, 1995. 4-methyl-5-nonanol (ferrugineol) as an aggregation pheromone of the coconut pest, Rhynchophorus ferrugineus F. (Coleoptera: Curculionidae): Synthesis and use in a preliminary field assay. J. Natural Sci. Council of Sri Lanka, 23: 71-79. Gupta, J.J., B.P.S. Yadav and H.K. Gupta, 1995. Nutritional value of jack bean for broiler. Indian J. Poult. Sci., 30(2): 112-116. Gupta, S.C., T.D. Leathers, G.N. El-Sayed and C.M. Ignoffo, 1991. Production of degradative enzymes by Metarhizium anisopliae during growth on defined media and insect cuticle. Exp. Mycol., 15: 310-315. Gurr, G.M., S.D. Wratten and M.A. Altieri, 2004. Ecological engineering for pest management: habitat manipulation for arthropods. CSIRO Publishing, Collingwood, Australia. Hagley, E., 1965. Test of attractant for the palm weevil. J. Econ. Entomol., 58: 1002-1003. Hajek, A.E. and R.J. St-Leger, 1994. Interactions between fungal pathogens and insect host. Ann. Rev. Entomol., 39: 293-322. Hajek, A.E., 1997. Ecology of terrestrial fungal entomopathogens. Adv. Microbiol. Ecol., 15: 193-249. Hallet, R.H., G. Gries, J.H. Borden, E. Czyzewska, A.C. Oehlschlager, H.D. Pierce Jr., N.P.D. Angerilli and A. Rauf, 1993. Aggregation pheromones of two Asian palm weevils, Rhynchophorus ferrugineus and R. vulneratus. Naturwissen, 80: 328-331. Hallett, R.H., B.J. Crespi and J.H. Borden, 2004. Synonymy of Rhynchophorus ferrugineus (Olivier), 1790 and R. vulneratus (Panzer), 1798 (Coleoptera, Curculionidae, Rhynchophorinae). J. Natural History, 38(22): 2863-2882. Hanounik, S.B., 1998. Steinemematids and Heterorhabditids as biological control agents for the red palm weevil, Rhynchophorus ferrugineus. Sultan Qaboos Uni. J. Sci. Res. Agri. Sci., 3: 95-102. Hanounik, S.B., M.M.E. Saleh, R.A. Abuzuairah, M. Alheji, H. Aldhahir and Z. Aljarash, 2000. Efficacy of entomopathogenic nematodes with antidesiccants in controlling the red palm weevil Rhynchophorus ferrugineus on date palm trees. Inter. J. Nematol., 10: 131-134. Haseeb, M. and H. Murad, 1997. Susceptibility of the predator, Coccinella septempunctata to the entomogenous fungus, Beauveria bassiana. Ann. Plant Prot. Sci., 5: 188-219. Hassan, A.E.M. and A.K. Charnley, 1989. Ultrastructural study of the penetration by Metarhizium anisopliae through Dimilin affected cuticle of Manduca sexta. J. Inver. Pathol., 54: 117-124. Heckel, D.G., L.J. Gahan, S.W. Baxter, J.Z. Zhao, A.M. Shelton, F. Gould and B.E. Tabashnik, 2007. The diversity of Bt resistance genes in species of Lepidoptera. J. Inver. Pathol., 95(3): 192-197. Hedgecock, S., D. Moore, P.M. Higgins and C. Prior, 1995. Influence of moisture content on temperature tolerance and storage of Metarhizium flavoviride conidia in an oil formulation. Biocontrol Sci. Technol., 5: 371-377.

Hegazy, G., O. Al-Muhanna , S.B. Hanounik , T.S. Al-Gumaiah and A.A. Aldossary, 2007. Efficacy of new isolates of the entomopathogenic fungus Beauveria bassiana against RPW Rhynchophorus ferrugineus in Saudi Arabia. Egyp. J. Agri. Res., 85(1): 61-71. Herms, C.P., D.G. McCullough, L.S. Bauer, R.A. Haack, D.L. Miller and N.R. Dubois, 1997. Susceptibility of the endangered Karner blue butterfly (Lepidoptera: Lycaenidae) to Bacillus thuringiensis var. kurstaki used for gypsy moth suppression on Michigan. Great Lakes Entomol., 30: 125-141. Hilgenboecker, K., P. Hammerstein, P. Schlattmann, A. Telschow and J.H. Werren, 2008. How many species are infected with Wolbachia? a statistical analysis of current data. Fems. Microbiol. Lett., 281: 215-220. Hofmann, C., H. Vanderbruggen, H. Hofte, J.V. Rie, S. Jansens and H.V. Mellaert, 1988. Specificity of Bacillus thuringiensis δ-endotoxins is correlated with the presence of high- affinity binding sites in the brush border membrane of target insect midguts. Proc. Nati. Acad. Sci. USA., 85: 7844-7848. Hong, T.D., R.H. Ellis and D. Moore, 1997. Development of a model to predict the effect of temperature and moisture on fungal spore longevity. Annal. Botany, 79: 121-128. Hosokawa, T., R. Koga, Y. Kikuchi, X.Y. Meng and T. Fukatsu, 2010. Wolbachia as a bacteriocyte-associated nutritional mutualist. Proc. Nat. Acad. Sci., 107: 769-774. Hu, Y., S.B. Georghiou, A.J. Kelleher and R.V. Aroian, 2010. Bacillus thuringiensis Cry5B protein is highly efficacious as a single-dose therapy against an intestinal roundworm infection in mice. PLOS Negl. Trop. Dis., 4(3): e614. Hussain, A., M. Rizwan-Ul-Haq, H. Al-Ayedh, S. Ahmed and A.M. Al-Jabr, 2015. Effect of Beauveria bassiana infection on the feeding performance and antioxidant defence of red palm weevil, Rhynchophorus ferrugineus. BioControl, 60: 849-859. Hussain, A., M.R.U. Haq, A.M. Al-Jabr and H.Y. Al-Ayied, 2013. Managing Invasive Populations of Red Palm Weevil: A Worldwide Perspective. J. Food Agri. Envir., 11: 456-463. Ibargutxi, M.A., D. Muñoz, I.R.D. Escudero and P. Caballero, 2008. Interactions between Cry1Ac, Cry2Ab, and Cry1Fa Bacillus thuringiensis toxins in the cotton pests Helicoverpa armigera (Hübner) and Earias insulana (Boisduval). Biol. Control, 47(1): 89-96. Ibrahim, L., T.M. Butt, A. Beckett and S.J. Clark, 1999. The germination of oil-formulated conidia of the insect pathogen, Metarhizium anisopliae. Mycol. Res., 103(7): 901-907. Inglis, D.G., D.L. Johnson and M.S. Goettel, 1996. Effect of bait substrate and formulation on infection of grasshopper nymphs by Beauveria bassiana. Biocontrol Sci., Tech., 6: 35-50. Inglis, G.D., G.M. Duke, P. Kanagaratnam, D.L. Johnson and M.S. Goettel, 1997. Persistence of Beauveria bassiana in soil following application of conidia through crop canopies. Mem. Entomol. Soci. Canada, 171: 253-263. Inglis, G.D., M.S. Goettel, M.T. Butt and A. Strasser, 2001. Use of hyphomycetous fungi for managing insect pests. In: Butt, T.M., C. Jackson and N. Magan (Eds.). Fungi as Biocontrol Agents. CAB International, Wallingford, UK. Pp. 24-69. Inglis, G.D., S.T. Jaronski and S.P. Wraight, 2002. Use of spray oils with entomopathogens. In: Beattie, G.A.C., D.M. Watson, M.L. Stevens, D.J. Rae and R.N. SpoonerHart (Eds.). Spray Oils Beyond 2000-Sustainable Pest and Disease Management. University of Western Sydney Press. Pp. 302-312.

Iriarte, J., Y. Bel, M.D. Ferrandis, R. Andrew, J. Murillo, J. Ferré and P. Caballero, 1998. Environmental distribution and diversity of Bacillus thuringiensis in Spain. System. Appl. Microbiol., 21: 97-106. Ishiwata, S., 1901. On a kind of severe flacherue (sotto disease). Dainihan Sanshi, Kaiho, 114: 1- 5. Jalinas, J., B. Güerri-Agulló, R.W. Mankin, R. López-Follana and L.V. Lopez-Llorca, 2015. Acoustic assessment of Beauveria bassiana (Hypocreales: Clavicipitaceae) effects on Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) larval activity and mortality. J. Econ. Entomol., 108(2): 444-453. James, R.R. and B. Lighthart, 1994. Susceptibility of the convergent lady beetle (Coleoptera: Coccinellidae) to four entomogenous fungi. Environ. Entomol., 23: 190-192. James, R.R., J.S. Buckner and T.P. Freeman, 2003. Cuticular lipids and silverleaf whitefly stage affect conidial germination of Beauveria bassiana and Paecilomyces fumosoroseus. J. Inver. Pathol., 84(2): 67-74. Jatoi, M.A., N. Solangi and Z. Markhand, 2010. Dates in Sindh: facts and figures. In: Markhand G.S. and A.A. Abul-Soad (Eds.). Proceedings international dates seminar, 28 July 2009, Khairpur. Pp. 59-71. Jeffs, L.B. and G.G. Khachatourians, 1997. Toxic properties of Beauveria pigments on erythrocyte membranes. Toxicon, 35: 1351-1356. Joshi, L., R.J. St. Leger and M.J. Bidochka, 1995. Cloning of a cuticle-degrading protease from the entomopathogenic fungus, Beauveria bassiana. FEMS Microbiol. Lett., 125: 211- 217. Ju, R.T., F. Wang, F.H. Wan and B. Li, 2011. Effect of host plants on development and reproduction of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). J. Pest Sci., 84: 33-39. Juárez, M.P., 1994. Inhibition of cuticular lipid synthesis and its effect on insect survival. Arch. Insect Biochem. Physiol., 25: 177-191. Jurat-Fuentes, J. and T. Jackson, 2012. Bacterial entomopathogens. In: Vega, F. and H. Kaya (Eds.). Insect Pathology, 2nd edition. Edited by Elsevier. Pp. 266-349. Kaakeh, W., 2005. Longevity, fecundity, and fertility of the red palm weevil, Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) on natural and artificial diets. Emirates J. Agri. Sci., 17: 23-33. Kaakeh, W., A. Khamis and M.M. Aboul-Nour, 2001. The Red Palm Weevil. The most dangerous agricultural pest. UAE University Press. Pp. 163. Kalantari, M., R. Marzban, S. Imani and H. Askari, 2014. Effects of Bacillus thuringiensis isolates and single nuclear polyhedrosis virus in combination and alone on Helicoverpa armigera. Archi. Phytopathol. Plant Prot., 47(1): 42-50. Kanaoka, M., A. Isogai, S. Murakoshi, M. Ichione, A. Suzuki and S. Tamura, 1978. Bassianolide, a new insecticidal cylcodepsipeptide from Beauveria bassiana and Verticillium lecanii. Agric. Biol. Chem., 42: 629-635. Kanzaki, N., F. Abe, R.M. Giblin-Davis, K. Kiontke, D.H.A. Fitch, K. Hata and K. Soné, 2008. Teratorhabditis synpapillata Sudhaus, 1985 (Rhabditida: Rhabditidae) is an associate of the red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae). Nematol., 10: 207-218. Kanzaki, N., R.M. Giblin-Davis, Y. Zeng, W. Ye and B.J. Center, 2009. Acrostichus rhynchophori n. sp. (Rhabditida: Diplogastridae): a phoretic associate of Rhynchophorus

cruentatus Fabricius and R. palmarum L. (Coleoptera: Curculionidae) in the Americas. Nematol., 11: 669 688. Kaur, G. and V. Padmaja, 2008. Evaluation of Beauveria bassiana isolates for virulence against Spodoptera litura (Fab.) (Lepidoptera; Noctuidae) and their characterization by RAPD- PCR. Afri. J. Microbiol. Res., 2: 299-307. Kaya, H.K. and R. Gaugler, 1993. Entomopathogenic nematodes. Ann. Rev. Entomol., 38: 181- 206. Kaya, H.K., 1990. Soil ecology. In: Gaugler, R., H.K. Kaya (Eds.). Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, FL. Pp. 93-115. Kehat, M., 1999. Threat to date palms in Israel, Jordan and the Palestinian Authority by the red palm weevil, Rhynchophorus ferrugineus. Phytoparasitica, 27: 107-108. Keller, S. and G. Zimmermann, 1989. Mycopathogens of soil insects. In: Wilding, N., N.M. Collins, P.M. Hammond and J.F. Webber (Eds.). Insect-Fungus Interactions. Academic Press, London, UK. Pp. 239-270. Kendrick, M., 2000. The Fifth Kingdom, 3rd Edition. Mycologue Publications, Sydney, Australia. Kenis, M., M.A. Auger-Rozenberg, A. Roques, L. Timms, C. Péré, M.J.W. Cock, J. Settele, S. Augustin and C. Lopez-Vaamonde, 2009. Ecological effects of invasive alien insects. Biol. Invasi., 11: 21-45. Kershaw, M.J., E.R. Moorhouse, R. Bateman, S.E. Reynolds and A.K. Charnley, 1999. The role of destruxins in the pathogenicity of Metarhizium anisopliae for three species of insects. J. Inver. Pathol., 74: 213-223. Khachatourians, G.G., 1986. Production and use of biological pest control agents. Trends Biotech., 4: 120-124. Khachatourians, G.G., 1991. Physiology and genetics of entomopathogenic fungi. In: Arora, D.K., L. Ajello and K.G. Mukerji (Eds.). Handbook of Applied mycology, vol 2: humans, animals, and insects. Marcel Dekker Inc, New York. Pp. 613-661. Kiewnick, S., 2006. Effect of temperature on growth, germination, germ-tube extension and survival of Paecilomyces lilacinus strain 251. Biocontrol Sci. Technol., 16: 535-546. Klass, J.I., S. Blanford and M.B. Thomas, 2007. Development of a model for evaluating the effects of environmental temperature and thermal behavior on biological control of locusts and grasshoppers using pathogens. Agri. Forest Entomol., 9: 189-199. Knowles, B.H. and J.A.T. Dow, 1993. The crystal δ-endotoxins of Bacillus thuringiensis: models for their mechanism of action on the insect gut. BioEssays, 15: 469-476. Kontodimas, D.C., P. Milonas, V. Vassiliou, N. Thymakis and D. Economou, 2006. The occurrence of Rhynchophorus ferrugineus in Greece and Cyprus and the risk against the native Greek palm tree Phoenix theophrasti. Entomol. Hellenica, 16: 11-15. Kooyman, C, P.R.P. Bateman, J.R. Langewald, C.J. Lomers, Z. Ouambama and M.B. Thomas, 1997. Operational-scale application of entomopathogenic fungi for control of Sahelian grasshoppers Proceedings of Royal Society of London, The Royal Soc. Printed in Great Britain, 264: 541-546. Koppenhöfer, A.M. and E.M. Fuzy, 2004. Effect of white grub developmental stage on susceptibility to entomopathogenic nematodes. J. Econ. Entomol., 97: 1842-1849. Koppenhöfer, A.M. and E.M. Fuzy, 2008. Early timing and new combinations to increase the efficacy of neonicotinoid-entomopathogenic nematode (Rhabditida: Heterorhabditidae)

combinations against white grubs (Coleoptera: Scarabaeidae). Pest Manag. Sci. 64: 725- 735. Kovács, Á.M., E. Téglás and A.D. Endress, 2010. The social sense: susceptibly to others’ beliefs in human infants and adults. Science, 330: 1830-1834. Krattiger, A.F., 1997. Insect resistance in crops: a case study of Bacillus thuringiensis (Bt) and its transfer to developing countries. ISAAA Briefs No. 2. ISAAA: Ithaca, NY. Pp. 42. Kreutzweiser, D.P., S.B. Holmes, S.S. Capell and D.C. Eichenberg, 1992. Lethal and sublethal effects of Bacillus thuringiensis var. kurstaki on aquatic insects in laboratory bioassays and outdoor stream channels. Bull. Environ. Contam. Toxicol., 49(2): 252-258. Kryukov, V.Y., V.P. Khodyrev, O.N. Yaroslavtseva, A.S. Kamenova, B.A. Duisembekov and V.V. Glupov, 2009. Synergistic action of entomopathogenic hyphomycetes and the bacteria Bacillus thuringiensis sp. morrisoni in the infection of colorado potato beetle Leptinotarsa decemlineata. Appl. Bioch. Microbiol., 45: 511-516. Kubilay, M.E.R., H. Tunaz, A.A. Isikber, S. Satar, C. Mart and N. Uygun, 2008. Pathogenicity of entomopathogenic fungi to Coccinella septempunctata L. (Col.: Coccinellidae) and a survey of fungal diseases of coccinellids. KSU J. Sci. Eng., 11(1): 118-122. Kumar, K., K.S. Yashaswini and N. Earanna, 2013. Molecular characterization of Lepidopteran specific Bacillus thuringiensis strains isolated from hilly zone soils of Karnataka, India. Afri. J. Biotech., 2(20): 2924-2931. Kurian, C. and K. Mathen, 1971. Red palm weevil Hidden enemy of coconut palm. Ind. Farmi., 21: 29-31. Lacey, L.A. and D.I. Shapiro-Ilan, 2008. Microbial control of insect pests in temperate orchard systems: Potential for incorporation into IPM. Ann. Rev. Entomol., 53: 121-144. Lacey, L.A. and M.S. Goettel, 1995. Current developments in microbial control of insect pests and prospects for the early 21st century. Entomophaga, 40: 3-27. Lacey, L.A., A.A. Kirk, L. Millar, G. Mereadier and C. Vidal, 1999. Ovicidal and larvicidal activity of conidia and blastospores of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) against Bemisia argentifolii (Homoptera: Aleyrodidae) with a description of a bioassay system allowing prolonged survival of control insects. Biocontrol Sci. Technol., 9: 9-18. Lacey, L.A., R. Frutos, H.K. Kaya and P. Vail, 2001. Insect pathogens as biological control agents: Do they have a future? Biol. Control, 21: 230-248. Lacey, L.A. and H.K. Kaya, 2007. Field Manual of Techniques in Invertebrate Pathology: Application and Evaluation of Pathogens for Control of Insects and Other Invertebrate Pests. (Springer, The Netherlands). Lawo, N.C., R.J. Mahon, R.J. Milner, B.K. Sarmah, T.J.V. Higgins and J. Romeis, 2008. Effectiveness of Bacillus thuringiensis-transgenic chickpeas and the entomopathogenic fungus Metarhizium anisopliae in controlling Helicoverpa armigera (Lepidoptera: Noctuidae). Appl. Environ. Microbiol., 74(14): 4381-4389. Lee, D.R., 1963. Date cultivation in the Coachella Valley California. The Ohio J. Sci., 63(2): 82- 87. Lefroy, H.M., 1906. More Important Insects Injurious to Indian Agriculture, Govt. Press, Calcutta. Lewis, L.C., E.C. Berry, J.J. Obrycki and L.A. Bing, 1996. Aptness of insecticides (Bacillus thuringiensis and carbofuran) with endophytic Beauveria bassiana, in suppressing the larval populations of the European corn borer. Agri. Ecosy. Environ., 57: 27-34.

Lewis, R.S., N.Y. Weekes and T.H.Y. Wang, 2006. The relationship among a naturalistic stressor, frontal asymmetry, stress, and health, Manuscript submitted for publication. Llácer, E., M. Negre and J.A. Jacas, 2012. Evaluation of an oil dispersion formulation of Imidacloprid against Rhynchophorus ferrugineus (Coleoptera, Curculionidae) in young palm trees. Pest Manag. Sci., 68(6): 878-882. Llácer, E., M.M. Martínez de Altube and J.A. Jacas, 2009. Evaluation of the efficacy of Steinernema carpocapsae in a chitosan formulation against the red palm weevil, Rhynchophorus ferrugineus, in Phoenix canariensis. BioControl, 54(4): 559-565. Lord, J.C., 2009. Beauveria basssiana infection of eggs of stored-product beetles. Entomol. Res., 39: 155-157. MacIntosh, S.C., T.B. Stone, S.R. Sims, P.L. Hunst, J.T. Greenplate, P.G. Marrone, F.J. Perlak, D.A. Fischhoff and R.L. Fuchs, 1990. Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects. J. Inver. Pathol., 56: 258- 266. MacLeod, D.M., 1954. Investigations on the genera Beauveria Vuill. and Tritirachium Limber. Canad. J. Bot., 32: 818-890. Mahmoud, M.F., 2009. Pathogenicity of three commercial products of entomopathogenic fungi, Beauveria bassiana, Metarhizum anisopliae and Lecanicillium lecanii against adults of olive fly, Bactrocera oleae (Gmelin) (Diptera: Tephritidae) in the laboratory. Plant Prot. Sci., 45(3): 98-102. Malumphy, C. and H. Moran, 2009. Red palm Weevil, Rhynchophorus ferrugineus. Plant Pest Factsheet. Available online at: www.fera.defra.gov.uk/plants/publications/ documents/factsheets/redPalmWeevil.pdf. Accessed on 7 June 2011. Manachini, B., D. Schillaci and V. Arizza, 2013. Biological responses of Rhynchophorus ferrugineus (Coleoptera: Curculionidae) to Steinernema carpocapsae (Nematoda: Steinernematidae). J. Econ. Entomol., 106: 582-1589. Manachini, B., P. Lo-Bue, E. Peri and S. Colazza, 2009. Potential effects of Bacillus thuringiensis against adults and older larvae of Rhynchophorus ferrugineus. IOBC/WPRS Bull., 45: 239-242. Manachini, B., V. Arizza and N. Parrinello, 2008a. Sistema immunitario del Punteruolo Rosso (Rhynchophorous ferrugineus). In: La ricerca scientifica sul punteruolo rosso e gli altri fitofagi delle palme in Sicilia, Regione Siciliana-Assessorato Agricoltura e Foreste Dipartimento Interventi Infrastrutturali, Servizi allo Sviluppo. Pp. 133-136. Manachini, B., V. Arizza, D. Parrinello and N. Parrinello, 2011. Hemocytes of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces cerevisiae and Bacillus thuringiensis. J. Inver. Pathol., 106(3): 360-365. Manachini, B., V. Mansueto, V. Arizza and N. Parrinello, 2008b. Preliminary results on the interaction between Bacillus thuringiensis and red palm weevil. In: 41st Annual Meeting of Society for Invertebrate Pathology and 9th International Conference on Bacillus thuringiensis, Warwick, UK. Pp. 45. Manjula, K. and K. Padmavathamma, 1996. Effect of microbial insecticides on the control of Maruca testulalis and on the predators of redgram pest complex. Entomon., 21: 269-271. Marshall, J., 1931. Mohenjo-Daro and the Indus Civilization. Arthur Probsthain, London. Matsuura, H., 1993. Weevils associated with palms. Kobe Plant Prot., 901: 46-47.

Mazza, G., A. Cini, R. Cervo and S. Longo, 2011b. Just phoresy? reduced lifespan in red palm weevils Rhynchophorus ferrugineus (Coleoptera: Curculionidae) infested by the mite Centrouropoda almerodai (Uroactiniinae: Uropodina). Ital. J. Zool., 78: 101-105. Mazza, G., V. Arizza, D. Baracchi, G.P. Barzanti, C. Benvenuti, V. Francardi, A. Frandi, F. Gherardi, S. Longo, B. Manachini, B. Perito, P. Rumine, D. Schillaci, S. Turillazzi and R. Cervo, 2011a. Antimicrobial activity of the red palm weevil Rhynchophorus ferrugineus. Bull. Insectol., 64: 33-41. McCoy, C.W., R.A. Samson and D.G. Boucias, 1988. Entomopathogenic fungi. In: Ignoffo, C.M. (Ed.). CRC Handbook of Natural pesticides; Vol. 5-Microbial Insecticides: Part A- Entomopathogenic Protozoa and Fungi. CRC, Press, Boca Raton, FL. Pp.151-243. McGaughey, W.M. and M.E. Whalon, 1992. Managing insect resistance to Bacillus thuringiensis toxins. Science, 258: 1451-1455. Meadows, M. P. 1993. Bacillus thuringiensis in the environment: ecology and risk assessment. In: Entwistle, P.F., J.S. Cory, M.J. Bailey and S. Higgs (Eds.). Bacillusthuringiensis, an Environmental Biopesticide: Theory and Practice. John Wiley and Sons, New York. Pp. 193-220. Melifronidou-Pantelidou, A., 2009. Eradication campaign for Rhynchophorus ferrugineus in Cyprus. Bull. OEPP., 39(2): 155-160. Merghem, A., 2011. Susceptibility of the red palm weevil, Rhynchophorus ferrugineus (Olivier) to the green muscardine fungus, Metarhizium anisopliae (Metsch.) in the laboratory and in palm tree orchards. Egyp. J. Biol. Pest Control, 21: 179-183. Merghem, A., 2011. Susceptibility of the red palm weevil, Rhynchophorus ferrugineus (Olivier) to the green muscardine fungus, Metarhizium anisopliae (Metsch.) in the laboratory and in palm tree orchards. Egyp. J. Biol. Pest Control, 21: 179-183. Meyling, N.V. and J. Eilenberg, 2006. Occurrence and distribution of soil borne entomopathogenic fungi within a single organic agroecosystem. Agri. Ecosys. Environ., 113: 336-341. Mietkiewski, R.T., J.K. Pell and S.J. Clark, 1997. Influence of pesticide use on the natural occurrence of entomopathogenic fungi in arable soils in the UK: field and laboratory comparisons. Biocontrol Sci., Tech., 7: 565-575. Milne, D., 1918. The Date Palm and Its Cultivation in the Punjab. The Punjab Government. Pp. 153. Milne, R. and H. Kaplan, 1993. Purification and characterization of a trypsin like digestive enzyme from spruce budworm (Christoneura fumiferana) responsible for the activation of δ-endotoxin from Bacillus thuringiensis. Insect Biochem. Molecul. Biol., 23: 663-673. Milner, R.J., 1989. Ecological considerations in the use of Metarhizium for control of soildwelling pests. In: Robertson L.N. and P.G. Allsopp (Eds.). Proceedings of a Soilinvertebrate Workshop, Queensland Department of Primary Industries Conference and Workshop series QC 89004, Indooroopilly, Queensland. Pp. 10-13. Milner, R.J., 1997. Insect pathogens-how effective are they against soil insect pests? In: Allsopp, P.G., D.J. Rogers and L.N. Robertson (Eds.). Soil Invertebrates in 1997. Proceedings of the 3rd Brisbane Workshop on Soil Invertebrates. Bureau of Sugar Experiment Station, Brisbane Paddington, Australia. Pp. 63-67. Mitani, K. and J. Watari, 1916. A new method to isolate the toxin of Bacillus sotto Ishiwata by passing through a bacerial filter and a preliminary report on the toxic action of this toxin to the silkworm larvae. Archi. Gensanshu Serzojo Hokoku, 3: 33-42.

Mochizuki, M., A. Kawanishi, H. Sakamoto, S. Tashiro, R. Fujimoto and M. Ohwaki, 1993. A calicivirus isolated from a dog with fatal diarrhoea. Veter. Record, 132: 221-222. Mohan, L.M., 1917. Rept. Asst. Prof. Entomol; Rept. D Sagr. Punjab, for the year ended 30th June, 1917. Molina, A.J.P., R.I. Samuels, I.R. Machado and C. Dolinski, 2007. Interactions between isolates of the entomopathogenic fungus Metarhizium anisopliae and the entomopathogenic nematode Heterorhabditis bacteriophora JPM4 during infection of the sugar cane borer Diatraea saccharalis (Lepidoptera: Pyralidae). J. Inver. Pathol., 96(2):187-92. Mollier, P., J. Lagnel, B. Fournet, A. Aïoun and G. Riba, 1994. A Glycoprotein highly toxic for Galleria melonela larvae secreted by the entomopathogenic fungus Beauveria sulfurecens. J. Inver. Pathol., 64: 200-207. Mommaerts, V., K. Jans and G. Smagghe, 2010. Impact of Bacillus thuringiensis strains on survival, reproduction and foraging behaviour in bumblebees (Bombus terrestris). Pest Manag. Sci., 66(5): 520-525. Monzer, A.E. and R. Abd El-Rahman, 2003. Effect on Heterorhabditis indica of substance occurring in decomposing palm tissues infested by Rynchophorus ferrugineus. Nematol., 5(5): 647-657. Monzer, M.A., 2004. Response of Heterorhabditis indica infective juveniles to host diffusates in a modified laboratory bioassay. Egyp. J. Biol. Pest Control, 14: 309-313. Morton, A. and F. Garcia-Del-Pino, 2011. Possible interaction of the phoretic mite Centrouropoda almerodai on the control of Rhynchophorus ferrugineus by entomopathogenic nematodes. IOBC-WPRS Bull., 66: 363-366. Müller-Kögler, E., 1965. Pilzkrankheiten bei Insekten. Paul Parey, Berlin. Pp. 186. Murphy, S.T. and B.R. Briscoe, 1999. The red palm weevil as an alien invasive: Biology and the prospects for biological control as a component of IPM. Biocontrol New. Info., 20(1): 35- 45. Mwamburi, L.A., M.D. Laing and R. Miller, 2009. Interaction between Beauveria bassiana and Bacillus thuringiensis var. israelensis for the control of house fly larvae and adults in poultry houses. Poult. Sci., 88: 2307-2314. Nägeli, K.W., 1857. Uber die neue Krankheit der Seidenraupe und verwandte Organismen. Bot. Z., 15: 760-761. Namatame, I., H. Tomoda, N. Tabata, S.Y. Si, S. Omura, 1999. Structure elucidation of fungal Beauveriolide-III, a novel inhibitor of lipid droplet formation in mouse macrophages. J. Antibiot., 52: 7-12. Nardi, S., E. Ricci, R. Lozzi, F. Marozzi, E. Ladurner, F. Chiabrando, L. Granchelli, E. Verdolini, N. Isidoro and P. Riolo, 2011. Control of Rhynchophorus ferrugineus (Olivier, 1790) according to EU Decision 2007/365/EC in the Marche region (Central-Eastern Italy) Bull. OEPP., 41(2): 103-115. Nassar, M., and M.A. Abdllahi, 2001. Evaluation of Azadiractin fo the control of red palm weevil, Rhynchophorus ferrugineus (Oliever) (Curculionidae: Coleoptera) J. Egypt., 6: 163-173. Nester, E.W., L.S. Thomashow, M. Metz and M. Girdon, 2002. 100 years of Bacillus thuringiensis: a critical scientific assessment. American Society for Microbiology, Washington DC, USA. Pp. 1-22. Nirula, K.K., 1956. Investigations on the pests of coconut palm. Part. Rhynchophorus ferrugineus. Ind. Coc. J., 9: 229-247.

Nixon, R.W., 1951. The date palm: ‘‘Tree of Life’’ in the subtropical deserts. Econ. Bot., 5: 274- 301. Oehlschlager, A.C., C.M. Chinchilla and L.M. Gonzalez, 1993. Optimization of a pheromone- based trap for the American palm weevil, Rhynchophorus palmarum. In: Proceedings, International Oil Palm Congress, 20-25 September 1993. Palm Oil Research Institute of Malaysia, Kuala Lumpur, Malaysia. OEPP/EPPO, 2005. Data sheets on quarantine pests Rhynchophorus palmarum. EPPO Bull., 35: 468-471. Onofre, S.B., C.M. Miniuk, N.M. Barros and J.L. Azevedo, 2001. Pathogenicity of four strains of entomopathogenic fungi against the bovine tick Boophilus microplus. Amer. J. Vet. Res., 63: 1478-1480. Oreste, M., F. De Luca, E. Fanelli, A. Troccoli and E. Tarasco, 2013. New nematodes associated to Rhynchophorus ferrugineus (Coleoptera: Curculionidae): preliminary description. IOBC-WPRS Bull., 90: 271. Ortiz-Urquiza, A. and N.O. Keyhani, 2013. Action on the surface: Entomopathogenic fungi versus the insect cuticle. Insects, 4: 357-374. Ovchinnikov, Yu. A., A.A. Kiryushkin and I.V. Kozhevnikova, 1971. Gen. Chem. USSR., 41: 2105-2116. Padmavathi, J., K.U. Devi and C.U.M. Rao, 2003. The optimum and tolerance PH range is correlated to colonial morphology in isolates of the entomopathogenic fungus Beauveria bassiana, a potential biopesticide. World J. Microbiol. Biotech., 19: 469-477. Paoli, F., R. Dallai, M. Cristofaro, S. Arnone, V. Francardi and P.F. Roversi, 2014. Morphology of the male reproductive system, sperm ultrastructure and cirradiation of the red palm weevil Rhynchophorus ferrugineus Oliv. (Coleoptera: Dryophthoridae). Tissue and Cell, 46 (4): 274-285. Pasha, S.A., A. Hussain and I.B. Gajani, 1972. Date Palm of Sindh. Punjab Fruit. J., 33(4): 9-14. Pasteur, L., 1874. Observations (au sujet des conclusions de M. Dumas) relatives au phylloxera. Comptes rendus hebdomadaires des séances de l’Académie des Sci., 79: 1233-1234. Pell, J.K. and J.D. Vandenberg, 2002. Interactions among the aphid Diuraphis noxia, the entomopathogenic fungus Paecilomyces fomosoroseus and the coccinellid Hippodamia convergens. Biocontrol Sci. Technol., 12: 217-224. Peng, D.H., X.H. Xu, L.F. Ruan, Z.N. Yu and M. Sun, 2010. Enhancing Cry1Ac toxicity by expression of the Helicoverpa armigera cadherin fragment in Bacillus thuringiensis. Research in Microbiol., 161(5): 383-389. Pereira, E.J.G., H.A.A. Siqueira, M. Zhuang, N.P. Storer and B.D. Siegfried, 2010. Measurements of Cry1F binding and activity of luminal gut proteases in susceptible and Cry1F resistant Ostrinia nubilalis larvae (Lepidoptera: Crambidae). J. Inver. Pathol., 103(1): 1-7. Pérez, L., N. André, C. Gutleben, J. Vendeville, A.I. Lacordaire, A. Maury and E. Chapin, 2010. Palmier, efficacité curative du nématode Steinernema carpocapsae contre le papillon palmivore Paysandisia archon: résultats d'essais conduits dans des jardins et espaces verts. Phytoma La Défense des Végétaux, 637: 14-17. Pérez-García, G., R. Basurto-Ríos and J.E. Ibarra, 2010. Potential effect of a putative σHdriven promoter on the over expression of the Cry1Ac toxin of Bacillus thuringiensis. J. Inver. Pathol., 104(2): 140-146.

Peterson, A.T., J. Soberón and V. Sánchez-Cordero, 1999. Conservatism of ecological niches in evolutionary time. Science, 285: 1265-1267. Peveling, R. and S.A. Demba, 1997. Virulence of the entomopathogenic fungus Metarhizium flavoviride Gams and Rozsypal and toxicity of diflubenzuron, fenitrothionesfenvalerate and profenofos-cypermethrin to nontarget arthropods in mauritania. Archi. Environ. Contami. Toxicol., 32: 69-79. Plattner, R.D. and P.E. Nelson, 1994. Production of beauvencin by a stram of Fusarium proliferatum Isolated from corn fodder for Swine. Applied and Environmental MicrobIology, 60(10): 3894-3896. Poinar Jr., G.O., 1990. Taxonomy and biology of Steinernematidae and Heterorhabditidae. In: Gaugler, R. and H.K. Kaya (Eds.). Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, FL. Pp. 23-61. Poinar, Jr. G.O., 1969. Praecocilenchus rhaphidophorus n. gen., n. sp. (Nematoda: Aphelenchoidea) parasitizing Rhynchophorus bilineatus (Montrouzier) (Coleoptera: Curculionidae) in New Britain. J. Inver. Pathol., 1: 227-231. Popenoe, P., 1924. ‘The date palm in antiquity’. Sci. Month., 19: 313-325. Poprawski, T.J. and l. Majchrowicz, 1995. Effects of herbicides on in vitro vegetative growth and sporulation of entomopathogenic fungi. Crop Prot., 14(1): 81-87. Porter, J.R., 1973. Agostino Bassi bicentennial (1773-1973). Bacteriol. Rev., 37: 284-288. Prabhu, S.T. and R.S. Patil, 2009. Studies on the biological aspects of red palm weevil, Rhynchophorus ferrugineus (Oliv.), Karnataka J. Agri. Sci., 22: 732-733. Prior, C. and M. Arura, 1985. The infectivity of Metarhizium anisopliae to two insect pests of coconuts. J. Inver. Pathol., 45: 187-194. Purwar, J.P. and G.C. Sachan, 2006. Synergistic effect of entomogenous fungi on some insecticides against Bihar hairy caterpillar, Spilarctia obliqua (Lepidoptera: Arctiidae). Microbiol. Res., 161: 38-42. Qasim, M. and S.A. Naqvi, 2012. A fruit from heaven. In: Manickavasagan, A., M.M. Essa and E. Sukumar (Eds.). Dates: production, processing, food, and medicinal values, CRC Press, Taylor & Francis Group, 6000 Broken Sound Parkway NW, Suite 300, Boca Raton. Pp. 341-349. Quesada-Moraga, E., I. Martín-Carballo, I. Garrido-Jurado and C. Santiago-Álvarez, 2008. Horizontal transmission of Metarhizium anisopliae among laboratory populations of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Biol. Control, 47: 115-124. Quesada-Moraga, E., R. Santos-Quiros, P. Valverde-Garcia and C. Santiago-Álvarez, 2004. Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (anamorphic fungi) on the German cockroach (Blattodea: Blattellidae). J. Inver. Pathol., 87: 51-58. Rahalkar, G.W., M.R. Harwalkar and H.D. Rananvare, 1972. Development of red palm weevil, Rhynchophorus ferrugineus Oliv. on sugarcane. Ind. J. Ent., 34: 213-215. Rajagopal, R., N. Arora, S. Sivakumar, N.G.V. Rao, S.A. Nimbalkar and R.K. Bhatnagar, 2009. Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus thuringiensis is due to improper processing of the protoxin. Biochem. J., 419: 309-316. Rajamanickam K., J.S. Kennedy and A. Christopher, 1995.Certain components of integrated management for red palm weevil, Rhynchophorus ferrugineus F. (Curculionidae: Coleoptera) on coconut. Mededelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen Universiteit Gent., 60: 803-805.

Ramachandran, C.P., 1998. Biotypic variability among four populations of red palm weevil, Rhynchophorus ferrugineus Oliv. from different parts of India. Coc. Res. Develop. (CORD), 14: 26-41. Ramachandran, C.P.,1991. Effect of gamma radiation on various stages of red palm weevil, Rhynchophorus ferrugineus F. J. Nuclear Agri. and Biol., 20: 218-221. Ramathilaga, A., A.G. Murugesan and C. Sathesh Prabu, 2012. Biolarvicidal activity of Peanibacillus macerans and Bacillus subtilis isolated from the dead larvae against Aedes aegypti. Vector for Chikungunya. Proc. Inter. Acad. Ecol. Environ. Sci., 2(2): 90-95. Rao, P.N. and Y.N. Reddy, 1980. Description of a new nematode Praecocilienchus ferruginophorus n. sp. from weevil pests (Coleoptera) of coconut palms in South India. Rivista di Parassitologia, 44: 93-98. Rath, A.C., 1992. Metarhizium anisopliae for control of the Tasmanian pasture scarab Adoryphorus couloni. In: T.A. Jackson and T.R. Glare (Eds.). Use of Pathogens in Scarab Pest Management. Intercept, Andover. Pp. 217-227. Reddy, N.P., A.P.A Khan, U.D. Koduru, S.V. John and H.C. Sharma, 2008. Assessment of the suitability of Tinopal as an enhancing adjuvant in formulations of the insect pathogenic fungus Behauveria bassiana (Bals.) Vuillemin. J. Pest Manag. Sci., 3: 15-19. Reid, T.W. and I.B. Wilson, 1971. Enzymes. 3rd Ed. 4: 373-415. Riad, M., 2006. ‘The date palm sector in Egypt’. CIHEAM- Options Mediterraneennes. Pp. 45- 53. Riba, G. and C. Silvy, 1989. Combattre les ravageurs des cultures enjeux et perspectives, INRA, Paris. Ricaño, J., B. Güerri-Agulló, M.J. Serna-Sarriás, G. Rubio-Llorca, L. Asensio, P. Barranco and L.V. Lopez-Llorca, 2013. Evaluation of the pathogenicity of multiple isolates of Beauveria bassiana (Hypocreales: Clavicipitaceae) on Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) for the assessment of a solid formulation under simulated field conditions. Florida Entomol., 96(4): 1311-1324. Roberts, D.W. and A.S. Campbell, 1977. Stability of entomopathogenic fungi. Miscellaneous Publications of the Entomol. Soc. Ameri., 10: 19-76. Roberts, D.W., 1989. World picture of biological control of insects by fungi. Memoirs Institute Oswaldo Cruz Rio de Janeiro, 84: 89-100. Roh, J.Y., Y.S. Kim, Y. Wang, Q. Liu, X. Tao, H.G. Xu, H.J. Shim, J.Y. Choi, K.S. Lee, B.R. Jin and Y.H. Je, 2010. Expression of Bacillus thuringiensis mosquitocidal toxin in an antimicrobial Bacillus brevis strain. J. Asia-Pacific Entomol., 13(1): 61-64. Ruiu, L., 2013. Brevibacillus laterosporus, a Pathogen of Invertebrates and a Broad-Spectrum Antimicrobial Species. Insects, 4: 476-492. Sabbour, M. and M. Abdel-Raheem, 2014. Evaluations of Isaria fumosorosea isolates against the Red Palm Weevil Rhynchophorus ferrugineus under laboratory and field conditions. Curr. Sci. Inter., 3(3): 179-185. Sabbour, M.M. and A.F. Sahab, 2005. Efficacy of some microbial control agents against cabbage pest in Egypt. Pak. J. Biol. Sci., 8(10): 1351-1356. Sabbour, M.M. and N.Y. Solieman, 2014. Preliminary investigations into the biological control of Red Palm Weevil Rhynchophorus ferrugineus by using three isolates of the fungus Lecanicillium (Verticillium) lecanii in Egypt. Inter. J. Sci. Res., 3(8): 2016-2066.

Salama, H., M. Hamdy and M. Magd El-Din, 2002. The thermal constant for timing the emergence of the red palm weevil, Rhynchophorus ferrugineus (Oliv.), (Coleoptera, Curculionidae). J. Pest Sci., 75: 26-29. Salama, H.S. and M.M. Abd-Elgawad, 2001. Isolation of heterorhabditid nematodes from palm tree planted areas and their implications in the red palm weevil control. Anzeiger für Schädlingskunde, 74: 43-45. Salama, H.S. and M.M. Saker, 2002. DNA fingerprints of three different forms of red palm weevil collected from Egyptian date palm orchards. Archi. Phytopathol. Plant Prot., 35: 299-306. Salama, H.S., M.S. Foda, M.A. El-Bendary and A. Abdel-Razek, 2004. Infection of red palm weevil Rhynchophorus ferrugineus, by spore-forming bacilli indigenous to its natural habitat in Egypt. J. Pest Sci., 77: 27-31. Salamitou, S., F. Ramisse, M. Brehelin, D. Bourguet, N. Gilois, M. Gominet, E. Hernandez and D. Lereclus, 2000. The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects. Microbiol., 146(11): 2825- 2832. Saleh, M.M.E. and M. Alheji, 2003. Biological control of red palm weevil with entomopathogenic nematodes in the eastern province of Saudi Arabia. Egyp. J. Biol. Pest Control, 13: 55-59. Saleh, M.M.E., M.A. Alheji, M.H. Alkhazal, H. Alferdan and A. Darwish, 2011. Evaluation of Steinernema sp. SA a native isolate from Saudi Arabia for controlling adults of the red palm weevil. Rhynchophorus ferrugineus (Oliver). Egyp. J. Biol. Pest Control, 21: 277- 282. Sample, B.E., L. Butler, C. Zivkovich, R.C. Whitmore and R. Reardon, 1996. Effects of Bacillus thuringiensis Berliner var. kurstaki and defoliation by the gypsy moth [Lymantria dispar L. (Lepidoptera: Lymantriidae)] on native arthropods in West Virginia. Canad. Entomol., 128: 573-592. Samson, R.A., H.C. Evans and J.P. Latg, 1988. Atlas of entomopathogenic fungi. Springer, Berlin Heidelberg New York. Samuels, R.I., A.K. Charnley and S.E. Reynolds, 1988. The role of destruxins in the pathogenicity of 3 strains of Metarhizium anisopliae for the tobacco hornworm Manduca sexta. Mycopathol., 104: 51-58. Santhi, V.S., L. Salame, Y. Nakache, H. Koltai, V. Soroker and I. Glazer, 2015. Attraction of entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora to the red palm weevil (Rhynchophorus ferrugineus). Biol. Control, 83: 75-81. Sayyed, A.H., B. Raymond, M.S. Ibiza-Palacios, B. Escriche and D.J. Wright, 2004. Genetic and biochemical characterization of field-evolved resistance to Bacillus thuringiensis toxin Cry1Ac in the diamondback moth, Plutella xylostella. Appl. Environ. Microbiol., 70(12): 7010-7017. Schnepf, E., N. Crickmore, J.V. Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Zeigler and D.H. Dean, 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Molecul. Biol. Rev., 62: 775-806. Screen, S.E. and R.J. St. Leger, 2000. Cloning, expression, and substrate specificity of a fungal chymotrypsin. Evidence for lateral gene transfer from an actinomycete bacterium. J. Biol. Chem., 275: 6689-6694.

Sewify, G.H., M.H. Belal and S.A. Al-Awash, 2009. Use of the entomopathogenic fungus, Beauveria bassiana for the biological control of the red palm weevil, Rhynchophorus ferrugineus Olivier. Egyp. J. Biol. Pest Control, 19(2):157-163. Shah, P.A. and M. S. Goettel, 1999. Directory of Microbial Control Products and Services, 2 nd edn. Division on Microbial Control. Society for Invertebrate Pathology, Division on Microbial Control, Gainesville, USA. Pp. 81. Shahid, M., S. Hameed, A. Imran, S. Ali and J.D. Van Elsas, 2012. Root colonization and growth promotion of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter sp. Fs-11. World J. Microbiol. Biotech., 28: 2749-2758. Shahina, F., J. Salma, G. Mehreen, M.I. Bhatti and K.A. Tabassum, 2009. Rearing of Rhynchophorus ferrugineus in laboratory and field conditions for carrying out various efficacy studies using EPNs. Pak. J. Nematol., 27: 219-228. Shaiju-Simon, R.K.K. and C. Gokulapalan, 2003. Occurrence of Beauveria sp. on red palm weevil, Rhynchophorus ferrugineus (Oliv.) of coconut. Insect Environ., 9: 66-67. Shamseldean, M.M. and A.A. Atwa, 2004. Virulence of Egyptian steinernematid nematodes used against the red palm weevil, Rhynchophorus ferrugineus (Oliv.). Egyp. J. Biol. Pest Control, 14: 135-140. Shamseldean, M.M., 2002. Laboratory trails and field applications of Egyptian and foreign entomopathogenic nematodes used against the red palm weevil Rhynchophorus ferrugineus Oliv., In: Proceedings of the First International Workshop on Entomopathogenic Nematodes, Sharm ElSheikh, Egypt. Pp. 57-68. Shapiro-Ilan, D.I., D.H. Gouge and A.M. Koppenhöfer, 2002. Factors affecting commercial success: case studies in cotton, turf and citrus. In: Gaugler, R. (Ed.). Entomopathogenic nematology. Wallingford: CABI Publishing. Pp. 333-355. Sharma, H.C., M.K. Dhillon and R. Arora, 2008. Effects of Bacillus thuringiensis deltaendotoxin-fed Helicoverpa armigera on the survival and development of the parasitoid Campoletis chlorideae. Entomol. Experi. et Appli., 126(1): 1-8. Sharma, P., V. Nain, S. Lakhanpaul and P.A. Kumar, 2010. Synergistic activity between Bacillus thuringiensis Cry1Ab and Cry1Ac toxins against maize stem borer (Chilo partellus Swinhoe). Lett. Appl. Microbiol., 51(1): 42-47. Shawir, M.S. and A.M. Al-Jabr, 2010. The infectivity of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to Rhynchophorus ferrugineus (Olivier) stages under laboratory conditions. Proceeding of the 4th International Date Palm Conference. Acta Horti., 882: 431-436. Shu, C., G. Yan, R. Wang, J. Zhang, S. Feng, D. Huang and F. Song, 2009. Characterization of a novel cry8 gene specific to Melolonthidae pests: Holotrichia oblita and Holotrichia parallela. Appl. Microbiol. Biotech., 84(4): 701-707. Silva-Filha, M.H., L. Regis, C. Nielsen-LeRoux and J.F. Charles, 1995. Lowlevel resistance to Bacillus sphaericus in a field-treated population of Culex quinquefasciatus (Diptera: Culicidae). J. Econ. Entomol., 88: 525-530. Simberloff, D., J.L. Martin, P. Genovesi, et al., 2013. Impacts of biological invasions: what’s what and the way forward. Trends Ecol. Evol., 28: 58-66. Sims, S.R., 1997. Host activity spectrum of the cryIIa Bacillus thuringiensis subsp. kurstaki protein: effects on Lepidoptera, Diptera, and non-target arthropods. Southwestern Entomol., 22: 395-404.

Singh, G., B. Sachdev, N. Sharma, R. Seth and R.K. Bhatnagar, 2010. Interaction of Bacillus thuringiensis vegetative insecticidal protein with ribosomal S2 protein triggers larvicidal activity in Spodoptera frugiperda. Appl. Environ. Microbiol., 76(21): 7202-7209. Sivasithamparam, K., 1998. Root cortex the final frontier for the biocontrol of root-rot with fungal antagonists: a case study on a sterile red fungus. Ann. Rev. Phytopathol., 36: 439- 452. Sivasupramaniam, S., G.P. Head, L. English, Y.J. Li and T.T. Vaughn, 2007. A global approach to resistance monitoring. J. Inver. Pathol., 95: 224-226. Soberón, M., 2005. Bacillus thuringiensis mechanisms and use. In: Comprehensive Molecular Insect Science. Elsevier BV, Amsterdam. Pp. 175-206. Soper, R.S., 1974. The genus massospore, entomopathogenic for cicadas, Part I, taxonomy of the genus mycotaxon, 1: 13-40. St. Leger, R.J., L. Joshi, M.J. Bidochka, N.W. Rizzo and D.W. Roberts, 1996. Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles. Appl. Environ. Microbiol., 62: 1257- 1264. St. Leger, R.J., R.M. Cooper, and A.K. Charnley, 1986. Cuticle degrading enzymes of entomopathogenic fungi: regulation of production of chitinolytic enzymes. J. Gen. Microbiol., 132: 1509-1517. Starnes, R.L., C.L., Liu and P.G., Marrone, 1993. History and future of microbial insecticides. Amer. Entomol., 38-40: 83-91. Steinhaus, E.A., 1949. Principals of Insect Pathology. Mc Graw Hill Book co. Inc., New York. Pp. 757. Steinhaus, E.A., 1956. Microbial control: The emergence of an idea. Hilgardia, 26: 107-160. Steinhaus, E.A., 1975. Disease in a Minor Cord. Ohio State Univ. Press, Columbus, OH. Stevens, C.B., D.A. Cameron and D.L. Stenkamp, 2011. Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure. BMC. Dev. Biol., 11: 51. Storey, G.K. and W.A. Gardner, 1987. Vertical movement of commercially formulated Beauveria bassiana conidia through four Georgia soil types. Environ. Entomol., 16: 178- 181. Strand, M.R., 2008. The insect cellular immune response. Insect Sci., 15: 1-14. Strasser, R.J., A. Srivastava and M. Tsimilli-Michael, 2000. The Fluorescence Transient As a Tool to Characterize and Screen Photosynthetic Samples. In: Yunus, M., U. Pathre and P. Mohanty (Eds.). Probing Photosynthesis: Mechanisms, Regulation and Adaptation. Taylor & Francis: London. Pp. 445-483. Sudhaus, W., K. Kiontke, K. and R.M. Giblin-Davis, 2011. Description of Caenorhabditis angaria n. sp. (Nematoda: Rhabditidae), an associate of sugarcane and palm weevils (Coleoptera: Curculionidae). Nematol., 13: 61-78. Suzuki, T., S. Tominaga, S. Standgaad and T. Nakamura, 1975. Fluorescein cineangiography of the pial microcirculation in the rat in acute angiotensin-induced hypertension. Blood Flow and Metabolism in the Brain. In: Harper, A.M. et al. (Eds.). Churchill Livingstone, Edinburgh, London and New York. Pp. 8-5. Tanada, Y. and H.K. Kaya, 1993. Insect pathology. Academic Press, London. Pp. 319-385.

Tapia, G., M.A. Ruiz and M.M. Téllez, 2011. Recommendations for a preventive strategy to control red palm weevil (Rhynchophorus ferrugineus, Olivier) based on the use of insecticides and entomopathogenic nematodes. OEPP/EPPO. Bull., 41: 136-141. Tarasco, E., F. Porcelli, M. Poliseno, E. Quesada-Moraga, C. Santiago Álvarez and O. Triggiani, 2008. Natural occurrence of entomopathogenic fungi infecting the red palm weevil Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera, Curculionidae) in Southern Italy. IOBC/WPRS Bull., 31: 195-197. Tefera, T. and K. Pringle, 2003. Germination, radial growth, and sporulation of Beauveria bassiana to Chilo partellus (Lepidoptera: Pyralidae) at different temperatures. Biocontrol Sci. Technol., 13: 699-704. Thakur, R. and S.S. Sandhu, 2010. Distribution, occurrence and natural invertebrate hosts of indigenous entomopathogenic fungi of Central India. Ind. J. Microbiol., 50(1): 89-96. Then, C., 2009. Risk assessment of toxins derived from Bacillus thuringiensis synergism, efficacy, and selectivity. Environ. Sci. Pollut. Res., 17(3): 791-797. Thungrabeab, M. and S. Tongma, 2007. Effect of entomopathogenic fungi, Beauveria bassiana (Balsam) and Metarhizuim anisopliae (Metsch). KMITL Sci. Technol., 7(1): Torta, L., V. Leone, G. Caldarella, G. Lo-Verde and S. Burruano, 2009. Microrganismi fungini associati a Rhynchophorus ferrugineus (Olivier) in Sicilia e valutazione dell’efficacia entomopatogena di un isolato di Beauveria bassiana (Bals.) Vuill, Ossevazioni preliminari. Micologia Italiana, 2: 49-56. Triggiani, O. and E. Tarasco, 2011. Evaluation of the autochthonous and commercial isolates of Steinernematidae and Heterorhabditidae on Rhynchophorus ferrugineus. Bull. Insectol., 64: 175-180. Triggiani, O. and P. Cravedi, 2011. Entomopathogenic nematodes. Redia, XCIV: 119-122. Ugine, T.A., S.P. Wraight and J.P. Sanderson, 2005. Acquisition of lethal doses of Beauveria bassiana conidia by western flower thrips, Frankliniella occidentalis, exposed to foliar spray residues of formulated and unformulated conidia. J. Inver. Pathol., 90: 10-23. Van-Driesche, R.G., R.I. Carruthers, T. Center, M.S. Hoddle, J. Hough-Goldstein, L. Morin, L. Smith, D.L. Wagner, et al. 2010.Classical biological control for the protection of natural ecosystems. Biol. Control, 54: 2-33. Vey, A., J.M. Quiot, I. Mazet and C.W. McCoy, 1993. Toxicity and pathology of crude broth filtrate produced by Hirsutella thompsonii var. thompsonii in shake culture. J. Inver. Pathol., 61:131-137. Vey, A., R. Hoagland and T.M. Butt, 2001. Toxic metabolites of fungal biocontrol agents. In: Butt, T.M., C. Jackson and N. Magan (Eds.). Fungal Biocontrol Agents: Progress Problem and Potential. CABI, Wallingford. Pp. 311-346. Vidal, C., J. Fargues and L.A. Lacey, 1997. Intraspecific variability of Paecilomyces fumosoroseus: effect of temperature on vegetative growth. J. Inver. Pathol., 70: 18-26. Vitale, A., V. Leone, L. Torta, S. Burruano and G. Polizzi, 2009. Prove preliminari di lotta biologica con Beauveria bassiana e Metarhizium anisopliae nei confronti del punteruolo rosso. In: La ricerca scientifica sul punteruolo rosso e gli altri fitofagi delle palme in Sicilia, Vol.1. Regione Siciliana, Assessorato Agricoltura e Foreste Dipartimento Interventi Infrastrutturali, Italy. Pp. 169-172. Wakil, W., M.U. Ghazanfar, T. Riasat, M.A. Qayyum, S. Ahmed and M. Yasin, 2013. Effects of interactions among Metarhizium anisopliae, Bacillus thuringiensis and

chlorantraniliprole on the mortality and pupation of six geographically distinct Helicoverpa armigera field populations. Phytoparasitica, 41(2): 221-234. Walter, C., M. Fladung and W. Boerjan, 2010. The 20-year environmental safety record of GM trees. Nature Biotech., 28(7): 656-658. Wattanapongsiri, A.L., 1966. Revision of the genera Rhynchophorus and Dynamis (Coleoptera: Curculionidae),. Bulletin 1, Department of Agriculture Science, Bangkok, Thailand. Pp. 328. Whaley, W.H., J. Anhold and G.B. Schaalje, 1998. Canyon drift and dispersion of Bacillus thuringiensis and its effects on select nontarget Lepidopterans in Utah. Environ. Entomol., 27: 539-548. Whalon, M.E. and B.A. Wingerd, 2003. Bt: mode of action and use. Arch. Insect Biochem. Physiol., 54: 200-211. Wolfersberger, M.C., 1989. Neither barium nor calcium prevents the inhibition by Bacillus thuringiensis S-endotoxin of sodiumor potassium-gradient-dependent amino acid accumulation by tobacco hornworm midgut brush border membrane vesicles, Arch. Insect Biochem. Physiol., 12: 267-277. Wraight, S.P. and M.E. Ramos, 2005. Synergistic interaction between Beauveria bassianaand Bacillus thuringiensis tenebrionis based biopesticides applied against field populations of Colorado potato beetle larvae. J. Inver. Pathol., 90: 139-150. Xu, L., Z. Wang, J. Zhang, K. He, N. Ferry and A.M.R. Gatehouse, 2010. Cross-resistance of Cry1Ab-selected Asian corn borer to other Cry toxins. J. Appl. Entomol., 134(5): 429- 438. Zhang, G.L., W.D. Fu and K. Liu, 2008. Agricultural invasive pest in China. Science Press, Beijing. Pp. 172. Zhong, C.H., D.J. Ellar, A. Bishop, C. Johnson, S.S. Lin and E.R. Hart, 2000. Characterization of a Bacillus thuringiensis delta-endotoxin which is toxic to insects in three orders. J. Inver. Pathol., 76(2): 131-139. Zimmermann, G., 1993. The entomopathogenic fungus Metarhizium anisopliae and its potential as a biocontrol agent. Pesti. Sci., 37: 375-379. Zimmermann, G., 2007. Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii. Biocontrol Sci. Tech., 17: 553-596.

CHAPTER 3

Genetic variation among populations of Red Palm Weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) from the Punjab and Khyber Pakhtunkhwa provinces of Pakistan

Abstract The red palm weevil (RPW) Rhynchophorus ferrugineus is a voracious pest of various palm species. In recent decades its geographic range has expanded greatly, particularly impacting the date palm industries in the countries of the Middle East. This has led to conjecture regarding the origins of invasive RPW populations. For example, in parts of the Middle East, RPW is commonly referred to as the “Pakistani weevil” in the belief that it originated there. We sought evidence to support or refute this belief. The first reports of the weevil in Pakistan were from the Punjab region in 1918, but it is unknown whether RPW is native or invasive there. We estimated genetic variation across 5 populations of RPW from the Punjab and Khyber Pakhtunkhwa provinces of Pakistan, using sequences of the mitochondrial cytochrome oxidase subunit I gene. Four haplotypes were detected, of which, two (H1, H5) were abundant (accounting for >88% of specimens) across all five sampled populations. There was no geographic overlap in the distribution of the remaining “rare” haplotypes (H51 and H52) which were restricted to three (Bahawalpur, Muzaffargarh and Dera Ismail Khan) and two (Dera Ghazi Khan and Layyah) populations respectively. Levels of mitochondrial haplotype diversity reported herein were much lower than those previously recorded in accepted parts of the native range of RPW, suggesting that the weevil may indeed be invasive in Pakistan. In a “global” analysis, the close affinity of Pakistani haplotypes to those reported from India (and of course the geographical proximity of the two countries), make the latter a likely “native” source. With regards the validity of the name “Pakistani weevil”, we found little genetic evidence to justify the name.

Key words: Rhynchophorus ferrugineus, Middle East, Pakistani weevil, Punjab, Khyber Pakhtunkhwa

3.1 Introduction The Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae: Rhynchophorinae) has been recognized as a major economically important pest of palm species for more than a century. It has been found devastating > 40 different commercial and ornamental palm tree species, belonging to 23 different genera and 3 families (Faleiro et al. 2012; Giblin-Davis et al., 2013). These include date palm Phoenix dactylifera L. (Mukhtar et al., 2011), oil palm Elaeis guineensis (Murphy and Briscoe, 1999), coconut Cocos nucifera (Faleiro, 2006) and Canary Island date palm P. canariensis, (El-Mergawy and Al-Ajlan, 2011). The larval stages of RPW typically reside within the trunk of an infested palm tree, destroying the vascular system and boring into the heart of the host. Voracious feeding by these larvae may subsequently lead to tree collapse (Ju et al., 2011). In favorable climates, the reproductive biology of RPW is such that an infestation of just a single female has the potential to turn into about ﬁve million weevils in just four generations (about one year) (Nirula, 1956; Rahalkar et al., 1972; Avand- Faghih, 1996; Esteban-Durán et al., 1998; Cabello, 2006). In India, yield loses of 10-25% have been reported in coconut plantations (Murphy and Brisco, 1999). In the Arabian Peninsula (an area that accounts for 30% of global date production), RPW is estimated to damage up to 5% of the date palm plantations, resulting in losses of 5-20 million US dollars (El-Sabea et al., 2009). In Pakistan, production losses of 10-20% have been reported in different varieties of dates (Baloch et al., 1992). Around the world, RPW is also variously referred to as the Asiatic palm weevil, coconut weevil, sago palm weevil, and red stripe weevil (although the actual specific identity of many reported populations, particularly those in SE Asia, is likely to be wrong; Rugman-Jones et al., 2013). Furthermore, because of the cryptic, internal nature of the beetle’s attack, and the resulting slow death of the palm tree, it has also been referred to as “the hidden enemy” and even date palm AIDS (Khamiss and Abdel-Badeea, 2013). In the Middle East, RPW is often referred to as the “Pakistani weevil” in the belief that it invaded the former from Pakistan. However, this is somewhat controversial nomenclature, since there is little empirical evidence supporting a causative link (Rugman-Jones et al., 2013). Furthermore, RPW is generally considered to be invasive in Pakistan, although it was first formerly reported in what are now the Multan, Muzaffargarh and Dera Ghazi Khan Districts of the Pakistani province of Punjab, and the neighboring Indian state of Punjab, almost a century ago (Lal, 1917; Milne, 1918). Wattanapongsiri (1966) has since defined the native range of RPW as an area stretching east from India throughout SE Asia (although its occurrence in Indonesia has recently been thrown into doubt; Rugman-Jones et al., 2013), and in his detailed revision of the genus Rhynchophorus, based on several extensive museum collections, he recorded only a single un-dated specimen from modern day Pakistan. However, it remains a possibility that RPW was “always” present in Pakistan. The objective of this study was to characterize genetic diversity within and between different RPW populations in Pakistan with the hope of identifying: 1) whether Pakistan forms part of the native range of RPW; or if not, 2) the most likely origin of Pakistani RPW populations; and finally, 3) whether there is any conclusive evidence that Middle Eastern populations of RPW originated from Pakistan. We used sequences of the mitochondrial cytochrome oxidase I (COI) gene to investigate genetic variation in RPW from 5 different geographically isolated populations in the Punjab and Khyber Pakhtunkhwa (KPK) provinces of Pakistan, and also compared these with publically available sequences from RPW populations from around the world. If Pakistan is part of the native range, we expect to find relatively high

62 levels of diversity (i.e. a large number of haplotypes; see Rugman-Jones et al., 2013). In contrast, relatively low levels of diversity are typical of invasive populations and by comparison with other populations, can be used to make inferences about their potential origins.

3.2 Materials and methods

3.2.1 Specimen collections During February and March, 2015, RPW were collected from five districts spread across the Punjab and KPK provinces of Pakistan (Table 3.1; Figure 3.1). Live insects were collected from infested or fallen date palm plants, with the permission of the orchard’s owners or farmers, and permission from the Director of the Regional Agricultural Research Institute (RARI) (Bahawalpur). A total of 80 RPW adults were collected (Table 3.1), stored collectively in plastic jars containing 95% ethanol (one per location), and maintained at -20 °C in the Microbial Control Laboratory, University of Agriculture, Faisalabad. At the end of March 2015, the ethanol-preserved insects were transported to the laboratory of Dr. Richard Stouthamer (University of California Riverside, USA [UCR]). During transport, the insects were not kept under controlled temperatures. At UCR each weevil was transferred to an individual plastic vial, labelled accordingly, and kept in the freezer at -20 °C until processing.

3.2.2 DNA extraction and amplification Each specimen was extracted using the protocol detailed in Rugman-Jones et al. (2013). Specifically, a small piece (2-5 mm3) of muscle tissue was dissected from a single tibia using flame-sterilized scissors and forceps, and allowed to air dry for 1 min on sterile filter paper. The tissue was then transferred to a sterile 0.6 ml microcentrifuge tube and ground up in 6 µl proteinase-K (>600mAU/mL; Qiagen, Valencia, CA) using a glass pestle. To this was added 120 µl of a 5% (w/v) suspension of Chelex® 100 resin (Bio-Rad Laboratories, Hercules, CA) and the reaction was incubated at 55 °C for 1 h followed by 10 min at 99 °C. After this the tubes were centrifuged for 4 minutes at 14000 rpm to pellet the Chelex. Subsequently, 80 µl of supernatant was carefully transferred to a new eppendorf tube. The polymerase chain reaction (PCR) was used to amplify a section of the mitochondrial gene (mtDNA) cytochrome oxidase subunit 1 (COI) from each specimen. PCR was performed in 25 µl reactions containing 2 µl of DNA template (concentration not determined), ddH2O,1X ThermoPol PCR Buffer (New England BioLabs, Ipswich, MA), an additional 1 mM MgCl2, 400 µM dUTP, 200 µM each dATP, dCTP and dGTP, 10 µg BSA (NEB), 1 U Taq polymerase (NEB), and 0.2 µM of each PCR primer. Initial reactions utilized the primers C1-J-1718 and C1- N-2329 (Simon et al., 1994). Reactions were performed in a Mastercycler® ep gradient S thermocycler (Eppendorf North America Inc., New York, NY) programmed for an initial denaturing step of 2 min at 94 °C; followed by five cycles of 30 s at 94 °C, 1 min 30 s at 45 °C, and 1 min at 72 °C; followed by a further 35 cycles of 30 s at 94 °C, 1 min 30 s at 51 °C, and 1 min at 72°C; and, a final extension of 5 min at 72 °C. Amplification was verified by standard agarose gel electrophoresis, and samples that failed to amplify were subject to two further attempted amplifications, this time using the primer sets SIMON and BRON (El-Mergawy et al., 2011) or BRON and C1-N-2329 (see Rugman-Jones et al., 2013). The integrity of the DNA extracted from any specimen that still failed to yield a COI amplicon was tested in a 4th PCR, this time targeting the 28S rRNA region with the primers (28sF3633 and 28sR4076) and protocol detailed in Rugman-Jones et al. (2013).

3.2.3 Cleaning and sequencing PCR products were purified using ExoSAP-IT® (Affymetrix, Santa Clara, CA), and sequenced in both directions at the Institute for Integrative Genome Biology core instrumentation facility (UCR). Sequences were aligned manually and trimmed to 528 bp (removing the primers and ambiguous tails) using BioEdit version 7.0.9.0 (Hall, 1999), and then translated using the EMBOSS-Transeq website (http://www.ebi.ac.uk/Tools/emboss/transeq/index.html) to confirm the absence of nuclear pseudogenes (Song et al., 2008). All sequences were deposited in GenBank (Benson et al., 2008) (accession numbers KU696489-KU696537).

3.2.4 Genetic analysis Sequences of the COI gene generated in this study were collapsed into haplotypes, and the number and nature of polymorphic sites was characterized, using DnaSP v5.10.01 (Librado and Rozas, 2009). Genetic variation within each Pakistani population (Table 3.2), was characterized by calculating the number of COI haplotypes, haplotype diversity (Hd; the probability that two randomly sampled haplotypes are different), and the average number of nucleotide differences in pairwise comparisons among COI sequences (k) using DnaSp. Since most estimators of population differentiation can be highly unreliable when using a single locus and relatively small sample sizes, genetic variation between topographical populations was also investigated simply by obtaining population-pairwise estimates of k, again in DnaSP. In order to put variation within Pakistan into a global context, we then combined our sequences with those from three earlier studies (El-Mergawy et al., 2011; Rugman-Jones et al., 2013; Wang et al., 2015; GenBank accessions GU581319-GU581628, KF311358-KF311740, KF413063- KF413073, respectively). All sequences were trimmed to a uniform length, resulting in a matrix of 539 sequences, each 528bp long. Sequences were again collapsed into haplotypes using DnaSP v5.10.01 and a “global” haplotype network was constructed using the statistical parsimony method of Templeton et al. (1992) in the software program TCS, version 1.21 (Clement et al., 2000).

3.3 Results Using various combinations of four PCR primers, sequences of the COI gene were successfully obtained from 50 of our 80 RPW specimens collected from the Punjab and KPK provinces of Pakistan. For the remaining 30 “failed” specimens, attempts to amplify the highly conserved 28S rRNA also failed, suggesting that our extractions from those specimens had yielded no amplifiable DNA. Among the 50 specimens successfully sequenced, we found four haplotypes. The four haplotypes were very closely related, with only three polymorphic nucleotides (positions 63, 156, and 174) (see GenBank accessions). All substitutions were synonymous. Just two haplotypes accounted for the majority (88%) of the specimens and corresponded to haplotypes H1 (n=20) and H5 (n=24) previously encountered by El-Mergawy et al. (2011) and Rugman-Jones et al. (2013). These haplotypes were common in all five Pakistani populations (Table 3.2; Figure 3.2). The remaining two haplotypes had restricted, and non- overlapping distributions, being found in three and two populations, and our “global” haplotype analysis revealed that neither had been encountered in the earlier studies of El-Mergawy et al. (2011), Rugman-Jones et al. (2013), or Wang et al. (2015). Hereafter they are referred to as H51 (n=4) and H52 (n=2), respectively. Given the very similar nature of the four Pakistani haplotypes, and the prevalence of just two of those haplotypes across the five Pakistani RPW populations, estimates of genetic

64 variation within and between populations were very low (k was <1 in all comparisons; Table 3.3). In global terms, the haplotypes detected in our Pakistani samples were most similar to native haplotypes from other parts of the Indian sub-continent; haplotypes H9-16 from Rugman- Jones et al. (2013) (Figure 3.3). Outside of the native range, the most common Pakistani haplotypes (H1 and H5) were also invasive in the United Arab Emirates, Oman, and Syria (Figure 3).

3.4 Discussion In the Middle East, RPW is often referred to as the “Pakistani weevil” in the belief that it invaded from Pakistan. However, strong empirical evidence to justify this belief has not been forthcoming (e.g., Rugman-Jones et al., 2013). Furthermore, there is some doubt as to the actual status (invasive or native) of RPW in Pakistan, although it has typically been considered an invasive pest there. In this study we found four haplotypes across 50 specimens, from five sampled populations located in the Pakistani provinces of Punjab and KPK. Of these haplotypes, two (H1 and H5) were common in all the populations sampled. The other two were relatively rare, and non-overlapping, with one (H51; Fig 3.3) represented by only four specimens (two from Bahawalpur and one form Muzaffargarh and DI Khan), and the other (H52; Fig 3.3) by two specimens (one from Layyah and another from DG Khan). In global terms, the four Pakistani haplotypes were very similar to native haplotypes in the remainder of the Indian sub-continent (Fig 3.3), and H1 and H5 were also common in invasive populations in parts of the Middle East (UAE, Oman, and Syria).

Is the red palm weevil native to Pakistan? Low levels of genetic diversity are atypical of RPW populations across its described native range, but very characteristic of invasive populations of this species around the globe (see Rugman-Jones et al., 2013). Levels of genetic diversity detected in our study lay somewhere between the two extremes. If we consider RPW to be an invasive, this suggests that the RPW populations in the Punjab and KPK provinces of Pakistan have resulted from: a) the influx of a large number of weevils during a single invasion event; and/or b) multiple invasions from one or more sources. The genetic similarity between our Pakistani haplotypes and those previously reported from the Indian state of Goa suggests that India would have been the most likely source of any invasion. This is also the most likely scenario in a biogeographical sense. Commercial cultivation of dates in India is focused largely in the western states of Gujarat, Rajasthan and Punjab, adjacent to the border with Pakistan, and (to a much lesser extent) the southernmost states of Tamil Nadu and Kerala. RPW is a strong flyer and it is easy to imagine that Pakistan may have been invaded by weevils from one or more of these Indian states. However, convincing support for such a hypothesis will require a much bigger sample from India, which sadly remains a genetic “black hole” since the country does not allow researchers to collect and export specimens, due to Indian claims of intellectual property rights over genetic resources. In contrast to the invasive “argument”, our results could also be interpreted as evidence that the Punjab and KPK provinces of Pakistan actually fall within the native range of the weevil. At least three known hosts of RPW are native to Pakistan [Nannorhops ritchiana, Phoenix loureirii and P. sylvestris (Champion et al., 1960; Mughal, 1992; Malumphy and Moran, 2007; Malik, 2015), and the date palm Phoenix dactylifera has been cultivated in the Sindh province of Pakistan for more than a thousand years (http://edu.par.com.pk/wiki/dates/).

Furthermore, the presence of RPW in the Punjab province of Pakistan, and what is now the neighboring Indian state of Punjab, was first documented almost a century ago (Lal, 1917; Milne, 1918). Therefore, we must consider the possibility that small populations of RPW have always been present in Pakistani Punjab, but have simply gone ignored, or unnoticed, because of the relative isolation of the region, and/or because their economic impact was (at that time) not significant. In light of this information, it is perhaps surprising that Wattanapongsiri (1966), in his revision of the genus Rhynchophorus, considered only a single R. ferrugineus specimen from anywhere in Pakistan (a specimen from the Kalat District of the modern Balochistan province, held in the Bavarian State Collection of Zoology, Munich). Prior to the Partition of India in 1947, the two “Punjabs” were considered a single province under the governance of the British Raj, and British collectors described vast numbers of insects from the entire Indian sub-continent (including Pakistan), depositing the bulk of their specimens at the British Museum of Natural History, London. Had RPW been abundant at that time, it seems unlikely that such a conspicuous insect would have escaped collection. However, despite having access to the BMNH collections (among many others), Wattanapongsiri (1966) included only the single “Balochistan” specimen, in his work. Unfortunately, that specimen was without a collection date, and so sheds little further light on the history of RPW in Pakistan. Whether native or invasive, RPW has certainly been present in Pakistan for some time. The recent “rise” of RPW in the Punjab and KPK provinces has likely been exacerbated by anthropogenic movement of date palm germplasm from the neighboring provinces of Sindh and Baluchistan where date palms have been cultivated for centuries, and/or the rise of date cultivation in neighboring Indian states. Again, this is difficult to substantiate without sampling of those areas, and that should be a priority for further genetic work.

The Pakistani weevil? It has been claimed by some Middle Eastern countries that RPW originally crossed into Arabia in ornamental plants imported from Pakistan in 1985 (Dawn News 2003). While our data cannot completely refute this hypothesis it cannot fully support it either. Although both of the abundant Pakistani haplotypes detected in our study have been recorded in the Middle Eastern countries of UAE and Oman (and H1 only also in Syria), a third haplotype H8, has not been detected in Pakistan, but was found to be widespread in Saudi Arabia (El Mergawy et al. 2011; Rugman-Jones et al., 2013). Indeed, El Mergawy et al. (2011) detected three further haplotypes from Oman and UAE. If the Middle East was invaded solely by RPW from Pakistan it is hard to explain why there are additional haplotypes in the Middle East that have not been detected in Pakistan. One answer, originally put forward by Rugman-Jones et al. (2013), is that whether or not the Middle East has been invaded from Pakistan, it has also been invaded from somewhere else in the native range of RPW (most likely Thailand). It should also be noted that RPW-like damage was recorded in Iraq around the same time RPW was first recorded in the Punjab, although no specimens were collected to confirm this (Buxton, 1920). There is currently no genetic data available for Iraqi populations of RPW, but given its relative proximity to the Middle East, it is possible that the latter (and indeed Pakistan) were invaded by RPW from Iraq. Intensive sampling of Iraq should be a priority for future genetic work.

Conclusions The present study showed that the red palm weevil is native to Pakistan and had been present in Pakistan for a long time. It may have been invaded from India and Sri Lanka. The population present in KSA is called the "Pakistani Weevil’’ but in my study I have indicated that this weevil might have been invaded from Thialand or Vietnam instead of Pakistan. In Pakistan four different groups of haplotypes are present which are commonly found in al the collection areas of the country. Only the haplotype H52 was the rare haplotype that was only present in the populations collected from Layyah and Dera Ghazi Khan districts of Punjab.

Acknowledgements This research work was supported by the scholarship from Higher Education Commission (HEC), Islamabad, Pakistan (112-30536-2AV1-263) under Indigenous Ph.D. Fellowship Program.

3.5 References Baloch, H.B., M.A. Rustamani, R.D. Khuhro, M.A. Talpur and T. Hussain, 1992. Incidence and abundance of date palm weevil in different cultivars of date palm. Proc. Pak. Cong. Zool., 12: 445-447. Benson, D.A., I. Karsch-Mizrachi, D.J. Lipman, J. Ostell and D.L. Wheeler, 2008. GenBank. Nucleic Acids Res., 36: 25-30. Buxton, P.A., 1920. Insect pests of dates and the date palm in Mesopotamia and elsewhere. Bull. Entomol. Res., 11: 287-304. Champion, H.G., S.K. KSeth and G.M. Khattak, 1960. Manual of silviculture for Pakistan. Clement, M., D. Posada and K.A. Crandall, 2000. TCS: a computer program to estimate gene genealogies. Mol. Ecol., 9: 1657-1660. DAWN News, 2003. Insect pests ravage red date palm trees. Available at: http://www.dawn.com/news/89129/insect-pests-ravage-red-date-palm-trees. El-Mergawy, R.A.A.M. and A.M. Al-Ajlan, 2011. Red palm weevil, Rhynchophorus ferrugineus (Olivier): economic importance, biology, biogeography and integrated pest management. J. Agric. Sci. Tech., 1: 1-23. El-Mergawy, R.A.A.M., M.I. Nasr, N. Abdallah and J.F. Silvain, 2011. Mitochondrial genetic variation and invasion history of red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae), in Middle-East and Mediterranean Basin. Inter. J. Agric. Biol., 13: 631-637. El-Sabea, A.M.R., J.R. Faleiro and M.M. Abo-El-Saad, 2009. The threat of red palm weevil Rhynchophorus ferrugineus to date plantations of the Gulf region in the Middle-East: an economic perspective. Outlooks on Pest Manag., 20: 131-134. Esteban-Durán, J., J.L. Yela, F. Beitia-Crespo and A. Jiménez-Alvarez, 1998. Biology of red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae: Rhynchophorinae), in the laboratory and field, life cycle, biological characteristics in its zone of introduction in Spain, biological method of detection and possible control. Boletin de Sanidad Vegetal Plagas, 24: 737-748. Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm during the last one hundred years. Inter. J. Trop. Insect Sci., 26: 135-154. Faleiro, J.R., A. Ben Abdullah, M. El-Bellaj, A.M. Al-Ajlan and A. Oihabi, 2012. Threat of red palm weevil, Rhynchophorus ferrugineus (Olivier) to date palm plantations in North Africa. Arab J. Plant Prot., 30: 274-280. Giblin-Davis, R.M., J.R. Faleiro, J.A. Jacas, J.E. Peña and P.S.P.V. Vidyasagar, 2013. Coleoptera: Biology and management of the red palm weevil, Rhynchophorus ferrugineus pp. 1-34 In: J.E. Peña (Ed.). Potential Invasive Pests of Agricultural Crop Species. CABI. Wallingford, UK. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Sym. Ser., 41: 95-98. Ju, R.T., F. Wang, F.H. Wan and B. Li, 2011. Effect of host plants on development and reproduction of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). J. Pest Sci., 84: 33-39. Khamiss, O. and A. Abdel Badeea, 2013. Initiation, characterization and karyotyping of a new cell line from red palm weevil Rhynchophorus ferrugineus adapted at 27 °C in AFPP - palm pest Mediterranean conference 16, 17 and 18 January 2013, NICE.

Lal, M.M., 1917. Report of assistant professor of entomology, department of agriculture Punjab for year ended 30th June 1917. Librado, P. and J. Rozas, 2009. DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics, 25: 1451-1452. Malik, K.A., 2015. Flora of Pakistan. Available at: http://www.efloras.org/florataxon.aspx?flora_id=5&taxon_id=20427. Malumphy, C. and H. Moran, 2007. Red palm weevil Rhynchophorus ferrugineus. Plant Pest Notice, Central Sci. Lab., 50: 1-3. Milne, D., 1918. The date palm and its cultivation in the Punjab. The Punjab government, Lyallpur. Pp. 153. Mughal, M.S., 1992. Spotlight on species: Nannorrhops ritchieana. Pak. J. For., 42: 162-166. Mukhtar, M., K.G. Rasool, M.P. Parrella, Q.I. Sheikh, A. Pain, L.V. Lopez-Llorca, Y.N. Aldryhim, R.W. Mankin and A.S. Aldawood, 2011. New initiatives for management of red palm weevil threats to historical Arabian data palms. Florida Entomol., 94: 733-736. Murphy, S.T. and B.R. Briscoe, 1999. The red palm weevil as an alien invasive: biology and the prospects for biological control as a component of IPM. Biocontrol News and Info., 20: 35-46. Nirula, K.K., 1956. Investigations on the pests of coconut palm. Part-IV. Rhynchophorus ferrugineus. Ind. Coc. J., 9: 229-247. Rahalkar, G.W., M.R. Harwalkar and H.D. Rananavare, 1972. Development of red palm weevil Rhynchophorus ferrugineus (Oliv.) on sugarcane. Ind. J. Entomol., 34: 213-215. Rugman-Jones, P.F., C.D. Hoddle, M.S. Hoddle and R. Stouthamer, 2013. The lesser of two weevils: molecular-genetics of pest palm weevil populations confirm Rhynchophorus vulneraturs (Panzer 1798) as a valid species distinct from R. ferrugineus (Olivier 1790), and reveal the global extent of both. PLoS ONE, 8: 1-15. Simon, C., F. Frait, A. Bechenback, B. Crespi, H. Liu and P.K. Flook, 1994. Evolution, weighting and phylogentic utility of mitochondrial gene sequence and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Amer., 87: 651-701. Song, H., J.E. Buhay, M.F. Whiting and K.A. Crandall, 2008. Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified. Proc. Nati. Acad. Sci. USA., 105: 13486-13491. Tamura, K., G. Stecher, D. Peterson, A. Filipski, and S. Kumar, 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol., 30: 2725-2729. Templeton, A.R., K.A. Crandall and C.F. Sing, 1992. A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data III. Cladogram estimation. Genetics, 132: 619-633. Wang, G., X. Zhang, Y. Hou and B. Tang, 2015. Analysis of the population genetic structure of Rhynchophorus ferrugineus in Fujian, China, revealed by microsatellite loci and mitochondrial COI sequences. Entomol. Exp. et Appli., 155: 28-38. Wattanapongsiri, A., 1966. A revision of the genera Rhynchophorus and Dynamis (Coleoptera: Curculionidae). Dept. Agric. Sci. Bull., 1: 1-328.

Table 3.1 Sampling information for RPW populations collected from date palm Phoenix dactylifera in Punjab and KPK provinces of Pakistan

Collection No. of Geographical Characteristic Population Location Province date specimens Alt. (m) Lat. Long. LY Layyah 27-Feb-2015 8 Punjab 143 30°58'N 70°56'E

BWP Bahawalpur 14-Mar-2015 17 Punjab 252 29°59′N 73°15′E

DGK D.G. Khan 10-Feb-2015 21 Punjab 150 29°57'N 70° 29'E

MG Muzaffargarh 8-Mar-2015 18 Punjab 114 30°50'N 71°54'E

DIK D.I. Khan 1-Feb-2015 16 KPK 166 31°49'N 70°52'E

Table 3.2 Genetic characterization of five RPW populations from the Punjab and KPK provinces of Pakistan based on a 528 bp section of the mitochondrial COI gene. For population abbreviations, see Table 3.1.

Population N No. of haplotypes Haplotypes Haplotype diversity (Hd) LY 9 3 H1, H5, H52 0.556 BWP 11 3 H1, H5, H51 0.691 DGK 8 3 H1, H5, H52 0.464 MG 15 3 H1, H5, H51 0.590 DIK 7 3 H1, H5, H51 0.668

Table 3.3 Variation in a 528 bp segment of the cytochrome c oxidase subunit I (COI) region of mitochondrial DNA (mtDNA) of Rhynchophorus ferrugineus. Average number of pairwise nucleotide differences (k) within (diagonal element) and between (below diagonal) populations in the Punjab and KPK provinces of Pakistan. For population abbreviations, see Table 3.1.

LY BWP DGK MG DIK LY 0.722 - - - - BWP 0.747 0.836 - - - DGK 0.833 0.909 0.500 - - MG 0.689 0.733 0.667 0.667 - DIK 0.778 0.792 0.714 0.686 0.857

Figure 3.1 Map of collection sites in Punjab and KPK provinces of Pakistan.

Figure 3.2 Distribution of mitochondrial haplotypes across five populations of RPW from the Punjab and KPK provinces of Pakistan.

Figure 3.3 Relationships between four Pakistani COI haplotypes and 48 others occurring around the world. Haplotype network constructed from 539 COI sequences (each 528 bp long) generated by the present study and three earlier studies (see text). Each haplotype is represented by an oval or for that with the highest outgroup probability, a rectangle. Size of each haplotype is indicative of the number of specimens sharing that haplotype; also given inside each haplotype. H1-43 are numbered according to El Mergawy et al. (2011) and Rugman-Jones et al. (2013); H44-50 correspond to additional haplotypes from Wang et al. (2015); and H51-52 are new to this study.

CHAPTER 4

Resistance to commonly used insecticides and phosphine (PH3) against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) in Punjab and Khyber Pakhtunkhwa, Pakistan

Abstract In the first ever survey of insecticide resistance in field populations of Red Palm Weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) in Pakistan were collected from seven date palm growing areas across Punjab and Khyber Pakhtunkhwa (KPK), Pakistan, and assessed by the diet incorporation method against the formulated commonly used chemical insecticides profenophos, imidacloprid, chlorpyrifos, cypermethrin, deltamethrin, spinosad, lambda-cyhalothrin and a fumigant phosphine (or hydrogen phosphide) (PH3). Currently, there is no IRAC approved bioassay method for R. ferrugineus, so this study aimed to develop a suitable susceptibility test. Elevated levels of resistance were recorded for cypermethrin, deltamethrin and PH3 in R. ferrugineus after a long history of use in Pakistan. Resistance Ratios (RRs) documented for PH3 were 63- to 79-fold for cypermethrin 16- to 74-fold for deltamethrin 13- to 58-fold for profenophos 2.6- to 44-fold for chlorpyrifos 3- to 24-fold for lambda-cyhalothrin 2- to 12-fold and for Spinosad 1- to 10-fold as compared to the control. Resistant populations of R. ferrugineus mainly belonged to southern Punjab and to some extent from the KPK populations. The populations from Bahawalpur, Vehari, Layyah and Dera Ghazi Khan were found most resistant to chemical insecticides, while all populations exhibited high levels of resistant to phosphine. Of the eight agents tested, lower LC50 and LC90 values were recorded for spinosad and lambda-cyhalothrin. Resistance levels were very low to low against imidacloprid, very low to moderate against profenophos and chlorpyrifos, low to high against cypermethrin and deltamethrin and high against phosphine. These results suggest that spinosad and lambda- cyhalothrin exhibit unique modes of action and given their better environmental profile could be used in insecticide rotation or assist in discarding the use of older insecticides.

Keywords: Rhynchophorus ferrugineus, insecticide resistance, profenophos, imidacloprid, chlorpyrifos, cypermethrin, deltamethrin, spinosad, lambda-cyhalothrin, phosphine

4.1 Introduction The Red Palm Weevil’s (RPW), Rhynchophorus ferrugineus (Olivier) (Col., Curculionidae) invasive potential is a consequence of the elevated female fecundity (Faleiro, 2006), the ability to complete several generations in a year even in the same tree (Rajamanickam et al., 1995; Avand Faghih, 1996). It is one of the most destructive pests of ornamental and economically important palms, which is currently present in 50% of date growing countries and 15% coconut producing countries of the world. The weevil is concealed in nature and all their life stages remain inside the tree, usually found up to 1m in the tree trunk (Azam et al., 2001). The beetles quite often interbreed and reproduce within the same plant and continue devastating their host to death. The aboriginal home of this pest is South and Southeast Asia and Melanesia where it has been found destructing coconut palms (Lefroy, 1906; Brand, 1917; Viado and Bigornia, 1949; Nirula, 1956). Lately in 1918 the beetle was found inflicting date palm in India, during the same year weevil was found from some southern districts of Punjab, Pakistan (Multan, Muzaffargarh and Dera Ghazi Khan) (Milne, 1918). Two year latter Buxton (1920) reported this pest on date palm plantation from Mesopotamia (Iraq). It was only during the mid-1980s that RPW attained a major pest status on date palms, in the Middle Eastern region (Abraham et al., 1998). Subsequently, the weevil moved from North Africa into Europe, where it was reported for the ﬁrst time in the South of Spain (Cox, 1993; Barranco et al., 1995). So far pest has been distributed to many areas worldwide; its range now includes much of Asia, regions of Oceania, the Middle-East and North Africa, southern Europe, the Caribbean, and most recently it has been found in southern California in 2010 from the canary Island. To combat this voracious pest synthetic pesticide remained the mainstay since decades but this offers a challenge due to the cryptic nature of the pest, moreover insecticidal treatments with fumigants, soil treatments with insecticides, frond axil filling, trunk injections, wound dressing and crown drenching remains the main strategy for R. ferrugineus control (Hussain et al., 2013). Different scientists evaluated insecticidal potential of various chemical insecticides against this pest successfully through different application methods and their combinations (Cabello et al., 1997; Azam et al., 2000; Ajlann et al., 2000; Khalifa et al., 2001; AboEl-Saad et al., 2001; Abdul-salam et al., 2001; Al-Rajhy et al., 2005; Kaakeh, 2006; Llácer and Jacas, 2010). In Pakistan, the application of insecticides on date palms has an ancient history to combat R. ferrugineus infestation because more than hundred year history of R. ferrugineus presence, but their published records was not available. Shar et al. (2012) evaluated ten different insecticides against R. ferrugineus under field conditions and found fipronil, spirotetramat, chlorpyrifos and methidathion the most effective among all the tested insecticides. Al-Jabr et al. (2013) tested ten different chemical insecticides against R. ferrugineus midgut cell line, emamectin benzoate was a highly potent that significantly reduce the growth inhibition and increased the mortality. In Pakistan effectiveness of commercially used insecticides has complained by the date palm grower, reduced effectiveness may be because of the development of resistance. Although, resistance mechanism in R. ferrugineus against commonly used insecticides is poorly understood. The limited exploration of resistance mechanism in R. ferrugineus is focused in the design of the current investigation. We selected seven commonly used insecticides and phosphine (PH3), and checked their effectiveness against different distinct populations of R. ferrugineus collected from seven different areas of the Pakistan.

4.2 Materials and methods

4.2.1 RPW collection and rearing Different developmental stages of R. ferrugineus were collected from fallen and infested date palm trees from various areas of Punjab and KPK during 2014-2015 (Table 1). The areas were selected on the basis of ancient history of date palm cultivation and long term use of insecticides and phosphine. During collection adults, larvae and pupae were kept in separate plastic jars for each location until brought to the laboratory. In the laboratory larvae were provided with sugarcane (Saccharum officinarum L.; Poales: Poaceae) stems for feeding and pupation, while adults were offered shredded sugarcane pieces for both feeding and as an oviposition substrate. Pupae were kept in separate boxes for adult emergence in incubators (Sanyo Corporation Japan) at 27±2 oC, 60±5 RH and photoperiod of 12: 12 (D: L) hours. On emergence adults were transferred to jars for feeding and mating. The colonies were maintained in plastic boxes (15×30×30 cm) having a lid whose center (8 cm diameter covered with mesh wire gauze (60 mesh) for aeration. Rearing was carried out in IPM laboratory, Department of Entomology, University of Agriculture, Faisalabad, Pakistan. The adult food was changed after every three days and replaced sugarcane pieces were kept in separate jars for egg hatch. After egg hatching neonate larvae were transferred to sugarcane pieces for feeding until freshly molted 4th instar larvae were recovered. The laboratory strain was used as reference strain which was reared in the IPM laboratory since year 2009. The strain was maintained for more than 25 generations in the laboratory without any insecticidal exposure before these tests commenced. Preliminary laboratory bioassays showed high susceptibly of tested insecticides, being as susceptible as used by Ahmad et al. (2003).

4.2.2 Test chemicals For bioassay, commercial formulations of Curacron® (profenophos, 500 g/liter, 500 EC; Syngenta Pakistan Ltd., Karachi, Pakistan); Confidor® (jmidacloprid, 700g/kg, 70 WG; Bayer Crop Sciences, Pakistan Pvt. Ltd., Karachi, Pakistan); Lorsban® (chlorpyrifos, 400 g/liter, 40 EC; Arysta Life Science Pakistan Pvt. Ltd., Karachi, Pakistan); Arrivo® (cypermethrin, 100 g/liter, 10% EC; FMC United Pvt. Ltd., Lahore, Pakistan), Deltamethrin® (deltamethrin, 25 g/ liter, 2.5% EC; Target Agro Chemicals, Lahore, Pakistan); Tracer® (spinosad, 240 g/liter, 240 SC; Arysta LifeScience, Pakistan Pvt. Ltd.), Karate® (lambda-cyhalothrin 50g/liter, 5 EC; Syngenta Pakistan Ltd., Karachi, Pakistan). Phosphine (PH3) was generated by using aluminum phosphide (Celphos 56%; Jaffer Brothers (PVT) Ltd., Lahore, Pakistan) tablets.

4.2.3 Generation of phosphine gas The PH3 gas was generated using the FAO method (Anonymous, 1975). The apparatus for generation of PH3 gas consisted of a 5 liter beaker, a collection tube (cylinder), an inverted funnel, Aluminum phosphide tablets and muslin cloth. The tube for collection of gas was sealed from one side with an air-tight rubber stopper and then was filled with 5% sulphuric acid (H2SO4) solution. Half of the beaker was also filled with 5% H2SO4 solution. The gas collecting tube was placed carefully into the beaker over the inverted funnel in such a way that there is no loss of H2SO4 solution from the collection tube, while dipping into the beaker. Before generating PH3 gas all air in collection tube was removed within collection tube. Then aluminum phosphide tablets (wrapped in muslin cloth) were placed under inverted funnel. PH3 gas was then collected in the gas collecting tube inverted over the funnel. As the funnel filled with generated gas, the

76 level of solution went down. When the collecting tube was filled, 5 ml gas were sucked out with the help of an air tight syringe and was injected into sealed desiccators of known volume, then 50 ml of gas was taken out from the desiccators and injected into a Phosphine meter to measure gas concentration allowing the required concentrations of PH3 gas to be obtained.

4.2.4 Bioassay From each population freshly molted F1 fourth-instar (L4) larvae of R. ferrugineus were challenged with the test insecticides. The F1 generation was obtained by mass mating of the field collected beetles and the beetles emerged from field-collected larvae and pupae. Toxicity bioassays were performed using artificial diet (Martín and Cabello, 2006). For each bioassay artificial diets were prepared by diluting the respective concentrations of commercial products in distilled water (mg a.i./liter of water) previously determined for each bioassay. In the control treatment diet was prepared using distilled water. A piece of artificial diet from each diet was offered to ten freshly molted L4 larvae of R. ferrugineus individually in plastic cups measuring (6×6 cm) with 40 mesh/inch screen lids for aeration and the avoid insects escape. All the treatments were replicated three time and incubated at 27±2 °C with 65±5% RH and a photoperiod of 12: 12 (L: D) hours. FAO Method No. 16 (FAO 1975) was used on 4th instar larvae of R. ferrugineus with slight modifications. For each population, 10 larvae were placed in each of 10 glass cups and were placed in 4 liter air tight glass boxes (serving as fumigation chambers) before PH3 fumigation. A small quantity of artificial diet (10 g) was added to each cup. The boxes were centrally equipped with a port on the metal screw on lid which was fitted with a rubber injection point which served as an entry point for PH3. Before the lid was screwed onto the box, a rubber gasket was placed in it, and a thin layer of vacuum grease applied for a tight seal between the metal lid and the top edge of the box to increase gas tightness. Five phosphine doses (mg l-1) were measured in preliminary toxicity assays against 4th instar larvae of R. ferrugineus. Another 4 liter box without any treatment served as control. The gas was introduced into each box containing R. ferrugineus larvae through the rubber septum by using a gas tight syringe after first removing an equivalent volume of air from the jar using a syringe. Two drops of water were added to each box using a syringe in order to maintain 70% RH inside the boxes. Boxes were then placed in an incubator maintained at 27±2 °C. The boxes were opened 24 hours after application, and all larvae from each treatment were placed on damp filter paper and maintained at 27±2 °C and 65±5% RH for an extra 5 days to allow recovery.

4.3 Statistical Analysis For insecticidal treatments three days post-release of insects to treated diets, mortality counts were made in each treatment and at 24h +(5 recovery days) after PH 3 application. Results for mortality were converted into percentages, corrected using mortality in the untreated check using Abbott’s (1925) formula and analyzed statistically in Probit analysis by Polo-Plus (LeOra, 2003). LC50, LC90 and 95% fiducial limits were estimated for each insecticide at each location. Resistance ratios (RRs) were determined at LC50 and LC90 by dividing the lethal concentration (LC) values of each insecticide by the respective LC values determined for the laboratory susceptible strain. The distinction devised by Ahmad and Arif (2009) was used for categorizing the RRs, which were summarized as “no resistance” if (RR≤ 1), “very low” (RR = 2–10), “low” (RR = 11-20), “moderate” (RR = 21-50), “high” (RR = 51-100), and “very high” (RR> 100).

4.4 Results

4.4.1 Imidacloprid Resistance levels varied among the seven distinct field populations of R. ferrugineus. The degree of resistance varied from very low to low levels i.e RR 2.10- to 17.68-fold for all the tested populations (Figs. 4.1 and 4.2; Table 42). A very low resistance level was recorded in Bahawalpur (2.10-fold), Dera Ismail Kahn (2.86-fold), Vehari (4.20-fold) and Muzaffargarh (5.60-fold) populations at LC50 level. Low level of resistance were observed in populations of R. ferrugineus from Rahim Yar Khan (11.70-fold), Dera Ghazi Khan (15.12-fold) and Layyah (17.68-fold) at LC50 values.

4.4.2 Spinosad Very low level (1.02- to 9.77-fold) resistance ratios were recorded to spinosad among different field populations of R. ferrugineus (Fig. 4.2; Table 4.2). The resistance levels were detected in Dera Ghazi Khan (1.02-fold), Bahawalpur (2.03-fold), Layyah (2.37-fold), Rahim Yar Khan (3.41-fold), Muzaffargarh (4.40-fold), Vehari (7.15-fold) and Dera Ismail Khan (9.77- fold) populations of R. ferrugineus at LC50 values.

4.4.3 Lambda cyhalothorin Very low to low levels (1.96- to11.88-fold) RR to Lambda-cyhalothrin were recorded (Fig. 4.2; Table 4.2). A very low level of resistance was found in Bahawalpur (1.96-fold), Dera Ghazi Khan (2.01-fold), Layyah (2.77-fold), Rahim Yar Khan (3.76-fold), Muzaffargarh (4.72- fold) and Vehari (9.21-fold) populations, while low level of resistance was recorded in Dera Ismail Khan (11.88-fold) populations of R. ferrugineus at LC50 values.

4.4.4 Chlorpyrifos Very low and low to moderate levels (3.03- to 24.47-fold) RR to chlorpyrifos were recorded among tested populations of R. ferrugineus (Fig. 4.2; Table 4.2). A very low level of resistance was found in Dera Ghazi Khan (3.03- fold) and Bahawalpur (5.45-fold) populations, while low level of resistance was recorded in Layyah (10.65-fold), Muzaffargarh (17.91-fold) and Rahim Yar Khan (15.98-fold), and moderate level of resistance was recorded in Dera Ismail Khan (21.84-fold) and Vehari (24.47-fold) populations of R. ferrugineus at LC50 values.

4.4.5 Porfenophos Very low and low to moderate levels (RR 2.65- to 44.10-fold) were recorded for profenophos among different field populations of R. ferrugineus (Fig. 4.2; Table 4.2). A very low level of resistance was found in Bahawalpur (2.65-fold), Dera Ismail Khan (4.57-fold) and Vehari (6.90-fold) populations, while low level of resistance was recorded in Muzaffargarh (10.50-fold), and moderate level of resistance was recorded in Rahim Yar Khan (30.31-fold), Dera Ghazi Khan (35.22-fold) and Layyah (44.10-fold) populations of R. ferrugineus at LC50 values.

4.4.6 Deltamethrin Low and moderate to high levels (RR 12.90- to 57.97-fold) of resistance to deltamethrin were recorded among different field populations of R. ferrugineus (Fig. 4.2; Table 4.2). A low level of resistance was found in Dera Ghazi Khan (12.90-fold) and Dera Ismail Khan (16.42-

78 fold) populations, while moderate level of resistance was recorded in Layyah (26.76-fold), Rahim Yar Khan (34.52-fold), and high level of resistance was recorded in Muzaffargarh (51.37- fold), Bahawalpur (53.81-fold) and Vehari (57.97-fold) populations of R. ferrugineus at LC50 values.

4.4.7 Cypermethrin Low and moderate to high levels (RR 15.89- to 73.82-fold) of resistance to cypermethrin were recorded among different field populations of R. ferrugineus (Fig. 4.2; Table 4.2). A low level of resistance was found in Dera Ghazi Khan (15.89-fold) population, while moderate level of resistance was recorded in Dera Ismail Khan (22.59-fold), Layyah (31.11-fold), Rahim Yar Khan (39.41-fold), and high level of resistance was recorded in Muzaffargarh (64.29-fold), Bahawalpur (69.49-fold) and Vehari (73.82-fold) populations of R. ferrugineus at LC50 values.

4.4.8 Phosphine High levels of resistance were recorded against PH3 in all the tested populations of R. ferrugineus, ranging from 63.09- to 79.46-fold (Fig. 4.2; Table 4.2) with very little difference between populations. The highest levels of resistance were recorded in Rahim Yar Khan (63.09- fold), Muzaffargarh (63.78-fold), Bahawalpur (68.76-fold), Dera Ghazi Khan (72.29-fold), Dera Ismail Khan (73.64-fold), Vehari (76.30-fold) and (79.46-fold) in Layyah populations of R. ferrugineus at LC50 values. The slope of regression line was >2 in all the insect populations, indicating significant differences in RR from the reference strain.

4.5 Discussion Knowledge of the resistance status of pests of economic importance is imperative for researchers to guide the farming community in combating pest problems. It could be helpful for the farming community in partially reduce or completely suspend the use of particular chemical in their farming system. In our study we considered seven populations of R. ferrugineus collected from different areas of Punjab and Khyber Pakhtunkhwa provinces of Pakistan and tested resistance to seven commonly used chemical insecticides and PH3 against laboratory reared F1 of these populations. These areas are reported to be the major date producing areas of the country and contribute the major share of the in country’s date production. Among these areas most have the history of R. ferrugineus infestation stretching back almost 100 years (Milne, 1918). To combat this voracious pest, farmers have mainly used conventional insecticides and fumigants, particularly PH3, throughout the country over decades, resulting in R. ferrugineus. This is the very first report of a resistance investigation in R. ferrugineus against conventional insecticides and PH3 in Pakistan. Seven laboratory populations of R. ferrugineus were established from field collections and all tested strains exhibited significantly different susceptibility levels to all tested insecticides through dose-mortality bioassays. In our study laboratory population was considered reference strain which exhibited no resistance to the tested chemicals. Among the tested chemicals spinosad and lambda-cyhalothrin were the most effective and revealed very low level of resistance in any tested population. The LC50 value of spinosad and lambda-cyhalothrin was significantly lower for all the tested populations as compared to rest of the insecticides. The chemical insecticides fed with artificial diet to R. ferrugineus larvae caused mortality in a dose-dependent manner. The most consistent resistance across seven populations was recorded for deltamethrin and cypermethrin. High level of resistance against PH3 were observed for all the seven populations. The current study provides a base line of

79 resistance to cypermethrin, deltamethrin and PH3 against R. ferrugineus. In the strains examined low and moderate to high level of resistance was due to the excessive application of these chemical insecticides to manage R. ferrugineus in Pakistan. The cryptic habit of R. ferrugineus facilitates almost year-round activity in date palm plantations which has forced the farmers to expose field populations to different chemical insecticides and fumigants in order to successfully control pest infestations. The repeated application of these chemical insecticides could then lead to the resistance in R. ferrugineus populations. Resistance to cypermethrin and deltamethrin is common amongst the arthropod pests worldwide (Mueller-Beilschmidt, 1990). In Pakistan resistance against these commonly used insecticides in various crop pests such as in Spodoptera exigua, Brevicoryne brassicae, Spodoptera litura, Helicoverpa armigera and Bemisia tabaci has been reported by many scientists (Ahmad 2008, 2009; Ahmad and Akhtar, 2013; Ahmad and Mehmood, 2015; Ahmad et al., 2001; Sayyed et al., 2008; Ishtiaq et al., 2012; Qayyum et al., 2015). So far only one published record of cypermethrin resistance against R. ferrugineus is reported from Saudi Arabia by Al-Ayedh et al. (2015). Resistance to PH3 is not surprising because deployment of PH3 in the form of aluminum phosphide tablets is common practice among date palm farming community in the country. Resistance to PH3 is common in stored grain insets such as Tribolium castaneum, Rhyzopertha dominica and psocid species worldwide e.g. Opit et al. (2012) reported 119 fold and 1500 fold resistance in T. castaneum and R. dominica strains respectively, collected from Oklahoma. Many other researchers have reported PH3 resistance in stored product pests from all over the world. It is advised not to use cypermethrin and deltamethrin against this pest and alternative chemicals such as spinosad and lambda-cyhalothrin should be utilized in control strategies. So far, there is no understanding of the molecular mechanisms in this species not any understanding of cross-resistance pattern. This gap should be filled in order to design strategies to mitigate the resistance problem. Spinosad and lambda-cyhalothrin possesses high efficacy against the devastating stage of R. ferrugineus in laboratory assays (Abo-El-Saad et al., 2001). Treatment strategies with chemical insecticides which include spinosad in control programs would be worth considering. Moreover, integrated use of microbial control agents with newer chemistry insecticides with novel modes of action could successfully replace the resisted chemistries in field control programs.

Conclusions The present study showed that the population of red palm weevil in Pakistan has resistance against commonly used chemical insecticides and phosphine. The unwise use of these insecticides can lead to this problem. Almost all the insecticides exhibited resistance which was varied form very low to, low and moderate to high for all the population tested. Delatmethrin, cypermethrin and phosphine exhibited moderate to high resistance to almost all the populations. Populations from Layyah Dera Ismail Khan and Vehari showed most resistance as compared to the other populations tested.

Acknowledgements This research work was supported by the scholarship from Higher Education Commission (HEC), Islamabad, Pakistan (112-30536-2AV1-263) under Indigenous Ph.D. Fellowship Program.

4.6 References Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol., 18: 265-267. Abdulsalam, K.S., M.S. Shawir, M.M. Abo-El-Saad, M.A. Rezk and A.M. Ajlan, 2001. Regent (fipronil) as a candidate insecticide to control red palm weevil (Rhynchophorus ferrugineus Olivier). Ann. Agric., 46: 841-849. Abo-El-Saad, M.M., A.M. Ajlan, M.S. Shawir, K.S. Abdul-salam and M.A. Rezk, 2001. Comparative toxicity of four pyrethroid insecticides against red palm weevil, Rhynchophorus ferrugineus (Olivier) under laboratory conditions. J. Pest Cont. Environ. Sci., 9: 63-76. Abo-El-Saad, M.M., H.A. Elshafie, J.R. Faleiro and I.A. Bou-Khowh, 2011. Toxicity evaluation of certain insecticides against the red palm weevil, Rhynchophorus ferrugineus (Olivier), under laboratory conditions. ESA Annual Meeting, 2011. Abraham, A., M.A. Al-Shuhaibi, J.R. Faleiro, R.A. Abozuhairah and P.S.P. Vidyasagar, 1998. An integrated management approach for red palm weevil Rhynchophorus ferrugineus Oliv. as key pest of date Palm in the Middle East .Agri. Sci., 3: 77- 83. Ahmad, M., 2008. Potentiation between pyrethroid and organophosphate insecticides in resistant field populations of cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) in Pakistan. Pesti. Biochem. Physiol., 91: 24-31. Ahmad, M., 2009. Observed potentiation between pyrethroid and organophosphorus insecticides for the management of Spodoptera litura (Lepidoptera: Noctuidae). Crop Prot., 28: 264- 268. Ahmad, M. and M.I. Arif, 2009. Resistance of Pakistani field populations of spotted bollworm Earias vittella (Lepidoptera: Noctuidae) to pyrethroid, organophosphorus and new chemical insecticides. Pest Manag. Sci., 65: 433-439. Ahmad, M. and R. Mehmood, 2015. Monitoring of resistance to new chemistry insecticides in Spodoptera litura (Lepidoptera: Noctuidae) in Pakistan. J. Econ. Entomol., 108(3): 1279- 1288. Ahmad, M. and S. Akhtar, 2013. Development of insecticide resistance in field populations of Brevicoryne brassicae (Hemiptera: Aphididae) in Pakistan. J. Econ. Entomol., 106(2): 954-958. Ahmad, M., M.I. Arif, Z. Ahmad and I. Denholm, 2001. Cotton whitefly (Bemisia tabaci) resistance to organophosphate and pyrethroid insecticides in Pakistan. Pest Mang. Sci., 58: 203-208. Ahmad, M., M. I. Arif, and Z. Ahmad, 2003. Susceptibility of Helicoverpa armigera (Lepidoptera: Noctuidae) to new chemistries in Pakistan. Crop Prot., 22: 539-544 Ajlann, A.M., M.S. Shawir, M.M. Abo-El-Saad, M.A. Rezk and K.S. Abdulslam, 2000. Laboratory evaluation of certain organophosphorus insecticides against the red palm weevil, Rhynchophorus ferrugineus (Oliver). Sci. J., 1: 15-16. Al-Ayedh, H., A. Hussain, M. Rizwan-ul-Haq and A.M. Al-Jabr, 2015. Status of insecticide resistance in field-collected populations of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). Int. J. Agric. Biol., 17(6): 1-9. Al-Jabr, A.M., M. Rizwan-ul-Haq, A. Hussain, A.I. Al-Mubarak and H.Y. Al-Ayed, 2013. Establishing midgut stem cell culture from Rhynchophorus ferrugineus (Olivier) and toxicity assessment against 10 different insecticides. In Vitro Cell. Dev. Biol. Anim., 50: 296-303.

Al-Rajhy, D.H., H.I. Hussein and A.M.A. Al-Shawaf, 2005. Insecticidal activity of carbaryl and its mixture with piperonylbutoxide against red palm weevil Rhynchophorus ferrugineus (Olivier) (Curculionidae: Coleoptera) and their effects on acetylcholinesterase activity. Pak. J. Biol. Sci., 8: 679-682. Anonymous, 1975. Recommended methods for detection and measurement of resistance in agricultural pests to pesticides. Tentative methods for adults of some major pest species to stored cereals with methyl bromide and phosphine. FAO Plant Prot. Bull., 23:12-35. Avand-Faghih, A., 1996. The biology of red palm weevil, Rhynchophorus ferrugineus Oliv. (Coleoptera, Curculionidae) in Saravan region (Sistan &Balouchistan Province, Iran). Appl. Entomol. Phytopathol., 63: 16-18. Azam, K.M., S.A. Razvi and I. Al-Mahmuli, 2000. Management of red date palm weevil, Rhynchophorus ferrugineus Oliver on date palm by Prophylactic measures. In proceedings of first workshop on control of date palm red weevil. Ministry of Higher Education, King Faisal University, Date Palm Research Centre, Kingdom of Saudi Arabia. Pp. 26-34. Azam, K.M., S.A. Razvi and I. Al-Mahmuli, 2001. Survey of red palm weevil, Rhynchophorus ferrugineus Oliver. Infestation in date palm in Oman. 2nd International Conf. on Date Palms. (Al-Ain, UAE). Pp. 25-27. Barranco, P., J.P. DeLa and T. Cabello, 1995. Un Nuevo Curculiónido tropical para la fauna Europa, Rhynchophorus ferrugineus (Olivier 1790), (Curculionidae: Coleoptera). Boletin de la Asociaction Espanola de Entomol., 20: 257-258. Brand, E., 1917. Coconut red weevil. Some facts and fallacies. Tropic. Agric. Mag. Ceylon Agricul. Soc., 49: 22-24. Buxton, P.A., 1920. Insect pests of dates and the date palm in Mesopotamia and elsewhere. Bull. Entomol. Res., 11: 287-304. Cabello, T.P., J. de la Peña, P. Barranco and J. Belda, 1997. Laboratory evaluation of imidacloprid and oxamyl against Rhynchophorus ferrugineus. Tests of Agrochem. Culti., 18: 6-7. Cox, M.L. 1993. Red palm weevil, Rhynchophorus ferrugineus, in Egypt. FAO Plant Prot. Bull., 41: 30-31. European and Mediterranean Plant Protection Organisation (EPPO), 2008. Rhynchophorus ferrugineus, EPPO Bulletin Volume 38, Issue 1, pages 55-59, April 2008. Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm during the last one hundred years. Inter. J. Trop. Insect Sci., 26: 135-154. Food and Agriculture Organization (FAO), 1975. Tentative method for adults of some major pest species of stored cereals with methyl bromide and phosphine, FAO method no. 16. FAO Plant Prot. Bull., 23: 12-25. Hussain, A., M.R.U. Haq, A.M. Al-Jabr and H.Y. Al-Ayied, 2013. Managing Invasive Populations of Red Palm Weevil: A Worldwide Perspective. J. Food Agric. Environ., 11: 456-463. Ishtiaq, M., M.A. Saleem and M. Razaq, 2012. Monitoring of resistance in Spodoptera exigua (Lepidoptera: Noctuidae) from four districts of the southern Punjab, Pakistan to four conventional and six new chemistry insecticides. Crop Prot., 33: 13-20.

Kaakeh, W., 2006. Toxicity of imidacloprid to developmental stages of Rhynchophorus ferrugineus (Curculionidae: Coleoptera): Laboratory and field tests. Crop Prot., 25: 432- 439. Khalifa, O., A.H. El-Assal, F.A.A. Ezaby, M.A. Murse, S.M.A. Nuaimi and N.S. Al-Zehli, 2001. Database for infestation of date palm by red palm weevil (Rhynchophorus ferrugineus (Oliver). In U.A.E. and Oman. 2nd International Conf. on Date Palms. (Al-Ain, UAE). Pp. 25-27. Lefroy, H.M., 1906. The more important insects injurious to Indian agriculture. Govt. Press, Calcutta. Pp. 151. LeOra, 2003. Polo plus, A User’s Guide to Probit and Logit Analysis. LeOra Software, Barkeley, CA. Llácer, E. and J.A. Jacas, 2010. Efficacy of phosphine as a fumigant against Rhynchophorus ferrugineus (Coleoptera: Curculionidae) in palms. Spanish J. Agri. Res., 8: 775-779. Martín, M.M. and T. Cabello, 2006. Manejo de la cría del picudo rojo de la palmera, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera, Dryophthoridae), en dieta artificial y efectos en su biometría y biología. Boletín de Sanidad Vegetal de Plagas, 32: 631-641. Milne, D., 1918. The Date Palm and Its Cultivation in the Punjab. The Punjab Government. Pp. 153. Mueller-Beilschmidt, D., 1990, "Resistance of insect pests and disease vectors to synthetic pyrethroids", J. Pesti. Ref., 10(4): 34-38. Nirula, K.K., 1956. Investigations on the pests of coconut palm. Part. Rhynchophorus ferrugineus. Ind. Coc. J., 9: 229-247. Opit, G.P., T.W. Phillips, M.J. Aikins, and M.M. Hasan, 2012. Phosphine resistance in Tribolium castaneum and Rhyzopertha dominica from stored wheat in Oklahoma. J. Econ. Entomol., 105(4): 1107-1114. Rajamanickam, K., J.S. Kennedy and A. Christopher, 1995. Certain components of integrated management for red palm weevil, Rhynchohphorus ferrugineus F. (Curculionidae: Coleoptera) on coconut. Mededelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen, 60: 803-805. Sayyed, A.H., M. Ahmad and M.A. Saleem, 2008. Cross-resistance and genetics of resistance to indoxacarb in Spodoptera litura (Lepidoptera: Noctuidae). J. Econ. Entomol., 101(2): 472-479. Shar, M.U., M.A. Rustamani, S.M. Nizamani and L.A. Bhutto, 2012. Red palm weevil (Rhynchophorus ferrugineus Olivier) infestation and its chemical control in Sindh province of Pakistan. Afri. J. Agri. Res., 11: 1666-1673. Viado, G.B.S. and A.E. Bigornia, 1949. A biological study of the Asiatic palm weevil, Rhynchophorus ferrugineus Oliv. (Curculionidae: Coleoptera). Philip. Agri., 33: 1-27.

Table 4.1 Geographical characteristics of the localities where R. ferrugineus populations were collected in the Punjab and Khyber Pakhtunkhwa, Pakistan

Geographical Characteristic Location Province Host plant Alt. (m) Lat. Long. Layyah Punjab Date palm 143 30°58'N 70°56'E Bahawalpur Punjab Date palm 252 29°59′N 73°15′E Dera Ghazi Khan Punjab Date palm 150 29°57'N 70°29'E Muzaffargarh Punjab Date palm 114 30°50′N 71°54′E Dera Ismail Khan KPK Date palm 166 31°49'N 70°52'E Vehari Punjab Date palm 135 29°58'N 71°58'E Rahim Yar Khan Punjab Date palm 83 27°40'N 60°45'E

Table 4.2 Resistance to commonly used insecticides and phosphine against susceptible strains and field-collected populations of R. ferrugineus

-1 -1 Insecticide Localities LC50 (mg liter ) LC90 (mg liter ) Slope Resistance (95% fiducial limits) (95% fiducial limits) Ratio (RR) Bahawalpur 16.59 (12.09-23.41) 250.87 (197.42-314.98) 1.12±0.18 2.10 Muzaffargarh 44.22 (36.31-51.39) 669.08.16 (581.35.793.45) 1.42±0.19 5.60 Layyah 139.52 (108.54-195.02) 2112.86 (1984.81-2358.44) 2.50±0.22 17.68 Imidacloprid Dera Ismail Khan 22.64 (18.76-29.78) 341.52 (285.16-459.49) 1.34±0.20 2.86 Dera Ghazi Khan 119.37 (94.62-137.87) 1806.54 (1685.43-2132.38) 2.21±0.23 15.12 Vehari 33.19 (26.14-42.81) 501.51 (435.32-651.32) 1.23±0.19 4.20 Rahim Yar Khan 92.35 (79.37-117.69) 1397.43 (1258.15-1606.42) 1.36±0.21 11.70 Laboratory 7.89 (5.81-12.25) 119.48(106.03-148.11) 0.74±0.14 - Bahawalpur 7.98 (3.03-10.08) 128.22 (114.34-165.10) 0.92±0.16 2.03 Muzaffargarh 17.26 (14.96-22.82) 276.34 (204.28-385.40) 1.14±0.18 4.40 Layyah 9.32 (4.67-13.92) 150.22 (101.34-236.10) 1.06±0.15 2.37 Spinosad Dera Ismail Khan 38.30 (30.57-46.74) 620.75 (502.22-844.95) 1.42±0.19 9.77 Dera Ghazi Khan 3.98 (2.80-5.55) 64.41 (42.16-97.84) 0.61±0.13 1.02 Vehari 28.06 (24.87-33.91) 454.23 (357.81-648.35) 1.12±0.17 7.15 Rahim Yar Khan 13.37 (10.75-17.21) 216.72 (141.38-321.18) 1.17±0.15 3.41 Laboratory 3.92 (2.89-5.63) 63.56 (49.34-88.32) 0.69±0.14 - Bahawalpur 13.85 (10.51-18.83) 221.59 (165.41-352.82) 0.72±0.17 1.96 Muzaffargarh 32.29 (26.54-42.12) 535.51 (448.32-679.32) 1.21±0.19 4.72 Layyah 19.64 (15.31-24.44) 313.82 (217.46-416.10) 1.07±0.17 2.77 Lambda Dera Ismail Khan 83.44 (73.39-95.72) 1347.16 (1081.84-1564.82) 1.61±0.21 11.88 cyhalothrin Dera Ghazi Khan 14.15 (10.97-19.21) 228.76 (151.39-318.30) 0.72±0.15 2.01 Vehari 64.76 (56.34-71.72) 1045.11 (803.22-1281.35) 1.41±0.21 9.21 Rahim Yar Khan 26.44 (20.31-75.63) 425.93 (319.32-620.47) 1.33±0.20 3.76 Laboratory 7.02 (5.21-11.27) 113.48(102.03-130.10) 0.90±0.15 - Bahawalpur 44.21 (37.57-53.40) 611.92 (499.40-881.55) 1.22±0.19 5.45 Muzaffargarh 145.36 (117.57-174.38) 2011.35 (1861.65-2351.88) 2.42±0.21 17.91 Layyah 86.42 (73.70-105.08) 1195.43 (996.15-1440.42) 1.37±0.20 10.65 Chlorpyrifos Dera Ismail Khan 177.25 (145.86-197.65) 2452.54 (2275.47-2865.36) 2.31±0.21 21.84 Dera Ghazi Khan 24.61 (20.48-30.22) 340.55 (249.42-481.64) 1.72±0.19 3.03 Vehari 198.52 (146.54-237.53) 2747.67 (2505.43-3018.38) 2.55±0.20 24.47 Rahim Yar Khan 129.64 (97.54-182.38) 1794.75 (1598.89-2121.52) 2.31±0.18 15.98 Laboratory 8.11 (4.01-11.92) 112.29 (78.34-208.10) 0.97±0.16 - Bahawalpur 23.31 (19.18-27.71) 307.01 (261.52-377.90) 1.25±0.18 2.65 Muzaffargarh 92.19 (78.12-115.08) 1220.43 (1161.15-1383.42) 1.30±0.21 10.50 Layyah 387.26 (330.65-424.59) 5125.41 (4871.21-5561.27) 3.22±0.23 44.1 Profenophos Dera Ismail Khan 40.20 (32.98-46.87) 531.87 (414.23-680.45) 1.41±0.18 4.57 Dera Ghazi Khan 309.37 (286.17-353.53) 4093.08 (3814.56-4405.82) 2.53±0.18 35.22 Vehari 60.64 (52.34-67.72) 801.21 (719.22-1029.35) 1.41±0.17 6.90 Rahim Yar Khan 266.28 (224.23-305.29) 3522.54 (3368.83-3917.76) 2.56±0.20 30.31 Laboratory 8.78 (4.44-12.35) 116.22 (90.45-174.59) 0.99±0.16 - Bahawalpur 348.28 (296.65-374.59) 5729.41 (5204.21-6480.27) 3.61±0.23 53.81 Muzaffargarh 332.43 (283.65-365.59) 5469.41 (5108.21-5911.27) 3.36±0.21 51.37 Layyah 173.29 (144.54-199.65) 2849.54 (2617.47-3227.36) 2.31±0.20 26.76 Deltamethrin Dera Ismail Khan 106.34 (96.12-121.05) 1748.22 (1553.08-2092.29) 1.73±0.19 16.42 Dera Ghazi Khan 83.54 (73.24-92.11) 1373.16 (1202.84-1591.82) 1.54±0.17 12.90 Vehari 375.95 (329.65-411.59) 6172.41 (5728.21-6798.27) 3.52±0.23 57.97 Rahim Yar Khan 223.41 (194.36-284.66) 3675.76 (3339.78-4105.55) 2.72±0.21 34.52 Laboratory 6.47 (4.97-9.12) 106.48(93.03-122.13) 0.71±0.14 - Bahawalpur 503.12 (454.22-583.48) 8127.65 (7875.63-8587.39) 3.72±0.23 69.49 Muzaffargarh 565.54 (391.43-605.66) 7519.76 (7256.32-7991.18) 3.83±0.22 64.29

Layyah 225.39 (201.57-267.29) 3638.02 (3425.25-4058.32) 2.59±0.20 31.11 Cypermethrin Dera Ismail Khan 163.65 (139.23-204.32) 2641.58 (2470.43-2911.43) 2.48±0.19 22.59 Dera Ghazi Khan 115.18 (94.12-128.11) 1859.64 (1715.76-2119.64) 2.16±0.21 15.89 Vehari 534.47 (489.22-622.48) 8633.65 (7247.63-9211.39) 3.65±0.23 73.82 Rahim Yar Khan 255.42 (233.23-311.23) 4609.54 (4266.83-5165.76) 2.81±0.21 39.41 Laboratory 7.24 (5.39-11.23) 116.96(103.77-134.12) 0.71±0.19 - Bahawalpur 4008.70 (3280.23- 51177.95 (43719.73- 4955.42) 59088.76) 5.46±0.27 68.76 Muzaffargarh 3718.37 (3011.23- 47679.37 (42165.65- 4604.56) 55248.54) 5.82±0.24 63.78 Layyah 4632.51 (3846.43- 59402.62 (51484.42- 5517.72) 64804.32) 6.37±0.34 79.46 Dera Ismail Khan 4293.21 (3618.43- 55048.84 (50342.23- 5180.41) 62268.54) 6.20±0.30 73.64 Phosphine Dera Ghazi Khan 4214.50 (3529.43- 54039.67 (5045534- 5065.54) 621180.23) 5.82±0.28 72.29 Vehari 4448.33 (3750.43- 57038.53 (50118.76- 5309.52) 63308.37) 6.02±0.31 76.3 Rahim Yar Khan 3678.14 (2972.23- 47163.47 (42540.65- 4545.56) 54540.84) 4.83±0.23 63.09 Laboratory 58.3 (52.45-64.54) 747.56 (692.54-894.89) 1.48±0.21 -

1. Bahawalpur 2. Rahim Yar Khan 3. Vehari 4. Dera Ghazi Kahn 5. Muzaffargarh 6. Layyah 7. Dera Ismail Khan

Figure 4.1 Map of collection sites in Punjab and Khyber Pakhtunkhwa provinces of Pakistan (1. Bahawalpur 2. Rahim Yar Khan 3. Vehari 4. Dera Ghazi Khan 5. Muzaffargarh 6. Layyah 7: Dera Ismail Khan)

Figure 4.2 Resistance ratios (RRs) of chemical insecticides and phosphine against susceptible strains and field-collected populations of R. ferrugineus populations of R. ferrugineus from various localities in Punjab and Khyber Pakhtunkhwa, Pakistan

CHAPTER 5

Insecticidal potential of Beauveria bassiana and Metarhizium anisopliae isolates against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

Abstract Entomopathogenic fungi are amongst the most common microbial control agents in nature against insect pests. Their efficacy against a variety of arthropod pests had been witnessed since for many years. The aim of this study was to screen 19 different isolates of Beauveria bassiana s.l. and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales), recovered from different soil samples (field crops, fruit orchards, vegetable fields and forests) and insect cadavers at two different spore concentrations (1×107 and 1×108 conidia ml-1). Three isolates of B. bassiana (WG-41, WG-42 and WG-43) and two isolates of M. anisopliae (WG-44 and WG-45) exhibited ˃88% larval and ˃75% adult mortality of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) on their highest dose rate. On the other hand more sporulating cadavers were observed at high dose rate compared to low dose on both life stages of R. ferrugineus. The current study confirmed the lethal action of B. bassiana, and M. anisopliae isolates with differential mortality levels, usually directly proportional to the conidial concentration. This study further confirmed that the isolates recovered from R. ferrugineus dead cadavers exhibited more mortality compared to the other sources. In virulence assay WG-41 and WG-42 caused highest percentage of both larval and adult mortality at all the exposure intervals which suggest that these two isolates may be the most promising for their use in sustainable management programs aimed at microbial control in date palm plantation.

Key words: Entomopathogens, Beauveria bassiana, Metarhizium anisopliae, Red Palm Weevil sporulating

5.1 Introduction The red palm weevil (RPW) Rhynchophorus ferrugineus (Olivier 1790) (Coleoptera: Curculionidae) is a devastating palm pest that has caused large economic losses in palm farming worldwide (Murphy and Briscoe, 1999; Faleiro, 2006). This beetle can affect a wide range of palms (Barranco et al., 2000) including economically important species such as the date palm (Phoenix dactylifera L.), Canary Islands date palm (P. canariensis Hort), coconut (Cocos nucifera L.), African oil palm (Elaeis guineensis Jacq.) and chusan palm (Trachycarpus fortunei) (Sabbour and Solieman, 2014). The weevil has been found devastating palm plantations almost 50% of the date palm growing countries of the world (Faleiro, 2006) resulting yield losses from 0.7-10 tons hac-1 (Singh and Rethinam, 2005), while in Pakistan it caused 10-20% production losses to different varieties of dates (Baloach et al., 1992). Presently its distribution is reported in Oceania, Asia, Africa and Europe and was found in Curaçao and Marruecos, in 2008, and USA, in 2010 (EPPO 2006, 2007, 2009a, 2009b, 2010). The females lay eggs at the base of the fronds in separate holes made with their rostrum. Neonate larvae bore into the palm core and upon completion of development move back to the base of the fronds to pupate. A new generation emerges and adults may remain within the same host and reproduce until the palm eventually dies and after that adults emerge out of the plant for infection to the new plants (Dembilio et al., 2010). Furthermore, R. ferrugineus is a strong flyer that increases the weevil’s ability to disperse, colonize and breed at new sites (Murphy and Briscoe, 1999). However, longevity, activity and behavior of adult weevils are greatly affected by humidity. The most commonly used control treatments for this voracious pest are chemical insecticides such as Diazinon, Imidacloprid, Phosmet and phosphine (Llácer and Jacas, 2010; Llácer et al., 2010). These pesticides are applied with numerous application methods, including wound dressing, frond axil ﬁling, fumigation, injection, and spraying, are being tried for the control of RPW infestations (Cabello et al., 1997; Abraham et al., 1998; Al-Rajhy et al., 2005). Undoubtedly, the use of synthetic pesticides will continue to reduce RPW infestations. However, several factors, including the evolution of resistance, residue persistence, applicator safety, environmental hazards and harms non-target organisms have urged researchers to explore alternatives to control RPW which can be compatible to human health and environment friendly (Gindin et al., 2006; Hussain et al., 2013; Jalinas et al., 2015). Alternatively, a number of biological control agents like predators, parasites, parasitoids, and microbial control agents (bacteria, fungi and nematodes) are deployed to combat this pest. In laboratory studies of the infection of RPW with different microorganisms, including entomopathogenic nematodes (EPNs) (Gerber and Giblin-Davis, 1990; Llácer et al., 2009; Dembilio et al., 2010a), entomopathogenic bacteria (Banerjee and Dangar, 1995; Salama et al., 2004; Manachini et al., 2009) and entomopathogenic fungi (EPFs) (Ghazavi and Avand-Faghih, 2002; Shaiju-Simon and Gokulapalan, 2003; Gindin et al., 2006; Dembilio et al., 2010b; Cito et al., 2014) have shown variable results in terms of larval and adult mortality. Among these microorganisms, EPFs are considered promising microbial control agents against RPW due to their epizootic potential, transmitted horizontally, natural dispersion and safe to non-target organisms, environment friendly and ability to maintain lasting control once established in the environment (Van Driesche et al., 2007; Hussain et al., 2015). Microbiological treatments with Beauveria bassiana s.l. (Ascomycota: Hypocreales) and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales) offer an alternative and ecologically compatible pest management strategy (Inglis et al., 2001). Several research programs have also been initiated for

90 studying the biological control of RPW. Specifically, by deploying these two agents, have been detected on the RPW and tested under laboratory and field conditions (Deadman et al., 2001; Gindin et al., 2006; El-Sufty et al., 2007; 2009; 2011; Sewify et al., 2009; Torta et al., 2009; Vitale et al., 2009; Dembilio et al., 2010; Güerri-Aguilló et al., 2010; 2011; Merghem, 2011; Francardi et al., 2012; Ricaño et al., 2013; Cito et al., 2014). The main advantage of using EPFs is their unique mode of action. Unlike other insect pathogens, fungi infect the host by contact, penetrating the insect cuticle. The host can be infected by direct treatment or by transmission of inoculum from treated insects/cadavers to untreated insects or to subsequent developmental stages via the new generation of spores (Quesada-Moraga et al., 2004). A number of studies were carried out to isolate the EPFs from different developmental stages of RPW. Entomopathogenic M. anisopliae was isolated from Rhynchophorus bilineatus (Montrouzier) (Coleoptera: Curculionidae) in New Guinea, after treatment of young palms against the Scapanes australis Boisduval (Coleoptera: Scarabaeidae: Dynastinae) with a formulation based on M. anisopliae spores (Murphy and Briscoe, 1999). Other published report (Ghazavi and Avand-Faghih, 2002) from Iran; showed the recovery of M. anisopliae and B. bassiana from RPW adults and pupae. The pupae of R. ferrugineus presumed to be infected with entomopathogenic fungi were collected in a date palm grove in Spain during 2007 which later on proved to be infected with the entomopathogenic fungus B. bassiana (Dembilio et al., 2010b). The aim of this study was to screen and identify pathogenicity of 19 isolates of M. anisopliae and B. bassiana recovered from different soils, stored grain insects and cadavers of infected R. ferrugineus. This study also aimed to determine the exposure time dose mortality relationships of virulent fungal isolates, and to conﬁrm infection against RPW under laboratory conditions.

5.2 Materials and Methods

5.2.1 RPW collection and rearing Different life stages of RPW were collected from fallen and infested date palm trees with the permission of farmers (owners) from date palm growing areas of west Punjab and Khyber Pakhtunkhwa (KPK), Pakistan. During collection adult, larvae and pupae were kept in 2 liter plastic jars until brought to the laboratory. After arriving to the laboratory larvae were provided with sugarcane (Saccharum officinarum L.; Poales: Poaceae) stems for feeding and pupation, while adult were offered with the shredded sugarcane pieces as both for feeding and substrate for oviposition. Pupae were kept in separate boxes (15×30×30 cm) for adult emergence in incubator. As the adults were emerged, they were transferred to the adult’s jars for feeding and mating. Colony was developed in plastic boxes (15×30×30 cm) having a lid covered with mesh wire gauze (60 mesh size) in the middle (7 cm diameter) for aeration. Rearing was carried out in Microbial Control Laboratory, Department of Entomology, University of Agriculture, Faisalabad, Pakistan. The rearing conditions were maintained at 25±2oC and 65±5% RH and 12:12 (D: L) h photoperiod in an incubator. Adults diet was changed after every three days and replaced sugarcane pieces were kept in separate jars for egg hatching. After egg hatching neonate larvae were transferred to the new sugarcane pieces for feeding and pupation.

5.2.2 Culture collection The virulence of 19 isolates of entomopathogenic fungi B. bassiana and M. anisopliae, 14 of them (WG-2, WG-3, WG-4, WG-5, WG-6, WG-7, WG-10, WG-11, WG-12, WG-13, WG- 15, WG-16, WG-17, WG-18) belonging to the culture collection of Microbial Control

Laboratory, Department of Entomology, University of Agriculture, Faisalabad, Pakistan. All these isolates were recovered from soils of different origin (field crops, fruit orchards, vegetable fields and forest) and stored grain insect pests (Wakil et al., 2013; 2014). Other five isolates, three of B. bassiana (WG-41, WG-42 and WG-43) and two of M. anisopliae (WG-44 and WG- 45) were isolated from infected RPW cadavers collected from Layyah, Bahawalpur, Dera Ismail Khan and Muzaffargarh districts (Table 4.1).

5.2.3 Isolation from RPW cadavers The B. bassiana and M. anisopliae isolates were obtained from naturally infected RPW adults collected from different areas of Punjab, Pakistan. All the dead cadavers were washed with 75% ethanol for 30 sec, rinse with distilled water and transferred to 3% NaClO for 1 min, followed by rinse in distilled water for 2 times. Cadavers were then dried on filter paper and placed in petri dishes containing either Sabouraud Dextrose Agar (Merck, Germany) or Potato Dextrose Agar (BD, France) supplemented with 0.1 g liter-1 of streptomycin sulfate. Plates were sealed with parafilm and incubated at 25°C for 7 days. Insects were examined under a microscope every 24 h for the appearance of any fungal outgrowth on the medium (Wakil et al. 2014). Where more than one fungal colony was present on the medium, the culture was purified following single spore method (Choi et al. 1999). The identification of the isolated fungi was done with the taxonomic keys (Barnett & Hunter 1999; Domsch et al. 2007). The culture was sub-cultured and stored in petri dishes at 4°C in refrigerator. Spore concentration was determined with an improved Neubauer haemocytometer and the conidial viability was confirmed <90% before each assay.

5.2.4 Screening assay Nineteen fungal isolates (Table 4.1) were assayed against larvae and adults of R. ferrugineus. Two conidial concentrations (1×107 and 1×108 conidia ml-1) were employed against 4th instar larvae and adult by dipping method. The larvae were immersed in conidial suspension for 60s and adults for 90s (Dembilio et al., 2010b). After treatment insects were transferred to the moistened filter paper for 24 h and shifted individually to 150 ml plastic cups (6×6 cm). The cups were covered with screening lids and treated larvae were offered with artificial diet (Martín and Cabello 2006) and adults with shredded sugarcane pieces (3×3 cm). The control individuals were treated with 0.01% Tween-80 solution. Each treatment consisted of 10 individuals and three replicates in each treatment, a total of 30 insects per treatment were used. Mortality was checked daily for 12 days and dead individuals were transferred to SDA medium for 10 days to observe mycosed individuals. The whole experiment was repeated twice.

5.2.5 Virulence assay Potential strains which showed high pathogenicity in preliminary screening assays (WG- 41, WG-42, WG-43, WG-44 and WG-45) were further tested against both larvae and adult of R. ferrugineus at 1×106, 1×107, 1×108 and 1×109 conidia ml-1. Ten 6th instar larvae were dipped in a conidial suspension for 60s and adults for 90s and air dried in sterile petri dish lined with damp filter paper for 24 h and transferred to the plastic cups individually (Dembilio et al., 2010b). The conditions in an incubator were adjusted to a photoperiod 12:12 h (L: D) and 65±5% RH at 25±2°C. Then the larvae were allowed to feed on artificial diet (Martín and Cabello, 2006) in 150 ml plastic cups (6×6 cm) individually and adults with shredded sugarcane pieces (3×3 cm). The larval mortality was recorded after 7, 14 and 21 days. For control treatment the individuals

92 were immersed in distilled water containing 0.01% Tween-80. Three replicates were used for each treatment and entire experiment was repeated thrice independently. Median lethal concentration LC50, LC90 and lethal time LT50, LT90 were calculated for different exposure intervals.

5.2.6 Statistical analysis Mortality for each treatment was corrected for control mortality using Abbott's (1925) formula and subjected to one way analysis of variance (ANOVA) in Minitab (Minitab, 2002) using Tukey’s Kramer test (HSD) (Sokal and Rohlf, 1995) at 5% significance level. Probit analysis was used to estimate the LC50, LC90, LT50 and LT90 of isolates using Confidence Limit 95% (CL).

5.3 Results

5.3.1 Screening assay The results revealed that all the tested isolates of B. bassiana and M. anisopliae were pathogenic to larvae and adult of R. ferrugineus under laboratory conditions. Overall both fungal isolates inflicted greater mortality of R. ferrugineus larvae (Table 5.3) compared to the adults (Table 5.4). For example, five isolates caused highest mortality ranging 65.55-88.33% at 1×108 conidia ml-1 after 12 days post incubation, whereas adult mortality was ranged between 46.03- 75.95% at the same concentration. All the fungal isolates differ significantly in their virulence 7 -1 8 against larvae (F18, 113 = 31.2, P≤0.05) at 1×10 conidia ml and (F18, 113 = 56.8, P≤0.05) at 1×10 -1 7 -1 conidia ml and for beetles (F18, 113 = 41.4, P≤0.05) at 1×10 conidia ml and (F18, 113 = 46.8, P≤0.05) at 1×108 conidia ml-1. More numbers of mycosed individuals were recorded from the treatments where lower spore concentration (1×107 conidia ml-1) was applied than higher concentration 8 -1 (1×10 conidia m l ). Mycosis in larvae was also significantly different (F18, 113 = 757, P≤0.05) at 7 -1 8 -1 1×10 conidia ml and (F18, 113 = 433, P≤0.05) at 1×10 conidia ml and for beetles (F18, 113 = 763, 7 -1 8 -1 P≤0.05) at 1×10 conidia ml and (F18, 113 = 423, P≤0.05) at 1×10 conidia ml . Main effects and their associated interactions for mortality and mycosis were significantly different (Table 5.2). Among five most virulent isolates three belonged to B. bassiana (WG-41, WG-42 and WG-43) and two to M. anisopliae (WG-44 and WG45). These five isolates were selected for further virulence assays.

5.3.2 Virulence assay In virulence assay the mortality of larvae and adult was recorded after 7, 14 and 21 days after application. Until 7th day of application no isolate caused 100% mortality in either larvae or adult, highest activity was recorded for WG-41 76.95% and 63.96% in larvae and adult respectively at highest spore concentration (Table 5.6). After last count all isolates caused 100% mortality both in larvae and adult at highest dose rate used except WG-43 and WG-45 which caused 98.41and 91.83 % adult mortality respectively. Overall WG-41 was the most virulent isolate followed by WG-42, WG-44, WG-43 and WG-45. The mortality of larvae were significantly affected by the dose rates of fungal isolates at 5days exposure (WG-41: F3, 35 = 71.1, P≤0.05; WG-42: F3, 35 = 101, P≤0.05; WG-43: F3, 35 = 63.3, P≤0.05; WG-44: F3, 35 = 105, P≤0.05 and WG-45: F3, 35 = 60.8, P≤0.05). Similarly significant difference was also recorded in case of adults at this exposure (WG-41: F3, 35 = 133, P≤0.05; WG-42: F3, 35 = 108, P≤0.05; WG-43: F3, 35 = 106, P≤0.05; WG-44: F3, 35 = 56.6, P≤0.05; WG-45: F3, 35 = 58.3, P≤0.05). After 14 days post

93 application, only WG-41 exhibited 100% larval mortality at highest dose rate used (Table 5.7), while highest adult mortality (93.22%) was also recorded for the same isolate at highest fungal dose rate (Table 5.8). Significant differences have been observed between the mortalities of the larvae at all the dose rates (WG-41: F3, 35 = 220, P≤0.05; WG-42: F3, 35 = 193, P≤0.05; WG-43: F3, 35 = 120, P≤0.05; WG-44: F3, 35 = 170, P≤0.05; WG-45: F3, 35 = 203, P≤0.05). Significant differences were also recorded in case of adults at this exposure (WG-41: F3, 35 = 166, P≤0.05; WG-42: F3, 35 = 162, P≤0.05; WG-43: F3, 35 = 112, P≤0.05; WG-44: F3, 35 = 172.0, P≤0.05; WG-45: F3, 35 = 135, P≤0.05). After last count, all tested isolates caused 100% mortality both in larvae and adult except WG-43 and WG-45, and significant differences were observed between all the tested isolates for larvae (WG-41: F3, 35 = 138, P≤0.05; WG-42: F3, 35 = 245, P≤0.05; WG-43: F3, 35 = 179, P≤0.05; WG-44: F3, 35 = 168, P≤0.05; WG-45: F3, 35 = 223, P≤0.05) and adults (WG-41: F3, 35 = 164, P≤0.05; WG-42: F3, 35 = 312, P≤0.05; WG-43: F3, 35 = 261, P≤0.05; WG-44: F3, 35 = 165, P≤0.05; WG-45: F3, 35 = 271.0, P≤0.05). Factorial analysis shows that the main effects were significant, while their associated interactions Isolate × Treatment and Isolate × Interval for larvae were non-significant and Isolate × Interval were also non-significant for adults (Table 5.5). The results of our study revealed that all isolates of B. bassiana and M. anisopliae are virulent to larvae and adults of R. ferrugineus at the dose rate of 106, 107, 108, and 109 conidia -1 th ml . The lethal concentrations LC50 and LC90 at the 15 day after treatment were assessed (Table 5.9). The isolates (WG-41 and WG-42) exhibited half of the larval mortality with a 5 -1 concentration of 10 conidia ml and isolates (WG-43, WG-44 and WG-45) revealed LC50 values at 106 conidia ml-1. While half of the adult mortality for all the tested isolates were recorded for a concentration of 106 conidia ml-1. The isolates WG-41 and WG-42 caused a high percentage of adult mortality, thus requiring a lower concentration of fungal conidia to cause an average percentage of adult or larval mortality; hence, these isolates were considered the most virulent among all the isolates investigated in this study. The increased virulence, shown by the reduction in LT50, increased mortality and proportion of mycosed cadavers were closely related to the conidial concentration. LT 50 6 9 -1 decreased when conidial concentration increased from 10 -10 conidia ml . Estimated LT50 of all fungal isolates against R. ferrugineus larvae varied from 17.75 to 27.92 days at 106 conidia ml-1, while less time 5.27 to 7.82 days were recorded in case of highest dose rate used (Table 5.10). In case of estimated LT50 of all fungal isolates against R. ferrugineus adults varied from 20.41 to 30.06 days at 106 conidia ml-1, while less time 4.93 to 9.57 days were recorded in case of 109 conidia ml-1 (Table 5.11). Overall WG-41 was considered most virulent isolate against both larvae and adult which inflicted highest adult and larval mortality at almost all the dose rate used within short period of time as compared to the other isolates used.

5.4 Discussion Fungal entomopathogens are important biological control agents against insect pests worldwide and have been the subject of intense research for more than 100 years (Vega et al., 2012). Some of the advantages offered by the use of entomopathogenic fungi in microbial control programs are their specificity, contact transmission, natural dispersion, safety for non- target organisms and the ability to maintain lasting control once established in the environment (Van Driesche et al., 2007). Laboratory screening of fungal isolates bring down to manageable number is a vital step in identifying virulent strains prior to field use (Cherry et al., 2005). The two stage approach to screening adopted in this study proved a robust mechanism that has been

94 used effectively by many other workers (Moino et al., 1998; Kassa et al., 2002). In our studies, a panel of 19 isolates that were never screened were selected and selected 5 isolates based on their pathogenicity potential for critical assays in vivo which exhibited ˃ 85% and ˃ 75% larval and adult mortality respectively. Via this hierarchical approach we ultimately identified three B. bassiana (WG-41, W-42 and WG-43) and two M. anisopliae (WG-44, WG-45) isolates, 5 -1 exhibited shortest LC50 of 10 conidia ml with potential as a novel bio-pesticide for use against R. ferrugineus. The conidial viability is very important in the host infection process because it permits success at the beginning of the early stages of fungal infection process of the host cuticle, followed by conidial germination and the formation of a germ tube (Schrank and Vainstein, 2010). In the present study, the conidia viability of the 19 isolates was 100%, which ensured the quality of conidia present in the fungal suspensions used to treat R. ferrugineus larvae. The re- isolation of fungal isolates after treatment confirmed the infection capacity of the studied isolates. Our results indicated the B. bassiana isolates (WG-41, WG-42) exhibited high larval and adult mortality, suggesting an enormous potential for this fungal species to be used for pest control. Moreover, five isolates presented the better results, with average almost 100% larval and adult mortality by the 21st day post treatment at highest dose rate used and they should be considered as ideal isolates to be used in formulations for field studies, exhibited LC50 of high virulence range within 6.47×105-3.66×106 conidia ml-1 for larvae and 1.04×106-9.30×106 conidia -1 ml . These LC50 values are practical for the development of a myco-insecticide aimed to control R. ferrugineus in integrated management programs. The individuation of virulent strains of entomopathogenic fungi towards the R. ferrugineus in the countries of introduction represent a precious opportunity to increase studies on the microbiological control efficacy in view of a possible field applications. Within fungal taxa, individual isolates can exhibit substantially restricted host range (Inglis et al., 2001) and isolates recovered from a target host and closely related species are generally more virulent than isolates from non-related species or from soil. Ricaño et al. (2013) reported high efficacy of B. bassiana isolates recovered from RPW as compared to the other sources (Insects and soil samples). Moreover, B. bassiana strains recovered from 30 day old RPW were most pathogenic strains among all tested isolates. These isolates were also the most virulent on RPW adults. Lo Verde et al. (2014) also reported the significantly more pathogenic action of RPW isolated B. bassiana strains. El-Sufty et al. (2009) obtained a mortality of 12.8-47.1% in adult R. ferrugineus population in field assays using a strain of B. bassiana isolated in the United Arab Emirates. These published reports are supportive to our findings as our results showed that B. bassiana isolates (WG-41, WG-42) recovered from RPW cadavers inflicted highest mortality. Further corroborating our results, an indigenous strain of B. bassiana obtained from mycosed RPW collected in field showed good results in laboratory and field tests (El-Sufty et al., 2007; Sewify et al., 2009). Our results are in accordance with Cherry et al. (2005) who reported, indigenous isolates that had been recovered from C. maculatus were more virulent in laboratory bioassays against C. maculatus than exotic isolates from other insects. Similarly Goettel et al. (1990) reported, a fungal isolate may be more pathogenic against the host from which it was obtained than to other novel hosts. Contrarily, Monteiro et al. (1998a, b) reported that an ant derived isolate was more effective against ticks than a tick derived isolate. Ángel-Sahagún et al. (2010) indicate that isolates with high pathogenicity against ticks could be found from samples derived from soil on the last instars of the great wax moth, Galleria mellonella. Fernandes et al. (2011), observed that

95 several B. bassiana isolates obtained from naturally infected ticks were not significantly more virulent against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) than isolates obtained from other arthropod orders. With regard to entomopathogenic fungi important factors in reaching these goals are: intra alia, the availability of an isolate highly virulent towards the target insect, suitability for mass spore production on an appropriate contamination substratum, an efficient delivery system and inoculum stability and germinability in field conditions (Ibrahim et al., 1999; Zhang et al., 2011). On contrary, Gindin et al. (2006) observed high mortality of R. ferrugineus adults treated with dry spores of M. anisopliae than the B. bassiana treatments after two weeks. Tests carried out with the experimental traps showed that M. anisopliae was the more virulent pathogen, causing 75% cumulative mortality in R. ferrugineus adults, while B. bassiana gave a 45% cumulative mortality (Francardi et al., 2103). Similar findings were reported by the Francardi et al. (2012) who evaluated field collected strain of B. bassiana and M. anisopliae isolated in Italy from naturally infected R. ferrugineus adults, and tested under laboratory conditions. M. anisopliae obtained from R. ferrugineus showed the highest efficacy against RPW larvae and adults which showed values of cumulative larval mortality of 100% and adult mortality of 90%. Our study indicated that the indigenous strain of B. bassiana recovered from R. ferrugineus were more infective to larvae and adult of R. ferrugineus. This study was further supported by the findings of Dembilio et al. (2010b) who reported potential effect of indigenous strain of B. bassiana against different developmental stages of R. ferrugineus. The use of entomopathogenic fungi, in particular indigenous strains of B. bassiana and M. anisopliae, obtained from naturally infected weevils, should be seriously considered for biological control because both have provided encouraging results for the control of certain economic pests (Jaronski, 2010). This suggests that the identification of indigenous entomopathogenic fungi already active on the weevil may offer better prospects for its biological control.

Conclusion The present study showed that B. bassiana and M. anisopliae isolates recovered from the RPW used in laboratory bioassays caused high mortality in larvae and adults compared to the other tested fungal isolates. More number of mycosed individuals was also observed from the same isolates. For these reasons, the use of entomopathogenic fungi can be considered to be useful tool as an integral part of successful IPM program. Moreover, the inoculum of entomopathogenic fungi may be transmitted by R. ferrugineus due to their low mortality which may further be helpful in reducing RPW population in date palm systems, further research is needed to support this thought.

Acknowledgements This research work was supported by Higher Education Commission, Islamabad (Pakistan) (2AV1-263) under Indigenous Ph.D. Fellowship Program.

5.5 References Abraham V.A., A.S., Mahmood, J.R. Faleiro, R.A. Abozuhairah and P.S.P.V. Vidyasagar, 1998. An integrated approach for the management of red palm weevil Rhynchophorus ferrugineus Oliv. A key pest of date palm in the Middle East. Sultan Qaboos University, J. Sci. Res. Agric. Sci., 3: 77-83. Al-Rajhy, D.H., H.I. Hussein and A.M.A. Al-Shawaf, 2005. Insecticidal activity of carbaryl and its mixture with piperonylbutoxide against red palm weevil Rhynchophorus ferrugineus (Olivier) (Curculionidae: Coleoptera) and their effects on acetylcholinesterase activity. Pak. J. Biol. Sci., 8: 679-682. Ángel-Sahagún, C.A., R. Lezama-Gutiérrez, J. Molina-Ochoa, A. Pescador-Rubio, S.R. Skoda, C. Cruz-Vázquez, A.G. Lorenzoni, E. Galindo-Velasco, H. Fragoso-Sánchez and J.E. Foster, 2010. Virulence of Mexican isolates of entomopathogenic fungi (Hypocreales: Clavicipitaceae) upon Rhipicephalus = Boophilus microplus (Acari: Ixodidae) larvae and the efﬁcacy of conidia formulations to reduce larval tick density under ﬁeld conditions. Vet. Parasitol., 170: 278-286. Baloach, H.B., M.A. Rustamani, R.D. Khuro, M.A. Talpur and T. Hussain, 1992. Incidence and abundance of date palm weevil in different cultivars of date palm. Proc. 12th Cong. Zool. Soc. Pak., 12: 445-447. Banerjee, A. and T.K. Dangar, 1995. Pseudomonas aeruginosa, a facultative pathogen of red palm weevil, Rhynchophorus ferrugineus. World J. Microbiol. Biotech., 11: 618-620. Barnett, H.L. and B.B. Hunter, 1999. Illustrated Genera of Imperfect Fungi, 4th Edn. APS Press, The American Phytopathological Society, St. Paul, MN. p 218. Barranco, P., J.A. De La Peña, M.M. Martin and T. Cabello, 2000. Rango de hospedantes de Rhynchophorus ferrugineus (Olivier, 1790) y diametro de la palmera hospedante. (Coleoptera, Curculionidae). Boletín de Sanidad Vegetalde Plagas, 26: 73-78. Cabello, T.P., J.A. De La Peña, P. Barranco and J. Belda, 1997. Laboratory evaluation of imidacloprid and oxamyl against Rhynchophorus ferrugineus. Test. Agrochem. Culti., 18: 6-7. Carruters, R.I. and R.S. Soper, 1987. Fungal Diseases. In Fuxa, J.R. and Y.T. Tanada, (eds.) Epizootology of Insect Diseases. Wiley and Sons, New York. pp. 357-416. Cherry, A.J., P. Abalo and K. Hell, 2005. A laboratory assessment of the potential of different strains of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) to control Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) in stored cowpea. J. Stored Prod. Res., 41: 295-309. Choi, I., R.E. Nisbett and A. Norenzayan, 1999. Causal attribution across cultures: Variation and universality. Psychol. Bull., 125: 47-63. Cito, A., G. Mazza, A. Strangi, C. Benvenuti, G.P. Barzanti, E. Dreassi, T. Turchetti, V. Francardi and P.F. Roversi, 2014. Characterization and comparison of Metarhizium strains isolated from Rhynchophorus ferrugineus. FEMS Microbiol. Lett., 355: 108-115. Deadman, M.L., K.M. Azam, S.A. Ravzi and W. Kaakeh, 2001. Preliminary investigation into the biological control of the red palm weevil using Beauveria bassiana. Proc. 2nd Inter. Conf. Date Palm, Al-Ain, UAE. March 25-27. pp. 225-232. Dembilio Ó., E. Llácer, M.M. Martínez de Altube and J.A. Jacas, 2010a. Field efficacy of imidacloprid and Steinernema carpocapsae in a chitosan formulation against the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) in Phoenix canariensis. Pest Manag. Sci., 66: 365-370.

Dembilio Ó., E. Quesada-Moraga, C. Santiago-Álvarez and J.A. Jacas, 2010b. Biocontrol potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana (Ascomycota; Hypocreales) against the red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae). J. Inver. Pathol., 104: 214-221. Domsch, K.H., W. Gams and T.H. Anderson, 2007. Compendium of soil fungi. 2. ed. London: Lubrecht & Cramer Ltd. El-Sufty, R., S. Al Bgham, S.A. Al-Awash, A.S. Shahdad and A.H. Al Bathra, 2011. A trap for auto-dissemination of the entomopathogenic fungus Beauveria bassiana by the red palm weevil adults in date palms plantations. Egyp. J. Biol. Pest Control, 21(2): 271-276. El-Sufty, R., S.A. Al-Awash, A.M. Al-Amiri, A.S. Shahdad, A.H. Al-Bathra and S.A. Musa, 2007. Biological control of red palm weevil, Rhynchophorus ferrugineus (Col.: Curculionidae) by the entomopathogenic fungus Beauveria bassiana in United Arab Emirates. Proc. of the 3rd Inter. Conf. Date Palm. Acta Horti., 736: 399-404. El-Sufty, R., S.A. Al-Awash, S. Al Bgham, A.S. Shahdad, A.H. Al-Bathra, 2009. Pathogenicity of the fungus Beauveria bassiana (Bals.) Vuill to the red palm weevil, Rhynchophorus ferrugineus (Oliv.) (Col.: Curculionidae) under laboratory and field conditions. Egyp. J. Biol. Pest Control, 19: 81-85. EPPO. (European and Mediterranean Plant Protection Organization), 2009a. EPPO Reporting Service. 2009/002 - First record of Rhynchophorus ferrugineus in Curaçao, Netherlands Antilles January 26, 2009. EPPO. (European and Mediterranean Plant Protection Organization) 2009b. Rhynchophorus ferrugineus found on Howea forsteriana in Sicilia, Italy. No. 3 2009/051. European and Mediterranean Plant protection Organization, Paris, France. Available at: https://archives.eppo.int/EPPOReporting/2009/Rse-0912.pdf. Accessed on: 12 June 2015. EPPO. (European and Mediterranean Plant Protection Organization) 2010. EPPO Reporting Service. 2010/176 - First record of Rhynchophorus ferrugineus in the USA, November 1, 2010. EPPO. (European and Mediterranean Plant Protection Organization) 2006. Reporting Service, No.112006-11-01/2006/225, First record of Rhynchophorus ferrugineus in France and 2006/226, First report of Rhynchophorus ferrugineus in Greece and 2006/226, Rhynchophorus ferrugineus found in Lazio region, Italy. Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm during the last one hundred years. Inter. J. Tropi. Insect Sci., 26: 135-154. Fernandes, E.K.K., I.C. Angelo, D.E.N. Rangel, T.C. Bahiense, A.M.L. Moraes, D.W. Roberts and V.R.E.P. Bittencourt, 2011. An intensive search for promising fungal biological control agents of ticks, particularly Rhipicephalus microplus. Vet. Parasitol., 182: 307- 318. Francardi, V., C. Benvenuti, P.F. Roversi, P. Rumine and G. Barzanti, 2012. Entomopathogenicity of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorokin isolated from different sources in the control of Rhynchophorus ferrugineus (Olivier) (Coleoptera Curculionidae). Redia, 95: 49-55. Francardi, V., C. Benventi, G.P. Barzanti and P.F. Rovers, 2013. Auto contamination trap with entomopathogenic fungi: A possible strategy in the control of Rhynchophorus ferrugineus (olivier) (Coleoptera Curculionidae). REDIA, XCVI: 57-67.

Gerber, K. and R.M. Giblin-Davis, 1990. Association of the red ring nematode and other nematode species with the palm weevil, Rhynchophorus palmarum. J. Nematol., 22: 143- 149. Ghazavi, M. and A. Avand-Faghih, 2002. Isolation of two entomopathogenic fungi on red palm weevil, Rhynchophorus ferrugineus (Olivier) (Col., Curculionidae) in Iran. Appl. Entomol. Phytopathol., 9: 44-45. Gindin, G., S. Levski, I. Glazer and V. Soroker, 2006. Evaluation of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana against the red palm weevil Rhynchophorus ferrugineus. Phytoparasitica, 34(4): 370-379. Goettel, M.S., T.J. Poprawski, J.D. Vandenberg, Z. Li and D.W. Roberts, 1990. Safety to non- target invertebrate of fungal bicontrol agents. p 209-232. In: Laird, M., L.A. Lacey and E.W. Davidson (eds.). Safty of Microbial insecticides. Boca Raton, CA: CRS Press. Güerri-Agulló, B., S. Gómez-Vidal, L. Asensio, P. Barranco and L.V. Lopez-Llorca, 2010. Infection of the red palm weevil (Rhynchophorus ferrugineus) by the entomopathogenic fungus Beauveria bassiana: a SEM study. Microscopy Res. Tech., 73: 714-725. Hussain, A., M. Rizwan-Ul-Haq, H. Al-Ayedh, S. Ahmed and A.M. Al-Jabr, 2015. Effect of Beauveria bassiana infection on the feeding performance and antioxidant defence of red palm weevil, Rhynchophorus ferrugineus. BioControl, 60: 849-859. Hussain, A., M.R.U. Haq, A.M. Al-Jabr and H.Y. Al-Ayied, 2013. Managing Invasive Populations of Red Palm Weevil: A Worldwide Perspective. J. Food Agri. Environ., 11: 456-463. Hussain, A., M.Y. Tian, Y.R. He and S. Ahmed, 2009. Entomopathogenic fungi disturbed the larval growth and feeding performance of Ocinara varians Walker (Lepidoptera: Bombycidae) larvae. Insect Sci., 16: 511-517. Hussain, A., M.Y. Tian, Y.R. He, J.M. Bland and W.X. Gu, 2010 Behavioral and electrophysiological responses of C. formosanus towards entomopathogenic fungal volatiles. Biol. Control, 55: 166-173. Hussain, A., S. Ahmed and M. Shahid, 2011. Laboratory and field evaluation of Metarhizium anisopliae var. anisopliae for controlling subterranean termites. Neotrop. Entomol., 40(2): 244-250. Inglis, D.G., D.L. Johnson and M.S. Goettel, 1996. Effect of bait substrate and formulation on infection of grasshopper nymphs by Beauveria bassiana. Biocontrol Sci. Tech., 6: 35-50. Inglis, G.D., M.S. Goettel, T.M. Butt and H. Strasser, 2001. Use of hyphomycetous fungi for managing insect pests. p 23-69. In: Butt, T.M., C.W. Jackson and N. Magan (eds), Fungi as Biocontrol Agents: Progress, Problems and Potential. CABI International/ AAFC, Wallingford, United Kingdom. Jalinas, J., B. Güerri-Agulló, R.W. Mankin, R. López-follana and L.V. Lopez-Llorca, 2015. Acoustic assessment of Beauveria bassiana (Hypocreales: Clavicipitaceae) effects on Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) larval activity and mortality. J. Eco. Entomol., 108(2): 444-453. James, R.R., J.S. Buckner and T.P. Freeman, 2003. Cuticular lipids and silverleaf whitefly stage affect conidial germination of Beauveria bassiana and Paecilomyces fumosoroseus. J. Inver. Pathol., 84(2): 67-74. Jaronski, S.T., 2010. Ecological factors in the inundative use of fungal entomopathogens. Biocontrol, 55: 159-185.

Kassa, A., G. Zimmermann, D. Stephan and S. Vidal, 2002. Susceptibility of Sitophilus zeamais (Motsch.) (Coleoptera: Curculionidae) and Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) to entomopathogenic fungi from Ethiopia. Biocontrol Sci. Tech., 12: 727- 736. Llácer, E. and J.A. Jacas, 2010. Short communication. Efficacy of phosphine as a fumigant against Rhynchophorus ferrugineus (Coleoptera: Curculionidae) in palms. Spanish J. Agri. Res., 8(3): 775-779. Llácer, E., Ó. Dembilio and J.A. Jacas, 2010. Evaluation of the efficacy of an insecticidal paint based on chlorpyrifos and pyriproxyfen in a microencapsulated formulation against Rhynchophorus ferrugineus (Coleoptera: Curculionidae). J. Econ. Entomol., 103: 402- 408. Lo Verde, G., L. Torta, V. Mondello, C.G. Caldarella, S. Burruano and V. Caleca, 2014. Pathogenicity bioassays of isolates of Beauveria bassiana on Rhynchophorus ferrugineus. Pest Manag. Sci., 71: 323-328. Manachini, B., P. Lo Bue, E. Peri and S. Colazza, 2009. Potential effects of Bacillus thuringiensis against adults and older larvae of Rhynchophorus ferrugineus. IOBC/ wprs Bull., 45: 239-242. Martín, M.M. and T. Cabello, 2006. Manejo de la cría del picudo rojo de la palmera, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera, Dryophthoridae), en dieta artificial y efectos en su biometría y biología. Boletín de Sanidad Vegetal de Plagas, 32: 631-641. Merghem, A., 2011. Susceptibility of the red palm weevil, Rhynchophorus ferrugineus (Olivier) to the green muscardine fungus, Metarhizium anisopliae (Metsch.) in the laboratory and in palm tree orchards. Egyp. J. Biol. Pest Control, 21: 179-183. Mohan, C.M., K.A. Lakshmi and K.U. Devi, 1999. Laboratory evaluation of the pathogenicity of three isolates of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin on the American cockroach (Periplaneta americana). Biocontrol Sci. Tech., 9(1): 29-33. Moino Jr, A., S.B. Alves and R.M. Pereira, 1998. Efficacy of Beauveria bassiana (Balsamo) Vuillemin isolates for control of stored grain pests. J. Appl. Entomol., 122: 301-305. Monteiro, A.C., A.C. Fiorin and A.C.B. Correia, 1998a. Pathogenicity of isolates of Metarhizium anisopliae (Metsch.) Sorokin towards the cattle tick Boophilus microplus (Can.) (Acari: Ixodidae) under laboratory conditions. Rev. Microbiol., 29: 109-112. Monteiro, S.G., W.R.E.P. Bittencourt, E. Daemon and J.L.H. Faccini, 1998b. Pathogenicity under laboratory conditions of the fungi Beauveria bassiana and Metarhizium anisopliae on larvae of the tick Rhipicephalus sanguineus (Acari: Ixodidae). Revista brasileira de parasitologia veterinaria, 7: 113-116. Murphy, S.T. and B.R. Briscoe, 1999. The red palm weevil as an alien invasive : Biology and the prospect for biological control as component of IPM. Bio-control News Info., 20: 35-46. Quesada-Moraga E, R. Santos-Quiros, P. Valverde-Garcia and C. Santiago-Álvarez, 2004. Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (anamorphic fungi) on the German cockroach (Blattodea: Blattellidae). J. Inver. Pathol., 87: 51-58. Ricaño J., B. Güerri-Agulló, M.J. Serna-Sarriás, G. Rubio-Llorca, L. Asensio, P. Barranco, L.V. Lopez-Llorca, 2013. Evaluation of the pathogenicity of multiple isolates of Beauveria bassiana (Hypocreales: Clavicipitaceae) on Rhynchophorus ferrugineus (Coleoptera:

100

Dryophthoridae) for the assessment of a solid formulation under simulated field conditions. Florida Entomol., 96: 1311-1324. Sabbour, M.M. and N.Y. Solieman, 2014. Preliminary Investigations into the Biological Control of Red Palm Weevil Rhynchophorus ferrugineus by using three isolates of the fungus Lecanicillium (Verticillium) lecanii in Egypt. Inter. J. Sci. Res., 3(8): 2016-2066. Salama, H.S., M.S. Foda, M.A. El-Bendary and A. Abdel-Razek, 2004. Infection of red palm weevil Rhynchophorus ferrugineus, by spore-forming bacilli indigenous to its natural habitat in Egypt. J. Pest Sci., 77: 27-31. Schrank, A. and M.H. Vainstein, 2010. Metarhizium anisopliae enzymes and toxins. Toxicon, 56: 1267-1274. Sevim, A., B.G. Donzelli, D. Wu, Z. Demirbaq, D.M. Gibson and B.G. Turqueon, 2012. Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence. Current Genetics, 58: 79-92. Sewify, G.H., M.H. Belal and S.A. Al-Awash, 2009. Use of the entomopathogenic fungus, Beauveria bassiana for the biological control of the Red Palm Weevil, Rhynchophorus ferrugineus Olivier. Egyp. J. Biol. Pest Control, 19(2):157-163. Shah, F.A., M.A. Ansari, M. Prasad and T.M. Butt, 2007. Evaluation of black vine weevil (Otiorhynchus sulcatus) control strategies using Metarhizium anisopliae with sublethal doses of insecticides in disparate horticultural growing media. Biol. Control. 40: 246-252. Shaiju-Simon, K.R.K. and C. Gokulapalan, 2003. Occurrence of Beauveria sp. on red palm weevil, Rhynchophorus ferrugineus (Oliv.) of coconut. Insect Environ., 9: 66-67. Shawir, M.S. and A.M. Al-Jabr, 2010. The infectivity of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to Rhynchophorus ferrugineus (Olivier) stages under laboratory conditions. Proceeding of the 4th International Date Palm Conference, Acta Horti., 882: 431-436. Singh, S.P., and P. Rethinam, 2005. Trapping-a major tactic of BEPM strategy of palm weevils. Cord., 21(1): 57-79. Torta, L., V. Leone, C.G. Caldarella, G. Lo Verde and S. Burruano, 2009. Microrganismi fungini associati a Rhynchophorus ferrugineus (Olivier) in Sicilia e valutazione dell’efficacia entomopatogena di 484 Ann Microbiol., 65: 477-485. Van Driesche, R.G., M.S. Hoddle and T.D. Center, 2007. Use of Insect Pathogens as Pesticides in Control of Pests and Weeds by Natural Enemies. p 443-462. In: Van Driesche, R.G. and M.S. Hoddle (eds.). Center TD Forest Health Technology Enterprise Team, New York. Vega, F.E., N.V., Meyling, J.J. Luangsa-Ard, and M. Blackwell, 2012. Fungal Entomopathogens. P. 171-220. In: Vega F.E. and H.K. Kaya (eds.). Insect Pathology London: Elsevier. Vitale, A, V. Leone, L. Torta, S, Burruano and G. Polizzi, 2009. Prove preliminari di lotta biologica con Beauveria bassiana e Metarhizium anisopliae nei confronti del punteruolo rosso. In Regione Siciliana - Assessorato Agricoltura e Foreste. La ricerca scientifica sul Punteruolo rosso e gli altri fitofagi delle palme in Sicilia, Palermo, Italy, 1:169-172. Wakil, W., M.U. Ghazanfar and M. Yasin, 2014. Naturally occurring entomopathogenic fungi infecting stored grain insect species in Punjab, Pakistan. J. Insect Sci., 14(182): 1-7.

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Zhang, H., W. Yin, J. Zhao, L. Jin, Y. Yang, S. Wu, B.E. Tabashnik and Y. Wu, 2011. Early warning of cotton bollworm resistance associated with intensive planting of Bt cotton in China. PLoS ONE, 6(8): e22874.

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Table 5.1 Characterization of B. bassiana and M. anisopliae isolates obtained from soils and insect cadavers

Species Isolate Host/substrate Location Geographical attributes No. Altitude (m)* Latitude* Longitude* Metarhizium anisopliae WG-02 Soil (Vegetables) Changa Manga 191 31°08′N 73°96′E Metarhizium anisopliae WG-03 Tribolium castaneum Murree 2300 33°56'N 73°28'E Metarhizium anisopliae WG-04 Soil (Vegetables) Chichawatni 159 30°53′N 72°70′E Metarhizium anisopliae WG-05 Rhyzopertha dominica Khanewal 128 30°71'N 71°55'E Metarhizium anisopliae WG-06 Soil (Forests) Lal Sohanra 114 29°28′N 71°58′E Metarhizium anisopliae WG-07 Soil (Forests) Bahawalpur 109 29°24'N 71°40'E Metarhizium anisopliae WG-10 Soil (Crop ﬁelds) Rawalpindi 497 33°58′N 73°08′E Beauveria bassiana WG-11 Soil (Crop ﬁelds) Lal Sohanra 114 29°28′N 71°58′E Beauveria bassiana WG-12 Soil (Fruits) Chichawatni 159 30°53′N 72°70′E Beauveria bassiana WG-13 Sitophilus oryzae Changa Manga 191 31°08'N 73°96'E Beauveria bassiana WG-15 Soil (Forests) Faisalabad 184 31°30′N 73°05′E Beauveria bassiana WG-16 Tribolium castaneum Sargodha 193 32°10'N 72°40'E Beauveria bassiana WG-17 Callosobruchus maculates Gujranwala 223 32°10'N 72°12'E Beauveria bassiana WG-18 Soil (Forests) Rawalpindi 497 33°58′N 73°08′E Beauveria bassiana WG-41 Rhynchophorus ferrugineus Layyah 143 30°58'N 70°56'E Beauveria bassiana WG-42 Rhynchophorus ferrugineus Dera Ismail Khan 166 31°49'N 70°52'E Beauveria bassiana WG-43 Rhynchophorus ferrugineus Bahawalpur 109 29°24'N 71°40'E Metarhizium anisopliae WG-44 Rhynchophorus ferrugineus Layyah 143 30°58'N 70°56'E Metarhizium anisopliae WG-45 Rhynchophorus ferrugineus Muzaffargarh 114 30°50'N 71°54'E *Geographical attributes based on web source indicating the nearest point

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Table 5.2 Factorial analysis of screening and mycosis of R. ferrugineus exposed to B. d M. anisopliae isolates

Mortality Mycosis S.O.V. Df Larvae Adult Larvae Adult F P F P F P F P Treatment 1 158.20 ≤0.05 733.72 ≤0.05 178.89 ≤0.05 733.72 ≤0.05 Isolate 18 146.69 ≤0.05 110.84 ≤0.05 18.28 ≤0.05 110.84 ≤0.05 Treatment × 18 29.90 ≤0.05 8.49 ≤0.05 1.87 0.02 8.49 ≤0.05 Isolate Error 185 ------Total 227 ------

Table 5.3 Percentage pathogenicity (%±SE) and mycosis (%±SE) of 19 isolates of B. bassiana and M. anisopliae isolates against R. ferrugineus larvae after 12 days post incubation

Isolate 1×107 Conidia ml-1 1×108 Conidia ml-1 % Mortality % Mycosis % Mortality % Mycosis WG-02 3.49±0.21j 0.00±0.00j 8.01±0.66k 17.8±0.87h WG-03 9.90±0.89ghij 3.50±0.42ij 25.23±1.12hi 47.16±1.68g WG-04 18.35±1.31fghi 12.50±0.56gh 48.25±1.48ef 87.33±1.42bc WG-05 21.69±1.17 33.33±0.71d 55.23±2.16de 93.66±1.35ab WG-06 9.90±0.85ghij 15.83±0.94g 27.61±1.55ghi 63.83±1.90e WG-07 13.75±1.09fghij 21.33±1.22f 35.63±1.62fgh 54.50±1.54f WG-10 15.88±0.98fghij 25.83±0.94e 41.50±1.19efg 73.66±2.04d WG-11 5.46±0.34ij 4.50±0.42i 24.12±1.38hij 42.50±1.17g WG-12 8.96±1.09ghij 10.66±0.84h 29.92±1.52ghi 56.66±1.42f WG-13 16.99±1.08fghij 31.33±1.22d 49.28±1.43ef 88.50±1.70bc WG-15 4.27±0.24j 3.50±0.42ij 9.04±0.39jk 15.33±1.04h WG-16 26.21±1.40def 56.66±1.38c 56.42±2.16cde 95.50±1.78a WG-17 19.46±1.21efgh 32.16±1.25d 46.98±1.70ef 93.33±1.71ab WG-18 7.68±0.53hij 9.50±0.76h 19.44±1.17ijk 41.66±1.28g WG-41 53.71±1.28a 72.50±2.78a 88.33±2.47a 96.50±1.99a WG-42 46.80±1.07ab 65.33±1.45b 79.04±1.54ab 95.66±1.62a WG-43 35.52±1.55bcd 56.66±1.88c 71.27±1.69bc 87.33±1.38bc WG-44 41.33±1.21abc 61.83±1.67b 74.68±2.56ab 84.66±1.67c WG-45 32.11±1.60cde 53.50±1.99c 65.55±1.47bcd 90.66±1.73abc

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Table 5.4 Percentage pathogenicity (%±SE) and mycosis (%±SE) of 19 isolates of B. M. anisopliae isolates against R. ferrugineus adults after 12 days post incubation

Isolate 1x107 Conidia ml-1 1x108 Conidia ml-1 % Mortality % Mycosis % Mortality % Mycosis WG-02 0.00±0.00i 0.00±00m 5.63±0.65k 4.83±0.30l WG-03 4.44±0.22ghi 2.33±0.33lm 20.65±1.12hij 47.50±1.23i WG-04 16.94±1.09def 8.50±0.42jk 32.06±1.37efgh 72.50±1.88ef WG-05 21.58±1.05de 29.66±1.26g 38.88±1.88ef 74.16±2.10rf WG-06 5.55±0.64ghi 6.66±0.66kl 21.82±1.00ghij 43.33±1.40i WG-07 8.96±1.06fghi 11.50±0.69j 23.96±1.63ghi 56.50±1.52h WG-10 10.15±1.10fghi 16.33±1.08i 27.54±1.42fghi 65.83±1.90g WG-11 0.00±0.00i 0.00±00m 17.06±1.09ijk 30.33±1.08j WG-12 3.33±0.37ghi 1.66±0.22m 21.74±1.29ghij 48.50±1.76i WG-13 11.34±0.81efgh 17.50±1.08hi 35.47±1.18efgh 71.33±1.54fg WG-15 0.00±0.00i 0.00±00m 8.968±0.88jk 16.16±1.10k WG-16 21.50±1.21de 36.66±1.14f 43.49±2.01de 78.66±2.52de WG-17 13.65±1.03efg 21.50±0.98h 36.58±1.85efg 77.50±1.85def WG-18 2.22±0.24hi 0.00±00m 13.65±1.07ijk 31.16±1.24j WG-41 41.90±1.88a 78.16±1.19a 75.95±2.78a 95.33±1.62a WG-42 36.19±1.36ab 62.83±1.42b 69.04±2.73ab 87.16±2.12bc WG-43 27.22±1.16bcd 55.83±1.37c 55.00±1.37bcd 91.50±2.14ab WG-44 32.93±1.65abc 49.66±1.54d 59.68±2.93bc 83.33±1.70cd WG-45 24.92±1.04cd 41.50±1.20e 46.03±2.07cde 76.83±1.44ef

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Table 5.5 Factorial analysis for virulence of B. bassiana and M. anisopliae isolates against larvae and adult of R. ferrugineus

S.O.V. df Larvae Adult F P F P Isolate 4 153.27 ≤0.05 163.71 ≤0.05 Treatment 3 2192.69 ≤0.05 2202.71 ≤0.05 Interval 2 2700.31 ≤0.05 2852.28 ≤0.05 Isolate × Treatment 12 1.24 0.25 1.94 0.02 Isolate × Interval 8 0.66 0.72 1.92 ≤0.05 Treatment × Interval 6 90.92 ≤0.05 78.87 ≤0.05 Isolate × Treatment × 24 6.26 ≤0.05 5.60 ≤0.05 Interval Error 472 - - - - Total 539 - - - -

Table 5.6 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 7 days of exposure treated with B. bassiana and M. anisopliae isolates

Stage Isolates Dose (Conidia ml-1) 106 107 108 109 WG-41 19.52±1.11Ca 26.57±1.36Ca 47.17±2.12Ba 76.95±2.41Aa WG-42 16.56±1.27Cab 21.23±1.41Cab 40.29±2.19Bab 65.73±2.28Ab Larvae WG-43 11.21±1.03Cab 15.82±1.25Cb 28.79±1.53Bcd 52.48±1.78Ac WG-44 13.43±1.32Cab 19.75±1.37Cab 36.60±1.26Bbc 58.62±1.90Abc WG-45 8.14±1.15Cb 13.49±1.20Cb 22.71±1.04Bd 43.54±1.50Ad WG-41 12.69±1.32Ca 19.47±1.23Ca 38.30±1.11Ba 63.96±1.78Aa WG-42 9.68±1.10Cab 15.02±1.28Cab 32.22±1.69Bab 54.76±1.66Ab Adult WG-43 5.92±1.03Cab 10.47±1.08Cbc 21.79±1.45Bcd 40.63±1.62 Acd WG-44 8.14±1.20Cab 12.69±1.19Cabc 26.98±1.17Bbc 47.35±2.09Abc WG-45 2.91±0.45Cb 7.46±0.76Cc 18.73±1.14Bd 35.23±1.56Ac

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Table 5.7 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 14 days of exposure treated with B. bassiana and M. anisopliae isolates

Stage Isolates Dose (Conidia ml-1) 106 107 108 109 WG-41 37.35±1.19Ca 58.83±2.22Ba 93.28±1.92Aa 100.0±0.00Aa WG-42 31.95±1.37Dab 49.68±2.06Cab 84.70±2.34Bab 98.46±2.01Aab Larvae WG-43 23.49±1.26Dbc 38.78±1.37Cc 73.12±2.58Bc 92.38±1.78Ab WG-44 27.46±1.18Dbc 43.49±1.73Cbc 78.46±2.84Bbc 95.45±1.59Aab WG-45 19.78±1.07Dc 35.02±1.26Cc 71.69±2.19Bc 84.60±1.34Ac WG-41 28.04±1.14Ca 47.40±1.60Ba 84.76±2.90Aa 93.22±1.97Aa WG-42 21.27±1.46Dab 41.16±1.34Cab 76.98±2.51Bab 86.87±2.26Aa Adult WG-43 14.44±1.08Cbc 35.82±1.34Bbc 67.19±1.98Abc 75.29±2.49Abc WG-44 17.46±1.19Db 38.14±1.48Cbc 70.05±1.54Bb 83.12±2.10Aab WG-45 8.30±0.78Dc 32.06±1.96Cc 57.24±1.46Bc 71.53±1.51Ac

Table 5.8 Mean mortality (%±SE) of larvae and adult of R. ferrugineus after 21 days of exposure treated with B. bassiana and M. anisopliae

Stage Isolates Dose (Conidia ml-1) 106 107 108 109 WG-41 57.67±2.00Ca 88.50±2.15Ba 100.0±0.00Aa 100.0±0.00Aa WG-42 52.16±1.56Cab 83.16±2.00Bab 100.0±0.00Aa 100.0±0.00Aa Larvae WG-43 37.54±1.34Cc 65.93±2.16Bc 94.70±1.86Aa 100.0±0.00Aa WG-44 46.88±2.09Cb 77.75±2.86Bb 98.41±1.64Aa 100.0±0.00Aa WG-45 31.40±1.31Dc 59.10±2.00Cc 85.49±2.47Bb 100.0±0.00Aa WG-41 49.78±2.05Ca 80.79±2.73Ba 100.0±0.00Aa 100.0±0.00Aa WG-42 41.48±1.34Cab 72.32±2.62Bab 96.98±2.13Aab 100.0±0.00Aa Adult WG-43 30.74±1.94Dcd 59.15±1.93Cc 85.55±2.27Bc 98.41±1.94Aa WG-44 38.30±1.55Dbc 68.62±2.32Cb 91.53±2.55Bbc 100.0±0.00Aa WG-45 22.22±1.29Dd 43.86±1.61Cd 76.08±2.15Bd 93.81±2.40Ab

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Table 5.9 LC50 and LC90 values of B. bassiana and M. anisopliae isolates tested against larvae and adult R. ferrugineus

-1 -1 2 Stage Isolate LC50 (Conidia ml ) (CI) LC90 (Conidia ml ) (CI) Slope Intercept ᵪ (df = 2) P WG-41 6.42×105 (3.61×105-9.49×105) 6.52×106 (4.51×106-1.03×107) 0.54±0.07 -6.84 0.53 <0.01 WG-42 9.46×105 (5.67×105-1.39×106) 1.34×107 (8.95×106-2.36×107) 0.47±0.05 -7.95 3.56 <0.01 Larvae WG-43 2.22×106 (1.50×106-3.11×106) 3.90×107 (2.52×107-6.81×107) 0.44±0.06 -10.11 6.98 <0.01 WG-44 1.24×106 (7.92×105-1.76×106) 1.81×107 (1.20×107-3.15×107) 0.46±0.05 -8.65 6.05 <0.01 WG-45 3.63×106 (2.41×106-5.22×106) 1.01×108 (6.21×107-1.80×108) 0.37±0.04 -10.81 4.07 <0.01 WG-41 1.04×106 (6.35×105-1.51×106) 1.53×107 (1.02×105-6.81×104) 0.47±0.05 -8.21 4.68 <0.01 WG-42 1.61×106 (1.02×106-2.35×106) 3.34×107 (2.16×107-5.95×107) 0.42±0.06 -9.44 3.27 <0.01 Adult WG-43 3.92×106 (2.61×106-5.64×106) 1.23×108 (7.55×107-2.30×108) 0.37±0.04 -10.9 4.62 <0.01 WG-44 2.13×106 (1.33×106-3.15×106) 6.25×107 (3.90×107-1.15×108) 0.37±0.03 -9.88 2.16 <0.01 WG-45 9.28×106 (6.51×106-1.25×107) 2.63×108 (1.60×108-5.02×108) 0.38±0.03 -12.24 10.55 <0.01

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Table 5.10 LT50 and LT90 values of B. bassiana and M. anisopliae isolates tested against larvae of R. ferrugineus

-1 -1 2 Isolate Dose LT50 (Conidia ml ) LT90 (Conidia ml ) Slope Intercept x (df =2) P (CI) (CI) 106 17.75 (15.90-20.33) 35.70 (30.34-45.82) 0.07± 0.01 -7.07 0.00 ˂0.01 WG-41 107 11.62 (10.41-12.69) 21.79 (20.04-24.26) 0.12± 0.01 -8.02 0.30 ˂0.01 108 7.14 (6.07-7.98) 12.89 (11.88-14.33) 0.22± 0.02 -6.15 0.16 ˂0.01 109 6.01* 7.66* 0.77±108.49 -0.01 0.00 0.99 106 19.64 (17.55-22.96) 38.08 (32.03-49.87) 0.06±0.01 -7.43 0.03 ˂0.01 WG-42 107 13.24 (12.09-14.35) 23.84 (21.86-26.69) 0.12±0.01 -8.70 0.57 ˂0.01 108 8.16 (7.10-9.03) 14.77 (13.66-16.28) 0.19±0.02 -7.01 1.39 ˂0.01 109 5.27 (3.61-6.26) 10.40 (9.45-11.95) 0.24±0.04 -3.74 0.00 ˂0.01 106 25.22 (21.64-32.94) 47.08 (37.54-69.54) 0.05±0.01 -7.59 0.01 ˂0.01 WG-43 107 16.35 (15.00-17.94) 29.66 (26.45- 34.79 0.09±0.01 -8.48 0.00 ˂0.01 108 10.07 (8.96-11.03) 18.41 (17.08-20.19) 0.15±0.01 -8.06 0.24 ˂0.01 109 6.53 (5.19-7.52) 12.95 (11.88-14.48) 0.19±0.02 -5.22 0.34 ˂0.01 106 21.53 (19.11-25.73) 40.25 (33.55-53.72) 0.06±0.01 -7.77 0.04 ˂0.01 WG-44 107 14.34 (13.15-15.57) 25.88 (23.53-29.39) 0.11±0.01 -8.67 0.94 ˂0.01 108 8.85 (7.72-9.79) 16.44 (15.23-18.08) 0.16±0.01 -7.33 0.44 ˂0.01 109 5.81 (4.30-6.84) 11.89 (10.85-13.42 0.21±0.02 -4.45 0.20 ˂0.01 106 27.92 (23.52-38.35) 50.31 (39.43-77.72) 0.05±0.01 -7.80 0.11 ˂0.01 WG-45 107 17.86 (16.35-19.85) 32.19 (28.33-38.66) 0.08±0.01 -8.46 0.02 ˂0.01 108 11.21 (9.98-12.29) 21.19 (19.54-23.50) 0.12±0.01 -7.87 7.41 ˂0.01 109 7.82 (6.66-8.75) 14.74 (13.61-16.30) 0.18±0.02 -6.51 1.66 ˂0.01 *Unable to estimate confidence limits from the data

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Table 5.11 LT50 and LT90 values of B. bassiana and M. anisopliae isolates tested against adult of R. ferrugineus

-1 -1 2 Isolate Dose LT50 (Conidia ml ) LT90 (Conidia ml ) Slope Intercept x (df = 2) P (CI) (CI) 106 20.41 (18.46-23.42) 36.38 (31.26-45.68) 0.08± 0.01 -8.43 0.00 ˂0.01 WG-41 107 13.89 (12.77-15.00 24.38 (22.37-27.26) 0.12±0.01 -9.09 0.24 ˂0.01 108 8.47 (7.49-9.29) 14.79 (13.71-16.25) 0.20±0.02 -7.49 1.15 ˂0.01 109 4.93 (2.82-6.29) 12.35 (11.19-14.02) 0.17±0.02 -3.41 0.91 ˂0.01 106 23.17 (20.65-27.56) 39.98 (33.70-52.11) 0.07±0.01 -8.61 0.031 ˂0.01 WG-42 107 15.55 (14.40-16.81) 26.73 (24.35-30.27) 0.11±0.01 -9.34 0.00 ˂0.01 108 9.53 (8.46-10.45) 17.20 (15.97-18.83) 0.16±0.01 -7.97 0.04 ˂0.01 109 6.36 (4.71-7.53) 14.12 (12.91-15.82) 0.16±0.02 -4.72 1.88 ˂0.01 106 26.96 (23.45-34.03) 44.57 (36.59-61.63) 0.07±0.01 -8.67 0.00 ˂0.01 WG-43 107 17.90 (16.55-19.61) 30.46 (27.25-35.54) 0.10±0.01 -9.23 1.03 ˂0.01 108 11.81 (10.66-12.84) 21.50 (19.87-23.76) 0.13±0.01 -8.43 4.45 ˂0.01 109 8.68 (7.43-9.69) 16.89 (15.59-18.66) 0.15± 0.01 -6.82 1.93 ˂0.01 106 24.25 (21.55-29.12) 40.79 (34.31-53.47) 0.07±0.01 -8.80 0.22 ˂0.01 WG-44 107 16.38 (15.20-17.72) 27.69 (25.15-31.48) 0.11±0.01 -9.52 0.04 ˂0.01 108 10.72 (9.59-11.71) 19.63 (18.19-21.58 0.14±0.01 -8.20 0.98 ˂0.01 109 7.52 (6.21-8.54) 14.99 (13.80-16.64) 0.17±0.01 -5.97 2.61 ˂0.01 106 30.06 (25.65-39.89) 47.23 (38.10-68.46) 0.07± 0.01 -8.60 0.03 ˂0.01 WG-45 107 21.17 (19.18-24.31) 36.51 (31.45-45.68) 0.08±0.01 -8.67 4.74 ˂0.01 108 13.54 (12.32-14.72) 24.90 (22.70-28.12) 0.11±0.01 -8.34 3.70 ˂0.01 109 9.57 (8.268-10.66) 18.97 (17.48-21.02) 0.13±0.01 -7.02 0.00 ˂0.01

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CHAPTER 6

Combined effectiveness of endophytically colonized Beauveria bassiana and Bacillus thuringiensis against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

Abstract Research study was carried out to investigate the insecticidal properties of endophytically colonized Beauveria bassiana and Bacillus thuringiensis var. kurstaki (Bt-k) against 2nd, 4th and 6th instar larva of red palm weevil (RPW) Rhynchophorus ferrugineus. Initially five isolates of B. bassiana (WG-11, WG-40, WG-41, WG-42 and WG-43) were screened by inoculating endophytically in date palm leaf petioles. Only one B. bassiana isolate (WG-41) recovered from up to the 10 cm after 30 days during both years was considered to be effective. Both agents were applied alone and in combination to tested instars and pupation, adult emergence and egg eclosion was recorded from survivers. Moreover development, diet consumption, frass production and weight gain were also observed. Mortality was low in sole treatments, while in combined treatments increase in mortality, decrease in pupation, adult emergence and egg eclosion found inversely correlated to toxic levels of both microbial agents. Second instar larvae exhibited more susceptibility followed by 4th and 6th instar larvae. Synergistic effect (CTF≥20) on the mortality was observed when larvae were exposed to simultaneous application of WG-41 with 40 µg ml-1 of Bt-k in case of all three larval instars tested. All the tested instars exhibited varying level of growth and development when exposed to the sub-lethal doses of palm petiole piece (6 cm away from inoculated point) inoculated with WG-41 and dipped in Bt-k concentrations; moreover significant variations were recorded for larval duration, larval weight, pupal duration, pupal weight, pre-pupal duration, pre-pupal weight, adult longevity (male and female) and adult weight (male and female). The toxic nature of microbial agents also influenced the frass production and diet consumption. Larvae treated with Bt-k gained more weight than the WG-41 and their combined application. Initial weight of larvae exerted its impact on the weight gain and diet consumption and the trend was found linked to pathogenicity of applied agents. It can be surmised from the findings that microbial agents exhibit a reliable level of mortality against R. ferrugineus. Hence it would be fruitful to replace the conventional reliance on chemical approaches.

Keywords: Rhynchophorus ferrugineus, Beauveria bassiana, Bacillus thuringiensis, development, diet consumption, frass production

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6.1 Introduction The Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) is considered one of the most destructive pests of variety of palm species, including date palms (Giblin-Davis, 2001). The weevil lives and breed into the tree trunk and devastate its vascular system which lead to the tree collapse and death of host plant. Until 1917, it was considered that RPW is the only pest of coconut palm but latter on it was found devastating date palms in Pakistan, India and Iraq (Mohan, 1917; Milne, 1918; Buxton, 1920). In Pakistan infestation of the RPW was observed during 1913 when researcher found the beetle attack on the date palm trees imported from Middle East and later on Milne an Economist of Punjab Agriculture College, Lyallpur (Presently University of Agriculture, Faisalabad, Pakistan) observed RPW infestation in date palms and collected insect specimens from Multan, Muzaffargarh and Dera Ghazi Khan Districts of Punjab (Milne, 1918). Since last 30 years RPW has caused huge economic losses and no effective control measure has been invented so for (Murphy and Briscoe, 1999; Faleiro, 2006). For the successful control of RPW trapping, deployment of chemical insecticides and fumigants has been considered a core component since long time. The unwise use of these chemical insecticides and fumigants lead the resistance against this voracious pest which lead the researchers to search for the alternative control strategies which are safer for human beings and compatible to environment (Abraham et al., 1998). Moreover, RPW live in the self-made tunnels which make them less vulnerable to Chemical and mechanical control. Thus, self-pathogens have been proposed for the successful control of RPW (Dangar, 1997). Entomopathogens are the safer, environment friendly and economic alternative to the chemical insecticides and getting serious attention against RPW control. Fungal entomopathogens play a key role in managing plant pathogens and herbivorous insects by improving the plant host defense mechanism or by directly affecting plant pathogens (Sivasithamparam, 1998; Arnold et al., 2003). Entomopathogenic fungi are preferred to the other entomopathogens due to its unique mode of action by infecting their host through contact action and penetrating into insect hemocoel by breaching the host cuticle. Fungal infection can be transformed by direct contact of infected individuals to the healthy ones or subsequent development via new generation of spores (Lacey et al., 1999; Quesada-Moraga et al., 2004). On the other hand, an entomopathogenic bacterium from the genus Bacillus is also an economical and potential alternative to the chemical insecticides. They are key antagonists of insect pests of economic importance, their products and by products (Salama et al., 2004). It is often an integral part of products used in biological control strategies worldwide, about 95% microbial pesticides being used globally are bacterial in origin with annual sale of about $100 million (Federici et al., 2006). A number of species of this genus particularly Bacillus thuringiensis (Berliner) are frequently used against vast array of insect pests from the order Coleoptera, Lepidoptera and Diptera etc, which exhibit specificity towards the host and specific stage of the host (Salama et al., 2004). It is a gram-positive soil bacterium, spore forming mesophile having ability to produce proteinaceous parasporal inclusions during sporulation. It produces δ-endotoxins in the form parasporal crystals which may vary from one to several types and formed from different δ-endotoxins that are related to each other (Aronson et al., 1986). So for more than 350 genes from B. thuringiensis has been discovered which are encoded for specific toxic proteins which are specific to the larvae of various orders (Schnepf et al., 1998). Both pathogens are widely distributed in the environment and found from the soil of different origin and insect cadavers (Martin and Travers, 1989). The intervention of more than

112 one biocontrol agent can enhance the effectiveness of the other partner, many studies have been conducted in this regard. The combined effect of B. bassiana and B. thuringiensis working synergistically delivers more harm to insect pests (Wraight and Ramos, 2005) and hence can be a hint for those willing to manage it. Combine effect of B. thuringiensis and Entomopathogenic fungi has synergistic effect performed by several researchers (Navon, 2000). Wraight and Ramos (2005) observed that combinations of B. thuringiensis and B. bassiana have been successful in increasing mortality in some insects. Sandner and Cichy (1967) applied a mixture of B. thuringiensis var. kurstaki (Bt-k) and B. bassiana against larvae of the Mediterranean flour moth. Results revealed that the two agents acted independently (mean mortalities from B. bassiana, B. thuringiensis and the mixture were 57, 44, and 71% respectively). The present study aiming at the endophytically colonizing B. bassiana isolates in date palm and their evaluation against R. ferrugineus alone and in integrated manners with B. thuringiensis

6.2 Materials and Methods

6.2.1 RPW collection and rearing Different developmental stages (larvae, pupae and adult) of RPW were collected from fallen and infested date palm trees with the permission of farmers (owners) from date palm growing areas of west Punjab, Pakistan. During collections, adult, larvae and pupae were kept in separate plastic jars until brought to the laboratory Microbial Control Laboratory, Department of Entomology, University of Agriculture, Faisalabad, Pakistan. After arriving to the laboratory larvae were provided with sugarcane (Saccharum officinarum L.; Poales: Poaceae) stems for feeding and pupation, while adults were offered shredded sugarcane pieces for feeding and oviposition substrates. On pupation, pupal cocoons were kept in separate boxes for adult emergence in incubator at 25±2 oC, 65±5% RH and 12:12 (D: L) hours photoperiod. As adults emerged, they were transferred to the adult’s jars for feeding and mating with shredded sugracne pieces. Colony was developed in plastic boxes (15×30×30 cm) having a lid covered with mesh wire gauze (60 mesh size) in the middle (7 cm diameter) for aeration. Rearing was carried out in Microbial Control Laboratory. The rearing conditions were maintained as mentioned above. Adult’s diet was changed after every three days and replaced sugarcane pieces were kept in separate jars (8×8×12 cm) for egg hatching. On egg hatching, neonate larvae were allowed to feed for 3 days in the same sugarcane set and then were transferred to the sugarcane sets for feeding and pupation. Larvae were shifted to the new sugarcane sets biweekly until pupation.

6.2.2 Fungal isolates Five isolates of B. bassiana used in the study belonging to the culture collection of Microbial Control Laboratory, originally isolated from soil samples from crop ﬁelds (WG-11), recovered endophytically from tomato plants (WG-40) and R. ferrugineus cadavers (WG-41, WG-42 and WG-43). The cultures were maintained continuously on slants and sub-cultured for 14 days at 25±2 °C under a 12:12 (D: L) hours, tubes were tightly sealed and the culture was stored at 4 °C. Mass culturing was done by inoculating Petri plates containing Sabourad Dextrose Agar (SDA, from BD, Becton, Dickinson and Company Spark, MD 21152 USA) media. Spore concentration of 1×106 spore ml-1 was determined with a Neubauer haemocytometer.

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6.2.3 Preparation of Bacillus thuringiensis spore-crystal mixtures The commercial formulation of B. thuringiensis var. kurstaki (Bt-k) was obtained from Microbial Control Laboratory, originally obtained from National Center for Genetic Engineering and Biotechnology (BIOTEC) in Thailand. This strain was then subjected to sporulation by culturing in 50 ml nutrient broth media. Harvesting of culture was carried out by centrifugation at 6000 rpm for 15 min (Crecchio and Stotzky, 2001; Hernández et al., 2005). The pellets formed resultantly were washed twice in cold 1M NaCl and thrice in sterile distilled water (SDW), re- suspended in distilled water (5 ml). From the suspension formed, 1 ml was centrifuged for 5 min at 10,000 rpm, dried for 4 hours at 37 °C and weighed (Wakil et al., 2013).

6.2.4 Screening of fugal isolates Date palm trees less than one year old were selected in the date palm plantations located at Faisalabad during 2014 and 2015. Inoculation of B. bassiana isolates were carried out following the method of Gómez-Vidal et al., (2006). From each palm, three petioles were rubbed with 70% ethanol and 30 µl of conidial suspension was injected using insulin syringe. To avoid sun drying and external contamination the inoculated area were wrapped with Parafilm. For re- isolation, after 15 and 30 days post inoculation, petioles were sampled from inoculation site, 2, 4, 6, 8 and 10 cm above or below the inoculation site with the help of sterilize cork borer (0.6 cm diameter). The samples were surface-sterilized with 1% sodium hypochlorite for 1 min, followed by rinsing three times with sterile distilled water (Gómez-Vidal et al., 2006). Samples were then placed on PDA for 8 days at 25±2 ͦ C for spore germination. Different fungi were recovered from petiole pieces that were further purified by sub-culturing on SDA medium, and fungal isolates were identified following the keys developed by Barnett and Hunter (1999).

6.2.5 Bioassay procedure To evaluate the effect of endophytically colonized B. bassiana WG-41, a piece of inoculated palm petiole (2×2 cm2), 2 cm away from the inoculated site was offered to the 2nd, 4th and 6th instar larvae of R. ferrugineus separately in 150 ml plastic cups measuring (6×6 cm) individually. While in control treatment untreated palm petiole was offered to the respective larval stages. The larvae were allowed to feed on palm piece for 48 hours and then shifted to artificial diet (Martín and Cabello, 2006) for rest of the period and provided with fresh diet every day. For Bt-k treatments, three concentrations (30, 40 and 50 µg ml-1) were prepared. An untreated piece of date palm petiole (2×2 cm2) immersed in respective doses of Bt-k suspension for 90s and offered to the respective larval stages individually. The larvae were allowed to feed on treated palm piece for 48 hours and shifted to the artificial diet for rest of the period individually. In combined treatments WG-41 inoculated date palm petiole piece was immersed in respective doses of Bt-k suspensions for 90s and then offered to the larvae. After last instar dry coir was provided for pupation to the last instar larvae. Larval mortality was counted daily until larvae pupated or died. Larvae that failed to respond on slight prodding by blunt needle were considered as dead. From the surviving individual percent pupation, adult emergence and egg eclosion were also recorded. Three replicates of ten larvae were used for each treatment and same count of larvae fed on normal petiole served as untreated check. All the treatments were incubated at 27±2 °C and 65±5 % RH and a 12: 12 (D: L) hours photoperiod in an incubator (Sanyo, Japan). Entire experiment was repeated thrice.

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6.2.6 Bioassay on development of R. ferrugineus To determine the effect of sub-lethal doses of WG-41 and Bt-k on developmental parameters viz. larval duration, larval weight, pre-pupal duration, pre-pupal weight, adult longevity (male and female) and adult weight (male and female) was recorded on 4th instar larvae of R. ferrugineus. The piece of endophytically colonized WG-41 (6 cm away from inoculation site) and Bt-k (10, 15 and 20 µg ml-1) were applied alone and in combination against 4th instar larvae and maintained at above motioned conditions. The larvae were allowed to feed on treated petiole piece for 48 hours and then shifted to the artificial diet for rest of the period. After last instar larvae was provided with dry coir (coconut coir) for pupal development. All the above mentioned parameters were observed hereafter.

6.2.7 Bioassay on larval development th A new batch of 10 instar larvae of R. ferrugineus (L10) were encountered with sub-lethal dose of WG-41 and Bt-k (10 µg ml-1). A piece of endophytically colonized palm petiole (6 cm away from inoculated area) alone and in combination with Bt-k was offered to the larvae. The larvae were allowed to feed for the whole 10th instar on the treated diet. Before exposure to the palm petiole each larval instar were weighed and transferred to rearing vial with palm piece. Every day until the larvae pupated or died; larvae were changed to new cups individually and provided with fresh diet every day. Frass produced during this period was separated from vials using tip of fine camel hair brush and weighed. Diet left unused in each vial was recovered, dried in drying oven at 80 °C. Prior to assay, diet in 30 cups was dried to obtain an estimate of the dry weight. Diet consumption of each larva was determined by subtracting the after feeding mass of diet from before feeding mass. Moreover, frass production and weight gains during this period were also determined.

6.2.8 Statistical analysis Mortality for each treatment was corrected for control mortality using Abbott's (1925) formula and subjected to one way analysis of variance (ANOVA) in Minitab (Minitab, 2002) and means were separated using Tukey’s Kramer test (HSD) (Sokal and Rohlf, 1995) at 5% significance level. Type of interaction among combined treatments of Bt-k and WG-41 was determined by equation; CTF = (Oc-Oe)/ Oe×100, where CTF is the co-toxicity factor, Oc is the observed mortality (%) in combined application, and Oe the expected mortality (%), that is the sum of individual mortality (%) encountered in each of the treatments used in the combination (Mansour et al., 1966). The interactions were categorized as additive, synergistic or antagonistic: CTF≥20 meaning synergism, CTF>20 - –20 meaning additive, and CTF<-20 meaning antagonism (Mansour et al., 1966; Wakil et al., 2012). To inspect the impact of microbial agents on the diet consumption, weight gain and frass production were analyzed by ANCOVA using initial larval weight and diet consumption as covariates (Janmaat et al., 2014).

6.3 Results

6.3.1 Fungal colonization of date palm petioles All the fungal isolates successfully colonized endophytically in date palm petioles more or less from the inoculation point during both years. At the injection site hydrophobic and necrotic lesions appeared above and below the injection site. Different entomopathogenic fungi were recovered from the site of injection up to 10 cm above and below. At the injection site all

115 the inoculated fungi were recovered (90-100%) from all the plants after 15 days of inoculation during 2014 and (80-100%) following year (Table 6.1). B. bassiana isolate WG-41 was recovered 10 cm above and below the injection site, while no entomopathogenic fungi were recovered from non-inoculating petioles. Non-entomopathogenic fungi were less near the inoculation site and gradually increased towards the ends in both years.

6.3.2 Toxicity of microbial agents Toxicity assay was conducted on 2nd, 4th and 6th instar larvae of R. ferrugineus by deploying B. bassiana inoculated date palm piece and Bt-k alone and in integrated manners. Significant differences (Table 6.2) were recorded for mortality among different treatments and larval instars (treatment: F7, 172 =201.87, P≤0.05; instar: F2, 161=63.34, P≤0.05) but non- significant interaction was recorded for (instar × treatment: F14, 161=1.56, P=0.093). Synergistic effect (CTF≥20) on the mortality was observed when larvae were exposed to simultaneous application of endophytic B. bassiana and 30 µg of Bt-k in case of all the three instars tested. Second instar larvae of R. ferrugineus were more susceptible to both pathogenic agents followed by 4th and 6th instar larvae in all the treatments tested. Highest level of larval mortality (83.17±2.28%) was observed in second instar larvae with the simultaneous application of B. bassiana and Bt-k (50 µg ml-1) followed by 71.01±2.39% when treated with B. bassiana and Bt-k (40 µg ml-1) and 54.63±2.26% when treated with B. bassiana and Bt-k (30 µg ml-1) (Table 6.3). Additive effect (CTF≤20) was recorded when tested instar were treated with low and high dose of Bt-k in integration with endophytic B. bassiana. A similar trend in mortality was recorded for 4th and 6th instar larvae of R. ferrugineus while mortality was found increasing with increase of concentration of Bt-k. Combined application of endophytic B. bassiana and Bt-k proved more fatal to all the instar larvae as compared to their sole application. Both microbial agents were found working additively and synergistically. Percent pupation, adult emergence and egg eclosion from surviving individuals was found inversely correlated to toxic level of microbial agents in all instar larvae. Increase in mortality, while decrease in pupation, adult emergence and egg eclosion was found in concentration dependent manner.

6.3.3. Development of R. ferrugineus Development of 4th instar larvae was adversely affected by the toxic effect of microbial agents. When larvae were exposed to different concentrations of Bt-mixed diets and B. bassiana, significant variations were recorded for larval duration, larval weight, pre-pupal duration, pre- pupal weight, pupal duration, pupal weight, adult longevity and adult and adult weight (larval duration: F7, 71 =72.73, P≤0.05; larval weight F7, 71 =47.54, P≤0.05; pre-pupal duration: F7, 71 =53.12, P≤0.05; pre-pupal weight: F7, 71 =33.80, P≤0.05; pupal duration F7, 71 =43.95, P≤0.05; pupal weight F7, 71 =26.61, P≤0.05; adult longevity (female F7, 71 =58.20, P≤0.05 and male F7, 71 =65.03, P≤0.05); adult weight (male F7, 71 =12.50, P≤0.05 and female F7, 71 =7.76, P≤0.05). Increase in larval and pupal duration while decrease in pupal weight and adult duration was recorded depending upon the lethal action of the applied agent. When compared to control, significantly increased larval and pupal duration was recorded for combined application of Bb and highest concentration of Bt-k followed by Bb and middle concentration of Bt-k. Similarly extended pre-pupal and pupal period and reduced adult life span was recorded in concentration dependent manner. While decrease in pre-pupal and adult weight was also recorded in concentration dependent manners (Table 6.5).

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6.3.4 Effect on larval development Diet consumption by 10th instar larvae was significantly influenced by the treatments applied diet consumption was low in combined treatments as compared to sole applications. Least diet consumption was recorded for combined treatments followed by B. bassiana and Bt-k. While, highest diet consumption was recorded for the control treatment (Fig 6.1a). Frass production was influenced by treatments applied with lowest frass production for combined treatments of Bt-k and B. bassiana from (0.61±0.05 to 0.00±0.00g) during experimental period. More frass production was recorded during the initial days of treatments which gradually decreased to zero before pupation. On the other hand, highest level of frass production was found in untreated larvae for all the period of last instar larvae till pupation (Fig 6.1b). Larvae treated with sub-lethal concentrations of B. bassiana and Bt-k gained more weight as compared to their combined application (Single agent versus combined treatment). Initial weight of larvae (10th instar: 4.35±0.12g) exerted its impact on the weight gain and among treatments, there was a trend of weight gain linked to pathogenicity. Combined application of B. bassiana and Bt-k had an adverse impact on the weight gain and lowest gain (-0.54±.04g) was recorded for the combined treatment while highest gain (-0.11±.02g) was recorded in untreated larvae (Fig 6.1c).

6.4 Discussion The endophytic colonization by B. bassiana is commonly practiced to combat voracious pests of different field crops. Endophytic fungi are also capable of colonizing date palm tissues after petiole wounding. The B. bassiana isolate WG-41 was recovered from up to 10 cm away from the inoculation site during both years. The results confirmed that entomopathogenic fungi survived endophytically and colonized petiole tissue of date palms. Gómez-Vidal et al., (2006) also confirmed the endophytic colonization of entomopathogenic fungi in live and detached palm petioles of date palm. They confirmed the movement in the parenchyma and sparsely within vascular tissue using microscopy techniques. An effective remedy to combat voracious insect pests is influenced by a number of factors which includes the toxic nature, compatibility to other control agents, speed of kill, feeding deterrence to insect, effect on development of insect, acceptance to insect and environmental persistence. Integration of two or more entomopathogens to fight against insect pests may brighten the chances of targeting multiple hosts (Pingel and Lewis, 1999). Marzban et al. (2009) reported that the integration of two or more myco-pathogens interact positively than their individual effect. In most combinations the virulence of an agent is rectified by the action of the other agent which resultantly increases the speed of kill, retard growth, feeding sesation, improve virulence and broaden host range. The findings of our study revealed higher mortality levels of RPW in combined treatments of B. bassiana inoculated date palm piece and Bt-k as compared to their sole application. Moreover, these deterrent effects increased with the increase of number of Bt spores. Similar findings were revealed by Khalique and Ahmad (2002), who reported the extended larval duration with the increase in Bt-k concentration. While, larvae in check treatments consumed more food which are in accordance with the findings of Marzban et al. (2009). The studies of Ma et al. (2008) and Marzban et al. (2009) also support our findings who reported the growth retardation of Ostrinia furnacalis and H. armigera when challenged with Cry1Ac from Bt- treated diets and combined action of Bt (Cry1Ac) and HaCPV respectively. The mode of action of entomopathogens is the key to success factor which make them fit for the use in IPM program to combat any insect pest. For infection to occur Bt toxins attach to

117 the specific bindings sites of the insect’s midgut which then leads to cell lysis. This lysis may result in the insect to stop feeding, become lethargic and ultimately die (Marzban et al., 2009). While B. bassiana exhibit novel mode of action by adhering to the insect cuticle; later on breaching the insect cuticle with the aid of enzyme complexes (beauvericin, bassianolide and oosporein) followed by infection to the hemocoel where fungal conidia germinate and proliferate in the insect body resultanting in insect death (Vey and Fargues, 1977). In combined treatments of Bt and B. bassiana both agents work synergistically weakening the insect and affecting the insect immune response to allow entomopathogens to infect the host more efficiently. Moreover, conidial concentrations of both agents are the key factor in the degree of disease severity. B. thuringiensis is rather quick in action which gets amplified when a larger number of fungal spores are available to target the insect host. When B. bassiana gains access to the insect gut, it boosts the infection of Bt toxins. In this way both agents help each other in retardation of normal physiological functions of an insect host. These findings are further supported by Allee et al. (1990) who found B. bassiana germinating and invading the insect favors the Bt toxins to increase severity in grubs of Colorado potato beetle. Synergistic interaction was reported by Wraight and Ramos (2005) when B. bassiana (GHA) and Bt-k were sprayed on potatoes to protect against potato beetle (Leptinotarsa decemlineata). Similar findings were also reported by Furlong and Groden (2003) who reported synergistic interaction between B. bassiana and B. thuringiensis. Other researchers have also observed the similar results when combined B. bassiana and B. thuringiensis withsynthetic insecticides (Fargues, 1975; Lewis and Bing, 1991; Sander and Chichy, 1967). Contrarily, no synergistic interaction was observed between B. bassiana and B. thuringiensis against 4th stage larvae of L. decemlineata (Costa et al., 2001). Here the method of fungal spore application may retard the synergistic effect and respond differently in terms of time and level of mortality. Different response of Chilo partellus was reported when fungal spores were applied on leaf disk (Tefera and Pringle, 2003a). In our present study synergistic interaction may be due to the treatment of RPW larvae with fungal spores by larval dip method in which dorso-ventral infection of fungal spores may weaken the larvae and render it more susceptible to Bt-k spores. Tefera and Pringle (2003b) observed that loss of feeding H. armigera after exposure to Bt mixed diet make it easier for B. bassiana to grab the physiology and hence proliferate rather easily. Similar loss of feeding was observed by Tefera and Pringle (2003b) when second and third instar C. partellus were exposed to simultaneous action of Bt (Cry1Ac) and B. bassiana as behavioral response elucidated reduced diet uptake. Jadhav et al. (2012) found similar retardation in growth and survival of H. armigera and Spodoptera litura after treatment with three flavonoids namely chlorogenic acid, quercetin and rutin. The feeding experiments on RPW exhibited that B. thuringiensis invades the hemolymph and the total circulating hemocytes decreased, mainly the plasmatocytes, after 19 h, and for the first time, many Bt vegetative forms were recorded in the hemolymph of RPW after Bt commercial product ingestion (Manachini et al., 2011). Integrated action of Bt and fungi may also hypothesized by delayed larval molts with Bt treatments which enhance the inter-molt period, thereby providing fungus extended period of time for infection before next molt. The reduced food consumption with Bt treatments (Nathan et al., 2005; Ramalho et al., 2011), food utilization (Prutz and Dettner, 2005; Ramalho et al., 2011) and reduced larval weight has been observed. On the other hand in healthy larvae loss of conidia may happen during molting, hence conferring interruption in fungal colonization (Wraight and Ramos, 2005). Contrarily, Slansky (1993) reported surprising results by observing the reduction in larval weight, food consumption and frass production in fungal treated larvae as compared to

118 the untreated control. Our findings suggest that assimilation activities were found related to toxic effect of pathogen as indicated by decreased quantity of frass produced. Our results are in accordance with the study of Janmaat et al. (2014) who reported reduced frass production in Trichoplusia ni with the increase of concentrations. This may be attributed to the fact that enhanced Bt concentrations may alter the protein to carbohydrate ratio of the diet as required optimally which resultantly disturb the growth response (Simpson and Raubenheimer, 1995). Moreover, the toxicant larvae try to repair its midgut lining and the lysis effect is directly proportional to the concentration of pathogen (Tanaka et al., 2012). Muñoz et al. (2014) reported reduced food intake, growth and weight gain in H. armigera exposed to the sublethal doses of Bt which in response did not allowed most of them to get the critical weight and pupate in time. This type of feeding behavior may be the result of metabolic interference of the entomopathogens with larva’s growth. A greater knowledge of RPW biology and, in particular, of the interaction between potential pathogens and immunocytes would be useful to improve RPW-IPM programs, which should focus on the identification of more virulent natural pathogen strains and on improving the virulence capacity of Bt (Manachini et al., 2011). While choosing microbial control agents to fight insect pests, several uncontrolled factors must be considered as the comprehensive nature of entomopathogens requires multidisciplinary targeted efforts. This study may help to apply laboratory study to field conditions covering almost all the questions required to be answered. However field study regarding the persistence of entomopathogens is required to have a detailed knowledge about some of the factors that cannot be imagined under laboratory or green house condition. This study could be a foundation and direction for the future researches.

Conclusions The present study showed that B. bassiana can colonize endophytically in date palm petiole even after 30 days post inoculation. WG-41 isolate was recovered up to 10 cm from the site of inoculation even after 30 days. Endophytic B. bassiana in integration with Bt-k can be effectively used against 2nd, 4th and 6th instar larvae of this pest under laboratory conditions. Moreover they also exert detrimental effect on their growth and development parameters such as diet consumption, frass production and weight gain.

Acknowledgements This research work was supported by the scholarship from Higher Education Commission (HEC), Islamabad, Pakistan (112-30536-2AV1-263) under Indigenous Ph.D. Fellowship Program.

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References Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol., 18: 265-267. Abraham, V.A., M.A. Al-Shuaibi, M.A. Faleiro, J.R. Abozuhairah, R.A. and P.S.P. Vidyasagar, 1998. An integrated management approach for red palm weevil Rhynchophorus ferrugineus Oliv. a key pest of date palm in the Middle East. Proc. Inter. Conf. on Integrated Pest Management (Muscat, Sultanate of Oman). Sultan Qaboos Uni. J. Sci. Res., 3: 77-83. Allee, L.L., R.S. Soper and D.W. Roberts, 1990. Germination and infection processes of the entomophthoralean fungus Erynia radicans on the potato leafhopper Empoasca fabae. J. Inver. Pathol., 56: 157-174. Arnold, A.E., L.C. Mejía, D. Kyllo, E.I. Rojas, Z. Maynard, N. Robbins and E.A., Herre, 2003. Fungal endophytes limit pathogen damage in a tropical tree. Proc. Nati. Acad. Sci. USA., 100: 15649-15654. Aronson, A.I., W. Beckman and P. Dunn, 1986. Bacillus thuringiensis and related insect pathogens. Microbiol. Rev., 50: 1-24. Barnett, H.L. and B.B. Hunter, 1999. Illustrated Genera of Imperfect Fungi, 4th Edn. APS Press, The American Phytopathological Society, St. Paul, MN. Pp. 218. Buxton, P.A., 1920. Insect pests of dates and the date palm in Mesopotamia and elsewhere. Bull. Entomol. Res., 11: 287-304. Costa, S.D., M.E. Barbercheck and G.G. Kennedy, 2001. Mortality of Colorado potato beetle (Leptinotarsa decemlineata) after sub-lethal stress with the CRYIIIA delta-endotoxin of Bacillus thuringiensis and subsequent exposure to Beauveria bassiana. J. Inver. Pathol., 77: 173-179. Crecchio, C. and G. Stotzky, 2001. Biodegradation and insecticidal activity of the toxin from Bacillus thuringiensis subsp. kurstaki bound on complexes of montmorillonite-humic acids-Al hydroxypolymers. Soil Biol. Biochem., 33: 573-581. Dangar, T.K., 1997. Infection of red palm weevil, Rhynchophorus ferrugineus, by a yeast. J. Plant. Crops, 25: 193-196. Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm during the last one hundred years. Int. J. Trop. Insect Sci., 26: 135-154. Fargues, T., 1975, Etude experimental dans la nature de Beauveria and Metarhizium a dose reduite contre Lephinotarsa decemlineata. Annal. Zool. Ecol. Ani., 7: 247-264. Federici, B.A., H.W. Park and Y. Sakano, 2006. Insecticidal protein crystals of Bacillus thuringiensis. In: Inclusions in Prokaryotes. Shively, J.M. (Ed.). Springer-Verlag, Berlin- Heidelberg. Pp. 195-235. Furlong, M.J. and E. Groden, 2003. Starvation induced stress and the susceptibility of the Colorado potato beetle, Leptinotarsa decemlineata, to infection by Beauveria bassiana. J. Inver. Pathol., 83: 127-138. Giblin-Davis, R.M., 2001. Borers of palms. In: Howard F.W., D. Moore, R.M. Giblin-Davis, R.G. Abad (Eds.). Insects on Palms. CABI Publishing, Wallingford, UK. Pp. 267-305. Gómez-Vidal, S., L.V. Lopez-Llorca, H.B. Jansson and J. Salinas, 2006. Endophytic colonisation of date palm (Phoenix dactylifera L.) leaves by entomopathogenic fungi. Micron, 37: 624-632.

120

Hernández, C.S., R. Andrew, Y. Bel and J. Ferre, 2005. Isolation and toxicity of Bacillus thuringiensis from potato-growing areas in Bolivia. J. Inver. Pathol., 88: 8-16. Jadhav, D.R., N. Mallikarjuna, A. Rathore and D. Pokle, 2012. Effect of some flavonoids on survival and development of Helicoverpa armigera (Hübner) and Spodoptera litura (Fab) (Lepidoptera: Noctuidae). Asi. J. Agric. Sci., 4(4): 298- 307. Janmaat, A.F., L. Bergmann and J. Ericsson, 2014. Effect of low levels of Bacillus thuringiensis exposure on the growth, food consumption and digestion efficiencies of Trichoplusia ni resistant and susceptible to Bt. J. Inver. Pathol., 119: 32-39. Khalique, F. and K. Ahmad, 2002. Retarding effect of spore-δ-endotoxin complex of Bacillus thuringiensis (Berliner) strains on the development of Helicoverpa armigera (Hubner). Pak. J. Biol. Sci., 5(8): 853-857. Lacey, L.A., A.A. Kirk, L. Millar, G. Mereadier and C. Vidal, 1999. Ovicidal and larvicidal activity of conidia and blastospores of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) against Bemisia argentifolii (Homoptera: Aleyrodidae) with a description of a bioassay system allowing prolonged survival of control insects. Biocontrol Sci. Tech., 9: 9-18. Lewis, L.C. and L.A. Bing, 1991. Bacillus thuringiensis Berliner and Beauveria bassiana (Balsamo) Vuillemin for European Corn Borer Control: Potential for Immediate and Season-long Suppression. Canad. Entomol., 123: 387-393. Lezama-Guttierez, R., J.J. Hamm, J. Molina-Ochoa, M. Lopez-Edwards, A. Pescador-Rubio, M. Gonzalez-Ramirez and E.L. Styer, 2001. Occurrence of entomopathogens of Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Mexican states of Michoacan, Colima, Jalisco, Tamaulipas. Florida Entomol., 84: 23-30. Ma, X.M., X.X. Liu, X. Ning, B. Zhang, F. Han, X-M. Guan, Y.F. Tan and Q.W. Zhang, 2008. Effects of Bacillus thuringiensis toxin Cry1Ac and Beauveria bassiana on Asiatic corn borer (Lepidoptera: Crambidae). J. Inver. Pathol., 99: 123-128. Manachini, B., V. Arizza, D. Parrinello and N. Parrinello, 2011. Hemocytes of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces cerevisiae and Bacillus thuringiensis. J. Inver. Pathol., 106:360-365. Mansour, N.A., M.E. Eldefrawi, A. Toppozada and M. Zeid, 1966. Toxicological studies on the Egyptian cotton leafworm, Prodenia litura Vl potentiation and antagonism of carbamate insecticide. J. Econ. Entomol., 59: 307-311. Martin, P. and R. Travers, 1989. Worldwide abundance and distribution of Bacillus thuringiensis isolates Appl. Environ. Microbiol., 55(10): 2437-2442. Marzban, R., Q. He, X. Liu and Q. Zhang, 2009. Effects of Bacillus thuringiensis toxin Cry1Ac and cytoplasmic polyhedrosis virus of Helicoverpa armigera (Hübner) (HaCPV) on cotton bollworm (Lepidoptera: Noctuidae). J. Inver. Pathol., 101: 71-76. Milne, D., 1918. The Date Palm and Its Cultivation in the Punjab. The Punjab Government. Pp. 153. Minitab, 2003. MINITAB Release 14 for Windows. Minitab Inc., State College, Pennsylvania, USA. Mohan, L.M., 1917. Rept. Asst. Prof. Entomol; Rept. D Sagr. Punjab, for the year ended 30th June, 1917. Murphy, S.T. and B.R. Briscoe, 1999. The red palm weevil as an alien invasive: biology and prospects for biological control as a component of IPM. BioControl, 20: 35-45.

121

Nathan, S.S., P.G. Chung and K. Murugan, 2005. Effect of biopesticides applied separately or together on nutritional indices of the rice leaffolder, Cnaphalocrocis medinalis. Phytoparasitica, 33: 187-195. Navon, A., 2000. Bacillus thuringiensis insecticides in crop protection reality and prospects. Crop Prot., 19: 669-676. Pingel, R.L. and L.C. Lewis, 1999. Effect of Bacillus thuringiensis, Anagrapha falcifera multiple nucleopolyhedrovirus, and their mixture on three Lepidoptera corn ear pests. J. Econ. Entomol., 92: 91-96. Prutz, G. and K. Dettner, 2005. Effects of various concentrations of Bacillus thuringiensis Corn leaf material on food utilization by Chilo partellus larvae of different ages. Phytoparasitica, 33: 467-479. Quesada-Moraga, E., R. Santos-Quiros, P. Valverde-Garcia and C. Santiago-Álvarez, 2004. Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (anamorphic fungi) on the German cockroach (Blattodea: Blattellidae). J. Inver. Pathol., 87: 51-58. Ramalho F.D., T.L. Azeredo, A.R.B. de Nascimento, F.S. Fernandes, J.L. Jr. Nascimento, J.B. Malaquias, C.A.D. da Sliva and J.C. Zanuncio, 2011. Feeding of fall armyworm, Spodoptera frugiperda, on Bt transgenic cotton and its isoline. Entomol. Exper. Appl., 139: 207-214. Salama, H.S., M.S. Foda, M.A. El-Bendary and A. Abdel-Razek, 2004. Infection of red palm weevil Rhynchophorus ferrugineus, by spore-forming bacilli indigenous to its natural habitat in Egyp. J. Pest Sci., 77: 27-31. Sandner, H. and D. Cichy, 1967. Research on the effectiveness of fungal and bacterial insecticides. Ekol. Pol. Ser., 15: 325-333. Schnepf, E., N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D.R. Zeigler and D.H. Dean, 1998. Bacillus thuringiensis and its pesticidal crystal proteins, Microbiol. Mol. Biol. Rev., 62: 775-806. Simpson, S.J. and D. Raubenheimer, 1995. The geometric analysis of feeding and nutrition: a user's guide. J. Insect Physiol., 41: 545-553. Sivasithamparam, K., 1998. Root cortex - the final frontier for the biocontrol of root-rot with fungal antagonists: a case study on a sterile red fungus. Ann. Rev. Phytopathol., 36: 439- 452. Slansky, F. Jr., 1993. Nutritional ecology: the fundamental quest for nutrients. In: Stamp, N.E. and T.M. Casey (Eds.). Caterpillars: ecological and evolutionary constraints on foraging. Chapman & Hall, London. Pp. 29-91. Sokal, R.R. and F.J. Rohlf, 1995. Biometry. Freeman, New York. Tanaka, S., Y. Yoshizawa and R. Sato, 2012. Response of midgut epithelial cells to Cry1Aa is toxin-dependent and depends on the interplay between toxic action and the host apoptotic response. FEBS J., 279: 1071-1079. Tefera, T. and K.L. Pringle, 2003a. Effect of exposure method to Beauveria bassiana and conidia concentration on mortality, mycosis, and sporulation in cadavers of Chilo partellus (Lepidoptera: Pyralidae). J. Inver. Pathol., 84: 90-95. Tefera, T. and K.L. Pringle, 2003b. Food consumption by Chilo partellus (Lepidoptera: Pyralidae) larvae infected with Beauveria bassiana and Metarhizium anisopliae and effects of feeding natural versus artificial diets on mortality and mycosis. J. Inver. Pathol., 84: 220-225.

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Vey, A. and J. Fargues, 1977. Histological and ultrastructural studies of Beauveria bassiana infection in Leptinotarsa decemlineata larvae during ecdysis. J. Inver. Pathol., 30: 207- 221. Wakil, W., M.U. Ghazanfar, T. Riasat, M.A. Qayyum, S. Ahmed and M. Yasin, 2013. Effects of interactions among Metarhizium anisopliae, Bacillus thuringiensis and chlorantraniliprole on the mortality and pupation of six geographically distinct field populations. Phytoparasitica, 41: 221-234. Wraight, S.P. and M.E. Ramos, 2005. Synergistic interaction between Beauveria bassianaand Bacillus thuringiensis tenebrionis-based biopesticides applied against field populations of Colorado potato beetle larvae. J. Inver. Pathol., 90: 139-150.

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Table 6.1 Percentage of petiole fragments colonized by entomopathogenic (E) and other (O) fungi in live palm petioles experiments

2014 2015 Isolate Site 15 days 30 days 15 days 30 days E O E O E O E O A5 - 100 - 100 - 100 - 100 A4 - 100 - 100 - 100 - 100 A3 20 80 20 80 15 85 25 75 A2 60 40 40 60 45 55 30 70 A1 100 - 60 40 90 10 50 50 INS 90 10 60 40 80 20 45 55 WG-11 B1 80 20 60 40 70 30 40 60 B2 40 60 20 80 33 66 15 85 B3 - 100 - 100 - 100 - 100 B4 - 100 - - - 100 - - B5 - 100 - - - 100 - - A5 - 100 - 100 - 100 - 100 A4 35 65 20 80 25 75 15 85 A3 66 33 40 60 50 50 30 70 A2 80 20 40 60 66 33 55 45 A1 100 - 60 40 90 10 60 40 INS 100 - 100 - 100 - 100 75 WG-40 B1 100 - 80 20 95 05 70 30 B2 75 25 80 20 66 33 66 33 B3 50 50 40 60 40 60 30 70 B4 25 75 15 85 20 80 - 100 B5 - 100 - 100 - 100 - 100 A5 50 50 25 75 40 60 20 80 A4 70 30 50 50 66 33 40 60 A3 75 25 50 50 70 30 40 60 A2 90 10 75 25 75 25 65 35 A1 100 - 90 10 90 10 80 20 INS 100 - 100 - 100 - 100 - WG-41 B1 100 - 80 20 90 10 70 30 B2 95 5 75 25 80 20 60 40 B3 80 20 66 33 70 30 50 50 B4 65 35 40 60 50 50 30 70 B5 40 60 33 66 40 60 20 80 A5 - 100 - 100 - 100 - 100 A4 33 66 - 100 25 75 - 100 A3 60 40 33 66 40 60 25 75 A2 85 15 55 45 70 30 40 60 A1 100 0 65 35 90 10 50 50 INS 100 0 85 15 100 0 85 15 WG-42 B1 100 0 60 40 85 15 50 50 B2 75 25 50 50 66 33 40 60 B3 66 33 20 80 50 50 15 85 B4 35 65 10 90 25 75 - 100 B5 - 100 - 100 - 100 - 100

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A5 - 100 - 100 - 100 - 100 A4 33 66 - 100 25 75 - 100 A3 55 45 40 60 40 60 30 70 A2 67 33 55 45 55 45 40 60 A1 90 10 75 25 75 25 60 40 INS 100 - 90 10 100 - 90 10 WG-43 B1 85 15 60 40 70 30 50 50 B2 67 33 45 55 50 50 35 65 B3 50 50 33 66 40 60 20 80 B4 15 85 - 100 - 100 - 100 B5 - - - 100 - - - 100 A5 - 100 20 80 - 100 - 100 A4 - 100 - 100 - 100 - 100 A3 10 90 - 100 - 100 20 80 A2 - 100 - 100 - 100 - 100 A1 - 100 - 100 20 80 10 90 INS - 100 - 100 - 100 - 100 Control B1 - 100 15 85 - 100 - 100 B2 - 100 - 100 - 100 - 100 B3 - 100 - 100 - 100 - 100 B4 - 100 - 100 - 100 - 100 B5 - 100 - 100 - 100 - 100 INS: site of injection; A1-A5 and B1-B5: site 2-10 cm above or below site of injection.

Table 6.2 Factorial analysis of mortality, pupation, adult emergence and egg eclosion of R. ferrugineus exposed to endophytically colonized B. bassiana and B. thuringiensis

Mortality Pupation Adult Egg Eclosion S.O.V. df Emergence F P F P F P F P Instar 2 63.34 ≤0.05 59.23 ≤0.05 51.45 ≤0.05 34.56 ≤0.05 Treatment 7 201.87 ≤0.05 229.10 ≤0.05 233.45 ≤0.05 249 ≤0.05 Instar × 14 1.56 0.093 0.68 0.625 0.61 0.635 0.78 0.515 Treatment Error 149 ------Total 172 ------

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Table 6.3 Mean mortality (%±SE) of 2nd, 4th and 6th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Treatments Second Instar Fourth Instar Sixth Instar Actual Expected CTF Actual Mortality Expected CTF Actual Mortality Expected CTF Type of Mortality (%) Mortality (%) Mortality (%) Mortality Interaction Bb 29.90±1.27de - - 23.36±1.18cde - - 20.67±1.17cde - - - Bt1 17.65±1.16e - - 12.53±1.09e - - 8.27±0.89e - - - Bt2 25.89±1.61de - - 18.58±1.12de - - 14.19±1.22de - - - Bt3 41.20±1.01cd - - 33.01±1.45cd - - 25.02±1.38cd - - - Bb + Bt1 54.63±2.26bc 47.55 14.88 42.13±1.32bc 35.89 17.38 34.07±1.50bc 28.94 17.72 Add. Bb + Bt2 71.01±2.39ab 55.79 27.28 54.07±1.65ab 41.94 28.92 47.87±1.42ab 34.86 37.32 Syn. Bb + Bt3 83.17±2.28a 71.1 16.97 65.86±1.50a 55.42 18.83 52.68±1.57a 44.96 17.17 Add. Control 2.4 - - 1.5 - - 1.00 - - - df 6 - - 6 - - 6 - - - F 33.3 - - 18.7 - - 26.1 - - - P ≤0.05 - - ≤0.05 - - ≤0.05 - - -

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Table 6.4 Pupation, adult emergence and egg eclosion (%±SE) of 2nd, 4th and 6th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Treatments Second Instar Fourth Instar Sixth Instar Pupation (%) Adult Egg Eclosion Pupation Adult Egg Eclosion Pupation Adult Egg Emergence (%) (%) Emergence (%) (%) Emergence Eclosion (%) (%) (%) (%) Bb 63.31±2.35c 57.72±2.77c 51.13±2.20c 71.16±2.60cd 64.46±2.93c 57.73±2.23b 76.64±3.33bc 70.06±2.88c 64.46±2.93b Bt1 78.82±3.15b 70.00±2.92b 63.37±2.48b 84.44±3.36ab 76.62±2.35b 65.97±3.01b 87.73±2.94ab 84.42±2.93ab 75.51±2.42b Bt2 66.67±2.87c 58.83±2.60bc 52.28±2.73bc 75.56±2.75bc 67.73±3.13bc 58.75±2.23b 80.10±2.88bc 73.36±3.12bc 66.65±2.88b Bt3 49.02±2.21d 37.96±1.53d 30.02±1.88d 58.81±2.60de 50.07±2.88d 43.31±2.35c 68.85±3.21c 52.24±2.77d 51.16±2.60c Bb + Bt1 32.24±2.12e 25.53±1.42e 18.84±1.15de 46.64±1.72e 38.83±2.09d 32.24±1.85c 53.36±2.40d 45.54±2.42d 37.72±2.77d Bb + Bt2 18.79±1.12f 11.10±1.02f 7.72±0.86ef 31.15±1.51f 17.96±1.11e 15.56±1.05d 44.42±2.93de 31.16±1.88e 24.46±2.42e Bb + Bt3 13.31±1.26f 6.66±0.63f 3.38±0.45f 24.41±1.34f 13.30±1.09e 8.83±0.69d 35.54±1.75e 22.27±1.23e 15.53±1.15e Control 94.48±1.75a 91.14±2.60a 85.52±2.93a 96.64±1.66a 92.18±2.24a 90.08±2.35a 95.49±1.91a 93.32±2.35a 87.72±2.22a df 7 7 7 7 7 7 7 7 7 F 119.0 117 122 83.1 103 90.1 46.0 73.6 100 P ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05

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Table 6.5 Growth parameters e.g. larval duration (days), larval weight (grams), pre-pupal duration (days), pre-pupal weight (grams), pupal duration (days), pupal weight (grams), adult longevity (days) and adult weight (grams) (%±SE) of 2nd instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-k (Bt1: 10 µg; Bt2: 15 µg; Bt3: 20 µg ml-1) alone and in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Larval Duration Larval Weight Pre-Pupal Pre-Pupal Pupal Pupal Weight Adult Longevity (days) Adult Weight (g) Treatments (days) (g) Duration Weight Duration (g) Male Female Male Female (days) (g) (days) Bb 109.16±3.56c 3.34±0.08bc 17.60±1.13de 3.20±0.06cd 24.16±1.12de 3.27±0.07cd 38.27±1.31a 42.16±1.15a 1.39±0.09abc 1.19±0.07bc Bt1 99.72±2.25d 4.07±0.08a 15.83±0.76fg 3.82±0.11ab 21.72±0.92fg 3.93±0.10ab 36.94±1.17ab 40.27±1.07ab 1.58±0.10ab 1.35±0.06ab Bt2 105.27±3.07cd 3.66±0.11b 16.48±0.57ef 3.52±0.12bc 22.94±1.18ef 3.65±0.10bc 35.50±1.05abc 39.16±1.25ab 1.50±0.11ab 1.28±0.06abc Bt3 111.71±3.10c 3.21±0.12cd 18.76±0.65cd 3.03±0.10de 25.83±1.04cd 3.12±0.11de 33.16±1.21bcd 37.94±1.03abc 1.37±0.08abcd 1.17±0.07bcd Bb + Bt1 118.62±2.80b 3.08± 0.10cde 20.16±1.38bc 2.77±0.06def 26.94±1.35bc 2.93±0.08def 30.94±0.91cde 35.61±1.14bcd 1.21±0.07bcd 1.02±0.06cde Bb + Bt2 121.27±2.56ab 2.96±0.09de 21.41±0.70ab 2.61±0.09ef 28.27±1.26ab 2.78±0.08ef 28.83±1.06de 33.00±0.98cd 1.07±0.05cd 0.91±0.04de Bb + Bt3 125.41±3.23a 2.74±0.07e 22.72±1.33a 2.38±0.04f 29.61±1.41a 2.54±0.09f 25.72±1.11e 30.38±1.03d 0.98±0.04d 0.88±0.03e Control 85.72±2.19e 4.42±0.15a 14.16±0.86g 4.02±0.11a 20.27±0.94g 4.14±0.11a 39.50±1.40a 43.72±1.40a 1.70±0.08a 1.51±0.06a Df 7 7 7 7 7 7 7 7 7 7 F 72.73 47.54 53.12 33.8 43.95 26.61 65.03 58.20 7.76 12.5 P ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05

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Table 6.6 Analysis of Co-variance for 10th instar larvae of R. ferrugineus regarding weight gain, frass production and diet consumption when treated with endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-k (Bt: 10 µg ml-1). Initial weight of larvae and diet consumption were taken as covariate

S.O.V. df F P Covariate: Diet Consumption 1 0.193 0.660 Covariate: Weight Gain 1 3.062 0.081 Frass Production × Diet Consumption 24 2.318 ≤0.05 Diet Consumption × Weight Gain 1 0.138 0.710 Frass Production × Weight Gain 27 2.733 ≤0.05 Frass Production × Diet Consumption × Weight 24 2.704 ≤0.05 Gain Error 687 - - Total 828 - -

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Figure 6.1 Mean mycosis (%±SE) in cadavers of R. ferrugineus treated with endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Figure 6.2 Sporulation (conidia ml-1) on R. ferrugineus cadavers treated with endophytic B. bassiana (Bb: 2 cm away from inoculation site) and Bt-k (Bt1: 30 µg; Bt2: 40 µg; Bt3: 50 µg ml-1) alone and in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

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Figure 6.3 Diet consumption (grams) in 10th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-k (Bt: 10 µg ml-1)

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Figure 6.4 Frass production (grams) in 10th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-k (Bt: 10 µg ml-1)

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Figure 6.5 Weight gain (grams) in 10th instar larvae of R. ferrugineus treated with endophytic B. bassiana (Bb: 6 cm away from inoculation site) and Bt-k (Bt: 10 µg ml-1)

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CHAPTER 7 Integrated Effect of Entomopathogenic fungi and Entomopathogenic Nematode against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

Abstract Research study was carried out to investigate the insecticidal properties of entomopathogenic fungi Beauveria bassiana s.l. (Ascomycota: Hypocreales) strain WG-11, Metarhizium anisopliae s.l. (Ascomycota: Hypocreales) strain WG-02 and the entomopathogenic nematode, Heterorhabditis bacteriophora Poinar (Heterorhabditidae) for their virulence against 2nd, 4th and 6th instar larva of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). Both agents were either applied alone or in combination, with H. bacteriophora 1 and 2 weeks after fungi application. Moreover, the complete spectrum of toxicity, development, diet consumption, frass production and weight gain were observed at sub lethal doses of both agents. In combined treatments, additive and synergistic interactions were observed for all the three instars and effects were not significantly different among the treatments either applied simultaneously or in sequential combinations with each other. Enhanced morality was recorded for the combined treatments when delayed application of H. bacteriophora was made 1 or 2 weeks after fungus treatment as compared to their sole application. Decrease in pupation, adult emergence and egg hatching were also found related to the toxic effect of treatments. Duration of different developmental stages was significantly affected by the treatments applied. Decreased larval weight, increased larval duration, increased pre-pupal and pupal period and decreased weight, decreased adult weight and life span were recorded and compared to the control. Larvae fed on sub lethal amounts of both agents revealed reduced food ingestion, reduced growth and weight gain, preventing most of them from achieving the critical weight. Initial weight of larvae exerted its impact on weight gain and diet consumption, and the trend was found linked to pathogenicity of applied agents. A result of the present study suggests that R. ferrugineus can be successfully managed by applying entomopathogenic fungi and H. bacteriophora. Additionally, their simultaneous and sequential application may offer enhanced mortality as compare to the application of either of them alone.

Keywords: Rhynchophorus ferrugineus, Beauveria bassiana, Metarhizium anisopliae, Heterorhabditis bacteriophora, diet consumption, frass production

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7.1 Introduction The coleopteran insects are ranked among the most voracious pests of economically important crops. Among these the Red Palm weevil (RPW) Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) is most destructive to 29 different palm species particularly date palms of economic importance in the Middle East, Africa and South East Asia (Malumphy and Moran, 2009). Synonymously it is known as Asiatic palm weevil, coconut weevil, red stripe weevil, hidden enemy and also called AIDS palm because of the damage and the slow death of the palm tree (Khamiss and Abdel-Badeea, 2013). The pest has cryptic nature and mostly damages the palm trees younger than 20 years (Nirula, 1956; Abraham et al., 1998) in which crown, trunk and bole are the natural sites of damage, while the crown is the site of infestation in older plantations. The larval stages destroy the vascular system while boring into the heart of the host leading to tree collapse (Ju et al., 2011). Insecticides and fumigants remained the mainstay of date palm growers for decades but the cryptic nature of RPW presented an access challenge to treatments (Hussain et al., 2013). Moreover, insecticides exert negative effects on the environment and human health and more importantly pests have developed resistance against these chemicals (Abraham et al., 1998). Alternatively entomopathogens can be used for suppression of this notorious pest in a wide array of management approaches in versatile manners. Among microbial control agents entomopathogenic fungi (EPFs) particularly, Beauveria bassiana s.l. (Ascomycota: Hypocreales) and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales) are considered promising alternatives to conventional chemical insecticides. They pose negligible detrimental effects on the environment and human health (Khan et al., 2012), and have been reported to be effective against a number of arthropod pests (Charnley and Collins, 2007; de Faria and Wraight, 2007). Several researchers have isolated and successfully deployed these two strains against different developmental stages of RPW as bio-control agents both under laboratory and field conditions (Deadman et al., 2001; Gindin et al., 2006; El-Sufty et al., 2007, 2009, 2011; Sewify et al., 2009; Torta et al., 2009; Vitale et al., 2009; Güerri-Aguilló et al., 2010; Merghem, 2011; Francardi et al., 2012; Ricaño et al., 2013; Cito et al., 2014). EPFs are preferred over the other microorganisms due to their novel mode of action by direct contact to the host cuticle instead of ingestion or engulfing and their ability to transfer inoculum from treated insects to untreated insects or to subsequent developmental stages via the new generation of spores (Quesada- Moraga et al. 2004). Similarly, entomopathogenic nematodes (EPNs) are also promising microbial control agents and declared efficient control agents against vast array of insect pests (Abbas et al., 2001; Llácer et al., 2009; Dembilio et al., 2010a). They are obligate parasites in the families Steinernematidae and Heterorhabditidae which kill insects with the aid of mutualistic bacterium, which is carried in their intestine (Xenorhabdus spp. and Photorhabdus spp. are associated with Steinernema spp. and Heterorhabditis spp., respectively) (Poinar, 1990). Both agents are considered safer to non-target organisms (vertebrates and invertebrates) and compatible to environment, and they are successfully integrated with each other exhibiting strong additive and synergistic interactions (Thurston et al., 1993, 1994; Koppenhöfer and Kaya, 1997; Koppenhöfer et al., 1999). This study aimed at the integration of B. bassiana, M. anisopliae and H. bacteriophora to examine the mortality; development and growth of R. ferrugineus under laboratory conditions to select the suitable application times of both agents for future field trials to successfully manage R. ferrugineus populations in Pakistan.

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7.2 Materials and Methods

7.2.1 RPW collection and rearing Survey was conducted for collection of R. ferrugineus in date palm growing areas of west Punjab, Pakistan. Different developmental stages (larvae, pupae and adults) were collected from fallen and infested date palm trees with the permission of farmers (owners). All the stages collected were kept separately in plastic jars until brought to the Microbial Control Laboratory, Department of Entomology, University of Agriculture, Faisalabad (UAF), Pakistan. Larvae were fed with sugarcane (Saccharum officinarum L.; Poales: Poaceae) sets and the same were used for pupation after last instar, while shredded sugarcane pieces were offered to adults for feeding and substrate for oviposition. After pupation pupal cocoon were kept in separate plastic jars for adult emergence in incubator set at 25±2 oC, 65±5% RH and 12:12 (D: L) hours photoperiod. After adult emergence beetles were shifted to the adult’s jar for feeding, mating and oviposition. Colony was developed in plastic boxes (30×60×60 cm) having a lid covered with mesh wire gauze (60 mesh size) in the middle (10 cm diameter) for aeration. Adult’s diet was changed after every three days and replaced sugarcane pieces were kept in separate jars (8×8×12 cm) for egg hatching. After egg hatching neonate larvae were allowed to feed for some time in the same set after 3 days larvae were transferred to the same sugarcane sets for feeding and pupation. Larvae were shifted to the new sugarcane sets after every week until pupation. The rearing conditions were maintained at 25±2 oC, 65±5% RH and 12:12 (D: L) hours photoperiod.

7.2.2 Entomopathogenic Nematode Infective juveniles (IJs) of H. bacteriophora culture was obtained from Microbial Control Laboratory which was used for the bioassay against 2nd, 4th and 6th instar larvae of R. ferrugineus. H. bacteriophora was maintained in 3rd instar Galleria mellonella L. (Lepidoptera: Pyralidae) following the procedure of Kaya and Stock (1997).

7.2.3 Entomopathogenic fungi Two isolates of entomopathogenic fungi B. bassiana (WG-11) and M. anisopliae (WG- 02) used in the study were taken from the culture collection of Microbial Control Laboratory, originally isolated from soils of vegetables and crop fields respectively. Mass culturing was done by inoculating Petri plates containing Potato Dextrose Agar (PDA) media (BD, France). Spore concentration of 1×106 spore ml-1 was determined with a Neubauer haemocytometer.

7.2.4 Treatment with entomopathogenic fungi A spore concentration of 1×106 spore ml-1 was prepared from conidial powder of B. bassiana and M. anisopliae using haemocytometer. Second, 4th and 6th instar larvae were directly immersed in 100 ml conidial suspension for 60s individually and control was treated in aqueous solution of 0.01% Tween-80 (Merck, KGaA, Darmstadt, Germany) (Dembilio et al., 2010b). The fungal isolate treated and control larvae were individually shifted to 150 ml cylindrical plastic cups, each measuring 6 cm in height with 6 cm diameter. The top of the cups were covered with a fine mesh in order to avoid the insects to escape. A piece of 2×2 cm2 artificial diet (Agar, brewer’s yeast, wheat germ, corn flour, ascorbic acid, benzoic acid, amino acid-vitamin mix, chloramphenicol and nipagin) (Martín and Cabello, 2006) were kept in the center of cups and incubated at 27±2 °C, 65±5 % RH and 12:12 (D: L) hours photoperiod in an incubator (Sanyo, Japan). Three replicates of 10 larvae were treated to the fungal suspension. Each cup was opened

136 daily and checked for mortality, and the old diet was replaced with fresh artificial diet until dying or pupation. After last instar dry coir (coconut coir) was provided to the surviving larvae for pupation. The bioassay was repeated thrice to avoid the pseudo-replication phenomenon.

7.2.5 Treatment with H. bacteriophora Nematode suspension was prepared with a concentration of 100 IJs ml-1 in glass jars and 1 ml of suspension was poured into the cylindrical plastic cups lined with Whatman filter paper. After pouring 30 minutes were given for evenly distribution of nematodes on filter paper. A small piece of artificial diet 2×2 cm2 was placed in middle of the cups as a food source. Ten larvae for each treatment were used separate in each cup and each treatment was replicated three times, while control treatment received 1 ml of distilled water. The cups were maintained at above mentioned conditions. Each cup was opened daily and checked for mortality, and the old diet was replaced with fresh artificial diet until dying or pupation. After last instar dry coir was provided to the surviving larvae for pupation. Whole bioassay was repeated thrice to avoid the pseudo replication phenomenon.

7.2.6 Treatment with entomopathogenic fungi and nematode In combined treatments both agents were applied simultaneously or at different time intervals as follows:

 B. bassiana, M. anisopliae and H. bacteriophora were applied simultaneously: larvae were immersed in fungal suspensions and transferred to the cylindrical plastic cups lined with moisten filter paper treated with H. bacteriophora IJs and maintained at 27±2 °C and 65±5% RH at 12: 12 (D: L) hours photoperiod.  Insects were first inoculated with B. bassiana and M. anisopliae, maintained at 27±2 °C and 65±5% RH for one week, transferred to cylindrical plastic cups lined with moisten filter paper treated with H. bacteriophora IJs and maintained at above mentioned conditions.  Insects were first inoculated with B. bassiana and M. anisopliae, maintained at 27±2 °C and 65±5% RH for two weeks, transferred to cylindrical plastic cups lined with moisten filter paper treated with H. bacteriophora IJs and maintained at above mentioned conditions.  Control insects were immersed in aqueous solution with 0.01% Tween-80 and maintained in cylindrical plastic cups lined with moistened filter paper using conditions stated above.

Larval mortality was recorded after one, two and three weeks post application. For all treatments, artificial diet was offered to the larvae as food source. Larvae that failed to respond on slight prodding by blunt needle were considered dead. After the last instar dry coir was provided for pupation. Percent pupation, adult emergence and egg eclosion were also recorded.

7.2.7 Effects of entomopathogens on R. ferrugineus development To check the effect of entomopathogens on development of RPW, 4th instar larvae were exposed to the sub-lethal dose of fungal entomopathogens (1×104 spore ml-1) and H. bacteriophora (50 IJs ml-1). The larvae were fed on artificial diet and transferred to the treatment cups with 1 ml of. Dry coir was provided to the each larva before pupation for cocoon formation. While adult on emergence were offered with shredded sugarcane pieces. Developmental

137 parameters of each stage (Larval duration, larval weight, pre-pupal duration, pre-pupal weight, pupal duration, pupal weight, adult longevity (male and female) and adult weight (male and female) was recorded.

7.2.8 Effects of entomopathogens on larval development For the larval development last instar larvae of RPW was exposed to sub-lethal doses of B. bassiana (1×104 spore ml-1) and H. bacteriophora (50 IJs ml-1). Before exposure in all the treatments larvae were weighed first and transferred to the rearing cups with artificial diet. Larvae continued to feed until pupated under experimental conditions maintained at 25±2 °C, 65±5% RH and L: D (12: 12) hours photoperiod. Every day until the larvae pupated, larvae were changed to a new clean cup and new piece of artificial diet was offered. Frass produced during this period was separated from vials using tip of fine camel hair brush and weighed. Diet left unused in each vial was recovered, dried in drying oven at 80 °C. Prior to assay, diet in fifteen vials was dried to obtain an estimate of the dry weight. Diet consumption of each larva was determined by subtracting after feeding mass of diet from before feeding mass. Three replicates of ten insects were used for each treatment and same count of larvae fed on normal diet served as untreated check while entire experiment was repeated thrice.

7.2.9 Statistical analysis The fungus nematode interactions (synergistic, additive or antagonistic) were calculated using formula devised by Nishimatsu and Jackson (1998). The type of interaction was determined through a comparison of expected and observed percentage mortality of RPW. Expected mortality was calculated using formula PE = P0 + (1- P0) (P1) + (1- P0) (1- P1) (P2), where PE is the expected mortality of the combination, P0 is the control mortality, P1 is the mortality from one pathogen treatment applied alone, and P2 is the mortality from the other pathogen applied alone. A X2 test was applied to the observed and expected results: X2 = 2 2 (L0 - LE) / LE + (D0 - DE) / DE, where L0 is the number of living larvae observed, LE the number of living larvae expected, D0 the number of dead larvae observed, and DE the number 2 2 of dead larvae expected. Interactions were additive if X < 3.84, antagonistic if X > 3.84 and PC 2 < PE, and synergistic if X > 3.84 and PC > PE, where PC is the observed mortality from the combination and PE is the expected mortality from the combination. Data for pupation, adult emergence, egg eclosion and developmental parameters were subjected to one way analysis of variance (ANOVA) in Minitab (Minitab, 2003) means were separated using Tukey’s Kramer test (HSD) (Sokal and Rohlf, 1995) at 5% significance level. To inspect the impact of microbial agents on the diet consumption, weight gain and frass production were analyzed by ANCOVA using initial larval weight and diet consumption as covariates (Janmaat et al., 2014).

7.3 Results

7.3.1 Entomopathogenic fungi and nematode interaction In integrated applications of H. bacteriophora with B. bassiana and M. anisopliae additive to synergistic interaction were observed when both agents were applied simultaneously or delayed nematode application for all the three instars tested (Table 7.1, 7.2 and 7.3). During simultaneous application, B. bassiana and H. bacteriophora produced additive lethality to 2nd instar larvae for first two weeks, while synergistic interactions were observed the third week after application. The degree of synergism increased with the delayed application of H.

138 bacteriophora one or two weeks after initial B. bassiana treatments. For M. anisopliae additive effects were recorded for simultaneous application, while interactions were shifted towards synergism when delayed nematode application was made after one and two weeks of fungal spore application (Table 7.1). Similar trends were recorded for 4th and 6th instar larvae but 2nd instars were less susceptible to treatments. Percent pupation, adult emergence and egg eclosion from surviving individuals was found inversely related to toxic level of microbial agents and delayed application of H. bacteriophora for all instars tested (Table 7.5). In factorial analysis main effects for pupation adult emergence and egg eclosion were significant while their interaction effects were non-significant except pupation (Table 7.4)

7.3.2 Development of R. ferrugineus Growth and development of 4th instar RPW larvae was adversely affected by the toxic effect of the microbial agents. When larvae were exposed to the sub-lethal doses of H. bacteriophora, B. bassiana and M. anisopliae, significant variations were recorded for larval duration, larval weight, pre-pupal duration, pre-pupal weight, pupal duration, pupal weight, adult longevity and adult weight (larval duration: F5, 53 =9.92, P≤0.05; larval weight F5, 53 =27.3, P≤0.05; pre-pupal duration: F5, 53 =6.59, P≤0.05; pre-pupal weight: F5, 53 =6.94, P≤0.05; pupal duration F5, 53 =5.15, P≤0.05; pupal weight F5, 53 =11.10, P≤0.05; adult longevity (female F5, 53 =3.93, P≤0.05 and male F5, 53 =5.58, P≤0.05 ); adult weight (female F5, 53 =4.26, P≤0.05 and male F5, 53 =12.7, P≤0.05). Increase in larval, pre-pupal and pupal duration while decrease in weight was recorded for all the treatments tested. On the other hand decrease in adult life span and weight (male and female) was also recorded. Highest detrimental effect on growth was recorded for combined application of B. bassiana and H. bacteriophora followed by M. anisopliae and H. bacteriophora, H. bacteriophora alone, B. bassiana and M. anisopliae (Table 7.6).

7.3.3 Effect on larval development Diet consumption by 10th instar larvae was significantly influenced by the treatments applied; diet consumption was low in combined treatments of H. bacteriophora and B. bassiana as compared to their individual applications (Fig 6.1). Similarly frass production was influenced by treatments applied with lowest frass production for combined treatments of H. bacteriophora and B. bassiana (0.57±0.04 to 0.00±0.00g) during the experimental period. After treatment frass production gradually decreased to zero before pupation. On the other hand, the highest frass production was found in untreated larvae during the last instar larvae until pupation (Fig 6.2). Larvae treated with sub-lethal concentrations of either B. bassiana or H. bacteriophora alone gained more weight compared to larval treated with a combined application. Initial weight of larvae (10th instar: 4.31±0.14g) exerted its impact on the weight gain and among treatments, there was a trend of weight gain was linked to pathogenicity. Combined application of B. bassiana and H. bacteriophora had an adverse impact on the weight gain. The lowest weight gain (-0.54±0.04g) was recorded for the combined treatment while the highest gain (- 0.12±0.02g) was recorded in untreated larvae (Fig 6.3).

7.4 Discussion This is very first study to investigate the combined effect of fungal isolates and H. bacteriophora against larvae of RPW. The results revealed that both agents can effectively control the larval stages, either applied simultaneously or delayed application of H.

139 bacteriophora 1 or 2 weeks of fungal treatment. Additive to synergistic interactions were recorded for combined applications of both agents. Greater additive or synergistic interactions were observed when fungi were applied 1 or 2 weeks before H. bacteriophora treatment. Our study corroborates the findings of Ansari et al. (2004, 2006) who reported similar results with combined application of H. megidis or S. glaseri with M. anisopliae CLO 53 against 3rd instar H. philanthus under laboratory and greenhouse conditions, and between H. bacteriophora and M. anisopliae CLO 53 under field conditions respectively. Similarly additive and synergistic effects were observed in combined treatments of H. bacteriophora and M. anisopliae isolate MM against barley chafer grub, C. curtipennis (Anbesse et al., 2008). They have suggested exposing grubs 3 or 4 weeks before addition of nematodes to get stronger synergistic interaction. However in our study enhanced efficacy and stronger interactions were recorded 1 or 2 weeks delayed application of H. bacteriophora. It is tempting to speculate that longer grubs were exposed to the fungus, the more debilitated they become and subsequently were more susceptible to the EPN. The debilitated insects respired more, attracting the EPNs, which followed a CO2 gradient to their hosts (Ansari et al., 2008). Steinhaus, (1958) also suggested that the stressed insects were more vulnerable to pathogen infection, hence enhancing insect mortality or facilitating the speed of kill and enhancing additive or synergistic effects in combined treatments. For example, Paenibacillus popilliae (Dutky) against scarab larvae acted as a stressor to nematode infection that caused elevated larval mortality (Thurston et al., 1993; Thurston et al., 1994). Other authors also have reported similar results during their studies (Kermarrec and Mauleon, 1989; Barbercheck and Kaya, 1990; Thurston et al., 1993, 1994; Koppenhöfer and Kaya, 1997; Koppenhöfer et al., 1999). Contrarily Shapiro-Ilan et al. (2004) found antagonism between EPNs and P. fumosoroseus. This antagonism may be due to pathogen interactions prior to or during infection. In case of Sternima marcescens and P. fumosoroseus, it is possible that these organisms are directly pathogenic to EPNs, therefore nematodes may have been killed or their fitness reduced prior to infection. The negative interactions may also be due to antagonistic toxins produced by the entomopathogens after infection was initiated. The synergy shown between fungi and H. bacteriophora provided an opportunity to reduce the cost of RPW control while increasing the overall efficacy of the control strategy. For performing normal daily functions, an insect needs to have proper growth and development and any delay may render the insect susceptible to biotic and abiotic factors such as natural enemies, and environmental regimes that ultimately influence the growth, development, diet consumption and frass production. In this regard larval stages are considered vulnerable towards these agents (Marzban et al., 2009). The extended larval and pupal period reduces time span left for adult stage that directly affects the insect’s fecundity and in other words threatens the survival in next generation. Outcomes of our present study indicate that entomopathogenic fungi and H. bacteriophora can be applied successfully against RPW but their efficacy varies depending upon the interval of nematode application. Larval development is greatly influenced by the lethal action of both entomopathogenic fungi and H. bacteriophora affecting the degree of diet consumption that establishes the foundation for insect control. The alone and combined concentrations of both agent offers a range of toxicity that exerts corresponding effect on the development and survival of target host. In present study detrimental effects imposed by EPFs and H. bacteriophora decreased larval, pre-pupal, pupation rate, pupal weight and prolonged the developmental period. It is generally accepted that major part of the insect energy consumed fighting pathogens lead to the

140 weakness and growth retardation in insects (Sikorowski and Thampson, 1979; Wiygul and Sikorowski, 1991). Similar observations have been made during the current research and the integrated application of H. bacteriophora and EPFs increased the larval mortality compared with their respective individual treatments. Therefore, in an IPM program they can be recommended for pest control where this entomopathogens are important natural enemies.

Conclusions The present study showed that B. bassiana and M. anisopliae isolates in integration with H. bacteriophora under laboratory conditions caused high mortality against larvae of red palm weevil. The pathogens exerted detrimental effects on growth and development of different developmental stages of R. ferrugineus. Hence, integrated application of H. bacteriophora in sequential manners with B. bassiana and M. anisopliae can be effectively used for the successful control of red palm weevil.

Acknowledgements This research work was supported by the scholarship from Higher Education Commission (HEC), Islamabad, Pakistan (112-30536-2AV1-263) under Indigenous Ph.D. Fellowship Program.

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7.5 References Abraham, V.A., M.A. Al-Shuaibi, M.A. Faleiro, J.R. Abozuhairah, R.A. and P.S.P. Vidyasagar, 1998. An integrated management approach for red palm weevil Rhynchophorus ferrugineus Oliv a key pest of date palm in the Middle East. Proc. Inter. Conf. on Integrated Pest Management (Muscat, Sultanate of Oman). Sultan Qaboos Uni. J. Sci. Res., 3: 77-83. Anbesse, S.A., B.J. Adge, W.M. and Gebru, 2008. Laboratory screening for virulent entomopathogenic nematodes (Heterorhabditis bacteriophora and Steinernema yirgalemense) and fungi (Metarhizium anisopliae and Beauveria bassiana) and assessment of possible synergistic effects of combined use against grubs of the barley chafer Coptognathus curtipennis. Nematol., 10: 701-709 Ansari, M.A., B.N. Adhikari, F. Ali and M. Moens, 2008. Susceptibility of Hoplia philanthus (Coleptera: Scarabaeidae) larvae and pupae to entomopathogenic nematodes (Rhabditida: Steinernematidae, Heterorhabditidae). Biol. Control, 47: 315-321. Ansari, M.A., F.A. Shah, L. Tirry and M. Moens, 2006. Field trials against Hoplia philanthus (Coleoptera: Scarabaeidae) with a combination of an entomopathogenic nematode and the fungus Metarhizium anisopliae CLO 53. Biol. Control, 39: 453-459. Ansari, M.A., S. Vestergaard, L. Tirry and M. Moens, 2004. Selection of a highly virulent fungal isolate, Metarhizium anisopliae CLO 53, for controlling Hoplia philanthus. J. Inver. Pathol., 85: 89-96. Barbercheck, M.E. and H.K. Kaya, 1990. Interactions between Beauveria bassiana and the entomogenous nematodes Steinernema feltiae and Heterorhabditis heliothidis. J. Inver. Pathol., 55: 225-234. Charnley, A. and S.A. Collins, 2007. Entomopathogenic fungi and their role in pest control. In: Howard, D.H. and J.D. Miller (Eds.), The Mycota IV: Environmental and Microbial Relationships, Springer-Verlag, Berlin, Heidelberg, pp.159-187. Cito, A., G. Mazza, A. Strangi, C. Benvenuti, G.P. Barzanti, E. Dreassi, T. Turchetti, V. Francardi and P.F. Roversi. 2014. Characterization and comparison of Metarhizium strains isolated from Rhynchophorus ferrugineus. FEMS Microbiol. Lett., 355: 108-115. de Faria, M.R. and S.P. Wraight, 2007. Mycoinsecticides and Mycoacaricides: a comprehensive list with worldwide coverage and international classification of formulation types. Biol. Control, 43: 237-256. Deadman, M.L., K.M. Azam, S.A. Ravzi and W. Kaakeh. 2001. Preliminary investigation into the biological control of the red palm weevil using Beauveria bassiana. Proceedings of the Second International Conference on Date Palm, Al-Ain, UAE. March 25-27: 225- 232. Dembilio, Ó., E. Quesada-Moraga, C. Santiago-Alvarez and J.A. Jacas, 2010b. Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the red palm weevil, Rhynchophorus ferrugineus. J. Inver. Pathol., 104(3): 214-221. El-Sufty, R., S. Al Bgham, S.A. Al-Awash, A.S. Shahdad and A.H. Al Bathra, 2011. A trap for auto-dissemination of the entomopathogenic fungus Beauveria bassiana by the red palm weevil adults in date palms plantations. Egyp. J. Biol. Pest Control, 21(2): 271-276. El-Sufty, R., S.A. Al-Awash, A.M. Al-Amiri, A.S. Shahdad, A.H. Al-Bathra and S.A. Musa, 2007. Biological control of red palm weevil, Rhynchophorus ferrugineus (Col.: Curculionidae) by the entomopathogenic fungus Beauveria bassiana in United Arab

142

Emirates. Proceeding of the 3rd International Conference on Date Palm. Acta Horticulturae. 736: 399-404. El-Sufty, R., S.A. Al-Awash, S. Al Bgham, A.S. Shahdad, A.H. Al-Bathra, 2009. Pathogenicity of the fungus Beauveria bassiana (Bals.) Vuill to the red palm weevil, Rhynchophorus ferrugineus (Oliv.) (Col.: Curculionidae) under laboratory and field conditions. Egyp. J. Biol. Pest Control, 19: 81-85. Francardi, V., C. Benvenuti, P.F. Roversi, P. Rumine and G. Barzanti, 2012. Entomopathogenicity of Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) Sorokin isolated from different sources in the control of Rhynchophorus ferrugineus (Olivier) (Coleoptera Curculionidae). Redia, 95: 49-55. Gindin, G., S. Levski, I. Glazer and V. Soroker, 2006. Evaluation of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana against the red palm weevil Rhynchophorus ferrugineus. Phytoparasitica, 34(4): 370-379. Güerri-Agulló, B., S. Gómez-Vidal, L. Asensio, P. Barranco and L.V. Lopez-Llorca, 2010. Infection of the red palm weevil (Rhynchophorus ferrugineus) by the entomopathogenic fungus Beauveria bassiana: a SEM study. Microsco. Res. Tech., 73: 714-725. Hussain, A., M.R.U. Haq, A.M. Al-Jabr and H.Y. Al-Ayied, 2013. Managing Invasive Populations of Red Palm Weevil: A WorldwidePerspective. J. Food Agri. Environ., 11: 456-463. Janmaat, A.F., L. Bergmann and J. Ericsson, 2014. Effect of low levels of Bacillus thuringiensis exposure on the growth, food consumption and digestion efficiencies of Trichoplusia ni resistant and susceptible to Bt. J. Inver. Pathol., 119: 32-39. Ju, R.T., F. Wang, F.H. Wan and B. Li, 2011. Effect of host plants on development and reproduction of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). J. Pest Sci., 84(1): 33-39. Kaya, H.K. and S.P. Stock, 1997. Techniques in insect nematology. Manual of Techniques in Insect Pathology (ed. by LA Lacey). Academic Press, London, UK. pp. 281-324. Kermarrec, A. and H. Mauleon, 1989. Synergy between chlordecone and Neoaplectana carpocapsae Weiser (Nematoda: Steinernematidae) in the control of Cosmopolites sordidus (Coleoptera: Curculionidae). Rev. Nematol., 12: 324-325. Khamiss, O. and A. Abdel Badeea, 2013. Initiation, characterization and karyotyping of a new cell line from red palm weevil rhynchophorus ferrugineus adapted at 27°c. AFPP - palm pest mediterranean conference nice - 16, 17 and 18 january 2013. Khan, S., L. Guo, Y. Maimaiti, M. Mijit and D. Qiu, 2012. Entom opathogenic fungi as microbial biocontrol agent. Mol. Plant Breed, 3(7): 63-79. Koppenhöfer, A.M. ans H.K. Kaya, 1997. Additive and Synergistic interactions between entomopathogenic nematodes and Bacillus thuringiensis for scarab grub control. Biol. Control, 8: 131-137. Koppenhofer, A.M., H.Y. Choo, H.K. Kaya, D.W. Lee and W.D. Gelernter, 1999. Increased field and greenhouse efficacy against scarab grubs with a combination of an entomopathogenic nematode and Bacillus thuringiensis. Biol. Control, 14: 37-44. Malumphy, C. and H. Moran, 2009. Red palm Weevil, Rhynchophorus ferrugineus. Plant Pest Factsheet. Available online at www.fera.defra.gov.uk/plants/publications/documents/factsheets/redPalmWeevil.pdf (accessed 19 September 2012).

143

Martín, M.M. and T. Cabello, 2006. Manejo de la cría del picudo rojo de la palmera, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera, Dryophthoridae), en dieta artificial y efectos en su biometría y biología. Boletín de Sanidad Vegetal de Plagas, 32: 631-641. Marzban, R., Q. He, X. Liu and Q. Zhang, 2009. Effects of Bacillus thuringiensis toxin Cry1Ac and cytoplasmic polyhedrosis virus of Helicoverpa armigera (Hübner) (HaCPV) on cotton bollworm (Lepidoptera: Noctuidae). J. Inver. Pathol., 101: 71-76. Merghem, A., 2011. Susceptibility of the red palm weevil, Rhynchophorus ferrugineus (Olivier) to the green muscardine fungus, Metarhizium anisopliae (Metsch.) in the laboratory and in palm tree orchards. Egyp. J. Biol. Pest Control, 21: 179-183. Minitab, 2003. MINITAB Release 14 for Windows. Minitab Inc., State College, Pennsylvania, USA. Mirza, I. 2007. Tomato paste plant to be set up at Killa Saifullah. Available at url; http://www.pakissan.com/english/news/newsDetail.php?newsid=15041. Accessed on August 31, 2007. Nirula, K.K., 1956. Investigations on the pests of coconut palm. Part IV. Rhynchophorus ferrugineus. Ind. Coc. J., 9: 229-247. Nishimatsu, T., J.J. Jackson, 1998. Interaction of insecticides, entomopathogenic nematodes, and larvae of the western corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol., 91: 410-418. Poinar Jr, G.O., 1990. Taxonomy and biology of Steinernematidae and Heterorhabditidae. In: Gaugler, R., H.K. Kaya (Eds.), Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, FL, pp. 23-61. Ricaño, J., B. Güerri-Agulló, M.J. Serna-Sarriás, G. Rubio-Llorca, L. Asensio, P. Barranco, L.V. Lopez-Llorca. 2013. Evaluation of the pathogenicity of multiple isolates of Beauveria bassiana (Hypocreales: Clavicipitaceae) on Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) for the assessment of a solid formulation under simulated field conditions. Florida Entomol., 96: 1311-1324. Sewify, G.H., M.H. Belal and S.A. Al-Awash, 2009. Use of the entomopathogenic fungus, Beauveria bassiana for the biological control of the red palm weevil, Rhynchophorus ferrugineus Olivier. Egyptian Journal of Biological Pest Control. 19(2):157-163. Shapiro-Ilan, D.I., M. Jackson, C.C. Reilly and M.W. Hotchkiss, 2004. Effects of combining an entomopathogenic fungi or bacterium with entomopathogenic nematodes on mortality of Curculio caryae (Coleoptera: Curculionidae). Biol. Control, 30: 119-126. Sikorowski, P.P. and A.C. Thampson, 1979. Effects of cytoplasmic polyhedrosis virus on diapausing Heliothis virescens. J. Inver. Pathol., 33: 66-70. Sokal, R.R. and F.J. Rohlf, 1995. Biometry. Freeman, New York. Steinhaus, E.A., 1958. Stress as a factor in insect disease. Proceedings of the Xth Inter. Cong. Entomol., 4: 725-730. Thurston, G.S., H.K. Kaya and R. Gaugler, 1994. Characterization of enhanced susceptibility of milky disease infected scarabaeid grubs to entomopathogenic nematodes. Biol. Control, 4: 67-73. Thurston, G.S., H.K. Kaya, T.M. Burlando and R.E. Harrison, 1993. Milky disease bacteria as a stressor to increase susceptibility of scarabaeid larvae to an entomopathogenic nematode. J. Inver. Pathol., 61: 167-172.

144

Torta, L., V. Leone, C.G. Caldarella, G. Lo Verde and S. Burruano, 2009. Microrganismi fungini associati a Rhynchophorus ferrugineus (Olivier) in Sicilia e valutazione dell’efficacia entomopatogena di 484 Ann Microbiol., 65: 477-485. Vitale, A, V. Leone, L. Torta, S, Burruano and G. Polizzi, 2009. Prove preliminari di lotta biologica con Beauveria bassiana e Metarhizium anisopliae nei confronti del punteruolo rosso. In Regione Siciliana - Assessorato Agricoltura e Foreste. La ricerca scientifica sul Punteruolo rosso e gli altri fitofagi delle palme in Sicilia, Palermo, Italy. pp. 1:169-172. Wiygul, G. and P.P. Sikorowski, 1991. Oxygen uptake in larval bollworm (Heliothis zea) infected with iridescent virus. J. Inver. Pathol., 58: 252-256.

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Table 7.1 Mean mortality (%±SE) of 2nd instar larvae of R. ferrugineus treated with B. bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae were used each @ 1×106 spore ml-1 and H. Bacteriophora was applied @ 100 IJs ml-1.

Treatments Intervalsa Weekb Observed Expected Chi Sq. Type of mortality mortality interaction - 1 11.22 - - - Bb - 2 14.28 - - - - 3 20.40 - - - - 1 8.16 - - - Ma - 2 12.24 - - - - 3 17.34 - - - - 1 14.28 - - - EPN - 2 21.42 - - - - 3 29.59 - - - 0 1 27.55 23.90 0.48 Additive Bb+EPN 0 2 43.87 32.65 2.87 Additive 0 3 61.22 43.96 4.86 Synergistic 0 1 23.71 21.28 0.24 Additive Ma+EPN 0 2 32.98 38.21 0.82 Additive 0 3 48.45 36.94 2.73 Additive 7 1 32.99 26.53 1.26 Additive Bb+EPN 7 2 51.54 37.46 3.84 Synergistic 7 3 73.19 51.86 6.21 Synergistic 7 1 28.86 24.78 0.57 Additive Ma+EPN 7 2 45.36 35.05 2.33 Additive 7 3 64.94 48.27 4.28 Synergistic 14 1 51.54 37.46 3.84 Synergistic Bb+EPN 14 2 69.07 51.86 4.28 Synergistic 14 3 88.65 62.51 7.70 Synergistic 14 1 44.32 35.05 1.93 Additive Ma+EPN 14 2 64.94 48.99 3.92 Synergistic 14 3 75.25 53.95 6.02 Synergistic a Intervals between the application of EPFs and EPNs. b Week after fungal application.

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Table 7.2 Mean mortality (%±SE) of 4th instar larvae of R. ferrugineus treated with B. bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae were used each @ 1×106 spore ml-1 and H. Bacteriophora was applied @ 100 IJs ml-1.

Treatments Intervalsa Weekb Observed Expected Chi Sq. Type of mortality mortality interaction - 1 9.18 - - - Bb - 2 11.22 - - - - 3 16.32 - - - - 1 6.12 - - - Ma - 2 9.18 - - - - 3 14.28 - - - - 1 12.24 - - - EPN - 2 17.34 - - - - 3 23.46 - - - 0 1 24.29 20.30 0.71 Additive Bb+EPN 0 2 34.69 26.62 1.87 Additive 0 3 51.02 35.96 4.44 Synergistic 0 1 19.58 17.61 0.19 Additive Ma+EPN 0 2 28.86 24.93 0.53 Additive 0 3 44.32 34.40 2.22 Additive 7 1 27.83 22.09 1.18 Additive Bb+EPN 7 2 43.29 30.84 3.58 Additive 7 3 61.85 42.99 5.75 Synergistic 7 1 23.71 20.30 0.48 Additive Ma+EPN 7 2 38.14 29.15 2.11 Additive 7 3 55.67 40.64 4.05 Synergistic 14 1 42.26 30.84 3.08 Additive Bb+EPN 14 2 58.76 42.99 4.23 Synergistic 14 3 80.41 57.39 6.58 Synergistic 14 1 36.08 29.15 1.33 Additive Ma+EPN 14 2 54.63 40.64 3.58 Additive 14 3 72.16 53.95 4.50 Synergistic a Intervals between the application of EPFs and EPNs. b Week after fungal application.

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Table 7.3 Mean mortality (%±SE) of 6th instar larvae of R. ferrugineus treated with B. bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae were used each @ 1×106 spore ml-1 and H. Bacteriophora was applied @ 100 IJs ml-1.

Treatmen Intervalsa Weekb Observed Expected Chi Sq. Type of ts mortality (%) mortality interaction - 1 7.14 - - - Bb - 2 9.18 - - - - 3 13.26 - - - - 1 4.081 - - - Ma - 2 7.14 - - - - 3 11.22 - - - - 1 9.18 - - - EPN - 2 14.28 - - - - 3 18.36 - - - 0 1 17.34 15.67 0.16 Additive Bb+EPN 0 2 28.57 22.15 1.43 Additive 0 3 42.85 29.19 4.35 Synergistic 0 1 13.40 12.89 0.01 Additive Ma+EPN 0 2 22.68 20.40 0.22 Additive 0 3 35.05 27.53 1.61 Additive 7 1 21.64 17.52 0.78 Additive Bb+EPN 7 2 35.05 25.65 2.51 Additive 7 3 50.51 35.02 4.74 Synergistic 7 1 17.52 15.67 0.19 Additive Ma+EPN 7 2 30.92 23.90 1.59 Additive 7 3 44.32 31.69 3.60 Additive 14 1 34.02 25.65 2.05 Additive Bb+EPN 14 2 48.45 35.02 3.72 Additive 14 3 71.13 51.86 5.22 Additive 14 1 28.86 23.90 0.85 Additive Ma+EPN 14 2 42.26 31.69 2.64 Additive 14 3 61.85 46.03 4.04 Additive a Intervals between the application of EPFs and EPNs. b Week after fungal application.

Table 7.4 Factorial analysis for pupation, adult emergence and egg eclosion of R. ferrugineus exposed to B. bassiana, M. anisopliae and H. Bacteriophora

S.O.V. df Pupation Adult emergence Egg eclosion F P F P F P Instar 2 18.78 ≤0.05 39.94 ≤0.05 35.0 ≤0.05 Treatment 9 114.28 ≤0.05 84.44 ≤0.05 90.89 ≤0.05 Instar × Treatment 18 10.49 ≤0.05 0.48 0.96 0.46 0.97 Error 232 ------Total 269 ------

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Table 7.5 Pupation, adult emergence and egg eclosion (%±SE) of 2nd, 4th and 6th instar R. ferrugineus larvae treated with B. bassiana, M. anisopliae and H. Bacteriophora. B. bassiana and M. anisopliae were used each @ 1×106 spore ml-1 and H. Bacteriophora was applied @ 100 IJs ml-1. Mean sharing the same letters are not significantly different. Means sharing the same letters within columns are not significantly different

Treatmen Inter Second instar Fourth instar Sixth instar ts val Pupation Adult Egg eclosion Pupation Adult Egg eclosion Pupation Adult Egg eclosion (%) emergence (%) (%) emergence (%) (%) emergence (%) (%) (%) (%) Bb - 62.22±2.23bc 57.77±2.77bc 53.33±2.33bc 71.11± 3.54b 66.66±3.08b 59.55±2.92b 80.00±2.88bc 74.44±3.37b 66.33±3.10b Ma - 67.77±2.64b 61.11±3.21b 58.88±2.23b 73.33±3.40b 68.88±3.51b 62.22±2.77b 83.33±2.35ab 78.88±3.88ab 72.22±3.22b EPN - 56.66±2.33bcd 50.55±2.69bcd 45.55±1.67bcd 62.22±2.77bc 57.77±2.33bc 51.11±2.51bc 69.44±3.42cd 65.55±2.75bc 59.33±2.44bc Bb+EPN 0 45.55±1.93def 40.33±2.35def 36.66±1.88def 48.88±2.09cd 43.33±3.08cd 38.88±1.60cd 54.44±2.76ef 49.44±2.57de 43.33±2.35de Ma+EPN 0 51.11±1.51cde 44.77±2.89cde 39.44±1.36cde 54.44±2.12cd 49.44±2.16cd 44.44±2.23cd 60.00±2.88de 56.66±2.72cd 51.11±2.51cd Bb+EPN 7 39.44±1.69ef 36.66±1.33def 31.11±1.51def 45.55±2.42d 40.55±1.93d 35.55±1.93d 51.11±2.51ef 45.55±2.93de 40.55±1.42de Ma+EPN 7 43.55±1.73def 38.88±1.51def 34.44±1.37def 51.11±2.88cd 46.11±2.32cd 41.11±1.51cd 57.77±2.77def 52.22±2.23cde 46.66±1.33cde Bb+EPN 14 32.22±1.27f 27.77±1.77f 22.22±1.46f 39.44±2.42d 36.66±1.33d 31.11±1.60d 44.44±2.93f 40.22±2.79e 34.44±1.93e Ma+EPN 14 35.55±1.43f 30.55±3.37ef 26.66±1.68ef 47.77±2.23cd 42.22±2.12cd 37.77±1.22cd 54.44±2.76ef 47.77±2.64de 42.22±1.22de Control 90.55±2.11a 86.66±2.88a 81.11± 2.60a 93.33±2.66a 90.55±2.11a 83.33±3.33a 95.55±1.75a 92.22±2.22a 85.55±2.93a df 9 9 9 9 9 9 9 9 9 F 35.7 31.3 31.0 23.4 23.6 28.8 30.6 28.1 32.4 P ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05

149

Table 7.6 Effect of B. bassiana, M. anisopliae and H. Bacteriophora on the development of R. ferrugineus. B. bassiana and M. anisopliae were used each @ 1×104 spore ml-1 and H. Bacteriophora was applied @ 50 IJs ml-1. Mean sharing the same letters are not significantly different

Treatmen Larval duration Larval Pre-pupal Pre-pupal Pupal duration Pupal weight Adult longevity (days) Adult weight (g) ts (days) weight duration weight (days) (g) (g) (days) (g) Male Female Male Female Bb 98.16±3.21b 4.01±0.12bc 15.16±0.58bc 4.02±0.20ab 22.94±1.20bc 3.92±0.18abc 39.05±1.54ab 42.83±1.78ab 1.411±0.12ab 1.14±0.11abc Ma 96.50±3.88bc 4.41±0.15ab 15.94±0.79bc 4.07±0.19ab 23.72±1.36abc 4.11±0.15ab 41.83±1.40a 43.61±1.67a 1.33±0.14ab 1.28±0.15ab EPN 101.16±3.63ab 3.72±0.12cd 16.72±0.95abc 3.85±0.14ab 24.16±1.41abc 3.67±0.10bcd 37.16±1.23ab 40.38±1.45ab 1.18±0.1bc 1.05±0.12abc Bb+EPN 109.27±4.16a 3.08±0.12e 20.16±1.14a 3.07±0.11c 27.61±1.24a 3.21±0.11d 34.94±1.29b 36.50±1.78b 0.84±0.10d 0.78±0.11c Ma+EPN 104.38±3.32ab 3.27±0.11de 18.50±0.98ab 3.48±0.12bc 25.50±1.41ab 3.43±0.13cd 36.16±1.17b 38.71±1.68ab 1.01±0.17cd 0.92±0.16bc Control 87.05±1.08c 4.87±0.14a 14.38±0.74c 4.17±0.25a 21.27±1.36c 4.24±0.10a 42.83±1.61a 45.27±1.43a 1.56±0.13a 1.37±0.11a df 5 5 5 5 5 5 5 5 5 5 F 9.92 27.3 6.59 6.94 5.15 11.1 5.58 3.93 12.7 4.26 P ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05 ≤0.05

150

S.O.V. df F P Covariate: Diet Consumption 1 0.191 0.62 Covariate: Weight Gain 1 3.060 0.078 Frass Production × Diet Consumption 24 2.312 ≤0.05 Diet Consumption × Weight Gain 1 0.132 0.73 Frass Production × Weight Gain 27 2.730 ≤0.05 Frass Production × Diet Consumption × Weight Gain 24 2.701 ≤0.05 Error 687 - - Total 828 - -

151

Figure 7.1 Diet consumption in last instar larvae of R. ferrugineus when treated with B. bassiana and H. bacteriophora

152

Figure 7.2 Frass production in last instar larvae of R. ferrugineus when treated with B. bassiana and H. bacteriophora

153

Figure 7.3 Weight gain in last instar larvae of R. ferrugineus when treated with B. bassiana and H. bacteriophora

154

CHAPTER 8

Combined toxicity of Beauveria bassiana, Bacillus thuringiensis and Heterorhabditis bacteriophora against red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

Abstract Laboratory studies were carried out to evaluate the insecticidal effect of Beauveria bassiana (Bb), Bacillus thuringiensis var. kurstaki (Bt-k) and an entomopathogenic nematode (EPN) Heterorhabditis bacteriophora against distinct populations of Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). Four populations of RPW were collected from different districts of Punjab, Pakistan including Layyah, Dera Ghazi Kahn, Muzaffargarh and Rahim Yar Khan. All the three agents were used alone and in all possible combinations (Bt-k+Bb, Bt-k+EPN, Bb+EPN and Bt-k+Bb+EPN) against 6th instar larvae and adults of RPW. The experiments were carried out at 25±2 °C and 70±5% RH and 12:12 (D: L) hours, mortality counts were taken after 7, 14 and 21 days post incubation. H. bacteriophora was more effective followed by B. bassiana and Bt-k in alone treatments, while in combined treatments increased mortality was recorded. Combined treatments of Bb+Bt-k exhibited lowest mortality followed by Bt-k+ EPN, Bb+EPN and BB+Bt-k+EPN. The maximum rate of mycosis and sporulation in the cadavers of RPW was observed where B. bassiana was applied alone and similar trend was recorded for nematode production. The results of the present study indicate that all three control measures may provide effective control against RPW. But need of the hour is to evaluate these agents under field conditions.

Keywords: Beauveria bassiana, Heterorhabditis bacteriophora, Bacillus thuringiensis Rhynchophorus ferrugineus

8.1 Introduction The Red Palm Weevil (RPW) is an important invasive pest which has almost been invaded and fully established in more than 50% of the date palm growing areas of the world which attributes to the high fecundity than the normal species (Faleiro, 2006), capability to live and interbreed in the same tree even for several generations (Rajamanickam et al., 1995; Avand- Faghih, 1996), adult flight capacity to a longer distance (Wattanapongsiri, 1966) and pest tolerance to a wide range of climatic conditions due to its hidden habit in palm tree. To combat RPW different control practices have been deployed among date palm growing areas of the world. Treatments revolve around the deployment of conventional chemical insecticides, sterile insect techniques, use of semio-chemicals (Paoli et al., 2014) and bio-control agents (Wattanapongsiri, 1966; Murphy and Briscoe, 1999; Faleiro, 2006). Most commonly used control treatments are insecticides such as Diazinon, Imidacloprid, and Phosmet (Abbas, 2010). However, heavy use of chemical treatments causes environmental damage and harms non-target organisms, and also leads to the development of insecticide resistance against RPW (Jalinas et al., 2015). Very few studies have been conducted on the natural entomophagous enemies of R. ferrugineus or other Rhynchophorus species (Murphy and Briscoe, 1999; Faleiro, 2006). Entomopathogenic fungi (EPFs) are commonly found in the nature and cause epizootics in insect populations, thus play significant role in regulating insect population. Mostly, the member of Entomophthorales and Hyphomycetes attack on terrestrial insects. EPFs from various strains of B. bassiana and M. anisopliae have been found in association with RPW and found among the most relevant biological agents suggested to control RPW (Faleiro, 2006). Unlike the other entomopathogens, entomopathogenic fungi infect the host by contact, then germinate and penetrate the insect cuticle. The host can be infected both by direct treatment and by horizontal transmission from infected insects or cadavers to healthy insects. Subsequently, infection can occur via the new generation of spores (Lacey et al., 1999; Quesada-Moraga et al., 2004). These unique characters make EPF especially important for the control of concealed insects such as RPW. Bacillus thuringiensis (Bt) is another important microbial control agent which holds a prominent position among commercial chemical compounds important for agricultural insect pests. Different researchers have evaluated the pathogenic potential of Bt against RPW and revealed successful control (Banerjee and Dangar, 1995; Alfazariy, 2004; Bauce et al., 2002; Sivasupramaniam et al., 2007; Birda and Akhursta, 2007; Manachini et al., 2008; Manachini et al., 2009). Studies reveled that feeding seasation and midgut damage were observed amongst the larvae survived after treatments. Entomopathogenic nematodes (EPNs) have been declared an efficient entomopathogen against variety of insect in integrated pest management program against RPW (Abbas et al., 2001; Llácer et al., 2009; Dembilio et al., 2010a). They are obligate parasites in the families Steinernematidae and Heterorhabditidae which kill their host with the aid of mutualistic bacterium present in their intestine (Poinar, 1990). As for as the life cycle is concerned the nematodes complete 2-3 generations within the host, after which free-living infective juveniles emerge to seek new hosts (Poinar, 1990). Several formulations have been developed to improve the activity of nematodes against insect pests (Georgis, 1990; Georgis and Kaya, 1998). In coleopteran pests larvae of several weevil species (Coleoptera: Curculionidae) such as the black vine weevil, Otiorhynchus sulcatus (F.), and the Diaprepes root weevil, Diaprepes abbreviatus (L.) was successfully controlled with EPNs (Shapiro-Ilan et al., 2002).

The intervention of more than one biocontrol agent can enhance the effectiveness of the other partner; many studies have been conducted in this regard. The combined effect of B. bassiana and B. thuringiensis working synergistically delivers more harm to insect pests (Wraight and Ramos, 2005). Similarly combined application of EPNs and EPFs have been evaluated against different insect pests (Thurston et al., 1993, 1994; Koppenhöfer and Kaya, 1997; Koppenhöfer et al., 1999; Yadav et al., 2004; Ansari et al., 2004, 2006, 2008). Hence integrated practices can be a hint for those willing to manage RPW.

8.2 Materials and Methods

8.2.1 RPW collection and rearing Four different populations of R. ferrugineus were collected from Layyah, Dera Ismail Khan (D.I. Khan), Muzaffargarh and Rahim Yar Khan (R.Y. Khan) districts of Punjab (Pakistan). Different developmental stages were collected from infested and fallen trees with the permission of farmer (owner). From each area insects were collected and kept in different plastic boxes assigned for a specific stage and brought to the laboratory until enough collection was done. Further multiplication for one generation was carried out in Microbial Control Laboratory, Department of Entomology, University of Agriculture, Faisalabad, Pakistan. Larvae were offered with pieces of sugarcane (Saccharum officinarum L.; Poales: Poaceae) stem for feeding and pupation, while shredded sugarcane pieces were offered to adults for feeding and substrate for oviposition. After pupation, pupal cocoon were kept in separate plastic jars for adult emergence in an incubator (Sanyo, Japan). After emergence beetles were shifted to the adult’s jar for feeding, mating and oviposition. Colony was developed in plastic boxes (30×60×60 cm) having a lid covered with mesh wire gauze (60 mesh size) in the middle (10 cm diameter) for aeration. Adult’s diet was changed after every three days and replaced sugarcane pieces were kept in separate jars for egg hatching. After egg hatching neonate larvae were allowed to feed for 3 days in the same set and then shifted to the sugarcane sets for feeding and pupation. Larvae were shifted to the new sugarcane sets after every week until pupation. The rearing conditions were maintained at 25±2 oC, 65±5% RH and 12:12 (D: L) hours photoperiod.

8.2.2 Preparation of B. thuringiensis spore-crystal mixtures The commercial formulation of B. thuringiensis var. kurstaki (Bt-k) was obtained from Microbial Control Laboratory, originally obtained from National Center for Genetic Engineering and Biotechnology (BIOTEC) in Thailand. This strain was then subjected to sporulation by culturing in 50 ml nutrient broth media. Harvesting of culture was carried out by centrifugation at 6000 rpm for 15 min (Crecchio and Stotzky, 2001; Hernández et al., 2005). The pellets formed resultantly were washed twice in cold 1M NaCl and thrice in sterile distilled water (SDW), re- suspended in distilled water (5 ml). From the suspension formed, 1 ml was centrifuged for 5 min at 10,000 rpm, dried for 4 hours at 37 °C and weighed (Wakil et al., 2013).

8.2.3 Entomopathogenic nematode The Infective Juveniles (IJs) of H. bacteriophora were obtained from the culture collection of Microbial Control Laboratory. Second instar larvae and adult of R. ferrugineus were encountered with 300 IJs under laboratory conditions. H. bacteriophora was maintained in 3rd instar Galleria mellonella L. (Lepidoptera: Pyralidae) following the procedure of Kaya and Stock (1997).

8.2.4 Preparation of fungi The fungal isolate of B. bassiana (WG-43) used in the study was taken from the culture collection of Microbial Control Laboratory, originally isolated from dead cadaver of RPW. Fungi were sub-cultured on Sabouraud Dextrose Agar (BD, Becton, Dickisonand Company sparks, MD 21152 USA). Conidial suspension was prepared with 0.01% Tween-80 (Merck, KGaA, Darmstadt, Germany) in sterile distilled water and conidial concentration of 1×107 conidia ml-1 determined using a Neubauer haemocytometer.

8.2.5 Treatment with B. bassiana Sixth instar larvae and adults of uniform age from each population were directly immersed into the conidial suspensions for 60 and 90s respectively and control was treated in aqueous solution with 0.01% Tween-80. Isolate-treated and control insects were individually shifted to 150 ml cylindrical plastic cups, each measuring 6 cm in height with 6 cm diameter. The top of cups were covered with fine mesh in order to avoid the insects to escape. A piece of 2×2 cm2 artificial diet (Agar, brewer’s yeast, wheat germ, corn flour, ascorbic acid, benzoic acid, amino acid-vitamin mix, chloramphenicol and nipagin) (Martín and Cabello, 2006) was kept in the center of the cups for larvae and a shredded sugarcane piece was offered to the adults. All the treatments were incubated at 25±2 °C, 70±5 % RH and a 12:12 (D: L) hours photoperiod and mortality counts were made after 7, 14 and 21 days post-incubation. The causal agent of dead larvae or adults were confirmed by shifting the cadavers into a Petri dish lined with wet filter paper and incubating them at 25±2 °C and 70±5 % RH for up to 15 days.

8.2.6 Treatment with B. thuringiensis var. kurstaki (Bt-k) Sixth instar larvae form each population was individually offered with artificial diets (Martín and Cabello, 2006), mixed with the diluted spore-crystal (70 µg g-1). To each larvae, Bt-k treated diet piece of (2×2 cm2) was provided to feed. For adults shredded sugarcane pieces were dipped in known concentration of Bt-k for 90s and offered to respective populations.

8.2.7 Treatment with H. bacteriophora H. bacteriophora suspension was prepared with a concentration of 300 IJs in glass jars and 1 ml of suspension was poured into the cylindrical plastic cups lined with damp Whatman filter paper. The top of the cups were covered with a fine mesh in order to avoid the insects to escape. After pouring nematodes 30 minutes time was given for their even distribution on filter paper (Atwa et al., 2014). A small piece of artificial diet 2×2 cm2 was placed in middle of the cups as a food source for larvae and provided with new food every day. In each cup one 6th instar larvae from each population was placed on top of the filter paper. Same procedure was repeated for adult population and shredded sugarcane pieces were offered as food source. Each treatment was replicated three times, while control treatment received 1 ml of distributed water. The cups were placed in an incubator at 25±2 °C and 70±5% RH at 12: 12 (D: L) hours photoperiod. Mortality data was recorded after 7, 14 and 21 days after treatment and whole bioassay was repeated thrice to avoid the pseudo-replication phenomenon. Dead individuals were transferred to the modified White traps (White, 1927) and left for 10 more days for IJs emergence. The insects exhibiting typical odor and color (signs for nematode infestation) were considered to be killed by nematodes (Woodring and Kaya, 1988).

8.2.8 Treatment with B. bassiana, Bt-k and H. bacteriophora In combined treatments of B. bassiana, Bt-k and H. bacteriophora 6th instar larvae and adults were directly immersed in 100 ml of conidial suspensions for 60 and 90s respectively and control was treated in aqueous solution with 0.01% Tween-80 solution. After fungal treatments larvae and adults were offered with Bt-k treated artificial diet and sugarcane pieces respectively on H. bacteriophora treated plastic cups lined with damp Whatman filter paper. Experimental conditions were maintained at 25±2 °C, 70±5% RH and 12:12 (L: D) hours photoperiod and mortality data was recorded after 7, 14 and 21 days. Three replicates of ten insects were used for each treatment and same count of larvae fed on normal diet served as untreated check while entire experiment was repeated thrice. Larvae that exhibiting fungal infection symptoms (hardening of the cadaver or emergence of conidiophores) were maintained as described above and production of spores on the cadavers were evaluated following 14 day of incubation at 25±2 °C. Larvae demonstrating symptoms of EPN infection (changes in pigmentation) were maintained in White traps for 10 days for production of IJs.

8.2.9 Sporulation and Nematode production Mycosed larvae after 14 days of incubation were vortexed for 30 minutes in distilled water containing 0.01% Tween-80 and number of spores was estimated in 1 ml from the suspension using a haemocytometer. Concentration of IJs was measured by 1 ml sample from the final solution and counting IJs with the help of a Peters’ slide and microscope.

8.2.10 Statistical analysis Mortality for each treatment was corrected for control mortality using Abbott's (1925) formula and subjected to one way analysis of variance (ANOVA) in Minitab (Minitab, 2003) means were separated using Tukey’s Kramer test (HSD) (Sokal and Rohlf, 1995) at 5% significance level.

8.3 Results

8.3.1 Mortality of larvae and adult The results of present study revealed that the larval and adult mortality was signiﬁcantly affected by the main effects and their associated interactions for all the population tested (Table 8.1). The mortality of both larvae and adult was non-signiﬁcant (P≤0.05) among all the population tested after each exposure interval, except the Bt-k+EPN for larvae after 7 days of exposure and EPN, Bt-k+EPN and Bb+EPN for larvae and Bb+EPN for adults after 14 days of exposure, after 21 days of exposure the treatments Bb and EPN for larvae and EPN, Bt-k+EPN and Bb+EPN for adult were significantly different (P≤0.05). Overall the mortality was higher on combined treatments as compared to individual applications of either B. bassiana, Bt-k or H. bacteriophora for both larvae and adult, while the larval mortality was higher as compared to the adult beetles in all the treatments applied at all the exposure intervals (Table 8.2, 8.3 and 8.4). The laboratory population was more susceptible followed by R.Y. Khan, D.G. Khan and Muzaffargarh at all the exposure intervals. After the last count Bb+EPN treatment exhibited 100% larval and adult mortality for all the populations tested, while Bt-k+EPN exhibited 100% mortality for the laboratory population after 21 days post incubation.

8.3.2 Mycosis and sporulation The maximum mycosed larvae (85.74%) and adults (69.07%), and sporulation in larvae and (189.22 conidia ml-1) adults (164.56 conidia ml-1) was observed in treatments where B. bassiana was applied alone against larvae and adults respectively in laboratory population (Figure 8.1a, b and 8.2a, b), however low rate of mycosis and sporulation was observed in the treatments where H. bacteriophora and B. bassiana were applied in combined manners. Similar trend was recorded for the R.Y. Khan, D.G. Khan and Muzaffargarh populations.

8.3.3 Insects affected by EPF and EPN and their production The maximum lethality in larvae affected by nematode was 92.40% and in adults 81.29%. The maximum number of nematode production on white trap was (178 IJs ml-1) adult (153 IJs ml-1) was observed in treatments where H. bacteriophora was applied alone against larvae and adult respectively in laboratory population (Figure 8.4a and 8.4b), however low rate of nematode affected and production was recorded in the treatments where H. bacteriophora and B. bassiana were applied in combined manners. Similar trend was recorded for R.Y. Khan, D.G. Khan and Muzaffargarh population.

8.4 Discussion Evidence suggests that entomopathogens play a key role in the host biology like production of essential nutrients (amino acids and vitamins) and indispensable compounds which influence some essential parameters such as growth, development, longevity, fertility, vector capability, immunological competences and deliver protection against natural enemies (Valzano et al., 2012). Numerous studies have documented sole and integrated applications of entomopathogens against a number of insect pests. Generally the combined treatment of entomopathogens exhibit enhanced mortality as compared to their individual application. Therefore, simultaneous use of these agents did not cause any harmful effects on the efficiency of the other agent. All agents have different modes of actions which enhance the disease severity in a short period of time, hence reduce the time span to inflict damage to the host crop. The entomopathogens that curtail RPW infestations are more effective in managing weevil a population as compared to the plant protection (Salama et al., 2004; Dembilio et al., 2010; El-Sufty et al., 2011). A number of entomopathogens are available worldwide that are very effective against RPW including entomopathogenic fungi, bacteria and nematodes. Among bacteria members of genus Bacillus like B. sphaericus, B. lentimorbus, B. popilliae are important antagonists of RPW. These bacteria produce insecticidal proteins that target specific developmental stages of RPW (Bulla et al., 1975; Salama et al., 2004). The entomopathogenic fungi M. anisopliae and B. bassiana inflict mortality in different developmental stages of RPW (Gindin et al., 2006; Dembilio et al., 2010). Moreover, the combined treatments of B. bassiana and Bt exhibit enhanced larval mortality as compared to their individual applications. Similar findings were observed by a number of researchers (Sander and Chichy, 1967; Kaliuga, 1968; Fargues, 1973, 1975; Kalvish and Krivstova, 1978; Lewis and Bing, 1991). Synergistic effects resulting from a combination of entomopathogenic nematodes with other entomopathogens have been reported in a number of studies (Thurston et al., 1993, 1994; Koppenhöfer and Kaya, 1997; Koppenhöfer et al., 1999). Contrarily Shapiro-Ilan et al. (2004) found antagonism between entomopathogenic nematodes and P. fumosoroseus. Such antagonisim may be due to pathogen interactions prior to or during infection. In case of Sternima marcescens and P. fumosoroseus, it is possible that these organisms are directly pathogenic to

entomopathogenic nematodes, and therefore the nematodes may have been killed or their fitness reduced prior to infection. These negative interactions may also be due to antagonistic toxins produced by the entomopathogens after infection was initiated. In our present study high mortality was recorded after different exposure intervals in combined treatments as compared to their sole application. Higher mortalities were recorded for (Bb+EPN) followed by (Bt-k+EPN) and (Bt-k+Bb) at all the exposure intervals. While in sole treatments H. bacteriophora was found more effective followed by B. bassiana and Bt-k. Our results are in accordance with the findings of Koppenhöfer and Kaya (1997) who reported additive or synergistic interactions among entomopathogens when applied simultaneously. Koppenhöfer and his collogues also recorded positive interaction between H. bacteriophora and B. thuringiensis against Cyclocephala pasadenae, C. hirt and Anomala orientalis after different exposure intervals (Koppenhöfer et al., 1999). Similar findings were reported by many other scientists (Barbercheck and Kaya, 1990; Kermarrec and Mauleon, 1989). Ansari et al. (2008) found synergistic interaction against black vine weevil larvae when applied M. anisopliae and EPNs simultaneously. They also found similar results with H. philanthus Füessly white grubs and revealed that type of interaction between EPN and fungal entomopathogens depends on the time of application and specie of EPNs (Ansari et al., 2004, 2006). Similarly additive or slight synergistic interaction was recorded between M. anisopliae and EPNs against Holotrichia consanguinea larvae (Yadav et al., 2004) and the larvae of pecan weevil (Shapiro-Ilan et al., 2004). A good knowledge of biological parameters of RPW and most importantly the interaction among entomopathogens could play a key role to expand RPW-IPM programs. This, calls for the isolation and identification of more virulent strains of entomopathogens (Manachini et al., 2011). Moreover, the field evaluation of these substances in combined manners can provide substantial information and help in developing new strategies by deploying IPM production systems (Neves et al., 2001). In summary, the results of the present study indicate that the integration of entomopathogens may be preferable to the use of a single agent. The integration takes advantage of the positive characteristics of each agent. For example, the Bt treatments lead the gut to septicemia causing the insect to stop feeding, and weakening the host immune system. This will favor the B. bassiana to work efficiently with very low resistant of the host immune system thereby increasing mortality.

Conclusions The present study showed that B. bassiana and Bt-k and H. bacteriophora can kill the larvae and adult of R. ferrugineus from different populations collected across Punjab and Khyber Pkhtunkhwa, Pakistan. They also exert the detrimental effect on their growth and development which can be use effectively against this pest.

Acknowledgements This research work was supported by the scholarship from Higher Education Commission (HEC), Islamabad, Pakistan (112-30536-2AV1-263) under Indigenous Ph.D. Fellowship Program.

8.5 References Abbas, M.S.T., S.B. Hanounik, S.A. Mousa and S.H. Al-Bagham, 2000. Soil application of entomopathogenic nematodes as a new approach for controlling Rhynchophorus ferrugineus on date palm. Inter. J. Nematol., 10: 215-218. Abbas, M.S.T., 2010. IPM of the red palm weevil, Rhynchophorus ferrugineus. In: Ciancio, A. and K.G. Mukerji (Eds.). Integrated management of arthropod pests and insect borne diseases, integrated management of plant pests and diseases 5, Springer, Berlin. Pp. 209- 233. Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol., 18: 265-267. Alfazariy, A.A., 2004. Notes on the survival capacity of two naturally occurring entomopathogens on the red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). Egyp. J. Biol. Pest Control, 14: 423. Ansari, M.A., F.A. Shah and T.M. Butt, 2008. Combined use of entomopathogenic nematodes and Metarhizium anisopliaeas a new approach for black vine weevil, Otiorhynchus sulcatus, control. Entomol. Experi. et Appli., 129: 340-347. Ansari, M.A., S. Vestergaard, L. Tirry and M. Moens, 2004. Selection of a highly virulent fungal isolate, Metarhizium anisopliae CLO 53, for controlling Hoplia philanthus. J. Inver. Pathol., 85: 89-96. Ansari, M.A., F.A. Shah, L. Tirry and M. Moens, 2006. Field trials against Hoplia philanthus (Coleoptera: Scarabaeidae) with a combination of an entomopathogenic nematode and the fungus Metarhizium anisopliae CLO 53. Biol. Control, 39: 453-459. Avand-Faghih, A., 1996. The biology of red palm weevil, Rhynchophorus ferrugineus Oliv. (Coleoptera, Curculionidae) in Saravan region (Sistan & Balouchistan Province, Iran).- Appl. Entomol. Phytopathol., 63: 16-18. Banerjee, A. and T.K. Dangar, 1995. Pseudomonas aeruginosa a facultative pathogen of red palm weevil Rhynchophorus ferrugineus. World J. Microbiol. Biotech., 11: 618-620. Barbercheck, M.E. and H.K. Kaya, 1990. Interactions between Beauveria bassiana and the entomogenous nematodes Steinernema feltiae and Heterorhabditis heliothidis. J. Inver. Pathol., 55: 225-234. Bauce, E., Y. Bidon and R. Berthiaume, 2002. Effects of food nutritive quality and Bacillus thuringiensis on feeding behaviour, food utilization and larval growth of spruce budworm Choristoneura fumiferana (Clem.) when exposed as fourth- and sixth instar larvae. Agri. Forest Entomol., 4: 57-70. Birda, L.J. and J.R. Akhursta, 2007. Variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) in Australia to two Bacillus thuringiensis toxins. J. Inver. Pathol., 94: 84-94. Bulla, Jr L.A., R.A. Rhodes and G.S. Julian, 1975. Bacteria as insect pathogens. Ann. Rev. Microbiol., 29: 163-190. Crecchio, C. and G. Stotzky. 2001. Biodegradation and insecticidal activity of the toxin from Bacillus thuringiensis subsp. kurstaki bound on complexes of montmorillonite-humic acids-Al hydroxypolymers. Soil Biol. Biochem., 33: 573-581. Dembilio, Ó., E. Quesada-Moraga, C. Santiago-Álvarez and J.A. Jacas, 2010. Potential of an indigenous strain of the entomopathogenic fungus Beauveria bassiana as a biological control agent against the red palm weevil, Rhynchophorus ferrugineus. J. Inver. Pathol., 104: 214-221.

El-Sufty, R., S. Al-Bgham, S. Al-Awash, A. Shahdad and A. Al-Bathra, 2011. A trap for auto dissemination of the entomopathogenic fungus Beauveria bassiana by red palm weevil adults in date palm plantations. Egyp. J. Biol. Pest Control, 21: 271-276. Faleiro, J.R., 2006. A review of the issues and management of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Rhynchophoridae) in coconut and date palm during the last one hundred years. Int. J. Trop. Insect Sci., 26: 135-154. Fargues, J., 1973. Susceptibility of Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) towards Beauveria bassiana (Bals.) Vuill. (Fungi Imperfecti, Moniaiales) in presence of reduced doses of insecticide. Ann. Zool. Ecol. Anim. 5, 231-246. Fargues, J., 1975. Field experiments on the combination of Beauveria bassiana with insecticides for the control of Leptinotarsa decemlineata. Ann. Zool. Ecol. Anim., 7: 247-264. Georgis, R., 1990. Formulation and application technology. In: Gaugler, R. and K.H. Kaya (Eds.). Entomopathogenic Nematodes in Biological Control. Boca Raton, Fl: CRC Press. Georgis, R. and H.K. Kaya, 1998. Advances in entomopathogenic nematode formulation. In: Burges, H.D. (Ed.). Formulation of microbial biopesticides, beneficial microorganisms nematodes and seed treatments. Kluwer Academic Publishers, Dordrecht, the Netherlands. Pp. 289-308. Gindin, G., S. Levski, I. Glazer and V. Soroker, 2006. Evaluation of the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana against the red palm weevil Rhynchophorus ferrugineus. Phytoparasitica, 34(4): 370-379. Hernández, C.S., R. Andrew, Y. Bel and J. Ferre, 2005. Isolation and toxicity of Bacillus thuringiensis from potato-growing areas in Bolivia. J. Inver. Pathol., 88: 8-16. Jalinas, J., B. Güerri-Agulló, R.W. Mankin, R. López-Follana and L.V. Lopez-Llorca, 2015. Acoustic assessment of Beauveria bassiana (Hypocreales: Clavicipitaceae) effects on Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) larval activity and mortality. J. Econ. Entomol., 108(2): 444-453. Kaliuga, M.V., 1968. Combined use of microbiological preparations in the control of pests. Ent. Obozr., 47: 450-453. Kalvish, T.K., and N.V. Krivtsova, 1978. Interaction of Muscardine fungi and Bacillus thuringiensis var. Galleriae in vitro and in vivo. Izv. Sib. Otd. Akad. Nauk. SSSR Ser. Biol. Nauk, 5: 40-46. Kaya, H.K. and S.P. Stock, 1997. Techniques in insect nematology. Manual of Techniques in Insect Pathology (ed. by LA Lacey). Academic Press, London, UK. pp. 281-324. Kermarrec, A. and H. Maul eon, 1989. Synergy between chlordecone and Neoaplectana carpocapsae Weiser (Nematoda: Steinernematidae) in the control of Cosmopolites sordidus (Coleoptera: Curculionidae). Rev. Nematol., 12: 324-325. Koppenhöfer, A.M. and H.K. Kaya, 1997. Additive and synergistic interaction between entomopathogenic nematodes and Bacillus thuringiensis for scarab grub control. Biol. Control, 8: 131-137. Koppenhöfer, A.M., H.Y. Choo, H.K. Kaya, D.W. Lee and W.D. Gelernter, 1999. Increased field and greenhouse efficacy with combination of an entomopathogenic nematode and Bacillus thuringiensis against scarab grubs. Biol. Control, 14: 37-44. Lacey, L.A., A.A. Kirk, L. Millar, G. Mereadier and C. Vidal, 1999. Ovicidal and larvicidal activity of conidia and blastospores of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) against Bemisia argentifolii (Homoptera: Aleyrodidae) with a description

of a bioassay system allowing prolonged survival of control insects. Biocontrol Sci. Tech., 9: 9-18. Lewis, L.C. and L.A. Bing, 1991. Bacillus thuringiensis and Beauveria bassiana (Balsamo) Vuillemin for European corn borer control: Program for immediate and season-long suppression. Can. Ent., 123: 387-393. Llácer, E., D.E. Martínez, M.M. Altube and J.A. Jacas, 2009. Evaluation of the efficacy of Steinernema carpocapsae in a chitosan formulation against the red palm weevil, Rhynchophorus ferrugineus, in Phoenix canariensis. BioControl, 54(4): 559-565. Manachini, B., V. Arizza, D. Parrinello and N. Parrinello, 2011. Hemocytes of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces cerevisiae and Bacillus thuringiensis. J. Inver. Pathol., 106: 360-365. Manachini, B., V. Mansueto, V. Arizza and N. Parrinellom, 2008. Preliminary results on the interaction between Bacillus thuringiensis and red palm weevil, In: 41st Annual Meeting of Society for Invertebrate pathology and 9th international Conference on Bacillus thuringiensis, Warwick, UK. Pp. 45 Manachini, B., P. Lo Bue, E. Peri and S. Colazza, 2009. Potential effects of Bacillus thuringiensis against adults and older larvae of Rhynchophorus ferrugineus. IOBC/WPRS Bull., 45: 239-242. Mansour, N.A., M.E. Eldefrawi, A. Toppozada and M. Zeid, 1966. Toxicological studies on the Egyptian cotton leafworm, Prodenia litura Vl potentiation and antagonism of carbamate insecticide. J. Econ. Entomol., 59: 307-311. Martín, M.M. and T. Cabello, 2006. Manejo de la cría del picudo rojo de la palmera, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera, Dryophthoridae), en dieta artificial y efectos en su biometría y biología. Boletín de Sanidad Vegetal de Plagas, 32: 631-641. Minitab, 2003. MINITAB Release 14 for Windows. Minitab Inc., State College, Pennsylvania, USA. Murphy, S.T. and B.R. Briscoe, 1999. The red palm weevil as an alien invasive: Biology and the prospects for biological control as a component of IPM. Biocontrol New. Info., 20(1): 35- 45. Neves, P.O.J., E. Hirose, P.T. Tchujo and A. Moino Jr, 2001. Compatibility of entomopathogenic fungi with neonicotinoid insecticides. Neotro. Entomol., 30: 263-268. Paoli, F., R. Dallai, M. Cristofaro, S. Arnone, V. Francardi and P.F. Roversi, 2014. Morphology of the male reproductive system, sperm ultrastructure and cirradiation of the red palm weevil Rhynchophorus ferrugineus Oliv. (Coleoptera: Dryophthoridae). Tissue and Cell, 46 (4): 274-285. Poinar Jr., G.O., 1990. Taxonomy and biology of Steinernematidae and Heterorhabditidae. In: Gaugler, R. and H.K. Kaya (Eds.). Entomopathogenic Nematodes in Biological Control. CRC Press, Boca Raton, FL. Pp. 23-61. Quesada-Moraga, E., R. Santos-Quiros, P. Valverde-Garcia and C. Santiago-Álvarez, 2004. Virulence, horizontal transmission, and sublethal reproductive effects of Metarhizium anisopliae (anamorphic fungi) on the German cockroach (Blattodea: Blattellidae). J. Inver. Pathol., 87: 51-58. Rajamanickam, K., J.S. Kennedy and A. Christopher, 1995.Certain components of integrated management for red palm weevil, Rhynchophorus ferrugineus F. (Curculionidae:

Coleoptera) on coconut. Mededelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen Universiteit Gent, 60: 803-805. Salama, H.S., M.S. Foda, M.A. El-Bendary and A. Abdel-Razek, 2004. Infection of red palm weevil, Rhynchophorus ferrugineus, by spore-forming bacilli indigenous to its natural habitat in Egypt. J. Pest Sci. 77: 27-31. Sander, H., and D. Cichy, 1967. Research on the effectiveness of fungal and bacterial insecticides. Ekolog. Polska Ser., 15: 325-333. Shapiro-Ilan, D.I., D.H. Gouge and A.M. Koppenhofer, 2002. Factors affecting commercial success, case studies in cotton, turf and citrus. In: Gaugler, R. (Ed.). Entomopathogenic Nematology, New York: CABI. Pp. 333-355. Shapiro-Ilan, D.I., M. Jackson, C.C. Reilly and M.W. Hotchkiss, 2004. Effects of combining an entomopathogenic fungi or bacterium with entomopathogenic nematodes on mortality of Curculio caryae (Coleoptera: Curculionidae). Biol. Control, 30: 119-126. Sivasupramaniam, S., G.P. Head, L. English, Y.J. Li and T.T. Vaughn, 2007. A global approach to resistance monitoring. J. Inver. Pathol., 95: 224-226. Sokal, R.R. and F.J. Rohlf. 1995. Biometry, 3rd edn. Freedman and Company, New York. Thurston, G.S., H.K. Kaya, T.M. Burlando and R.E. Harrison, 1993. Milky disease bacteria as a stressor to increase susceptibility of scarabaeid larvae to an entomopathogenic nematode. J. Inver. Pathol., 61: 167-172. Thurston, G.S., H.K. Kaya and R. Gaugler, 1994. Characterization of enhanced susceptibility of milky disease infected scarabaeid grubs to entomopathogenic nematodes. Biol. Control, 4: 67-73. Valzano, M.G., F. Achille, I. Burzacca, C. Ricci, P. Damiani, G. Scuppa and G. Favia, 2012. Deciphering microbiota associated to Rhynchophorus ferrugineus in Italian samples: a preliminary study. J. Entomol. Acarol. Res., 44: e16 Wakil, W., M.U. Ghazanfar, F. Nasir, M.A. Qayyum and M. Tahir, 2012. Insecticidal efficacy of Azadirachta indica, nucleopolyhedrovirus and chlorantraniliprole singly or combined against field populations of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae). Chil. J. Agricul. Res., 72(1): 53-61. Wakil, W., M.U. Ghazanfar, T. Riasat, M.A. Qayyum, S. Ahmed and M. Yasin, 2013. Effects of interactions among Metarhizium anisopliae, Bacillus thuringiensis and chlorantraniliprole on the mortality and pupation of six geographically distinct field populations. Phytoparasitica, 41: 221-234. Wattanapongsiri, A., 1966. A revision of the genera Rhynchophorus and Dynamis (Coleoptera: Curculionidae). Department of Agriculture Science Bulletin (Bangkok). Pp. 328. White, G.F., 1927. A method for obtaining infective nematode larvae from cultures. Science, 66: 302-303. Woodring, L. and H. K. Kaya, 1988. Steinernematid and heterorhabditid nematodes: a handbook of techniques. Series Bull. vol. 331. Arkansas Agricultural Experiment Station, Fayetteville, AR. Yadav, B.R., V. Singh and C.P.S. Yadava, 2004. Application of entomogenous nematode, Heterorhabditis bacteriophora and fungi, Metarhizium anisopliae and Beauveria bassiana for the control of Holotrichia consanguinea by soil inoculation method. Annal. Agri. Bio. Res., 9: 67-69.

Table 8.1 ANOVA parameters for the main effects and associated interactions for mortality levels of R. ferrugineus larvae and adults

S.O.V. Larvae Adult df F P F P Treatment 5 454.81 ≤0.05 437.56 ≤0.05 Interval 2 1546.71 ≤0.05 1099.79 ≤0.05 Location 4 25.54 ≤0.05 17.88 ≤0.05 Treatment × Interval 10 19.98 ≤0.05 26.11 ≤0.05 Treatment × Location 20 0.58 0.92 0.64 0.88 Interval × Location 8 0.93 0.49 1.24 0.27 Treatment × Interval × 40 0.73 0.89 0.41 0.99 Location Error 550 - - - - Total 647 - - - -

Table 8.2 Mean mortality (%±SE) of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination after 7 days of exposure (means followed by the same letter within each treatment and insect populations not significantly different; HSD test P≤0.05)

Stage Treatments Insect Populations Layyah D.G. Khan Muzaffargarh R.Y. Khan F P Bt-k 9.47±0.78e 6.47±0.55d 6.08±0.78d 7.93±0.71e 0.38 0.77 Bb 13.48±1.18de 10.47±1.12cd 8.39±1.01cd 11.68±1.16de 0.30 0.82 EPN 25.57±1.39cd 21.57±1.38bc 19.58±1.17bc 23.45±1.13cd 0.62 0.60 Larvae Bt-k + Bb 32.28±1.61bc 28.36±1.46ab 25.06±1.30b 30.17±1.65bc 1.28 0.29 Bt-k + EPN 45.37±2.15ab 33.73±1.50ab 28.08±1.57ab 42.11±2.36ab 6.46 ≤0.05 Bb + EPN 51.68±2.34a 42.19±2.51a 39.40±2.14a 46.82±2.54a 2.05 0.12 F 24.8 30.5 18.8 14.4 - - P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - - Bt-k 6.54±0.70d 4.69±0.49d 4.02±0.53c 5.46±0.74d 0.24 0.87 Bb 10.46±1.12d 8.41±0.87cd 7.56±1.04bc 9.29±1.09cd 0.16 0.92 EPN 19.18±1.24cd 14.90±1.01bcd 13.22±1.35bc 16.51±1.21cd 0.72 0.54 Adult Bt-k + Bb 24.70±1.65bc 21.24±1.96abc 19.69±1.56ab 22.06±1.56bc 0.31 0.81 Bt-k + EPN 35.41±2.35ab 28.84±2.11ab 27.09±1.75b 31.52±2.13ab 0.90 0.45 Bb + EPN 39.73±2.09a 32.37±2.03a 30.89±2.05a 36.08±2.45a 1.38 0.26 F 19.2 8.90 12.4 14.4 - - P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -

Table 8.3 Mean mortality (%±SE) of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination after 14 days of exposure (means followed by the same letter within each treatment and insect populations not significantly different; HSD test P≤0.05)

Stage Treatments Insect Populations Layyah D.G. Khan Muzaffargarh R.Y. Khan F P Btk 22.50±1.58c 20.67±1.39c 17.70±1.08c 19.50±1.23c 0.43 0.73 Bb 31.02±1.81c 27.76±1.85c 23.39±1.92c 25.64±1.08c 0.59 0.62 EPN 60.38±2.71b 55.66±2.44b 49.22±2.65b 51.57±2.18b 1.38 0.26 Larvae Bt-k + Bb 65.24±3.10b 59.10±2.18b 52.55±2.77b 55.09±3.07b 3.39 ≤0.05 Bt-k + EPN 89.66±3.26a 83.10±3.72a 74.57±3.14a 78.42±3.87a 4.07 ≤0.05 Bb + EPN 97.37±2.73a 91.55±3.27a 82.68±3.86a 86.17±3.03a 3.67 ≤0.05 F 86.2 73.1 46.3 60.3 - - P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - - Btk 17.40±1.46c 15.78±1.04c 12.01±0.88c 13.76±1.07c 0.73 0.54 Bb 24.07±1.50c 21.85±1.28c 17.85±1.33cd 20.52±1.32c 0.37 0.77 EPN 45.06±2.48b 40.40±1.65b 34.54±2.03bc 37.62±1.99b 0.93 0.43 Adult Bt-k + Bb 52.54±2.49b 48.01±2.14b 39.98±2.19b 43.27±2.75b 2.56 0.07 Bt-k + EPN 72.37±3.21a 67.77±3.10a 61.64±2.93a 65.80±2.57a 1.48 0.23 Bb + EPN 81.29±3.37a 75.74±3.34a 64.43±3.06a 70.18±2.28a 3.87 ≤0.05 F 50.5 42.1 27.9 41.8 - - P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -

Table 8.4 Mean mortality (%±SE) of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination after 21 days of exposure (means followed by the same letter within each treatment and insect populations not significantly different; HSD test P≤0.05)

Stage Treatments Insect Populations Layyah R.Y. Khan Muzaffargarh D.G. Khan F P Bt-k 58.36±4.55d 54.66±4.46c 46.86±4.53d 49.78±2.45c 1.55 0.22 Bb 72.63±3.88c 66.79±3.45c 57.27±2.75cd 55.55±2.92c 6.04 ≤0.05 EPN 84.54±2.71bc 81.06±3.14b 70.45±3.19bc 73.92±2.74b 4.75 ≤0.05 Larvae Bt-k + Bb 87.01±3.04b 82.17±2.90b 75.77±4.75b 78.24±2.39b 2.09 0.12 Bt-k + EPN 100.00±0.00a 98.41±1.58a 93.35±3.06a 95.57±2.85a 1.75 0.17 Bb + EPN 100.0±0.00a 100.0±0.00a 100.0±0.00a 100.0±0.00a - - F 29.8 34.9 35.5 69.0 - - P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - - Bt-k 39.04±2.41d 32.56±3.80d 26.79±3.68f 30.74±2.96f 2.45 0.08 Bb 47.55±2.60d 43.07±4.15d 38.13±4.09e 36.12±3.31e 2.03 0.12 EPN 61.13±3.81c 58.15±3.25c 52.37±2.20d 54.35±1.82d 1.83 0.16 Adult Bt-k + Bb 80.53±2.66b 76.69±3.11b 67.96±3.92c 71.04±3.18c 2.99 ≤0.05 Bt-k + EPN 94.24±2.28a 90.03±2.82a 81.27±2.28b 86.48±1.46b 5.88 ≤0.05 Bb + EPN 100.0±0.00a 100.0±0.00a 100.0±0.00a 100.0±0.00a - - F 95.3 70.9 80.5 130 - - P ≤0.05 ≤0.05 ≤0.05 ≤0.05 - -

Figure 8.1a Mean mycosis (%±SE) in larvae of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Figure 8.1b Mean mycosis (%±SE) in adults of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Figure 8.2a Sporulation (conidia ml-1) in larvae of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Figure 8.2b Sporulation (conidia ml-1) in adult of R. ferrugineus populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Figure 8.3a R. ferrugineus larvae affected by H. bacteriophora (%±SE) from different populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Figure 8.3b R. ferrugineus adult affected by H. bacteriophora (%±SE) from different populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Figure 8.4a Nematode production (IJs ml-1) in larvae of R. ferrugineus affected by H. bacteriophora from different populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Figure 8.4b Nematode production (IJs ml-1) in adult of R. ferrugineus affected by H. bacteriophora from different populations collected from Layyah, D.G. Khan, Muzaffargarh and R.Y. Khan treated with Bt-k (70 µg g-1), B. bassiana (1×107 conidia ml-1) and H. bacteriophora (300 IJs) applied alone or in combination (means followed by the same letter within each treatment are not significantly different; HSD test P≤0.05)

Summary The Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) is one of the major and destructive insect pests of 29 different palm species all around the world. It is an important invasive pest that has invaded and established in more than 50% of the date palm growing areas of the world attributed to the high fecundity of this species (Faleiro, 2006), ability to live and interbreed in the same tree for several generations (Rajamanickam et al., 1995; Avand-Faghih, 1996), adult flight capacity (Wattanapongsiri, 1966) and pest tolerance to a wide range of climatic conditions due to its hidden habit in palm tree. To combat RPW different control practices has been deployed among date palm growing areas of the world. Treatments revolve around the deployment of conventional chemical insecticides, sterile insect techniques, use of semio-chemicals (Paoli et al., 2014) and bio-control agents (Wattanapongsiri, 1966; Murphy and Briscoe, 1999; Faleiro, 2006). Integration of RPW associated microbial control agents with other control practices such as entomopathogens, chemical insecticides and attract-and-kill techniques. Various management strategies have been adopted for controlling this pest mostly relying upon the use of broad-spectrum insecticides, but the injudicious use of such chemicals raises various environmental and human health related issues that necessitates review of prevailing control measures and evaluation of the new and alternative control methods. The utilization of entomopathogenic microorganisms such as entomopathogenic fungi, entomopathogenic bacteria and entomopathogenic nematodes are considered to be promising alternatives to conventional insecticides in managing this voracious pest. Prior to the application of any control strategy, sampling or monitoring of the pest population and their genetic analysis can give a better idea of the exact status of the insect populations and can facilitate the adaptation of appropriate curative measures. In order to have base line data about the genetic diversity of R. ferrugineus from local populations and their comparison with the rest of the world populations can give the idea of their native and invaded range and their distribution pattern. Moreover, local populations of R. ferrugineus have gained resistance to commonly used chemical insecticides and phosphine due to the excessive and unwise use of these chemical insecticides. Resistance against seven different populations of R. ferrugineus was determined from very low to low and moderate to high level against agents commonly used insecticides. Phosphine, cypermethrin and deltamethrin exhibited highest resistance against almost all populations of this insect pest. Entomopathogenic fungi are potent alternatives to these chemical insecticides. Screening of 19 different fungal isolates of B. bassiana and M. anisopliae exhibited variable ranges of mortality against larvae and adults. Five best isolates that caused highest mortality against larvae and adult after 5, 10 and 15 days of incubation were screened by virulence assays. WG-41 and WG-42 were the best isolates that caused highest mortality and significantly reduced the developmental parameters. B. bassiana are capable of colonizing endophytically in live date plam petioles even after 30 days of inoculation and can significantly reduce the weevil population when exposed to the endophytically colonized date palm pieces. Moreover Bt-k is also an effective agent that can also cause detrimental effects of larval and adult survival alone and in combination with endophytically colonized date palm pieces. Both agents also had great influence on the developmental parameters such as larval duration, larval weight, prepupal duration, prepupal weight, pupal duration, pupal weight, adult longevity and adult weight etc. the agents also affect the developmental parameters like, diet consumption, frass production and weight gain.

The alone and integrated use of entomopathogenic fungi, Bt-k and nematodes can also cause to suppress the weevil population collected from 4 different areas of Punjab and Khyber Pkhtunkhwa, Pakistan under laboratory conditions. Hence we can use microbial entomopathogens against this voracious pest which are safer to environment and compatible to environment.