Isolation, Molecular Identification and Lab Evaluation of ... · Utilizing morphological analysis &...

I

Isolation, Molecular Identification and Lab

Evaluation of the Entomopathogenic Fungi;

(Metarhizium sp. and Beauveria sp.) against the

Red Palm Weevil Rhynchophorus ferrugineus

وانتقييى انخبري نهفطريبث انرضت اندزيئيانعزل, انتشخيص

Metarhizium sp. وفطر Beauveria sp نهحشراث يثم فطر .

واستخذايهب ضذ حشرة سىست اننخيم انحراء

Mahmoud W. I. El-Hindi

Supervised by

Prof. Abboud Y. El Kichaoui

(Ph.D. Botany and Mycology)

A thesis submitted in partial fulfillment

of the requirements for the degree of

Master of Biotechnology

Dec/2016

زةــغ – تــالييــــــت اإلســـــــــبيعـاند

شئى انبحث انعهي وانذراسبث انعهيب

انعهــــــــــــــــــــــــــــــــــــىوت هيــــــك

انتكنىنىخيب انحيىيـــــــــــت يبخستير

The Islamic University–Gaza

Research and Postgraduate Affairs

Faculty of Science

Master of Biotechnology

II

إقــــــــــــــرار

أنب انىقع أدنبه يقذو انرسبنت انتي تحم انعنىا:

Isolation, Molecular Identification and Lab Evaluation

of the Entomopathogenic Fungi; (Metarhizium sp. and

Beauveria sp.) against the Red Palm Weevil

Rhynchophorus ferrugineus

نهحشراث يثم وانتقييى انخبري نهفطريبث انرضت اندزيئيانعزل, انتشخيص

واستخذايهب ضذ حشرة سىست .Beauveria sp وفطر .Metarhizium spفطر

اننخيم انحراء

ج اإلشبسة إن حزب سد، أ أقش بأ يب اشخهج ػه ز انشسبنت إب خبس صذ انخبص، ببسخزبء يب ح

ز انشسبنت ككم أ أ صضء يب نى قذو ي قبم االخش نم دسصت أ نقب ػه أ بحز نذ أ يؤسست

حؼهت أ بحزت أخش.

Declaration

The work provided in this thesis, unless otherwise referenced, is the researcher's own

work, and has not been submitted by others elsewhere for any other degree or

qualification.

:Student's name يحد نذ إبشاى انذ اسى انطبنب:

:Mahmoud Signature انخقغ:

:Date 14/12/2016 انخبسخ:

IV

Abstract

Background: Plant diseases generate challenging problems in commercial, agriculture and

pose real economic threats. The red palm weevil (Rhynchophorus ferrugineus) (RPW) is

one of the most destructive pests of palms in the world. Nowadays, control methods revolve

around treatments based on chemicals, biotechnological systems using semichemicals or

the development of the sterile insect technique and Biological control.

Objectives: Our aim was to evaluate the entomopathogenicity of indigenous Beauveria

bassiana and Metarhizium anisopliae against larvae and adults of R. ferrugineus.

Methodology: B. bassiana & M. anisopliae taken from dead adults and dead larvae of R.

Ferrugineus. Utilizing morphological analysis & molecular identification test by using

PCR technique. Evaluation the efficiency of the isolated fungi under lab conditions and

optimize it as biological control agent product after divided all adults and larvae into 4

groups. Incubation the adults RPW groups for 28 days and 6 days for larvae RPW groups.

On another hand, the ability of treated RPW male to infect if the females was examined. All

Data was examined by (Abbott's Formula) in this study.

Results: Our results showed that the B. bassiana and M. anisopliae exhibited a good

biological control agent against larvae and adults of RPW. The pathogenicity of the two

most virulent isolates and the toxicity assay on larvae showed the highest mortality

percentage which reached to 100% against the larvae with B. Bassiana, but reaches to 90%

after spraying the larvae with M. anisopliae, and reaches 43.3% after treated by pesticide.

The bioassay on the adults of RPW and the maximum mortality of weevils reaches 100%

on 28th day after spraying the adult with B. bassiana, while the mortality was up to 90%

after spraying the adult with M. anisopliae. The mortality for the adults treated with

pesticide arrives to 50% and the control group 10% at the same time. Also, our results

revealed that the infection males of RPW by EPF can be disseminated into the healthy

population, after treatment the male adults of the RPW by B. bassiana and M. anisopliae.

The highest mortality of up to 90% for two isolated fungi compare with the group which

was treated with pesticide (20%) after incubation for 28 day.

Conclusions: Our research concludes that B. bassiana and M. anisopliae locally isolated

can be used as biological control agents with great efficacy.

Keywords: B. bassiana, M. anisopliae, Red Palm Weevil, Molecular identification,

Biocontrol.

V

نهخصا

حضبست صساػت طؼبت حشكم حذذاث اقخظبدت حققت سست انخم خسبئش أيشاع انببحبث خش ػب خهفيت انذراست:

. احذة ي افبث األكزش حذيشا ألشضبس انخم ف انؼبنى، خبطت ف قطبع غضة (R. ferrugineus)انحشاء

ػه اناد انكبئت، أظت انخكنصب انحت ببسخخذاو طشق انكبفحت حذس حل انؼالصبث انقبئت

semichemicals حتششة انؼقت انكبفحت انأ حطش حقت انح.

M. anisopliae فطش B. bassianaحقى انفطشبث انشضت يزم ي ز انذساست ذفب األهذاف:

. ة سست انخم انحشاءاسخخذايب ضذ انشقبث انببنغ ي حشش

ي انفبت ( ي انشقبث انببنغ M. anisopliae فطش B. bassianaحى ػضل انفطشبث )قذ انطرق واألدواث:

فطشبث اسخخذاو أسبط غزائت خبطت ن ان حى ػضنب حخب ػه أسبط غزائت ػبيتحذ .سست انخم انحشاء

حقى كفبءة انفطشبث انؼضنت حى انخبطت ببنفطشبث. نضبػفت انضبث PCRرت ببسخخذاو حقت حى ححذذ انبظت انسا

4 بؼذ حقسى كم ي انببنغ انشقبث إن حتف ظم ظشف انخخبش رنك ػه انص األيزم كخش نهكبفحت ان

نهشقبث ي سست انخم أبو 6اث انببنغت يب نهحشش 28فخشة انحضبت نسست انخم انحشاء نذة . يضػبث

انزكس سخظب اإلبد ببنفطشبث انشضت كبج انحشاء. ػالس انببنغ انزكس ي سست انخم انحشاء نخقى يب إرا

ببسخخذاو قب كبفت انبببث حى فحض يضػبث كب يب أػال. 4ال، بؼذ حقسى كم ي انحششاث إن وأ

(Abbott's Formula) .

بشكم صذ ك اسخخذايب( B. bassiana & M. anisopliaeأظشث خبئضب أ انفطشبث انخ حى ػضنب ) اننتبئح:

ضذ انشقبث انببنغ ي سست انخم انحشاء. قذ أظشث ست اضحت ػه انشقبث حذ حتكؼايم يكبفحت

.Bفطش ي أبو ي سش انشقبث 6٪ خالل 100سبت يث طهج إن كبج أكزش ضشاة ػهب كبج أػه

bassiana بؼذ سش انشقبث ي فطش 90، نك سبت انث طهج إن ٪M. anisopliae ف ح طهج سبت .

انحششاث ٪ ف فس انقج. انخقى انخبش ػه 43.3انث ف انشقبث انخ حى يؼبنضخب ببنبذاث انكبئت إن

يب بؼذ سش انحششاث انببنغت انكببس 28٪ خالل 100 انببنغت ي سست انخم انحشاء أػه يؼذل نهث طم إن

.M٪ بؼذ سش انحششاث انببنغت ي فطش 90 , ف ح أ يؼذل انفبث طم إنB. bassianaي فطش

anisopliae بؼذ أ حى يؼبنضخب ببنبذاث انكبئت انضػت 50. نك كبج سبت انفبث ي انحششاث انببنغت ٪

٪ ف فس انقج. أضب، كشفج خبئضب أ 10كبج سبت انث حظم فب إن انخ حى سشب ببنبء انقطش انضببطت

س ي سست انؼذ ك أ حشش ي ركس سست انخم انحشاء إن اإلبد أربء انخضاس، بؼذ يؼبيهت انببنغ انزك

٪ نكال 90. حذ طم أػه يؼذل نهفبث إن M. anisopliaeفطش B. bassianaانخم انحشاء ي فطش

٪ بؼذ فخشة 20فب سبت انث إن جطه انخ انخ حى يؼبنضخب كبئب ببسخخذاو انبذاث تبنضػبانفطش يقبست

يب. 28انحضبت انخ كبج نذة

يحهبانخ حى ػضنب , B. bassiana & M. anisopliae)سخخش ي رنك أ كال ي انفطشبث انؼضنت ) تبج:االستن

.حسخخذو كؼايم يكبفحت حت بصحتك أ

, سست انخم انحشاء, انخشخض انضضئ, انقبيت انحت.B. Bassiana ,M. anisopliae :انكهبث انفتبحيت

VI

Dedication

It gives me pleasure to dedicate this thesis to my beloved parents, all my wonderful

sisters (Nieven, Nihal & Nissren) and my lovely brothers (Mohammed, Ibrahim &

Nahed) who have always supporting me since the beginning of my studies.

The thesis is also dedicated to Al-Aqsa Mosque, beloved Jerusalem and all

Palestine’s martyrs, especially my friend's martyrs (Salah El Khairy, Ahmed El Dalo,

Hasan El saqqs and Fadi Hasanin).

Last but not least, this thesis is dedicated to all those who supported me and were

beside me all the way and those who believe in the richness of learning

VII

Acknowledgment

All praises and thanks are for Almighty Allah the most gracious and merciful, for

helping me in completion of this study.

My deepest and profound acknowledgments are to my supervisors Dr. Abboud Y.

El Kichaoui for his professionalism, continuous support, generous helps, fruitful and

constructive suggestions. I could not have imagined having a better supervisor and

mentors for my master thesis.

Many heartfelt thanks for Interpal Foundation for their generous funding of this

research.

I am especially indebted to the outstanding staff of Department of Biotechnology

and Biological Control Unit at IUG for their useful assistance, valuable advice, and

permissions.

Many special heartful thanks and sincere gratitude to Mis: Bara'a A. Abu Asaker

Department of Biotechnology at IUG for her great help.

Special thanks and greatly gratitude to the staff of Ministry of Agriculture and Mr.

Nahed El Sabe for their endless help.

Special thanks and greatly gratitude to Prof. Adnan El Hindi and his wife Mis.

Maryam Qanou for their endless help.

Finally, I thank the countless people who contributed to this research and anyone

who helped me in any way.

VIII

List of content

Declaration ................................................................................................................. II

Abstract ...................................................................................................................... IV

Dedication ................................................................................................................. VI

Acknowledgment ...................................................................................................... VII

List of Tables ............................................................................................................. XI

List of Tables ..................................................................... .خطأ! اإلشبسة انشصؼت غش يؼشفت

List of Figures .......................................................................................................... XII

List of Abbreviations ................................................................................................ XV

Chapter 1 Introduction ............................................................................................... 1

Chapter 1 Introduction ............................................................................................... 2

1.1 Background and Context .................................................................................... 2

1.2 Objectives ............................................................................................................. 3

1.2.1 General objective .............................................................................................. 3

1.2.2Specific objective: .............................................................................................. 3

1.3 Signification .......................................................................................................... 3

1.4 Limitations of study ............................................................................................ :4

1.5 Overview of Thesis ............................................................................................... 4

Chapter 2 Literature Review ...................................................................................... 5

Chapter 2 Literature Review ....................................................................................... 6

2.1 Importance of date palm trees: ........................................................................... 6

2.2 Scientific Taxonomy of Date Palm (Phoenix dactylifera) ................................. 8

2.2.1 Scientific Classification .................................................................................... 8

2.3Distributions and Ecology of Date palm ............................................................ 8

2.4 Description of palm trees ................................................................................... 9

2.5 Importance of the Date palm trees .................................................................. 10

2.6 Pest and disease of palm trees .......................................................................... 11

2.6.1 Red Palm Weevil (RPW) ............................................................................... 11

2.7 Biological Control of RPW by B.bassiana & M. anisopliae ............................ 19

IX

2.6.1 Importance of Beauveria bassiana: .............................................................. 19

2.7.2 Importance of Metarhizium anisopliae ......................................................... 21

2.8 Molecular identification of Entomopathogenic Fungi: ................................. 23

Chapter 3 Materials and Methods ............................................................................ 25

3.1 Materials .............................................................................................................. 26

3.1.1 Chemicals and Reagents ................................................................................ 26

3.1.2 Equipments ...................................................................................................... 26

3.2 Methodology: ...................................................................................................... 27

3.2.1 Isolation of B. bassiana & M. aneosiplaia fungi ......................................... . 27

3.2.2 Purification of fungi by using selective medium ........................................ . 27

3.2.3 Sub-culturing: ................................................................................................. 27

3.2.4Spore suspension: ............................................................................................. 27

3.2.5Morphological Identification of Fungal Isolates ........................................... 28

3.2.6Molecular Identification of fungi .................................................................... 28

3.2.7Laboratory Rearing ......................................................................................... 29

3.2.8Bioassay (Contact application of fungi): ........................................................ 29

3.2.9Data Collection and Statistical analysis. ........................................................ 30

Chapter 4 Results and Discussion ............................................................................ 32

4.1 solation of B. bassiana & M. aneosipliae fungi: ............................................... 32

4.2Morphological Characterization & Microscopic Examination for B. bassiana

& M. aneosipliae fungi: ........................................................................................... 33

4.3 Enrichment for two fungi and Spore Suspension ........................................... 35

4.4 Molecular Characterization for B. bassiana & M. anisopliae fungi. ............. 35

4.4.1PCR amplification of ITS ............................................................................... 35

4.4.2PCR amplification of β-tubulin ...................................................................... 38

4.5Laboratory Rearing for RPW. .......................................................................... 40

4.6Bioassay (Contact application of fungi): ........................................................... 42

4.6.1Pathogenicity of entomopathogenic fungi to R. ferrugineus eggs:............... 42

4.6.2Pathogenicity of entomopathogenic fungi to R. ferrugineus larvae: ........... 42

4.6.3Pathogenicity of entomopathogenic fungi to R. ferrugineus Adult:

................................................................................................................................... 44

X

4.6.4Pathogenicity of entomopathogenic fungi to R. ferrugineus Adult male as

vector to transfer infections into female: ............................................................... 47

Chapter 5 Conclusions and Recommendations ....................................................... 53

Chapter 5 Conclusions and Recommendations ....................................................... 54

5.1Conclusion ........................................................................................................... 54

5.2Recommendations for improving this study: ................................................... 54

The Reference List .................................................................................................... 56

Appendix 1: Protocol for DNA Extraction Kit ......................................................... 69

Appendix 2: Formula for Fungi media.................................................................... 74

XI

List of Tables

Table (2.2): Types, Numbers and Percentage of the Date palm in Gaza strip.

(Ministry of Agriculture, 2013) ................................................................................. 9

Table (2.3): Show the number infected, healthy, burned, and examined of RPW,

addition for palm tree was treated in 2012, 2013 and 2014 in Gaza strip. (MOA,

2014) .......................................................................................................................... 16

Table (3.1): Chemicals, reagents and cultures mediums that were used in this

work ........................................................................................................................... 26

Table (3.2): Major Equipments used in the present study. .................................. 26

Table (3.4): The primers sequences used in ITS, β-tubulin and SCAR analysis.

................................................................................................................................... 28

Table (4.1): Fragment sizes of the PCR-amplified of different genes for isolated

fungi. (bp). ................................................................................................................ 39

Table (4.2): The larvae of RPW treated with EPF. .............................................. 43

Table (4.3): The mortality percentage for adults of RPW treated with 3.4x108

spores/ml of B.bassiana. ........................................................................................... 44

Table (4.4): The mortality percentage for adults of RPW treated with 3.6x108

spores/ml of M. anisopliae. ...................................................................................... 45

Table (4.5): Male of RPW contaminated with 3.4x108

spores/ml and 3.6x108

spores/ml of B.bassiana and M.anisopliae, respectively. ...................................... 48

XII

List of Figures

Figure (2.1): Date palm Phoenix dactylifera. (MOA, 2014) .................................... 8

Figure (2.2): R. ferrugineus, Red Palm Weevil. (Tofailli, 2010). ......................... 13

Figure (2.4): Plate of B. bassiana. (Zambrano et l., 2013) .................................... 19

Figure(2.7): M. anisopliae culture and spores.

(http://www.naro.affrc.go.jp/org/fruit/epfdb/Deutte/Metarh/plate_M.htm) ..... 22

Figure (2.8): Dead of Larvae and adult from R. ferrugineus by sporulation of M.

anisopliae (Gindin, 2006). ........................................................................................ 23

Figure (4.1): B.bassiana was covered adult & larvea of RPW ............................. 32

Figure (4.2): Soil samples that collected for B. bassiana & M. anesipliae

isolation. .................................................................................................................... 32

Figure (4.3): Culture of M. aneosipliae on OMA Selective Medium and PDA

Medium. .................................................................................................................... 33

Figure (4.4): Microscopic examinations for M. anesipliae 100X. ........................ 33

Figure (4.5): Culture of B. bassiana on DOC2 Selective Medium and PDA

Medium. .................................................................................................................... 34

Figure (4.6): Microscopic examinations for B. bassiana 40X & 100X. ............... 34

Figure (4.7): Spore suspension of fungi in PDB media. A: B. bassiana & B: M.

anisopliae. .................................................................................................................. 35

Figure (4.8): Fragment sizes of the PCR-amplified ITS1 regions as obtained by

gel electrophoresis. In the peripheral of the photograph, bands from a DNA ladder

200bp scale (M) are shown. L1: negative control, L2: ITS1 gene for M.

anisopliae 215 bp & L3: ITS1 gene for B. bassiana 230 bp. ................................. 36

Figure (4.9): Fragment sizes of the PCR-amplified ITS2 regions as obtained by

gel electrophoresis. In the peripheral of the photograph, bands from a DNA

ladder 200bp scale (M) are shown. L1: negative control, L2: ITS2 gene for B.

bassiana 380 bp & L3: ITS2 gene for M. anisopliae ranging between 360 bp to

1000 bp. ..................................................................................................................... 37

Figure (4.10): Fragment sizes of the PCR-amplified of whole ITS regions as

obtained by gel electrophoresis. In the peripheral of the photograph, bands

from a DNA ladder 200bp scale (M) are shown. L1: negative control, L2: ITS

XIII

regions gene for B. bassiana 640 bp & L3: ITS regions for M. anisopliae ranging

between 630 bp. ........................................................................................................ 37

Figure (4.11): Fragment sizes of the PCR-amplified of whole Bt regions as

obtained by gel electrophoresis. In the peripheral of the photograph, bands

from a DNA ladder 200bp scale (M) are shown. L1: negative control, L2: Bt

gene for B. bassiana 500 bp & L3: Bt gene for M. anisopliae ranging between

380 bp. ....................................................................................................................... 38

Figure (4.12): Fragment sizes of the PCR-amplified of SCAR region as obtained

by gel electrophoresis. In the peripheral of the photograph, bands from a DNA

ladder 100bp scale (M) are shown. L1: negative control, L2 & L3: SCAR gene

for B. bassiana 96 bp. ............................................................................................... 39

Figure (4.13): Rearing of the RPW under Lab Conditions.................................. 41

Figure (4.14): Stages of the Red Palm Weevil in rearing box. A: Eggs stage, B:

Cocoons collected from the sugarcane stems, C: Adualt stage of RPW & D:

larva of RPW. ........................................................................................................... 41

Figure (4.15): All eggs of RPW with & without treatment. A: Eggs killed after

treatment with B.bassiana and M.anisopliae. B: Eggs without treatment. ......... 42

Figure (4.16): Larvae of RPW treated with 3.4x108 spores/ml of B.bassiana. ... 43

Figure (4.17): Larvae of RPW treated with 3.6x108 spores/ml of M. anisopliae. 43

Figure (4.18): RPW without treatment after 28 day. ........................................... 45

Figure (4.19): RPW treated with 3.4x108 spores/ml of B.bassiana. ..................... 46

Figure (4.20): RPW treated with 3.6x108 spores/ml of M.anisopliae. ................. 46

Figure (4.21): Sporulation of B. bassiana on the R. ferrugineus (Olivier).

Dissecting Microscopy. ............................................................................................ 47

Figure (4.22): RPW treatment of male with 3.4x108spores/ml of B.bassiana and

the mortality after 28 days of treating. .................................................................. 49

Figure (4.23): RPW treatment of male with 3.6x108spores/ml of M. anisopliae

and the mortality after 28 days of treating. ........................................................... 49

Figure (4.24): Mortality percentage for groups of the larvae of RPW after

treated with 3.6X108 spores/ml of M. anisopliae, 3.4 X 10

8 sopres/ml of B.

bassiana, chemical pesticide & negative control. .................................................. 50

XIV

Figure (4.25): Mortality percentage for groups of the adult of RPW after treated

with 3.6X108 spores/ml of M. anisopliae, 3.4 X 10

8 sopres/ml of B. bassiana,

chemical pesticide & negative control. ................................................................... 51

Figure (4.26): Mortality percentage for R. ferrugineus. After treatment the Male

adult of RPW with 3.6X108 spores/ml of M. anisopliae, 3.4 X 10

8 sopres/ml of B.

bassiana & chemical pesticide. ................................................................................ 52

XV

List of Abbreviations

% Percentage

AFLP Amplified Fragment Length Polymorphism

AOAD Arab Organization for Agriculture Development

BCP Biological Control Project

Bp Base pair

Bt2 β-tubulin

Co Celsius

DNA Deoxyribonucleic acid

DOC2 Dodine acetate selective media

EPF Entomopathogenic Fungi

Fo Fahrenheit

FAO Food and Agriculture Organization

FAOSTAT Food and Agriculture Organization Statistical

Fig. Figure

G Gram

GHA Beauveria bassiana strain GHA

IPM Integrated pest management

ITS Internal Transcribed Spacer

ml millilitre

MOA Ministry of Agriculture

OMA Oatmeal agar

PCR Polymerase Chain Reaction

PDA Potato Dextrose Agar

PDB Potato Dextrose Broth

RAPD Random Amplified Polymorphic DNA

rDNA Ribosomal DNA

RFLP Restriction Fragment Length Polymorphism

RNA Ribonucleic acid

RPM Round per minutes

RPW Red Palm Weevil

SCAR Strain-specific sequence-Characterized Amplified

Region

SDA Sabouraud Dextrose Agar

SDAY Sabouraud Dextrose Agar containing Yeast Extract

SIT Sterile Insect Technique

SPSS Statistical package for social sciences

UAE United Arab Emirates

USA United States of America

Chapter 1

Introduction

2

Chapter 1

Introduction

1.1 Background and Context

Plant diseases generate challenging problems in commercial, agriculture and pose

real economic threats to both conventional and organic farming systems, so this all

diseases need to be controlled to maintain the quality and abundance of food, feed,

and fiber produced by growers around the world (Abdollahi, 2004). Different

approaches may be used to prevent plant diseases. Beyond good agronomic and

horticultural practices, growers often rely heavily on chemical fertilizers and

pesticides.

Excessive use of chemicals has resulted rise in significant and dangerous in the

proportion of soil and groundwater contamination, as well as a clear increase in the

incidence of diseases of cancer among consumers in general and farmers in

particular, where the studies suggest that the increase in cancer on the one hand and

environmental pollution on the other hand is still on the rise continuously (Abdollahi,

2004).

The Gazan economy is largely dependant on agriculture, however due to closures

and landrazing, this sector has been greatly affected. For many people, the date palm

(Phoenix dactylifera L.) is more than just a fruit tree; it is a symbol of their religious,

cultural, and economic heritage. Saudi Arabia is home to more than 23 million date

palms and ranks third worldwide in fruit yield and area under cultivation (Erskine et

al., 2004), (Mukhtar, 2011)

Date-palm (Phoenix dactylifera L.) is attacked by a large number of pests, including

fungi, insects, and nematodes (Carpenter and Elmer, 1978). Some of these pests are

serious and difficult to control such as red palm weevil (Rhynchophorus ferrugineus

Oliv, Coleoptera: Curculionidae) (El-Sufty et al., 2007), (Arab, 2012).

The red palm weevil (RPW) R. ferrugineus is one of the most destructive pests of

palms in the world. This weevil affects more than 20 palm species (Barranco et al.,

2000) including the date palm (Phoenix dactylifera L.). R. ferrugineus was

introduced in Spain mainland in 1995 (Barranco et al., 1996a) and then spread to all

palm growing areas in the Mediterranean and recently also to the Canary Islands.

The pest has caused large economic losses in date palms worldwide for the last 30 yr

(Murphy and Briscoe, 1999; Faleiro, 2006; Güerri, 2010).

The weevils develop within the tree trunk, destroying its vascular system and

eventually causing the collapse and death of the tree. The pest is widely distributed in

Oceania, Asia, Africa and Europe (Gindin, 2006).

Nowadays, control methods revolve around treatments based on chemicals,

biotechnological systems using semichemicals or the development of the sterile

insect technique (hardly sustainable at this time) (e.g. Paoli et al., 2014), and

biological control (Murphy and Briscoe, 1999; Faleiro, 2006; Paoli et al., 2014;

Mazza, 2014).

3

Additionally, the spread of plant diseases in natural ecosystems may preclude

successful application of chemicals, because of the scale to which such applications

might have to be applied. Consequently, some pest management researchers have

focused their efforts on developing alternative inputs to synthetic chemicals for

controlling pests and diseases. Among these alternatives are those referred to as

biological control.

Biological control as the use of natural microorganisms, crude extract from

microorganisms or genetically improved to resist or eliminate of pathogens. It is

performed by using microorganisms from the environment itself directly or makes

some changes in their properties, to increase their effectiveness or use one of their

products.

The advantage of using of this method is to reduce the costs of pest control.

Additionally it preserves human health and environment from pollution, which

caused by chemical pesticides usage. Also it minimizes the formation of insecticides

resistance in some pest.

1.2 Objectives

1.2.1 General objective:

We work within a general project that aims to solve health and environmental

problems by reduction of organic pesticide and fertilizer. The present study aims to

develop and optimize an effective biological control product from Beauveria

bassiana and Metarhizum anisopliae against the RPW forward a successful market

introduction.

1.2.2 Specific objective:

Isolation of B. bassiana and M. anisopliae from fields of Gaza strip.

Utilizing morphological properties and simple molecular technique for

identification of these fungi.

Isolation, rearing and bioassay application of RPW under laboratory

conditions.

1.3 Signification

It is very important to prove scientifically that the biological control that considered

an alternative to chemical fertilization and pesticide. The importance of this idea is

reflected in a distinct area like Gaza, where the density of population is high and

there is an intensive agriculture in a narrow agricultural area. This situation forcing

the farmers to excessive use of chemical fertilizers and pesticide to compensate the

deficiency of the agricultural land used. Moreover, the sandy nature of the soil

4

facilitate the arrival of these chemicals into the groundwater causing a significant

pollution through the irrigation. Putting effective alternatives for these farmers is of

utmost importance to begin to develop a strategy aimed to reduce the use of chemical

fertilizers and pesticide, while maintaining appropriate agricultural production

(MOA, 2014). In addition, it is worth mentioning also that the only source of

drinking water in this region of the world is the groundwater wells that are directly

affected by the use of high amount of chemical fertilizers and pesticide. This is the

ultimate goal of such researches that fall within an integrated system aimed at

preserving the environment, drinking water, health and at the same time providing a

high agricultural productivity.

1.4 Limitations of study:

The limitations of current study can be seen in many facets, these include:

Overcome contamination during isolation of fungi.

Problems in purchasing RPW.

1.5 Overview of Thesis

The potential of this research is providing our farmers with well-researched and

developed biological solutions for agricultural challenges. Developing and

introducing biological control products into our region will improve and sustain plant

production “ vital sector for Gazans to guarantee the minimum limit of food

security”, protect our lands and the limited drinking water resources from chemical

pesticide residues, biologically based products are eco-friendly and safe for both

growers and consumers, last but not least, the findings will offer a unique

opportunity and a great deal for the private sectors to invest in such local industry.

These products represent one of the fastest growing sectors of the pest control

industry.

5

Chapter 2

Literature Review

6

Chapter 2

Literature Review

In order to show the importantce of Date palm in our regions we tried to collect some

important information about the palm tree in Gaza strip.

2.1 Importance of date palm trees:

The date palm, Phoenix dactylifera (Palmae) its fruit has provided the staple food of

local people for thousands of years. The main agricultural crop in Oman, occupying

83% of the total area grown under fruits and 50% of the total cultivated land. Date

palm cultivated in over 40 countries with approximately 930,000 hectares under

production annually producing some seven million metric tonnes of fruit, (FAO,

2006). However, the greatest production is in Iraq, Iran and Saudi Arabia

(Purseglove, 1972) in (Murphy, 1999).

Global date production is almost exclusively centered on North Africa and the Arab

States. Egypt, Saudi Arabia, Iran, UAE, Pakistan and Algeria are the biggest

producers (FAOSTAT, 2007). Much of this production is for local consumption,

while Iran, Pakistan, Tunisia, Saudi Arabia, UAE, Iraq and Algeria are the major

exporters by volume. The USA and Israel are smaller producers but achieve the

highest export unit value (Reilly, 2010).

Date palm is a multi-purpose tree. It provides food, shelter, timber products and all

parts of the palm can be used. Because of these qualities, and its tolerance to harsh

environmental desert conditions, areas under cultivation have increased

tremendously in recent years. Improvement in marketing and export efficiency are

priorities for date palm growers (Alabdulhadi et al., 2004).

Date palm is mainly grown for its fruits, but the whole tree is utilized. Dates are a

highly nutritious source of sugars, minerals, vitamins and antiatherogenic nutrients.

Dates are used as a sweetener in numerous traditional desserts and contemporary

baked goods. (Manickavasagan et al., 2013).

Propagation of date palm is traditionally by offshoots; however, increased demands

for offshoots to expand agricultural areas have necessitated the use of tissue culture.

Remarkable progress has been made in date palm micropropagation since it was first

achieved in the early 1970s. At present, commercial micropropagation is becoming

commonplace for commercial date palm production. Researchers continue to

improve this process through empirical assessment of various tissue culture factors in

relation to the wide array of available cultivars. (Ibraheem et al., 2013).

In the Palestinian territories, there has been an increase in date production to 4,688

metric tons in 2011 (FAO, 2013).

7

In the Gaza Strip, most of the date palms are planted in the coastal area and in many

cases the plantations utilize mixed farming systems. The main date palm cultivars in

the Gaza Strip are Hayani and to a lesser extent Bentaisha. In 1999 other types such

as Barhi, Zahedi, Ameri and Halawy were introduced (Banna, 2007), and the Table

2.1 showed a summary of the morphological characteristics of six date palm cultivars

present in the Gaza Strip (El Kichaoui, et al., 2013 ).

Table (2.1): A summary of the morphological characteristics of six date palm

cultivars present in the Gaza Strip (El Kichaoui, et al., 2013).

The total area of the West Bank is about 6 million donums (1 donum = 0.1 hectare),

of which 30% is cultivated, while the total area of the Gaza Strip is 365,000 donums,

of which 55% is cultivated. Date palm is cultivated in a small area in the West Bank

(about 500 donums in Jericho) and 2,200 donums in Gaza. The total annual

production of date palm in those areas is 3,000 tons. This amount is about 10% of the

dates consumed in the West Bank and Gaza (Abu-Qaoud, 1996).

According Ministry of Agriculture 2013, the palm tree is one of the most important

tributaries of the agricultural sector, accounting for 2.5% of gross domestic product

in the Gaza Strip. The total area of the Gaza strip is about 6222 donums, and the

number of palm trees in the Gaza Strip, about (150,000) trees of which 120,000) fruit

trees, with an estimated annual production of about (10,000 tons). (MOA, 2013).

8

2.2 Scientific Taxonomy of Date Palm (Phoenix dactylifera)

The date palm Phoenix dactylifera L is the most important fruit tree in the Arab

region and it is extensively cultivated for its edible sweet fruit.

2.2.1 Scientific Classification

Kingdom – Plantae

Order – Arecales

Family – Arecaceae

Genus – Phoenix

Species – dactylifer (Ateeq, et al., 2013)

Figure (2.1): Date palm Phoenix dactylifera. (MOA, 2014)

2.3 Distributions and Ecology of Date palm

The date palm requires rise temperatures and low humidity to set fruit and ripen to

maturity. The date palm grows best in temperatures above 20°F (-7°C). However,

they can survive into the mid to lower teens for short periods of time. For pollen

germination, a temperature of 95°F (35°C) is needed. As with most palms, research

has shown that warm to hot night temperatures also promotes faster growth. Deep

soils is the better growing conditions for palms and preferably sand 3 to 5 feet deep,

and a good supply of either sub-surface or irrigation water. Date palms grow

naturally between 15 and 35 degrees north latitude in the Sahara, and in the southern

fringe of the Near East. This area is nearly rainless. The date palm is distributed in

the Middle East, and in the northern, eastern, and southern areas of Africa. They are

also found in North America, Southern Europe, and Central and South America. Yet

with this great distribution, there are still vast areas of the world where this palm

could adapt to the harsh climates and provide badly required food crops. The date

palm is adaptable to large and small production in arid and semi-arid regions.

(Robinson, 2012).

The distribution according to latitude for both northern and southern hemispheres is

illustrated in Tables 1 and 2. The extreme limits of date palm distribution are

between 10°N (Somalia) and 39°N (Elche/Spain or Turkmenistan). Favourable areas

9

are located between 24° and 34°N (Morocco, Algeria, Tunisia, Libya, Israel, Egypt,

Iraq, Iran,.). In USA date palm is found between 33° and 35°N. Because of climatic

factors, the date palm will grow, but will not fruit properly outside the above defined

geographical limits. (Zohary and Hopf 2000)

Nevertheless, date palms are being grown in traditional oases or modern-day

plantations in many countries around the world, including Iraq, Kuwait, Bahrain,

Saudi Arabia, United Arab Emirates, Oman, Yemen, Jordan, Syria, Palestine,

Mauretania, Senegal, Sudan, Somalia, Spain, Canary Islands, USA, Australia and

New Caledonia. However, south of the great Sahara Desert of North Africa,

increasing rainfall imposed a barrier to any extension of date palm culture which has

been limited to small plantings along the northern edge of the equatorial rain belt

from Senegal and the Upper Niger to Sudan (Darfur and the Blue Nile provinces).

(Jaradat, 2011) in (Zabar, 2012).

Most of the date palms planted in Gaza strip, Hayani and to a lesser extent Bentaisha,

Barhi, Zahedi, Ameri and Halawy and (Table 2.2) showed the number of trees and

the percentage for each type in Gaza strip. (Ministry of Agriculture, 2013).

Table (2.2): Types, Numbers and Percentage of the Date palm in Gaza

strip. (Ministry of Agriculture, 2013)

Species Numbers Percentage %

Hayani 14,3250 95.5

Bentaisha 2,250 1.5

Ameri 750 0.5

Barhi 3,000 2

Others 750 0.5

Total 150,000 100

2.4 Description of palm trees

Palm stems are tree trunks of very tall palm trees. The trunk elongate by loses its

leaves at the bottom of the canopy and grow new ones from the top of canopy. The

stem which located below the canopy is covered in scars from leave were attached.

Palm trees vary in thickness; date palms have a very thick, tree–like stem. (Al-Mana,

2010; Robinson, 2012).

The leaves of the palms arranged in more or less intervals along the stem. In the

young condition, while still unfolded they arise from the succulent end of the stem.

Under normal growth conditions an average of 12 to 15 new leaves are formed by the

palm every year. The shape of palm tree flowers are small and green or white, its

born on a spike and are symmetrical in shape. Some palms produce both male and

10

female flowers in the same tree and others may produce one type in one year and

other type in the next year (Robinson, 2012).

The date fruit is a berry contain a single seed surrounded by a fibrous, it takes up to

about 200 day from pollination to reach full maturation (tamr stage). The fruit passes

through a number of distinct phases during its formation and ripening, each of them

distinguished by different characteristic both physiognomically and chemically.

(Robinson, 2012; Sulieman et al., 2012; & Hamad et al., 2015).

Palm trees have a tap root or a few large primary roots that grow from the base of the

trunk. These roots have a large diameter, they branch outward and downward

forming a network under the tree. Palm trees don’t have a woody tap root. There are

a large number of roots from its base, that’s form a root ball which supports the tree.

These are called adventitious. (Hodel, 2005).

2.5 Importance of the Date palm trees

Date palm trees have been growing for the last 5000 years in harshest climatic

condition and feeding people as source of energy, nutrition security, and as a healthy

fruit. The Arab countries produce the majority of the world’s total date crop

(FAOSTAT, 2009). The importance of date palm not considered as a concentrated

energy food only, it also a more amenable habitat for the people to live in by

providing shade and protection from the desert winds. In addition the date palm

yields a variety of products for use in agricultural production and for domestic

containers.

Now a day all directions look at the palm as a row material source of industrial

purposes. Particularly all parts of date palm except the roots are useful for a purpose

best suited to them like for example, the trunk usually used for roofing and can be

burned for fuel, the leaves are important to the production of paper and cartons, date

seeds can be soaked in water until soft and then fed to horses, cattle, camels, sheep

and goats. (El-Juhany, 2010).

Dates have proved to be the best resource to ensure food security during food

insufficiency. The date palm help to establishing a sustainable system in subsistence

agricultural areas and thus plays an important social role in supporting the

subsistence base of large population group by helping them to settle in rural area and

decrease the migration to urban center (Sawaya, 2000) in (El-Juhany, 2010).

The trees are an amazing palm for landscaping large area. It also prevents soil

degradation and desertification, thus protecting the environment. In fact, the date

palm represented a significant example of integrated sustainable use of renewable

material resources. The most commonly used parts of the date palm are its fruit, bark

and leaves and they have the commercial and medicinal applications. Date, as one of

the product of the palm, is rich in protein, vitamins and mineral salts. So that it

considered as an essential element of diet for cultivars himself and his animal (EI-

Mously, 1998) in (El-Juhany, 2010).

11

Earlier studies have shown that constituents of dates act as potent antioxidant, anti-

tumour as well as anti-inflammatory, provide a suitable alternative therapy in various

diseases cure. In this review, dates fruits has medicinal value are summarized in

terms of therapeutic implications in the diseases control through anti-oxidant, anti-

inflammatory, anti-tumour and ant-diabetic effect. (Rahmani et al., 2014).

The detailed information on nutritional and health promoting components of P.

dactylifera enhances our knowledge and appreciation for the use of date palm fruits

in our daily diet and as a functional food ingredient. P. dactylifera fruits are

characterized by high carbohydrate content and relatively reasonable amounts of K,

Na, P, Mg and Ca. They are also rich in leucine, glutamic acid, argine, aspartic acid

and alanine. Thus these fruits could be of high nutritional value serving as a good

source of these nutrients for man and his animals. (Shaba et al., 2015).

Date palm help diversify the economic base of participating rural communities and

provide added value with import replacement and export earning as well as

stimulating agri-tourism opportunities. This hardy plant species also provides a high

tolerance to salinity, drought and extremes in temperature thus ensuring better food

security outcomes.

2.6 Pest and disease of palm trees

An increasing number of insects and diseases are destroying palm trees of high

economic and aesthetic value throughout the world is threatened by an increasing

number of insect and disease pests, and overall production inefficiency (El-Juhany,

2010). Date palms are affected by large number of pests, including insects,

nematodes and diseases caused by fungi, bacteria and phytoplasma. (Downer et al.,

2009). Date palm trees were affected by major pests such as: Fusarium disease,

Phytophthora palmivora, Phytoplasmas & Red Palm Weevil.

In the following we highlights the red palm weevil, classification, size of the losses

caused by RPW, the methods which used in controlling and what is the reality of red

palm weevil in the Gaza Strip?..

2.6.1 Red Palm Weevil (RPW)

2.6.1.1 Introduction of the RPW.

The RPW, R. ferrugineus Olivier (Coleoptera: Curculionidae), is an economically

important, tissue-boring pest of date palm in many parts of the world and one of

highest damaging pests in palm plantation, which appeared in the Gaza Strip in late

2011, and arrived to Gaza strip from the Sinai Peninsula several years ago (MOA,

2014). RPW is one of the most important pests of several palm species native to

southern Asia and Melanesia.

R. ferrugineus was introduced in Spain mainland in 1995 and then spread to all palm

growing areas in the Mediterranean and recently also to the Canary Islands. It attacks

12

more than 20 palm species worldwide (Barranco et al., 2000), including date (Giblin

Davis, 2001) and coconut palm (Malumphy and Moran, 2007).

For the first time in Saudi Arabia was recorded in 1986 in Al-Katif Region (Al-

Abdulmohsin, 1987), from United Arab Emirates in 1986, and from the Republic of

Iran in 1992. It spread to North Africa in Egypt in 1993 (Cox 1993). Today red palm

weevil is widely distributed in Europe, Africa, Oceania and Asia (Yuezhong et al.,

2009). And in 1999 in Israel, Jordan and the Palestinian Authority Territories.( RPW

redistributed by movement of infested live palms into the Mediterranean and the

Caribbean (Longo et al., 2011).

Until this day only the American continent was free from the pest, but since

December 2008 the RPW has been found in the Island of Curaçao, Netherlands

Antilles, and more in Orange County, California (CDFA, 2010).

The agro-climatic conditions of the country that planting Date palm along with

monoculture and intensive modern date palm-farming practices, favored the

establishment of this pest (Faleiro, 2006).

RPW were officially detected when an adult was recovered from a heavily damaged

Canary Island date palm in a private garden (Hoddle, 2011). The RPW is attracted by

kairomones of damaged palms, in the trunk of which larvae develop. As a result, the

central tissue of the palm is destroyed and the tree eventually collapses and dies. As

RPW is a hidden tissue borer it is difficult to detect of its attack at an early stage of

infestation. Preventative measures are thus very important for the success of any

RPW-Integrated Pest Management (IPM) programme (Faleiro, 2006).

2.6.1.2 Taxonomy of the RPW

The taxonomy has changed multiple times in the past. Recent molecular research

suggests that R. ferrugineus may actually be a species complex composed of two or

more species (Rugman et al., 2013).

The second species, R. vulneratus, is currently synonymized under R. ferrugineus.

Phylum: Arthropoda.

Class: Insecta.

Order: Coleoptera.

Family: Curculionidae.

Genus: Rhynchophorus.

Full Name: Rhynchophorus ferrugineus (Olivier).

Preferred Common Name: Red Palm Weevil.

Synonyms: Rhynchophorus signaticollis Chevrolat, 1882, Curculio ferrugineus

Olivier, 1790, Calandra ferruginea Fabricius, 1801 (CABI 2009), R. vulneratus

(Panzer), 1798 (Hallett et al. 2004; El-Mergawy, 2011).

13

Figure (2.2): R. ferrugineus, Red Palm Weevil. (Tofailli, 2010).

2.6.1.3 Life Cycle of the RPW

Generally, takes about three to four months to complete the life cycle. RPW female

chews a hole into the palm tissue by using long beak. Eggs are laid singly into these

holes and also in wounds caused by the Rhinoceros beetle in the palm trunk. We

have recorded the maximum of 349 eggs laid by a single female during 47 days at

28ºC. (El-Bakl, 2014)

Neonate larvae bore into the palm core making tunnels and feeding on its inner

contents. As larvae molt, their appetite increases and they tend to feed primarily on

the soft tissues surrounding the apical meristem and it is destroyed resulting in the

palm death. Subsequently, adults will fly away and look for new hosts. (Hussain,

2013).

All stages (egg, larvae, pupa and adult) are spent inside the palm itself and the life

cycle cannot be completed in some other place.

Egg:

The females deposit about 300-500 eggs in separate holes they produced while

searching for food or injuries on the palm. They will also lay eggs in wounds caused

by the beetle Oryctes rhinoceros. Initially ovipostion rate was low but progressed

rapidly and peaked after 2 weeks, remaining stable for about a month (EPPO, 2008).

After laying, the female protects and secures the eggs with a secretion that rapidly

hardens around the eggs.

On average, females produce 210 eggs per clutch, most of which hatch over a period

of 2-5 days. The eggs are white, cylindrical, glossy, oval shaped, and measure 1 to

2.5 mm. Efficiency of egg laying is variable depending on different hosts. Host plant

food quality plays a major role in fertility of insects (Awmack & Leather, 2002).

Larvae:

Characteristics of larvae:

Up to 35mm long.

Brown head.

White body composed of 13 segment.

14

Mouth parts developed well and strongly chitinised.

Average length of fully grown larvae 50mm.

Width in middle 20mm.

Larval development averages around two months, freshly hatched larvae have

legless which bore into the interior of the palms, moving by peristaltic muscular

contractions of the body and feed on the soft succulent tissues, discarding all fibrous

material. The development period of larvae varies from 1 to 3 months. (Murphy and

Briscoe, 1999; Dembilio & Jacas, 2011).

Pupa:

Pre-pupal stage of 3 days and pupal period of 12-20 days, and the pupa was

characterized by:

Pupae have a creamy color, and then brown, with shiny surface, greatly furrowed and

reticulated; average size 35 mm x 15 mm.

Upon completion of larval development, the larva will emerge from the trunk of the

palm tree, and build up a pupa which consists of fiber extracted from the inside of the

palm trees. Pupa will then undergo transformation into an adult. (Dembilio, et al.,

2009)

Adult:

Adults are reddish-brown and about 35 x 12 mm in size. After hatching into an adult,

the weevil emerges from the pupal case, but remains in the pupa stage for several

days before exiting, during this time, the weevil is completing sexual maturity. Adult

weevils live for about 2 to 3 months, feeding on palms and cause economic loss of

date palm trees, mating multiple times, and laying eggs (Murphy and Briscoe 1999).

The sex ratio found in a study in the United Arab Emirates was 1 male: 1.5 females

(Abbas et al., 2006), in Egypt, 1:2 (El-Garhy 1996), and in Israel, 1:2.5 (Soroker et

al. 2005). Adult weevils are mainly active during the day and are capable of long

distance flight (> 900 meters) from the location of hosts or breeding sites (USDA-

APHIS, Marked and released weevils migrated up to 7 km during a period of 3 to 5

days (Abbas et al., 2006).

15

A B

C D

Figure (2.3): The four life stages of the Red Palm Weevil (Al-Saqer & Hassan,

2011). (a) Eggs (b) Larva; (c) Pupa fibrous cocoom removed and (d) Adult RPW.

2.6.1.4 Damage

RPW is among the most highly destructive pest of palms. It has been reported to

infest ≥ 29 different palm species belonging to Agavaceae and Arecaceae. In Spain,

Canary Palm (Phoenix canariensis) is being reported as the most susceptible palm

species. However, the infestation of RPW in the Arabian Peninsula is mainly

responsible for the destruction of date palm plantations. Their creamy white color

larvae (grubs) are the most destructive stage. These legless larvae feed on the

succulent plant tissues that create feeding galleries and move towards the center of

the infested palms. Such feeding pattern disrupts the vascular system of the infested

palm resulting toppling, collapse and death of the infested palm under severe attack

(Dembilio, 2013).

2.6.1.5 RPW in Gaza Strip

For the first time in Gaza strip was recorded in 18/09/2011 in the Middle

Governorate, through observation the injury of the trunk for palm tree, and after the

examination noting all stages for RPW are present in palm tree, after these RPW

observed in other governorates such as Khan-Yonus and Rafah, but it's not viewing

in North Gaza and Gaza governorate at the same year, and this indicate on the source

of infection coming from Egyptian border. (MOA, 2014).

16

In 2012, recorded for one palm tree infected with RPW in Gaza governorate, but it's

not viewing in North Gaza at the same year. In 2013, some injuries for palm tree was

reported in North Gaza governorate and increased of RPW number in Gaza strip.

(MOA, 2014).

Table (2.3): Show the number infected, healthy, burned, and examined of

RPW, addition for palm tree was treated in 2012, 2013 and 2014 in Gaza

strip. (MOA, 2014)

# Year

No. of

examined

Palm tree

No. of

Healthy

Palm tree

No. of

infected

Palm tree

No. of

treated

Palm tree

No. of

burned

Palm tree

1 2012 10658

- 2178 518 1660

2 2013 35675

30001 5674 4906 768

3 2014 125517

109636 20950 17606 3344

Total

171850 139637 28802 23030 5772

2.6.1.6 Management of the RPW

Because the red palm weevil is a hidden tissue borer and difficult to eradicate, early

detection is essential for an effective eradication or management program. When a

population of red palm weevil becomes determined, efforts must be put into an

(IPM) system. Different strategies have been adopted against different life stages of

RPW. The previous investigations have reported the control of adult RPW by

adopting different tactics such as the use of Sterile Insect Technique (SIT), insect

pheromones and insecticidal applications to prevent the adult entry into the tree

trunk. The use of SIT to control RPW was considered for the first time during 1970s,

suggested that the 1-2 d exposure of X-rays to the newly emerged male populations

of RPW at a dose of 1.5 Krad greatly (~90) induced the sterility. In another study,

field trials were conducted to investigate the effect of radiations on the growth of

RPWs and the viability of eggs laid by the females. They reported that the sterile

RPW males remained live till 100 days post exposure. Pheromones usage into the

management strategy of RPW started with the identification of aggregation

pheromones (ferrugineol {4-Methyl-5-nonanol} and ferrugineone {4-methyl-5-

nonanone} during 1993. Later on, the work on the use of pheromones to enhance

their trapping potential started in different parts of the world (Dembilio, 2012).

The most common and practical measure it is Chemical control. In chemical control

is mainly based on the repeated application of large quantities of synthetic

insecticides employed in a range of preventive and curative procedures designed to

17

contain the infestation (Hussain, 2013). So the following is some strategies for

controlling of the RPW.

2.6.1.7 Physical treatment can be including:

Sanitation:

Carry out sanitation in plantations, gardens, landscapes, and other establishments

where hosts are present within the buffer areas. Sanitation includes the following

technique depending on the available equipment:

Burning:

All infested palms should be destroyed at the first sign of larval weevil infestation by

cutting down into small pieces and burning. This practice will prevent larvae from

hatching and put back to the same area (Alhudaib, 2009).

Burning the top of the tree alone does not kill the stages in the middle of the trunk,

so heavily infested trees should be eradicated, split open to expose the different

stages of the pest inside, and burned (Soroker et al., 2005).

Pruning:

When green leaves are cliped, they should be cut 120 cm from the base (Alhudaib,

2009).

Treating Palm Injuries:

According to the fact that female weevils have an ability to lay eggs in any opining

region within the palm tree, all injuries to palms must be treated immediately with an

insecticide (Alhudaib, 2009). Wounds should be quickly covered to stop the release

of kairomones, which attract the weevils.

Encourage ground cover:

Promoting higher levels of natural enemies and fewer pest problems can be

accomplished by Encourage ground covers around areas with high palm populations

(Murphy and Briscoe, 1999).

2.6.1.8 Chemical treatment:

After detection of RPW, insecticide should be used even in area that does not

show significantly of infestation. For these prophylactic treatments, spraying

should be done during times when weevils disperse using a foliar insecticide

(Faleiro, 2006).

All cuts and injuries of palms must be treated immediately with insecticide, so

insecticides that are currently used to control red palm weevil include:

Acephate (O, S-Dimethyl acetylphosphoramidothioate) (Beevi et al., 2004).

Azinphos-methyl (O, O-Dimethyl-S-4-oxo-1, 2, 3-benzotriazin-3(4 H)-

ylmethyl phosphorodithioate (Soroker et al., 2005).

Carbaryl (1-naphthyl n-methylcarbamate) (Murphy and Briscoe 1999).

Chlorpyriphos (diethyl O-(3, 5, 6-trichloro-2-pyridyl) phosphorothioate)

(Abraham et al., 2000).

18

Diazinon (O,O-Diethyl O-(2-isopropyl-6-methyl-4-pyrimidinyl

)Phosphorothioate) (Ferry and Gómez 2002).

Endosulfan(1,2,3,4,7,7-Hexachlorobicyclo(2.2.1)Hepten-5,6

Bioxymethylenesulfite) (Abraham et al., 2000).

Imidacloprid (1-((6-chloro-3-pyridinyl)methyl)-4,5-dihydro-Nnitro- 1H-

imidazol-2-amine) (Kaakeh, 2006).

Malathion (Dicarboethoxyethyl O,O-Dimethyl Phosphorodithioate) (El

Ezaby et al., 1998).

Methidathion (O,O-dimethyl-s-(2-methoxy-1,3,4-thiadiazol-5(4H)-onyl-(4)-

methyl)phosphorodithioate) (Ferry and Gómez, 2002).

Dimethoate (O,O-Dimethyl S-(N-Methylcarbamoylmethyl Dithiophosphate)

(Murphy and Briscoe, 1999).

Trichlorfon ((1-Hydroxy-2,2,2-trichloroethyl )phosphonic acid, dimethyl

ester) (Murphy and Briscoe, 1999).

Application of this pesticide should be repeated to avoid an increase in RPW

population density (Conti et al., 2008). Systemic insecticides such as imidacloprid

are favored over organophosphate and carbamate insecticides for control of the red

palm weevil (Kaakeh, 2006). Imidacloprid can be applied through soil-soak

irrigation and can be detected for up to 4 months in the foliage (Dembilio et al.,

2009).

Insecticide can be applied to control RPW by different ways and techniques:

Dust the leaf axils after clipping (Murphy and Briscoe, 1999).

Seal slow-release aluminum phosphide tablets inside the tree (Kaakeh, 2006).

Spray or soak the tree trunk (Giblin, 2001).

Inject directly into the trunk (Kaakeh, 2006).

Apply systemic insecticide through irrigation water (Kaakeh, 2006).

2.6.1.9 Mass trapping:

Mass trapping uses a mixture of materials including a trap, food material, and a

pheromone; these materials are available to all stage of insect. Adult weevils are the

most highly attracted to the combination of aggregation pheromone and the volatile

compound that excreted from infected palm trees, which can be natural or synthetic

(Soroker et al., 2005). Food bait should be used along with the synthetic pheromone

to maintain the overall efficiency of the trapping system (Faleiro and Satarkar, 2003).

The pheromone-food bait is often supplemented with pesticides to prevent weevils

from escaping. Mass trapping as a part of an integrated pest management program not

only reduces the pest population, but also assists in the detection of newly infested

trees (Soroker et al., 2005).

2.6.1.10 Biological treatment of RPW:

Biological pest control, by using of microorganisms to control pests is one alternate to

reverse the use of hazardous synthetic insecticides, Naturally occurring biological

19

control agents are known for high degree of host specificity because of their unique

ability to search the host.

The incorporation of bio-control agents is advantageous and led to a number of

benefits such as environmentally safe, cheap, safe to non-target organisms and self-

perpetuation. Either entomophagous arthropods (predators and parasitoids) or

entomopathogenic microorganisms (nematodes, bacteria, fungi and viruses). Few

studies have been conducted on the natural entomophagous enemies of R. ferrugineus

or other Rhynchophorus species. 12

The control of RPW by using environmental

friendly bio-control agents has become the most demanding research in many parts of

the world (El-Bakl, 2014).

Many researches and studies focusing only on the use of pathogens such as

entomopathogenic nematodes, bacteria and entomopathogenic fungi in controlling

RPW. Naturally occurring bio-control agents are alternative to reverse the use of

hazardous synthetic insecticides. Among these microorganisms, the use of

entomopathogenic fungi was found to be promising alternate for insect's control.

According to an estimate, more than 700 species of fungi belonging to different genera

are known to infect insects. In the past, the potential of entomopathogenic fungi

especially B. bassiana, M. anisopliae and Isaria fumosorosea have been valuated

against different pests including Aphis craccivora, Aedes aegypti, Bemisia argentifolii,

Coptotermes formosanus, Melanoplus sanguinipes, Ocinara varians Walker,

Odontotermes obesus, Periplaneta americana, R. ferrugineus, Scolytus scolytus,

Thrips tabaci (Dembilio, 2012).

2.7 Biological Control of RPW by B.bassiana & M. anisopliae

2.6.1 Importance of Beauveria bassiana:

B. bassiana is an ascomycotic filamentous fungus of order hypocreales and genus

Beauveria. A broad range of Beauveria species have been isolated from a variety of

insects worldwide that are of medical or agricultural significance. Beauvericin is a

famous mycotoxin produced by many fungi, such as B. bassiana, which used as an

insecticide, the spores are sprayed on affected crops as an emulsified suspension or

wettable powder or applied to mosquito nets as a mosquito control agent (El-Sufty,

2009).

Figure (2.4): Plate of B. bassiana. (Zambrano et l., 2013)

http://en.wikipedia.org/wiki/Mosquito_net

20

2.7.1.1 Previous Study for using B. bassiana as biocontrol agent against RPW

Resent study showed the successful control of red palm weevils mainly depends on

the host pathogen interactions. So, there is a constant struggle between host and

pathogen that ultimately lead to the success or failure of pathogens. In case of

compatible interaction, the pathogen must have high number of conidia with strong

adhesion that ultimately penetrate into the host through directly penetrating

structures. Moreover, the invading pathogen must have the capacity to bypass or

overcome the host immune system by producing toxins (Gindin, 2006).

Figure (2.5): Sporulation of B. bassiana on the cadaver of R. ferrugineus (Olivier)

(Hussain, 2013).

Another study showed the first report of the use of the local strain UAE-B2 of B.

bassiana against RPW in date palm plantations, and used this fungus in an integrated

biological control program for RPW in United Arab Emirates (El-Sufty, 2006).

Many research showed the B. bassiana used an effective method against eggs, larvae

and adults of RPW. Among these stages, adults withstand relatively more time (4-5

weeks) upon exposure with spore suspensions, while dried formulations impart 100%

mortality (Hussain, 2013).

Biological control agent from B. bassiana by direct injection with two rates of B.

bassiana spore suspension (0.5 x 107 or 1.5 x 10

7 spores/ml), was used in a

laboratory diet for the larvae of the RPW. The larval mortality during 14 days

achieved 80.3 %, under laboratory conditions (Arab, 2012).

More study appear the B. bassiana, killing the treated larvae relatively quickly and

the lethal time 50 (LT50: 3.5 days), in comparison with the B. bassiana strains that

began to affect the larvae only after 5.6 days (LT50: 5.8 - 6.5 days). Figure 2.6

showed the larvae killed by B. bassiana change color from pale-yellow to pink (here

appear darker than control) (Gindin, 2006).

21

Figure (2.6): Larvae killed by B. bassiana (Gindin, 2006).

Some studies show the potential of a strain of the entomopathogenic fungus B.

bassiana were evaluated in laboratory, semi-field and field assays, and resulted

highly efficient against RPW (El-Sufty, 2007).

In 2001, the study determine that of B. bassiana used against RPW during their

residence in the trap, each adult has high probability of receiving sufficient B. spp. to

cause death in approximately 4 days. There was also a significant probability of

horizontal transfer from infected individuals to other insects coming into contact with

treated individual, but away from the bait site (Deadman, 2001).

Recently (Güerri, 2010) showed that B. bassiana caused 70-85% R. ferrugineus

mortality. B. bassiana solid formulation with high RPW pathogenicity and

persistence could be applied as a preventive as well as curative treatment for RPW

control. Our B. bassiana formulation can be a significant component of an IPM

strategy for RPW control.

New Saudi Arabia isolates of the entomophathogenic fungi B. bassiana (BSA 3

Saudi isolate) showed some result in a field experiment, B.bassiana fungi codacide

oil suspension was sprayed at a concentration of 5x108 conidia/ ml on infested date

palm trees. One month following the first spraying in December the number of

weevils was reduced from 16.5 to 6 weevils/ trap giving a reduction of 63.64%.

(Hegazy, 2009).

2.7.2 Importance of Metarhizium anisopliae

M. anisopliae, a formerly known as Entomophthor anisopliae (basionym), is

a fungus that grows naturally in soils throughout the world and causes disease in

various insects by acting as a parasitoid, and is the most intensively studied species

of the genus Metarhizium. The reproductive structures of M. anisopliae (the

anamorph, the most commonly encountered form) comprise conidiophores and

conidia. Leveduriform structures or blastospores and appressoria are produced by M.

anisopliae through mycelial differentiation. The disease caused by the fungus is

sometimes called green muscardine disease because of the green colour of its spores.

When these mitotic (asexual) spores (called conidia) of the fungus come into contact

with the body of an insect host, they germinate and the hyphae that emerge penetrate

the cuticle. The fungus then develops inside the body eventually killing the insect

after a few days; this lethal effect is very likely aided by the production of

insecticidal cyclic peptides (destruxins) (Tiago, 2014).

http://en.wikipedia.org/wiki/Basionym

http://en.wikipedia.org/wiki/Fungus

http://en.wikipedia.org/wiki/Insect

http://en.wikipedia.org/wiki/Parasitoid

http://en.wikipedia.org/wiki/Spore

http://en.wikipedia.org/wiki/Conidium

http://en.wikipedia.org/wiki/Hypha

http://en.wikipedia.org/wiki/Cuticle

http://en.wikipedia.org/wiki/Peptide

22

Figure (2.7): M. anisopliae culture and spores.

(http://www.naro.affrc.go.jp/org/fruit/epfdb/Deutte/Metarh/plate_M.htm)

(http://www.forestryimages.org/browse/detail.cfm?imgnum=1276025)

2.7.2.1 Previous Study for using M. anisopliae as biocontrol agent against RPW

Previous study showed the M. anisopliae (M.08/I05), which isolated from Italy, was

appeared to be an indigenous virulent strain which provided an effective control

against RPW and its efficacy could be supported and/or enhanced by suitable insect

host treatment. (Francardi, 2012).

Another study appear the effective of M. anisopiae after tested on RPW eggs and

adults, incubation in a substrate treated with M. anisopliae spores increased egg

mortality their hatchability and the total percentage mortality of eggs and hatched

resulted by 80-82%, compared with 34% in the control (Gindin, 2006).

In order to evaluate the biocontrol agent potential of M. anisopliae and B. bassiana

against RPW, (El-Bakl, 2014) showed that these fungi can be effective against eggs

and larvae of RPW. Among these stages, adults withstand relatively more time (4-5

weeks) upon exposure with spore suspensions, while dried formulations impart 100%

mortality within 2-3 weeks. Their results further suggested that M. anisopliae is more

effective in causing 100% mortality of the larvae between 6 and 7 days.

A new study show that all of the screened M. anisopliae strains exhibited

pathogenicity to all development stages of RPW, causing up to 80–100% mortality of

larvae and adult weevils under laboratory conditions. When eggs were exposed to

sawdust previously sprayed with M. anisopliae spores, the total survival of both the

eggs and the hatched larvae was reduced by a factor of approximately two to three,

relative to control (Sabbour, 2014).

http://www.naro.affrc.go.jp/org/fruit/epfdb/Deutte/Metarh/plate_M.htm

http://www.forestryimages.org/browse/detail.cfm?imgnum=1276025

23

Figure (2.8): Dead of Larvae and adult from R. ferrugineus by sporulation

of M. anisopliae (Gindin, 2006).

2.8 Molecular identification of Entomopathogenic Fungi:

When fulfillment biological control using EPF, it is very significant to have an

efficient system of identification to identify the applied fungal isolates (Coates et al.,

2002a). The identification is necessary for measuring their efficacy and detecting

multiple isolate infections in the host.

The morphology-based identification has been shown a non-reliable method in some

cases. For example, (Glare et al., 1996) found that the morphology could vary

depending on the growth medium. Recent evidence suggests that morphological

features are often vague and that the genera contain invisible species, both in

Beauveria (Rehner, 2005) and Metarhizium (Bischoff et al., 2006). So,

morphological and molecular studies have shown that the broad patterns of variety in

Beauveria and Metarhizium have been accurately predicted in previous

morphological studies. However, DNA-based techniques for identifying species and

varieties are accurate and widely used (Driver et al., 2000; Entz et al., 2005).

Several types of molecular techniques have been used to study genetic diversity have

been proposed to assess genetic variability as a complementary strategy to more

traditional approaches in genetic resources management. A number of markers, such

as RFLPs, RAPDs, AFLPs, DNA barcoding, and microsatellites, are now available

to detect polymorphisms in nuclear DNA. These different techniques have been

successfully used for genotyping approaches. They show differences in their ability

to reveal polymorphism between related individuals of the same species.

The nuclear-encoded ribosomal RNA genes (rDNA) of fungi exist as a multiple-copy

gene family comprised of highly similar DNA sequences (typically from 8-12 kb

each). ITS region is perhaps the most widely sequenced DNA region in fungi.

Comparisons of rDNA nucleotide sequence provide a tool for analyzing phylogeny

over high and low taxonomic levels. The rDNA consists of highly conserved regions

interspersed with variable regions, making it an ideal candidate for molecular

evolutionary studies (White et al., 1990). The ITS region evolves fast and may vary

among species within a genus or among populations (Jorgensen and Cluster, 1988;

24

Gardes et al., 1991). This region was found to be highly variable at the intra species

as detected by (Hirata and Takamatsu, 1996).

These probes should be useful in taxonomic, ecological, and population-level studies.

Nuclear ribosomal DNA sequences, especially the two internal transcribed spacers

(ITS1 and ITS2), have demonstrated differential rates of nucleotide changes that

allow intra-specific comparison in fungi (Neuvéglise et al., 1994; Buscot et al.,

1996).

The β-tubulin gene codes for tubulins that constitute the structural components of

microtubules. Microtubules have straight, hollow tubes structure. Their wall consists

of 13 columns of globular tubulin molecules. Each tubulin molecule consists of two

similar polypeptide subunits, α-tubulin and β-tubulin. Microtubules which provide

the molecular basis for chromosome segregation, cell division, generation and

maintenance of cell shape, intracellular transport, and cell motility by flagellar-ciliar

movement. Tubulins are amongst the most highly conserved eukaryotic proteins

(Wade, 2007).

The genes for tubulins, especially for β-tubulin, are receiving growing attention in

the investigation of evolutionary relationships at all levels: (i) in kingdom level

phylogenetic analyses (Keeling & Doolittle 1996, Baldauf et al., 2000), and (ii) in

studies of complex species groups within protists, animals, fungi and plants (Mages

et al., 1995, Keeling et al., 1998, Schutze et al., 1999, Ayliffe et al., 2001, Edgcomb

et al., 2001). The modulation of organisms in phylogenetic analyses depends on the

reliable amplification of β-tubulin genes.

25

Chapter 3

Materials and Methods

26

Chapter 3

Materials and Methods

3.1 Materials

3.1.1 Chemicals and Reagents

Chemicals, cultures medium and reagents used in this study are shown in Table 3.1

Table (3.1): Chemicals, reagents and cultures mediums that were used in

this work

# Reagents & Cultures Media Manufacture Country

1 PDA media HiMedia India

2 SDA media HiMedia India

3 PDB media HiMedia India

4 Choloromphenicol tablets HiMedia India

5 Tween 20 Sigma-Aldrich USA

6 Yeast Extract HiMedia India

7 Peptone HiMedia India

9 SDAY media HiMedia India

11 Crystal Violet HiMedia India

12 Methylene Blue HiMedia India

13 Qiagen DNA Extraction Kit Qiagen Germany

14 Primers Invitrogen, USA

15 PCR Master Mix 2X Kit Invitrogen, USA

16 DNA Ladder 200bp Invitrogen, USA

3.1.2 Equipments

The major equipments that will be used are listed in Table 3.2.

Table (3.2): Major Equipments used in the present study.

Instrument Manufacturer Country

Centrifugation DRE USA

Shaker Grant UK

Incubator IKS International USA

Microscopic Olympus Japan

PCR biometra Germany

Gel Electrophoresis biometra Germany

27

3.2 Methodology:

3.2.1 Isolation of B. bassiana & M. aneosiplaia fungi.

B. bassiana was isolated from dead larvae of red palm weevil from South of

Gaza strip. The small larval segment were externally sterilized in 100 % ethanol

for about one minute and allowed to air dry for another minute. Sterilized surface

segments were put into PDA medium in Petri-dishes (Arab & El-Deeb, 2012).

M. aneosipliae was isolated from soil. Soil sample was also collected from Gaza

strip. The sample was placed into plastic bags and stored at 4–8°C. (NouriAiin et

al., 2014).

3.2.2 Purification of fungi by using selective medium.

Selective medium is generally required for isolation of B. bassiana and M.

aneosipliae from soil.

DOC2 medium for B. bassiana, autoclaved and poured into 15 cm Petri

dishes (Shin et al., 2010).

Oatmeal agar medium (OMA) for M. aneosipliae, autoclaved and poured into

15 cm Petri dishes (Liu et al., 2015).

Soil sample (1g) from a corn field in Gaza strip was suspended in sterile

distilled water (200 ml) containing Tween 80 as surfactant.

Suspensions were applied at a concentration of 0.2 ml/plate and spread using

a glass rod and plates were incubated at 25°C in the total darkness.

3.2.3 Sub-culturing:

Increasing of quantity of B. bassiana and M. aneosipliae by using of Potato

Dextrose Agar (PDA) medium.

Incubation at 25°C in the total darkness.

3.2.4 Spore suspension:

Preparation of spore suspension from fungi in a liquid medium (PDB) media.

Liquid media (PDB) was used for production of spores required for

experiments.

Liquid mediums were autoclaved and inoculated with fungal spores

propagated on PDA.

Spores were harvested from 2 – 3 week old surface cultures by scraping and

used to inoculate the liquid medium in flasks.

The flasks were held on a shaker (110 rpm) for 5 days at 25˚C.

The suspensions were stirred and filtered through a single layer of linen to

remove culture debris and mycelia.

After this time the blast spore concentrations were determined using a

haemocytometer and were calibrated to 3.4×108 spores/ml for B. bassiana

and 3.6×108 spores/ml M. aneosipliae respectively.

These suspensions represented the primary stock suspensions to making the

spore product. (Gindin et al., 2006).

28

3.2.5 Morphological Identification of Fungal Isolates

Cultures were examined periodically and identified when they sporulated. The

cultures were separated into groups based on their morphological characteristics

including growth pattern, colony texture, pigmentation, and growth rate of the

colonies on PDA (Promputtha et al., 2005). When fungal colonies sporulated on

PDA, small plaques from the edge and the center of each growing colony were

transferred onto glass slides, and then were examined using a compound light

microscope, for characteristics of their vegetative and reproductive structures such as

hyphal color and structures, shape and size of conidia and conidiophores (Yu, 2010).

3.2.6 Molecular Identification of fungi

3.2.6.1 DNA extraction

Fungal genomic DNA was extracted from the hyphae using a partially modified

chemical lysis method, Approximately 50 mg of crushed mycelium was used for

DNA extraction, and the rest of the sample was stored at –20 °C until needed. DNA

extraction was done using the DNeasy Plant Mini Kit (QIAGEN, Germanny) and the

Nucleo Spin Plant Kit (Clontech) according to the manufacturers’ recommendations.

The extracted DNA was stored at –20 °C until use as a template for PCR. (Shin et

al., 2010; Sevim & DEMİRBA, 2012).

3.2.6.2 Primer design and PCR conditions for M. anisopliae & B. bassiana:

The nuclear rDNA region spanning the ITS1, ITS2, 5.8S rRNA gene for isolated

fungi and SCAR fragment for B. bassiana only were amplified by polymerase chain

reaction (PCR) from two strains and all primers were presented in table 3.4

additionally to PCR conditions for each primer.

Table (3.4): The primers sequences used in ITS, β-tubulin and SCAR

analysis.

Primer

Name

Sequences 5'...3' Amplified

region

References

ITS1 5'-TCCGTAGGTGAACCTGCGG-3'

ITS1 (White et al.,

1990)

ITS2 5'-GCTGCGTTCTTCATCGATGC-3'

ITS3 5'-GCATCGATGAAGAACGCAGC-3' ITS2

ITS4 5'-TCCTCCGCTTATTGATATGC-3'

ITS5 5'-GGAAGTAAAAGTCGTAACAAGG-3' ITS1+5.8

S+ITS2 P3 5'-GCCGCTTCACTCGCCGTTAC-3' (Kusaba &

Tsuge, 1995) Bt2a 5'-GGTAACCAAATCGGTGCTGCTTTC-3'

β-tubulin

(Glass and

Donaldson,

1995) Bt2b

5'-ACCCTCAGTGTAGTGACCCTTGGC-3'

GHTqF1 5'-TTTTCATCGAAAGGTTGTTTCTCG-3' SCAR

(Castrillo et al.,

2008) GHTqR1 5'- CTGTGCTGGGTACTGACGTG-3'

29

In each amplification reaction, the final volume of 25 µl consisted of 3 µl of total

genomic DNA, 0.5 µl of each primer (forward and reverse), and 21 µl of ultra-pure

distilled water (Biological Industries).

Then, all components were added to AccuPower® PCR PreMix tube (Bioneer

Corporation - Hylabs). For each isolate, PCR amplification of ITS1, ITS2 and the

whole region of ITS (ITS1+5.8S+ITS2) were performed in a thermocycler

(Biometra, Germany) with the following conditions (Hirata and Takamatsu, 1996):

an initial denaturing step at 95◦C for 2 min; thermocycling for 30 cycles, where each

cycle consisted of 30 s at 95◦C followed by 30 s at 52◦C for annealing, and 30 s at

72◦C for extension, and a final extension cycle of 7 min at 72◦C.

For each isolate, PCR amplification of β-tubulin gene was performed in a

thermocycler with the following conditions successfully used by (Devi et al., 2006):

an initial denaturing step at 94◦C for 3 min; thermocycling for 35 cycles, where each

cycle consisted of 1 min at 94◦C followed by 1 min at 57◦C for annealing, and 2 min

at 72◦C for extension and a final extension period of 5 min at 72◦C.

For each isolate, PCR amplification of SCAR gene was performed in a thermocycler

with the following conditions successfully used by (Castrillo et al., 2008): for initial

denaturation at 94 0C for 4 min; 30 cycles of denaturation at 94

0C for 1 min,

annealing at 55 0C for 1 min; and extension at 72

0C for 1 min.

3.2.7 Laboratory Rearing

RPW were reared to provide samples for the research which required large numbers

of RPW of various stages. The rearing process was carried out in a rearing room with

the mean of 30 ± 2ºC and 60-80% RH. The photoperiod was approximately 12:12

L:D (AL-Ayedh, 2011). The room was also used for handling and preparing food for

the samples as well as storage room for stems of sugarcane. Equipment and materials

required for rearing on sugarcane are as follow: machete, medium-sized and large

rectangular plastic container, glass petri dish, forceps, test tube racks, and water

spray bottle. (Fiaboe et al., 2012)

3.2.8 Bioassay (Contact application of fungi):

Application on the development stages of R. ferrugineus.

Divided all insects into 4 groups (Control sample, insect's treatment with

pesticide, third group from insect's treatment with biological control agent

from B. bassiana and the last group form insect's treatment with biological

control agent from M. aneosipliae) and data examined after 28 days to adults

and 6 days for larvae from incubation.

Treatment the male adult to evaluate if the male infection the female by fungi

or no, so divided all insects into 4 groups (Control sample, insect's treatment

with pesticide, third group from insect's treatment with biological control

30

agent from B. bassiana and the last group form insect's treatment with

biological control agent from M. aneosipliae) and data examined after 28

days from incubation.

Spraying of spore's suspension for isolated fungi by using hand sprayer.

3.2.9 Data Collection and Statistical analysis.

The data was subjected to statistical analysis, virulence was expressed by cumulative

mortality (%), treatment efficacy (Abbott’s formula) (ABBOTT, 1925), within 28

days after treatment. The bar chart tested by using SPSS Sttatistics 17.0 (SPSS Inc.

2009).

31

Chapter 4

Results and Discussion

32

Chapter 4

Results and Discussion

4.1 Isolation of B. bassiana & M. aneosipliae fungi:

B. bassiana was isolated in current study, and presented in figure 4.1, but M.

anesipliae found in the soil. Were done and these samples shown in Figure 4.2

Figure (4.1): B.bassiana was covered adult & larvea of RPW

Figure (4.2): Soil samples that collected for B. bassiana & M.

anesipliae isolation.

Filamentous fungal isolates from different positions in Gaza strip were screened on

different media on different Petri dishes. All of these strains were purified and

transferred to PDA for further identification. As a result, all isolates were identified as

B. bassiana & M. anisopliae.

33

4.2 Morphological Characterization & Microscopic Examination for B. bassiana & M.

aneosipliae fungi:

According to the macroscopic examination for M. aneosipliae, we found two distinct

strains based on the differences in colony morphology. After four days of incubation,

the colonies of all isolates on PDA were nearly completely covered with mycelium,

but the colonies of this fungus grow rather slowly on PDA and sometimes are a pale

luteous to citrine in the centre with yellow pigment diffusing into the medium. After

10 days of incubation, the culture produces a white mycelial margin with clumps of

more or less verticillate branching conidiophores. The colors vary from olivaceous

buff to cream color to dark green (Fig. 4.3). This is akin to the observations of (Bridge

et al., 1993). However, there were founded the conidial shape and size of the two

kinds of isolates: cylindrical with obtuse ends, and the conidial width (1.5 to 3 μm)

and length (4 to 8 μm), and this result presented in figure 4.4.

Figure (4.3): Culture of M. aneosipliae on OMA Selective Medium and

PDA Medium.

Figure (4.4): Microscopic examinations for M. anesipliae 100X.

34

The cultural characteristics of the suspected B. bassiana isolates were examined.

Generally, in culture, B. bassiana grows as a white mould. It produces many dry,

powdery conidia in distinctive white spore balls. Each spore ball is composed of a

cluster of conidiogenous cells (fig. 4.5). This result supported by (Elkichaoui et al.,

2016).

Figure (4.5): Culture of B. bassiana on DOC2 Selective Medium and PDA

Medium.

Microscopic characters observation of B. bassiana was shape, size, color and

thickness of hyphae, conidiophore, and conidium. Microscopic observation result

show that hyphae size about 1-2 μm which grouped on conidiogene cells with 3-6

μm in size. Hyphae then branched and formed conidiogene cells with bottle like

form, small neck, and branch long were up to more than 20 μm and 1 μm wide.

Fertile hyphae was found on branch, circular and normally thicken or swollen. While

mycelium which is hyphae aggregate of B. bassiana was white and insulated. This

result agree with that estimated by (Elkichaoui et al., 2016), and was examined in

figure 4.6.

Figure (4.6): Microscopic examinations for B. bassiana 40X & 100X.

35

4.3 Enrichment for two fungi and Spore Suspension:

Liquid medium Potato-dextrose- broth (PDB) was used for production of spores

required for experiments. Spores were harvested from 2 – 3 week old surface

cultures by scraping and used to inoculate the liquid medium in flasks (fig. 4.7).

After this time the blastospore is the lethal concentration for RPW. Based on

previous study for killing the RPW such as (beigi, & Port, 2013; Malik et al., 2016)

the concentrations were determined using a haemocytometer and were calibrated to

3.4×108 and 3.6×10

8 spores/ml for B. bassiana and M. anisopliae respectively. These

suspensions represented the primary stock suspensions of blastospore.

Figure (4.7): Spore suspension of fungi in PDB media. A: B. bassiana & B:

M. anisopliae.

4.4 Molecular Characterization for B. bassiana & M. anisopliae fungi.

4.4.1 PCR amplification of ITS

Molecular techniques are accurate and widely used for identifying species and

varieties. The PCR techniques have been used in the current study. The ITS1, ITS2

as well as the whole ITS region (ITS1 + 5.8S + ITS2) were successfully amplified

for two fungal species. There was a difference in fragment size of ITS1, ITS2 and

(ITS1+5.8S+ITS2) between two fungal species. For example, the length of the ITS1

region in B. bassiana was larger than that of the M. anisopliae (230bp and 215 bp,

respectively). Whereas, the ITS2 fragment in M. anisopliae was greater than that of

the B. bassiana (375bp compared to 360 bp). Our results were confirming by

(Unpublished Data). The fragment sizes of all ITS regions, as obtained by gel

electrophoresis, are shown in figure 4.8, 4.9, 4.10 and summerized in table 4.1. Cruz

A B

36

et al., 2006 indicated that the fragment size of ITS1 of B.bassiana was 570 bp. In

another study, the fragment size of ITS1-5.8S-ITS2 region was 481 bp for

B.bassiana and 540 bp for M.anisopliae.

Figure (4.8): Fragment sizes of the PCR-amplified ITS1 regions as

obtained by gel electrophoresis. In the peripheral of the photograph,

bands from a DNA ladder 200bp scale (M) are shown. L1: negative

control, L2: ITS1 gene for M. anisopliae 215 bp & L3: ITS1 gene for B.

bassiana 230 bp.

M 1 2 3

200 bp

37

Figure (4.9): Fragment sizes of the PCR-amplified ITS2 regions as



control, L2: ITS2 gene for B. bassiana 380 bp & L3: ITS2 gene for M.

anisopliae ranging between 360 bp to 1000 bp.

Figure (4.10): Fragment sizes of the PCR-amplified of whole ITS regions

as obtained by gel electrophoresis. In the peripheral of the photograph,


control, L2: ITS regions gene for B. bassiana 640 bp & L3: ITS regions

for M. anisopliae ranging between 630 bp.

M 1 2 3

M 1 2 3

640 bp

38

4.4.2 PCR amplification of β-tubulin

Part of β-tubulin gene was amplified successfully for tow fungal species. The size of

this part in B. bassiana isolate was found to be greater than the corresponding one in

M. anisopliae isolates (500 bp and 380 bp), respectively, figure 4.11. B-tubulin was

developed for sequencing purposes as described by (Bischoff et al., 2006).

Figure (4.11): Fragment sizes of the PCR-amplified of whole Bt regions as



control, L2: Bt gene for B. bassiana 500 bp & L3: Bt gene for M.

anisopliae ranging between 380 bp.

Standard PCR examines utilizing primers GHTqF1 and GHTqR1 against strain of B.

bassiana created a 96-bp, recouped from tainted RPW grown-ups taking after shower

application. The PCR item produced was of the anticipated length in view of the

SCAR part whereupon the preliminaries were based. The fragment sizes of SCAR

regions, as obtained by gel electrophoresis, were presented in figure 4.12.

M 1 2 3

39

Figure (4.12): Fragment sizes of the PCR-amplified of SCAR region as



control, L2 & L3: SCAR gene for B. bassiana 96 bp.

Table (4.1): Fragment sizes of the PCR-amplified of different genes for

isolated fungi. (bp).

Fungi ITS1 ITS2 ITS region β-tubulin SCAR

B. bassiana 230 380 640 500 96

M. ansopliae 215 360-1000 630 380 -

This study provides general information about the genetic diversity of

entomopathogenic fungi B. bassiana and M. anisopliae strains in the Gaza strip of

Palestine. Many of molecular markers were used as a modern technique to discussion

the genetic variability and to identify distinct isolates of M. anisopliae and B.

bassiana. Genetic materials based technique may allow distinguishing between

isolates that are very similar in morphology.

The evolution and using of PCR amplification from different rDNA regions has

greatly facilitated the fungi classification studies. Alignments and molecular analyses

confirmed the B. bassiana and M. anisopliae strains taxonomic identity.

However, since some conserved sites were found in the ITS regions, the conserved

sites of the ITS regions and the 5.8S rRNA gene were used for current analysis.

M 1 2 3

40

Investigation of ITS-rDNA sequences have been applied to determine the genetic

diversity of M. anisopliae and B. bassiana (Entz et al., 2005; Becerra et al., 2007;

Freed et al., 2011). Thus, (Bautista-Galvez et al. 2012), made the genetic

characterization of M. anisopliae strains obtaining fragments of 600 to 800 bp by

PCR amplification from the ITS1-ITS4 rDNA regions. Our ITS1 – 5.8S – ITS2

sequencing data showed variations which allowed us to design specific primers

which could not only detect and identify M. anisopliae but also to differentiate

between M. anisopliae and B. bassiana.

The ITS regions and 5.8S rDNA of Metarhizium were amplified using the ITS1 and

ITS2 primers that was a unique fragment of approximately 630 bp for Gaza isolate.

(Destéfano et al. 2004) analyzed at the same region with 540 bp fragment for M.

anisopliae var. anisopliae strain E9, B/Vi and C isolated in Brazil and 600 bp for M.

anisopliae strain 14 isolates in Australia.

The ITS and BT markers have the different level of informativeness in

discriminating Beauveria & Metarhizium isolates. While ITS regions was more

informative than BT in discriminating as ITS marker distinguished.

PCR assays of B. bassiana which isolated from adults of RPW with SCAR primers

resulted in DNA fragments of the same size as the B. bassiana GHA amplicons

which done by Castrillo et al., 2008 as expected based on primer design. For field

studies, the accuracy of detection from any samples may be improved by increasing

the number of subsamples taken and the number of PCR assays for different genes

per DNA extraction (Dionisi et al., 2003).

4.5 Laboratory Rearing for RPW.

Samples of male and female adults of RPW were collected from the infesting palm

trees in several selected areas in Gaza strip with the help of MOA. Samples were

adult weevils of RPW that provide the initial samples for rearing as show in figure

4.13. The insects of RPW were successfully reared in the laboratory on sugarcane

which is one of the natural food of the pest weevils. Resh and Carde, 2009 stated that

sugarcane is the best medium for the development of all larval stages to pupa in lab

conditions. The availability of sugarcane in Gaza strip also contributes to the

possibility of it to be used in the rearing process for RPW under lab conditions.

Sugarcanes also are more rot resistance, hence able to provide good quality of food

for much longer period compared to the young shoots of palms. In addition,

sugarcane was naturally contained with fiber which needed for RPW to make their

cocoon, and the sugarcane ensure adequate and timely accurate supply of fiber in the

final stage of RPW instar (Norzainih et al., 2015).

41

Figure (4.13): Rearing of the RPW under Lab Conditions.

The complete life cycle of the weevil, from egg to adult emergence, takes an average

120 days under lab conditions; result revealed is in line with the works of Ajlan,

2008. In contrast, (Prabhu and Patil, 2009) were recorded that the life cycle of RPW

takes about three months to complete. Different climate, food materials and other life

parameter between countries may cause the life cycle of RPW to slightly change.

Based on our result obtained, the egg takes about 2-5 days to hatch while the larva

takes about 80-90 days before molted to pupa. The pupal stage takes about 3 weeks

for the emergence of the adult weevils these results show in figure 4.14 and agree

with some study, which that estimated by (Kaakeh et al., 2001; Sharaby and Al-

Dhafar, 2013).

Figure (4.14): Stages of the Red Palm Weevil in rearing box. A: Eggs stage, B:

Cocoons collected from the sugarcane stems, C: Adualt stage of RPW & D: larva of

RPW.

C

A

D

B

42

4.6 Bioassay (Contact application of fungi):

4.6.1 Pathogenicity of entomopathogenic fungi to R. ferrugineus eggs:

The pathogenicity of the two most virulent isolates of M. anisopliae and B. bassiana,

selected in the initial screening on adult and larvea, was tested against R. ferrugineus

eggs. Both isolates killed all the eggs during 3 days, without preliminary colonization

on the egg surface. The characteristic symptoms which appeared on treated eggs, e.g.

loss of tumefy lethargy and darkening of the eggs, appeared 2-3 days after treatment;

subsequently the eggs were destroyed and disappeared in the substrate and as shown

in figure 4.15.

Figure (4.15): All eggs of RPW with & without treatment. A: Eggs killed

after treatment with B.bassiana and M.anisopliae. B: Eggs without

treatment.

4.6.2 Pathogenicity of entomopathogenic fungi to R. ferrugineus larvae:

To verify the effect of EPF on the growth of last-instar R. ferrugineus larvae, the

larval mortality was measured by Bottle equation. Significant difference in growth

were recorded between treated and untreated larvae, the toxicity assay on larvae were

treated with the M. anisopliae & B. bassiana isolate, which proved to be the most

virulent to the larvae. The mortality of larvae was recorded for 6 days after contact

with spraying (Hand Sprayer) for spore suspension which was presented in figure

4.16 & 4.17, which presented the larvae was treated by 3.4x108 spores/ml of

B.bassiana and 3.6x108 spores/ml of M. anisopliae.

A B

43

Figure (4.16): Larvae of RPW treated with 3.4x108 spores/ml of

B.bassiana.

Figure (4.17): Larvae of RPW treated with 3.6x108 spores/ml of M.

anisopliae.

The highest percentage mortality of the larvae reached 100% by 6 days after spraying

with B. bassiana, but 90% after spraying with M. anisopliae at the same time (Table

4.2).

Table (4.2): The larvae of RPW treated with EPF.

Groups Total

number

Mortality

number

Number of

alive

insects

% of morality

Larvae treated

with B.bassiana 30 30 0 100%

Larvae treated

with M. anisopliae 30 27 3 90%

Larvae treated

with chemical

pesticide

30 13 17 43.3%

RPW without

treatment 30 2 28 6.6%

44

This result supported by Lo Verde et al., 2015 which shows that the pathogenicity of

wild isolates of B. bassiana differs among the tested R. ferrugineus instars and the

mortality of treated larvae was 88 and 92% during 10 days. Similar differences were

found by Dembilio et al., 2009 and by Francardi et al., 2012 using isolates obtained

from pupae and adults of R. ferrugineus respectively, and by Ricano et al., 2013 who

tested multiple isolates of B. bassiana obtained from R. ferrugineus, another five

insect species and soil.

4.6.3 Pathogenicity of entomopathogenic fungi to R. ferrugineus Adult:

The bioassays on adults were treated with the M. anisopliae & B. bassiana isolate,

which proved highest percentage for mortality. Results of the first experiment

indicated that the mortality of R. ferrugineus adults differed according to the fungus

application method. The mortality of adult weevils was recorded for 28 day after

contact with spraying with spore suspension. The adults treated by 3.4x108 spores/ml

of B.bassiana and 3.6x108 spores/ml of M. anisopliae.

After repeating the experiment 3 times the maximum mortality of weevils reached

100% by 28 day after spraying with B. bassiana (Table 4.3), but 90% after spraying

with M. anisopliae at the same time (Table 4.4). Results of the persent work

supported by investigated the effect of M. anisopliae isolated from Italy on mortality

of RPW. He found that mortality is influenced the type of infected substratum. This

results indicated higher mortality (100%) after larcal infection, while mortality of

infected adults was 90% (Francardi, 2012). In this worke, mortality among control

group (aqueous D.W) was 10% (Figure 4.18), while mortility resulted from chemical

pesticide treatment reached up to 35% by 28 day.

Table (4.3): The mortality percentage for adults of RPW treated with

3.4x108 spores/ml of B.bassiana.

Groups Total

number

Mortality

number

Number

of alive

insects

% of

mortality

RPW treated with

B.bassiana 30 30 0 100%

RPW treated with

chemical pesticide 20 7 13 35%

RPW without

treatment 20 2 18 10%

45

Table (4.4): The mortality percentage for adults of RPW treated with

3.6x108 spores/ml of M. anisopliae.

Figure (4.18): RPW without treatment after 28 day.

Larvae showed a higher susceptibility than adults in terms of both mortality and

speed of infection, because the larval body does not have chitin as adults, and

because the isolated fungi needs a long time to analyse the chitin content of the

adults exsoskeleton. Adults killed by the fungus did not change color, whereas dead

adults in the control treatment darkened. After incubation of cadavers under moist

conditions, fungi emerged on the dorsal and ventral surfaces of the weevil and

formed conidiophores with conidia (Figure 4.19 & 4.20).

Groups Total

number

Mortality

number

Number

of alive

insects

% of mortality

RPW treated with

M.anisopliae 30 27 3 90%

RPW treated with

chemical pesticide 20 7 13 35%

RPW without

treatment 20 2 18 10%

46

Figure (4.19): RPW treated with 3.4x108 spores/ml of B.bassiana.

Figure (4.20): RPW treated with 3.6x108 spores/ml of M.anisopliae.

The current examination showed that the tested M. anisopliae and B. bassiana

isolates infect the adult and larvae are fully completed their life cycles by

forming conidiophores with conidia on RPW. B. bassiana and M. anisopliae are

entomopathogens fungi which are characterized by difference in virulence toward

different insect species. Fungal virulence is determined by different intrinsic

characteristics in the strains. (Hall & Papierok, 1982).

Good results in control of R. ferrugineus in lab and field test obtained from

indoginous strain of B. bassiana isolated from mycosed RPW collected from the

filed in Egypt (EL-Sufty et al., 2007; Sewify et al., 2009). Shawir & AL-Jabr,

2010, studying the infectivity of M. anisopliae and B. bassiana isolates against R.

ferrugineus, observed different mortality values according to the infecting

method, with higher mortality of both larvae and adults infected with fungal

spore suspensions (1x107 spores/ml) by injection than by the dipping technique.

In the treatment by injection, B. bassiana caused 80-85% larval and adult

mortality, while M. anisopliae caused 70% larval and adult mortality. In the

dipping method, the mortality values were lower: B. bassiana caused 60% larval

mortality and 40-55% adult (male-female) mortality, while M. anisopliae caused

60% larval and 35%-50% adult (male-female) mortality within 10 days after

treatment, but using the spraying spores suspension it the best method for killing

all stages of RPW because the injection and dipping method were difficult in

47

application under field conditions but the spraying spores of isolated fungi easy

in application under field conditions.

The arrival of entomopathogenic fungi to infest the host is through the cuticle is

considered successfully control of RPW, which that involves complex

biochemical interactions between the host and the pathogen (fungus) such as B.

bassiana & M. anisopliae before germination, penetration, growth, and

reproduction of the fungus (figure 4.21). Before to host invasion, there are certain

characteristics of fungi that designate them virulent or avirulent strains. So, there

is a constant struggle between host and pathogen that ultimately lead to the

success or failure of pathogens.

Figure (4.21): Sporulation of B. bassiana on the R. ferrugineus (Olivier).

Dissecting Microscopy.

In case of compatible interaction, the pathogen must have high number of conidia

with strong adhesion that ultimately penetrate into the host through directly

penetrating structures. Moreover, the invading pathogen must have the capacity

to bypass or overcome the host immune system by producing toxins (Hussain,

2013a).

4.6.4 Pathogenicity of entomopathogenic fungi to R. ferrugineus Adult male

as vector to transfer infections into female:

The results shown in Table 4.5 demonstrated that all entomopathogenic fungal

strains caused significantly increased mortality, which was investigated in the

laboratory for male of RPW contaminated with entomopathogenic fungal

conidia can transfer the inoculum to female during copulation. The results

appeared the male of RPW which contaminated by B. bassiana & M. anisopliae

as a vector of indirectly infected into female which were death after 28 day.

48

Table (4.5): Male of RPW contaminated with 3.4x108 spores/ml and

3.6x108 spores/ml of B.bassiana and M.anisopliae, respectively.

Groups Male

Number

Female

Number

Total

Number

Mortality

Number

Number of survival

insects % of

Mortality Male Female

Male of RPW

treated with

B.bassiana

4 6 10 9 1 0 90%

Male of RPW

treated with

M.anisopliae

4 6 10 9 0 1 90%

Male of RPW

treated with

chemical pesticide

4 6 10 2 2 6 20%

RPW without

treatment 10 10 20 2 9 9 10%

The mating behavior of the RPW resulted in transferring the spores from infected

male insects to the uninfected female insects. The pathogenic efficacy of B. bassiana

& M. anisopliae indirectly infecting females is illustrated in Figure 4.22 & 4.23. The

efficacy of indirect infection was high.

The period required to obtain the maximum mortality of 90% was extended to

compare the directly infected females or males, but the maximum mortality of 20%

for male adult was treated by chemical pesticide. However, the time to death of

indirectly infected females from mating with the infected male insects ranged from 3

to 6 day after mating. These results supported by Hajjar et al., 2015.

The pathogenic efficacy of the indirect infection was high, as all the indirectly

infected insects of both sexes were killed after 3 days of mating. The early deaths of

the directly infected insects could be due to the high load of B. bassiana spores on

the body as compared with indirectly infected RPW.

Recently Llacer et al., 2012b advanced the possibility of using sterile irradiated

males as a vector of B. bassiana for microbiological control of R. ferrugineus. In

laboratory bioassays, the transmission system successfully attracted, infected and

released weevil adults after they contacted cereal substrata inoculated with

indigenous strains of B. bassiana and M. anisopliae (Francardi, 2012).

49

Figure (4.22): RPW treatment of male with 3.4x108spores/ml of

B.bassiana and the mortality after 28 days of treating.

Figure (4.23): RPW treatment of male with 3.6x108spores/ml of M.

anisopliae and the mortality after 28 days of treating.

All our results showed in above suggest that the entomopathogenic fungi (EPF)

against R. ferrugineus can also provide an excellent alternative to chemical control.

The entomopathogenicity of B. bassiana and M. anisopliae strains obtained from

different sources. Figure 4.24 as a bar chart and it shows the mortality percentage for

using the EPF as biological control agent against the larvae of RPW.

50

Figure (4.24): Mortality percentage for groups of the larvae of RPW after


8 sopres/ml of B.

bassiana, chemical pesticide & negative control.

All of the screened EPF strains exhibited pathogenicity to larvae stages of RPW,

causing up to 90–100% mortality of larvae weevils under laboratory conditions. The

fungal spores had a significantly reduced survival in comparison with larvae which

emerged in the control treatment. In this study the effects of B. bassiana treatments

on activity and survival of larvae correlate well with previous studies conducted

outside of hidden environments (Gindin et al. 2006; Nussenbaum and Lecuona,

2012).

In such studies, live spores or conidia germinated when they touch the insect cuticle.

After germination, the fungus penetrated into the insect cuticle and expanded within

the body of its host which, once infected, reduced its feeding and movement

activities. High doses of fungal treatments such as the 6.3 X 107 to 3.0 X10

9

conidia/ml treatments in Dembilio et al. 2011 have been shown to cause 100% larval

mortality in R. ferrugineus within 6 – 7 days and these studies supported our results.

In other side the bioassays, significantly higher mortalities of RPW adult were

observed at use of B. bassiana as biocontrol agent against the R. ferrugineus. After

spraying RPW adult with spore suspensions (3.4x108 ml-1) of B. bassiana isolates

from dead adult, but in the second term the M. anisopliae caused the death for RPW

and the mortality percentage 86.6% after treatment the R. ferrugineus adult with

spore suspensions (3.6x108 ml-1). While the lower mortalities of RPW adult were

observed at use of chemical pesticide at the same time and was presented in figure

4.25

51

Figure (4.25): Mortality percentage for groups of the adult of RPW after


8 sopres/ml of B.

bassiana, chemical pesticide & negative control.

The host can be infected both by direct treatment and by horizontal transmission

from infected insects or cadavers to healthy insects. The infection of

entomopathogenic fungi can be disseminated into the healthy population through the

post-mortal sporulation and mycosis in the dead ones (Riasat et al., 2001). In the

current bioassays, maximum number of sporulation and mycosis in cadavers of R.

ferrugineus was recorded at the highest and sole concentration of B. bassiana & M.

anisopliae as compared to all other treatments. Similar trend in mycosis and

sporulation were reported by Ramakrishnan, 1999; Riasat et al., 2001). Figure 4.26

shows the mortality percentage for RPW.

52

Figure (4.26): Mortality percentage for R. ferrugineus. After treatment

the Male adult of RPW with 3.6X108 spores/ml of M. anisopliae, 3.4 X 10

8

sopres/ml of B. bassiana & chemical pesticide.

Efficacies up to 90% were obtained compare with chemical pesticide 35%, and these

results are indicative that contact infection of adults actually occurred and confirm

the potential of this strain as a biological control agent against R. ferrugineus.

Consequently, adults should be considered as the targets of any treatment involving

this entomopathogenic fungus because they are actually the only free-living stage.

This information is of great importance to continue in such projects. Given that

partial replacement of chemical control by biological is becoming increasingly

interesting to the crop protection industry, especially as regulations on the use of

chemical control become ever more stringent while a dynamic growth of the share in

global pesticide market holds promise for continuous growth of use of biocontrol in

the nearest future and in light of the high selectivity of biocontrol agents, long-term

reproduction and persistence of BCAs in the environment, public acceptance for

fulfilling special demands of consumers (e.g. organic food )and environmental

protection (e.g. drinking water supply ), we will take up this work for the first time in

Gaza strip to develop an effective and reliable biological based formulation to

control RPW as a step forward supporting the development in agro-industry

investment in our region.

53

Chapter 5

Conclusions and

Recommendations

54

Chapter 5

Conclusions and Recommendations

5.1 Conclusion

The study conducted has yielded some conclusions based on the findings that were

summarized in the previous section. It is now possible to derive several conclusions

based on the objectives presented in the first chapter. These conclusions are the

following:

This work fall within general project that aims to solve some of

environmental and health problems by reducing the use of chemical

fertilizers, pesticides and drugs and replace them by natural material or

organisms. (Biocontrol).

The current study presented that B. bassiana & M. anisopliae locally isolates

used in laboratory bioassays caused high mortality in larvae and adult and can

be an alternative to chemical pesticide.

Natural infections by B. bassiana and other entomopathogenic fungi found in

the present study were very high, considering the instar. For these reasons,

the use of entomopathogenic fungi can be considered to be useful as a

preventive and control tool in Gaza strip palm protection.

Moreover, the high mortality of treated adults suggests that their use as

vectors of B. bassiana & M. anisopliae can represent a potential tool for

reducing R. ferrugineus populations in Gaza strip.

5.2 Recommendations for improving this study:

The following recommendations are offered as possible ways to improve this study.

Further studies are needed in order to define the nature of inhibitory

substance(s), which are produced by B. bassiana & M. anisopliae against R.

ferrugineus.

It is recommended to use B. bassiana & M. anisopliae in the control of RPW

in agriculture, and this will help reduce the use of chemical fungicides, and

therefore reduce its risks on the environment and health .

55

It is required to improve & develope a bio-fungicide from native isolates of

B. bassiana and M. anisopliae against the red palm weevil, the major wilt

pathogen in Palestine, espacillaly Gaza strip.

Based on previous studies, the safety of entomopathogenic fungi for humans,

and the environment and non- target organisms are clearly an important

criterion for consideration and each insect–fungus system must again be

considered on a case-by-case basis.

Increase awareness among farmers by MOA and Biotechnologist to

importance of these products and using bio-fungicide, which have highly

efficiency and do not have any negative effects on health, environment and

groundwater.

56

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Appendix 1: Protocol for DNA Extraction Kit

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Appendix 2: Formula for Fungi media

1. DOC2 media

3g peptone.

0.2g Cucl2.

2mg crystal Violet.

15 g agar.

0.5 g Chloramphenicol.

Suspend the ingredients in 1000ml cold distilled water. Heat to boiling to

dissolve completely. Dispence into appropriate containers and autoclave for 15

minutes at 121 0C. The medium should be pH 7.4 at room temperature.

2. OMA (Oatmeal Agar) media

60g Oat Meal.

12.5g Agar.

Suspend the ingredients in 1000ml cold distilled water. Heat to boiling to

dissolve completely. Dispence into appropriate containers and autoclave for 15

minutes at 121 0C. The medium should be pH 7.4 at room temperature.

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