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