EFFICACY OF THE TWO FUNGI, Beauveria bassiana (Balsamo)Vuillemin AND Metarhizium anisopliae (Metschnikoff) Sorokin ON SOME STORED GRAIN INSECTS By MAGDA HANNA NAROZ HANNA B.Sc. (Economic Entomology), Fac. Agric., Cairo Univ., Egypt, 2000 M.Sc. (Economic Entomology), Fac. Agric., Cairo Univ., Egypt, 2006 THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY In AGRICULTURAL SCIENCES (ECONOMIC ENTOMOLOGY) Department of Economic Entomology and Pesticides Faculty of Agriculture Cairo University EGYPT 2012
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EFFICACY OF THE TWO FUNGI,
Beauveria bassiana (Balsamo)Vuillemin AND
Metarhizium anisopliae (Metschnikoff) Sorokin
ON SOME STORED GRAIN INSECTS
By
MAGDA HANNA NAROZ HANNA B.Sc. (Economic Entomology), Fac. Agric., Cairo Univ., Egypt, 2000
Rhyzopertha dominica, Sitophilus oryzae, Oryzaephilus suranmensis and albino rats.
v
ACKNOWLEDGMENT
Firstly, Ultimate thanks to Our GOD
I wish to express my deepest appreciation and thanks to Dr. Hamdy, A. El-Shabrawy, Professor of Economic Entomology, Department of Economic Entomology and Pesticides, Faculty of Agriculture, Cairo University, for supervision, careful guidance, valuable suggestions and reading the manuscript. Also, I express my thanks and deepest gratitude to Dr. Essam, E. A. Eweis, Professor of Pesticides, in the same Department, for his fruitful supervision, careful guidance continuous advice, reading the manuscript and constructive criticism which made this work possible.
Also, my greatest thanks to Dr. Gamal, E. Sewify, Professor of Biological control, in the same Department, for suggesting the research subject and providing the two fungi species.
Thanks are also due to Dr. Ibtisam A. Hemeida, Professor of Economic Entomology, in the same Department and Dr. Ramadan A. K. Salama, Professor of Economic Entomology, in the same Department, for their guidance for laboratory rearing of all tested insects.
Thanks are also due to Dr. Mohamed H. Belal, Professor of pesticides, in the same Department and Dr. Mansour M. Rabie Professor of Pesticides, in the same Department, for their gave laboratory of fungi.
Also thank goes to Dr. Mohamed A. Kandeel, Professor of Pesticides, in the same Department for his gave toxicology laboratory.
Also thank goes to Dr. Sobhy Abd EL- Shafi, Researcher Professor of Parasitology, NRC, for his help in the statistical analysis of data.
Finally, I would also like to express my deep sense of gratitude to my parents and husband for their kind help and encouragement during the period of these studies.
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CONTENTS
Title Page
INTRODUCTION 1
REVIEW OF LITERATURE 4
1. Effect of entomopathogenic fungi, B. bassiana and
M. anisopliae on some stored grain insects …….…
5
a. Mode of action of the entomopathogenic fungi…… 5
1. The life cycle of B. bassiana……………………………. 5
2. The infection process ……………………….……………. 6
a. Adhesion and germination of conidia…………….. 8
b. Infection structure formation …………….……………. 9
c. The cuticle penetration ……………………….…………… 9
d. Colonization of host tissue and fungal emergence… 10
b. Studies of entomopathogenic fungi on some stored
grain insect……………………………….………………..……….
11
1. Bioassay procedure………………………..…………………….. 11
2. Effect of entomopathogenic fungi on some stored
grain insects under different temperatures and relative
humidity ………………………..…………………………………..
17
3. Influence of entomopathogenic fungi combined with
the formulations on some stored grain insects…….
19
2. Safety levels of entomopathogenic fungi, B. bassiana and M.
anisopliae on albino rat males………………………………….…. 28
MATERIALS AND METHODS……………………………….. 35
1. Effect of entomopathogenic fungi, B. bassiana and M.
anisopliae on certain stored grain insects…………………
35
a. Preparation for all experiments……………….…………….. 35
1. Insects rearing…………………………………………..………… 35
2. Fungal inoculums………………………………………...……. 37
b. Technical of each experiment.………………………………..
43
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Title Page
1. Microscopic studies of B. bassiana and M.
anisopliae spores on all tested insects ………….……
43
a. Electron microscope (EM)……………………………….………. 43
1. Preparation for Scanning Electron Microscope (SEM) 43
2. Germination percentage of spores…….…………………. 44
b. Light microscope (LM)……………………………………….. 44
2. Bioassay procedure…………………………………….……………. 44
3. Pathogenicity of B. bassiana and M. anisopliae under
different temperatures and relative humidity on
tested insects……………………………………………………………
45
4. Influence of B. bassiana and M. anisopliae combined
with different materials on tested insects………………….
46
c. Statistical analysis……………………………………………………. 47
2. Safety levels of entomopathogenic fungi, B. bassiana
and M. anisopliae towards albino rat males for two
months……………………………………………………………………
47
a. Test Animal……………………………………………… 47
b. Entomopathogenic fungi…………………………………….. 48
c. Effect of B. bassiana and M. anisopliae on certain
enzymes activities of albino rats………………………………
48
1. Determination of protein – Biuret method 48
2. Determination of total lipids………………………… 49
3. Determination of glucose-6-phosphate dehydrogenase 51
4. Determination of acid phosphatase……………… 53
5. Determination of alkaline phosphatase …. 54
d. Statistical analysis………………………………………….. 56
RESULTS AND DISCUSSION………………………………. 57
1. Effect of entomopathogenic fungi, B. bassiana and M.
anisopliae on all tested insects………………………………
57
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Title Page
a. Investigation on tested insects infected with two fungi,
B. bassiana and M. anisopliae using microscopy………….
57
1. Electron microscopy (EM)…………………………………...…… 57
a. Investigation of Infected insects with B. bassiana …….. 57
b. Investigation of Infected insects with M. anisopliae…… 60
c. Germination rate of spores…………………………………….. 62
2. Light microscopy…………………..………………………………… 66
a. Growing of B. bassiana spores on tested insects…..…… 67
b. Growing of M. anisopliae spores on tested insects……… 67
b. Bioassay studies on the efficacy of fungal species on
tested insects.………………………..………………………..
70
1. Callosobruchus maculatus ………………….…………………. 70
2. Rhyzopertha dominica……………………….…………………. 75
3. Sitophilus oryzae……………………………………………………. 80
4. Oryzaephilus suranmensis…………………………………… 85
c. Pathogenicity of B. bassiana and M. anisopliae under
different temperatures and relative humidity on tested
insects………………………………………………….….……………….
92
1. C. maculatus…………………………..……………………………… 92
2. R. dominica…………………………….……………………………. 95
3. S. oryzae………………………………………………………………. 98
4. O. suranmensis……………………………………….…………. 101
d. Influence of the two fungi, B. bassiana and M.
anisopliae combined with three materials on tested
insects……………………………………….……………………....
103
1. C. maculatus……………………………………………….…………. 103
2. R. dominica……………………………………………………………. 106
3. S. oryzae………………………………………………………………. 109
4. O. suranmensis………………………………………………………. 112
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Title Page
2. Safety levels of entomopathogenic fungi, B.
bassiana and M. anisopliae on albino rats…………
115
a. Effect of B. bassiana concentrations on body weight 115
b. Effect of M. anisopliae concentrations on body
weight …………………………………………..……………….
117
c. Effect of the two fungi, B. bassiana and M.
anisopliae concentrations on different organs weight
of the body………………………………………..………………
119
1. Liver weight……………………………………..………………. 119
2. Brain weight……………………………………..………………. 121
3. Kidney weight……………………………………….……………. 122
d. Biochemical effects of the two fungi, B. bassiana and
M. anisopliae ……………………………………………………
124
1. Total protein……………………………………………………...… 124
2. Total lipids………………………………………………………. 126
3. G-6-P DH activity…………………………………………………. 128
4. Acid phosphates activity………………………………………… 129
5. Alkaline phosphates activity…………………………………… 131
SUMMARY……………………………………………………………….. 134
REFERENCES………………………………………………………… 150
ARABIC SUMMARY
INTRODUCTION
Stored grains are subject to attack by pests which may result in
damage to stored-products and subsequent economic losses (Talukder
et al., 2004 and Haq et al., 2005). It has been estimated that 10 – 25%
of the worldwide grain crop is lost each year during storage; much of
this is due to infestation. Losses caused by insects include not only the
direct feeding damage resulting in loss of weight, but also severely
reduced nutrients, lowering percentage of seeds germination, reducing
grade and lowering their marketing value due to accumulations of
wastes. The favorable climatic conditions and poor storage system in
Egypt favor growth and development of these pests, resulting in
considerable crop losses. The rice weevil, Sitophilus oryzae L.