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
APPROVAL SHEET
EFFICACY OF THE TWO FUNGI,
Beauveria bassiana (Balsamo)Vuillemin AND
Metarhizium anisopliae (Metschnikoff) Sorokin
ON SOME STORED GRAIN INSECTS
Ph.D. Thesis
In
Agric. Sci. (Economic Entomology)
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
APPROVAL COMMITTEE
Dr. FAWZY FAIEK SHALABY........................................... Professor of Economic Entomology, Fac. Agric., Benha University
Dr. EZZ EL-DEN ABD EL-SAMEA EL-SHAZLY…………. Professor of Economic Entomology, Fac. Agric., Cairo University
Dr. ESSAM ELDIN ABDELRAOUF EWEIS………………… Professor of Pesticides, Fac. Agric., Cairo University
Dr. HAMDY ABD ELSAMED ELSHABRAWY ……………. Professor of Economic Entomology, Fac. Agric., Cairo University
Date: 26 /12 / 2012
SUPERVISION SHEET
EFFICACY OF THE TWO FUNGI,
Beauveria bassiana (Balsamo) Vuillemin AND
Metarhizium anisopliae (Metschnikoff) Sorokin
ON SOME STORED GRAIN INSECTS
Ph.D. Thesis
In
Agric. Sci. (Economic Entomology)
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
SUPERVISION COMMITTEE
Dr. HAMDY ABD ELSAMED ELSHABRAWY Professor of Economic Entomology, Fac. Agric., Cairo University
Dr. ESSAM ELDIN ABDELRAOUF EWEIS Professor of Pesticides, Fac. Agric., Cairo University
Name of Candidate: Magda Hanna Naroz Hanna Degree: Ph.D.
Title of Thesis: Efficacy of the Two Fungi, Beauveria bassiana (Balsamo)
Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin on
Some Stored Grain Insects
Supervisors: Dr. Hamdy Abdel Samed Elshabrawy
Dr. Essam Eldin Abdelraouf Eweis
Department: Economic Entomology and Pesticides
Branch: Economic Entomology Approval: 26 /12 /2012
ABSTRACT
The study aimed to evaluate the efficacy of Beauveria bassiana and Metarhizium
anisopliae against some stored product insects such as Callosobruchus maculatus,
Rhyzopertha dominica, Oryzaephilus suranmensis and Sitophilus oryzae. Also, safety
levels of the two fungi on albino rat males were demonstrated. The data recorded in this
investigation could be summarized as follows: By scanning electron microscopy, there
were differences between numbers of attached and germinating conidia of the two fungi on
insect bodies; the attachment and germination of B. bassiana and M. anisopliae spores
were the highest on C. maculatus. Bioassay tests revealed that B. bassiana was more
virulent to C. maculatus than R. dominica, where it caused highest mortality at lowest
concentration in shortest time. However, M. anisopliae spores were more virulent to C.
maculatus followed by S. oryzae. Pathogenicity of two fungi under three temperatures
(20, 25 and 30±1°C) and relative humidity (40 and 70±5%R.H) on tested insects,
maximum mortality percentages of C. maculatus treated with B. bassiana and M.
anisopliae were 100% (25±1°C&70±5%R.H) and 100%(30±1°C&40±5% RH),
respectively, 8days after treatment. While maximum mortality percentages of R. dominica
treated with two fungi were 98% (25±1°C&40±5% R.H) and 76% (30±1°C&70±5% R.H),
respectively, 15days after treatment. High record of mortality percentages of S. oryzae
treated with two fungi were 80 and 95%, respectively, at 30±1°C & 40±5%, 15days after
treatment, also, high record mortality percentages of O. suranmensis treated with two
fungi were 100 and 88 %, respectively, at the same conditions. Influence of M. anisopliae
and B. bassiana combined with three mixtures (clay, talc powder and diatoma powder) on
tested insects. Statistical analysis showed that mortality percentages of C. maculatus were
non-significant difference between treatments with the fungi combined with three mixtures
and fungi alone. However, the mortality percentages of R. dominica, S. oryzae and O.
suranmensis showed significant differences between treatments with the fungi combined
with three mixtures and fungi alone. The study proved that two fungi combined with
diatoma powder gave high record mortality percentages of treated insects.
Safety levels of B. bassiana and M. anisopliae on albino rats, when tested five
concentrations of each fungus. The five concentrations of B. bassiana increased feeding of
rats that reflected on the body weight. However, the different concentrations of M.
anisopliae had no effect on the body weight in last two weeks in second month. Tested
animals with different concentrations of B. bassiana and M. anisopliae showed less effect
on brain weight for two months. Also, the liver weight of treated animals with different
concentrations of M. anisopliae was less effect than treated animals with B. bassiana. On
the other hand, the kidney weight of treated animals with different concentrations of M.
anisopliae was more effective than treated animals with B. bassiana. The experiment was
tested on biochemical effects of B. bassiana and M. anisopliae on albino rats. The study
proved that B. bassiana was more effective than M. anisopliae on total protein, total lipids,
G-6-P DH activity, acid phosphates activity and alkaline phosphates activity. Key words: Beauveria bassiana, Metarhizium anisopliae, Callosobruchus maculatus,
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.
viii
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
ix
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
x
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
xi
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.
(Coleoptera: Curculionidae), Lesser Grain Borer, Rhyzopertha
dominica F. (Coleoptera: Bostrichidae), Sawtoothed grain beetle,
Oryzaephilus suranmensis (L.) (Coleoptera: Silvanidae) and cowpea
beetle, Callosobruchus maculatus F. (Coleoptera: Bruchidae) are the
most destructive of stored grains and seeds in the tropics, as they were
regarded as cosmopolitan (Abd El Aziz, 2011). The use of pesticides
was one of means for preventing some loss during storage. However,
caused problems such as insecticide resistance, chemical residues in
foodstuffs, increasing cost and environmental pollution (Sullivan, 2002
and Estévez et al., 2008).
These conditions are necessary to find out safer alternative
control strategies such as the use of microbial control agents against
stored-product insect pests. Using fungal biocontrol agents and selected
2
insecticides can potentially reduce the use of chemical insecticides and
their subsequent side effects in agriculture. Beauveria bassiana and
Metarhizium anisopliae are naturally occurring entomopathogenic
fungi with a wide host range (Wakil and Ghazanfar, 2010). Several
studies have shown that B. bassiana and M. anisopliae are effective on
stored grain insects such as S. oryzae (L.), R. dominica (F.),
Acanthoscelides obtectus and C. maculatus Say (Sheeba et al., 2001;
Lord, 2001; Lord, 2005; Murad, et al., 2006 and Lord, 2007).
B. bassiana was isolated from different insect hosts in
Egypt (El-Safty et al., 1994). There are a variety of factors may
determine or influence the host range and specificity of fungal
pathogens. These include the fungal strain, the host's
physiological state, nutrition, defense mechanisms, cuticle and
epicuticular microorganisms (Wan, 2003). The fungi are mostly
non-toxic or infective to mammals, exposure to the public and
the environment will be minimal to non-existent (DeFrancesco,
2010). On basis of the present knowledge, M. anisopliae was
considered to be safe with minimal risks to vertebrates, humans
and the environment (Zimmermann, 2007a and b). On the other
hand, he evaluated risks related to human exposure to B.
bassiana. Like any micro-organism, B. bassiana has the
potential to act as an opportunistic pathogen, but B. bassiana
infections are extremely rare events. Therefore, the present
work aimed at the following:
3
1. Effect of entomopathogenic fungi, B. bassiana and M.
anisopliae on tested insects (Rhyzopertha dominica,
Sitophilus oryzae, Oryzaephilus suranmensis (L.) and
Callosobruchus maculatus) include:
a. Microscopic studies of B. bassiana and M. anisopliae spores
on all tested insects.
b. Bioassay studies on the efficacy of fungal species on tested
insects.
c. Pathogenicity of B. bassiana and M. anisopliae under
different temperatures and relative humidity on tested
insects.
d. Influence of B. bassiana and M. anisopliae combined with
three mixtures on tested insects.
2. Safety levels of entomopathogenic fungi, B. bassiana and M.
anisopliae on albino rat males for two months include:
a. Effect of different concentrations of two fungi on body
weight of albino rat males
b. Effect of different concentrations of two fungi on organs;
e.g., liver, brain and kidney weight of albino rat males.
c. Biochemical effects of entomopathogenic fungi, B. bassiana
and M. anisopliae spores on albino rat males.
xii
LIST OF TABLES
No. Title Page
1. Attachment of B. bassiana spores on different body regions of
maculatus, O. suranmensis, R. dominica and S. oryzae………… 61
2. Germination of B. bassiana spores on different body regions of C.
maculatus, O. suranmensis, R. dominica and S. oryzae……… 61
3. Attachment of M. anisopliae spores on different body regions of
C. maculatus, O. suranmensis, R. dominica and S. oryzae…………… 63
4. Germination of M. anisopliae spores on different body regions of
C. maculatus, O. suranmensis, R. dominica and S. oryzae………. 63
5. Germination percentages of B. bassiana and M. anisopliae spores
on bodies of different insects………………………………….…………….. 65
6. Cumulative mortality percentages of C. maculatus adults treated
with two fungi B. bassiana and M. anisopliae at different
concentrations…..……………………………….………………………………... 71
7. Mortality percentages of C. maculatus treated with two fungi, B.
bassiana and M. anisopliae at different concentrations…………….. 72
8. LC50, LC90 and LT50 (days)a
values of C. maculatus, R.
dominica, O. suranmensis and S. oryzae treated with two
fungi, B. bassiana and M. anisopliae…………………………… 73
9.
Cumulative mortality percentages of R. dominica treated with two
fungi, B. bassiana and M. anisopliae at different concentrations… 76
10. Mortality percentages of R. dominica treated with two fungi, B.
bassiana and M. anisopliae at different concentrations…….…….. 77
11. Cumulative mortality percentages of S. oryzae treated with two
fungi, B. bassiana and M. anisopliae at different concentrations 81
12. Mortality percentages of S. oryzae treated with two fungi, B.
bassiana and M. anisopliae at different concentrations……………… 82
13. Cumulative mortality percentages of O. suranmensis treated with
two fungi, B. bassiana and M. anisopliae at different
concentrations…..……………………………….………………………………... 86
14. Mortality percentages of O. suranmensis treated with two fungi,
B. bassiana and M. anisopliae at different concentrations……….… 88
xiii
No.
Title
Page
15
Cumulative mortality percentages of C. maculatus adults treated
with concentration 0.26 ×107 of B. bassiana and 0.25 ×10
7 of M.
anisopliae under different temperatures and relative humidity 93
16. Cumulative mortality percentages of R. dominica adults treated
with concentration 0.26×107of B. bassiana and 0.25 ×10
7 of M.
anisopliae under different temperatures and relative humidity… 96
17. Cumulative mortality percentages of S. oryzae adults treated with
concentration 0.26 ×107 of B. bassiana and 0.25 ×10
7 of M.
anisopliae under different temperatures and relative humidity 99
18. Cumulative mortality percentages of O. suranmensis adults
treated with concentration 0.26×107of B. bassiana and 0.25×10
7of
M. anisopliae under different temperatures and relative humidity 102
19. Cumulative mortality percentages of C. maculatus adults treated
with two fungi, B. bassiana and M. anisopliae combined with
three formulations …………………………………………………………….… 104
20 Co-toxicity pathogenicity factor (CF) of C. maculatus due
to B. bassiana and M. anisopliae combined with different
materials ……………………………………………………………….... 105
21 Cumulative mortality percentages of R. dominica adults treated
with two fungi, B. bassiana and M. anisopliae mixed with three
materials …..…………………………………………………………..………. 106
22 Co-toxicity pathogenicity factor (CF) of R. dominica adults
due to B. bassiana and M. anisopliae combined with
different materials ……………………………………………………… 107
23 Cumulative mortality percentages of S. oryzae adults treated with
two fungi, B. bassiana and M. anisopliae mixed with three
materials …………………………………………………………………………. 110
24 Co-toxicity pathogenicity factor (CF) of S. oryzae adults due to B.
bassiana and M. anisopliae combined with different materials 111
25 Cumulative mortality percentages of O. suranmensis adults
treated with two fungi, B. bassiana and M. anisopliae
combined with three materials. …………………………………………… 113
xiv
No.
Title
Page
26 Co-toxicity pathogenicity factor (CF) of O. suranmensis adults
due to B. bassiana and M. anisopliae combined with different
materials.. 114
27 Means of body weight (g) of treated albino rats with different
concentrations of B. bassiana for two months………………………… 116
28 Means of body weight (g) of treated albino rats with different
concentrations of M. anisoplia for two months………………..……… 118
29 Means of liver weight (g) of treated albino rats with different
concentrations of B. bassiana and M. anisopliae for two
months….…………………………………………………………………………. 119
30 Means of brain weight (g) of treated albino rats with different
concentrations of B. bassiana and M. anisopliae for two
months……………………………………………………………………………. 121
31 Means of kidney weight (g) of treated albino rats with different
concentrations of B. bassiana and M. anisopliae for two months 123
32 Effect of B. bassiana and M. anisopliae concentrations on total
protein concentration (g/dl) for two months………………..………….. 125
33 Effect of B. bassiana and M. anisopliae concentrations on total
lipids concentration (mg/dl) for two months……………………….. 127
34 Effect of B. bassiana and M. anisopliae concentrations on G-6-P
DH activity (the number of erythrocytes per ml blood) for two
months……………………………………………………………………….………. 128
35 Effect of B. bassiana and M. anisopliae concentrations on acid
phosphates activity (U/L) for two months…………………………. 130
36 Effect of B. bassiana and M. anisopliae concentrations on
alkaline phosphates activity (U/L) for two months………………… 131
35
MATERIALS AND METHODS
This study was carried out in the Biological Control
Laboratory at the Department of Economic Entomology and
Pesticides, Faculty of Agriculture, Cairo University.
1. Effect of entomopathogenic fungi, B. bassiana and M. anisopliae
on certain stored grain insects
a. Preparation for all experiments
1. Insects rearing
The tested stored-product insects in this study
included; the rice weevil, Sitophilus oryzae L. (Coleoptera:
Curculiondae), Lesser Grain Borer, Rhyzopertha dominica F.
(Coleoptera: Bostrichidae), Sawtoothed grain beetle,
Oryzaephilus suranmensis (L.) (Coleoptera: Silvanidae) and the
cowpea beetle, Callosobruchus maculatus F. (Coleoptera:
Bruchidae). All tested insect species (Fig. 3) were taken from
laboratory culture reared for several generations without any
exposure to pesticides. R. dominica, S. oryzae and O.
suranmensis were reared on the whole wheat grains (Fig. 4),
while C. maculatus F. was fed on blacked eye cowpea. Insect
cultures were maintained in glass jars (1 Lit. volume) covered
with muslin cloth. All insects were reared under laboratory
conditions of 25 + 5 ºC and 75 + 5% RH.
36
a. Adult of S. oryzae b. Adult of R. dominica
c. Adult of C. maculatus. d. Adult of O. suranmensis
Fig. 3 (a, b, c and d). Adults of S. oryzae, R. dominica, C.
maculatus and O. suranmensis
.
37
Fig. 4. The culture of R. dominica, S. oryzae and
O. suranmensis
2-Fungal inoculums
The used entomopathogenic fungi, B. bassiana and M.
anisopliae in the experiments were originally isolated from red
palm weevil, Rhynchophorus ferrugineus Olivier that were
obtained from the soil at Ismailia Governorate in Egypt by Dr.
Gamal H. Sewify (Dept. Econom. Entomol. and Pesticides, Fac.
Agric. Cairo Univ.). These fungi were grown on autoclaved
Sabouraud and dextrose yeast agar (SDYA), containing 1%
peptone, 0.2% yeast extract, 4%dextrose and 1.5% agar in
distilled water and incubated for two weeks under 26 ±1°C
(Figs. 5 and 6).