EFFICACY OF THE TWO FUNGI, Beauveria bassiana … · EFFICACY OF THE TWO FUNGI, Beauveria bassiana (Balsamo)Vuillemin AND Metarhizium anisopliae (Metschnikoff) Sorokin ON SOME STORED

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


Beauveria bassiana (Balsamo)Vuillemin AND



Ph.D. Thesis

In

Agric. Sci. (Economic Entomology)

By



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


Beauveria bassiana (Balsamo) Vuillemin AND



Ph.D. Thesis

In

Agric. Sci. (Economic Entomology)

By



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


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


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).

38

a b

c d

e f

Fig. 5 (a, b, c, d, e and f). All inoculation stages of two fungi

39

B. bassiana

M. anisopliae

Fig. 6. Plates inoculated with two fungi B. bassiana and M. anisopliae

Documents

EFFICACY OF THE TWO FUNGI, Beauveria bassiana … · EFFICACY OF THE TWO FUNGI, Beauveria bassiana (Balsamo)Vuillemin AND Metarhizium anisopliae (Metschnikoff) Sorokin ON SOME STORED