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Ph.D Thesis
“ANALYSIS OF THE PATHOGENIC APPLICATIONS OF
ASPERGILLUS SPECIES AGAINST ACRIDID GRASSHOPPERS OF
AGRICULTURE IMPORTANCE IN SINDH”
THESIS SUBMITTED TOWARDS THE PARTIAL FULFILLMENT OF THE
REQUIREMENT OF THE UNIVERSITY OF SINDH, FOR THE AWARD OF
DOCTOR OF PHILOSOPHY DEGREE IN ZOOLOGY
SANTOSH KUMAR
Department of Zoology,
UNIVERSITY OF SINDH, JAMSHORO, SINDH-PAKISTAN
2016
In the Name of Allah
The Most Gracious
The Most Merciful
Who’s Help We Solicit
AALLLL PPRRAAIISSEESS TTOO AALLMMIIGGHHTTIILLYY AALLLLAAHH,, WWHHOO GGIIVVEE MMEE AANN OOPPPPOORRTTUUNNIITTYY
TTOO CCOONNTTRRIIBBUUTTEE AA LLIITTTTLLEE BBIITT IINN TTHHEE OOCCEEAANN OOFF SSCCIIEENNCCEE
CERTIFICATE
Certified that the research work embodied in this thesis entitled “ANALYSIS OF THE
PATHOGENIC APPLICATIONS OF ASPERGILLUS SPECIES AGAINST ACRIDID
GRASSHOPPERS OF AGRICULTURE IMPORTANCE IN SINDH” has been carried
out by Mr. Santosh Kumar (Research Scholar) under our joint supervision and guidance in
the Department of Zoology, University of Sindh, Jamshoro (Pakistan). The work is original
and suitable to submit for the award of Ph.D Degree in Zoology.
Supervisor,
Dr. Riffat Sultana,
Assistant Professor,
Department of Zoology,
University of Sindh,
Jamshoro.
Co-Supervisor,
Dr. Muhammad Saeed Wagan, Honorary Prof. & Ex-Chairman,
Department of Zoology,
University of Sindh,
Jamshoro.
Co-Supervisor,
Prof. Dr. Abdul Rassol Abbasi,
Department of Fresh Water Biology and Fisheries,
University of Sindh,
Jamshoro.
DEDICATED
“TO MY RESPECTABLE SUPERVISOR DR. RIFFAT SULTANA FROM WHOM
I HAVE LEARNT THE ART OF STRUGGLE”
CONTENTS
ACKNOWLEDGMENTS……....................................................................... vii
ABSTRACT ………………………………………………………………… ix
LIST OF TABLES ………………………………………………………….. xi
LIST OF FIGURES …………………………………………………………. xii
LIST OF PLATES …………………………………………………………... xiv
CHAPTER NO. 1 ………………………………………………………….. 1
INTRODUCTION …………………………………………………………. 1
1.1 Acridids as Pest ……………………………………………………………... 1
1.2 Pathogenicity of Insects …………………………………………………….. 1
1.3 Entomopathogenic Fungi (EPFs) ………………………………………...... 2
1.4 Integrated Pest Management (IPM) ………………………………………… 3
1.5 Brief Geographical Feature of Sindh ……………………………………….. 4
1.6 The objectives of this study were .........………………………...…................ 5
CHAPTER NO. 2 …………………………………………………………... 6
REVIEW OF LITERATURE …………………………………………….. 6
CHAPTER NO. 3 …………………………………………………………... 16
MATERIAL AND METHODS …………………………………………… 16
3.1 Insect’s Sampling ………..…………………………………………………... 16
3.2 Collection of Infected Samples ……………………………………………... 16
3.3 Incubation in Laboratory ……………………………………………………. 16
3.4 Fungal Isolation and Sporulation Test ……………………………………… 17
3.5 Identification of Fungal Isolates …………………………………………….. 17
3.6 Pathogenic Bioassay ………………………………………………………… 17
3.7 Formulation of Aspergillus conidia ………………………………………… 17
3.8 Bio-pesticides application ……..…….....…………………………………… 18
3.9 Observations under Scanning Electron Microscopy (EDS) ……………….. 18
3.10 Experimental procedure …………………………………………………….. 19
3.11 Statistical analysis …………………………………………………………... 19
CHAPTER NO. 4 ………………………………………………………….. 20
RESULTS …………………………………………………………………. 20
4.1 Pest status of Acrididae ……………………………………………………... 20
4.2 Key to the sub-families of Acrididae occurring in Sindh ………………….... 21
4.3 Prevalence of Acridids in field ……………………………………………… 21
4.4 Lethal infection of entomopathogenic fungi (EPFs) ………………………... 22
4.5 Isolation and association of entomopathogenic fungi (EPFs) ………………. 24
4.6 Food consumptions and faecal production of infected insects ……………… 25
4.7 Variation in conidium shape of entomopathogenic fungi ………………….... 27
4.8 Reproductive activities of H. oryzivorus after pathogenic treatment ……….. 28
4.9 Behavioural activity of insect after pathogenic treatment …………………... 29
4.10 Elements concentration under Scanning Electron Microscopy (SEM) in
various treated entomopathogenic fungi (EPFs)...............................................
30
CHAPTER NO. 5 …………………………………………………………... 81
DISCUSSION, CONCLUSION AND RECOMMENDATIONS …..…… 81
5.1 Discussion …………………………………..…………………………... 81
5.2 Conclusion ………………………………………………………..…..... 92
5.3 Recommendations ………………………………………………….... 97
REFERENCES ……………………………………………………….......... 99
Appendixes ……………………………………………........ 130
Map ………………………………………………………………….............. 138
C.V …………………………………………………………………............... 139
Publications………………………………………………………................... 144
ACKNOWLEDGMENTS
I owe great depth of gratitude to my worthy supervisor Dr. Riffat Sultana Assistant Professor,
Department of Zoology, University of Sindh, Jamshoro for her kind, loving, positive and
thoughtful criticism, friendly attitude, keen personal interest, sincere advice, vital instructions
and close supervision throughout the period of my study and research. I shall always regard
my supervisor in high esteem for shouldering a task which would have been impossible for
me to execute single handed. I wish to thanks my co-supervisor Prof. Dr. Muhammad Saeed
Wagan for his in species identification, literature search, and for commenting on the
manuscript. Thanks are also due to Prof. Dr. Abdul Rasool Abbasi my 2nd co-supervisor
Department of Fresh Water Biology and Fisheries for his kind encouragement and support
during entire period of my study.
I am highly thankful to the Worthy Vice Chancellor, Late Prof. Dr. Abida Tahirani for her
kind cooperation and physical facilities provided for the smooth running of my research.
Although, she is not with us but she has rendered invaluable educational services and played
core role for the promotion of higher education and research in campus for this she will live
in our hearts. I am also thankful to our honourable, Acting Vice Chancellor Prof. Dr.
Muhammad Siddique Kalhoro for his keen interest for the promotion of research culture in
the University Campus. I also extent my thanks to Prof. Dr. Akhtar Hussian Mughal, Dean,
Faculty of Natural Science, University of Sindh, Jamshoro for all necessary facilities.
I wish to express my grateful thanks to Prof. Dr. Tahir Rajput Ex. Dean, Faculty of Natural
Science, University of Sindh, Jamshoro for providing fungi literature and identification of
many fungi taxa and offered comments on the culturing of entomopathogenic fungi. I am
thankful to Prof. Dr. Nasreen Memon Chairperson, Department of Zoology, University of
Sindh, Jamshoro for departmental facilities. Profound thanks are also due to Dr. Muhammad
Irshad (Principal Scientific Officer) NARC Islamabad, for providing important references
regarding my work.
I am also thankful to Mr. Aqeel Bhutto, Lecturer, Institute of Biotechnology and Genetic
Engineering, University of Sindh, Jamshoro for accessing to his laboratory for rearing,
culturing and isolation of fungi media. I am extremely thankful to Mr. Muhammad Kashif
Samoon, Lecturer and Mr. Irfan Ahmed, Assistant Professor, Centre for Pure and Applied
Geology, University of Sindh, Jamshoro for helping me in operating Scanning Electron
Microscope (SEM), Telescope images and compiling the results through spectrum graphs and
tables.
I also offer my sincere thanks to all my friends especially Dr. Waheed Ali Panhwar, Assistant
Professor, Department of Entomology, Sindh Agriculture University, Tandojam who
provided me his charming and positive company during field’s trips and always extended the
helping hands in maintaining the atmosphere friendly and delightful. Beside this, I would be
very ignorant if I forgot to pay thanks to my all Entomology and Bio-Control Research Lab.
(EBCRL) fellows from whom gathering, I feel home atmosphere at laboratory.
I am also thankful to Dr. Javed Khokhar, Mr. Aftab Hussain Khaskali and Mr. Noor Ahmed
Junejo, Institute of Biotechnology and Genetic Engineering, University of Sindh, Jamshoro
for stabilizing temperature and relative humidity in the fungus culturing process which was
very crucial for my work. I am also thankful to all Faculty, Department of Zoology,
University of Sindh, Jamshoro but particular my thanks extend to Dr. Barkat Ali Bughio for
his care and encouragement. I am also thanks to non-teaching staff Mr. Muhammad Fazal and
Mr. Akhtar Hussian who always care my experiments during off-days of University.
Thanks are also due to my friends Mr. Ajeet Kumar Maheshwari, Mr. Murli Das Manglani,
Mr. Bhagwan Das Manglani and Mr. Darshan Lal Mandhwani for their endless cooperation
care and love during my stay at Hyderabad. I would be very ignorant if I forget to pay thanks
to my family members who allow spending their time for this study but affection and love for
my little daughter Tirsha Rani is uncountable, I miss her a lot during my study.
Finally, I am deeply thankful to all those who have always wished to see me glittering high
on the skies of success. May Allah Almighty bless them with great health and prosperous
long lives and be a source of constant prayers (Ameen).
Santosh Kumar
ABSTRACT
Aspergillus species registered world-wide for grasshoppers and locusts control. This practice
is currently under consideration as a potential alternative to chemical insecticides for
grasshopper control in Pakistan. Grasshoppers are major agricultural pests. They destroyed
the rice, sugarcane, wheat, maize and fodder crops in everywhere. For control of this pest
several pesticides of billion rupees are used indiscriminately in every year. On the other hand,
these chemicals are injurious and health hazardous effects on living creature and their
environment. So, there should be suitable, beneficial and cheap alternative of these poisonous
chemical. For this purpose the biological control is very important therefore, an attempt was
made to introduce pathogenic fungi, against the reduction of acridid population in Sindh.
During the present study, a total of 2520 specimens pertaining to 06 sub-families of
Acrididae were collected from different ecological zones of Sindh. The isolation percentage
of entomopathogenic fungi and their association with pest species of grasshopper has been
presented in comparative manner. Total No. of isolated percentage of Aspergillus niger was
reported 6.77% and 5.64% on Acrida exaltata and A. gigantea respectively, in sub-family
Acridinae, 6.21% in Acrotylus humbertianus of Oedipodinae and 5.08% Truxalis exmia
exmia contaminated with A. fumigatus and Oxya fuscovittata infected 5.08% with A. flavus.
The order of prevalence of grasshopper’s species varying in both selected region. Lethal
infection level of entomopathogenic fungi from lower Sindh, site-I indicates that significant
highest No. of sporulation was recorded for A. gigantea and A. exaltata i.e 71.42% and
68.42% respectively, while unknown fungal infection was 36.36% followed by 31.57% on
Phlaeoba tenebrosa and A. exaltata respectively.
It was observed that infestation ratio of entomopathogenic fungi vary species to species in
different localities. Beside this, observations taken under Scanning Electron Microscopy
(SEM) showed that there is significant difference in coloration and phialides pattern of three
Aspergillus species including two unknown fungi. SEM results regarding spectrum
acquisition indicate that normal weightage % of Oxygen (O2) was highest i.e 56.19%
followed by 42.60% for Carbon (C) and very least ratio i.e 1.21% for Sodium (Na) was
observed in A. niger. As far as, chemical composition of A. flavus is concerned the normal
weightage % for Carbon (C) was 52.33% followed by Oxygen (O2) i.e 46.84% opposing to
this, least percentage was calculated for Sodium (Na) 0.83%. In case of A. fumigatus the
greater normal weightage % was found for Oxygen (O2) i.e 54.61% followed by 43.92% for
Carbon (C).
Food consumption and faecal production by the insects treated with different formulation of
the Aspergillus species were analyzed under laboratory conditions. Three replicates i.e
A. flavus, A. fumigatus and A. niger excluding control. It seems that greater reduction in
faecal production was noticed after the treatment of oil formulation. Reduction in feeding of
the infected insects stages (N1 to N3) was started after treatment of 1st to 2nd day. Significant
reduction in faecal production was noted from 1st to 4th day after that all immature consists on
(N1 to N3) stages were died except few individuals. However, mortality of insects on day first
was noted significantly highest i.e [F0.48 = 84.65, P < 0.05] followed by [F0.35 = 61.96, P <
0.05] and [F0.27 = 48.00, P < 0.05] on day 4th and 2nd respectively. Beside this, it was
extremely low [F0.17 = 30.54, P < 0.05] on day 3rd. As for as developmental stage of (N4 to
N6) are concerned their faecal production was significantly reduce on 2nd day i.e [F0.18 =
32.29, P < 0.05]. The faecal production of Acridid (adults) when treated with conidial
concentration in H2O was found maximum on first day. Mortality of Acridid adults suggest
that maximum casualties were noted on day 7th i.e [F13.7 = 23.56, P < 0.05] followed by [F12.5
= 21.82, P < 0.05] on 6th day and minimum mortality i.e [F0.44 = 77.67, P < 0.05] was on day
1st followed by [F0.77 = 35.26, P < 0.05] on 3rd day.
The results showed that cumulative percent of faecal material of the treated insect with
various pathogenic fungi was significantly differ with control whereas other three treatments
having significant impact on the food consumption and feeding behaviour, even as the
mortality of Acridid when treated with H2O formulation, indicate that maximum mortality of
individual was record on day 8th i-e [F1.00 = 02.62, P < 0.05] whilst, it was significantly low
i.e [F0.06 = 11.34, P < 0.05] and [F0.02 = 04.36, P < 0.05] on day 1st and 2nd. However,
mortality of these adult individual were non-significant from day 3rd to 7th respectively.
During the present investigation, it was noted that insect pathogen unlike chemical
insecticides don’t have quick response on pest feeding but, after 2nd day insect gradually
reduce its feeding.
Sexual reproductive activities of Hieroglyphus oryzivorus were also affected by the infection
of Aspergillus. It was noticed that infected insects altered their thermoregulatory response
and showed very interesting behavioral changing that include: insect’s feeding stopped
completely, poor coordination, jerky movements, excessive grooming, loss of orientation,
confuse during mating, short mating , drop egg without searching oviposition site, ecdysis
process slow or complete stop, Behavioral fever (body temperature raised) and body fat
accumulation was also reduced.
Present study recommends that exploration and screening must be conducted to provide
additional pathogens for evaluation as potential biological control against grasshoppers and
locusts.
LIST OF TABLES
S.NO. CAPTION OF TABLES PAGE
NO.
1 Table. I. Showing the collected number of pest species from upper Sindh
during the year 2013-2015.
31
2 Table. II. Showing the collected number of pest species from lower Sindh
during the year 2013-2015.
32
3 Table. III. Showing the total No. of grasshopper’s species caught from three
sites of upper Sindh in the year 2013-2015.
33
4 Table. IV. Showing the total No. of grasshopper’s species caught from three
sites of lower Sindh in the year 2013-2015.
34
5 Table. V. Lethal infection level of entomopathogenic fungi in various species
of grasshoppers collected from three sites of upper Sindh in the year
2013-2015.
35
6 Table.VI. Lethal infection level of entomopathogenic fungi in various species
of grasshoppers collected from three sites of lower Sindh in the year
2013-2015.
38
7 Table.VII. Showing the insect along with their major and minor target
habitats.
41
8 Table. VIII. Showing the isolated percentage of entomopathogenic fungi and
their association with pest species of grasshoppers during the year 2014 from
Sindh.
42
9 Table. IX. Identification of entomopathogenic fungi isolated from acridid
population.
43
10 Table. X. Showing the list of ecological association between
entomopathogenic fungi and insects recorded in the year 2013-2015 from
three selected sites of Sindh province.
44
11 Table. XI. Showing the association between Aspergillus species (EPFs) and
pest species of grasshoppers recorded by earlier workers (Shah et al., 1994,
1998) and original data.
45
12 Table. XII. Sowing the faecal production of immature Acridid culture in
small jars under laboratory conditions (after treatment of Aspergillus oil
formulation).
46
13 Table. XIII. Sowing the faecal production of adult Acridid culture in small
jars under laboratory conditions (after treatment of Aspergillus oil
formulation).
47
14 Table. XIV. Showing the faecal production of Acridid (Nymphs) population
treated with conidial concentration in H2O cultured maintained in the large
cage.
48
15 Table. XV. Showing the faecal production of Acridid (Adults) population
treated with conidial concentration in H2O cultured maintained in the large
cage.
48
16 Table. XVI. Showing the mortality of Acridid (Nymphs) population cultured
in small jars under laboratory conditions (after treatment of Aspergillus oil
formation).
49
17 Table. XVII. Showing the mortality of Acridid (Adults) population cultured
in small jars under laboratory conditions (after treatment of Aspergillus oil
formation).
50
18 Table. XVIII. Showing the mortality of Acridid (Nymphs) population treated
with conidial concentration in H2O cultured maintained in the large cage.
51
19 Table. XIX. Showing the mortality of Acridid (Adults) population treated
with conidial concentration in H2O cultured maintained in the large cage.
51
20 Table. XX. Reproductive activities of healthy and unhealthy samples of
H. oryzivorus under laboratory conditions.
52
21 Table. XXI. Fecundity rate of healthy and unhealthy samples of H. oryzivorus
under laboratory conditions.
52
22 Table. XXII. Showing the spectrum acquisition under scanning electron
microscope (SEM) of Aspergillus niger.
53
23 Table. XXIII. Showing the spectrum acquisition under scanning electron
microscope (SEM) of Aspergillus flavus.
53
24 Table. XXIV. Showing the spectrum acquisition under scanning electron
microscope (SEM) of Aspergillus fumigatus.
53
25 Table. XXV. Showing the spectrum acquisition under scanning electron
microscope (SEM) of unknown Fungi I.
54
26 Table. XXVI. Showing the spectrum acquisition under scanning electron
microscope (SEM) of unknown Fungi II.
54
LIST OF FIGURES
S.NO. CAPTION OF FIGURES PAGE
NO.
1 Fig. I. Showing the collected number of pest species from upper Sindh during
the year 2013-2015.
55
2 Fig. II. Showing the collected number of pest species from lower Sindh
during the year 2013-2015.
56
3 Fig. III. Showing the total No. of grasshopper’s species caught from three
sites of upper Sindh in the year 2013-2015.
57
4 Fig. IV. Showing the total No. of grasshopper’s species caught from three
sites of lower Sindh in the year 2013-2015.
58
5 Fig. V. Showing the lethal infection level of entomopathogenic fungi in
various species of grasshoppers collected from site-I of upper Sindh in the
year 2013-2015.
59
6 Fig.VI. Showing the lethal infection level of entomopathogenic fungi in
various species of grasshoppers collected from site-II of upper Sindh in the
year 2013-2015.
60
7 Fig. VII. Showing the lethal infection level of entomopathogenic fungi in
various species of grasshoppers collected from site-III of upper Sindh in the
year 2013-2015.
61
8 Fig. VIII. Showing the lethal infection level of entomopathogenic fungi in
various species of grasshoppers collected from site-I of lower Sindh in the
year 2013-2015.
62
9 Fig. IX. Showing the lethal infection level of entomopathogenic fungi in
various species of grasshoppers collected from site-II of lower Sindh in the
year 2013-2015.
63
10 Fig. X. Showing the lethal infection level of entomopathogenic fungi in
various species of grasshoppers collected from site-III of lower Sindh in the
year 2013-2015.
64
11 Fig. XI. Showing the isolated percentage of entomopathogenic fungi and their
association with pest species of grasshoppers during the year 2014 from
Sindh.
65
12 Fig. XII (a). Showing the faecal production of immature Acridid (N1-N3)
culture in small jars under laboratory conditions (after treatment of
Aspergillus oil formulation).
66
13 Fig. XII (b). Showing the faecal production of immature Acridid (N4-N6)
culture in small jars under laboratory conditions (after treatment of
Aspergillus oil formulation).
67
14 Fig. XIII. Showing the faecal production of adult Acridid culture in small jars
under laboratory conditions (after treatment of Aspergillus oil formulation).
68
15 Fig. XVI. Showing the faecal production of Acridid (Nymphs) population
treated with conidial concentration in H2O cultured maintained in the large
cage.
69
16 Fig. XV. Showing the faecal production of Acridid (Adults) population
treated with conidial concentration in H2O cultured maintained in the large
cage.
70
17 Fig. XVI (a). Showing the mortality of Acridid (N1-N3) population cultured in
small jars under laboratory conditions (after treatment with Aspergillus oil
formation).
71
18 Fig. XVI (b). Showing the mortality of Acridid (N4-N6) population cultured
in small jars under laboratory conditions (after treatment with Aspergillus oil
formation).
72
19 Fig. XVII. Showing the mortality of Acridid (Adults) population cultured in
small jars under laboratory conditions (after treatment with Aspergillus oil
formation).
73
20 Fig. XVIII. Showing the mortality of Acridid (Nymphs) population treated
with conidial concentration in H2O cultured maintained in the large cage.
74
21 Fig. XIX. Showing the mortality of Acridid (Adults) population treated with
conidial concentration in H2O cultured maintained in the large cage.
75
22 Fig. XX. Showing the element concentration under scanning electron
microscope (SEM) of Aspergillus niger.
76
23 Fig. XXI. Showing the element concentration under scanning electron
microscope (SEM) of Aspergillus flavus.
77
24 Fig. XXII. Showing the element concentration under scanning electron
microscope (SEM) of Aspergillus fumigatus.
78
25 Fig. XXIII. Showing the element concentration under scanning electron
microscope (SEM) of unknown Fungi I.
79
26 Fig. XXVI. Showing the element concentration under scanning electron
microscope (SEM) of unknown Fungi II.
80
LIST OF PLATES
S.NO. CAPTION OF PLATES PAGE
NO.
1 Plate. I. Important insect pest of sub-family Acridinae. 114
2 Plate. II. Important insect pest of sub-family Calliptaminae. 116
3 Plate. III. Important insect pest of sub-family Gomphocerinae. 117
4 Plate. IV. Important insect pest of sub-family Hemiacridinae. 118
5 Plate. V. Important insect pest of sub-family Oedipodinae. 119
6 Plate. VI. Important insect pest of sub-family Oxyinae. 121
7 Plate. VII. Showing the horizontal transmission of Aspergillus niger on the
host body (a) Head, Pronotum (b) Thorax (c) Tegmina, all were infected by
pathogenic fungi.
122
8 Plate. VIII. Showing the transmission of Aspergillus flavus on the host body
(a) Pronotum (b-c) Thorax and Tegmina, significantly effective by fungi.
123
9 Plate. IX. (a-c) Showing the significant infection of Aspergillus fumigatus on
the host body image has been taken after 72 hrs of treatment.
124
10 Plate. X. (a-c) Showing the infection of unknown species on the host body
image clearly showed that insect become hard and Aspergillus slight spreed
and cover whole the body.
125
11 Plate. XI. Scanning Electron Microscopy of Aspergillus conidia (a) A. niger
(b) A. flavus (c) A. fumigatus.
126
12 Plate. XII. Scanning Electron Microscopy of Unknown Fungi conidia
(a) Unknown Fungi I (b) Unknown Fungi II.
127
13 Plate. XIII. (a) Collection of infected sample (b) Fungal isolation
(c) Culturing of pathogen media (d) Insertion of prepare medium in
incubation for 24hrs.
128
14 Plate. XIV. (a-b) Smooth cutting of core chips (c) Fixing of core chips
(d) Placement of Aspergillus samples on conductive double side’s carbon
solution taps.
129
CHAPTER 1
INTRODUCTION
1.1. Acridids as pest
Grasshoppers are major agricultural pests throughout world including Pakistan and India
Roonwal (1978), COPR (1982), Steedman (1990), Riffat (2008), Riffat and Wagan (2010,
2011, 2012, 2015) and Riffat et al., (2013), they are polyphagous badly damage rice,
sugarcane, wheat, maize and fodder crops in Pakistan. For their control farmers expends
millions of rupees per year when their population increased, many species exhibit migratory
and gregarious behavior. This behaves lead to the formulation of spectacular swarm. Various
species of grasshoppers have been control by insecticides and pesticides i.e (Solutions of
Dieldrin, Chlordane, DDT, Parathion, Malathion, Dimetilan and Sevin), billion rupees are
used indiscriminately for buying these products. On the other hand, these chemicals are
injurious and health hazardous effects, on living creature and their environment. So, there
should be suitable, beneficial and cheap alternative instead of chemicals. For this purpose the
biological control is very important it is cheap safe for all organisms as well for their
environment. It is one of the oldest and most effective means of achieving insect’s pest
control. Therefore, an attempt was made to introduce pathogenic fungi, against the reduction
of grasshopper’s population.
Earlier, many authors carried work on this subject Aldrovandi (1923), Christie (1936),
Balfour-Browne (1960), Greathead (1963, 1992), Nickel (1972), Poinar (1975), Roonwal
(1976), Henry et al., (1985), Prior and Greathead (1989), Bidochka and Khatchatourias
(1992), Hermandez Crespo and Santigo Alvarez (1997), Balogun and Fagade (2004), Paraiso
et al., (1992), Shah et al., (1994, 1998) and Riffat et al., (2013). Additon to this, there are
many recent examples of utilization of different pathogens, given by Kassa (2003), Tounou
(2007), Cummings (2009) and Mouatcho (2010) against different insect pests but
introduction of this bio-control agents is yet unknown from this region, therefore, present
study was designed to implement this pathogen against acridid population in Sindh.
1.2. Pathogenicity of Insects
Insects, through their diversity in type, numbers, life-cycle and habitat expose themselves to
wide range of pathogens i.e (virus, fungi, bacteria, nematodes and protozoa etc.) whatever;
goes wrong with an insect may be considered a pathology or disease, after applying different
formulation of entomopathogenic fungi. In agriculture sector, population of various insect’s
pests can be devastated by natural outbreak of pathogens. Entomologists, as early as the mid-
19th century, were aware of outbreak of different diseases caused by pathogens, and they
attempted to control the insect pests with the use of pathogens. There are many recent
example of the effectiveness of pathogens when used against insect pest in many countries of
the world like USA, Mexico, India, Australia, Greece, Italy, Spain, Denmark, Sweden,
Switzerland etc. but nothing was available regarding this subject in Pakistan, and there is no
any product of entomopathogenic fungi registered still as bio-pesticides on national level,
although nematodes are utilized commercially in Karachi, Pakistan (http://www.pjn.com.pk).
The principal application of this study was utilization of many entomopathogenic fungi in
pest reduction; species rapidly decline when fungi develop in dramatic epizootics.
1.3. Entomopathogenic fungi (EPFs)
Entomopathogenic fungi are regarded as bio-pesticides and expected to have a significant and
increasing role for the control of locust and grasshopper in world including Pakistan Riffat et
al., (2013). These microbial agents are commonly famous as myco-insecticides that have
great potential to kill locust and grasshopper species. Beside this; it is also beneficial to
control flies, beetles and aphids in field Roditakis et al., (2000). Pathogenic fungi penetrate
into host’s external surface after utilization of pathogenic fungi large number of grasshoppers
and locusts were killed, this finding suggests that this microbial agent is very useful against
many pest species. Microbial agents that include: bacteria, virus, nematodes, protozoan and
pathogenic fungi are good bio-control agents. Lomer et al., (2001) stated that pathogenic
fungi are very important and interesting bio-control agent due to its observed capacity that
lead to formation of epizootics.
Earlier, many workers have done research on this i.e Goettel et al., (2005, 2008), Pell (2007),
Vega et al., (2009), Hajek (2009), Jaronski (2009), Pell et al., (2010) and Riffat et al., (2013).
About 35 genera comprise on 400 species/sub-species of pathogenic fungi have been
identified. These identified species have close association with more than 1800 insect species
in field and mostly killed the wide varieties of insect’s population in their favorable season
Jenkevica (2004).
Pathogenic fungi are cosmopolitan in their distribution and diversity; they put cruel attack on
the insect’s population. Due to their eco-friend and bio-persistence behavior and easily
preference to kill pest species at different developmental stages, their utilization is increasing
day-by-day. Now large numbers of pathogenic micro-organisms are available for evaluation
against grasshopper and locust in the world. Micro-organism’s priority is given to the
entomopathogenic fungi and entomopoxvirus that are stable for prolonged period of storage
and application. This microbial agent is considered very useful in IPM program.
After application of different insecticides and pesticides large number of scale insect are
killed in the field but at the other hand it cause health and environmental issues, this problem
has led to renewed interest in the development of eco-friendly microbial agents that are now
incorporated into IPM strategy. Utilization of entomopathogenic fungi for practical pest
control is very limited due to lack of scientific literature on the epidemiology of infection in
particular the host parasitic system. Therefore, efforts made in the investigation to isolate,
identify and characterize mycoflora associated with natural mortality of various pest species
of grasshopper and to study the prospect of bio-control from this area. Utilization of different
chemicals put very harsh impact on the environment and frequent use of chemicals enhance
the resistance power in insect’s body. As a result in 1987 outbreak of locust was not
controlled by pesticides.
Consequently, present attempt has been made to adopt biological control measures against
pest by using the myco-insecticides from this region. Although, majority of studies have been
done by Driver et al., (2000), Moore et al., (1992), Inglis et al., (1996) and Blanford et al.,
(1998) to assess the mortality ratio of target pest after treating with various entomopathogenic
fungi. But still, nothing has been published with exception of Johnson and Pavlikova (1986),
Olfert and Erlandson (1991) and Fargues et al., (1991) who carried work on the infection of
feeding of insect after pathogenic treatment. But, mostly these scientists worked under
environment constant regions that are condition for more infection and could not consider
how this behavior and the overall impact of pathogen might change under more realistic,
variable condition of experiment in the field. The present study was aimed to improve the
effeteness of pathogenic doses on the feeding and incubation of insects under controlled
conditions where temperature was optimum.
1.4. Integrated Pest Management (IPM)
The data obtained during present survey is not only important for academic forum but it is
also utilized as applied approach. Through, Integrated Pest Management (IPM) cultivators
can save their crops by adopting different biological methods, such as biological control,
mechanical control, physical control, field burning, hunting and space fumigation. Among
these all enlisted methods the most effective and alterative way is the biological control that
is considered sustainable method in reduction of pest species in field by application of virus,
protozoan, pathogenic fungi, nematode and different bacteria. Keeping in view, its economic
importance and effectiveness of biological control present attempt has been carried out on
the utilization of fungi along with application of some opportunistic fungi on acrididae, most
important family of group califera, its large No. of species are considered as major pests of
valued crops in agriculture. Beside this, pathogenic fungi also put effect on the survival-ship
of individual. It is also likely that, this research will be basic guideline for future researchers
who intend to commercialize this bio-product on large scale. During this study, different
species of Aspergillus were documented along with element concentration under scanning
electron microscope (SEM). This will certainly provide the firm base for rather more
promising field biological control.
1.5. Brief geographical feature of Sindh
Sindh Province forms the lower Indus basin it lies between (27.5562No) Latitude and
(68.2141Eo) Longitude. It is located on the western corner of South Asia. Geographically, it
is the third largest province of Pakistan with stretching range of 579km from North to South
and about 442km from East to West. Beside this, Sindh has the vast desert, Khirthar
Mountain along with beautiful Arabian Sea while center covered with fertile plan around the
Indus River, its area was composed on 140,915km of Pakistan territory, average rainfall
about 7ʺ per annum, mostly falling in July to August. Monsoon wind start in mid- February to
end of September in south west. However, cold wind below from north in winter season
during the month of October to mid-January.
Climatically, Sindh is divided into 3 regions Siro (the upper region, center on Jacobabad),
Wicholo (the middle region, center on Hyderabad), and Lar (the lower region, center
on Karachi). Upper Sindh is very dry due to thermal equator. Opposing to this, central Sindh
has low temperature compared to upper Sindh and very high than lower Sindh. During the
summer season there are very long and dry days while the nights are cool. Maximum
temperature range in Sindh is 43-44oC and 109-111oF. Damper and humid climate occurred
in lower Sindh that affect the winds begins in southwestern part in summer and northeastern
wind in winter, while there is low rainfall than central part of region. Temperate range in
lower Sindh is about 35-38oC and 95-100oF. Beside this, Kirthar range is 1800mm about
(5900ft) and higher at Gorakh hill along with other peaks print in Dadu and Larkana districts,
where temperature recorded is near to freezing point and brief snowfall is also received in the
winters, extremely low temperature was noted in Gorakh hills.
1.6. The objectives of this study were:
1. Collection and building of grasshoppers through the extensive surveys from
various districts of Sindh.
2. Rearing of collected material under laboratory conditions in order to know the
impact of pathogenic fungi on the Acridid species.
3. To note the physical behavior of grasshoppers after pathogenic treatment.
4. Assessment of faecal material in order to know the reduction in feeding of insects
after treatments.
5. Preparation of oil formulation of different fungi and utilization of range of
pathogen doses on feeding and observe mortality rate of insects.
6. To document the proportional cumulative survival of treated and untreated
insects under laboratory conditions.
CHAPTER 2
REVIEW OF LITERATURE
Extensive work has been done on the utilization of different microbial agents against
reduction of pest population in the world. The efforts of many co-workers in this field are
being discussing below:
The bulk of information was available on the bio-control agent in all around the world mostly
work has been done by Berger (1991) and Ritchie and Dobson (1995) after this Murphy et
al., (1994) carried comprehensive work on the toxicities of different pesticides and stated that
pesticides are responsible for causing 45% to 55% mortality of insects. A part from
pathogenic infection of entomopathogenic fungi, Murai (1959) carried work on the egg
parasites of rice grasshoppers Oxya japonica (Thunberg) and O. velox (F.) and recommended
that Scelio muraii and S. tsuruokensis are excellent natural enemies for the embryonic stages
of grasshoppers and these agents can be utilized to control grasshoppers and locusts
population in the fields.
In addition to this, Mongkolkiti and Hosford (1971) also worked on the biological control of
the grasshopper by utilization of Mermis nigrescens and stated that decline and disappearance
of natural population of the many grasshopper i.e Hesperotettix viridis pratensis was related
to sever infection by M. nigrescens they further, reported that wet habitats where the
grasshoppers fed primarily on Solidago missouriensis were significantly infected and infected
individual failed to develop ovaries and testes in H. viridis pratensis, its population was
totally destroyed prior to egg-laying by parasitism of M. nigrescens.
Ashrafi et al., (1968) while working on the energy producing enzyme after the treatment of
various insecticides and reported that acetone stopped the phosphatases activity. Beside this,
they stated that about 30% inhibition was reported in solution of Malathion, Petkolin,
Parathion and Acetone when insect’s treats with this they also recommended that above
mentioned insecticides helped in restoring the enzyme activity to some extent but not more
than normal. Naqvi et al., (1969) studied the effect of varying doses of insecticides on the
phosphor monoesterases of the desert locust Schistocerca gregaria and reported that various
does of DDT, Dieldrin, Chlordane, Dimetilan, Sevin, Parathion and Malathion when injected
into the body of the desert locust after this it was noticed that two insecticides i.e chlorinated
and carbonate inhibited the enzyme in in-vivo condition while organophosphorus insecticides
activated these enzymes.
Ferron (1975) stated that fungal pathogens cause disease in insects which at time regulate the
population of insect pest in nature. Carl (1982) stated that biological control is most
applicable to native as well as introduced pests. He further, reported that native natural
enemies that have adapted introduced species as hosts. The first recorded instance of
successful biological control of a native species by an introduced natural enemy is that of
Oryctes tarandus which was known as sugarcane pest. Beside this, coconut moth, Levuana
iridescens which is serious pest of copra industry also successfully control by biological
agent’s i.e Bessa remota. In addition to this, CIBC in Barbados has also led to an outstanding
success against the sugarcane moth borer i.e Diatraea saccharalis.
Further, he also reported that high degree of specificity of a parasite is considered because it
prevents the insect from being “distracted” to other host. He recommend that promising
natural enemies used against native pests would have to show the same desirable attributes as
those introduced against exotic species i.e ecological compatibility high super and multi-
parasitism, short developmental period, broad ecological range (preferably the same breath as
that of the host), possibly non-random searching and so on.
Henary et al., (1985) studied isolation of a yeast type fungus from 3 species of grasshopper
i.e Oedaleus senegalensis (Krauss), Aiolopus thalassinus (Fab.) and Anacridium species from
West Africa they reported that these fungi cause highly infection to grasshopper in the
laboratory. Fungi are highly pleiomorphic and very difficult to grow on defined media;
therefore, their taxonomic status has not been fully determined. Nevertheless, they should be
tested against locusts and grasshoppers. Nnakumusana (1985) also mentioned that in
laboratory bioassay a non-identified Pythium species proved pathogen to early instar of Aedes
aegypti, A. africanus, A. simpsoni, Culerx quinquefascians, C. tigripes, Charmoy and
Anopheles ganbiae.
Samson et al., (1988) noticed that fungal disease significantly destroyed the colony of insects.
Similarly, Hajek and Leger (1994) recorded that pathogenic fungi gave excellent results it
should be implement on world-wide for suppression of many pest species in agriculture
sector.
Gunnarsson (1988) carried work on the immune response of many insects that were treated
with different pathogens. He observed that after 12 hour of treatment insect show significant
change in its immune activity. Beside this, Gotz and Vey (1974) studied the humeral
encapsulation in Beauveria bassiana hyphae even within the cuticle and insect alerted to
infection at early stage in the alteration include behavioral fever, raise in body temperature
above the optional range. Additionally, Moore et al., (1992) and Seyoum et al., (1994) also
reported that after the treatment of insect with any pathogenic fungi they reduce their feeding.
Beside this, they also stated that host thermoregulation was restricted under control condition
and infection of pathogenic fungi limit this attribute directly to colonization and production
of metabolites followed by invasion of tissue and nutrient depletion.
Streett and Henry (1990) worked on several microbial control agents that effect locusts and
grasshoppers in the semi-arid tropical regions. They stated that viral and protozoan pathogen
including entomopathogenic fungit gave significant results to reduce pest population. They
also observed demonstration of lethal disease in locust by a Lepidopteran nuclear
polyheelrosis virus. Further, they recommend that pathogenic isolated were very impotence
in reduction of Zonocerus variegatus. Similarly, Paraiso et al., (1992) also obtained some
results while testing Fusarium sp. and B. bassiana on Z. variegatus. Goettel and Roberts
(1992) stated that Entomophaga grylli is very difficult to produce in bulk therefore, its use in
native biological control agent is limited. Johnson and Goettel (1993) also recommend the
B. bassiana as myco-pesticides in reduction of insect population.
Shah et al., (1994) observed that grasshopper infected by 03 hyphomycetes fungi viz:
Deuteromcotina, Metarhizium and B. bassiana they reported average incidence of
Metarhizium flavoviride with ratio of 2.9% in population on most grasshopper cadavers with
10 days of collection. Besides this, they also stated that smaller grasshopper i.e Pyrgomorpha
cognata (Krauss) and Acorypha glaucopsis (Walker) were infected significantly compared to
large size insects. Mortality of smaller grasshopper is also greater followed by large size
insect this dispirty might be due to volume of body.
Shah et al., (1994) reported that three hyphomycete fungi i.e M. flavoviride, B. bassiana and
Sorosporella sp. significantly infect grasshoppers population they further analysied that small
grasshopper that include Chrotogonus senegalensis, P. cognate, P. vignaudii when infected
with M. flavoviride die earlier compare to large size species i.e Acrido deresstrenus (Walker),
Ornithacris cavroisi (Finot), Hieroglyphus daganensis (Krauss), Spathosternum pygmaeum
(Karsch), Acorypha clara (Walker) and Trilophida replete (Walker). They stated that smaller
individual contaminations ratio is high than large size. They carried comprehensive work on
the 02 major families i.e Acrididae and Pyrgomorphidae. Acrididae comprising on 10 sub-
families viz: Acridinae, Calliptaminae, Catantopinae, Cyrtacanthacridinae,
Eyprepocenemidinae, Gomophocerinae, Hemiacridinae, Oedopdinae, Tropidopolinae and
Truxalinae. They also enlisted the natural incidence of M. flavoviride infection in 02 sites
Prpim and Bodjekali from Northern Benin Communities and reported that majority of
infected species had adult quiescence of continuously reproducing life-cycle and were either
arboricolous or terricolous.
Lactin and Johnson (1995) reported that feeding in 5 th instar of M. sanguinipes was optimal
to the preferred thermoregulatory temperature on 38-40oC. However, cure around this
temperature was in asymmetrical. Beside this, they also showed that low temperature is
responsible for decline in feeding rate, while on 46oC feeding causing abruptly in this result
behavior fever response after that fever shifted to other body parts where feeding inhibited.
Similarly, Inglis et al., (1996) reported that body temperature suddenly increase in the
M. sanguinipes F. when this species contaminated with B. bassiana and some observations
were also reported by Boorstein and Ewald (1987) when M. sanguinipes infected by Nosema
acridophagus.
Hermendez Cerspo and Santiago Alvarez (1997) worked out on the infection level of
B. bassiana in Moroccan locust and calculated 2.8 and 8.6% infection ratio in this pest
population. Many workers i.e Milner (1997), Milner et al., (1997) and Driver et al., (2000)
carried significant work on the ecology, distribution and pest status of many grasshopper
species viz: Chortoicetes terminifera and Phaulacridium viltatum commonly known as
plague locusts could be control by potential biological control. Addition to this, Baker (1993)
also outlines the preventative control strategy for C. terminifera.
Matthew et al., (1997) reported that effect of M. flavoviride on feeding of Z. variegatus. They
observed that significant reduction in feeding was indicated by faecal assessment. All
infected individual died by 7th day of treatment on the last day they noted that faecal
production of treated individual was found equivalent to less than 2 day faecal assessment by
individual untreated with spores. They stated that reduction in feeding actually was
associated with behavioral response of grasshopper. They further stated that, insect display
full signs of mycosis on day 4th and 5th when treated with any pathogenic fungi while
maximum mortality was noted on 7th day, opposing to this, control group has attempted
singnificat low mortality.
Peveling et al., (1997) carried numerous experiments in order to show harm effect of
different pesticides on non-target animals and stated that apart from environmental
persistence pesticides caused long lasting population decline in field. In addition to this,
Van Der Valk and Niassy (1997) carried work on the impact of insecticide on terrestrial
acridids and discussed the possibility of various insecticides application to decline the
grasshopper population. Moore and Caudwell (1997) recommend that use of bait formulation
of myco-pesticides against grasshopper and locust is very useful in Africa.
Shah et al., (1998) reported that natural incidence of M. flavoviride from two sites in northern
Benin they observerd 1030 individual belonging to 38 species. The majority of infected
species were A. blondeli, C. senegalensis, P. cognata and Stenohippus sp., Cryptocatantops
haemorrhoidalis, C. stramineus, D. axillaris and Stylifer. However, K. amabile and H.
tenuicornis were phytophilous they also reported that grasshopper infected by M. flavoviride
were commonly lowest i.e 3.2% in 1994 due to high amount of rainfall end flooding which
would have been conducive for epizootic level of M. flavoviride to develop.
Houndekon and De Groote (1998) carried few attempts to external costs concerned with
locust and grasshopper control. Martin et al., (1998) also utilize the most effective
insecticides i.e Pyrethroid deltamethrin against grasshoppers. Similarly, Johnson et al.,
(1992) used low level of insecticides i.e Carbarly in wheat bran and get more than 70%
reduction in post treatment population of 1-5 grasshoppers per square meter.
Karim and Riazuddin (1999) stated that insect’s pathogen offer an alternate and important
control strategy to chemical insecticides they further highlighted that microbial control agents
are easy to manipulate for aerial spray than predators and parasites and their augmentation is
also easy. Insect pathogens are safe to humans and non-target species could be used
harmoniously with other control agents. The only major dis-advantage of pathogens is their
slow speed of action in comparison with chemical. Regarding the entomopathogenic fungi,
they recommend that Metarhizium, Beauveria, Hirsutella, Nanuraea and Paecilomyces also
have great potential as biological pesticides. More than 150 species of natural enemies are
known to attack the rice stem borers about 90% to 98% mortalities of eggs and pupa in stem
borer caused by parasitoids and predators. Further, they also reported that 100% egg
parasitism along with sever outbreak of stem borer from those regions where
pesticides/insecticides were sprayed but where natural enemies were available there was no
any significant harm. Awareness of the insecticides and pesticides regarding the human
health and environment has been enhanced in the people therefore; cultivators avoid ignoring
this practice.
Roditakis et al., (2000) carried work on secondary pick-up of fungal pathogen conidia against
insect and investigated different methods for increasing conidia acquisition by enhancement
of host movement through utilization of E-β-farnescne that increase mortality of aphids.
Morever they suggest that more practical approach to increase conidia pick-up appears to be
the use of sub-lethal doses of the choronicotinyl insecticide imidacloprid they recommend
that significant mortality was active when aphids population were exposed to insecticide-
treated leaf discs that were sprayed with fungal conidia.
Mensah (2000) analyzed the susceptibility of 02 grasshoppers viz: S. gregaria and
Z. varigatus to aqueous and oil formulations of some strains of Metarhizium species, he
reported that both species of grasshoppers responses to increasing concentrations of the
pathogens but it was notify that S. gregaria was more susceptible to M. flaviride then
Z. varigatus he further indicated that infection occurred at all humidifies studied. In addition
to above observation, he also added that infection of susceptible host insects following field
application of myco-insecticides has for a long time been considered as dependent on weather
condition particularly relative humidity and temperature, while Ferron (1977) and Marcandier
and Khachatourians (1987) pointed out that Beauveria infection may proceed independently
of anbient.
Blanford and Thomas (2001) carried work on the adult survival, maturation and reproductive
activities of desert locust they reported that S. gregaria adults were infected with pathogenic
fungi show significantly high mortality at high temperature and there was reduce mortality
when permit to low thermoregulation.
Richard and David (2001) reported large scale field trials of bio-pesticides (fungi) against
Locusta migratoria that gave significant result and doses of 50-75 g/ha were considered more
effective. It was also noted that Austracris guttulosa were found very susceptible in the
laboratory but in the field they have proved difficult to assess because of the high death ratio
of adult and the absence of hoppers bands at the nymphal stages. They further stated that B.
bassiana are more effective against the wingless grasshopper than the locust and small
insects which don’t started to disperse were easily infective with this. Beside this, their initial
bioassay revealed that isolates obtained from field infected grasshopper both wingless and
other species in the complex, were found highly virulent than the other isolates tested.
Evans and Shah (2002) studied the occurrence of disease associated with the spread of genus
Sorosporella on many grasshoppers and locusts from Africa. They enlisted infected hosts
belonging to 10 genera within 05 subfamilies of Acridoid i.e Oedopodinae,
Crytacanthacridinae, Catantopinae, Hemiacridinae and Pyrgomorphidae. They observed that
infected individual characterized by red, thick-walled chlamydospores which completely
filled the cadaver. They also described Syngliocladium acridiorum as new species to science
before this, Sorosporella is treated as a synonym of Syngliocladium. Further, they reported
that African migratory locust occurs as two distinct sub-species but significantly, no
infections were recorded on the mainland type Locusta migratoria migratorioides which is
endemic to the Niger flood plain.
Jenkevica (2004) studied the occurrence of entomopathogenic fungi and their host range from
central and western parts of Lativa. He highlighted the registration of association between
pathogenic fungi and various insects. He found that B. bassiana, M. anisopliae and Vertici
lliumlecanii on 05 important host species i.e Conidiobolus obscurus, C. thromoboides,
Entomophthora muscae, E. aphidis and Entomophthora sp. He also indicated that B.
brongniartii was first identified in Lativa and he enlisted 09 associations between B. bassiana
and insects. Similarly, Balogun and Fagade (2004) carried work on the Z. variegates infected
with entomopathogenic fungi. They described 08 fungi species viz. Fusarium sp., B.
bassiana, Metarhizium sp., A. flavus, A. niger, Pencillium sp. and Mucor sp. with significant
high rate fungal pathogens on insect population and discussed the relationship of host species
with their environmental ecology.
Jan Scholte et al., (2004) while stadying occurrence of disease in insect and mosquito group
reported that virtually all insect susceptible to fungal attacked. Similarly, Robert (1974) stated
that most common fungal pathogens i.e Lagenidium, Coelomomyces and Culicinomyces
affect the mosquito population he analyzed that these fungus cause maximum mortality at the
larval and adult stage.
Devarajan and Suryanarayanan (2006) isolated the fungal endophytes from Catctropis
gigantea. Poekilocerus pictus (painted grasshopper) fed on the leaves of Calotropis gigantea
neither avoided preferred milkweed leaves coated with a spore suspension of C.
gloeosporicides they suggest that these phytophagous insects serve as an important biological
agent for the dispersal of non-grass fungal endophytes in tropical forest. Assaf (2007) also
reported that most common insect species i.e Eurygaster integriceps a sun pest in Iraq could
be easily control by B. bassiana. Ehrlich (2007) reported that there are 132-200 species of
Aspergillus were associated with the production of mycotoxins.
Magalhaes et al., (2001) introduced the fungus M. anisopliae var. acridum as most promising
bio-control candidate against Rhammatocerus schistocercoides in Brazil and verified its
effect on Orthoptera, Diptera and Hymenoptera including other non-target organisms. They
also highlighted that fungi and nematodes particular mermithids infecting grasshoppers in
their natural environment. They reported that 07 isolates of M. anisopliaevar, Acridum and 05
isolated of B. bassiana infecting S. pallens and R. schistocercoides.
Diba et al., (2007) identified 205 Aspergillus isolates among 153 with 75% environmental
Aspergillus and 52 with 25% clinical isolates they also reported that in nature A. niger, A.
flavus and A. fumigates were the most common Aspergillus isolates from all of the
specimens. They further, reported that morphological differential media is the most reliable
and sensitive to isolate Aspergillus species. Varga et al., (2011) record 04 new species viz:
Aspergillus eucalypticola, A. neoniger, A. fijiensis and A. indologenus from Australia. They
concluded that these 04 species are very important to study the food mycology, medical
mycology and bio-technology regarding genetic relationships.
Selouane et al., (2009) carried work on the natural occurrence of Ochratoxigenic Aspergillus
species in grapes they analyzed 360 strains of Aspergillus and stated that most abundant
species is A. niger with aggregation ration of (82.5%) whereas A. carbonari occurrence is
occasionally recorded. Inglis et al., (1996) worked out on the impact of temperature in B.
bassiana in many colonies of insects. They reported that low prevalence of mycosis (≤ 7%)
was analyzed in inoculated nymphs exposed to a colonies temperature of 35oC and 40oC
whereas colonies exposure to 30oC did not have a significant effect on disease development.
Similarly, Douglas et al., (1996) uses the combination of different pathogen to overcome the
constraints of temperature on entomopathogenic hyphamycetes against grasshopper they
recorded that temperature showed great influences on the mortality of grasshoppers.
Angela et al., (2009) reported the predator, mediate the effect of fungal pathogen on prey.
They performed many field trails to observed interaction of grasshopper with predatory
spider with combination of lethal fungi. Pathogenic effect on the population of grasshoppers
they notified that fungal pathogens were abundant in favorable weather i.e (day time,
temperature, relative humidity and total precipitation) effect the growth rate of fungi and
abundant presence of spider reduce the fungal pathogen in field. They further stated that large
No. of death caused by pathogen lead to trend of enhancement in the soil as more dominate
spore that leading to a slight trend of increased grasshoppers densities. They recorded that
Entomophaga grylli over winters in the soil as dormant spores that emerge in late spring or in
early summer season. They further said that, 50 individual of grasshoppers with 20% were
died from E. grylli by this maximum population of grasshopper were die. E. grylli increased
when spider population decreased in field but they recommend that there was no effect of
insect densities on the number of death from E. grylli.
Divya et al., (2010) reported that like Entomopathogenic fungi (EPFs) and
Entomopathogenic nematodes (EPNs) has great potential tendency as biological control
agents (BCAs) and could be used at the infection juvenile stage. Beside this, they also
observed that foliar application of nematode are very useful to control the leaf eating
caterpillar on various crops and having the great potential in controlling many other insect
pest. In addition to this, while conducting the laboratory bioassay they reported that nematode
cause great mortality in all larval and adult stage of Helicoverpa armigera, Spodoptera litura
and Galleria mellonella all were die 100% when their larva was exposed for 12hrs, 18hrs and
24hrs to this nematode.
Assaf et al., (2011) carried extensive field survy and observed that Aspergillus and Beauveria
have close association with 07 species of grasshopper and isolated ratio of these
entomopathogenic fungi was significantly high. Shehu and Bello (2011) reported that fungi
play major role in the storage of cereals and environmental factors i.e temperature relative
humidity and light have influence on the growth of Aspergillus species. They observed that
light put no significant effect on the growth of mycelia. As far as relative humidity is
concerned, 85% to 100% was consider most favorable for the mycelial growth of Aspergillus
species and poor growth was recorded between range of 32.5% to 50.5% relative humidity
(RH), while the significant growth ratio was obtained on the 30oC to 35oC.
Gautam and Bhadauria (2012) analyzed about 82 samples of triphala powder for the
association of various fungi species they stated that Aspergillus has significant difference in
band patterns and number of band obtained after Polymerase Chain Reaction/Amplification
(PCR). They carried PCR Amplification on 06 species of Aspergillus i.e A. niger, A. flavus,
A. fumigatus, A. terreus, A. nidulans and A. amstelodami. On the bases of PCR test they
recommend that only A. flavus showed amplification with all the three aflatoxigenic and other
Aspergillus species as non-toxigenic after PCR analysis. Geetha et al., (2012) carried work on
the interaction of important entomopathogenic fungi i.e B. bassiana, B. brongniartii and
M. anisopliae are most important opportunistic soil fungi of ecosystem in sugarcane
i.e Fusarium saechari, Aspergillus and Penecillium species were assayed in vivo against
Galleria mellonella larvae. They stated that insect species affected by sporulation of
M. anisopliae with the least treatment of B. bassiana applications following M. anisopliae
and the same interaction was also seen in soil fungi when this was combine with pathogenic
fungi they caused high mortality on the first day while 24 on the second day on fourth instars
larva of Galleria mellonella and got significant results.
Riffat et al., (2013) studied the susceptibility of 03 Hieroglyphus species to some strains of
the entomopathogenic fungi and isolated 03 pathogenic fungi i.e M. flavoviride, B. bassiana
and Aspergillus sp. from 03 destructive pests of Hieroglyphus i.e (H. perpolita, H.
nigrorepletus and H. oryzivorus) and stated that cumulative survival of Hieroglyphus in the
different treatment of fungi was significant low when treated with entomopathogenic fungi all
began to die with full signs of mycosis on day 5th and complete mortality was noted on 6th
day. Beside this, they showed that application of M. flavoviride proved more effective against
Hieroglyphus population compare to other treated fungi.
Ortiz-Urquiza and Keyhani (2013) stated that entomopathogenic fungi having adhesion and
identification of host surface for direct response for the production of detoxifying and
hydrolytic enzyme. Beside this, they also reported that insect have evolved number of
different mechanism to carry pathogen at bay i.e cuticular production, antimicrobial fats,
protein and metabolites. Beside this, cuticle shedding in development process and
environmental behavior adaptation that include: fever, burrowing and growing.
More recent, while working on the nematodes Soomro (2014) highlighted the infection of
Mermis nigrescens on the different species of grasshoppers. She reported that M. nigrescens
significantly reduce the survival-ship and reproductive activitie of insects. She collected 983
specimens of various grasshoppers from different regions all were belong to 8 sub-families of
Acrididae. She further, analyses that infestation percentage was significantly highest on
flooded rice fields compare to other grassland and maximum infection of Mermis was
observed in population of Oxya species. Addition to this, Riffat et al., (2014) also
recommended that super parasitism M. nigrescens caused in different host sub-families of
many grasshoppers was significantly greater in month of June.
Lacey et al., (2015) reported that entomopathogenic fungi caused epizootics in host body and
recommend that mass production delivery and formulation system should be devised to
supply an ever flourishing market.
CHAPTER 3
MATERIAL AND METHODS
3.1. Insect’s sampling
The stock of grasshoppers both mature and immature were collected from various districts of
Sindh (Map-I). Specimen were captured with swept net having (25×25cm) diameter while,
82cm in length (without diameter). Some specimen’s were also captured by hand picking,
sweeping, trapping, night trap, aerial netting and black light pan traps when-ever found.
Collected insects took to the laboratory where two cages of different measurement i.e (42cm
in length, 30cm in width) and (35cm in length, 32.5cm in width) were maintained. All
collected individuals equally divided and put into cages. Fresh leaves of Zea mays serve to
rearing insects before this leaves and twigs were sterilized in 5% solution of Sodium
hypochlorite (NaOCl). This methodology has been adopted from (Prior et al., 1995 and Riffat
et al., 2013). For identification of samples scheme given by Riffat and Wagan (2015) was
followed.
3.2. Collection of infected samples
For capturing of insects contaminated with pathogenic fungi carefully observation has been
made in field and only those insects were collected which having clear symptoms of mycoses
viz: (i) insect don’t move fast, (ii) de-coloration not original (iii) fungal mycelia fully spread
on cuticle (iv) insects look sluggish/inactive and very easy to capture. Infected specimens
were easy captured with large forceps after collection material transferred into glass jars and
brought to laboratory for further analysis. All were sorted out into different host species and
kept in clean cages. Fresh Zea mays leaves were provided to insects. Food plant change daily
and food consumption, through analysis of faecal material and mortality of insect after every
24hrs were noted.
3.3. Incubation in laboratory
Different species of Acrididae divided into group of about 50 individuals for each treatment.
However, there was no differentiation in age, sex and developmental stage. All collection
placed into wooden cages under laboratory conditions where temperature range between
(28±2oC to 41±2oC) and Relative humidity (RH) was (26.5% to 60.5%). Population of
grasshoppers were comprised on all developmental stages which were collected from field
maintained in the laboratory, Entomology and Bio-Control Research Lab. (EBCRL),
Department of Zoology, University of Sindh, Jamshoro (25o-23/N, 68o-24/E).
3.4. Fungal isolation and sporulation test
The sporulating fungi separated in pure culture on SDA (Sabouraud Dextrose Agar), after this
it was formulated into oil (coconut) after preparing the oil formulation this fresh suspension
was kept in sonicator for 60 sec to break the conidial chain. After breaking conidial was
counted with the help of haemocytometer, this method has been adopted from (Poinor and
Thomas 1984, Kumar et al., 2013) Plate XIII, a-d.
3.5. Identification of fungal isolates
Various species of Aspergillus have been identified on the basis of conidia shape and size.
Beside this, for detail and authentic identification element concentration has been determined
under scaning electron microscope (SEM). For reorganization of fungi terminology given by
(Hoog 1972, Domsch et al., 1980, IMI 1983, Balazy 1993 and Humber 2012) was followed
(Table IX, Plate XI, a-c XII, a-b).
3.6. Pathogenicity Bioassay
Different fungi species were isolated and then isolates was grown at 28oC where photoperiod
ratio: 12hrs light, and 12hrs darkness about 15 days (Balogun and Fagade (2004) and Kumar
et al., 2013). Sterile spatula after incubation was used to harvest the conidia from fungal
culture. This harvested conidia shifted into small McCartney bottle (fully sterilize and
contained coconut oil) fungal spores suspension prepared in oil and spore concentration
measured with Neuberger Haemocytometer (Lomer and Lomer 1996).
3.7. Formulation of Aspergillus conidia
Two different formulations were selected in order to know that which formulation is more
effected. Before starting the experiment different part of Zea mays (consist on leaves and
stem) were broken shake into tap water than dire and put vertically into cage as well as in jars
before this, weight of food plants were taken i.e (2.5gm) put in small jars (26gm) kept in
cages respectively. The insects were reared into small jars as well as in captivity. 10 insects
were reared in 4 liters plastic jars, while 50 individuals were kept in different cages.
1. Formulation for small jars
5×106 (Conidial concentration) + 20ml (Coconut oil) = Oil formulation
2. Formulation for colony
5×1030 (Conidial concentration) + 100ml (Distal water) = Water formulation
The conidial oil distilled water formulation was sprayed on the insects using a hard held
sprayer. Each insect was directly and individually sprayed with 3.5ml of the appropriate
concentration. After 15 to 20 minutes the treated insects were transferred to the jars as well as
in cages. Control groups received the water formulation but, without conidia. The insect in
each replicate were fed on Zea mays (30gm every 48hrs).
3.8. Bio-pesticides application
Before the commencement of bioassay test insects were reared in cage for one week. After
that 0.1ml of conidial oil suspension was carefully applied beneath the pronotum shield of the
insect by the help of (Sterile Pasteur Pipette). Beside this, in control replicant blank oil with
spores was applied on the pronotum shield of hopper that was reared in jars individually
while in second replicant the conidial (mix in distilled water) formulation were sprayed on
the insects (reared in captivity) using a hard held sprayer. Each insect was directly and
individually sprayed with 3.5ml of the appropriate concentration. After 15 to 20 minutes the
treated insects were transferred to the cages. Control groups received the same water
formulation without conidia. The insect in each replicate were fed on Zea mays (30gm after
every 48hrs).
Insect feeding was assessed by measuring consumption of food and then assessing their
faecal production. Food consumption of insets for every 48hrs was measured after treatment
the faeces production from each cage and jars were also collected every 48hrs. After this
insect contaminated with Aspergillus and healthy grasshoppers were shifted into separate
cages place in laboratory. After transferring the insect their detail mortality was recorded
daily.
3.9. Observations under Scanning Electron Microscopy (EDS)
Analyzing of various elements concentration occurring in tested fungi was done under
scanning electron microscope (SEM). This parameter has been proformed at the Centre for
Pure and Applied Geology, University of Sindh, Jamshoro. For this experiment 05 samples of
different entomopathogenic fungi that include: Aspergillus niger, A. flavus and A. fumigatus
and 02 unidentified fungi i.e (Uk FI and Uk FII) were taken. Their spores were isolated from
insects cadavers brought to Geology laboratory as described by Kumar et al., (2013). After
this it kept for 12-14 hrs photoperiod under the sun for evaporation of water when these spore
become fully dry up then they were analysed under scaning electron microscope for noting
the higher and lower chemical composition of different types of elements present in their
spores, through this finging spore could be identified their this differenciation method has
been reported for this first time under SEM.
3.10. Experimental procedure
The procedure was initialized with the smooth cutting of core chips and their mounting
sample stub of Scanning Electron Microscopy conductive double side carbon solution tape
was used in SEM. All the samples were mounted in (JEOL JSM-6490 LV Model) sample
chamber. Scanning Electron Microscope also equipped with (Energy Dispersive X Ray) an
extra accessory component of Bruker EDS. Scanning Electron Microscope take about 15-20
minutes to develop vacuum, when development of vacuum shown on screen of computer then
start to select the portion of sample for magnification and for EDS one by one.
All Aspergillus samples were placed on conductive double sided carbon solution tape which
was placed on sample stub and numbers were given to all samples. After numbering, to all
samples magnified images and elemental composition in each Aspergillus species was
analyzed. The suitable operational parameters of (SEM) were put to record the fine focusing
on maximum and desire magnification of the samples. In the end the analysis was followed
by the elemental determination of the samples, both qualitative and quantitative. After getting
an ideal magnification i.e (X80) all the samples chemically analyzed and various element
compositions was calculated in the form of different peaks height for qualitative data and the
same time the result of sample analysis were finalized in tabulate and graphic formats for the
quantitative analysis as well. (Table XXII to XXVI, Fig. XX to XXIV)
3.11. Statistical analysis
Data was analyzed using statistical software (SPSS version 16.0). Obtained data from
experimental groups was subjected to one-way analysis of variance (ANOVA), with repeated
measures and significant means were determined using Least Significant Difference (LSD) to
identify the infected and uninfected samples of grasshoppers.
CHAPTER 4
RESULTS
4.1. Pest status of Acrididae
During the present study siginificant large numbers of grasshoppers were captured from
defferent climatic regions of Sindh the collected material was sorted out into 32 species
belong to 06 sub-families i.e Acridinae, Calliptaminae, Gomphocerinae, Hemiacridinae,
Oedipodinae and Oxyinae of family Acrididae. Four dominant species i.e Hieroglyphus
nigrorepletus Bolivar, 1912, Oxya velox (Fabricius, 1787), Acrida exaltata (Walker, 1859)
and O. hyla hyla Serville, 1831 accounted for maximum numbers compare to other collection
Appendix (I).
It was noticed that representatives of family Acrididae, including grasshoppers and locusts
are coming the most voracious pests known, with the fifth or sixth instars and adults capable
of eating their own body weight in various vegetation. In result of extensive survey a total of
2520 specimens have been collected from different districts of Sindh. Grasshopper having
great economic important due to its geographically distribution and wide pest status
numerous species Acrida exaltata, Duroniella laticornis, Gelastorhinus semipictus, Acorypha
glaucopsis, Chorthippus indus, Gonista rotundata, Ochrilidia geniculata, Oxypterna
afghana, Spathosternum prasiniferum, Locusta migratoria and Oxya species were reported as
major pest of earning crops like rice, sugar-cane, maize, wheat and cotton, they destroy the
important vegetables, fruits and fodder crops as well. Beside this, their targeted habitats were
also highlighted and pest status of 6 sub-families was also discussed in Table (VII)
Plate (I to VI).
Acridid is the largest family of Orthoptera, and indeed, of all Orthopteroids. It includes all the
true locusts and grasshoppers. It comprise on twelve sub-families i.e Acridinae,
Calliptaminae, Catantopinae, Cyrtacanthacridinae, Eyprepocnemidinae, Gomphocerinae,
Hemiacridinae, Oedipodinae, Oxyinae, Spathosterinae, Teratodinae and Tropidopolinae but,
I have worked out on six sub-families. The Acrididae include an enormous assemblage of
grasshopper into which the majorty of species is groups. The family cannot be defined on
presence or absence of prosternal tubercle, apical outer tibial spine, stridulatory mechanisim,
etc., such characteristics being present or absent in various sub-families of the Acrididae.
Studied sub-families were identified by observing the important morphological characters
presented in the key:
4.2. Key to the sub-families of Acrididae occurring in Sindh
1. Lower lobes of hind knee pointed, spine-like hind tibiae often flattend, outer apical
spine usually present ……………………….………….………..………….... Oxyinae
_. Not as above ……………………………………………………………....………..... 2
2. Prosternal tubercle present …...……………………………………………………… 3
_. Prosternal tubercle absent ……………………………...……………………………. 4
3. Prosternal tubercle wedge shaped male cercus simple ………………. Hemiacridinae
_. Prosternal tubercle bend at apix lateral cercus well developed ……..... Calliptaminae
4. Antennae always filliform, face approximately or fairly vertical……......Oedipodinae
_. Antennae usually ensiform, face typically very slanted .............................................. 5
5. Body small to large size, fastigial foveolae absent ……………...………… Acridinae
_. Body small to medium size, fastigial foveolae present....................... Gomphocerinae
4.3. Prevalence of Acridids in field
It can be seen from (Table I-II, Fig. I-II) that maximum No. of species belonging to sub-
family Acridinae and Oedipodinae (which comprise on 08 species) followed by
Gomphocerinae and Hemiacridinae with 05 and 04 species to Oxyinae while Calliptaminae
was reported with less numbers. It was noticed that fair No. of specimens i.e 556 with
22.06% have been captured from site-1st that comprise on Ghotki, Sukkur and Shikarpur and
least numbers i.e 269 with 10.64% were collected from Khairpur, Nawabshah and
Nausheroferoz while in case of site-1st and 3rd from lower Sindh shows the abundance of
species with 494 and 449 numbers respectively and least No. i.e 378 were collected from
Jamshoro, Hyderabad and Dadu districts.
The order of prevalence of grasshopper species was varying in both upper and lower selected
regions. The data presented in (Table III Fig. III) showed that: Acrida exaltata, A. gigantea,
Hieroglyphus banian and H. nigrorepletus dominant and prevalent species at site-1st while
H. orzivorus was quite prominent at site-2nd this showed its potential economic status in this
area opposing to this A. exaltata, A. gigantea, Chorthippus dorsatus and H. perpolita were
reported in moderate range. As regards the result of lower Sindh, 05 species of grasshoppers
among 03 i.e Locusta migratoria, Acrotylus humbertianus, Aiolopus thalassinus thalassinus
of Oedipodinae and 02 species of Oxyinae i.e O. hyla hyla and O. velox revealed the
dominant status at site-1st and H. oryzivorus and O. hyla hyla at site-2nd while
H. nigrorepletus, A. humbertianus, O. velox and O. hyla hyla at site-3rd (Table III-IV, Fig. III-
IV).
Ochrilidia geniculata and Chorthippus indus species fall into group (D). That is occasionally
of substantial importance pest addition Phlaeoba tenebrosa, H. perpolita, Spathosternum
prasiniferum and O. velox recorded as occasionally importance pest (E). Similarly, species
belonging to group (F) and (G) were A. longipes longipes, A. thalassinus thalassinus, O.
fuscovittata and A. exaltata, Truxalis exmia exmia, T. fitzgeraldi, Acorypha glaucopsis,
Gonista rotundata respectively. These are considered regular or occasional minor pest
opposing to this some species grouped as (H) and (K) i.e Oedaleus rosescens and P.
infumata, A. humbertians, Hilethera aelopoides, Trilophidia annulata respectively were
minor importance and cause less damage with negligible economic significance.
4.4. Lethal infection of entomopathogenic fungi (EPFs)
Lethal infection level of entomopathogenic fungi from upper Sindh site-I was presented in
the (Table V, Fig. V to VII). The data indicated that significant high No. of sporulation was
recorded for A. gigantea and A. exaltata i.e (71.42% and 68.42%) respectively of subfamily
Acridinae, while unknown fungal infection i.e (36.36%) followed by (31.57%) was recorded
on P. tenebrosa and A. exaltata respectively. Opposing to this, Calliptaminae was
significantly affected by unknown sporulation. As for as G. rotundata of Gomphocerinae was
concerned significantly affected by Aspergillus sporulation while infestation was not reported
on Chorthippus indus and Oxypterna afghana the sporulation ratio on sub-family
Hemiacridinae was observed 100% followed by 76.47% and 57.14% for H. oryzivorus,
H. banian and H. perpolita respectively (Table V, Fig. V).
Beside this, infection of unknown sporulation was noted i.e 66.66% for S. prasiniferum,
Aspergillus sporulation on Oedipodinae was calculated significantly highest i.e (100%, 75%
and 71.42%) on L. migratoria, A. humbertianus and A. longipes longipes respectively and
while single collected individual T. annulata was not affected. Most important rice pest
O. fuscovittata was significantly affected by Aspergillus sporulation followed by 77.77% and
71.42% for O. hyla hyla and O. velox respectively maximum ratio of unknown sporulation
was noted on the O. bidentata i.e 40% and it was 60% on Aspergillus.
In case of site-II maximum sporulation of Aspergillus was calculated for A. exaltata,
D. laticornis and P. tenebrosa while G. semipictus was sporulated by unknown fungi single
O. geniculata of Gamophocerinae was captured was not affected by any sporulation.
Maximum Aspergillus sporulation i.e 77.77% for H. banian while 100% infection of
unknown fungi was recorded on S. prasiniferum, A. thalassinus thalassinus and O. rosescens
of Oedipodinae were fully sporulated with Aspergillus i.e 90.90% for O. hyla hyla and 100%
on O. velox was reported by unknown fungi. From the site-III greater percentage of
sporulation i.e 66.66% was observed on A. exaltata and S. undulatus undulates was 100%
infected with Aspergillus, while it was not affected by Aspergillus in remaining 02 sites. In
addition to this, greater ratio of Aspergillus i.e 100% was analyzed for O. senegalensis of
Oedipodinae and O. hyla hyla of Oxyinae and unknown sporulation 100% was also recorded
for O. fuscovittata.
Further, lethal infection of entomopathogenic fungi from lower Sindh site-I was presented in
(Table VI, Fig. VIII-X). It seems from this data that maximum sporulation for P. infumata
and T. exmia exmia were recorded 100% and 90% respectively and greater sporulation i.e
100% was noted from cadavers of A. gigantea and single member of sub-family
Calliptaminae i.e Sphodromerus undulatus undulatus was significantly affected by unknown
fungi and Chorthippus dorsatus, G. rotundata and O. afghana was also affected by unknown
fungi, while 100% infection of Aspergillus was noted on H. nigrorepletus and H. perpolita
both were equally affected by sporulation. L. migratoria the most destructive pest of
Oedipodinae was affected (82.60%) with Aspergillus sporulation and (17.39%) with some
unknown fungi in sub-family Oxyinae (100%) sporulation was noted for O. bidentata and
least i.e (43.75%) was noted on O. velox. As far as site-II is concerned maximum sporulation
was calculated for A. exaltata i.e (100%) with Aspergillus sporulation and infection of
unknown fungi i.e (100%) was noted for A. gigantea and P. infumata and T. exmia exmia and
A. glaucopsis were equally affected by Aspergillus and with two unknown fungi.
Only single specimen of Ch. dorsatus was come in collection but it was not contaminated
with any sporulation and maximum ratio of unknown infection was also observed on two
species of Gomphocerinae Chorthippus indus and Oxypterna afghana, while H. nigrorepletus
was affected (69.23%) with Aspergillus and around (63.15%) with unknown sporulation was
noted on H. oryzivorus. As H. perpolita is very sluggish in nature their hoppers and adults
hide themselves under roots of vegetations due to their protective behavior, their collection
and sporulation isolation was too different. Further, Aspergillus greater sporulation ratio was
noted (63.63% and 60%) for A. humbertianus and A. thalassinus thalassinus, while 6
specimens of L. migratoria was incubated among these 100% was affected by some unknown
sporulation in this site and infection percentage for Oxyinae was noted i.e (66.66%) for O.
velox affected by Aspergillus and i.e (42.10%) with O. hyla hyla. As far as site-III is
concerned, maximum infection ratio was obtained (100%) for A. gigantea and (66.66%) for
A. glaucopsis and single species of Calliptaminae i.e S. undulatus undulatus was (100%)
affected by unknown fungi least No. of Gomphocerinae was captured from this site and
(100%) sporulation of unknown fungi was affected with C. indus and (100%) Aspergillus
sporulation was recorded on O. afghana, maximum Aspergillus infection was recorded on H.
nigrorepletus i.e (90.32%) and least infection by unknown fungi i.e (9.67%) was recorded on
this pest. A. humbertianus was significantly affected by Aspergillus and greater ratio of
unknown fungi i.e (66.66%) was recorded for H. aelopoides and L. migratoria. Maximum
infection of Aspergillus i.e (68.42% and 65.21%) was also noted for O. hyla hyla and
O. velox respectively.
4.5. Isolation and association of entomopathogenic fungi (EPFs)
During present investigation it was noted that about 15 important species of Aspergillus occur
in Pakistan (Appendix-III). Among these 3 species i.e A. niger, A. fumigatus and A. flavus
along with 2 unknown species (their taxonomic status is still uncertain possibly they were
noted opportunistic fungi against many grasshopper species) were reported on 6 sub-families
of acrididae. Most probably they were noted opportunistic fungi against large numbers of
grasshopper’s species. The isolated percentage of entomopathogenic fungi and their
association with pest species of grasshopper was presented in (Table VIII, Fig. XI).
According to this table total No. of isolated percentage of A. niger was (6.77% and 5.64%) on
A. exaltata and A. gigantea in sub-family Acridinae with (6.21%) in A. humbertianus of
Oedipodinae and (5.08%) on Truxalis exmia exmia contaminated with A. fumigatus and
O. fuscovittata infected (5.08%) with A. flavus.
During the present study, about 73 associations of entomopathogenic fungi with different
species of grasshoppers were analyzed. Amongst these associations original associations (O)
were counted 47, while earlier finding was 09 indicated with (E). Beside this, earlier and
recent recorded associations were 17 and showed with (OE). Table (XI) concise the
association between pathogenic fungi and pest species of grasshoppers it was, noted that
Truxalis exmia exmia, T. fitzgeraldi, Acrida gigantea, A. exaltata, Phlaeoba tenebrosa,
P. infumata, Gelastorhinus semipictus, Duroniella laticornis pertaining to Acridinae was
infected with entomopathogenic fungi for the first time and infection of A. fumigates was
noted for the first time on grasshopper population. As for as sub-family Oxyinae is concerned
infection of A. niger and A. fumigatus was observed for first time on two species i.e Oxya
velox and O. bidentata. However, infection of these fungi on the Oedipodinae was also
calculated in Acrotylus longipes longipes, Oedaleus senegalensis and Aiolopus thalassinus
thalassinus. During the present study, a total 10 species were recorded with infection of A.
niger, 11 species with A. fumigatus while 12 were significantly contaminated with A. flavus.
Addition to this, few species have the conidia of two unknown fungi with minimum ratio.
Table (XI) suggests that maximum numbers of Acridinae species were contaminated with
Aspergillus followed by Hemiacridinae with 05 species and 04 species to Oxyinae and
Oedipodinae. This wide range showed that many important grasshoppers sub-families
directly contamination with Aspergillus species. Table (XI) indicated that maximum infection
of A. niger was reported on the most dominant species of grasshopper followed by A. flavus
and A. fumigatus and 02 unidentified fungi.
4.6. Food consumptions and faecal production of infected insects
Food consumption and faecal production by the insects treated with different formulation of
the Aspergillus species were analyzed under laboratory conditions. Three replicate i.e A.
flavus, A. fumigatus and A. niger excluding control. It seems from (Table XII, Fig. XII a, b)
that greater reduction in faecal production was noticed after the treatment of oil formulation.
Reduction in feeding of the infected insects stages (N1-N3) was started after treatment of 1st to
2nddays. Significant reduction in faecal production was noted from 1st to 4th days after that all
immature stage consists on (N1 to N3) were died; only few individual were survive. However,
mortality of insects on day first was noted significant highest i.e [F0.48 = 84.65, P < 0.05]
followed by [F0.35 = 61.96, P < 0.05] and [F0.27 = 48.00, P < 0.05] on day 4th and 2nd
respectively. Beside this, it was extremely low i.e [F0.17 = 30.54, P < 0.05] on day 3rd (Table
XVI, Fig XVI a, b). As for as developmental stages (N4-N6) are concerned their faecal
production was significantly reduced on 2nd day i.e [F0.18 = 32.29, P < 0.05]. However, its
maximum value i.e [F0.03 = 68.94, P < 0.05] was noted on 1st day and there was no significant
difference in the faecal production of insect deposited i.e [F0.20 = 35.78, P < 0.05] on day 3rd
and 4th respectively. In control replicate the mortality ratio for stage (N4-N6) was maximum
on day 2nd i.e [F10.7 = 18.33, P < 0.05] followed by [F4.20 = 07.85, P < 0.05] and [F3.77 = 06.11,
P < 0.05] on 4th and 3rdday respectively. Similarly, it was minimum i.e [F0.48 = 84.65, P <
0.05] on day 1st.
In comparison with oil formulations the rate of faecal production of Acridid (nymphs) treated
with conidial concentration in H2O maintain in cages was shown in (Table XVI, Fig. XIV a,
b), indicate that the maximum faecal production was obtained on day 2nd i.e [F 0.24 = 42.76, P
<0.05] followed by [F 0.23 = 41.02, P < 0.05] on 5th and 6th day. However, least amount of
faecal material was obtained on day 1st i.e [F 0.08 = 14.84, P < 0.05]. Beside this, mortality of
Acridid (nymphs) population kept in large cage when treated with conidial concentration
formed in H2O was maximum on day 6th i.e [F 0.82 = 43.99, P < 0.05] and it was non-
significant i.e [F 8.5 = 14.84, P < 0.05] and [F 7.25 = 13.09, P < 0.05] on 2nd and 3rd day
respectively, while it was significant low on 5th day i.e [F 3.32 = 06.11, P < 0.05]
(Table XVIII, Fig. XVIII).
Table (XV) showed the faecal production of Acridid (adults) when treated with conidial
concentration was maximum in H2O. It was seem that greater ratio of feacal material was
obtained on day 8th i.e [F 0.22 = 39.27, P < 0.05] and it was non-significant on 2nd to 7th day
(Table XV, Fig. XV), and it was significantly low i.e [F 0.10 = 18.33, P < 0.05] on day 1st.
Beside this, faecal production of adult Acridid, cultured in small jars when treated with oil
formulation their faecal production was maximum i.e [F0.21 = 37.52, P < 0.05] was on day 6th
while, significantly least value i.e [F0.09 = 16.58, P < 0.05] was observed on 1st day while,
observation for day 2nd to 5th and 7th were non-significant (Table XIII, Fig. XIII), whereas,
mortality of Acridid adult suggests that maximum mortality was observed on day 7th i.e [F13.7
= 23.56, P < 0.05] followed by [F12.5 = 21.82, P < 0.05] on 6th day and minimum mortality i.e
[F0.44 = 77.67, P < 0.05] was noted on day 1st followed by [F0.77 = 35.26, P < 0.05] on 3rd day.
Similarly, a mortality ratio for 4th and 5th day was non-significant (Table XVII, Fig. XVII).
The result showed that cumulative percentage of feacal material of the treated insects with
various pathogenic fungi was significantly differ with control and other three treatments have
significant impact on the food consumption, feeding behavior and the mortality of acridids
when treated with H2O formulation this indicate that maximum mortality of individual was
record on day 8th i.e [F1.00 = 02.62, P < 0.05] (Table. XIX), it was significantly low i.e [F0.06 =
11.34, P < 0.05] and [F0.02 = 04.36, P < 0.05] on day 1st and 2nd. However, mortality of these
adult individual were found non-significant from day 3rd to 7th respectively (Table XIX, Fig
XIX). During the present study it was noted that insect pathogen unlike chemical insecticides
don’t have quick response on pest feeding but, after 2ndday insect gradually reduce it feeding.
However, during earlier 2nd to 3rd days insect continuous feed and consume large portion of
food supplied to them. Reduction in feeding due to pathogenic effect may affect body fat
accumulation therefore, insect become thin and sluggish day by day.
4.7. Variation in conidium shape of entomopathogenic fungi
Although, conidial ontogeny can be reduce to a comparatively small number of distinct
patterns, there is an enormous range in the form of the fully developed conidium. Conidia
may be unicellular, bicellular and multicellular conidia may be divided by septa in one to
three planes. The shape of the conidium may be varied e.g. globose, elliptical, ovoid,
cylindrical, branched, and spirally coiled. The color of the conidia (and the mycelium and
conidiophores) may be hyaline, i.e colorless, brightly colored (e.g. pink, green) or dark. The
dark pigments are probably melanins. The color of the conidiophores and conidia are
important feature used in classification that showed scaning electron microscope (SEM)
variation in conidium shape and some of them used to describe these variants. An artificial
system of classification of conidial fungi has been devised by saccardo, making use of
conidial color and from to group together similar from-genera. Such a system has its uses as
an aid to cataloguing the large number of conidial fungi, but studies of development and
information about the perfect states of some of these conidial fungi, show that the grouping
based only on conidial form and color are very artificial.
Beside this, Table (IX), Plate (XI, a-c XII, a-b) also indicated general characters of studied
fungi and two unknown fungi. This data showed that there is significant difference in
coloration and phialides pattern while the spores formulation is also different and growth
morphology of five studied fungi are differ with each other. But, for further isolation of these
Aspergillus species element concentration was also done under Scanning Electron
Microscopy (SEM).
4.8. Reproductive activities of H. oryzivorus after pathogenic treatment
During present study, some observations were also carried on reproductive activities of
H. oryzivorus which is one of important rice pest belong to acridid Table (XX-XXI) indicated
that sexual reproductive activities of H. oryzivorus was also affected by the infection of
Aspergillus. The healthy range of 6th instar maturation was given i-e 6.00±1.3 days by (Riffat
and Wagan 2010) on opposing to this, the normal individual treated with A. flavus under
laboratory condition took (8.01±1.02) days for maturation. Average normal maturation period
for adult was reported (10.93±2.6) days on contrast to this, infected individual took prolong
time for converting into adult stage. I had failed to observe the total matings time in insects
because of their less survival. (Table XX).
Beside this, healthy individual showed maximum copulation duration that remains together
for prolong time however, in case of infected samples they close together for about 7.6±3.95
hrs then, immediately leave each other and never attempt again for copulation. Similarly,
only single mating was observed in contaminated individual while there was maximum
mating i.e (12.17±4.12) observed by (Riffat, 2008) in the healthy individual of H.
oryzivorus.10 healthy females which were ready for oviposition after 24 hrs were picked
from rearing stock of grasshoppers colonies, maintained under laboratory conditions. They all
were treated with prepared medium of Aspergillus in order to know the fecundity activity of
unhealthy individual (Table XX-XXI). This suggests that oviposition time of unhealthy
samples were (21.02±0.21 mints) and female deposit only single and broken egg pods with
lesser No. of eggs i.e (17.23±0.2) and size of egg pods was (16.30±0.01 mm) this size was
reduced compare to its normal range. However, there was no significant difference in the
length of eggs.
It was very interesting to note that female secrete less quantity of brownish foamy mass
instead of yellowish and took just (3.42±0.23 minutes) for foamy mass secretion while in
case of normal individual it take (14.53±3.39 mints) for secretion of foamy mass and covered
the whole opening. Present observation showed that, A. flavus had a broad infection range.
Current investigation recommends that A. flavus not only influence the survival-ship of H.
oryzivorus but it also infect the other reproductive activities of this pest and hence could be
exploited as a microbial control agents of the H. oryzivorus in rice producing areas
(Table XXI).
4.9. Behavioral activity of insect after pathogenic treatment
It was observed that virtually all insect found susceptible to fungal disease. During this study,
following observations were taken out. Thermoregulatory behavior of acridid species was
observed in the laboratory following a spray application of oil- and water based formulation
of Aspergillus and (unsprayed) individual under laboratory conditions. All treated
grasshoppers maintained in (jars and cages) were monitored for 3 days from the second day
after application. During present study, it was noticed that infected insects altered their
thermoregulatory behavior and showed a behavioral fever response to the pathogen their
body temperature were raised as a means of literally toasting a fungal invader. Further, these
behavioral responses may result in enhanced spore diffusion and fungal fitness. This is first
indication to a microbial infection for any natural population. After the pathogenic
applications it was also noticed that the production of cuticulur antimicrobial lipids, protein,
and metabolites. Shedding of the cuticle during development and behavior environmental
adaptation that include: fever, burrowing and growing was also effective significantly.
Behavioral fever is the elevation of body temperature in infected insects above that normally
range. This can achive by infected insects by seeking out specificlocations in the environment
that are at a greater temperature, and the upshot is death or destruction of the pathogen and a
suspension in the time till death. Fever is a communal host response to many pathogens. It is
an actively costly process and is not unavoidably valuable to hosts, but there are many
examples in which the onset of fever does suppress pathogens and so decreases or deferrals
host mortality during the present study behavioral fever was observed in many species of
grasshopper. It was noticed that infected insects altered their thermoregulatory response and
showed very interesting behavioral changing that include: insect’s feeding stopped
completely, poor coordination, jerky movements, excessive grooming, loss of orientation,
confuse during mating, short mating , drop egg without searching oviposition site, ecdysis
process slow or complete stop, Behavioral fever (body temperature raised) and body fat
accumulation was also reduced. (Plate VII-X, a-c).
4.10. Elements concentration under Scanning Electron Microscopy (SEM) in various
treated entomopathogenic fungi (EPFs)
During the present study identification of fungi species has been carried out under (SEM)
according to SEM observation spectrums acquisition of A. niger presented in (Table XXII-
XXIV, Fig. XX-XXII) indicates that normal weightage % of Oxygen (O2) was highest i.e
56.19% (with 17.5 error) followed by 42.60% (with 13.1 error) Carbon (C) and very least
ratio of Sodium (Na) i.e 1.21% (with 0.1 error) was analyzed in A. niger. As far as chemical
composition of A, flavus is concerned the normal weightage % value of Carbon (C) was
found highest i.e 52.33% (with 16.1 error) followed by Oxygen (O2) 46.84% (with 14.5 error)
opposing to this, least percentage was calculated for Sodium (Na). Similarly, in case of A.
fumigatus the greater normal weightage % was noted for Oxygen (O2) i.e 54.61% followed
by (with 17.1 error), 43.92% Carbon (C) (with 13.6 error). However, remaining elements
concentration values for Sodium (Na), Sulphur (S) and Phosphorus (P) were recorded i.e
0.92%, 0.35% and 0.20% respectively with very minimum error value.
Beside this, normal weightage % of 2 unknown fungi was presented in (Table XXV-XXVI,
Fig. XXIII-XXIV). According to these observations unknown fungi I (Uk FI) having greater
value of carbon (C) i.e 62.82 with error of 19.2%, while Oxygen (O2) value was calculated
36.82% with 11.4 error and Sodium (Na) contributed minimum concentration. In case of
unknown fungi II (Uk FII) 05 elements were deducted through spectrum acquisition in
Carbon (C) this ratio was higher i.e 54% with 16.6% error value, followed by Oxygen (O2)
43.53% with 13.6% error count. Opposing to this, Sulphur (S) and Sodium (Na) values were
minimum i.e 0.79% and 0.1% error value. Over all, it was noticed that elements
concentration values of these three entomopathogenic fungi including two unknown fungi
was significantly differ with each other the elements concentration analysis, may also led to
the correct identification of fungi in future ( Plate XIV, a-d).
Table .I. Showing the collected number of pest species from upper Sindh during the
year 2013-2015.
Sub-family/Species/Sub-species
Localities with number of specimens collected
1st
Sites (556) 2nd
Sites (374) 3rd
Sites (269)
GHT SKR SHK LKA JBA KSM KHP NBS NSF
Acrid
ina
e
Acrida exaltata A. gigantea Duroniella laticornis Gelastorhinus semipictus Phlaeoba infumata P. tenebrosa Truxalis exmia exmia
T. fitzgeraldi
19 32 - - 7
17 4
2
27 7 - 3 2
21 5
1
13 23 3 2 3 - 7
1
9 5 - 4 - 9 1
-
9 7 4 2 3 1 -
2
6 7 5 - - 1 2
-
7 31 9 3 - 1 2
1
6 21 - - 7 2 -
-
7 11 4 7 2 - 1
1
Ca
llip
tam
ina
e
Acorypha glaucopsis
Sphodromerus undulatus undulatus
-
2
-
1
10
-
7
2
9
-
7
3
2
2
-
1
-
1
Gom
ph
ocer
ina
e
Chorthippus indus
Ch. dorsatus Gonista rotundata Ochrilidia geniculata Oxypterna afghana
1
- 1 - 2
1
- 2 1 3
2
- 4 1 5
-
2 3 - -
1
1 - 1 4
-
3 2 - 6
1
5 - 1 -
1
6 - - -
2
11 1 - 2
Hem
iacri
din
ae
Hieroglyphus banian H. nigrorepletus H. oryzivorus H. perpolita Spathosternum prasiniferum
- 21 7 9 2
30 19 5
32 3
21 20 - 5 5
- -
19 - 1
11 -
31 - 2
19 -
21 - 1
1 9 -
17 3
- - - 7 2
- 8 - 5 -
Oed
ipo
din
ae
Acrotylus humbertianus A. longipes longipes Aiolopus thalassinus thalassinus
Hilethera aelopoides Locusta migratoria Oedaleus rosescens O. senegalensis Trilophidia annulata
7 13 1
1 7 2 1 -
5 - 2
- 8 - 3 -
2 6 -
- - 1 2 1
2 - 4
1 - 3 2 1
3 3 3
- 13 - - 1
- 2 1
- 8 2 1 -
1 1 -
1 11 1 2 1
2 - 2
- - - 1 -
6 - 1
1 - 1 2 1
Oxy
ina
e
Oxya bidentata O. fuscovittata O. hyla hyla O. velox
3 1 5 -
2 3
11 2
13 9
12 19
8 9
17 5
9 10 8 2
9 6 7 1
- - 1 -
- 1 1 -
3 9 8 -
Note: GHT= Ghotki, SKR= Sukkur, SHK= Shikarpur, LKA= Larkana, JBA= Jacobabad, KSM= Kashmore, KHP= Khairpur, NBS= Nawabshah (Benazirabad), NSF= Nausheroferoz
Table.II. Showing the collected number of pest species from lower Sindh during the
year 2013-2015.
Sub-family/Species/Sub-species
Localities with number of specimens collected
1st
Sites (494) 2nd
Sites (378) 3rd
Sites (449)
THR UKT MPK JAM HYD DDU SNG BDN MTR
Acrid
ina
e
Acrida exaltata A. gigantea Duroniella laticornis Gelastorhinus semipictus Phlaeoba infumata P. tenebrosa Truxalis exmia exmia
T. fitzgeraldi
13 7 1 - 1 -
17
6
7 1 - 1 2 - 9
3
3 1 1 - 1 1 7
2
1 2 3 1 3 1 1
-
3 - - - 2 2 -
-
2 1 - 1 2 1 6
-
7 2 1
10 - - 3
2
6 1 3
13 1 - 3
1
3 5 2 9 1 - 1
2
Ca
llip
tam
ina
e
Acorypha glaucopsis Sphodromerus undulatus undulatus
7 2
3 1
6 2
7 -
2 -
1 -
3 -
5 3
4 2
Gom
ph
ocer
ina
e
Chorthippus indus Ch. dorsatus Gonista rotundata Ochrilidia geniculata Oxypterna afghana
- 1 1 3 3
- - 1 - 2
- 2 1 - 3
1 - - - 2
- 1 - - 1
3 - 1 - -
2 2 - 1 1
- 1 1 - 4
1 1 - - -
Hem
iacri
din
ae
Hieroglyphus banian H. nigrorepletus H. oryzivorus
H. perpolita Spathosternum prasiniferum
- 1 -
3 -
- 2 -
2 -
- - -
1 -
- 7 -
2 -
- 13 -
3 1
- 23 63
- 2
1 31 -
- -
5 43 -
3 -
3 10 -
2 1
Oed
ipo
din
ae
Acrotylus humbertianus A. longipes longipes Aiolopus thalassinus thalassinus
Hilethera aelopoides Locusta migratoria Oedaleus rosescens O. senegalensis Trilophidia annulata
27 2
21
13 37 - 2 -
13 7
23
10 28 - 1 -
26 3
17
7 9 2 - -
20 6
19
6 2 1 - 1
4 -
10
3 1 - 1 -
13 5 6
4 3 - 1 2
24 4 8
5 5 - 1 -
13 3 7
2 7 1 - 1
17 9 3
7 3 - - -
Oxy
ina
e
Oxya bidentata O. fuscovittata O. hyla hyla O. velox
- 1
17 23
2 -
10 19
1 -
32 10
- 1 8 7
- -
17 23
1 1
37 10
1 - 7 8
- -
42 53
- - 7 5
Note: THR= Tharparkar, UKT= Umerkot, MPK= Mirpurkhas, JAM= Jamshoro, HYD= Hyderabad,
DDU= Dadu, SNG= Sanghar, BDN= Badin, MTR= Matiari
Table.III. Showing the total No. of grasshopper’s species caught from three sites of
upper Sindh in the year 2013-2015.
Sub-family/Species/Sub-species
Site – I
(n= 556)
Site – II
(n= 374)
Site – III
(n= 269)
No. of
Caught
Species
Rank
No. of
Caught
Species
Rank
No. of
Caught
Species
Rank
Acrid
ina
e
Acrida exaltata 59 DS+++ 24 MS
++ 20 MS++
A. gigantea 62 DS+++ 19 LS
+ 63 MS++
Duroniella laticornis 3 LS+ 9 LS
+ 13 LS+
Gelastorhinus semipictus 5 LS+ 6 LS
+ 10 LS+
Phlaeoba infumata 12 LS+ 3 LS
+ 9 LS+
P. tenebrosa 38 MS++ 11 LS
+ 3 LS+
Truxalis exmia exmia 16 LS+ 3 LS
+ 3 LS+
T. fitzgeraldi 4 LS+ 2 LS
+ 2 LS+
Ca
llip
tam
ina
e
Acorypha glaucopsis 10 LS+ 23 MS
++ 2 LS+
Sphodromerus undulatus undulatus 3 LS+ 5 LS
+ 4 LS+
Gom
ph
ocer
ina
e
Chorthippus indus 4 LS+ 1 LS
+ 4 LS+
Ch. dorsatus --- LS+ 6 LS
+ 22 MS++
Gonista rotundata 7 LS+ 5 LS
+ 1 LS+
Ochrilidia geniculate 2 LS+ 1 LS
+ 1 LS+
Oxypterna afghana 10 LS+ 10 LS
+ 2 LS+
Hem
iacri
din
ae Hieroglyphus banian 51 DS
+++ 30 MS++ 1 LS
+
H. nigrorepletus 60 DS+++ - LS
+ 17 LS+
H. oryzivorus 12 LS+ 71 DS
+++ - LS+
H. perpolita 46 MS++ - LS
+ 29 MS++
Spathosternum prasiniferum 10 LS+ 4 LS
+ 5 LS+
Oed
ipo
din
ae
Acrotylus humbertianus 14 LS+ 5 LS
+ 9 LS+
A. longipes longipes 19 LS+ 5 LS
+ 1 LS+
Aiolopus thalassinus thalassinus 3 LS+ 8 LS
+ 3 LS+
Hilethera aelopoides 1 LS+ 1 LS
+ 2 LS+
Locusta migratoria 15 LS+ 21 MS
++ 11 LS+
Oedaleus rosescens 3 LS+ 5 LS
+ 2 LS+
O. senegalensis 6 LS+ 3 LS
+ 5 LS+
Trilophidia annulata 1 LS+ 2 LS
+ 2 LS+
Oxy
ina
e
Oxya bidentata 18 LS+ 26 MS
++ 3 LS+
O. fuscovittata 13 LS+ 25 MS
++ 10 LS+
O. hyla hyla 28 MS++ 32 MS
++ 10 LS+
O. velox 21 LS+ 8 LS
+ - LS+
Note: Key to the species rank according to it pest status is under: Dominant Status (DS+++), Moderate Status (MS++) and Low Status (LS+)
Table.IV. Showing the total No. of grasshopper’s species caught from three sites of
lower Sindh in the year 2013-2015.
Sub-family/Species/Sub-species
Site – I
(n= 494)
Site – II
(n= 378)
Site – III
(n= 449)
No. of
Caught
Species
Rank
No. of
Caught
Species
Rank
No. of
Caught
Species
Rank
Acrid
ina
e
Acrida exaltata 23 MS++ 6 LS
+ 16 LS+
A. gigantea 9 LS+ 3 LS
+ 8 LS+
Duroniella laticornis 2 LS+ 3 LS
+ 6 LS+
Gelastorhinus semipictus 1 LS+ 2 LS
+ 32 MS++
Phlaeoba infumata 4 LS+ 7 LS
+ 2 LS+
P. tenebrosa 1 LS+ 4 LS
+ - LS+
Truxalis exmia exmia 33 MS++ 7 LS
+ 7 LS+
T. fitzgeraldi 11 LS+ - LS
+ 5 LS+
Ca
llip
tam
ina
e
Acorypha glaucopsis 16 LS+ 10 LS
+ 12 LS+
Sphodromerus undulatus undulatus 5 LS+ - LS
+ 5 LS+
Gom
ph
ocer
ina
e
Chorthippus indus - LS+ 4 LS
+ 3 LS+
Ch. dorsatus 3 LS+ 1 LS
+ 4 LS+
Gonista rotundata 3 LS+ 1 LS
+ 1 LS+
Ochrilidia geniculate 3 LS+ - LS
+ 1 LS+
Oxypterna afghana 8 LS+ 3 LS
+ 5 LS+
Hem
iacri
din
ae Hieroglyphus banian - LS
+ - LS+ 9 LS
+
H. nigrorepletus 3 LS+ 43 MS
++ 84 DS+++
H. oryzivorus - LS+ 63 DS
+++ - LS+
H. perpolita 6 LS+ 5 LS
+ 5 LS+
Spathosternum prasiniferum - LS+ 3 LS
+ 1 LS+
Oed
ipo
din
ae
Acrotylus humbertianus 66 DS+++ 37 MS
++ 54 DS+++
A. longipes longipes 12 LS+ 11 LS
+ 16 LS+
Aiolopus thalassinus thalassinus 61 DS+++ 35 MS
++ 18 LS+
Hilethera aelopoides 30 MS++ 13 LS
+ 14 LS+
Locusta migratoria 74 DS+++ 6 LS
+ 15 LS+
Oedaleus rosescens 2 LS+ 1 LS
+ 1 LS+
O. senegalensis 3 LS+ 2 LS
+ 1 LS+
Trilophidia annulata - LS+ 3 LS
+ 1 LS+
Oxy
ina
e
Oxya bidentata 3 LS+ 1 LS
+ 1 LS+
O. fuscovittata 1 LS+ 2 LS
+ - LS+
O. hyla hyla 59 DS+++ 62 DS
+++ 56 DS+++
O. velox 52 DS+++ 40 MS
++ 66 DS+++
Note: Key to the species rank according to it pest status is under: Dominant Status (DS+++), Moderate Status (MS++) and Low Status (LS+)
Table.V. Lethal infection level of entomopathogenic fungi in various species of
grasshoppers collected from three sites of upper Sindh in the year 2013-2015.
Site – I
Sub-family/Species/Sub-species No. of
Incubated
No. of
Sporulation
No. of
Aspergillus
Sporulation
Unknown
Sporulation
% of Infection
Asp. Uk
Acrid
ina
e
Acrida exaltata 59 19 13 6 68.42 31.57
A. gigantea 62 21 15 6 71.42 28.57
Duroniella laticornis 3 - - - - -
Gelastorhinus semipictus 5 1 1 - 100 -
Phlaeoba infumata 12 1 - 1 - 100
P. tenebrosa 38 11 7 4 63.63 36.36
Truxalis exmia exmia 16 4 3 1 75 25
T. fitzgeraldi 4 - - - - -
Ca
llip
tam
ina
e
Acorypha glaucopsis 10 2 - 2 - 100
Sphodromerus undulatus undulatus 3 1 - 1 - 100
Gom
ph
ocer
ina
e
Chorthippus indus 4 - - - - -
Ch. dorsatus - - - - - -
Gonista rotundata 7 2 2 - 100 -
Ochrilidia geniculate 2 - - - - -
Oxypterna afghana 10 4 - - - -
Hem
iacri
din
ae Hieroglyphus banian 51 17 13 4 76.47 23.52
H. nigrorepletus 60 21 9 12 42.85 57.14
H. oryzivorus 12 3 3 - 100 -
H. perpolita 46 4 8 4 57.14 42.85
Spathosternum prasiniferum 10 3 1 2 33.33 66.66
Oed
ipo
din
ae
Acrotylus humbertianus 14 4 3 1 75 25
A. longipes longipes 19 7 5 2 71.42 28.57
Aiolopus thalassinus thalassinus 3 - - - - -
Hilethera aelopoides 1 - - - - -
Locusta migratoria 15 4 4 - 100 -
Oedaleus rosescens 3 - - - - -
O. senegalensis 6 2 1 1 50 50
Trilophidia annulata 1 - - - - -
Oxy
ina
e
Oxya bidentata 18 5 3 2 60 40
O. fuscovittata 13 3 3 - 100 -
O. hyla hyla 28 9 7 2 77.77 22.22
O. velox
21 7 5 2 71.42 28.57
Site – II
Sub-family/Species/Sub-species No. of
Incubated
No. of
Sporulation
No. of
Aspergillus Sporulation
Unknown
Sporulation
% of Infection
Asp. Uk
Acrid
ina
e
Acrida exaltata 24 7 7 - 100 -
A. gigantea 19 5 3 2 60 40
Duroniella laticornis 9 2 2 - 100 -
Gelastorhinus semipictus 6 1 - 1 - 100
Phlaeoba infumata 3 - - - - -
P. tenebrosa 11 2 2 - 100 -
Truxalis exmia exmia 3 - - - - -
T. fitzgeraldi 2 - - - - -
Ca
llip
tam
ina
e
Acorypha glaucopsis 23 6 5 1 83.33 16.66
Sphodromerus undulatus undulatus 5 1 - 1 - 100
Gom
ph
ocer
ina
e
Chorthippus indus 1 - - - - -
Ch. dorsatus 6 1 1 - 100 -
Gonista rotundata 5 2 - 2 - 100
Ochrilidia geniculate 1 - - - - -
Oxypterna afghana 10 2 - 2 - 100
Hem
iacri
din
ae
Hieroglyphus banian 30 9 7 2 77.77 22.22
H. nigrorepletus - - - - - -
H. oryzivorus 71 22 13 9 59.09 40.90
H. perpolita - - - - - -
Spathosternum prasiniferum 4 1 - 1 - 100
Oed
ipo
din
ae
Acrotylus humbertianus 5 2 1 1 50 50
A.longipes longipes 5 - - - - -
Aiolopus thalassinus thalassinus 8 2 2 - 100 -
Hilethera aelopoides 1 - - - - -
Locusta migratoria 21 6 5 1 83.33 16.66
Oedaleus rosescens 5 1 1 - 100 -
O. senegalensis 3 - - - - -
Trilophidia annulata 2 - - - - -
Oxy
ina
e
Oxya bidentata 26 9 7 2 77.77 22.22
O. fuscovittata 25 7 4 3 57.14 42.85
O. hyla hyla 32 11 10 1 90.90 9.09
O. velox 8 2 - 2 - 100
Site – III
Sub-family/Species/Sub-species No. of
Incubated
No. of
Sporulation
No. of
Aspergillus Sporulation
Unknown
Sporulation
% of Infection
Asp. Uk
Acrid
ina
e
Acrida exaltata 20 6 4 2 66.66 33.33
A. gigantea 63 20 13 7 65 35
Duroniella laticornis 13 4 2 2 50 50
Gelastorhinus semipictus 10 1 - 1 - 100
Phlaeoba infumata 9 1 - 1 - 100
P. tenebrosa 3 - - - - -
Truxalis exmia exmia 3 - - - - -
T. fitzgeraldi 2 - - - - -
Ca
llip
tam
ina
e
Acorypha glaucopsis 2 - - - - -
Sphodromerus undulatus undulatus 4 1 1 - 100 -
Gom
ph
ocer
ina
e
Chorthippus indus 4 1 1 - 100 -
Ch. dorsatus 22 7 5 2 71.42 28.57
Gonista rotundata 1 - - - - -
Ochrilidia geniculate 1 - - - - -
Oxypterna afghana 2 - - - - -
Hem
iacri
din
ae Hieroglyphus banian 1 - - - - -
H. nigrorepletus 17 4 3 1 75 25
H. oryzivorus - - - - - -
H. perpolita 29 8 7 1 87.5 12.5
Spathosternum prasiniferum 5 1 1 - 100 -
Oed
ipo
din
ae
Acrotylus humbertianus 9 2 1 1 50 50
A. longipes longipes 1 - - - - -
Aiolopus thalassinus thalassinus 3 - - - - -
Hilethera aelopoides 2 - - - - -
Locusta migratoria 11 2 1 1 50 50
Oedaleus rosescens 2 - - - - -
O. senegalensis 5 1 1 - 100 -
Trilophidia annulata 2 - - - - -
Oxy
ina
e
Oxya bidentata 3 - - - - -
O. fuscovittata 10 2 - 2 - 100
O. hyla hyla 10 3 3 - 100 -
O. velox - - - - - -
Note: Overall it was found that small size insect contaminated easily as compare to large size insects. Asp. = Aspergillus, Uk. = Unknown fungi
Table.VI. Lethal infection level of entomopathogenic fungi in various species of
grasshoppers collected from three sites of lower Sindh in the year 2013-2015.
Site – I
Sub-family/Species/Sub-species No. of
Incubated
No. of
Sporulation
No. of
Aspergillus
Sporulation
Unknown
Sporulation
% of Infection
Asp. Uk
Acrid
ina
e
Acrida exaltata 23 7 5 2 71.42 28.57
A. gigantea 9 1 - 1 - 100
Duroniella laticornis 2 - - - - -
Gelastorhinus semipictus 1 - - - - -
Phlaeoba infumata 4 1 1 - 100 -
P. tenebrosa 1 - - - - -
Truxalis exmia exmia 33 10 9 1 90 10
T. fitzgeraldi 11 3 2 1 66.66 33.33
Ca
llip
tam
ina
e
Acorypha glaucopsis 16 5 1 4 20 80
Sphodromerus undulatus undulatus 5 1 - 1 - 100
Gom
ph
ocer
ina
e
Chorthippus indus - - - - - -
Ch. dorsatus 3 1 - 1 - 100
Gonista rotundata 3 1 - 1 - 100
Ochrilidia geniculate 3 - - - - -
Oxypterna afghana 8 2 - 2 - 100
Hem
iacri
din
ae Hieroglyphus banian - - - - - -
H. nigrorepletus 3 1 1 - 100 -
H. oryzivorus - - - - - -
H. perpolita 6 2 1 1 50 50
Spathosternum prasiniferum - - - - - -
Oed
ipo
din
ae
Acrotylus humbertianus 66 21 15 6 71.42 28.57
A. longipes longipes 12 3 2 1 66.66 33.33
Aiolopus thalassinus thalassinus 61 19 15 4 78.94 21.05
Hilethera aelopoides 30 8 6 2 75 25
Locusta migratoria 74 23 19 4 82.60 17.39
Oedaleus rosescens 2 - - - - -
O. senegalensis 3 - - - - -
Trilophidia annulata - - - - - -
Oxy
ina
e
Oxya bidentata 3 1 1 - 100 -
O. fuscovittata 1 - - - - -
O. hyla hyla 59 18 12 6 66.66 33.33
O. velox 52 16 7 9 43.75 56.25
Site – II
Sub-family/Species/Sub-species No. of
Incubated
No. of
Sporulation
No. of
Aspergillus Sporulation
Unknown
Sporulation
% of Infection
Asp. Uk
Acrid
ina
e
Acrida exaltata 6 2 2 - 100 -
A. gigantea 3 1 - 1 - 100
Duroniella laticornis 3 - - - - -
Gelastorhinus semipictus 2 - - - - -
Phlaeoba infumata 7 1 - 1 - 100
P. tenebrosa 4 - - - - -
Truxalis exmia exmia 7 2 1 1 50 50
T. fitzgeraldi - - - - - -
Ca
llip
tam
ina
e
Acorypha glaucopsis 10 2 1 1 50 50
Sphodromerus undulatus undulatus - - - - - -
Gom
ph
ocer
ina
e
Chorthippus indus 4 2 - 2 - 100
Ch. dorsatus 1 - - - - -
Gonista rotundata 1 - - - - -
Ochrilidia geniculate - - - - - -
Oxypterna afghana 3 1 - 1 - 100
Hem
iacri
din
ae
Hieroglyphus banian - - - - - -
H. nigrorepletus 43 13 9 4 69.23 30.76
H. oryzivorus 63 19 7 12 36.84 63.15
H. perpolita 5 1 - - - -
Spathosternum prasiniferum 3 - - - - -
Oed
ipo
din
ae
Acrotylus humbertianus 37 11 7 4 63.63 36.36
A. longipes longipes 11 3 1 2 33.33 66.66
Aiolopus thalassinus thalassinus 35 10 6 4 60 40
Hilethera aelopoides 13 3 1 2 33.33 66.66
Locusta migratoria 6 1 - 1 - 100
Oedaleus rosescens 1 - - - - -
O. senegalensis 2 - - - - -
Trilophidia annulata 3 - - - - -
Oxy
ina
e
Oxya bidentata 1 - - - - -
O. fuscovittata 2 - - - - -
O. hyla hyla 62 19 8 11 42.10 57.89
O. velox 40 12 8 4 66.66 33.33
Site – III
Sub-family/Species/Sub-species No. of
Incubated
No. of
Sporulation
No. of
Aspergillus Sporulation
Unknown
Sporulation
% of Infection
Asp. Uk
Acrid
ina
e
Acrida exaltata 16 5 3 2 60 40
A. gigantea 8 2 2 - 100 -
Duroniella laticornis 6 2 - 2 - 100
Gelastorhinus semipictus 32 9 4 5 44.44 55.55
Phlaeoba infumata 2 - - - - -
P. tenebrosa - - - - - -
Truxalis exmia exmia 7 1 - - - -
T. fitzgeraldi 5 1 - - - -
Ca
llip
tam
ina
e
Acorypha glaucopsis 12 3 2 1 66.66 33.33
Sphodromerus undulatus undulatus 5 1 - 1 - 100
Gom
ph
ocer
ina
e
Chorthippus indus 3 1 - 1 - 100
Ch. dorsatus 4 1 - 1 - 100
Gonista rotundata 1 - - - - -
Ochrilidia geniculate 1 - - - - -
Oxypterna afghana 5 1 1 - 100 -
Hem
iacri
din
ae Hieroglyphus banian 9 2 1 1 50 50
H. nigrorepletus 84 31 28 3 90.32 9.67
H. oryzivorus - - - - - -
H. perpolita 5 - - - - -
Spathosternum prasiniferum 1 - - - - -
Oed
ipo
din
ae
Acrotylus humbertianus 54 17 13 4 76.47 23.52
A. longipes longipes 16 5 3 2 60 40
Aiolopus thalassinus thalassinus 18 5 1 4 20 80
Hilethera aelopoides 14 3 1 2 33.33 66.66
Locusta migratoria 15 3 1 2 33.33 66.66
Oedaleus rosescens 1 - - - - -
O. senegalensis 1 - - - - -
Trilophidia annulata 1 - - - - -
Oxy
ina
e
Oxya bidentata 1 - - - - -
O. fuscovittata - - - - - -
O. hyla hyla 56 19 13 6 68.42 31.57
O. velox 66 23 15 8 65.21 34.78
Note: Overall it was found that small size insect contaminated easily as compare to large size insects. Asp. = Aspergillus, Uk. = Unknown fungi
Table.VII. Showing the insect along with their major and minor target habitats.
Taxonomic status of insects
Targeted Habitat Total No.
of insects
(N= 2520) Major Minor
Acrid
ina
e
Acrida exaltata Maize, Wheat Fodder crops 148
A. gigantea Grass, Alfalfa Cotton 164
Duroniella laticornis Maize Sugarcane 36
Gelastorhinus semipictus Wheat Millet 56
Phlaeoba infumata Maize Sun hemp 37
P. tenebrosa Alfalfa Grass, Fodder crops 57
Truxalis exmia exmia Meadow grass Thorn weed 69
T. fitzgeraldi Millet, Maize Maize 24
Ca
llip
tam
ina
e
Acorypha glaucopsis Rice Mustard 73
Sphodromerus undulatus undulatus Jawar, Millet Bahamas grass 22
Gom
ph
ocer
ina
e
Chorthippus indus Rice, Cotton Crab grass 16
Ch. dorsatus Grasses Fodder crops 36
Gonista rotundata Sugarcane Vegetable 18
Ochrilidia geniculate Maize Fodder crops 8
Oxypterna afghana Rice Cabbage 38
Hem
iacri
din
ae
Hieroglyphus banian Rice, Sugarcane, Maize Thorny vegetation 91
H. nigrorepletus Rice, Sugarcane Maize, Wheat 207
H. oryzivorus Rice, Cotton Sugarcane 146
H. perpolita Surrkanda Grass, Maize 91
Spathosternum prasiniferum Maize, Rice, Sugarcane Vegetable, Fruits 23
Oed
ipo
din
ae
Acrotylus humbertianus Grasses, Vegetable Maize 185
A. longipes longipes Vegetable Cotton 64
Aiolopus thalassinus thalassinus Bajra Wheat 128
Hilethera aelopoides Jawar, Maize Cauliflower 61
Locusta migratoria Rice, Sugarcane Wheat, Cotton 142
Oedaleus rosescens Grain Vegetable 14
O. senegalensis Grasses, Maize Flower 20
Trilophidia annulata Rice, Grasses Bermuda grass 9
Oxy
ina
e
Oxya bidentata Rice, Maize Bind weed 52
O. fuscovittata Rice, Maize Cereal plant 51
O. hyla hyla Rice, Maize Wheat, Grasses 247
O. velox Rice, Maize Maize, Jower 187
Table.VIII. Showing the isolated percentage of entomopathogenic fungi and their
association with pest species of grasshoppers during the year 2014 from Sindh.
Pest species of grasshopper
Associated fungi
species on
host species
No. of isolated
fungi
(N=177)
Isolation
percentage (%)
Acrid
ina
e
Acrida exaltata Aspergillus niger 12 6.77%
A. gigantea A.niger 10 5.64%
Duroniella laticornis A. flavus 03 1.69%
Gelastorhinus semipictus A.niger 02 1.12%
Phlaeoba infumata A. flavus 05 2.82%
P. tenebrosa A.niger 03 1.69%
Truxalis exmia exmia A.fumigatus 09 5.08%
T. fitzgeraldi A.niger 05 2.82%
Ca
llip
tam
ina
e Acorypha glaucopsis A. flavus 08 4.51%
Sphodromerus undulatus undulatus A.niger 06 3.38%
Gom
ph
ocer
ina
e Chorthippus indus A. flavus 07 3.95%
Ch. dorsatus A.fumigatus 06 3.38%
Gonista rotundata A.niger 05 2.82%
Ochrilidia geniculate A.niger 02 1.12%
Oxypterna afghana A.niger 03 1.69%
Hem
iacri
din
ae Hieroglyphus banian A. flavus 01 0.56%
H. nigrorepletus A.niger 03 1.69%
H. oryzivorus A. flavus 03 1.69%
H. perpolita A.niger 04 2.25%
Spathosternum prasiniferum A.fumigatus 02 1.12%
Oed
ipo
din
ae
Acrotylus humbertianus A.niger 11 6.21%
A. longipes longipes A. flavus 03 1.69%
Aiolopus thalassinus thalassinus A.fumigatus 10 5.64%
Hilethera aelopoides A.niger 02 1.12%
Locusta migratoria A. flavus 10 5.64%
Oedaleus rosescens A.niger 02 1.12%
O. senegalensis A.niger 04 2.25%
Trilophidia annulata A.niger 06 3.38%
Oxy
ina
e Oxya bidentata A.niger 06 3.38%
O. fuscovittata A. flavus 09 5.08%
O. hyla hyla A.fumigatus 07 3.95%
O. velox A.niger 08 4.51%
Table.IX. Identification of entomopathogenic fungi isolated from acridid population.
Growth
Morphology Color Phialides Spores
Probable Organisms
Fast growing and
heavily sporing Dirty Green
Typically radiate
(Splitting to several
poorly defined
column)
Typically
globose to
subglobose
Aspergillus flavus
Fast growing and
heavily sporing
Black to dark
brown
Globose, Tangled
(Splitting into
columns)
Rough
echinulated
globose conidia
A. niger
Fast growing and
moderately
sporing
Grey-Green Chainbasipetally
Conidia
(air borne
spores)
A. fumigatus
Dense
sporangiophores Ash-coloured Not seen Not seen Unknown fungus
Note: International Mycological Institute (IMI) manual of pathogenic fungi and bacteria (1983).
Table.X. Showing the list of ecological association between entomopathogenic fungi and
insects recorded in the year 2013-2015 from selected sites of Sindh province.
Entomopathogenic Fungi Original host Place of detection
(District) Year
Aspergillus niger Acrida exaltata Umerkot 2013
A. flavus Duroniella laticornis Jamshoro 2014
A. niger Gelastorhinus semipictus Sukkur 2014
A. niger Phlaeoba infumata Khairpur 2013
A. fumigates P. tenebrosa Ghotki 2015
A. fumigates Truxalis exmia exmia Tharparkar 2014
A. flavus Acorypha glaucopsis Badin 2014
A. niger Ch. dorsatus Sanghar 2015
A. flavus Sphodromerus undulatus undulatus Sukkur 2014
A. fumigates Chorthippus indus Mirpurkhas 2013
A. flavus Gonista rotundata Matiari 2014
A.niger Oxypterna afghana Sukkur 2015
A. niger Hieroglyphus nigrorepletus Kashmore 2015
A. niger H.oryzivorus Larkana 2014
A. niger H. perpolita Jacobabad 2014
A.flavus Spathosternum prasiniferum Shikarpur 2013
A. niger Acrotylus humbertianus Umerkot 2015
A. niger Aiolopus thalassinus thalassinus Tharparkar 2013
A. niger Hilethera aeolopoides Khairpur 2014
A. niger Locusta migratoria Umerkot 2015
A. fumigates Oedaleus rosescens Mirpurkhas 2013
A. niger O. senegalensis Khairpur 2014
A. niger Trilophidia annulata Umerkot 2013
A. flavus Oxya fuscovittata Larkana 2015
A. niger O. hyla hyla Ghotki 2013
A. niger O. velox Sukkur 2015
Table.XI. Showing the association between Aspergillus species (EPF) and pest species of
grasshoppers recorded by earlier workers (Shah et al., 1994, 1998) and original data.
Host Entomopathogenic fungi A.
niger
A.
fumigatus
A.
Flavus
uknown
fungi I
Uknown
fungi II Sub-family/Species/Sub-species
Acrid
ina
e
Acrida exaltata O N O N N
A. gigantea O N N O N
Duroniella laticornis N O N N O
Gelastorhinus semipictus N N O N N
Phlaeoba infumata O N N N N
P. tenebrosa N O N O N
Truxalis exmia exmia N N O N O
T. fitzgeraldi OE OE O N N
Ca
llip
tam
ina
e Acorypha glaucopsis O N N E O
Sphodromerus undulatus undulatus E O OE N N
Gom
ph
ocer
ina
e Chorthippus Indus OE OE OE N N
Ch. dorsatus OE E OE N O
Gonista rotundata O O N N N
Ochrilidia geniculate OE O OE O N
Oxypterna afghana O N O N N
Hem
iacri
din
ae Hieroglyphus banian N O N N O
H. nigrorepletus N N E N N
H. oryzivorus E OE E OE N
H. perpolita N O N N N
Spathosternum prasiniferum N N O N O
Oed
ipo
din
ae
Acrotylus humbertianus O N O N N
A. longipes longipes O N N O N
Aiolopus thalassinus thalassinus N O O N N
Hilethera aelopoides N O N N O
Locusta migratoria N N O N N
Oedaleus rosescens O N N N N
O. senegalensis N O N O N
Trilophidia annulata N N O N O
Oxy
ina
e Oxya bidentata OE OE O N N
O. fuscovittata O N N E O
O. hyla hyla OE E OE N O
O. velox E O OE N N
Note: O = Original data, E = Earlier finding, OE = Earlier & Recent finding N= Not recorded.
Table.XII. Showing the faecal production of immature Acridid culture in small jars under laboratory conditions (after treatment of
Aspergillus oil formulation).
a) Nymphs Stages 1st to 3
rd
Treatments Days of observation (Mean ± SE)
1st 2
nd 3
rd 4
th 5
th 6
th 7
th
A. flavus 0.020±3.242b 0.031±4.374b 0.033±2.827c 0.028±2.733c 0.00±0.00 0.00±0.00 0.00±0.00
A.fumigatus 0.019±2.598c 0.029±4.995c 0.031±3.550b 0.030±3.099b 0.00±0.00 0.00±0.00 0.00±0.00
A.niger 0.017±2.766c 0.030±3.181b 0.035±2.820c 0.00±0.00d 0.00±0.00 0.00±0.00 0.00±0.00
Control 0.692±0.033a 0.641±0.040a 0.794±0.040a 0.715±0.026a 0.00±0.00 0.00±0.00 0.00±0.00
F.(0.05) (0.18) 32.29 (0.18) 32.29 (0.22) 39.27 (0.19) 34.03 ------- ------- -------
b) Nymphs Stages 4th
to 6th
Treatments Days of observation (Mean ± SE)
1st 2
nd 3
rd 4
th 5
th 6
th 7
th
A. flavus 0.022±5.042c 0.026±3.643b 0.032±2.874b 0.031±3.181c 0.00±0.00b 0.00±0.00b 0.00±0.00b
A.fumigatus 0.032±4.395d 0.025±3.562c 0.030±2.947c 0.032±2.582c 0.00±0.00b 0.00±0.00b 0.00±0.00b
A.niger 0.026±5.740b 0.024±2.283c 0.031±3.137b 0.033±2.769b 0.00±0.00b 0.00±0.00b 0.00±0.00b
Control 0.077±7.781a 0.657±0.047a 0.745±0.044a 0.707±0.030a 0.032±3.501a 0.714±0.031a 0.778±0.040a
F.(0.05) (0.03) 68.94 (0.18) 32.29 (0.20) 35.78 (0.20) 35.78 ------- ------- -------
Note: Mean in the same column followed by the same letters is not significantly different from one another at 5% level of probability
Table.XIII. Showing the faecal production of adult Acridid culture in small jars under laboratory conditions (after treatment of
Aspergillus oil formulation).
Treatments Days of observation (Mean ± SE)
1st 2
nd 3
rd 4
th 5
th 6
th 7
th
A. flavus 0.061±3.115b 0.032±6.535b 0.030±2.427c 0.031±2.773c 0.033±3.273b 0.027±4.633d 0.030±5.049d
A.fumigatus 0.060±5.498b 0.031±2.759c 0.033±2.680b 0.036±3.772b 0.031±2.424c 0.031±3.315c 0.033±2.840b
A.niger 0.039±0.011c 0.055±4.565d 0.031±3.173c 0.032±3.247c 0.035±2.827b 0.033±2.769b 0.034±3.116c
Control 0.227±0.113a 0.642±0.038a 0.700±0.031a 0.722±0.033a 0.715±0.041a 0.711±0.032a 0.732±0.033a
F.(0.05) (0.09) 16.58 (0.19) 34.03 (0.19) 34.03 (0.20) 35.78 (0.20) 35.78 (0.21) 37.52 (0.20) 35.78
Note: Mean in the same column followed by the same letters is not significantly different from one another at 5% level of probability.
Table.XIV. Showing the faecal production of Acridid (Nymphs) population treated with conidial concentration in H2O cultured
maintained in the large cage.
Treatments Days of observation (Mean ±SE)
1st 2
nd 3
rd 4
th 5
th 6
th 7
th 8
th 9
th 10
th
A. flavus 0.075±3.106b 0.068±3.419d 0.063±5.158c 0.068±2.605b 0.073±2.314b 0.070±2.608d 0.073±2.356b 0.00±0.00b 0.00±0.00b 0.00±0.00b
A.fumigatus 0.062±3.496c 0.074±4.571c 0.070±2.656b 0.059±5.328c 0.067±3.102d 0.072±2.149c 0.00±0.00c 0.00±0.00b 0.00±0.00b 0.00±0.00b
A.niger 0.073±3.279b 0.083±3.077b 0.072±2.150b 0.067±2.582b 0.070±3.229c 0.074±2.959b 0.00±0.00c 0.00±0.00b 0.00±0.00b 0.00±0.00b
Control 0.125±5.217a 0.736±0.036a 0.709±0.031a 0.704±0.026a 0.713±0.024a 0.731±0.024a 0.739±0.022a 0.731±0.019a 0.714±0.026a 0.721±0.023a
F.(0.05) (0.08) 14.84 (0.24) 42.76 (0.22) 39.27 (0.22) 39.27 (0.23) 41.02 (0.23) 41.02 ------- ------- ------- -------
Table.XV. Showing the faecal production of Acridid (Adults) population treated with conidial concentration in H2O cultured
maintained in the large cage.
Treatments Days of observation (Mean ± SE)
1st 2
nd 3
rd 4
th 5
th 6
th 7
th 8
th 9
th 10
th
A. flavus 0.082±3.177d 0.067±2.769c 0.063±5.537c 0.069±2.477d 0.068±3.101d 0.071±2.415c 0.072±2.499c 0.074±2.695b 0.075±2.624b 0.072±3.492b
A.fumigatus 0.085±5.740c 0.057±6.099d 0.060±4.790d 0.071±2.385c 0.072±2.354c 0.070±2.357d 0.075±2.413b 0.073±2.356b 0.00±0.00c 0.00±0.00c
A.niger 0.089±4.600b 0.085±3.969b 0.064±4.061b 0.073±1.800b 0.076±2.793b 0.072±2.417b 0.074±2.207b 0.00±0.00c 0.00±0.00c 0.00±0.00c
Control 0.163±3.667a 0.753±0.071a 0.733±0.026a 0.741±0.029a 0.732±0.023a 0.766±0.031a 0.739±0.025a 0.734±0.027a 0.695±0.026a 0.768±0.023a
F.(0.05) (0.10) 18.33 (0.24) 42.76 (0.23) 41.02 (0.23) 41.02 (0.23) 41.02 (0.24) 42.76 (0.24) 42.76 (0.22) 39.27 ------- -------
Note: Mean in the same column followed by the same letters is not significantly different from one another at 5% level of probability.
Table.XVI. Showing the mortality of Acridid (Nymphs) population cultured in small jars under laboratory conditions (after treatment
of Aspergillus oil formation).
a) Nymphs Stages 1st to 3
rd
Treatments Days of observation (Mean ± SE)
1st 2
nd 3
rd 4
th 5
th 6
th 7
th
A. flavus 0.45±0.23c 0.35±0.21a 0.20±0.21a 0.8±0.23a 0.00±0.00 0.00±0.00 0.00±0.00
A.fumigatus 0.62±0.01a 0.22±0.01c 0.16±0.01b 0.2±0.01c 0.00±0.00 0.00±0.00 0.00±0.00
A.niger 0.55±0.2b 0.32±0.23b 0.13±0.1c 0.00±0.00d 0.00±0.00 0.00±0.00 0.00±0.00
Control 0.3±0.1d 0.2±0.1d 0.2±0.23a 0.4±0.2b 0.00±0.00 0.00±0.00 0.00±0.00
F.(0.05) (0.48) 84.65 (0.27) 48.00 (0.17) 30.54 (0.35) 61.96 ------- ------- -------
b) Nymphs Stages 4th
to 6th
Treatments Days of observation (Mean ± SE)
1st 2
nd 3
rd 4
th 5
th 6
th 7
th
A. flavus 0.29±0.1c 42±0.2a 13±0.2a 16±0.1a 0.00±0.00b 0.00±0.00b 0.00±0.00b
A.fumigatus 0.43±0.1b 0.38±0.1c 0.7±0.32d 0.12±0.2d 0.00±0.00b 0.00±0.00b 0.00±0.00b
A.niger 0.62±0.2a 0.28±0.1d 0.8±0.2c 0.2±0.1c 0.00±0.00b 0.00±0.00b 0.00±0.00b
Control 0.6±0.1a 0.4±0.3b 0.6±0.2b 0.5±0.3b 0.7±0.1a 0.6±0.1a 0.5±0.1a
F.(0.05) (0.48) 84.65 (10.7) 18.33 (3.77) 06.11 (4.20) 07.85 ------- ------- -------
Note: Mean in the same column followed by the same letters is not significantly different from one another at 5% level of probability.
Table.XVII. Showing the mortality of Acridid (Adults) population cultured in small jars under laboratory conditions (after treatment of
Aspergillus oil formation).
Treatments Days of observation (Mean ± SE)
1st 2
nd 3
rd 4
th 5
th 6
th 7
th
A. flavus 0.35±0.32b 0.00±0.00d 1.5±0.47a 6.9±1.41a 11.0±2.10a 27.8±1.30a 3.4±2.00a
A.fumigatus 0.00±0.00c 2.5±1.00a 0.61±0.32c 3.8±1.32b 5.8±0.43b 9.8±1.20c 27.0±3.9b
A.niger 1.42±0.31a 1.00±0.58b 1.00±0.43b 4.5±0.53b 4.9±1.02c 11.42±1.30b 22.8±1.90c
Control 0.00±0.00c 0.75±0.31c 0.00±0.00d 1.9±0.46c 0.00±0.00d 1.00±0.57d 1.8±0.00d
F.(0.05) (0.44) 77.67 (1.06) 02.62 (0.77) 35.26 (4.27) 07.85 (5.42) 09.60 (12.5) 21.82 (13.7) 23.56
Note: Mean in the same column followed by the same letters is not significantly different from one another at 5% level of probabili ty.
Table.XVIII. Showing the mortality of Acridid (Nymphs) population treated with conidial concentration in H2O cultured maintained in
the large cage.
Treatments Days of observation (Mean ± SE)
1st 2
nd 3
rd 4
th 5
th 6
th 7
th 8
th 9
th 10
th
A. flavus 13±0.1c 11±0.2b 7±0.1c 6±0.2b 5±0.2a 2±0.1a 6±0.2a 0.00±0.00 b 0.00±0.00 b 0.00±0.00 b
A.fumigatus 15±0.1b 12±0.1a 9±0.2b 7±0.2a 5±0.1a 1.0±0.1b 0.00±0.00c 0.00±0.00 b 0.00±0.00 b 0.00±0.00 b
A.niger 21±0.1a 7±0.2c 10±0.1a 7±0.2a 3±0.1b 0.2±0.1c 0.00±0.00c 0.00±0.00 b 0.00±0.00 b 0.00±0.00 b
Control 0.2±0.1d 4±0.2d 3±0.1d 6±0.1b 0.3±0.00c 0.1±0.00c 0.1±0.3b 0.43±0.1a 0.21±0.2a 0.22±0.1a
F.(0.05) (12.3) 21.82 (8.5) 14.84 (7.25.) 13.09 (6.5) 11.34 (3.32) 06.11 (0.82) 43.99 ------- ------- ------- -------
Table.XIX. Showing the mortality of Acridid (Adult) population treated with conidial concentration in H2O cultured maintained in the
large cage.
Treatments Days of observation (Mean ± SE)
1st 2
nd 3
rd 4
th 5
th 6
th 7
th 8
th 9
th 10
th
A. flavus 0.02±0.32b 0.01±0.02b 1.2±0.01a 0.23±0.15b 1.5±0.03a 1.5±0.01a 1.2±0.32b 1.2±0.13c 1.3±0.14b 1.5±0.01a
A.fumigatus 0.01±0.21c 0.00±0.00c 0.3±0.04c 1.6±0.01a 1.2±0.03b 0.5±0.04c 1.3±0.01a 1.5±0.02a 0.00±0.00c 0.00±0.00b
A.niger 0.2±0.23a 0.1±0.12a 0.1±0.15d 0.23±0.1b 0.5±0.7d 0.2±0.15d 0.01±0.2c 0.00±0.00d 0.00±0.00c 0.00±0.00b
Control 0.01±0.02d 0.00±0.00c 0.5±0.23b 0.02±0.1c 0.6±0.22c 0.7±0.23b 0.00±0.00d 1.3±0.01b 1.4±0.01a 1.5±0.04a
F.(0.05) (0.06.) 11.34 (0.02) 04.36 (0.52) 91.63 (0.52) 91.63 (0.95) 16.58 (0.72) 26.54 (0.62) 09.08 (1.00) 02.62 ------- -------
Note: Mean in the same column followed by the same letters is not significantly different from one another at 5% level of probability.
Table. XX. Reproductive activities of healthy and unhealthy samples of H. oryzivorus
under laboratory conditions
Life History Statistics Healthy Range
(n= 15 )
Unhealthy Range
(n= 10 )
6th instar duration of maturation 6.00±1.3e(days) 8.01±1.02 b (days)
Maturation of adult 10.93±2.6 c(days) 13.02±1.00 b (days)
Total mating time during entire life 139.06±55.3) a (hrs.) 0.00±0.00c
Duration of copulation 33.26±13.9 b (hrs.) 7.6±3.95 b (hrs.)
No. of mating 12.17±4.12d 1.00±0.00d
Table. XXI. Fecundity rate of healthy and unhealthy sample of H. oryzivorus under
laboratory conditions.
Life History Statistics Healthy Range
(n= 15 )
Unhealthy Range
(n= 10 )
Oviposition time 47.26±6.0 a(mints) 21.02±0.21 a(mints)
No. of egg pods 3.26±0.96c 1.00±0.1 d (Broken)
No. of egg 35.65±14.64b 17.23±0.2b
Size of egg pods 34.68±0.84 b(mm) 16.30±0.01 bmm (Broken)
Size of egg 4.52±0.07 d(mm) 3.10±0.01 c (mm)
Weight of egg pods 1.30±0.03 e(gm) 0.20±0.01egm
Secretion of foamy mass 14.53±3.39 c (mints) 3.42±0.23c (mints)
Note: Mean in the same column followed by the same letters is not significantly different from one another at 5% level of probability.
Table.XXII. Showing the spectrum acquisition under scanning electron microscope
(SEM) of Aspergillus niger.
Element Series unn. C [wt. %] Norm. C [wt. %] Atom. C Error [%]
Carbon (C) K-series 42.60b 42.60b 49.88a 13.1b
Oxygen (O2) K-series 56.19a 56.19a 49.88a 17.5a
Sodium (Na) K-series 1.21c 1.21c 0.74c 0.1c
Total 100.00 100.00 100.00
Table.XXIII. Showing the spectrum acquisition under scanning electron microscope
(SEM) of Aspergillus flavus.
Element Series unn. C [wt. %] Norm. C [wt. %] Atom. C Error [%]
Carbon (C) K-series 52.33a 52.33a 59.88a 16.1a
Oxygen (O2) K-series 46.84b 46.84b 39.88b 14.5b
Sodium (Na) K-series 0.83c 0.83c 0.49c 0.1c
Total 100.00 100.00 100.00
Table.XXIV. Showing the spectrum acquisition under scanning electron microscope
(SEM) of Aspergillus fumigatus.
Element Series unn. C [wt. %] Norm. C [wt. %] Atom. C Error [%]
Carbon (C) K-series 43.92b 43.92b 51.31a 13.6b
Oxygen (O2) K-series 54.61a 54.61a 47.89b 17.1a
Sulfur (S) K-series 0.35d 0.35d 0.15d 0.0d
Sodium (Na) K-series 0.92c 0.92c 0.56c 0.1c
Phosphorus (P) K-series 0.20d 0.20d 0.09d 0.0d
Total 100.00 100.00 100.00
Note: Mean in the same column followed by the same letters is not significantly different from one another at 5% level of probability
Table.XXV. Showing the spectrum acquisition under scanning electron microscope
(SEM) of unknown fungi I.
Element Series unn. C [wt. %] Norm. C [wt. %] Atom. C Error [%]
Carbon (C) K-series 62.82a 62.82a 69.30a 19.2a
Oxygen (O2) K-series 36.82b 36.82b 30.49b 11.4b
Sodium (Na) K-series 0.36c 0.36c 0.21c 0.1c
Total 100.00 100.00 100.00
Table.XXVI. Showing the spectrum acquisition under scanning electron microscope
(SEM) of unknown fungi II.
Element Series unn. C [wt. %] Norm. C [wt. %] Atom. C Error [%]
Carbon (C) K-series 54.00a 54.00a 69.38a 16.6a
Oxygen (O2) K-series 43.53b 43.53b 30.14b 13.6b
Fluorine (F) K-series 1.17c 1.17c 0.84c 0.5d
Sodium (Na) K-series 0.52e 0.52e 69.31a 0.1c
Sulfur (S) K-series 0.79d 0.79d 30.34b 0.1c
Total 100.00 100.00 100.00
Note: Mean in the same column followed by the same letters is not significantly different from one another at 5% level of probability.
Fig.I. Showing the collected numbers of pest species from upper Sindh during the year
2013-2015.
0
5
10
15
20
25
30
35
No
. o
f co
llec
ted
sam
ple
s
Various species of Acridid
GHT
SKR
SHK
LKA
JBA
KSM
KHP
NBS
TMK
Fig.II. Showing the collected numbers of pest species from lower Sindh during the year
2013-2015.
0
10
20
30
40
50
60
70
No
. of
coll
ecte
d s
am
ple
s
Various species of Acridid
THR
UKT
MPK
JAM
HYD
DDU
SNG
BDN
MTR
Fig.III. Showing the total No. of grasshopper’s species caught from three sites of upper
Sindh in the year 2013-2015.
0
20
40
60
80
100
120
140
160
No
. o
f co
llec
ted
sam
ple
s
Various species of Acridid
Site – III
Site – II
Site – I
Fig.IV. Showing the total No. of grasshopper’s species caught from three sites of lower
Sindh in the year 2013-2015.
0
10
20
30
40
50
60
70
80
90
No
. o
f co
llec
ted
sam
ple
s
Various species of Acridid
Site – I
Site – II
Site – III
Fig.V. Showing the lethal infection level of entomopathogenic fungi in various species of
grasshoppers collected from site-I of upper Sindh in the year 2013-2015.
0
20
40
60
80
100
120
Various species of Acridid
No. of Incubated
No. of Sporulation
No. of Aspergillus Sporulation
Unknown Sporulation
% of Infection Asp.
% of Infection Uk
Fig.VI. Showing the lethal infection level of entomopathogenic fungi in various species of
grasshoppers collected from site-II of upper Sindh in year 2013-2015.
0
20
40
60
80
100
120
Various species of Acridid
No. of Incubated
No. of Sporulation
No. of Aspergillus Sporulation
Unknown Sporulation
% of Infection Asp.
% of Infection Uk
Fig.VII. Showing the lethal infection level of entomopathogenic fungi in various species of
grasshoppers collected from site-III of upper Sindh in the year 2013-2015.
0
20
40
60
80
100
120
No
. of
coll
ecte
d s
am
ple
s
Various species of Acridid
No. of Incubated
No. of Sporulation
No. of Aspergillus Sporulation
Unknown Sporulation
% of Infection Asp.
% of Infection Uk
Fig.VIII. Showing the lethal infection level of entomopathogenic fungi in various species of
grasshoppers collected from site-I of lower Sindh in the year 2013-2015.
0
20
40
60
80
100
120
No
. of
coll
ecte
d s
am
ple
s
Various species of Acridid
No. of Incubated
No. of Sporulation
No. of Aspergillus Sporulation
Unknown Sporulation
% of Infection Asp.
% of Infection Uk
Fig.IX. Showing the lethal infection level of entomopathogenic fungi in various species of
grasshoppers collected from site-II of lower Sindh in the year 2013-2015.
0
20
40
60
80
100
120
No
. of
coll
ecte
d s
am
ple
s
Various species of Acridid
No. of Incubated
No. of Sporulation
No. of Aspergillus Sporulation
Unknown Sporulation
% of Infection Asp.
% of Infection Uk
Fig.X. Showing the lethal infection level of entomopathogenic fungi in various species of
grasshoppers collected from site-III of lower Sindh in the year 2013-2015.
0
20
40
60
80
100
120
No
. of
coll
ecte
d s
am
ple
s
Various species of Acridid
No. of Incubated
No. of Sporulation
No. of Aspergillus Sporulation
Unknown Sporulation
% of Infection Asp.
% of Infection Uk
Fig.XI. Showing the isolated percentage of entomopathogenic fungi and their association
with pest species of grasshoppers during the year 2014 from Sindh.
0
2
4
6
8
10
12
14
No
. o
f co
llec
ted
sam
ple
s
Various species of Acridid
No. of isolated fungi
Isolation percentage
Fig.XII (a). Showing the faecal production of immature Acridid (N1-N3) culture in small jars
under laboratory conditions (after treatment of Aspergillus oil formulation)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1st 2nd 3rd 4th 5th 6th 7th
Days of observation
Tre
atm
ents
of
EP
Fs
Control
A.niger
A.fumigatus
A. flavus
Fig.XII (b). Showing the faecal production of immature Acridid (N4-N6) culture in small jars
under laboratory conditions (after treatment of Aspergillus oil formulation)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1st 2nd 3rd 4th 5th 6th 7th
Days of observation
Tre
atm
ents
of
EP
Fs
Control
A.niger
A.fumigatus
A. flavus
Fig.XIII. Showing the faecal production of adult Acridid culture in small jars under
laboratory conditions (after treatment of Aspergillus oil formulation).
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1st 2nd 3rd 4th 5th 6th 7th
Days of observation
Tre
atm
ents
of
EP
Fs
Control
A.niger
A.fumigatus
A. flavus
Fig.XIV. Showing the faecal production of Acridid (Nymphs) population treatment with
conidial concentration in H2O cultured maintained in the large cage.
0
0.2
0.4
0.6
0.8
1
1.2
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
Days of observation
Tre
atm
ents
of
EP
Fs
Control
A.niger
A.fumigatus
A. flavus
Fig. XV. Showing the faecal production of Acridid (Adults) population treatment with
conidial concentration in H2O cultured maintained in the large cage.
0
0.2
0.4
0.6
0.8
1
1.2
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
Days of observation
Tre
atm
ents
of
EP
Fs
Control
A.niger
A.fumigatus
A. flavus
Fig.XVI (a). Showing the mortality of Acridid (N1-N3) population cultured in small jars
(after treatment with Aspergillus oil formation).
0
0.5
1
1.5
2
2.5
1st 2nd 3rd 4th 5th 6th 7th
Days of observation
Tre
atm
ents
of
EP
Fs
Control
A.niger
A.fumigatus
A. flavus
Fig.XVI (b). Showing the mortality of Acridid (N4-N6) population cultured in small jars
(after treatment with Aspergillus oil formation).
0
5
10
15
20
25
30
35
40
45
50
1st 2nd 3rd 4th 5th 6th 7th
Days of observation
Tre
atm
ents
of
EP
Fs
Control
A.niger
A.fumigatus
A. flavus
Fig.XVII. Showing the mortality of Acridid (Adults) population cultured in small jars (after
treatment with Aspergillus oil formation).
0
10
20
30
40
50
60
1st 2nd 3rd 4th 5th 6th 7th
Days of observation
Tre
atm
ents
of
EP
Fs
Control
A.niger
A.fumigatus
A. flavus
Fig.XVIII. Showing the mortality of Acridid (Nymphs) population treatment with conidial
concentration in H2O cultured maintained in the large cage.
0
10
20
30
40
50
60
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
Days of observation
Tre
atm
ents
of
EP
Fs
Control
A.niger
A.fumigatus
A. flavus
Fig.XIX. Showing the mortality of Acridid (Adults) population treatment with conidial
concentration in H2O cultured maintained in the large cage.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th
Days of observation
Tre
atm
ents
of
EP
Fs
Control
A.niger
A.fumigatus
A. flavus
Fig.XX. Showing the element concentration under scanning electron microscope (SEM) of
Aspergillus niger.
1 2 3 4 5 6keV
0
10
20
30
40
50
cps/eV
C O Na
Fig.XXI. Showing the element concentration under scanning electron microscope (SEM) of
Aspergillus flavus.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0keV
0
10
20
30
40
50
60
cps/eV
C O Na
Fig.XXII. Showing the element concentration under scanning electron microscope (SEM) of
Aspergillus fumigatus.
2 4 6 8 10 12keV
0
5
10
15
20
25
30
35
40
cps/eV
C O S S
Na P
Fig.XXIII. Showing the element concentration under scanning electron microscope (SEM) of
Unknown Fungi I.
0.5 1.0 1.5 2.0 2.5 3.0keV
0
10
20
30
40
50
60
70
80
cps/eV
C O Na
Fig.XXIV. Showing the element concentration under scanning electron microscope (SEM) of
Unknown Fungi II.
0.5 1.0 1.5 2.0 2.5 3.0keV
0
10
20
30
40
50
cps/eV
C O F Na S S
CHAPTER 5
DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1 DISCUSSION:
Aspergillus is one of the most fascinating groups of fungi exhibiting immense ecological and
metabolic diversity Machida and Gomi (2010). Beside this, it is a large group with 180
accepted species belonging to different genera Pitt et al., (2000). Uvarov (1977)
recommended that Orthopteran species are classified according to their reproductive
strategies, micro-habitat and micro-humidity niche preference. Phipps (1968) describe
Xerophilous, Mesophilous and Hydrophilous preferences for dry, medium and humid habitat.
Shah et al., (1998) suggested that Hieroglyphus daganensis and C. fuscocreruleipes were
reported as more dominant species infesting millet, sorghum and rice crops in the Malanville
area Benin. They treated these species with entomopathogenic fungi and got significant
results, the investigations were carried out during present study are closely related with
earlier findings.
With a view to determine prevalence of grasshopper’s species in certain areas of Sindh,
investigations were undertaken in the upper Sindh, that comprise on 9 regions offering
different ecological condition with varities of the host plants. From control point of view,
these investigations provided useful information’s in establishing prevalence of grasshopper’s
species in different seasons in afore said areas which did not seem to have been reported from
Sindh. Keeping the objectives in view, studies on seasonal incidence of grasshoppers were
initiated in the Sindh province. This province is known to have precaution topography. The
major portion of the province was a part of desert areas. Present study recommends that,
information pertaining to seasonal occurrence of grasshopper in a given habitat with different
ecological conditions i.e cropped/non-cropped area will be useful in understanding
population development in these areas.
During the present study, it was noticed that order of prevalence of grasshopper species was
varying in both selected upper and lower regions of Sindh. It can be seen from the data that
majority of species having dominant and moderate pest status. The population pattern of
these species suggest that grasshoppers are available throughout the season both in cropped
and non-cropped areas due to their polyphagous habits wide range of host plants affected by
this. The activities of grasshopper, however, vary season to season in both selected regions. It
is recommended that control measures during monsoon (i.e June to July) may prove effective
when grasshopper population starts rising. It is suggests that adjoining non-cropped areas of
wild-flora be treated, where development of grasshopper population is continued in parallel
cropped areas. The specimens have been captured during present survey, all having great
importance. Earlier, COPR, (1982) gave overall assessment of many importance species of
locust and grasshopper in Agricultural Manual; they indicated their pest status by rating
different letters (see Appendix II). Presently all minor and major important insects were
encountered in collection.
As everyone knows that, lucusts and grasshoppers damage valued crops of million of rupees
in each year throughout world including; Pakistan (Biodochka and Khachatourian 1992,
Riffat et al., 2013). During current survey, it was noticed that representatives of family
Acrididae i.e grasshoppers and locust are found sever pests adult and 5th instar are noted
voracious eater, they can consume their own body weight in various vegetations. Pickford
(1963) while reporting the outbreak of Camnula pellucida (clear-winged grasshopper) stated
that this pest has destroyed as large area 2000 square miles, comparing on small grains and
granes. Usually, for controlling grasshoppers and locust farmers and cultivators mostly used
the different types of insecticides and pesticides. But, unfortunately frequent and misuses of
different chemicals on crops enhance the resistance among the pest population that rendered
insecticide efficacy, self-defense and short-live. Therefore, present attempt is made in order
to resolve this problem. Bio-pesticide should be introduced at commercial level against pest
to minimize the harmful effects on surrounding environment.
Magalhaes et al., (2001) recommended that utilization of pathogenic fungi as bio-pesticide
against grasshopper in Brazil reduced the lack of a consistent production system, short self-
life and very low action in killing the host. Effort is being directed to optimize the production
system for M. anisopliae var. acridum and to increase its storage capacity at room
temperature. The slow action in killing the host is minimized by the apparent reduction in
mobility and food consumption presented by the infected insects, and by the fact that young
nymphs of Rhammatocerus schistocercoides usually occurs in natural vegetations instead of
cultivated areas. Another problem is that R. schistocercoides is univoltine. This allow only
one field trail with small nymphs per year, in the period between November to December.
The lack of serious grasshopper problem in Brazil in recent years has hampered the
development of research in this area, since funds were reduced. The isolate CG 423 of
Metarhizium anisopliae is highly virulent against the grasshopper’s R. schistocercoides and
S. robusta, and possibly other species, present study recommends that the accessibility of this
good candidate as a mycoinsecticide may be valuable in the upcoming era.
Pathogenic fungi are cosmopolitan in nature having rich diversity, and play most of important
role in (IPM) program. Because of their, eco-friend and bio-persistence they are prepared to
kill many developmental stages. Three important pathogenic fungi B. bassiana, M. anisoplila
and Isaria fumosorosea are used on commercial level. All of these belong to Oomucota,
Ascomycota, Chytridiomycota and Zygomocota respectively. But Aspergillus species have
been treated on Acridid population for the first time from this region.
During the present study, it was observed that pathogenic fungi have direct contact to many
insects, it direct infect the cuticle and slowly penetrate into the host body. Slowly reduce the
toxin level and lastly kill the insect. When the insect is contaminated with pathogenic fungi
reduce, the feeding but, in same case feeding rate suddenly increased. In behavioral fever and
reproductively activity such as aberrant mating and changing in ovipositional preference. The
spore spread from the insect cadaver through air, soil and water resources. Present finding are
strongly co-relate with earlier findings of Hafiza et al., (2014).
According to FAO (1997) use of chemical pesticides put harmful effects on the environment,
people, livestock, birds, terrestrial creature and aquatic organisms particular on the fish. Pell
et al., (2010) stated that biological control mostly developed on the modification of the
environment and management. This practice promotes and encourages natural enemies that
exit in the system that enhanced their ability to control pest population in field. For
implementation of best biological control understanding of ecology of the species is
necessary, present study is agreed with this. Simon and Thomas (2001), stated that M.
anisoplilae not gave good result for the reduction of S. gregaria adult but this pathogen is
very important for reduction of different immature stages of this species. They further,
reported that, infection of pathogenic put effects on the fecundity and mortality of insects.
Therefore, it should be implemented against pest; similar results have been obtained during
present study.
During this research, rice fields were also inspected time to time and it was noticed that rice
ecosystem, is one of the most important approache to biological control for conservation of
the natural enemies complex. It is considered very key component of biological control, this
indicate the factor which include: effectiveness of specific natural enemies that reduce the
pest population in field.
Disruption of various natural enemies could be minimized by reducing the use of different
pesticides and insecticides. Beside this, selection of resistance plant varieties and changing in
cultured control via use of strip-planting, field borders or cover crops and alternation of
regional area.
In short, conservation, importation and augmentation of natural predators and parasites in
numerous agro ecosystems, but further application have enhanced biological control and
refinements of these technologies are needed in Pakistan. Earlier, Hernandez Crespo and
Santiago Alvarez (1997), Gunde-Cimerman et al., (1998), Balogun and Fagade (2004)
reported significant incidence rates in insect’s colonies. Beside this, Fusarium species were
also reported as pathogenic agent in larvae and adult form of insects. Further, Sur et al.,
(1999), Shtayeh et al., (2002), Sun and Liu (2008) and Abdullah and Amin (2009) reported
some opportunistic pathogenic on the cadavers of various grasshopper species. In this study,
three Aspergillus species i.e A. flavus, A. fumigatus and A. niger along with two unidentified
species were obtained from the cadavers of insects, these overall observation showed that
these grasshoppers act as secondary colonizers till proven their pathogenicity. Addition to
this, isolates of Pencillium, Trichoderma and Mucor genera mostly effected with different
opportunistic pathogenic fungi. Abdullah and Amin (2009) reported high infection rate of B.
bassiana and other opportunistic fungi isolated from sunn pests. They also suggest that
possibly these fungi play important role in regulation of insect population in field when their
population is increased during their dormancy at hibernation sites.
In this study, I have reported greater incidence of Aspergillus niger, present study
recommends that it might be greater due to availability of favourable climatic condition.
Apart from, the utilization of entomopathogenic fungi on the colonies of grasshoppers and
locusts, earlier co-worker also carried the work on the other group of insects. Nauen (1995)
studied sub-lethal dose of imidaclopoid on aphid population, he suggested that aphids
increase their movement after the treatment of insecticides. It was observed that aphid is
mobile throughout the 12hrs, compared to the movement recording on control. His study
recommends that imidaclopoid stopped the setting of insect. He further suggested that, this
pesticides increase the mortality of aphids that were allowed to take this conidia. Beside this,
he also recommended that aphid cover greater distance compared to untreated aphid.
Shehu and Bello (2011) isolated the 05 species of entomopathogenic fungi stored grains their
isolated species include: A. candidus, A. fumigatus, A. flavus, A. oryzae and A. niger they
further, reported that A. flavus and A. niger frequently spread on the host body and they also
reported 29.5-30% infestation of these fungi on the host body. But at the present, I have
reported three species of Aspergillus from many important pest species of grasshoppers.
Concerned African countries and several donor agencies have expressed interest in the
potential use of biological control strategies in recent years. The interest is probably an
outcome of our inability to manage locust with traditional chemical insecticides and has
prompted the initiation of several projects to explore the potential value of microbial
organisms. Pathogenic fungi are known as more predominant natural suppressing agent in
arthropod population. Studies regarding epizooties showed that insect population having
great potential of this bio-pesticide. Pathogenic fungi penetrate into cuticle and can damage
both hard and soft tissue of insects. Besides, causing destruction in the Acrididae species, this
pathogen also affect the wide range of tick and mites belong to Acari. Addition to this,
cuticular invasion also promote the fungal to infect the other insect like whiteflies, psyllids
and aphid (Burges 2007, McCoy et al., 2009, Lacey et al., 2011). Present study fully supports
the view of earlier workers.
During different experiments it was noticed that fungi significantly suppress the insect’s
population and pathogenic fungi pose minimal risk to beneficial creature’s i.e honey bee,
collembola and earthworms which are consider key ecosystem regulators Goettel et al.,
(2001), Traugott et al., (2005), Callaghan and Browbridge (2009). This indicates their
significant role in (IPM). Different natural enemies permit them to make maximum
contribution in regulation of pest species and maintained of bio-diversity. This new trend
attributes the possibilities of use of multiple roles in control of arthropod pest species.
Chandler et al., (2008) while studying the anamorphic fungi i.e B. bassiana and M. anisoplila
stated that “industrial” pathway mass production systems provide the large quantities of
inoculation that could be formulated and spray granules etc. (Shah and Pell 2003,
Brownbridge 2006, Charnley and Collins 2007). Research so far conducted on utilization of
entomopathogenic fungi (FPFs) from last 150 years stated that, this is very beneficial bio-
control agent and all the earlier workers stressed on the utilization of this microbial agent
against pest.
Davidson (2012) stated that much of the effort has failed to lead commercially successful
microbial pesticide products, while some of the issues are related to biological constraints; a
major factor is the absence of a clearly understood model for the commercialization of
(EPFs). A variety of factors contribute to the potential for market success, which is
essentially a measure of cost and benefits including expected protection of the crop and crop
value, and efficiency of competing products (Black et al., 1997, Shapiro-Ilan et al., 2002 a,b,
2012 a,b, Ravensberg 2011, Glare et al., 2012).
Present study recommends that more awareness should be created among the scientific
community and agricultural sectors in order to improve this technique in biological control. A
number of pathogenic microorganisms are available for evaluation against grasshoppers and
locusts in world. Priority should be given to the entomopathogenic fungi because they
appears to be moderately virulent end are stable for prolonged period of storage and
application. However, priority should also be given to an extensive survey of grasshoppers
and locusts in Pakistan for other useful microbial. Previous efforts have demonstrated that
these insects serve as natural host to many pathogenic organisms. Particular emphasis should
be placed on the isolation of Aspergillus strains which might be improved genetically and
used as microbial insecticides against gregarized locust. During present study, I have tried to
give little bit information on development of pathogenic fungi that will promote opportunity
for application of entomopathogenic fungi and identification of some factors that still needed
to investigate for their wide promotion. It was observed that collection of such large number
of species along with these infection levels of entomopathogenic will be very beneficial for
the utilization of these mycopesticide against grasshopper. The basic aim of this study was
also determined the natural occurring infection level of entomopathogenic fungi so that
proper planning could be made possible for future. Furthermore, leading to modern insight
aimed towards increasing the efficacy of myco-insecticides as parts of IPM practices present
study has been designed for the first time from this area.
During present study, greater and lesser samples of grasshoppers were collected in all
seasons. Thus, the results of present studies revealed that grasshoppers were present
throughout the year but their population level varied species to species. On the basis of peak
seasonal activities of abundant and prevalent species it can be suggest that control measures
would be effective during May and early June when their population started building up.
Since monsoon rains are uncertain in Sindh, a careful over population build-up of
grasshopper is also necessary as it was observed that the population of some occasional
species was higher during monsoon month (June to September) as compared to have direct
influence in relation to the amount and extent of rainfall in the areas.
Street and Henry (1990) stated that efforts to artificially propagate the fungus in grasshopper
have largely been disappointing undoubtedly because of moisture level requirements but in
recent year several commercial forms in the U.S and Europe have developed improved
culture techniques which have led to the selection of strains that are more active against
grasshoppers. Henry et al., (1985) reported some unidentified fungi from Oedaleus
senegalensis (Krauss), Aiolopus thalassinus (Fab.) and Anacridium sp. from West Africa. At
the present, I have reported the infection of Aspergillus along with 02 unidentified fungi on
32 species of acridid.
Jenkevica (2004) reported 66 associations between different fungi and important pest of
agriculture crop i.e flies, aphid, thrips, butterflies, moth, grasshopper etc. In addition to this;
he also identified 16 species of entomopathogenic fungi 10 of Zygymycotes and 06 belong to
Deuteromycetes. However, during the present study all observed fungi i.e Aspergillus and 02
unidentified fungi belonging to class Deuteromycetes. My results, correlate with finding of
Henery et al., (1985). Because, this is a first lab demonstration of effectiveness of Aspergillus
and other fungi that act as biological control agents that significantly suppress the Acrididae
densities and collection of large numbers of individual contaminated with fungi has
confirmed that Aspergillus spores significantly destroyed the insect population. During the
present study, it was noticed that entomopathogenic fungi has lethal effect on the insect that
reduce their feeding. By reduction in their feeding we can save our agricultural crops. Present
study recommends that, this microbial agent must be cultured and utilized on commercial
level.
Currently, I have reported a vast population of the grasshoppers occurring in most important
crops such as rice, wheat, cotton, sugarcane, vegetations as well as fruits and consumed their
large portion. Similarly, many fruits, vegetables, fodder crops, important grasses which are
food items for many animals, were also attacked by numerous grasshoppers’ species as
enlisted in Appendix (I) are considered very serious in causing quantitative loss of crops.
Biological control is the combination of utilization of predators, parasitoids, pathogens,
antagonists or competitor population to suppress a pest population, making it less abundant
and less damaging than it would (Irshad and Stephen 2012) present study is strongly agreed
on this account.
Occureness of entomopathogenic fungi has been shown in Table (X) it could be suggested as
biopesticide against the Orthoptera insects, it can be an economical and environmentally
friendly solution to reduce pest population. But unfortunately, in Pakistan advocating this
type of approach since many decades, it is urgent need for the registration of this biopesticide
against Orthopteran pest; it might be proved new turn with the management of insect pests by
biological methods. Earlier, some successful experiences are also conducted by Irshad (2008)
and Haq and Irshad (2011). Present study is the basic step in the IPM because through this
technique many problem of pest could be solved permanently. Shah et al., (1994, 1998) when
carried out the surveys in Madagascan gave a similar discussion on the African migratory
locust and Welling et al., (1995) also urged that, this locust produced conidial anamorph in
vitro process and can be utilized against grasshoppers population. Presently, I got significant
results after pathogenic applications of Aspergillus. During this study, infection of many
Aspergillus species particular A. flavus has been reported on the cadavers of H. nigrorepletus
while infection through A. niger and A. fumigatus earlier reported by Riffat at al., (2013) on
three Hieroglyphus species.
During the present survey, I have observed infestation of acridid species pertaining to 06 sub-
families i.e Acridinae, Calliptaminae, Gomphocerinae, Hemiacridinae, Oedipodinae and
Oxyinae were severely attacked by 03 Aspergillus species including 02 unknown pathogenic
fungi. Earlier, many workers, i.e Ozols (1963), Cinovskij and Jegina (1972), Strazdinja
(1972), Jegina et al., (1976, 1977) and Cudare (1998) also highlighted the effect of
entomopathogenic fungi on aphids, flies, moth, weevils, colorado beetles and thrips from
different region of the world. Shah et al., (1994) carried work on the 10 sub-families with 43
species of Acrididae and 03 species of family Pyrgomorphidae as far as infestation of
entomopathogenic fungi on different sub-families of grasshoppers is concerned, these bio-
pesticides is found very effective for reduction of pests in the field. Although, Shah et al.,
(1994) reported large numbers of grasshopper species from Northern Benin but they failed to
describe any single species of Oxyine from this region at the present, I have reported the
infection of Aspergillus on the 04 species of Oxyinae i.e Oxya hyla hyla, O. veox,
O. fusovittata and O. bidentata it was also observed that due to smaller size of Oxyinae they
got quick infection and can die earlier compared to other large and medium size insects.
Surveys for mycopathogens of Orthoptera pest were conducted between 1990 and 1993 in
Africa and Asia as a main project of biological control of grasshoppers and locust (Kooyman
and Shah 1992). In this project, they highlighted the world’s most destructive agricultural
pests, with the most damaging, the plague of migratory pest. Studies on the enzootic levels of
Aspergillus infection in Orthoptera have received less attention in Pakistan. Although, this
subject has widely studies in rest of world as test treatment against many pest species of
Orthoptera and there are many references are available i.e Shah et al., (1994, 1998), Lomer et
al., (2001), Prior et al., (1995). However, such studies are necessary because they provide the
indication of background of entomopathogenic infection level which should be surpassed by
an application of released of artificially production. Additionally, this study also give the
knowledge how an entomopathogenic fungi present between the different species of
grasshoppers in field.
Jankevica (2004) stated that entomopathogenic fungi cause lethal infections of insects and
can regulate their population in nature by epizootics fungi having broad host range. About
1800 association between fungi and different insects were recorded. During the present study,
I have analyzed 73 associations of Aspergillus and other fungi on different species of
grasshoppers. A host range is a set of species that allow survival-ship and reproduction of the
pathogen Onstad and McManus (1996). The implementation of entomopathogenic fungi as
bio-pesticides against grasshoppers in Pakistan is greatly limited by the lack of a consistent
production system, short self-life, and their slow action in killing the host. The lack of serious
grasshopper’s problems in Pakistan in recent years has hampered the development of research
in this area. Efforts are being directed to optimize the production system.
Current finding suggests that, research method, mass production along with formulation and
application technique should be revised /improved. Beside this, strain selections that only kill
the host without distribution/damaging non-target insect. To meet the challenges of
promoting bio-control agent for global pest species that include: animal and plant disease,
control of vector causing harm to human being that growing on global level and utilization of
bio-control agent’s need detail introduction for these targets organism. Pathogenic fungi have
been known to cause drastic decline among acridid population from many centuries. The
major groups of fungi that infect grasshoppers are Entomophaga grylli, Metarhizium,
Beauveria, Verticillium and Aspergillus are very common Entomopathogenic fungi causing
significant loss in insect’s population.
Pathogenic fungi is key component in the regulation of natural population it plays vital role in
IMP. It is found very effective against pest population and its infection significantly reduces
the arthropods numbers in fields. Inglis et al., (2000) and Carruthers et al., (1997) suggest
that it is very important integral part of myco-insecticides in horticulture, agriculture and
forestry. Califeran species cause significant loss to the agricultural sector in grassland
biomass Lomer et al., (2001). Riffat and Wagan (2015) reported that about 97 pest species of
grasshoppers and locusts occur in Sindh they cause maximum damage to valued crops among
these 19 species are known as minor pest. But present study, recommends that if this pest
would not be controlled at their earlier stage they may cause considerable damage to crops
and become major pest with advancement of developmental stages.
Varieties of fungal product occupied niche market, within the different countries or on the
geographical regions. Fungi used as active microbial pesticides in field of pest management.
Despite the significant use of fungi as bio-control agent their active utilization in now under
way. According to Insect Pathology Manual (1983) the genera and species with powdery
conidia with lipophilic cell walls viz: Metarhizium, Beauveria, Nomuraea and Paecilomyces
are encountered as entomopathogenic fungi while the Verticillium lecanii and Entomophaga
grylli have less potential or development as ULV mycopesticide formulation against pest
opposing to this saprophytic species not apparently useful for control purpose. Review of
literature showed that, applications of bio-control agents as active pesticides is neglected but
recent data on this subject will filled certain gabs in IPM about the utilization of bio-control
agents.
My findings are consistent with the work done by Carruther et al., (1988, 1997) they suggest
that E. grylli epizootics are linked to high densities of susceptible nymphs. However,
additional research is needed to understand how host dynamics affect acridid population
responses to entomopathogens. Current knowledge on the bio-pesticides of Acridid
population reveled that bio-pesticides are now international commodity subject to the
regulations of different countries governing their marketing and applications. Most of the
developed countries have regulated by law, they use distribution and sale of pesticides in
their countries for which proper legislation have been made. Moreover, they can also be
spewed with the prevailing distribution systems and used in broad scale in agriculture sector
whereas water-based formulation is principal formulation. In Pakistan utilization of bio-
control agent as potential use is under consideration. During the present study, detailed data
was provided in the light of this finding, registration of biological control as bio-pesticides
are required. Therefore, present attempt is being made to introduce pathogenic fungi in IPM
as more active agents.
Present study suggests that these microbial insecticides are harmless to non-target organisms
which are available in field. This research is an initiative step towards the utilization of
pathogenic fungi in Pakistan. It is recommends that grasshoppers that are contaminated with
fungi assist the raising of body temperature because basking for longer time condensed
pathogen induced mortality. Earlier, same observations were also taken by (Carruthers et al.,
1992, and Marikovsky 1962) they reported fever response in many taxonomic varied hosts
pathogenic. But in some cases there is very difficult to identify the active behavior fever and
other advantages that put side effect on the natural thermoregulatory specific basking
response of insects.
5.2 CONCLUSION:
Entomopathogenic fungi commonly known as (EPFs) have wide range of potential existence
and diversity in the IPM program this microbial agent prefers to kill varieties of insects
because of its eco-friendly and bio-persistence behavior. They found very effective to control
immature stage of insect. Yet, three pathogenic fungi i.e B. bassiana, M. anisopliae and I.
fumosorosea were commercially utilized in world. At the present, I have selected Aspergillus
group due to its cosmopolitan existence and fast growth rate. Introduction of this product in
(IPM) particularly in Sindh and generaly in Pakistan is being introduced for the first time.
Present study suggests that, utilization of microbial agent as bio-pesticide instead of chemical
used contribute a lot in various fields such as agriculture, forestry, horticulture culture. It is
also recommends that while making its formulation special care should be taken so that
beneficial creature in the environment may not be affected. Beside this, series of strategies
are needed. For mass production of conidia on smaller and large level as important part of
integral part of IPM in many ecological zones.
Presently, it was observed that high rates of disease mortality in the acridid population due to
entomopathogen system with response to disease ultimately depend upon host developmental
stage. Clearly, age demographic was significant in this experiment. During experimental
procedure, I have found that all treated entomopathogenic fungi can reduce grasshopper’s
numbers, but this impact varies across all life stages. The grasshopper pose constant threat to
agricultural valued crops of both irrigated and rainfall area in Sindh. It seems no work has so
far been reported on seasonal occurrence in these areas on grasses and other host plants. This
is an important aspect of assessment of acridid population in developing forecasting methods
indicating time, place and population density for their preventive/curative measures with this
objective in view; preliminary investigations were carried out to study the utilization of
different bio-pesticides against pest species of Acridid.
Thirty two species pertaining to 06 sub-families of Acrididae were captured in the province
of Sindh and eighteen districts were visited time to time. An index of abundance of this
species was prepared in population to population level of abundant species i.e Acrida
gigantea, Hieroglyphus nigrorepletus, Acrotylus humbertianus, Oxya hyla hyla and O. velox.
Presence of various species during season was categorized as major pest and indicating by
(A) symbol, prevalent and occasion according to their index of abundance. The population
pattern of both abundant and prevalent species indicating that Acrida gigantea and Locusta
migratoria were grouped as (A), while Duroniella laticornis was considered in group (B),
and Gelastorhinus semipictus, Sphodromerus undulatus undulatus, Oxypterna afghana,
Hieroglyphus banian, H. nigrorepletus, H.oryzivorus, Oedaleus senegalensis, Oxya bidentata
and O. hyla hyla were cataloged as (C).
Whereas Chorthippus indus and Ch. dorsatus species are fall into (D) category. These are
occasionally of substantial importance pest addition to this Phlaeoba tenebrosa,
Hieroglyphus perpolita, Spathosternum prasiniferum and Oxya velox recorded as
occasionally importance pest (E). Similarly, species belonging to group (F) and (G) are
Acrotylus longipus longipus, Aiolopus thalassinus thalassinus, Oxya fuscovittata and Acrida
exaltata, Truxalis exmia exmia, T. fitzgeraldi, Acorypha glaucopsis, Gonista rotundata
respectively. These are considered regular or occasional minor pest opposing to this some
species grouped as (H) and (K) i.e Oedaleus rosescens and Phlaeoba infumata, Acrotylus
humbertianus, Hilethera aelopoides and Trilophidia annulata respectively were reported as
minor importance and cause minor damage and negligible economic significance.
During the present study, it was observed that infection of Aspergillus caused a significant
reduction in host feeding well before death. Further, this study also revealed that pest status
of infected insects is markedly reduced soon after application of the pathogen. This study
demonstrate that Aspergillus species among all the isolated entomopathogenic fungi are
major factors of mortality in grasshopper population and hence could be exploited as
microbial control agents of the grasshopper in Pakistan. It was observed that three studied
entomopathogenic fungi were found infecting grasshopper. A significant difference was
noted in the time to death of small grasshopper individual infected with Aspergillus niger
compared to large individual. Furthermore, as a result of examination of dead samples of
grasshopper, fungal contamination was found significantly higher in lower Sindh i.e 1321
followed by 1199 in upper Sindh. The proportional cumulative survivals of these treated
grasshoppers species in the different treatments of fungi suggest that, survival time of treated
insects was significantly low compare to control. The associations of pest species along with
infected percentage of pathogenic fungi have been presented in comparative manner.
According to this comparison, total No. of isolated percentage of A. niger i.e 6.77% and
5.64% on A. exaltata and A. gigantea in sub-family Acridinae, 6.21% in A. humbertianus of
Oedipodinae and 5.08% Truxalis exmia exmia contaminated with A. fumigatus and O.
fuscovittata infected 5.08% with A. flavus. The order of prevalence of grasshopper species
was varying in both selected regions.
Lethal infection level of entomopathogenic fungi from lower Sindh site-I indicate that
significant highest No. of sporulation was recorded for Acrida gigantea and A. exaltata i.e
71.42% and 68.42% respectively of subfamily Acridinae, while unknown fungal infection
was 36.36% followed by 31.57% on Phlaeoba tenebrosa and A. exaltata respectively.
Opposing to this, Calliptaminae was significantly affected by unknown sporulation. As for as
Gonista rotundata of Gomphocerinae is concerned, it affected by Aspergillus sporulation. It
was observed that infestation ratio of entomopathogenic fungi was vary species to species in
different localities it might be due to presence of moisture level.
Beside this, observation taken under Scanning Electron Microscopy (SEM) showed that there
is significant difference in coloration and phialides pattern of three Aspergillus species
including two unknown fungi. SEM observation regarding spectrum acquisition showed that
normal weightage % of Oxygen (O2) was highest i.e 56.19% followed by 42.60% for Carbon
(C) and very least ratio i.e 1.21% of Sodium (Na) was observed for A. niger.As far as
chemical composition of A, flavus is concerned the normal weightage % value of Carbon (C)
was noted highest i.e 52.33% followed by Oxygen (O2) i.e 46.84% opposing to this least
percentage was calculated for Sodium (Na). In case of A. fumigatus the greater normal
weightage % was counted for Oxygen (O2) i.e 54.61% followed by 43.92% for Carbon (C),
while other elements i.e Sodium (Na), Sulphur (S) and Phosphorus (P) were noticed
minimum concentration i.e 0.92%, 0.35% and 0.02% respectively.
Close association of pest with pathogenic fungi stated that Acrida gigantea, A. exaltata,
Truxalis exmia exmia, T. fitzgeraldi, Phlaeoba tenebrosa, P. infumata Gelastorhinus filatus
and Duroniella laticornis all were belong to Acrididae significantly infected with pathogenic
fungi this observation has been taken for the first time. This wide range of infection showed
that many important grasshoppers’ sub-families have direct contamination with Aspergillus
species. Particularly, maximum infection of A.niger was reported on the most dominant
species of grasshoppers followed by A.flavus and A.fumigatus and with two unknown fungi.
Food consumption and faecal production by the insects treated with different formulation of
the Aspergillus species were analysed under laboratory conditions. Three replicates i.e
A. flavus, A. fumigatus and A. niger excluding control. Present finding indicates that there is
greater reduction in faecal production after the treatment of oil formulation. Reduction in
feeding of the infected insects (N1-N3) was started after treatment of 1st and 2nd days.
Significant reduction in faecal production was noted from 1stto 4th day after that all immature
stages (consist on N1 to N3) were died. In comparison with oil formulation the rate of faecal
production of Acridid (nymphs) treated with conidial concentration in H2O maintain in cages
showed that the maximum faecal production was obtain on day 2nd i.e [F 0.24 = 42.76, P <
0.05] followed by [F 0.23 = 41.02, P < 0.05] on 5th and 6th day. However, least amount of
faecal material was obtained on day 1st i.e [F 0.08 = 14.84, P < 0.05]. Beside this, mortality of
Acridid (nymphs) population kept in large cage when treated with conidial concentration
formed in H2O was found maximum on day 6th.
It was observed that virtually all insects found susceptible to fungal disease. It was also
noticed that thermoregulatory behavior of acridid species was observed in the laboratory
following a spray application of oil and water based formulation of Aspergillus and
(unsprayed) individual under laboratory conditions. All treated grasshoppers maintained in
(jars and cages) were monitored for 3 days from the second day after application. During
present study, it was noticed that infected insects altered their thermoregulatory behavior and
showed a behavioral fever response to the pathogen their body temperature were raised as a
means of literally toasting a fungal invader. Further, these behavioral responses may result in
enhanced spore diffusion and fungal fitness.
Fair numbers of entomopathogenic species along with their targeted insects have been
enlisted by various workers from all over the world but, any single reference is not avaible
from Pakistan present attempt is being carried out for first time Appendix (IV). Earlier, Faria
and Wraight (2007) complied the comprehensive list of fungal species from Asia, Africa,
Europe, America and Oceania etc. They provided the different bio-pesticides which are
registered as commercial products. But there was no work has been carried out from Pakistan.
Present attempt regarding Aspergillus has been carried out for the first time from this region
and soon it will be marked/introduce as registered bio-product in national level
Appendix (V).
This is first indication to a microbial infection for any natural population. After the
pathogenic application it was also noticed that the production of cuticulur antimicrobial
lipids, protein, and metabolites. Shedding of the cuticle during development and behavior
environmental adaptation that include: fever, burrowing and growing was also effective
significantly. It was also noted that after the application of oil and water based formulation of
Aspergillus acridid species undergo in very interesting behavior insects altered their
thermoregulatory response and showed very interesting behavioral changing that include:
insect’s feeding stopped completely, poor coordination, jerky movements, excessive
grooming, loss of orientation, confuse during mating, short mating , drop egg without
searching oviposition site, ecdysis process slow or complete stop, Behavioral fever (body
temperature raised) and body fat accumulation was also reduced.
Present study recommends that exploration and screening must be conducted to provide
additional pathogens for evaluation as potential biological control against grasshoppers and
locusts. Finally it could be concluded that, entomopathogenic fungi (EPFs) play vital role to
implement the IPM techniques in the field and it can give sustainable pest control when
conjugated with other techniques.
5.3 RECOMMENDATIONS:
Actually, varieties of pathogenic fungi are registered as bio-control agent that commercially
used against many species of Califera and Ensifera in all over the world with exception of
Pakistan. Although, during the this study I have tried to work on collection of pest, resources
of collection, culturing of Aspergillus species, bio-pesticides applications, host fecundity and
survivability but still a detail attention is needed from scientific community on:
1. The use of molecular techniques based on the Random Amplification
of Polymorphic DNA (RAPD) analysis of the Internal Transcribed Spacer (ITS)
region of the ribosomal DNA (rDNA) of Aspergillus should be studied.
2. For the significant and effective use of entomopathogenic fungi a detail and
comprehensive investigation is needed on the ecological behavior of fungi within
their surroundings. Beside this, efficient mass production, formulation of different
pathogen and effective delivery system is required for its disposal at commercial
level.
3. Extensive biosystematics and pathogenic research is required to confirm the
identification and sustainability of individual pathogen along with its host range in
different crops. As various species of fungi are mass producing this aspect should be
studied in detail so that mass production of pathogenic fungi could be used on large
extent.
4. Effect of biotic and abiotic factor on the efficiency, persistence and potential
capacities of difference bio-control agent including natural enemies should be studied
in detail in order to improve the environment stability cost effectiveness and efficacy.
There are many untouched areas where we can continue to derive new knowledge
toward the advancement and development of pathogenic fungi.
5. Comprehensive testing of toxicology, environment impact, invention performance and
other phases are must undertaken in future.Addition to this, several temporal, genetic
and spatial factors that regulate the disease dynamics of entomopathogenic fungi
(EPFs) and conjunction with pest life should be work out in detail.
6. “Bio-pesticides Awareness Program” for safety of environment should be introduce
through electronic and print media.
7. Efforts on the large scale should be taken to engage stock- holders, producers,
regulators, farmers, cultivators, retailers and consumers to ensure the acceptance of
this microbial agent as effective bio-pesticides in IPM.
8. However, priority should also be given to an extensive survey of grasshoppers and
locusts in remaining districts of Sindh Pakistan for search of other useful microbial so
that in the light of comprehensive data proper planning could be made.
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Acrida exaltata Walker, 1859 (G)
Acrida gigantea Herbst, 1794 (A)
Duroniella laticornis Krauss, 1909 (B)
Phlaeoba tenebrosa Walker, 1871 (E)
Truxalis exmia exmia Eichwald, 1830 (G)
Truxalis fitzgeraldi Drish, 1951 (G)
PLATE. I. Important insect pest of sub-family Acridinae.
Acorypha glaucopsis (Walker, 1870) (G)
Sphodromerus undulatus undulatus (Kirby, 1914) (C)
PLATE. II. Important insect pest of sub-family Calliptaminae.
Ochrilidia geniculata (Bolivar, 1913) (K)
Oxypterna afghana Ramme, 1952 (C)
PLATE. III. Important insect pest of sub-family Gomphocerinae.
Hieroglyphus banian Fabricius, 1798 (C)
Hieroglyphus nigrorepletus I. Bolivar, 1912 (C)
Hieroglyphus orzivorus Carl, 1916 (C)
Hieroglyphus perpolita (Uvarov, 1932) (E)
PLATE. IV. Important insect pest of sub-family Hemiacridinae.
Acrotylus humbertianus Saussure, 1884 (K)
Aiolopus thalassinus thalassinus Fabricius, 1781 (F)
Hilethera aelopoides (Uvarov, 1922) (K)
Locusta migratoria (Linnaeus, 1758) (A)
Oedaleus senegalensis Krauss, 1877 (C)
Trilophidia annulata Thunberg, 1815 (K)
PLATE. V. Important insect pest of sub-family Oedipodinae.
Oxya bidentata Willemse, 1925 (C)
Oxya fuscovittata Marschall, 1836 (F)
Oxya hyla hyla Serville, 1831 (C)
Oxya velox Fabricius, 1787 (E)
PLATE. VI. Important insect pest of sub-family Oxyinae.
Note: Capital letter showing pest status of species according to COPR (1982).
(a)
(b) (c)
PLATE. VII. Showing the horizontal transmission of Aspergillus niger on the host body (a) Head,
Pronotum (b) Thorax (c) Tegmina, all were infected by pathogenic fungi (Bar-line 1 mm).
(a)
(b)
(c)
PLATE. VIII. Showing the transmission of Aspergillus flavus on the host body (a) Pronotum (b-c)
Thorax and Tegmina significantly effective by fungi (Bar-line 1mm).
(a)
(b)
(c)
PLATE. IX. (a-c) Showing the significant infection of Aspergillus fumigatus on the host body images
have been taken after 72hrs of treatment (Bar-line 1 mm).
(a)
(b)
(c)
PLATE. X. (a-c) Showing the infection of uknown fungi species on the host body image clearly
showed that insect become hard and Aspergillus slight spreed and cover whole the body
(Bar-line 1 mm).
(a) Aspergillus niger
(b) Aspergillus flavus
(c) Aspergillus fumigatus
PLATE. XI. Scanning Electron Microscopy of Aspergillus conidia (a) Aspergillus niger
(b) A. flavus (Bar-line 100µm) (c) A. fumigatus (Bar-line 200µm).
(a) Unknown Fungi I
(b) Unknown Fungi II
PLATE. XII. Scanning Electron Microscopy of unknown Fungi conidia (a) Unknown Fungi I (b) Unknown Fungi II (Bar-line 100µm).
(a) (b)
(c) (d)
PLATE. XIII. (a) Collection of infected sample (b) Fungal isolation (c) Culturing of
pathogen media (d) Insertion of prepare medium in incubation for 24hrs.
(a) (b)
(c) (d)
PLATE. XIV. (a-b) Smooth cutting of core chips (c) Fixing of core chips (d) Placement of
Aspergillus samples on conductive double side’s carbon solution taps.
Appendix-I
Important Pest species of Acrididae occurring in Sindh
Sub-family Species
Acridinae
Acrida exaltata (Walker, 1859)
A. gigantea (Herbst, 1786)
Duroniella laticornis (Krauss, 1909)
Gelastorhinus semipictus (Walker, 1870)
Phlaeoba infumata Brunner von Wattenwyl, 1893
P. tenebrosa Walker, 1871
Truxalis exmia exmia Eichwald, 1830
T. fitzgeraldi Drish, 1950
Calliptaminae Acorypha glaucopsis (Walker, 1870)
Sphodromerus undulatus undulatus (Kirby, 1914)
Gomphocerinae
Chorthippus indus Uvarov, 1942
Ch. dorsatus (Zetterstedt, 1821)
Gonista rotundata Uvarov, 1933
Ochrilidia geniculata (Bolivar, 1913)
Oxypterna afghana Ramme, 1952
Hemiacridinae
Hieroglyphus banian (Fabricius, 1798)
H. nigrorepletus Bolivar, 1912
H. oryzivorus Carl, 1916
H. perpolita (Uvarov, 1933)
Spathosternum prasiniferum (Walker, 1871)
Oedipodinae
Acrotylus humbertianus Saussure, 1884
A. longipes longipes (Charpentier 1845)
Aiolopus thalassinus thalassinus (Fabricius, 1781)
Hilethera aeolopoides (Uvarov, 1922)
Locusta migratoria (Linnaeus, 1758)
Oedaleus rosescens Uvarov, 1942
O. senegalensis (Krauss, 1877)
Trilophidia annulata (Thunberg, 1815)
Oxyinae
Oxya bidentata (Willemse, 1925)
O. fuscovittata (Marschall, 1836)
O. hyla hyla Serville, 1831
O. velox (Fabricius, 1787)
Appendix-II
Explanation of rating letter to asses the importance of species according to
(CORP 1982)
A Major pest of many crops.
B Major pest of few crops.
C Pest regularly of substantial importance.
D Pest occasionally of substantial importance.
E Pest occasionally of localized importance.
F A regular minor pest
G An occasional minor pest
H Of very minor importance at times.
K Few records of minor damage; negligible economic significance.
A-E would usually justify control measure whereas F-K would not.
Appendix-III
List of important Aspergillus species occurring in Sindh
S.No. Species
01 Aspergillus candidus Link, 1809.
02 A. crystallinus Kwon & Fennell, in Raper, K.B. and Fennell D. I., 1965.
03 *A. flavus Friedrich link 1809
04 *A. fumigates Fresenius, 1863.
05 A. melleus Yukawa, 1911.
06 A. nidulans (Eidam) Wint. 1884.
07 *A. niger Van Tieghem 1867.
08 A. niveus Blochwitz, 1929.
09 A. oryzae (Ahlburg) Cohn, 1883.
10 A. ostianus Wehmer, 1899.
11 A. puniceus Kwon & Fennell, in Raper, K.B. and Fennell D. I., 1965.
12 A. sclerotiorum Huber, 1933.
13 A. terreus Thom, and Church, 1918.
14 A. unilateralis Thrower, 1954.
15 A. ustus Thom, and Church, 1926.
Note: *Showing the species were tested during this study.
Appendix-IV
Showing an overview of the entomopathogenic fungi that have been developed for
microbial control of insects pests in world-wide (after Lacey et al., 2015).
Species name Targeted insects Produced in Selected reference
Aschersonia aleyrodis Hemiptera (Aleyrodidae)
Russia Fransen (1990), Meekers et al., (2002), Lacey et al., (2008 a,b), McCoy et al., (2009)
Aspergillus sp. Orthoptera Pakistan Kumar and Riffat (2015), Kumar et al., (2013, 2014 a,b, 2015, 2016)
Beauveria bassiana sensu
lato
Acari, Coleoptera,
Diplopoda, Diptera, Lepidoptera, Orthoptera, Siphonoptera, Thysanoptera
Africa, Asia,
Australia, Europe, South & North America
Rosa et al., (2000), Wraight et al.,
(2000, 2007 b), Brownbridge et al., (2001, 2006), Chandler et al., (2005), Wekesa et al., (2005), Brownbridge, et al., (2006), Labbe et al., (2009)
Beauveria brongniartii Coleoptera (Scarabaeidae)
Europe, Colombia, Reunion Island
Zimmermann (1992), Keller et al., (2000, 2003), Dolci et al., (2006), Townsend et al., (2010)
Conidiobolus thrombodies Acari Hemiptera,
Thysanoptera
Colombia,
India, South Africa
Papierok and Hajek (1997), Nielsen
and Hajek (2005), Hajek et al., (2012)
Hirsutella thompsonii Acari India McCoy et al., (2009), Chandler et al., (2000, 2005)
Isaria fumosorosea Acari, Diptera, Coleoptera, Hemiptera, Thysanoptera
Belgium, Colombia, Mexico, USA, Venezuela
Wraight et al., (2000, 2007 a,b), Lacey et al., (2008 a,b, 2011), Zimmermann (2008)
Lagenidium giganteum Diptera (Culicidae)
USA Kerwin and Petersen (1997), Skovmand et al., (2007)
Lecanicillium longisporum Hemiptera Brazil,
Netherland
Bird et al., (2004), Down et al.,
(2009), Kim et al., (2009)
Lecanicillium muscarium Acari, Hemiptera, Thysanoptera
Netherland, Russia
Chandler et al., (2005), Cuthbertson and Walters (2005), Burges (2007), Goettel et al. (2008)
Metarhizium anisopliae sensu lato
Acari, Blattoidea, Diptera, Coleoptera, Hemiptera, Isoptera, Lepidoptera, Orthoptera
Africa, Asia, Australia, Europe, South Central & North America
Rosa et al., (2000), Chandler et al.,(2005), Wekesa et al., (2005), Jaronski and Jackson (2012), Lacey et al., (2011)
Metarhizium acridum Orthoptera Australia, South Africa, USA
Lomer et al., (1999, 2001), Thomas (2000
Nomuraea rileyi Lepidoptera Columbia, India
Moscardi and Sosa-Gomez (2007), Thakre et al., (2011)
Note: Fair No. of entomopathogenic species along with their targeted insects have been enlisted by various workers from all over the world but, any single reference is not yet avaible from Pakistan
present attampt is being carried out for the first time.
Appendix-V
List organized by fungal species and within each species by region (Europe, Africa,
Asia, Oceania, North America, Central America and South America). Within each
region, countries are listed in alphabetical order after de Faria and Wraight (2007).
Country(ies)
Where
Undergoing
Registration,
Registration or
Marketed
Trade name Propagule(s)
Formulation
Claimed Target(s)
(Orders and Families) Manufacturer
Beauveria bassiana
Mexico Bio-Fung A/NI Orthoptera Centro de Sanidad
Vegetal de Guanajuato (CESAVEG), Mexico
USA, Mexico, Denmark, Italy, Sweden
Mycotrol ES C/OD Coleoptera, (Chrysomelidae, Curculionidae, Scarabaeidae), Hemiptera (Miridae, Cicadellidae, Fulgoridae, Aleyrodidae, Aphididae, Pseudococcidae, Psyllidae), Lepidoptera (Crambidae), Orthoptera
(Acrididae, Tettigoniidae), Thysanoptera (Thripidae)
Laverlam International Corporation, USA (Previously: Emerald Bio-Agricultural Corp., USA;
Mycotech Corp., USA)
USA, Mexico,
Denmark, Italy, Sweden
Mycotrol O C/OD Coleoptera, (Chrysomelidae, Curculionidae, Scarabaeidae),
Hemiptera (Miridae, Cicadellidae, Fulgoridae, Aleyrodidae, Aphididae, Pseudococcidae, Psyllidae), Lepidoptera (Crambidae, Noctuidae, Pieridae, Plutellidae) Orthoptera (Acrididae,
Tettigoniidae), Thysanoptera (Thripidae)
Laverlam International
Corporation, USA (Previously: Emerald Bio-Agricultural Corp., USA; Mycotech Corp., USA)
USA Mycotrol OF C/SU Orthoptera (Acrididae, Tettigoniidae)
Mycotech Crop., USA
USA Mycotrol OF C/OD (also for ULV application)
Orthoptera (Acrididae, Tettigoniidae)
Mycotech Crop., USA
USA, Mexico, Denmark, Italy, Sweden
Mycotrol WP C/WP Coleoptera, (Chrysomelidae, Curculionidae, Scarabaeidae), Hemiptera (Miridae, Cicadellidae, Fulgoridae, Aleyrodidae, Aphididae, Pseudococcidae, Psyllidae), Thysanoptera
(Thripidae) Lepidoptera (Crambidae, Orthoptera (Acrididae, Tettigoniidae)
Emerald Bio-Agriculture Corp., USA (Previously: Mycotech Corp., USA)
USA,
Mexico, Greece, Italy, Spain, Switzerland
Naturalis L
(Fermone Natural L-225)
C/OD Coleoptera (Chrysomelidae,
Curculionidae, Scarabaeidae), Diptera (Ephydridae, Mycetophilidae, Sciaridae, Tipulidae), Hemiptera (Lygaeidae, Miridae, Cercopidae, Cicadellidae, Aleyrodidae, Aphididae, Pseudococcidae, Psyllidae), Hymenoptera
(Formicidae), Lepidoptera (Crambidae, Gelechiidae, Geometridae, Noctuidae, Tortricidae), Orthoptera (Acrididae, Gryllotalpidae), Thysanoptera (Thripidae) + Acari (Eriophyidae, Tetranychidae) +
Crustacea + Diplopoda
Troy Bio-sciences
Inc., USA
USA Naturalis L Home & Garden
C/OD Coleoptera (Chrysomelidae, Curculionidae, Scarabaeidae), Hymenoptera (Formicidae), Diptera (Tipulidae), Hemiptera
(Lygaeidae, Cercopidae, Cicadellidae, Aleyrodidae, Aphididae, Pseudococcidae, Psyllidae), Lepidoptera (Crambidae, Geometridae, Noctuidae), Orthoptera (Acrididae, Gryllotalpidae),
Thysanoptera (Thripidae) + Acari (Tarsonemidae, Tetranychidae) + Crustacea + Diplopoda
Troy Bio-sciences Inc., USA
USA Organigard
Emulsifiable Suspension Mycoinsecticides
C/OD Coleoptera (Chrysomelidae,
Curculionidae, Scarabaeidae), Hemiptera (Miridae, Cicadellidae, Aleyrodidae, Aphididae, Pseudococcidae, Psyllidae), Thysanoptera (Thripidae), Orthoptera (Acrididae, Tettigoniidae), Lepidoptera (Crambidae)
Emerald Bio-
Agriculture Corp., USA (Previously: Mycotech Corp., USA)
Lecanicillium sp. (formerly V. lecanii)
India Biovert Rich A/NI
(powder)
‘‘Insects’’ + Nematoda Plantrich
Chemicals & Biofertilizers Ltd, India
Metarhizium anisopliae
Mexico Fitosan-M A/NI Coleoptera (Scarabaeidae),
Orthoptera
Centro de Sanidad
Vegetal de Guanajuato (CESAVEG), Mexico
Mexico Meta-Sin C/WP Coleoptera (Curculionidae,
Scarabaeidae), Hemiptera (Cercopidae), Orthoptera
Agrobiologicos
del Noroeste S.A. de C.V.
(Agrobionsa),
Mexico
Mexico Meta-Sin C/OD Coleoptera (Curculionidae, Scarabaeidae), Hemiptera (Cercopidae), Orthoptera
Agrobiologicos del Noroeste S.A. de C.V. (Agrobionsa),
Mexico
Metarhizium anisopliae var. acridum
Mozambique, Namibia, Tanzania, South Africa, Sudan, Zambia
Green Muscle OF
C/OF Orthoptera (Acrididae, Pyrgomorphidae)
Biological Control Products SA (Pty) Ltd, South Africa (under license from CABI, UK
Australia Green Guard ULV
C/OF Orthoptera (Acrididae) Becker Underwood Inc., USA-Australian division (under licence from CSIRO, Australia)
Australia
Green Guard SC
C/TC (dry spores, surfactant solution and emulsifiable
oil are sold together, but not mixed)
Orthoptera (Acrididae) Becker Underwood Inc., USA-Australian division (under licence from
CSIRO, Australia)
Note: This comprehensive table showing that lot of work has been done on the utilization of entomopathogenic fungi from abroad but there was no work has been carried out from Pakistan. Present attempt regarding Aspergillus is being carried out for the first time from Pakistan and soon it will be marked as registered bio-product at national level.
Appendix-VI
List of abbreviations used during this study.
S.No. Abbreviations Explanations
01 ANOVA Analysis of variance
02 BCAs Biological Control Agents
03 EPFs Entomopathogenic fungi
04 EPNs Entomopathogenic nematodes
05 Fig. Figure
06 LSD Least significant difference
07 N1/N3 First and third nymph stages
08 N4/N6 Fourth and Sixth nymph stages
09 No. Number of samples/insects/replicates
10 RH Relative humidity
11 SE Standard error of the mean
12 sp/spp Species/sub-species
13 Uk FI Unkown fungi I
14 Uk FII Unkown fungi II
15 ULV Ultra low volume
16 UV Ultra violet light
Map. I. Showing the various districts of Sindh from where sampling has been made during
the year 2013-2015.
SANTOSH KUMAR Ph.D Scholar
Department of Zoology, University of Sindh, Jamshoro.
Email: [email protected], [email protected]
URL: https://www.riffatumar.com Cell: +92-333-7133666
PERSONAL INFORMATION
Name: Santosh Kumar Father’s Name: Mithu Mal Date of Birth: 13th October 1986 Religion: Hindu Domicile: Sukkur CNIC No: 45504-9198877-5
ACADEMIC QUALIFICATION
Qualification Grade/Division Institution Year
Ph.D. (Zoology) ------- University of Sindh, Jamshoro ---
M. S (Zoology) First Division University of Sindh, Jamshoro 2013 B.S (Zoology) First Division Shah Abdul Latif University, Khairpur 2009 Intermediate (Pre-medical) A Grade B.I.S.E Sukkur 2005 Matriculation B Grade B.I.S.E Sukkur 2003
GRE Score 69 with 93.62 Percentile NTS 2015
EXPERIENCE
Job title/ Profession
Department/ Status
From To Duration on Post
Job Responsibilities
Research Scholar
Department of Zoology, University of Sindh, Jamshoro.
9th January 2012
Update 4 Years 8 Months 24 Days
Conducting B.S-IV project with supervisor. Inchage (Sindh Entomological Museum) at Department of Zoology, University of Sindh, Jamshoro.
PUBLICATIONS
Scientific Papers: Published 17 quality research papers in peer reviewed Journals of international repute, while 04 in process of writing.
MEMBER OF PROFESSIONAL SOCIETIES
Member of Zoological Society of Pakistan. Member of Pakistan Entomology Society of Pakistan. Member National Academy of Young Scientists Pakistan.
MUSEUM VISITED
Natural History Museum NARC Islamabad.
Natural History Museum PSF Islamabad. Entomology Museum University of Karachi.
PARC Museum Karachi. NIA Museum Tandojam.
SPECIALIZED TRAINING/ WORKSHOP
1. Attended three days’ workshop on “6th National Workshop on Bio-Control Technology” held from 17 to 19th November 2015 at NIA Tandojam.
2. Attended three days’ workshop on “5th National Workshop on Bio-Control Technology” held from 18 to 20thJune 2013 at NIA Tandojam.
3. Attended seminar on “Ecosystem, Health, Soil Plant, Microbial interactions in
Natural and Artificial Ecosystem” on 9th May 2012 at University of Sindh, Jamshoro.
4. Attended three days’ workshop on “4th National Workshop on Bio-Control Technology” held from 2nd to 4th May 2012 at NIA Tandojam.
Total Impact Factor Score
All since 2012 t0 update Total Impact Factor Google Scholar: Citation: 05 H-index: 01
18.544 (IFS)
PUBLICATIONS BY MR. SANTOSH KUMAR
Research Articles (ISI Impact Factor)
1. Kumar, S. and Riffat, S. (2016). Lethal effect of entomopathogenic fungi on the
Grasshoppers (Acrididae: Orthoptera) with special reference to its body size. Sindh
Univ. Res. Jour. (Sci. Ser.). 48 (1): 49-52. [HEC category ‘X’]
2. Riffat, S., Imran, K., Bughio, A., Panhwar, W., Kumar, S. and Soomro, I. (2015). Studies on the importance of common Calotropis procera (asclepiadaceae) and close association of poekilocerus pictus (Fabricus, 1775). Pak. J. Entomol. 30 (2): 161-164. [HEC category ‘Z’]
3. Kumar, S. and Riffat, S. (2015). Investigation on entomopathogenic fungi an effective microbial agent against locusts and grasshoppers in Pakistan. Pak. J. Entomol. 30 (2): 171-174. [HEC category ‘Z’]
4. Zamir, M., Rifft, S., Ali, R., Panhwar, W. and Kumar, S. (2015). Study on the threshold conditions for infection of Visceral leishmaniasis. Sindh Univ. Res. Jour. (Sci. Ser.). 47 (3): 493-496. [HEC category ‘X’]
5. Kumar, S., Riffat, S. and Wagan, M.S. (2015). Impact of entomopathogenic fungi Aspergillus flavus on life history statistics of Hieroglyphus oryzivorus (Orthoptera: Acrididae) Sindh Univ. Res. Jour. (Sci. Ser.). 47 (3): 619-622. [HEC category ‘X’]
6. Kumar, S., Riffat, S. and Wagan, M.S. (2014). Role of Entomopathogenic fungi in suppressing of grasshopper population from Sindh Pakistan.Pak. J. Entomol. 29 (1): 15-20. [HEC category ‘Z’]
7. Soomro, I., Riffat, S., Wagan, M.S. and Kumar, S. (2014). Mating strategies of Poekilocerus pictus (Fabricus, 1775) (Pyrgomorphidae: Acridoidea: Orthoptera). Pak. J. Entomol. 29 (1): 21-25. [HEC category ‘Z’]
8. Panhwar, W.A., Riffat, S., Wagan, M.S., Khatari, I. and Kumar, S. (2014). Systematic study on the various tribes of Phaneropterinae (Tettigonioidea: Orthoptera) occurring in Pakistan. Pak. J. Zool. 46(1): 203-213. [Impact Factor 0.404]
9. Kumar, S., Riffat, S. and Wagan, M.S. (2014). Entomopathogenic fungi in population of acridid grasshopper from Sindh, Pakistan. Int. J. Adv. Res. 2(8): 227-231. [Impact Factor 5.336]
10. Waheed, A.P., Riffat, S., Wagan, M.S., Wagan, Y.S., Kumar, S. and Solangi, F.H. (2014). Taxonomy and Ecology of Genus Euconocephalus Karny, 1907 (Orthoptera: Tettigonioidea: Conocephalinae) from Pakistan. Int. J. Adv. Res. 2(2): 268-277. [Impact Factor 5.336]
11. Kumar, S., Riffat, S., Wagan, M.S., Waheed, A.P. and Solangi, F.H. (2014). Reduction in faeces production and food consumption by three rice grasshopper after infection with Aspergillus species from Badin, Sindh. Inter. J. Bio. Sci. 2220-6655: 10-17. [Impact Factor 0.076]
12. Waheed, A.P., Riffat, S., Wagan, M.S. and Kumar, S. (2013). Notes on the distribution and morphological description of Glyphonotus sinensis Uvarov, 1939 (Orthoptera: Tettigoniinae: Glyphonotini) from Pakistan. Int. J. Adv. Res. 1: 679-682. [Impact Factor 5.336]
13. Kumar, S., Riffat, S. and Wagan, M.S. (2013). Pathogenic application of Aspergillus species for the control of agricultural important grasshoppers. J. Biodiv. Enviro. Sci. (12): 223-229. [Impact Factor 1.028]
14. Waheed, A.P., Riffat, S., Wagan, M.S., and Kumar S. (2013). On the distribution and taxonomy of Conocephalus species (Orthoptera: Tettigonioidea: Conocephalinae) from Pakistan. J. Biodiv. Enviro. Sci. 3(11): 171-176. [Impact Factor 1.028]
15. Khan, M., Riffat, S., Bughio, B.A., Ali, A., Solongi, B.K., and Kumar, S. (2013). Studies on the population dynamics of sugarcane stem borer, Chilo infuscatellus (Lepidoptera: Pyralidae) and its Parasitoid Cotesia flavipes (Hymenoptera: Braconidae) in sugarcane in Hyderabad Region of Sindh. Sind. Uni. Res. J. (Sci-Ser)., 45(3): 542-545. [HEC category ‘X’]
16. Riffat, S., Panhwar, G.R., Panhwar, W.A., Kumar S. and Bhugio, B.A. (2013). Effect of excessive irrigation on the breakdown of Root Rot diseases in cotton crop from sakrand Sindh. Uni. Res. J. (Sci-Ser). 45(1): 15-16. [HEC category ‘X’]
17. Riffat, S., Waseem, A., Bughio, A.B., Waheed, A.P. and Kumar S. (2013). Preliminary studies on the occurrence of Orthoptera from district Jamshoro. Fed. Urdu Uni. Art. Sci. Tech. J. Biol. 3(2): 111-116
PAPER PRESENTATION IN INTERNATIONAL CONGRESS BY MR. SANTOSH KUMAR
1. Riffat, S and Kumar, S. “Natural control of grasshopper by entomopathogenic fungi in Pakistan” has been presented in fundamental and applied aspects of forest soil science, VI All- Russian Scientific Conference with International Participation at Russia September 14-18, 2015. pp. 176.
2. Kumar, S and Riffat, S. “Entomopathogenic fungi associated with natural population of Hieroglyphus perpolita (Uvarov) in Pakistan” has been presented in Fundamental and Applied aspects of Forest Soil Science, VI All- Russian Scientific Conference with International Participation at Russia September 14-18, 2015. pp. 151-152.
3. Kumar, Sand Riffat S and Wagan M.S. “Reduction in the faces production and food
consumption by three rice grasshoppers after infection with Aspergillus species from Badin Sindh, Pakistan”. 3rd Internation Science Congress at Karunya University, Karunya Nagar, Coimbatore, Tamil Nadu, India December 8th-9th, 2013. ISCA-ISC-2013-3BS-68.
PAPER PRESENTATION IN NATIONAL CONGRESS BY MR. SANTOSH KUMAR
1. Kumar, S., Riffat, S. and Wagan, M.S. “Contribution of Aspergillus species in biological control to reduce rice pest population”. 36th congress of Zoology (International) at University of Sindh, Jamshoro. February 2016. pp. 134.
2. Kumar, S., Riffat, S. and Wagan, M.S. “Conservation of biological control using entompathogenic fungi against acridid fauna from Sindh”. 35th congress of Zoology (International) at University of Karachi, Karachi. March 2015. pp. 83.
3. Kumar, S., Riffat, S. and Wagan, M.S. “Studies on the natural level of fungal
infection in grasshopper colonies in lower Sindh, Pakistan”. 2nd International Conference on Agriculture, food and Animal Sciences. 25th-26th February 2015. pp. 30.
4. Kumar, S., Riffat, S. and Wagan, M.S. “Studies on the natural level of fungal infection in grasshoppers colonies in upper Sindh” has presented in 34th congress of Zoology at Bahauddin Zakariya University, Multan 25th-27th February 2014. pp. 172.
5. Kumar, S., Riffat, S. and Wagan, M. S. Susceptibility of developmental stages of Oxya velox (Fabricius) (Orthoptera: Acrididae) to Aspergillus species from Sindh has presented in 33th congress of Zoology at Islamabad 2nd -4th April 2013. pp. 173.
POSTER PRESENTATION IN NATIONAL CONGRESS BY MR. SANTOSH KUMAR
1. Kumar, S. and Riffat, S. “Study on the conservation biological control using entomopathogenic fungi against pest”. 35th congress of Zoology (International) at University of Karachi, Karachi. March 2015.
Updated: October 3rd , 2016
