الفهرس 
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Abstract
UMMARY
Medicinal and aromatic plants play a valuable and important role
in economic, social, cultural and aspects of local communities all over the
world. Medicinal and aromatic plants are infested with several insect
pests including leafhoppers. Leafhoppers are considered as piercing and
sucking pests. Many species are economically important pests of
agricultural crops in worldwide. The present work aims to survey and
identify different leafhopper species infesting medicinal and aromatic
plants in Egypt as well as fingerprinting of each species to detect
phylogenetic relationships among different surveyed species.
1. Surveying of leafhopper species infesting medicinal and aromatic
plants in Egypt:
Leafhoppers adults and nymphs were collected from these plants
at different localities in Egypt using both sweeping net and aspirator
throughout 2011 to 2015.
Fifteen leafhopper species belonging to four subfamilies and
fourteen genera were surveyed.
Synonyms, diagnostic morphological characters, host plants and
geographical distribution of each species were provided. Results showed
that Empoasca decipiens is the most widespread among different species
where it was collected from 32 host plants at Sharqyia, Fayoum,
Qalyubyia and Giza Governorates in Egypt.
The surveyed fifteen leafhopper species:
1.1.Aconurella prolixa (Lethierry)
This species was collected from three plant species; horse mint,
rosemary and roselle at Fayoum and Qalyubyia Governorates during 2013
to 2015.
1.2.Exitianus pondus Ross
This species was collected from eleven plant species including
geranium, marjoram, jasmine, evening primrose and bunchflower daffodil
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Heba E. Ibrahim, M. Sc. Thesis, 2016
at Sharqyia, Fayoum, Qalyubyia and Giza Governorates during 2011 to
2015.
1.3.Nephotettix modulates Melichar
During the present work, only females of this species were
collected from roselle at Qalyubyia Governorate during 2013 to 2015.
1.4.Parabolocratalis sp. Evans
As far as the available literature of leafhoppers, this genus is
considered as a new record in Egypt. During the present work, neither
adult males nor females were collected, only few nymphs were found. So
some specimens were sent to Prof. Dr. christopher H. Dietrich of the
Illinois Natural History Survey, USA. for identification. During the
present work, nymphs of this species were collected from wormseed at
only Qalyubyia Governorate during 2014.
1.5.Balclutha frontalis (Ferrari)
This species was collected from seventeen plant species mainly
ginger, evening primrose and red pepper at Sharqyia, Fayoum, Qalyubyia
and Giza Governorates during 2011 to 2015.
1.6.Cicadulina bipunctella zeae China
This species was collected from eleven plant species mainly
including wormwood plants, ginger and rosemary at Sharqyia, Fayoum,
Qalyubyia and Giza Governorates during 2011 to 2015.
1.7.Cicadulina chinai Ghauri
This species was collected from eleven plant species mainly
wormwood plants, ginger and rosemary at Sharqyia, Fayoum, Qalyubyia
and Giza Governorates during 2011 to 2015.
1.8.Macrosteles sexnotatus (Fallen)
This species was collected from two plant species named, horse
mint and spearmint at both Fayoum and Qalyubyia Governorates during
2014 to 2015.
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1.9.Orosius albicinctus Distant
This species was collected from six plant species; pot marigold,
geranium, roselle, rosemary, jasmine, and ginger at Sharqyia, Fayoum and
Qalyubyia Governorates during 2011 to 2015.
1.10.Psammotettix alienus (Dahlbom)
This species was collected from three plant species; evening
primrose, rosemary and roselle at Qalyubyia Governorate during 2013 to
2015.
1.11.Neolimnus aegyptiacus (Matsumura)
This species was collected from six plant species; roselle,
geranium, rosemary, sweet basil, spearmint and wormseed at Sharqyia,
Fayoum, Qalyubyia and Giza Governorates during 2011 to 2015.
1.12.Empoasca decipiens Paoli
This species was the most widespread among these species and
collected from thirty two plant species mainly pot marigold, wormseed
plant, chamomile, horse mint, water mint, common sage, liquorice, indian
abutilon, or indian mallow, hollyhock or marshmallow, celery, ginger, red
pepper, evening primrose and bunchflower daffodil at Sharqyia, Fayoum,
Qalyubyia and Giza Governorates during 2011 to 2015.
1.13.Eupteryx cypria Ribaut
This species was collected from nine plant species; wild ment,
spearmint, peppermint, horse mint, rosemary, common sage, marjoram,
sweet basil and roselle at Sharqyia, Fayoum, Qalyubyia and Giza
Governorates during 2011 to 2015.
1.14.Megulopa sahlbergorum Lindberg
During the present work, females only of this species were
collected from two host plants; geranium and ginger at Qalyubyia
Governorate during 2014 and 2015.
1.15.Austroagallia sp. Evans
During the present work only females of this species were
collected from horse mint at Fayoum Governorate during 2013 to 2015.
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2-An identification key based on morphological characters designed
to identify different surveyed species.
3- Molecular genetic analysis of surveyed species:
This molecular analysis was carried out by using two techniques;
ISSR-PCR and mtDNA COI based on polymerase chain reaction (PCR)
as follow:
3.1. Inter Simple Sequence Repeat-Polymerase Chain Reaction
(ISSR-PCR):
Inter Simple Sequence Repeat Polymerase Chain Reaction
technique was carried out using seven random primers that were selected
to apply with the fifteen leafhopper species. These primers could be used
to differentiate between different leafhopper species and leafhopper
genera as well. These primers produced a total of sixty nine bands, fifty
six bands were polymorphic while 9 bands were positive markers, 3
common bands and 3 bands were negative markers as follow:
3.1. 1. Primer 14A
This primer produced seven bands with molecular sizes ranged
between 1337-342bp. All of them were polymorphic (100%). While band
with molecular size of 1337 bp. can be assigned as a positive marker for
C. chinai. No common band was detected by this primer.
3.1. 2. Primer 44B
This primer amplified ten bands with molecular sizes ranged
between 919 – 224bp. Nine of them were polymorphic (90%). On the
other hand, band with molecular size of 384 bp. was monomorphic
(common). The band with molecular size of 307bp. can be assigned as a
negative marker for N. aegyptiacus..
3.1. 3. Prime HB-08
This primer amplified ten bands with molecular sizes ranged
between 734 – 174bp. Nine of them were polymorphic (90%). Band with
molecular size of 689 bp. was unique and can be assigned as a positive
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marker for A. prolixa. While bands with molecular sizes of 332 and 281
bp. was assigned as a negative markers for Austroagallia sp. and C.
bipunctella zeae, respectively. No common band was detected by this
primer.
3.1. 4. Primer HB10
This primer amplified nine fragments with molecular sizes ranged
between 946 – 288bp. All of them were polymorphic (100%). The highest
number of bands were 6 bands detected in A. prolixa, E. decipiens, N.
modulates, P. alienus and M. sahlbergorum.
3.1. 5. Primer HB12
This primer amplified 12 bands with molecular sizes ranged
between 1672 – 167bp. Six of them were polymorphic. Band with
molecular sizes of 1672bp. can be assigned as a positive marker and
unique band for Parabolocratalis sp. Also three bands with molecular
sizes of 759, 670 and 613bp can be assigned as a positive markers and
unique bands for C. chinai. Each of Bands with molecular sizes of 364
and 276 bp. could be assigned as positive markers and unique bands for
A. prolixa. and P. alienus, respectively.
3.1. 6. Primer HB14
This primer amplified ten bands with molecular sizes ranged
between 922 – 252bp. Eight of them were polymorphic (80%) while
bands with molecular sizes of 922 and 294bp. were unique where these
bands can be assigned as positive markers for Austroagallia sp. and C.
bipunctella zeae respectively. No common bands were detected.
3.1. 7. Primer HB15
This primer amplified eleven bands with molecular sizes ranged
between 1542 – 315bp. Nine of them were polymorphic while bands with
molecular sizes of 980 and 538bp. were monomorphic (common) bands.
On the other hand, band with molecular size of 315bp. can be assigned as
a negative marker for Austroagallia sp.
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3. 2. Mitochondrial Cytochrome Oxidase I (mtCOI)
These primers were designed and registered at GenBank and sent
abroad for Sequencing then find the percentages of similarity matrix.
Only 8 species interacted with COI primers and also registered at
GenBank. The phylogenetic dendrogram was drawn.
4.Phylogenetic relationships and similarity analysis among different
surveyed leafhoppers:
Phlogenetic relationships among different surveyed leafhopper
species in Egypt were based on three ciriteria; diagnostic morphological
characters, ISSR-PCR and the combined relationship between diagnostic
morphological characters and ISSR-PCR. Results showed that the
molecular and morphological analysis together provided more conclusive
results among different leafhopper species.
4.1. Phylogenetic relationships based on morphological characters
analysis:
The phylogenetic relationships among different leafhopper species
based on 65 diagnostic morphological characters were recorded as absent
and present (0/1).
The closest relationship was scored between C. chinai and C.
bipunctella zeae with similarity percentage of 89.9 %. While no similarity
percentage (0 %) was recorded between Parabolocratalis sp. and the
other leafhopper species except for M. sahlbergorum with similarity
percenage of 15.4%.
Result showed that the phylogenetic dendrogram was divided into
two main clusters. The first cluster included species of Parabolocratalis
sp. and M. sahlbergorum which were grouped together. The second
cluster was divided into two sub-clusters, the first sub-cluster contained
Eupteryx cypria and Empoasca decipiens which were grouped together.
The second sub-cluster was separated into two sub-sub-clusters; within
the first sub-sub-cluster, species of Austroagallia sp. and N. modulates
were grouped together. Meanwhile, the second sub-sub-cluster contained
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M. sexnotatus, N. aegyptiacus, E. pondus, P. alienus, A. prolixa, O.
albicinctus, B. frontalis, C. chinai and C.bipunctella zeae which were
grouped together.
4.2. Phylogenetic relationships based on molecular (ISSR-PCR)
analysis:
This analysis was based on seven primers. The closest relationship
was scored among E. cypria and M. sexnotatus. The highest similarity
percentage was 92.5 % while the lowest similarity value was 32.7 %
among Austroagallia sp. and M. sahlbergorum.
Result showed that the phylogenetic dendrogram branched into
two main clusters that E. decipiens, A. prolixa, M. sahlbergorum, N.
modulates, Parabolocratalis sp. and P. alienus were grouped at the first
cluster and species Austroagallia sp., N. aegyptiacus, E. pondus, C.
chinai, C. bipunctella zeae, O. albicinctus, B. frontalis, M. sexnotatus
and E. cypria were grouped at the second cluster.
4.3.Phylogenetic relationships based on both morphological
characters and molecular (ISSR-PCR) analysis:
This analysis was based on seven primers and 65 morphological
characters. The most closest relationship was scored among C.
bipunctella zeae and C. chinai. The highest similarity value was 80.3%
and the lowest similarity value was 26.3% among C. bipunctella zeae and
M. sahlbergorum,individuals.
Result showed that the phylogenetic dendrogram branched into
two main clusters that the first cluster divided into two sub-cluster. The
first sub-cluster separated into two sub-sub-clusters. The first sub-subcluster,
E. decipiens was found alone. The second sub-sub-cluster divided
into two clades within the first clade A. prolixa was located alone while
the second clade included P. alienus and N. modulates. The second subcluster
included M. sahlbergorum and Parabolocratalis sp. which were
grouped together. The second cluster separated into two sub-clusters. The
first sub-cluster Austroagallia sp. was located alone. Meanwhile, the
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second sub- cluster separated into two sub-sub-clusters. The first sub-subcluster
N. aegyptiacus and E. pondus were grouped together. The second
sub-sub-cluster contained M. sexnotatus and E. cypria, O. albicinctus
and B. frontalis, C. chinai and C. bipunctella zeae which were grouped
together.
4.4.Phylogenetic relationships based on the Mitochondrial
Cytochrome Oxidase Gene (COI):
Eight cicadellid species only were successfully amplified, the
identity percentages between the partial sequences from each species
obtained and the highest similarity value was 84.5% between M.
sexnotatus and O. albicinctus and the lowest similarity value was 38.7%
between E. decipiens and the rest of species.
Result showed that the phylogenetic dendrogram indicated two
main clusters in the first cluster Empoasca decipiens was found alone.
The second cluster separated into two sub-cluster, within the first subcluster
N. modulates was alone. Meanwhile, the second sub- cluster
Austroagallia sp., Eupterxy cypria, P. alienus, O. albicinctus, M.
sexnotatus and E.pounds were grouped