الفهرس 
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Abstract
The present study dealt with the survey of native entomopathogenic
species and strains effecting lepidopteran species. Laboratory assays of the
entomopathogenic nematode species and strains against lepidopteran species
were carried out. Production of Heterorhabditis bacteriophora infective
nematodes of three strains from Galleria mellonella larvae and pupae was
determined in the laboratory . Effect of storage of entomopathogenic nematodes
in distilled water and sandy soil at 15° C & 25° C on their viability was
also studied. Also, field applications of some promising local and other
entomopathogenic nematode species and strains against the apple stem borer
were carried out. Obtained results may be summarized as follows:
1. Laboratory Experiments :
A. Survey of Indigenous Nematode Isolates:
Soil samples were collected from three different regions, i. e. , Salhiya
(Ismailia Governorate) , Siwa Oasis, (Mersa Matrouh Governorate) and North
Sinai Governorate. Three H. bacteriophora isolates were recovered from the
soil collected from Tanaweesh ( under olive trees, Olea europaes), Talloh and
Tammoussi ( under palm trees, Phoemix dactylifera) districts of Siwa . No
nematode isolates were recovered from soil samples collected from the other
two Governorates . All the treated Galleria larvae showed the dark reddish
colour indicating that the nematodes belong to the genus Heterorhabditis.
Nematode preparations were examined for their efficacies against different
lepidopteran pests. Three strains of H. hacteriophora were identified and
nominated as strain GNI , strain GN2 and strain GN3 .
B. Nematode Identification :
Morphological and morphometric characters of the infectivejuveniles
of each of the three isolates ( GNI , GN2 and GN3 ) of H. hacteriophora in
the present study were determined. Some morphometries of the three isolates
were found different such as body length, greatest width, head to excretory
pore and head to pharynx base. The differences were considered as depended
mainly on the habitats and localities of isolates.
c. Molecular Work
* RAPD - peR Optimization:
Similarity matrix for the results of I Js of entomopathogenic
nematode isolates ranged between 0.69 -1.00 . H. hacteriophora (strain
GN 2 ) showed the lowest level of similarity compared to the other two
strains( GN [ and GN3) • H. hacteriophora (strain GNI ) showedthe highest
level of similarity than strain GN3 . Random amplified polymorphic DNA
(RAPD) fragment analysis showed that the species also has a distinct genetic
pattern in RAPD bands relative to the other 3 strains or isolates of
Heterorhahditis that were compared. Cluster analysis of the populations using
scorable markers revealed that the 3 populations could be grouped into 2
clusterswith genetic similarity ranging from 69 to 80 % . Differences
observed in the RAPD profiles of different isolates could be used for
developingmolecular markers specific to a particular population .
D . Infectivity of Some H. bacteriophora Isolates in the Control
of Some Insect Pests Related to order Lepidoptera
In these studies, each of the three tested isolates of H.
bacteriophora was applied in each of six concentrations ( 10 , 20 , 40 , 80 ,
160 and 320 infective juveniles / em? of soil surface) on the sand surface
containing the desired stage which was placed either every 5 individuals
( first method) , or single individual ( second method) in sand in a 100 cc half
filled plastic cup. The efficacy of all of the three entomopathogenic
nematode strains, on the full - grown larvae and one and three day old pupae
of all of the tested species was a concentration dependent; i. e., the mortality
percentages increased as the applied concentrations were increased. Individual
treatments ( second method) of either of the used stages led to more efficacy
of the applied nematode strains ( higher mortality rates and lower LC50 s)
than application of the same nematode strains and concentrations by using
the first method ( 5 individuals/ cup) . Lower infectivity ( lower mortality rates)
of H. bacteriophora isolates ( GNI , GN2 and GN3) was observed for all used
nematode dosages at temperature 25° C as compared to 30° C which exhibited
higher infectivity of nematode isolates against the targeted pest stages. Among
the tested stages , G. mellon ella full- grownlarvae were the highest
susceptible ( highest mortality rate and lowest LC50) compared to the one and
three day old pupae of the highest pest species . On the contrary, the
three day old pupae showed highest resistance.
Generally, H. bacteriophora strain GN3 gave the highest efficacy
against G. mellon ella treated larvae and one & 3 day old pupae at 25° C
showing low LC50 s , being 22.32,28.08 and 33.38 I Js /cm ? of soil
surface, respectively, in first method, opposed to 17.71 ,21.58 and 26.38
I Js / cm2 of soil surface, respectively, in the second method. While, at 30° C ,
in first method, strain GN, showed highest efficacy as the LC 50 ’ s were
11.38 and 14.54 I Js / em” against full- grown larvae and one day old
pupae, respectively, and the efficacy was the same between ONI and ON3
in second method, as the LC50 against G. mellonella full- grown larva was
5.38IJs / cm2
•
H. hacteriophora (strain ON3) exhibited the highest efficacy against
S. littoralis stages, showing lowest LC50 values ( 5.38 , 6.67 and 12.45I J s /
cm2 ) on treated larvae, and one & three day old pupae, respectively,
followed by strain GN, . While, H. hacteriophora (strain ON2 ) was the
least effective one on S. littoralis stages.
In the first method at 25° C, H. hacteriophora strainGN2 gave the
highest efficacy against full - grown larvae and 3 day old pupae of A . ipsilon
(lowest LC50. s; 25.82 and 38.10 I Js /cm’’ ,respectively) , while GN3gave
highest efficacy against one day old pupae (LCsO = 34.56 I Js / em 2) • While,
at 30° C , GN2gave highest efficacy against full- grown larvae and GN3 gave
high efficacy against one and three day old pupae. In the second method
at 25° C , higher efficaciesresulted from GNI strain against full - grown larvae
and from ON2 against one and three day old pupae. At 30° C ,ONI gave
highest efficacy against A. ipsilon stages.
Generally at 25° C , in first method H. hacteriophora strainGN2
gave the highest efficacy against full - grown larvae and same efficacy as
GN,against one day old pupae of P. operculella, and ON! gave highest
efficacy against three day old pupae, but in second method GN, gave
highest efficacy against all stages. At 30° C , in the first method, H.
bacteriophora GN2 was the highest effective against full - grown larvae
and one day old pupae, and GN[gave highest efficacy against three day old
pupae. In second method, the three strains were of the same efficacy.
H. bacteriophora strainGN2 gave the highest efficacy against E.
cautella stages at 25° C , but at 30° C in first method GN 2 was highest
effective against full - grown larvae and one day old pupae, and GN 3
gave highest efficacy against three day old pupae. In second method, GN2
gave high efficacy against full - grown larvae and one day old pupae, but
GNI gave high efficacy against three day old pupae.
By exposure of 5 individuals / cup, H. bacteriophora strainGN2
gave the highest efficacy against all inspected stages of E. calidella at 25
and 30° C . While, in second method at 25° C , GN3 gave highest efficacy
against E. calidella stages, but at 30° C , GN2 was the highest effective
against full - grown larvae and one day old pupae, and gave the same
efficacy as GN [and GN3 in second method against three day old pupae.
Generally, H. bacteriophora strain GN2 was the highest effective
againstA. sabellae stages in second method (individual exposure) at 25° C ,
but in first method, GN3 gave highest efficacy against full - grown larvae
and same efficacy as GN2 against one day old pupae while GN2 gave highest
efficacy against three day old pupae. In first method at 30° C , GN 3 gave
highest efficacy against A. sabellae stages, while by using the second
method at 30° C , GN[and GN2 were higher in efficacy against full - grown
larvae, but the three strains of H. bacteriophora were of the same efficacy
against one and three day old pupae.
E.!. Production of infective juveniles from G. mellonella larvae
infected at concentrations of 2000 and 4000 I Js / cm 2 of soil
surface:
H. bacteriophora (strain GN [) was, generally, the highestreproductive
strain from G. mellonella full-grown larvae infected at two tested
concentrations 2000 and 4000 I Js / em’ of soil surface. While, the other two
strains ( GN2 and GN3 ) were of lower productivity of I Js compared to
strain( GN [ ) . A single full - grown larva of G. mellonella infectedwith
H. bacteriophora (strain GNI) produced an average of 143060 (133800-
155500) I Js / larvae when infected with a concentration of2000 I Js / cm2
of soil surface, opposed to 171080 (150300 - 183350) I Js / larvae when
infected at 4000 I Js / em2 of soil surface.
E.2. Production of infective juveniles from G. mellonella pupae
infected at concentrations of 2000 and 4000 I Js / em2 of soil
surface:
H. bacteriophora (strain GNI ) was, also, the highest reproductive
strain at the two tested concentrations 2000 and 4000 I Js / em 2 of soil
surface. Production of Lis by H. bacteriophora (strains GN2 and GN3 )
was less compared to strain ( GNI ) . A single one day - old pupa of G.
mellonella infected with H. bacteriophora (strain GNI ) at concentrations
2000 and 4000 I Js / em” of soil surface, produced the averages of 73295
(66900 - 80750) and 87005 (76900 - 95600) I Js / pupa, respectively.
F. Storage:
F.l. Storage in distilled water:
The persistence of H. bacteriophora I is in distilled water was
determined for either of the three isolates at four different concentrations
( 1000,000; 500,000 ; 250,000 and 125,000 I is / cup) and at temperatures
( 15 and 25° C ) . The comparison between different treatments was based
on time elapsed until mortality of I is .
Prolongation of the storage period for 50 or 60 days at 15° C in
distilled water had harmful effect on juveniles as the majority of these
I is ( 64 - 100 %) died. It could be also deduced from the obtained results
that the mortality percentages among the stored juveniles of GNI strain were,
generally, lower than those of GN2 , while GN3 juveniles manifested the
highest mortality percentages .
It was clear from data of treatments that most of the stored juveniles
survived in distilled water at the two lower concentrations ( 125 and 250
thousands of juveniles / cup) , while on the contrary, all the juveniles died
by storage at the two higher concentrations (5000000 and 1000000juveniles /
cup) for 30 days.
It could be, generally, stated the H. bacteriophora juveniles of the
three identified strains are better to be stored in distilled water at 15° ethan
25 ° C in order to insure more surviving juveniles for a longer period
of storage .
F.2. Storage in sterile sand:
The persistence of different H. bacteriophora isolates stored at the
same mentioned four concentrations for different periods was estimated at
two temperatures (15 and 25 ° C) . The comparison between the different
treatments was based on the infectivity of! Js to G. mellonella larvae after
different periods of storage.
from the obtained data, it could be stated that, in order to obtain
highest infectivity of H. bacteriophora juveniles after storage in moistened
sandy soil at 15° C , it was better store the juveniles for a period not more
than 2 months. To store for 3 months, it was better to use a low
concentration of 125000 I Js / cup.
It was clear from data of treatments that storage of juveniles for
the longest period ( 240 days) was the highest in causing deleterious effect
on the capability of juveniles to infect G. mellonella larvae at 15° C as the
two lower concentrations of GN I and the lowest concentration of GN2 showed
20 % infectivity. While at 25° C the longest period of storage was 60 days
for the three strains when stored at the two lower concentrations ( 125 and
250 thousands of I Js / cup) . It could be , generally, stated that H.
bacteriophora juveniles of the three identified strains are better to be stored
in sterile sand at 15° C than 25° C in order to insure highest infectivity of
juveniles to target insects after a longer period of storage .
G. Histopathological Study of the Greater Wax Moth G.
mellonella After infection With 11. bacteriophora (strains
GN” GNz and GN3)
G.
The. hIiStopath01ogi.ca1patterns revealed variable effects on
mellonella. larvae after infection with the entomopathogenic nematode, H.
b
. h ( strains GNI , GN2 and GN3) . In all cases, muscles and
acteriop ora
fat tissues of the infected larvae were greatly affected in comparison with
those of control tissues ( untreated larvae) . When treated with
H.
. ( t . ONI ON2 d ON3 ) , muscles suffered destruction bacteriophora s rams , an
and fattissues showing high
in the fibrillate with some fragmentation
vacuolation.
H.Infectivity of Different Entomopathogenic Nematodes to Full-
Grown Larvae and One Day Old Pupae of Clear Wing
Moth ,Synonthedon myopaeformis :
The effrcacy f h f Steinernema carpocapsae , S. abbasi, 0 eac 0
H b . h against the full-grown larvae Heterorhabditis indica or . acteriop ora
and one day old pupae of the clear - wing moth were studied in the
1 b 2 0 C Exposure of larvae or pupae to I Js of the desired
a oratory at 5
nematode species were made by placing 5 individuals / cup after treatment of
soil by either of the three concentrations ( 10,20, or 40 juveniles / em 2 of
’1surface) . With all treatments, the efficacy was a concentration
SOl
dependent. By application of either of the mentioned nematode 4 species,
lowest mortality rates resulted after treatments by the lowest concentration
278
( 35 ,35 ,50 & 35 % mortality among treated larvae opposed to 30 , 25 , 35
& 35 % among treated pupae, respectively) . While, the highest mortality
percentages ( 85 , 90 , 90 & 90 % among larvae and 85 , 80 , 90 & 90 % ,
respectively among treated pupae) resulted one week after treatment by
the 4 nematode species at 40 I Js / cm’’ of soil
2. Field Experiments :
A field experiment was conducted to find out the role of the
same mentioned 4 entomopathogenic nematode species in suppressing
population of Zeuzera pyrina larvae and pupae infesting apple and olive
trees. Orchards were chosen in Kalubia (Apple trees) and North Saini
( Olive trees) Governorates. The rierrratocle species used were S.
carpocapsae, S. abbasi, H. indica and H. bacteriophora. Theinjection
method was adopted. The highest efficacy rate on olive trees was
recorded from S. carpocapsae treatments ( 63.62 % ) mortality while H.
bacteriophora causedthe highest mortality rate ( 64.06) on apple trees.