الفهرس 
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Abstract
Pomegranate (Punica granatum L.) is an important fruit in many tropical and subtropical regions. In Egypt, pomegranate is one of the most important fruit crops. Unfortunately, pomegranate fruits are attacked with a range of insect pests. The most serious pests are the lepidopteran insects.
The present work was initiated generally to identify species composition of lepidopteran insect pests attacking pomegranate and inhabiting different host plants and the eggs spatial distribution of pomegranate butterfly. Also, the study of entomophagous insects associated with pomegranate butterfly and evaluate the efficacy of egg parasitoids Trichogramma on it. The study was conducted in the Experimental Farm, Faculty of Agriculture, Assiut University, Assiut, Egypt 31° 11’ 21.4188’’ E Longitude; “27° 10’ 48.4824’’ N” Latitude during two successive growing seasons (2020 and 2021). A total number of 130 trees (12 years old) of Manfaloty cultivar in pomegranate orchard were chosen for the present study. The other host plants: sweet acacia and date palm trees were distributed in clusters around pomegranate orchards. Inspection of lepidopteran insect pests started from pomegranate fruit setting tell harvesting. Also, pomegranate lepidopteran insect pests were inspected from sweet acacia and date palm. Insects inspected were picked out to laboratory for identification. Reviewing literature concerning species composing leads our work forward to the main insect pest attacking pomegranate fruits is pomegranate butterfly which only attacking severely pomegranate fruits.
The main goal of this study is to identify pomegranate butterfly inhabiting different host plant by morphological and molecular genetics. Also, to study spatial distribution pattern of pomegranate butterfly eggs and entomophagous insects associated it. Achieving such goals helps to plan and design an integrated pest control program for such a dangerous pest. Results can be summarized as follow:
1. The Species composition of lepidopteran insect pests attacking pomegranate and inhabiting different host plants
1.1. Morphological and molecular identification of pomegranate butterfly
1.1.1. Morphological identification
The body coloration, wing venation, legs, and body morphometric were the morphological criteria used for identifying pomegranate butterflies. By using light microscopy and scanning electron microscopy (SEM), some morphological structures of male and female pomegranate butterfly were illustrated.
1.1.1.1. General body coloration
General color of body scales for the three specimens are mix dark light grey in dorsal side and white in ventral side in both sexes.
1.1.1.2. The head
1.1.1.2.a. Compound eyes
The color of compound eyes was brown and coppery in the three specimens. For the first time study by focusing on one scanned eye, the interfacial hairs were found distributed across the eye in a corner between three facets.
1.1.1.2.b. Antennae:
The antenna of the three specimens is clavate type. The number of segments 39 segments in both sexes. The color of terminal segments is orange and black, but other segments are black and white.
1.1.1.2.c. Mouthparts:
Mouthparts of sucking type in the three specimens were found with a pair of three-segmented labial palps and a brown coiled proboscis (galea).
1.1.1.3. The thorax
In both sexes, the coloration of the thorax is shiny dark grey.
1.1.1.3.a. Legs
The apical tarsomere bears two claws with pulvill and empodium in all legs for both sexes except the foreleg of the male consist of one claw.
1.1.1.3.b. Wings
Wing coloration
Both sexes of the three collected pomegranate butterfly are differed in their coloration.
• Upper side of female and male wing:
Female forewing upper side scaled, dark brown in the apical angle and apical margin, brown color in the post-discal and discal area, and a bluish tint becoming darker in the humeral angle. Female hind wing upper side covered in brown scales except for the anal margin and humeral angle, which are squirrel color. Also, two spotted patches were recognized nearby the anal angle. The upper side of the male forewing is covered by dark orang scales, except for the costal margin, apical angle, and apical margin, which are brown in color. Except for the humeral angle, anal margin, and part of, the male hind wing upper side is covered by dark orang scales.
• Lower side of female and male wing
The lower sides of wings were similar in both sexes. The fore and hind wings lower side covered with grey scales with different bands scattered.
Wing Venation
The forewing consists of 11 veins: Sc, R1, R2, R3, R4+5, M1, M2, M3, Cu1, Cu2 and A1+2. The hind wing consists of 9 veins, include Sc + R1, Rs, M1, M2, M3, Cu1, Cu2, A1+2 and A3.
1.1.1.4. The abdomen
The number of abdominal segments is 10 segments in both sexes. The last three segments are greatly modified to form the external genitalia.
1.1.2. Morphometrics for three pomegranate butterfly specimens
Statistical analysis indicated that the differences between measured morphological features include BL, AntL, and WL were non-significant in the three specimens of pomegranate butterfly.
In general, the morphological full description and morphometric measurements can be considered of great importance in studying the pomegranate butterfly as a serious pest attacking fruits of three host plantation. Comparing our results with other morphological studies emphasized the importance of molecule identification for the pomegranate butterfly collected to submit the real scientific name which transferred Virachola livia to D. livia.
1.1.3. Molecular genetic identification
The first-time record of Deudorix livia, in The GenBank for the partial nucleotide sequences of CO1 were deposited under accession numbers (MW463927, MW463928, and MZ429317 for pomegranate, date Palm, and sweet acacia, respectively). The average nucleotide frequencies of adenine (A), thymine (T), cytosine (C) and guanine (G) were 33.5, 37.9, 14.7, and 13.9 %, respectively. The average A+T content (71.4%), was higher than the average of C+G content (28. 6%). Among the understudied D. livia were pomegranate butterfly specimens with pairwise genetic distances ranged from 0.0017 to 0.0035. Among the understudied D. livia and other related species the pairwise genetic distances ranged from 0.0017 to 0.0153. The importance of real nomenclature for the pest one of the most important steps for conducting appropriate control approach to reduce the population density of pomegranate butterfly bellow the economic injury level.
2. Distribution patterns of D. livia inhabiting different host plants
2.1. Phenology of host plants and incidence of D. livia during 2020 and 2021 seasons
Results of our present study indicated that sweet acacia plantations harbored D. livia eggs early before pomegranate fruit setting. For that acacia can be considered a source of infestation for pomegranate fruits. Also, it is of interest to point herein that the presence of this pest on these alternative plant hosts helps in monitoring its arrival and starting planning to control it on pomegranate and date fruits by safe alternative control methods such as inundation by biological control agents.
2.2. Population fluctuation of D. livia eggs on Manfaloty pomegranate cultivar in relation with a biotic factor during the growing seasons
D. livia egg numbers increased in October during two seasons, proved that egg deposition during this period of the pest can be seriously favorable time for damaging pomegranate fruits. Therefore, increasing the population in this period might led to a great economic loss in pomegranate orchards. The total numbers of D. livia eggs encountered on pomegranate fruits in 2020 was (3816 eggs) higher than 2021 (2902 eggs). The statistical analysis between the differences of both seasons were highly significant (f value= 26.11**). However, the populations of eggs fluctuated with three peaks during 2020 more than 2021 (two peaks).
The maximum, minimum wind speed, and relative humidity were highly significant (f value= 9.72**, 129.58**, and 92.09**); respectively. However, the r value between eggs average numbers and maximum wind speed was negatively and highly significant in 2020, but in 2021 was negatively and non-significant. This difference is one reason for the differentiation in the total numbers of D. livia eggs counted during the two seasons. On the other hand, the coefficient of determination for all weather factors was R2 = 68% and 20.31% in 2020 and 2021, respectively during the growing seasons. The differences effects of the ambient climatic factors on D. livia egg numbers, explains the differences in fluctuation dynamics of encountered between two seasons.
3. Spatial distribution patterns of D. livia on pomegranate Manfaloty cultivar during 2020 and 2021growing seasons
The spatial distribution of D. livia eggs in cardinal directions and preferable direction side on pomegranate orchard are decisive factors that have important implications for regulation of D. livia population on pomegranate fruits and should be considered for designing the management program.
The population density of eggs deposited by D. livia in pomegranate orchards in both seasons was the West direction. Also, the preferable side direction was the Western South side. The pattern of cardinal spread of D. livia eggs in the orchard may provide good information about the native any system of pest activity in the orchard. So, control measures should be applied and concentrated in west south direction. Such an application should give successful management saving money, effect, and time.
4. Entomophagous insects associated D. livia in pomegranate Manfaloty cultivar during 2020 and 2021growing seasons
4.1. Entomophagous Insects recovered by different methods in pomegranate orchard
Predacious insect’s data revealed the existence of 19 insect species belonging to 15 genera under 9 families of 7 orders. Moreover, unidentified parasitoids species belonging to 2 genera under 2 families 1order and 2 species identified belonging to 2 genera under 2 families of 2 orders.
Thirteen insect species, represented by (68% of predacious insects) were collected by means of pitfall traps. However, 11 insect species were collected by yellow sticky traps and represented by (58%). Intensive and extensive observations indicated that pitfall traps can be considered as one of the most important methods for collecting predacious insects especially adult stages which flight on pomegranate orchards such insect predators include Labidura riparia, Geocoris punctipes, Coccinella undecimpunctata, Camponotus maculatus maculatus, and Syrphus corollae. Also, yellow sticky traps can be considered an important collecting device for monitoring and collecting arboreal predacious insects such as Scymnus suturalis, S. syriacus, chrysoperla carnea, and S. corollae. Concerning parasitoids species recovered needs more future studies for collecting and monitoring on pomegranate butterfly stages.
For monitoring entomophagous insects, the direct observation and picked-up flower methods can be used. Species collected by the direct observation and picked-up flower methods reached 10 and 4 species, respectively.
4.2. Dominance and abundance of certain predacious insects collected by two devices
Yellow sticky traps can be considered the best trap for monitoring and collecting arboreal species and pitfall traps the best device in case of terrestrial species.
Dominance and abundance percentages values of predacious insect’s inhabiting pomegranate orchards encourage researchers to maximize role as naturally occurring biological control agents (NOBCA). from our point of view, the more abundant predacious insects in pomegranate orchards are of great importance. In this situation, coleopteran predacious insects such as C. undecimpunctata, S. syriacus, and S. suturalis were more abundant by pitfall and yellow sticky traps. These coleopters insects can play active role not only on pomegranate orchards but also in any given agroecosystem.
4.3. Population dynamics of pomegranate butterfly eggs in relation to biotic factors
Predaceous insect’s population collected by pitfall and yellow sticky traps ware reached (88 and 782 insects) during 2020 growing season respectively. However, populations in both devices reached (105 and 2172 insects) during 2021 growing season respectively. The above-mentioned numbers referred to the differences in number of predaceous insects collected by each pitfall or yellow sticky traps. The population fluctuations for predaceous insects in yellow sticky trap was fluctuated more than in pitfall traps in both seasons.
The Correlation coefficient (r) values between the average number of eggs and predaceous insects collected by pitfall traps negative and significant during the first season. But in the second season was negative and non-significant with values (r = -0.01379*, p= 0.0599 and -0.01379ns, p= 0.8463), respectively. However, in both seasons were positive and highly significant in yellow sticky traps with values (r = 0.22979**, p= 0.0019 and 0.22091**, p= 0.0017) in 2020 and 2021 growing seasons, respectively. The coefficient determination for the biotic and a biotic factor was 92.5 and 44.1% during 2020 and 2021 growing seasons, respectively. These findings means that there is another unknown factor (7.5 and 55.9%) responsible about the population density of D. livia in pomegranate orchards.
4.4. Spatial distribution patterns of predaceous insects collected by yellow stick trap on pomegranate Manfaloty cultivar
4.4.1. Distribution of predaceous insects collected by yellow stick trap in cardinal directions and center
The maximum percentages of predators collected in 2020 season was 23% in West direction. It was followed by 22, 19.2, 18.4, and 17.1% in east, south, center, and north directions, respectively. During 2021 The maximum percentages of predators was 25% in north direction followed by 22.3, 21.8, 19.5, and 11.2% in center, east, south, and west directions, respectively.
Two seasons analysis revealed that the averages encountered in all directions were in 2021 season higher than 2020 season. F value between two seasons was significant (2.37*). Generally, the maximum average number of predators in both years was in north followed by east, center, south, and west (8.81, 8.49, 8.21, 7.53, and 5.81), respectively.
Two seasons analysis revealed that the average encountered in all directions in 2021 season higher than 2020 season. F value between two seasons was significant (2.37*). Generally, the maximum average number of predators in both years was in north followed by east, center, south, and west (8.81, 8.49, 8.21, 7.53, and 5.81insect), respectively.
4.4.1. Preferable spatial distribution in cardinal directions for predaceous insects collected by yellow stick trap on pomegranate Manfaloty cultivar
Most preferable direction for predators was the Southwestern side during 2020 growing season. However, during 2021 growing season was East Northern side. The pomegranate plants in the Southwestern side making angle (27º10´54”) during 2020 growing season and (27º11´02”) in East Northern side during 2021 growing season.
The density of predators differed in different directions during two growing seasons, and this may be due to different a biotic factors or other unknown factors.
It is important to point herein that, knowing that there are no natural enemies in different directions encourages the best use of control methods by increasing the number of natural enemies by releasing egg parasitoids Trichogramma or suitable predatory such as green lacewing and reducing the use of pesticides in directions with an abundance of natural enemies. This improves the knowledge in applying the best integrated management strategy to combat such a dangerous pest D. livia.
In conclusion, this knowledge about spatial distribution of predacious insects in pomegranate orchards is firstly discussed in the present work.
5. Efficacy of Trichogramma turkestanica on eggs of D. livia and Achroia grisella under laboratory conditions
The biological parameters to determine the efficacy of egg parasitoid T. turkestanica on pomegranate butterfly eggs, D. livia in comparison to lesser wax moth, A. grisella were studied under laboratory conditions. The biological criteria include percentages of successive parasitized eggs, adults emerged from parasitized eggs, female emerged parasitoids, failed in emergence from parasitized eggs, adults failed in emergence, and female failed in emergence.
Statistical of the data indicated that the parasitism percentage on D. livia egg higher than A. grisella was (84± 14.1 and 80±16.7%), respectively, and f value= 0.29ns. Also, the emergence eggs on D. livia egg higher than A. grisella was (85± 22.4 and 66±34.4%), respectively, and f value= 0.92 ns. On other hand, the adults emerged from parasitized eggs percentage of D. livia compared with A. grisella were (358 ±101 and 100±0 %), consequently and f value= 32.69**. However, adults failed in emergence from D. livia eggs were higher than A. grisella with an average (205±204.9 and 40±54.8%), consequently and f value= 3.26 ns.
Egg parasitoid Trichogramma was more efficient on D. livia eggs as a biological control agent under laboratory conditions. Also, this parasitoid is considered non-discriminating parasitoid. This increases the difficulty after releasing in the orchard. So, for using egg parasitoid as a biological control agent to reduce the population density of D. livia below the economic threshold of injury level (ETL) must be considered this phenomenon. In conclusion, for using egg parasitoid in controlling pomegranate butterfly must be used good tactics and strategy under orchard conditions.
from the foregoing, the importance of this study is evident in improving knowledge when applying the best integrated management strategy to control such a serious pest as pomegranate butterfly on pomegranates and alternative hosts.