116 Egypt. J. Chem. Vol. 64, No. 3 pp. 1239 - 1255 (2021) Egyptian Journal of Chemistry http://ejchem.journals.ekb.eg/ Nuclear polyhedrosis virus DNA identification and first evaluation against Strawberry pest, Pentodon algerinum (Coleoptera: Scarabaeidae) 1* 2 2 Samah, M.M. Abd El-Aziz ; Magda, H. Rady ; Bouthina, A. Merdan ; Hany, M. Hussein1; El-Sayed, M.A. El-Saiedy1 ; Abd El-Salam,A.M.E1. 1. Pests &Plant Protection-National Research Centre , Dokki ,Giza, Egypt. 2. Department of Entomology, Faculty of Science, Ain- Shams University, Cairo, Egypt Abstract In Egypt, The white grub, Pentodon algerinum (Coleoptera: Scarabaeidae) damages various economic important plants recently strawberry. This study aimed to DNA identification and first evaluation of the Spodopteda littoralis Nuclear polyhedrosis virus (SpliNPV) and Pentodon algerinum Nuclear polyhedrosis virus (PNPV) against Pentodon algerinum third instar larvae in the laboratory to find successful safe alternative control method against this pest. Bioassay was made by two experiments, one used five SpliNPV concentrations, 6.7X106, 3.11X107, 1.1X108, 3.64X109 and 3.64X1010 Polyhedral inclusion bodies (PIB/ml) achieved 80, 100, 100, 80 & 90% mortality respectively during thirteen days. Another experiment used four PNPV concentrations, 8.2x107, 8.2x 108, 2.6x109 and 8.2x109 PIB/ml caused 100, 70, 67 & 72.7% mortality respectively during ten 14 9 9 days. SpliNPV Lethal concentrations LC50 6.2x10 & LC90 8.9x10 PIB/ml were more than PNPV LC50 2.8X10 & LC90 1.8X108 PIB/ml. Thus PNPV and SpliNPV were effective against Pentodon larvae but PNPV was effective and specific more than SpliNPV. DNA of SpliNPV and PNPV was identified by RAPD PCR using four RAPD primers, Operon A9, A20, B17 & B7 (Operon Technologies, Inc., Alameda, CA, USA) using automated (Bio Rad, USA) thermal cycler. Total 33 bands were produced including 24 PNPV bands with molecular weights between 152-1204 base pairs (bp) included between resulted 9 SpliNPV bands having molecular weights between 138 - 1767 bp. Therefore PNPV is similar to SpliNPV in almost its DNA sites and also is a polymorphic copy from SpliNPV. Thus PNPV and SpliNPV can be recommended against this pest. Keywords: the white grubs; Pentodon algerinum; larval stage ; strawberry plants; RAPD PCR; SpliNPV; PNPV Introduction small fruit plants, shrubs and trees in many parts of Strawberry yield (Fragaria x ananassa Duch.) is the world [6, 7]. Strawberries producers faced a major economic vegetable crop for local problems with declining the efficacy of the chemical consumption and exportation in Egypt. Recently in insecticides and developing resistance against these Egypt, the white grub Pentodon algerinum compounds, [8]. Pathogenic virus for controlling of (Coleoptera: Scarabaeidae) extensively destroys insect pests has the advantage of tolerance to strawberry. The larvae are known as the waste extreme climatic conditions of heat& humidity and organic manure larvae (White grubs) and the adult that one time use is enough to reduce the number of stage is named as the hard black beetle. [1, 2]. The insect pests. Viruses are tiny obligate intracellular larvae live in the soil and feed on organic matters parasites being either RNA or DNA genome and plant roots [3]. The larvae go near to the soil involved by a protective, virus coded protein surface, looking for any roots or tubers to feed on. envelope. They are of cellular origin and These larvae cluster around the roots when soil is distinguished by a long co-evolution of virus and removed. The infection causes plant wilt and death. host. Infection by viruses depends on specialized The larvae prefers living in the sandy soil (newly host cells providing the complex metabolic and reclaimed area).White grubs are basic pests of biosynthetic machinery of eukaryotic or prokaryotic different agricultural crops and the most damaging cells. [9, 10]. The entomopathogenic Baculovirus group for the turf grass, nurseries and ornamentals in cannot be transmitted to human because this virus the worldwide [4,5, 2]. The white grubs cause requires an alkaline based cell structure to replicate extensive damage to the roots of grasses, legumes, itself while Human is acidic based. Intracellular _________________________________________________________________________________________________ *Corresponding author e-mail: [email protected].; (Samah M.M. Abd El-Aziz). Receive Date: 19 January 2021, Revise Date: 04 February 2021, Accept Date: 07 February 2021 DOI: 10.21608/EJCHEM.2021.58765.3268 ©2021 National Information and Documentation Center (NIDOC) 1240 S., M.M. Abd El-Aziz et.al. _____________________________________________________________________________________________________________ pathogenic viruses need to complement with its the region needed to be amplified. Once that is specific receptor sequence on the target cell surface known, one can make large quantities of that region for their entry to initiate infection. Virus receptor starting with tiny amounts of material, such as the binding is too specific, and this specificity marks DNA within a single human hair. With the both the species and the cell type that can be infected availability of almost complete or complete by a given virus. Baculovirus receptor on insect host sequences of genomes from many species, the range cells is not found in human cells. Entomopathogenic of genes to which it can be applied is enormous. baculoviruses are considered as safe alternative [21]. method for insect pest control such as the Nuclear This study aimed to DNA identification and first Polyhedrosis Virus (NPV) being a double stranded evaluation of the Spodopteda littoralis Nuclear DNA baculovirus and highly specific to their host polyhedrosis virus (SpliNPV) and Pentodon insects. [9,10]. Today Nuclear polyhedrosis virus algerinum Nuclear polyhedrosis virus (PNPV) (NPV) (Baculoviridae) is an ideal tool in the pest against Pentodon algerinum third instar larvae in the management programs, as it is highly specific to its laboratory to find successful safe alternative control host insects, safe to the environment, humans, other method against this pest. plants and beneficial natural enemies[11,12,13]. The NPV has large polyhedron shaped structures Experimental: called polyhedral containing many virions [14]. The occlusion of the virus inside a protein coat is 1- Rearing of Pentodon algerinum important to protect the infective particles in the The first and second instars of Pentodon algerinum transmission of the virus from insect to insect [15, larvae were collected from infested organic 16]. The Baculoviridae family contains the strawberry fields at Tokh town; Qalubia Nucleopolyhedrovirus such as the multiple and Governorate. Infestation was firstly recorded at single Nuclear polyhedrosis viruses (MNPV, SNPV) Septamber and Novamber 2018. These fields were and the Granulovirus such as granulosis viruses [17]. not treated with insecticides nor entomopathogenic During larvae infection, occlusion bodies (OBs) agents during the previous year. Reared collected P. consisting of enveloped virions enclosed in algerinum larvae fed on a mixture of sandy soil, polyhedrin protein are dissolved in the alkaline organic manure, and potato pieces with roots of grass insect host midgut releasing virions making a plant under laboratory conditions (Temp. of primary infection. [18,19]. They replicate when 22.0±3.0ºC & RH of 70.0±5.0) according to [2]. ingested by the insect host larvae by biphasic cycle These larvae have three larval instars. Third instar containing budded virus produced in the primary larvae were used for laboratory experiments. infection, and later, when viral particles are produced, they become occluded virus ( polyhedral 2. Production and isolation of Nuclear inclusion bodies). The budded virus is responsible Polyhedrosis virus for virus infection within the host while the The original Nuclear Polyhedrosis virus (NPV) occlusion bodies are responsible for spreading of types were produced and isolated from infected virus in the environment between susceptible larvae. Spodoptera littoralis larvae as (SpliNPV) and from This is achieved through cell lysis of infected larvae infected Pentodon algerinum larvae as (PNPV) by resulting in contamination of the feeding plant parts (Samah M.M. Abd EL-Aziz) according to [9,10] at such as leaf surfaces eaten by healthy larvae the laboratory of Pests & Plant Protection becoming infected by virus. Other important Department, National Research Centre and stored at advantages of baculovirus for pest control are a lack -20o C till use. of toxic residues, allowing farmers to treat their crops even shortly before harvest, with low 3-Preparation of SpliNPV and PNPV viral possibility to develop stable resistance [20]. The concentration identification of the DNA of cells can be carried out The virus stocks of SpliNPV and PNPV were used by Random Amplification of Polymorphic DNA to prepare different dilutions for each of them. (RAPD PCR) which is a type of Polymerase chain The number of polyhedral inclusion bodies (PIB /ml) reaction (PCR). The segments of DNA are randomly was counted under light microscope by amplified by using short primers (8–12 nucleotides) haemocytometer according to [22]. to make DNA fingerprint. This method is now one of the most commonly used assays for obtaining a 4- Bioassay tests: particular segment of DNA or RNA. It is rapid and Bioassay was made by two experiments, one used extremely sensitive. This method does