. J. Plant Prot. Res. Inst. (2019), 2 (2): 378 - 386

Egyptian Journal of Plant Protection Research Institute www.ejppri.eg.net

Histopathological and ultrastructural impacts of nuclear polyhedrosis virus on infected tissues of the leopard pyrina (: ) using transmission electron microscopy Merghem, A.1 and Hassan, K.2 ¹Plant Protection Research Institute, Agricultural Research Centre, Dokki, Giza, Egypt. 2Plant Pathology Research Institute, Agricultural Research Centre, Giza, Egypt. ARTICLE INFO Article History Abstract: Received: 2 / 4 / 2019 Leopard moth, (L.) (Lepidoptera: Accepted: 4 / 6 /2019 Cossidae) is considered one of the most dangerous and ______destructive wood boring pests that attack several varieties Keywords of the horticultural fruit trees mainly the apple trees, Zeuzera pyrina, viral domestica B. (Rosales: Rosacease). In Egypt, this borer infection, nuclear causes severe crop loss resulting in serious detrimental and polyhedrosis virus, economic damage thus continuous control trials are taken histopathology and place aiming to avoid these key problems. Through the transmission electron ongoing study surveying visits were done to collect and microscopy. sample the natural infected stage of this cossid borer. Then a series of experiments was performed to isolate and trace of the viral pathogen which is identified as the Zeuzera pyrina nuclear polyhedrosis virus (ZPNPV). Then testing the susceptibility of Z. pyrina larvae to this virus was done at three different controlled temperatures (25±5°C) in the laboratory. Screening tests revealed high mortality rates for the larval stage at 20°C recording 85.7±0.38 %. Ultimate sequence of experiments was applied to conduct histopathological and ultrastructural studies using transmission electron microscopy. Gained results showed severe malformation and distortion of the mid-gut cells confirming the susceptibility of Z. pyrina to the nuclear polyhedrosis viral infection and possibility of ZPNPV to be applied within the control programs of this cossid borer. It is concluded that Z. pyrina revealed a definite susceptibility to the infection with ZPNPV and it is highly provoked to be involved in the integrated pest management programs against this dangerous moth. Introduction Leopard moth, Zeuzera pyrina (L.) (Rosales: Rosacease) in several global (Lepidoptera: Cossidae) is a highly regions especially the Mediterranean destructive wood boring pest attacking area including Egypt causing serious several horticultural fruit trees mainly damage and considerable crop loss the apple trees, Malus domestica B. (Navon, 1977; Katlabi, 1989; Ismail et

378

Merghem and Hassan, 2019 al., 1988 and 1992; Guario et al., 2000; Present study was undertaken to Hegazi and Khafagi, 2005; Kutinkova et explore the susceptibility of the wood al., 2006; Merghem, 2012; Mani et al., boring pest Z. pyrina to the viral disease 2014 and Manja and Aoun, 2019). Thus as a biocontrol agent for it. Moreover, it was of great importance to combat this focusing on the isolation of viral diseases devastating boring pest, control measures that naturally infect the stages of this for this borer are mainly focused cossid borer was undertaken. Ultimately chemical applications. concern with the viral impacts on the In Egypt, the control and larval tissues of Z. pyrina, through management programs against this borer histopathology and electron microscopy are depending mainly on pesticides investigations was studied. applications for many years and still up Materials and methods till now. Excessive chemical sprays lead 1. Field surveyed visits: to high hazard risk, resistance problems During the current study, different and dangerous health effects for human, localities of the host horticultural trees of livestock and beneficial (Tadros Z. pyrina, mainly the apple trees, had et al., 1993; Haniotakis et al., 1999; been visited throughout 2016 to 2017 Sarto, 2001; Osuna and Patanita, 2006; searching for the naturally diseased Patanita et al., 2009 and Almanoufi et individuals in Qalubeia and Beheira al., 2012). Governorates. Consequently, searching for safe 2. Sampling, preservation and and non-chemical or alternative agents is identification: encouraged aiming at maximizing the Collected intact, health and efficiency of that boring pest’s moribund specimens of this target boring control program and preventing the pest were collected and preserved detrimental, residual and adverse effects separately in glass vials containing resulting from chemical pesticide 0.55% sterile saline solution. Then applications such as biocontrol agents. samples were subjected to a preliminary Unfortunately, biological control identification for the collected stages of elements were restrictedly applied the boring insect done at the Department against Z. pyrina such as entomogenous of Wood Borers and Termite in the Plant nematodes and ectoparasitoids in Protection Research Institute (PPRI) addition to some scanty trials using followed by a confirmation of this borer microbial bioagents such as bacteria and identity undertaken at the Entomological fungi (Abdel-Kawy et al., 1992; Collection of the Classification and Nashnosh et al., 1993; Tawfik and Research Department of the Ramadan, 2006; Lawrence et al., 2007; same research institute (i.e. PPRI) and Japoshvili and Hansen, 2013; Merghem finally it was revealed that it is the and Hassan, 2014 and Labaude and leopard moth, Z. pyrina. Griffin, 2018). Diseased specimens were collected Viral disease infections, especially from the surveyed localities of the fore- the nuclear polyhedrosis viruses (NPV), mentioned Governorates. Then the viral- as microbial entomopathogenic agents shown specimens were separated in the are still used on a limited scale for the saline solution for the further control of the wood boring pests pathological experiments and the especially this cossid borer thus there moribund symptoms were recognized was a need to estimate the efficiency of according to the identification keys after such biocontrol agents against this Vlak and Grőner (1980) and Evans and boring pest. Shapiro (1997). Dissection of infected larvae was done and the digestive guts

379

Egypt. J. Plant Prot. Res. Inst. (2019), 2 (2): 378 - 386 were obtained for the following 4.1. Histopathological study: microscopic examination elucidating the To achieve the purpose of the internal symptoms as the presence of the histopathological study and the further virions and inclusion bodies virus (IBV) investigation with electron microscope, a in the infected tissues with considerable sequence of laboratory steps was numbers, dispersion of air sacs and followed beginning with the dissection vacuolation within cells and destruction of the larval body, removal of visceral of the nuclear membrane of target cells. and fat tissues and getting the gut (i.e. Both histological and electron the mid-gut). Then, a procedure of microscopy studies confirmed the micro-technique preparation was identity of a NPV infection to this cossid provided following by the dehydration, borer called Zeuzera pyrina nuclear clearing, embedding, sectioning, polyhedrosis virus (ZPNPV). staining, and fixation processes till the 3. Laboratory screening tests: full examination, this procedure was On identifying the viral pathogen according to Hamm (1999). type, a second series of experiments 4.2. Electron microscopy study: dealing with the susceptibility of this IBVs were randomly selected then leopard moth borer to the infection of suspended in a 50:50 glycerin/water ZPNPV, was undergone. Firstly, a stock solution and measured at x1,250 (p