Turk J Biol 25 (2001) 419-425 © TÜB‹TAK Viral Control of the European Pine Sawfly, Neodiprion sertifer (Geoffroy) in Turkey Mustafa YAMAN, Remziye NALÇACIO⁄LU, Zihni DEM‹RBA⁄ Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, 61080, Trabzon - TURKEY Received: 23.10.2000 Abstract: The European pine sawfly, Neodiprion sertifer (Geoffr.) (Hymenoptera: Diprionidae), is a serious pest of pine plantations and ornamentals. Chemical pesticides used to control this pest have a detrimental effect on the environment. The Neodiprion sertifer nuclear polyhedrosis virus (NsNPV) frequently causes considerable decreases in the populations but doses not harm other animals or plants. In this study, we tested five different concentrations of NsNPV against the larvae of N. sertifer and showed viral infection using Giemsa staining. When a viral concentration of 5x109 PIB/ml (Polyhedral Inclusion Body) was utilized, larval mortality started on the fourth day and reached 90.4% on the ninth day of the bioassay. However, lower viral concentrations, 5x109, 5x108 and 5x107 PIB/ml, showed the same mortality, but the infection periods occurred on different days. This study shows that NsNPV can be effectively used against N. sertifer as a safe biological control agent in Turkey. Key Words: Neodiprion sertifer, Nuclear Polyhedrosis Virus, Biological Control Neodiprion sertifer (Geoffr.) (Hymenoptera: Diprionidae)’in Viral Kontrolü Özet: K›rm›z›mt›rak sar› çal› antenli yaprakar›s› Neodiprion sertifer (Geoffr.) (Hymenoptera: Diprionidae) çam fidanl›klar›n›n önemli zararl›lar›ndan biridir. Bu zararl›y› kontrol etmek için kullan›lan kimyasal pestisidler çevrede zararl› etkilere sahiptir. N. sertifer nüklear polihedrozis virüsü (NsNPV) bu zararl›n›n populasyonunda önemli bir azalmaya neden olurken, çevredeki di¤er bitki ve hayvanlar› etkilememektedir. Bu makalede, N. sertifer larvalar›na karfl› NsNPV’nin farkl› konsantrasyonlar› test edildi ve viral enfeksiyon giemsa boyama metoduyla gösterildi. Larval ölüm test süresinin dördüncü gününde bafllarken, 5x109 PIB/ml konsantrasyonunda 9. günde % 90.4’e ulaflt›. 5x109, 5x108 and 5x107 PIB/ml konsantrasyonlar› ayn› ölüm oran›n› gösterirken, enfeksiyon süreleri farkl› günlerde geliflti. Bu çal›flma NsNPV’nin N. sertifer’e karfl› Türkiye’de etkili bir biyolojik mücadele ajan› olarak kullan›labilece¤ini göstermektedir. Anahtar Sözcükler: Neodiprion sertifer, Nüklear Polihedrozis Virüs, Biyolojik Kontrol 419 Viral Control of The European Pine Sawfly, Neodiprion sertifer (Geoffroy) in Turkey Introduction The European pine sawfly, Neodiprion sertifer (Geoffroy), is a pest of most species of twoneedled pines the following places in Turkey: ‹stanbul, Bal›kesir-Dursunbey, Ankara-Beynam, Burdur-Kemer, Bucak, Mu¤la-Y›lanl›, Eskiflehir-Çatac›k, K›z›lcahamam-Çamkoru (1) and Trabzon. It is particularly serious in Scotch pine, Pinus sylvestris (L.), and red pine (P. resinosa (Ait.)) plantations (2,3). Watershed areas are particularly vulnerable to attack, because the use of chemical insecticides is restricted to avoid the risk of water contamination. As the chemical pesticide question is a social issue, the objectives of nutrition, health and environmental quality can be addressed more efficiently by the implementation of integrated pest management techniques (IPM) rather than through current crop protection practices (4). Biological control of this pest is especially desirable in Turkey, because many of the hosttree species occur in watershed plantings. Fortunately, N. sertifer populations are reduced by a natural nuclear polyhedrosis virus (NPV) (Baculovirus), first reported by Escherich (5) and later described in detail by Bird and Whalen (6). This virus is capable of causing disease in larvae of N. sertifer, but does not harm other animals or plants. Nuclear polyhedrosis viruses represent an effective, selective and safe biological control agent (7). The best results with polyhedrosis viruses have been achieved against conifer sawflies, especially in Europe. Many factors such as temperature, moisture, acid rains and dosage influence the effect of the Neodiprion sertifer nuclear polyhedrosis virus (NsNPV). This insect can have resistance against the virus in different geographic regions (8-10). Therefore, many scientists have studied the effect of NsNPV in several countries (8,11-13). In pest control strategies for environmental protection, the use of viruses is relatively new in Turkey, although there are rapid developments in the bacterial control of plant pests (1418). This study will encourage Turkish scientists to investigate and use insect viruses against plant pests. Materials and Methods Collecting and rearing larvae of N. sertifer Larvae of N. sertifer were collected on young pine foliage in Çamburnu-Sürmene, Trabzon, and brought to the laboratory in appropriate boxes. The larvae were reared in specially prepared jars (Figure 1). Each group was fed with an equal amount of pine foliage. Viral suspension and bioassays The viral suspension (Virox) was provided by Dr. Ziemnicka, Institute of Plant Protection, Department of Biological Control and Quarantine 60-318, Poznan, Poland. The concentration of polyhedra in the suspension was counted in a hemacytometer. Fresh pine foliage was dipped in five different concentrations of the virus suspension: 5x109, 5x108, 5x107, 5x106 and 5x105. 420 M. YAMAN, R. NALÇACIO⁄LU, Z. DEM‹RBAG Figure 1. General view experiment. of the The foliage was air dried on filter paper and placed in a small jar containing water, which was put into a large jar (Figure 1). For the control, 20 larvae received leaves dipped in water. The larval mortalities were recorded daily and dead larvae were removed. All larvae tested were kept at 21 ± 2ºC and 60% RH on a 12:12 h photoperiod until they spun cocoons (8). All bioassays were repeated 3 times. Data were evaluated by using Abbott’s formula (19). Determination of viral infection The dead larvae were dissected, and the infection was observed under light microscopy. In addition, occurrence of viral infection was proved with Giemsa staining. For this, a thin smear of infected insect tissue slide was prepared under a microscope and dried in air. The smear was immersed for 1-2 min in Giemsa’ fixative and rinsed under running tap water for 5-10 s. Then the smear was stained for two hours in 10% Giemsa stain in 0.02 M phosphate buffer. After staining, the dye was rinsed off in running tap water for 5-10 s and gently blotted with absorbent paper, and then it was dried and examined with an oil immersion objective (20). Results and Discussion In all bioassays carried out to determine the effect of NsNPV on the larvae of the European pine sawfly, Neodiprion sertifer (Geoffroy), larval mortalities were found in different ratios and periods for different viral concentrations (Figure 2). As seen in Figure 2, the viral concentrations, 5x109, 5x108 and 5x107 showed the same mortality, but the infection periods occurred on different days. The mortal period of larvae decreased as the doses increased. At 421 — Figure 2. — 1 — 2 — 3 — 4 — 5 Effect of NsNPV on the second instar larvae of N. sertifer. (1= 5 x 109; 2= 5 x 108; 3= 5 x 107; 4= 5 x 106; 5= 5 x 105) — — — 1 2 3 4 — — — — — — — — — — — — — — — 100 90 80 70 60 50 40 30 20 10 0 — Mortality (%) Viral Control of The European Pine Sawfly, Neodiprion sertifer (Geoffroy) in Turkey 5 6 7 8 9 10 11 12 13 14 15 Infection period (day) the 5x109 PIB concentration, larval mortality started on the fourth day and reached 90.4% on the ninth day of the bioassay. The highest daily mortality during this period was determined to be 40% on the sixth day at 5x109 PIB concentration. Dead larvae showed common symptoms of viral infection such as hanging from foliage, liquefied body content, and delicate, rupturing integument. The infected larvae lost appetite, were less active and died 4 to 14 days after infection. The dead larvae had a faint-green color (Figure 3), and their cuticule was easily broken, liberating fluid in which a large number of polyhedral bodies was present under the microscope. Many dead larvae hung on pine foliage, characteristically attached by the abdominal prolegs (Figure 4). Some uninfected or low infected older larvae reached the pupal stage. In the Giemsa stain, performed to show viral infection, stained PIBs of NPV appear as clear, round objects (Figure 5). Figure 3. 422 Healthy (left) and infected (right) larvae of N. sertifer. M. YAMAN, R. NALÇACIO⁄LU, Z. DEM‹RBAG Figure 4. NsNPV-Infected N. sertifer larvae hunging on pine foliage. Figure 5. Polyhedral Inclusion Bodies (PIBs) stained with Giemsa. In different countries, scientists have found different effects of NsNPV on the larvae of N. sertifer at different periods using similar viral dosages. Glowacka et al. (13) found that the larval mortality reached 100% on the 9th day of the laboratory tests at all doses used (1x 1021 x 106 PIB/ml). Podgwaite et al. (11) determined that a dose rate of 2.5 x 109 PIB/ha of NsNPV provided acceptable control. Many factors such as temperature (8), dosage (9) moisture (10) and acid rains (21) influence the effect of Neodiprion sertifer nuclear polyhedrosis virus (NsNPV) and, also, this insect can have resistance against the virus in different geographic regions. 423 Viral Control of The European Pine Sawfly, Neodiprion sertifer (Geoffroy) in Turkey The speed and high rate of deaths of larvae infected with NsNPV indicate that it is a very promising agent for biological control of the European pine sawfly. The nuclear polyhedrosis virus of N. sertifer represents an effective, selective and safe biological control agent. However, Greathead et al. 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