Pathogenicity of Steinernema brazilense (Rhabditida: Steinernematidae) to Gonipterus platensis (Coleoptera: Curculionidae) prepupae

aDepartamento de Produção Vegetal, Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP, CEP 18603-970, Botucatu, SP, Brasil bDepartamento de Fitotecnia, Universidade Federal de Viçosa – UFV, CEP 36570-900, Viçosa, MG, Brasil cCentro Experimental, Instituto Biológico, CEP 13001-970, Campinas, SP, Brasil dDepartamento de Entomologia, Instituto de Biotecnologia Aplicada à Agropecuária – BIOAGRO, Universidade Federal de Viçosa – UFV, CEP 36570-900, Viçosa, MG, Brasil


Introduction
The genus Gonipterus Marelli, 1927 (Gonipterinae: Curculionidae) has about 20 species known as Eucalyptus weevils or Eucalyptus snout-beetles (Mapondera et al., 2012). The weevil G. platensis was the first introduced eucalyptus pest in New Zealand (1890), Africa (1916), South America (1925, Europe (1975) and North America (1994) (Mapondera et al., 2012). The introduction of key pests as G. platensis and Thaumastocoris peregrinus Carpintero & Dellapé (Hemiptera: Thaumastocoridae) is a risk to forest plantations (Reis et al., 2012;Almeida et al., 2018), such as those of eucalypts (Bhattacharya et al., 2003;Clarke et al., 1998). Adults and larvae of the eucalyptus weevil feed preferably on young leaves, buds and developing shoots causing high damage (Clarke et al., 1998). G. platensis is the main eucalypt pests in many regions (Reis et al., 2012) and its impact and that of other eucalyptus weevils is poorly studied, mainly due to the difficulties of identifying control factors influencing this crop productivity (Reis et al., 2012).
The egg parasitoid Anaphes nitens (Girault, 1928) (Hymenoptera: Mymaridae) has been used for the biological control of G. platensis (Hanks et al., 2000), but this pest has been causing high damage levels (Echeverri-Molina and Santolamazza-Carbone, 2010). The life cycle of entomopathogenic nematodes in the soil make them promising biological control agents of insects with part of its life cycle in the soil as eucalyptus weevils (Barbosa-Negrisoli et al., 2010;Grewal, 2000) including G. platensis.
The objective of this study was to evaluate the pathogenicity of S. brazilense to G. platensis prepupae.

Material and Methods
The experiment was conducted at the Laboratory of Agricultural Nematology of the São Paulo State University (UNESP) in Botucatu, São Paulo state, Brazil. Steinernema brazilense was obtained from the Entomopathogenic Nematode Collection of the Entomopathogens "Oldemar Cardim Abreu" Bank of the Biological Institute of São Paulo, São Paulo, Brazil.
Infective juveniles (IJs) of S. brazilense IBCBn 06 were multiplied in D. saccharalis (third to fifth instar) larvae (Woodring and Kaya, 1988). Five larvae per Petri dish (9-cm diam) lined with filter paper moistened with a suspension of nematodes at the concentration of 500 IJs/cm 2 of Petri dish surface was used. Dead D. saccharalis larvae were transferred to white trap (White, 1927) and stored in chamber at 25 °C, 70 ± 80% RH, in the dark. IJs were obtained from this host and collected in a water film (1 cm deep) in Erlemeyers maintained in BOD chamber at 18 °C, 70 ± 80% RH and used two days after collection. The G. platensis prepupae were collected in a eucalyptus plantation in Itatinga (23°11'35.1"S 48°38'32.5"W) São Paulo state, Brazil.
The nematode S. brazilense IBCBn 06 pathogenicity to G. platensis prepupae (Woodring and Kaya, 1988) was evaluated in two treatments with five replications, each with four prepupae of this insect per Petri dish (9 cm). Two mL of the suspension of this isolate was applied in aqueous suspension with a pipette, equivalent to a dose of 500 IJ/insect (125 IJ/cm 2 ) per dish with bottom lined with two filter paper sheets. The control treatment had 2 mL of distilled water per dish. The nematode pathogenicity was evaluated with D. saccharalis pupae (Woodring and Kaya, 1988). Petri dishes were sealed with transparent PVC plastic film and stored in BOD chamber at 26 °C and 70% RH in the dark. Dead and live insects were counted after 4 d. Prepupae of G. platensis and D. saccharalis were rinsed in tap water and individually kept in new Petri dishes (5 cm) with a moistened filter paper for dissection. The number of insect adults, dead prepupae and IJs second generation was quantified under a stereomicroscope.

Results and Discussion
Steinernema brazilense IBCBn 06 killed all G. platensis prepupae at 4 d after inoculation with infection rates of 10.3 IJs, and a total of 1275.7 IJs produced in the second generation. The D. saccharalis pupae parasitism was 100% with infection rate of 15.5, and a total of 7201.9 second generation IJs, proving the high entomopathogenic viability of this nematode. No mortality of G. platensis prepupae and D. saccharalis pupae was recorded in the controls (Table 1, Figure 1).
The lower production of S. brazilense IBCBn 06 IJs in G. platensis pupae than in those of D. saccharalis may be due to the larger size of this nematode. This may have caused space and food limitations and a lower offspring number in the first host. The 7201.9 IJs of this nematode produced per D. saccharalis pupae confirms results for H. indica IBCBn 05 in Mahanarva fimbriolata (Stål) (Hemiptera: Cercopidae) nymphs (Leite et al., 2003). S. brazilense was pathogenic and to G. platensis prepupae, however, with a lower production of IJs per host compared to that found with G. mellonella larvae with up to 200000 IJs of this microorganism (Dutky et al., 1964).
The nematode S. brazilense IBCBn 06 parasitized and killed 100% of G. platensis prepupae, showing potential for integrated management of this eucalyptus weevil.