Typical baculovirus infection symptoms were observed in Chrysodeixis includens Walker (Lepidoptera: Noctuidae) larvae from a laboratory rearing in Tucumán, Argentina. Scanning electron microscopy revealed the occurrence of polyhedral occlusion bodies displaying a heterogeneous morphology. Amplification, sequencing, and phylogenetic analysis of conserved polyhedrin, lef-8 and lef-9 genes identified the pathogen, for the first time in Argentina, as a variant of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV). The biocontrol potential of this new isolate is worthy of future research.
Chrysodeixis (= Pseudoplusia) includens Walker (Lepidoptera: Noctuidae) is a polyphagous Neotropical pest, especially on soybean and cotton. Control failures due to insecticide resistance are a major concern (Owen et al. 2013), partially overcome by new-generation insecticides, transgenic crops, and natural enemies (Ramos et al. 2017). Also, a highly virulent baculovirus, Chrysodeixis includens nucleopolyhedrovirus (ChinNPV), has been proposed as a promising bioinsecticide candidate for the specific control of C. includens (Alexandre et al. 2010).
In 2017, baculovirus infection symptoms (sluggish movement, body color change, and tissue liquefaction) were recorded in laboratory-reared C. includens larvae in Tucumán, Argentina. The colony originated from specimens collected on soybean and bean in the northwest of the country. Light microscopy of larval homogenates revealed presumed occlusion bodies (proteinaceous structures that protect the virions contained within them from environmental factors). Based on this observation, additional analyses were conducted to elucidate the disease etiology.
Symptomatic larvae were ground in water + 0.5% sodium dodecyl sulfate (SDS) and centrifuged at low speed. The supernatant was filtered, centrifuged at 7,000 rpm for 20 min, and the occlusion bodies were resuspended in water. Some occlusion bodies were dehydrated, metallized, and observed in a scanning electron microscope Quanta 250 (FEI Co., Eindhoven, The Netherlands). Occlusion bodies were variable in shape and size, with a quite irregular appearance, compared to other baculovirus species. Their diam ranged from about 0.7 µm to about 1.6 µm, and adopted distinct polyhedral morphologies (Fig. 1A-C). For molecular characterization, occlusion bodies were dissolved with sodium carbonate. The sample was adjusted to 10 mM Tris-HCl, 10 mM EDTA, 0.25% SDS, and 500 µg per ml proteinase K, and incubated overnight at 37 °C. Viral DNA was extracted with chloroform/isoamyl alcohol (24:1) (12,000 rpm, 10 min, 3 times) and precipitated at 12,000 rpm for 15 min with isopropanol + 0.2 M NaCl. The taxonomically informative polhyedrin, lef-8 and lef-9 baculovirus genes were partially amplified by PCR with primers prPH1/prPH2, prL8-2/prL8-1.b, and prL9-1/prL9-2, respectively, as mentioned elsewhere (Jehle et al. 2006). Purified products were directly sequenced (both strands) in an ABI PRISM 3500 XL genetic analyzer (Applied Biosystems, Foster City, California, USA) at Instituto de Biotecnología-INTA (Hurlingham, Argentina). Each gene was amplified independently and sequenced twice. BLAST searches for NCBI databases identified the virus under study as a new variant of ChinNPV, hereafter ChinNPV-Tuc. Sequences exhibited ≥ 99% nucleotide identity to those from ChinNPV isolates infecting C. includens in other Latin American countries (Rowley et al. 2011; Craveiro et al. 2016). Polyhedrin, lef-8 and lef-9 partial sequences of ChinNPV-Tuc were deposited at Gen-Bank under accession numbers MG865665, MG865666, and MG865667, respectively. They were aligned with ClustalW, concatenated (1,463 nt in total) and compared to all known ChinNPV isolates by maximum-likelihood phylogenetic analysis (1,000 bootstrap replicates) using MEGA7 (Kumar et al. 2016). Chrysodeixis chalcites nucleopolyhedrovirus served as the outgroup. The phylogeny resulted in 2 major ChinNPV clades. One clade grouped ChinNPV-Tuc from Argentina with 3 additional Brazilian isolates (ChinNPV-IE, -IF and -IG), whereas the other included isolates from Guatemala (ChinNPV-IA), Colombia (ChinNPV-458), and Brazil (ChinNPVIB, -IC and -ID) (Fig. 1D).
The persistent agricultural damage caused by C. includens has shifted attention toward alternative control measures. Differentially virulent ChinNPV isolates have been characterized (Alexandre et al. 2010; Craveiro et al. 2016), supporting the search for novel, genetically diverse variants that may be suitable for controlling C. includens. This is the first description of a ChinNPV isolate from Argentina. The pathogenicity of ChinNPV-Tuc and its potential use for pest management should be assessed.
This work was funded by Agencia Nacional de Promoción Científica y Tecnológica (PICT-2016-1949 to JDA) and Instituto Nacional de Tecnología Agropecuaria, Argentina.