In this study, three SpliNPV isolates were derived from S. littoralis-infected larvae from cultivated field of cabbage, pepper and eggplant crops in different districts in Egypt. Their DNA were purified and subjected to REN analysis. Results of the REN analyses showed that all three isolates, Cab2, P2 and P3, exhibited slightly different number and pattern of gel fragments demonstrating the existence of genetic differences among them. The difference in the three SpliNPV isolate profiles demonstrated that the isolates comprised genotypic variants in different proportions. Hence, the variations in the genomes of baculoviruses could result in relative difference in their virulence and pathogenicity (Behle and Popham 2012).
Previously, REN analysis was performed on nine SpltNPV isolates from sick Spodoptera litura and an Egyptian isolate of Spodoptera littoralis NPV. Six of the ten isolates had new submolar bands, which indicated a combination of genotypes (Takatsuka et al. 2003). Cherry and Summers (1985) investigated the genetic diversity of NPVs that infected S. littoralis and also discovered two different viruses, SpliNPV-A and SpliNPV-B, which were equally dispersed throughout Israel. In addition, REN profile analysis of SpliNPV Egyptian isolates named Cab-3 and Spli-7 showed a number of submolar bands where these isolates comprised genotypic variants (Elmenofy et al. 2020).
The NPV isolates in the current study showed a significant homology with 100 NPV isolates based on GenBank database. They showed 93.1% identity with S. littoralis NPV polyhedrin gene of both SpliNPV-Tun2 and SpliNPV-AN1956 strains under accession number of MG958660.1 and NC_038369.1, respectively, which were isolated from Tunisia and Egypt.
Bioassays of the Cab2, P2 and P3 in this study revealed a distinct pattern of virulence among them. The variant Cab2 presented a high virulence on the first instar larvae of S. littoralis with LC50 value of 1.59 × 104 OB/ml, than the P2 and P3 variants that represent lower LC50 value of 1.23 × 106 and 2.7 × 104 OBs/ml, respectively. Moreover, it was shown that Cab2 was faster to kill the larvae with ST50 value of 120 h compared to both other isolates P2 and P3, which showed ST50 value of 132 h for both isolates.
The selection of native isolates is crucial for developing a virus-based insecticide against S. littoralis. Elmenofy et al. (2020) studied a new field collected from SpliNPV-Cab3 genotypic variant to determine its toxic effect against S. littoralis, as a novel, environmentally safe candidate virus-based bioinsecticide. SpliNPV-Cab3 indigenous isolate was identified as a variation isolate of the reference strain SpliNPV-AN1956 based on the REN pattern and phylogenetic analysis.
The virulence parameters of the three NPV isolates of the current study showed that the LC50 values were higher than the LC50 observed for the first instar S. littoralis of some SpliMNPV isolates presented by Toprak et al. (2005). The LC50 value of both Cab2 and P3 (1.59 × 104 and 2.7 × 104 OBs/ml) was slightly higher than the calculated LC50 values for the S. littoralis first instar using Spli-6 and Spli-7 isolates (3 × 104 and 9.5 × 104 OBs/ml, respectively) that was studied by Elmenofy et al. (2020). On the other hand, the speed of mortality by both isolates Spli-6 and Spli-7 based on ST50 value was 96 h, which showed to be faster than that the current isolates Cab2, P2 and P3 against S. littoralis larvae with ST50 values of 120, 132 and 132 h for the three isolates, respectively. On the other hand, the ST50 value of the isolates under the current study was less than the ST50 value for SpliMNPV isolates calculated by Lacey et al. (2015), which ranged from 125 to 360 h. Hence, the genetic variations observed in SpliNPV isolates Cab2, P2 and P3 might have influenced the viral potency of S. littoralis larvae.
The variability of LC50 and ST50 is probably due to different factors such as the technique of surface treatment, homogeneously treated diet and feeding habit of the insect species or due to difference in larval instars. It might possibly be also related to differences in the host's sensitivity to NPV isolates, the total number of virions for each OB, the virus strain's pathogenicity and/or the cycles used for the viral isolate propagation (Seufi 2008).
Therefore, the development of novel and environmentally safe alternatives has become an urgent need for integrated control programs for the cotton leafworm, with emphasis on biological control through entomophagous plant extracts and microorganisms (Matloub et al. 2021).
Based on the virulence characteristics (LC50 and ST50) of the three isolates described in this study, their potential as a promising viral-based bioinsecticide is suggested, specially isolate Cab2, which showed the best virulence values against S. littoralis larvae. As long as baculoviruses are isolated in Egypt from different districts, it is expected to discover much more virus isolates in virgin districts where no chemical pesticides are used. These observations suggested that the Egyptian environment is suitable for not only gathering novel baculovirus isolates but also for their potential use as viral-based pesticides in integrated pest management schemes (IPM).