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ABSTRACT: The insecticidal characteristics of two Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) strains produced on two different S. exigua colonies were measured using the same two host colonies. These strains constitute the active ingredients of the biological insecticides Vir-ex and Spexit and were produced on insect colonies from Spain and Switzerland. Demographic characteristics of insects from each colony were examined before infection. Larval developmental time, larval survival, and adult sex ratio did not differ between the colonies, whereas mean pupal weight was significantly higher in the Spanish colony insects. After infection, susceptibility to virus occlusion bodies (OBs), time to death, larval weight at death, and total production of OBs/larva varied significantly depending on virus strain and the colony used. Vir-ex OBs produced in Spanish colony larvae had improved insecticidal characteristics in terms in lethal dose and speed of kill metrics than other strain-colony combinations. OB production was significantly higher in Spanish colony insects infected with Spexit compared with Vir-ex infected insects from the Swiss colony, with intermediate values for the other two strain-colony combinations. Virus strain and host colony origin were highly influential in determining the insecticidal characteristics of OBs and should be considered as key parameters that require optimization during the production of SeMNPV-based insecticides.
Journal of Economic Entomology 02/2013; 106(1):50-6. · 1.70 Impact Factor
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Biological Control 01/2013; · 2.00 Impact Factor
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ABSTRACT: A natural Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) isolate from Florida shares a strikingly similar genotypic composition to that of a natural Spodoptera frugiperda MNPV (SfMNPV) isolate from Nicaragua. Both isolates comprise a high proportion of large deletion genotypes that lack genes that are essential for viral replication or transmission. To determine the likely origin of such genotypically similar population structures, we performed genomic and functional analyses of these genotypes. Nucleotide homology of the deleted regions was as high as 79%, similar to that of other colinear genomic regions, although some SfMNPV genes were not present in SeMNPV. In addition, no potential consensus sequences were shared between the deletion flanking sequences. These results indicate an evolutionary mechanism that generates and sustains deletion mutants within each virus population independently. Functional analyses with different proportions of complete and deletion genotypes were performed using the two viruses in mixtures of occlusion bodies (OBs) or co-occluded virions. Ratios greater than 3:1 of complete:deletion genotypes resulted in reduced pathogenicity (expressed as median lethal dose), but no significant changes in speed of kill. In contrast, OB yields increased in the 1:1 mixture. The three phenotypic traits analyzed provide a broader picture of the functional significance of the most largely deleted SeMNPV genotype and further contribute to elucidate the role of such mutants in baculovirus populations.
Applied and environmental microbiology 11/2012; · 3.69 Impact Factor
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ABSTRACT: Three vip3 genes were identified in two Bacillus thuringiensis Spanish collections. Sequence analysis revealed a novel Vip3 protein class (Vip3C). Preliminary bioassays of larvae from 10 different lepidopteran species indicated that Vip3Ca3 caused more than 70% mortality in four species after 10 days at 4 μg/cm(2).
Applied and environmental microbiology 08/2012; 78(19):7163-5. · 3.69 Impact Factor
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ABSTRACT: An isolate of the Spodoptera frugiperda multiple nucleopolyhedrovirus comprises a stable proportion of deletion genotypes (e.g., SfNIC-C), that lack pif1 and pif2 rendering them noninfectious per os, and that survive by complementation with a complete genotype (SfNIC-B) in coinfected cells. To determine whether selection
for particular ratios of complete and deletion genotypes occurs mainly during the establishment of the primary infection in
insect midgut cells or during subsequent systemic infection, we examined genotype frequencies in insects that fed on OBs comprising
different co-occluded mixtures of genotypes. Dramatic changes in genotype frequencies were observed between the OB inoculum
and budded virus (BV) samples taken from larvae inoculated with OBs comprising 10% SfNIC-B + 90% SfNIC-C indicating that a
marked reduction of SfNIC-C genotype had occurred in the insect midgut due to the immediate elimination of all OBs that originated
from cells that had been infected only by SfNIC-C. In contrast, immediate changes were not observed in OBs comprising mixtures
of 50% SfNIC-B + 50% SfNIC-C or those comprising 10% SfNIC-B + 90% SfNIC-C as most of the OBs in these mixtures originated
from cells that had been infected by both genotypes. Subsequent changes in genotypic frequencies during five days of systemic
infection were fairly small in magnitude for all genotypic mixtures. We conclude that the prevalence of defective genotypes
in the SfNIC population is likely determined by a balance between host selection against OBs produced in cells infected by
SfNIC-C alone and within-host selection for fast-replicating deletion genotypes. The strength of intra-host selection is likely
modulated by changes in MOI during the infection period.
Virologica Sinica 04/2012; 24(4):350-358.
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ABSTRACT: The complete genomic sequence of a Nicaraguan plaque purified Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV) genotype SfMNPV-B was determined and compared to previously sequenced isolates from United States (SfMNPV-3AP2) and Brazil (SfMNPV-19). The genome of SfMNPV-B (132,954bp) was 1623bp and 389bp larger than that of SfMNPV-3AP2 and SfMNPV-19, respectively. Genome size differences were mainly due to a deletion located in the SfMNPV-3AP2 egt region and small deletions and point mutations in SfMNPV-19. Nucleotide sequences were strongly conserved (99.35% identity) and a high degree of predicted amino acid sequence identity was observed. A total of 145 open reading frames (ORFs) were identified in SfMNPV-B, two of them (sf39a and sf110a) had not been previously identified in the SfMNPV-3AP2 and SfMNPV-19 genomes and one (sf57a) was absent in both these genomes. In addition, sf6 was not previously identified in the SfMNPV-19 genome. In contrast, SfMNPV-B and SfMNPV-19 both lacked sf129 that had been reported in SfMNPV-3AP2. In an effort to identify genes potentially involved in virulence or in determining population adaptations, selection pressure analysis was performed. Three ORFs were identified undergoing positive selection: sf49 (pif-3), sf57 (odv-e66b) and sf122 (unknown function). Strong selection for ODV envelope protein genes indicates that the initial infection process in the insect midgut is one critical point at which adaptation acts during the transmission of these viruses in geographically distant populations. The function of ORF sf122 is being examined.
Journal of Invertebrate Pathology 01/2011; 107(1):33-42. · 2.06 Impact Factor
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ABSTRACT: A new cell line was established from the embryos of the insect Chrysodeixis chalcites (Lepidoptera, Noctuidae, Plusiinae). The cell line contains several morphologically different cell types and was distinguished from three other lepidopteran cell lines propagated in the laboratory by DNA amplification fingerprinting. The cultured cells, which we officially named WU-CcE-1 cells, were permissive for infection by C. chalcites nucleopolyhedrovirus (ChchNPV) and large numbers of occlusion bodies were produced that retained their infectivity for C. chalcites larvae. The CcE-1 cells were also permissive for Trichoplusia ni single nucleopolyhedrovirus (TnSNPV). ChchNPV could be passaged in these cells for at least four passages indicating that budded virus production was supported. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Helicoverpa armigera (Hear) NPV both induced apoptosis in these cells. The results obtained indicate that the CcE-1 cell line will be a useful tool in the study of both ChchNPV and TnSNPV.
Journal of Invertebrate Pathology 09/2010; 105(1):56-62. · 2.06 Impact Factor
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ABSTRACT: Recombinant insect nucleopolyhedroviruses lacking the egt gene generally kill their hosts faster than wild-type strains, but the response of insects to mixtures of virus genotypes is less well known. Here, we compared the survival time, lethal dose and occlusion body yield in third instar larvae of Helicoverpa armigera (Hübner) after challenge with wild-type H. armigera SNPV (HaSNPV-wt), a strain with a deletion of the egt gene, HaSNPV-LM2, and a 1:1 mixture of these two virus strains. A range of doses was used to determine whether the total number of OBs influenced the response to challenge with a mixture of virus strains versus single strains. At high virus doses, HaSNPV-LM2 killed H. armigera larvae significantly faster (ca. 20 h) than HaSNPV-wt, but at low doses, there was no significant difference in survival time between the viruses. The survival time after challenge with mixed virus inoculum was significantly different from and intermediate between that of the single viruses at high doses, and not different from that of the single viruses at low doses. No differences in lethal dose were found between single and mixed infections or between virus genotypes. The number of occlusion bodies produced per larva increased with time to death and decreased with virus dose, but no significant differences among virus types were found.
Journal of Invertebrate Pathology 05/2010; 104(1):44-50. · 2.06 Impact Factor
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ABSTRACT: A low-cost simplified diet has been successfully developed for rearing Spodoptera exigua larvae under laboratory conditions. The cost of ingredients was lower than that of the standard diet based on a modified tobacco hornworm, Manduca sexta (L.), diet. The simplified diet fulfilled larval nutritional requirements without apparent adverse effects on the reproductive capacity of the insect. Survival, pupal sex ratio, and fecundity registered in insects that were reared on the simplified diet did not differ from those observed on the standard diet. The mean larval development period of insects that consumed the simplified diet was also similar to that of insects that consumed the standard diet, whereas weight of pupae and adult longevity were significantly higher in insects reared on simplified diet. Larvae consumed approximately 11% more of the standard diet compared with the simplified diet and a corresponding increase was observed in the number of larvae that could be reared through to pupation on each liter of simplified diet. The production of S. exigua multiple nucleopolyhedrovirus (SeMNPV) occlusion bodies (OBs) in insects grown on each type of diet was also evaluated. Weights of larvae at inoculation and at death, OB yields and biological activity of OBs did not differ significantly for each type of diet. The simplified low-cost diet appears suitable for large-scale in vivo production of SeMNPV OBs.
Journal of Economic Entomology 02/2010; 103(1):17-24. · 1.70 Impact Factor
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ABSTRACT: An insect nucleopolyhedrovirus naturally survives as a mixture of at least nine genotypes. Infection by multiple genotypes results in the production of virus occlusion bodies (OBs) with greater pathogenicity than those of any genotype alone. We tested the hypothesis that each OB contains a genotypically diverse population of virions. Few insects died following inoculation with an experimental two-genotype mixture at a dose of one OB per insect, but a high proportion of multiple infections were observed (50%), which differed significantly from the frequencies predicted by a non-associated transmission model in which genotypes are segregated into distinct OBs. By contrast, insects that consumed multiple OBs experienced higher mortality and infection frequencies did not differ significantly from those of the non-associated model. Inoculation with genotypically complex wild-type OBs indicated that genotypes tend to be transmitted in association, rather than as independent entities, irrespective of dose. To examine the hypothesis that virions may themselves be genotypically heterogeneous, cell culture plaques derived from individual virions were analysed to reveal that one-third of virions was of mixed genotype, irrespective of the genotypic composition of the OBs. We conclude that co-occlusion of genotypically distinct virions in each OB is an adaptive mechanism that favours the maintenance of virus diversity during insect-to-insect transmission.
Proceedings of the Royal Society B: Biological Sciences 11/2009; 277(1683):943-51. · 5.41 Impact Factor
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ABSTRACT: The insecticidal potency of a nucleopolyhedrovirus population (SfNIC) that infects Spodoptera frugiperda (Lepidoptera) is greater than the potency of any of the component genotypes alone. Occlusion bodies (OBs) produced in mixed infections comprising the complete genotype and a deletion genotype are as pathogenic as the natural population of genotypes from the field. To test whether this increased potency was due to the deletion or to some other characteristic of the deletion variant genome, we used the SfNIC-B genome to construct a recombinant virus (SfNIC-B Delta 16K) with the same 16.4-kb deletion as that observed in SfNIC-C and another recombinant (SfNIC-B Delta pifs) with a deletion encompassing two adjacent genes (pif1 and pif2) that are essential for transmission per os. Mixtures comprising SfNIC-B and SfNIC-B Delta 16K in OB ratios that varied between 10:90 and 90:10 were injected into insects, and the progeny OBs were fed to larvae in an insecticidal potency assay. A densitometric analysis of PCR products indicated that SfNIC-B was generally more abundant than expected in mixtures based on the proportions of OBs used to produce the inocula. Mixtures derived from OB ratios of 10, 25, or 50% of SfNIC-B Delta 16K and the corresponding SfNIC-B proportions showed a significant increase in potency compared to SfNIC-B alone. The results of potency assays with mixtures comprising various proportions of SfNIC-B plus SfNIC-B Delta pifs were almost identical to the results observed with SfNIC-B Delta 16K, indicating that deletion of the pif gene region was responsible for the increased potency observed in mixtures of SfNIC-B and each deletion recombinant virus. Subsequently, mixtures produced from OB ratios involving 10 or 90% of SfNIC-B Delta 16K with the corresponding proportions of SfNIC-B were subjected to four rounds of per os transmission in larvae. The composition of each experimental mixture rapidly converged to a common equilibrium with a genotypic composition of approximately 85% SfNIC-B plus approximately 15% SfNIC-B Delta 16K. Nearly identical results were observed in peroral-passage experiments involving mixtures of SfNIC-B plus SfNIC-B Delta pifs. We conclude that (i) the deletion of the pif1 and pif2 region is necessary and sufficient to explain the increased potency observed in mixtures of complete and deletion genotypes and (ii) viral populations with decreased ratios of pif1- and pif2-deficient genotypes in the virus population increase the potency of genotypic mixtures and are likely to positively influence the transmission of this pathogen.
Journal of Virology 04/2009; 83(10):5127-36. · 5.40 Impact Factor
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ABSTRACT: Larvae of the tomato fruitworm, Helicoverpa armigera, were surveyed for nucleopolyhedrovirus (NPV) infection (referred to as HearNPV) in three different locations from the Iberian Peninsula: Olivenza and Toledo in Spain and the Oeste region in Portugal. Twenty HearNPV isolates were obtained from single field-collected larval cadavers. Restriction endonuclease (REN) profiles of the collected isolates with BglII and PstI allowed identification of six different H. armigera single-embedded NPV strains in Spain (referred to as HearSP3, HearSP4, HearSP5, HearSP6, HearSP7, and HearSP8) and two in Portugal (referred to as HearPT1 and HearPT2). No strains were shared by isolates from different geographical regions except HearSP5, which was found in isolates from Olivenza and Toledo. Cluster analysis based on the restriction fragment length polymorphisms of these strains in relation to two previously identified strains from Badajoz (HearSP1) and Cordoba (HearSP2) in Spain, showed no correlation among the strains and their geographical origin. The biological activity of HearSP2, HearSP4, HearSP7, HearSP8, HearPT1, and HearPT2 was compared in terms of pathogenicity (50% lethal concentration, LC50) and virulence (mean time to death). HearPT2 and HearSP7 were significantly more pathogenic than HearSP2, with LC50 values 2.8 and 2.6-fold higher than the latter, respectively, on H. armigera second instars. HearSP4 and HearPT2 killed larvae significantly faster than HearSP8, whereas HearSP2, HearSP7, and HearPT1 showed intermediate mean time to death values.
Biological Control. 01/2009;
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ABSTRACT: Quality control during mass production of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV), and studies on environmental fate following the use of this virus as a biological pesticide, would be facilitated by a rapid method for the detection and identification of isolates. A molecular biology tool was developed that combined the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) to differentiate SeMNPV isolates. Oligonucleotide primers were designed to amplify five variable SeMNPV genomic regions (V01, V02, V03, V04, V05). Four wild-type SeMNPV strains isolated from the United States (US2) and Spain (SP1, SP2, and SP3), and a laboratory cloned genotype (US1A), were analyzed with 36 different primer-endonuclease combinations. BglII digestion of the variable region 1 (V01) amplicon was the only combination that differentiated each of the five virus isolates tested, although genetic heterogeneity limited the discriminatory power of the technique. Six novel SeMNPV isolates originating from greenhouse soils in southern Spain were successfully identified using this method. As judged by sequence analysis, the V01 region, which comprises the homologous region 1 (hr1), is the most variable genomic region among the genotypes present in the Spanish isolates. This method constitutes a useful tool for processing large number of environmental samples and could be used to address environmental biosafety concerns.
Journal of Virological Methods 08/2006; 135(1):1-8. · 2.01 Impact Factor
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ABSTRACT: The influence of an optical brightener, Tinopal LPW, on the activity of a purified genotype of the nucleopolyhedrovirus (SeMNPV) of the beet armyworm, Spodoptera exigua (Hübner), was determined in second to fifth instar (L2-L5) S. exigua. When mixed with viral occlusion bodies (OB) 1% Tinopal LPW significantly reduced the median lethal dose (LD50) of the virus in all instars compared with insects treated with SeMNPV alone. Levels of enhancement, as determined by LD50 values, ranged from 2.6- to 580-fold, depending on the instar. The greatest enhancement occurred on the two later instars, L4 (70-fold) and L5 (580-fold), which show a much higher resistance to SeMNPV infection than earlier instars. The median time to death (MTD) values were not significantly different in any instar among larvae treated with SeMNPV + Tinopal LPW and those treated with SeMNPV alone. Larval development in SeMNPV + Tinopal LPW treated larvae was retarded, in second and fourth instars, compared with controls or larvae treated with SeMNPV alone. The OB yields from SeMNPV treated larvae were almost 1.6-fold greater in second instars (9.3 x 10(6) OBs/larvae), and 1.9-fold greater in fourth instars (1.9 x 10(8) OBs/larvae), than those obtained in larvae treated with SeMNPV + Tinopal LPW. The addition of 1% Tinopal LPW to the virus suspension did not alter the genotypic composition of viral progeny during four successive passages of the virus.
Journal of Economic Entomology 01/2004; 96(6):1668-74. · 1.70 Impact Factor
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ABSTRACT: US2A, US2D, and US2F are three in vivocloned genotypic variants from the wild-type strain of a Spodoptera exiguanucleopolyhedrovirus (SeMNPV) isolated in Florida (USA) and is the active component of the commercial bioinsecticide Spod-X. These variants were compared in terms of pathogenicity (LD50), speed of kill (expressed as mean time to death) and viral progeny productivity (OBs/larva). LD50values were similar for the three cloned genotypes. The mean time to death value for US2D (113.7h) was significantly higher than those of US2A (31.7h) and US2F (27.8h). Virus yield was determined for L4larvae infected with the estimated LD99. US2D infected larvae attained higher weight than those infected with US2A and US2F, and produced a higher OB yield than larvae infected with US2A or US2F. An outstanding feature of US2F, in contrast to US2A and US2D, was its inability to disrupt the teguments of NPV-killed larvae. To study the relative proportion of the three genotypic variants throughout successive passages, S. exigualarvae were originally infected with a viral inoculum containing a 1:1:1 mixture of the three genotypes. After three successive passages, US2D was no longer detected in either of the three replicate experiments performed, while US2A was the predominant genotype in all of them, and US2F remained at similar proportions throughout the three passages. The influence of the phenotypic characteristics of the three variants on their relative proportions in mixed infections is discussed.
Entomologia Experimentalis et Applicata 01/2000; 97(3):275-282. · 1.53 Impact Factor
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ABSTRACT: Four genotypes named SP2A, SP2B, SP2C and SP2D were obtained in vivo by infecting S. exigua larvae with limiting dilutions of the Spanish field isolate Spodoptera exigua Nucleopolyhedrovirus (Se-SP2) of SeMNPV. The cloning of variants SP2A, SP2B and SP2C took 1, 6, and 3 passages, respectively, before the DNA profiles showed all bands in equimolar concentrations, and they remained constant for at least six further passages indicating the stability of their genotypes. The SP2D variant isolation took over ten passages and it was genetically less stable. Physical maps of their genomes were constructed for the restriction enzymes BamHI, BglII, PstI, and XbaI. The region between 8–10 m.u. was highly variable and characteristic of each cloned genotype and, hence, can be used as RFLP markers for all four genotypic variants. This region, included in the PstI-MB fragment, was cloned and sequenced showing that all the Se-SP2 variants contained a homologous region (hr) with a variable number of 98 bp sequences tandemly repeated, which were used to distinguish genotypic variants from each other. The biological activity of the genotypic variants SP2A, SP2B, and SP2C when compared in terms of LD50 and LT50, were not significantly different. However, the SP2D genotypic variant was found to be significantly less infective (higher LD50). The emergence of new genotypes in the Se-SP2 field populations is discussed.
Virus Research 02/1999; · 2.94 Impact Factor
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ABSTRACT: One of the most effective strategies recommended to delay insect resistance to Bt-plants is concurrent expression of several toxins in the same plant. A new generation of Bt-cotton, including Bollgard II and WideStrike, has been developed to simultaneously express two different Cry toxins, Cry1Ac and Cry2Ab, and Cry1Ac and Cry1Fa, respectively. The aim of this study was to determine the individual and combined toxic effect of Cry1Ac, Cry2Ab, and Cry1Fa in the cotton pests Helicoverpa armigera and Earias insulana, as well as the nature of the interactions between these toxins, as determined by mean lethal concentration (LC50) values and larval growth inhibition studies. Singly, all three assayed toxins were more toxic against E. insulana than against H. armigera larvae. Toxin Cry1Ac was significantly more toxic than the other two on H. armigera, while toxin Cry1Fa was the least toxic and caused no significant mortality. When combined, Cry1Ac and Cry1Fa showed an additive interaction in all proportions analyzed for both pest species, whereas Cry1Ac and Cry2Ab interacted synergistically in mixtures comprising 1:1 or 1:4 of each toxin against H. armigera. In E. insulana, there was no synergism between Cry1Ac and Cry2Ab but both these toxins showed a high insecticidal activity when administered individually and in mixtures. This study suggest that each particular toxin or toxin combination expressed in transgenic Bt-cotton should be carefully selected depending on the most important pest species present in each geographical area.
Biological Control.
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ABSTRACT: The incorporation of certain stilbene optical brighteners into virus-based formulations has been demonstrated to increase viral pathogenicity (as indicated by reduced LD/LC50 values) but their effect on Bacillus thuringiensis activity has been scarcely investigated. We determined the effect of nine optical brighteners on the insecticidal activity of B. thuringiensis ser. kurstaki HD-1 strain (Bt HD-1) on Helicoverpa armigera and also compared the effect of two optical brighteners on the insecticidal activity of Bt HD-1 and occlusion bodies (OBs) of a Spanish isolate of H. armigera single nucleocapsid nucleopolyhedrovirus (HearNPV-SP1). Blankophor CLE, Blankophor DRS, Blankophor ER, and Leucophor SAC significantly increased the pathogenicity of Bt HD-1. In contrast, Tinopal UNPA-GX, Tinopal CBS, Blankophor BA, Leucophor AP, and Leucophor UO had an adverse or no effect on its insecticidal activity. Mixtures of HearNPV-SP1 OBs with Tinopal UNPA-GX or Leucophor UO resulted in 31.4- and 11.4-fold increases in pathogenicity, respectively, at 1%, and 11.4- and 6.3-fold increases in pathogenicity, respectively, at 0.1%, compared to the OBs alone. However, none of these brighteners increased Bt HD-1 activity. These results appear consistent with the hypothesis that the enhancement of HearNPV-SP1 pathogenicity and the null or antagonistic effects observed in Bt HD-1 against H. armigera were due to optical brightener-mediated degradation of the peritrophic membrane, but additional systematic studies involving a broad range of brighteners and electron microscope observations are required to confirm this premise.
Biological Control.
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ABSTRACT: Genotypic and phenotypic variation of SeMNPV was examined in seven isolates of SeMNPV originating from occlusion body (OB) populations in the soil of greenhouses in Spain. Semi-quantitative PCR indicated that some of the isolates were composed of a single dominant genotype, whereas other isolates were composed of two or three genotypes in equal proportions. The coexistence of genotypes could be explained by trade-offs among the three phenotypic traits analyzed, namely pathogenicity (LD50), speed of kill (mean time to death), and OB yields, so that increases in one trait were accompanied by decreases in another. Mixed genotype isolates tended to behave differently to single genotype isolates. Two of the genotypic mixtures were significantly more pathogenic (lower LD50 values) for Spodoptera exigua (Hübner) larvae than the single genotypes that they comprised. OB yield/insect was greater for single genotype compared to mixed genotype isolates, despite genotype-specific differences in mean times to death. Total OB production/insect was positively correlated with time to death. Two out of three of the mixed genotype isolates had lower OB yield/mg insect weight at death compared to single genotype isolates. Each genotypic combination appeared to interact to produce a unique phenotype. This suggests the existence of trade-offs between traits leading to the coexistence of distinct genotypes and genotypic mixtures with similar transmissibility.
Biological Control.
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ABSTRACT: The abundance and genetic structure of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) occlusion body (OB) populations in greenhouse substrates in southern Spain was determined during an 18 month period over an area of 426 km2. To detect the presence of OBs, substrate samples were incorporated into a semi-synthetic diet and fed to first instar S. exigua. SeMNPV OBs were detected in 34% of substrate samples (N = 267). The prevalence of substrates positive for OBs did not differ significantly between samples from different crops or from four different geographical zones. Seasonally, Spring and Summer samples had significantly greater OB concentrations than samples taken in Autumn and Winter, concurrent with changes in the host insect population. Restriction fragment length polymorphism analysis revealed at least nine different variants. Substrate pH was identified as a major factor influencing the abundance and genetic composition of the soil OB population. Certain variants were more prevalent in substrates of more alkaline pH suggesting genotype-specific differences in survival outside the host. The prevalence of mixed variant infections was inversely related to an increasing substrate pH. Greenhouse substrates of southern Spain represent an abundant and diverse reservoir for the survival of SeMNPV. Studies are required to determine the degree to which agropractices that conserve soil OB populations would favor long-term microbial control of lepidopteran pests in greenhouse crops.
Biological Control.
