Molecular and Morphological characterization of multiple nucleopolyhedrovirus from Mexico and their insecticidal activity against Spodoptera frugiperda (Lepidoptera: Noctuidae)

The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera: Noctuidae), is the most important pest of maize in many countries. Entomopathogenic viruses mainly Baculoviruses family are excellent biological control agents and therefore a viable alternative for managing this pest. The aim of this study was to determine the biological activity of eight native nucleopolyhedrovirus (NPVs) against FAW larvae. Additionally, two of the most virulent isolates (SfCH32 and SfCH15) were characterized biologically by bioassays to estimate their median lethal dose (LD₅₀) and median lethal time (LT₅₀), morphologically by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and molecularly by restriction enzymes. Three (SfCH15, SfCH18 and SfCH32) of the eight tested native nucleopolyhedrovirus isolates caused mortalities ˃98% at 168-hr post-inoculation (hpi) with a dose of 9.2 × 10⁴ OBs/larva. SfCH15 and SfCH32 isolates showed occlusion bodies (OBs) of irregular shape and size (1.02–2.24 μm). The SfCH15 and SfCH32 isolates showed similar median lethal dose (5.6 × 10²–6.4 × 10² OBs/larva). The lowest median lethal time (114.5 hpi) was observed with the SfCH15 isolate at the highest concentration (2.5 × 10⁶ OBs/larva). The DNA restriction profiles for SfCH15 and SfCH32 were different, with their genome size being ~128,000 bp and 132,000 bp, respectively. SfCH15 and SfCH32 isolates showed similar morphological characteristics and the highest virulence against fall armyworm. This study showed that native isolates were highly virulent against S. frugiperda larvae, being similar to other reported strains; however, field studies are required to confirm their insecticidal effect.     

Potential of betabaculoviruses to control the tomato leafminer Tuta absoluta (Meyrick)

The tomato leafminer Tuta absoluta (Meyrick 1917) (Lepidoptera:Gelechiidae) is a devastating pest, causing losses of up to 100%. An interesting tool for its control is the use of the insect specific viruses of Baculoviridae family. Despite its high potential, its use on T. absoluta larvae has been poorly studied. In this work, Colombian granuloviruses VG013 and VG003 isolated from T. absoluta and Tecia solanivora (Lepidoptera:Gelechiidae) larvae sampled in tomato and potato crops, respectively, were morphologically, biologically and molecularly characterized. Occlusion bodies showed ovoid shape containing one nucleocapsid. Restriction endonuclease analysis revealed a pattern similar to Phthorimaea operculella granulovirus and bioinformatics studies showed that both isolates are variants of that baculovirus specie. Similar mean lethal concentrations (LC₅₀) on T. absoluta larvae were estimated for both viruses, although VG013 exhibited shorter mean time to death than VG003. The latter reached a higher OBs yield in comparison with VG013. These results demonstrated an interesting potential of evaluated betabaculoviruses to control T. absoluta populations and pointed key features to its use under field conditions.     

Microencapsulated Spodoptera frugiperda nucleopolyhedrovirus: insecticidal activity and effect on arthropod populations in maize

Excessive use of chemical insecticides to control Spodoptera frugiperda in maize (Zea mays L.) crops in Colombia has resulted in pest resistance and environmental contamination. A Colombian multicapsid nucleopolyhedrovirus (SfMNPV) was produced in laboratory reared larvae and formulated as a wettable powder by microencapsulating the viral occlusion bodies (OBs) with a methacrylic acid polymer. Microencapsulated OBs were tested for insecticidal activity in laboratory, greenhouse and field bioassays, and were then sprayed onto a commercial crop where their effect on arthropod populations was estimated. In laboratory bioassay, microencapsulated and non-microencapsulated OBs caused a larval mortality of 98 and 96%, respectively. The percent of plants showing recent feeding damage in a greenhouse experiment oscillated between 2.5 and 7.5% when OBs were applied compared to 70% observed in the control. In two field trials, the highest dose of 1.5×10¹² OBs/ha (1500 g/ha) was selected as the recommended rate for field application in maize crops, with efficacies higher than 70%. Total arthropod populations were negatively affected by chemical pesticides; whereas the OBs formulation did not affect the diversity of these organisms. Our results indicate that microencapsulated SfMNPV has the potential to be commercialised as an alternative to substitute or complement chemical insecticides in fall armyworm integrated management programmes.     

Insecticidal Activity of the Granulosis Virus in Combination with Neem Products and Talc Powder Against the Potato Tuberworm Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae)

The potato tuberworm Phthorimaea operculella (Zeller) is an important agricultural pest that causes significant economic losses to potato growers worldwide. The addition of an effective method of biological control for the potato tuberworm is greatly needed, and is currently unavailable in Brazil. The granulosis virus (Baculoviridae) is a promising biological control agent to protect post-harvest potatoes and in storage from the potato tuberworm. However, the control measure must be economically feasible. Liquid suspensions of a granulosis virus applied alone or in mixture with two commercial neem oil-based products (DalNeem? and NeemAzal?), and a dry powder formulation of viral granules were evaluated for control of potato tuberworm larvae by treating potato tubers under laboratory conditions. High larval mortality (86.7%) was achieved when DalNeem and virus were applied together at 4?mg of azadirachtin/L and 10⁴ occlusion bodies (OBs)/mL, respectively. This combination resulted in ??50% efficacy in relation to their counterparts alone. Conversely, NeemAzal did not enhance virus effectiveness against larvae of the potato tuberworm. The talc-based virus formulation was used for dusting seed tubers at different concentrations and resulted in 100% larval mortality at 5?×?10⁸ OBs/g. Formulated and unformulated virus provided 50% mortality at 166 OBs/g and at 5.0?×?10⁵ OBs/mL, respectively. As a result, talc-based virus formulation had a better control efficiency on potato tuberworm than the aqueous virus suspension. The granulosis virus combined with DalNeem at low rates or formulated with talc powder is a viable option to control the potato tuberworm under storage conditions.     

Occurrence and characterization of a nucleopolyhedrovirus from <Emphasis Type="Italic">Maruca vitrata</Emphasis> (Lepidoptera,Pyralidae) isolated in Taiwan

A nucleopolyhedrovirus (MaviMNPV) was isolated from diseased larvae of legume pod borer (LPB), Maruca vitrata, at Tainan in Taiwan. Electron microscopical studies on the ultrastructure of MaviMNPV occlusion bodies (OBs) showed several virions (up to 19) with multiple nucleocapsids (up to 6) packaged within a single viral envelope. The diameter of OBs was 0.9 to 1.3 μm with a mean of 1.152±0.116 μm. The complete sequence of the MaviMNPV polyhedrin (Polh) gene contained 735 nucleotides (GenBank accession number DQ399596). Phylogenetic analyses using the complete sequence of the Polh gene of MaviMNPV indicated that this virus clusters with Group I NPVs. The genome size of MaviMNPV estimated with restriction enzymes viz., HindIII, EcoRI, BglII and PstI was 113.41 ± 1.50 kbp. First instar LPB larvae were the most susceptible stage (LC₅₀ 2.053 × 10² OBs/ml) followed by second, third and fourth instars with the median lethal concentrations (LC₅₀s) 1.410 × 10³, 2.390 × 10³ and 2.636 × 10³ OBs/ml, respectively. This is the first record of this virus from this region. The first and second authors have equal contributions in this paper     

Fitness-related traits of entomopoxviruses isolated from Adoxophyes honmai (Lepidoptera: Tortricidae) at three localities in Japan

Three entomopoxviruses (EPVs) isolated from diseased Adoxophyes honmai larvae at different localities (Tsukuba, Itsukaichi, and Miyazaki) in Japan were compared for biochemical identity and key parameters of virus fitness, fatal infection, speed of kill, and virus yield. When the structural peptides of occlusion bodies (OBs) and occlusion-derived viral particles were compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no difference in banding patterns was observed. However, DNA restriction endonuclease analysis showed that the three isolates were genotypically different, but many commonly sized DNA fragments were observed. Five tortricid species, A. honmai, Adoxophyes orana, Adoxophyesdubia, Homona magnanima, and Archips insulanus were susceptible to all isolates. No significant differences in the key viral fitness parameters were detected among the isolates in A. orana. However, the Miyazaki isolate had a different effect on H. magnanima; it allowed infected insects to survive longer and develop to a larger size, but had a lower yield of OBs per larva at any given time to death. OB yields per unit cadaver weight for the Miyazaki isolate, which indicate the conversion rate of the insect to virus, were lower over time compared to the other two isolates. The implications for selecting a candidate isolate to control tortricid pests are discussed.     

Insecticidal efficacy of two isolates of Beauveria bassiana (Bals.) (Vuill.), on the second larval stage of Leptinotarsa decemlineata (Say) (Col.: Chrysomelidae)

Leptinotarsa decemlineata is a serious potato pest throughout the world that has developed resistance to many insecticides. This study investigated the virulence of two indigenous isolates of Beauveria bassiana, consisting of DEBI007 and IR1217C. Five different concentrations of each isolate, 1 × 10⁷, 3.1 × 10⁷, 1 × 10⁸, 3.1 × 10⁸ and 1 × 10⁹ conidia ml^?1, were applied in bioassay using dipping method on second instar larvae. Control insects were treated with distilled water containing 0.01% Tween-80 (Sigma, Germany) as surfactant. The results showed that mortality percentages due to DEBI007 and IR1217C isolates in the highest concentration were 60% and 57.77%, respectively. There was a significant difference between efficacies of different conidial concentrations; therefore, with rise in conidial concentration, the mortality percentage was increased. The DEBI007 isolate that was previously isolated from the soil was more effective than IR1217C isolate. Calculated LC₅₀ for this effective isolate was 1.4 × 10⁷ conidia ml^?1, which, respectively, is the highest concentration.     

Characterisation of three Alphabaculovirus isolates from the gypsy moth,Lymantria dispar dispar (Lepidoptera: Erebidae), in Turkey

In this study, Lymantria dispar dispar larvae, collected from three different localities in Turkey, were examined for the presence of inclusion bodies under phase contrast and electron microscopes. Inclusion bodies from infected larvae were subjected to polymerase chain reaction using the conserved primers for polyhedrin (polh), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes. Sequence analysis confirmed that larvae collected from the three different localities contained multiple nucleopolyhedrosis viruses (MNPVs). These isolates were designated LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3. Phylogenetic analyses of these isolates were performed using target genes polh, lef-8 and lef-9. Restriction endonuclease analysis of the three geographic isolates with EcoRI and PstI enzymes demonstrated some differences existed among the isolates. According to the EcoRI profile, the mean estimated size for the complete genome of each isolate (LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3) was calculated to be approximately 170, 153 and 170?kb, respectively. Insecticidal activities of each isolate were tested on L. d. dispar larvae using four different viral concentrations between 10³ and 10⁶?OBs/ml. Results showed that the mortalities for LdMNPV-T1, -T2 and -T3 ranged between 13–53%, 47–100% and 46–93%, respectively. The LC₅₀ and LC₉₅ values of LdMNPV-T2 were not significantly different from the respective corresponding values of the other two isolates. However, isolate LdMNPV-T2 killed larvae with a LC₅₀ value that was lower than the other two isolates. Our results suggested there are promising LdMNPV isolates in Turkey that can be used for microbial control of L. d. dispar larvae.     

Evaluation of Paenibacillus spp. isolates for the biological control of black rot in Brassica oleracea var. capitata (cabbage)

The ability of isolates of Paenibacillus spp. to protect Brassica oleracea var. capitata (cabbage) against the black rot pathogen, Xanthomonas campestris pv. campestris (Xcc),was evaluated. Twenty-four isolates of Paenibacillus spp., isolated from New Zealand-grown brassica hosts or soil, were evaluated for in vitro antagonism towards six Xcc isolates. Seven Paenibacillus spp. isolates with different levels of in vitro suppressive activity against Xcc were screened in pot experiments for their capacity to reduce black rot symptoms on cabbage. Two Paenibacillus isolates (P10 and P16) exhibited biocontrol activity against Xcc, and four isolates (P1, P6, P9, and P24) reduced cabbage seed germination and seedling emergence. The dependence of bioactivity on inoculum rate was investigated with three Paenibacillus isolates (P6, P10, and P16) at three different concentrations (5?×?10⁸, 5?×?10⁹, and 5?×?10¹⁰?CFU?ml^?1). Negative effects on seedling emergence were detected with isolate P6 at concentrations?≥5?×?10⁹?CFU?ml^?1. All three isolates applied at the three concentrations reduced black rot symptoms on the cotyledons and true leaves. There was poor or no relationship between the inhibitory effect of Paenibacillus spp. isolates on the growth of Xcc in vitro, and their biocontrol activity in vivo. Paenibacillus isolate P16 was identified as a potential biological control of black rot in cabbage.     

Morpho‐pathological and Molecular Characterization of Bipolaris oryzae in Rice (Oryzae sativa)

Fifty isolates of Bipolaris oryzae from rice were characterized morpho‐pathologically and molecularly. Based on colony morphology and growth pattern on PDA, these isolates were grouped into four categories: black with suppressed growth (21 isolates), black with cottony growth (16 isolates), black with fluffy growth (12 isolates) and white with cottony growth (1 isolate). The frequency of the black and suppressed type was the highest (42%) with maximum aggressiveness (mean spore count of 1854/cm²), whereas the white and cottony growth isolate had lowest frequency (2%) and aggressiveness (548/cm²). Thirteen B. oryzae isolates (four isolates from Groups I, II and III and one isolate from Group IV) were further tested for their variability with random amplified polymorphic DNA (RAPD) primers. Twenty RAPD primers were screened, of which 10 gave amplification; however, only six primers gave reproducible results. Based on the molecular similarity of the RAPD profiles, the isolates were grouped in to three major clusters and maximum linkage distance between them was determined as 0.29 units. This study establishes the variability among B. oryzae isolates.     

Biochemical analysis and pathogenicity of entomopathogenic fungi to Diaphorina citri Kuwayama (Hemiptera: Liviidae)

Asiatic citrus psylla, Diaphorina citri, Kuwayama (Hemiptera: Liviidae) is an economic pest of citrus groves and a vector of the bacterium, Candidatus Liberibacter spp., one of the causative agents of citrus greening. In order to estimate the infectivity of six different isolates of Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea, fungal bioassay was performed on the adults of D. citri. Adults of D. citri were treated individually with 1 × 10⁵, 1 × 10⁶, 1 × 10⁷, 1 × 10⁸, 2 × 10⁸ spores/mL fungal concentrations by the immersion method. Subsequent to fungal bioassay, treated D. citri were used to determine the levels of esterase and glutathione S‐transferases (GST) enzymes over a period of 3–7 days. The mortality results suggest that I. fumosorosea isolates (If‐02) caused 82.2% mortality on the seventh day of treatment. However, B. bassiana isolate (Bb‐08) with lowest LC₅₀ (1.4 × 10⁷ spores/mL) proved to be highest potential isolate against D. citri. Biochemical determination of esterase and GST activity assay showed significant differences in activities after infection of fungi. Significantly high activity of esterase was observed by Bb‐01 (27.0 unit per mg protein) on the seventh day, while Ma‐11.1 and If‐2.3 (16.9 and 36.3 unit per mg protein) on the third day post treatment. However, maximum GST's activity was showen by isolates Bb‐08,Ma‐M₂ and If‐2.3 (37.6, 1.40 and 10.9 unit per mg protein) on the third day. The current investigation will help to explore the relations between the insect defense system and entomopathogenic fungi. Moreover, the determination of enzymatic activities will be useful for selecting the most pathogenic isolates.     

A new nucleopolyhedrovirus strain (LdMNPV-like virus) with a defective fp25 gene from Lymantria xylina (Lepidoptera: Lymantriidae) in Taiwan

A new multiple nucleopolyhedrovirus strain was isolated from casuarina moth, Lymantria xylina Swinhoe, (Lepidoptera: Lymantriidae) in Taiwan. This Lymantria-derived virus can be propagated in IPLB-LD-652Y and NTU-LY cell lines and showed a few polyhedra (occlusion bodies) CPE in the infected cells. The restriction fragment length polymorphism (RFLP) profiles of whole genome indicated that this virus is distinct from LyxyMNPV and the virus genome size was approximately 139 kbps, which was smaller than that of LyxyMNPV. The molecular phylogenetic analyses of three important genes (polyhedrin, lef-8 and lef-9) were performed. Polyhedrin, LEF-8 and LEF-9 putative amino acid analyses of this virus revealed that this virus belongs to Group II NPV and closely related to LdMNPV than to LyxyMNPV. The phylogenetic distance analysis was further clarified the relationship to LdMNPV and this virus provisionally named LdMNPV-like virus. A significant deletion of a 44 bp sequence found in LdMNPV-like virus was noted in the fp25k sequences of LdMNPV and LyxyMNPV and may play an important role in the few polyhedra CPE. In ultrastructural observations, the nuclei of the infected LD host cells contained large occlusion bodies (OBs), and few OBs, which presented as one or two OBs in a nucleus that was otherwise filled with free nuclocapsids and virions. We concluded that this LdMNPV-like virus is a new LdMNPV strain from L. xylina.     

Cosmopolites sordidus (Germar) susceptibility to indigenous Cameroonian Beauveria bassiana (Bals.) Vuill. and Metarhizium anisopliae (Metsch.) isolates

Management of the banana root borer (BRB), Cosmopolites sordidus (Germar; Coleoptera: Curculionidae), remains a challenge in banana and plantain production worldwide. Synthetic pesticides remain the most widely used solution while mycoinsecticides are increasingly being recommended. In this study, we selected indigenous isolates of Beauveria bassiana and Metarhizium anisopliae collected from plantain fields in Cameroon, and tested them in the laboratory for their viability, pathogenicity and virulence against all C. sordidus life stages. Of 13 isolates initially screened for spore germination and pathogenicity to adult weevils in conidial suspension of 3.2 × 10⁸ conidia/ml, eight isolates with high to moderate germination and highest weevil mortality were selected for dose–response bioassays with four concentrations per isolate: 3.2 × 10², 3.2 × 10⁴, 3.2 × 10⁶ and 3.2 × 10⁸ conidia/ml. The virulent isolates from adult bioassays were tested with eggs, larva and pupae in conidial suspension of 3.2 × 10⁸ conidia/ml. Isolates performance depended on insect life stage with significantly high pathogenicity and virulence against larval, pupa and adult stages. The Beauveria isolate BIITAC6.2.2 caused the highest mortality rates followed by MIITAC1.1.5. Lethal times and lethal concentrations were relatively low for the three M. anisopliae isolates and three B. bassiana isolates which were the best isolates in almost all insect life stages. Apart from being effective in multiple life stages, these isolates were transmitted horizontally from one stage to another when eggs and pupae were treated. The implication of these findings for integrated management of the BRB, and potential biopesticides development and commercialization are discussed.     

Production of microsclerotia of the fungal entomopathogen Lecanicillium lecanii (Hypocreales: Cordycipitaceae) as a biological control agent against soil-dwelling stages of Frankliniella occidentalis (Thysanoptera: Thripidae)

Abstract

Microsclerotia (MS) production by two isolates of Lecanicillium lecanii on various culture media is described, and the efficacy of MS against western flower thrips is evaluated. High concentrations of MS (2.9–3.1×10⁵·mL^?1) were produced in media with C:N ratios of 7.4:1 and 10.3:1 by isolate SN21. Bioassays using soil-incorporated MS resulted in significant infection and mortality of thrips.     

Enhancing effect of proteins derived from Xestia c-nigrum granulovirus on Mamestra brassicae nucleopolyhedrovirus infection in larvae of Autographa nigrisigna (Lepidoptera: Noctuidae) on cabbage

The insecticidal effect of Mamestra brassicae nucleopolyhedrovirus (MabrNPV) and the enhancing activity of proteins derived from occlusion bodies (OBs) of Xestia c-nigrum granulovirus (GVPs) on the infectivity of MabrNPV were evaluated in a bioassay with second-instar larvae of Autographa nigrisigna (Walker) fed virus-applied cabbage plants. The lethal concentrations of MabrNPV achieving 50 and 95% mortality for A. nigrisigna were estimated to be 1.4 × 10⁵ and 3.1 × 10⁶ OBs/ml, respectively. When larvae were fed cabbage plants treated with MabrNPV and various concentrations of GVPs, the requisite concentration of GVPs achieving 95% mortality of A. nigrisigna was estimated to be 26.2–138.8 μg/ml in combination with 10⁴ OBs/ml MabrNPV and 8.46–24.09 μg/ml with 10⁵ OBs/ml MabrNPV. Increases in the concentration of MabrNPV or GVPs caused larval death at younger instars. A. nigrisigna has lower susceptibility to MabrNPV than M. brassicae and Helicoverpa armigera reported in Mukawa and Goto (J Econ Entomol 103:257–264, 2010). We estimated that the requisite concentration of MabrNPV for the control of A. nigrisigna was 10⁵ OBs/ml, which is a tenfold higher concentration than that for M. brassicae and H. armigera, with the increase achieved by adding 10 μg/ml GVPs.     

Effects of a nucleopolyhedrovirus in mixtures with azadirachtin on Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) larvae and viral occlusion body production

Abstract

The combined and alone effects of azadirachtin (AZA) and Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV) on the mortality of S. frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) were evaluated in the laboratory. For this, diet surface contamination bioassays were performed on S. frugiperda in the third instar. LC₅₀ values for SfMNPV alone were determined to be 430 and 373 viral occlusion bodies (OBs)/mm² at 192 and 216 h after treatment, respectively. LC₅₀ values for AZA alone were estimated for two periods of continuous exposure (4 or 5 days). In this case, LC₅₀ values were 45.5 and 16.8 mg L^?1 at 216 h after treatment (4 or 5 days of larval exposition to insecticide, respectively). We observed that although the interaction of AZA with SfMNPV increased viral pathogenicity, such improvements were of greater magnitude and more consistent at the lower OB concentration used (177 OBs/mm²). Application of SfMNPV (430 OBs/mm²), AZA (26.4 mg L^?1) or SfMNPV–AZA mixtures resulted in a significant reduction in the mean weight of larvae treated in the third instar across the experiment, by 23–41%, 17–95% and 26–97%, respectively, compared to control. The duration of larval development during the third and fourth instars increased significantly in larvae exposed to SfMNPV–AZA mixtures and AZA alone compared to SfMNPV alone and control treatments. The yield of OBs/mg weight of larvae treated with SfMNPV alone was 1.8-fold higher than OB yields from insects inoculated with SfMNPV–AZA mixtures. We conclude that AZA + SfMNPV mixtures are unlikely to be useful for the mass production of this virus and laboratory observations on the value of AZA + SfMNPV mixtures as a potentiator of biological insecticides require validation in field studies under commercial growing conditions.     

Isolation and virulence of entomopathogenic fungi against the great spruce bark beetle,Dendroctonus micans (Kugelann) (Coleoptera: Scolytidae)

Twelve fungal strains including Lecanicillium muscarium (Petch.) Zare and Gams, Isaria farinosa (Holmsk.) Fr., Fusarium sp., Beauveria bassiana Sensu Lato and Beauveria sp. were isolated from larvae and adults of D. micans. In addition, virulence of these isolates against this pest was determined. Conidia suspensions of 1×10⁶ conidia mL^–1 were applied to larvae and adults. The highest mortality and mycosis for larvae were obtained from isolate ARSEF 9271 (Beauveria bassiana) with 90% mortality and mycosis within 10 days. ARSEF 9271 also produced 93% mortality and mycosis in adults. On the other hand, the highest mortality and mycosis for adults were obtained with isolate ARSEF 9272 (Beauveria sp.), with 100% mortality and 80% mycosis within 10 days. These results indicate that isolates ARSEF 9271 and ARSEF 9272 seem to be the most promising potential fungal biocontrol agents against D. micans.     

Fluorescent Brighteners Improve Anticarsia gemmatalis (Lepidoptera: Noctuidae) Nucleopolyhedrovirus (AgMNPV) Activity on AgMNPV-Susceptible and Resistant Strains of the Insect

Fluorescent (optical) brighteners are known for their characteristics of protecting baculoviruses against deactivation by ultraviolet (UV) light and enhancing the activity of these agents as microbial insecticides on hosts and semipermissive hosts. These substances were evaluated in combination with the velvetbean caterpillar, Anticarsia gemmatalis Hübner, multiple-embedded nucleopolyhedrovirus (AgMNPV). The first trial involved 4 fluorescent brighteners (Blankophor BBH, Blankophor HRS, Blankophor RKH, and Tinopal LPW) obtained from the United States. The second trial was conducted with 11 fluorescent brighteners (Tinopal UNPA-GX, Tinopal DMS, Tinopal CBS, Leukophor DUB, Leukophor BSBB, Hostalux KS-N, Hostalux ETBN, BRY 10 D2 100, BRY 10 D2 150, Uvitex BHT, and Uvitex NFW) available in Brazil in combination with the AgMNPV to determine the degree of enhancement of viral activity. These brighteners were also evaluated with regard to AgMNPV protection against deactivation by UV light. Combinations of the virus with selected fluorescent brighteners were tested against both AgMNPV-susceptible and resistant strains of A. gemmatalis. In the first trial, brighteners obtained from the United States promoted increases in AgMNPV activity from 5.2-fold (Blankophor HRS) to 76.6-fold (Blankophor RKH) and reduced the mean time to death by 2.8 to 3.5 days. In the second trial, the most effective brightener (Tinopal UNPA-GX) reduced the LC₅₀ in A. gemmatalis larvae from 7083 occlusion bodies (OBs)/ml (virus alone) to 77.8 OBs/ml (≈90-fold). When 4 selected brighteners were tested in combination with the AgMNPV in resistant insects, the LC₅₀ was reduced by ca. 10,000-fold (Leukophor DUB) to ca. 62,000-fold (Tinopal UNPA-GX), in comparison to the LC₅₀ of 3.7 × 10⁷ OBs/ml observed for the virus alone. Therefore, mortality of highly resistant A. gemmatalis larvae to the AgMNPV increased dramatically when the virus was combined with some fluorescent brighteners. UV protection measured by original activity remaining (OAR) varied from <30% OAR (Uvitex NFW) to >90% OAR (Tinopal UNPA-GX and BRY 10 D2 100). All efficacious brighteners were stilbene disulfonic acid derivatives and, when used alone, none showed negative effects against A. gemmatalis larvae.     

Selection of promising fungal biological control agent of the western flower thrips Frankliniella occidentalis (Pergande)

Aims: Larval stages of Frankliniella occidentalis are known to be refractory to fungal infection compared with the adult stage. The objective of this study was to identify promising fungal isolate(s) for the control of larval stages of F. occidentalis. Methods and Results: Ten isolates of Metarhizium anisopliae and eight of Beauveria bassiana were screened for virulence against second‐instar larvae of F. occidentalis. Conidial production and genetic polymorphism were also investigated. Metarhizium anisopliae isolates ICIPE 7, ICIPE 20, ICIPE 69 and ICIPE 665 had the shortest LT₅₀ values of 8·0–8·9 days. ICIPE 69, ICIPE 7 and ICIPE 20 had the lowest LC₅₀ values of 1·1 × 10⁷, 2·0 × 10⁷ and 3·0 × 10⁷ conidia ml^?1, respectively. Metarhizium anisopliae isolate ICIPE 69 produced significantly more conidia than M. anisopliae isolates ICIPE 7 and ICIPE 20. Internally transcribed spacers sequences alignment showed differences in nucleotides composition, which can partly explain differences in virulence. Conclusion: These results coupled with the previous ones on virulence and field efficacy against other species of thrips make M. anisopliae isolate ICIPE 69 a good candidate. Significance and Impact of the Study: Metarhizium anisopliae isolate ICIPE 69 can be suggested for development as fungus‐based biopesticide for thrips management.     

Selection of Antonospora locustae (Protozoa: Microsporidae) with higher virulence against Locusta migratoria manilensis (Orthoptera:Acrididae)

Antonospora locustae is a microsporidian parasite of grasshopper insects that is used as a biological control agent. We report on laboratory selection of isolates from different regions with increased virulence. Bioassays were conducted against third instar nymphs of Locusta migratoria manilensis. AL2008L01 was originally imported from the USA in 1986, AL2008M01 was isolated from Melanoplus differentialis in USA and AL2008F01 was isolated from infected Fruhstorferiola tonkinensi collected in Guangdong, China. The results showed that all three isolates can infect the locust and that pathogenicity increased gradually with increased dose. The LD₅₀ values of the original isolates at the highest dose (5×10⁶ spores/nymph) were 19, 23 and 22 days and LD₅₀ values were 3.2×10⁵, 3.4×10⁶ and 0.7×10⁶ spores/g, respectively. After selecting for three generations, the virulence of all isolates increased significantly. LT_50s were reduced to 17, 20 and 21 days at the highest dose (5×10⁶ spores/nymph) and LD_50s were reduced to 1.4×10⁵, 2.5×10⁵ and 1.7×10⁵ spores/g.