草坪地灰翅夜蛾的发生与防治药剂筛选

灰翅夜蛾Spodoptera mauritia是危害草坪的重要害虫。药剂测定试验表明,抑太保,氯氰菊酯,灭多威等药剂对灰翅夜蛾有较高毒力。对3龄初幼虫的LC50为0.4895-2.3672mg/L。4龄以上害虫耐药性显著增强。     

Co-Occlusion and Persistence of a Baculovirus Mutant Lacking the Polyhedrin Gene

A co-occlusion process was evaluated as a commercially and ecologically acceptable strategy for the development of genetically improved baculovirus insecticides. Coinfection of Spodoptera frugiperda (IPLB-SF-21) tissue culture cells with Autographa californica nuclear polyhedrosis virus (AcMNPV) and an AcMNPV mutant (Ac-E10) lacking the polyhedrin gene resulted in occlusion of both virus types within polyhedra. The amount of occluded Ac-E10 virions in progeny polyhedra populations during serial passage in Trichoplusia ni larvae was evaluated. Maintenance of the mutant in progeny polyhedra required polyhedra inocula containing equal numbers of the two virus types at a high dose. A significant reduction in occluded mutant nucleocapsids occurs with inoculum levels below a 100% lethal dose. At inoculum levels below a 30% lethal dose, the majority of fourth-instar larvae were infected with only one type of virus. The commercial application and ecological advantages of the co-occlusion process are discussed.     

Transmission agents of the nuclear polyhedrosis virus ofNeodiprion sertifer [Hym.: Diprionidae]

Colonies ofNeodiprion sertifer (Geoffroy) larvae infected by nuclear polyhedrosis virus were visited by parasitoids, predators, and scavengers. Six transmission pathways were studied in detail. Viable virus polyhedra (PIB) were isolated from external surfaces of parasitic wasps and faeces of tachinid flies, coccinellid larvae, and scavenging flies. Faeces of the scavenging flies remained infectious 9 months on foliage in the field. A bird fed on earthworms from contaminated soil passed infectious faeces. Raindrops on foliage below diseased larval colonies contained more than 10⁸ PIB/ml. It was concluded that although there are numerous transmission pathways, only a few may be available at any one time. The stochastic nature of many pathways implies that the overall transmission rate is highly variable. These results are consistent with frequent observations of low transmission levels between larvae of the same generation.      

Activation of latent virus infections in larvae of Adoxophyes orana (Lepidoptera: Tortricidae) and Barathra brassicae (Lepidoptera: Noctuidae) by foreign polyhedra

The number of larvae containing polyhedra increased when larvae of Adoxophyes orana and Barathra brassicae were fed on polyhedra of nuclear polyhedrosis virus (NPV) of the reciprocal species. Comparison of restriction endonuclease EcoRI cleavage patterns of DNA isolated from polyhedra used as inocula and from polyhedra obtained after cross-inoculation showed that cross infection did not occur. The observations indicate that latent viruses were activated in both insects. Activation of the A. orana latent NPV with polyhedra of a cytoplasmic polyhedrosis virus (CPV) of B. brassicae, and cross-inoculation with an extract prepared from healthy larvae indicated that an activating agent does not have to be a component of nuclear polyhedra.     

Comparison of wild type and genetically engineered nuclear polyhedrosis viruses of Autographa californica for mortality, virus replication and polyhedra production in Trichoplusia ni larvae

Virus replication and polyhedra production of two polyhedron-positive recombinant nuclear polyhedrosis viruses of Autographa californica, AcJHE.KK and AcAaIT which encode juvenile hormone esterase and scorpion toxin, respectively, were compared with those of a plaque purified wild-type nuclear polyhedrosis virus, AcMNPV-C6, in Trichoplusia ni larvae. Though average times required to kill the T. ni larvae increased with the age of the larvae, killing time by either recombinant virus was significantly shorter than that by wild-type virus. Killing time was reduced ca. 30% for AcAaIT-infected larvae and 5 to 8% for AcJHE.KK-infected larvae as compared to that for AcMNPV-C6-infected larvae. The average weight of larvae infected with AcAaIT was significantly lower than that of larvae infected with AcJHE.KK and AcMNPV-C6. The mean numbers of polyhedra produced in each larva inoculated with AcAaIT and AcJHE.KK were ca. 20% and 60%, respectively, of those for AcMNPV-C6. Total virus titers in AcMNPV-C6-infected larvae were significantly higher than those in AcJHE.KK- and AcAaIT-infected larvae until 2 days post infection.     

Aspects of the cross transmission to Galleria mellonella of a Baculovirus from the alfalfa looper, Autographa californica

The nuclear polyhedrosis virus originally isolated from the alfalfa looper, Autographa californica, was successfully transmitted to the greater wax moth, Galleria mellonella. Both the many polyhedra per nucleus (MP) and the few polyhedra per nucleus (FP) plaque variants of this virus were found to be infective when injected intracoelomically. When polyhedra of each plaque variant were fed to G. mellonella larvae, a difference in response was observed; the MP plaque variant was estimated to be 30 times more infective than the FP variant.     

A Comparison of the Efficacy of Nuclear Polyhedrosis and Granulosis Viruses in Spray and Bait Formulations for the Control of Agrotis segetum (Lepidoptera: Noctuidae) in Maize

Agrotis segetum nuclear polyhedrosis virus (AsNPV) and granulosis virus (AsGV), propagated in laboratory cultures of A. segetum in England and A. ipsilon in Spain, respectively, were applied to plots of maize plants at the one‐ to four‐leaf stage of growth. Plots were arranged in a 6 x 6 Latin square design and infested with second‐instar A. segetum larvae (the common cutworm). Each virus was applied in separate treatments by two application methods; as an aqueous spray containing 0.1% Agral as a wetting agent, and as a bran bait. The NPV was applied at a rate of 4 X 10¹² polyhedra/ha, and the GV at 4 X 10¹³ granules/ha. Soil and plants were sampled for larvae on three occasions following virus treatment: 24 h, 4 days and 11 days. The larvae were reared on diet in the laboratory, until death or pupation, to examine the rate and level of viral infection. Infection data showed 87.5% and 91% NPV infection and 12.5% and 55% GV infection in spray and bait treatments, respectively, in larvae sampled 24 h after treatment. In larvae sampled 4 days after treatment, the results were 78% and 100% NPV infection, and 13% and 6% GV infection. A total of only six larvae were retrieved on day 11. In both treatments larvae infected with AsNPV died significantly more rapidly and at an earlier instar than those infected with AsGV, indicating that AsNPV appears to have better potential as a control agent for A. segetum.     

Quantitative and qualitative aspects in the production of a nuclear polyhedrosis virus in Spodoptera exigua larvae

Spodoptera exigua nuclear polyhedrosis virus was produced in late fourth instar S. exigua larvae, reared on semi-artificial diet. A maximum amount of virus, 1–2 × 10⁹ polyhedra/larva, was produced in individually-reared larvae after 7 days of incubation, with an inoculum of 7–5 × 10⁴ polyhedra/cm² diet surface. Virus yield was slightly reduced to 9 × 10⁸ polyhedra/larva when production was carried out in groups of 400 and 600 larvae per container. Biological activity of virus harvested from living larvae and from dead cadavers was similar. Microbial contaminants, predominantly bacteria, in the virus product numbered 1–6% of the number of polyhedra. Tests for the presence of vertebrate-pathogenic bacteria in the virus product were all negative.     

The effects of cations on the activity of the gypsy moth (Lepidoptera: Lymantriidae) nuclear polyhedrosis virus

Fourteen cations were tested at a 1% concentration (wt:wt), as chlorides, for their effects on the biological activity of the gypsy moth, Lymantria dispar (L.), nuclear polyhedrosis virus (LdMNPV). Cupric chloride was toxic to gypsy moth larvae. Ferrous and ferric chloride were inhibitory to larval growth and development as well as to virus activity. Strontium chloride was inhibitory to virus activity but had no apparent effects on gypsy moth larvae. Six cations had little or no effect on virus activity (i.e., calcium, lanthanum, magnesium, nickel, potassium, sodium), whereas four cations (i.e., cobalt, manganese, ruthenium, zinc) acted as viral enhancers, as indicated by reductions in LC50s.     

Residual susceptibility of the red imported fire ant (Hymenoptera: Formicidae) to four agricultural insecticides

The red imported fire ant, Solenopsis invicta Buren, is an abundant predator in cropping systems throughout its range. It has been documented to be an important predator of numerous crop pests, as well as being an agricultural pest itself. Information on the impact of insecticides on natural enemies such as fire ants is necessary for the integration of biological and chemical control tactics in an effective pest management program. Therefore, a residual vial bioassay was developed to determine the concentration-mortality responses of S. invicta workers to four commonly used insecticides: acephate, chlorpyrifos, methomyl and lambda-cyhalothrin. Fire ant workers showed a mortality response to serial dilutions to all four chemicals. Methomyl (LC50 0.04 microg/vial, fiducial limits 0.03-0.06) was the most toxic, followed by chlorpyrifos (LC50 0.11 microg/vial, fiducial limits 0.07-0.17) and acephate (LC50 0.76 microg/vial, fiducial limits 0.50-1.04). Of the chemicals assayed, it took a much higher concentration of lambda-cyhalothrin (LC50 2.30 microg/vial, fiducial limits 1.57-3.59) to kill 50% of the workers compared with the other three chemicals. The results of this study demonstrate the wide range in responses of fire ants to four insecticides that are labeled and commonly used on numerous agricultural crops throughout the United States. These results further suggest the possibility of using a discriminating dose of lambda-cyhalothrin to control the target pest species while conserving fire ants in the agricultural systems in which their predatory behavior is beneficial to the integrated pest management program.     

Effect of larval age and dosage of nuclear polyhedrosis virus on the susceptibility of diamondback moth, Plutella xylostella

A laboratory experiment was conducted to study the efficacy of nuclear polyhedrosis virus (NPV) with nine concentrations against all stadia of Plutella xylostella (L). The susceptibility of the larvae was correlated negatively with the period of development of the larvae and positively with the virus concentrations. The highest mortality of 84% was recorded in first stadium larvae compared to lowest mortality of 38% in fourth stadium larvae. The LC₅₀ was 5.5×10¹ and 4.0×10⁴ polyhedral inclusion bodies (PIB)/ml for first and fourth stadium larvae, respectively.     

Strain selection during serial passage of Trichoplusia in nuclear polyhedrosis virus.

Two strains of a nuclear polyhedrosis virus (NPV) of Trichoplusia ni were isolated on the basis of plaque morphology. They are designated as MP (having greater than 30 polyhedra per nucleus) and FP (having fewer than 10 polyhedra per nucleus). Serial, undiluted passage of plaque, purified MP nonoccluded. Virus (NOV) in tissue culture led to the production of the FP phenotype detectable at passage 9. With continued serial, undiluted passage, FP became the predominant strain. Comparative growth curves showed that FP NOV are released faster than MP NOV. MP morphology was not observed after 14 serial, undiluted passages of plaque-purified FP. By the plaque neutralization assay, NOV from both strains of virus was neutralized by the homologus and heterologous antisera. The FP phenotype was observed when FP virus was grown in culture at 17, 22, and 27 C. Hence, the FP phenotype was not considered to be the result of temperature-inhibited crystallization of polyhedrin under standard tissue culture conditions. The NOV of both strains killed insects when injected directly into the hemocoele of T. ni larvae. Only MP inclusion bodies were virulent per os. The FP inclusion bodies fed to cabbage looper larvae did not kill, and no infectious agent could be detected in the hemolymph. Electron micrographs of MP polyhedra showed bundles of nucleocapsids of normal length within the polyhedra, whereas FP polyhedra contained heterogeneous, electron-dense material, which could account for their lack of pathogenicity.     

Pathogenicity of a nuclear polyhedrosis virus of the almond moth, Cadra cautella

Tests were conducted with neonate Cadra cautella larvae to determine the pathogenicity of a nuclear polyhedrosis virus. A bioassay on an agar base diet showed that concentrations of 0.25, 0.50, 2.00, and 4.00 polyhedra/mm² killed 27, 55, 87, and 92% of the test larvae, respectively. A study of the time of death showed that most larvae died on the 9th or 10th day after exposure to 4 polyhedra/mm² at 27°C. When larvae were exposed to 8, 16, 32, and 64 × 10³ polyhedra/g of bran diet, recorded mortalities were 18, 22, 48, and 80%, respectively. All the samples of virus in bran diet which were incubated at various temperatures for 7, 14, and 28 days remained stable at all test conditions except the sample incubated at 42°C for 14 days, and those held at 37° and 42° for 28 days. Larvae of C. cautella, Plodia interpunctella, Ephestia elutella, and Paramyelois transitella placed on a diet with 40 × 10³ polyhedra/g had mortalities of 75, 59, 16, and 4%, respectively. Light and electron microscopical examination of P. interpunctella cadavers showed that they were infected with a multiply occluded nuclear polyhedrosis virus.     

Dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by the digestive fluid of Trichoplusia ni (Lepidoptera:Noctuidae) larvae

The dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus by digestive fluid collected from 5th stage Trichoplusia ni larvae was studied in vitro. Observations were made at timed intervals using phase contrast microscopy, and scanning and transmission electron microscopy. Dissolution occurred rapidly and in a detectable sequence. Under phase contrast, most polyhedra lost their refringence by 0.5 min. The polyhedra became rounded in appearance with small protuberances on the surface and Brownian movement was observed within. After 1 min, the envelope of most polyhedra had ruptured, releasing the enclosed virions. The protuberances were also observed under the scanning electron microscope after digestion for 0.5 min. Many shell fragments devoid of internal contents were seen after more lengthy digestion. Internal structural changes were revealed by electron microscopy. After 1 min of exposure, polyhedra were observed in all stages of dissolution. By 3 min, only virions, scattered about in heterogeneous material, could be distinguished.     

Virulence of cloned variants of Autographa californica nuclear polyhedrosis virus.

The relative virulence of five different genotypic variants of Autographa californica nuclear polyhedrosis virus was tested by determining the 50% lethal dose of occluded virus for larvae of Trichoplusia ni. The 50% lethal dose values of uncloned virus and the five cloned genotypic variants ranged between 10 and 21 polyhedra per larva, and no statistically significant differences were observed. Cloning has therefore neither enhanced nor decreased the virulence of this potential microbial pesticide.     

A synopsis of the obligate and facultative insect parasitic nematodes

A study of the pathogenicity of a cytoplasmic polyhedrosis virus of the gypsy moth, Porthetria dispar, was conducted. Third- and fourth-instar larvae were highly susceptible to the pathogen even when fed low dosages of polyhedra. Only 30–50% of the treated insects died of polyhedrosis, but the debilitating effects of the virus on larval and postlarval stages were remarkable. Pupal weights were consistently reduced. An inverse and, respectively, a direct, relationship was found between the size of diseased specimens and the developmental periods of larvae and pupae surviving to lethal infection. The highest infected male adults showed a lighter wing coloration. Structural abnormalities and melanotic abdominal inclusions, recorded in diseased adults, were more frequent in females than in males. The presence of melanotic inclusions in diseased females obtained from early larval infection experiments was associated with a greater pupal weight. Lower dosages were the most effective in producing larval mortality and reducing pupal weights and adult emergence. An interference phenomenon due to a mixed virus infection is suspected.     

A plaque assay for titration of alfalfa looper nuclear polyhedrosis virus in a cabbage looper (TN-368) cell line

This is the first report of plaque formation by a pathogenic insect virus. Trichoplusia ni (TN-368) cells overlaid with medium containing 0.6% methyl cellulose continued to multiply, developed into monolayers, and produced plaques after infection with alfalfa looper nuclear polyhedrosis virus. Viral polyhedral inclusion bodies were first observed 24 hr after exposure of cells to virus, and plaques continued to increase in size for 72 hr. Two different types of plaques were observed: one in which all cells had many polyhedra in their nuclei, and another in which few cells had inclusion bodies. When virus from either plaque was injected into T. ni larvae, they died of typical nuclear polyhedrosis virus disease. The assay was reproducible, and plaque numbers were related to virus concentration.     

Functional expression of lepidopteran-selective neurotoxin in baculovirus: potential for effective pest management

Recombinant baculovirus expressing insect-selective neurotoxins derived from venomous animals are considered as an attractive alternative to chemical insecticides for efficient insect control agents. Recently we identified and characterized a novel lepidopteran-selective toxin, Buthus tamulus insect-selective toxin (ButaIT), having 37 amino acids and eight half cysteine residues from the venom of the South Indian red scorpion, Mesobuthus tamulus. The synthetic toxin gene containing the ButaIT sequence in frame to the bombyxin signal sequence was engineered into a polyhedrin positive Autographa californica nuclear polyhedrosis virus (AcMNPV) genome under the control of the p10 promoter. Toxin expression in the haemolymph of infected larvae of Heliothis virescens and also in an insect cell culture system was confirmed by western blot analysis using antibody raised against the GST-ButaIT fusion protein. The recombinant NPV (ButaIT-NPV) showed enhanced insecticidal activity on the larvae of Heliothis virescens as evidenced by a significant reduction in median survival time (ST50) and also a greater reduction in feeding damage as compared to the wild-type AcMNPV.     

Changes in growth and virulence of a nuclear polyhedrosis virus from Choristoneura fumiferana after passage in Trichoplusia ni and Galleria mellonella

An isolate of nuclear polyhedrosis virus from Choristoneura fumiferana was fed to neonate larvae of Trichopulsia ni and Galleria mellonella. It caused infection and mortality in both of these species. After passage in the alternate hosts, the isolate became increasingly virulent for these hosts. The passaged virus retained its infectivity for Choristoneura but diseased larvae did not wilt and at death they were found to contain only a few polyhedra indicating the virus had been changed.