James R. Fuxa

DNA restriction polymorphism in wild isolates of Spodoptera frugiperda nuclear polyhedrosis virus

Restriction endonuclease analysis was used to examine variation in DNA of 22 wild isolates of Spodoptera frugiperda nuclear polyhedrosis virus (SfNPV). Eleven of the 15 isolated from Louisiana were distinguishable based on restriction fragment profiles from the enzymes BamHI, HindIII, and EcoRI. There was significant genetic variation in SfNPV isolates within single agricultural fields. Nucleotide sequence divergence values, based on restriction fragment profiles, indicated that genetic variation among isolates foreign to Louisiana (Ohio, Ecuador, Mexico, Georgia, Colombia, and Venezuela) was greater than that among the Louisiana isolates. However, certain foreign isolates were similar to or identical with Louisiana isolates. Genetic variation of the viral DNA was not influenced by the insects host plan species.

Survey of entomopathogenic nematodes and fungi endemic to pecan orchards of the Southeastern United States and their virulence to the pecan weevil (Coleoptera: Curculionidae).

Abstract The pecan weevil, Curculio caryae (Horn), is a major pest of pecans in the Southeastern United States. Entomopathogenic nematodes and fungi are potential alternatives to chemical insecticides for C. caryae control. Our objective was to survey pecan orchards in the southeastern United States for entomopathogenic nematodes and fungi and determine the virulence of the new isolates to C. caryae larvae. Soil was collected from 105 sites in 21 orchards in Arkansas, Georgia, Louisiana, and Mississippi. Entomopathogens were isolated by exposing soil to C. caryae and greater wax moth larvae, Galleria mellonella, (L.). We isolated entomopathogenic fungi and nematodes from 16 and 6 of the 21 orchards surveyed, respectively. The entomopathogenic fungi included Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin, and nematodes included Heterorhabditis bacteriophora Poinar, Steinernema carpocapsae (Weiser), Steinernema glaseri (Steiner), and Steinernema rarum (Doucet). This is the first report of Steinernema rarum in the United States. Soil characteristics in orchards were analyzed for pH, organic matter, and nutrients; we detected a negative relationship between fungal occurrence and manganese levels in soil and a positive relationship between M. anisopliae occurrence and calcium or magnesium levels. In laboratory assays, virulence of 15 nematode and 22 fungal isolates to C. caryae larvae was tested in small plastic cups containing soil. Results indicated poor susceptibility of the C. caryae larvae to entomopathogenic nematodes. Several fungal isolates that caused significantly higher mortality in C. caryae larvae than other strains (including a commercial strain of B. bassiana) should be investigated further as potential control agents of C. caryae.

Reversion of resistance by spodoptera frugiperda to nuclear polyhedrosis virus

A laboratory colony of fall armyworm, Spodoptera frugiperda, was selected for resistance by feeding larvae of every generation the LD80 of its nuclear polyhedrosis virus (NPV). The LD50 was 5.9 polyhedral inclusion bodies (PIB)/insect in a control (susceptible) colony and 18.7 PIB/insect in the resistant insects. These LD50s were significantly different based on nonoverlap of 95% fiducial limits. The NPV-resistant colony was divided into two: one colony (V +) continued to be fed the LD80 of the NPV in every generation, and the other resistant colony (V −) was no longer exposed to NPV. Within one generation the LD50 of NPV against the V − colony (17.0 PIB/insect) was no longer significantly different, based on 95% fiducial limits, from that of the control colony (5.6 PIB/insect). Within three generations of dividing the resistant colony, the LD50 in the V − colony (10.1 PIB/insect) was significantly different from that in the V + colony (19.8 PIB/insect). After eight generations, the LD50s were 5.8, 19.5, and 7.3 PIB/insect in the control, V +, and V − colonies, respectively. Thus, fall armyworm populations can lose their resistance to NPV when they are not exposed to the virus. There were no differences between the control and resistant (V +) colonies in mean weights of larvae or pupae, but the NPV-susceptible insects had a significantly (P < 0.01) longer life span, more eggs/female, and a greater percentage of eggs hatch than the resistant (V +) insects.

Effects of virulence, sporulation, and temperature on Metarhizium anisopliae and Beauveria bassiana laboratory transmission in Coptotermes formosanus

Sporulation characteristics and virulence of Metarhizium anisopliae and Beauveria bassiana were examined in relation to laboratory transmission in Coptotermes formosanus. Fungal isolates significantly affected disease prevalence in termite populations. Sporulation of M. anisopliae played a more important role than virulence in producing epizootics within small groups of termites, but this was not the case for B. bassiana. Isolates characterized by quick sporulation (day 2 after death) did not exhibit better transmission in termites than those with high total sporulation (day 11 after death) in either fungal species. An isolate of M. anisopliae ranking highly in all three categories (virulence, quick sporulation, and total sporulation) produced better epizootics than an isolate that was inferior in all three characteristics. High temperatures (35 degrees C) significantly reduced fungal germination rates, leading to significant reduction of epizootics. M. anisopliae was better than B. bassiana in producing epizootics at 27 degrees C. Thus, fungal characteristics other than virulence should be considered for the seasonal colonization approach to termite microbial control.

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.

Susceptibility ofHeliothis zea (Boddie) larvae toNomuraea rileyi (Farlow) Samson

To determine the impact of α-tomatine at the third trophic level, the following model was developed:Nomuraea rileyi (Farlow) Samson, the secondary consumer, acting onHeliothis zea (Boddie), the primary consumer, fed an artificial diet modified with α-tomatine. In vitro, the allelochemical inhibited colony formation and growth of the fungus. The in vivo test revealed that larval growth and developmental time were affected by α-tomatine andN. rileyi. Detrimental effects on pupal development were observed in larvae fed diet containing α-tomatine and also treated withN. rileyi (LC90). The fungus was detected in the hemolymph and tissue of larvae treated with two lethal concentrations (LC50 and LC90) ofN. rileyi, including those fed α-tomatine. At the LC50, α-tomatine protected larvae againstN. rileyi and increased survivorship; at the LC90, it inhibited the development ofN. rileyi, thereby reducing production of conidia. Thus, the allelochemical α-tomatine retains its antifungal qualities beyond the second trophic level, inhibiting the development ofN. rileyi inH. zea.

Sporulation of Metarhizium anisopliae and Beauveria bassiana on Coptotermes formosanus and in vitro.

The sporulation of 22 total isolates of Metarhizium anisopliae and Beauveria bassiana was quantified on cadavers of the Formosan subterranean termite, Coptotermes formosanus. Conidial production increased significantly over 11 days post-death. Effects of isolates of M. anisopliae and B. bassiana on in vivo sporulation were significant. Although the overall effects of fungal species on in vivo sporulation were not significant, the interactions between fungal species and certain times post-death were significant, indicating different sporulation patterns between the two fungal species. B. bassiana isolates could be categorized into a group with high total sporulation (day 11) and low quick sporulation (on days 2 and 3), while M. anisopliae isolates fell into another group with high quick sporulation and low total sporulation. This could give M. anisopliae an advantage over B. bassiana in termite microbial control due to termite defensive social behaviors. Conidial production was significantly higher in vitro than in vivo. In vitro and in vivo sporulation differed by as much as 89x and 232x among the selected isolates of B. bassiana and M. anisopliae, respectively. Correlation between in vivo and in vitro conidial production was positive and significant. This may allow preliminary in vitro screening of a large number of isolates for high in vivo sporulation.

Vertical transmission of TnSNPV, TnCPV, AcMNPV, and possibly recombinant NPV in Trichoplusia ni

Four viruses were tested for vertical transmission in Trichoplusia ni: T. ni nucleopolyhedrovirus (TnSNPV), T. ni cypovirus (TnCPV), Autographa californica nucleopolyhedrovirus (AcMNPV), and AcMNPV engineered to express a scorpion toxin (AcMNPV.AaIT). Fifth instars were exposed to each virus, the survivors were reared and mated, and second-generation (F(1)) insects were examined for infection. TnSNPV was transmitted to offspring at a prevalence rate of 15.4%, TnCPV at 10.2%, and AcMNPV at 10.1%. Only one of 2484 F(1) insects was infected with AcMNPV.AaIT; this experiment was repeated, and none of 4774 insects was infected. Thus, vertical transmission is unlikely to contribute to AcMNPV.AaIT contacting non-target organisms after its field release. There was evidence that TnCPV and possibly TnSNPV were activated to overt infections by ingestion of a different virus. TnCPV, but not the NPVs, routinely infected 0.3-1.7% of non-treated insects, probably indicating that it is vertically transmitted at enzootic levels.

Polyhedra without virions in a vertically transmitted nuclear polyhedrosis virus

Abstract Polyhedra were observed in larvae, pupae, and adults (f1 generation) of Spodoptera frugiperda whose parents had survived exposure to the S. frugiperda nuclear polyhedrosis virus (NPV). These polyhedra were isolated from the f1 insects and fed to first instars. The polyhedra from f1 larvae and pupae infected the first instars (86–98 and 69–88% infection, respectively), but most of those recovered from adults of the f1 generation did not (0–11% infection). Electron microscopy indicated that the infectious polyhedra from adults contained virions, but the noninfectious polyhedra from adults did not. A monoclonal anti-polyhedrin antibody used in conjunction with fluorescence microscopy reacted positively with the empty, noninfectious polyhedra, indicating that the polyhedrin protein was present. Thus, when the S. frugiperda NPV is vertically transmitted, the infections in the f1 adults are nonlethal and are manifested mainly as noninfectious, empty polyhedra which are composed of the polyhedrin protein.

Spore-Toxin Interactions and Sublethal Effects of Bacillus thuringiensis in Spodoptera frugiperda and Pseudoplusia includens (Lepidoptera: Noctuidae)

Interactions between the spores and δ-endotoxins of Bacillus thuringiensis Berliner were tested in a low-susceptibility insect, Spodoptera frugiperda (J. E. Smith). The spores (formulation MYD™) an...