Rational Design of an Epstein-Barr Virus Vaccine Targeting the Receptor-Binding Site.

Epstein-Barr virus (EBV) represents a major global health problem. Though it is associated with infectious mononucleosis and ∼200,000 cancers annually worldwide, a vaccine is not available. The major target of immunity is EBV glycoprotein 350/220 (gp350) that mediates attachment to B cells through complement receptor 2 (CR2/CD21). Here, we created self-assembling nanoparticles that displayed different domains of gp350 in a symmetric array. By focusing presentation of the CR2-binding domain on nanoparticles, potent neutralizing antibodies were elicited in mice and non-human primates. The structurally designed nanoparticle vaccine increased neutralization 10- to 100-fold compared to soluble gp350 by targeting a functionally conserved site of vulnerability, improving vaccine-induced protection in a mouse model. This rational approach to EBV vaccine design elicited potent neutralizing antibody responses by arrayed presentation of a conserved viral entry domain, a strategy that can be applied to other viruses.

Efficacy of detergent-based cleaning and wiping against SARS-CoV-2 on high-touch surfaces.

Efficacy of cleaning methods against SARS-CoV-2 suspended in either 5% soil load (SARS-soil) or simulated saliva (SARS-SS) was evaluated immediately (hydrated virus, T0) or 2 hours post-contamination (dried virus, T2). Hard water dampened wiping (DW) of surfaces, resulted in 1.77-3.91 log reduction (T0) or 0.93-2.41 log reduction (T2). Incorporating surface pre-wetting by spraying with a detergent solution (D + DW) or hard water (W + DW) just prior to dampened wiping did not unilaterally increase efficacy against infectious SARS-CoV-2, however, the effect was nuanced with respect to surface, viral matrix, and time. Cleaning efficacy on porous surfaces (seat fabric, SF) was low. W + DW on stainless steel (SS) was as effective as D + DW for all conditions except SARS-soil at T2 on SS. DW was the only method that consistently resulted in > 3-log reduction of hydrated (T0) SARS-CoV-2 on SS and ABS plastic. These results suggest that wiping with a hard water dampened wipe can reduce infectious virus on hard non-porous surfaces. Pre-wetting surfaces with surfactants did not significantly increase efficacy for the conditions tested. Surface material, presence or absence of pre-wetting, and time post-contamination affect efficacy of cleaning methods.

Evaluation of surface disinfection methods to inactivate the beta coronavirus Murine Hepatitis Virus.

The list of EPA-approved disinfectants for coronavirus features many products for use on hard, non-porous materials. There are significantly fewer products registered for use on porous materials. Further, many common, high-touch surfaces fall in between non-porous materials such as glass and porous materials such as soft fabrics. The objective of this study was to assess the efficacy of selected commercially available disinfectant products against coronaviruses on common, high-touch surfaces. Four disinfectants (Clorox Total 360, Bleach solution, Vital Oxide, and Peroxide Multi-Surface Cleaner) were evaluated against Murine Hepatitis Virus A59 (MHV) as a surrogate coronavirus for SARS-CoV-2. MHV in cell culture medium was inoculated onto four materials: stainless steel, latex-painted drywall tape, Styrene Butadiene rubber (rubber), and bus seat fabric. Immediately (T0) or 2-hr (T2) post-inoculation, disinfectants were applied by trigger-pull or electrostatic sprayer and either held for recommended contact times (Spray only) or immediately wiped (Spray and Wipe). Recovered infectious MHV was quantified by median tissue culture infectious dose assay. Bleach solution, Clorox Total 360, and Vital Oxide were all effective (>3-log10 reduction or complete kill of infectious virus) with both the Spray Only and Spray and Wipe methods on stainless steel, rubber, and painted drywall tape when used at recommended contact times at both T0 and T2 hr. Multi-Surface Cleaner unexpectedly showed limited efficacy against MHV on stainless steel within the recommended contact time; however, it showed increased (2.3 times greater efficacy) when used in the Spray and Wipe method compared to Spray Only. The only products to achieve a 3-log10 reduction on fabric were Vital Oxide and Clorox Total 360; however, the efficacy of Vital Oxide against MHV on fabric was reduced to below 3-log10 when applied by an electrostatic sprayer compared to a trigger-pull sprayer. This study highlights the importance of considering the material, product, and application method when developing a disinfection strategy for coronaviruses on high-touch surfaces.

Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies.

Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process constrained by dated technology and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here we use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited haemagglutination inhibition antibody titres more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin-nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.

Structural and genetic basis for development of broadly neutralizing influenza antibodies.

Influenza viruses take a yearly toll on human life despite efforts to contain them with seasonal vaccines. These viruses evade human immunity through the evolution of variants that resist neutralization. The identification of antibodies that recognize invariant structures on the influenza haemagglutinin (HA) protein have invigorated efforts to develop universal influenza vaccines. Specifically, antibodies to the highly conserved stem region of HA neutralize diverse viral subtypes. These antibodies largely derive from a specific antibody gene, heavy-chain variable region IGHV1-69, after limited affinity maturation from their germline ancestors, but how HA stimulates naive B cells to mature and induce protective immunity is unknown. To address this question, we analysed the structural and genetic basis for their engagement and maturation into broadly neutralizing antibodies. Here we show that the germline-encoded precursors of these antibodies act as functional B-cell antigen receptors (BCRs) that initiate subsequent affinity maturation. Neither the germline precursor of a prototypic antibody, CR6261 (ref. 3), nor those of two other natural human IGHV1-69 antibodies, bound HA as soluble immunoglobulin-G (IgG). However, all three IGHV1-69 precursors engaged HA when the antibody was expressed as cell surface IgM. HA triggered BCR-associated tyrosine kinase signalling by germline transmembrane IgM. Recognition and virus neutralization was dependent solely on the heavy chain, and affinity maturation of CR6261 required only seven amino acids in the complementarity-determining region (CDR) H1 and framework region 3 (FR3) to restore full activity. These findings provide insight into the initial events that lead to the generation of broadly neutralizing antibodies to influenza, informing the rational design of vaccines to elicit such antibodies and providing a model relevant to other infectious diseases, including human immunodeficiency virus/AIDS. The data further suggest that selected immunoglobulin genes recognize specific protein structural 'patterns' that provide a substrate for further affinity maturation.

H5N1 Vaccine-Elicited Memory B Cells Are Genetically Constrained by the IGHV Locus in the Recognition of a Neutralizing Epitope in the Hemagglutinin Stem.

Because of significant viral diversity, vaccines that elicit durable and broad protection against influenza have been elusive. Recent research has focused on the potential of highly conserved regions of the viral hemagglutinin (HA) as targets for broadly neutralizing Ab responses. Abs that bind the highly conserved stem or stalk of HA can be elicited by vaccination in humans and animal models and neutralize diverse influenza strains. However, the frequency and phenotype of HA stem-specific B cells in vivo remain unclear. In this article, we characterize HA stem-specific B cell responses following H5N1 vaccination and describe the re-expansion of a pre-existing population of memory B cells specific for stem epitopes. This population uses primarily, but not exclusively, IGHV1-69-based Igs for HA recognition. However, within some subjects, allelic polymorphism at the ighv1-69 locus can limit IGHV1-69 immunodominance and may reduce circulating frequencies of stem-reactive B cells in vivo. The accurate definition of allelic selection, recombination requirements, and ontogeny of neutralizing Ab responses to influenza will aid rational influenza vaccine design.

Coinfection with Streptococcus pneumoniae modulates the B cell response to influenza virus.

Pathogen-specific antibodies (Abs) protect against respiratory infection with influenza A virus (IAV) and Streptococcus pneumoniae and are the basis of effective vaccines. Sequential or overlapping coinfections with both pathogens are common, yet the impact of coinfection on the generation and maintenance of Ab responses is largely unknown. We report here that the B cell response to IAV is altered in mice coinfected with IAV and S. pneumoniae and that this response differs, depending on the order of pathogen exposure. In mice exposed to S. pneumoniae prior to IAV, the initial virus-specific germinal center (GC) B cell response is significantly enhanced in the lung-draining mediastinal lymph node and spleen, and there is an increase in CD4(+) T follicular helper (TFH) cell numbers. In contrast, secondary S. pneumoniae infection exaggerates early antiviral antibody-secreting cell formation, and at later times, levels of GCs, TFH cells, and antiviral serum IgG are elevated. Mice exposed to S. pneumoniae prior to IAV do not maintain the initially robust GC response in secondary lymphoid organs and exhibit reduced antiviral serum IgG with diminished virus neutralization activity a month after infection. Our data suggest that the history of pathogen exposures can critically affect the generation of protective antiviral Abs and may partially explain the differential susceptibility to and disease outcomes from IAV infection in humans. Importance: Respiratory tract coinfections, specifically those involving influenza A viruses and Streptococcus pneumoniae, remain a top global health burden. We sought to determine how S. pneumoniae coinfection modulates the B cell immune response to influenza virus since antibodies are key mediators of protection.

Flow cytometry reveals that H5N1 vaccination elicits cross-reactive stem-directed antibodies from multiple Ig heavy-chain lineages.

UNLABELLED: An understanding of the antigen-specific B-cell response to the influenza virus hemagglutinin (HA) is critical to the development of universal influenza vaccines, but it has not been possible to examine these cells directly because HA binds to sialic acid (SA) on most cell types. Here, we use structure-based modification of HA to isolate HA-specific B cells by flow cytometry and characterize the features of HA stem antibodies (Abs) required for their development. Incorporation of a previously described mutation (Y98F) to the receptor binding site (RBS) causes HA to bind only those B cells that express HA-specific Abs, but it does not bind nonspecifically to B cells, and this mutation has no effect on the binding of broadly neutralizing Abs to the RBS. To test the specificity of the Y98F mutation, we first demonstrated that previously described HA nanoparticles mediate hemagglutination and then determined that the Y98F mutation eliminates this activity. Cloning of immunoglobulin genes from HA-specific B cells isolated from a single human subject demonstrates that vaccination with H5N1 influenza virus can elicit B cells expressing stem monoclonal Abs (MAbs). Although these MAbs originated mostly from the IGHV1-69 germ line, a reasonable proportion derived from other genes. Analysis of stem Abs provides insight into the maturation pathways of IGVH1-69-derived stem Abs. Furthermore, this analysis shows that multiple non-IGHV1-69 stem Abs with a similar neutralizing breadth develop after vaccination in humans, suggesting that the HA stem response can be elicited in individuals with non-stem-reactive IGHV1-69 alleles. IMPORTANCE: Universal influenza vaccines would improve immune protection against infection and facilitate vaccine manufacturing and distribution. Flu vaccines stimulate B cells in the blood to produce antibodies that neutralize the virus. These antibodies target a protein on the surface of the virus called HA. Flu vaccines must be reformulated annually, because these antibodies are mostly specific to the viral strains used in the vaccine. But humans can produce broadly neutralizing antibodies. We sought to isolate B cells whose genes encode influenza virus antibodies from a patient vaccinated for avian influenza. To do so, we modified HA so it would bind only the desired cells. Sequencing the antibody genes of cells marked by this probe proved that the patient produced broadly neutralizing antibodies in response to the vaccine. Many sequences obtained had not been observed before. There are more ways to generate broadly neutralizing antibodies for influenza virus than previously thought.

Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin.

Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocket on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.

A multi-gene phylogeny provides additional insight into the relationships between several Ascosphaera species.

Ascosphaera fungi are highly associated with social and solitary bees, with some species being pathogenic to bees (causing chalkbrood) while others are not, and proper identification within this genus is important. Unfortunately, morphological characterizations can be difficult, and molecular characterizations have only used one genetic region. We evaluated multiple phylogenies of the Ascosphaera using up to six loci: the Internal Transcribed Spacer (ITS) region, 18S rRNA, 28S rRNA, Elongation Factor-1α (EF-1α) the RNA polymerase II largest subunit (RPB1), and the second largest subunit (RPB2). The ITS sequence alone produced an inadequate phylogeny, and the addition of both the 18S and 28S rRNA loci to the ITS sequence produced a phylogeny similar to that based on all six genetic regions. For all phylogenies, Ascosphaera torchioi was in a separate clade that was the most basal, with a strong genetic similarity to Eremascus albus, introducing the possibility of paraphyly within Ascosphaera. Also, based on this new phylogeny, we now suggest that the Apis mellifera (honey bee) pathogens arose within a group of saprophytes, and the Megachile (leafcutting bees) pathogens arose separately.

Mechanisms by which pesticides affect insect immunity.

The current state of knowledge regarding the effect of pesticides on insect immunity is reviewed here. A basic understanding of these interactions is needed for several reasons, including to improve methods for controlling pest insects in agricultural settings, for controlling insect vectors of human diseases, and for reducing mortality in beneficial insects. Bees are particularly vulnerable to sublethal pesticide exposures because they gather nectar and pollen, concentrating environmental toxins in their nests in the process. Pesticides do have effects on immunity. Organophosphates and some botanicals have been found to impact hemocyte number, differentiation, and thus affect phagocytosis. The phenoloxidase cascade and malanization have also been shown to be affected by several insecticides. Many synthetic insecticides increase oxidative stress, and this could have severe impacts on the production of some antimicrobial peptides in insects, but research is needed to determine the actual effects. Pesticides can also affect grooming behaviors, rendering insects more susceptible to disease. Despite laboratory data documenting pesticide/pathogen interactions, little field data is available at the population level.

Potential of ozone as a fumigant to control pests in honey bee (Hymenoptera: Apidae) hives.

Ozone is a powerful oxidant capable of killing insects and microorganisms, and eliminating odors, taste, and color. Thus, it could be useful as a fumigant to decontaminate honey comb between uses. The experiments here are intended to determine the exposure levels required to kill an insect pest and spore forming bee pathogens. Ozone was effective against greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae), even on naturally infested comb. Neonates and adults were the easiest life stages to kill, requiring only a few hours of exposure, whereas eggs required a 48-h exposure (at 460-920 mg O3/m3). Two honey bee, Apis mellifera L. (Hymenoptera: Apidae), pathogens, Ascosphaera apis (a fungus that causes chalkbrood) and Paenibacillus larvae (a bacterium that causes American foulbrood), also were killed with ozone. These pathogens required much higher concentrations (3200 and 8560 mg O3/m3, respectively) and longer exposure periods (3 d) than needed to control the insects. P. larvae was effectively sterilized only when these conditions were combined with high temperature (50 degrees C) and humidity (> or =75% RH). Thus, ozone shows potential as a fumigant for bee nesting materials, but further research is needed to evaluate its acceptability and efficacy in the field. The need for a reliable method to decontaminate honey bee nesting materials as part of an overall bee health management system is discussed.

Architectural nucleoporins Nup157/170 and Nup133 are structurally related and descend from a second ancestral element.

The nuclear pore complex (NPC) constitutes one of the largest protein assemblies in the eukaryotic cell and forms the exclusive gateway to the nucleus. The stable, approximately 15-20-MDa scaffold ring of the NPC is built from two multiprotein complexes arranged around a central 8-fold axis. Here we present crystal structures of two large architectural units, yNup170(979-1502) and hNup107(658-925) x hNup133(517-1156), each a constituent of one of the two multiprotein complexes. Conservation of domain arrangement and of tertiary structure suggests that Nup157/170 and Nup133 derived from a common ancestor. Together with the previously established ancestral coatomer element (ACE1), these two elements constitute the major alpha-helical building blocks of the NPC scaffold and define its branched, lattice-like architecture, similar to vesicle coats like COPII. We hypothesize that the extant NPC evolved early during eukaryotic evolution from a rudimentary structure composed of several identical copies of a few ancestral elements, later diversified and specified by gene duplication.

Evaluation of ultrafiltration cartridges for a water sampling apparatus.

AIMS: To determine the efficiency of various ultrafiltration cartridges (UFC) in concentrating test micro-organisms from drinking water. METHODS AND RESULTS: Replicate drinking water samples from three potable water supplies were dosed with Bacillus anthracis Sterne, Francisella tularensis LVS, Yersinia pestis CO92, bacteriophages MS2 and phi-X174, and Cryptosporidium parvum. The test micro-organisms were dosed together in 100 l of water, which was then recirculated through one of five different UFC until the retentate volume was reduced to c. 500 ml. The micro-organisms were assayed before and after ultrafiltration concentration and per cent recoveries were calculated. There were nine statistically significant differences among pairs of filters out of a possible 180 different combinations of UFC, test micro-organisms, and water types. CONCLUSIONS: No filter consistently performed better or worse than the others for each test micro-organism in all water samples tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides performance data on the ability of several different UFC to concentrate a panel of test micro-organisms from three sources of potable water. Water utilities and first responders may use these data when selecting UFC for use in emergency response protocols. This study also provides additional data as to the efficacy of ultrafiltration for recovering bacteria, virus-like particles, and protozoan oocysts from water samples.

Long-term storage of Ascosphaera aggregata and Ascosphaera apis, pathogens of the leafcutting bee (Megachile rotundata) and the honey bee (Apis mellifera).

Survival rates of Ascosphaera aggregata and Ascosphaera apis over the course of a year were tested using different storage treatments. For spores, the storage methods tested were freeze-drying and ultra-low temperatures, and for hyphae, freeze-drying, agar slants, and two methods of ultra-low temperatures. Spores of A. aggregata and A. apis stored well at -80 degrees C and after freeze-drying. A. aggregata hyphae did not store well under any of the methods tested while A. apis hyphae survived well using cryopreservation. Spores produced from cryopreserved A. apis hyphae were infective. Long-term storage of these two important fungal bee diseases is thus possible.

Fungicide tests on adult alfalfa leafcutting bees (Hymenoptera: Megachilidae).

Chalkbrood is a serious disease of alfalfa leafcutting bee Megachile rotundata (F.) (Hymenoptera: Megachilidae) larvae, causing upward of 20% infection in the field. The causative agent is the fungus Ascosphaera aggregata. This bee is used extensively for alfalfa seed pollination in the United States. Using laboratory bioassays, we previously demonstrated that fungicides can reduce chalkbrood levels in the larvae. Here, we evaluate the toxicity of four fungicides, Benlate, Captan, Orbit, and Rovral, to adult bees by using three different bioassays. In the first test, fungicides were applied to bees' thoraces. In the second test, mimicking foliage residue, a piece of filter paper soaked in fungicide was placed on the bottom of a container of bees. The third test evaluated oral toxicity by incorporating fungicides into a sugar-water solution that was fed to the bees. The filter paper test did not discriminate among the fungicides well, and the oral test resulted in the greatest mortality. Toxicity to males was greater than to females. The use of fungicides for chalkbrood control is a logical choice, but caution should be used in how they are applied in the presence of bees.

Laboratory bioassays to evaluate fungicides for chalkbrood control in larvae of the alfalfa leafcutting bee (Hymenoptera: Megachilidae).

Chalkbrood, a fungal disease in bees, is caused by several species of Ascosphaera. A. aggregata is a major mortality factor in populations of the alfalfa leafcutting bee, Megachile rotundata (F.) (Hymenoptera: Megachilidae) used in commercial alfalfa seed production. Four formulated fungicides, Benlate 50 WP, Captan, Orbit, and Rovral 50 WP were tested in the laboratory for efficacy against hyphal growth of A. aggregata cultures. The same fungicides, with the addition of Rovral 4 F, were tested for their effects on incidence of chalkbrood disease, and toxicity to M. rotundata larvae. Benlate, Rovral 50 WP, and Rovral 4 F reduced incidence of chalkbrood with minimal mortality on larval bees. Benlate and Rovral 50 WP also reduced hyphal growth. Orbit was effective in reducing hyphal growth, but it did not reduce incidence of chalkbrood and was toxic to bee larvae. Captan was not effective in reducing hyphal growth or chalkbrood incidence, and it was toxic to bee larvae. Fungicides that reduce incidence of chalkbrood and larval mortality in this laboratory study are candidates for further study for chalkbrood control.

The Similarity and Appropriate Usage of Three Honey Bee (Hymenoptera: Apidae) Datasets for Longitudinal Studies.

Honey bee (Apis mellifera L., Hymenoptera: Apidae) colonies have experienced profound fluctuations, especially declines, in the past few decades. Long-term datasets on honey bees are needed to identify the most important environmental and cultural factors associated with these changes. While a few such datasets exist, scientists have been hesitant to use some of these due to perceived shortcomings in the data. We compared data and trends for three datasets. Two come from the US Department of Agriculture's National Agricultural Statistics Service (NASS), Agricultural Statistics Board: one is the annual survey of honey-producing colonies from the Annual Bee and Honey program (ABH), and the other is colony counts from the Census of Agriculture conducted every five years. The third dataset we developed from the number of colonies registered annually by some states. We compared the long-term patterns of change in colony numbers among the datasets on a state-by-state basis. The three datasets often showed similar hive numbers and trends varied by state, with differences between datasets being greatest for those states receiving a large number of migratory colonies. Dataset comparisons provide a method to estimate the number of colonies in a state used for pollination versus honey production. Some states also had separate data for local and migratory colonies, allowing one to determine whether the migratory colonies were typically used for pollination or honey production. The Census of Agriculture should provide the most accurate long-term data on colony numbers, but only every five years.

Impact of disinfecting nesting boards on chalkbrood control in the alfalfa leafcutting bee.

The alfalfa leafcutting bee, Megachile rotundata (F.), is a solitary, cavity-nesting bee that has been managed in large numbers to pollinate alfalfa, Medicago spp., seed crops since the 1960s. Propagation of these bees from 1 yr to the next has been seriously hampered by chalkbrood, a larval disease caused by the fungus Ascosphaera aggregata Skou. In the United States, attempts to control the disease have been fairly unsuccessful, but include removing nests from the nesting boards and then disinfecting the boards with heat treatments or a fumigant. The problem is that many boards are made of polystyrene (so heat cannot be used), and very few fumigants are registered for this use. In this study, ozone was tested as a fumigant and compared with heat treatments and methyl bromide fumigation. Ozone was found to be inadequate for killing A. aggregata spores and for reducing chalkbrood levels in the field. Methyl bromide and heat treatments did greatly reduce spore viability in the boards, but did not reduce chalkbrood levels in the field. Surprisingly, larvae in new nesting boards (boards free of contamination) and chalkbrood infection levels were similar to those from nests in contaminated, used boards. Disinfecting nesting boards may be necessary for controlling chalkbrood, but the results reported here indicate that it is not sufficient in and of itself. Some other source of spores was present in the field that was greater than the effect of contamination from the boards, but the source still needs to be determined.

Combining azadirachtin and Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) to control Bemisia argentifolii (Homoptera: Aleyrodidae).

Both azadirachtin and Paecilomyces fumosoroseus (Wize) Brown & Smith have been used to control the whitefly Bemisia argentifolii Bellows & Perring, but with only moderate effectiveness. Azadirachtin is a botanical insecticide derived from the neem tree, and P. fumosoroseus is an entomopathogenic fungus. To test whether these two agents might be more effective for whitefly control if used together, different rates of each were combined in laboratory bioassays in factorial treatment. Both tank mixes and separate sprays were tested. Up to 90% nymphal mortality was obtained when both the fungus and azadirachtin were combined, a significant increase over the 70%, or less, mortality obtained when only one agent was used; however, the combined effects were less than additive. Azadirachtin had moderately inhibitory effects on growth and germination of P. firmosoroseus, which may explain this antagonism.