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Summary: Bacteria belonging to the genera Photorhabdus and Xenorhabdus participate in a trilateral symbiosis in which they enable their nematode hosts to parasitize insect larvae . The bacteria switch from persisting peacefully in a nematode''s digestive tract to a lifestyle in which pathways to produce insecticidal toxins, degrading enzymes to digest the insect for consumption, and antibiotics to ward off bacterial and fungal competitors are activated. This study addresses three questions: (1) What molecular signal triggers antibiotic production in the bacteria? (2) What small molecules are regulated by the signal? And (3), how do the bacteria recognize the signal? Differential metabolomic profiling in Photorhabdus luminescens TT01 and Xenorhabdus nematophila revealed that L-proline in the insect''s hemolymph initiates a metabolic shift. Small molecules known to be crucial for virulence and antibiosis in addition to previously unknown metabolites are dramatically upregulated by L-proline, linking the recognition of host environment to bacterial metabolic regulation. To identify the L-proline-induced signaling pathway, we deleted the proline transporters putP and proU in P. luminescens TT01. Studies of these strains support a model in which acquisition of L-proline both regulates the metabolic shift and maintains the bacterial proton motive force that ultimately regulates the downstream bacterial pathways affecting virulence and antibiotic production. [Copyright &y& Elsevier]

The sigma virus (DMelSV), which is a natural pathogen of Drosophila melanogaster, is the only Drosophila-specific rhabdovirus that has been described. We have discovered two new rhabdoviruses, D. obscura and D. affinis, which we have named DObsSV and DAffSV, respectively. We sequenced the complete genomes of DObsSV and DMelSV, and the L gene from DAffSV. Combining these data with sequences from a wide range of other rhabdoviruses, we found that the three sigma viruses form a distinct clade which is a sister group to the Dimarhabdovirus supergroup, and the high levels of divergence between these viruses suggest that they deserve to be recognized as a new genus. Furthermore, our analysis produced the most robustly supported phylogeny of the Rhabdoviridae to date, allowing us to reconstruct the major transitions that have occurred during the evolution of the family. Our data suggest that the bias towards research into plants and vertebrates means that much of the diversity of rhabdoviruses has been missed, and rhabdoviruses may be common pathogens of insects. [ABSTRACT FROM AUTHOR]

Background: The entomopathogenic fungi of the genus Beauveria are cosmopolitan with a variety of different insect hosts. The two most important species, B. bassiana and B. brongniartii, have already been used as biological control agents of pests in agriculture and as models for the study of insect host - pathogen interactions. Mitochondrial (mt) genomes, due to their properties to evolve faster than the nuclear DNA, to contain introns and mobile elements and to exhibit extended polymorphisms, are ideal tools to examine genetic diversity within fungal populations and genetically identify a species or a particular isolate. Moreover, mt intergenic region can provide valuable phylogenetic information to study the biogeography of the fungus. Results: The complete mt genomes of B. bassiana (32,263 bp) and B. brongniartii (33,920 bp) were fully analysed. Apart from a typical gene content and organization, the Beauveria mt genomes contained several introns and had longer intergenic regions when compared with their close relatives. The phylogenetic diversity of a population of 84 Beauveria strains -mainly B. bassiana (n = 76) - isolated from temperate, sub-tropical and tropical habitats was examined by analyzing the nucleotide sequences of two mt intergenic regions (atp6-rns and nad3-atp9) and the nuclear ITS1-5.8SITS2 domain. Mt sequences allowed better differentiation of strains than the ITS region. Based on mt and the concatenated dataset of all genes, the B. bassiana strains were placed into two main clades: (a) the B. bassiana s. l. and (b) the "pseudobassiana". The combination of molecular phylogeny with criteria of geographic and climatic origin showed for the first time in entomopathogenic fungi, that the B. bassiana s. l. can be subdivided into seven clusters with common climate characteristics. Conclusions: This study indicates that mt genomes and in particular intergenic regions provide molecular phylogeny tools that combined with criteria of geographic and climatic origin can subdivide the B. bassiana s.l. entomopathogenic fungi into seven clusters with common climate characteristics. [ABSTRACT FROM AUTHOR]

Although pigments produced by pathogenic microbes are generally hypothesized as essential virulence factors, the role of red pigment prodigiosin in the pathogenesis of entomopathogenic Serratia marcescens is not clear. In this study, we analyzed the pathogenicity of different pigmented S. marcescens strains and their non-pigmented mutants in silkworms. Each pigmented strain and the corresponding non-pigmented mutants showed very similar LD 50 value (statistically no difference), but caused very different symptom (color of the dead larva). Our results clearly indicated that the red pigment prodigiosin is not an essential virulence factor in entomopathogenic S. marcescens . [ABSTRACT FROM AUTHOR]

Under laboratory conditions of 27 ± 2 °C, 80 ± 5% R. H. and 12:12 L:D, we performed a characterization of 16 strains of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin, through bioessays by immersion. The strains were isolated from different hosts and their pathogenicity was evaluated on Ceratitis capitata (Wiedemann) females. Results showed that mortality of flies ranged from 9.1 to 91.9%. Average lethal time (LT50) of the most pathogenic strain was 3.8 days; while that the less pathogenic strain took 17.6 days. In a second series of experiments, the strains that registered the highest biological activity were evaluated as mentioned above. These strains showed high levels of mortality, spore production and spore recovery from the infected insects. According with these results, we discuss the potential that five strains of B. bassiana have be tested under field conditions to determinate if can be used in the control of this pest. [ABSTRACT FROM AUTHOR]

Abstract: Chagas’ disease is the most important endemic arthropod–zoonosis in Argentina with an estimated 1.6 million people infected with the causative agent Trypanosoma cruzi. Triatoma infestans is the main vector of Chagas’ disease in Argentina. A survey for parasites and pathogens of Triatominae was conducted from August 2002 to February 2005. Collections of insects were made in domiciles, peridomiciles, and in the natural habitats of the Triatominae. Insects from these collections were dissected and their organs and tissues examined for flagellates. Frass from these insects was collected and examined for detection of the entomopathogenic virus Triatoma virus (TrV) using AC-ELISA and PCR. Triatominae belonging to four species, T. infestans (n =1646), Triatoma guasayana (n =4), Triatoma platensis (n =1) and Triatoma sordida (n =5) were collected from 62 sites located in 13 provinces of Argentina. Triatoma virus and two protozoan species, Blastocrithidia triatomae and T. cruzi, the etiological agent of Chagas disease, were found infecting Triatominae. The total prevalence of TrV in 1646 T. infestans analyzed by ELISA was 9.66% (159/1646) from 7 to 13 provinces where collections were made. Triatoma virus positive triatomines were found in 17 of 62 populations when examined by AC-ELISA but in 38 of 62 populations when PCR was used for detection. The prevalence of B. triatomae in T. infestans was 0.43% (7/1646), while the prevalence of T. cruzi was 1.3% (21/1646). This is the first study on the diversity, distribution and prevalence of flagellated protozoa and TrV of Triatominae in endemic Chagas’ disease regions of Argentina. [Copyright &y& Elsevier]

The aim of this research was to identify pathogens in field populations of the genus Pityokteines from Croatia with special reference to their spatial distribution throughout the country. Pathogens occurrence was studied in 5,968 adult specimens of three silver fir bark beetle species: Pityokteines curvidens, P. spinidens and P. vorontzovi. Four pathogen species were observed in different tissues of their hosts. The pathogens Canningia spinidentis and Menzbieria sp. were found only in P. spinidens, while Chytridiopsis typographi and Gregarina sp. were also present in P. curvidens and P. vorontzovi. Multiple infections were observed in all three beetle species where gregarines occurred together with C. typographi and in combination with C. spinidentis in the specimens of P. spinidens. All pathogen species found in this research are first descriptions on host Pityokteines spp., except Canningia spinidens. Data presented in this study show possible candidates for further tests to utilize pathogens as microbial control agents against fir bark beetles in the future. [ABSTRACT FROM AUTHOR]

Abstract: Helicosporidium sp. is a pathogenic alga that replicates in the hemolymph of various invertebrate hosts. Morphogenesis of the infectious life stage, the cyst, occurs in the infected host, but to date cannot be induced in vitro. Using larvae of the heterologous host Helicoverpa zea, we examined potential factors influencing pathogenicity and in vivo cyst production of the alga and the impact of infection on host survival. Factors tested were cyst dosage administered per os (ranging from 102 to 105 cysts per larva) and host age at exposure (third, fourth, and fifth larval instar). Cyst production occurred between 7 and 13days after treatment, regardless of host age at treatment. Increasing dosage increased both percent infection and mortality, but cyst production did not track the total infection response. Increasing host age at exposure mitigated dosage effects on infection and mortality and also elevated cyst production in later-treated larvae. Only the highest dosage produced a significant decrease in the overall time to death. Moderate cyst dosages and later host ages were most effective at regenerating Helicosporidium cysts. [Copyright &y& Elsevier]

Abstract: Termites encounter a diverse array of potentially useful and harmful fungi in their subterranean habitats. These vary from symbiotic to harmful species with varying levels of virulence. How these hemiedaphic insects survive in habitats with infective fungi is not well understood. Possible mediation of olfactory signals in avoiding contact with entomopathogenic fungi has been explored by a number of workers. In the present study, we initially found that Macrotermes michaelseni detected a virulent isolate of Metarhizium anisopliae from some distance and avoided direct physical contact. We hypothesized that there may be a relationship between virulence and repellency of different isolates of M. anisopliae and Beauveria bassiana to the termite. We compared these for selected isolates of the two fungi. Positive correlations between the two parameters for both sets of isolates of the fungi were obtained. The results show an interesting co-evolutionary phenomenon in which the termite''s response to either M. anisopliae or B. bassiana is directly related to potential harm these fungi can inflict on the insect and that the virulent strains are more likely to be recognized from some distance and avoided. [Copyright &y& Elsevier]

The extracellular protease from the entomopathogenic fungus, Beauveria bassiana in the presence of Eurygaster integriceps cuticle was isolated, purified and characterized. Isolate B1 of B. bassiana that shows high virulence against E. integriceps was examined for the production of the cuticle-degrading proteases. Results showed that both subtilisin-like (Pr1) and trypsin-like (Pr2) cuticle-degrading proteases were produced and the enzyme kinetic properties showed better activity of Pr1 in comparison with Pr2. The proteases were purified using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 5.09-fold increase in specific activity and 21.86% recovery. The enzyme molecular weight was estimated to be 47 kDa and the optimal pH and temperature were 8 and 45°C, respectively. The purified protease was activated by divalent cations, Ca2 + and Mg2 +, and inhibited by NaCl, KCl and determined as a serine protease by inhibition of its activity due to using PMSF, EDTA, mercaptoethanol and SDS. Studies on the timing of the protease secretion in the presence of cuticular substrates could provide information about the role of the accumulated hydrolytic enzymes during pathogenesis to better understand these processes. [ABSTRACT FROM AUTHOR]

Abstract: The simultaneous use of two biocontrol agents for the concurrent control of three pest species was investigated. Leaf disc bioassays were conducted to establish a suitable method (surface dosing) for the dissemination of an entomopathogenic fungus (Lecanicillium longisporum or L. muscarium) by the predatory bug Orius laevigatus. Predatory bugs surface dosed with fungal conidia successfully disseminated conidia onto sweet pepper leaf discs. Most (98%) of the peach-potato aphids (Myzus persicae) that were subsequently maintained on the leaf discs became infected with the pathogen and died within 5days. However, fungal conidia disseminated by surface dosed predatory bugs did not infect and kill the western flower thrips (Frankliniella occidentalis) or the sweetpotato whitefly (Bemisia tabaci). Plant trials were performed to assess the efficacy of using predatory bugs surface dosed with L. longisporum as an effective means of controlling M. persicae and F. occidentalis populations. The results indicated that the number of aphids and thrips were significantly lower (66% and 95%, respectively) on the plants where surface dosed predatory bugs were used as a control measure compared with plants where the fungal pathogen alone was used and were statistically comparable to the numbers on plants where the predatory bug alone was used. The potential for using this dual approach is discussed in the context of improved biological control of glasshouse pests. [Copyright &y& Elsevier]

Abstract: Fiber bands impregnated with entomopathogenic fungi (=fungal bands) provide an effective method for controlling the invasive Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae). In this study we investigated the effective longevity of fungal bands for use against A. glabripennis, using several fungal strains. In the laboratory, adult beetles were immobilized on squares of fungal band impregnated with Metarhizium anisopliae (Metsch.) Sorokin isolate F 52 (ARSEF 7711) cultures to establish the median lethal concentration of 3.08×106 conidia/cm2. During the summers of 2001–2004, bands impregnated with strains of M. anisopliae (2002–2004), Beauveria bassiana (2002), and Beauveria brongniartii (2001) were attached to trunks of trees in Queens, New York City to evaluate changes in densities of viable conidia over intervals of up to 119days. The viability of conidia from bands was not associated with time in the field although total conidial density and density of viable conidia decreased with increasing time in the field. Bioassays conducted using samples of M. anisopliae F 52 bands in field sites in 2004 demonstrated that adult A. glabripennis died more quickly when exposed to higher concentrations of viable conidia. Although densities of viable conidia on bands decreased over time for all trials, they never decreased below the LC50 estimates calculated in this study for M. anisopliae F 52. Our results demonstrate the pathogenicity of M. anisopliae F 52 fungal bands against A. glabripennis adults and show that the conidia from fungal bands can retain virulence in the field for at least 112days. [Copyright &y& Elsevier]

Abstract  The control of arthropod vectors of pathogens that affect human and animal health is important for the eradication of vector-borne diseases. The ortholog of the tick-protective antigen, subolesin, was identified in Aedes albopictus and found to have conserved epitopes in ticks and mosquitoes. RNA interference with the tick and mosquito double-stranded RNA in three tick species resulted in significant gene knockdown and decreased tick weight and/or survival. Feeding Anopheles atroparvus, Aedes caspius, and Culex pipiens female mosquitoes on an A. albopictus subolesin hyperimmune serum resulted in 11 ± 5% to 29 ± 6% survival inhibition when compared to controls fed on preimmune serum. Feeding sand flies, Phlebotomus perniciosus, on antimosquito subolesin ortholog protein antibodies inhibited female survival and the number of larvae and adults obtained after hatching by 28 ± 22% and 16 ± 3%, respectively, when compared to controls. Vaccination with tick and mosquito subolesin ortholog proteins significantly reduced Ixodes scapularis tick infestation and weight in a similar way. However, vaccination with the recombinant mosquito subolesin ortholog antigen did not protect against Amblyomma americanum and Rhipicephalus sanguineus tick infestations. Collectively, these preliminary results provided the first evidence that development of vaccines may be possible for control of multiple arthropod vectors using subolesin orthologs but suggested that multiple antigens may be required to produce an effective vaccine. [ABSTRACT FROM AUTHOR]

Abstract: Reticulitermes flavipes workers were topically inoculated with ≈10,000 conidia of the entomopathogenic fungus Metarhizium anisopliae. After being kept in groups of 20 individuals for 1–9 d, histopathological examination showed that termites had an individual immune reaction. The nodule formation at the point of entrance of the fungal hyphae was identified as a cellular encapsulation and the different steps in the nodule formation are described. The relative number of hemocytes per termite increased 24h after fungal exposure and remained high in the hemolymph for at least 3 d before decreasing back to pre-exposure levels. The role of an individual immune cellular reaction in social insects is discussed. [Copyright &y& Elsevier]

Pathogenicity of the entomopathogenic fungus, Beauveria bassiana (Bals.) Vuill. to the red palm weevil, Rhynchophorus ferrugineus (Oliv.) was studied using a local strain "UAE-B2" in United Arab Emirates. For adult weevils, the calculated LC50 was 6.3x106 conidia /ml. Duration of the parasitic phase of the fungus was 9.6 days. Most of insects died between the 8th and 13th days. In the first 6 days after contamination, no symptoms were observed. In the 7th day, diseased adults appeared sluggish in their movement and the insects were unable to get up of their back. Larvae differed in their susceptibility to infection due to their age. The young instars were more susceptible than the old ones. In adult and larval stages, the fungus remains dormant inside the cadavers and starts to continue its saprophytic development when RH approaching 100%. For adults, duration of the saprophytic phase was 14.1 days and the fungus successfully developed on 81.5% of the dead insects. Complete mycosed cadaver produced 2.12 x 109 conidia. For young larval instars, duration of the saprophytic phase was 6.6 days and the fungus successfully developed on 85.7% of the dead larvae. Complete mycosed cadaver produced 4.3 x 107 conidia. Release of the fungus in a date palm plantation by two application methods caused a mortality of 12.8-21.2% and 23-47.1% in adult population in 2005 and 2006 seasons, respectively. The results lead to better understanding of the biology and pathogenicity of Beauveria bassiana as a biological control agent against the red palm weevil. [ABSTRACT FROM AUTHOR]

"Lure and kill" technology has been used for several decades in pest management and eradication of invasive species. In lure and kill, the insect pest attracted by a semiochemical lure is not "entrapped" at the source of the attractant as in mass trapping, but instead the insect is subjected to a killing agent, which eliminates affected individuals from the population after a short period. In past decades, a growing scientific literature has been published on this concept. This article provides the first review on the potential of lure and kill in long-term pest management and eradication of invasive species. We present a summary of lure and kill, either when used as a stand-alone control method or in combination with other methods. We discuss its efficacy in comparison with other control methods. Several case studies in which lure and kill has been used with the aims of long-term pest management (e.g., pink bollworm, Egyptian cotton leafworm, codling moth, apple maggot, biting flies, and bark beetles) or the eradication of invasive species (e.g., tephritid fruit flies and boll weevils) are provided. Subsequently, we identify essential knowledge required for successful lure and kill programs that include lure competitiveness with natural odor source; lure density; lure formulation and release rate; pest population density and risk of immigration; and biology and ecology of the target species. The risks associated with lure and kill, especially when used in the eradication programs, are highlighted. We comment on the cost-effectiveness of this technology and its strengths and weaknesses, and list key reasons for success and failure. We conclude that lure and kill can be highly effective in controlling small, low-density, isolated populations, and thus it has the potential to add value to long-term pest management. In the eradication of invasive species, lure and kill offers a major advantage in effectiveness by its being inverse density dependent and it provides some improvements in efficacy over related control methods. However, the inclusion of insecticides or sterilants in lure and kill formulations presents a major obstacle to public acceptance. [ABSTRACT FROM AUTHOR]

Abstract: Beauveria bassiana is a facultative entomopathogen with an extremely broad host range that is used as a commercial biopesticide for the control of insects of agricultural, veterinary and medical significance. B. bassiana produces bassianolide, a cyclooligomer depsipeptide secondary metabolite. We have cloned the bbBsls gene of B. bassiana encoding a nonribosomal peptide synthetase (NRPS). Targeted inactivation of the B. bassiana genomic copy of bbBsls abolished bassianolide production, but did not affect the biosynthesis of beauvericin, another cyclodepsipeptide produced by the strain. Comparative sequence analysis of the BbBSLS bassianolide synthetase revealed enzymatic domains for the iterative synthesis of an enzyme-bound dipeptidol monomer intermediate from d-2-hydroxyisovalerate and l-leucine. Further BbBSLS domains are predicted to catalyze the formation of the cyclic tetrameric ester bassianolide by recursive condensations of this monomer. Comparative infection assays against three selected insect hosts established bassianolide as a highly significant virulence factor of B. bassiana. [Copyright &y& Elsevier]

Insects are not only the most diverse group of animals on our planet, but also a huge reservoir for unusual microorganism which are a rich source of pharmaceutically interesting natural products. This review focuses on recent advances in the understanding of secondary metabolism of Bacillus thuringiensis, Pseudomonas entomophila, and Xenorhabdus and Photorhabdus bacteria all of which are entomopathogenic. Genome-sequencing projects revealed the capacity of these bacteria to produce several different secondary metabolites including peptides, polyketides, and hybrids of both. This richness for interesting compounds is reflected by an increasing number of compounds that have been identified from these bacteria as discussed in this review. [Copyright &y& Elsevier]

Abstract: The scale insect pathogens Hypocrella s. lat. and their Aschersonia anamorphs, collected at various locations in Thailand, were surveyed for their productivity of three hopane triterpenes, zeorin (6α,22-dihydroxyhopane), dustanin (15α,22-dihydroxyhopane), and 3β-acetoxy-15α,22-dihydroxyhopane, when cultured in a liquid medium (potato–dextrose broth) under static conditions. Among 53 strains of Aschersonia species, 48 strains (91%) produced at least one of these compounds. Hypocrella and Moelleriella species (43 strains) also frequently produced these triterpenoids; only two strains lacked all of these triterpenes. The results demonstrate that hopane triterpenes may be suitable for use as chemotaxonomic markers for Hypocrella and Moelleriella species and their Aschersonia anamorphs. [Copyright &y& Elsevier]

An ongoing debate in biological control consists of whether interference between biological agents can disrupt pest control. This study investigated the outcome of interactions between the entomopathogen Beauveria bassiana with the whitefly predator Dicyphus hesperus and the parasitoid Encarsia formosa, as well as their effect on the control of the greenhouse whitefly Trialeurodes vaporariorum on greenhouse tomato crops. Our objective was to determine whether the generalist B. bassiana would disrupt biological control by interfering with D. hesperus or E. formosa. In experimental greenhouses, whitefly, parasitoid and predator populations were established, and over 27 days, tomato plants were sprayed with three applications of the B. bassiana based product BotaniGard® (5.13×103 conidia/mm2) or water (control). Populations of greenhouse whitefly and biological control organisms were regularly monitored in control and B. bassiana-treated compartments. Overall, 10.6% of all whiteflies in treated compartments were infected, and 0.98% were both infected and parasitized. There were 31.7 and 22.3% fewer immature and adult whiteflies, respectively, on B. bassiana-treated plants relative to controls. Parasitism by E. formosa and predation by D. hesperus occurred at rates of 7.5 and 2.5%, respectively, in B. bassiana-treated compartments, and 5 and 6%, respectively in control compartments. Our study suggests that applications of B. bassiana for short-term biological control of greenhouse whiteflies are compatible with the concurrent use of E. formosa and D. hesperus on greenhouse tomato crops. [ABSTRACT FROM AUTHOR]

A complex of Fusarium spp., including F. pseudograminearum, F. culmorum, F. avenaceum, F. equiseti, and F. acuminatum, was isolated from field-collected larval cadavers of wheat stem sawfly at two locations for 2 yr. The Fusarium spp. isolates caused mortality in both diapausing larvae in a topical bioassay and in developing larvae feeding in infected stems in a greenhouse experiment. Larval mortality was >90% in both experiments at the highest dose. The pattern of correlation between integument discoloration, hyphal growth, and larval mortality showed that the Fusarium spp. isolates actively infect larvae and kill them, rather than colonizing larval tissue as secondary postmortem invaders. The versatility of Fusarium spp. as plant and insect pathogens enables colonization that results in disease in wheat plants and subsequent mortality of the wheat stem sawfly larvae developing within the same tissue. [ABSTRACT FROM AUTHOR]

• High-severity fire effects in recent fires exceeded those of the early 20th century. • Multi-aged open-canopy forests, once dominant, were not restored by recent fires. • Peak-season wildfire reduced forest, ponderosa pine, and medium to large tree cover. • Current management hinders widespread recovery of climate- and fire-adapted forests. • Uncharacteristic high-severity fire reduces options for people and ecosystems. In fire-dependent forest landscapes, frequent low- to moderate-severity fire maintained vegetation patterns that limited the severity of droughts, wildfires, and insect and pathogen activity. More than a century of fire exclusion, in combination with intensive timber management, has altered these spatial patterns and eroded resistance. Today's much denser forests and highly connected fuel loads are increasingly vulnerable to drought and fire, especially under a rapidly warming climate. Current policies favor continued fire exclusion and suppression. Consequently, most area burns at the peak of the fire season when hot, dry, and often windy conditions foster uncontrollable high-intensity wildfires in today's fire-excluded forests. As a result, high-severity fire effects are replacing the once abundant influence of low- to moderate-severity fire. For three subwatersheds in central Oregon, we assessed the vulnerability of contemporary conditions to drought and fire by quantifying their departure from the conditions that continue to demonstrate greater resistance and resilience today. We also compared contemporary conditions on recently (1984–2016) burned and unburned sites to evaluate the potential of contemporary peak-season wildfires to foster a diversity of forest and nonforest conditions in fire-excluded landscapes. In the early 20th-century, extensive high-severity fire effects were relatively common (4–20% of area) in mesic forests, but essentially absent (0–1% of area) in intermediate and dry forests. In recent wildfires, however, high-severity fire effects were evenly distributed (6–7% of area) across forest zones. After these fires, nonforest cover was more abundant and in larger patches than in the early to mid-20th century. Until at least the mid-20th century, open-canopy overstories with medium and/or large (>40 cm dbh) trees strongly dominated forest cover (>75% of area) in mesic to dry forests. By 2016, overstory cover dominated by medium and/or large trees was less than half as common, and trees > 63.5 cm dbh were essentially absent on recently burned sites. Open-canopy forest dominated by medium and large ponderosa pine trees has declined substantially since the mid-20th century, especially on recently burned sites. These changes demonstrate the magnitude and extent of departure of fire-excluded forests and landscapes from the conditions that continue to foster resistance and resilience to drought and fire today. In fire-excluded landscapes, peak-season wildfires compound the legacy of past management and further reduce options for conserving or restoring cultural, social, and ecological functions of multi-aged forests. [ABSTRACT FROM AUTHOR]

The repat gene family encodes midgut proteins overexpressed in response to pathogen infection in the lepidopteran Spodoptera exigua. Up-regulation of repat genes has been observed after challenging the larvae with both Bacillus thuringiensis toxins and after infection with the baculovirus Autographa californica multiple nucleopolyhedrovirus. In our study, PCR amplification of the genomic region and genome walking were used to obtain the genomic structure and the sequence of the 5′-upstream region of repat1 and repat2, two of the most phylogenetically distant members of the repat family. A similar gene structure between repat1 and repat2 has been found, with conserved exon-intron positions and junction sequences, suggesting a common origin for these genes. Recombinant baculoviruses carrying the firefly luciferase gene under the control of different 5′-upstream regions of the repat1 gene were constructed to elucidate the influence of these regions in gene expression. Infection of Helicoverpa zea gut-derived cells with the recombinant baculoviruses revealed the upstream regions of the repat1 gene which are involved in gene transcription and demonstrated the role of an intron located in the 5′-untranslated region in the enhancement of gene expression. [ABSTRACT FROM AUTHOR]

A cDNA library construction method was established that allows gene expression of the entomopathogenic fungus Metarhizium anisopliae var. acridum to be studied during its growth on Locusta migratoria wings. Before inoculation, locust wings were treated with an RNA fragmentation buffer to degrade host nucleic acid. Reverse transcription-PCR analysis of several constitutively expressed locust genes was used to confirm the degradation of locust nucleic acid. A normalized cDNA library of M. anisopliae var. acridum germinating and differentiating on locust wings was constructed and analyzed. Analysis of 162 unigenes out of 221 sequenced inserts from the cDNA library revealed that the cDNA library was not contaminated with locust transcripts, and contained previously characterized genes known to be expressed during infection. Further studies of the expressed sequence tags from this library will provide insights into the molecular basis of infection of this fungus. [ABSTRACT FROM AUTHOR]

A new species of Hirsutella, H. proturicola, isolated from a subterranean proturan ( Baculentulus densus; Protura, Hexapoda), is described and illustrated. Hirsutella proturicola is characterized by producing monoblastic phialides of 24–51.5 × 2.5–5 μm with a slightly roughened neck, fusiform and curved conidia of 9–18 × 2.5–4 μm that have a truncate base and a papillate projection often capped with sheath-like mucilage, and pluricellular, globose to subglobose chlamydospores of 21–48 × 21–41.5 μm. This species is morphologically and phylogenetically close to H. rostrata, an acaropathogenic species, but can be distinguished from the size of the phialides and the size and shape of the conidia. [ABSTRACT FROM AUTHOR]

Beauveria bassiana is an insect pathogen with a significant host range and specificity. A biopesticide was developed as an emulsifiable concentrate (EC) using B. bassiana strain Bv060 to be integrated into soybean production systems. The aim of this study was to assess the storage stability and in vitro compatibility of this biopesticide with recommended fungicides, insecticides, and herbicides commonly used in soybean cropping. In vitro compatibility was evaluated using response variables conidia germination (%) and viability (CFU/mL). The biopesticide was compatible with several insecticides and herbicides (except Gramoxone® after 6 contact hours) but was incompatible with all the fungicides evaluated. The EC storage stability was evaluated at 8°C, 18°C and 28°C for six months. Conidial germination was maintained at >80% for 6 months under all temperatures assessed. Germination kinetics were used to estimate the shelf-life of the biopesticide by using different mathematical models. The results obtained can be used in the implementation of this biopesticide based in B. bassiana (Bv060) in an integrated pest management program. • Biopesticide based on B. bassiana show compatibility with herbicides and insecticides. • Viability of B. bassiana biopesticide stored at 8°C, 18°C and 28°C was maintained at >80% for 6 months. • Biopesticide based on B. bassiana is promising candidates for integrated pest management strategies in soy crop. • This study can serve as a guide to commercializing the entomopathogenic fungus B. bassiana strain Bv060. [ABSTRACT FROM AUTHOR]

Background: Temperature is a critical determinant of the development of malaria parasites in mosquitoes, and hence the geographic distribution of malaria risk, but little is known about the thermal preferences of Anopheles. A number of other insects modify their thermal behaviour in response to infection. These alterations can be beneficial for the insect or for the infectious agent. Given current interest in developing fungal biopesticides for control of mosquitoes, Anopheles stephensi were examined to test whether mosquitoes showed thermally-mediated behaviour in response to infection with fungal entomopathogens and the rodent malaria, Plasmodium yoelii. Methods: Over two experiments, groups of An. stephensi were infected with one of three entomopathogenic fungi, and/or P. yoelii. Infected and uninfected mosquitoes were released on to a thermal gradient (14 - 38°C) for "snapshot" assessments of thermal preference during the first five days post-infection. Mosquito survival was monitored for eight days and, where appropriate, oocyst prevalence and intensity was assessed. Results and conclusion: Both infected and uninfected An. stephensi showed a non-random distribution on the gradient, indicating some capacity to behaviourally thermoregulate. However, chosen resting temperatures were not altered by any of the infections. There is thus no evidence that thermally-mediated behaviours play a role in determining malaria prevalence or that they will influence the performance of fungal biopesticides against adult Anopheles. [ABSTRACT FROM AUTHOR]

Abstract: Microbial biocontrol agents, which are selective and non-polluting measures for controlling insect pests, are sensitive to environmental factors, notably solar UV radiation. To overcome the constraint of photoinstability and provide adequate field performance, the photostabilizing potential of organo-clay matrices was studied. The clay mineral surfaces were modified by adsorption of chitosan, a polycationic biopolymer. The positively charged clay–chitosan complexes served for adsorption of potent photoprotective anionic dyes. A variety of clay–chitosan–dye matrices were prepared and examined as to their potential to photostabilize UV-irradiated fungal conidia of the entomopathogen Beauveria bassiana. The matrices were composed of palygorskite, montmorillonite, bentonite and kaolinite and of 6 anionic dyes: Acid Yellow 25 (AY), Chrysophenine G (Chrys.), Direct Yellow 50 (DY), Mordant Blue (MB), Fast Green (FG) and Naphthol Yellow S (NYS). The adsorption isotherms of dyes and their level of adsorption to the organo-clays were established. Clay–chitosan complexes per se were able to bestow significant measures of photoprotection, apparently due to their UV light scattering property. High and relatively consistent levels of photostabilization were observed with clay–chitosan matrices containing Chrys., AY, NYS and DY following prolonged irradiation. Depending on the clay-based matrix, MB and FG provided less persistent photostabilization levels. In contrast to the other clay–chitosan complexes, the high photoprotection bestowed by kaolinite–chitosan per se obscured to a great extent any contribution of the anionic dyes. Apparently, the photostabilization of the clay-based matrices is a combination of UV light scattering capacity of clay minerals and the UV screening effect due to the adsorbed dyes. [Copyright &y& Elsevier]

The article examines the effectiveness of entomopathogens Beauveria bassiana and Steinernema carpocapsae as biological control agents of bark-feeding moth Indarbela dea on litchi trees in two separate trials. Researchers report on the achievement of complete or nearly complete mortality of the larvae in both trials 3 weeks after application of S. carpcapsae alone and 2 weeks following the combined application of entomopathogens. Meanwhile, they found that the single applications of B. bassiana were only effective on young larvae.

Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides. [ABSTRACT FROM AUTHOR]

B. thuringiensis is a naturally occurring insect pathogen, genetically closely related to the human pathogen B. cereus. Commercial B. thuringiensis biopesticides have a long track record of safe use. Still, concerns are raised on the potential for enterotoxin production by biopesticide strains, especially since B. thuringiensis found in salad was a hypothesized cause of a foodborne outbreak in the EU. This review uses the basic steps of a risk assessment to collect available knowledge relevant to B. thuringiensis biopesticides and their impact on food safety of fresh produce. Subsequently, some directions for effective risk management strategies are provided, reflecting on various aspects that might impact decision-making on the use of B. thuringiensis as a biopesticide. Phylogenetic studies show that B. thuringiensis biopesticide strains are part of another clade compared to highly pathogenic B. cereus group strains. Although they contain enterotoxin genes, their ability to produce these toxins in the human gastrointestinal tract may be more limited. Furthermore, surveys show that it is unlikely to find elevated levels of B. thuringiensis on ready-to-eat fresh produce, higher than the established action limit of 105 CFU/g for presumptive B. cereus on foods. Finally, the B. cereus diarrheal syndrome is generally mild and self-limiting. Therefore, the use of B. thuringiensis biopesticides is expected to pose a low food safety risk. [ABSTRACT FROM AUTHOR]

• Bigleaf maple decline has been reported across the Pacific Northwest region. • We examined the role of abiotic and biotic factors on bigleaf maple decline. • No plant pathogen or insect herbivore was implicated in the decline. • Site conditions, especially hotter urban sites, are predisposing stems to decline. Acer macrophyllum is a prominent component of the western Washington landscape where it performs ecological, economic, and cultural functions. Reports of its decline and increased mortality in the Pacific Northwest were documented beginning in 2011. Symptoms of this decline include a systemic loss of vigor, loss of transpiration, and reduced photosynthesis due to leaf loss. We conducted a preliminary study of A. macrophyllum decline across western Washington in 2014–2015 and observed decline symptoms across the region, but we did not detect any specific biotic causative agents. We subsequently conducted a multi-approach study in 2017 to quantify the spatial and temporal patterns of A. macrophyllum decline in western Washington, and to examine biotic and abiotic associations with its decline. We sampled in urban and suburban areas, and in wildland forests, and collected site-specific data to test for associations with decline. We also measured elemental concentrations in foliar and soil samples to determine their association with decline. Lastly, we conducted a dendrochronological analysis to ascertain the spatial and temporal patterns of decline. We report that A. macrophyllum decline is a recent phenomenon, particularly since 2011, that was positively associated with sites closer to roads and with increased development, and with increases in summer temperatures. Site conditions, especially hotter urban sites, are predisposing A. macrophyllum to mortality. We did not detect a consistent biotic agent that could be implicated in A. macrophyllum decline. We contend that abiotic factors are either causing direct mortality to A. macrophyllum , or making then vulnerable to opportunistic biotic agents. The results of this study inform mitigating management strategies for A. macrophyllum in the forest of the Pacific Northwest. [ABSTRACT FROM AUTHOR]

Combined use of the entomopathogenic nematodes (EPNs), Heterorhabditis bacteriophora Poinar (Heterorhabditidae), Steinernema feltiae Bovien, and Steinernema kraussei Steiner (Steinernematidae) and the insect-pathogenic fungus, Metarhizium anisopliae (Metsch.) Sorokin (Clavicipitaceae) was evaluated for control of third-instar black vine weevil, Otiorhynchus sulcatus Fabricius (Coleoptera: Curculionidae). Black vine weevil larvae were exposed to various concentrations of M. anisopliae and EPNs and mortality was assessed weekly or at 3-day intervals under laboratory and greenhouse conditions. The EPNs were added simultaneously, or 1 or 2 weeks after application of M. anisopliae. Throughout the experiments, the combined application of EPNs with M. anisopliae resulted in increased efficacy against black vine weevil. When the EPNs were applied 1 or 2 weeks after application of the fungus, 100% larval mortality was obtained, even when the biocontrol agents were used at reduced rates. The interactions observed suggest that EPN and M. anisopliae work together synergistically in potted Euonymus fortunei Blondy (Celastraceae) under greenhouse conditions and may provide a powerful and economically feasible approach for black vine weevil larval control. [ABSTRACT FROM AUTHOR]

The occurrence of deuteromycetous entomopathogenic fungi was determined by examining 224 soil samples from 19 locations in three climatic zones of Mauritius. Three sites were sampled per location: one site under vegetables cultivation, one site under sugar cane plantation and one natural site each within 1 km of each other. Soil samples were baited with the waxmoth larvae Galleria mellonella L. and incubated in the dark at 15, 20, 25 or 30°C for 7, 14 and 21 days. Entomopathogenic fungi were isolated from 77 out of 224 (38.6%) soil samples. Metarhizium anisopliae was isolated from 42 (18.8%) samples, Beauveria bassiana from 24 (10.7%), Metarhizium spp. and Paecilomyces fumosoroseus from 5 (2.2%) each and Beauveria spp. from 1 (0.4%). It was observed that M. anisopliae was isolated more frequently from soils under vegetables as compared to soils under sugarcane or habitat with natural vegetation. Beauveria bassiana was isolated more frequently at the lowest incubation temperature (15°C) while M. anisopliae isolates were recovered more frequently at higher temperatures (25 and 30°C). The pathogenicity of seven isolates of M. anisopliae, five isolates of B. bassiana and two isolates of P. fumosoroseus towards the adults of Bactrocera zonata and Bactrocera cucurbitae was tested by topical application of conidial suspension of 1 × 106 conidia/ml. All the isolates tested were pathogenic to the two fruit fly species. Mortality of B. zonata varied between 12.0 and 98.0% and between 2.0 and 94.0% in B. cucurbitae at 5 days post-treatment. Our results suggest that entomopathogenic fungi present locally, could be integrated for the control of B. zonata and B. cucurbitae. [ABSTRACT FROM AUTHOR]

Dengue fever vectored by the mosquito Aedes aegypti is one of the most rapidly spreading insect-borne diseases, stimulating the search for alternatives to current control methods. Screening assays using a range of Metarhizium anisopliae and Beauveria bassiana isolates were performed against adult female Ae. aegypti. Four virulent isolates were selected for detailed study. Adult female mosquitoes were exposed to supports previously inoculated with fungal suspensions. Fungal isolates were suspended in Tween 80+8% vegetable oil. The isolates caused between 70 and 89% mortality as a result of fungal infection over the 7-day test period. Mean survival times varied between 3 and 5 days for treated insects, whilst control survival exceeded 40 days. The most promising isolate, M. anisopliae LPP133, based not only on virulence but facility for mass production, was used for lethal exposure time determinations. An exposure time of only 3.5 h was necessary to cause 50% mortality. Large cage trails were also carried out and mean survival time of insects exposed to fungus impregnated black cloths was significantly reduced. These results show that entomopathogenic fungi could be promising biological control agents for use against adult Ae. aegypti, by inoculating fungi onto surfaces on which the mosquitoes tend to rest. The subsequent mortality caused by the fungi could potentially reduce the populations of this insect thus reducing the incidence of Dengue. [ABSTRACT FROM AUTHOR]

The entomopathogen Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) survives as an endophyte in a wide range of plants, offering substantial protection against tunneling insect pests. Although current research indicates that the fungus can live as an endophyte in tissue-cultured banana plants, there is no information on the efficacy of endophytic B. bassiana against the banana weevil, Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae). Our study aimed at determining the effects of endophytic B. bassiana on oviposition rate and populations of the banana weevil, and their plant damage. Screenhouse studies were undertaken in which 8-week-old B. bassiana-inoculated tissue-cultured banana plants [cultivar Kibuzi, Musa spec. (Musaceae), genome group AAA-EA] were infested with female banana weevils. Five days after plant infestation with the banana weevil, there was no effect of B. bassiana on oviposition rate and egg hatchability. However, after 15 weeks, the presence of B. bassiana as an endophyte in banana tissues greatly reduced banana weevil populations and their damage to plants. Between 53.4 and 57.7% of the banana weevil adults died because of B. bassiana infection, resulting in a reduction of plant damage by 29.1–62.7% depending on plant part. Beauveria bassiana was re-isolated from different plant parts, but to a greater extent from the roots and rhizomes than from pseudostem bases. Artificial inoculation of B. bassiana in banana plants caused no negative impact on banana growth. Our study demonstrated for the first time that endophytic B. bassiana reduced populations of C. sordidus and the plant damage they cause, and can thus be an alternative method for effective delivery of this fungus to banana plants for the management of C. sordidus. [ABSTRACT FROM AUTHOR]

Photorhabdus are entomopathogenic members of the family Enterobacteriaceae. In addition to killing insects Photorhabdus also have a mutualistic association with nematodes from the family Heterorhabditidiae. Therefore, the bacteria have a complex life cycle that involves temporally separated pathogenic and mutualistic associations with two different invertebrate hosts. This tripartite Photorhabdus–insect–nematode association provides researchers with a unique opportunity to characterize the prokaryotic contribution to two different symbioses, i.e. pathogenicity and mutualism while also studying the role of the host in determining the outcome of association with the bacteria. In this review I will outline the life cycle of Photorhabdus and describe recent important advances in our understanding of the symbiology of Photorhabdus. Finally, the contribution made by this model to our understanding of the nature of symbiotic associations will be discussed. [ABSTRACT FROM AUTHOR]

Second instar nymphs of African migratory locust, Locusta migratoria migratorioides, and desert locust, Schistocerca gregaria, were tested for their susceptibility to the microsporidium pathogen Johenrea locustae (Lange et al. 1996, Journal of Invertebrate Pathology, 68, 28) in the laboratory. Spores of J. locustae were produced from live L.m. migratorioides, a conspecific to L. migratoria capito. Locusta m. migratorioides and S. gregaria were exposed to wheat seedlings sprayed with 20 mL of three concentrations (106, 107 and 108 spores mL-1). Both second-instar nymphs of L.m. migratorioides and S. gregaria were susceptible to J. locustae infection at the three concentrations. There was no effect of concentration of the microsporidium on mortalities of S. gregaria (92-98%) 19 days postinfection. Adjusted mortality in L.m. migratorioides at 19 days post-exposure was 24, 43 and 80% at the corresponding treatment concentrations. The effect of infection on fecundity was tested on both L.m. migratorioides and S. gregaria. Spinach leaf discs were treated with different concentrations (0, 104, 105 and 106 spores mL-1) of J. locustae and presented to female insects for 24 h. Female L.m. migratorioides surviving infection as nymphs laid significantly fewer egg pods than untreated controls at all levels of exposure. The number of eggs per female was also significantly lower in treated lots than in the controls. Higher spore concentrations also adversely affected egg hatching rate. The effect of J. locustae infection on feeding was tested on S. gregaria. There was a significant decrease in food intake among S. gregaria nymphs treated only at the high concentration (106 spores mL-1). [ABSTRACT FROM AUTHOR]

The article reports on the assessment of the suitability of Tinopal as an enhancing adjuvant in formulations of the insect pathogenic fungus Beauveria bassiana. It discusses that the resistance to azinphosmethyl, methoxyfenozide and lambda-cyhalothrin was related with reduced residual action in the field. It infers that the codling moth is one of the primary pests in the world. It is also inferred that the broad-spectrum resistance introduces serious problems for management of the codling moth in Michigan. In addition, apple plots were dealt with marked field rate doses of four insecticides.

Effects of entomopathogenic fungus Verticillium lecanii on biological characteristics and life table of Serangium japonicum, a predator of whiteflies against five different conidial concentrations (1×104, 1×105, 1×106, 1×107, and 1×108 conidia/mL) were studied under laboratory conditions. The developmental periods for all immature stages (from eggs, 1st, 2nd, 3rd, 4th instar nymph and pupae up to emergence) among the treatments were significantly different when compared to that of control, and the longest development period was observed as treated with 1×108 spore/mL. However, no significant difference on the percent survival of all immature stages was observed among the treatments and control. Also, there were no significantly different effects of V. lecanii on mean generation time, intrinsic rate, the finite rate of increase and longevity of S. japonicum among the treatments and control. [ABSTRACT FROM AUTHOR]

We tested the laboratory susceptibility of four endemic and two exotic species of North American Coccinellidae to a single rate (2.5 * 105 conidia/ml) of Beauveria bassiana derived from different sources. The endemic species Olla v-nigrum, Cycloneda munda and Hippodamia convergens were susceptible to B. bassiana Isolate B which was originally isolated from O. v-nigrum and the rate assayed was known to represent the LC50 against O. v-nigrum. However, neither the endemic C. maculata nor the exotic species Harmonia axyridis and Coccinella septempunctata were susceptible to this isolate at this rate. Additionally, all species of endemic and exotic Coccinellidae used in these assays were resistant to B. bassiana strain GHA and BbAR1 at that rate. We discuss the implications of these results in regard to the establishment of exotic species in new habitats. [ABSTRACT FROM AUTHOR]

The role of predators and parasitoids in the regulation of insect populations is widely reported in the context of both pest and non-pest insects. However, this is not the case for pathogens (entomopathogens). Indeed, most studies on insect life history refer only to predators and parasitoids when considering natural enemy guilds, even though naturally occurring entomopathogens are undoubtedly more diverse and widespread. This is certainly the case with the Coccinellidae; the natural enemies of coccinellids have been the subject of a number of review articles but pathogens receive only brief mention. In this review we attempt to address the balance and consider the interactions of natural populations of Coccinellidae with entomopathogenic fungi. Most research on entomopathogens and Coccinellidae focuses on the non-target impact of biorational insecticides against coccinellids and the impact of fungal infections upon aggregations of overwintering coccinellids; with the former overwhelmingly dominating the literature. Given the prominence of coccinellids in classical and conservation biological control, it is surprising that studies have not measured the natural impact of pathogenic fungi upon introduced species or natural populations of Coccinellidae, as has been done with numerous insect pest species. Here we review the literature on the intriguing interactions between coccinellids and fungal pathogens. We examine the literature on direct infection (sub-lethal and lethal) of coccinellids by pathogenic (Beauveria bassiana, Metarhizium anisopliae, Paecilomyces fumosoroseus) or parasitic (Hesperomyces virescens). We also consider the role of intra-guild predation (on the aphid-specific fungus Pandora neoaphidis and Beauveria bassiana) and finally indirect interactions such as coccinellids dispersing P. neoaphidis. We suggest that fungal pathogens are all too often forgotten natural enemies and future research should address the profound absence of knowledge in this field. [ABSTRACT FROM AUTHOR]

Abstract: Fungal endophytes are quite common in nature and some of them have been shown to have adverse effects against insects, nematodes, and plant pathogens. Our research program is aimed at using fungal endophytes-mediated plant defense as a novel biological control mechanism against the coffee berry borer, the most devastating pest of coffee throughout the world. A survey of fungal endophytes in coffee plants from Hawaii, Colombia, Mexico, and Puerto Rico has revealed the presence of various genera of fungal entomopathogens, including Acremonium, Beauveria, Cladosporium, Clonostachys, and Paecilomyces. Two of these, B. bassiana and Clonostachys rosea, were tested against the coffee berry borer and were shown to be pathogenic. This paper reviews the possible mode of action of entomopathogenic fungal endophytes. [Copyright &y& Elsevier]

Abstract: The recent revision of Verticillium sect. Prostrata led to the introduction of the genus Lecanicillium, which comprises the majority of the entomopathogenic strains. Sixty-five strains previously classified as Verticillium lecanii or Verticillium sp. from different geographical regions and hosts were examined and their phylogenetic relationships were determined using sequences from three mitochondrial (mt) genes [the small rRNA subunit (rns), the NADH dehydrogenase subunits 1 (nad1) and 3 (nad3)] and the ITS region. In general, single gene phylogenetic trees differentiated and placed the strains examined in well-supported (by BS analysis) groups of L. lecanii, L. longisporum, L. muscarium, and L. nodulosum, although in some cases a few uncertainties still remained. nad1 was the most informative single gene in phylogenetic analyses and was also found to contain group I introns with putative open reading frames (ORFs) encoding for GIY–YIG endonucleases. The combined use of mt gene sequences resolved taxonomic uncertainties arisen from ITS analysis and, alone or in combination with ITS sequences, helped in placing uncharacterised Verticillium lecanii and Verticillium sp. firmly into Lecanicillium species. Combined gene data from all the mt genes and all the mt genes and the ITS region together, were very similar. Furthermore, a relaxed correlation with host specificity—at least for Homoptera—was indicated for the rns and the combined mt gene sequences. Thus, the usefulness of mt gene sequences as a convenient molecular tool in phylogenetic studies of entomopathogenic fungi was demonstrated. [Copyright &y& Elsevier]

The present research is aimed at a formulation of the entomopathogenic fungi, Beauveria bassiana (Bal.) Vuillemin and Metarhizium anisopliae (Metch.) Sorokin, in two types of diatomaceous earth dusts, fossil shield and Silico-Sec, are then applied against the adults of three species of stored-grain insects: Sitophilus oryzae L., Rhyzopertha dominica Fab. and Tribolium castaneum Herbs. Effect of the treatment was assessed by comparing the mortality percentage of the adults of the three insect species exposed to the formulated fungi with that of the adults exposed to the unformulated fungi or the diatomaceous earth dusts or the undisturbed control. Results obtained from these exposures have indicated that treatment of the adults with the formulated fungi resulted in a significantly higher mean mortality percentage compared to the treatment with the unformulated fungi or the diatomaceous earth dusts or the undisturbed control. A synergistic interaction between the effect of fungal species and the diatomaceous earth dusts was shown. Viability of conidia of both fungal species in diatomaceous earth dusts was assessed by calculating the germination percentage of the conidia over time. Results indicated a small loss of mean germination percentage for formulated conidia of both fungal species versus a high loss of mean germination percentage for the unformulated conidia, thus the diatomaceous earth dusts used in the formulation of both fungi demonstrated a negligible effect on the viability of formulated conidia compared to the unformulated. [ABSTRACT FROM AUTHOR]

Bacteria can control pest insects that damage food crops, vector diseases and defoliate trees. Conventionally, isolation of these bacteria has been from soil and sporadically from dead insects. A simplified approach for isolating insecticidal bacteria from soil using the target insect as the selective agent was employed in this study. Instead of isolating single strains of bacteria from soil and testing each individual strain for insect toxicity, mixtures of bacteria present in each soil sample were tested together directly for toxicity using Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae) as a model insect. Thirty-five soil suspensions or bacterial suspensions of the 40 suspensions tested killed at least one M. sexta larva. All but one bacterial culture isolated from dead larvae and retested for toxicity, killed at least one M. sexta larva. Nineteen bacterial strains isolated from larvae killed in the first test, were identical to the bacteria fed to the retested larvae. Of the 19 strains isolated, 14 were identified by 16S rDNA sequencing as belonging to the Bacillus cereus group including three strains that formed crystals that were identified as B. thuringiensis. Of the three other spore-forming strains, two were identified as psychrotrophic B. weihenstephanensis and the third as Lysinibacillus fusiformis. Two others were identified as Enterococcus faecalis. This approach, microbial combinatorics, reduces the number of insects necessary for toxicity screening and associated time and resources compared to conventional methods that first isolate bacteria and then individually test for toxicity as well as a means of discovery of new pathogens using the insect as the selective agent. [ABSTRACT FROM AUTHOR]

Aim:  To examine whether isolates of the entomopathogenic fungus Beauveria bassiana are more closely associated to their summer hosts compared with overwintering hosts, with recently developed molecular tools based on mitochondrial regions. Methods and Results:  Primers for the traditional ITS1-5·8S-ITS2 region and two mitochondrial intergenic regions, namely, nad3- atp9 and atp6- rns, were used. All amplified products were sequenced, aligned and Neighbour-Joining (NJ), parsimony and Bayesian phylogenetic inference analyses were performed. The isolates examined were grouped with very good support into three distinct groups, two of them showed geographical correlation, but no clear association to their host. Conclusions:  The mitochondrial intergenic regions used were more informative than the nuclear ITS1-5·8S-ITS2 sequences. The sequence variability observed, that allowed the phylogenetic placement of the isolates into distinct groups, depended on the geographical origin of the isolates and can be exploited for designing group-specific and isolate-specific primers for their genetic fingerprinting. No clear associations with summer Sunn Pest populations were observed. Significance and Impact of the Study:  Studies on the genetic variability of biocontrol agents like B. bassiana are indispensable for the development of molecular tools for their future monitoring. [ABSTRACT FROM AUTHOR]

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The ability to produce cuticle-degrading proteases to facilitate host penetration does not distinguish per se entomopathogenic fungi from saprophytes. However, adapted pathogens may produce host-protein specific enzymes in response to cues. This possibility prompted an investigation of the regulation of isoforms of the subtilisin Pr1-like proteases from five aphid-pathogenic isolates of Lecanicillium spp. Significant differences were found in substrate specificity and regulation of Pr1-like proteases between isoforms of the same isolate and between different isolates. For example, the pI 8.6 isoform from KV71 was considerably more active against aphid than locust cuticle and was induced specifically by N-acetylglucosamine (NAG). Isoform pI 9.1 from the same isolate was only produced on insect cuticle while most other isoforms were more prominent on chitin containing substrates but not induced by NAG. The ability to regulate isoforms independently may allow production at critical points in host penetration. Appearance of proteases (not subtilisins) with pI 4.2 and 4.4 only on aphid cuticle was a possible link with host specificity of KV71. The absence of C or N metabolite repression in subtilisins from KV42 is unusual for pathogen proteases and may help to account for differences in virulence strategy between aphid-pathogenic isolates of Lecanicillium longisporum (unpublished data). [ABSTRACT FROM AUTHOR]

Photorhabdus luminescens is a gram-negative insect pathogen that enters the hemocoel of infected hosts and produces a number of secreted proteins that promote colonization and subsequent death of the insect. In initial studies to determine the exact role of individual secreted proteins in insect pathogenesis, concentrated culture supernatants from various P. luminescens strains were injected into the tobacco hornworm Manduca sexta. Culture supernatants from P. luminescens TTO1, the genome-sequenced strain, stimulated a rapid melanization reaction in M. sexta. Comparison of the profiles of secreted proteins from the various Photorhabdus strains revealed a single protein of approximately 37 kDa that was significantly overrepresented in the TT01 culture supernatant. This protein was purified by DEAE ion-exchange and Superdex 75 gel filtration chromatography and identified by matrix-assisted laser desorption ionization-time of flight analysis as the product of the TTO1 gene plu1382 (NCBI accession number NCõ005126); we refer to it here as PrtS. PrtS is a member of the M4 metalloprotease family. Injection of PrtS into larvae of M. sexta and Galleria mellonella and into adult Drosophila melanogaster and D. melanogaster melanization mutants (Bc) confirmed that the purified protein induced the melanization reaction. The prtS gene was transcribed by P. luminescens injected into M. sexta before death of the insect, suggesting that the protein was produced during infection. The exact function of this protease during infection is not clear. The bacteria might survive inside the insect despite the melanization process, or it might be that the bacterium is specifically activating melanization in an attempt to circumvent this innate immune response. [ABSTRACT FROM AUTHOR]