Role of the exoprotease InA in the pathogenicity of Bacillus thuringiensis in pupae of Hyalophora cecropia

Two geographical biotypes of Nomuraea rileyi (from Ecuador and the United States) were topically bioassayed against seven lepidopteran species, i.e., Anticarsia gemmatalis, Heliothis zea, Heliothis virescens, Heliothis subflexa, Pseudoplusia includens, Spodoptera exigua, and Trichoplusia ni. There was an average difference of 1.7-fold in mortality in how cultures of the same insect species from different sources responded to topical applications of either biotype of N. rileyi. Regression equations and LC₅₀ values were obtained for each insect species and fungal biotype combination. Larvae of S. exigua were equally susceptible to both biotypes of N. rileyi. Although larvae of A. gemmatalis were moderately susceptible to the Ecuadoran biotype, they were relatively nonsusceptible to the Mississippian biotype. Species of Heliothis (H. zea, H. virescens, and H. subflexa) were about equally susceptible to the Mississippian biotype. Larvae of H. subflexa and H. virescens, however, were significantly less susceptible than H. zea to the Ecuadoran biotype. When the integumental barrier was breached via intrahemocoelic injections, larvae of H. virescens were as susceptible as H. zea larvae to blastospores of either biotype of N. rileyi.     

Susceptibility of the cabbage looper, Trichoplusia ni, and the velvetbean caterpillar, Anticarsia gemmatalis, to several isolates of the entomopathogenic fungus Nomuraea rileyi

Cabbage looper larvae, Trichoplusia ni, were equally susceptible to isolates of the entomogenous fungus Nomuraea rileyi from Missouri, Mississippi, and Brazil; an isolate from Florida was 7–17 times less active than the other isolates. Velvetbean caterpillars, Anticarsia gemmatalis, were either only slightly susceptible or not susceptible to the isolates of N. rileyi from Missouri, Florida, and Mississippi. In contrast, larvae of A. gemmatalis cultured from three locations (Missouri, Florida, and Brazil) all were equally susceptible to a Brazilian isolate of N. rileyi.     

The infectivity of a nuclear polyhedrosis virus of the velvetbean caterpillar for eight noctuid hosts

Eight species of noctuid larvae were tested for susceptibility to a nuclear polyhedrosis virus of the velvetbean caterpillar, Anticarsia gemmatalis. Velvetbean caterpillar larvae were highly susceptible to crude preparations of polyhedral inclusion bodies (PIBs; LD₅₀ = 4.7 PIBs/larva), but preparations of purified polyhedra were much less effective against these larvae (LD₅₀ = 319.7 PIBs/larva). Of seven other noctuid species tested, only Heliothis virescens was as susceptible to the virus as A. gemmatalis. High dosages were required to kill Heliothis zea, Trichoplusia ni, Pseudoplusia includens, and Spodoptera ornithogalli. Plathypena scabra and Spodoptera frugiperda were not susceptible.     

Differential susceptibility of parasitized and nonparasitized larvae of Trichoplusia ni to a nuclear polyhedrosis virus

Nonparasitized second-instar larvae of Trichoplusia ni were twice as susceptible (at the LD₅₀ level) to the singly enveloped T. ni nuclear polyhedrosis virus as those parasitized by Hyposoter exiguae (Hymenoptera: Ichneumonidae). The LD₅₀ values for nonparasitized and parasitized larvae were 1.58 × 10³ and 3.16 × 10³ polyhedra/ml of diet, respectively. The LD₉₅ value for parasitized larvae was approximateely 5 times higher than that for nonparasitized larvae. The slopes (b values) were 1.2 for parasitized larvae and 1.7 for nonparasitized larvae. The LT₅₀ values for parasitized larvae also were significantly longer than those for nonparasitized larvae. No significant difference was found between the food consumption of parasitized and nonparasitized T. ni larvae.     

Laboratory studies of the entomopathogenic fungus Nomuraea rileyi: Soil-borne contamination of soybean seedlings and dispersal of diseased larvae of Trichoplusia ni

Germinating soybean seedlings were contaminated by soil-borne conidia of the entomogenous fungus Nomuraea rileyi. Although N. rileyi was detected on the unifoliate and trifoliate leaflets, most of the inoculum was found on the cotyledons. Diseased larvae of Trichoplusia ni released on the first trifoliate leaves of soybean plants dispersed to all trifoliates and died. In this way, inoculum is produced that infects other larvae feeding on these leaves.     

Effect of temperature on germination,growth, and infectivity of the mosquito pathogen Tolypocladium cylindrosporum (deuteromycotina; hyphomycetes)

The mosquito pathogen Tolypocladium cylindrosporum was examined with regard to its response to temperature. Similar temperature ranges were found for growth, germination, and infectivity of blastospores and conidia. Germination occurred at 8° and 33°C but not at 6° and 35°C. Optimal germination and growth was noted between 24° and 27°C for both spore types. Infectivity of blastospores and conidia at different temperatures was examined by exposing L₂Aedes sierrensis larvae to concentrations of 5 × 10⁵ blastospores/ml or 5 × 10⁶ conidia/ml. Larvae were incubated at 12°, 15°, 25°, and 30°C. Infection occurred at all temperatures tested with LT₅₀ values ranging from 22.7 days (12°C) to 5.6 (25°C) days for conidia and 4.7 days (12°C) to 0.6 day (25°C) for blastospores. These results confirmed earlier findings that blastospores infected and killed host larvae more rapidly than conidia and suggested that this difference is largely due to the more rapid germination rate of blastospores. These experiments demonstrated that T. cylindrosporum can be active against mosquito larvae over a broad range of temperatures encompassing both the cold-water habitat of certain temperate mosquito species as well as the habitat of tropical vector species.     

Effects of successivein vitro andIn vivo passages on the virulence of the entomopathogenic fungus,Nomuraea rileyi

There was no significant decrease or increase in the activity of conidia ofNomuraea rileyi after 12_{in vivo} serial passages in larvae ofTrichoplusia ni (Hübner) or after 12in vitro serial passages on a semi-synthetic medium. The LC-50 at the start of the serial passage was 16.8±4.0 conidia/mm²; after 12 serial passages the LC-50 for thein vitro- andin vivo-produced conidia was 16.2±2.5 conidia/mm² and 12.0±1.9 conidia/mm², respectively.

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Résumé Il n'y a pas d'augmentation ou de réduction significative dans l'activité des conidies deNomuraea rileyi après 12 passages en sériein vivo chez les larves deTrichoplusia ni (Hübner) ou après 12 passages en sériein vitro sur un milieu semi-synthétique. La concentration léthale 50 au début de ces passages était de 16,8±4,0 conidies/mm², après 12 passages la CL 50 des conidies produitesin vitro etin vivo fut respectivement de 16,2±2,5 conidies/mm² et de 12,0±1,9 conidies/mm².

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Mention of a proprietary product in this paper does not constitute a recommendation for use by the USDA.     

Insecticidal and feeding deterrent activities of essential oils in the cabbage looper,Trichoplusia ni (Lepidoptera: Noctuidae)

Ten essential oils were tested against the cabbage looper, Trichoplusia ni larvae for contact, residual and fumigant toxicities and feeding deterrent effects. Against third instar T. ni, Syzygium aromaticum (LD₅₀ = 47.8 μg/larva), Thymus vulgaris (LD₅₀ = 52.0 μg/larva) (the two positive controls) and Cinnamomum glanduliferum (LD₅₀ = 76.0 μg/larva) were the most toxic via topical application. Litsea pungens (LD₅₀ = 87.1 μg/larva), Ilex purpurea (LD₅₀ = 94.0 μg/larva), Cinnamomum cassia (LD₅₀ = 101.5 μg/larva) and Litsea cubeba (LD₅₀ = 112.4 μg/larva) oils were equitoxic. Thymus vulgaris (LC₅₀ = 4.8 mg/ml) and S. aromaticum (LC₅₀ = 6.0 mg/ml) oils were the most toxic in residual bioassays. Cymbopogon citratus (LC₅₀ = 7.7 mg/ml) and C. cassia (LC₅₀ = 8.5 mg/ml) oils were equitoxic followed by Cymbopogon nardus (LC₅₀ = 10.1 mg/ml) in this bioassay. The remaining five oils showed little or no residual effects. In a fumigation bioassay, L. cubeba (LC₅₀ = 16.5 μl/l) and I. purpurea (LC₅₀ = 22.2 μl/l) oils were the most toxic. Cinnamomum glanduliferum (LC₅₀ = 29.7 μl/l) and Sabina vulgaris (LC₅₀ = 31.2 μl/l) oils were equitoxic. Interestingly, S. aromaticum did not exhibit any fumigant toxicity. Cymbopogon citratus, C. nardus and C. cassia strongly deterred feeding by third instar T. ni (DC₅₀s = 26.9, 33.8 and 39.6 μg/cm², respectively) in a leaf disc choice bioassay. The different responses of T. ni larvae to the oils in different bioassays suggest that these essential oils exhibit different modes of action. Based on their comparable efficacy with essential oils already used as active ingredients in many commercial insecticides (i.e. clove oil and thyme oil), some of these essential oils may have potential as botanical insecticides against T. ni.     

Ultrastructural studies on the fungus,Nomuraea rileyi,infecting the velvetbean caterpillar,Anticarsia gemmatalis

Combined scanning and transmission electron microscopy was used to study the fine structure of the developmental stages of Nomuraea rileyi infecting host larvae of Anticarsia gemmatalis. Larvae were dusted with large numbers of fungal conidia, which germinated and penetrated the cuticle within 6 hr post-treatment. Within 24 hr, penetration hyphae had reached the cuticular epidermis and, via a budding process, initiated the hyphal body stage in the hemocoel. The hyphal bodies, suspended in hemolymph, multiplied and spread throughout the host larvae. By 6–7 days post-treatment, the majority of larvae were mummified. Within 12 hr postmortem numerous conidiophores emerged producing a confluent mycelial mat over the entire cuticular surface. Numerous hydrophobic conidia were formed on phialides present on the aerial conidiophores.     

Soybean leaf consumption by Nomuraea rileyi (Fungi: Deuteromycotina)-infected Plathypena scabra (Lepidoptera: Noctuidae) larvae

To determine the impact of Nomuraea rileyi on consumption by the green cloverworm, Plathypena scabra, larvae were reared from eggs obtained from field-collected moths, inoculated with conidia, and placed individually in separate plastic vials with a piece of surface-sterilized soybean leaflet. No significant differences in consumption rates were found between N. rileyi-inoculated and control larvae until after 144 hr post-treatment. After this period, consumption by larvae inoculated as first, second, third, or fourth instars was significantly less than that of control larvae because of mortality. First or second instars inoculated with N. rileyi conidia had significantly longer subsequent stadia than did control larvae. The average LT₅₀ of all inoculated instar groups was 6.5 days, and there was little evidence of significantly different N. rileyi susceptibility among instar groups. In general during the N. rileyi infection period leading to their deaths, inoculated P. scabra larvae had consumption patterns that were very similar to those of healthy larvae.     

Activity of oil-formulated conidia of the fungal entomopathogens Nomuraea rileyi and Isaria tenuipes against lepidopterous larvae

The fungi Nomuraea rileyi and Isaria tenuipes (=Paecilomyces tenuipes) are ecologically obligate, widespread pathogens of lepidopterans. Bioassays were carried out to evaluate the activity of oil-suspended conidia of N. rileyi and I. tenuipes against larvae of Spodoptera frugiperda, Spodoptera exigua, Helicoverpa zea, and Heliothis virescens. The tests consisted of two bioassay sets. In the first set, conidia of N. rileyi and I. tenuipes were suspended in water + Tween 80, and in vegetable (canola, soybean) and mineral (proprietary mixture of alkanes and cyclic paraffins) oils, and tested against S. frugiperda. Both fungi were highly compatible with oils and caused mortalities near 100% in all oil treatments; the lowest LT₅₀ values were 4.7 days for N. rileyi in mineral oil and 6.0 days for I. tenuipes in soybean oil. The second set included additional fungal strains and oil formulations (mineral, canola, sunflower, olive and peanut oils) tested against larvae of S. exigua, S. frugiperda, H. zea and H. virescens. The highest activity was that of N. rileyi in mineral oil against Spodoptera spp., with LT₅₀ values of 2.5 days (strain ARSEF 135) and 3 days (strain ARSEF 762) respectively. For two different isolates of I. tenuipes the lowest LT₅₀ values (5.1-5.6 days respectively) were obtained with mineral oil formulations against Spodoptera spp. and H. zea respectively. Additionally, we tested both fungi against prepupae of all four lepidopteran species. Mortalities with I. tenuipes against S. exigua ranged from 90% to 100% (strains ARSEF 2488 and 4096); N. rileyi caused 95% mortality on S. frugiperda. The activity of formulations depended on host species and oil used; Spodoptera spp. was more susceptible to these fungi than Heliothis and Helicoverpa. The results indicate that a comprehensive evaluation of these entomopathogens in agriculture using oil application technologies is advisable, particularly, in organic and sustainable settings.     

Molecular epidemiology ofCladosporium carrionii

The effects of cuticle from larvae ofTrichoplusia ni andHelicoverpa (=Heliothis)zea on expression of proteases and chitinases by germinating conidia ofNomuraea rileyi in submerged cultures were studied. Increasing the concentration ofT. ni orH. zea cuticle resulted in a 13- and 15-fold increase in protease activity, respectively. Endochitinase and N-acetylglucosaminidase activity on theT. ni andH. zea substrates increased as cuticular concentrations increased to 2.5%, then stabilized or decreased thereafter. The simultaneous expression of both proteases and chitinases suggests that they are controlled by a multiple-regulatory system.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by US Department of Agriculture.     

Pathogenesis of infection by the hyphomycetous fungus,Tolypocladium cylindrosporum inAedes sierrensis andCulex tarsalis [Dip.: Culicidae]

The mode of infection and cycle of development ofTolypocladium cylindrosporum Gams was examined inAedes sierrensis andCulex tarsalis. Larvae were found to be infected through the external cuticle, the pharynx and the midgut. Blastospores and conidia were both infective although for equal numerical concentrations blastospores proved more virulent causing high mortality within the first 48 h after inoculation (80 % for L2 larvae exposed to 5×10⁵ spores/ml), while conidia generally took 7–10 days to produce the same results. Sporulation did not occur on submerged cadavers. Conidia were produced only on floating cadavers in contact with air. Conidial production on floating 4th instar larvae was found to average 1.8×10⁷ conidia/larva. Invasion of the haemocoele and fairly extensive growth of the fungus almost invariably occurred before larvae were killed. This was particularly true forAedes sierrensis larvae. Details are presented of growth within the host and post-mortem penetration of the fungus out of the cadaver. AdultA. sierrensis sprayed with a conidial suspension proved susceptible to infection with 100 % mortality being recorded at 10 days. Infections originated in the thorax, suggesting, the integument or possibly the thoracic spiracles to be the most probable site of infection.     

Modified Adamek's medium renders high yields of Metarhizium robertsii blastospores that are desiccation tolerant and infective to cattle-tick larvae

Blastospores are yeast-like cells produced by entomopathogenic fungi that are infective to arthropods. The economical feasible production of blastospores of the insect killing fungus Metarhizium spp. must be optimized to increase yields. Moreover, stabilization process is imperative for blastospore formulation as a final product. In this sense, our goal was to increase blastospore production of two Metarhizium isolates (ESALQ1426 and ESALQ4676) in submerged liquid cultures. A modified Adamek's medium was supplemented with increased glucose concentrations and the fermentation time was accelerated by using a blastospore pre-culture as inoculum. Virulence of air-dried stable blastospores was compared with conidia toward larvae of the cattle tick, Rhipicephalus microplus. Our results revealed that blastospore production of Metarhizium is isolate- and species-dependent. Glucose-enriched cultures (140 g glucose/L) inoculated with pre-cultures improved yields with optimal growth conditions attained for Metarhizium robertsii ESALQ1426 that rendered as high as 5.9 × 10⁸ blastospores/mL within 2 d. Resultant air-dried blastospores of ESALQ1426 were firstly proved to infect and quickly kill cattle tick larvae with comparable efficiency to conidia. Altogether, we argue that both osmotic pressure, induced by high glucose titers, and isolate selection are critical to produce high yields of blastospores that hold promise to control cattle-tick larvae.     

Culture medium improvement for Isaria fumosorosea submerged conidia production

Submerged conidia and blastospores of the entomopathogenic fungus Isaria fumosorosea are produced in several liquid culture media. However, yields and the ecological fitness of these propagules vary according to culture media composition. In most culture media, hyphae, blastospores and submerged conidia are white but we found that in some media they develop a brown pigmentation. A dark pigment was extracted from brown-pigmented propagules and analyzed by IR spectroscopy. Adsorption bands coincided to those characteristics of melanins.Hadamard's matrices were employed in order to increase submerged conidia yields and brown pigmentation of fungal propagules. Media containing 20–30 mg/l of FeSO₄·7H₂O and 6–12 mg/l of CuSO₄·5H₂O allowed reaching the highest pigmentation (9 in a hedonic scale). A maximal concentration of submerged conidia of 1.0 (±1.2) × 10¹² cell/l was achieved after 120 h of liquid culture in a improved culture medium, containing 25 ml/l of Polyethylene glycol (MW 200), substance which enhanced submerged conidia production, reducing free mycelia or mycelial pellets formation. In the improved medium, it was estimated that more than 60% of produced biomass corresponded to submerged conidia and blastospores, while in other media, mycelia were the main product (80–97%).     

A synchronous peroral technique for the bioassay of insect viruses

A relatively fast and simple peroral technique for the bioassay of insect viruses is described in which newly hatched larvae ingest a uniform volume of virus suspension. Three isolates of the Autographa californica nuclear polyhedrosis virus (NPV) and one isolate of the Heliothis zea NPV were used to test the procedure with Trichoplusia ni and H. zea larvae, respectively. Within-assay and between-assay variation was very low with coefficients of variation averaging 0.012 ± 0.006 and 0.20 ± 0.04 for time-mortality and dose-mortality tests, respectively. The synchronous uptake of virus removed the acquisition-time component of the LT₅₀ values while the constant volume improved the accuracy of LD₅₀ values. The procedure was shown to be suitable for a wide variety of lepidopterous species, including Spodoptera frugiperda, S. eridania, Estigmene acrea, Plutella xylostella, Choristoneura fumiferana, Ostrinia nubilalis, Plodia interpunctella, and Pieris rapae.     

Effect of Host Plant on the Infectivity of Sl MNPV to Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) Larvae

The susceptibility of Spodoptera litura to SlMNPV infection was markedly affected by phyto-chemicals ingested during the acquisition of viral inoculum on foliage of tomato and cauliflower. The LD₅₀ values computed for second, third and fourth instar larvae assayed on tomato leaves were 254, 819 and 23395 PIBs/larva, respectively whereas, it was 326, 1719 and 43843 PIBs/larva for respective instars when assayed on cauliflower leaves. Thus LD₅₀ values for second, third and fourth instar larvae were 1.28-, 2.09- and 1.87- fold lower, respectively in tomato leaves. Similarly, LT₅₀ values for second, third and fourth instar larvae assayed on tomato leaves were 7.1 and 7.5 days, respectively at inoculum dose of 2.7×10⁴ PIBs/larva whereas, it was 7.7 and 8.0 days for respective instars when assayed on cauliflower leaves at same inoculum. This result also showed that the S. litura were more susceptible on tomato leaves in comparison to cauliflower leaves as the time required for mortality was lower in tomato leaves. The possible biochemical bases for differential level of mortality of S. litura larvae on tomato and cauliflower crops needs to be investigated.     

Effect of the interaction between Anticarsia gemmatalis multiple nucleopolyhedrovirus and Epinotia aporema granulovirus,on A. gemmatalis (Lepidoptera: Noctuidae) larvae

The bean shoot borer Epinotia aporema Wals. (Lepidoptera: Tortricidae) and the velvet bean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) are key pests of soybean and other legume crops in South America. They are often found simultaneously in certain regions. A. gemmatalis nucleopolyhedrovirus (AgMNPV) is widely used to control A. gemmatalis. More recently, E. aporema granulovirus (EpapGV) has been characterized and evaluated as a bioinsecticide for E. aporema. In order to increase its potential use and to design optimized strategies for the management of lepidopteran pests, we evaluated the interaction between EpapGV and AgMNPV on third instar A. gemmatalis larvae. Larvae fed with 50 AgMNPV OBs/larva showed an increase in the mortality rates (from 42% to 81%) and a decrease in the median survival time (from 7.7 days to 5.7 days) when these OBs were mixed with 6000 EpapGV OBs/larva. When 300 AgMNPV OBs/larva were used alone or in combination with EpapGV OBs no changes in biological parameters were observed. No mortality was detected in A. gemmatalis larvae treated with EpapGV alone. In larvae fed with the viral mixtures, only AgMNPV DNA was detected by PCR. A. gemmatalis peritrophic membranes (PMs) examined by SDS–PAGE and scanning electron microscopy showed signs of damage. Notably, we found the presence of spheroidal bodies associated with damaged areas in the PMs of larvae fed with EpapGV but not in those that were given AgMNPV alone. These results show that EpapGV increases the viral potency of AgMNPV, and thus the insecticidal efficiency, suggesting that the use of formulations including both viruses might be a valuable tool for pest management.     

Pathogenicity of entomopathogenic hyphomycetes, Paecilomyces fumoso-roseus and Nomuraea rileyi, to eggs of noctuids, Mamestra brassicae and Spodoptera littoralis

Bioassays were conducted to determine the susceptibility of egg masses of Mamestra brassicae and Spodoptera littoralis to different spore doses of Paecilomyces fumoso-roseus and Nomuraea rileyi at 20° and 25°C. P. fumoso-roseus was highly virulent against eggs, whereas N. rileyi provoked only a deferred mortality of larvae hatched from treated eggs. Nevertheless, larval mortality of S. littoralis caused by N. rileyi at 25°C was more effective after first-instar larval contamination than after egg mass treatment. The duration of the egg stage could explain differences of susceptibility between the two noctuids at 25°C. Scanning electron microscopical observations suggested two ways of contamination of newly hatched larvae. First, fungal germinations on the chorion surface suggested that newly hatched larvae might be infected by penetration of the egg integument before hatching. Second, conidia on the egg cuticle could be an entomopathogenic inoculum for newly emerging larvae which fed upon chorions. Results showed that pathogenicity of Hyphomycetes to noctuid eggs might be a promising area of investigation for biological control.     

Trichoplusia ni attacin A,a differentially displayed insect gene coding for an antibacterial protein

The mRNA differential display method was used to isolate antibacterial defense genes from Trichoplusia ni. The mRNA population in last-instar T. ni larvae injected with bacteria was compared to that of untreated larvae. Using a PCR amplified probe corresponding to an induced mRNA, we were able to clone an attacin homolog from a λ cDNA library from vaccinated larvae. The corresponding protein showed 63% identity to Hyalophora cecropia acidic attacin. The induction kinetics of T. ni attacin A gave optimal mRNA levels at 20 h post-infection. Genomic analysis showed this to be a single-copy gene with two introns.