Efficacy of focal applications of a mycoinsecticide to control Aedes aegypti in Central Brazil.

Entomopathogenic fungi can achieve important innovative outcomes for integrated mosquito control especially of Aedes aegypti, the key vector of arboviruses to humans in the tropics and subtropics. This study sought to design and to develop a simple dissemination device to attract and to infect gravid A. aegypti adults with a granular formulation of the ascomycete Metarhizium humberi IP 46, and to validate this device in the laboratory as well as in semi-field and field conditions. Hydrogel (polyacrylamide potassium polyacrylate) was confirmed to be a suitable substitute for water used in the device that attracted gravid females under field conditions. Females laid eggs on black polyethylene terephthalate carpet fixed in the device that also proved to be a suitable substrate for a granular formulation of fungal microsclerotia and/or conidia. The plastic device (29.5 cm high) was divided into a lower closed compartment with a water reservoir and an upper, laterally open but covered compartment with continuously hydrated gel and the fungal formulation attached to the carpet. The uppermost compartment permitted free circulation of mosquito adults. The device attracted both male and female A. aegypti. The fungal formulations of IP 46 propagules tested in the device were effective against adults in laboratory, semi-field, and field settings. Findings in the laboratory, semi-field, and especially in field conditions strengthen the value and utility of this innovative device for focal applications of a mycoinsecticide against this important mosquito vector.Key points• Low-cost and simple disseminating device for focal control of Aedes aegypti.• Granulized Metarhizium humberi IP 46 and hydrogel yield extended control.• Findings in field tests strengthen benefit of the device for focal application.

Optimization of granular formulations of Metarhizium humberi microsclerotia with humectants.

Granular microsclerotial formulations of entomopathogenic fungi deserve attention because of their post-application, in situ production of new conidia that enhance and prolong mycoinsecticidal efficacy against a target pest insect. Because high ambient moisture is a crucial condition to induce fungal development and conidiogenesis on granules, we tested the impacts of the additions of three humectants-glycerin, propylene glycol, and polyethylene glycol 400-on water absorption by pellets incorporating microsclerotia of Metarhizium humberi IP 46 with microcrystalline cellulose or vermiculite carriers, and on the production of infective conidia of IP 46 microsclerotia in ambient humidities suboptimal for routine conidiogenesis. Glycerin facilitated greater and faster absorption of water than the other humectants. Microcrystalline cellulose absorbed low quantities of water without any added humectant whereas vermiculite did not. IP 46 did not grow or sporulate on pellets prepared with or without glycerin at 86% relative humidity (RH) or on control pellets without glycerin at 91% RH; conidial production on pellets prepared with vermiculite or microcrystalline cellulose and 10% glycerin reached 1.1 × 105 conidia/mg and 1 × 105 conidia/mg, respectively, after 20 days of exposure at 91% RH. Hence, these results strongly support glycerin as a suitable humectant for granular microsclerotial formulations of this fungus.

Efficacy of collars with allomones on dogs to control Rhipicephalus sanguineus sensu lato infestations under field conditions.

Benzaldehyde and 2-hexanone are allomones produced by beagle dogs that reduce infestation by Rhipicephalus sanguineus sensu lato on these animals and on susceptible dogs which artificially release these repellents. These observations were obtained in previous laboratory tests or artificial infestations of susceptible dogs under controlled conditions. Here we evaluated the efficacy of collars delivering these repellents for suppressing the loads of R. sanguineus s.l. on naturally infested mixed-breed dogs under field conditions. Thirty dogs naturally infested with R. sanguineus s.l. were separated into two groups with 15 dogs each. The dogs from the treatment group received the collars with slow-release formulations of the allomones, and the dogs from the control group received collars without such compounds. Collar effectiveness tests were carried out over 30 days. All ticks found were removed from each dog, identified, and counted every collection day. The density of the different life stages of R. sanguineus s.l. varied greatly between groups and across evaluation times. Adult ticks were the stage most abundant and prevalent on infested dogs. The counts for larvae, nymphs and adults were similar between the control and the treatment groups within each tick collection day. Conversely, when we considered the total number of ticks over 30 days and gathered all life stages collected in the same animal, the dogs from the control group had significantly higher loads (35.3 ± 4.27) of ticks than the dogs from the treatment group (21.8 ± 2.96) (P < 0.01). In addition, the efficacy of treatment with repellent collars was high for adult ticks (30.8%) but was extremely low for reducing larval or nymphal infestation (0 or 2.6%, respectively). The present study demonstrates, for the first time, the efficacy of these volatile compounds on naturally infested dogs under field conditions. The development of a technologically enhanced device for slow release of the allomones tested here can be an advantageous alternative for reducing R. sanguineus s.l. infestation on dogs in residential environments.

Tolerance to Abiotic Factors of Microsclerotia and Mycelial Pellets From Metarhizium robertsii, and Molecular and Ultrastructural Changes During Microsclerotial Differentiation.

Metarhizium species fungi are able to produce resistant structures termed microsclerotia, formed by compact and melanized threads of hyphae. These propagules are tolerant to desiccation and produce infective conidia; thus, they are promising candidates to use in biological control programs. In this study, we investigated the tolerance to both ultraviolet B (UV-B) radiation and heat of microsclerotia of Metarhizium robertsii strain ARSEF 2575. We also adapted the liquid medium and culture conditions to obtain mycelial pellets from the same isolate in order to compare these characteristics between both types of propagules. We followed the peroxisome biogenesis and studied the oxidative stress during differentiation from conidia to microsclerotia by transmission electron microscopy after staining with a peroxidase activity marker and by the expression pattern of genes potentially involved in these processes. We found that despite their twice smaller size, microsclerotia exhibited higher dry biomass, yield, and conidial productivity than mycelial pellets, both with and without UV-B and heat stresses. From the 16 genes measured, we found an induction after 96-h differentiation in the oxidative stress marker genes MrcatA, MrcatP, and Mrgpx; the peroxisome biogenesis factors Mrpex5 and Mrpex14/17; and the photoprotection genes Mrlac1 and Mrlac2; and Mrlac3. We concluded that an oxidative stress scenario is induced during microsclerotia differentiation in M. robertsii and confirmed that because of its tolerance to desiccation, heat, and UV-B, this fungal structure could be an excellent candidate for use in biological control of pests under tropical and subtropical climates where heat and UV radiation are detrimental to entomopathogenic fungi survival and persistence.

Carnauba wax enhances the insecticidal activity of entomopathogenic fungi against the blowfly Lucilia sericata (Diptera: Calliphoridae).

Blowfly, Lucilia sericata (Diptera: Calliphoridae), is a problematic synanthropic insect pest, a vector of microbial pathogens, and the causal agent of secondary myiasis. Fungal biopesticides are considered eco-friendly tools, alternative to synthetic pesticides, for the control of arthropod pests; however, to date, little is known about their bioactivity against blowflies. In this study, we assessed the insecticidal activity of three well-known entomopathogenic fungi, Beauveria bassiana, Beauveria pseudobassiana and Akanthomyces muscarius against L. sericata. In addition, we tested powdered carnauba wax as an electrically charged dust carrier in an attempt to enhance the virulence of fungal spores. Pathogenicity tests on adult flies, by adult immersion in conidial suspension (108 conidia mL-1), showed that the median lethal time (LT50) was 5.3, 5.9, and 6.2 days for B. bassiana, A. muscarius and B. pseudobassiana, respectively. In topical tests, when 108 dry conidia were mixed with or without carnauba wax, the LT50 was 7.7, 10.2, and 14 days without this carrier and 6.9, 8.6, and 13.8 days with it for B. bassiana, B. pseudobassiana and A. muscarius, respectively. Overall, our findings showed that, among the tested fungi, B. bassiana was the most virulent when formulated as a dry powder with carnauba wax, which greatly improved fungal efficacy against the blowfly. We discuss the utility of carnauba wax for electrostatic formulation powder of fungal spores in the integrated management of blowflies as an environmentally sustainable tool to reduce the over-reliance on chemical insecticides and their risk of resistance.

Novel combination of a biosurfactant with entomopathogenic fungi enhances efficacy against Bemisia whitefly.

BACKGROUND: Microbial surfactants are multifunctional surface-active molecules that have been overlooked in formulating microbial biopesticides. We report a novel approach using the biosurfactant rhamnolipid (RML) against the destructive cosmopolitan insect pest Bemisia tabaci, as well as the combined action of RML with aerial conidia of two entomopathogenic fungi, Cordyceps javanica and Beauveria bassiana. RML was also tested as a suspension agent to improve the recovery rate of conidia from solid substrate for fungal preparations. RESULTS: The recovery rate of conidia increased dramatically (two to five times) with RML compared with a standard surfactant (Tween 80). Spraying solutions of 0.075% and 0.1% (w/v) RML on B. tabaci third instar nymphs induced 100% mortality within 4 days. Conidial suspensions at 5 × 106 conidia/mL amended with RML at 0.01% or 0.05% markedly increased nymphal mortalities and considerably reduced LC50 . Conidial suspensions of B. bassiana with 0.05% RML added were more effective against whitefly nymphs (87.3% mortality) than C. javanica + RML (51.4% mortality). CONCLUSION: Our results show that this bacterium-based RML improved the recovery rate of hydrophobic conidia, and that mixtures of RML with fungal spore suspensions increased their insecticidal activity. © 2019 Society of Chemical Industry.

The entomopathogenic fungus Beauveria bassiana produces microsclerotia-like pellets mediated by oxidative stress and peroxisome biogenesis.

Several filamentous fungi are known to produce macroscopic pigmented hyphal aggregates named sclerotia. In recent years, some entomopathogenic fungi were reported to produce small sclerotia termed 'microsclerotia', becoming new potential propagules for biocontrol strategies. In this study, we described the production of microsclerotia-like pellets by the entomopathogenic fungus Beauveria bassiana. The carbon: nitrogen ratio equal to or higher than 12.5:1 amended with Fe2+ induced the germination of conidia, producing hyphal aggregate that formed sclerotial structures in submerged liquid cultures. These aggregates were able to tolerate desiccation as they germinated and subsequently produced viable conidia. Conidia derived from microsclerotial aggregates formulated with diatomaceous earth effectively kill Tribolium castaneum larvae. Optical and transmission microscopical imaging, qPCR and spectrophotometric analysis revealed that an oxidative stress scenario is involved in conidial differentiation into microsclerotia-like pellets, inducing fungal antioxidant response with high peroxidase activity - mainly detected in peroxisomes and mitochondria - and progress with active peroxisome proliferation. The results provide clues about B. bassiana microsclerotial differentiation and indicate that these pigmented aggregates are promising propagules for production, formulation and potentially application in the control of soil-inhabiting arthropod pests.

Rapid discrimination of Isaria javanica and Isaria poprawskii from Isaria spp. using high resolution DNA melting assays.

The current study evaluates the potential of using high resolution DNA melting assays to discriminate species in the genus Isaria. The study utilizes a previously identified 103 base pair PCR amplicon, which was reported to be selective for Isaria fumosorosea. Our study finds the amplicon selective for Isaria javanica and Isaria poprawskii when assayed against all members of the genus. In addition, the high resolution melting profile of this amplicon can be used to discriminate between I. javanica, I. poprawskii and a 1:1 mixture of the two species. The practical application of this technique was confirmed using a bioassay on whitefly nymphs (Bemisia tabaci biotype B) inoculated with I. javanica, I. poprawskii or a 1:1 mixture of the two species. This assay provides a simple assay to identify these two species of entomopathogenic fungi.

Effect of heat stress and oil formulation on conidial germination of Metarhizium anisopliae s.s. on tick cuticle and artificial medium.

The effect of heat stress (45°C) versus non-heat stress (27°C) on germination of Metarhizium anisopliae sensu stricto (s.s.) isolate IP 119 was examined with conidia formulated (suspended) in pure mineral oil or in water (Tween 80, 0.01%), and then applied onto the cuticle of Rhipicephalus sanguineus sensu lato (s.l.) engorged females or onto culture medium (PDAY). In addition, bioassays were performed to investigate the effect of conidia heated while formulated in oil, then applied to blood-engorged adult R. sanguineus females. Conidia suspended in water then exposed to 45°C, in comparison to conidia formulated in mineral oil and exposed to the same temperature, germinated less and more slowly when incubated on either PDAY medium or tick cuticle. Also, conidial germination on tick cuticle was delayed in comparison to germination on artificial culture medium; for example, germination was 13% on tick cuticle 72h after inoculation, in contrast to 61.5% on PDAY medium. Unheated (27°C) conidia suspended in either water or oil and applied to tick cuticle developed appressoria 36h after treatment; whereas only heat-stressed conidia formulated in oil developed appressoria on tick cuticle. In comparison to conidia heated in mineral oil, there was a strong negative effect of heat on germination of conidia heated in water before being applied to arthropod cuticle. Nevertheless, bioassays [based primarily on egg production (quantity) and egg hatchability] exhibited high percentages of tick control regardless of the type of conidial suspension; i.e., water- or oil-formulated conidia, and whether or not conidia were previously exposed to heat. In comparison to aqueous conidial preparations, however, conidia formulated in oil reduced egg hatchability irrespective of heat or no-heat exposure. In conclusion, mineral-oil formulation protected conidia against heat-induced delay of both germination and appressorium production when applied to the cuticle of R. sanguineus.

Reproduction of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) B biotype in maize fields (Zea mays L.) in Brazil.

BACKGROUND: Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae) was observed to have completed its reproductive cycle from the egg to the adult on maize (Zea mays L.). Field and screenhouse studies were carried out to investigate the durability of this putative and unprecedented adaptation to a grass host. RESULTS: Analysis of the mitochondrial COI gene sequence identified the maize-associated B. tabaci as the exotic B biotype (major clade North Africa-Mediterranean-Middle East). Results showed that whiteflies migrated from soybean crops and successfully established in maize plants. Females exhibited a preference for oviposition primarily on the first and second leaves of maize, but were also able to colonise developing leaves. A high, natural infestation on maize (193.3 individuals, all developmental stages) was observed within a 7.1 cm2 designated 'observation area'. Whiteflies collected from naturally infested maize leaves and allowed to oviposit on maize seedlings grown in a screenhouse developed from egg to adulthood in 28.6 ± 0.2 days. CONCLUSION: This is the first report of the B biotype completing its development on maize plants. This surprising anomaly indicates that the B biotype is capable of adapting to monocotyledonous host plants, and importantly, broadens the host range to include at least one species in the Poaceae. © 2016 Society of Chemical Industry.

Liquid culture production of microsclerotia and submerged conidia by Trichoderma harzianum active against damping-off disease caused by Rhizoctonia solani.

Media and culturing protocols were identified that supported the formation of submerged conidia and microsclerotia (MS) by Trichoderma harzianum Rifai strain T-22 using liquid culture fermentation. Liquid media with a higher carbon concentration (36 g L(-1)) promoted MS formation at all C:N ratios tested. Hyphae aggregated to form MS after 2 d growth and after 7 d MS were fully melanized. This is the first report of MS formation by T. harzianum or any species of Trichoderma. Furthermore, submerged conidia formation was induced by liquid culture media, but yields, desiccation tolerance, and storage stability varied with C:N ratio and carbon rate. Air-dried MS granules (<4% moisture) retained excellent shelf life under cool and unrefrigerated storage conditions with no loss in conidial production. A low-cost complex nitrogen source based on cottonseed flour effectively supported high MS yields. Amending potting mix with dried MS formulations reduced or eliminated damping-off of melon seedlings caused by Rhizoctonia solani. Together, the results provide insights into the liquid culture production, stabilization process, and bioefficacy of the hitherto unreported MS of T. harzianum as a potential biofungicide for use in integrated management programs against soilborne diseases.

Thermal requirements and development of Herpetogramma phaeopteralis (Lepidoptera: Crambidae: Spilomelinae).

ABSTRACT The tropical sod webworm, Herpetogramma phaeopteralis Guenée is a major turfgrass pest in the southeastern United States. We evaluated larval development on five artificial diets and at six temperatures (15, 20, 25, 30, 32.5, 35 +/- 1 degree C) on St. Augustinegrass (Stenotaphrum secundatum (Walter) Kuntze). Only larvae fed St. Augustinegrass and soy-wheat germ diets completed their lifecycles. None of the artificial diets tested (corn-based, soy-wheat germ, corn cob-wheat germ, corn cob-soy flour, or pinto bean) were suitable for rearing this species, because of high mortality and slower developmental time. Total developmental time (oviposition to adult) on S. secundatum significantly decreased from 47.8 d at 20 degrees C to 21.1 d at 30 degrees C, and then increased to 32.6 d at 32.5 degrees C. Tropical sod webworm failed to complete larval development at 15 and 35 degrees C. The relationship between temperature and developmental rate was described using linear (common and polynomial) and nonlinear models (Briere-1, Briere-2, and Lactin-2). The estimated lower temperature thresholds using a linear model for eggs, first, second, third, fourth, fifth, and sixth instars, prepupa, pupa, and total development were 10.1, 6.9, 12.3, 10.5, 15.3, 13.9, 9.1, 13.1, 12.0, and 13.1 degrees C, and the thermal constant of these stages were 62.9, 66.2, 38.2, 40.3, 24.9, 32.3, 51.9, 106.4, 109.9, and 370.4 degree-days, respectively. The Briere-1 model provided the best fit with estimated lower, upper, and optimum thresholds for total development of 14.9, 34.3, and 29.4 degrees C, respectively. The developmental requirements of H. phaeopteralis can be used to help predict the distribution and seasonal phenology of this pest.