Repellent activity of the non-host semiochemical (E)-2-octenal against Amblyomma sculptum and Amblyomma dubitatum ticks under field conditions.

Amblyomma ticks pose a significant public health threat due to their potential to transmit pathogens associated with rickettsial diseases. (E)-2-octenal, a compound found in donkeys (Equus asinus), exhibits strong repellent properties against Amblyomma sculptum nymphs under laboratory conditions. This study assessed the effectiveness of the (E)-2-octenal in wearable slow-release devices for personal human protection against Amblyomma ticks under natural conditions. Slow-release devices treated with (E)-2-octenal and untreated controls were prepared and tested on two volunteers walking through a tick-infested area in Goiania, Brazil. The experiment was conducted twice daily for three series of 10 days, with each volunteer wearing two devices attached to each leg, one on the ankle and one just above the thigh. Volunteers with control and treated devices exchanged them between rounds. Also, the daily release rate of (E)-2-octenal from the slow-release devices was determined in the laboratory, increasing significantly from 0.77 ± 0.14 µg/day on the first day to 9.93 ± 1.92 µg/day on the 4th day and remaining constant until the 16th day. A total of 5409 ticks were collected from both volunteers. Treated devices resulted in recovering fewer ticks (n = 1,666; 31%) compared to untreated devices (control: n = 3,743; 69%). (E)-2-octenal effectively repelled Amblyomma spp. larvae, A. sculptum adults, and exhibited pronounced repellency against A. dubitatum nymphs and adults. These findings suggest the potential of (E)-2-octenal delivered by wearable slow-release devices as a green-based repellent. Further improvements, however, are necessary to provide better protection for humans against A. sculptum and A. dubitatum in field conditions.

Akanthomyces diversity in Brazil and their pathogenicity to plant-sucking insects.

Currently, species within the genus Akanthomyces are poorly studied and explored compared to other hypocrealean entomopathogenic fungi employed as commercial biocontrol agents. This study aimed to molecularly identify 23 Brazilian Akanthomyces strains, most originally isolated from aphids and scales (n = 22), and one from the coffee leaf rust, and further investigate their pathogenicity to six plant-sucking insects as a means to better understand their host spectra. We also explored the capacity of A. muscarius CG935 for blastospore production via liquid fermentation. Akanthomyces dipterigenus, A. muscarius, A. lecanii, and two unidentified species were recognized as naturally occurring in Brazil. Akanthomyces dipterigenus CG829 and A. muscarius CG935 were highly virulent to nymphs of Bemisia tabaci (67.5-85.4% confirmed mortality) and the aphid Aphis fabae (74.6-75.3%), but only the first strain was virulent to the mealybug Planococcus sp. (80.9%). Akanthomyces lecanii CG824 was weakly virulent to all tested insects. None of the strains were pathogenic to the thrips Caliothrips phaseoli, and all strains showed low virulence to the wooly whitefly Aleurothrixus floccosus and the scale Duplachionaspis divergens. Submerged liquid fermentation yields varied from 1.72 × 109 (day 2) to 3.90 × 109 (day 5) blastospores mL-1. Blastospores or aerial conidia from A. muscarius CG935, at a single concentration of 1 × 107 viable propagules mL-1, resulted in 67.5-83.1% mortality of B. tabaci nymphs within 8 days post-treatment. Overall, these results encourage additional studies that could lead to the development of new mycopesticides based on Akanthomyces strains.

Impact of osmotic stress on production, morphology, and fitness of Beauveria bassiana blastospores.

Beauveria bassiana is a cosmopolitan entomopathogenic fungus that can infect over 1000 insect species. During growth inside the host, B. bassiana transitions from hyphal to yeast-like unicellular growth as blastospores. Blastospores are well suited as an active ingredient in biopesticides due to their ease of production by liquid fermentation. Herein, we investigated the impact of hyperosmotic growth environments mediated by ionic and non-ionic osmolytes on two strains of B. bassiana (ESALQ1432 and GHA) relevant to growth morphology, blastospore production, desiccation tolerance, and insecticidal activity. Polyethylene glycol (PEG200) increased osmotic pressure in submerged cultures leading to decreased blastospore size but higher blastospore yields for one strain. Morphologically, decreased blastospore size was linked to increased osmotic pressure. However, smaller blastospores from PEG200 supplemented cultures after air-drying exhibited delayed germination. Ionic osmolytes (NaCl and KCl) generated the same osmotic pressure (2.5-2.7 MPa) as 20% glucose and boosted blastospore yields (> 2.0 × 109 blastospores mL-1). Fermentation performed in a bench-scale bioreactor consistently promoted high blastospore yields when using NaCl (2.5 MPa) amended media within 3 days. Mealworm larvae (Tenebrio molitor) were similarly susceptible to NaCl-grown blastospores and aerial conidia in a dose-time-dependent manner. Collectively, these results demonstrate the use of hyperosmotic liquid culture media in triggering enhanced yeast-like growth by B. bassiana. Understanding the role of osmotic pressure on blastospore formation and fitness will hasten the development of viable commercial fungal biopesticides. KEY POINTS: • Osmotic pressure plays a critical role in submerged fermentation of B. bassiana. • Ionic/non-ionic osmolytes greatly impact blastospore morphology, fitness, and yield. • Desiccation tolerance and bioefficacy of blastospores are affected by the osmolyte.

On-farm Production of Microbial Entomopathogens for use in Agriculture: Brazil as a Case Study.

In Brazil, the production of beneficial microorganisms by growers exclusively for their own use is a practice known as "on-farm production". Regarding on-farm bioinsecticides, they were initially deployed for pests of perennial and semi-perennial crops in the 1970s but, since 2013, their use has extended to pests of annual crops such as maize, cotton, and soybean. Millions of hectares are currently being treated with these on-farm preparations. Local production reduces costs, meets local needs, and reduces inputs of environmentally damaging chemical pesticides, facilitating establishment of more sustainable agroecosystems. Critics argue that without implementation of stringent quality control measures there is the risk that the on-farm preparations: (1) are contaminated with microbes which may include human pathogens or (2) contain very little active ingredient, impacting on field efficacy. The on-farm fermentation of bacterial insecticides predominates, especially that of Bacillus thuringiensis targeting lepidopteran pests. However, there has been a rapid growth in the past 5 years in the production of entomopathogenic fungi, mostly for the control of sap-sucking insects such as whitefly (Bemisia tabaci (Gennadius)) and the corn leafhopper (Dalbulus maidis (DeLong and Wolcott)). In contrast, on-farm production of insect viruses has seen limited growth. Most of the ca. 5 million rural producers in Brazil own small or medium size properties and, although the vast majority still do not practice on-farm production of biopesticides, the topic has aroused interest among them. Many growers who adopt this practice usually use non-sterile containers as fermenters, resulting in poor-quality preparations, and cases of failure have been reported. On the other hand, some informal reports suggest on-farm preparations may be efficacious even when contaminated, what could be explained, at least partially, by the insecticidal secondary metabolites secreted by the pool of microorganisms in the liquid culture media. Indeed, there is insufficient information on efficacy and mode of action of these microbial biopesticides. It is usually the large farms, some with > 20,000 ha of continuous cultivated lands, that produce biopesticides with low levels of contamination, as many of them possess advanced production facilities and have access to specialized knowledge and trained staff. Uptake of on-farm biopesticides is expected to continue but the rate of adoption will depend on factors such as the selection of safe, virulent microbial strains and implementation of sound quality control measures (compliance with emerging Brazilian regulations and international standards). The challenges and opportunities of on-farm bioinsecticides are presented and discussed.

Microsclerotial pellets of Metarhizium spp.: thermotolerance and bioefficacy against the cattle tick.

The cattle tick, Rhipicephalus microplus (Acari: Ixodidae), is a multi-billion dollar ectoparasite of global importance affecting beef and milk production. Submerged cultures of cosmopolitan entomopathogenic fungal species of the genus Metarhizium typically produce microsclerotia that provide both long-term survival and environmental resistance. Microsclerotia hold great potential as an unconventional active propagule to control this tick under laboratory and semi-field conditions. However, heat stress caused especially by elevated temperatures poses a critical environmental constraint for the successful development and efficacy of microsclerotia under tropical conditions. First, we screened six strains of Metarhizium anisopliae, Metarhizium robertsii and Metarhizium humberi for their ability to produce microsclerotia by submerged liquid cultivation. In addition, we assessed the biological fitness and bioefficacy of dried microsclerotial pellets under amenable (27 °C) and heat-stressed (32 °C) incubation against engorged adult females of R. microplus. Microsclerotia in pelletized formulation prepared with carriers based on diatomaceous earth and microcrystalline cellulose exhibited conidial production at different extents according to the fungal strain and the incubation temperature, but most strains displayed reduced sporogenesis when exposed to 32 °C. Engorged tick females exposed to sporulated microsclerotia from pelletized M. anisopliae CG47 or IP 119 had fewer number of hatching larvae in comparison to the control group, irrespective of the incubation temperature tested. The minimum dosage of microsclerotial pellets that effectively reduced hatchability of tick larvae was estimated to be 2 mg per plate (equivalent to 6.0 kg per hectare). Metarhizium microsclerotial pellets exhibited significant tolerance to 32 °C and pronounced acaricidal activity against this economically important ectoparasite of cattle, even under simulated environmental heat stress. KEY POINTS: • Heat stress affects conidial production by microsclerotia of most pelletized Metarhizium strains • Heat stress does not impair the acaricidal performance of pelletized microsclerotia • Pellet formulation of Metarhizium microsclerotia is a promising mycoacaricide.

From the laboratory to the field: efficacy of entomopathogenic nematodes to control the cattle tick.

BACKGROUND: The control of ticks is challenged by the resistance of tick populations to chemical acaricides. In this study, we evaluated, under laboratory conditions, the efficacy of Heterorhabditis bacteriophora against Rhipicephalus (Boophilus) microplus engorged females with varying body weights (150, 200, 250, 300 or 350 mg per female) or from eight different geographical populations. We also determined the efficacy of H. bacteriophora for tick control under field conditions. RESULTS: R. microplus engorged females with varying body weights exposed to 150 juveniles of H. bacteriophora resulted in a high control efficacy (97.5% to 98.4%). Tests with females from different geographical populations comprised eight tick strains treated with H. bacteriophora and their respective control groups. The biological parameters of females exposed to nematode treatments did not differ significantly and resulted in 89% to 99% of control efficacy. Trials conducted under field conditions were performed in field plots with Megathyrsus maximus grass. Treatment groups received eight cadavers of Tenebrio molitor fully colonized with H. bacteriophora at 1 week prior to the release of female ticks, whereas control groups were untreated. On the first day of the experiment, six engorged females were distributed in each plot. On day 42 and day 63, the apical portion of the grasses with R. microplus larvae were collected and quantified. The population of R. microplus larvae was reduced up to 73.1% in plots treated with H. bacteriophora at day 63 after treatment. CONCLUSION: R. microplus engorged females with varying body weights or from different geographical populations were highly susceptible to H. bacteriophora. The field test demonstrated the efficacy of H. bacteriophora in reducing R. microplus larvae in infested pastures. © 2022 Society of Chemical Industry.

Dual functionality of Trichoderma: Biocontrol of Sclerotinia sclerotiorum and biostimulant of cotton plants.

Microbial crop protection products based on Trichoderma have the ability to display multifunctional roles in plant protection, such as pathogen parasitism, enhance nutrient availability and stimulate plant growth, and these traits can be used to enhance the overall agronomic performance of a variety of crops. In the current study, we explored the multifunctional potential of two indigenous Brazilian strains of Trichoderma (T. asperelloides CMAA 1584 and T. lentiforme CMAA 1585) for their capability of controlling Sclerotinia sclerotiorum, a key plant pathogen of cotton, and for their ability of growth promotion in cotton plants (Gossypium hirsutum). Both strains were able to solubilize mineral phosphorus (CaHPO4), to release volatile organic compounds that impaired the mycelial growth of S. sclerotiorum, and to promote the growth of cotton plants under greenhouse conditions. In dual culture, Trichoderma strains reduced the growth rate and the number of sclerotia formed by S. sclerotiorum. By treating sclerotia with conidial suspensions of these Trichoderma strains, a strong inhibition of the myceliogenic germination was observed, as a result of the marked mycoparasitic activity exerted on the sclerotia. The parasitism over S. sclerotiorum was more effective with T. asperelloides CMAA 1584, whilst the biostimulant effects on cotton growth were more pronounced with T. lentiforme CMAA 1585, which also showed a higher capacity of phosphate solubilization. Thus, T. asperelloides CMAA 1584 displays higher efficiency in controlling S. sclerotiorum, while T. lentiforme CMAA 1585 is more suitable as a biostimulant due to its ability to promote growth in cotton plants. Overall, these Trichoderma strains may be used in mixture to provide both pathogen control and promotion of plant growth, and this strategy will support growers in minimizing the use of synthetic fertilizers and fungicides against white mold in cotton crops.

Natural occurrence of Beauveria caledonica, pathogenicity to Cosmopolites sordidus and antifungal activity against Fusarium oxysporum f. sp. cubense.

BACKGROUND: Entomopathogenic fungi can provide a set of ecological services, such as suppressing arthropod pests and plant pathogens. In this study, novel indigenous Beauveria caledonica (Bc) strains were isolated from naturally infected banana weevils (Cosmopolites sordidus) occurring in commercial banana plantations in Brazil. RESULTS: The prevalence of infection by Bc strains on field-caught C. sordidus ranged from 1.3% to 12.9%. Similar to the Beauveria bassiana strains tested, none of the Bc strains caused more than 50% weevil mortality at a concentration of 1 × 108 conidia ml-1 . Bc strain CMAA1810 caused the highest mortality in C. sordidus and had enhanced insecticidal activity when formulated with an emulsifiable oil. In paired co-culture assays, this same strain showed a significant growth-inhibitory effect on the causal agent of Fusarium banana wilt (Fusarium oxysporum f. sp. cubense, Foc) of twofold magnitude compared with the control. Cell-free crude filtrates derived from the red-pigmented culture broth of Bc (CMAA1810) strongly reduced Foc conidial viability, and this inhibitory activity was inversely related to the age of the Bc culture. Crude concentrated filtrates from 4-day-old cultures exhibited the strongest antifungal activity (13-fold) compared with untreated Foc conidia. The abundant compound identified in the crude filtrate of Bc was oosporein (1,4-dibenzoquinone) present at a concentration of 0.829 ± 0.018 mg g-1 dry matter, and the antifungal activity of the filtrate was demonstrated. CONCLUSION: These results indicated that Bc strains might have the potential to manage both C. sordidus and Foc, two of the major phytosanitary problems in banana crops worldwide. Further research under field conditions using suitable formulations of virulent Bc strains in combination with the metabolite oosporein is needed to evaluate their efficacy in the management of C. sordidus and Foc in banana plantations. © 2022 Society of Chemical Industry.

Production of Purpureocillium lilacinum and Pochonia chlamydosporia by Submerged Liquid Fermentation and Bioactivity against Tetranychus urticae and Heterodera glycines through Seed Inoculation.

Pochoniachlamydosporia and Purpureocilliumlilacinum are fungal bioagents used for the sustainable management of plant parasitic nematodes. However, their production through submerged liquid fermentation and their use in seed treatment have been underexplored. Therefore, our goal was to assess the effect of different liquid media on the growth of 40 isolates of P. lilacinum and two of P. chlamydosporia. The most promising isolates tested were assessed for plant growth promotion and the control of the two-spotted spider mite (Tetranychus urticae) and the soybean cyst nematode (Heterodera glycines). Most isolates produced > 108 blastospores mL−1 and some isolates produced more than 104 microsclerotia mL−1. Microsclerotia of selected isolates were used to inoculate common bean (Phaseolus vulgaris L.) seeds in greenhouse trials. All fungal isolates reduced the T. urticae fecundity in inoculated plants through seed treatment, while P. chlamydosporia ESALQ5406 and P. lilacinum ESALQ2593 decreased cyst nematode population. Purpureocillium lilacinum was more frequently detected in soil, whereas P. chlamydosporia colonized all plant parts. Pochonia chlamydosporia ESALQ5406 improved the root development of bean plants. These findings demonstrate the possibility of producing submerged propagules of P. chlamydosporia and P. lilacinum by liquid culture, and greenhouse trials support the applicability of fungal microsclerotia in seed treatment to control P. vulgaris pests.

Clonostachys rosea: Production by Submerged Culture and Bioactivity Against Sclerotinia sclerotiorum and Bemisia tabaci.

Among the prospective biocontrol agents, the saprophytic filamentous fungus Clonostachys rosea is an excellent necrotrophic mycoparasite of numerous plant pathogenic fungi. However, its commercial development has been hampered by mass production difficulties during solid-state fermentation. Conversely, the submerged liquid fermentation shortens the cultivation time while increasing yields of fungal propagules. However, this method has been overlooked for C. rosea. In this work, we investigated the impact of liquid pre-culture inoculum on the spore production by the two-stage fermentation process using rice grains in comparison to the traditional solid-state fermentation. In parallel, we studied the submerged cultivation of C. rosea by manipulating carbon-to-nitrogen (C:N) ratio and nitrogen source, with the further optimization of spore production in a benchtop bioreactor. Additional bioassays included assessing the bioactivity of water-dispersible microgranules (that contained a submerged conidia) against the whitefly (Bemisia tabaci biotype B) and Sclerotinia sclerotiorum (causal agent of the white mold). Our results showed a maximum concentration of 1.1 × 109 conidia/g-dry-matter after 7 days of cultivation by two-stage fermentation process. The liquid fermentation yielded 1.4 × 109 submerged conidia/ml after 7 days using a medium with a 50:1 C:N ratio, and it also induced the production of microsclerotia (MS) up to 1.35 × 104/ml within 6 days with 10:1 C:N ratio; both media were supplemented with dextrose monohydrate and soybean meal. The fermentation batches carried out in a benchtop bioreactor with medium 50:1 C:N ratio and amended with soybean meal rendered a production peak on the fourth day, corresponding to 1.11 × 109 conidia/ml and 4.35 × 108 colony forming units (CFU)/ml. Following air-drying, the conidia production from air-dried microgranules of C. rosea biomass was estimated at 3.4 × 1010 conidia/g of formulated product upon re-hydration for 7 days. Both submerged conidia and MS of C. rosea inhibited 100% germination of S. sclerotiorum sclerotia by direct parasitism. The air-dried submerged conidia exhibited a suppressive activity on sclerotia (88% mycoparasitism) and early whitefly nymphs (76.2% mortality) that rendered LC50 values of 3.2 × 104 CFU/g soil and 1.5 × 107 CFU/ml, respectively. Therefore, the submerged liquid culture of C. rosea may offer a feasible and cost-effective method for its large-scale production, alleviating critical constraints to their commercial use while providing an additional tool for management of B. tabaci and S. sclerotiorum.

Development of novel spray-dried and air-dried formulations of Metarhizium robertsii blastospores and their virulence against Dalbulus maidis.

The present research addressed spray-drying and air-drying techniques applied to Metarhizium robertsii blastospores to develop wettable powder (WP) formulations. We investigated the effect of co-formulants on blastospore viability during drying and assessed the wettability and stability of formulations in water. The effect of oxygen-moisture absorbers was studied on the shelf life of these formulations stored at 26 °C and 4 °C for up to 90 days. Additionally, we determined the virulence of the best spray-dried and air-dried formulations against the corn leafhopper Dalbulus maidis. While sucrose and skim milk played an essential role as osmoprotectants in preserving air-dried blastospores, maltodextrin, skim milk, and bentonite were crucial to attain high cell survival during spray drying. The lowest wettability time was achieved with spray-dried formulations containing less Ca-lignin, while charcoal powder amount was positively associated with formulation stability. The addition of oxygen-moisture absorbers inside sealed packages increased from threefold to fourfold the half-life times of air-dried and spray-dried formulations at both storage temperatures. However, the half-life times of all blastospore-based formulations were shorter than 3 months regardless of temperature and packaging system. Spray-dried and air-dried WP formulations were as virulent as fresh blastopores against D. maydis adults sprayed with 5 × 107 blastospores mL-1 that induced 87.8% and 70.6% mortality, respectively. These findings bring innovative advancement for M. robertsii blastospore formulation through spray-drying and underpin the importance of adding protective matrices coupled to oxygen-moisture absorbers to extend cell viability during either cold or non-refrigerated storage. KEY POINTS: • Cost-effective wettable powder formulations of M. robertsii blastospores were developed. • Bioefficacy of formulations against the corn leafhopper was comparable to fresh blastospores. • Cold storage and dual oxygen-moisture absorber are critical for extended shelf life.

Inorganic pellets containing microsclerotia of Metarhizium anisopliae: a new technological platform for the biological control of the cattle tick Rhipicephalus microplus.

This study was sought to devise pellets containing inorganic materials and microsclerotia of Metarhizium anisopliae strain IP 119 for biological control of Rhipicephalus microplus, the most economically important tick in Brazilian cattle industry. In addition, we evaluated the storage stability of the pellets, their tolerance to ultraviolet radiation (UV-B), and efficacy against ticks under laboratory conditions. Fungal microsclerotia were produced by liquid culture fermentation and mixed with pre-selected inorganic matrices: vermiculite powder, diatomaceous earth, and colloidal silicon dioxide (78:20:2, w/w/w). The microsclerotial pellets were then prepared by a two-stage process involving extrusion and spheronization. Pellet size averaged 525.53 ± 7.74 µm, with a sphericity index of 0.72 ± 0.01, while biomass constituents did not affect the wet mass properties. Conidial production from microsclerotial pellets upon rehydration ranged from 1.85 × 109 to 1.97 × 109 conidia g-1 with conidial viability ≥ 93%. Conidial production from pellets stored at 4 °C was invariable for up to 21 days. Unformulated microsclerotia and microsclerotial pellets were extremely tolerant to UV-B compared with aerial conidia. Engorged tick females exposed to conidia from sporulated pellets applied to soil samples and upon optimal rehydration exhibited shorter oviposition time length, shorter life span, and reduced number of hatched larvae. In summary, microsclerotial pellets of M. anisopliae IP 119 effectively suppressed R. microplus and showed outstanding UV-B tolerance in laboratory tests. Prospectively, this formulation prototype is promising for targeting the non-parasitic stage of this tick on outdoor pasture fields and may offer a novel mycoacaricide for its sustainable management. KEY POINTS: • Pellets with microsclerotia and inorganic materials are innovative for tick control. • Metarhizium microsclerotia show superior UV-B tolerance in relation to conidia. • Pellets of Metarhizium microsclerotia produce infective conidia against ticks.

First record of a new microsporidium pathogenic to Gonipterus platensis in Brazil.

Microsporidia are naturally occurring fungal-related parasites that can infect nearly all animal hosts, but their biocontrol potential of insect pests is routinely overlooked in agriculture and forestry. This research brings the first report describing the natural occurrence of a microsporidium causing disease in field-collected populations of the invasive eucalyptus snout beetle, Gonipterus platensis (Coleoptera: Curculionidae), a major destructive pest of eucalyptus plantations in Brazil. Adult beetles were collected during field surveys in commercial eucalyptus plantations in southern Brazil to be examined and dissected with typical symptoms to verify presence of microsporidian spores in haemolymph. From 14 plantations in different sites, the natural infection occurrence in these populations ranged from 0 to 65%, while a lab colony exhibited an infection incidence of 70%. Spore density in haemolymph of symptomatic insects averaged 2.1 (± 0.4) × 107 spores/beetle. Symptoms in infected adults were identified by an abnormal abdomen with malformation of the second pair of wings, impairing their flight activity. Electron transmission microscopy of the pathogen showed morphological features similar to species belonging to the genus Nosema or Vairimorpha. Phylogenetic analysis of the full-length small subunit ribosomal RNA gene suggests this pathogen's placement in the genus Vairimorpha, but with a sequence identity of ~ 94% with the nearest neighbours. The low level of sequence identity suggests this pathogen may represent a novel taxon in the genus and further requires whole genome sequencing for definitive taxonomic resolution. These findings provide insights on the natural occurrence of this novel pathogen of this invasive pest in Eucalyptus plantations in Brazil. Further studies are needed to determine potential of this microsporidium in the design of conservative or augmentative biological control programs for this invasive pest.

Entomopathogenic fungi as the microbial frontline against the alien Eucalyptus pest Gonipterus platensis in Brazil.

The eucalyptus snout beetle (ESB), Gonipterus platensis, is endemic to Australia but has become a major invasive, destructive pest of Brazilian eucalyptus plantations. Efforts to develop insecticides based on entomopathogenic fungi against ESB are limited by the lack of known virulent strains. We therefore explored the virulence of indigenous Brazilian strains of major entomopathogenic fungi-Beauveria spp. and Metarhizium anisopliae-against ESB adults. We found widely varying virulence and later capacities for conidial production on infected adult cadavers. Two strains stood out, B. bassiana IBCB-240 and M. anisopliae IBCB-364, as especially lethal for ESB adults under laboratory conditions, sporulated abundantly on infected insects, and also outperformed comparable strains used in commercial mycoinsecticides. Notably, B. bassiana IBCB-240 exhibited lower LT50 values at low inoculum levels (≤ 107 conidia mL-1) and smaller LC50 values than M. anisopliae IBCB-364. Taken together, this study emphasizes natural variation in virulence among indigenous Beauveria and Metarhizium strains against ESB adults and identifies fungal strains with superior lethality to existing commercialized strains for managing this eucalyptus pest in Brazil.

Transcriptional Responses of Beauveria bassiana Blastospores Cultured Under Varying Glucose Concentrations.

Culturing the entomopathogenic fungus, Beauveria bassiana, under high glucose concentrations coupled with high aeration results in a fungal developmental shift from hyphal growth to mostly blastospores (yeast-like cells). The underlying molecular mechanisms involved in this shift remain elusive. A systematic transcriptome analysis of the differential gene expression was preformed to uncover the fungal transcriptomic response to osmotic and oxidative stresses associated with the resulting high blastospore yield. Differential gene expression was compared under moderate (10% w/v) and high (20% w/v) glucose concentrations daily for three days. The RNAseq-based transcriptomic results depicted a higher proportion of downregulated genes when the fungus was grown under 20% glucose than 10%. Additional experiments explored a broader glucose range (4, 8, 12, 16, 20% w/v) with phenotype assessment and qRT-PCR transcript abundance measurements of selected genes. Antioxidant, calcium transport, conidiation, and osmosensor-related genes were highly upregulated in higher glucose titers (16-20%) compared to growth in lower glucose (4-6%) concentrations. The class 1 hydrophobin gene (Hyd1) was highly expressed throughout the culturing. Hyd1 is known to be involved in spore coat rodlet layer assembly, and indicates that blastospores or another cell type containing hydrophobin 1 is expressed in the haemocoel during the infection process. Furthermore, we found implications of the HOG signaling pathway with upregulation of homologous genes Ssk2 and Hog1 for all fermentation time points under hyperosmotic medium (20% glucose). These findings expand our knowledge of the molecular mechanisms behind blastospore development and may help facilitate large-scale industrial production of B. bassiana blastospores for pest control applications.

Relative humidity impacts development and activity against Aedes aegypti adults by granular formulations of Metarhizium humberi microsclerotia.

The impact of ambient relative humidity (RH) on conidial production of Metarhizium humberi IP 46 microsclerotia (MS) formulated in pellets or granules was investigated, and a promising granular formulation was tested against Aedes aegypti adults to confirm its efficacy. Microcrystalline cellulose (MC) and diatomaceous earth (DE) or a combination of vermiculite (VE), DE and silicon dioxide (SD) were tested as carriers in granular formulations containing MS. A range of 93-96.5% RH was critical for fungal development, and at least 96.5-98.5% RH was required for high conidial production on pellets or granules. Conidial production was clearly higher on pellets and granules prepared with VE than MC as the main carrier. VE granules containing MS were highly active against A. aegypti adults. Most mosquitoes were killed within 6 days after treatment regardless of the exposure time of adults to the formulation (1 min-24 h) or ambient humidity (75 or >98%). Production of conidia on dead adults varied between 7.3 × 106 and 2.2 × 107 conidia/individual, when exposed to MS granules for 12 h and 1 min, respectively. Granular formulations containing VE as the main carrier and MS as the active ingredient of M. humberi have strong potential for use against A. aegypti. KEY POINTS: • High conidial production on granular microsclerotial formulations at >96.5% RH • Vermiculite is more appropriate as a carrier than microcrystalline cellulose • Granules with IP 46 microsclerotia are highly active against Aedes aegypti adults.

Innovative granular formulation of Metarhizium robertsii microsclerotia and blastospores for cattle tick control.

The tick Rhipicephalus microplus poses a serious threat to the cattle industry, resulting in economic losses aggravated by tick resistance to chemical acaricides. Strains of Metarhizium spp., a well-known group of entomopathogenic fungi, can contribute to managing this ectoparasite. We explored two novel granular, microsclerotia- or blastospores-based formulations of Metarhizium robertsii for R. microplus control under semi-field conditions. Fungal persistence in soil was also observed for 336 days. The experiment used pots of Urochloa decumbens cv. Basilisk grass, treated with 0.25 or 0.5 mg of granular formulation/cm2 (25 or 50 kg/ha) applied to the soil surface prior to transferring engorged tick females onto the treated soil. The fungal granules yielded more conidia with subsequent sporulation under controlled indoor conditions than in the outdoor environment, where the levels of fungus rapidly declined over time. Metarhizium-root colonization ranged from 25 to 66.7% depending on the propagule and rate. Fungal formulations significantly reduced the number of tick larvae during the humid season, reaching at least 64.8% relative efficacy. Microsclerotia or blastospores-granular formulations of M. robertsii can reduce the impact of R. microplus, and thus prove to be a promising tool in the control of ticks.

Multispectral and X-ray images for characterization of Jatropha curcas L. seed quality.

BACKGROUND: The use of non-destructive methods with less human interference is of great interest in agricultural industry and crop breeding. Modern imaging technologies enable the automatic visualization of multi-parameter for characterization of biological samples, reducing subjectivity and optimizing the analysis process. Furthermore, the combination of two or more imaging techniques has contributed to discovering new physicochemical tools and interpreting datasets in real time. RESULTS: We present a new method for automatic characterization of seed quality based on the combination of multispectral and X-ray imaging technologies. We proposed an approach using X-ray images to investigate internal tissues because seed surface profile can be negatively affected, but without reaching important internal regions of seeds. An oilseed plant (Jatropha curcas) was used as a model species, which also serves as a multi-purposed crop of economic importance worldwide. Our studies included the application of a normalized canonical discriminant analyses (nCDA) algorithm as a supervised transformation building method to obtain spatial and spectral patterns on different seedlots. We developed classification models using reflectance data and X-ray classes based on linear discriminant analysis (LDA). The classification models, individually or combined, showed high accuracy (> 0.96) using reflectance at 940 nm and X-ray data to predict quality traits such as normal seedlings, abnormal seedlings and dead seeds. CONCLUSIONS: Multispectral and X-ray imaging have a strong relationship with seed physiological performance. Reflectance at 940 nm and X-ray data can efficiently predict seed quality attributes. These techniques can be alternative methods for rapid, efficient, sustainable and non-destructive characterization of seed quality in the future, overcoming the intrinsic subjectivity of the conventional seed quality analysis.

Optimizing mass production of Trichoderma asperelloides by submerged liquid fermentation and its antagonism against Sclerotinia sclerotiorum.

Commercial products based on Trichoderma are obtained mainly from solid-state fermentation. Submerged liquid fermentation is the most appropriate method compared to the solid medium for large-scale production of Trichoderma spp. The present study aimed to optimize the combination of key variables that influence the liquid fermentation process of Trichoderma asperelloides LQC-96 for conidial production coupled with its efficiency in the control of Sclerotinia sclerotiorum. In addition, we verified whether the optimized culture conditions can be used for the conidial production of Trichoderma erinaceum T-12 and T-18 and Trichoderma harzianum T-15. Fermentation studies were performed in shake flasks following a planned experimental design to reduce the number of tests and consumable costs. The effect of temperature, pH, photoperiod, carbon:nitrogen ratio and water activity on conidial production were assessed, which of pH was the only meaningful factor contributing to increased conidial production of T. asperelloides LQC-96. From the five variables studied initially, pH and C:N ratio were further used in the second design (rotational central composite design-RCCD). Hence, the best conditions for the production of T. asperelloides LQC-96 conidia by liquid fermentation consisted of initial pH of 3.5, C:N ratio of 200:1 at 30 °C, without glycerol, and under 24 h photoperiod. The highest conidial concentration was observed after seven days of fermentation. Under these optimal conditions, T. erinaceum T-12 and T-18, and T. harzianum T-15 were also cultivated, but only LQC-96 efficiently parasitized S. sclerotiorum, precluding sclerotium myceliogenic germination. Our findings propose optimal fermentation conditions that maximize conidial production of T. asperelloides as a potential biofungicide against S. sclerotiorum.

Microsclerotia production of Metarhizium spp. for dual role as plant biostimulant and control of Spodoptera frugiperda through corn seed coating.

The fungal genus Metarhizium comprises entomopathogenic species capable of producing overwintering structures known as microsclerotia. These structures offer many advantages in pest control due to the formation of infective conidia in situ and their persistence in the environment under adverse conditions. In addition, the in vitro production of Metarhizium microsclerotia under controlled liquid fermentation is faster and with greater process control than the production of aerial conidia. However, the potential of Metarhizium microsclerotia to control pests from the orders Lepidoptera and Hemiptera is unexplored. In this study, we examined the ability of Metarhizium spp. microsclerotia to promote corn growth and to provide plant protection against Spodoptera frugiperda (Lepidoptera: Noctuidae) and Dalbulus maidis (Hemiptera: Cicadellidae), through seed coating using microsclerotial granules. A screening to find higher microsclerotia producers was conducted by culturing 48 native Brazilian isolates of Metarhizium spp. (Metarhizium anisopliae, Metarhizium robertsii, Metarhizium humberi and Metarhizium sp. indeterminate). The best microsclerotia producers, M. anisopliae ESALQ1814, M. robertsii ESALQ2450 and M. humberi ESALQ1638 improved the leaf area, plant height, root length, and dry weight of plants compared to un-inoculated plants. Significant reduction in S. frugiperda survival (mortality > 55% after 7 days) was observed when larvae were fed on corn plants treated with any of the three Metarhizium species. Conversely, survival of D. maidis adults were unaffected by feeding on fungus-inoculated plants. Our results suggest that microsclerotia of Metarhizium spp. may act as biostimulants and to provide protection against S. frugiperda in corn through seed coating, thus adding an innovative strategy into the integrated management of this major worldwide pest.