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Entomopathogenic fungi (EPF) can be defined as beneficial multifunctional eukaryotic microorganisms that display pivotal ecological services in pest management, with some species possessing the special ability to establish mutualistic relationships with plants. Mass production of these fungi is critical to support affordable widespread commercialization and worldwide field application. Among the mass production methods explored mainly by industry, submerged liquid fermentation is a robust and versatile technology that allows the formation of different types of propagules designated for various applications in pest control. Many hypocrealean EPF are easily culturable on artificial substrates by producing single-celled structures (hyphal bodies, blastospores, and submerged conidia) or multicellular structures (mycelium and microsclerotia). Less frequently, some EPF may form environmentally resistant chlamydospores, but these structures have almost always been overlooked. A continued research pipeline encompassing screening fungal strains, media optimization, and proper formulation techniques aligned with the understanding of molecular cues involved in the formation and storage stability of these propagules is imperative to unlock the full potential and to fine-tune the development of robust and effective biocontrol agents against arthropod pests and vectors of diseases. Finally, we envision a bright future for the submerged liquid fermentation technology to supplement or replace the traditional solid substrate fermentation method for the mass production of many important EPF. KEY POINTS: • Submerged liquid fermentation (SLF) allows precise control of nutritional and environmental factors • SLF provides a scalable, robust, and cost-effective platform for mycopesticide production • Enhancing formulation, shelf life, and field efficacy of submerged propagules remain crucial • Understanding the molecular mechanisms behind submerged propagule formation is key to advancing SLF technology.

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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.

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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.

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The filamentous fungus Beauveria bassiana is an economically important pathogen of numerous arthropod pests and is able to grow in submerged culture as filaments (mycelia) or as budding yeast-like blastospores. In this study, we evaluated the effect of dissolved oxygen and high glucose concentrations on blastospore production by submerged cultures of two isolates of B. bassiana, ESALQ1432 and GHA. Results showed that maintaining adequate dissolved oxygen levels coupled with high glucose concentrations enhanced blastospore yields by both isolates. High glucose concentrations increased the osmotic pressure of the media and coincided with higher dissolved oxygen levels and increased production of significantly smaller blastospores compared with blastospores produced in media with lower concentrations of glucose. The desiccation tolerance of blastospores dried to less than 2.6 % moisture was not affected by the glucose concentration of the medium but was isolate dependent. Blastospores of isolate ESALQ1432 produced in media containing 140 g glucose L(-1) showed greater virulence toward whitefly nymphs (Bemisia tabaci) as compared with blastospores produced in media containing 40 g glucose L(-1). These results suggest a synergistic effect between glucose concentration and oxygen availability on changing morphology and enhancing the yield and efficacy of blastospores of B. bassiana, thereby facilitating the development of a cost-effective production method for this blastospore-based bioinsecticide.

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A major constraint to the commercial use of fungal biocontrol agents is the availability of low-cost production media and processes. Previous attempts in producing Beauveria blastospores using liquid culture fermentation processes required long fermentation times (6-8days) and produced cells that had poor survival after desiccation and storage. In this study, isolates of Beauveria bassiana and Isaria fumosorosea were evaluated for blastospore yield, desiccation tolerance, storage stability, and biocontrol efficacy using fermentation media containing acid hydrolyzed casein or cottonseed flour as the nitrogen source. Cultures of B. bassiana and I. fumosorosea grown in media containing cottonseed flour produced high blastospore concentrations (>1×10(9)mL(-1)) after 3days which is comparatively less expensive nitrogen source than acid hydrolyzed casein. The resultant air-dried blastospores (<3% moisture) of all fungal isolates survived drying (61-86% viability), irrespective of the nitrogen source tested. Storage stability at 4°C varied with nitrogen source and fungal strain. Air-dried blastospores of B. bassiana strains showed half-lives >14months in contrast to 9.2-13.1months for I. fumosorosea. Blastospores of B. bassiana and I. fumosorosea killed Bemisia tabaci whitefly nymphs faster and required lower concentrations compared with aerial conidia. Our findings support the use of liquid culture fermentation as a cost-effective process to rapidly produce high yields of stable and infective blastospores of either B. bassiana or I. fumosorosea. These results support further evaluation of blastospore sprayable formulations for the control of soft-bodied insects.

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We investigated the potential production and desiccation tolerance of microsclerotia (MS) by Brazilian strains of Metarhizium anisopliae (Ma), M. acridum (Mc) and M. robertsii (Mr). These fungi were grown in a liquid medium containing 16 g carbon l⁻¹ with a carbon:nitrogen ratio of 50:1. One hundred milliliters cultures were grown in 250 ml Erlenmeyer flasks in a rotary incubator shaker at 28 °C and 200 rpm for 5 days. Five-day-old MS were harvested, mixed with diatomaceous earth (DE) and air-dried for 2 days at 30 °C. The air-dried MS-DE granular preparations were milled by mortar + pestle and stored in centrifuged tubes at either 26 or -20 °C. Desiccation tolerance and conidia production were assessed for dried MS granules by measuring hyphal germination after incubation for 2 days on water agar plates at 26 °C and for conidia production following 7 days incubation. Yields of MS by all strains of Metarhizium were 6.1-7.3 × 106 l⁻¹ after 3 days growth with maximum MS yields (0.7-1.1 × 107 l⁻¹) after 5 days growth. No differences in biomass accumulation were observed after 3 days growth, whereas Ma-CG168 showed the highest biomass accumulation after 5 days growth. Dried MS-DE preparations of all fungal strains were equally tolerant to desiccation (≥93 % germination) and the highest conidia production was obtained by MS granules of Mc-CG423 (4 × 109 conidia g⁻¹). All MS granules showed similar stability after storage at either 26 or -20 °C for 3.5 months.

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