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.

Liquid culture fermentation for rapid production of desiccation tolerant blastospores of Beauveria bassiana and Isaria fumosorosea strains.

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.

Production of microsclerotia by Brazilian strains of Metarhizium spp. using submerged liquid culture fermentation.

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.