Advances in >i<Metarhizium>/i< blastospores production and formulation and transcriptome studies of the yeast and filamentous growth

Abstract

Advances in Metarhizium blastospores production and formulation and transcriptome studies of the yeast and filamentous growth Biological control of pests is a growing market in the world. It is expected that the use entomopathogenic fungi to control pests will take an important share of this market. Most fungal products in the world are based on aerial conidia produced by solid fermentation using cereal grains. An alternative for aerial conidia is the use of blastospores, yeast-like hydrophilic cells that can be produced in large amounts by liquid fermentation in a short time (<4 days), in a small space and with low hand labor compared to the solid fermentation method. Therefore, the main objectives of the present studies were first to optimize a liquid culture medium for low cost production of Metarhizium blastopores; second: to assess the bioactivity of air-dried blastospores against the cattle-tick Rhipicephalus microplus; third: to develop an airdried and spray-dried Metarhizium blastospore formulation with bioactivity against the corn-leafhopper Dalbulus maidis; fourth: to improve the shelf-life of the best air-dried and spray-dried formulations stored in refrigerated (± 4°C) and in ambient conditions (28°C) using oxygen and moistures absorbrs or vacuum and fifth: to use comparative genome-wide transcriptome analyses to determine changes in gene expression between the filamentous and blastospore growth phases in vitro to characterize physiological changes and metabolic signatures associated with M. anisopliae and M. rileyi dimorphism. We showed that blastospore production of Metarhizium is isolate-and species-dependent. Glucose-enriched cultures inoculated with pre-cultures improved yields reaching optimal growth for Metarhizium robertsii ESALQ1426 (5.9 × 10 8 blastospores/mL) within 2 d. Resultant air-dried blastospores of ESALQ1426 were proved to quickly kill R. micropulus larvae with an efficiency comparable to that of conidia. 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 R. microplus larvae. Fermentation experiments based on growth kinetics defined a low-cost medium using 80 g/L corn steep liquor as the most suitable nitrogen source for inducing blastospore growth in M. robertsii (4.7 × 10 8 cells/mL) in only 2 days of cultivation at a total cost of $0.30 USD per L. The dried blastospores were as high virulence as fresh cells to D. maidis adults fed on maize plants. Co-formulants were selected to compose formulations that allowed to keep M. roberstii blastospore viability after drying. The most promising resulting spray-dried and air-dried were as infective as fresh blastopores to the corn leafhopper inducing mortality rates ranging from 60.3 to 78.2% after spraying with 5 × 10 7 blastospores/mL. The LC 50 was significantly higher for spray-dried formulation (2.42 × 10 7) than for the air-dried formulation (4.65 × 10 6), suggesting a possible detrimental effect of the former tech...