Advances in Metarhizium blastospores production and formulation and transcriptome studies of the yeast and filamentous growth

Author(s)

Natasha Sant'Anna Iwanicki

• Document type: Doctoral Thesis
• Press: Piracicaba.
• Institution: Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC)
• Defense date: 2020-03-31

Examining board members

Gilberto José de Moraes; Gilberto Ubida Leite Braga; Tariq Mahmud Butt; Nicolai Vitt Meyling; Claudia Barros Monteiro Vitorello

• Advisor: Italo Delalibera Junior

Abstract

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 air- dried 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 x 108 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 x 108 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 x 107 blastospores/mL. The LC50 was significantly higher for spray-dried formulation (2.42 x 107) than for the air-dried formulation (4.65 x 106) suggesting a possible detrimental effect of the former technology in fungal virulence. Comparative genome- wide transcriptome of M. anisopliae showed a clear molecular distinction between the blastospore and mycelial phases. The main physiological processes associated with up-regulated gene content in blastospores during liquid fermentation were oxidative stress, amino acid metabolism, respiration processes, transmembrane transport and production of secondary metabolites. In contrast, the up-regulated gene content in hyphae was associated with increased growth metabolism and cell wall re- organization, which underlines the specific functions and altered growth morphology of M. anisopliae blastospores and hyphae, respectively. Conversely, it was observed that the M. rileyi yeast-like cells produced in liquid medium activated a series of specific genes related to signal transduction, and specific membrane transporters related to iron acquisition which was not observed in hyphae. Oxidative stress and activation of specific heat shock proteins were key factors involved in formation of yeast-like cells. On the other hand the yeast-like phase grown in solid medium activates a set of unique genes, not found in other Metarhizium spp., specific membrane proteins and several virulence factors. Significant transcriptomic differences between the metabolism of blastospores and of hyphae were demonstrated. These findings illustrate important aspects of fungal morphogenesis in M. anisopliae and highlight the main metabolic activities of each propagule under in vitro growth conditions. The genomic studies laid the foundation for understanding the main metabolism required for blastospores growth in liquid medium and identified candidate genes that will serve as a basis for future research on optimizing M. rileyi blastospore production. (AU)

• FAPESP's process: 16/20610-6 - Advances in Metarhzium blastospores production and formulation and transcriptome study of metacyclic yeast growth
• Grantee: Natasha Sant Anna Iwanicki
• Support Opportunities: Scholarships in Brazil - Doctorate