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Natasha Final 1202.Pdf (4.528Mb) University of São Paulo “Luiz de Queiroz” College of Agriculture Advances in Metarhizium blastospores production and formulation and transcriptome studies of the yeast and filamentous growth Natasha Sant´Anna Iwanicki Thesis presented to obtain the degreee of Doctor in Science. Area: Entomology Piracicaba 2020 UNIVERSITY OF COPENHAGEN FACULTY OF SCIENCE Advances in Metarhizium blastospores production and formulation and transcriptome studies of the yeast and filamentous growth PhD THESIS 2020 – Natasha Sant´Anna Iwanicki Natasha Sant´Anna Iwanicki Agronomic Engineer Advances in Metarhizium blastospores production and formulation and transcriptome studies of the yeast and filamentous growth Advisors: Prof. Dr. ITALO DELALIBERA JUNIOR Prof. PhD and Dr. agro JØRGEN EILENBERG Co-advisor for transcriptomic studies: Associate professor PhD HENRIK H. DE FINE LICHT Thesis presented to obtain the double-degreee of Doctor in Science of the University of São Paulo and PhD at University of Copenhagen. Area: Entomology Piracicaba 2020 2 Dados Internacionais de Catalogação na Publicação DIVISÃO DE BIBLIOTECA – DIBD/ESALQ/USP Iwanicki, Natasha Sant´Anna Advances in Metarhizium blastospores production and formulation and transcriptome studies of the yeast and filamentous growth / Natasha Sant´Anna Iwanicki. - - Piracicaba, 2020. 248 p. Tese (Doutorado) - - USP / Escola Superior de Agricultura “Luiz de Queiroz”. 1. Blastosporos 2. Fermentação líquida 3. Dimorfismo fúngico 4. Fungos entomopatogênicos I. Título 3 4 ACKNOWLEDGMENTS First, I would like to thank my supervisors, Prof. Italo Delalibera Júnior and Prof. Jørgen Eilenberg for their confidence in my potential as a student, for the opportunities they gave me and the knowledge they shared, for their guidance and friendship over these years. I also thank my co-advisors, Prof. Henrik H. De Fine Licht and Dr. Gabriel Moura Mascarin for their friendship, guidance, help in data analysis, in writing the articles, patience and motivation during my doctorate. I am grateful to have had the opportunity to work with them. I would like to extend a special thanks to Dr. Gabriel Mascarin. Gabriel became a person very close to me during my doctoral journey. His enthusiasm, positivism, determination, professionalism, unconditional support and motivation helped me a lot in times of discouragement and changed the way I faced the challenges in academia. Thank you very much Gabriel. To the endless support, patience understanding and collaboration of my parents, Ivania Sant´Anna and Jacek Lech Iwanicki, my wife Ana Luisa Martins Attie and my siblings Lara Sant´Anna Iwanicki and André Sant´Anna Iwanicki during these years. To my aunt and uncle, Danuta and Prof. Edward Potworowski (Edek) for providing me with the opportunity to participate in the Workshop on Scientific Communication in Poland and to the family support during these years. I also thanks to my Polish family for hosting me during the Workshop in Poland. A special thanks to my uncle Edek for teaching me scientific communication; for helping me to improve my writing of the scientific articles presented in this thesis and for the enormous support in the final moment of writing the thesis. I thank to the team of the Laboratory of Pathology and Microbial Control of Insects - ESALQ / USP for all the support and moments shared. A special thank for Bruna Oliveira and Sara Giro Moreno for all help, moments shared and friendship. You were my left arm during my research time in Brazil and I thank you very much. I thank the technical assistance of Marcela Paduan for helping during RNA extraction protocols and sequencing, to the spray dryer team: Fernanda Ramos, 5 always very helpful and Bianca Correa; to Anderson Silva for providing insects for bioassays; to Larissa Bueno for the friendship and support with oily formulation experiments; To the technician Lenita Maria and Professor Marcos Yassuo Kamogawa for the help in the extraction and quantification of nitrogen in liquid culture media. I thank the University of São Paulo (ESALQ) and University of Copenhagen (SOBI) for the academic structure and support offered so that I could carry out my research. To all the professors from the department of Entomology and Acarology ESALQ / USP for the sharing their knowledge during all these years. To the funding institutions: FAPESP and CAPES for the scholarship that sponsored my Ph.D research. This work was supported by FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo)[Grant: 2017/048700 and 2016/206106], CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and by Projeto Universal CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), Grant: 421629/2016-9 6 “Nie ten mocny, kto nie upada, ale kto ma siły sie podniesc” Not the strong who does not fall, but who has the strength to rise Não é o mais forte aquele que não cai, mas sim aquele que tem forças para se levantar Sentence written in the 600 years history book of the Iwanicki´s family. 7 SUMMARY RESUMO ............................................................................................................................................... 11 ABSTRACT ........................................................................................................................................... 13 SAMMENDRAG .................................................................................................................................... 15 1. Introduction...................................................................................................................................... 17 1.1. The biopesticide market .............................................................................................................. 17 1.2. The genus Metarhizium ............................................................................................................... 18 1.3. Genetics of Metarhizium spp. ..................................................................................................... 21 1.4. Study of the transcriptome and its applications ....................................................................... 22 1.5. Liquid culture fermentation and formulation of Metarhizium .................................................. 23 1.6. Corn leafhopper Dalbulus maidis ............................................................................................... 26 1.7. Cattle-tick, Rhipicephalus (Boophilus) microplus .................................................................... 27 1.8. Objectives and hypotheses ......................................................................................................... 29 1.9. Obtained results and future perspectives ................................................................................. 31 References ........................................................................................................................................... 37 2.MODIFIED ADAMEK'S MEDIUM RENDERS HIGH YIELDS OF Metarhizium robertsii BLASTOSPORES THAT ARE DESICCATION TOLERANT AND INFECTIVE TO CATTLE-TICK LARVAE ................................................................................................................................................ 48 Abstract ................................................................................................................................................ 49 2.1 Introduction.................................................................................................................................... 49 2.2. Materials and methods ................................................................................................................ 52 2.2.1. Isolate selection ........................................................................................................................ 52 2.2.2. Screening isolates for blastospores production in Adamek´s medium .............................. 52 2.2.3. Blastospores production in modified adamek’s medium ..................................................... 53 2.2.4. Glucose-enriched medium with and without preculture ....................................................... 54 2.2.5. Stabilization of blastospores in air-drying system ................................................................ 54 2.2.6. Bioassay with stabilized blastospores against cattle tick larvae ........................................ 55 2.2.7 Statistical analyses .................................................................................................................... 56 2.3. Results ........................................................................................................................................... 56 2.3.1. Screening isolates for blastospore production in Adamek’s medium ................................ 56 2.3.2. Blastospore production on glucose-enriched medium ........................................................ 58 2.3.3. Blastospore production by pre-culture followed by cultivation in glucose-enriched modified adamek’s medium ............................................................................................................... 59 2.3.4. Bioassay with stabilized blastospores against cattle tick larvae ........................................ 61 2.4. Discussion .................................................................................................................................... 64 References ..........................................................................................................................................
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