PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS BIOLÓGICAS (ÁREA: MICROBIOLOGIA APLICADA)

IFELOJU DAYO-OWOYEMI

TAXONOMIC ASSESSEMENT AND BIOTECHNOLOGICAL POTENTIAL OF YEASTS HOLD AT THE UNESP - CENTRAL FOR MICROBIAL RESOURCES

Rio Claro 2012

TAXONOMIC ASSESSEMENT AND BIOTECHNOLOGICAL POTENTIAL OF YEASTS HOLD AT THE UNESP –CENTRAL FOR MICROBIAL RESOURCES

IFELOJU DAYO-OWOYEMI

Thesis presented to the Institute of Biosciences, Universidade Estadual Paulista ´´Julio de Mesquita Filho``- Rio Claro, in fulfilment of requirements for the award of Doctor of Philosophy in Biological Sciences (Applied Microbiology)

Supervisor: Prof. Dr. Fernando Carlos Pagnocca

Co-supervisor: Prof. Dr. André Rodrigues

Rio Claro

2012

DEDICATION

To the memory of my loving father, Victor Adedayo Owoyemi

´If I have seen further it is by standing on the shoulders of

giants``

Sir Isaac Newton

Acknowledgement

Completing my PhD was a long and challenging task. Many people supported and encouraged me in so many different ways during the process; it is therefore my pleasure to thank those who helped to see my dream come true.

First, I thank the Almighty God, the true source of wisdom and knowledge, for his immense love and infinite mercy towards me. ´´A man has gotten nothing except he be given from above``; I appreciate the rare door of opportunity He opened for me and also for the strength and inspiration given to me for the successful completion of this work.

I thank my supervisor Prof. Dr. Fernando Carlos Pagnocca for the wonderful opportunity he gave me in his laboratory. I am grateful to him for believing in me. I appreciate the fatherly role he also played during my stay and studies. I am also indebted to Dr Andre Rodrigues, who apart from being my co-supervisor also played the role of a colleague and brother. ``If I have seen further it is by standing on the shoulders of giants``, together, I thank him and my supervisor very much; for investing their time, resources and wealth of knowledge in me. Most importantly, I appreciate them for contributing to securing a promising future for me.

I thank the Brazilian government through the National Council of Technological and Scientific Development (CNPq) as well as the Third World Academy of Science (TWAS) for the PhD fellowship awarded me and for the sponsorship of my education. I also aknowledge Universidade Estadual Paulista “Julio de Mesquita Filho” for the quality education I received and for all the material and infrastructural resources that were made available during the course of my work. I wish to equally use this opportunity to thank the good people of Brazil whose hard earned resources were used to give me an opportunity of quality education.

I have been fortunate to have worked with kind and generous group of people whose emotional support and enthusiasm have contributed to the success of my work. Space will not permit me to mention all their names. However, I thank all of them for their generosity, especially, Virginia Elina Masiullionis, Silvio Lovato Arcuri (and his fiancée Clara), Thais Demarchi Mendes, Weilan Paixão Gomes (and her fiance Thiago Gazone), Paula Sanchez de Sousa, Tatiana de Carvalho, Aline da Silva Cruz, Aline Bartelolochi Pinto, Dr. Francisco Eduardo de Carvalho Costa, Samuel, Dirce, Fabio, Liu, Lydia (Berinjela), Sadala and Lucas and Rafael.

Several other lecturers and non lectures as well as colleagues also contributed directly or indirectly to the success of my career and their contributions are well appreciated. They include Dra. Sandra Mara Martins Franchetti, Dr. Lara Durães Sette, Prof. Dr. Vanderlei Martins, Prof. Dr. Jonas Conteiro, Necis Lima, Rosemary D. Oliveira S. Cardoso, Josiele Fernanda Magri, Dr. Mauricio Bacci Jr., Joaquim Martim Jr., Mileni Ferro, Cynara, Lusiana, Alexandre, Alex and Paulo, Dr. Bolatito Boboye, Dr. Victor Oyetayo, Engineer and Mrs Taiwo Mogaji, Dr Mathias C. Ahii, Dr. Adebayo Adeyemo and Dr Olubunmi Adebayo.

This note of acknowledgement would be incomplete if I fail to appreciate Dra. Derlene Attili de Angelis and her wonderful family. I am immensely grateful and specially thank them for being a family away from home. I appreciate Dra. Dejanira de Franceschi de Angelis, my Brazilian grandmother, for her constant love and concern for me.

A special appreciation goes to my husband Olusegun Folaring Jonah. I would not have successfully completed this work without his support and encouragement. I greatly appreciate his unwavering love, trust, patience and understanding. I am grateful to him for standing solidly by me throughout the course of this work.

Finally, my utmost appreciation goes to my parents Victor Adedayo Owoyemi (Late) and Philomena Dayo-Owoyemi. I am forever grateful to both of them for the sacrifice they made to give me a good moral and educational foundation. I thank them for their financial and spiritual supports, without which I would not have been able to attain this height. I am deeply grateful to my brother and sister Bolaji and Omolola respectively, and also to my uncle Kolade Ogunmolade for their love, care and encouragements.

RESUMO Atualmente, existe um crescente interesse em explorar diversos habitats, a fim de revelar a biodiversidade microbiana, incluindo as leveduras. Tal diversidade ainda não acessada guarda a descoberta de novas espécies para ciência, provavelmente muitas das quais com potencial para aproveitamento em processos biotecnológicos. Com o objetivo de explorar e conservar a diversidade de fungos, o Central de Recursos Microbianos da UNESP (CRM – UNESP) mantém em seu acervo várias estirpes de leveduras isoladas de ecossistemas diversos, sendo alguns deles pouco explorados. No início deste trabalho sabíamos que muitas das leveduras depositadas no acervo do CRM – UNESP não estavam totalmente caracterizadas tanto em nível taxonômico, quanto em relação ao potencial biotecnológico que poderiam apresentar. Portanto, o presente estudo foi desenhado para caracterizar e identificar taxonomicamente leveduras depositadas no CRM – UNESP, bem como selecionar estirpes que produzem enzimas extracelulares degradadoras de polissacarídeos como amilase, celulase, xilanase, pectinase e ligninase. Usando uma abordagem polifásica, um total de 340 isolados de leveduras foi identificado, sendo que 71,2% compreendem 43 taxa de ascomicetos e os restantes 28,8% foram classificados em 27 taxa de basidiomicetos. O estudo também levou à descoberta de 8 prováveis novas espécies. Baseado nesta constatação, a classificação taxonômica e análise filogenética foi realizada para duas espécies anamórficas de ascomicetos e uma espécie teleomórfica de basidiomiceto. A descrição destas três espécies é apresentada neste estudo. Os resultados demonstraram que Wickerhamiella kiyanii FB1-1DASPT e W. pindamonhangabaensis H10YT pertencem à clade Wickerhamiella da ordem Saccharomycetales (Ascomycota: Saccharomycetes), enquanto que a espécie Bulleromyces texanaensis ATT 064T pertence à clade Bulleromyces / Papiliotrema / Auriculibuller da ordem Tremellales (Basidiomycota: Agaricomycotina). Num outro estudo, demonstramos a variabilidade intraespecífica em onze (11) isolados de Hannaella kunmingensis (incluindo a type strain CBS 8960T). Essas onze estirpes foram obtidas de substratos e locais diferentes e analisamos sua variabilidade fisiológica e genética. Ainda, usando uma combinação de análise filogenética e de rede parcimônia, demonstramos o grau da divergência genética (região espaçadora intergênica (ITS) e o gene do citocromo b) dentro desta espécie. Os nossos resultados revelaram variabilidade elevada das características morfológicas e bioquímicas, assim como a existência de três haplótipos genéticos em H. kunmingensis. Uma das estirpes (CBS 8356) apresentou uma divergência de 27,3% das outras linhagens no gene citocromo b, sugerindo a possibilidade de especiação

desta estirpe. Este trabalho mostrou características que não foram previamente descritos em H. kunmingensis, e assim pudemos contribuir para a emenda referente à descrição desta espécie, procedimento necessário para acomodar as novas descobertas. Além disso, a partir da triagem das enzimas extracelulares de 312 estirpes, foi detectada a atividade de amilase em 28 estirpes (8,95% do total), celulase em 64 estirpes (20,51%), xilanase em 87 estirpes (27,88%), poligalacturonase em 45 estirpes (14,42%), pectina liase em 59 estirpes (18,91%) e ligninolítica em 2 estirpes (0,64%). As enzimas celulase, amilase, xilanase foram as mais encontradas entre as leveduras basidiomicetas; enquanto que as leveduras ascomicetas foram maiores produtores de pectinases. A determinação da produção de endoxilanase e β- xilosidase de 73 estirpes degradadoras de xilana levou à descoberta de três estirpes que demonstraram elevada produção de amilase, celulase, xilanase e pectinase na presença de bagaço de cana como substrato, indicando que elas são boas candidatas para as pesquisas envolvendo a produção de enzimas úteis na conversão de biomassa vegetal em bioetanol. No geral, este estudo revelou que o CRM – UNESP abriga um acervo de leveduras diversas, com capacidade de produzir várias enzimas industrialmente úteis. Estas leveduras poderiam ser aproveitadas para futuras aplicações biotecnológicas. Além disso, o acervo do CRM – UNESP também provou ser uma fonte de conservação de várias espécies novas para ciência, o que reflete a importância desse tipo de conservação ex-situ para o estudo da biodiversidade microbiana.

Palavras chaves: biodiversidade, coleção de cultura, ascomicetos, basidiomicetos, polissacarídeos, enzimas.

ABSTRACT

In recent time, there has been an increasing interest in exploring diverse ecological habitats in order to reveal the yeast biodiversity. The increased awareness in the biotechnological potentials of yeasts has also spurred attempts to search for new species with novel biotechnological capabilities. Aiming to explore and conserve the fungal diversity from various ecosystems, the UNESP – Central for Microbial Resources (UNESP – CMR) harbors various strains of ecologically diverse yeasts isolates, some of which were yet to be identified. Therefore, this study was designed to identify and characterize some yeasts from the UNESP – MRC and to select strains possessing extracellular plant polysaccharide degrading enzymes namely amylase, cellulase, xylanase, pectinase and ligninase. Using a polyphasic approach, a total of 340 strains were identified. Taxonomic classification grouped 71.2% of these isolates into 43 ascomycetous taxa while the remaining 28.8% were classified in 27 basidiomycetous taxa. The study also led to the discovery of 8 putative new species. As a result, we classified two anamorphic species in the Ascomycota and one teleomorphic species in the Basidiomycota. In this study we provide the description of both species. Our results demonstrated that the two ascomycetous species proposed as Wickerhamiella kiyanii FB1-1DASPT and W. pindamonhangabaensis H10YT belong to the Wickerhamiella clade of the Saccharomycetales (Saccharomycetes) while the basidiomycetous species proposed as Bulleromyces texanaensis ATT064T belong to the Bulleromyces / Papiliotrema / Auriculibuller clade of the Tremellales (Agaricomycotina). In order to show the significance of intraspecific diversity in yeasts, in one of our studies, we subjected 11 strains, (including the type strain CBS 8960T) of Hannaella kunmingensis, obtained from different substrates and geographic locations, to detailed physiological and genetic characterization. Using a combination of phylogenetic and parsimony network analysis, we demonstrated the extent of genetic (internal transcribed spacer region (ITS), D1/D2 domains of the large subunit rDNA (LSU), and cytochrome b gene) divergence within this species. Our findings revealed the high variability of morphological and biochemical characteristic as well as the existence of 3 genetic haplotypes in H. kunmingensis. One of the strains (CBS 8356T) exhibited a 27.3 % divergence from the other strains in the cytochrome b gene; hence, we concluded the possibility of speciation of this strain. This work led to the discovery of additional strains and characteristics not previously reported in H. kunmingensis, therefore, the emendation of H. Kunmingensis was done to accommodate the new discoveries. Furthermore, from the screening of 312 yeast strains for secreting extracellular enzymes, amylase activity was detected in 28 strains representing 8.97% of the total isolates screened; cellulase activity in 64 strains (20.51%), xylanase activity in 87 strains

(27.88%), polygalacturonase activity in 45 strains (14.42%), pectin lyase activity in 59 strains (18.91%) and lignolytic activity in 2 strains (0.64%). This study further revealed that amylase, cellulase and xylanase are the major enzymes found among the basidiomycetous yeasts while ascomycetous yeasts are producers of pectinases. Determination of extracellular endoxylanase and β-xylosidase activities in culture supernatants of 73 xylanase positive strains led to the discovery of three strains which demonstrated high amylase (endo- and exomylase), cellulase, xylanase and pectinase activities in presence of sugar cane bagasse; therefore are good candidates for research involving production of enzymes useful in biomass conversion. Overall, this study revealed that UNESP – MRC possess metabolically diverse yeasts with ability to produce various industrially useful enzymes. Such strains could be harnessed for future biotechnological applications. In addition, the UNESP – MRC proved to harbors new species to science that are now preserved ex-situ of long-term maintenance.

Keywords: biodiversity, culture collection, ascomycetes, basidiomycetes, polysaccharides, enzyme

TABLE OF CONTENTS

INTRODUCTION 19 Aims and objectives 21

STUDY OUTLINE 23

CHAPTER 1 25 BACKGROUND OF STUDIES AND LITERATURE REVIEW 1 BACKGROUND OF STUDIES AND LITERATURE REVIEW 26 1.1 UNESP – Central for Microbial Resource (UNESP-CMR) 26 1.2 Importance of culture collections to microbiology and biotechnology 30 1.3 Yeast: introduction and definition 32 1.4 Evolution of yeast identification methods 32 1.5 Yeast classification 36 1.6 Yeast ecology and diversity 42 1.7 Biotechnological importance of yeasts 47 1.8 Biodegradation of starch, lignocelluloses and pectin 47 REFERENCES 57

CHAPTER 2 69 TAXONOMIC STUDIES OF YEASTS HOLD AT UNESP – CENTRAL FOR MICROBIAL RESOURCES 2.1 Abstract 70 2.2 Introduction 71 2.3 Material and methods 72 2.3.1 Cultural Characterization 72 2.3.2 Molecular Identification 74 2.4 Results 76 2.5 Discussion 79 REFERENCES 85

CHAPTER 3 88

SCREENING FOR AMYLOLYTIC, LIGNOCELLOLYTIC AND PECTINOLYTIC YEASTS 3.1 Abstract 89 3.2 Introduction 90 3.3 Materials and methods 91 3.3.1 Screening procedures for extracellular enzymatic activities 91 3.3.2 Amylase activity 93 3.3.3 Cellulases 93 3.3.4 Xylanases 93 3.3.5 Pectinases 94 3.3.6 Ligninase 94 3.3.7 Xylanase enzymes assays 94 3.3.8 Fermentation of sugarcane bagasse 96 3.3.9 Enzyme assays 96 3.3.10 Statistical analysis 98 3.4 Results 98 3.4.1 Screening for enzymatic activities 98 3.4.2 Xylanase (endoxylanase and β-xylosidase) assays 106 3.4.3 Extracellular enzyme production from sugar cane bagasse fermentation 110 3.5 Discussion 112 3.5.1 Enzymatic activity profile 112 3.5.2 Enzyme production from sugar cane baggase 113 REFERENCES 115

CHAPTER 4 118 WICKERHAMIELLA KIYANII SP. NOV. AND W. PINDAMONHANGABAENSIS SP. NOV., TWO ANAMORPHIC YEASTS ISOLATED FROM NATIVE PLANTS OF THE SOUTH EASTERN ATLANTIC RAINFOREST OF BRAZIL 4.1 Abstract 119 4.2 Introduction 120 4.3 Material and methods 121 4.4 Results and discussion 123 4.5 Description of Candida kiyanii Pagnocca, Rosa, Dayo-Owoyemi and Rodrigues sp. nov. 128

4.6 Description of Candida pindamonhangabaensis Pagnocca, Rosa, Dayo-Owoyemi and Rodrigues sp. nov. 129 REFERENCES 131

CHAPTER 5 134 DESCRIPTION OF BULLEROMYCES TEXANAENSIS SP. NOV., ISOLATED FROM FUNGUS GARDEN OF THE LEAFCUTTER ANT ATTA TEXANA AND LEAVES OF BROMELIAD NEOREGELIA CRUENTA (BROMELIACEAE) 5.1 Abstract 135 5.2 Introduction 136 5.3 Materials and methods 137 5.3.1 Strain information 137 5.3.2. Morphological and phenotypic characterization 138 5.3.3 DNA extraction, Sequence and phylogenetic analyses 138 5.4 RESULTS AND DISCUSSION 139 5.4.1 DNA sequence and phylogenetic analysis 139 5.5 Description of Bulleromyces texanaensis Dayo-Owoyemi, Rodrigues, Garcia, Hagler and Pagnocca sp. nov. 144 5.5.1 Growth on YM broth 144 5.5.2 Growth on YM agar 144 5.5.4 Dalmau plate culture on corn meal agar 144 5.5.5 Formation of ballistoconidia 144 5.5.6 Sexual reproduction 144 5.6 PHENOTYPIC DESCRIPTION 147 5.7 Origin of the strains studied 147 5.8 Systematics and Ecology of Bulleromyces texanaensis 147 References 151

CHAPTER 6 154 Intraspecific variation and emendation of Hannaella kunmingensis CONCLUSIONS AND PERSPECTIVES 164

ABBREVIATIONS

°C – Degree centigrade

CBS – Centraalbureau voor Schimmelcultures

CMCase – Carboxymethyl cellulase

DBB – Diazonium blue B

DNA – Deoxyribo nucleic acid g. – Gravitational force g.L – Gram per Liter gm – gram

ITS – internal transcribed spacers

LSU – Large subunit

MCase- Microcrystalline cellulase

M – Molar min – minutes mM – Milimolar

MSP-PCR – Microsatellite Primed Polymerase chain reaction.

NCBI – National Center for Biotechnology Information

SNA – Synthetic nutrient agar

TCS – Parsimony network analysis

UNESP – CMR – Universidade Estadual Paulista – Central for Microbial Resources

UV – Ultraviolet

YMA – Yeast malt agar

LIST OF FIGURES

Figure 1.1 Map of fungi rRNA gene showing the internal transcribed spacer (ITS) region, intergenic spacer (IGS) regions,18S small subunit (SSU) and 25-28S large subunit (LSU) 34

Figure 1.2 Phylogeny of the phylum Ascomyceota showing the classification of Ascomycetous yeasts . 38

Figure 1.3 Phylogeny of the phylum Basidiomycota showing the classification of Basidiomycetous yeasts 40

Figure 1.4 Plant cell wall structure. 48

Figure 1.5 Action of major cellulase enzymes 51

Figure 1.6 Action of major enzymes involved in the depolymerization of Xylan 52

Figure 1.7 Action of major enzymes involved in the deconstruction of pectin. 53

Figure 1.8 Scheme showing the actions of lignin degrading enzymes 55

Figure 1.9 Scheme showing the actions of starch degrading enzymes 56

Figure 2.1 Growth (assimilation) test on carbon compounds 77

Figure 2.2 PCR fingerprinting patterns of some identified strains 77

Figure 3.1 Degradation halos around yeast strains producing 100 amylase (A), cellulose (B), xylanase (C), polygalacturonase (D), pectin lyase (E) and ligninase(F).

Figure 3.2 Enzymatic activity profiles of the ascomycetous and basidiomycetous 103 yeasts screened

Figure 3.3 Comparisons of enzyme activity profiles of ascomycetous and basidiomycetous yeasts respectively 104

Figure 3.4 Extracellular enzyme production from sugar cane bagasse fermentation by Aureobasidium pullulans strain CG5-5BY, Aureobasidium pullulans strain PBM1and Pseudozyma hubeiensis strain MP2-2CB 111

Figure 4.1 Phylogenetic placement of Candida kiyanii and C. pindamonhangabaensis in the Wickerhamiella clade

(Saccharomycetes, Saccharomycetales) determined from Neighbor-joining analysis of sequences from LSU rRNA gene. Bootstrap values are from 1000 replicates. T = type species. 131

Figure 4.2 Candida kiyanii (A) and Candida pindamonhangabaensis (B and C). Phase contrast micrograph showing budding cells (A and B) with pseudomycelium after 3 days at 25 °C on YM agar (A) and on corn meal agar 25 °C (C). 126

Figure 4.3 Lipase activity test of Cpindamonhangabaensis (upper colonies) and C. kiyanii (lower colonies). 128

Figure 5.1 Evolutionary tree showing the relationships of Bulleromyces texanaensis and related species based on combined LSU and ITS sequences. 141

Figure 5.2 Phylogenetic relationships of strain Bulleromyces texanaensis and other closely related species based on LSU D1/D2 rRNA gene sequences. 142

Figure 5.3 Phylogenetic relationships of strain Bulleromyces texanaensis and other closely related species based on ITS sequences. 143

Figure - 5.4 Growth phases of Bulleromyces texanaensis 145

LIST OF TABLES

Table 1.1 Some major international microbial culture collections, their acronyms and type of culture holding 27

Table 1.2 Quality control procedures recommended for microorganisms (OECD, 2007) 29

Table 1.3 Industrial applications of some enzymes 49

Table 3.1 Number and origin of yeasts and dimorphic fungi profiled for enzymatic activity 92

Table 3.2 Ascomycetous yeasts screened for amylolytic and lignocellulolytic and pectinolytic activities 100

Table 3.3 Basidiomycetous yeasts screened for amylolytic and lignocellulolytic and pectinolytic activities 102

Table 3.4 Xylanase activities in extracellular and cell wall associated cell free supernatant of selected strains 107

Table 3.5 Enzyme yield per gram of substrate 112

Table 4.1 Extent of D1/D2 LSU rDNA and ITS sequence divergences of C. kiyanii and close relatives pairwise some based on alignment. 125

Table 4.2 Physiological characteristics differentiating C. kiyanii from closely related strains 125

Table 5.1 Phenotypic characteristics of strain Bulleromyces texanaensis CBS 11955T 148

LIST OF APPENDIX

Appendix 1 Identities of yeasts and dimorphic fungi maintained at UNESP – CMR 166

Appendix 2 Result of extracellular enzyme screening with some yeasts in the UNESP - Central for Microbial Resources 179

Appendix 3 Statisitical (one-way anova) analysis of reducing sugars (RS) produced from sugar cane bagasse fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM 1 and Aureobasidium pullulans strain CG5-5BY 192

Appendix 4 Statisitical (one way anova) analysis of activitiy of endoamylase produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM 1and Aureobasidium pullulans strain CG5-5BY (using 0.5% starch) 194

Appendix 5 - Statisitical (one way anova) analysis of activitiy of exoamylase produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM 1 and Aureobasidium pullulans strain CG5-5BY (using 1.0% starch) 196

Appendix 6 Statisitical (one way anova) analysis of activitiy of pectinase produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM 1and Aureobasidium pullulans strain CG5-5BY 198

Appendix 7 Statisitical (one way anova) analysis of activitiy of xylanase produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM1and Aureobasidium pullulans strain CG5-5BY 200

Appendix 8 Statisitical (one way anova) analysis of activitiy of carboxymethyl cellulase (CMCase) produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM 1 and Aureobasidium pullulans strain CG5-5BY 202

Appendix 9 Statisitical (one way anova) analysis of activitiy of microcrystaline cellulase (MCase) produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM 1 and Aureobasidium pullulans strain CG5-5BY 204 19

INTRODUCTION

20

INTRODUCTION

Since the last decade, the destruction of the natural ecosystem by human activities and changing global climatic conditions has raised concerns to microbiologists and ecologist as to the endangering of microorganisms. Such events are leading to gradual microbial species extinction, hence reduction of microbial diversity. According to Hibbett et al. (2011) more than 100,000 fungal species have been described to date. Although, the exact number of extant fungal species is not known, several estimatives were raised by mycologists including an an estimate of 712,000 extant species elaborated by Schmit and Mueller (2007). However, recent estimates based on high-throughput sequencing methods suggest that as many as 5.1 million fungal species exist (BLACKWELL, 2011). Authors trying to estimate the number of extant yeast species usually conclude that more than 98% of yeasts are yet undiscovered. Given the average number of fungal species described each year since 1999 to be about 1200, Hibbett et al. (2011) predicted that at the current rate of discovery, it may take up to 4000 years to describe all fungal species. Since, local extinctions (i.e. strong reductions in the abundance of microbial species) occur quite frequently due to clearing of forests, agricultural activity or erupting volcanoes, it was predicted that microbial extinction rate might soon surpass the recovery rate of extant undescribed species. Effort towards microbial diversity recovery has included sampling of microorganisms from various ecological habitats, especially those whose biodiversity are been endangered and development of culture collections by private laboratories, institutions and universities for ex situ conservation. The urgent need therefore arises for biodiversity and taxonomic studies with emphasis on the discovery, classification and description of novel species. Due to their wide application in various industrial processes, the demands for plant polysaccharide degrading enzymes including amylases, cellulases, xylanases, pectinases, and ligninases, have greatly increased in recent times. For example, the primary enzymes used in animal feed are xylanases, β-glucanases (and phytases) because they aid in digestion of polysaccharides in monogastric animals. Hydrolysis of carbohydrate polymers such as cellulose, xylan and starch is used to produce fermentable sugars for bio-ethanol production. Ethanol production from lignocellulosic has been identified as a cheaper alternative for the sustainable fuel ethanol. The complete degradation of the cell wall of lignocellulosic materials to fermentable sugars requires the contribution of lignocellulolytic enzymes namely cellulases, hemicellulases and ligninase enzymes. Furthermore, in the fermentative production of ethanol from starch, amylolytic enzymes are routinely added as pretreatment of the 21

starch to convert it to linear oligomers and ultimately to glucose units, that are then fermented by yeasts. Xylan is the most abundant hemicellulose and xylanases are one of the major hemicellulases which hydrolyse the β-1,4 bond in the xylan to short xylooligomers which are further hydrolysed into single xylose units by β-xylosidase. Microbial xylanases are becoming more demanding due to their wide application in various industrial sectors. In the energy sector, one area of considerable importance is the enhanced production of ethanol through the release of substantial amount of fermentable feedstock (PÉREZ et al., 2002; ALMEIDA et al., 2007). In the step involving the conversion of hemicelluloses to fermentable sugars, some of the hemicelluloses, mostly xylan, remain associated with the cellulosic-rich water insoluble fraction (CHANDRA et al., 2007). Because, effective enzymes capable of digesting these woody materials are still lacking, in order to improve cellulose accessibility, hence, enhance substrate digestibility, cellulose enzymes are often supplemented with xylanases as ‘accessory enzymes’ (KUMAR; WYMAN, 2009). Hence, the demand for xylanase producing microorganisms has increased. One of the main areas of research in enzyme biotechnology has been driven by the need to isolate and identify organisms which are good producers of plant polysaccharides (lignocelluloses) degrading enzymes. In contrast to fungi and bacteria, few types of yeast are known to be capable of degrading lignocelluloses. Ability of yeasts to utilize plant polysaccharides is important because such information could be useful for taxonomic classification as well as biotechnological applications. Therefore, more information is needed about yeasts possessing these characteristics. However, it is known that yeasts are highly diverse in terms of nutrition, exploitation of ecological niches and secondary metabolism (this diversity reflects in their wide biotechnological applications in various industrial sectors such as food, beverages, chemicals, industrial enzymes, pharmaceuticals agriculture and environment) and that microbial diversity is the foundation for biotechnology; the basis for the discovery of new products, secondary metabolites and genes. Microbial culture collection serves as a pool where metabolically and genetically diverse yeast strains with unique properties and applications could be discovered.

Aims and objectives

This study was designed to meet the general objective of providing accurate taxonomic identification to some unidentified yeasts hold at the UNESP – Central for Microbial Resources (UNESP – CMR) situated at the Institute of Biosciences, Rio Claro, 22

State of São Paulo as well as to predicting biotechnological utility of newly discovered taxa and already existing taxa making up this collection.

The specific objectives of this research therefore were:

(i) to identify and characterize some yeast and yeast-like organisms at the UNESP – Central for Microbial Resources of the Institute of Biosciences, Rio Claro. (ii) to describe novel species in the culture collection (iii) to identify yeast and yeast-like organisms with ability to produce lignocellulose degrading enzymes namely: cellulases, hemicellulases and ligninase enzymes. (iv) to quantitatively determine the endo-xylanase and β-xylosidase activities of selected xylanase producing strains discovered in this study and determination afterwards of polysaccharide degrading enzymes produced from sugar cane bagasse fermentation. 23

STUDY OUTLINE

The primary objectives of this study were to provide accurate identification of some yeasts hold at the UNESP – CMR with emphasis on classifying and describing new strains; as well as to provide information about the applicability in enzyme technology of some yeasts in this collection, particularly plant polysaccharide degrading enzymes namely amylases, cellulases, xylanases and pectinases. Here we organized the study into chapters with the first one being a brief background while the subsequent chapters present investigations and findings relating to the theme of the study. The chapter opener begins with a brief introduction of the UNESP – CMR and how its activities have contributed to broadening the knowledge of fungal diversity. It also present reviews of literature about the definition of yeasts and how advancements in molecular biology have shaped the methods used for yeast identification. Because the yeasts examined in this work were isolated from different habitats during different ecological studies, effort was laid on briefly reviewing some roles played by yeasts in these habitats. Furthermore, this chapter highlights the enzyme systems involved in the degrading of plant polysaccharides and yeasts found from previous studies to possess these enzymes. Chapter 2 presents taxonomic studies of yeasts hold at UNESP – CMR. Basically, this study involved the identification and classification of 340 yeast isolates obtained from various ecological studies embarked upon by the UNESP – CMR research team using a polyphasic approach. The identification process led to the discovery of several undescribed fungal species including two ascomycetous species whose novel status were proposed in the genus Candida (n = 2) and a basidiomycetous species proposed in the genus Bulleromyces. The findings of this study also revealed that the UNESP – CMR collection holds many metabolically and genetically diverse types of yeasts that could be harnessed for further biotechnological studies. Chapter 3 reports screening of the UNESP – CMR for yeasts possessing enzyme systems for the degradation of plant polysaccharides specifically starch, cellulose, xylan, pectin and lignin. These enzymes are useful in various industrial applications including the production of biofuels. Because few types of yeast are still known to produce plant polysaccharidases, the study aimed at screening to cover a wide taxonomic range (93 taxa) of yeasts. Three hundred and twelve strains were screened out of which 192 were strains identified in the previous study (Chapter 2). This study led to the discovery of many types of yeast possessing enzyme systems for the degradation of plant polymers. It also revealed 24

among other things that xylanase and cellulase activities are characteristics more expressed by basidiomycetous yeasts whereas, pectin degrading activities is more linked to ascomycetous yeasts. In addition, this study revealed three strains capable of producing amylases, celluloses, and pectinases at high levels from sugarcane bagasse fermentation. Chapter 4 and 5 presents the description of three novel species discovered in this study. Two anamorphic ascomycetous species namely Wickerhamiella kiyanii (strain FB1- 1DASPT) and Wickerhamiella pindamonhangabaensis (strains H10YT and H10-10AY) were proposed and their taxonomic descriptions as well as systematic classifications are provided in chapter 4. The presence of lipase enzyme systems was demonstrated in the latter species. These two species were found to phylogenetically belong to the Wickerhamiella clade (Saccharomycetes, Saccharomycetales). On the other hand, chapter 5 presents the description of a teleomorphic species proposed in the genus Bulleromyces. These two chapters contribute to our knowledge of yeast diversity. The description of these species will permit the deposition of their holotypes as well as their nomenclatural information in internationally recognized and publicly accessible culture collections as well as the official publication of their names. Finally, chapter 6 presents a study on the intraspecific variation and emendation of Hannaella kunmingensis. Our study so far with yeasts has been revealing the existence of intraspecific genetic and phenotypic variations among different strains of yeasts belonging to the same species. In one of such scenarios, three strains of yeasts from the UNESP – CMR were found to differ by 11 nucleotide substitutions in the internal transcribed spacer (ITS) region from H. kunmingensis (CBS 8960T), but based on their conspecificity in the D1D2 domains of the large subunit ribosomal DNA gene, the 3 strains were considered as H. kunmingensis species. The later discovery of sequences of 7 other similar strains deposited in the GenBank offered the opportunity to intensively study the intraspecific variation in genetic as well as phenotypic properties of this species. Using parsimony network analysis, the presence of three genetic haplotypes in H. kunmingensis was demonstrated. The study also revealed variations in morphological characteristics as well as biochemical characteristics among the 11 strains studied. Based on these findings, an emendation of H. kunmingensis species was carried out. Besides contributing to the knowledge of the intraspecific diversity, the study contributed to increasing the number of strains of H. kunmingensis, which was formely described based on a single strain.

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CHAPTER 1

BACKGROUND OF STUDIES AND LITERATURE REVIEW 26

1. BACKGROUND OF STUDIES AND LITERATURE REVIEW

The increasing awareness of the importance of biodiversity and the hidden genetic potential has resulted in a rise in recognition of the value of microbial culture collections. From the early days of biodiversity surveys and fieldtrip collections, microbiologists have been gathering samples and evidences related to their discoveries. In order to identify and perform more advanced investigations and analyses on their collected specimens, they had to be kept alive and maintained in a condition as close as possible to their original states. When properly preserved, microbial strains can maintain the same properties found in nature and can therefore be reused in many different types of studies, such as physiology, genetics or applied biotechnology. This is where the idea of culture collections came into being. Microbial culture collections are living libraries and reference sources of microorganisms. In the past, due to the lack of adequately functioning and reliable culture banks, many microbial cultures were lost (MAHILUM-TAPAY, 2002). The first scientist to realize the importance of culture collection was Professor Frantisek Kral (1846-1911) who collected cultures and made it available for free to other researchers. His collection was later transferred to Vienna in 1915 (MALIC; CLAUSE, 1987). The Centraalburreau voor Schmmelcultures (CBS) culture collection, Netherlands, is the next oldest collection been founded in 1906. A list of some major culture collections in various countries is given in Table 1.1.

1.1 UNESP Central for Microbial Resources (UNESP- CMR),

In year 2006, the Microbiology laboratory (LAM) of the Institute of Biosciences, Rio Claro, developed a private collection of microorganisms, mainly yeasts, filamentous fungi and actinomycetes formerly known as Center for the Study of Social Insects (CEIS) UNESP campus of Rio Claro. Recent institutionalization of this culture collection led to its renaming as “Central for Microbial Resources (UNESP- CMR)", according to the Ordinance of the Institute of Biosciences / São Paulo State University (IB / UNESP) No. 4/2013. The UNESP - CMR's mission is to act as a Centre of Biological Resources, as defined by the OECD (OECD 2007). Currently, the UNESP- CMR holds a total of approximately 5,100 cultures, consisting of 4000 yeasts and filamentous fungi derived from samples of nests of ants and

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Table 1.1 - Some major international microbial culture collections, their acronyms and type of culture holding

Culture collection (acronym) Patent institute Type of culture Location Web address Agricultural Research Service Culture National Center Actinomycetes, Bacteria, Fungi Peoria, Illinois USA http://nrrl.ncaur.usda.gov Collection (NRRL) for Agricultural Utilization Research Moscow Region, All-Russian Culture Collection (VKM) Russian Academy of Sciences Bacteria, Archaea, Fungi http://www.vkm.ru Pushchino, Russia American Type Culture Collection (ATCC) American Type Culture Collection Bacteria, Archaea, Fungi, viruses Manassas, Virginia USA http://www.atcc.org Brazilian Collection of Environmental and Bacteria, Filamentous fungi and http://webdrm.cpqba.unicamp.br University of Campinas, São Paulo Campinas, Brazil Industrial Microorganisms Yeast /cbmai/ Belgian Coordinated Collections of Micro- Belgian Coordinated Collections of Bacteria and Fungi Various cities, Belgium http://bccm.belspo.be/index/php Organisms (BCCM) Micro-Organisms Centraalbureau voor Schimmelcultures Institute of Royal Academy, Art and Utrecht, Fungi http://www.cbs.knaw.nl (CBS) Science The Netherlands German Collection of Microorganisms and Leibniz-Institute Bacteria, Fungi and Plant Viruses Braunschweig, Germany http://www.dsmz.de/home.html Cell Culute (DSMZ)

Japan Collection of Microorganisms (JCM) RIKEN BioResource Center Bacteria, Archaea, Fungi Saitama, Japan http://www.jcm.riken.jp

National Collection of Yeast Cultures Food Research Institute, Colney Lane, Yeasts and other known Norwich, http://www.NCYC.co.uk (NCYC) Norwich NR47UA, Norfolk, UK pathogens United Kingdom National Institute of Technology Evaluation National Institute of Technology and Actinomycetes, archaea and Chiba, Japan http://www.nbrc.nite.go.jp _ Biological Resource Center (NBRC) Evaluation Fungi Phaff Yeast Culture Collection, University University of Carlifonia, Davis Yeasts Davis, California, USA http://www.phaffcollection.org of California, Portuguese Yeast Culture Collection Faculdade de Ciências e Tecnologia Yeast http://[email protected] Caparica, Portugal (PYCC) Universidade Nova de Lisboa

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environments impacted with petroleum and its products, 1,000 fungi associated with marine environments (coastal Antarctica and Brazil), and about 100 bacteria used as reference strains; and the number of cultures is rapidly growing due to the many biodiversity studies embarked upon by researchers affiliated with this culture collection. The collection was formed from research projects or collaborative research coordinated with the participation of researechers of UNESP and has been used for diversity studies and prospecting compounds of biotechnological interest, such as, enzymes, antibiotics, biofuels and bioremediation agents. UNESP - CMR is divided into two main parts namely (i) the main collection and (ii) the research collection. The main collection comprises isolates with biotechnological potential or are representatives of the biodiversity of a given environment and the reference strains and which have been correctly identified based on molecular taxonomy (ribosomal DNA sequencing, genetic fingerprinting and phylogenetic analysis) and conventional taxonomy (morphological and biochemical) while the strains contained in the research collection comprises isolates recovered from different environmental samples still under study and are potential candidates to be incorporated into the main UNESP- CMR collection after being subjected to necessary preliminary bioprospective studies as well taxonomic characterization. All the microorganisms in the UNESP - CMR are stocked and preserved according to the recommended guidelines of the Organization for Economic co-operation and Development OECD Best Practice Guidelines for microbial recource centers (Table 1.2) for microorganisms. Before starting this study, some of the yeasts in this collection were conventionally identified using physiological, biochemical or phenotypic methods, while many were yet to be identified. This offered an opportunity to apply DNA sequencing and molecular taxonomic methods for their proper identification as well as exploitation for biotechnological applications. Initially, the culture collection was majorly focused on yeast biodiversity studies; this biodiversity assessment led to the discovery of many previously unidentified yeast species namely Cryptococcus haglerorum isolated from the floor of a nest of the leaf-cutting ant Atta sexdens (MIDDELHOVEN et al. 2003), Blastobotrys attinorum formerly Sympodiomyces attinorum from the nests of the leaf-cutting ants (CARREIRO et al. 2004), Candida leandrae from fruit of Leandra reversa (Melastomataceae) from Atlantic rainforest in Brazil (RUIVO et al. 2004), Candida bromeliacearum and Candida ubatubensis known from Canistropsis seidelii collected from the Atlantic rainforest of south-eastern Brazil (RUIVO et al. 2005), Candida

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Table 1.2 Quality control procedures recommended for microorganisms (OECD, 2007)

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heliconiae, Candida picinguabensis and Candida saopaulonensis from flowers of Heliconia (RUIVO et al. 2006) and Trichosporon chiarellii from a nest of the fungus-growing ant Myrmicocrypta (PAGNOCCA et al. 2010). In addition, the culture collection contributed to the description of a new yeast genus, Bandoniozyma and seven new species inside (VALENTE et al., 2012). Due to the knowledge accumulated over the years, from various physiological and taxonomic studies, about the vast metabolic activities of the microorganisms making up the UNESP – CMR, bioprospecting of the collection has also been extended to screening of yeasts for biotechnologically important enzymes. Furthermore, currently, a research involving the bioprospection of the yeasts from this collection for the conversion of lignocellulosic materials (sugarcane bagasse) to ethanol is being implemented. Hence, UNESP – CMR, not only serve as a biotechnology-based bank of valuable microorganisms that could be harnessed for biotechnological applications, teaching and research and other purposes, but also as microbial repository for the preservation of Brazilian microbial biodiversity which constitute the heritage of the country.

1.3 Importance of culture collections to microbiology and biotechnology i. Depository of Microbial Diversity

The primary role of a culture collection is the preservation of biological diversity. This is achieved through providing repositories for microorganisms of scientific and industrial interests, as well as of regional and international interests. Microbial culture collections provide services, which include accession, maintenance, preservation, documentation and cataloguing of microorganisms. Such microorganisms include strains of newly discovered taxa; type strains, neotype, unique biotype and selected reference strains; strains utilized in patent and novel applications; genetically modified strains and other strains of special educational, agricultural, biotechnological and medical significance (MALIK; CLAUSE, 1987). The importance of these functions could be emphasized from examples of microbial species that have not been re-isolated since their first discovery but whose biotypes are still maintained in public collections. For example, Candida tanzawaensis was described 22 years ago by Nakase et al. (1988) and has not

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been isolated since then. Furthermore, many recent species descriptions and publications are based on materials collected in the past but preserved in culture collections with the hope that new technologies that could facilitate their description would later be developed (LACHANCE, 2006). ii. Screening and exploitation of microbial diversity

Culture collections play important roles in providing lage microbial resources for screening for important biotechnological products. Introducing screening programs to collections allow biodiversity rich countries to benefit from exploitation of the microbial diversity they have (SMITH, 2003). For scientists who are interested in screening for novel products, culture collections provide an opportunity of access to large numbers of authenticated microorganisms. For example, in a search for L-arabinose fermenting yeasts for the bioconversion of biomass to ethanol, Dien et al. (1996) carried out an extensive screening of 116 yeast strains, from the ARS Culture Collection (National Center for Agricultural Utilization Research, Peoria, Illinois) in which four species namely Candida auringiensis, Candida succiphila, Ambrosiozyma monospora, and Candida sp. (YB-2248) were found to be able to ferment the sugar. In the same vein, Hou and Johnston (1992) screened 1229 selected microbial cultures (including 508 bacteria, 479 yeasts, 230 actinomycetes and 12 fungi) obtained from the ARS Culture Collection for lipase activities in which 25 % were lipase positive.

ii. Development and training of new preservation methods and skills

Successful maintenance of microbial cultures in such a way as to ensure their long term viability, stability and accessibility is very crucial. Methods used for culture preservation should be those that cause little or no damage to cells while at the same time still retaining their genetic and phenotypic characteristics as well as viability over a long period of storage. In most cases these require personal experience and a good understanding of individual microorganism coupled with familiarity with modern methods of preservation (KIRSOP, 1983). Therefore, culture collections test and develop new methods of culture preservation suitable for individual and

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group of microorganisms. They also provide specialized training in culture handling and taxonomic studies. In addition, the identification of organisms is generally a specialized activity; hence, many collections provide identification services for people who have do not have the required skills or facilities required.

1.3 Yeast: introduction and definition

Yeasts are microscopic fungi, which reproduce asexually by budding or fission and by the production of forcefully ejected ballistoconidia on stalks termed sterigmata; resulting in growth made up of single cells and whose sexual states are not formed within or upon a fruiting body (SUH et al. 2006; KURTZMAN; FELL; BOEKHOUT, 2011a). These characteristics differentiate them from other filamentous fungi and mushrooms that are predominantly multicellular, and whose sexual structures are enclosed within complex fruiting bodies (KURTZMAN; FELL; BOEKHOUT, 2011a). In contrast to the yeasts, filamentous and dimorphic fungi grow by means of hyphae that extend at their apices while branching sub-apically, thereby resulting in an interconnected network of hypha known as mycelium. Several yeasts however exhibit pseudohyphae made up of chains of elongated buds that remain attached to the parent cell after formation. In addition to ascomycete and basidiomycete yeasts, some fungi are dimorphic and exhibit a yeast stage that shifts to mycelial growth under certain cultural conditions. The term ‘‘yeast-like’’ has also been used to represent the cellular phase of dimorphic members of the zygomycete genus Mucor (FLEGEL, 1977), the black yeasts (HOOG, 1999), which comprise diverse pigmented ascomycete genera such as Aureobasidium, Fonsecaea and Phaeococcomyces as well as certain achlorophyllous algae in the genus Prototheca (KURTZMAN; FELL; BOEKHOUT, 2011a).

1.4 Evolution of yeast identification methods

The approaches used in yeast taxonomy have rapidly evolved over the years. Before the current era of species identification by DNA comparisons, yeasts were normally delineated based on differences in observable characteristics, i.e. morphological characteristics and phenotypic attributes such as presence or absence of a sexual state, type of cell division, presence or absence

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of hyphae and pseudohyphae, fermentation of simple sugars and growth on various carbon and nitrogen compounds (KURTZMAN, 2011). Chemotaxonomic characteristics such as cell wall carbohydrate composition and coenzyme Q are also sometimes used in yeast taxonomy (PRILLINGER et al. 2011). The need for more accurate diagnostic tools for yeast species identification soon became apparent after various works began to reveal variations in phenotypic characteristics displayed by yeast strains of the same species (KURTZMAN; FELL, 2006; KURTZMAN, 2011). The shift from phenotypic identification of yeasts to molecular identification began with DNA reassociation techniques. In this technique, single-stranded DNAs of two isolates are mixed and allowed to repair as a double strand. According to Price, Fuson and Phaff (1978), strains that showed 80% or greater nuclear DNA relatedness on the basis of shared phenotype, as measured by reassociation, are members of the same species. Although, DNA reassociation technique provided the first opportunity of genetic based yeast species delimitation, it is limited by the fact that only closely related species can be resolved using the technique. The use of DNA sequence comparisons for yeast identification soon became widely embraced after Peterson and Kurtzman (1991) and Kurtzman and Robnett (1998), studied the variable domains (D1 and D2) of the large subunit (LSU) rRNA gene of ascomycetous yeasts (Figure 1.1) and revealed that these regions offer the opportunity to resolve most closely and distantly related species. Kurtzman and Robnett (1998) predicted that strains showing six or more nucleotides differences (1% substitution) in the D1/D2 nucleotide domains of the ribosomal DNA (Figure 1.1) represent different species. A complementary D1/D2 database was created for basidiomycetous yeasts by Fell et al. (2000). Molecular analysis of the small and large subunit of the rRNA gene led to significant progress in systematic of basidiomycetous yeasts while various works such as Sugita et al. (1999, 2000) and Scorzetti et al. (2002) contributed to the development of databases for the internal transcribed spacers (ITS) 1 and 2 regions of the rDNA (figure 1) for basidiomycetous yeasts. The ITS region is highly substituted and provides a higher resolution for some closely related species that could not be separated by D1/D2 region (SCORZETTI et al. 2002). Several other genes have been successfully used for yeast taxonomic classification and they include the intergenic spacer (IGS) region, translation elongation factor-1α (TEF-1α), actin-1, mitochondrial small subunit (MtSm 5) rRNA, Cytochrome b gene and the cytochrome oxidase II (COX II) gene (KURTZMAN; FELL; BOEKHOUT, 2011b). Other methods used for rapid identification of species and for detection of polymorphisms in nuclear

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Figure 1.1 Map of fungi rRNA gene showing the internal transcribed spacer (ITS) region, intergenic spacer (IGS) regions, 18S small subunit (SSU) and 25-28S large subunit (LSU).

Source: http://biology.duke.edu/fungi/mycolab/primers.htm

DNA include restriction-enzyme fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified polymorphic length polymorphism (AFLP) and microsatellites (OLIVE; BEAN, 1999). The estimation of genetic and historical relationships among yeast species have relied mostly on trees derived from phylogenetic analyses of sequences of target genes. Some of the methods used for phylogenetic estimation of sequence data include distance based methods namely Neighbor-Joining and UPGMA (Unweighted Pair-Group Method with Arithmetic mean); in addition to the algorithms of Parsimony, Maximum likelihood and Bayesian (WEIß; GÖKER, 2011). According to Heath, Hedtke and Hillis (2008) accurate phylogenetic estimates of true historical relationships among species are determined by four main factors: (1) appropriate selection of target genes for analysis; (2) collection of enough sequence data to obtain a robust and repeatable estimate; (3) use of accurate analytical methods; and (4) sufficient taxon sampling. Although, single gene sequence offers the advantage of rapid species identification, nucleotide substitution rates of diagnostic genes often vary within lineages, hence, species inference using single gene analysis usually suffer limitations such as the inability to clearly defined species boundaries as well as weak support for basal branches. On the other hand, multigene analysis

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offers the opportunity of detecting nucleotide substitution rates and basal lineages are generally well supported (KURTZMAN; ROBNETT, 2003; KURTZMAN; FELL, 2006). Scorzetti et al. (2000) proposed the analysis of combined sequences of the D1/D2 and ITS regions for species identification after examining the resolution provided by these rRNA regions for the identification of basidiomycetous yeasts in the Pucciniomycotina and Agaricomycotina. Using a multigene approach of combined sequences of SSU, LSU, ITS, mitochondrial small subunit rDNAs with elongation factor 1-α and cytochrome oxidase II, species of the Saccharomyces complex were phylogentically resolved into 14 clades including 4 (Saccharomyces sensu stricto, Zygosaccharomyces sensu stricto, Torulaspora and Eremothecium) well-supported monophyletic clades (KURTZMAN; ROBNETT, 2003). By analyzing the combined sequences of SSU, LSU, ITS region and mitochondrial cytochrome b gene, Wang and Bai (2008) clearly separated three monophylectic clades namely, Dioszegia clade, Derxomyces mrakii and Hannaella sinensis from Cryptococcus luteolus lineage of the Tremellales. A similar approach by Kurtzman, Robnett and Basehoar-Powers (2008) found species formerly assigned to Issatchenkia to belong to the Pichia membranifaciens clade after the analysis of concatenated gene sequences from EF-1α and the LSU and SSU rRNA genes, hence, were consequently transferred to the genus Pichia. Phylogenetic tree constructing methods do not accurately measure gene genealogies of haplotypes resulting from intraspecific polymorphisms (CLEMENT; POSADA; CRANDALL, 2000). The extent of intraspecific genetic and phenotypic (morphological and physiological) variations among yeast strains that share a common gene pool have been continuously revealed by several studies (LACHANCE et al., 2010, 2011; DAYO-OWOYEMI et al., 2012) and this phenomenon is more pronounced in basidiomycetous yeasts as was exemplified in the work of Scorzetti et al. (2002). The use of Parsimony network analysis (TCS) have been shown to offer accuracy for the circumscription of phylogenetic species in the light of correctly measuring genetic discontinuities between species while at the same time revealing where such discontinuities only represent alleles of a locus or haplotypes within a species (CLEMENT; POSADA; CRANDALL, 2000; POSADA; CRANDALL, 2001; HART; SUNDAY, 2007). The accuracy of the TCS analysis for differentiating species based on barcoding sequences in higher organisms was thoroughly reviewed by Hart and Sunday (2007) and they reported the method as having a high positive rate to identify known species boundaries as well as for discovering new species from sequence data

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especially where such taxa were adequately sampled. The inter-specific sequence variation and high discriminative power of the ITS region have assisted in solving many taxonomic and systematic problems relating to separation of species, hence it has served the role of a DNA barcode marker for some fungi identification (BEGEROW, 2010). However, the ITS region is often highly variable within species (BEGEROW, 2010), consequently, members of the same species may be treated as being separate when pairwise ITS sequence divergence is used as a means of species delimitation. Nevertheless when combined with the D1/D2 region for parsimony network analysis, intraspecific discontinuities can be identified (LACHANCE et al. 2010, 2011).

1.5 Yeast classification

Yeasts are classified under two broad taxonomic groups, i.e. ascomycetes and basidiomycetes; each comprising anamorphic and teleomorphic states. These two groups of yeast differ in their cell wall composition and molecular structure and also in their mode of bud formation (i.e. asexual reproduction) and spore formation (sexual reproduction). Yeast cellular compositions vary with their phylogenetic diversity, as observed by the variety in the biochemical composition of the cell walls, ultrastructural organization and morphology of the septa (VAN DER KLEI et. al. 2011). However, cell walls of ascomycetous yeasts consist of two layers: an inner skeletal layer consisting of load-bearing polysaccharides and an outer layer consisting of glycoproteins that are covalently linked to the inner layer (YAMAGUCHI et al. 2002; SUH et al. 2006, VAN DER KLEI et al. 2011). In basidiomycetous yeasts, the walls are often, but not always, multilayered with alternating regions of electron-dense and electron- translucent material. While the cell walls of ascomycetous and basidiomycetous fungi contain a similar β-1,3-glucan, cell wall polysaccharide composition is dominated by chitin in the basidiomycetes (SUH, 2006, VAN DER KLEI et. al., 2011). In the ascomycetous yeasts, budding (vegetative reproduction) is holoblastic, in which budding results from the stretching out of the entire cell wall of the mother cell; the bud separates from the narrow base leaving a scar through which no further budding occurs. On the other hand, in the basidiomycetous yeasts, budding is enteroblastic, in which newly formed bud cell rupture the cell wall of the mother cell, resulting in the formation of collaret due

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to recurrent formation and separation of a succession of buds (KURTZMAN; FELL; BOEKHOUT, 2011a). In ascomycetous yeasts, sexual reproduction occurs through spore formation whereas; sexual states of basidiomycetous yeasts are characterized by formation of septate dikaryotic hyphae with clamp connections. Basidiomycetous yeasts can also be differentiated from ascomycetous yeasts based on Diazonium Blue B (DBB) test. A dark red reaction is observed on the former when a buffered solution of Diazonium Blue B (DBB) is applied to cultures left overnight at 60 °C (BOEKHOUT et al., 2011).

i. Phylogenetic classification of ascomycetous yeasts

The relationships among the ascomycetes are becoming clearer since the introduction of multigene sequence analysis for the estimation of phylogeny; and consequently have resulted in changes in the classification of the ascomycetous yeasts. For instance, the reassignment of some formerly Pichia species (e.g P. anomala) to a new genus Wickerhamomyces after the polyphylectic nature of Pichia species was further confirmed by phylogenetic analyses (KURTZMAN; ROBNETT; BASEHOAR-POWERS, 2008; KURTZMAN, 2011). Another important change is the re-assignment of the saturn spored genus Williopsis into four distinct genera namely Barnettozyma, Lindnera, Ogatae and Wickerhamomyces. Based on the latest phylogenetic classification by Hibbett et al. (2007), the phylum Ascomycota is divided into three subphyla (their respective classes are provided in parenthesis): Subphylum Taphrinomycotina (classes Taphrinomycetes, Neolectomycetes, Pneumocystidomycetes, Schizosaccharomycetes) Subphylum Saccharomycotina (class Saccharomycetes), Subphylum Pezizomycotina (classes Arthoniomycetes, Dothideomycetes, Eurotiomycetes, Arthoniomycetes, Dothideomycetes, Eurotiomycetes, Laboulbeniomycetes, Lecanoromycetes, Leotiomycetes, Orbiliomycetes, Pezizomycetes, Sordariomycetes) (Figure 1.2). This phylogenetic classification was previously revealed by a single gene analysis based on 5S rRNA gene sequence (WALKER, 1985). Multigene sequence of concatenated data of SSU, LSU, 5.8S rRNA, elongation factor 1-α (EF1α), and two RNA polymerase II subunits (RPB1 and

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Figure 1.2 Phylogeny of the phylum Ascomyceota showing the classification of Ascomycetous yeasts (Hibett et al. 2007).

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RPB2) (JAMES et al., 2006) confirmed that ascomycetous yeasts belong to a single lineage, Saccharomycotina. Yeasts in the subphylum Taphrinomycotina are classified in the order Schizosaccharomycetales (Schizosaccharomycetes) while the subphylum Pezizomycotina consists majorly of filamentous fungi. The teleomorphic genus Schizosaccharomyces Linder is the only genus of yeasts currently known in the Schizosaccharomycetales and currently includes 3 species namely Schiz japonicas, Schiz. pombe and Schiz.octosporus. Some distinct characteristics of this genus is their cylindrical shape and asexual form of reproduction which is by fission (VAUGHAN-MARTINI; MARTINI, 2011).

ii. Phylogenetic classification of basidiomycetous yeasts

Basidiomycetous yeasts including sexual and asexual forms are currently classified into three subphyla namely (i) Subphylum Pucciniomycotina, (formerly class Urediniomycetes), (ii) Subphylum Ustilaginomycotina (formerly class Ustilaginomycetes) and (iii) Subphylum Agaricomycotina (formerly Hymenomycetes) (Figure 1.3) (HIBBETT et al. 2007). The presence of three main lineages within the Basidiomycota was earlier demonstrated by many phylogenetic studies including sequence analyses small-subunit SSU (FELL et al. 2000), LSU and ITS (SCORZETTI et al. 2002). Basidiomycetous yeasts accommodated in the Pucciniomycotina are distributed in the class Agaricostilbomycetes (e.g. Kondoa spp., Chionosphaera sp. and the asexual genera Bensingtonia, Kurtzmanomyces, Sporobolomyces and Sterigmatomyces); class Cystobasidiomycetes (e.g. some pink-colored asexual genera: Bannoa, Cyrenella, Rhodotorula, Sporobolomyces, Erythrobasidium and some sexual and dimorphic species such as Cystobasidium, Occultifur, Naohidea and Sakaguchia) and class Microbotryomycetes (e.g Rhodosporidium and Sporidiobolus and their asexual counterparts, Rhodotorula and Sporobolomyces, also the teliospore-forming genera Leucosporidium and Mastigobasidium, and their asexual state Leucosporidiella (BOEKHOUT et al. 2011a). The subphylum Ustilaginomycotina are the smut fungi and their relatives and whose sexual states are characterized by transversely septate auricularoid basidium. They are distributed in the class

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Figure 1.3. Phylogeny of the phylum Basidiomycota showing the classification of Basidiomycetous yeasts (Hibbett et al. 2007).

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Ustilaginomycetes and class Exobasidiomycetes. The Exobasidiomycetes include anamorphic species such as Tilletiopsis and its teleomorph Tilletiaria, Acaromyces, Meira, Malassezia and Sympodiomycopsis while only two species namely Farysizyma and Pseudozyma are currently classified in the Ustilaginomycetes. The subphylum Agaricomycotina formerly known as Hymenomycetes comprises three classes, namely Agaricomycetes, Dacrymycetes and Tremellomycetes (HIBBETT et al. 2007). Yeast states are found only in the Tremellomycetes and are classified in four distinct orders namely Tremellales, Trichosporonales, Filobasidiales and Cystofilobasidiales (SCORZETTI et al. 2002). While ballistoconidia may be produced by members of the Tremellales (e.g., Bullera, Dioszegia, Kockovaella) and the Cystofilobasidiales (e.g., Udeniomyces), distinct morphological trait of the Trichosporonales (e.g Trichosporon chiarellii, T. ashaii, T. insectorum) is the formation of arthroconidia. Some sexual states of the Tremellales such as Auricullibuller fuscus, Bulleribasidium and Papiliotrema are characterized by the formation of conspicuous basidiocarps and tremelloid haustoria branches that are adapted to their mycoparasitic mode of life (BOEKHOUT; FONSECA; BATENBURG-VAN DER VEGTE, 1991; SAMPAIO et al. 2002). The teleomorphic genus Tremella and the anamorphic genera Bullera and Cryptococcus are polyphyletic and latter is distributed in all the four lineages of the Tremellales (SCORZETTI et al. 2002). Three teleomorphic genera are dinstincted in the Cystofilobasidiales namely Mrakia, Cystofilobasidium and Xanthophyllomyces. The first two genera are characterized by the formation of teleospores. Examples of species found in this lineage include species of the cold tolerant genus Mrakia (e.g M. frigida and M. gelida) and the carotenoid pigmented species of Cystofilobasidium e. g Cystofilobasidium infirmominiatum. Only one species is recognized in the genus Xanthophyllomyces, namely X. dendrorhous (anamorph Phaffia rhodozyma). The Xanthophyllomyces have a unique mode of sexual reproducition involving cell to cell mating to produce basidiospores on the apex of an elongate basidium (GOLUBEV, 1995). They are important for their carotenoid pigment, astaxanthin (JOHNSON, 2003). The monotypic anamorphic species Guehomyces pullulans and arthroconidia forming genus Tausonia pamirica are also phylogenetically placed within the Cystofilobasidiales (BOEKHOUT et al. 2011).

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1.6 Yeast ecology and diversity

Yeasts are typically known as decomposers of dead organic matter in which they bring about nutrient transformation. They can engage in intimate relationships with other organisms as mutualists, competitors, parasites, or pathogens (STARMER; LACHANCE, 2011). Yeasts are widely distributed in almost all biomes of the world and their ubiquity is complemented by their diversity (KURTZMAN; FELL; BOEKHOUT, 2011c). They grow well in moist environments where there is availability of simple sugars, amino acids. Some are however able to degrade complex polysaccharide such as starch, cellulose, hemicellulose and pectin (ALONSO et al. 2010; BIELY; KREMNICKÝ, 1998). The following discussions of diversity of yeasts in various habitats relate to some of the isolation sources of the yeasts used in this study. i. Yeasts associated with plants (flowers and fruits)

Due to their rich content of easily utilizable carbon, fruits serve as natural habitats for a variety of yeasts. Yeasts particularly ascomycetes are distributed on surfaces of fruits, exudates of leaves, flowers and tree trunks. Prada and Pagnocca (1997) reported the isolation of two hundred and two strains of yeasts and yeast-like fungi from naturally occuring fruits in the tropical rain forest of Juréia-Itatins Ecological Reserve, with 38 species constituting 74 % of the total isolates been ascomycetous. The finding that naturally occurring apple yeasts can protect fruit against postharvest diseases spurred interest in the isolation of yeasts from various fruits with the aim of discovering new yeast antagonists against postharvest diseases (JANISIEWICZ, 1987). Certain yeasts associated with fruits can produce sugar-derived prebiotics such as fructooligosaccharides. According to Maugeri and Hernalsteens (2007) apud Johnson and Echavarri-Erasun (2011), yeasts from fruits and flowers in Brazilian tropical forests including Candida, Rhodotorula, and Cryptococcus produced substantial quantities of fructooligosaccharides. ii. Yeasts associated with leaf phylloplane

Plant surfaces harbor numerous and diverse microbial communities in which yeasts, particularly basidiomycetous yeasts, are among the most frequently membered (INÁCIO, 2002;

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FONSECA; INÁCIO, 2006). Basidiomycetous yeasts particularly species of the genera Sporobolomyces, Rhodotorula, Cryptococcus, Bensingtonia, Cystofilobasidium, Leucosporidium and Pseudozyma are dominant on leaf phylloplanes (MAKSIMOVA; CHERNOV, 2004; STAMER; LACHANCE, 2011). Species of Bullera, Sporobolomyces and Tilletiopsis are particularly adapted to this environment due to the production of forcibly ejected ballistospores (FONSECA; INÁCIO, 2006). A survey on the phylloplane yeasts from Mediterranean plants (Acer monspessulanum, Quercus faginea, Cistus albidus and Pistacia lentiscus) collected at the ‘Arrabida Natural Park,’ Portugal revealed the high frequency of occurrence of species of Taphrina and Lalaria including five previously unknown species of the latter (INÁCIO, 2004). In an investigation studying the yeasts community colonizing the leaf surfaces of various fruit trees in southwest Slovakia, Slávicová, Vadkertiová and Vránová (2009) isolated 150 strains belonging to seventeen yeast species out of which Aureobasidium pullulans, Cryptococcus laurentii, and Metschnikowia pulcherrima were the most abundant species isolated. Yeasts are also found living in fruiting bodies of mushrooms, which has also been the source of many ascomycetous and basidiomycetous yeast species (NAKASE et al. 1999; BABJEVA et al., 2000; MIDDELHOVEN, 2004). Yurkov et al. (2012) isolated various yeasts species of Rhodotorula, Rhodosporidium, Mastigobasidium, Cryptococcus, Cystofilobasidium, Holtermanniella, Trichosporon and the ascomycetous Kluyveromyces from Boletales fruiting bodies truffle ascocarps. Nakase et al. (1999) isolated three new species of yeasts namely Candida fungicola, C. sagamina and C. fukazawae from fruiting bodies of unidentified mushrooms collected from Tanzawa Mountains, Kanagawa Prefeitura.

iii. Yeasts associated with insects

Yeasts have been isolated from insects in many different families. While such insects serve the primary role of dispersing yeasts to new habitats, many of these yeasts have also been shown to improve insect nutrition and to detoxify plant chemicals to which insects are exposed (SUH et al., 2005; LACHNACE et al., 2001, ROSA et al., 2003).

Nests of fungus-growing ants provide a suitable habitat for yeasts (CRAVEN et al. 1970; CARREIRO, 1997; 2000). Fungus-growing ants of the higher-attini lineages (Atta and

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Acromyrmex) exclusively cut fresh leaves and plant material to cultivate their mutualistic fungus (WEBER, 1972; MUELLER; REHNER; SCHULTZ, 1998). Breakdown and transformation of the protein and starch rich plant materials by the cultivated fungus make nutrients available within the fungus garden matrix and this is a major target for exploitation by other microorganisms including yeasts (CARREIRO et al., 1997; RODRIGUES et al., 2009; SCOTT et al., 2010).

Yeasts associated with fungus-growing ant nests contribute to the stability of the complex microbiota found in the leaf-cutting ant nests through nutrients generation and removal of potentially toxic compounds (MENDES et al., 2012). Carreiro (1997) identified yeasts species of the genera Candida, Pichia, Cryptococcus, Rhodotorula, Sporobolomyces, Tremella and Trichosporon from the nests of the leaf-cutting ant Atta sexdens. Rodrigues et al. (2009) investigated the diversity of yeasts in Atta texana gardens and isolated ascomycetous yeasts belonging to the genera Aureobasidium, Candida, Kodamaea, Saccharomyces and basidiomycetous yeasts of the genera Bullera, Bulleromyces, Cryptococcus, Pseudozyma, Rhodosporidium, Rhodotorula, Sporidiobolus and Trichosporon.

The dispersal of yeasts by leaf-cutting ants was studied by Pagnocca et al. (2008) and it was revealed that these insects harbor various species of yeasts in their body including several opportunistic human pathogens e.g. Candida parapsilosis and C. metapsilosis, hence, may serve as vectors of these pathogens. Another groups of yeasts commonly found associated with fungus- growing ants are black yeasts in the genus Phialophora. Black yeast is the term used to refer to groups of yeasts characterized by melanized cell wall (STERFLINGER, 2006). These yeasts grown on the cuticle of the ants and are considered symbionts that play antagonistic roles in the fungus-growing ant mutualism (LITTLE; CURRIE, 2008).

Although the association of yeasts with the wasps Polybia ignobilis has not been extensively studied, socials wasps were identified as vector and natural reservoir of S. cerevisiae (STEFANINI et al., 2012). Yeasts in Starmerella and neighbouring clades are mostly associated with bees (ROSA et al., 2003). Study involving the characterization of yeasts associated with the wasp Polybia ignobilis revealed the presence of various species including Candida, Cryptococcus, Hanseniaspora and Rhodotorula with Candida azyma, Candida chrysomelidarum,

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Cryptococcus liquefaciens and Rhodotorula mucilaginosa being most frequently encountered (SANCHEZ DE SOUSA, 2011). Ascomycetous yeasts associated with flowers and exudates are usually found in specialized niches involving interactions with insects or other invertebrate animals that they rely upon for dispersal (MORAIS et al., 1992; SUH et al., 2005; LACHNACE et al., 2001; ROSA et al., 2003). Lachance et al. (2001) reported the affiliation of yeasts in the Metschnikowia, Kodamaea, Wickerhamiella, and Starmerella clades with these floricolous insects that visit Hibiscus flowers and some flowers in the families Convolvulaceae and Cactaceae. Beetles may have a yet unclear strong symbiotic relationship with yeasts; possibly, the yeasts may carry out the transformation of scarce and poorly digestible components of flowers into a richer diet for the insects (STARMER; LACHANCE, 2011). iv. Yeasts associated with honey

Ability of some yeast to tolerate low temperature, low oxygen concentration, high acidic conditions, high osmotic pressure or high salinity are important adaptive properties that determine their ability to survive under restricted habitats. Zygosaccharomyces rouxii and Z. bailii are often implicated in the spoilage of honey and jam because of their unique abilities to tolerate the high osmotic stresses and low water activity (MARTORELL et al. 2007). Other yeasts such as Z. bisporus, Z. mellis, Schizosaccharomyces pombe, Torulaspora delbrueckii, Debaryomyces hansenii, and various Candida and Moniliella species are commonly associated with foods containing high concentrations (40-70%) of sugar such as honey (FLEET, 2011). v. Yeasts found in soil Many studies on the biological diversity of various soil types have shown that soil is a diverse habitat dominated by invertebrates, prokaryotes and fungi including yeasts (BOTHA, 2006, VISHNIAC, 2006a). In fact, majority of described yeasts species were isolated in the soil environment. Recently seven new yeast taxa including Clavispora reshetovae, Barnettozyma vustinii and Leucosporidium drummii were discovered during an investigation on yeast diversity in soils (YURKOV; SCHÄFER; BEGEROW, 2012). Soil yeasts play important roles, which include soil aggregation formation and maintenance of soil structure, mineralization of organic

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materials and carbon cycling, whereby they serve as mutrient sources for bacteria and other predators in the soil (FITTER et al. 2005; BOTHA, 2006; 2011). Ecological soil surveys revealed the most abundant yeast soils to include Cryptococcus albidus, Cr. curvatus, Cr. gastricus, Cr. gilvescens, Cr. humicolus, Cr. laurentii, Cr. podzolicus. Cr. erreus, Filobasidium uniguttulatum, Cystofilobasidium capitatum, Leucosporidius scottii, Mrakia frigida, Rhodotorula aurantiaca, R. glutinis, R. mucilaginosa, Sporobolomyces roseus, Trichosporon cutaneum and Schizoblastosporion starkeyi-henricii (BOTHA, 2006). Recently, Yurkov; Schäfer and Begerow (2012b) investigated the diversity of cultivable yeasts in soils under different land use and isolated 40 yeast species, 11 of which had earlier been reported from soil, i.e. the basidiomycetous Cryptococcus aerius, Cr. laurentii, Cr. terreus, Cr. terricola, Cr. podzolicus, Geotrichum pullulans and the ascomycetous Barnettozyma californica, B. pratensis, Schwanniomyces (Debaryomyces) occidentalis, Lindnera (Williopsis) saturnus and Schizoblastosporion starkeyi-henricii. vi. Yeasts in Antarctic environments Many species of yeasts have been found to successfully colonize the Antarctic continent. While some of these yeasts are Antarctic indigenes and obligate psychrophiles that do not survive when subjected to temperatures different from those obtainable in their natural habitat (i.e., > 20 °C), several could be considered as non-indigenes that were brought in by wind and ocean currents as well as by birds, humans and other animals who occasionally visit this habitat, and became adapted to Antarctic habitat (VISHNIAC, 1996; 2006b). The latter groups are mostly psychroptrophs and mesophiles, which equally grow and multiply at room temperature (25 ± 2 °C), although may remain dormant for a long time at low temperatures. The ability to tolerate low temperature, high salinity, high radiation and other extreme conditions are fundamental adaptations of yeasts found in Antarctic environments (RAY et al., 1989; ROBINSON, 2001; SHIVAJI; PRASAD, 2009). Yeasts such as Candida psychrophila, Leucosporidium antarcticum, Cr. vishniacii, Mrakia frigida, Mrakia robertii and Mrakia blollopis are obligate psychrophiles are not able to grow at temperatures above 20 °C (VISHNIAC, 2006b; THOMAS-HALL et al., 2010). Other yeasts that have been isolated from various antartic samples include Candida sake, Cryptococcus antarcticus, Cr. victoriae, Cr. watticus, Cr adeliensis, Dioszegia hungarica and

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Leucosporidium scottii (VISHNIAC, 2006b; VAZ et al., 2011). Some of them are mesophilic yeasts that became adapted to Antarctic habitat. The extreme environmental conditions obtainable in the Antartica means that microorganisms found there would have evolved unique characteristics for survival that could be exploited for biotechnological applications. Hence, yeast biodiversity of the Antarctica has raised interest for bioprospection for novel enzymes and biomolecules (SHIVAJI; PRASAD, 2009; VAZ et al., 2011).

1.7 Biotechnological importance of yeasts

The biotechnological potentials of yeasts have been exploited by man in many industrial processes ranging from food industries to the biofuel industries where yeast is used for the production of bioethanol. Yeasts, especially Saccharomyces cerevisiae are used for making various fermented products such as beer, wine, bakery products, cheese etc. They are also used in the production of enzymes, biocatalysts, pigment, flavours, and pharmaceutical prouducts as well as as biocontrol agents (JOHNSON; ECHAVARRI-ERASUN, 2011). Meyerozyma (Pichia) guilliermondii is known as a hyper producer of riboflavin (SIBIRNY; BORETSKY, 2009). Due to the ease of growth and genetic manipulation, yeasts such as S. cerevisiae Schizosaccharomyces pombe are used as model organisms for genetic studies (TAKEGAWA, et al., 2009). Other biotechnological potentials been investigated in yeasts include phenol and alkane degradation by Candida maltosa and C. tropica, production of biosurfactants by Pseudozyma spp., production of heterologous protein by Schizosaccharomyces pombe, fermentation of xylose to ethanol by Scheffersomyces stipitis and production of lipids and single cell oil by Yarrowia lipolytica (SATYANARAYANA; KUNZE, 2009).

1.8 Biodegradation of starch, lignocelluloses and pectin

Plant cell walls are the planet`s dominant form of lignocellulose biomass. The main structural polymers are: cellulose, a homopolymer of β-(1,4)-linked cellobiose residues which make up 40 %; hemicelluloses, a branched cross-linking heteropolymer of varied compositions comprising an average of 33 % and 23 % lignins and are strongly intermeshed and chemically linked by non-covalent forces and by covalent cross-linkages (Figure 1.4) (LODISH.; BERK;

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Figure 1.4. Plant cell wall structure. Source: Lodish et al. (2000)

ZIPURSKY et al., 2000; PÉREZ et al., 2002; HOWARD et al., 2003). Hemicelluloses are polysaccharides consisting of xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan present along with cellulose in plant cell walls (LODISH et al. 2000; SCHELLER; ULVSKOV, 2010). Ability of yeasts and yeast-like fungi to degrade plant polysaccharide is of great importance in ecology and physiology as well as in biotechnology. This is due to the ample information that can be obtained about their biotechnological potentials such as their usefulness in the production of industrially important enzymes as well as in the conversion of complex polymers to useful products e.g biological fuels. Table 1.3 gives a summary of industrial applications of some enzymes.

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Table 1.3. Industrial applications of some enzymes (KIRK, BORCHERT, FUGISANG, 2002)

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i. Cellulose degradation

Cellulose is the most abundant renewable organic polymer in the biosphere and is highly crystalline, water insoluble and relatively resistance to depolymerization. The degradation of cellulose to glucose requires the synergistic action of three distinct classes of enzymes namely, (i) endoglucanases, (ii) exoglucanases and (iii) β-glucosidases (cellobiases) (EC 3.2.1.21) (KARMAKAR; RAY, 2011) (i) The ´´endo-1,4-β-glucanase`` or 1,4-β-D-glucan 4- glucanohydrolases (EC 3.2.1.4), breaks internal bonds of cellulose i.e., β(1→ 4) linkages, to disrupt the crystalline structure and expose individual cellulose polysaccharide chains. (ii) The ´´exo-1,4- β-D-glucanases`` include both the 1,4- β-D-glucan glucohydrolases (EC 3.2.1.74) which liberate D- glucose from 1,4-β-D-glucans and 1,4- β-D-glucan cellobiohydrolase (EC 3.2.1.91) which liberates D-cellobiose from 1,4- β-glucans; resulting in tetrasaccharides or disaccharides, such as cellobiose. (iii) The ´´β-D-glucosidases`` or β-D-glucosidase glucohydrolases (EC 3.2.1.21) hydrolyses the exoglucanase product into individual monosaccharides i.e. release of D-glucose units from cellobiose (Figure 1.5) (KARMAKAR; RAY, 2011). Cellulose activities have been found in yeasts such as Candida glabrata, C. stellata, C. sheatae, Kloeckera apiculata (STRAUSS et al., 2001) Aureobasidium pullulans (KUDANGA; MWENJE, 2005; THONGEKKAEW; KHUMSAP; CHATSA-NGA, 2012). Trichosporon cutaneum, T. pullulans (STEVENS; PAYNE, 1977) and Gueomyces pullulans (SONG et al. 2010). Cellulases have wide industrial applications including in the pulp and paper industry for enhancement of drainage and beatability of pulp, in the textile industry for finishing of cellulose- based textiles, biostoning of denim garments and softening and defribillation of garments; in the bioethanol industry for the fermentation of biomass into Biofuels. In the wine and brewry industry, cellulose is added to malt to reduce the viscosity of wort, hence, improve the filterability. Other industrial uses include the manufacture of detergents; extraction and clarification of vegetable and fruit juices as well as for improvement of digestibility of animal feed.

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Figure 1.5. Action of major cellulase enzymes. Endoglucanases cleave internal β-1,4-linkages; exoglucanases cleaves two to four units from the ends of the exposed chains produced by endoglucanase while β-glucosidases cleave cellobiose to glucose units (KARMAKAR; RAY, 2011).

ii. Xylan degradation

The major enzymes responsible for the complete depolymerization of xylan are collectively known as xylanases and are composed of various hydrolases namely β-1,4- endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-glucuronidase, acetyl xylan esterase, and phenolic acid (ferulic and p-coumaric acid) esterase. Endoxylanases (EC3.2.1.8) act on the back bone of xylan specifically β-(1,4)-xylopyranose polymers to produce xygooligosaccharides which after the action of other debranching enzymes are finally converted by β-xylosidase (EC 3.2.1.37) to subunits of xylose (Figure 1.6) (BEG et al. 2001, JEFFRIES, 1994, SUN et al. 2012). Xylanases of yeasts such as Aureobasidium pullulans (LI et al. 1993), Pseudozyma hubeiensis (ADSUL; BASTAWDE; GOKHALE, 2009) and Trichosporon cutaneum (LIU et al. 1998) have been extensively studied while the cold adapted endoxylanase of Cryptococcus adeliensis (CBS 8351) was characterized by Petrescu et al. (2000).

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Figure 1.6 - Action of major enzymes involved in the depolymerization of xylan (SUN et al., 2012).

Xylanases are important in the bleaching of pulp in the paper industry. Potential applications of xylanases also include bioconversion of lignocellulosic material and agro-wastes to fermentative products, clarification of juices, improvement in consistency of beer and the digestibility of animal feed stock. Xylanases are also used in addition with proteases and cellulases directly or indirectly for improvement improve of the strength of the gluten network hence, the quality of bread. Application of xylanase in the saccharification of xylan in agrowastes and lignocellulose biomass for the production of bio-energy intensifies the need of exploiting the potential role of them in biotechnology (SUBRAMANIYAN; PREMA, 2002) iii. Pectin degradation

Pectin, the major constituent of plant cell walls, is a complex heteropolysaccharide mainly composed of d-polygalacturonic acid residues (Figure 1.7) (VORAGEN et al. 2009). Pectin degrading enzymes are known as pectic enzymes, pectinases, or pectinolytic enzymes. Diverse enzymes are required for pectin hydrolysis and are classified in two main groups, namely

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pectinesterases (PE) and depolymerases. Pectinesterases de-esterify pectin by removal of methoxyl residues while depolymerases split the main chain of pectin by hydrolysis of α-(1,4) linkages (BLANCO; SIEIRO; VILLA, 1999, JAYANI; SAXENA; GUPTA, 2005). The depolymerising enzymes are divided into polygalacturonases (PG) [Exo-PG (EC 3.2.1.67), Endo-PG (EC 3.2.1.15) and Polymethylgalacturonases (PMG) (EC 3.2.1.15)], enzymes that cleave the glycosidic bonds by hydrolysis, and lyases (PL) [Exo-PL (EC 4.2.2.9), Endo-PL (EC 4.2.2.2) and Pectin methyl-lyase (PML) (EC 4.2.2.10)] which break the glycosidic bonds by a β-elimination process (Figure 1.7) (BLANCO; SIEIRO; VILLA, 1999; JAYANI; SAXENA; GUPTA, 2005; HIMMEL et al. 2007).

Figure 1.7. Action of major enzymes involved in the deconstruction of pectin. Arrows indicate the point of action of pectinase enzymes. PMG, polymethylgalacturonases; PG, polygalacturonases (EC 3.2.1.15); PE, pectinesterase (EC 3.1.1.11); PL, pectin lyase (EC-4.2.2.10) (JAYANI, SAXENA, GUPTA, 2005).

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Pectinases find their application in the extraction and clarification of fruit juices, grape must, wine technology, maceration of vegetables and fruits, extraction of vegetable oils as well as in coffee and tea fermentation (JAYANI; SAXENA; GUPTA, 2005). Some yeasts are able to use pectin as carbon source hence, are pectin degrading enzyme producers, however, pectin degrading activity is reported in few genera of yeasts including Candida, Pichia, Zygosaccharomyces, Kluyveromyces, Rhodotorula, Cryptococcus and Trichosporon (BIELY; KREMNCKÝ, 1998). iv. Lignin degradation

Structurally, lignin is a non-water soluble amorphous heteropolymer that consist of phenyl propane units joined together by different types of linkages (LODISH et al. 2000; SANCHÉZ, 2009). Coniferyl alcohol is the principal component of softwood lignins, whereas guaiacyl and syringyl alcohols are the main constituents of hardwood lignins. Enzymes that degrade lignin are named lignin-modifying enzymes (LMEs) (Figure 1.8). Four types of LMEs have been characterized namely lignin peroxidases (EC 1.11.1.14), manganese peroxidases (EC 1.11.1.13), laccases (EC 1.10.3.2) and versatile peroxidases (EC 1.11.1.16) (MARTINEZ et al. 2005; DOSORETZ; REDDY, 2007). Only few lignin degrading yeasts have been identified and they include Trichosporon cutaneum (GEORGIEVA et al. 2006), Trichosporon pullulans (SLÁVIKOVÁ et al. 2009) and Rhodotorula glutinis (GUPTA et al. 1990). Basidiomycetous fungi that cause white rot decay of wood are the most efficient degraders of ligninand show complete mineralization of lignin to carbon dioxide and water (DOSORETZ; REDDY, 2007). v. Starch degradation

Starch which is a primary photosynthetic product in leaves of plants is a mixture of amylose (α-1,4-linked D-glucose residues) and amylopectin polysaccharide polymers containing both α-1,4-and α-1,6-linked D-glucose residues (HOSTINOVÁ, 2002). Enzymes that bring about the breakdown of the internal α-1,4-glycosidic linkages in starch to low molecular weight compounds, such as glucose, maltose and maltotriose are α-amylases. β-amylase catalyzes the

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Figure 1.8. Scheme showing the actions of lignin degrading enzymes (MARTINEZ et al. 2005).

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hydrolysis of the second α-1,4 glycosidic bond cleaving off two glucose units (maltose) at a time while γ-amylase cleaves α-(1-6) glycosidic linkages, in addition to cleaving the last α-(1-4) glycosidic linkages at the nonreducing end of amylose and amylopectin, yielding glucose (Figure 1.9). Yeasts possessing amylolytic systems are of biotechnological interest due to their application in food and energy industries. Some yeasts known to produce amylolytic enzymes include Schwanniomyces occidentalis, and species in the genera Cryptococcus, Candida, Dioszegia, Sporobolomyces, Pichia, Schwanniomyces, Saccharomycopsis, Aureobasidium, and Galactomyces (STRAUSS et al. 2001; BUZZINI; MARTINI, 2002; BRIZZIO et al. 2007). Amylases have potential application in a number of industrial processes such in bread making, manufacture of glucose and fructose syrups, detergents, fuel ethanol from starches, fruit juices, alcoholic beverages and sweeteners (HOSTINOVÁ, 2002; KIRK, BORCHERT, FUGISANG, 2002).

Figure 1.9. Scheme showing the actions of starch degrading enzymes (SIGMA, 2007)

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THOMAS-HALL, S. R.; TURCHETTI, B.; BUZZINI, P.; BRANDA, E.; BOEKHOUT, T.; THEELEN, B.; WATSON, K. Cold-adapted yeasts from Antarctica and the Italian Alps— description of three novel species: Mrakia robertii sp. nov., Mrakia blollopis sp. nov. and Mrakiella niccombsii sp. nov. Extremophiles, Tokyo, v. 14, n. 1, p. 47-59, jan. 2010

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CHAPTER 2

TAXONOMIC STUDIES OF YEASTS HOLD AT UNESP – CENTRAL FOR MICROBIAL RESOURCES

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2.1 ABSTRACT

Three hundred and forty strains of yeasts from a variety of natural substrates were identified. This amount of strains is 17% of the total number of strains deposited at the UNESP –Central for Microbial Resources (UNESP – CMR) of the Institute of Biosciences. The isolates were characterized using standard phenotypic (carbon and nitrogen assimilation) tests coupled with molecular markers. The molecular methods used were (i) PCR fingerprinting using primer (GTG)5 for microsatellite regions and (ii) determination of nucleotide sequences of D1/D2 domains of the LSU ribosomal DNA gene and the internal transcribed spacer (ITS) region. The 340 strains were classified into 70 species. A total of 242 strains making up 71.2% of the studied strains were identified as ascomycetous yeasts and are classified in 43 taxa while 98 strains (28.8%) were classified in 27 taxa of the phylum basidiomycota. The genera Candida and Cryptococcus predominated among the ascomycetous and basidiomycetous yeasts, respectively. A total of 19 strains classified in 8 species differ in at least 1.3 % nucleotide substitutions (LSU) from their closest relative and may represent new species. Analysis of LSU of these strains showed that Candida sp. FB1-1DASP (96.5%), Candida sp. H10Y (86.5%), Candida sp. H10-10AY (86.5%), Lecytophora sp. W3a2 (96.5%) and Lecytophora sp. W7 (96.5%) did not match with sequences of their closest relatives existing in the GenBank and MycoBank, and are well separated from other known yeast species; therefore, they represent putative new taxa while others such as Bulleromyces sp. ATT 064 and ATT 067 have counterparts already depositied in the genbank. This study reveals the importance of the CMR because it holds several putative new fungal species. In addition, due to the diverse ecological and geographical origins of strains, the CMR collection is a “tool-in-hand” for bioprospecting for metabolically active compounds as well as for future barcoding studies on yeasts and yeast-like organisms.

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2.2 INTRODUCTION

The aims of fungal taxonomy are to discover, describe, and classify species of fungi and provide tools for their identification. Correct identification of microorganisms using a combination of molecular as well as cultural based methods is important for the proper assignment of strains to species level and classification in respective phylogenetic groups. Correct identification of species is also necessary for many other fields of biological sciences, including ecology, biotechnology and animal and disease diagnosis. Furthermore, in ecological studies, the concept of biodiversity is fundamentally based on the species unit (i.e. alpha diversity) because it is the base from which the higher levels of biodiversity (i.e.beta and gamma diversity) are derived (BEGEROW et al. 2010). While cultural based methods rely on phenotype, i.e., cell morphology, ability to ferment sugars and growth on various carbon and nitrogen compounds, molecular methods employs sequencing of conserved genes or genetic barcodes that ensures rapid species identification (BEGEROW, 2010). The two major ribosomal DNA regions commonly used for yeast taxonomy are the D1/D2 domains of the LSU and the internal transcribed spacer (ITS) region located between the small subunit (SSU) and LSU rRNA genes of the rDNA repeat (Fig. 1.1, Chapter 1) (KURTZMAN; ROBNETT, 1998; FELL et al. 2000; SCORZETTI et al., 2002). Most yeast species can be identified from sequence divergence in the D1/D2 domain but greater species resolution could be achieved through the combination of these genetic regions (SCORZETTI, 2002; KURTZMAN; FELL; BOEKHOUT, 2011). Recently, ITS gained the status of the universal DNA barcode for fungi because it has the highest probability of successful identification for the broadest range of fungi (SCHOCH et al., 2012). Genetic regions other than rRNA genes such as cytochrome b, elongation factor-1α gene (EF-1α), cytochrome oxidase II (COX II) gene can be additionally analyzed to delimit species inseparable by the two conventionally used genetic regions (KURTZMAN; FELL; BOEKHOUT, 2011b; SCHOCH et al., 2012). Here, we provide the identities and taxonomic classification of 340 previously unidentified yeasts (and fungi with yeast phases) deposited in the UNESP – CMR. Using a polyphasic approach for species determination we unravel the identities of several yeast strains. Particularly, to accomplish this task we used multiple molecular markers including sequencing of the ITS region, D1/D2 LSU and cytochrome b genes. Overall, we found a broad taxonomic range

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(70 taxa) within the strains identified including several yeast taxa representing potential new species. With the growing interest of biotechnological research, this work provides ready available and valuable information (about the identities and biochemical characteristics) for future studies that may involve exploration of these strains for biotechnological applications.

2.3 MATERIAL AND METHODS

A total of 340 yeasts including yeast-like organisms and dimorphic fungi isolated from various natural substrates were selected for this study (Appendix 1). Of the 340 yeasts analyzed, a total of 98 strains constituting strains isolated from Antarctic environments were studied using conventional characterization methods and this study constitute part of the objectives of the doctorate research of Alysson Wagner Duarte titled ´´Yeasts isolated from the Antarctic marine and terrestrial ecosystems: Diversity and screening of cold-active lipases`` All strains are maintained in the UNESP – CMR, The strains are preserved in 15 % glycerol at -85 °C. Taxonomic assessment of the Antarctic yeasts was performed as a collaborative work with the Prof. Lara Sette research group.

2.3.1 Cultural Characterizations i. Colony morphological characterization

Colonial characteristics studied were colour (such as white, cream, yellow, orange, peach, and red), texture (mucoid, fluid or viscous, butyrous, friable, or membranous), surface (whether glistening or dull, smooth, rough, sectored, folded, ridged, or hirsute), elevation (flat, depressed in the center, raised and dome-like, or conical) and margin (such as entire, undulating, lobed, erose, or fringed with hyphae or pseudohyphae) as described by Kurtzman et al. (2011). To examine the colony morphology all strains were cultured in YM agar at 25 ° C for 3-5 days at 15 °C for 3-7 days for strains isolated from the Antarctic environments.

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ii. Cellular characteristics

Cell morphologies were examined from cultures grown on 2% (w/v) Glucose-peptone- yeast extract agar (in g.L-1: glucose 20, peptone 10, yeast extract 5 and agar 15) or YM agar (in g.L-1: yeast extract 3, malt extract 3, peptone 5, glucose 10 and agar 15). Incubation was carried out for 3–5 days at 25°C, but for strains isolated from the Antarctica, incubation was done at 15 °C for 3-7 days. Cellular morphologies were determined by observation using phase contrast microscope (Leica DM-1000) of cell shapes and sizes, types and modes of vegetative reproduction (i.e. budding, fission and ballistoconidia) (KURTZMAN et al., 2011).

iii. Slide culture technique

The presence or absence of pseudohyphae or true hyphae where verified from 3 to 7 days old cultures on corn meal agar using slide culture technique. Molten corn meal agar was poured onto the glass slides to form a thin layer. After the agar has solidified, the yeasts were lightly inoculated in either one or two lines along each slide and a sterile cover glass was placed over the lines of streak. The Petri dish was humidified to prevent agar from drying. Incubation was carried out for 3 to 21 days and microscopic examinations were done at intervals of a few days, for formation of hyphae and pseudohyphae along the edges of the streak and under the coverglass. iv. Formation of sexual structures

Induction of ascospores (for yeasts in the ascomycetous taxa) and hyphae, clamp connections, basidia and teleospores (for yeasts in the basidiomycetous taxa) was performed by inoculating young single cultures or by mixing actively growing cultures of different strains on malt extract agar, acetate agar, synthetic nutrient agar (SNA) and corn meal agar followed by incubation at 15 and 25 ⁰C (BOEKHOUT; FONSECA; BATENBURG-VAN DER VEGTE, 1991; SAMPAIO et al., 2004, KURTZMAN et al., 2011). Cultures were observed for up to 3 months for ascospore morphology such as shape, size and number of ascospores per ascus as well as ascus ornamentation (ascomycetes) as well as hyphae and basidium (basidiomyctes).

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v) Biochemical and Physiological Tests

Biochemical and physiological characterizations were based on ability to assimilate various carbon and nitrogen compounds using replica plating method (Figure 3.1); as well as ability to ferment some sugars (as measured by the production of carbon dioxide) in Durham tubes according to methods described in Kurtzman et al. (2011). The carbon compounds tested include hexoses such as glucose and galactose, some dissacharides such as maltose, sucrose and lactose, pentoses such as D-xylose, L-arabinose and D-arabinose, alcohols (ethanol, methanol, and glycerol), organic acids (lactic acids, citric acid and succinic acid), glycosides (D-glucoside and salicine) and amines (such as D-Glucosamine and N-Acetyl-D-glucosamine). For the carbon assimilation test, cells initially grown on starvation medium (0.1% glucose) were inoculated on medium containing 0.5 % of the carbon compounds and 0.67 % yeast Nitrogen Base (Difco). For sugar fermentation tests, tubes separately containing 2 % carbon sources (namely glucose, maltose, galactose, sucrose, lactose and treahalose) and 0.5 % yeast extract were slightly inoculated with 24 h old cell suspension and incubated at 25 °C for at least 3 weeks. Fermentation ability was confirmed by collection of carbon dioxide gas in inserted tubes. Other tests used for yeast identification included Diazonium blue B (DBB) test, urease test, formation of starch-like compounds and gelatin liquefaction tests. Growth on cycloheximide 0.1 and 0.01%, high sugar and salt concentration as well as vitamin free medium were also tested (YARROW, 1998; KURTZMAN et al., 2011).

2.3.2 Molecular Identification i. DNA extraction

Genomic DNA extractions were carried out using 3-5days old cells incubated at 25 °C, on Potato Dextrose Agar (PDA), Saboraud Dextrose agar or glucose-yeast extract-peptone agar (YPGA). Two loop-full of cell culture were suspended in 500 µL of lysing buffer containing [50 mmol Tris L –1; 250 mmol NaCl L–1; 50 mmol EDTA L–1; 0.3% (w/v) sodium dodecyl sulfate; pH 8] (ALMEIDA, 2005) and approximately 200 µL of 425–600 µm glass beads (Sigma Aldrich). After being vortexed for 3 min, the tubes were incubated for 1 h at 65 °C. This step was

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repeated after which the tubes were vortexed for another 3 min and centrifuged for 15 min at 13.000 rpm. Supernatants were collected and stored at -20 °C. ii. PCR fingerprinting analysis

In order to group the isolates at species level, PCR fingerprinting analysis was performed using microsatellite-primed PCR technique (MSP-PCR) and primers (GTG)5 (5’GTG GTG GTG

GTG GTG 3’) (Prodimol) according to the method described by Gadanho and Sampaio (2002). PCR reactions were performed in 25 µL reaction volumes containing 2.5 µL of 10 X PCR buffers, 1.25 mmol l-1 of each of the four dNTPs (Prodimol), 0.8 mmol l-1 of primer, 50 mM

MgCl2, and 5 µL of the diluted supernatant containing the diluted genomic DNA (1:750) and 5U / µL of Taq DNA polymerase (Invitrogen). Amplification was performed in a GenePro Thermal Cycler (Bioer Technology). Reaction steps consisted of an initial denaturation step at 95 °C for 3 min, followed by 40 cycles at 93 °C for 45 sec, 50 °C for 1 min and 72 °C for 1 min, and a final extension step at 72 °C for 6 min. A negative control in which DNA was replaced by sterile distilled water was also included. Amplified DNA fragments were separated by electrophoresis in 1.4% (w/v) agarose gel, in 0.5 X TBE (TrisBorate-EDTA) buffer at 90 V for 3.5 h and stained with ethidium bromide or gel red. DNA 1Kb (Prodimol) was used as the reference molecular size marker. DNA banding patterns were visualized under UV and acquired using Kodak Digital Science EDA 120 System and the Kodak Digital Science 1D Image Analysis Software. One to two isolates per PCR fingerprinting group were selected for sequence-based identification. iii. DNA amplification, sequencing and identification

For DNA sequence analysis, D1/D2 domains of the large subunit of ribosomal DNA (LSU rDNA) was amplified using primers NL1 (5′-GCA TAT CAA TAAGCG GAG GAA AAG-3′) and NL4 (5′-GGT CCG TGTTTC AAGACG G-3′), and internal transcribed spacer (ITS) region was amplified using the forward primer ITS1 (5′-TCCGTAGGTGAACCTGCGG- 3′) and the reverse primer ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (WHITE et al., 1990).

Twenty-five µL of polymerase chain reactions were carried out using 1 µL of 50 mM MgCl2, 4 µL of 1.25 mM of dNTP Mix, 2.5 µL of 10X PCR buffer, 2 µL of 10 lM of each primer, 0.2 µL

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of 5 U/ µL of Taq polymerase and 5 µL of DNA template (diluted 1:750) and 8.3 µL PCR water. The amplification protocol consisted in an initial step of 96 °C for 3 min, followed by 35 cycles: 96 °C for 30s, 61 °C for 45s and 72 °C for 1 min (PAGNOCCA et al. 2008). Amplified products were purified with illustra PCR DNA and Gel Band Purification Kit (GE Healthcare UK Limited, Buckinghamshire, UK) or with Nucleospin Gel and PCR Clean-up kit (Macherey-nagel-MN). Reaction sequencing was carried out with the same forward and reverse primers used in cycle sequencing reactions using the BigDye terminator chemistry (Applied Biosystems) and sequenced on an ABI 3130 sequencer.

2.4 Results

All the isolates were initially characterized and classified by cultural and molecular approach that included, in a first stage, grouping based on morphological and phenotypic characteristics (Figure 2.1) followed by fingerprinting using MSP PCR (e.g. Figure 2.2). The results of phenotypic / biochemical characterization of the isolates are not presented in this work, however, they are provided for the three novel species described in subsequent chapters. Except for the novel strains that are described. Identification was achieved by comparing the sequences with those of related sequences in the GenBank http://blast.ncbi.nlm.nih.gov/Blast.cgi and Mycobank http://www.mycobank.org/ databases. A total of 340 strains were identified in this study. Except for the four strains of Exophiala dermatitidis, PCR amplification and sequencing of the D1/D2 LSU ribosomal DNA gene, using primers NL1 and NL4, was successful and correctly provides the identities of the strains. Appendix 1 shows the list of the strains with their respective identities (in %) to type species or most closely related phylogenetic sequences (in the case of putative new strains), isolation sources and geographic regions of isolation. The majority of the isolates (71.2%) were grouped in the ascomycetes while 28.8% are basidiomycetes. DNA sequence analyses placed the isolates in 18 ascomycetous genera and 13 basidiomycetous genera. The yeast strains in the ascomycetous and basidiomycetous genera were assigned to 43 and 27 species respectively. Three black yeasts namely Aureobasidium pullulans, A. pullulans var. melanigenum and Exophiala dermatitidis as well as yeasts with dimorphic states such as Lecythophora sp. and Sporothrix schenkii were among the species identified (Appendix 1).

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Figure 2.1. Growth (assimilation) test on carbon compound

Figure 2.2. PCR fingerprinting patterns of some identified strains. Strains that represent the same species gave rise to distinct PCR fingerprints: A, B, C, D. (M = 1kb)

A B C D

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Majority of the strains identified as Candida metapsilosis and C. parapsilosis were similar in the D1/D2 LSU ribosomal DNA gene sequences, hence, their classification were based in the differences observed in their morphological and phenotypic characteristics, MSP-PCR fingerprints and analysis of ITS regions. On the other hand, strains of Meyerozyma caribbica and M. guilliermondii presented almost similar morphological and phenotypic characteristics, but were resolved by the slight differences observable in their MSP-PCR fingerprint patterns and LSU nucleotide differences ranging between 0–3. Except for Aureobasidium pullulans, A. pullulans var. melanigenum, Cadophora luteo- olivacea, Exophiala dermatitidis and Lecytophora sp.that are classified in the Pezizomycotina, all the ascomycetous yeasts belong to the Saccharomycetales (subphylum Saccharomycetes) and were distributed in 9 families namely: Debaryomycetaceae (Candida aaseri, C. albicans, C. chickasaworum , C. metapsilosis, C. parapsilosis, C. glaebosa, C. jaroonii, C. zeylanoides, C. sake, Debaryomyces macquariensis, D. nepalensis, D. polymorphus, D. hansenii, Meyerozyma caribbica, M. guilliermondii, Schwanniomyces polymorphus); Lipomycetaceae (Myxozyma geophila); Metschnikowiaceae (C. hawaiiana, C. picinguabensis, C. rancensis, Hyphopichia burtonii, Kodamaea ohmeri, Metschnikowia australis); Pichiaceae (Candida silvae, Pichia manshurica and P. terricola); Saccharomycetaceae (Saccharomyces cerevisiae, Zygosaccharomyces mellis and Zygosaccharomyces siamensis); Saccharomycodaceae (Hanseniaspora clermontiae and H. uvarum); Saccharomycopsidaceae (Saccharomycopsis crataegensis); Trichomonascaceae (Wickerhamiella sp. strain H10Y (=Wickerhamiella sp. strain H10-10AY) and Wickerhamiella sp. strain FB1-1DASP); Wickerhamomycetaceae (C. boidinii, Sporopachydermia sp. and Wickerhamomyces anomalus). The Basidiomycetes were distributed in the Subphyla Pucciniomycotina (Cystobasidiomycetes), Agaricomycotina (Tremellomycetes), and Ustilaginomycotina (Ustilaginomycetes) and are classified in 8 orders namely Cystofilobasidiales (Cystofilobasidium capitatum, Cystofilobasidium infirmo-miniatum, Guehomyces pullulans, Rhodotorula laryngis); Sporidiobolales (R. glacialis, R. mucilaginosa, Rhodotorula sp. CRUB 1484); Leucosporidiales (Leucosporidium scottii); Tremellales (Bandoniozyma complexa, Bullera pseudoalba, Bulleromyces sp. strain ATT 046 (=ATT 067), Cr. cf.flavescens, Cr. flavus, Cr. laurentii, Cr. sp. cf.laurentii, Cr. nemorosus, Cr. victoriae, Hannaella kunmingensis and Tremella indecorata); Filobasidiales (Cryptococcus adeliensis, Cr. albidosimilis); Trichosporonales (Trichosporon

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chiarellii); Exobasidiales (Meira nashicola); and Ustilaginales (Pseudozyma hubeiensis, P. jejuensis, P. tsukubaensis).

2.5 Discussion

The combined analysis of the cultural and fingerprinting methods (MSP-PCR) corroborated for species grouping of most strains identified in this work. However, genetic heterogeneity was found among some species that were previously grouped together as the same species based on cultural or phenotypic characteristics. This was the case of Meyerozyma caribbica and Meyerozyma guilliermondii; both were indifferenciable morphologically and physiologically but varied slightly in their MSP fingerprint patterns. However, the opposite scenario was the case for Candida davisiana strains LANTA 101, LANTA 107 and LANTA 114 that were initially separated based on morphological characteristics but MSP-PCR fingerprints showed that the strains are members of the same species. Colonies of the former two strains are white with entire margin while colonies formed by the latter are orange and filamentous, thereby demonstrating intraspecific variation in this species. Except in few cases such as the strains of Exophiala dermatitidis which could only be identified based on ITS, sequence analyses of LSU and ITS proved to be useful in delimiting the previously unknown isolates as was earlier demonstrated by Kurtzman and Robnett, (1998); Fell et al. (2000) and Scorzetti et al. (2002). Due to its high rate of success for identification for the broadest range of fungi, ITS has been proposed for adoption as the primary fungal barcode marker (Schoch et al., 2012). The fact that, the black yeast Exophiala dermatitidis which could not be identified based on LSU was identifiable using ITS supports this proposal. Furthermore, high degree of of DNA relatedness (D1/D2 LSU rDNA gene sequences) was observed among strains of Candida metapsilosis and C. parapsilosis. According to TAVANTI et al. (2005), Candida metapsilosis represents the former C. parapsilosis group III and cannot be distinguished morphologically from C. parapsilosis and C. orthopsilosis (also a member of the C. parapsilosis complex) but according to Lachance et al. (2011), C. parapsilosis can be distinguished from other species of the Loddermyces clade by growth on L-arabinose (positive) and erythritol negative. This key was not useful for separating strains of C.

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metapsilosis and C. parapsilosis identified in this work, as the growth of some strains of the former were also positive on L-arabinose and negative on erythritol. Rather, MSP-PCR fingerprint proved useful for separating this two species in this study. Tavanti et al. (2005) separated C. orthopsilosis and C. metapsilosis from C. parapsilosis by multi-gene (COX3, L1A1, SADH and SYA1) analysis. Based on its high discrimination power, Mirhendi et al. (2010) proposed the use of the secondary alcohol dehydrogenase encoding gene (SADH) PCR-RFLP followed by digestion by restriction enzyme NlaIII for the separation of C. parapsilosis, C. metapsilosis and C. orthopsilosis. In most cases, sequences were compared with type strain sequences. Most of the strains exhibited LSU sequences almost similar (98-100%) to already existing species when compared with sequences deposited at the GenBank or MycoBank. However analysis of D1/D2 LSU rRNA gene sequences showed that strains namely Candida sp. strain FB1-1DASP (96.5%), Candida sp. strain H10Y (86.5%), Candida sp. H10-10AY (86.5%), Lecytophora sp. strain W3a2 (96.5%) and Lecytophora sp. strain W7 (96.5%) did not match closely with sequences existing in the GenBank and MycoBank, and are well separated from other known yeasts species; hence they represent putative new species based on the prediction that ´strains showing greater than 1% substitutions in the ca. 600-nucleotide D1/D2 domain are likely to be different species and that strains with 0–3 nucleotide differences are either conspecific or sister species’ (KURTZMAN; ROBNETT 1998). Candida sp. cf. aaseri (strains 29b, 39b, 53b), Bulleromyces sp. (strain ATT 064, ATT 067), Cryptococcus sp. cf. laurentii (LANTA 35, 59, 62, 63, 64, 70), Rhodotorula sp. CRUB 1484 strain FB1-1AASPb and Sporopachydermia sp. strain exudato M also represent putative undescribed species because the LSU sequences of these strains were not similar to those of their closest described relative but identical (except for Sporopachydermia sp.) with sequences of undescribed species existing in the GenBank. The 340 fungi isolates identified in this work were classified in 70 different taxa and only 12.3% of the identified strains represent strains isolated from geographic regions other than Brazil. The majority of the identified ascomycetous yeasts belong to the Debaryomycetaceae. Genera of the Debaryomycetaceae include numerous anamorphic species of clinical and economic importance e.g Candida albicans, C. dubliniensis, C. parapsilosis, C. metapsilosis, C. orthopsilosis and C. tropicalis. In addition, Debaryomycetaceae includes members of the Lodderomyces clade and the genus Meyerozyma. In the present study the Debaryomycetaceae

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was represented by the genus Candida was the most prominent followed by Meyerozyma (formerly Pichia) and Pichia. The genus Candida accommodates yeasts with clear ascomycetous affinity but which do not produce ascospores and are found in essentially all teleomorphic clades (SUH et al., 2006; LACHANCE et al., 2011). Despite the many multigene phylogenies that have been instrumental in revising the classification of many taxonomic groups, the genus Candida has not been reviewed yet. Hence, many types of ascomycetous yeast that have their teleomorph relatives classified in different families still have their anamorphs classified in the genus Candida, thereby, concealing biological information about the key characteristics of such groups of yeasts. Notable for their high frequency of occurrence in this work are C. metapsilosis, C. parapsilosis and C. silvae. C. metapsilosis and C. parapsislosis have long been recognized as human pathogens, accounting for the majority of cases of candidosis and have been isolated from various clinical materials. The majority of strains of these two species (identified in this work) were isolated from the leaf-cutting ant Atta sexdens and a few from soil. Suh et al. (2005) and Pagnocca et al. (2008) also reported the isolation of C. parapsilosis from Attini ants. This suggests that insects could serve as vectors for disseminating and spreading these yeast species to new habitats. Candida silvae is also widely abundant in nature and has been isolated from many miscellaneous substrates (LACHANCE et al., 2011). Although, the pool (19 strains) of C. silvae strains identified came from a single geographical region, the recordance from Drosophilid, Siphocampylus sp., Hedychium coronarium, mushroom and Bromeliad reflects substrate heterogeneity of this species. M. guilliermondii (n= 48 isolates) and M. caribbica (n= 14) were among the most prominent species in the present study. These two species formerly classified in the genus Pichia were transferred to the new genus Meyerozyma after phylogenetic analysis based on combined LSU and SSU showed that some coenzyme Q-9 producing Pichia and Candida species formed well supported clades (KURTZMAN; SUZUKI, 2010). Strains of both species were recorded from a variety of substrates and habitats as well as ecosystem including insect, anthropogenic dark earth and the extreme Antarctic environment, thereby revealing that they are metabolically and geographically diverse (KURTZMAN, 2011). Meyerozyma guilliermondii has wide biotechnological applications such as a model organism for the production of riboflavin and also for the production of xylitiol, due to its ability to convert xylose to this sugar. They are also killer yeasts used as biocontrol agents against moulds (SIBIRYNY; BETSKY, 2009).

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Basidiomycetouss yeasts are distributed in three lineages as supported by molecular phylogenetic studies based on D1/D2 domains of the LSU rRNA gene (FELL et al. 2000; HIBBETT et al, 2007) and ITS region (SCORZETTI et al., 2002). These are the Subphylum Pucciniomycotina, Subphylum Ustilaginomycotina and Subphylum Agaricomycotina. Among, the basidiomycetous yeasts identified in this study, species in the Tremellales (Agaricomycotina) were the most represented. Species in the genus Cryptococcus accounted for the highest number of basidiomycetous yeasts. Approximately 95 species of the genus Cryptococcus have been described to date. Their heterogeneity in nutritional requirements and ability to survive in harsh environmental conditions supported by the protective capsule normally formed around their cell walls account for their ecological success (FONSECA; BOEKHOUT; FELL, 2011). Species of the genus Rhodotorula were represented in almost all samples and locations as well. Ribosomal nucleotide sequences of species in this genus are divergent; hence they are polyphyletic and stretch across various classes (SCORZETTI, 2002). Five strains, isolated from fungus gardens (n= 3) and corroding aluminum screw of energy transmission tower (n= 2) were classified in the newly created genus Bandoniozyma in the Bulleromyces clade of the Tremellales. In addition to Bandoniozyma complexa (which was described based on the later two strains and 6 additional strains), six other taxa namely B. noutii (type species of the genus), B. aquatica, B. fermentans, B. glucofermentans, B. tunnelae and B. visegradensis were recently described in this genus (VALENTE et al. 2012). An important taxonomic key to species classified in this genus is the ability to ferment glucose, a rare trait among basidiomycetous yeasts. Trichosporon chiarellii was the only species classified in the Trichosporonales. This species was fomerly described based on strains isolated from nests of a fungus growing ant Myrmicocrypta camargoi by Pagnocca et al. (2010). The two strains studied in the present work (A4-D8 and A4-D9) also originated from fungus gardens but from an Acromyrmex sp. ant nest located in the state of Tocantins, Brazil. Repeated isolation of this species from fungus growing ant gardens suggests that it may be well adapted to this habitat (FERNANDO CARLOS PAGNOCCA, personal observation). Identification of some strains (n= 10) revealed that they belong to the group of fungi known as dimorphic fungi (in the genus Aureobasidium, Lecytophora, Exophiala and Sporothrix). Two species of the dimorphic euascomycete genus Aureobasidium namely A. pullulans and A. pullulans var. melanogenum and a species of the black "yeast-like" fungi

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Exophiala which were isolated from the fungus growing ant Atta sexdens (Tocantins, Brazil) were also among the yeasts identified. Aureobasidium phylogenetically belong to Ascomycota, Dothideales order, family Dothideaceae (SCHOCH et al. 2006), however, this group of fungi is reffered to as yeast-like because they usually produce abundant yeast states and often appear white to light pink in color on isolation plates that later turns blackish due to production of a specific melanin at chlamydospore production stage (HOOG; YURLOVA, 1994). A. pullulans are associated with a wide range of terrestrial and aquatic habitats. They are also commonly associated with fungus growing ants (PAGNOCCA et al. 2008). They are of high biotechnological importance due to their ability to secrete various metabolites, enzymes, antibiotics, single cell protein (SCP) and polysaccharides (GAUR et al., 2010). Exophiala species are commonly associated with decaying wood and soil enriched with organic wastes; however, they have also been reported from clinical cases from both normal and immunosuppressed patients (HOOG; HERMANIDES-NIJHOF, 1977). An isolate (Exudato 1) of the species of the achlorophyllic yeast-like algal genus Prototheca provided identical (100%) sequences to Prototheca ulmea. Other fungi that produced cells or yeast-like forms were identified including Cadophora lutea-olivacea, Exophiala dermatitidis, Lecythophora sp. and Sporothrix schenckii. The dimorphic fungi Sporothrix schenckii is classified in the ascomycetous family Ophiostomataceae (Class Sordariomycetes, Order Ophiostomatales). S. schenckii is the etiological agent of sporotrichosis. This dimorphic fungi exists as saprotrophic when grown at moderate temperatures (22°C –28°C), but convert to a yeast phase upon tissue invasion or in vitro culture. There are no records of previous isolation of this species from Attini fungus gardens and it means that they can be dispersed by these ants. This study reveals that the UNESP – CMR holds a high number of metabolically, ecologically and geographically diverse yeasts. Hence, could be explored by researchers searching for yeast strains with important biotechnological properties or activities including the presence of a particular enzyme, pigments, aromatic compounds, antibiotics etc. as well as for the presence of a particular gene of interest. Natural products from these yeasts can be applied in many commercial processes including biofuel production, cosmetics, food, and pharmaceutical processes. Ribosomal sequence analysis has revealed many unpublished species. Some of the identified species such as Trichosporon chiarellii are currently not available from any other source. At this period when fungal taxonomy faces serious challenges of species extinction, the

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description of these species will contribute to the recovery of extant fungal species as well as the cataloguing of fungal diversity.

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PAGNOCCA, F. C.; RODRIGUES, A.; NAGAMOTO, N. S.; BACCI, M. JR. Yeast and filamentous fungi carried by the gynes of leaf-cutting ants. Antonie van Leeuwenhoek, Amsterdam, v. 94, n. 4, p. 517-526, nov. 2008.

PAGNOCCA, F. C.; LEGASPE, M. F. C.; RODRIGUES, A.; RUIVO, C. C. C.; NAGAMOTO, N. S.; BACCI JR, M.; FORTI, L. C. Yeasts isolated from a fungus-growing ant nest, including the description of Trichosporon chiarellii sp. nov., an anamorphic basidiomycetous yeast. International Journal of Systemic and Evolutionary Microbiology, Reading, v. 60, n. 6, 1454-1459, jun. 2010.

SAMPAIO, J. P.; INACIO, J.; FONSECA, A.; GADANHO, M.; SPENCER-MARTINS, I.; SCORZETTI, G.; FELL, J. W. Auriculibuller fuscus gen. nov., sp. nov. and Bullera japonica sp. nov., novel taxa in the Tremellales. International Journal of Systematic and Evolutionary Microbiology, Reading. v. 54, n. 3, p. 987-993, may. 2004.

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SCORZETTI, G.; FELL, J. W.; FONSECA, A.; STATZELL-TALLMAN, A. Systematics of basidiomycetous yeasts: a comparison of large subunit D1D2 and internal transcribed spacer rDNA regions. FEMS Yeast Research, Amsterdam, v. 2, n. 4, p. 495-517, dec. 2002.

SIBIRNY, A. A.; BORETSKY, Y. R. Pichia guilliermodii. In: SATYANARAYANA, T.; KUNZE, G. Yeast biotechnology: diversity and applications. Berlin: Springer, 2009, p. 114- 132.

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TAVANTI, A.; DAVIDSON, A. D.; GOW, N. A. R.; MAIDEN, M. C. J.; ODDS, F. C. Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis Groups II and III. Journal of Clinical Microbiology, Washington, v. 43, n. 1, 284-292, jan. 2005.

VALENTE, P.; BOEKHOUT, T.; LANDELL, M. F,; CRESTANI, J.; PAGNOCCA, F. C. ET AL. Bandoniozyma gen. nov., a Genus of Fermentative and Non-Fermentative Tremellaceous Yeast species. PLoS One, California, v. 7, n. 10, e46060, 2012. (doi:10.1371/journal.pone.0046060)

WHITE, T. J.; BRUNS, T.; LEE, S.; TAYLOR, J. W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: INNIS, M. A.; GELFAND, D. H.; SNINSKY, J. J.; WHITE T. J. (Eds.) PCR Protocols: A Guide to Methods and Applications. New York: Academic Press, 1990. p. 315-322.

YARROW, D. Methods for the isolation, maintenance and identification of yeasts. In: KURTZMAN, C. P.; FELL, J. W. (Eds.) The yeasts: a taxonomic study, 4th edn. Amsterdam: Elsevier, 1998. P. 77–100.

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CHAPTER 3

SCREENING FOR AMYLOLYTIC, LIGNOCELUOLYTIC AND PECTINOLYTIC YEASTS AND DIMORPHIC FUNGI

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3.1 ABSTRACT

Plant polysaccharide degrading enzymes are useful in various biotechnological applications, especially in the production of biofuels and microorganisms have been recognized as the basis for the discovery of novel enzymes. In this study, a total of 312 yeast isolates (a subset of yeasts examined in chapter 2) comprehending 188 ascomycetes (49 species) and 124 basidiomycetes (44 species) were screened for extracellular enzymes (amylase, cellulase, xylanase, pectinase and ligninase) activities. Overall, amylase activity was detected in 28 strains representing 8.97% of the total isolates screened; cellulase activity was found in 64 strains (20.51%), xylanase activity in 87 strains (27.88%), polygalacturonase activity in 45 strains (14.42%), pectin lyase activity in 59 strains (18.91%) and ligninase activity in 2 strains (0.64%). Among the ascomycetous species pectin degradation (pectin lyase) was the ability most displayed, been obsevered among 37 strains making up 16 species. On the other hand, xylanase (71 strains of 28 species) followed by cellulase (38 strains of 17 species) were the enzyme activities most exhibited among the basidiomycetous yeasts. Quantitative xylanase enzymes assay was determined for 105 strains of yeasts constituting 32 xylanase positive strains isolated from Antarctic terrestrial and marine environments and 73 strains selected from the 87 total xylanase positive strains obtained from the screening studies, based on their diameter degradation halo (≥ 5 mm diameter). Two strains (Pseudozyma hubeiensis strain MP2-2CB and Aureobasidium pullulans strain CG5-5BY) were found to produce maximum endoxylanase (0.70U/L) and β-xylosidase (1.13 U/L) activites, respectively. Extracellular enzyme production from sugarcane bagasse fermentation was determined for these strains in addition to Aureobasidium pullulans strain PBM 1. Amylase, endoamylase, cellulase, xylanase and pectinase enzyme were detected in the three strains. Our findings revealed that the UNESP - CMR harbors interesting strains with ability to produce various industrially useful enzymes. Such strains could be explored for future biotechnological applications.

KEY WORDS: Plant polysaccharides, xylanase, sugarcane bagasse, Pseudozyma hubeiensis

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3.2 INTRODUCTION

Starch based feed stock have been used for bio-ethanol production (HOSTINOVÁ, 2002) but plant (lignocellulosic) biomass, such as wood chips, corn cobs, sugar cane bagasse, grass etc., contain high contents of sugar polymers. Plant biomass is abundant and renewable; ands offers sustainable alternantive for bioethanol production for future generation (SANCHEZ, 2009; SIVAKUMAR et al. 2010). However, the conversion of plant biomass to fuels relies on enzymatic digestion of plant polysaccharides to fermentable feedstocks (ALMEIDA et al. 2007); therefore, enzymes capable of plant polysaccharide depolymerization are of industrial importance. The breakdown of lignocellulose, the major component of plant biomass, is contributed not only by environmental factors but also by the degradative capacity of a microbial population. Lignocellulases are applicable in different stages of various industrial processes such as chemicals, fuel, food, brewery and wine, animal feed, textile and laundry, pulp and paper, and agriculture. Lignocellulases are also useful in wastewater treatment (HOWARD et al. 2003; KUHAD, 2011). Other promising areas for application of lignocellulases are in the de-inking of recycled fibers and the release of toners from office wastes (PÉREZ et al. 2002). Ability of yeasts to degrade complex polysaccharides is of high taxonomic, ecological and biotechnological importance (BIELY et al. 1998). In contrast to filamentous fungi and bacteria, few yeasts are known to be capable of degrading plant polysaccharides. This is partly contributed by the fact that the majority of previous screenings have been often limited to a few numbers or narrow range of yeast species. Screening of microorganisms continues to be an important primary step in biotechnology, for the discovery and selection afterwards of not only potentially useful strains, but also for the discovery of new genes applicable in physiological as well as genetic manipulation studies. In this work, an extensive screening of selected yeasts from the UNESP – CRM was carried out. The strains were tested for plant polysaccharides (starch, cellulose, xylan, pectin and lignin degrading enzymes activities. Xylanases are receiving more attention as ´accessory enzymes` in ethanol production from biomass, whereby they are used for dissociating xylan from the cellulosic-rich water insoluble fraction in order to improve cellulose accessibility (ALMEIDA, 2007; CHANDRA et al. 2007).

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In this section, special attention was paid to xylan degrading yeasts with the purpose of selecting strains applicable for the production of enzymes useful in the bioconversion of plant residues such as sugar cane bagasse to higer value products. The complete degradation of xylan requires the cooperative actions of a variety of hydrolytic enzymes, two major of which are endo-1,4-β- xylanase (EC 3.2.1.8) and xylan 1,4-β-xylosidase (EC 3.2.1.37). Although endo-xylanases are more common than β-xylosidases, the latter are necessary for the complete breakdown of xylan. These two groups of xylanases were also investigated in this work. Hence, the specific objectives of this work were to screen yeasts with ability to produce cellulases, hemicellulases and ligninases, to quantitatively determine the endo-xylanase and β- xylosidase activities of selected xylanase producing strains discovered in this study and previous studies as well as to determine afterwards, the production of polysaccharide degrading enzymes using sugar cane bagasse as substrate.

3.3 Materials and Methods

3.3.1 Screening procedures for extracellular enzymatic activities

A total of 312 yeasts from the UNESP – CMR were used in the present study (Table 3.1). The strains are preserved in 15% glycerol at -85 °C. The tested strains were reactivated on Sabouraud-dextrose agar or Potato-dextrose agar and not more than 3 days old culture were used for the entire test. Starch, cellulose, xylan, pectin and lignin degrading activities were investigated on specific polysaccharide substrates on solid medium. All media were sterilized in autoclave at 121 °C for 15 min. Inoculum from 3 days-old cultures, standardized to 106 cells.mL- 1, were inoculated on the test media with the aid of a point inoculator devise. All tests were carried out in triplicate. Pseudozyma tsukubaensis strain MG270406-2B56a (UNESP – CMR) was used as the positive control for amylase, cellulase, xylanase, polygalacturonase and pectin lyase activites (MENDES et al., 2012). Growth medium lacking the inducing substrates for the respective enzymes tested was used as the negative control. Polysaccharides were purchased from Sigma-Aldrich and all other chemicals used were reagent-grade. Autoclaving were carried out for 15 min at 121 °C.

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Table 3.1 - Number and origin of yeasts and dimorphic fungi profiled for enzymatic activity Substrate Sampling site and location Number of isolate

Soil Corumbataí, SP, Brazil, (S22°17’49.13’’; 17 W47°39’16.60’’) Botucatu, SP, Brazil, (S22°50.579’; W48°26.131’ 4 Fungus garden of ant (Atta sexdens Corumbataí, SP, Brazil, GPS: S22°17’49.13’’ and 11 rubropilosa) W47°39’16.60’’) Fungus gardens of ant (Cyphomyrmex 4 wheeleri) Fungus gardens of ant (Atta texana) Bastrop County/TX/USA, 44 (N30°13. 973’, W97°39.101’; N30° 13.94’), (W97°39.18’; N30°14.008’, W97°39.039’) Garden of an unidentified fungus growing ant Toccantins, Brazil 14

Ant (Atta sexdens) ,, 62 Adult Wasp (Polybia ignobilis) Rio Claro, São Paulo, Brazil. (22º23’49.13’’S; 38 47º32’36.87’’W) Pupa of wasp (Polybia ignobilis) ,, 2 Honey (food) from wasp (Polybia ignobilis) ,, 14 Honey from bee Apis melifera Rio Grande do Sul, Brazil 12 Drosophilid Atlantic Rainforest, Pindamonhangaba municipal – 4 SP (latitude 22o 44’ 28’’; longitude 45º 28’ 19’’). Exudate from Palm plant (Euterpe sp.) ,, 10 Exudate from Banana plant (Musa sp.) 5 Unidentified mushroom ,, 11 Water accumulated in tank of Bromeliad ,, 24 (Vriesea sp.) Flower bracts of Siphocampylus sp. ,, 12 (Campanulaceae) Flower of Hedychium coronarium Koening ,, 5

Fruit of Hedychium coronarium Koening ,, 13

Corroding aluminum screw of energy 6 transmission tower Sao Paulo, Brazil

Total 312

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Inoculum from 3 days old cultures, standardized to 106 cells.mL-1, were inoculated on the test media with the aid of a point inoculator devise.

3.3.2 Amylase activity

Amylolytic activity was determined on medium containing 2% starch (as inducing substrate) and 1.5% agar and adjusted to pH 6 according to Buzzini and Martini (2002) with few modification which included the addition of 0.5% ammonium sulphate [(NH4)2SO4] and 0.1% potassium hydrogen phosphate (KH2PO4). After the plates were incubated for 7 days at 25 °C, they were flooded with Lugol`s iodine (Gram iodine solution: 0.1% I2 and 1% KI) for 5 min and rinsed with deionized water. A clear zone around the colony indicates strain having amylolytic activity.

3.3.3 Cellulases

Cellulase activity was determined in medium containing 5 g/L carboxymethylcellulose (CMC) as substrate; 0.67g/L Difco Yeast Nitrogen base (YNB), and 1.5 g/L agar. After incubation for 7 days, plates were flooded with Congo red solution (1 g/L) prepared by dissolving 100 mg Congo red in 99 ml of water for 15 minutes followed by subsequent de-staining with NaCl (1 M) solution for 5 minutes. Positive strains were detected by formation of clear halo around colonies indicating the degradation of carboxymethylcellulose (CMC) (STRAUSS et al., 2001).

3.3.4 Xylanases

The method of Pointing (1993) was used for the detection of xylanase-producing strains. Assay medium contained Birchwood xylan (Sigma), 1g/L; YNB, 0.67g/L and agar 1.5g/L. After incubation for 7 days, plates were flooded with lugol for 15 minutes followed by rinsing with deionized water. Xylanase activity was detected by the formation of clear degradation haloaround positive strains. Xylanase-producing strains that exhibited degradation halos above 5 mm were selected for quantitative enzymes assay.

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3.3.5 Pectinases

Pectin degrading activity was investigated in medium containing polygalacturonic acid (for polygalacturonase activity), 1.25g/L or apple pectin (for pectin lyase activity), 1.0 g/L; YNB, 6.7 g/L; glucose, 2g/L and agarose (Sigma, A-953), 10 g/L in 50 mM potassium phosphate buffer at pH 5.5. Agarose was autoclaved separately to prevent acid hydrolysis and then mixed with the remaining medium upon cooling to about 55 °C. After 5 days of incubation at 25 °C, the plates were flooded with a 0.1% ruthenium red (Sigma, R-2757) solution and incubated for 1 hour at room temperature (OLIVEIRA et al. 2006). After this period the plates were rinsed with deionized water. Polygalacturonase activity was detected by the formation of a purple halo around positive strains on polygalacturonic acid medium while pectin lyase activity is evident by the formation of a clear halo around colonies on apple pectin medium (DA SILVA et al. 2005; OLIVIERA, 2006).

3.3.6 Ligninases

Dye decolorization of Remazol Brilliant Blue R (RBBR) was used to evaluate lignin degrading ability according to the method described by Kiiskinen; Rättö; Kruus (2004) and Machado et al. (2005) with little modification. The medium contained RBBR, 0.3g/L; glucose, 10g/L; peptone, 2g/L, yeast extract, 1g/L and agar 1,5g/L. Plates were incubated at 25 °C for 14 days. Formation of a clear halo around the colony indicates production of lignin degrading enzymes.

3.3.7 Xylanase enzymes assays i. Xylanase production in shake flask cultures A total of 105 strains xylanase-producing strains were selected for the quantitative determination of xylanase activities. The one hundred and five strains constitute 73 strains that exhibited degradation halos above 5 mm in the previous plate screening test and 32 xylanase positive strains obtained from a previus study carried out on yeasts isolated from Antarctic terrestrial and marine environments. Crude enzyme preparations were obtained by inoculating

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xylanase positive strains in medium containing birchwood xylan as the xylanase inducing substrate. One mL of cell suspension prepared to a concentration of 106 cells.mL-1 were used to inoculate 25 mL of Vogel`s medium (VOGEL, 1956) containing 0,5 % birchwood xylan in a 150 mL capacity Erlenmeyer flask. The inoculated flasks were incubated at 25 °C under agitation (120 rpm) in a rotary shaker. Samples were retrieved after 3 days of incubation. Slurry from each sample was centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatants were used as extracellular crude enzymes source. ii. Estimation of endoxylanase activity

Xylanase activity was assayed using 1.0 % (w/v) birchwood xylan as a substrate. The 300 µL reaction mixture contained 100 µL each of diluted enzyme and 0.5 % xylan in 50mM acetate buffer at pH 5.5. The mixture was incubated at 25 °C for 30 min. After incubation, xylanase activity was determined by measuring the amount of reducing sugars released from birchwood xylan according to dinitrosalicylic acid method of MILLER (1959) using xylose as standard. Absorbance readings of the resulting samples were measured against a blank at 575 nm using spectrophotometer (Beckman DU 640). One unit of enzyme activity was defined as the enzyme amount that releases 1 µmol of reducing sugar per min. iii. Estimation of β-xylosidase activity

β-xylosidase activity was estimated using cell-associated enzyme according to Ohta et al. (2010) with modifications. Two hundred µL of homogenized culture suspension was centrifuged and washed twice with sterilized distilled water. The cells were later resuspended in 200 µL of 50 mM, pH 5.5, sodium acetate buffer and ground with sterile glass beads. The slurry was centrifuged at 16,000 g at 4 °C and the supernatant was used as intracellular enzymes source. The assay mixture contained 200 µL 50mM sodium acetate buffer, pH 5.5, 200 µL 4mg/mL of p- nitrophenyl-β-D-xylopyranoside (6892-58-6) and 100 µL of enzyme preparation. The reaction mixtures were incubated at 40 °C for 30 min and the reaction was stopped by adding 1 mL of 1M

Na2CO3 solution and amount of p-nitrophenol released was determined spectrophotometrically by measuring the absorbance at 410 nm.

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3.3.8 Fermentation of sugarcane bagasse

Three strains exhibiting the highest xylanase (endoxylanase and β-xylosidase) activities were selected for the determination of enzymes (endoamylase, exoamylase, cellulase, xylanase and pectinase) production fom sugar cane bagasse fermentation. Cell suspensions of Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans PBM1 and A. pullulans CG5-5BY standardized to 106 cells / mL were separately used to inoculate 2.5g of sugar cane bagasse in Vogel`s medium (VOGEL, 1956) and incubated for 72 hours at 25 °C without agitation. After cultivation the culture filtrate was centrifuged at 16,000 g for 10 min at 4 °C and the supernatant was used for the determination of enzyme activities. Supernatant of uninoculated sugar cane bagasse was used for quantification as the negative control.

3.3.9 Enzyme assays

Dinitrosalicylic acid (DNS) method (MILLER, 1959) was used for the assay of amylolytic (endoamylase and exoamylase), endoglucanase (FROM carboxymethyl cellulose and microcrystalline cellulose), xylanase and pectinase activities in culture filterate obtained from sugarcane bagasse fermentation. Reaction mixture contained 80 µL of inducing substrate and 20 µL of culture filtrate. The amount of sugar released was measured spectrophotometrically at 575 nm and the enzyme activity was calculated according to standard curves that correlated with the reducing sugars released. Uninoculated sugarcane bagasse suspension was used as blank. i. Endoamylase (amylolytic) assay

For endoamylase (endo-1,4-α-D-glucan glucanohydrolase) and exoamylase activities detection, substrates containing 0.5 and 1% starch in 50 mM acetate buffer, pH 5.0 were used respectively and the reaction mixtures were incubated for 30 mins at 40 °C (ROBERTSON et al. 2006). The amount of glucose released was measured spectrophotometrically at 575 nm and enzyme activities were calculated according to standard curves of glucose; one unit of activity was defined as the amount of enzyme that converted 1 µmole of substrate per minute.

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ii. Pectinase assay

Pectinase activity was determined in a reaction mixture containing 0.2 % citric pectin in 50 mM acetate buffer, pH 5.0, and incubated for 1 hour at 25 °C. Pectinase activity was inferred from a standard curve of polygalacturonic acid. The amount of galacturonic acid released was measured spectrophotometrically at 575 nm and one unit of pectinase activity was defined as the amount of enzyme that liberated 1 µmol of galacturonic acid per min (SURESH; VIRUTHAGIR, 2010). iii. Xylanase assay

Xylanase (endo-1,4-β-xylanase) activity was performed in a reaction mixture containing 0.2% (w/v) birchwood xylan in 0.1 M citrate/phosphate buffer (pH 6.0), and incubated for 2 hours at 25°C. Xylose was used as reference reducing sugar for the preparation of standard curve. One unit of Xylanase activity was defined as the amount of enzyme that liberated 1 µmol of xylose per min. iv. Endoglucanase

Endoglucanase (endo-1,4-β-glucanase) activities from carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC) were respectively assayed in 1 hr reactions, containing 0.625% (w/v) of each substrates in 50 mM citrate/phosphate buffer (pH 6.0) solution at 25 °C. One endoglucanase unit is the amount of enzyme necessary to produce 1 µmol of glucose as glucose equivalents per min under the standard assay conditions (OPPERT et al., 2010).

v. Determination of total reducing sugars Total reducing sugars released in the fermented sugarcane bagasse hydrolysate were quantified by dinitrosalicylic acid (DNS) method according to Miller (1985). One hundred microlitre of culture filterate was added to DNS in equal proportion

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3.3.10 Statistical analysis

Statistical one-way anova was performed in R version 2.13.1 (2011-07-08) software package (The R Foundation for Statistical Computing) using Tukey T test. All the data are presented as the mean ± S.D. of triplicates (n = 3) except for enzyme assays from sugar cane bagasse fermentation where mean S.D was deduced from 15 replicates. Differences at p< 0.10 were regarded as statistically significant at 95% confidence limit. With respect to xylanase assays, multiple comparations of tested strains are presented based on results of endoxylanase activities.

3.4 Results

3.4.1 Screening for enzymatic activities

A total of 312 strains of yeasts were tested for starch, lignocellulose (cellulose, xylan, and lignin) and pectin degrading abilities. Positive results were inferred from the production of clear zones (halo) of degradation or pinkish/purple coloured halo (in the case of polygalacturonase activity) around growth colonies as are exemplified in Figure 3.1. The 312 strains constitute 188 ascomycetes and 124 basidiomycetes. Detailed information about the isolates, source of isolation and their enzyme profiles are presented in appendix 2. The ascomycetous yeasts are made up of 49 different species (Tables 3.2), while the basidiomycetous yeasts consist of 44 species (Tables 3.3). Overall, amylase activity was detected in a total of 28 strains representing 8.95% of the total isolates screened; cellulase activity was found in 64 strains (20.51%), xylanase activity in 87 strains (27.88%), polygalacturonase activity in 45 strains (14.42%), pectin lyase activity in 59 strains (18.91%) and ligninase activity in 2 strains (0.64%) (Tables 3. 2 and 3.3, Appendix 2, Fig. 3.2). The high number of Xylanase positive strains of species of Aureobasidium pullulans, Bandoniozyma complexa, Cryptococcus flavescence, Cryptococcus flavus and Trichosporon jirovecii among others contributed to the high number of xylanase producing yeast discovered. The ascomycetous species Aureobasidium pullulans was the best producer of amylase followed by Cryptococcus flavus.

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Figure 3.1. Degradation halos around yeast strains producing amylase (A), cellulose (B), xylanase (C), polygalacturonase (D), pectin lyase (E) and ligninase (F).

A B C

D E F

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Table 3.2. Ascomycetous yeasts screened for amylolytic and cellulolytic and pectinolytic activities

No. of Number of positive strains strains Yeast tested Amy Cel Xyl P. galact P. lyase Aureobasidium pullulans 6 4 6 5 2 1 Aureobasidium pullulans var. melanigenum 1 1 1 1 1 0 Candida cf. aaseri 1 0 0 0 0 1 C. albicans 7 0 1 0 0 0 C. boidinii 4 0 0 0 0 0 C. apicola 2 0 0 1 0 0 C. azyma 9 0 0 0 0 0 C. chickasaworum 1 0 0 0 1 0 C.chrysomelidarum 7 0 0 0 0 0 C. glaebosa 1 0 0 0 0 0 C. hawaiiana 1 0 0 0 0 0 C. jaroonii 1 0 0 0 0 0 C. melibiosica 1 0 0 0 0 0 C. membranifaciens 3 0 0 0 0 0 C. metapsilosis 20 0 0 0 0 0 C. oleophila 4 0 0 0 1 0 C. parapsilosis 17 0 0 0 0 0 C. picinguabensis 2 0 0 0 0 0 C. restingae 2 0 0 0 0 0 C. shehatae var. insectosa 3 0 0 0 0 2 C. shehatae var. shehatae 2 0 0 0 0 0 C. silvae 24 1 4 0 16 14 Candida sp. strain H10Y 2 0 1 0 0 1 Candida sp. strain FB1-1DASP 1 0 0 0 0 0 Candida sp. strain larva 5a 1 0 0 0 0 0 C. zyelanoides 1 0 0 1 0 0 Debaryomyces hansenii 3 0 0 0 1 1 Exophiala dermatitidis 4 0 0 4 0 0 Hanseniaspora clermontiae 1 0 0 0 0 0 H. guilliermondii 2 0 0 0 0 2 H. opuntiae 2 0 0 0 0 0 H. uvarum 2 0 1 0 0 0 Haneniaspora sp. strain S18a 1 0 0 0 0 1 Hyphopichia burtonii 2 2 2 0 0 0 Kodamaea ohmeri 1 0 0 0 1 0 Lecytophora sp. strain W3 2 2 2 2 0 2 Metschnikowia reukaufii 6 0 2 0 1 2 M. koreensis 1 0 1 0 0 0

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Table 3.2 - Ascomycetous yeasts screened for amylolytic and cellulolytic and pectinolytic activities (continuation) No. of Number of positive strains strains Yeast tested Amy Cel Xyl P. galact P. lyase Meyerozyma caribbica 2 0 0 0 0 0 Meyerozyma guilliermondii 8 0 0 1 0 4 Pichia kudriavzevii 3 0 2 0 0 0 Scheffersomyces spartinae 6 0 0 0 2 1 Saccharomyces paradoxus 1 0 0 1 0 0 Saccharomycopsis crataegensis 1 0 1 0 0 1 Sporopachydermia sp.strain exudato M 1 0 0 0 0 1 Sporothrix schenckii 2 2 2 0 2 2 Torulaspora delbrueckii 2 0 0 0 1 1 Zygosaccharomyces mellis 10 0 0 0 0 0 Zygosaccharomyces sp. 1 0 0 0 0 0 Total 188 12 26 16 29 37

Amy = amylase, Cel = Cellulase, Xyl = Xylanase, P. galact = polygalacturonase, P. lyase = pectin lyase

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Table 3.3. Basidiomycetous yeasts screened for amylolytic and cellulolytic and pectinolytic activities

Number Number of positive strains of strains Yeast tested Amy Cel Xyl P. galact P. lyase Bandoniozyma complexa 3 1 3 0 0 0 Bullera sinensis 1 1 1 1 0 0 Bulleromyces albus 1 0 1 1 0 0 Bulleromyces sp. 2 0 2 2 0 0 Cryptococcus albidosimilis 3 0 1 2 0 2 Cr.aff cylindricus 1 0 0 0 0 0 Cr. dimennae 2 0 0 2 0 0 Cr. flavescens 8 0 0 6 0 2 Cr. flavus 4 3 3 4 0 0 Cr. laurentii 28 3 12 22 7 2 Cr. liquefaciens 4 0 0 3 0 1 Cr. magnus 3 0 2 3 0 0 Cr. nemorosus 2 1 0 1 0 0 Cr. podzolicus 3 1 1 2 0 1 Cryptococcus sp PYCC 4949 1 0 0 0 0 1 Cryptococcus sp.CBS 681.93 1 0 1 1 0 0 Cryptococcus sp. CBS 8372 1 0 0 0 0 0 Cryptococcus sp. CBS 7944 1 1 0 1 1 0 Cryptococcus sp. CBS 8369 2 0 2 1 0 0 Cr. taibaiensis 1 0 1 0 0 0 Farysizyma taiwaniana 1 0 0 1 0 0 Hannaella kunmingensis 3 0 0 2 0 0 Meira nashicola 1 0 0 0 0 0 Moniliella suaveolens var. suaveolens 1 0 0 0 0 0 Pseudozyma hubeiensis 1 1 1 1 0 0 P. jejuensis 2 0 2 2 0 1 P. tsukubaensis 1 0 1 0 1 1 Pseudozyma sp. Strain ATT 068 1 1 1 1 1 1 Pseudozyma sp. BCRC 34227 3 0 0 2 0 1 Rhodotorula javanica 1 0 0 1 0 0 R. lactosa 1 0 0 0 0 0 R. marina 1 0 0 1 0 0 R. minuta 1 0 0 0 0 0 R. mucilaginosa 15 0 0 0 0 3 R. nothofagi 1 1 0 1 1 0 Rhodotorula sp. CBS 8885 1 0 0 0 0 0 Rhodotorula sp. CRUB 1484 2 0 0 0 0 2 Rhodosporidium sp. APSS 849 1 0 0 1 0 0

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Table 3.3. Basidiomycetous yeasts screened for amylolytic and cellulolytic and pectinolytic activities (continuation) Number Number of positive strains of strains Yeast tested Amy Cel Xyl P. galact P. lyase Sporidiobolus ruineniae 3 0 0 0 3 0 Sporisorium penniseti 2 0 0 1 2 0 Sympodiomycopsis paphiopedili 2 0 0 2 0 0 Trichosporon chiarellii 2 0 0 0 0 2 T. jirovecii 3 0 3 3 0 0 Ustilago spermophora 2 2 0 0 0 2 Total 124 16 38 71 16 22

Amy = amylase, CMC cel = Cellulase, Xyl = Xylanase, P. galact = polygalacturonase, P. lyase = pectin lyase

Figure 3.2. Enzymatic activity profiles of the ascomycetous and basidiomycetous yeasts screened

30 Basidiomycetes Ascomycetes

25

20

15

10 Number of strains (%)

5

0

Amylase Cellulase Xylanase Ligninase Pectin lyase

Polygalacturonase Enzyme activities

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Figure 3.3 - Comparisons of enzyme activity profiles of ascomycetous and basidiomycetous yeasts respectively. Bars represent percentage of species in which positive activities were recorded for the various enzymes in the two respective phyla.

Amylase

Cellulase

Xylanase

Polygalacturonase

Pectin lyase

Ascomycetes Ligninase

Amylase

Cellulase

Xylanase

Polygalacturonase

Pectin lyase Basidiomycetes Ligninase

0102030405060 Species of ascomycetes and basidiomycetes (%)

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The presence of cellulase was also detected in all the strains of A. pullulans (n=7) tested, including many strains of C. laurentii (n=12 of 28), Bandoniozyma complexa (n=3) and C. flavus (n=3 of 4). Candida silvae, Sporothrix schenkii, Pseudozyma tsukubansis and Pseudozyma sp. strain ATT068 among others produced both polygalacturonase and pectin lyase enzymes. Of the 188 strains of ascomycetous yeasts screened for amylase activity, 12 strains constituting A. pullulans (n= 4), A. pullulans var. melanigenum (n= 1), C. silvae (n= 1), Hyphopichia burtonii (n= 2), Lecytophora sp. (n= 2) and S. schenckii (n= 2) demonstrated these activities (Table 3.2). The ascomycetous species Aureobasidium pullulans was the best producer of this enzyme. Cellulase activity was detected in 13.82% of the ascomycetous strains screened while xylan degrading ability was found in only 16 strains constituting 8.51% of the total ascomycetous yeasts (Fig. 3.2). With respect to polygalacturonase activity, 29 strains making up 15.42% of the ascomycetous strains demonstrated ability to produce this enzyme while pectin lyase activity was detected in 19.68% strains. Moreover, ligninase activity was not detected in any of the ascomycetous yeasts. Pectin lyase activity was detected in the highest number (16 species constituting 32.7%) of ascomycetous species. This was followed by cellulase (13 species) and polygalacturonase (11 species) activities (Table 3.2, Fig. 3.3). On the other hand 12.2% and 16.9% of the ascomycetous yeasts specied produced amylase and xylanase enzymes respectively. Except for C. silvae with majority of strains showing positive polygalacturonase and pectin lyase activities, enzyme activities were not detected on majority of the species of Candida tested. However, except for ligninases all the enzymes screened were detected in most of the strains of A. pullulans tested. These species including Lecytophora sp., Metschnokowia reukauufii and Sporothrix schenkii produced at least 3 classes of enzymes (Table 3.2). With respect to the basidiomycetous taxa, 124 strains covering 44 taxa were screened. The presence of amylase was confirmed in 12.90% of the 124 basidiomycetous strains investigated. Approximately 31 and 57 % of the strains showed capacities to degrade cellulose and xylan respectively (Figure 3.2). Whereas, polygalacturonase activity was detected in 16 strains making up 12.9% of the screened basidiomycetous cultures, 22 strains making up 17. 74% where positive for pectin lyase enzyme while only 1.61% showed the capacity to produce ligninase enzyme (Table 3.3, Figure 3.3). Xylanses and cellulases were the enzymes most produced by the basidiomycetous yeast species being detected in 28 species making up 63.6% and 17 species making up 38.6% repectively. Eleven species constituting 25% the basidiomycetous yeasts demonstrated amylase activities (Figure 3.3). While polygalacturonase activity was detected in only 7 species, pectin

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lyase activity was detected in 14 species constituting 31.8% of the basidiomycetous yeasts being produced by majority of the Pseudozyma species. At least 3 classes of enzymes were detected in 10 species the basidiomycetous species tested. Except for ligninase enzyme, all the other enzymes screened were found in Pseudozyma sp. strain ATT068. Xylanase enzyme was detected in majority of the Cryptococcus species screened while the 3 out of 4 strains of Cryptococcus flavus tested (ATT259, ATT269 and ATT268) demonstrated capacity to degrade starch, cellulose and xylan. On the other hand, ligninase activity was detected in only 2 strains of Trichosporon jirovecii.

3.4.2 Xylanase (endoxylanase and β-xylosidase) assays

Endoxylanase and β-xyloside activities were quantified for 105 strains. The highest endoxylanase activity was produced by Candida davisiana strain LANTA 101 (0.75±0.07 U/mL), C. davisiana strain LANTA 107 (0.73±0.10 U/mL) and Pseudozyma hubeiensis strain MP2-2CB (0.7±0.04 U/mL) respectively. The 3 strains were not significantly different (p< 0.01). About 8.5% of the tested strains exhibited endoxylanase activities above 0.5 U/mL. Extracellular β-xylosidase activity was detected in 94.5% of the tested strains with the highest activities (0.44.1±0.56 U/mL and 0.40±0.02 U/mL) being detected in Pseudozyma sp. strain ATT 068 and P. hubeiensis strain MP2-2CB, respectively. Cell bound β-xylosidase enzyme was detected in a total of 87.6% of the tested strains. A. pullulans strain CG5-5BY was the most active with activity of 1.13 ±0.25 U/mL, followed by and P. hubiensis strain MP2-2CB (1.03±.07) and A. pullulans strain PBM 1 (0.96±0.057) (Table 3.4). However the performances of the three strains were statistically rated equal (p< 0.01). About 22.8% of the strains showed cell associated β-xylosidase activities above 0.5 U/mL while 29.5% showed activities ranging between 0.1 – 0.5 U/mL (Table 3.4). Based on their performances in plate screening test and the xylanase quantitative assays, strains MP2-2CB, CG5-5BY and PBM 1 were selected for further investigation.

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Table 3.4. Mean xylanase activities in extracellular and cell wall associated cell free supernatant of selected strains

β-xylosidase (U/mL) Endoxylanase Extracellular Cell-wall Species Strain (U/mL) S.D. enzyme S.D. enzyme S.D. A. pullulans ATT263 lmop 0.30 0.01 0.12 0.03 0.73 0.05 A. pullulans ATT269 mopq 0.25 0.01 0.08 0.04 0.70 0.08 A. pullulans PBM1 bcd 0.60 0.01 0.07 0.01 0.96 0.06 A. pullulans CG5-5BYcdf 0.53 0.05 0.01 0.003 1.13 0.30 A. pullulans var. melanigenum PBM3ijkp 0.35 0.03 0.13 0.004 0.63 0.08 Aureobasidium pullulans ATT262 dfgij 0.41 0.04 0.08 0.01 0.65 0.04 Bullera sinensis ATT074 mop 0.24 0.05 0.05 0.02 0.68 0.09 Bulleromyces albus ATT078 mnop 0.21 0.01 0.01 0.01 0.60 0.05 Bulleromyces sp. ATT064st 0.06 0.02 0.008 0.01 0.01 0.01 Bulleromyces sp. ATT067klmop 0.32 0.04 0.12 0.01 0.52 0.02 Cadophora lutea-olivacea L50 lnopq 0.33 0.06 0.05 0.03 0.11 0.03 Candida apícola RN1bs 0.07 0.01 0.49 0.01 nd nd Candida davisiana L101a 0.75 0.07 nd nd 0.02 0 Candida davisiana L107 ab 0.73 0.10 0.03 0.02 0.02 0 Candida davisiana L114jko 0.36 0.04 nd nd 0.04 0 Candida zyelanoides PBM26 nd nd 0.07 0.002 0.02 0.002 Cryptococcus adelienses L95jkm 0.34 0.01 0.08 0.02 0.31 0.03 Cryptococcus adelienses L108dfg 0.49 0.05 0.03 0.002 0.3 0.04 Cryptococcus adelienses L110 lmopq 0.30 0.05 0.07 0.03 0.25 0.25 Cryptococcus sp. CBS 9007 L35 dfg 0.51 0.02 0.18 0.01 0.65 0.04 Cryptococcus sp. CBS 9007 L59flnp 0.42 0.05 0.10 0.02 0.35 0.05 Cryptococcus sp. CBS 9007 L62gijk 0.42 0.05 0.06 0.01 1.09 0.13 Cryptococcus sp. CBS 9007 L63cdef 0.53 0.02 0.03 0.02 0.54 0.06 Cryptococcus sp. CBS 9007 L64hijkl 0.38 0.05 0.06 0.02 0.81 0.14 Cryptococcus sp. CBS 9007 L70bcf 0.61 0.01 0.14 0.01 0.37 0.02 Cryptococcus albidosimilis CG2-2BY-aijmn 0.36 0.03 0.06 0.02 0.47 0.02 Cryptococcus albidosimilis L94gijk 0.40 0.01 0.06 0.02 0.48 0.05 Cryptococcus albidosimilis L112s 0.17 0.02 0.14 0.06 0.16 0.03 Cryptococcus dimennae CY080st 0.03 0.003 nd nd 0.02 0.01 Cryptococcus dimennae CY08 st 0.04 0.01 0.05 0.001 0.64 0.01 Cryptococcus flavescens BR3-2AASP m 0.13 0.04 0.08 0.004 0.14 0.06 Cryptococcus flavescens ATT120mnop 0.23 0.01 0.04 0.01 0.001 0.03 Cryptococcus flavescens BR2-1Ym 0.14 0.01 0.08 0.006 0.06 0.001 Cryptococcus flavescens BR2-4A m 0.21 0.02 0.18 0.01 0.55 0.07 Cryptococcus flavescens BR3-2Irs 0.16 0.05 0.97 0.002 0.12 0.01 Cryptococcus flavescens BR3-3CYst 0.26 0.04 0.05 0.001 nd nd

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Table 3.4. Mean xylanase activities in extracellular and cell wall associated cell free supernatant of selected strains continued

β-xylosidase (U/mL) Endoxylanase Extracellular Cell-wall Species Strain (U/mL) S.D. enzyme S.D. enzyme S.D. Cryptococcus flavus ATT259 cdfg 0.44 0.02 0.19 0.01 0.60 0.06 Cryptococcus flavus ATT260 cdf 0.56 0.02 0.22 0.04 0.73 0.01 Cryptococcus flavus ATT268m 0.22 0.15 0.30 0.03 0.09 0.03 Cryptococcus laurentii BR7-3BM m s 0.13 0.02 0.09 0.02 0.20 0.04 Cryptococcus laurentii FB2-2BM st 0.05 0.01 0.08 0.04 0.02 0.01 Cryptococcus laurentii FH9Tmt 0.11 0.01 0.09 0.01 0.18 0.01 Cryptococcus laurentii ATT253 dfgij 0.42 0.01 0.07 0.03 0.32 0.02 Cryptococcus laurentii BD107lmop 0.29 0.04 0.05 0.08 0.19 0.10 Cryptococcus laurentii B2-3m 0.20 0.02 0.04 0.04 0.23 0.01 Cryptococcus laurentii B2-1mnopq 0.27 0.04 0.03 0.01 0.21 0.04 Cryptococcus laurentii PBM 21mq 0.23 0.04 0.02 0.001 0.39 0.01 Cryptococcus laurentii PBM 27mopq 0.25 0.02 0.01 0.003 0.33 0.06 Cryptococcus laurentii JA009jq 0.34 0.06 0.01 0.01 0.28 0.08 Cryptococcus laurentii JA012 moq 0.24 0.01 0.02 0.01 0.06 0.01 Cryptococcus laurentii SA043 mopq 0.25 0.004 0.05 0.01 0.07 0.04 Cryptococcus laurentii SA046hijk 0.38 0.09 0.02 0.01 0.19 0.01 Cryptococcus laurentii SA048s 0.28 0.004 0.04 0.004 0.05 0.01 Cryptococcus laurentii SA049 lmopq 0.29 0.01 .0.06 0.05 0.18 0.04 Cryptococcus laurentii SA050 mopq 0.25 0.08 0.03 0.01 0.02 0.01 Cryptococcus laurentii SC042 lmopq 0.28 0.02 0.03 0.02 0.02 0.01 Cryptococcus luteolus ATT122m 0.18 0.01 0.05 0.02 0.55 0.01 Cryptococcus magnus ATT069mst 0.08 0.002 0.02 0.01 0.01 0.004 Cryptococcus magnus ATT121lmno 0.33 0.02 0.08 0.01 0.26 0.004 Cryptococcus magnus ATT148mnopq 0.24 .0547 0.02 0.01 0.86 0.06 Cryptococcus nemorosus ATT178mnopq 0.24 0.01 0.02 0.002 nd nd Cryptococcus podzolicus ATT076lmno 0.27 0.02 0.01 0.01 0.76 0.01 Cryptococcus podzolicus ATT204t 0.03 0.02 0.07 0.04 0.014 0.01 Cryptococcus sp. CBS 681.93 ATT079 mnopq 0.21 0.02 0.02 0.01 0.28 0.02 Cryptococcus sp. CBS 7944 ATT176kmop 0.24 0.001 0.03 0.01 0.21 0.01 Cryptococcus sp. CBS 8369 SA095 ms 0.15 0.004 0.08 0.01 0.34 0.02 Crytococcus victoriae L92 mopq 0.27 0.00 0.07 0.02 0.14 0.05 Crytococcus victoriae L122klmn 0.37 0.08 0.03 0.02 0.47 0.01 Crytococcus victoriae L123mnop 0.23 0.03 0.02 0.02 0.23 0.08 Cystofilobasidium capitatum L45b s 0.03 0.00 nd nd nd nd Cystofilobasidium capitatum L4 6 s 0.06 0.00 nd nd nd nd

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continued

β-xylosidase (U/mL) Endoxylanase Extracellular Cell-wall Species Strain (U/mL) S.D. enzyme S.D. enzyme S.D. Cystofilobasidium capitatum L47b s 0.08 0.01 nd nd nd nd Cystofilobasidium infirmo-miniatum L44 st 0.03 0.00 nd nd nd nd Cystofilobasidium infirmo-miniatum L96 s 0.04 0.01 nd nd nd nd Cystofilobasidium infirmo-miniatum L111 s 0.06 0.01 nd nd nd nd Exophiala dermatitidis A1M-A7 lmop 0.29 0.05 0.30 0.06 0.04 0.03 Gueomyces pullulans L88eflnp 0.44 0.03 0.10 0.01 nd nd Guehomyces pullulans L86efghl 0.48 0.03 0.13 0.01 nd nd Guehomyces pullulans L109aefkq 0.42 0.03 0.13 0.04 nd nd Hannaella kunmingensis ATT265pr 0.25 0.003 0.11 0.02 0.41 0.09 Hannaella kunmingensis ATT066 lmop 0.28 0.004 0.06 0.02 0.50 0.01 Lecythophora sp. W7 mopq 0.26 0.02 0.05 0.01 0.28 0.04 Lecythophora sp. W3a2 st 0.03 0.02 0.01 0.01 0.09 0.05 Leucosporidium scotii L115s 0.05 0.00 nd nd nd nd Pichia guilliermondii PBM52 s 0.04 0.005 0.04 0.01 0.03 0.02 Pseudozyma hubeiensis MP2-2CB ab 0.70 0.04 0.40 0.02 0.10 0.07 Pseudozyma sp. BCRC 34227 SA045 lmopq 0.28 0.01 0.03 0.02 0.10 0.03 Pseudozyma sp. BCRC 34227 SC038 m 0.19 0.01 0.02 0.01 0.03 0.01 Pseudozyma sp. strain ATT068 ATT068 dfg 0.46 0.03 0.44 0.06 0.60 0.02 Rhodosporidium sp. APSS 849 SA041 s 0.06 0.001 0.02 0.01 nd nd Rhodotorula marina CY079 st 0.04 0.004 0.03 0.08 0.31 0.03 Rhodotorula nothofagi ATT177 t 0.01 0.01 0.04 0.002 0.93 0.05 Rhodotorula taiwaniana ATT070t 0.02 0.003 0.01 0.004 0.02 0.01 Sporisorium penniseti ATT255lmoq 0.26 0.03 0.05 0.03 0.14 0.01 Sympodiomycopsis paphiopedili ATT264t 0.03 0.01 0.01 0.01 nd nd Sympodiomycopsis paphiopedili ATT271st 0.05 0.00 nd nd nd nd Tremella indecorata L99st 0.06 0.00 nd nd nd nd Trichosporon jirovecii JA008 st 0.05 0.002 0.01 0.001 nd nd Trichosporon jirovecii JA011 mst 0.10 0.008 nd nd 0.10 0.02 Wickerhamomyces anomalus L36 s 0.04 0.00 nd nd nd nd Wickerhamomyces anomalus L37a s 0.06 0.00 nd nd nd nd Wickerhamomyces anomalus L55 s 0.07 0.01 nd nd 0.08 0.02

S.D. = standard deviation from mean of three replicates, nd = not detected

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3.4.3 Extracellular enzyme production from sugar cane bagasse fermentation

Extracellular enzyme production from sugar cane bagasse fermentation by A. pullulans strain CG5-5BY, A. pullulans strain PBM 1 and P. hubeiensis strain MP2-2CB was determined. These 3 strains were selected based on their good performances in the xylanase enzymes (endoxylanase and β-xylosidase) assay. All the ezymes assayed were detected at various concentrations in sugar cane bagasse free cell extract (Fig. 3.4). Enzyme yield per gram of sugar cane bagasse is also presented in Table 3.5 and was determined by dividing enzyme activities by the gram of substrate used (2.5 g) for fermentation. The highest endoamylase production was observed in A. pullulans strain CG5-5BY (1574 ± 217 U/mL) but this performance was not statistically different from that of A. pullulans strain PBM 1. However, endoamylase activity was detected at values above 500 U/mL in all the 3 strains (p<0.10). Maximum exoamylase activity of 1563 ± 350 U/mL was expressed by P. hubeiensis strain MP2-2CB whereas; the performances of the two strains of A. pullulans were statistically rated as the same. Cellulase production by P. hubeiensis strain MP2-2CB was detected at 1772 ± 330 U/mL in CMCellulose but at a lower level of 948 ± 123U/mL in microcrystalline cellulose. In the same vein, cellulase activities of 845 ± 148 U/mL and 366 ± 93 U/mL were exhibited by A. pullulans strain PBM 1 in CMcellulose and microcrystalline cellulose, respectively. On the other hand while a low activity of 351 ± 111 U/mL was observed in A. pullulans strain CG5-5BY when CMCellulose was used as cellulose inducing substrate, a high activity of 896 ± 246 U/mL was detected when crystalline cellulose was used as the inducing substrate. In all the 3 tested strains, xylanase activitiy was detected at values lower than 500 U/mL, however xylanase activites of A. pullulans strain CG5-5BY and P. hubeiensis strain MP2-2CB were not statistically different (p < 0.1). Pectinase activity was generally high for the 3 strains with A. pullulans strains CG5-5BY (2463 ± 353 U/mL) and PBM 1 (2206 ± 393 U/mL) exhibiting the highest activities (p < 0.1). The total reducing sugar was analyzed to determine the quantity of sugar liberated upon hydrolysis of the sugarcane bagasse by the tested isolates. Reducing sugar was not detected in any of the bagasse substrate by the assay method (MILLER, 1959) used.

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Figure 3.4 - Extracellular enzyme production from sugar cane bagasse fermentation by Aureobasidium pullulans strain CG5-5BY, Aureobasidium pullulans strain PBM 1 and Pseudozyma hubeiensis strain MP2-2CB

Table 3.5 Enzyme yield per gram of sugarcane bagasse fermentation Endoamylase Exoamylase CMCase MCase Xylanase Pectinase (U/mL) (U/mL) (U/mL) (U/mL) (U/mL) (U/mL) A. pullulans 629.5a 373.7b 140.4c 358.2a 117.6 b 882.5a CG5-5BY A. pullulans 616a 453.1ab 338.2b 146.4c 70.2 c 985.3a PBM1 P. hubeiensis 311.6b 625.3a 709.0a 379.1a 154.6 a 414.3b MP2-2CB CMCase, Carboxymethyl celluase; MCase, Microcrystalline cellulase

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3.5 Discussion

3.5.1 Enzymatic activity profile

Microbial hydrolytic enzymes are in high demand due to their useful applications in many biotechnological and industrial processes, therefore, microbial strains that produce polysaccharide-degrading enzymes, may be beneficial for industrial applications. Three hundred and twelve yeast strains were subjected to an extensive screening for the selection of some plant polysaccharide degrading enzymes (amylase, cellulose, xylanase, pectinases and ligninase). According to Fleet (2011) ´´yeasts do not degrade cellulose, and only a few species are known to degrade xylan. Pectin- and starch-degrading yeasts are more common``. This work revealed xylanase as the most produced enzyme by the strains tested followed by cellulase (Figure 3.2). These strains could be further investigated for future industrial applications. Although, there was no very strong correlation of enzyme activities with source of isolation of the tested yeasts, however, it was observed that apart from A. pullulans, most of the xylanase and cellulase producers were basidiomycetous plant associated yeasts belonging to the Tremellales, especially yeasts in the genus Cryptococcus, Bandoniozyma, Bullera and Bulleromyces. Most of these strains were isolated from fungus growing ants and and fungus gardens (Table 3.3, Appendix 2). Fungus growing ants cut fresh plants to nuture their mutualistic fungi (PAGNOCCA, 2008). Yeasts found in the ant garden also participate in the breadown of the plant materials through their efficient polysaccharide degrading enzyme systems (MENDES, 2012). Hence, ant fungus garden is a promising environment for the obtention of yeasts with biotechnological potentials. Furthermore, most of the pectinase enzyes producing strains such Candida silvae and Cryptococcus laurentii originated from plants. Most of the strains originating from honey did not exhibit capacity to produce the enzymes screened for. This is probably due to the fact that their habitat is rich in simple sugars that are assimilable directly. Amylase was also detected in high percentage of the yeasts screened. However, most of the xylanase positive yeasts were yeasts isolated from fungus growing ants and their nests, being detected in only one strain of the yeasts isolated from the Atlantic rain forest. Buzzini and Martini (2002), using a similar methodology used in this work also detected low percentage of amylolytic yeasts from strains obtain from Antlantic rain forest of Brazil.

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This work also revealed that β-D-xylosidase enzymes are generally more associated with cell wall in yeasts, judging by the low activities detected in extracellular extracts when compared with the cell wall associated β-D-xylosidase extracts. Findings of this work however revealed that P. hubeiensis strain MP2-2CB, A. pullulans strains PBM 1 and CG5-5BY are good producers of endo-1,4-β-xylanase. These strains in addition to Rhodotorula nothofagi strain ATT177 were also the highest producers of β-xylosidase having activities above 900 mU/mL. Hence, they could be explored for better performances in biotechnological applications. The β-D-xylosidase enzymes of these strains are primarily associated with their cell walls. Information about xylanase enzyme production in yeasts is still limited. A report on the xylanase activity of purified enzyme of a strain of Cryptococcus adeliensis revealed a 400 nkat.mL-1 (i.e., 23.99U/mL) activity under optimized conditions of 24.2 g/L of xylan and 10,2 g/L of yeast extract at pH of 7.5 and growth temperature of 4 °C (GOMES et al. 2000). In another work investigating the β–xylosidase activity of a strain of A. pullulans, activity of 82 U/mL was obtained when the yeast was cultivated in 1 % xylan medium at 28 °C (LI et al. 1993). Two xylanases from P. hubeiensis NCIM3574 have been characterized by Adsul, Bastawade and Gokhale which found to have molecular masses of 33.3 kDa (PhX33) and 20.1 kDa (PhX20). The authors did not report the xylanase activity of this yeast. The generally low values of xylanase activities obtained from the strains tested in this work could be due to the culture conditions used in the process of fermentation or in the enzymatic reactions (temperature, pH, substrate type, substrate concentration, etc.) as well as the presence of other proteins which may disrupt the reactions. It is believed that higher xylanase activities could be obtained when these conditions are optimized and after further purification of the enzymes.

3.5.2 Enzyme production from sugar cane baggase

The convertion of biomass like sugarcane bagasse to high value product such as ethanol requires several key step including hydrolysis to fermentable sugars after the pretreatment stage (ALMEIDA, 2007; CHANDRA et al. 2007), therefore, the discovery of enzymes capable of efficient hydrolysis of plant biomass are desirable. Generally the yeasts tested were efficient in producing enzymes useful in the saccharification of sugar cane bagasse. Pectinase activity was generally high for the tree strains investigated but was more pronounced in the two strains of

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A. pullulans while cellulose (CMCase) was more pronounced in P. hubeiensis strain MP2-2CB suggesting that these strains are promising yeasts for these enzymes, respectively. Interestingly, these strains did not produce halo of degradation on polygalacturonic acid, suggesting that polygalacturonase was not secreted into the test medium. The medium used for polygalacturonase assay contained 1 % glucose. It is most probable that the yeasts metabolized glucose which was a more easily metabolisable sugar. However, the high pectinase activity obtained for these strains render them as good candidates for future biotechnological applications. Endoamylase and exoamylase were detectable in all the three strains at high levels of activity. However, endoamylase activities of the two tested strains of A. pullulans were higher than that of P. hubeiensis strain MP2-2CB. However, an opposite scenario was observed when exoamylase activity is considered. Xylanase activities were generally low among the tested strains when compared with other enzyme activities. When grown on two percent (2%) sugar cane bagasse, CMCase and xylanase activities of 0.977 and 9.280 U/ml were obtained from a Trichoderma species (MAHAMUD; GOMES, 2012). The wide differences when compared with the results obtained from this work may be accounted for by the differences in sugar cane bagasse used. Cellulose activity of A. pullulans strain CG5-5BY was higher when assayed using microcrystalline cellulose as the cellulose inducing substrate than when carboxymethyl cellulose was the inducing substrate. Microcrystalline cellulose is structurally more similar to plant-derived cellulose than carboxymethyl cellulose (OPPERT et al. 2010), hence, the discovery of A. pullulans strain CG5-5BY that produce enzymes efficient in degrading complex forms of cellulose would be advantageous for efficient hydrolysis of plant polysaccharide. Considering the high activites of enzymes produced in the sugarcane bagasse when fermented with A. pullulans strain CG5-5BY, A. pullulans strain PBM1 and P. hubeiensis strain MP2-2CB, it is expected that reducing sugars should be detected at relatively high concentrations too. The most obvious explanation for the absence or lack of detection of reducing sugars in the fermenting substrates could be that the yeasts metabolized the sugars at a faster rate as soon as they were released. In conclusion, prospecting the UNESP – CMR collection has helped to identify some biotechnologically useful yeast strains and their lignicellulolytic capacities. More investigations such as, purification, cloning and characterization of novel polysaccharide degrading enzymes, are still needed to ensure proper harnessing of the biotechnological potentials of these yeasts.

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BIELY, P.; KREMNICKÝ, L. Yeasts and their enzyme systems degrading cellulose, hemicellulose and pectin. Food Technology and Biotechnology, Zagreb, v .36, n. 4, p. 305-312, nov. 1998.

BUZZINI, P., MARTINI, A. Extracellular enzymatic activity profiles in yeast and yeast-like strains isolated from tropical environments. Journal of Applied Microbiology, Oxford, v. 93, n. 6, p. 1020–1025, dec. 2002.

CHANDRA, R. P.; BURA, R.; MABEE, W. E.; BERLIN, A.; PAN, X SADDLER, J. N. Substrate pretreatment: the key to effective enzymatic hydrolysis of lignocellulosics? Advances in Biochemical Engineering and Biotechnology, v. 108: 67–93, 2007.

DA SILVA. E. G.; BORGES, M.; MEDINA, C.; PICCOLI, R. H., FREITAS SCHWAN, R. F. Pectinolytic enzymes secreted by yeasts from tropical fruits. FEMS Yeast Research, Amsterdam, v. 5, n. 9, p. 859-865, jun. 2005

GOMES, J.; GOMES, I.; STEINER W. Thermolabile xylanase of the Antarctic yeast Cryptococcus adeliae: production and properties. Extremophiles, Tokyo, v. 4, n. 4, p. 227-35, aug. 2000.

HOSTINOVÁ, E. Amylolytic enzymes produced by the yeast Saccharomycopsis buligera. Biologia, Bratislava, 57/Suppl. 11: 247-251, 2002.

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KUHAD, R. C.; GUPT, R.; SINGH, A. Microbial cellulases and their industrial applications. Enzyme Research, New York, v. 2011, (2011) Article ID 280696, 10 p. doi:10.4061/2011/280696

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LI, X. L.; ZHANG, Z.; DEAN, I. F. D.; ERIKSSON, K.L.; LJUNGDAHL, L. G. Purification and characterisation of a new xylanase (APX-II) from the fungus Aureobasidium pullulans Y-2311-1. Applied and Environmental Microbiology, Washignton, v. 59, p. 3212-3218, oct. 1993.

MAHAMUD, M. R; GOMES, D. J. Enzymatic Saccharification of Sugar Cane Bagasse by the Crude Enzyme from Indigenous Fungi. Journal of Scientific Research, Bangladesh, v. 4, n. 1, p. 227-238, jan. 2012.

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CHAPTER 4

WICKERHAMIELLA KIYANII SP. NOV. AND WICKERHAMIELLA PINDAMONHANGABAENSIS SP. NOV., TWO ANAMORPHIC YEASTS ISOLATED FROM NATIVE PLANTS OF THE SOUTH EASTERN ATLANTIC RAINFOREST OF BRAZIL

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4.1 ABSTRACT

Two novel species namely one lipase producing strain Wickerhamiella kiyanii (FB1-1DASPT) and Wickerhamiella pindamonhangabaensis (H10YT, H10-10AY) are proposed in the Wickerhamiella clade (Saccharomycetes, Saccharomycetales) to accommodate three strains isolated from flower and fruits of plants typical of the Brazilian Atlantic rainforest. W. kiyanii sp. nov. differs from its nearest phylogenetic neighbors Wickerhamiella pagnoccae CBS 12178T, C. jalapaonensis CBS 10935T and C. drosophilae CBS 8459T by 2.2–4.2% in the nucleotide sequence of the D1/D2 domains large subunit (LSU) rRNA gene and by 6.9–12.5% in the internal transcribed spacer (ITS) region. On the other hand, W. pindamonhangabaensis sp. nov. did not match closely with known yeast species, however, phylogenetic analysis of D1/D2 large subunit rDNA sequences also indicate an affinity with the Wickerhamiella clade with its closest described relative as Candida kazoui. Morphological, physiological and molecular characteristics of the two proposed species are hereby discussed.

Keywords: Wickerhamiella clade, Atlantic rainforest, LSU, ITS.

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4.2 INTRODUCTION Biodiversity studies focusing on yeasts inhabiting the Brazilian Atlantic rainforest have revealed the rich yeast diversity of this ecosystem (BRITO DA CUNHA et al., 1957, MORAIS et al., 1992, PRADA; PAGNOCCA, 1997, ARAUJO et al. 1998, RUIVO et al. 2005, LANDELL et al., 2006, PIMENTA et al., 2009). Although, this biome is not intensively studied, some reports have led to the discovery of many new species. Examples include Saturnispora hagleri (MORAIS et al., 2005), Candida leandrae (RUIVO et al. 2004), Candida ubatubensis (RUIVO et al., 2005) and C. saopaulonensis (RUIVO et al., 2006). Some of the species discovered in this ecosystem may be considered as rare or to be habitat specific because they have not been reportedly isolated elsewhere since their first discovery. During a survey of yeasts associated with diverse plant substrates in São Sebastião do Ribeirao Grande agricultural land, located at the município of Pindamonhangaba, south-eastern Brazil, various species, majorly of ascomycetous affinity, including three strains of two putative new yeast species were identified. Based on analysis of D1/D2 LSU rRNA gene, the strains phylogenetically clustered with species in the Wickerhamiella clade. Asexual states of species in the Wickerhamiella clade were formerly classified in the genus Candida, but are now re-classified in the genus Wickerhamiella in conformity with the provisions of the 124 Melbroune code (Norvell, 2011). Yeasts characterized in this genus which includes yeasts with spherical to ellipsoidal small cells that reproduce by multilateral budding, on either a narrow or a broad base. Hyphae or pseudohyphae are not formed, sugars are not fermented but nitrate is assimilated by most species (LACHANCE; KURTZMAN, 2011). In addition, cells of the sexual states and asexual states in this clade are generally small when compared with other yeast species (LACHANCE; KURTZMAN, 2011). Two converging characteristics among species in the Wickerhamiella clade are their physiological restrictions (in terms of carbon assimilation) and strong associations with flowers and floricolous insects; particularly, Drosophila and Nitidulid beetles (LACHANCE et al., 1998; LACHANCE; KURTZMAN, 2011). Because of the strong associations of W. lipophila (anamorph: Candida lipophila) and W. occidentalis with drosophilids found in morning glories, it was suggested that the yeasts may be involved in enhancing the nutritional values of the flies’ diet by converting flower lipids into a nutritionally richer biomass ((LACHANCE; KURTZMAN, 2011). The physiological and ecological monophyly of species in the Wickerhamiella clade made

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W. domercqiae to still be retained, despite the fact that sequence analyses of the small subunit (SSU) rRNA gene (SUZUKI et al. 1999) as well as the combined LSU, SSU and cytochrome oxidase II (KURTZMAN; ROBNETT, 2007) demonstrated that it may be separated from this clade. Based on the low degrees of sequence relatedness observed among some described species in the Wickerhamiella clade Lachance and Kurtzman (2011) predicted that many unknown species might yet be discovered in this clade. In this work, we provide the description of two additional species, Candida kiyanii and Candida pindamonhangabaensis to be accommodated in the Wickerhamiella clade using a polyphasic approach of species determination.

4.3 Material and methods i) Yeast isolation and maintenance

The proposed new species were isolated in 2006 from flowers and fruits collected at the São Sebastião do Ribeirão Grande agriculture land in the Atlantic rainforest of Pindamonhangaba municipal area of São Paulo State, Brazil (W 22o 44’ 28” and lS 45º 28’ 19”). Strain FB1- 1DASPT was isolated from flower bracts of Siphocampylus sp. (Campanulaceae) while strains H10YT and H10-10AY were recovered from fruits of white garland-lily (Hedychium coronarium Koening). Nectar juice of the flowers were aseptically collected using a sterile inoculating loop and streaked on the surface of YM agar medium containing (in g.L-1): yeast extract (3), malt extract (3), mycological peptone (5), glucose (1), agar (2) and chloramphenicol (150 mg.L-1). The plates were then incubated at 22 °C for 5 days. Yeast colonies were purified on YM agar and maintained at 4 °C and by cryopreservation in 15 % glycerol at -80 °C. For yeast isolation from fruits of Hedychium coronarium, mature fruits were inoculated in YM broth; after incubation for 5 days, aliquots of 100 µL were plated on YM agar. Isolation was also carried out by scrapping the surface of fruits with swab followed by streaking on YM agar.

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ii) Morphological and phenotypic characterization

The yeast isolates were characterized by procedures described in Kurtzman et al. (2011). The isolates were initially characterized based on colony morphological characteristics of 3 - 5 days old cultures grown on YM agar at 25 °C while cell morphologies were studied with phase contrast microscope (Leica DM-1000) using cultures grown in YM broth. Formation of pseudohyphae was investigated by slide culture on corn meal agar. Ascosporulation was investigated by inoculating the yeast strains on YM, 5% malt extract and acetate agar and incubating at 15 and 25 °C. The cultures were examined weekly for up to 2 months. Physiological and biochemical tests were performed by replica plating on solid media and in liquid media according to Kurtzman et al. (2011). Test samples were incubated at 25 °C and results were read weekly for up to 28 days. For the determination of lipase activity, medium containing yeast nitrogen base 0.67 %, yeast extract 0.05 % and agar 1.5 % were initially sterilized in autoclave (121 oC for 15 min) and cooled to 60 °C. Then, 630 and 200 µL respectively of olive oil and rhodamine B (dissolved in 95% ethanol) were added with vigorous shaking and then poured into agar plates. The yeast isolates were point inoculated on the surfaces of the solidified medium and incubated at 25 °C for 5 days. Lipase activity was detected as an orange fluorescent halo when plates were viewed under UV (350 nm) light (KOUKER; JAEGER, 1987; HOU; JOHNSTON, 1992). iii) DNA amplification and sequencing

The primer pair NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′- GGTCCGTGTTTCAAGACGG-3′) was used to amplify the approximately 600 bp fragment of the large subunit (LSU) rRNA gene. A 25 μl polymerase chain reaction (PCR) was performed using Ready-to-Go™ beads (GE Healthcare) using 5.0 μl of diluted DNA template (1:750). The PCR thermal cycler conditions consisted of a denaturation step of 3 min at 96 °C; 35 cycles of 30s at 96 °C, 45s at 61 °C and 1 min at 72 °C followed by a final extension of 4 min at 72 °C (PAGNOCCA et al., 2008). Amplification and sequencing of the internal transcribed spacer, which includes ITS1-5.8S-ITS2 used primers ITS1 and ITS4 (WHITE et al. 1990). Each PCR

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product was subsequently purified using the Nucleospin Gel and PCR Clean-up (Macherey- nagel-MN) and sequenced on an ABI 3130 genetic analyzer (Applied Biosystems) using the Big Dye Terminator v. 3 sequencing chemistry. Contig comparisons with other sequences were carried out in the databases of MycoBank (www.mycobank.org) and the NCBI-GenBank database (http://www.ncbi.nlm.nih.gov/).

iv) Phylogenetic analysis

Sequences alignments were performed in Muscle v. 3.8 (EDGAR, 2004) and trimmed in Bioedit v.7.0.5.3 (HALL, 1999) to remove primers sequences. LSU neighbor-joining tree construction (based on 1000 bootstrap iteration) was performed in Mega v. 5.03 (TAMURA et al., 2011). The tree was generated from 26 nucleotide sequences and consisted of a 562 base-pair dataset. Evolutionary distances were computed using the Kimura 2-parameter model (KIMURA, 1980). All positions containing gaps were excluded from the analyses. The GenBank accession numbers of sequences from previously described species and those obtained in this study are provided on the phylogenetic tree (Fig. 4.1).

4.4 Results and discussion

The domains D1 and D2 (D1/D2) of large-subunit (26S) rRNA were used in the phylogenetic analysis to infer evolutionary relationships among the novel species with their relatives (Fig. 4.1), while the ITS region was also sequenced to confirm their distinct statuses. Phylogenetic analysis of sequences of the LSU placed the two proposed new species in positions well separated from previously described species in the Wickerhamiella clade (Saccharomycetes, Saccharomycetales) (Fig 4.1). Wickerhamiella kiyanii clustered with three species, Wickerhamiella pagnoccae, C. jalapaonensis and C. australiensis in a subclade with a strong bootstrap support (99%). The D1/D2 domains of W. kiyanii differed in twelve (12) substitutions of a total of 549 nucleotide sites from that of W. pagnoccae its closest relative. However, with respect to the ITS region, W. kiyanii was genetically closer to C. jalapaonenesis (93.1 %

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Figure 4.1. Phylogenetic placement of Wickerhamiella kiyanii and W. pindamonhangabaensis in the Wickerhamiella clade (Saccharomycetes, Saccharomycetales) determined from Neighbor- joining analysis of sequences from LSU rRNA gene. Bootstrap values are from 1000 replicates. T = type species.

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Table 4.1 - Extent of D1/D2 LSU rDNA and ITS sequence divergences of W. kiyanii and close relatives pairwise some based on alignment.

W .kiyanii W. kiyanii W. kiyanii W. pagnoccae W. pagnoccae rRNA region W. pagnoccae C. jalapaonesis C. drosophilae C. jalapaonesis C. drosophilae LSU (549a) 2.2% 3.2% 4.2% 1.4% 2.6% ITS (400a) 12.5% 6.9 % 7.8% 5.3% 8.5% a number of nucleotide in aligned sequences.

Table 4.2 - Physiological characteristics differentiating W. kiyanii from closely related strains

Species

Characteristics W. W. C. C.

kiyanii pagnoccae jalapaonensis drosophilae

Growth on:

Galactose - - + s/-

Sucrose l - - -

Ethanol - - w +

Glycerol w - - s

D-Gluconate w - - -

Succinic acid - + + s

Cadaverine + - + s/-

Ethylamine w - + w/-

Cycloheximide + - - +

0.01%

Cycloheximide 0.1% w - - +

Growth at 30 °C - + + s

+, positive; -, negative; l, latent (rapid development of a positive reaction after a lag period); w, weak; s, positive but slo;, w/-, weak or negative; s/-, slow or negative.

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Figure 4.2 - Wickerhamiella kiyanii (A) and Wickerhamiella pindamonhangabaensis (B and C). Phase contrast micrograph showing budding cells (A and B) with pseudomycelium after 3 days at 25 °C on YM agar (A) and on corn meal agar 25 °C (C).

A B C

similarity overlap of 97.3 %) than W. pagnoccae (87.5% similarity) (Table 4.1). Due to its limited physiological capacity, which is also a common characteristic among species in the Wickerhamiella clade, it was almost difficult to separate W. kiyanii from related species based on phenotypic characteristics; however, few phenotypic differences were observed. W. kiyanii differed from W. pagnoccae in sucrose, glycerol, D-gluconate, ethylamine and cadaverine utilization as well as growth in the presence of 0.1% cycloheximide. Table 4.2 summarizes phenotypic tests where differences were observed with respect to related species. Differences were also observed between the cellular morphologies of W. kiyanii and related species. Whereas cells of W. pagnoccae are ovoid but do not form pseudohyphae, W. kiyanii forms elongated to ovoid cells and produce extensive pseudohyphae even without induction on a nutritionally poor medium. (Fig. 4.2a). This is a distinct characteristic considering that most species in the Wickerhamiella clade do not form pseudomycelia. The cells of W. kiyanii are also unusually larger when compared with those of related species. In addition, while W. kiyanii could not grow at 30°C, conversely, growth was recorded for W. pagnoccae at 37°C. Both the LSU and ITS sequences generated for W. pindamonhangabaensis did not match closely with those deposited in the Mycobank and GenBank. Although distantly related, blast results of the LSU in these databases revealed Candida kazuoi JCM 12559, a yeast strain that was isolated from insect frass in Thailand (NAKASE et al., 2007), as the closest described

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relative of W. pindamonhangabaensis with a similarity index of 86.8% (query coverage = 99.4 %). Wickerhamiella pindamonhangabaensis formed a subclade with Candida kazuoi, Candida hasegawae and two other yet undescribed strains (Fig. 4.1). Bootstrap value (78%) supported the clustering of W. pindamonhangabaensis with W. kazuoi on the LSU phylogenetic tree. The distant phylogenetic position from the closest relative could be as a result of missing sequences of species yet undiscovered. W. pindamonhangabaensis also exhibited a narrow range of carbon and nitrogen assimilation profile characteristics of species in the Wickerhamiella clade. Despite the low number of strains from which W. kiyanii and W. pindamonhangabaensis were known from, their isolation sources were consistent with the habitat characteristics of other members of the Wickerhamiella clade; which are frequently found in association with flowers and floricolous insects (LACHANCE; KURTZMAN, 2011). Lachance et al. (1998) described five species in the Wickerhamiella clade including two asexual taxa namely Candida drosophilae and Candida lipophila, all isolated from flower bracts of Heliconia psittacorum including nitidulid (sap) beetles and drosophilids. W. pagnoccae (BARBOSA et al., 2011) and its closest relative, C. jalapaonensis (ROSA et al., 2009) were also isolated from flowers of Heliconia psittacorum and Centropogon cornutus (Campanulaceae), respectively. It is likely that W. kiyanii and W. pindamonhangabaensis will also be associated with floricolous insects that visit the plants from which they were isolated. Discovery of additional strains in the near future may reveal ascosporic states of these species as well as provide information on intraspecific physiological and genetic variations. W. kiyanii was found to produce extracellular lipase when olive oil was used as a substrate (Fig. 4.3). Lipase activity was not shown by W pindamonhangabaensis. Lachance et al. (1998) reported lipase activity on Tween 80 for five species in the Wickerhamiella clade including C. drosophilae and W. lipophilae. This characteristic seems to be a common feature among species in the Wickerhamiella clade.

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Figure 4.3. Lipase activity test of Wickerhamiella pindamonhangabaensis (upper colonies) and W. kiyanii (lower colonies). The orange fluorescent halo around colonies of C. kiyanii indicate positive lipase activity

4.5 DESCRIPTION OF WICKERHAMIELLA KIYANII PAGNOCCA, ROSA, DAYO- OWOYEMI AND RODRIGUES SP. NOV.

The epithet kiyanii is in honour of Dr. Choiti Kiyan, a retired professor of the Departament of Biochemistry and Microbiology of the Institute of Biosciences – UNESP, Rio Claro, São Paulo State, Brazil Growth on YM agar: After 3 days at 25 °C, colony growth is white, convex, butyrous with entire margine. After 21 days, colonies are off-white, raised, butyrous, folded or smooth colonies with entire margins. Growth on YM broth: Cells are oval and elongated. The cells divide by multilateral budding occurring in budded pairs or chains of peseudohyphae and measuring 3.5–7.6 x 7.6–28.3 µm (Fig. 2). After 7 days, a white, creeping pellicle is formed. Dalmau plate culture on corn meal agar: Growth under the cover glass showed abundant pseudohyphae that consist of long, branched chains of elongated cells but no mycelium. No

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sexual structures were observed in pure cultures plated on cornmeal agar, 5% malt extract and acetate agar. Fermentation and growth reactions: Glucose is not femented. Glucose, L-sorbose, sucrose, glycerol (weak), D-mannitol, D-glucitol (weak), and D-gluconate (weak) are assimilated. Carbon compounds not assimilated include: galactose, maltose, cellobiose, trehalose, lactose, melibiose, raffinose, melezitose, inuline, soluble starch, D-xylose, L-arabinose, L-arabinose, D-ribose, L- rhamnose, D-glucosamine, N -acetyl-D-glucosamine, methanol, ethanol, erythritol, ribitol, galactitol, Methyl-α-D-glucoside, salicin, DL-lactic acid, succinic acid, citric acid, myo-inositol, 2-Keto-D-gluconate, 5-Keto-D-gluconate, saccharate, D-glucuronate, glucono-δ-lactone, xylitol, L-arabinitol. Nitrogen compounds assimilated are cadaverine and lysin. Nitrate, nitrite, creatine, creatinine and ethylamine are not assimilated. Extracellular amyloid compound is absent. Growth did not take place in the presence of 0.01 or 0.1% cycloheximide. Growth was not observed in medium containing 10% NaCl/5% glucose and in vitamins free medium. Diazonium blue B and urease reactions are negative. Positive for lipase production. Growth occurred at 25 and 28°C but not at 30°C. Type strain FB1-1DASPT, the only strain for this species, was isolated from flowers of a Siphocampylus sp. (Campanulaceae) collected at the Pindamonhangaba municipal area of São Paulo State, Brazil.

4.6 DESCRIPTION OF WICKERHAMIELLA PINDAMONHANGABAENSIS PAGNOCCA, ROSA, DAYO-OWOYEMI AND RODRIGUES SP. NOV.

Wickerhamiella pindamonhangabaensis was named after Pindamonhangaba municipal area of São Paulo State in Brazil, where the two strains of this species were isolated.

Growth on YM agar: After 3 days at 25 °C, Colony growth is white, raised, butyrous with entire margine. After 21 days, colonies are cream, raised, butyrous, smooth with entire margins. Growth on YM broth: Cells are oval, occurring in single budded pairs and measuring 1.6–2.9 x 1.9–6.7 µm in size. The cells divide by multilateral budding (Fig. 2b). After 7 days, a white, creeping pellicle is formed.

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Dalmau plate culture on corn meal agar: Growth under the cover glass showed rosettes of joined cells that form short pseudomycelium but no mycelium (Fig. 2c). No sexual structures were observed in pure cultures plated on cornmeal agar, 5% malt extract and acetate agar. Fermentation and growth reactions: Glucose is not femented. Glucose, sucrose (weak), ethanol (weak), D-mannitol, D-glucitol, D-gluconate (weak) and Glucono-δ-lactone (weak) are assimilated. Carbon compounds not assimilated include: galactose, L-sorbose, maltose, cellobiose, trehalose, lactose, melibiose, raffinose, melezitose, inuline, soluble starch, D-xylose, L-arabinose, L-arabinose, D-ribose, L-rhamnose, D-glucosamine, N -acetyl-D-glucosamine, methanol, glycerol, erythritol, ribitol galactitol, Methyl-α-D-glucoside, salicin, DL-lactic acid, succinic acid, citric acid, myo-inositol, 2-Keto-D-gluconate, 5-Keto-D-gluconate, saccharate, D- glucuronate, xylitol, L-arabinitol. Nitrogen compounds assimilated are cadaverine, lysine and ethylamine (weak). Nitrate, nitrite, creatine and creatinine are not assimilated. Extracellular amyloid compound is absent. Growth did not take place in the presence of 0.01 or 0.1% cycloheximide. Growth in medium containing 10% NaCl/5% glucose and 50% glucose is weak. Growth in vitamins free medium is negative. Diazonium blue B and urease reactions are negative. Lipase production is negative. Growth occurred at 25 ° and weakly at 28°C at 30°C. Type strain H10YT, was isolated from fruits of Hedychium coronarium collected at the Pindamonhangaba municipal area of São Paulo State, Brazil.

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REFERENCES

ARAUJO, F. V.; MEDEIROS, R. J.; MENDONCA-HAGLER, L. C.; HAGLER, A. N. A preliminary note on yeast communities of bromeliad-tank waters of Rio de Janeiro, Brazil. Revista de Microbiologia, São Paulo, v. 29, p. 18-121, 1998.

BARBOSA, C.; MORAIS, G. C.; MORAIS, P. B.; ROSA, L. H.; PIMENTA, R. S.; LACHANCE, M. A.; ROSA, C. A. Wickerhamiella pagnoccae sp. nov. and Candida tocantinsensis sp. nov., two ascomycetous yeasts from flower bracts of Heliconia psittacorum (Heliconiaceae). International Journal of Systemic and Evolutionary Microbiology, Reading, v. 62, n. 2, p. 459-464, feb. 2011.

BRITO DA CUNHA, A. B.; SHEHATA, A. M. E. T.; OLIVEIRA, W. A study of the diets and nutritional preferences of tropical species of Drosophila. Ecology, Tempe, Arizonia, v. 38, n. 1. p. 98-106, jan. 1957.

EDGAR, R. C. MUSCLE: multiple sequence alignment with high 545 accuracy and high throughput. Nucleic Acids Research, Oxford, v. 32, n.5, p. 1792-1797, mar. 2004.

HALL, T. A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Serius, Oxford, v. 41, n. 1 p. 95-98, 1999.

HOU, C. T.; JOHNSTON, T.M. Screening of Lipase Activities with Cultures from the Agricultural Research Service Culture Collection. Journal of the American Oil Chemists' Society, Chicago. v. 69, n. 11, p. 1088-1097, nov. 1992.

KIMURA, M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, New York, v. 16, n. 2, p. 111-120, dec. 1980.

KOUKER, G.; JAEGER, K. E. Specific and sensitive plate assay for bacterial lipases. Applied and Environmental Microbiology, Washington, v.53, n. 1, p. 211-213, jan. 1978.

KURTZMAN, C. P.; ROBNETT, C. J. Multigene phylogenetic analysis of the Trichomonascus, Wickerhamiella and Zygoascus yeast clades, and the proposal of Sugiyamaella gen. nov. and 14 new species combinations. FEMS Yeast Research, Amsterdam, v. 7, n. 1, p. 141–151, jan. 2007.

KURTZMAN, C. P.; FELL, J. W.; BOEKHOUT, T.; ROBERT, V. Methods for isolation, phenotypic characterization and maintenance of yeasts. In: KURTZMAN, C. P.; FELL, J. W.; BOEKHOUT, T. (Eds) The yeasts: a taxonomic study, 5th edn. San Diego: Elsevier, 2011. p. 88-110.

LACHANCE, M. A.; ROSA, C. A.; STARMER, W. T.; SCHLAG-EDLER, B.; BARKER, J. S.; BOWLES, J. M. Wickerhamiella australiensis, Wickerhamiella cacticola, Wickerhamiella occidentalis, Candida drosophilae and Candida lipophila, five new related yeast species from

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flowers and associated insects. International Journal of Systematic Bacteriology, Washington, v. 48, n. 4. p. 1431-1443, oct. 1998.

LACHANCE, M. A.; KURTZMAN, C. P. Wickerhamiella van der Walt (1973). In: KURTZMAN, C. P.; FELL, J. W.; BOEKHOUT, T. (Eds) The yeasts: a taxonomic study, 5th edn. San Diego: Elsevier, 2011. p. 891-898.

LANDELL, M. F. MAUTONE, J. N.; VALENTE, P. Biodiversity of yeasts associated to bromeliads in Itapuã park, Viamão/RS. Biociências, Porto Alegre, v. 14, n. 2, p. 144-149, dez. 2006.

MORAIS, P. B.; HAGLER, A. N.; ROSA, C. A.; MENDONÇA-HAGLER L. C.; KLACZKO, L. B. Yeasts associated with Drosophila in tropical forests of Rio de Janeiro, Brazil. Canadian Journal of Microbiology, Ottawa, v. 38, n. 11, p. 1150-1155, nov. 1992.

MORAIS, P. B.; LACHANCE, M. A.; ROSA, C. A. Saturnispora hagleri sp. nov., a yeast species isolated from Drosophila flies in Atlantic Rain Forest in Brazil. International Journal of Systemic and Evolutionary Microbiology, Reading, v. 55, n. 4, p. 1725-1727, jul. 2005.

NAKASE, T.; JINDAMORAKOT, S.; LIMTONG, S.; AM-IN, S.; KAWASAKI, H.; IMANISHI, Y.; POTACHAROEN, W.; TANTICHAROEN, M. Candida kazuoi sp. nov. and Candida hasegawae sp. nov., two new species of ascomycetous anamorphic yeasts isolated from insect frass in Thailand. Journal of General and Applied Microbiology, Tokyo, v. 53, n. 4, p. 239-245, aug. 2007.

NORVELL, L. L. Fungal nomenclature. Melbourne approves a new code. Mycotaxon, Ithaca, v. 116, p. 481-490, apr. – jun. 2011.

PAGNOCCA, F. C.; RODRIGUES, A.; NAGAMOTO, N. S.; BACCI, M. JR. Yeast and filamentous fungi carried by the gynes of leaf-cutting ants. Antonie van Leeuwenhoek, Amsterdam, v. 94, n. 4, p. 517-526, nov. 2008.

PIMENTA, R. S.; ALVES, P. D. D.; ALMEIDA, G. M. F.; SILVA, J. M. F.; MORAIS, P. B.; CORRÊA JR, A.; ROSA, C. A. Yeast communities in two Atlantic rain forest fragments in southeast Brazil. Brazilian Journal of Microbiology, São Paulo, v. 40, n. 1, 90-95, jan.- mar. 2009.

PRADA, G. M. M.; PAGNOCCA, F. C. Ascomycetous yeasts associated with naturally occurring fruits in a tropical rain forest. Folia Microbiology, Praha, v. 42, n. 1, p. 39-46, 1997.

ROSA, C. A.; MORAIS, P. B.; LACHANCE, M. A.; SANTOS, R. O.; MELO, W. G. P.; VIANA, R. H. O.; BRAGANCA, M. A. L.; PIMENTA, R. S. Wickerhamomyces queroliae sp. nov. and Candida jalapaonensis sp. nov., two yeast species isolated from Cerrado ecosystem in North Brazil. Journal of Systemic and Evolutionary Microbiology, Reading, 59, n. 5, p. 1232- 51236, may. 2009.

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RUIVO, C. C. C.; LACHANCE, M. A.; BACCI JR., M.; CARREIRO, S. C.; ROSA, C. A.; PAGNOCCA, F. C. Candida leandrae sp. nov., an asexual ascomycetous yeast species isolated from tropical plants. International Journal of Systemic and Evolutionary Microbiology, Reading, v. 54, n. 6, p. 2405-2408, nov. 2004.

RUIVO, C. C. C.; LACHANCE, M. A.; ROSA, C. A.; BACCI, M.; PAGNOCCA, F. C. Candida bromeliacearum sp. nov. and Candida ubatubensis sp. nov., two yeast species isolated from the water tank of Canistropsis seidelii (Bromeliaceae). International Journal of Systemic and Evolutionary Microbiology, Reading, v. 55, n. 5, p. 2213-2217, sept. 2005.

RUIVO, C. C. C.; LACHANCE, M. A.; ROSA, C. A.; BACCI, M.; PAGNOCCA, F. C. Candida heliconiae sp. nov., Candida picinguabensis sp. nov. and Candida saopauloensis sp. nov., three ascomycetous yeasts from Heliconia velloziana (Heliconiaceae). International Journal of Systemic and Evolutionary Microbiology, Reading, v. 56, n. 5, 1147-1151, may, 2006.

SUZUKI, M.; SUH, S. O.; SUGITA, T.; NAKASE, T. A phylogenetic study on galactose- containing Candida species based on 18S ribosomal DNA sequences. Journal of General and Applied Microbiology, Tokyo, v. 45, n. 5, p. 229-238, oct, 1999.

TAMURA K.; PETERSON D.; PETERSON N.; STECHER G.; NEI M.; KUMAR S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, Chicago, v. 28, n. 10, p. 2731-2739, out. 2011.

WHITE, T. J.; BRUNS, T.; LEE, S.; TAYLOR, J. W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: INNIS, M. A.; GELFAND, D. H.; SNINSKY, J. J.; WHITE T. J. (Eds.) PCR Protocols: A Guide to Methods and Applications. New York: Academic Press, 1990. p. 315-322.

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CHAPTER 5

DESCRIPTION OF BULLEROMYCES TEXANAENSIS SP. NOV., ISOLATED FROM FUNGUS GARDEN OF THE LEAFCUTTER ANT ATTA TEXANA AND LEAVES OF BROMELIAD NEOREGELIA CRUENTA (BROMELIACEAE)

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5.1 ABSTRACT Bulleromyces texanaensis, a novel teleomorphic yeast species is described based on four isolates obtained from two independent studies. Two strains (ATT064T and ATT067) were recovered from fungus gardens in a nest of the leafcutter ant Atta texana (Texas, USA) while the remaining two strains (UMFRJ 51951T and UMFRJ 51952) were endophytes of the bromeliad Neoregelia cruenta collected in Rio de Janeiro, Brazil. Molecular and phylogenetic analyses based on sequences of the large subunit (LSU) D1/D2 domains and internal transcribed spacer (ITS) region showed that the strains represent a novel species in the Bulleromyces / Papiliotrema / Auriculibuller lineage of the Tremellales (Tremellomycetes, Agaricomycotina). Strains ATT064T and UMFRJ 51951T were found to be compatible mating types whose conjugation result in the formation of hyphae with complete clamp connections and non-septate basidia. The species is separate from its closest relative Cryptococcus mangaliensis based on genetic differences at 6 base positions in the D1/D2 domains and 11 bp in the ITS region. Phenotipically, Bulleromyces texanaensis can also be distinguished from Cryptococcus mangaliensis by ability to grow on D-arabinose, methyl-α-D-glucoside, DL-lactic acid, soluble starch and lack of growth on ethanol, erithritol and 10% NaCl/Glucose 5%. Additionally, formation of extracellular amyloidal compound was observed on B. texanaensis but not on Cryptococcus mangaliensis. The systematic discussion of Bulleromyces texanaensis sp. nov. is provided in this work.

Keywords: Teleomorphic, Tremellomycetes, systematic.

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5.2 INTRODUCTION

An important taxonomic key for species categorized in the anamorphic genus Bullera has been the formation of rotationally symmetrical or occasionally bilaterally symmetrical ballistoconidia; although, the separation of the genera Cryptococcus and Bullera based on this characteristic has been criticized based on outcomes of various molecular phylogenetic studies (TAKASHIMA; NAKASE, 1999; FELL et al., 2000; SCORZETTI et al., 2002). With the better isolation techniques for ballistoconidium-forming yeasts described by Nakase & Takashima (1993), coupled with improved molecular taxonomic methods as well as increased isolation effort, the genus Bullera has witnessed a tremendous increase in the number of species described to date. This genus is polyphyletic and has expanded from various lineages of the Tremellales to the Filobasidiales as well as the Trichosporonales (BOEKHOUT et al., 2011).

Recent phylogenetic revision of the Luteolus lineage in Tremelalles by Wang and Bai (2008) led to a clear separation of the B. mrakii, B. sinensis and the Dioszegia clade as morphologically and phylogenetically monophyletic groups. Consequently, they proposed the genera Derxomyces and Hannaella to accommodate species in the B. mrakii and B. sinensis clades, respectively. While the genus Derxomyces consists of majorly ballistoconidium-forming, whitish to yellowish with butyrous to matted texture colony-forming species, the genus Hannaella accommodated some species (formerly classified in Bullera and Cryptococcus) that form highly mucoid white colonies and may or may not form ballistoconidia. For the ballistoconidia-forming species, ballistospores are rotationally symmetrical, flabelliform, turbinate, globose or subglobose. In the same vein, anamorphs of the genus Bulleribasidium Sampaio, Weiss & Bauer (SAMPAIO et al., 2002) formerly assigned to Bullera have been recently reclassified to a new genus Mingxiaea after molecular phylogenetic analysis revealed the monophyly of species in the Bulleribasidium clade (WANG et al., 2011).

Two teleomorphic genera namely Bulleromyces Boekhout and Fonseca (BOEKHOUT et al., 1991) and Auriculibuller Sampaio and Fonseca (SAMPAIO et al., 2004) currently represent sexual states of Bullera species. In addition to Papiliotrema bandonii Sampaio, Gadanho, Weiss & Bauer whose sexual phase has only been observed

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under natural conditions, they belong to the Bulleromyces / Papiliotrema / Auriculibuller clade of the Tremellales (Tremellomycetes, Agaricomycotina). In Bulleromyces albus (typus of Bulleromyces) and Auriculibuller fuscus (typus Auriculibuller), the teleomorphic states are heterothallic but self-sporulation was also observed in Bulleromyces albus (BOEKHOUT et al., 2011). Basidia morphology is of high taxonomic value for generic level classification among the Tremellales. In Bulleromyces albus, Tremella-type basidia that are longitudinally or obliquely cruciate-septate are formed (BOEKHOUT et al., 1991) whereas basidia formed by Auriculibuller and Papiliotrema are clavate to cylindrical and obliquely to transversely septate (SAMPAIO et al., 2002, 2004). Both types of basidia septation are seen in Bulleribasidium (SAMPAIO et al., 2002). Furthermore, hyphae are hyaline and clamped with tremelloid haustoria in Papiliotrema and Auriculibuller, respectively, while the hyphae states of Bulleromyces have incomplete clamp connections but form haustorial branches as well (BOEKHOUT et al., 2011). During a survey of yeasts present in the cultivated gardens of the fungus growing ants Atta texana, two ballistoconidia forming strains of a previously undescribed species were isolated. Another separate study which investigated the endophytic yeasts of the bromeliad Neoregelia cruenta led to the discovery of two strains also belonging to the novel species. Subsequent mating experiments revealed sexual compatibility between the two strains; hence, a new taxon designated as Bulleromyces texanaensis to accommodate this teleomorphic species.

5.3 MATERIALS AND METHODS

5.3.1 Strain information

Strains ATT064T (CBS 11955) and ATT067 were isolated from fungus garden specimens collected from a Atta texana nest located at the Hornsby Bend environmental Research Centre, Austin, Texas (N30º13.937 , W97º39.101 , see Rodrigueset al., 2009 for details). One gram of the fungus garden sample was diluted tenfold in 0.05 % Tween 80 and 0.2 % peptone water followed by homogenization on a vortex for one minute. For yeast isolation, 150 µL from the dilution was inoculated by surface spreading on yeast-malt agar medium (YMA in g.L-1: yeast extract 3.0, malt extract 3.0, peptone 5.0, glucose 10 and agar 15) supplemented with 150 µg ml-1.

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Strains IMUFRJ 51951 (CBS 12532) and IMUFRJ 51952 were isolated from leaves of the bromeliad plant Neoregelia cruenta collected from the sand-bank of Barra de Marica, Rio de Janeiro (S22º57.37 , W42º50.43 ). Isolation was carried out applying techniques used for the recovery of endophytes with little modification. Eleven grams of leaves of Neoregelia cruenta were surface sterilized using 75 % ethanol for 1 min followed by 2.5 % sodium hypochlorite for 3 min and 75 % ethanol for 30 sec. After rinsing twice with sodium chloride 0.85% (w/v) saline solution, a final rinse was done in a solution containing 0.5 % glucose, 0.04 % chloramphenicol and 0.0 5% amoxicillin. The leaves were then blended in 100 ml of the same solution used for the final rinse and 100 µL of the resulting suspension was inoculated on YM agar (GARCIA, 2007).

5.3.2. Morphological and phenotypic characterization

Morphological observations were made on cultures grown on YM medium (in g.L-1: yeast extract 3.0, malt extract 3.0, peptone 5.0, glucose 10, with and without 2.0 agar) at 25°C and cornmeal agar at 15°C. Phenotypic tests were carried out on solid media as recommended for yeast characterization (KURTZMAN et al., 2011a). Presence of sexual structures in pure and mixed cultures was tested on MYP agar at 25 °C and 15 °C (SAMPAIO et al., 2002). All micro- morphological observations were carried out using phase contrast optics (Leica DM-1000) photomicroscope.

5.3.3 DNA extraction, Sequence and phylogenetic analyses

Genomic DNA extraction was carried out according to Almeida (2005) while amplification reactions followed protocols elaborated in Pagnocca et al. (2008). The primers pairs NL1 (5′- GCATATCAATAAGCGGAGGAAAAG-3′) and NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) was used for the amplification of the D1/D2 domains of the nuclear large subunit (26S) rRNA gene while the primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′- TCCTCCGCTTATTGATATGC-3′) were used for the amplification of the ITS region, including the 5.8S rDNA. A 25 µL polymerase chain reaction was performed for each DNA sample in

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a GenePro Thermal Cycler (Bioer Technology) using 1 µL of 50 mM MgCl2, 4 µL of 1.25mM of each dNTPs, 2.5 µL of 10X PCR buffer, 2 µL of 10 µM of each primer, 0.2 µL of 5 U/ µL of Taq polymerase with 5 µL DNA template (diluted 1:750). PCR products were purified with GE Healthcare DNA purification kit. Amplification scheme for the LSU rRNA gene D1/D2 domains consist of an initial step of 96 ºC for 3 min, followed by 35 cycles: 96 ºC for 30s, 61 ºC for 45s and 72 ºC for 1 min, while for the amplification of the ITS region, a scheme consisting of an initial denaturation step of 94 ºC for 3 min, followed by 35 cycles: 94 ºC for 1 min, 55 ºC for 1 min and 72 ºC for 2 min was used. PCR products were purified using the illustra PCR DNA and Gel Band Purification Kit (GE Healthcare UK Limited, Buckinghamshire, UK). The purified products were used as templates for sequencing using ABI Genetic analyzer BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems). Sequences of the D1/D2 and ITS region were obtained using the same sets of primers used for the amplification of both regions. Forward and reverse sequences were aligned and with the program ClustalW provided in BioEdit (Hall, 1999). The sequences obtained for both regions were compared with sequences of other yeasts obtained by BLASTN search from the National Center for Biotechnology Information (NCBI)- GenBank (http://www.ncbi.nml.nih.gov). Sequences of related species were acquired from the GenBank; the sequences were aligned using MUSCLE v3.6 (EDGAR, 2004) and trimmed to remove primer sequences. Phylogenetic and molecular evolutionary analyses were conducted in MEGA v. 5 (TAMURA et al., 2011) using Neighbor-Joining method with Kimura`s two- parameter substitution as the evolutionary model for calculating distances. Bootstrap values were calculated with 1000 replicates.

5.4 RESULTS AND DISCUSSION

5.4.1 DNA sequence and phylogenetic analysis

Strains ATT064T (= CBS 11955T) and ATT067 have identical ITS sequences but differ at one nucleotide position in the D1/D2 domains of the LSU rRNA gene. The same accounts for strains IMUFRJ 51951T (= CBS 12533T) and IMUFRJ 51952. In order to confirm these differences, PCR reactions, sequencing and analysis were repeated.

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Nucleotide differences in the D1/D2 domain between the two type strains CBS 11955T and CBS 12533T are 2 and 3 respectively; hence, we conclude that they belong to the same species. The closest neighbors to Bulleromyces texanaensis CBS 11955T according to BLAST results are Cryptococcus mangaliensis (CBS 10870T), Bullera pseudoalba (CBS 7227T) and Bullera hoabinhensis (JCM 10835T). B. texanaensis CBS 11955T differs from C. mangaliensis by 6 base pair substitutions in the D1/D2 domains (transitions: transversions, ti : tv 3:3) and by 11 bp (ti : tv 8:3), in the ITS regions. Comparison of the DNA sequences of B. texanaensis CBS 11955T with Bullera pseudoalba revealed a total of 8 (ti : tv 5:3) and 13 (ti : tv 6:4 and 3 gaps) base pair differences in the D1/D2 and the ITS regions, respectively; while for Bullera hoabinhensis, nucleotide differences of 6 (ti : tv 3:3) and 22 (ti : tv 13:3 and 6 gaps) were recorded in these two regions, respectively. Phylogenetic tree inferred based on concatenated data set of the D1/D2 domains and ITS sequences placed C. mangaliensis as the closest relative of B. texanaensis while clustering with B. hoabinhensis and B. pseudoalba within the Bulleromyces / Papiliotrema / Auriculibuller clade of the Tremellales (Fig. 5.1). The branching of the subclade formed by this cluster is supported by a bootstrap value of 99 %. Within the subclade, B. texanaensis occupies a phylogenetic position, which depicts it as a separate species. The NJ trees separately constructed using sequence data of the D1/D2 domains and the ITS region also confirm B. texanaensis as a separate species with bootstrap support of 99 % and 64 % respectively (Figures 5.2 and 5.3). The closest teleomorphic species to B. texanaensis is Papiliotrema bandonii with 96.2 % and 88 % similarity indices in the LSU and ITS region, respectively.

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Figure 5.1 Evolutionary tree showing the relationships of Bulleromyces texanaensis and related species based on combined LSU and ITS sequences. The branching pattern was generated by the neighbour-Joining method with heuristic search and stepwise addition. Bootstrap values (expressed as percentages of 1000 replications) of above 50% are shown at branch points. Outgroup is Tremella aurantia CBS 6965T.

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Figure 5.2. Phylogenetic relationships of strain Bulleromyces texanaensis and other closely related species based on LSU D1/D2 rRNA gene sequences. The branching pattern was generated by the Neighbor-Joining methods with heuristic search and stepwise addition. Bootstrap values (expressed as percentages of 1000 replications) of above 50% are shown at branch points.

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Figure 5.3 Phylogenetic relationships of strain Bulleromyces texanaensis and other closely related species based on ITS sequences. The branching pattern was generated by the Neighbor- Joining methods with heuristic search and stepwise addition. Bootstrap values (expressed as percentages of 1000 replications) of above 50% are shown at branch points.

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5.5 DESCRIPTION OF BULLEROMYCES TEXANAENSIS DAYO-OWOYEMI, RODRIGUES, GARCIA, HAGLER AND PAGNOCCA SP. NOV.

5.5.1 Growth on YM broth: After 5 days at 25 °C, cells are mostly ellipsoidal (2.7-4.1 x 3-5.7 µm) to elongate and oval (2-3.9 x 3.1-6 µm). Reproduction is by monopolar budding, with cells occurring singly or in parent-bud pairs (Fig. 5.4 A). After one month ring or a pellicle are absent but abundant sediment of cells are formed. 5.5.2 Growth on YM agar: Colonies formed after 5 days at 25 °C, are entire, convex shaped, butyrous with glossy appearance and tarnish white pigmentation. After 21 days on YM agar, colonies turn light pink to peach with pasty texture, they are entire without hyphae and with dull appearance. 5.5.4 Dalmau plate culture on corn meal agar: After 1 week at 25 °C, rudimentary pseudomycelium may or may not be formed. 5.5.5 Formation of ballistoconidia: After 2 weeks at 15 °C on cornmeal agar, globose (7.9 x 5.6 µm) to napiform (3.5-5.6 x 1.9-2.5 µm) ballistoconidia are formed on short denticles (conidiophores) that measure 1.2 to 3.8 µm in length (Fig. 5.4 B). 5.5.6 Sexual reproduction: Bulleromyces texanaensis includes self-sterile (heterothallic) and self-fertile (homothallic) strain(s). Upon mixing compatible strains namely IMUFRJ 51951 (= CBS 12533) and ATT 064 (=CBS 11955) on MYP agar at 25 °C, conjugation tubes are formed and mating takes place (Fig. 5.4 C). Self fertility; formation of clamped hyphae as well as basidium was observed in one of the mating strains (IMUFRJ 51951). Following conjugation, hyphae formation (visible at the edge of the colony) initiates on 2 week-old crosses of the conjugated cells and haustorial branches and globose haustorial cells as well as clamp connections becomes visible. Within 3 weeks, the hyphae become more extensive, with diameter ranging between 1.7 to 5.6 µm, and develop complete clamp connections; in some cases, the clamps are formed at the branching of the hyphae. Subsequently, basidia develop laterally or terminally on the hyphae; such basidia are lageniform (8.7–13 x 19–23.5 µm), globose (9.7–18 x 9.8–20 µm) to subglobose (12.2–14.5 x 15.5–20 µm) in shape. The lageniform basidia are normally attached by short hyphae stalks to the main hyphae (measuring between 6–11 µm in length) and often occur singly while the globose and sub-globosed shaped basidia may occur singly, in pairs or in clusters, directly on hyphae and are non-septate (Figs. 5.4 D, E, F). Germination of basidia takes place by hyphae formation on which ovoid to ellipsoidal yeast cells,

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Figure - 5.4. Growth phases of Bulleromyces texanaensis

(A) (B)

(C) (D)

(E) (F)

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H (G)

Legend: (A) Budding cells after 5 days at 25 ºC in yeast malt broth, (B) ballistoconidium-forming cells after 2 week at 15 °C on cornmeal agar, (C) conjugating cells (cross of CBS 11955T and CBS 12533T), (D-F) hyphae with haustorial branches with clamp connection and basidia [note arrows (black) indicating clamp connections formed on hyphae branches as well as short hyphae stalk bearing a largeniform basidium and (white) basidia cells], (G, H) hyphae formed by germinated basidium on which yeast cells grow.

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measuring between 2-5 x 5-8.3 μm grow laterally (Figs. 5.4 G, H). The yeast cells further propagate by budding.

5.6 PHENOTYPIC DESCRIPTION

Phenotypic characteristics of B. texanaensis CBS 11955T are presented in Table 5.1. Phenotypic traits were similar in all the four strains tested except for assimilation of L-sorbose and ribitol which were negative in strains CBS 11955T and ATT 067 but positive in strains IMUFRJ 51951 (CBS 12533T) and IMUFRJ 51952 (data not shown). These characteristics should be considered variable within this species. Furthermore, it was possible to differentiate B. texanaensis from its closest described relative C. mangaliensis by 8 phenotypic properties (Table 5.1). While C. mangaliensis grows on ethanol, erithritol (w), and 10% NaCl + 5% Glucose (FELL et al. 2010), the novel species does not grow on these carbon compounds. Moreover, formation of extracellular starch like compounds as well as growth on D-arabinose, methyl-α-D- glucoside (w), DL-lactate and soluble starch was observed on B. texanaensis but not on C. mangaliensis.

5.7 Origin of the strains studied: The type strain of B. texanaensis CBS 11955T was isolated from a nest of the leafcutter ant Atta texana in Texas, USA; while strain CBS 12533T, the complementary mating type of CBS 11955 T was isolated from a bromeliad plant Neoregelia cruenta in Rio de Janeiro, Brazil.

5.8 Systematics and Ecology of Bulleromyces texanaensis i) Systematics

Sequence analyses based on the ITS and LSU rRNA gene confirmed that B. texanaensis is a member of the Tremelalles. Sequence variabilities were observed between the D1/D2 domains (ti : tv 0:2) and ITS region (ti : tv 0:3 and 1 gap) of the two compatiable mating types (CBS 11955T and CBS 12533T) of B. texanaensis. While such observation was not found in the mating strains of B. albus and Auriculibuller fuscus, smaller to higher nucleotide differences have been found between mating strains of many basidiomycetous teleomorphs such as Rhodosporidium

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Table 5.1: Phenotypic characteristics of strain Bulleromyces texanaensis CBS 11955T Assimilation of carbon compounds D-Glucose + Citrate + D-Galactose + myo-Inositol + L-Sorbose - Hexadecane - Sucrose + 2-Keto-D-gluconic acid w Maltose + 5-Keto-D-gluconic acid + Cellobiose + Saccharate - Trealose w D-glucoronate + Lactose + Xylitol w Melibiose - L-arabinitol - Raffinose w+ Arbutin n Melezitose + Propane 1,2 Diol - Inulin - Butan 2, 3 Diol - Soluble starch + Assimilation of nitrogen compounds D-Xylose + Nitrate l L-Arabinose + Nitrite - D-Arabinose + Cadaverine + D-Ribose - Creatinine - L-Rhamnose + Creatine - D-Glucosamine + Imidazol - N-acetyl-D-glucosamine + L-lysin + Methanol - Ethylamine - Ethanol - D-glucosamine (N source) + Glycerol + 50% Glucose - Erithritol - 10% NaCl/5% Glucose - Ribitol - Starch synthesis + Continuation of Table 5.1

Galactitol + Other tests D-Manitol + DBB +

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Table 5.1: Phenotypic characteristics of strain Bulleromyces texanaensis CBS 11955T Continued D-Glucitol + Urease activity +

Methyl-α-D-glucoside w Growth in vitamin free medium w Salicin - Growth in 0.01 % cycloheximide - D-Gluconic acid - Growth in 0.1 % cycloheximide -

D-Glucuronic acid + Growth at 25 °C + D-Glucono-δ-lactone + Growth at 30 °C w DL-Lactate + Growth at 35 °C -

Succinic acid w Growth at 37 °C -

+, positive growth; - , no growth; w, weak growth; l, delayed positive growth; v, positive and negative results; n, not tested.

T T diobovatum, D1/D2 (1); ITS (2) for mating strains CBS 6085 and CBS 9081 , Filobasidium capsuligenum, D1/D2 (3); ITS (11) for mating strains CBS 2206 T and CBS 2425 T, CBS 2630 (KURTZMAN et al., 2011b). The novel species phylogenetically clustered with some anamorphic ballistoconidia- forming and non-forming species in the Bulleromyces / Papiliotrema / Auriculibuller clade (Figure 5.1). Formation of ballistoconidia was not recorded in C. mengaliensis (CBS 10870T) the closest sister species of B. texanaensis, and this characteristic could however be predicted for subsequent isolates of this species. A converging characteristic among the teleomorphic taxa in the Tremellales is their septate (tremella type) basidia (BOEKHOUT et al., 2011); the characteristically non-septate basidia produced by B. texanaensis is rarely found among this group, hence making it to be morphologically distinct. Although more distantly related, B. texanaensis demonstrated a mode of basidia germination almost similar to B. albus but different from that of P. bandonii its closest teleomorphic relative. Sampaio et al. (2002) established P. bandonii to accommodate basidocarp forming yeast whose basidiospores are borne on sterigmata

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produced from transversely septate basidia. However, basidicarp formation was not observed in B. texanaensis. ii) Ecology

A primary habitat of B. texanaensis could be plants. Besides the strains isolated from bromeliads, ecological origin of strains isolated from A. texana nests could be traced to plant substrate foraged by these ants because leaf-cutting attine ants fetch leaves from trees to nurture their fungus symbiont (see MUELLER et al., 1998 and PAGNOCCA et al., 2008 for more about leaf-cutting ant foraging). Common habitats for most of the species in the Bulleromyces / Papiliotrema / Auriculibuller clade are plant materials or invertebrates associated with plants. Cryptococcus mengaliensis, the closest species to B. texanaensis was recovered from mangroves of the Florida everglades, USA (FELL et al., 2010) while B. hoabinhensis was isolated from a leaf of Anadendrum montanum (LUONG et al., 2005). Bullera pseudoalba was first isolated from dead leaves of Oryza sativa and Miscanthus sinensis in Japan (NAKASE; SUZUKI, 1986) but its presence was also recorded on decaying woods (NAKASE et al., 1996). Species of Bullera alba, anamorph of B. albus, have been isolated from various sources, this species as well as Auriculibuller fuscus are distributed on plants surfaces, hence are considered as phylloplane yeasts (SAMPAIO et al. 2004, BOEKHOUT et al., 2011). The clustering of B. texanaensis with plant associated yeasts could be said to be in line with its ecological origin.

iii) Biotechnology

B. texanaensis CBS11955T possess enzyme systems for the breakdown of xylan and cellulose but not starch Chapter 3, Appendix 2).

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BOEKHOUT, T.; FONSECA, A.; BATENBURG-VAN DER VEGTE, W. H. Bulleromyces genus novum (Tremellales), a teleomorph for Bullera alba, and the occurrence of mating in Bullera variabilis. Antonie van Leeuwenhoek, Amsterdam, v. 59, n. 2, p. 81-93, feb. 1991.

BOEKHOUT, T.; FONSECA, A.; SAMPAIO, J. P.; BANDONI, R. J.; FELL, J. W.; KWON- CHUNG, K. J. Discussion of Teleomorphic and Anamorphic Basidiomycetous Yeasts. In: KURTZMAN, C. P.; FELL, J. W.; BOEKHOUT, T. (Eds.) The yeasts: a taxonomic study, 5th edn. San Diego: Elsevier, 2011. p. 1339-1371.

FELL, J. W.; STATZELL-TALLMAN, A.; SCORZETTI, G.; GUTIÉRREZ, M. H. Five new species of yeasts from fresh water and marine habitats in the Florida Everglades. Antonie van Leeuwenhoek, Amsterdam, v. 99, n. 3, p. 533-549, mar. 2010.

EDGAR, R. C. MUSCLE: multiple sequence alignment with high 545 accuracy and high throughput. Nucleic Acids Research, Oxford, v. 32, n.5, p. 1792-1797, mar. 2004.

FELL, J. W.; BOEKHOUT, T.; FONSECA, A.; SCORZETTI, G.; STATZELL-TALLMAN, A. Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. International Journal of Systemic and Evolutionary Microbiology, Reading, v. 50, n. 33, p. 1351-1371, may. 2000.

HALL, T. A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Serius, Oxford, v. 41, n. 1 p. 95-98, 1999.

KURTZMAN, C. P.; FELL, J. W.; BOEKHOUT, T.; ROBERT, V. Methods for isolation, phenotypic characterization and maintenance of yeasts. In: KURTZMAN, C. P.; FELL, J. W.; BOEKHOUT, T. (Eds.) The yeasts: a taxonomic study, 5th edn. San Diego: Elsevier, 2011a. p. 88-110

LUONG, D. T.; TAKASHIMA, M.; TY, P. V.; DUNG, N. L.; NAKASE, T. Bullera hoabinhensis sp. nov., a new ballistoconidiogenous yeast isolated from a plant leaf collected in Vietnam. Journal of General and Applied Microbiology, Tokyo, v. 51, n. 335-342, dec. 2005.

MUELLER, U. G.; REHNER, S. A.; SCHULTZ, T. R. The evolution of agriculture in ants. Science, Washington, v. 281, n. 5385, p. 2034-2038, sep. 1998.

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NAKASE T.; Suzuki, M. Bullera derxii sp. nov. and Bullera pseudoalba sp. nov. isolated from dead leaves of Oryza sativa and Miscanthus sinensis. Journal of General and Applied Microbiology, Tokyo, v. 32, n. 2, p. 125-135, apr. 1986.

NAKASE, T.; TAKASHIMA, M. A simple procedure for the high frequency isolation of new taxa of ballistosporous yeasts living on the surfaces of plants. RIKEN Review, 3, 33-34, http://www.riken.go.jp/lab-www/library/publication/review/html/No03/03-16/03-16.html. 1993.

NAKASE, T.; SUZUKI, M.; HAMAMOTO, M.; TAKASHIMA, M.; HATANO, T.; FUKUI, S. A taxonomic study on cellulolytic yeasts and yeast-like microorganisms isolated in Japan. II. The genus Cryptococcus. Journal of General and Applied Microbiology, Tokyo, v. 42, n. 1, 7-15, 1996.

PAGNOCCA, F. C.; RODRIGUES, A.; NAGAMOTO, N. S.; BACCI JÚNIOR, M. Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants. Antonie van Leeuwenhoek, Amsterdam, v. 94, n. 4, p. 517-526, nov. 2008.

RODRIGUES, A.; CABLE, R. N.; MUELLER, U. G.; BACCI JÚNIOR, M.; PAGNOCCA, F. C. Antagonistic interactions between garden yeasts and microfungal garden pathogens of leaf- cutting ants. Antonie van Leeuwenhoek, Amsterdam, v. 96, n. 3, p. 331-342, out. 2009

SAMPAIO, J. P.; WEIß, M.; GADANHO, M.; BAUER, R. New taxa in the Tremellales: Bulleribasidium oberjochense gen. et sp. nov., Papiliotrema bandonii gen. et sp. nov. and Fibulobasidium murrhardtense sp. nov. Mycologia, Lawrence, v. 94, n. 5, p. 873-887, sep. - oct. 2002.

SAMPAIO, J. P.; INACIO, J.; FONSECA, A.; GADANHO, M.; SPENCER-MARTINS, I.; SCORZETTI, G.; FELL, J. W. Auriculibuller fuscus gen. nov., sp. nov. and Bullera japonica sp. nov., novel taxa in the Tremellales. International Journal of Systemic and Evolutionary Microbiology, Reading, v. 54, n. 3, p. 987-993, may. 2004

SCORZETTI, G.; FELL, J. W.; FONSECA, A.; STATZELL-TALLMAN, A. Systematics of basidiomycetous yeasts: a comparison of large subunit D1D2 and internal transcribed spacer rDNA regions. FEMS Yeast Research, Amsterdam, v. 2, n. 4, p. 495-517, dec. 2002.

TAKASHIMA, M. & NAKASE, T. Molecular phylogeny of the genus Cryptococcus and related species based on the sequences of 18S rDNA and internal transcribed spacer regions. Microbiol Culture Collection, v. 15, 33-45, 1999.

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WANG, Q. M.; BAI, F. Y. Molecular phylogeny of basidiomycetous yeasts in the Cryptococcus luteolus lineage (Tremellales) based on nuclear rRNA and mitochondrial cytochrome b gene sequence analyses: proposal of Derxomyces gen.nov. and Hannaella gen.nov., and description of eight novel Derxomyces species. FEMS Yeast Research, Amsterdam v. 8, n. 5, p. 799-814, aug. 2008.

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CHAPTER 6 Intraspecific variation and emendation of Hannaella kunmingensis

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CONCLUSIONS AND PERSPECTIVES

This study aimed at the taxonomic and biotechnological assessment of some yeasts in the UNESP – CMR. Various conclusions could be drawn from the findings of this study:

The identities of a total of 340 yeast isolates from the UNESP – CMR were determined using morphological and physiological assimilation tests as well as genetic molecular makers (microsatellite fingerprints and D1/D2 LSU ribosomal DNA gene sequencing). The finding of this work demonstrated the significance of these two yeast identification methods for yeast species delineation. In some cases such as Candida davisiana where morphologically variant strains were found, the molecular markers used in this study proved useful for the classification of these variants, which otherwise would have been considered as separate species if identification were based only on phenotypic characteristics. On the other hand, cultural and phenotypic characterization methods also proved useful for separating strains of different species exhibiting little or no genetic differences e.g Meyerozyma carribbica and M. guilliermondii. Thus, it is suggested that combination of both phenotypic and genetic markers should be applied for correct yeast identification, especially in cases where either of both methods cannot clearly resolved yeast identities.

The taxonomic assessment of yeasts from the UNESP – CMR led to the classification of 70 different species. This is high species diversity, considering that only 1.7% of yeasts in this culture collection were studied. The identification of a high number of species is therefore a pointer to yeast biodiversity as well as the high taxonomic and genetic diversity of yeasts in this collection. Furthermore, from this proportion of yeasts that were identified, strains representing 8 previously unknown species were revealed. Three out of 8 species were described in the present study. This further supports the hidden yeast diversity with the implication that many yeasts species still lies to be discovered. Therefore, more ecological sampling is needed in order to discover more species.

Biotechnological assessment of yeasts in the UNESP – CMR led to the discovery of many yeasts possessing plant polysaccharide degrading enzymes. Most of which are characterized by substrate and genetic diversity, thus revealing the high biotechnological potential of yeasts in this culture collection. More investigations are required to unravel as well as exploit the

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biotechnological potential of yeasts in this culture collection. The present study led to the discovery of three yeast strains namely Aureobasidium pullulans strain CG5-5BY, Aureobasidium pullulans strain PBM 1 and Pseudozyma hubeiensis strain MP2-2CB capable of producing enzymes involved in plant biomass degradation, especially xylanases, hence could be good candidates for subsequent investigations and biotechnological applications.

From the findings of this study, it was also possible to draw some taxonomical conclusions. Enzymes attacking plant polysaccharides such as starch, cellulose and xylan are found mostly among basidiomycetous yeasts. Enzyme systems degrading the polysaccharide pectin were found in a slightly higher proportion in the ascomycetes but in minor proportion in the basidiomycetes, whereas yeasts possessing ligninase enzymes are scarce in nature (Figure 3.2). Furthermore, from the data obtained from the quantitative determination of xylanase enzyme, it was observed that β-xylosidase activities obtained from extracellular extract of the tested strains were generally lower from those obtained from cell wall extracts, thereby implying that this enzyme may be cell wall associated in most yeasts.

Findings of this work revealed that while most strains of Cryptococcus flavescens are capable of producing xylan degrading enzymes, most strains of Cryptococcus flavus are able to produce amylolytic, cellulase and xylanase enzymes. Whereas, species such as Bandoniozyma complexa, Bullera sinensis, Bulleromyces sp. possess enzyme systems for cellulase degradation, most strains of Cryptococcus laurentii and Pseudozyma sp. are able to produce cellulase and xylanase enzymes. Furthermore, most strains of Candida silvae are able to produced pectinase (polygalacturonase and pectin lyase) enzyme. Although this pattern was not strict for all the strains of the species, however, these characteristics could serve as a useful key for classifying this species.

By examining the morphological, phenotypic and genetic characteristics of 11 strains of H. kunmingensis, it was possible to observe intraspecific variability among the yeast screened. This was also reflected in the results obtained from examining polysaccharide degrading activity in many strains belonging to the same yeast species, thereby, suggesting that intraspecific variability is an intrinsic property of yeasts.

– Appendix 1- Identities of yeasts and dimorphic fungi maintained at UNESP CMR (continuation)

Species name Pi (% nd) Strain code Isolation source Isolation region Isolates obtained from soil samples collected Amazonia, Brazil Candida parapsilosis 2AN Antropogenic soil (Caldeirão) Amazonia, Brazil 99.3% (4) Candida parapsilosis 33AN Antropogenic soil (Caldeirão) Amazonia, Brazil 99.3% (4) Candida sp. aff aaseri 29b Antropogenic soil (Caldeirão) Amazonia, Brazil 98.7% (7) Candida sp. aff aaseri 39b Antropogenic soil (Caldeirão) Amazonia, Brazil 98.7% (7) Candida sp. aff aaseri 53b Antropogenic soil (Caldeirão) Amazonia, Brazil 98.7% (7) Cryptococcus allantoinivorans 12A Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100% (0) Cryptococcus laurentii 87AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100% (0) Cryptococcus laurentii 24 Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100% (0) Cryptococcus laurentii 13A Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100% (0) Cryptococcus laurentii 43A Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100% (0) Cryptococcus laurentii 45A Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100% (0) Debaryomyces hansenii 33TH Antropogenic soil (Hatahara) Amazonia, Brazil 100% (0) Debaryomyces hansenii 1ATH Antropogenic soil (Hatahara) Amazonia, Brazil 100% (0) Debaryomyces nepalensis 54TH Antropogenic soil (Hatahara) Amazonia, Brazil 99.1% (5) Debaryomyces polymorphus 26AN Antropogenic soil (Caldeirão) Amazonia, Brazil 100% (0) Meyerozyma caribbica 81AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100 % (0) Meyerozyma caribbica 100AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100 % (0) Meyerozyma caribbica 106AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100 % (0) Meyerozyma caribbica 107AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100 % (0) Meyerozyma caribbica 82AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100 % (0) Meyerozyma caribbica 86AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100 % (0) Meyerozyma caribbica 88AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100 % (0) Meyerozyma caribbica 97AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100 % (0) Meyerozyma caribbica 22TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Meyerozyma caribbica 30TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Meyerozyma guilliermondii 102AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100 % (0) Meyerozyma guilliermondii 103AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100 % (0) Meyerozyma guilliermondii 96AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100 % (0)

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– Appendix 1- Identities of yeasts and dimorphic fungi maintained at UNESP CMR (continuation)

Species name Strain code Isolation source Isolation region Pi (% nd) Meyerozyma guilliermondii 106TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia manshurica 7 Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 1 Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 4 Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 16 Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 22 Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 45 Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 15A Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 24AC Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 25A Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 26A Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 33A Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 37A Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 38A Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 39A Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia manshurica 40A Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Pichia terricola 52b Antropogenic soil (Caldeirão) Amazonia, Brazil 100% (0) Pichia terricola 13TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 1TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 23TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 24ATH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 24TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 35THa Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 35THb Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 3TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 41TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 42TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 42TH(2) Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 46TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0)

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Appendix 1- Identities of yeasts and dimorphic fungi maintained at UNESP – CMR (continuation)

Species name Strain code Isolation source Isolation region Pi (% nd) Pichia terricola 47TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 48TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 4TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 32TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Pichia terricola 7TH Antropogenic soil (Hatahara) Amazonia, Brazil 100 % (0) Rhodotorula mucilaginosa 113AN Antropogenic soil (Caldeirão) Amazonia, Brazil 100% (0) Rhodotorula mucilaginosa 43TH Antropogenic soil (Hatahara) Amazonia, Brazil 100% (0) Rhodotorula mucilaginosa 58TH Antropogenic soil (Hatahara) Amazonia, Brazil 100% (0) Rhodotorula mucilaginosa 60TH Antropogenic soil (Hatahara) Amazonia, Brazil 100% (0) Rhodotorula mucilaginosa 119AH Antropogenic soil (adjacente Hatahara) Amazonia, Brazil 100% (0) Saccharomyces cerevisiae 2 Antropogenic soil (TPI-Cult. Caldeirão) Amazonia, Brazil 100 % (0) Schwanniomyces polymorphus 16AN Antropogenic soil (Caldeirão) Amazonia, Brazil 100% (0)

Isolates obtained from samples collected at the Atlantic rain forest, Pindamonhangaba, São Paulo, Brazil Candida silvae DC7M Drosophilid Pindamonhangaba, São Paulo, Brazil 100% (0) Sporopachydermia sp. Exudato M Exudate from Banana plant (Musa sp.) Pindamonhangaba, São Paulo, Brazil 98.8% (7) Candida boidinii FB1-1AYb Flower bract of Siphocampylus sp.(Campanulaceae) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida boidinii FB2-2AMa Flower bract of Siphocampylus sp.(Campanulaceae) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida boidinii FB2-2CM Flower bract of Siphocampylus sp.(Campanulaceae) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae FB1-1AYa Flower bract of Siphocampylus sp.(Campanulaceae) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae FB1-1CASP Flower bract of Siphocampylus sp.(Campanulaceae) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida sp. strain FBI-1DASP FBI-1DASP Flower bract of Siphocampylus sp.(Campanulaceae) Pindamonhangaba, São Paulo, Brazil 96% (12) Hanseniaspora aff. clermontiae FB1-1BYa Flower bract of Siphocampylus sp.(Campanulaceae) Pindamonhangaba, São Paulo 98.8% (6) Rhodotorula sp. CRUB 1484 FB1-1AASPb Flower bract of Siphocampylus sp. (Campanulaceae) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida rancensis H2-2BM Fruit of Hedychium coronarium Koening Pindamonhangaba, São Paulo, Brazil 100 (0)% Candida silvae H10-10BY Fruit of Hedychium coronarium Koening Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae H1-1AY Fruit of Hedychium coronarium Koening Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae H12-12BM Fruit of Hedychium coronarium Koening Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae H12-12BY Fruit of Hedychium coronarium Koening Pindamonhangaba, São Paulo, Brazil 100% (0)

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Appendix 1- Identities of yeasts and dimorphic fungi maintained at UNESP – CMR (continuation)

Species name Strain code Isolation source Isolation region Pi (% nd) Candida silvae H12Y Fruit of Hedychium coronarium Koening Pindamonhangaba, São Paulo, Brazil 100% (0) Candida sp. aff aaseri H1ASP Fruit of Hedychium coronarium Koening Pindamonhangaba, São Paulo, Brazil 98% (10) Candida sp. strain H10-10AY H10-10AY Fruit of Hedychium coronarium Koening Pindamonhangaba, São Paulo, Brazil 86.5% (68) Candida sp. strain H10Y H10Y Fruit of Hedychium coronarium Koening Pindamonhangaba, São Paulo, Brazil 86.5% (68) Candida silvae FH11T Flower of Hedychium coronarium Koening Pindamonhangaba, São Paulo, Brazil 100% (0) Cryptococcus laurentii FH9T Flower of Hedychium coronarium Koening Pindamonhangaba, São Paulo, Brazil 100% (0) Kodamaea ohmeri HFL1-1AY Flower of Hedychium coronarium Koening Pindamonhangaba, São Paulo, Brazil 100 % (0) Candida silvae CG2-2BYb Unidentified mushroom Pindamonhangaba, São Paulo, Brazil 100% (0) Cryptococcus albidosimilis CG2-2BASPa Unidentified mushroom Pindamonhangaba, São Paulo, Brazil 100% (0) Pseudozyma tsukubaensis CG7-7AYb Unidentified mushroom Pindamonhangaba, São Paulo 100% (0) Candida hawaiiana BR6-3AASP Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo 100% (0) Candida picinguabensis BR3-3AY Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100 (0) % Candida silvae BR1-2AM Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae BR1-2BM Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae BR1-2BMf Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae BR1-2I Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae BR1-4M Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae BR2-2Y Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae BR3-3BY Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae BR4-1M Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100% (0) Candida silvae BR5-1AM Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100% (0) Cryptococcus aff. flavescens BR7-1AI Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 98% (11) Cryptococcus laurentii BR7-3BM Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100% (0) Debaryomyces hansenii BR2-4Y Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100% (0) Myxozyma geophila BR5-4BI Water accumulated in tank of Bromeliad (Vriesea sp.) Pindamonhangaba, São Paulo, Brazil 100% (0)

Isolates obtained from Atta sexdens collected in Palmas, Tocantins State, Brazil Aureobasidium pullulans PBM1 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 96.6% (7) Aureobasidium pullulans var. melanigenum PBM 3 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 99.8% (1)

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Species name Strain code Isolation source Isolation region Pi (% nd) Candida albicans PBM 34 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 99.6% (2) Candida albicans PBM 41 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 99.6% (2) Candida albicans PBM 42 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 99.6% (2) Candida albicans PBM 50 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 99.6% (2) Candida albicans PBM 51 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 99.6% (2) Candida albicans PBM 7 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 99.6% (2) Candida glaebosa PBM 40 Exosceleton of Atta sexdens Palmas, Tocantins State, Brazil 99.5% (3) Candida metapsilosis PBM 38 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 10 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 11 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 13 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 15 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 16 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 17 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 20 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 23 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 25 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 29 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 36 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 37 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 45 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 46 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 48 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 49 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 61 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida metapsilosis PBM 63 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 12 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 14 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 18 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0)

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Appendix 1- Identities of yeasts and dimorphic fungi maintained at UNESP – CMR (continuation)

Species name Strain code Isolation source Isolation region Pi (% nd) Candida parapsilosis PBM 19 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 24 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 28 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 32 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 33 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 35 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 47 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 55 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 57 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 58 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 59 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 60 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 62 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 64 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida parapsilosis PBM 9 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Candida zeylanoides PBM 26 Exosckeleton of Atta sexdens Palmas, Tocantins State, Brazil 99.4% (4) Cryptococcus aff. nemorosus PBM56 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 98.8% (7) Cryptococcus flavus PBM 4 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 99.2% (4) Cryptococcus laurentii PBM 21 Exosckeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Cryptococcus laurentii PBM 27 Exosckeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Debaryomyces hansenii PBM 22 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Debaryomyces hansenii PBM 53 Exoskeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Hanseniaspora uvarum PBM 43 Exosceleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Hyphopichia burtonii PBM 30 Exosceleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Hyphopichia burtonii PBM 31 Exosceleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Meira nashicola PBM 39 Exosceleton of Atta sexdens Palmas, Tocantins State, Brazil 99.2% (4) Meyerozyma caribbica PBM 44 Exosceleton of Atta sexdens Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii PBM 52 Exosceleton of Atta sexdens Palmas, Tocantins State, Brazil 100 % (0) Pseudozyma jejuensis PBM 5 Exosckeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0)

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Appendix 1- Identities of yeasts and dimorphic fungi maintained at UNESP – CMR (continuation)

Species name Strain code Isolation source Isolation region Pi (% nd) Pseudozyma jejuensis PBM8 Exosckeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0) Rhodotorula mucilaginosa PBM54 Exosckeleton of Atta sexdens Palmas, Tocantins State, Brazil 100% (0)

Isolates obtained from ant fungus garden collected in Palmas, Tocantins State, Brazil Bandoniozyma complexa A4M-D14 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100% (0) Bandoniozyma complexa A4M-D15 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100% (0) Bandoniozyma complexa A4M-D17 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100% (0) Candida jaroonii A1-A4 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100% (0) aExophiala dermatitidis A1M-A15 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100% (0) aExophiala dermatitidis A1M-A16 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100% (0) aExophiala dermatitidis A1M-A6 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100% (0) aExophiala dermatitidis A1M-A7 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100% (0) Meyerozyma caribbica A1M-A12 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma caribbica A1M-A17 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 101 % (0) Meyerozyma caribbica A1-N2 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A2-B2 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A3-C2 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A1F-A9 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A4M-D13 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A2M-B6 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A3M-C7 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A4M-D4 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A1-A3 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A2M-B3 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A3F-C6 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A2M-B4 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A4M-D12 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A4-D1 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A4M-D2 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0)

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Appendix 1- Identities of yeasts and dimorphic fungi maintained at UNESP – CMR (continuation)

Species name Strain code Isolation source Isolation region Pi (% nd) Meyerozyma guilliermondii A2M-C1 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A2-B1 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A1F-A10 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A2M-B3 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A1F-A11 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A1M-A12 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A2-B2 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A3F-C5 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A4M-D18 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A4M-D5 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A2-B3 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii AIM-A5 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii AIM-N1 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A1F-A8 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii AIM-A1 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Meyerozyma guilliermondii A2M-B8 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100 % (0) Sporothrix schenckii A1F-A13 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100% (0) Sporothrix schenckii A1F-A14 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100% (0) Trichosporon chiarelii A4-D8 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100% (0) Trichosporon chiarelii A4-D9 Fungus garden of Acromyrmex sp. Palmas, Tocantins State, Brazil 100% (0)

Isolates obtained from ant fungus garden collected in Texas, USA Bulleromyces sp. ATT 067 Fungus garden of Atta texana Bastrop County, Texas, USA 98.6% (7) Bulleromyces sp. ATT 064 Fungus garden of Atta texana Bastrop County, Texas, USA 98.7% (6) Hannaella kunmingensis ATT 066 Fungus garden of Atta texana Bastrop County, Texas, USA 100% (0) Hannaella kunmingensis ATT 082 Fungus garden of Atta texana Bastrop County, Texas, USA 100% (0) Hannaella kunmingensis ATT 265 Fungus garden of Atta texana Bastrop County, Texas, USA 100% (0)

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Appendix 1 - Identities of yeasts and dimorphic fungi maintained at UNESP – CMR (continuation) Species name Strain code Isolation source Isolation region Pi (% nd) Isolates obtained from Wasp (Polybia ignobilis) and honey Lecythophora sp. W7 Nest of Wasp (Polybia ignobilis) Rio Claro, São Paulo State, Brasil 96.5% ((18) Lecythophora sp. W3a2 Nest of Wasp (Polybia ignobilis) Rio Claro, São Paulo State, Brasil 96.5% ((18) Pseudozyma hubiensis MP2-2CB Honey Mato-Nova Andradina, Mato Grosso do Sul 100% (0) Zygosaccharomyces mellis MP12-12A Honey Nova Andradina, Mato Grosso do Sul 100% (0) Zygosaccharomyces mellis MP13-13B Honey Cerrado Nova Andradina, Mato Grosso do Sul 100% (0) Zygosaccharomyces mellis MP14-14A Honey Nova Andradina, Mato Grosso do Sul 100% (0) Zygosaccharomyces mellis MP2-2CA Honey Mato-Nova Andradina, Mato Grosso do Sul 100% (0) Zygosaccharomyces mellis MP6-6A Honey Belo Visto, Mato Grosso do Sul 100% (0) Zygosaccharomyces mellis MP7-7A Honey Nova Andradina, Mato Grosso do Sul 100% (0) Zygosaccharomyces mellis MP7-7B Honey Nova Andradina, Mato Grosso do Sul 100% (0) Zygosaccharomyces mellis MP8-8B Honey Aroeira Niooque, Mato Grosso do Sul 100% (0) Zygosaccharomyces mellis MP9-9A Honey Niooque, Mato Grosso do Sul 100% (0) Zygosaccharomyces mellis MP9-9B Honey Niooque, Mato Grosso do Sul 100% (0) Zygosaccharomyces siamensis MP14-14B Honey Nova Andradina, Mato Grosso do Sul 100% (0)

Isolates obtained from aluminium screw of energy transmission tower Bandoniozyma complexa A2-1 Corroding aluminum screw of energy transmission tower São Paulo, Brazil 100% (0) Bandoniozyma complexa A2-4 Corroding aluminum screw of energy transmission tower São Paulo, Brazil 100% (0) Candida albicans BD-106 Corroding aluminum screw of energy transmission tower São Paulo, Brazil 99.7% (1) Cryptococcus laurentii B2-1 Corroding aluminum screw of energy transmission tower São Paulo, Brazil 100% (0) Cryptococcus laurentii B2-3 Corroding aluminum screw of energy transmission tower São Paulo, Brazil 100% (0) Cryptococcus laurentii BD 107 Corroding aluminum screw of energy transmission tower São Paulo, Brazil 100% (0)

Isolates obtained from samples collected from Antarctic environments Candida glaebosa LANTA 77 Antarctic (Penguin) soil Antarctica 99.6% (2) Candida glaebosa LANTA 79 Antarctic (Penguin) soil Antarctica 99.6% (2) Candida glaebosa LANTA 84 Antarctic (Penguin) soil Antarctica 99.6% (2) Candida glaebosa LANTA 76 Antarctic (Penguin) soil Antarctica 99.6% (2)

174 174

Appendix 1 - Identities of yeasts and dimorphic fungi maintained at UNESP – CMR (continuation) Species name Strain code Isolation source Isolation region Pi (% nd) Candida glaebosa LANTA 75 Antarctic (Penguin) soil Antarctica 99.6% (2) Candida glaebosa LANTA 78 Antarctic (Penguin) soil Antarctica 99.6% (2) Candida glaebosa LANTA 83 Antarctic (Penguin) soil Antarctica 99.6% (2) Debaryomyces macquariensis LANTA 80 Antarctic (Penguin) soil Antarctica 99.8% (1) Debaryomyces macquariensis LANTA 81 Antarctic (Penguin) soil Antarctica 99.8% (1) Debaryomyces macquariensis LANTA 82 Antarctic (Penguin) soil Antarctica 99.8% (1) Debaryomyces macquariensis LANTA 74 Antarctic (Penguin) soil Antarctica 99.8% (1) Cystofilobasidium infirmo-miniatum LANTA 98 Lichen Antarctica 100% (0) Crytococcus victoriae LANTA 92 Lichens Antarctica 100% (0) Guehomyces pullulans LANTA 86 Lichen Antarctica 100% (0) Guehomyces pullulans LANTA 88 Lichen Antarctica 100% (0) Leucosporidium scottii LANTA115 Lichen Antarctica 100 % (0) Rhodotorula laryngis LANTA 29 Lichen Antarctica 100% (0) Rhodotorula laryngis LANTA 25 Lichen Antarctica 100% (0) Rhodotorula laryngis LANTA 27 Lichen Antarctica 100% (0) Rhodotorula laryngis LANTA 87 Lichen Antarctica 100% (0) Rhodotorula mucilaginosa LANTA 26 Lichen Antarctica 100% (0) Rhodotorula mucilaginosa LANTA 28 Lichen Antarctica 100% (0) Tremella indecorata LANTA 99 Lichen Antarctica 99% (6) Debaryomyces hansenii LANTA 6 Marine algae Antarctica 100% (0) Meyerozyma guilliermondii LANTA 13 Marine algae Antarctica 100 % (0) Meyerozyma guilliermondii LANTA15 Marine algae Antarctica 100 % (0) Meyerozyma guilliermondii LANTA 5 Marine algae Antarctica 100 % (0) Meyerozyma guilliermondii LANTA 9 Marine algae Antarctica 100 % (0) Rhodotorula mucilaginosa LANTA 7 Marine algae Antarctica 100% (0) Cryptococcus adeliensis LANTA 108 Marine sea star Antarctica 100% (0) Cryptococcus adeliensis LANTA 110 Marine sea star Antarctica 100% (0) Cryptococcus adeliensis LANTA 95 Marine sea star Antarctica 100% (0) Cryptococcus albidosimilis LANTA 112 Marine sea star Antarctica 100% (0)

175 175

Appendix 1 - Identities of yeasts and dimorphic fungi maintained at UNESP – CMR (continuation) Species name Strain code Isolation source Isolation region Pi (% nd) Cryptococcus albidosimilis LANTA 94 Marine sea star Antarctica 100% (0) Cystofilobasidium infirmo-miniatum LANTA 96 Marine sea star Antarctica 100% (0) Guehomyces pullulans LANTA 109a Marine sea star Antarctica 100% (0) Meyerozyma guilliermondii LANTA 4 Marine sea star Antarctica 100 % (0) Meyerozyma guilliermondii LANTA11 Marine sediment Antarctica 100 % (0) Cryptococcus adeliensis LANTA121 Marine sediment Antarctica 100% (0) Crytococcus victoriae LANTA 122 Marine sediment Antarctica 100% (0) Crytococcus victoriae LANTA123 Marine sediment Antarctica 100% (0) Crytococcus victoriae LANTA 24 Marine sediment Antarctica 100% (0) Crytococcus victoriae LANTA 32 Marine sediment Antarctica 100% (0) Debaryomyces hansenii LANTA 3 Marine sediment Antarctica 100% (0) Leucosporidium scottii LANTA 116 Marine sediment Antarctica 100 % (0) Leucosporidium scottii LANTA 117 Marine sediment Antarctica 100 % (0) Leucosporidium scottii LANTA 118 Marine sediment Antarctica 100 % (0) Metschnikowia australis LANTA 2 Marine sediment Antarctica 100 % (0) Metschnikowia australis LANTA 33 Marine sediment Antarctica 100 % (0) Meyerozyma guilliermondii LANTA10 Marine sediment Antarctica 100 % (0) Meyerozyma guilliermondii LANTA119 Marine sediment Antarctica 100 % (0) Bullera pseudoalba LANTA 71 Marine sponge Antarctica 100% (0) Cryptococcus sp. aff laurentii LANTA 70 Marine sponge Antarctica 98.1% (11) Debaryomyces hansenii LANTA 72 Marine sponge Antarctica 100% (0) Rhodotorula mucilaginosa LANTA 73 Marine sponge Antarctica 100% (0) Cadophora lutea-olivacea LANTA 61 Marine squirt Antarctica 99% (3) Cadophora lutea-olivacea LANTA 50 Marine squirt Antarctica 99.4% (3) Rhodotorula mucilaginosa LANTA 34 Nacella concinna Antarctica 100% (0) Cryptococcus sp. aff laurentii LANTA 35 Nacella concinna Antarctica 98.1% (11) Rhodotorula mucilaginosa LANTA 38 Nacella concinna Antarctica 100% (0) Wickerhamomyces anomalus LANTA 36 Nacella concinna Antarctica 100% (0) Wickerhamomyces anomalus LANTA 37a Nacella concinna Antarctica 100% (0)

176 176

Appendix 1 - Identities of yeasts and dimorphic fungi maintained at UNESP – CMR (continuation) Species name Strain code Isolation source Isolation region Pi (% nd) Crytococcus victoriae LANTA 43 Salpa sp. Antarctica 100% (0) Crytococcus victoriae LANTA 42 Salpa sp. Antarctica 100% (0) Cystofilobasidium capitatum LANTA 45b Salpa sp. Antarctica 99.8% (1) Cystofilobasidium capitatum LANTA 47b Salpa sp. Antarctica 99.8% (1) Cystofilobasidium capitatum LANTA 46 Salpa sp. Antarctica 99.8% (1) Cystofilobasidium capitatum LANTA 47a Salpa sp. Antarctica 99.8% (1) Cystofilobasidium infirmo-miniatum LANTA 111 Salpa sp. Antarctica 100% (0) Cystofilobasidium infirmo-miniatum LANTA 44 Salpa sp. Antarctica 100% (0) Metschnikowia australis LANTA 48b Salpa sp. Antarctica 100 % (0) Rhodotorula mucilaginosa LANTA 49a Salpa sp. Antarctica 100% (0) Meyerozyma guilliermondii LANTA 19 Sea isopod Antarctica 100 % (0) Meyerozyma guilliermondii LANTA 20 Sea isopod Antarctica 100 % (0) Meyerozyma guilliermondii LANTA 21 Sea isopod Antarctica 100 % (0) Meyerozyma guilliermondii LANTA 17 Sea snail Antarctica 100 % (0) Meyerozyma guilliermondii LANTA 18 Sea snail Antarctica 100 % (0) Candida sake LANTA 51 Sea squirt Antarctica 99.6% (2) Candida sake LANTA 53 Sea squirt Antarctica 99.6% (2) Candida sake LANTA 58 Sea squirt Antarctica 99.6% (2) Rhodotorula mucilaginosa LANTA 52 Sea squirt Antarctica 100% (0) Rhodotorula mucilaginosa LANTA 56 Sea squirt Antarctica 100% (0) Wickerhamomyces anomalus LANTA 55 Sea squirt Antarctica 100 % (0) Rhodotorula mucilaginosa LANTA 69 Sea urchin Antarctica 100% (0) Cryptococcus sp. aff laurentii LANTA 59 Sea urchin Antarctica 98.1% (11) Cryptococcus sp. aff laurentii LANTA 62 Sea urchin Antarctica 98.1% (11) Cryptococcus sp. aff laurentii LANTA 63 Sea urchin Antarctica 98.1% (11) Cryptococcus sp. aff laurentii LANTA 64 Sea urchin Antarctica 98.1% (11) Rhodotorula laryngis LANTA 60 Sea urchin Antarctica 100% (0) Rhodotorula mucilaginosa LANTA 65 Sea urchin Antarctica 100% (0) Rhodotorula mucilaginosa LANTA 67a Sea urchin Antarctica 100% (0)

177 177

Appendix 1 - Identities of yeasts and dimorphic fungi maintained at UNESP – CMR (continuation) Species name Strain code Isolation source Isolation region Pi (% nd) Rhodotorula mucilaginosa LANTA 67b Sea urchin Antarctica 100% (0) Candida davisiana LANTA 101 Stone with lichen Antarctica 100% (0) Candida davisiana LANTA 114 Stone with lichen Antarctica 100% (0) Candida davisiana LANTA 107 Stone with lichen Antarctica 100% (0) Rhodotorula glacialis LANTA 106 Stones with lichens Antarctica 100% (0) Rhodotorula laryngis LANTA 104 Stones with lichen Antarctica 100% (0) Rhodotorula laryngis LANTA 103 Stones with lichen Antarctica 100% (0)

178 178

Appendix 2 – Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources

Yeast species P. Isolation source Strain code Amylase Cellulase Xylanase P. galact lyase Ligninase Aureobasidium pullulans Honey of Polybia ignobilis M 10 + + - + + - Aureobasidium pullulans Exoskeleton of Atta sexdens PBM1 + + + + - - Aureobasidium pullulans Fungus gardens of Atta texana ATT262 - + +++ - - - Aureobasidium pullulans Fungus gardens of Atta texana ATT263 - + +++ - - - Aureobasidium pullulans Fungus gardens of Atta texana ATT269 + + +++ - - - Aureobasidium pullulans Unidentified mushroom CG5-5BY + + + - - - Aureobasidium pullulans var. melanigenum Exoskeleton of Atta sexdens PBM3 + + + + - - Bandonizyma complexa Energy transmission tower A2-1 - + - - - - Bandonizyma complexa Energy transmission tower A2-4 - + - - - - Bandonizyma complexa Fungus garden of Acromyrmex sp. A4M-D17 + + - - - - Bullera sinensis Fungus gardens of Atta texana ATT074 + + w+ - - - Bulleromyces albus Fungus gardens of Atta texana ATT078 - + w - - - Bulleromyces sp. Fungus gardens of Atta texana ATT064 - + + - - - Bulleromyces sp. Fungus gardens of Atta texana ATT067 - + ++ - - - Candida aff. aaseri Fruit of Hedychium coronarium Koening H1ASP - - - - + - Candida albicans Energy transmission tower BD106 - + - - - - Candida albicans Exoskeleton of Atta sexdens PBM 34 ------Candida albicans Exoskeleton of Atta sexdens PBM 7 ------Candida albicans Exoskeleton of Atta sexdens PBM 41 ------Candida albicans Exoskeleton of Atta sexdens PBM 42 ------Candida albicans Exoskeleton of Atta sexdens PBM 50 ------Candida albicans Exoskeleton of Atta sexdens PBM 51 ------Candida boidinii Flower bracts of Siphocampylus sp. (Campanulaceae) FB1-1AY-b ------Candida boidinii Flower bracts of Siphocampylus sp. (Campanulaceae) FB2-2CM ------

179

Appendix 2– Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources continuation

P. Yeast species Isolation source Strain code Amylase Cellulase Xylanase P. galact lyase Ligninase Candida boidinii Flower bracts of Siphocampylus sp. (Campanulaceae) FB2-2AM-a ------Candida boidinii Exudate from Banana plant (Musa sp.) BN2-2AASP ------Candida cf. apícola Honey of Polybia ignobilis M 6b2 ------Candida cf. apícola Polybia ignobilis RN1b - - + - - - Candida cf. azyma Polybia ignobilis W19 ------Candida cf. azyma Polybia ignobilis B22a2 - ng - - - - Candida cf. azyma Polybia ignobilis B22b - ng - - - - Candida cf. azyma Polybia ignobilis B27b ------Candida cf. azyma Polybia ignobilis S16b ------Candida cf. azyma Polybia ignobilis S16b1 ------Candida cf. azyma Polybia ignobilis S16b2 ------Candida cf. azyma Polybia ignobilis RN10 - ng - - - - Candida cf. azyma Polybia ignobilis B29 ------Candida chickasaworum Drosophilid DC4M - - - + - - Candida chrysomelidarum Polybia ignobilis W1b ------Candida chrysomelidarum Polybia ignobilis W1Bb ------Candida chrysomelidarum Polybia ignobilis W12a ------Candida chrysomelidarum Polybia ignobilis B25 ------Candida chrysomelidarum Polybia ignobilis B27a1 ------Candida chysomelidarum Honey of Polybia ignobilis M 1 ------Candida chysomelidarum Honey of Polybia ignobilis M 11a ------Candida glaebosa Exoskeleton of Atta sexdens PBM 40 ------Water accumulated in tank of Bromeliad (Vriesea Candida hawaiiana sp.) BR6-3AASP ------Candida jaroonii Fungus garden of Acromyrmex sp. A1-A4 ------Candida melibiosica Fungus gardens of Atta texana AT003b ------Candida membranifaciens Fungus gardens of Atta texana AT001 ------Candida membranifaciens Fungus gardens of Atta texana AT002 ------Candida membranifaciens Fungus gardens of Atta texana AT004 ------

180

Appendix 2 – Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources continuation Yeast species P. Isolation source Strain code Amylase Cellulase Xylanase P. galact lyase Ligninase Candida metapsilosis Exoskeleton of Atta sexdens PBM 10 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 11 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 12 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 13 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 14 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 15 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 16 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 17 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 20 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 23 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 25 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 29 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 36 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 37 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 38 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 45 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 46 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 48 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 49 ------Candida metapsilosis Exoskeleton of Atta sexdens PBM 63 ------Candida oleophila Drosophilid DC1M ------Candida oleophila Flower bracts of Siphocampylus sp. (Campanulaceae) FB2-2BY - - - + - - Water accumulated in tank of Bromeliad (Vriesea Candida oleophila sp.) BR5-2AY ------Candida oleophila Exudate from Banana plant (Musa sp.) BN2Y ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 9 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 18 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 19 ------

181

Appendix 2 – Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources continuation Yeast species P. Isolation source Strain code Amylase Cellulase Xylanase P. galact lyase Ligninase Candida parapsilosis Exoskeleton of Atta sexdens PBM 24 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 28 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 32 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 33 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 35 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 47 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 55 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 57 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 58 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 59 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 60 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 61 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 62 ------Candida parapsilosis Exoskeleton of Atta sexdens PBM 64 ------Water accumulated in tank of Bromeliad (Vriesea Candida picinguabensis sp.) BR3-3AY ------Candida picinguabensis Exudate from Palm leaves (Euterpe sp.) PT1-1M ------Candida restingae Flower bracts of Siphocampylus sp. (Campanulaceae) FB1-1AASP-a ------Candida restingae Flower bracts of Siphocampylus sp. (Campanulaceae) FB1-1BY-b ------Candida shehatae var Water accumulated in tank of Bromeliad (Vriesea insectosa sp.) BR6-2AI - - - - + - Candida shehatae var Water accumulated in tank of Bromeliad (Vriesea insectosa sp.) BR6-2AY ------Candida shehateae var. insectosa Unidentified mushroom CG8-8BY - - - - + - Candida shehateae var. shehatae Exudate from Palm leaves (Euterpe sp.) PT1-1AASP ------Candida shehateae var. shehatae Exudate from Palm leaves (Euterpe sp.) PT1-1BASP ------

182

Appendix 2 – Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources continuation Yeast species P. Isolation source Strain code Amylase Cellulase Xylanase P. galact lyase Ligninase Candida silvae Drosophilid DCAY - - - + + - Candida silvae Fruit of Hedychium coronarium Koening H12-12BM - + - - - - Candida silvae Fruit of Hedychium coronarium Koening H12Y - - - + + - Candida silvae Fruit of Hedychium coronarium Koening H2-2AASP + - - + - - Candida silvae Fruit of Hedychium coronarium Koening H1-1AY - w+ - - - - Candida silvae Fruit of Hedychium coronarium Koening H10-10BY - w+ - - - - Candida silvae Unidentified mushroom CG2-2BY-b - - - + + -

Candida silvae Unidentified mushroom CG5-5AY - - - + + - Water accumulated in tank of Bromeliad (Vriesea Candida silvae sp.) BR1-2BM ------Water accumulated in tank of Bromeliad (Vriesea Candida silvae sp.) BR1-2I - - - + - - Water accumulated in tank of Bromeliad (Vriesea Candida silvae sp.) BR4-1M - - - + + - Water accumulated in tank of Bromeliad (Vriesea Candida silvae sp.) BR5-1AM - - - + + - Candida silvae Drosophilid DC7M ------Candida silvae Exudate from Palm leaves (Euterpe sp.) PT1-1Y - w+ - - - - Candida silvae Flower bracts of Siphocampylus sp. (Campanulaceae) FB1-1AY-a - - - + + - Candida silvae Flower bracts of Siphocampylus sp. (Campanulaceae) FB1-1CASP - - - + + - Candida silvae Flower of Hedychium coronarium Koening FH11T - - - + + - Candida silvae Fruit of Hedychium coronarium Koening H2-2AM - - - - + - Candida silvae Fruit of Hedychium coronarium Koening H3-3AASP - - - + + - Candida silvae Fruit of Hedychium coronarium Koening H3-3CASP - - - + - - Water accumulated in tank of Bromeliad (Vriesea Candida silvae sp.) BR1-2AM - - - + + - Water accumulated in tank of Bromeliad (Vriesea Candida silvae sp.) BR1-2BM-F - - - + + -

183

Appendix 2 – Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources continuation Yeast species P. Isolation source Strain code Amylase Cellulase Xylanase P. galact lyase Ligninase Water accumulated in tank of Bromeliad (Vriesea Candida silvae sp.) BR2-2Y - - - - + - Water accumulated in tank of Bromeliad (Vriesea Candida silvae sp.) BR3-3BY - - - + - - Candida sp. 1 Fruit of Hedychium coronarium Koening H10Y - - - - + - Candida sp. 1 Fruit of Hedychium coronarium Koening H10-10AY - w+ - - - - Candida sp. 2 Flower bracts of Siphocampylus sp. (Campanulaceae) FB1-1DASP ------Candida sp. 3 Larva of Polybia ignobilis larva 5a ------Candida zyelanoides Exoskeleton of Atta sexdens PBM 26 - - + - - - Water accumulated in tank of Bromeliad (Vriesea Cryptococcus aff cylindricus sp.) BR6-4BASP ------Cryptococcus albidosimilis Unidentified mushroom CG2-2BASP-c - + - - - - Cryptococcus albidosimilis Unidentified mushroom CG2-2BY-a - - ++ - + - CG2- Cryptococcus albidosimilis Unidentified mushroom 2BAASP-a - - w - + - Cryptococcus cf. podzolicus Soil SC081 - - - - + - Cryptococcus dimennae Fungus gardens of Cyphomyrmex wheeleri CY080 - - + - - - Cryptococcus dimennae Fungus gardens of Cyphomyrmex wheeleri CY081 - - + - - - Water accumulated in tank of Bromeliad (Vriesea Cryptococcus flavescens sp.) BR3-2AASP - - + - + - Water accumulated in tank of Bromeliad (Vriesea Cryptococcus flavescens sp.) BR7-1AI ------Cryptococcus flavescens Fungus gardens of Atta texana ATT120 - - + - - - Water accumulated in tank of Bromeliad (Vriesea Cryptococcus flavescens sp.) BR2-1Y - - + - - - Water accumulated in tank of Bromeliad (Vriesea Cryptococcus flavescens sp.) BR2-4AI - - + - - - Water accumulated in tank of Bromeliad (Vriesea Cryptococcus flavescens sp.) BR3-21 - - + - + - Water accumulated in tank of Bromeliad (Vriesea Cryptococcus flavescens sp.) BR3-3CY - - + - - -

184

Appendix 2 – Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources continuation P. Yeast species Isolation source Strain code Amylase Cellulase Xylanase P. galact lyase Ligninase Water accumulated in tank of Bromeliad (Vriesea Cryptococcus flavescens sp.) BR7-2I ------Cryptococcus flavus Fungus gardens of Atta texana ATT259 + + +++ - - - Cryptococcus flavus Fungus gardens of Atta texana ATT260 + + +++ - - - Cryptococcus flavus Fungus gardens of Atta texana ATT268 + + +++ - - - Cryptococcus laurentii Flower of Hedychium coronarium Koening FH9T - - + - - - Cryptococcus laurentii Energy transmission tower BD107 - + + - - - Cryptococcus laurentii Energy transmission tower B2-1 - + + - - - Cryptococcus laurentii Energy transmission tower B2-3 - + + - - - Cryptococcus laurentii Exoskeleton of Atta sexdens PBM 21 - + + - - - Cryptococcus laurentii Exoskeleton of Atta sexdens PBM 27 - - + - - - Cryptococcus laurentii Flower bracts of Siphocampylus sp. (Campanulaceae) FB2-2BM - - + - - - Cryptococcus laurentii Fungus garden of Atta sexdens rubropilosa JA012 - + + - - - Cryptococcus laurentii Fungus garden of Atta sexdens rubropilosa JA003 ------Cryptococcus laurentii Fungus garden of Atta sexdens rubropilosa JA009 - w + - - Cryptococcus laurentii Fungus gardens of Atta texana ATT073 + + +++ - - - Cryptococcus laurentii Fungus gardens of Atta texana ATT075 + + - - - - Cryptococcus laurentii Fungus gardens of Atta texana ATT253 + w +++ - - - Cryptococcus laurentii Soil SA048 - - + + - - Cryptococcus laurentii Soil SA049 - - + + - - Cryptococcus laurentii Soil SA050 - + + + - - Cryptococcus laurentii Soil SC041 ------Cryptococcus laurentii Soil SC042 - - + - - - Cryptococcus laurentii Soil SC039 - + - - - - Cryptococcus laurentii Soil SA042 - - + - + - Cryptococcus laurentii Soil SA092 - - - + - - Cryptococcus laurentii Soil SC040 - + - - - - Cryptococcus laurentii Soil SA046 - - + - - -

185 185

Appendix 2 – Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources continuation P. galact P. Yeast species Isolation source Strain code Amylase Cellulase Xylanase lyase Ligninase Cryptococcus laurentii Soil SA047 - - + + - - Cryptococcus laurentii Soil SA094 - - + + - - Cryptococcus laurentii Soil SA043 - - + + - - Water accumulated in tank of Bromeliad (Vriesea Cryptococcus laurentii sp.) BR7-3BM - - + - + - Water accumulated in tank of Bromeliad (Vriesea Cryptococcus laurentii sp.) BR3-4AY - - + - - - Cryptococcus liquefaciens Fungus gardens of Atta texana ATT122 - - w+ - - - Cryptococcus liquefaciens Honey of Polybia ignobilis M 16 - - w - w - Cryptococcus liquefaciens Honey of Polybia ignobilis M 28 ------Cryptococcus liquefaciens Polybia ignobilis W14a - - w - - - Cryptococcus magnus Fungus gardens of Atta texana ATT069 - + ++ - - - Cryptococcus magnus Fungus gardens of Atta texana ATT121 - + + - - - Cryptococcus magnus Fungus gardens of Atta texana ATT148 - - w+ - - - Cryptococcus nemorosus Exoskeleton of Atta sexdens PBM 56 + - - - - Cryptococcus nemorosus Fungus gardens of Atta texana ATT178 - - +++ - - - Cryptococcus podzolicus Fungus gardens of Atta texana ATT076 - + + - - - Cryptococcus podzolicus Fungus gardens of Atta texana ATT204 + - ++ - - - Cryptococcus sp PYCC 4949 Unidentified mushroom CG1ASP - - - - + - Cryptococcus sp. 4 Fungus gardens of Atta texana ATT080 ------Cryptococcus sp. 7 (CBS 7944) Fungus gardens of Atta texana ATT176 + - +++ + - - Cryptococcus sp. CBS 8369 Soil SA093 - + - - - - Cryptococcus sp. CBS 8369 Soil SA095 - + + - - - Cryptococcus sp.CBS 681.93 Fungus gardens of Atta texana ATT079 - + + - - - Cryptococcus taibaiensis Fungus gardens of Atta texana ATT065 - + - - - - Crytococcus flavus Exoskeleton of Atta sexdens PBM 4 - - w - - - Water accumulated in tank of Bromeliad (Vriesea Debaryomyces hansenii sp.) BR2-4Y - - - + + -

186

Appendix 2 – Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources continuation P. Yeast species Isolation source Strain code Amylase Cellulase Xylanase P. galact lyase Ligninase Debaryomyces hansenii Exoskeleton of Atta sexdens PBM 22 ------Debaryomyces hansenii Exoskeleton of Atta sexdens PBM 53 - - - - - Exophiala dermatitidis Fungus garden of Acromyrmex sp. A1M-A7 - - + - - - Exophiala dermatitidis Fungus garden of Acromyrmex sp. A1M-A15 - - + - - - Exophiala dermatitidis Fungus garden of Acromyrmex sp. A1M-A16 - - + - - - Exophiala dermatitidis Fungus garden of Acromyrmex sp. A1M-A6 - - + - - - Hannaella kunmingensis Fungus gardens of Atta texana ATT066 - - w+ - - - Hannaella kunmingensis Fungus gardens of Atta texana ATT082 ------Hannaella kunmingensis Fungus gardens of Atta texana ATT265 - - w - - - Hanseniaspora clermontiae Flower bracts of Siphocampylus sp. (Campanulaceae) FB1-1BY-a ------Hanseniaspora guilliermondii Polybia ignobilis W2b1 - - - - + - Hanseniaspora guilliermondii Polybia ignobilis W2b2.2 - - - - + - Hanseniaspora opuntiae Polybia ignobilis S20a ------Hanseniaspora opuntiae Polybia ignobilis S20b ------Hanseniaspora sp Polybia ignobilis S18a - - - - + - Hanseniaspora uvarum Polybia ignobilis S14 - + - - - - Hanseniaspora uvarum Exoskeleton of Atta sexdens PBM 43 ------Kodamaea ohmeri Flower of Hedychium coronarium Koening HFL1-1AY - - - + - - Lecythophora sp. Polybia ignobilis W3a2 + + + - + - Lecythophora sp. Polybia ignobilis W7 + + + - + - Meira nashicola Exoskeleton of Atta sexdens PBM39 ------Metchnikowia reukaufii Flower of Hedychium coronarium Koening HFL5-5BASP - + - - - - Metchnikowia reukaufii Fruit of Hedychium coronarium Koening H3M - + - - - - Metschnikowia koreensis Polybia ignobilis W3 - + - - - - Metschnikowia reukaufii Fruit of Hedychium coronarium Koening H2-2BM - - - w + - Metschnikowia reukaufii Polybia ignobilis W12b ------Metschnikowia reukaufii Polybia ignobilis W16a - - - - + -

187

Appendix 2 – Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources continuation P. Yeast species Isolation source Strain code Amylase Cellulase Xylanase P. galact lyase Ligninase Metschnikowia reukaufii Polybia ignobilis B30 ------Moniliella suaveolens Polybia ignobilis B22a1 - ng - - - - Pichia burtonii Exoskeleton of Atta sexdens PBM 30 + + - - - - Pichia burtonii Exoskeleton of Atta sexdens PBM 31 + + - - - - Pichia caribbica Fungus garden of Acromyrmex sp. A1M-A12 ------Pichia caribbica Exoskeleton of Atta sexdens PBM 44 ------Pichia guilliermondii Fungus garden of Acromyrmex sp. A3-C2 ------Pichia guilliermondii Fungus garden of Acromyrmex sp. A2-B2 ------Pichia guilliermondii Fungus garden of Acromyrmex sp. AIM-A5 ------Pichia guilliermondii Polybia ignobilis W13a1a - - - - + - Pichia guilliermondii Polybia ignobilis W13a1b - - - - + - Pichia guilliermondii Polybia ignobilis W13a1c - - - - + - Pichia guilliermondii Polybia ignobilis W14 - - - - + - Pichia guilliermondii Exoskeleton of Atta sexdens PBM 52 - - + - - - Pichia kudriavzevii Fungus garden of Atta sexdens rubropilosa JA004 ------Pichia kudriavzevii Fungus garden of Atta sexdens rubropilosa JA010 - + - - - - Pichia kudriavzevii Fungus garden of Atta sexdens rubropilosa JA013 - + - - - - Pichia spartinae Exudate from Palm leaves (Euterpe sp.) PT1-1AI ------Pichia spartinae Exudate from Palm leaves (Euterpe sp.) PT1-1BI ------Pichia spartinae Exudate from Palm leaves (Euterpe sp.) PT1-1CASP ------Pichia spartinae Exudate from Palm leaves (Euterpe sp.) PT1-1CI ------Pichia spartinae Exudate from Palm leaves (Euterpe sp.) PT2-2AY - - - + - - Pichia spartinae Exudate from Palm leaves (Euterpe sp.) PT2-2BY - - - + + - Pseudozyma hubiensis Honey MP2-2CB ++ +++ +++ - - - Pseudozyma jejuensis Exoskeleton of Atta sexdens PBM 5 - ++ ++ - + - Pseudozyma jejuensis Exoskeleton of Atta sexdens PBM 8 - ++ ++ - - -

188

Appendix 2 – Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources continuation P. Yeast species Isolation source Strain code Amylase Cellulase Xylanase P. galact lyase Ligninase Pseudozyma sp. Fungus gardens of Atta texana ATT068 + + +++ + + - Pseudozyma sp. BCRC 34227 Soil SC080 - - - - + - Pseudozyma sp. BCRC 34227 Soil SA045 - - + - - - Pseudozyma sp. BCRC 34227 Soil SC038 - - + - - - Pseudozyma tsukubaensis Unidentified mushroom CG7-7AY - + - + + - Rhodosporidium sp. APSS 849 Soil SA041 - - + - - - Rhodotorula javanica Fungus gardens of Atta texana ATT174 - - w - - - Rhodotorula lactosa Fungus gardens of Atta texana ATT258 ------Rhodotorula marina Fungus gardens of Cyphomyrmex wheeleri CY079 - - + - - - Rhodotorula minuta Fungus gardens of Cyphomyrmex wheeleri CY082 ------Rhodotorula mucilaginosa Fungus gardens of Atta texana ATT252 ------Rhodotorula mucilaginosa Honey of Polybia ignobilis M 19 ------Rhodotorula mucilaginosa Honey of Polybia ignobilis M 20 ------Rhodotorula mucilaginosa Honey of Polybia ignobilis M 24 ------Rhodotorula mucilaginosa Honey of Polybia ignobilis M 27 ------Rhodotorula mucilaginosa Larva of Polybia ignobilis larva 7 ------Rhodotorula mucilaginosa Larva of Polybia ignobilis larva 10 ------Rhodotorula mucilaginosa Larva of Polybia ignobilis PUP7 ------Rhodotorula mucilaginosa Polybia ignobilis W14b ------Rhodotorula mucilaginosa Polybia ignobilis RN8 - - - - + - Rhodotorula mucilaginosa Polybia ignobilis RN9 ------Rhodotorula mucilaginosa Pupa of Polybia ignobilis PUP8 - - - - + - Rhodotorula mucilaginosa Pupa of Polybia ignobilis PUP9 ------Rhodotorula mucilaginosa Exoskeleton of Atta sexdens PBM 54 - - - - - Rhodotorula mucilaginosa Unidentified mushroom CG3-3BM - - - - + - Rhodotorula nothofagi Fungus gardens of Atta texana ATT177 + - +++ + - - Rhodotorula sp. CBS 8885 Flower of Hedychium coronarium Koening HFL1-1AASP ------

189

Appendix 2 – Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources continuation P. Yeast species Isolation source Strain code Amylase Cellulase Xylanase P. galact lyase Ligninase Rhodotorula sp. CRUB 1484 Flower bracts of Siphocampylus sp.(Campanulaceae) FB1-1AASP-b - - - - + - CG2-2BASP- Rhodotorula sp. CRUB 1484 Unidentified mushroom b - - - - + - Rhodotorula taiwaniana Fungus gardens of Atta texana ATT070 - - + - - - Saccharomyces paradoxus Polybia ignobilis W4 - - w - - - Saccharomycopsis crataegensis Polybia ignobilis S11 - + - - + - Sporidiobolus ruineniae Fungus gardens of Atta texana ATT254 - - - + - - Sporidiobolus ruineniae Fungus gardens of Atta texana ATT256 - - - + - - Sporidiobolus ruineniae Soil SA040 - - - + - - Sporisorium penniseti Fungus gardens of Atta texana ATT255 - - +++ + - - Sporisorium penniseti Fungus gardens of Atta texana ATT257 - - - + - - EXSUDATO Sporopachydermia sp. Exudate from Banana plant (Musa sp) M - - - + - Sporothrix schenckii Fungus garden of Acromyrmex sp. A1F-A13 + + - + w - Sporothrix schenckii Fungus garden of Acromyrmex sp. A1F-A14 + + - + w - Sympodiomycopsis paphiopedili Fungus gardens of Atta texana ATT264 - - ++ - - - Sympodiomycopsis paphiopedili Fungus gardens of Atta texana ATT271 - - + - - - Torulaspora delbrueckii Exudate from Banana plant (Musa sp.) BN2-2BASP ------Torulaspora delbrueckii Exudate from Banana plant (Musa sp.) BN2-2AM - - - + + - Trichosporon chiarelii Fungus garden of Acromyrmex sp. A4-D8 - - - - + - Trichosporon chiarelii Fungus garden of Acromyrmex sp. A4-D9 - - - - + - Trichosporon jirovecii Fungus garden of Atta sexdens rubropilosa JA005 - + + - - + Trichosporon jirovecii Fungus garden of Atta sexdens rubropilosa JA008 - + + - - + Trichosporon jirovecii Fungus garden of Atta sexdens rubropilosa JA011 - + + - - - Ustilago spermophora Fungus garden of Atta sexdens rubropilosa JA001 + - - - w+ - Ustilago spermophora Fungus garden of Atta sexdens rubropilosa JA007 + - - - w+ -

190

Appendix 2 – Result of extracellular enzyme screening with some yeasts in the UNESP – Central for Microbial Resources continuation P. Yeast species Isolation source Strain code Amylase Cellulase Xylanase P. galact lyase Ligninase Zygosaccharomyces mellis Honey MP2-2CA ------Zygosaccharomyces mellis Honey MP6-6A ------Zygosaccharomyces mellis Honey MP7-7A ------Zygosaccharomyces mellis Honey MP7-7B ------Zygosaccharomyces mellis Honey MP8-8B ------Zygosaccharomyces mellis Honey MP9-9A ------Zygosaccharomyces mellis Honey MP9-9B ------Zygosaccharomyces mellis Honey MP12-12A ------Zygosaccharomyces mellis Honey MP13-13B ------Zygosaccharomyces mellis Honey MP14-14A ------Zygosaccharomyces siamensis Honey MP14-14B ------+, positive result; -, ++, diameter of halo ≥ 20 mm; +++, diameter of halo ≥ 30 mm; -, negative result; ng, no gowth; P. galact, polygalacturonase; P. lyase, pectin lyase

191 192

Appendix 3 - Statisitical (one-way anova) anlysis of reducing sugars (RS) produced from sugar cane bagasse fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM1, Aureobasidium pullulans strain CG5-5BY

trat RS

1 CG5-5BY 0.47 23 PBM1 0.48

2 CG5-5BY 0.41 24 PBM1 0.57

3 CG5-5BY 0.37 25 PBM1 0.63

4 CG5-5BY 0.36 26 PBM1 0.58

5 CG5-5BY 0.53 27 PBM1 0.53

6 CG5-5BY 0.44 28 PBM1 0.75

7 CG5-5BY 0.55 29 PBM1 0.64

8 CG5-5BY 0.49 30 PBM1 0.82

9 CG5-5BY 0.50 31 MP2-2CB 1.14

10 CG5-5BY 0.59 32 MP2-2CB 1.24

11 CG5-5BY 0.50 33 MP2-2CB 1.32

12 CG5-5BY 0.63 34 MP2-2CB 1.52

13 CG5-5BY 0.65 35 MP2-2CB 1.22

14 CG5-5BY 0.74 36 MP2-2CB 1.34

15 CG5-5BY 0.91 37 MP2-2CB 1.58

16 PBM1 0.83 38 MP2-2CB 1.62

17 PBM1 0.86 39 MP2-2CB 1.74

18 PBM1 0.76 40 MP2-2CB 1.43

19 PBM1 0.59 41 MP2-2CB 1.51

20 PBM1 0.58 42 MP2-2CB 1.68

21 PBM1 0.65 43 MP2-2CB 1.51

22 PBM1 0.57 44 MP2-2CB 1.27

193

45 MP2-2CB 1.41

46 CONTROL 1.60

47 CONTROL 1.54

48 CONTROL 1.53

49 CONTROL 1.50

> summary.aov(model1)

Df Sum Sq Mean Sq F value Pr(>F) trat 3 8.7074 2.90245 137.64 < 2.2e-16 ***

Residuals 45 0.9489 0.02109

---

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

> TukeyHSD(model1)

Tukey multiple comparisons of means

95% family-wise confidence level

Fit: aov(formula = AR ~ trat)

$trat

diff lwr upr p adj

CONTROL-CG5-5BY 0.9998333 0.78184039 1.2178263 0.0000000

MP2-2CB-CG5-5BY 0.8926667 0.75121417 1.0341192 0.0000000

PBM1-CG5-5BY 0.1133333 -0.02811916 0.2547858 0.1569894

MP2-2CB-CONTROL -0.1071667 -0.32515961 0.1108263 0.5606036

PBM1-CONTROL -0.8865000 -1.10449294 -0.6685071 0.0000000

PBM1-MP2-2CB -0.7793333 -0.92078583 -0.6378808 0.0000000

194

Appendix 4 - Statisitical (one way anova) analysis of activitiy of endoamylase produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM1, Aureobasidium pullulans strain CG5-5BY (using 0.5% starch)

trat amylase(0.5%) 23 PBM1 2074.61

1 CG5-5BY 1691.02 24 PBM1 2042.64

2 CG5-5BY 1595.12 25 PBM1 1467.25

3 CG5-5BY 1978.71 26 PBM1 1563.15

4 CG5-5BY 1978.71 27 PBM1 1435.29

5 CG5-5BY 1754.95 28 PBM1 955.79

6 CG5-5BY 1275.45 29 PBM1 1595.12

7 CG5-5BY 1499.22 30 PBM1 1754.95

8 CG5-5BY 1595.12 31 MP2-2CB 540.23

9 CG5-5BY 1595.12 32 MP2-2CB 859.89

10 CG5-5BY 1499.22 33 MP2-2CB 1211.52

11 CG5-5BY 1499.22 34 MP2-2CB 636.13

12 CG5-5BY 1275.45 35 MP2-2CB 476.30

13 CG5-5BY 1275.45 36 MP2-2CB 859.89

14 CG5-5BY 1563.15 37 MP2-2CB 1019.72

15 CG5-5BY 1531.18 38 MP2-2CB 795.96

16 PBM1 1275.45 39 MP2-2CB 604.16

17 PBM1 1211.52 40 MP2-2CB 763.99

18 PBM1 1339.39 41 MP2-2CB 1211.52

19 PBM1 1627.08 42 MP2-2CB 795.96

20 PBM1 1435.29 43 MP2-2CB 763.99

21 PBM1 1627.08 44 MP2-2CB 572.20

22 PBM1 1691.02 45 MP2-2CB 572.20

195

> summary.aov(model1)

Df Sum Sq Mean Sq F value Pr(>F) trat 2 6059174 3029587 48.833 1.1e-11 ***

Residuals 42 2605692 62040

---

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

> TukeyHSD(model1)

Tukey multiple comparisons of means

95% family-wise confidence level

Fit: aov(formula = amilase0.5 ~ trat)

$trat

diff lwr upr p adj

MP2-2CB-CG5-5BY -794.89533 -1015.8596 -573.9311 0.0000000

PBM1-CG5-5BY -34.09733 -255.0616 186.8669 0.9255915

PBM1-MP2-2CB 760.79800 539.8337 981.7623 0.0000000

196

Appendix 5 - Statisitical (one way anova) analysis of activitiy of exoamylase produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM1, Aureobasidium pullulans strain CG5-5BY trat amilase1% 23 PBM1 1531.18

1 CG5-5BY 1435.29 24 PBM1 668.09

2 CG5-5BY 856.70 25 PBM1 3449.16

3 CG5-5BY 1818.88 26 PBM1 572.20

4 CG5-5BY 1051.69 27 PBM1 572.20

5 CG5-5BY 700.06 28 PBM1 668.09

6 CG5-5BY 476.30 29 PBM1 700.06

7 CG5-5BY 540.23 30 PBM1 1786.91

8 CG5-5BY 476.30 31 MP2-2CB 1019.72

9 CG5-5BY 1051.69 32 MP2-2CB 1019.72

10 CG5-5BY 476.30 33 MP2-2CB 2586.07

11 CG5-5BY 732.03 34 MP2-2CB 1691.02

12 CG5-5BY 668.09 35 MP2-2CB 1307.42

13 CG5-5BY 923.82 36 MP2-2CB 987.76

14 CG5-5BY 2138.54 37 MP2-2CB 2234.44

15 CG5-5BY 668.09 38 MP2-2CB 572.20

16 PBM1 1051.69 39 MP2-2CB 1818.88

17 PBM1 1051.69 40 MP2-2CB 1435.29

18 PBM1 859.89 41 MP2-2CB 1307.42

19 PBM1 1051.69 42 MP2-2CB 2106.58

20 PBM1 1531.18 43 MP2-2CB 1563.15

21 PBM1 955.79 44 MP2-2CB 2841.80

22 PBM1 540.23 45 MP2-2CB 955.79

197

> summary.aov(model1)

Df Sum Sq Mean Sq F value Pr(>F) trat 2 3100857 1550429 3.7633 0.03138 *

Residuals 42 17303387 411985

---

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

> TukeyHSD(model1)

Tukey multiple comparisons of means

95% family-wise confidence level

Fit: aov(formula = amilase1 ~ trat)

$trat diff lwr upr p adj

MP2-2CB-CG5-5BY 628.8833 59.47204 1198.2946 0.0274701

PBM1-CG5-5BY 198.4027 -371.00863 767.8140 0.6765076

PBM1-MP2-2CB -430.4807 -999.89196 138.9306 0.1701797

198

Appendix 6 - Statisitical (one way anova) analysis of activitiy of pectinase produced through sugarcane baggase fermentation by Pseudozyma hubeiensis stra

trat pectinase 23 PBM1 1698.50

1 CG5-5BY 1985.30 24 PBM1 2622.64

2 CG5-5BY 2431.44 25 PBM1 2527.04

3 CG5-5BY 2877.57 26 PBM1 2463.30

4 CG5-5BY 1443.57 27 PBM1 2399.57

5 CG5-5BY 1953.43 28 PBM1 3100.64

6 CG5-5BY 1443.57 29 PBM1 2622.64

7 CG5-5BY 2176.50 30 PBM1 2240.24

8 CG5-5BY 2080.90 31 MP2-2CB 1252.36

9 CG5-5BY 2144.64 32 MP2-2CB 1188.63

10 CG5-5BY 2527.04 33 MP2-2CB 869.96

11 CG5-5BY 2527.04 34 MP2-2CB 1029.30

12 CG5-5BY 2303.97 35 MP2-2CB 1156.76

13 CG5-5BY 2272.10 36 MP2-2CB 710.63

14 CG5-5BY 2367.70 37 MP2-2CB 710.63

15 CG5-5BY 2558.90 38 MP2-2CB 1379.83

16 PBM1 2463.30 39 MP2-2CB 1316.10

17 PBM1 2176.50 40 MP2-2CB 1443.57

18 PBM1 1953.43 41 MP2-2CB 615.03

19 PBM1 2558.90 42 MP2-2CB 1443.57

20 PBM1 2463.30 43 MP2-2CB 583.16

21 PBM1 2941.31 44 MP2-2CB 1124.90

22 PBM1 2718.24 45 MP2-2CB 710.63

199

summary.aov(model1)

Df Sum Sq Mean Sq F value Pr(>F) trat 2 17372288 8686144 69.336 4.938e-14 ***

Residuals 42 5261613 125276

---

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

> TukeyHSD(model1)

Tukey multiple comparisons of means

95% family-wise confidence level

Fit: aov(formula = pectinase ~ trat)

$trat

diff lwr upr p adj

MP2-2CB-CG5-5BY -1170.5740 -1484.56698 - 856.5810 0.0000000

PBM1-CG5-5BY 257.0587 -56.93432 571.0516 0.1274337

PBM1-MP2-2CB 1427.6327 1113.63968 1741.6256 0.0000000

>

200

Appendix 7 - Statisitical (one way anova) analysis of activitiy of xylanase produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM1, Aureobasidium pullulans strain CG5-5BY

trat xilanase 23 PBM1 211.178

1 CG5-5BY 206.454 24 PBM1 248.973

2 CG5-5BY 215.902 25 PBM1 215.902

3 CG5-5BY 178.107 26 PBM1 121.415

4 CG5-5BY 258.421 27 PBM1 201.729

5 CG5-5BY 272.594 28 PBM1 97.794

6 CG5-5BY 116.691 29 PBM1 277.319

7 CG5-5BY 494.638 30 PBM1 248.973

8 CG5-5BY 211.178 31 MP2-2CB 414.324

9 CG5-5BY 64.723 32 MP2-2CB 376.530

10 CG5-5BY 107.242 33 MP2-2CB 338.735

11 CG5-5BY 206.454 34 MP2-2CB 414.324

12 CG5-5BY 300.940 35 MP2-2CB 518.260

13 CG5-5BY 650.541 36 MP2-2CB 338.735

14 CG5-5BY 532.433 37 MP2-2CB 414.324

15 CG5-5BY 593.849 38 MP2-2CB 296.216

16 PBM1 192.281 39 MP2-2CB 414.324

17 PBM1 145.037 40 MP2-2CB 352.908

18 PBM1 173.383 41 MP2-2CB 419.049

19 PBM1 93.069 42 MP2-2CB 423.773

20 PBM1 97.794 43 MP2-2CB 343.459

21 PBM1 93.069 44 MP2-2CB 404.876

22 PBM1 215.902 45 MP2-2CB 329.286

201

> summary.aov(model1)

Df Sum Sq Mean Sq F value Pr(>F) trat 2 335639 167820 12.258 6.41e-05 ***

Residuals 42 575019 13691

---

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

> TukeyHSD(model1)

Tukey multiple comparisons of means

95% family-wise confidence level

Fit: aov(formula = xilanase ~ trat)

$trat

diff lwr upr p adj

MP2-2CB-CG5-5BY 92.59707 -11.20393 196.39806 0.0887591

PBM1-CG5-5BY -118.42327 -222.22426 -14.62227 0.0220860

PBM1-MP2-2CB -211.02033 -314.82133 -107.21934 0.0000381

>

202

Appendix 8 - Statisitical (one way anova) analysis of activitiy of carboxymethyl cellulase (CMCase) produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM1, Aureobasidium pullulans strain CG5-5BY

trat CMCase 23 PBM1 781.575

1 CG5-5BY 318.064 24 PBM1 605.761

2 CG5-5BY 334.047 25 PBM1 989.356

3 CG5-5BY 605.761 26 PBM1 989.356

4 CG5-5BY 318.064 27 PBM1 861.491

5 CG5-5BY 334.047 28 PBM1 781.575

6 CG5-5BY 238.149 29 PBM1 605.761

7 CG5-5BY 238.149 30 PBM1 989.356

8 CG5-5BY 254.132 31 MP2-2CB 1532.782

9 CG5-5BY 381.997 32 MP2-2CB 1612.698

10 CG5-5BY 366.014 33 MP2-2CB 1468.850

11 CG5-5BY 334.047 34 MP2-2CB 1916.377

12 CG5-5BY 286.098 35 MP2-2CB 2331.939

13 CG5-5BY 318.064 36 MP2-2CB 1532.782

14 CG5-5BY 334.047 37 MP2-2CB 1612.698

15 CG5-5BY 605.761 38 MP2-2CB 1468.850

16 PBM1 989.356 39 MP2-2CB 1916.377

17 PBM1 861.491 40 MP2-2CB 2331.939

18 PBM1 781.575 41 MP2-2CB 1532.782

19 PBM1 605.761 42 MP2-2CB 1612.698

20 PBM1 989.356 43 MP2-2CB 1468.850

21 PBM1 989.356 44 MP2-2CB 1916.377

22 PBM1 861.491 45 MP2-2CB 2331.939

203

> summary.aov(model1)

Df Sum Sq Mean Sq F value Pr(>F) trat 2 15621419 7810709 163.15 < 2.2e-16 ***

Residuals 42 2010713 47874

---

Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

> TukeyHSD(model1)

Tukey multiple comparisons of means

95% family-wise confidence level

Fit: aov(formula = CMCase ~ trat)

$trat

diff lwr upr p adj

MP2-2CB-CG5-5BY 1421.4331 1227.3287 1615.5376 0e+00

PBM1-CG5-5BY 494.4117 300.3073 688.5162 6e-07

PBM1-MP2-2CB -927.0214 -1121.1259 -732.9169 0e+00

>

204

Appendix 9 - Statisitical (one way anova) analysis of activitiy of microcrystaline cellulase (MCase) produced through sugarcane baggase fermentation by Pseudozyma hubeiensis strain MP2-2CB, Aureobasidium pullulans strain PBM1, Aureobasidium pullulans strain CG5-5BY

trat Cellulase 23 PBM1 397.9800

1 CG5-5BY 1021.3221 24 PBM1 302.0812

2 CG5-5BY 957.3895 25 PBM1 270.1149

3 CG5-5BY 573.7944 26 PBM1 525.8450

4 CG5-5BY 1133.2040 27 PBM1 334.0474

5 CG5-5BY 541.8281 28 PBM1 397.9800

6 CG5-5BY 589.7775 29 PBM1 302.0812

7 CG5-5BY 1005.3389 30 PBM1 270.1149

8 CG5-5BY 637.7269 31 MP2-2CB 813.5414

9 CG5-5BY 1213.1196 32 MP2-2CB 941.4064

10 CG5-5BY 749.6088 33 MP2-2CB 989.3558

11 CG5-5BY 637.7269 34 MP2-2CB 845.5076

12 CG5-5BY 1021.3221 35 MP2-2CB 1149.1871

13 CG5-5BY 1005.3389 36 MP2-2CB 813.5414

14 CG5-5BY 1213.1196 37 MP2-2CB 941.4064

15 CG5-5BY 1133.2040 38 MP2-2CB 989.3558

16 PBM1 525.8450 39 MP2-2CB 845.5076

17 PBM1 334.0474 40 MP2-2CB 1149.1871

18 PBM1 397.9800 41 MP2-2CB 813.5414

19 PBM1 302.0812 42 MP2-2CB 941.4064

20 PBM1 270.1149 43 MP2-2CB 989.3558

21 PBM1 525.8450 44 MP2-2CB 845.5076

22 PBM1 334.0474 45 MP2-2CB 1149.1871

205

> summary.aov(model1)

Df Sum Sq Mean Sq F value Pr(>F) trat 2 3108250 1554125 55.051 1.834e-12 ***

Residuals 42 1185677 28230

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Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1

> TukeyHSD(model1)

Tukey multiple comparisons of means

95% family-wise confidence level

Fit: aov(formula = Cellulase ~ trat)

$trat

diff lwr upr p adj

MP2-2CB-CG5-5BY 52.21156 -96.84248 201.2656 0.6737268

PBM1-CG5-5BY -529.57440 -678.62844 - 380.5204 0.0000000

PBM1-MP2-2CB -581.78596 -730.84000 - 432.7319 0.0000000

>