Isolation of acidophilic and acid tolerant fungi from diverse environments in vietnam (khóa luận tốt nghiệp)

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  THESIS TITLE: ISOLATION OF ACIDOPHILIC AND ACIDTOLERANT FUNGI FROM DIVERSE ENVIRONMENTS IN VIETNAM Student : Nguyen Bao Ngoc Faculty : Biotechnology Supervisors : Nguyen Van Giang, Assoc Prof PhD Vu Nguyen Thanh, Assoc Prof PhD Hanoi, February 2021 COMMITMENT I hereby declare that: This is my study, which was conducted under the guidance of the supervisors; All data provided are true and accurate; All published data and information have been duly cited Hanoi, February 2021 Student Nguyen Bao Ngoc i ACKNOWLEDGEMENTS First of all, I would like to express my sincere gratitude to the Food Industries Research Institute (FIRI), especially to the Center for Industrial Microbiology for admitting and supporting me to conduct my thesis Besides, special thanks have to be given to the Department of Biotechnology, the Vietnam National University of Agriculture for teaching me the useful knowledge and experience to conduct this thesis Secondly, I am grateful to my supervisors Assoc Prof., Nguyen Van Giang for his priceless guidance and knowledge all the time I should also state my gratitude to my major Assoc Prof., Dr Vu Nguyen Thanh for allowing me to conduct my project in FIRI and providing me with the logistic support and his valuable suggestion to carry out my research successfully Above ground, I am indebted to my family for their love, caring, understanding, supporting and sacrifices for educating and my future Thank you very much! Nguyen Bao Ngoc ii TABLE OF CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi ABBREVIATIONS vi ABSTRACT viii INTRODUCTION LITERATURE REVIEW 2.1 Introduction of acidophilic fungi 2.1.1 Origin and characteristics of acidophilic fungi 2.1.2 Some representative group of acidophilic fungi Acidomyces acidophilus 2.2 Lignocellulose hydrolysis enzyme and enzyme from acid-tolerant fungi .10 2.2.1 Lignocellulose hydrolysis enzyme .10 2.2.2 Enzyme from acidophilic fungi 10 2.3 Research on acidophilic fungi in the world and in Vietnam 13 2.3.1 Research on acidophilic fungi in the world 13 2.3.2 Research on acidophilic fungi in Vietnam .15 MATERIALS AND METHODS .16 3.1 Materials 16 3.2 Chemicals, equipment and machines 16 3.2.1 Chemicals 16 3.2.2 Equipment .17 3.2.3 Media 18 3.3 Research methods 18 3.3.1 Method of isolation 18 3.3.2 Purification and maintenance of strains 18 3.3.3 Observation of colonies and cells .19 3.3.4 DNA extraction and purification method for mold cells 19 3.3.5 Methods of PCR fingerprinting (Maheshwari, 2011) 19 3.3.6 Electrophoresis method 20 iii 3.3.7 Staining the gel and read the result 20 3.3.8 Method to classify based on rDNA sequencing (Maheshwari, 2011) 20 3.3.9 Growth at different acid concentrations 21 3.3.10 Enzyme production and extraction (Maheshwari, 2011) 21 3.4.11 Determination Enzyme Activity Assays by agar diffusion method (Maheshwari, 2011) 22 3.3.12 Protein electrophoresis by SDS-PAGE method and Zymogram method SDS-PAGE method (Maheshwari, 2011) 23 RESULTS AND DISCUSSION 27 4.1 Isolation results 27 4.2 Observation of colonies and cells .28 4.3 PCR fingerprinting 30 4.4 Classification of acidophilic fungi strains based on rDNA sequence analysis .33 4.5 The growth of strains at different acid concentrations .36 4.6 Qualitative lignocellulose hydrolysis enzyme 38 4.6.1 Determination of starch hydrolysis and cellulose degradation by disk diffusion method 38 4.6.2 Determination of protein, CMCase and xylanase 40 CONCLUSION AND PROPOSAL 43 5.1 Conclusion 43 5.2 Proposal 43 REFERENCES 44 ANNEX 47 iv LIST OF TABLES Table The list of the acidophilic fungi, the fungi originally described as indigenous inhabitants of highly acidic habitats (pH < 3) (Hujslová et al., 2019) Table 2 An overview of applications of acidophilic fungal enzymes in various industries (Hassan et al., 2019) 11 Table Experimental content 16 Table Classification of the collected samples .28 Table Groups of PCR fingerprinting .32 Table The similarity between isolated strains and announced species 33 Table 4 The results of the CMC resolution 39 v LIST OF FIGURES Figure Extreme acidic environments Figure 2 Morphological features of the Acidomyces acidophilus WKC-1 Figure Acidothrixacidophila .7 Figure Acidea extrema Figure Microscopy of Hortaea acidophila, CBS 113389 Hyphae with annellated zones, and conidia Bar represents 10 µm Figure Some places collecting the samples 27 Figure Some pictures of isolation on Malt-Glucose 2Bx 1% H2SO4 medium agar plate 28 Figure Morphological characteristics of colonies and conidiophores on PDA (left) and Malt 2Bx pH1 (right) of strains AS 565-2, AS 612-3, ASS 3589 Bar, 10 µm .29 Figure 4 Electrophoresis images of PCR fingerprinting products of 67 strains .31 Figure Phylogenetic tree on the basis of their sequences 35 Figure The growth of strains on different acid concentration 37 Figure Resolution ring showing CMC hydrolysis capacity 40 Figure SDS-PAGE electrophoresis .41 vi ABBREVIATIONS CMC Carboxymethyl cellulose DNA Deoxyribonucleic Acid dNTPs Deoxyribonucleotide triphosphates PCR Polymerase Chain Reaction PDA Potato dextrose agar SDSPAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis TAE Tris-acetate-EDTA ITS Internal transcribed spacer vii ABSTRACT In the present work, we aimed to explore the biodiversity of acidophiles and acid- tolerance, especially fungi isolated in Vietnam Firstly, 103 different samples were collected to isolate acidic strains to be able to grow in extremely acidic conditions (pH 1.0) There are 109 strains were isolated and maintained before 20 representative strains were sequenced Strains belong to Acrodontium griseum, Aspergillus flavus, Aspergillus terreus, Aspergillus turcosus, Penicillium chermesinum, Penicillium citreonigrum, Penicillium georgiense, Talaromyces atroroseus, and Talaromyces diversus have been identified Two new species of Talaromyces and one new species of Penicillium also were detected By using untreated rice straw as the sole carbon source, some lignocellulolytic activities of 20 representative strains were determined Xylanase, CMCase and amylase were detected through the disk diffusion method, SDS-PAGE electrophoresis as well as zymogram electrophoresis Most of strains demonstrated strong CMCase, xylanase activities, meanwhile amylase activity was low viii INTRODUCTION All over the world, there are over 100,000 different species of fungi They exist at various extremes, including natural and man-made environments Fungi able to tolerate acidic conditions are frequently encountered in nature, and several species are capable of growing at very low pH levels There is no clear demarcation between acidophilic and acidophilic fungi, but it is often assumed that acidophilic fungi are those that can grow at pH 1.0 and have optimum growth at pH 3.0 or below In 1943, a strain of Acontium velatum and a “Fungus D” were shown to be capable of growing in a glucose medium containing 1.25 M sulphuric acid at pH Unfortunately, the strain of Acontium velatum appears to have been lost since the initial publication, but “Fungus D” is now believed to be a strain of Acidomyces acidophilus which is commonly found in extremely acidic environments According to Thanh et al (2019), acidophility has been shown for only fungal species, including Acidomyces acidophilus (=Scytalidium acidophilum = Acidomyces Acidomyces richmondensis = Fungus D), acidothermus, Acidothrix acidophila, Acidea extrema, Acontium velatum (no living specimen available) and Hortaea acidophila (=Neohortaea acidophila) Phylogenetically, all acidophilic species are Ascomycota, and the teleomorphic state is known only for Acidomyces acidothermus (described as Teratosphaeria acidotherma) However, studies on acidophilic species have not been published much, and the range of acidic tolarance in almost fungi remains a mystery Acidophilic fungi have received considerable attention, as their thermostable enzymes can be employed in industrial processes at elevated temperatures Increasing the process temperature can have advantages, for example, increasing the rate of chemical reactions, decreasing the viscosity of substrates and reducing the risk of contamination by mesophilic microorganisms For example, the strain Bispora sp MEY-1, well-known for the production of a range of thermophilic and acidophilic lignocellulolytic enzymes REFERENCES Aguilera, A et al (2007) ‘Distribution and seasonal variability in the benthic eukaryotic community of Rio Tinto (SW, Spain), an acidic, high metal extreme environment’, Systematic and Applied Microbiology, 30(7), pp 531–546 Aguilera, A and González-Toril, E (2019) ‘Eukaryotic Life in Extreme Environments: Acidophilic Fungi’, in Fungi in Extreme Environments: Ecological Role and Biotechnological Significance Springer, pp 21–38 Antranikian, G et al (2017) ‘Diversity of bacteria and archaea from two shallow marine hydrothermal vents from Vulcano Island’, Extremophiles, 21(4), pp 733–742 Baker, B J et al (2004) ‘Metabolically active eukaryotic communities in extremely acidic mine drainage’, Applied and environmental microbiology, 70(10), pp 6264– 6271 Brake, S S and Hasiotis, S T (2010) ‘Eukaryote-dominated biofilms and their significance in acidic environments’, Geomicrobiology Journal, 27(6–7), pp 534–558 Chan, W K et al (2019) ‘Acidomyces acidophilus: Ecology, Biochemical Properties and Application to Bioremediation’, in Fungi in Extreme Environments: Ecological Role and Biotechnological Significance Springer, pp 505–515 González-Toril, E et al (2015) ‘Pyrosequencing-based assessment of the microbial community structure of Pastoruri glacier area (Huascarán National Park, Perú), a natural extreme acidic environment’, Microbial ecology, 70(4), pp 936–947 Hamelinck, C N., Van Hooijdonk, G and Faaij, A P C (2005) ‘Ethanol from lignocellulosic biomass: techno-economic performance in short-, middle-and longterm’, Biomass and bioenergy, 28(4), pp 384–410 Hassan, N et al (2019) ‘Fungi in acidic fire: A potential source of industrially important enzymes’, Fungal Biology Reviews, 33(1), pp 58–71 10 Hölker, U et al (2004) ‘Hortaea acidophila, a new acid-tolerant black yeast from lignite’, Antonie van Leeuwenhoek, 86(4), pp 287–294 11 Hua, H et al (2014) ‘A thermostable glucoamylase from Bispora sp MEY-1 with stability over a broad pH range and significant starch hydrolysis capacity’, PLoS One, 9(11), p e113581 12 Huhndorf, S M et al (2009) ‘Amplistroma gen nov and its relation to Wallrothiella, two genera with globose ascospores and acrodontium-like anamorphs’, Mycologia, 101(6), pp 904–919 13 Hujslová, M et al (2014) ‘Three new genera of fungi from extremely acidic soils’, Mycological progress, 13(3), pp 819–831 14 Hujslová, M et al (2019) ‘Fungi, a neglected component of acidophilic biofilms: they have a potential for biotechnology?’, Extremophiles, 23(3), pp 267–275 15 Isobe, K and Yamada, M (2019) ‘β-Galactosidases from an Acidophilic Fungus, 44 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Teratosphaeria acidotherma AIU BGA-1’, in Fungi in Extreme Environments: Ecological Role and Biotechnological Significance Springer, pp 419–440 Kambura, A K et al (2016) ‘Bacteria and Archaea diversity within the hot springs of Lake Magadi and Little Magadi in Kenya’, BMC microbiology, 16(1), pp 1–12 Krueger, A et al (2018) ‘Towards a sustainable biobased industry–Highlighting the impact of extremophiles’, New biotechnology, 40, pp 144–153 Kurtzman, C P and Robnett, C J (1997) ‘Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5’end of the large-subunit (26S) ribosomal DNA gene.’, Journal of clinical microbiology, 35(5), pp 1216–1223 Li, X.-L et al (1993) ‘Purification and characterization of a new xylanase (APX-II) from the fungus Aureobasidium pullulans Y-2311-1.’, Applied and Environmental Microbiology, 59(10), pp 3212–3218 Lieckfeldt, E., Meyer, W and Börner, T (1993) ‘Rapid identification and differentiation of yeasts by DNA and PCR fingerprinting’, Journal of Basic Microbiology, 33(6), pp 413–425 Luo, H et al (2009) ‘A thermophilic and acid stable family-10 xylanase from the acidophilic fungus Bispora sp MEY-1’, Extremophiles, 13(5), pp 849–857 Maheshwari, R., Bharadwaj, G and Bhat, M K (2000) ‘Thermophilic fungi: their physiology and enzymes’, Microbiology and molecular biology reviews, 64(3), pp 461– 488 Ohta, K et al (2001) ‘Purification and characterization of an acidophilic xylanase from Aureobasidium pullulans var melanigenum and sequence analysis of the encoding gene’, Journal of bioscience and bioengineering, 92(3), pp 262–270 Pandey, A., Soccol, C R and Mitchell, D (2000) ‘New developments in solid state fermentation: I-bioprocesses and products’, Process biochemistry, 35(10), pp 1153– 1169 Rothschild, L J and Mancinelli, R L (2001) ‘Life in extreme environments’, Nature, 409(6823), pp 1092–1101 Schulze-Makuch, D., Airo, A and Schirmack, J (2017) ‘The adaptability of life on Earth and the diversity of planetary habitats’, Frontiers in microbiology, 8, p 2011 Selbmann, L et al (2008) ‘Drought meets acid: three new genera in a dothidealean clade of extremotolerant fungi’, Studies in mycology, 61, pp 1–20 Sigler, L and Carmichael, J W (1974) ‘A new acidophilic Scytalidium’, Canadian Journal of Microbiology, 20(2), pp 267–268 Skorupa, D J et al (2013) ‘Cyanidiales diversity in Yellowstone national park’, Letters in applied microbiology, 57(5), pp 459–466 Sletten, O and Skinner, C E (1948) ‘Fungi capable of growing in strongly acid media and in concentrated copper sulfate solutions’, Journal of bacteriology, 56(5), p 679 Starkey, R L and Waksman, S A (1943) ‘Fungi tolerant to extreme acidity and high concentrations of copper sulfate’, Journal of Bacteriology, 45(5), p 509 45 32 Thanh, V N et al (2019) ‘Surveying of acid-tolerant thermophilic lignocellulolytic fungi in Vietnam reveals surprisingly high genetic diversity’, Scientific Reports, 9(1), p 3674 doi: 10.1038/s41598-019-40213-5 33 Tiquia-Arashiro, S and Rodrigues, D F (2016) Extremophiles: applications in nanotechnology Springer 34 Tiquia, S M and Mormile, M R (2010) ‘Extremophiles–a source of innovation for industrial and environmental applications’ Taylor & Francis 35 White, T J et al (1990) ‘Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics’, PCR protocols: a guide to methods and applications, 18(1), pp 315–322 36 Zettler, L A A et al (2002) ‘Eukaryotic diversity in Spain’s River of Fire’, Nature, 417(6885), p 137 46 ANNEX Annex List of isolated results of 109 strains No Strain Place of collection Method of classification Species (Latin name) ASS 326-1 Sông Công, Thái Nguyên rDNA sequencing Penicillium sp1 ASS 327-1 Sông Công, Thái Nguyên Colony morphology Talaromyces sp1 ASS 328-3 Tân Ước, Thanh Oai, Hà Nội rDNA sequencing Talaromyces sp2 ASS 328-4 Tân Ước, Thanh Oai, Hà Nội Fingerprinting Talaromyces sp1 ASS 330-2 Tân Ước, Thanh Oai, Hà Nội Colony morphology Talaromyces sp1 ASS 330-5 Tân Ước, Thanh Oai, Hà Nội Colony morphology Penicillium citreonigrum ASS 332-3 Tân Ước, Thanh Oai, Hà Nội Fingerprinting Talaromyces sp1 ASS 332-5 Tân Ước, Thanh Oai, Hà Nội Colony morphology Talaromyces atroroseus ASS 333-2 Tân Ước, Thanh Oai, Hà Nội rDNA sequencing Aspergillus flavus 10 ASS 335-2 Tân Ước, Thanh Oai, Hà Nội Fingerprinting Aspergillus turcosus 11 ASS 336-1 Tân Ước, Thanh Oai, Hà Nội Fingerprinting Penicillium citreonigrum 12 ASS 338-1 Tân Ước, Thanh Oai, Hà Nội rDNA sequencing Penicillium citreonigrum 13 ASS 342-2 Tân Ước, Thanh Oai, Hà Nội rDNA sequencing Talaromyces diversus 14 ASS 342-4 Tân Ước, Thanh Oai, Hà Nội Fingerprinting Talaromyces sp1 15 ASS 343-4 Sông Công, Thái Nguyên rDNA sequencing Talaromyces sp1 16 ASS 344-1 Trâu Quỳ, Gia Lâm, Hà Nội Fingerprinting Talaromyces sp1 17 ASS 345-1 Văn Lâm, Hưng Yên Colony morphology Talaromyces sp1 18 ASS 346-1 Long Biên, Hà Nội Colony morphology Talaromyces atroroseus 19 ASS 346-2 Long Biên, Hà Nội Fingerprinting Talaromyces diversus 20 ASS 346-3 Long Biên, Hà Nội Fingerprinting Talaromyces diversus 21 ASS 346-4 Long Biên, Hà Nội Fingerprinting Talaromyces sp1 22 ASS 346-7 Long Biên, Hà Nội Fingerprinting Talaromyces atroroseus 23 ASS 347-3 Ninh Giang, Hải Dương Colony morphology Talaromyces sp1 24 ASS 348-1 Đông Anh, Hà Nội Colony morphology Penicillium citreonigrum 25 ASS 348-2 Đông Anh, Hà Nội rDNA sequencing Talaromyces sp1 26 ASS 348-3 Đông Anh, Hà Nội rDNA sequencing Penicillium sp2 27 ASS 348-4 Đông Anh, Hà Nội Colony morphology Talaromyces diversus 28 ASS 348-5 Đông Anh, Hà Nội Colony morphology Talaromyces sp1 29 ASS 350-1 Đông Anh, Hà Nội rDNA sequencing Aspergillus turcosus 30 ASS 350-3 Đông Anh, Hà Nội Fingerprinting Talaromyces sp2 31 ASS 351-2 Đông Anh, Hà Nội Colony morphology Talaromyces sp1 32 ASS 351-4 Đông Anh, Hà Nội Fingerprinting Talaromyces sp1 33 ASS 353-1 Đơng Hưng, Thái Bình Colony morphology Talaromyces sp1 34 ASS 353-3 Đơng Hưng, Thái Bình Colony morphology Talaromyces sp1 35 ASS 353-7 Đơng Hưng, Thái Bình Fingerprinting Talaromyces sp1 47 36 ASS 353-8 Đơng Hưng, Thái Bình Colony morphology Talaromyces sp1 37 ASS 353-9 Đơng Hưng, Thái Bình Fingerprinting Talaromyces sp1 38 ASS 354-1 Thanh Xuân, Hà Nội Fingerprinting Talaromyces sp1 39 ASS 354-2 Thanh Xuân, Hà Nội Colony morphology Talaromyces sp1 40 ASS 354-6 Thanh Xuân, Hà Nội Fingerprinting Talaromyces sp2 41 ASS 354-7 Thanh Xuân, Hà Nội Colony morphology Talaromyces sp1 42 ASS 355-1 Đông Hưng, Thái Bình Colony morphology Aspergillus turcosus 43 ASS 355-2 Đơng Hưng, Thái Bình Fingerprinting Talaromyces sp1 44 ASS 355-3 Đơng Hưng, Thái Bình Colony morphology Talaromyces sp1 45 ASS 355-4 Đơng Hưng, Thái Bình Fingerprinting Talaromyces sp1 46 ASS 356-2 Thanh Xuân, Hà Nội Colony morphology Talaromyces sp1 47 ASS 356-3 Thanh Xuân, Hà Nội Fingerprinting Talaromyces sp1 48 ASS 356-4 Thanh Xuân, Hà Nội Colony morphology Talaromyces sp1 49 ASS 357-1 Thanh Xuân, Hà Nội rDNA sequencing Talaromyces sp1 50 ASS 358-1 Mai Sơn, Sơn La Colony morphology Talaromyces sp1 51 ASS 358-10 Mai Sơn, Sơn La Colony morphology Talaromyces sp1 52 ASS 358-2 Mai Sơn, Sơn La Colony morphology Talaromyces sp1 53 ASS 358-3 Mai Sơn, Sơn La Colony morphology Talaromyces sp1 54 ASS 358-7 Mai Sơn, Sơn La Fingerprinting Talaromyces sp1 55 ASS 358-8 Mai Sơn, Sơn La Fingerprinting Talaromyces sp1 56 ASS 358-9 Mai Sơn, Sơn La rDNA sequencing Talaromyces sp1 57 ASS 359-1 Mai Sơn, Sơn La Fingerprinting Talaromyces sp2 58 ASS 359-4 Mai Sơn, Sơn La Fingerprinting Talaromyces sp1 59 ASS 360-2 Ứng Hòa, Hà Nội rDNA sequencing Talaromyces diversus 60 AS 564-3 Tân Ước, Thanh Oai, Hà Nội Fingerprinting Talaromyces diversus 61 AS 565-2 Tân Ước, Thanh Oai, Hà Nội rDNA sequencing Talaromyces diversus 62 AS 572-1 Sông Công, Thái Nguyên Colony morphology Talaromyces sp1 63 AS 581-2 Hà Đông, Hà Nội rDNA sequencing Penicillium chermesinum 64 AS 593-1 Sông Công, Thái Nguyên Colony morphology Penicillium chermesinum 65 AS 596-1 Sông Công, Thái Nguyên Colony morphology Talaromyces sp2 66 AS 600-4 Thanh Xuân, Hà Nội Colony morphology Penicillium chermesinum 67 AS 601-1 Thanh Xuân, Hà Nội Colony morphology Talaromyces atroroseus 68 AS 604-1 Thanh Xuân, Hà Nội Colony morphology Talaromyces atroroseus 69 AS 605-1 Thanh Xuân, Hà Nội Colony morphology Penicillium chermesinum 70 AS 606-1 Thanh Xuân, Hà Nội Fingerprinting Talaromyces atroroseus 71 AS 606-2 Thanh Xuân, Hà Nội Fingerprinting Penicillium chermesinum 72 AS 607-1 Thanh Xuân, Hà Nội Fingerprinting Penicillium chermesinum 73 AS 607-4 Thanh Xuân, Hà Nội Fingerprinting Penicillium chermesinum 74 AS 607-6 Thanh Xuân, Hà Nội rDNA sequencing Talaromyces diversus 48 75 AS 607-8 Thanh Xuân, Hà Nội Fingerprinting Penicillium chermesinum 76 AS 607-9 Thanh Xuân, Hà Nội Fingerprinting Talaromyces atroroseus 77 AS 609-3 Thanh Xuân, Hà Nội rDNA sequencing Aspergillus terreus 78 AS 612-1 Thanh Xuân, Hà Nội rDNA sequencing Acrodontium griseum 79 AS 612-3 Thanh Xuân, Hà Nội rDNA sequencing Talaromyces atroroseus 80 AS 613-2 Thanh Xuân, Hà Nội Colony morphology Talaromyces atroroseus 81 AS 614-1 Thanh Xuân, Hà Nội Fingerprinting Talaromyces sp1 82 AS 614-2 Thanh Xuân, Hà Nội rDNA sequencing Penicillium georgiense 83 AS 615-1 Thanh Xuân, Hà Nội Fingerprinting Talaromyces sp1 84 AS 615-2 Thanh Xuân, Hà Nội Colony morphology Talaromyces sp1 85 AS 615-5 Thanh Xuân, Hà Nội Fingerprinting Talaromyces sp1 86 AS 615-6 Thanh Xuân, Hà Nội Fingerprinting Talaromyces sp1 87 AS 616-1 Đơng Hưng, Thái Bình Colony morphology Talaromyces sp1 88 AS 616-2 Đơng Hưng, Thái Bình Colony morphology Talaromyces sp1 89 AS 616-3 Đơng Hưng, Thái Bình rDNA sequencing Talaromyces sp1 90 AS 616-6 Đơng Hưng, Thái Bình Fingerprinting Talaromyces sp1 91 AS 619-1 Thanh Xuân, Hà Nội Fingerprinting Talaromyces sp1 92 AS 619-2 Thanh Xuân, Hà Nội Colony morphology Talaromyces sp2 93 AS 619-3 Thanh Xuân, Hà Nội Colony morphology Talaromyces sp2 94 AS 620-1 Thanh Xuân, Hà Nội Colony morphology Talaromyces sp1 95 AS 620-2 Thanh Xuân, Hà Nội Fingerprinting Talaromyces sp2 96 AS 620-3 Thanh Xuân, Hà Nội Fingerprinting Talaromyces sp1 97 AS 620-4 Thanh Xuân, Hà Nội Colony morphology Penicillium chermesinum 98 AS 620-5 Thanh Xuân, Hà Nội Fingerprinting Talaromyces sp1 99 AS 623-1 Thanh Xuân, Hà Nội Colony morphology Talaromyces sp1 100 AS 625-3 Gia Lâm, Hà Nội Fingerprinting Talaromyces atroroseus 101 AS 627-1 Thanh Xuân, Hà Nội Colony morphology Talaromyces sp1 102 AS 627-2 Thanh Xuân, Hà Nội Colony morphology Talaromyces sp1 103 AS 627-3 Thanh Xuân, Hà Nội Fingerprinting Talaromyces sp1 104 AS 632-2 Gia Lâm, Hà Nội Colony morphology Penicillium citreonigrum 105 AS 633-3 Sông Công, Thái Nguyên Fingerprinting Penicillium chermesinum 106 AS 634-3 Gia Lâm, Hà Nội Fingerprinting Talaromyces atroroseus 107 AS 636-2 Gia Lâm, Hà Nội Colony morphology Talaromyces sp1 108 AS 637-1 Sông Công, Thái Nguyên Fingerprinting Talaromyces sp1 109 AS 637-2 Sông Công, Thái Nguyên Fingerprinting Talaromyces diversus 49 Annex Test the growth of strains on Malt 2Bx % H2SO4 (pH 1.0) medium No Symbol strains Ability of growth ASS 326-1 w ASS 327-1 w ASS 328-3 + ASS 328-4 + ASS 330-2 w ASS 330-5 w ASS 332-3 + ASS 332-5 + ASS 333-2 - 10 ASS 335-2 - 11 ASS 336-1 - 12 ASS 338-1 + 13 ASS 342-2 + 14 ASS 342-4 + 15 ASS 343-4 + 16 ASS 344-1 + 17 ASS 345-1 + 18 ASS 346-1 + 19 ASS 346-2 + 20 ASS 346-3 + 21 ASS 346-4 + 22 ASS 346-7 + 23 ASS 347-3 + 24 ASS 348-1 + 25 ASS 348-2 + 26 ASS 348-3 + 27 ASS 348-4 + 28 ASS 348-5 + 29 ASS 350-1 + 30 ASS 350-3 + 31 ASS 351-2 + 32 ASS 351-4 + 33 ASS 353-1 + 34 ASS 353-3 + 35 ASS 353-7 + 36 ASS 353-8 + 37 ASS 353-9 + 50 38 ASS 354-1 + 39 ASS 354-2 + 40 ASS 354-6 + 41 ASS 354-7 + 42 ASS 355-1 + 43 ASS 355-2 + 44 ASS 355-3 + 45 ASS 355-4 + 46 ASS 356-2 + 47 ASS 356-3 + 48 ASS 356-4 + 49 ASS 357-1 + 50 ASS 358-1 + 51 ASS 358-10 + 52 ASS 358-2 + 53 ASS 358-3 + 54 ASS 358-7 + 55 ASS 358-8 + 56 ASS 358-9 + 57 ASS 359-1 + 58 ASS 359-4 + 59 ASS 360-2 w 60 AS 564-3 - 61 AS 565-2 + 62 AS 572-1 + 63 AS 581-2 + 64 AS 593-1 + 65 AS 596-1 + 66 AS 600-4 - 67 AS 601-1 + 68 AS 604-1 + 69 AS 605-1 + 70 AS 606-1 + 71 AS 606-2 - 72 AS 607-1 + 73 AS 607-4 - 74 AS 607-6 + 75 AS 607-8 - 76 AS 607-9 - 51 77 AS 609-3 - 78 AS 612-1 w 79 AS 612-3 - 80 AS 613-2 + 81 AS 614-1 + 82 AS 614-2 + 83 AS 615-1 + 84 AS 615-2 + 85 AS 615-5 + 86 AS 615-6 + 87 AS 616-1 + 88 AS 616-2 + 89 AS 616-3 + 90 AS 616-6 + 91 AS 619-1 + 92 AS 619-2 + 93 AS 619-3 + 94 AS 620-1 + 95 AS 620-2 + 96 AS 620-3 + 97 AS 620-4 w 98 AS 620-5 + 99 AS 623-1 + 100 AS 625-3 - 101 AS 627-1 + 102 AS 627-2 + 103 AS 627-3 + 104 AS 632-2 + 105 AS 633-3 + 106 AS 634-3 + 107 AS 636-2 + 108 AS 637-1 + 109 AS 637-2 + Note: (+) positive (-) negative (w) weak 52 Annex The sequence of ITS1 – NL4 of the isolated strains ASS 343-4 ITS1-NL4 ITS4 VT3294 Talaromyces diversus 98.85% CCGTGGAAAATGTTGAGGGATGACCAACGCCCGCAGGCCCCTCCCGAGCGGGTGACAAAGCCCCATACGCTCGAGGAC CTAGGCGGGCGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCCGGGAAGGGGGGGGACCACACCCAACACACAAGCCGTG CTTGAGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTTCAAAGATTCGAT GATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCA TTGTTGAAAGTTTTGATTATTATCAAGACACTCAGACAGTCCATCTTCATAAGGGTTCACAGAGCGCTCTGGCGGGCG CGGGCCCGGGGGCGAACACGCCCCCCGGCGACCGGGGTGGCCCCGGTGGGCCCGCCAAAGCAACAGGGTATACAAGAG ACACGGGTGGGAGGTTGGACCCGCGAGGGGTCCGCACTCGGTAATGATCCTTCCGCAGGTT ASS 358-9 ITS1-NL4 ITS4 VT3295 Talaromyces diversus 98.87% CCGAGGTCAACCGTGGAAAATGTTGAGGGATGACCAACGCCCGCAGGCCCCTCCCGAGCGGGTGACAAAGCCCCATAC GCTCGAGGACCTAGGCGGGCGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCCGGGAAGGGGGGGGACCACACCCAACAC ACAAGCCGTGCTTGAGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTTCA AAGATTCGATGATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACC AAGAGATCCATTGTTGAAAGTTTTGATTATTATCAAGACACTCAGACAGTCCATCTTCATAAGGGTTCACAGAGCGCT CTGGCGGGCGCGGGCCCGGGGGCGAACACGCCCCCCGGCGACCGGGGTGGCCCCGGTGGGCCCGCCAAAGCAACAGGG TATACAAGAGACACGGGTGGGAGGTTGGACCCGCGAGGGGTCCGCACTCGGTAATGATCCTTCCGCAGGTTCA ASS 338-1 ITS1-NL4 ITS4 VT3296 Penicillium citreonigrum 99.61% CCTGGAAAAAAGATTGATGTGTCGGCAAGCGCCGGCCGGGCCTACAGAGCGAGTGACAAAGCCCCATACGCTCGAGGA CCGGACGCGGTGCCGCCGCTGCCTTTCGGGCCCGTCCCCCGGGAGGAGGACGGAGCCCAACACACAAGCCGTGCTTGA GGGCAGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGGGGGCGCAATGTGCGTTCAAAGACTCGATGATTC ACTGAATTCTGCAATTCACATTACGTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCGTTGTT GAAAGTTTTAAGTTATTTAACTAATTGCTCAGACTGCAATCTTCAGACAGCGTTCAATGGTGTCTTCGGCGGGCGCGG GCCAGAGGGCAGATGCCCCCCGGCGGCCTTGCGGCGGGCCCGCCGAAGCAACAAGGTACGATAAACACGGGTGGGAGG TTGGACCCAGAGGGCCCTCACTCGGTAATGATCCTTCCGCA ASS 350-1 ITS1-NL4 ITS4 VT3297 Aspergillus turcosus 99.82% CCGAGGTCAACCTTAGAAAATAAAGTTGGGGGGTCGGCTGGCGCCGGCCAGGCCTACAGAGCGGGTGACAAAGCCCCA TACGCTCGAGGACCGGACGCGGTGCCGCCGCTGCCTTTCGGGCCCGTCCCCGGGGAGAACCGGGGACGGGGGCCCAAC ACACAAGCCGTGCTTGAGGGCAGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGGGGGCGCAATGTGCGTT CAAAGACTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAA CCAAGAGATCCGTTGTTGAAAGTTTTAACTGATTATGGTAATCAAACTCAGACTGCATACTTTCAGAACAGCGTTCAT GTTGGGGTCTTCGGCGGGCGCGGGCCCGGGGGCGCGAGGGCCTCCCCGGCGGCCGTTCGAGAACGGCGGGCCCGCCGA AGCAACAAGGTACAATAGACACGGGTGGGAGGTTGGACCCAGAGGGCCCGCACTCGGTAATGATCCTTCCGCA AS 607-6 ITS1-NL4 ITS4 VT3298 53 Talaromyces diversus 100% CCGTGGAAAATGTTGAGGGATGACCAACGCCCGCAGGCCCCTCCCGAGCGGGTGACAAAGCCCCATACGCTCGAGGAC CTAGGCGGGCGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCCGGGATGGGGGGGACCACACCCAACACACAAGCCGTGC 54 TTGAGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTTCAAAGATTCGATG ATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCAT TGTTGAAAGTTTTGATTATTATCAAGACACTCAGACAGTCCATCTTCATAAGGGTTCACAGAGCGCTCTGGCGGGCGC GGGCCCGGGGGCGTGTTCGCCCCCCGGCGACCGGGGTGGCCCCGGTGGGCCCGCCAAAGCAACAGGGTACACAAGAGA CACGGGTGGGAGGTTGGACCCGCGAGGGGTCCGCACTCGGTAATGATCCTTCCGCAGGT ASS 342-2 ITS1-NL4 ITS4 VT3299 Talaromyces diversus 99.62% CGTGGAAAATGTTGAGGGATGACCAACGCCCGCAGGCCCCTCCCGAGCGGGTGACAAAGCCCCATACGCTCGAGGACC TAGGCGGGCGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCGGGATGGGGGGGACCACACCCAACACACAAGCCGTGCTT GAGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTTCAAAGATTCGATGAT TCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCATTG TTGAAAGTTTTGATTATTATCAAGACACTCAGACAGTCCATCTTCATAAGGGTTCACAGAGCGCTCTGGCGGGCGCGG GCCCGGGGGCGTGTTCGCCCCCCGGCGACCGGGGTGGCCCCGGTGGGCCCGCCAAAGCAACAGGGTACACAAAGAGAC ACGGGTGGGAGGTTGGACCCGCGAGGGGTCCGCACTCGGTAATGATCCTTCCGCAGGTTCACC AS 581-2 ITS1-NL4 ITS4 VT3300 Penicillium chermesinum strain CBS 117279 MT309662.1 99.81% CCGAGGTCAACCTGAAAAAAGGGGTTGAGGGGGTCGGCTGGCGCCGGCCGGGCCTGCGGAGCGGGTGACAGAGCCCCA TACGCTCGAGGACCGGACGCGGTGCCGCCGCTGCCTTTCGGGCCCGTCCCCGGGGGGACGGCGCCCAACACACAAGCC GGGCTTGAGGGCAGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGGGGGCGCAATGTGCGTTCAAAGACTC GATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGAT CCGTTGTTGAAAGTTTTAACTAATTTATGCGTATCGCTCAGACTGCAATCTTCAGACAGCGTTCAATGATGTCTTCGG CGGGCGCGGGCCCGGGGGCAGGTGCCCCCCGGCGGCCAGGCTGGCGGGCCCGCCGAAGCAACAAGGTACGGTATACAC GGGTGGGAGGTTGGACCCAGAGGGCCCGCACTCAGTAATGATCCTTCCGCAGGTTCACCTA ASS 348-2 ITS1-NL4 ITS4 VT3301 Talaromyces diversus isolate 6035 KX363453.1 99.03% GAAAAATGTTGAGGGATGACCAACGCCCGCAGGCCCCTCCCGAGCGGGTGACAAAGCCCCATACGCTCGAGGACCTAG GCGGGCGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCCGGGAAGGGGGGGGACCACACCCAACACACAAGCCGTGCTTG AGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTTCAAAGATTCGATGATT CACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCATTGT TGAAAGTTTTGATTATTATCAAGACACTCAGACAGTCCATCTTCATAAGGGTTCACAGAGCGCTCTGGCGGGCGCGGG CCCGGGGGCGAACACGCCCCCCGGCGACCGGGGTGGCCCCGGTGGGCCCGCCAAAGCAACAGGGTATACAAGAGACAC GGGTGGGAGGTTGGACCCGCGAGGGGTCCGCACTCGGTAATGATCCTTCCGCAGGTTCACCT AS 565-2 ITS1-NL4 ITS4 VT3302 Penicillium sp clone EF4 MF927536.1 100% GGTCAACCGTGGAAAAATGTTGAGGGATGACCAACGCCCGCAGGCCCCTCCCGAGCGGGTGACAAAGCCCCATACGCT CGAGGACCTAGGCGGGCGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCGGGATGGGGGGGACCACACCCAACACACAAG CCGTGCTTGAGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTTCAAAGAT TCGATGATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAG 55 ATCCATTGTTGAAAGTTTTGATTATTATCAAGACACTCAGACAGTCCATCTTCATAAGGGTTCACAGAGCGCTCTGGC GGGCGCGGGCCCGGGGGCGTGTTCGCCCCCCGGCGACCGGGGTGGCCCCGGTGGGCCCGCCAAAGCAACAGGGTACAC AAAGAGACACGGGTGGGAGGTTGGACCCGCGAGGGGTCCGCACTCGGTAATGATCCTTCCGCAGGTTCACCTA ASS 348-3 ITS1-NL4 ITS4 VT3303 Penicillium sp MMW-2015 strain OUCMBI110092 KP269004.1 99.82% TCCGAGGTCAACCGTGGAATAAATTCGTGAGGTGACCAACGCTCCGCGGGTCCATCCCGAGCGAGTGACAAAGCCCCA TACGCTCGAGGACCTGGGCGGGAACGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCGTTTCGGAGGGACCAGCGCCCAA CACACAAGCCGTGCTTGAGGGCAGAAATGACGCTCGGACAGGCATGCTCCCCGGAATGCCAGGGAGCGCAATGTGCGT TCAAAGATTCGATGATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGA ACCAAGAGATCCATTGTTGAAAGTTTTGATTATTATCAACGTTGCTCAGACAGTCCATCTTCACAAAGGGTTCAAGGG GCGCTCCGGCGGGCACGGGCCCGAGGACAGAGGCGTCCTCCGGCGACCGGGAGACCCGGTGGGCCCGCCAAAGCAACA CGGTATTCAAATAGACACGGGTGGGAGGTTGGACCGCGAGGGGTCCGCACTCGGTAATGATCCTTCCGCAGGTTCACC CTACG AS 609-3 ITS1-NL4 ITS4 VT3304 Aspergillus terreus 100% CCTGGAAAAAACAAGTTGCAAATAAATGCGTCGGCGGGCGCCGGCCGGGCCTACGGAGCGGAAGACGAAGCCCCATAC GCTCGAGGACCGGACGCGGTGCCGCCGCTGCCTTTCGGGCCCGTCCCCCGGGAGCCGGGGGACGAGGGCCCAACACAC AAGCCGGGCTTGAGGGCAGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGGGGGCGCAATGTGCGTTCAAA GACTCGATGATTCACTGAATTCTGCAATTCACATTAGTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAA GAGATCCATTGTTGAAAGTTTTAACTGATTGCAAAGAATCACACTCAGACTGCAAGCTTTCAGAACAGGGTTCATGTT GGGGTCTCCGGCGGGCACGGGCCCGGGGGCGAGTCGCCCCCCGGCGGCCAGCAACGCTGGCGGGCCCGCCGAAGCAAC AAGGTACAATAGTCACGGGTGGGAGGTTGGGCCATAAAGACCCGCACTCGGTAATGATCCTTCCGCAGG AS 612-1 ITS1-NL4 ITS4 VT3305 Acrodontium griseum strain CBS 554.90 MH873918.1 99.51% ATCAGGCGGCCTGACGAGCAGGCCTATGTTGGGGGTTTTACGGCTGGCACCCCGGCGCAGGTGCCCCTAAGCAGCTCA ACGTGAGAAGTTTACTGTGCTTGGGTCCTGATGCGAGGGCCGCGCAATGGTTTTGGGGGCGTAGGAAGCCTACTGCCC CAACACCGAGCACCACCTCCCCGGTGTGGAGAGGCGGGCTCGAGCGGTGTAATCGACGCTCGGACGGGCATGCCCGCC AGAGAGCTGGCGGGCGCTATGTGCGTTCAAAGATTCGATGACTCACTGGAGATCTGCAATTCGCATTACGTATCGCAT TTCGCTGCGTTCTTCATCGATGCCGGAGCCAAGAGATCCGTTGTTGCAGGTTGTAATGGGTTTTGATATTTTATATTG TTTGTTCTTTTGCAGTGGTATAACCCACTTACAATGCTTTTTAGTTTAGGCGCCACCGGCGGCGCCCGCCGCGCCGCG GGTTGCGGCTTAGCGAGCAGAGGCCCCCCCTGGGGCCGCCGCCGAGGCAGAGACATAAGTTTTCCGGCTAGGTACGCG GTATGGTTAAGAACAGTAGGTGGTCTCTCGACCCTCTAATGATCCCTCCGCAGGTTCACCTAC AS 614-2 ITS1-NL4 ITS4 VT3306 Penicillium georgiense isolate SRG7 MK748315.1 99.8% CATAAGAAGTTGTGCTTGAAGGGCAAGCGACCGGCCGGCCTACAAAGCGGGTGACAAAGCCCCATACGCTCGAGGACC GGACGCGGTGCCGCCATTGCATTTGAGGCCTGTCCCCCCGGAACGGAGGGACATAACCCAAGGACAAGCCGGGCTTGA GGGGTGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGGGGGCGCAATGTGCGTTCAAAGACTCGATGATTC ACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCGTTGTT 56 GAAAGTTTTAACTGATTAAAATTCCACTCAGACAACACTCACAGACAGAGTTCCAGGGTGTCTTCGGCGGGCGCACGC CCGGGGACAGCGTCCCCCGGCGGCCTGACAGCAGGCGGGCCCACCGAAGCAACAAGGTACAATAGACACGGGTGGGAG GTTAGACCCAGAGGGCCTGCACTCATTAATGATCCTTCCGCAG ASS 326-1 ITS1-NL4 ITS4 VT3307 Penicillium sp isolate FUN0092 MT832996.1 99.04% GAAAAAAGAAGGTTGGAGAGTCGGCAGACTGTCGGCCGTTCCTACCAGAGCGGAGTGACGAAGCCCCATACGCTCGAG GACCGGACGCGAAGTCGCCGCTGCCTTTCGGGCCCGTCCCCCCCGGGAAAGGAGGGGACGGCGACCCAACACACAAGC CGGGCTTGAGGGCAGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGGGGGCGCAATGTGCGTTCGAAGACT CGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGA TCCGTTGTTGAAAGTTTTTGATTGATTTGGAAAAAATCACTCAGACTGCAATCTTCAATAGAAGCGTTCACAGGTGTC TTCGGCGGGCGCGGGCCCGGAGGCCCAAGGGGCCATCCCGGCGGCCGCTGAGGGCGGGCCCGCCGAAGCAACAGGGTA TGGTAAACACGGGTGGGAGGTTGGACCCAGAGGGCCCTCACTCGGTAATGATCCTTCCGCAGGTTCACCTACGGA AS 612-3 ITS1-NL4 ITS4 VT3308 Talaromyces atroroseus strain DTO 390-I4 MN788119.1 99.82% CCGAGGTCAACGGTAAAAAAATATCGTGGGTGGCCAACGCCCGCAGGCCCCTCCCGAGCGGGTGACAAAGCCCCATAC GCTCGAGGACCTAGACGGGACGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCAACACCGGGGGGACCACACCCAACACA CAAGCCGCGCTTGAGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTTCAA AGATTCGATGATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCA AGAGATCCATTGTTGAAAGTTTTGATAATTCAAAATCACACTCAGACAGCCCATCTTCATTAGGGTTCACAGAGCGCT CTGGCGGGCGCAGGCCCGGGGGCGCAGTGCCCCCCGGCGACCGGGGCGACCCGGTGGGCCCGCCAAAGCAACAGGGTA TTCAAGAGACACGGGTGGGAGGTTGGACCCGCGAGGGGTCCGCACTCAGTAATGATCCTTCCGCAGGTTCACCCTACG G ASS 328-3 ITS1-NL4 ITS4 VT3309 Talaromyces diversus isolate GM73 MN108302.1 98.01% CGAGGTCAACCGTGGAAAAATGATGAGGGATGACCAACGCCCGCAGGCCCCTCCCGAGCGGGTGACAAAGCCCCATAC GCTCGAGGACCTAGGCGGGCGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCGGGGAAGGGGGGACCACACCCAACACAC AAGCCGTGCTTGAGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTTCAAA GATTCGATGATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAA GAGATCCATTGTTGAAAGTTTTGATTATTATCAAGACACTCAGACAGTCCATCTTCATAAGGGTTCACAGAGCGCTCT GGCGGGCGCGGGCCCGGGGGCGAACACGCCCCCCGGCGACCGGGGTGACCCCGGTGGGCCCGCCAAAGCAACAGGGTA GACAAGAGACACGGGTGGGAGGTTGGACCCGCGAGGGGGTCCGCACTCGGTAATGATCCTTCCGCAGGTTCACCTACG GA ASS 357-1 ITS1-NL4 ITS4 VT3310 Talaromyces diversus isolate 6035 KX363453.1 99.05% GAGGTCAACCGTGGAAAAATGTTGAGGGATGACCAACGCCCGCAGGCCCCTCCCGAGCGGGTGACAAAGCCCCATACG CTCGAGGACCTAGGCGGGCGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCCGGGAAGGGGGGGGACCACACCCAACACA CAAGCCGTGCTTGAGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTTCAA AGATTCGATGATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCA 57 AGAGATCCATTGTTGAAAGTTTTGATTATTATCAAGACACTCAGACAGTCCATCTTCATAAGGGTTCACAGAGCGCTC TGGCGGGCGCGGGCCCGGGGGCGAACACGCCCCCCGGCGACCGGGGTGGCCCCGGTGGGCCCGCCAAAGCAACAGGGT ATACAAGAGACACGGGTGGGAGGTTGGACCCGCGAGGGGTCCGCACTCGGTAATGATCCTTCCGCAGGTTCACCTACG G ASS 360-2 ITS1-NL4 ITS4 VT3311 Talaromyces diversus isolate 6035 KX363453.1 99.23% TGGAAAATGTTGAGGGATGACCAACGCCCGCAGGCCCCTCCCGAGCGAGTGACAAAGCCCCATACGCTCGAGGACCTA GGCGGGCGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCCCGGGATGGGGGGGGGACCACACCCAACACACAAGCCGTGC TTGAGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTTCAAAGATTCGATG ATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCAT TGTTGAAAGTTTTGATTATTATCAAGACACTCAGACAGTCCATCTTCATAAGGGTTCACAGAGCGCTCTGGCGGGCGC GGGCCCGGGGGCGTGATCGCCCCCCGGCGACCGGGGTGGCCCCGGTGGGCCCGCCAAAGCAACAGGGTATACAAGAGA CACGGGTGGGAGGTTGGACCCGCGAGGGGTCCGCACTCGGTAATGATCCTTCCGCAGGTTCAC ASS 333-2 ITS1-NL4 ITS4 VT3312 Aspergillus flavus clone SF_591 MT529867.1 100% CCTGGAAAAGATTGATTTGCGTTCGGCAAGCGCCGGCCGGGCCTACAGAGCGGGTGACAAAGCCCCATACGCTCGAGG ATCGGACGCGGTGCCGCCGCTGCCTTTGGGGCCCGTCCCCCCCGGAGAGGGGACGACGACCCAACACACAAGCCGTGC TTGATGGGCAGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGGGGGCGCAATGTGCGTTCAAAGACTCGAT GATTCACGGAATTCTGCAATTCACACTAGTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCA TTGTTGAAAGTTTTAACTGATTGCGATACAATCAACTCAGACTTCACTAGATCAGACAGAGTTCGTGGTGTCTCCGGC GGGCGCGGGCCCGGGGCTGAGAGCCCCCGGCGGCCATGAATGGCGGGCCCGCCGAAGCAACTAAGGTACAGTAAACAC GGGTGGGAGGTTGGGCTCGCTAGGAACCCTACACTCGGTAATGATCCTTCCGCAGGTCACCCTACGG AS 616-3 ITS1-NL4 ITS4 VT3313 Talaromyces diversus isolate 6035 KX363453.1 99.06% TCCGAGGTCAACCGTGGAAAAATGTTGAGGGATGACCAACGCCCGCAGGCCCCTCCCGAGCGGGTGACAAAGCCCCAT ACGCTCGAGGACCTAGGCGGGCGTCGCCGCTGCCTTTCGGGCAGGTCCCCCCCGGGAAGGGGGGGGACCACACCCAAC ACACAAGCCGTGCTTGAGGGCAGAAATGACGCTCGGACAGGCATGCCCCCCGGAATGCCAGGGGGCGCAATGTGCGTT CAAAGATTCGATGATTCACGGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAA CCAAGAGATCCATTGTTGAAAGTTTTGATTATTATCAAGACACTCAGACAGTCCATCTTCATAAGGGTTCACAGAGCG CTCTGGCGGGCGCGGGCCCGGGGGCGAACACGCCCCCCGGCGACCGGGGTGGCCCCGGTGGGCCCGCCAAAGCAACAG GGTATACAAGAGACACGGGTGGGAGGTTGGACCCGCGAGGGGTCCGCACTCGGTAATGATCCTTCCGCAGGTTCACCC TACGG Annex List of primers used in the study ITS1: 5'TCCGTAGGTGAACCTGCGG -3' NL4: 3'GGTCCGTGTTTCAAGACGG-5' 58