Characteristics of the enzymes produced by acid tolerant fungi

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  NGUYEN THI AN THESIS: CHARACTERISTICS OF THE ENZYMES PRODUCED BY ACID-TOLERANT FUNGI Hanoi, March 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  THESIS TITLE: CHARACTERISTICS OF THE ENZYMES PRODUCED BY ACID-TOLERANT FUNGI Student name: Nguyen Thi An Student code: 620649 Class: K62CNSHE Supervisors: Vu Nguyen Thanh, Assoc Prof PhD Nguyen Thi Thuy Hanh, PhD Hanoi, March 2022 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, March 2022 Student Nguyen Thi An 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, to teach me the useful knowledge and experience to conduct this thesis Secondly, I owe my deep gratitude to Dr Nguyen Thi Thuy Hanh for an opportunity to conduct my thesis at Food Industries Research Institute and her invaluable guidance during the past 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, support, and sacrifices for educating and my future Thank you very much! Nguyen Thi An ii TABLE OF CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF TABLES vi LIST OF FIGURES vii ABBREVIATIONS viii ABSTRACT ix INTRODUCTION LITERATURE REVIEW .3 2.1 Acid-tolerant fungi overview 2.2 General introduction to enzymes produced by acid-tolerant fungi 2.3 Research on acid-tolerant fungi in the world and in Vietnam 10 2.3.1 Research on acid-tolerant fungi in the world 10 2.3.2 Research on acid-tolerant fungi in Vietnam 12 2.4 Carbohydrate-active enzymes (CAZymes) .12 2.4.1 Lignocellulose and lignocellulose hydrolytic enzymes 12 2.4.2 α-Amylase and γ-Amylase 17 MATERIALS AND METHODS 20 3.1 Materials 20 iii 3.2 Chemicals, equipment, and machine 21 3.2.1 Chemicals .21 3.2.2 Equipment, instruments, and machine 21 3.3 Media .22 3.3.1 Fungi culture media .22 3.3.2 Malt 2Bx media (pH 5.5) 22 3.3.2 Low pH medium 22 3.3.3 Mineral media for fungi culture 22 3.3.4 Fungi enzyme culture media 22 3.3.5 Enzyme extraction buffer .22 3.3.6 Enzyme analysis buffer 23 3.4 Research methods 23 3.4.1 Purification and maintenance of strains 23 3.4.2 Enzyme extraction culture 23 3.4.3 Method for determination of cellulose-degrading activity 23 3.4.4 SDS-PAGE protein electrophoresis method 24 3.4.5 Zymogram electrophoresis method 24 3.4.6 Determination of enzyme activity by DNS method .24 3.4.7 Protein determination method (Lowry) 25 3.4.8 Determination of enzyme activity at different pH (pH 3, 5, 7) 26 iv RESULTS AND DISCUSSION 28 4.1 Survey on the growth ability of strains on different pH concentrations 28 4.2 Survey on the growth ability of strains on different carbohydrates 30 4.3 Analysis of protein content of fungal strains 32 4.4 CMCase, amylase, protease qualitative assay .33 4.5 CMCase, Xylanase quantitative assay 37 4.6 Analysis of the protein system and the lignocelulose hydrolytic enzyme system .39 4.7 CMCase, Xylanase quantitative assay at different pH 40 4.8 Thermostability qualitative assay 41 CONCLUSION PROPOSAL .43 5.1 Conclusion 43 5.2 Proposal 43 REFERENCES .44 ANNEX .46 v LIST OF TABLES Table 2.1 Enzyme-producing ability of some acid-tolerant fungal strains Table 2.2 Overview of the applications of acidophilic fungal enzymes in various industries Table 2.3 Classification table of the cellulase enzyme system 14 Table 3.1 42 strains are used in research…………………………….……………….20 Table 4.1 Growth of strains when grown on different substrates…….………………30 vi LIST OF FIGURES Figure 2.1 Lignocellulose structure 13 Figure 2.2 The molecular structure of cellulose 13 Figure 2.3 Cellulase reaction mechanism 15 Figure 2.4 The molecular structure of hemicellulose 16 Figure 2.5 Hemicellulose reaction mechanism 17 Figure 2.6 Amylose structure .17 Figure 3.1 Protein standard curve 25 Figure 3.2 D-xylose standard curve at pH 3, 5, 26 Figure 3.3 D-glucose standard curve at pH 3, 5, .27 Figure 4.1 Results of culturing fungi strains on different pH media 29 Figure 4.2 Results of culturing fungi strains on different substrate media 32 Figure 4.3 The graph shows the protein content of 42 fungi strains 33 Figure 4.4 Results of assessing the ability to degrade CMC substrates of 42 fungi strains 34 Figure 4.5 Results of assessing the ability to degrade Starch substrates of 42 fungi strains 35 Figure 4.6 Results of assessing the ability to degrade Skim milk substrates of 42 fungi strains 36 Figure 4.7 Xylanase activity of 42 fungi strains 37 Figure 4.8 CMCase activity of 42 fungi strains 38 Figure 4.9 SDS-PAGE, Zymogram CMC, and Xylan electrophoresis of 39 fungi strains 39 Figure 4.10 The graph shows the Xylanase activity of 42 fungi strains at pH 3, 5, 40 Figure 4.11 The graph shows the CMCase activity of 42 fungi strains at pH 3, 5, 41 Figure 4.12 Results of assessing the ability to degrade CMC substrates of heatresistant 42 fungi strains 42 vii ABBREVIATIONS CMC Carboxymethyl cellulose DNS 3,5 Dinitrosalicylic acid CMCase Carboxymethyl Cellulase SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis PDA Potato dextrose agar viii Figure 4.5 Results of assessing the ability to degrade Starch substrates of 42 fungi strains Represented by low pH tolerant fungal strains, the strains with very different starch degradation activities are reflected in the diameter of the digestion chamber Based on the figure, it can be seen that the resolution ring diameter of the enzyme extract ranges from 0-8mm Some strains with large hydrolytic ring diameters such as ASS 350-1, ASS 102-1, can be found that these strains have high enzyme activity The remaining strains have smaller hydrolytic rings, but range from 4-6mm A large number of strains such as ASS 104-2, ASM 122, ASM 137-2 not show hydrolysis It can be seen that they have no or very little enzymatic activity 35 Figure 4.6 Results of assessing the ability to degrade Skim milk substrates of 42 fungi strains Note: Control +: positive control (commercial enzyme cellulase), Control - : control negative (Na-citrate buffer 0.05M pH 5.0) Represented by low-pH tolerant fungal strains, strains with very different starch-degrading activities are reflected in the diameter of the digestion chamber Based on the figure, it can be seen that the resolution ring diameter of the enzyme extract ranges from 0-20mm It can be seen that some strains with large hydrolytic ring diameters such as ASS 89-1, AS 19, are strains with high enzyme activity The remaining strains have smaller hydrolytic rings, but range from 8-15mm A large number of strains like AST 152, AST 155-1, AST 157-3, ASS 80-2, AS 612-1, etc showed no hydrolysis It can be found that they have no or very little enzymatic activity 36 4.5 CMCase, Xylanase quantitative assay To evaluate the degradation ability of strong acid-tolerant fungal strains, we analyzed CMCase and Xylanase enzyme activities of crude enzyme extracts of strains obtained after culture Enzyme solution was diluted to appropriate concentrations with 0.05M Na-citrate buffer pH 5.0 and determined by DNS method  Xylanase The enzyme extract was reacted with 1% xylan substrate in 0.05 M Na-citrate buffer pH Figure 4.7 Xylanase activity of 42 fungi strains Analytical results showed that not all fungal strains have xylanase activity Strains belonging to Acidomyces acidothermus, Penicillum/Talaromyces sp1 nov., Sarocladium sp nov Neither had Xylanase activity Looking at the graph, it can be seen that the strains have very different activities The ASS 46-1 strain had a superior xylanase activity compared to other strains with an activity of 485.64 U/ml In each group of strains, a strain with the highest activity will be selected compared to the rest, while the remaining strains have relatively low activity such as AST 152 only 0.79 37 U/ml or ASS 89-1 only reached 0.24 U/ml  CMCase The crude enzyme solution was quantified for CMCase activity by DNS method with 1% CMC substrate mixed in 0.05M Na-citrate buffer pH Figure 4.8 CMCase activity of 42 fungi strains Through the analysis results, it can be seen that although all strains have CMCase activity, they are all very low, only about 0.22-4.81 U/ml Penicillum citreonigrum and Penicellim griseolum strains are two groups of strains capable of producing CMCase enzymes with high activity In each group of strains, it is possible to select strains with the highest activity Combined with the analysis of CMCase and xylanase enzymes, we found that not all fungi strains with high CMCase activity also have high xylanase activities because not all fungi strains are able to hydrolyze cellulose associated with hemicellulose This can be explained because the cellulase and xylanase enzyme systems of each strain are different and independent of each other To study more clearly the enzyme system of fungal strains, we conducted SDS-PAGE electrophoresis 38 and zymogram 4.6 Analysis of the protein system and the lignocelulose hydrolytic enzyme system For detailed evaluation of the extracellular proteins of the strains, their crude enzymes were electrophoresed on SDS-PAGE gel and Zymogram electrophoresis with CMC and Xylan substrates The results are shown in the figure below Figure 4.9 SDS-PAGE, Zymogram CMC, and Xylan electrophoresis of 39 fungi strains Note: (1) ASS 96-1, (2) AST 152, (3) AST 155-1, (4) AST 157-3, (5) AS 175-2, (6) ASS 46-1, (7) ASS 88-1, (8) ASS 99-1, (9) ASS 350-1, (10) AS 581-2, (11) ASS 80-1, (12) ASS 89-1, (13) ASS 102-1, (14) ASS 338-1, (15) ASS 45-1, (16) ASS 135-1, (17) ASS 125-1, (18) ASS 64-1, (19) ASM 122, (20) ASS 104-2, (21) ASS 71-1, (22) ASS 80-2, (23) AS 612-1, (24) AS 31, (25) ASM 139-1, (26) ASM 141-1, (27) ASM 137-2, (28) ASS 326-1, (29) ASS 40-3, (30) ASS 52-1, (31) ASM 115, (32) ASM 147, (33) ASM 153-1, (34) AS 19, (35) ASS 343-4, (36) ASS 358-9, (37) ASS 348-2, (38) AS 616-3, (39) ASS 342-2, (40) Control The results of SDS-PAGE electrophoresis and CMC, xylan zymogram of 39 low pH fungi strains are shown in the figure, clearly showing the xylanase and CMCase enzyme activities of the strains through the electrophoresis band However, the appearance of enzyme electrophoresis bands in the two substrates is not the same, 39 there are strains with CMCase heat stability but not xylanase heat stable strains such as Acidomyces acidothermus ASS 96-1 and AST 157-3 strains Penicillium griseolum strains ASS 45-1, ASS 97-2 on both Zymogram Xylan and CMC gels in wells of enzyme bands appeared similar, which proves that both strains have similar enzyme activities Two groups of strains Amplistroma sp and Acrodontium griseum are two groups of acid-resistant fungis with very diverse characteristics, both strains appear Enzymes are very clear and uniform in all wells, so it can be seen that fungi strains belonging to these two groups are active in both CMCase and Xylanase enzymes 4.7 CMCase, Xylanase quantitative assay at different pH The evaluation of the activity of enzymes at different pH is a parameter to evaluate the activity of enzymes, especially at low pH conditions Enzyme activity at pH3, pH5, and pH7 was experienced Enzyme activity was determined at different pH values using Britton Robinson buffer Figure 4.10 The graph shows the Xylanase activity of 42 fungi strains at pH 3, 5, 40 Figure 4.11 The graph shows the CMCase activity of 42 fungi strains at pH 3, 5, It can be seen that the active range of enzymes is in the range of pH 3-5 At pH 7, the enzyme activity of most strains was significantly reduced Some strains show higher enzymatic activity at pH such as xylanse activity of ASM 122 or CMCase activity of ASS 52-1 These strains are potentials for enzyme application under low pH conditions 4.8 Thermostability qualitative assay Heat stability is an important property of enzymes that many researchers are interested To test the heat stability of CMCase, we experimented to evaluate the ability to degrade CMC substrates with enzyme samples of low pH tolerant fungal strains, each 3-well strain corresponds to enzyme samples: non-enzymatic enzyme produce heat, the enzyme gives off a temperature of 70oC and the enzyme gives off a heat of 90oC in 20min We carried out 30µl of enzymes of fungal strains on a CMCagar medium The ability to resolve CMC was assessed by the formation of a hydrolytic ring diameter The following are the results of CMC resolution when treated at different temperatures 41 Figure 4.12 Results of assessing the ability to degrade CMC substrates of heat-resistant 42 fungi strains Note: T: enzyme without heating, T70: enzyme heated at 70 oC for 20min, T90: enzyme heated at 90oC for 20min, Control +: positive control (commercial enzyme cellulase), Control - : control negative (Na-citrate buffer 0.05M pH 5.0) The results of CMCase heat stability analysis showed that the strains with CMCase activity were relatively heat stable after heating at 70oC for 20min Especially, group of Amplistroma sp nov., Acrodonitium griseum show activity after heating at 70oC for 20min 42 CONCLUSION PROPOSAL 5.1 Conclusion From 42 strains of 15 different species: - I obtained results when testing on different pH media: strains grow well and are the same at Malt 2bx, pH 2.5 and pH 4.6 At pH 1.0 most strains grow slowly, colonies are filamentous and cannot form spores - I analyzed the protein content and found that the extracellular protein content of the isolates ranged from 1.0-3.5mg/ml strain Thyronectria sp.nov has the highest protein content of 3.55mg/ml AST 152 belongs to the Acidomyces acidothermus strain with the lowest protein content of 1.04mg/ml The average protein content of the 42 strains was 2.5mg/ml - I have qualitatively evaluated the activity of CMCase, it can be seen that the diameter of the resolution ring of the enzyme extract ranges from 4-12mm For amylase activity, the hydrolytic ring diameter ranges from 0-8 mm For protease activity, the hydrolysis ring diameter ranges from 0-20mm - I collected data when analyzing CMCase, Xylanase activities by DNS method on crude enzyme solution and on different pH 5.2 Proposal Hydrolytic enzymes produced by these strains should be further studied, for example, stability at low pH, heat resistance, etc These technological properties are important for exploitation in the feed industry 43 REFERENCES Aguilera, A et al (2006) ‘Eukaryotic community distribution and its relationship to water physicochemical parameters in an extreme acidic environment, Rio Tinto (southwestern Spain)’, Applied and Environmental Microbiology, 72(8), pp 5325–5330 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 et al (2010) ‘Eukaryotic microbial diversity of phototrophic microbial mats in two Icelandic geothermal hot springs’, Int Microbiol, 13(1), pp 21–32 Aguilera, A and González-Toril, E (2019) ‘Eukaryotic life in extreme environments: acidophilic fungi’, Fungi in extreme environments: ecological role and 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in Europe’, Fungal Diversity, 58(1), pp 33–45 11 Hujslová, M et al (2019) ‘Fungi, a neglected component of acidophilic biofilms: they have a potential for biotechnology?’, Extremophiles, 23(3), pp 267–275 12 Johnson, B.D and Aguilera, A (2016) ‘Environmental microbiology in acidophilic environments’, Manual of environmental microbiology, 4th edn.(MEM4) ASM Press, 44 Washington, DC, pp 34–47 13 Johnson, D.B (1998) ‘Biodiversity and ecology of acidophilic microorganisms’, FEMS microbiology ecology, 27(4), pp 307–317 14 Krüger, A et al (2018) ‘Towards a sustainable biobased industry–highlighting the impact of extremophiles’, New Biotechnology, 40, pp 144–153 15 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 16 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 17 Pérez, J et al (2002) ‘Biodegradation and biological treatments of cellulose, hemicellulose and lignin: an overview’, International microbiology, 5(2), pp 53–63 18 Shaikh, A.A et al (2011) ‘Electrochemical studies of the pH dependence of Cu (II) reduction in aqueous britton-robinson buffer solution’, Int J Electrochem Sci, 6, pp 2333– 2343 19 Thanh, V.N et al (2019) ‘Surveying of acid-tolerant thermophilic lignocellulolytic fungi in Vietnam reveals surprisingly high genetic diversity Sci Rep 9: 3674’ 20 Tiquia-Arashiro, S and Rodrigues, D.F (2016) Extremophiles: applications in nanotechnology Springer 45 ANNEX Annex Investigation of Xylanase and CMCase enzyme activities of acid-tolerant fungal strains Name Strain Xylanse CMCase ASS 96-1 Acidomyces acidothermus 19.17 0.77 AST 152 Acidomyces acidothermus 0.79 4.35 AST 155-1 Acidomyces acidothermus 2.23 0.42 AST 157-3 Acidomyces acidothermus 0 ASM 122 Acrodontium griseum 7.94 0.22 ASS 104-2 Acrodontium griseum 99.74 2.15 ASS 71-1 Acrodontium griseum 48.68 ASS 73-1 Acrodontium griseum 8.27 ASS 80-2 Acrodontium griseum 103.65 1.13 AS 612-1 Acrodontium griseum 362.91 AS 175-2 Amplistroma sp.nov 87.29 ASS 46-1 Amplistroma sp.nov 485.64 ASS 88-1 Amplistroma sp.nov 89.87 0.37 ASS 99-1 Amplistroma sp.nov 65.43 ASS 350-1 Aspergillus turcosus 6.3 2.32 AS 581-2 Penicillum chermesinum 111.1 3.1 ASS 80-1 Penicillum citreonigrum 1.57 4.81 ASS 89-1 Penicillum citreonigrum 0.24 ASS 102-1 Penicillum citreonigrum 8.49 3.7 ASS 338-1 Penicillum citreonigrum 2.41 1.77 ASS 45-1 Penicillum griseolum 272.59 1.25 ASS 97-2 Penicillum griseolum 11.06 3.87 AS 31 Penicillum sp1.nov 3.26 2.1 ASM 139-1 Penicillum sp1.nov 2.87 0.3 ASM 141-1 Penicillum sp1.nov 120.63 0.32 ASM 137-2 Penicillum sp1.nov 9.01 0.68 ASS 326-1 Penicillum sp2.nov 6.87 0.14 ASS 40-3 Penicillum sp2.nov 9.81 0.94 46 Name Strain Xylanse CMCase ASS 52-1 Penicillum sp3.nov 10.9 1.52 ASM 115 Penicillum sp4.nov 45.53 0.07 ASM 147 Penicillum sp4.nov 9.96 ASM 153-1 Penicillum sp4.nov 14.85 1.57 AS 19 Penicillum/Talaromyces 2.78 0.55 1.43 0.75 1.12 1.32 2.63 0.91 5.48 2.65 1.36 1.47 sp1.nov ASS 343-4 Penicillum/Talaromyces sp1.nov ASS 358-9 Penicillum/Talaromyces sp1.nov ASS 348-2 Penicillum/Talaromyces sp1.nov ASS 357-1 Penicillum/Talaromyces sp1.nov AS 616-3 Penicillum/Talaromyces sp1.nov ASS 135-1 Sarocladium sp.nov 1.01 ASS 125-1 Sarocladium sp.nov 6.26 ASS 342-2 Talaromyces diversus 1.67 2.59 ASS 64-1 Thyronectria sp.nov 8.05 0.72 Annex Investigation of Xylanase and CMCase enzyme activities of acid-tolerant fungal strains at pH 3, 5, Xylanase Name Strain Acidomyces ASS 96-1 pH 30.65 16.38 pH3 pH pH 1.39 6.1 0.43 7.57 6.11 3.57 0.4 0 9.86 10.17 4.78 0 0.11 acidothermus Acidomyces AST 155-1 18.15 pH acidothermus Acidomyces AST 152 pH3 CMCase acidothermus 47 Xylanase Acidomyces 2.46 4.47 CMCase 3.15 0.27 1.89 95.73 24.93 0.18 0.44 1.5 199.69 168.48 38.82 0.54 1.73 0.62 AST 157-3 acidothermus ASM 122 Acrodontium griseum ASS 104-2 Acrodontium griseum ASS 71-1 Acrodontium griseum 72.21 99.47 30.85 1.88 0.97 ASS 73-1 Acrodontium griseum 92.15 85.42 2.82 1.66 1.9 0.39 ASS 80-2 Acrodontium griseum 28.64 193.31 26.93 1.13 AS 612-1 Acrodontium griseum 349.87 363.76 59.87 1.62 1.83 1.08 AS 175-2 Amplistroma sp.nov 102.96 127.47 20.87 0.02 1.44 0.95 ASS 46-1 Amplistroma sp.nov 322.58 302.3 82.09 1.85 0.88 ASS 88-1 Amplistroma sp.nov 127.67 158.78 27.39 0.52 0.46 ASS 99-1 Amplistroma sp.nov 95.4 139.4 25.37 0 ASS 350-1 Aspergillus turcosus 8.24 18.26 2.62 3.62 1.69 134.15 170.63 3.51 2.6 0.87 Penicillum AS 581-2 chermesinum Penicillum AS 173-2 4.66 2.42 2.51 1.57 5.8 4.45 0.67 1.95 2.91 1.59 7.14 8.35 1.23 1.74 2.71 1.42 6.97 8.77 10.03 0.47 1.97 1.74 5.66 9.11 1.16 1.27 3.31 2.25 citreonigrum Penicillum ASS 102-1 10.87 citreonigrum Penicillum ASS 89-1 9.66 citreonigrum Penicillum ASS 80-1 76.64 citreonigrum Penicillum ASS 338-1 citreonigrum ASS 45-1 Penicillum griseolum 41.45 37.33 4.08 1.81 2.33 1.5 ASS 97-2 Penicillum griseolum 59.37 53.89 2.6 0.54 0.54 1.14 AS 31 Penicillum sp1.nov 0.92 11.78 3.49 3.99 2.19 ASM 139-1 Penicillum sp1.nov 8.29 8.24 2.69 2.08 3.97 0.24 48 Xylanase ASM 141-1 Penicillum sp1.nov ASM 137-2 Penicillum sp1.nov 115.23 142.79 CMCase 0.68 1.12 1.67 23.34 15.29 2.91 1.37 2.15 1.24 ASS 326-1 Penicillum sp2.nov 2.74 5.89 1.02 0.92 1.31 0.39 ASS 40-3 Penicillum sp2.nov 17.36 14.39 2.27 2.76 1.64 ASS 52-1 Penicillum sp3.nov 1.34 4.06 4.98 3.23 1.31 ASM 115 Penicillum sp4.nov 28.18 12.82 0.76 0.04 0.72 ASM 147 Penicillum sp4.nov 20.88 11.46 0.76 0.33 1.38 ASM 153-1 Penicillum sp4.nov 36.13 21.59 0.76 1.08 1.1 11.65 6.96 0.76 0 0.84 14.27 9.05 0 1.21 7.12 10.16 2.23 1.98 1.3 13.95 14.45 0.09 1.95 1.7 5.99 2.88 0.16 0.41 0.62 0.58 92.23 28.07 1.12 1.08 Penicillum/Talaromyces AS 19 sp1.nov Penicillum/Talaromyces ASS 343-4 sp1.nov Penicillum/Talaromyces ASS 358-9 sp1.nov Penicillum/Talaromyces ASS 348-2 sp1.nov Penicillum/Talaromyces ASS 357-1 sp1.nov Penicillum/Talaromyces AS 616-3 sp1.nov ASS 135-1 Sarocladium sp.nov 0 0 0.64 0.24 ASS 125-1 Sarocladium sp.nov 3.92 0.06 1.79 1.71 ASS 342-2 Talaromyces diversus 46.76 34.62 0.99 1.36 1.1 ASS 64-1 Thyronectria sp.nov 0 0.39 2.23 49