Isolation, identification and study of biological characteristics of the pathogenic fungi curvularia lunata causing leaf spot to bananas khóa luận tốt nghiệp

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  GRADUATION THESIS TITLE: ISOLATION, IDENTIFICATION AND STUDY OF BIOLOGICAL CHARACTERISTICS OF THE PATHOGENIC FUNGI CURVULARIA LUNATA CAUSING LEAF SPOT TO BANANAS Hanoi - 2023 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY  GRADUATION THESIS TITLE: ISOLATION, IDENTIFICATION AND STUDY OF BIOLOGICAL CHARACTERISTICS OF THE PATHOGENIC FUNGI CURVULARIA LUNATA CAUSING LEAF SPOT TO BANANAS STUDENT : NGO PHUONG HIEN STUDENT CODE : 637411 CLASS : K63-CNSHE FACULTY : BIOTECHNOLOGY MAJOR : MICROBIOLOGY INSTRUCTOR : Ph.D NGO THU HUONG Assoc Prof NGUYEN XUAN CANH Hanoi - 2023 COMMITMENT The research was conducted from 9/2022 to 2/2023 under the supervision of Ph.D Ngo Thu Huong, Assoc Prof Nguyen Xuan Canh at Department of Microbiotechnology – Faculty of Biotechnology – Vietnam National University of Agriculture I hereby declare that the data and results of this study are honest and have not been published in any scientific research at home or abroad References have been clearly cited Any help during the implementation of this thesis has been appreciated i i ACKNOWLEDGEMENTS Firstly, I want to express my sincere gratitude to all of the Executive Board, teachers, and lecturers of the Faculty of Biotechnology, Vietnam National University of Agriculture for helping, encouraging and providing the best working environment for me to complete the thesis Secondly, I would like to express my most sincere thank to Ph.D Ngo Thu Huong, Assoc Prof Nguyen Xuan Canh for guiding and giving valuable advice during my research so that I could complete my thesis successfully Thirdly, I would like to express my deep gratitude to the lecturers of the Department of Microbiotechnology, Assoc Prof Nguyen Van Giang, Msc Tran Thi Hong Hanh, Msc Nguyen Thanh Huyen, Msc Tran Thi Dao, Ph.D student Nguyen Thi Thu and researcher Duong Van Hoan, Ta Ha Trang for getting me through the difficulties during my final thesis course My completion of this project could not have been accomplished without their devotion Last but not least, I crave for expressing my appreciation to my beloved family members, my friends, and colleagues for their encouragement, motivation so that I could complete my graduation thesis Sincerely, Hanoi, February 3rd, 2023 Sincerely Ngo Phuong Hien ii CONTENTS COMMITMENT i ACKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS viii ABSTRACT ix I INTRODUCTION II LITERATURE REVIEW 2.1 Overview of Bananas 2.1.1 Bananas in food demanding 2.1.2 Bananas as economic importance 2.1.3 Diseases causing the banana production loss 2.1.3.1 Diseases from bacteria 2.1.3.2 Diseases from virus 2.1.3.3 Diseases from fungi 2.2 Overview of Curvularia lunata 2.2.1 Overview of Curvularia species 2.2.2 Overview of Curvularia lunata 2.2.2.1 Morphology of Curvularia lunata 2.2.2.2 Various hosts of Curvularia lunata 2.2.2.3 Infection mechanism of Curvularia lunata 12 III MATERIALS AND METHODS 14 3.1 Time and location for the research 14 iii 3.2 Materials 14 3.3 Methods 15 3.3.1 Samples collection method 15 3.3.2 Isolating and subculturing method 16 3.3.3 Pathogenicity test 17 3.3.4 Studying on some biological characteristics of the selected fungi strain 17 3.3.4.1 Studying on the morphological characteristics of conidia, mycelium and conidiophore of the selected fungi strain 17 3.3.4.2 Studying on the culturing features of the pathogenic fungi in different medium 18 3.3.4.3 Screening the ability to produce extracellular enzyme cellulase of the selected fungi strain 18 3.3.5 Indentification of the selected fungi strain 18 3.3.5.1 DNA extraction method 18 3.3.5.2 Amplification, sequencing and analyzing ITS gene sequence of isolated fungi strain method 20 3.3.6 Screening, detecting toxic genes of fungal strains 21 IV RESULTS AND DISCUSSION 23 4.1 Results of samples collection, isolation and subculturing the pathogenic pathogenic fungi strain 23 4.2 Pathogenicity test 25 4.3 Classification of pathogenic fungi strain HN 5.2 26 4.3.1 Amplification, sequencing and analyzing ITS gene sequence of fungi strain HN 5.2 26 4.3.2 Clg2P gene amplification 29 4.4 Screening the development of HN 5.2 strain in different medium 29 4.5 Testing the ability to extract cellulase of HN 5.2 strain 32 iv 4.6 Screening for the genes encoding virulence factors biosynthesis in pathogenic HN 5.2 fungi 33 V CONCLUSION AND PROPOSAL 36 5.1 Conclusion 36 5.2 Proposal 36 REFERENCES 37 APPENDIX 45 v LIST OF TABLES Table 3.1 Virulence factors primer pair sequences 21 Table 4.1 Features enumeration and growth comparison of different C lunata HN 5.2 fungi media 31 vi LIST OF FIGURES Figure 2.1 Symptoms Figure 2.2 Symptoms of crown rot in banana fruits Figure 2.3 Eumusa leaf spot in Southeast Asia Figure 2.4 Morphology of Curvularia canadensis with the scale bars = 10μm and 5μm respectively Figure 2.5 Conidia of C lunata CX-3 Figure 2.6 Characteristics of Curvularia leaf spot symptoms on a corn leaf 10 Figure 2.7 Leaf spot caused by Curvularia lunata in Aloe vera 11 Figure 4.1 Samples of banana leaves with typical disease symptoms 23 Figure 4.2 The morphology of HN 5.2 fungi strain in PDA medium (with A: Infected leaves isolation; B and C: Colony morphology of HN 5.2 after being cultured for days and days respectively; D: HN 5.2 strain mycelium; E: HN 5.2 strain condiophore; F: HN 5.2 strain conidia All of the figures were observed under the magnification of 40X) 24 Figure 4.3 Pathogenicity test of HN 5.2 fungi strain in banana leaves 25 Figure 4.4 Electrophoresis result of ITS gene region PCR product from HN 5.2 fungi strain 27 Figure 4.5 Phylogenetic tree based on ITS1/ITS4 sequences of HN 5.2 fungi strain 28 Figure 4.6 Electrophoresis result for the PCR product of HN 5.2 fungi strain using P1-F/P2-R primer pair 29 Figure 4.7 The growth and development of C lunata HN 5.2 colony 30 Figure 4.8 Extracellular cellulase activity of C lunata HN 5.2 33 Figure 4.9 Detecting pathogenic genes in C lunata HN 5.2 35 vii LIST OF ABBREVIATIONS Abbriviaions Full word C lunata Curvularia lunata MEA Malt Extract Agar PDA Potato Detrose Agar WA Water agar Xcm Xanthomonas campestris pv musacearum viii More specifically, mycelium of C lunata HN 5.2 in Richard is smooth, cotton-like with yellow-white colony From the 3rd day of culturing, it changes color to light gray with milky white edges Table 4.1 Features enumeration and growth comparison of different C lunata HN 5.2 fungi media Medium 5th day of culturing Description Smooth, cotton-like with yellow-white colony From the 3rd day of culturing, it changes Richard color to light gray with milky agar white edges After days, the colony turns to milky white with long, hairy aerial mycelium Initially white, cottony, smooth colony At the 3rd day of culturing, it begins to turn to PDA grey brown The thickness of outer white margin is about 0.2 mm Initially white, cottony, smooth colony At the 3rd day of culturing, it begins to turn to CZA light grey brown with the outer margin has paler color The inner colony morphology creates an oval like appearance 31 It has a round, smooth colony initially At the 3rd day of culturing, MEA the morphology begins to turn to dark grey look with long mycelium Later, it turns into jet black color It has a round, smooth colony WA initially Over time, the colony becomes light black and smooth 4.5 Testing the ability to extract cellulase of HN 5.2 fungi strain Research on the ability to produce extracellular enzymes in pathogenic fungi is very important Enzymes are one of the main factors that regulate the infection mechanism of fungi into plants For many fungi species, cellulose is the main component of plant cell wall Cellulase enzyme is known for its ability to break down and catalyze cellulose In Curvularia lunata, MAPK gene CLH1 takes responsibility to regulate cellulase synthesis (Wang et al , 2022) Nontheless, the cellulase enzyme, which destroys plant cell walls, is considered a major virulence factor and plays a fundamental role in fungal invasion (Wang et al , 2022) According to the pathogenic cycle of leaf spot fungi, during the infection of a host plant, pathogenic fungus face obstacles such as the cuticle layer and cell wall Cutinase, cellulase and other cell-wall-degrading enzymes 32 are secreted by pathogens Therefore, by doing experiment in screening the cellulase production ability of C lunata HN 5.2, we are able to state the theory about the infection pathway of this fungi strain into hosts Figure 4.8 Extracellular cellulase activity of C lunata HN 5.2 The fungi were inoculated in the CMC medium and incubated at 24°C for days Observing the result, it is very clear that, hydrolysis ring appears, proving that C lunata HN 5.2 fungi strain could degrade the amount of carboxymethyl cellulose-component of CMC medium As a result, C lunata HN 5.2 strain has been proven for the cellulase producing ability This result is similar to a previous study illustrating the occurence of cellulase in C lunata by testing the expression of endoglucanase and cellobiohydrolase – primary components of the cellulase combination (Aravinda & Manoharachary) 4.6 Screening for the genes encoding virulence factors biosynthesis in pathogenic HN 5.2 fungi We used virulence factors primer pairs of Clg2p, 3HNR, Clk1, Clh1, ClPKS18, Sod genes to detect whether or not the DNA sequence of HN 5.2 strain has regions may encode for the pathogenic pathway Fundamentally, Clg2p gene having huge roles in regulating the development of morphogenesis, conidia and appressoriums formation, and pathogenicity in fungi (Liu et al , 2016b) The 3HNR gene (1,3,8 33 trihydroxynaphthalene reductase) along with three other genes encoding for 1,3,6,8- tetra-HN reductase (4HNR), scytalone dehydratase (SCD) and polyketide synthase (PKS) take responsible for synthesizing DHN melanin – the integral virulence factors of C lunata (Lanisnik Rizner & Wheeler, 2003) Additionally, the selected Clk1 gene - Mitogen-activated protein kinase gene (MAPK) is the third candidate gene used in this experiment In other previous studies of this gene, scientists state that Clk1 might contribute to form conidia and infecting host plants through leisons (Gao et al , 2013) Morover, another MAPK gene, Clh1, has also been detected in this graduate thesis Clh1 has been proven being involved in synthesizing melanin and furan toxin synthetic pathway (Xuan et al , 2018) Nonetheless, Sod - another gene attributing to effect on the virulence producing melanin was also been chosen to be detected (Gao et al , 2017) Last but not least, the sixth primer pair used is to screen for polyketide synthase ClPKS18 Previous study has stated the roles of ClPKS18 in synthesizing 1,8dihydroxynaphthalene melanin synthesis and associating with biosynthetic pathway of secondary metabolite methyl 5-(hydroxymethyl) furan-2carboxylate (M5HF2C) toxin (Gao & Chen, 2017) 34 1500bp 500bp Figure 4.9 Detecting pathogenic genes in C lunata HN 5.2 As can be seen clearly from the figure, four out of six primers have been amplified sucessfully in C lunata HN 5.2 sequence The PCR products used 3HNR primer HNR-F/HNR-R, Clk1 primer Clk1_F/Clk1_R, Clh1 primer Clh1_u_f/ Clh1_u_r primer and Clg2p primer P1-F/P2-R having PCR product sizes of 900-950 bp; more than 1200 bp; 600-700 bp; about 900 bp respectively All of the PCR product results are analogous to the published papers In conclusion, the data prove that HN 5.2 fungi strain has a very high potential for virulence factors biosynthesis This is one of the important data for future research to figure out the actual infection mechanism of the fungi strain to banana leaves 35 V CONCLUSION AND PROPOSAL 5.1 Conclusion From infected banana leaf samples collected in Hung Yen province, HN 5.2 pathogenic strain causing leaf spot disease on banana was isolated Through the biological and molecular study of the isolated fungi strain, the HN 5.2 strain was intimately related to Curvularia lunata and named as Curvularia lunata HN 5.2 the HN 5.2 fungi strain has ability to produce cellulase enzymes Furthermore, four gene regions having ability to encode melanin and toxin biosynthetic pathway have been detected in C lunata HN 5.2 Therefore, this study has significant meanings in publishing and confirming the C lunata HN 5.2 strain causing leaf spot disease in Viet Nam for the first time Nonetheless, the results could be used as preliminaries for developing disease control measurements 5.2 Proposal From the research results which have been carried out, I have recommendations and directions for the further study listed below: ⁃ Conduct pathogenicity test of strain HN 5.2 in vivo conditions ⁃ Study the infection mechanism of HN 5.2 strain on bananas more deeply at the molecular level 36 REFERENCES AbdElfatah H S., Sallam N M A., Mohamed M S & Bagy H (2021) Curvularia lunata as new causal pathogen of tomato early 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PLoS Pathog 12(11): e1005940 62 Tripathi L., Ntui V O & Tripathi J N (2022) Control of Bacterial Diseases of Banana Using CRISPR/Cas-Based Gene Editing Int J Mol Sci 23(7) 63 Wang S., Lu Z., Lang B., Wang X., Li Y & Chen J (2022) Curvularia lunata and Curvularia Leaf Spot of Maize in China ACS Omega 7(51): 4746247470 64 Xu G., Zheng F., Ma R., Zheng F., Zheng L., Ding X & Xie C (2018) First report of Curvularia lunata causing leaf spot of Pennisetum hydridum in China Plant Disease 102(11): 2372-2372 43 65 Xuan N., GAO J., YU C., Meng W., SUN J., LI Y., & Jie C (2018) MAPKs and acetyl-CoA are associated with Curvularia lunata pathogenicity and toxin production in maize Journal of Integrative Agriculture 17(1): 139148 44 APPENDIX Table of the mycelium growth diameters of C lunata HN 5.2 in five medium (mm) Medium Richard PDA CZA MEA WA 33.6±0.24 31.7±1.24 30±0.81 26.3±0.47 22±0.81 46.6±0.94 48.6±0.94 43±0.81 39.6±0.47 34.3±0.47 64.3±3.09 67.6±1.24 62±1.63 84.6±0.47 76.7±0.47 71.6±1.24 66.6±0.47 53.6±1.69 86.3±1.24 81.3±1.69 81.3±0.94 77.3±0.94 89.6±0.47 85.6±0.94 87±0.81 Day 45 51±0.81 45±0.81 60±0.81 84.6±0.47 64.6±1.69