ANALYSIS OF CHITINASE ACTIVITY Maheshi Kukule Kankanamge A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE August 2017 Committee: Vipaporn Phuntumart, Advisor Paul Morris Raymond Larsen © 2017 Maheshi Kukule Kankanamge All Rights Reserved iii ABSTRACT Vipaporn Phuntumart, Advisor The oomycete Aphanomyces astaci infects crayfish, which can result in the mass mortality commonly referred to as “crayfish plague”. Additional oomycetes in the genera Aphanomyces and Saprolegnia also infect crayfish. In the present study, two distinct organisms were isolated and identified from infected marbled crayfish Procambarus fallax forma virginalis and two phylogenetic trees based on internal transcribed spacer I (ITSI) were constructed using MEGA 7 software and maximum likelihood method with 1000 bootstraps. It is known that crayfish pathogens that infect crayfish produce chitinases that enable them to penetrate the cuticle of the crayfish. Preliminary testing for chitinase activity of Aphanomyces sp. indicated that in vitro growth in terms of surface area of the plates covered by mycelia and dry weight of mycelia increased with increasing chitin concentration from 1%-3% and leveled off at 4% chitin. The effects of chitin on timing of sporangia formation and zoospore release of Apahanomyces sp. suggested that chitin plays a role in asexual reproduction of the pathogen. The time taken for Aphanomyces sp. to develop sporangia and zoospore release increased with the amount of chitin incorporated in the media. Based on these observations, isolates of Aphanomyces sp. and of Saprolegnia sp. were tested for their chitinase activity. Both isolates could utilize chitin as carbon and nitrogen source in their growth. Additional experiments suggested that the chitinase activity of Aphanomyces sp. and Saprolegnia sp. involved an unidentified acidic substance produced by both organisms. Dinitrosalicylic acid assay (DNS assay) indicated the presence of unidentified secondary metabolites and/or pigment produced by Saprolegnia sp. and Aphanomyces sp.in nutrient deprived media. In DNS assay, the media with chitin and water in which both pathogens were grown showed highest absorbance after 72 hours indicating the possibility of their maximum production of chitinase and other enzymes within 48-72 hours. iv Based on the average absorbance readings, Aphanomyces sp. could be producing significantly higher amount of enzymes that break down chitinous cuticle compared to Saprolegnia sp. Overall, the observations made in this study could indicate chitinase production in Aphanomyces sp. and Saprolegnia sp. v ACKNOWLEDGMENTS I would first like to thank my advisor, Dr. Vipa Phuntumart, for her mentorship and guidance. I would also like to thank my committee members Dr. Paul Morris and Dr. Ray Larsen for their time, help and advice. I thank Dr. Robert Huber for providing the marbled crayfish used in my project and thank other Biology faculty members for providing materials needed for my research. In addition, I would like to thank lab members in the Phuntumart lab who were always willing to lend a hand. Last but not least, I would like to acknowledge Bowling Green State University for providing a valuable graduate experience. vi TABLE OF CONTENTS Page INTRODUCTION…………………………………………………………………..……...... 1 The Class Oomycota………………………………………………………………… 1 Oomycete Life Cycle………………………………………………………………... 1 CHAPTER I. BACKGROUND……………………………………………….…………...... 4 Crayfish Pathogen- Aphanomyces astaci ……………………………………………. 4 Mechanism of Infection ……………………………………………………………… 6 Crayfish ………………………………………………………………………………. 7 Molecular Identification of the Oomycete Pathogens ………………...……………… 8 Chitinase ……………………………………………………………………………… 9 Roles of Chitin in Oomycete Cell Wall………………………………………………. 12 Hypothesis ……………………………………………………………………………. 12 Aims …………………………………………………………………………………… 13 CHAPTER II. MATERIALS & METHODS …………………………………………………. 14 Isolation of Crayfish Pathogen from Infected Crayfish …..…………………………... 14 Identification of the Isolated Organisms Using ITS Region …………………………... 15 Genomic DNA Isolation ……………………………………………………………….. 15 ITS Amplification and Sequence Analysis……………………………………... 16 Phylogenetic Analysis ……………………………………………………...….. 17 Chitinase Assay ……………………………………………………………………….. 18 Preparation of Colloidal Chitin ……………………………………………….. 18 vii The Effects of Chitin on Growth and Asexual Reproduction of Isolated Aphanomyces sp. ……………………………………………………..….…… 18 Measuring Surface Area of Mycelia ………………………..….……. 18 Wet and Dry Weight of the Mycelia …….…………………..….……. 19 Density of the Mycelia ……………………………………..…….…... 20 Zoospore Production and Quantification of Zoospores ..…….…....…. 20 pH Indicator Assay ...…………………………………………….….….……. 21 3, 5- Dinitrosalicylic Acid Assay ………………………………….…….…... 21 Construction of the Standard Curve Using N-Acetyl glucosamine .….………. 22 Assessing the Growth Capability of Aphanomyces sp. and Saprolegnia sp. on YPS Media with Different Strength and Composition ..……...……..…… 22 Performing Dinitrosalicylic Acid Assay .…………………..…………….…. 23 CHAPTER III. RESULTS ………………………………………………………………....... 25 Isolation of Crayfish Pathogen from Infected Crayfish ……………………………… 25 Molecular Identification of the Organism Using ITS Region ……………………….. 26 Genomic DNA Isolation ……………………………………………………… 26 PCR Amplification of ITS Region and Sequence Analysis ………………….. 29 Sequence Analysis ……………………………………………………………. 34 Phylogenetic Analysis ………………………………………………………… 38 Chitinase Assay …………………………………………………………………..…… 41 The Surface Area of Mycelial Growth ...……………………………………… 41 Dry Weight of Mycelia ......…………………………………………………… 44 Density of Mycelia ..…......…………………………………………………… 45 viii Timing for Sporangia Formation and Zoospore Release in the Presence of Varying Percentages of Chitin …………………….....................................… 47 The Zoospore Counts for Aphanomyces sp.………………..………………… 47 Observing the Morphology of Sporangia in the Presence of Varying Percentages of Chitin ……...…………………………………………………… 48 pH Indicator Assay ………………………...…………….…………………… 50 DNS Assay ……………………………………………………………………. 56 N-Acetyl glucosamine standard curve ……………………………..………….. 56 Assessing the Growth Capability on YPS Media with Different Nutrient Composition …………………………………………………………………… 58 Performing DNS Assay ……………………………………………………...… 58 CHAPTER IV. DISCUSSION & CONCLUSION ………………………………….……..... 66 Discussion ……………………………………………………………………………. 66 Conclusion …………………………………………………………………………….. 76 REFERENCES …………………………………………………………………….………….. 78 ix LIST OF FIGURES Figure Page 1.1 Lifecycle of A. astaci ................................................................................................. 5 1.2 ITS1 and ITS2 Regions and Primer Annealing ......................................................... 9 3.1 Growth #1 (after 3 days) (Later Identified as Aphanomyces sp.) .............................. 25 3.2 Growth #2 (after 3 days) (Later Identified as Saprolegnia sp.) ................................. 26 3.3 Genomic DNA from Growth #1 & Growth #2 Visualized on 0.8% Agarose Gel ….. 28 3.4 PCR Products of ITS Region for Growth #1 ............................................................. 30 3.5 PCR Products of ITS Region for Growth #2 ............................................................. 31 3.6 The Sequence Chromatogram for Growth #1 ITS Sequence with Reverse Primer ... 33 3.7 The Sequence Chromatogram for Growth #2 ITS Sequence with Reverse Primer ... 34 3.8 The Sequence Alignment on blastn for Growth #1 on FungiDB.............................. 35 3.9 The Sequence Alignment on blastn for Growth #2 on FungiDB............................... 37 3.10 The Phylogenetic Tree for Genus Aphanomyces ....................................................... 39 3.11 The Phylogenetic Tree for Genus Saprolegnia .......................................................... 40 3.12 The Average Surface Area (cm2 ) of Aphanomyces sp. Mycelia Grown on YPS with Varying Percentages of Chitin Over 4 days....................................................... 42 3.13 The Increase of Mycelial Surface Area (cm2 /day) of Aphanomyces sp. Grown on YPS with Varying Percentages of Chitin Over Four Days ........................................ 43 3.14 The Average Dry Weight (g) of Aphanomyces sp. Mycelia Grown on YPS with Varying Percentages of Chitin ................................................................................... 44 3.15 The Average Density of Aphanomyces sp. Mycelia .................................................. 46 3.16 Zoospore Count for Aphanomyces sp. ....................................................................... 48 x 3.17A Morphology of Sporangia of Aphanomyces sp. Grown on YPS Media with No Chitin ...………………...……………………………………………………… 49 3.17B Morphology of Sporangia of Aphanomyces sp. Grown on YPS Media Containing 1% Chitin ….……………………………………………………………………… 49 3.17C Morphology of Sporangia of Aphanomyces sp. Grown on YPS Media Containing 2% Chitin ….……………………………………………………………………… 49 3.17D Morphology of Sporangia of Aphanomyces sp. Grown on YPS Media Containing 3% Chitin ……………….…………………………...……………………………. 50 3.17E Morphology of Sporangia of Aphanomyces sp. grown on YPS media containing 4% Chitin ….……………………………………………………………………… 50 3.18A The Color of YPS Media with/ without 1.5% Chitin Media at pH 4.2, 4.7 and 5.2 …..……………………………………………………………………..... 51 3.18B The Color of YPS Media with/ without 1.5% Chitin Media at pH 7.1 ……....…… 52 3.18C Aphanomyces