2  Module 0: Getting Started and Data Management [Instructor Instructions]

2.1 Module 0.1 – Lab Materials and Equipment Overview

This document lists the materials and equipment required for implementing the Myco-Ed program.

2.1.1 Module 1- Fungal sampling and Culturing

  • 100x15mm Petri Dishes
  • Agar, Bacteriological
  • Autoclave
  • Laminar Flow Hood (not necessary, if not available)
  • Alcohol Lamp or Bunsen Burner
  • Forceps
  • Scalpel (make sure handle is metal, this will be flame sterilized, plastic will melt)
  • 150ml Beaker (or similar, 2 per student)
  • Parafilm
  • Gloves
  • Hole punch (all metal, for cutting leaf discs, scalpel will work in lieu of hole punch)
  • Kimwipes
  • Paper bags
  • 10% bleach
  • 70% ethanol
  • Paper towels

2.1.2 Module 2- Fungal Isolation

  • 60x15mm Petri Dishes
  • Potato Dextrose Agar
  • Autoclave
  • Laminar Flow Hood (not necessary, if not available)
  • Gloves
  • Sharpie (fine tip is preferable)
  • 70% ethanol
  • Kimwipes

2.1.3 Module 3 - Phenotypic Assays

Materials and equipment will vary based on the phenotypic assay choice.

2.1.4 Module 4 – DNA Sequencing and Species Identification

This list may vary depending on your preferred extraction, amplification, and sequencing protocols. - Gloves - Sharpie - Thermal cycler - Vortex - Centrifuge - Heat block - 1.5mL microcentrifuge tubes - 0.2 mL PCR tubes - Micro and PCR tube racks - Label tape - Extraction Buffer (EB) - 3% BSA solution - Parafilm - 70% ETOH - 1-10 and 20-200 µL micropipettors - 1-10 and 20-200 µL micropipette tips - Kimwipes - PCR Primers (ITS1f, ITS4, 5.8SR, fITS7) - Taq polymerase mix - PCR water

2.1.5 Module 5 - Tissue Preparation for Genome Sequencing

  • Cellophane
  • 60x15mm Petri Dishes
  • Potato Dextrose Agar
  • Silica beads
  • Gloves
  • Transfer hood/Bunsen burner
  • Incubator
  • Balance
  • Forceps
  • Scalpel
  • Small jar with lid
  • Pure fungal culture

2.1.6 Module 6 – Sample Submission

  • Shipping samples
  • Sample form printout

2.1.7 Module 7 – Genomic Analyses

  • Access to Mycocosm
  • Computer with internet access

2.2 Module 0.2 – Create iNaturalist Traditional Project Instructions

2.2.1 iNaturalist Introduction

iNaturalist is a tool that we can use to document the fungal isolates in Myco-Ed courses. This tool allows instructors and students to record the metadata associated with the fungal isolates. Metadata will include plant species, geographic location, elevation, fungal DNA sequences, growth assays, and any other data relevant for this project. This platform will also enable us to track growth of the MycoEd project. The goal of this long-term research project is to develop annotated fungal genomes to better understand fungal biology.

To begin, make sure that you have an iNaturalist account.

2.2.2 Project Overview

There are three kinds of projects: 1) Umbrella Projects 2) Collection Projects 3) Traditional Projects

The umbrella project for MycoEd is the MycoEd Fungal Genomics Education Project. Each course will establish a Traditional Project in iNaturalist. A traditional project allows us to control whose observations are included in the project, which enables better downstream data management. We ask that your class use the Traditional Project for MycoEd for only your MycoEd fungi. If your course includes a fungal collection using iNaturalist, please have a separate iNaturalist project for these fungal observations.

2.2.3 Make a MycoEd Traditional Project [Instructor Instructions]

  1. Log into iNaturalist.
  2. Go to Projects.
  3. This will take you to Your Projects.
  4. On the bottom right of the page, select Start a Project.
  5. Start a Traditional Project. Scroll to the bottom of the page to find the Traditional Project link for this option.

**Note: If you do not have this option, please contact Amy Honan (amy.honan@oregonstate.edu) or Andrew Wilson (andrew.wilson@BotanicGardens.org) for assistance.

  1. Complete the following criteria indicated in the lettered parts of the figure below:
    1. Add Title. Include Institution, term or semester, and year.
    2. Make this Invite Only. You will add your students to this project.
    3. Add images or logos as you see fit. This step is useful if you manage multiple projects.
  2. Other fields to consider:
    1. Location: You may choose to limit the locations of your observations. If your project is set to invite only, this is likely unnecessary.
    2. Observation Fields: Here you can link the fungal endophytes back to the host plants. In the future, we may have further instructions for data to add here.
  3. Complete the project by selecting the Create button.
  4. You may add your MycoEd concierges to your project if you wish (Andrew_wilson, ahonan).
  5. Email Andrew Wilson (andrew.wilson@BotanicGardens.org) to be added to the MycoEd Fungal Genomics Education Project.
  6. Add your project name to the text highlighted in yellow in copies of the following documents:
    1. Module 1 – Using iNaturalist
    2. Module 1 – Sampling Fungal Endophytes and Culturing Student Instructions

Adding your name to the copies of these documents will enable you to use them directly in your courses.

2.2.4 Add Participants (=Students) To The Project

Because we’ve made the Project “Invite Only” you will need to manually invite each of the students to join the course. They are instructed to join the course in Module 1.2.

  1. Open your Myco-Ed project.
  2. Select the “Members” section on the lower right. In this section you can manage membership and participation for the project.
  3. Click on “invite new members here” in the yellow box on the right.
  4. Once you’ve received the iNaturalist User IDs, start adding your students to the blank space under “Invite people…”. Their names/user ID should appear in the drop down menu.

If their names do not show up, it is because they have not completed their iNaturalist registration, or the User ID is different from what they’ve supplied.

  1. Instruct the students to look for their invitation and join the project. They must now add their course’s iNaturalist observations to the project for them to show up in the project.

Use this last step as a requirement for course credit to ensure the process is completed by students.

2.3 Module 0.3 – iNaturalist Checklist

2.3.1 iNaturalist Checklist

This checklist will help you track your progress in managing Myco-Ed project data using iNaturalist.

2.3.1.1 For Instructors

  • Create an iNaturalist Account. (Module 0.2)
  • Create an iNaturalist Traditional Project for your course. (Module 0.2) You may need to request the assistance of the Myco-Ed team to create this if you do not have the pre-requisite number of observations.
  • Tell your students the name of the iNaturalist Project, and request their iNaturalist user IDs.
  • Add the students to the course project once they’ve completed Module 1.2. (Module 0.2)
  • Instruct students on iNaturalist data management obligations for course credit (at the instructor’s discretion).

2.3.1.2 For Students/Participants

Homework Assignment 1 (Module 1.2) - Create an iNaturalist Account. - Get the iNaturalist App for your phone - Give your instructor your iNaturalist User Name. - Update your real name under your account settings. - Watch some videos and tutorials. - Join the course project once you receive an invitation from the instructor/TA.

Make an Observation (Module 1.2) - Take multiple images of the plant host (or habitat photos of the environment) and include at least one image with a unique field number. (e.g., MycoEd_004) - Add the observation to your course project. - Save and upload the observation to the cloud once you’re connected to wifi. - Add Observation Field for MycoEd ID. - Add Observation Field for DNA Barcode ITS once you have DNA sequence data. - Update the name of fungal isolates using DNA sequence BLAST results. - Add Observation Field for Host Observation for fungal isolates linking to the original host on iNaturalist.