Best Practices

1. Best Practices For researchers using Biotech Support Facility & Rightmire Hall Growth Chambers and Greenhouse

2. Introductions • Gary Posey, Greenhouse Superintendent • Day to Day at BSF & Rightmire • posey.8 292-9241 • Joan Leonard, Program Manager • Day to Day at Bio Sci Greenhouse • Oversee renovations and implementation of best practices at BSF & Rightmire • leonard.4 292-7904

3. Why? Reduce/ eliminate pest and disease issues & improve plant health Increase experiment success Improve uniformity and repeatability Abide by all regulations set by NIH, APHIS, EPA, ODA and Federal laws

4. Planting & Growing “Do’s” • DO use clean and sterile pots and trays • DO label all pots/flats with researcher name -unlabeled plants will be considered abandoned and will be disposed of. • DO surface sterilize seed (10% bleach, 10 min.) • DO follow order of entry to chambers/growth rooms/greenhouse • DO water and fertilize properly (use care not to overwater) • DO wear dedicated lab coat for each growing space • DO know how to ID thrips, fungus gnats and signs of INSV • Do make sure all staff and students have read the best practices and met with Gary for a training session.

5. Potting Room 100 BSF • No seeds are permitted in the potting area, room 100 BSF to prevent contamination of media • Fill pots in 100 BSF then plant seeds in room 160A, the greenhouse or your lab • For Arabidopsis, 3 scoops of media = 1 flat. Incorporate 2 TBL of osmocote for each flat prepared.

6. Photo courtesy of Purdue University

7. Proper Watering Arabidopsis IS NOT AQUATIC! Overwatering: #1 cause of plant diseases, pest problems and plant death! Too much-Too often. Overwatering weakens roots and allows pests and diseases an opportunity Never leave plants in a tray of standing water. Soil will reach saturation within 10 minutes—excess water in trays after this will only weaken the plants. Green algae growing on soil surface is sure indication of overwatering. Plants found standing in trays of water will have holes poked into the trays!

8. Quantity Watch nested flats: water can get “trapped” Only water pots/cells that are dry instead of whole flat. Know if plants need water—what is dry? If bottom watering whole flat, add only enough water to cover bottom of flat. This should get taken up within a few minutes. If not, then plants were not thirsty. Pour off excess water after 10 minutes! Never leave standing in water longer than 30 minutes. Water not used in 30 minutes will deprive roots of oxygen.

9. Frequency • Check daily BUT only water as needed! • Water individuals when dry rather than watering all plants in flat at same time • Don’t follow a rigid watering schedule • Avoid “watering insurance” • This is overwatering to compensate for not checking on plants daily. • Heavy watering on Friday will make sure plants don’t need any over weekend and possibly even until following Friday or longer! • No need to overwater to make it through the weekend. A well-watered plant on Friday will be fine on Monday. • Rarely have we seen underwatered plants! This is easily fixed by adding water. Overwatering cannot be easily fixed.

10. Disposal “DO’s” • DO cover all plants during transport and disposal • DO promptly dispose of unnecessary plants in yellow steam carts (no plastics, no stakes, etc) • DO keep growing spaces and adjacent areas, and potting room 100 clear of debris/dirty pots/dead plants • DO report infestations of pests and diseases to manager • DO immediately discard all INSV infected plants • DO make sure all lab staff are educated in procedures and are following them. This is the responsibility of the PI.

11. Handling “DON’Ts” • DON’T leave chamber/rooms doors hanging open • DON’T move plants from one chamber/room to another • “DON’T put all your eggs in one basket.” (Stagger germination dates and location of valuable plants to increase chances of success.)

12. Thrips ID • Western Flower Thrips Adults are less than 1/20th of an inch (<2mm) long Eggs are laid in epidermis on underside of leaf Only larval stages acquire INSV while feeding Pupal stages occur in soil Adults spread INSV when feeding

13. Fungus Gnat ID Larvae feed on plant roots & spread diseases Slows plant growth & can kill seedlings Thrive in wet soils Best control is prevention through proper watering—do not overwater

14. Powdery Mildew Common in crowded conditions with poor air circulation, low light and dampness—AKA a growth chamber! Spores are short-lived and need moisture to germinate. Sleeved plants are especially susceptible—cut holes in sleeves for better air flow. Water carefully!

15. INSV in Arabidopsis • Dead Leaves

16. INSV in Arabidopsis • Yellow Mid-Rib

17. INSV in Arabidopsis • Purple Leaves

18. INSV in Arabidopsis • Random Death

19. INSV Handling • Plants testing positive for INSV will be immediately removed from growth spaces and destroyed. • No new plants will be permitted in an infected space. • Any plant to be removed from an infected space must be bagged before removal. • Anyone working in an INSV infected space is to immediately exit the building when work is completed- they may return the next day.

20. Beneficials • N. cucumeris Feeds on Thrips larvae

21. Beneficials • Hypoaspis miles Feeds on Thrips pupae and Fungus Gnat larvae

22. Beneficials • Nematodes feeds on Fungus Gnat larvae

23. Space Rotation It is critical to break the thrips lifecycle by enforcing a chamber clean out every 3 months. • All chambers in 005 Rightmire and in Biotech Support Facility are common shared space. • Researchers requiring a chamber with special temp or daylength for a specific project will need to pre-arrange space with Gary. • There will no longer be any continuous cycling in chambers—all chambers will enter the rotation cycle.

24. Space Rotation Detail • Growth spaces will have a 6 week window for planting. This window may close sooner than 6 weeks IF space is full or INSV is detected. (Check for available space BEFORE planning to plant!) • Spaces will have posted on them the last date for new plants. No new plants will be allowed in the space after the end date —even if there is space available. • No plants are permitted to move from one space to another. • Once a space is empty, it will be cleaned by GH staff and heat treated for 4 days, then reopened for planting.

25. Order of Entry • Order of entry is posted on the dry erase boards in the BSF and Rightmire 005. Information is updated weekly based on actual pest counts. • Users should note these daily as they go about their work to make sure they are following the correct order of entry. • Users who have entered a space lower on the list cannot enter or re-enter a space above it on the list until the next day. • Dedicated lab coats are required for all growth spaces.