TITLE: Production of Rhizoctonia solani inoculum for sugar beet disease trials 
AUTHORS: bradley.sowder, Travis Vagher, nicholas.metz, Amy Nielson, kevin.dorn

[DESCRIPTION]
Rhizoctonia solani is an economically important plant pathogen resulting in substantial losses of crop production within the United States and worldwide. In order to properly study resistance to this pathogen, having a consistent, uniform disease pressure of correct intensity is essential to effectively screen plant cultivars for relevant disease traits. This procedure details the process of producing inoculum in order to create a uniform Rhizoctonia solani disease trials. 

[GUIDELINES]
always use aseptic technique.

[STEPS]
SECTION: Preparation of culture for long term storage
1. Obtain desired isolate from either fungal isolation techniques or material transfer from a third party resource.
SECTION: Preparation of culture for long term storage
2. Prepare petri plates containing 25ml of potato dextrose agar and place sterilized medium to be colonized for long term storage atop of petri plate.
SECTION: Preparation of culture for long term storage
2.2.  a.      Sterile concentration disks or sterile barley kernels used as medium to be colonized for long term storage.

SECTION: Preparation of culture for long term storage
2.3. 
 b.       Roughly 10 sterile kernels or Blank sterile concentration disks per petri dish.


 
SECTION: Preparation of culture for long term storage
3. Using aseptic technique, in a biological safety cabinet, transfer desired fungal isolates onto petri plate described in step two of preparation of culture for long term storage.
SECTION: Preparation of culture for long term storage
4. Incubate petri plates at 25°C with a photoperiod of 16 hours for 4-5 days to fully colonize plate. (Webb et al., 2011, doi:10.1111/j.1744-7348.2011.00464.x)


 

SECTION: Preparation of culture for long term storage
5. Once the petri plate is fully colonized, in a biological safety cabinet, using aseptic technique, transfer sterile medium colonized with isolate of interest into sterile cryogenic tubes and store at -80°C up to 21-26 months. (Sneh & Adams, 1996)
SECTION: Preparation of culture for long term storage
5.1. a.       This will be your stock (generation 1, G1) for long term storage.  Preparation of working cultures will be derived from this long term storage.
SECTION: Preparation of working cultures from long term storage
6. Obtain isolate of interest from long term storage.
SECTION: Preparation of working cultures from long term storage
7. Using aseptic technique, in a biological safety cabinet, transfer 5-10 pieces of colonized medium from long term storage onto a petri plate containing 25ml potato dextrose agar and incubate at 25°C with a photoperiod of 16 hours for 4-5 days to fully colonize plate.
SECTION: Preparation of working cultures from long term storage
7.1. a.       This will be the first generation colony. (G2)
SECTION: Preparation of working cultures from long term storage
7.2.  b.       These plates will be your working cultures for inoculum production.


 

SECTION: Preparation of working cultures from long term storage
7.4. i.      These plates should be kept in incubator at 25°C with a photoperiod of 16 hours for the duration of production.
SECTION: Preparation of working cultures from long term storage
8. If production requires more plates, using a sterile core borer, transfer G2 colony core onto a new PDA petri plate containing potato dextrose agar to form next generation (G3).
SECTION: Preparation of working cultures from long term storage
8.1. a.       On account of genetic drift, do not exceed past third generation (G3) (Lakshman et al., 2016)        
SECTION: Potato dextrose liquid broth inoculum production
9.  Prepare 400ml potato dextrose broth (PDB) according to manufactures specifications in a 500ml baffled cell culture Erlenmeyer flask with a vented Duocap.


 

SECTION: Potato dextrose liquid broth inoculum production
9.1. a.       Filter of the vented Duocap should be .22 microns.
SECTION: Potato dextrose liquid broth inoculum production
10. Autoclave flask with PDB at 250°F (121.1°C) for 30 minutes on liquid cycle.
SECTION: Potato dextrose liquid broth inoculum production
10.1. a.       Make sure filter cap is loose on the flask with the secondary cap for the filter tightened.
SECTION: Potato dextrose liquid broth inoculum production
11. Tighten the lid to the flask and allow flasks containing PDB to cool at room temperature.


  

SECTION: Potato dextrose liquid broth inoculum production
12. Once cool, using aseptic technique in a biological safety cabinet, inoculate each flask with several (2-4) cores from a fully colonized working culture petri plate.
SECTION: Potato dextrose liquid broth inoculum production
12.1. a.       Use a sterile core borer for cores.
SECTION: Potato dextrose liquid broth inoculum production
13. Remove the secondary cap to expose filter, while the cap is still tight onto the flask, and incubate on a shaker for 7 days at 200 RPM at 25°C.  


  

SECTION: Spawn bag production
14.  Fill a type 10B polypropylene spawn bag with 2L of hull-less barley (~1.7kg) and add 550ml NANOpure filtered water.


 
SECTION: Spawn bag production
14.2.   a.     Seal bag with impulse poly bag sealer.


  
SECTION: Spawn bag production
14.3. b.       Mix barley and water in bag and let rest horizontally with filter facing up for 1 hour.
SECTION: Spawn bag production
15. Place spawn bags into cotton cloth bags and place them into autoclave pans vertically.
SECTION: Spawn bag production
15.1. a.       Spawn bags are placed into cloth bags in order to not melt plastic spawn bags on metal autoclave pans.
SECTION: Spawn bag production
16. Autoclave spawn bags at 250°F (121.1°C) for 45min sterilization and 15min evacuation on liquid cycle.
SECTION: Spawn bag production
16.1. a.       Repeated two times in order to fully sterilize barley.
SECTION: Spawn bag production
17. Remove spawn bags and set out to cool horizontally with filter facing up until room temperature.
SECTION: Spawn bag production
17.1. a.       Bags should not exceed 24hours of cooling.
SECTION: Spawn bag production
18. Using aseptic technique, in a biological safety cabinet, add 4ml of antibiotic (10,000 ug/ml penicillin and 10,000 ug/ml streptomycin) to each colonized flask described in “potato dextrose broth inoculum production” step.


   

SECTION: Spawn bag production
18.1. a.       Let sit for 15min.
SECTION: Spawn bag production
19. Using aseptic technique, in a biological safety cabinet, open sterilized spawn bags and transfer one colonized flask of liquid inoculum into bag.
SECTION: Spawn bag production
19.1. a.       Seal spawn bag using impulse poly bag sealer.
SECTION: Spawn bag production
20. Mix sealed spawn bag of barley with liquid inoculum and incubate horizontally with filter facing up at 25°C with a photoperiod of 16 hours and humidity at 50-70%.


  

SECTION: Spawn bag production
21. After 24hours, mix spawn bag containing barley and liquid inoculum by hand and return to incubator vertically for 5-10 days or until fully colonized.


  

SECTION: Spawn bag production
21.1. a.       Check spawn bags daily for contamination.


 

SECTION: inoculum
22.  Once barley is fully colonized with desired isolate, open bags and let air dry on trays until completely dry.


 
SECTION: inoculum
22.2.  b.       This gives final colonized inoculum for application.


 

SECTION: inoculum
22.1.  a.      Further grinding of barley using a Wiley mill with a 4mm sieve to reduce particulate size for application may be performed after drying of colonized barley kernels.


  

SECTION: Preparation of working cultures from long term storage
7.3. c.    check plates daily for contamination. 

  
SECTION: Potato dextrose liquid broth inoculum production
13.1. a.    check flasks daily for contamination. 


  

None. 
SECTION: Preparation of culture for long term storage
1.1. For sugar beet, the Rhizoctonia solani strain R9 (AG2-2IIIB) has been used for identifying lines of sugar beet with strong resistance.
SECTION: Preparation of culture for long term storage
2.1. Suspend 39g of Difco™ Potato Dextrose Agar in 1L of purified water. Mix and heat with frequent agitation until the powder is completely dissolved. Autoclave at 121°C for 15 minutes.  Let cool and aseptically, in a biological safety cabinet, pipette out 25ml for each sterile petri plate. Let solidify and return to sleeve for future use.
SECTION: Spawn bag production
14.2. 
SECTION: Spawn bag production
14.1. NANOpure water is water purified using Barnstead/Thermolyne Nanopure lab water system which produces NCCLS, CAP, or ATSM type 1 water with having 18.2 megohm purity with little to no biologicals present. 
SECTION: Spawn bag production
21.3. Contaminated spawn bags should be autoclaved at 121°C for 30 minuets and disposed. 
SECTION: Spawn bag production
21.2. Bacterial contamination of spawn bags show a "soupy" non cohesive barley with an unpleasant "sour" smell.
Fungal contamination of spawn bags show differing colors and possible hyphae that form spores.
R9 should appear only white and cohesive throughout the barley without any fruiting or spore structures. 
SECTION: Spawn bag production
21.4. 
SECTION: Spawn bag production
21.3. If contamination is found, autoclave 
SECTION: Inoculum
22. Once barley is fully colonized with desired isolate, open bags and let air dry at room temperature (20°C) with circulating air and a relative humidity of ~15%  until completely dry. 
SECTION: Inoculum
22.1. Once Barley is completely dry using the proper PPE (mask, safety glasses/goggles, long shirt/pants, and hearing protection) grind the dry inoculum with a Wiley mill fitted with a 4mm sieve to reduce particulate size for application.
SECTION: Inoculum
22.2. This gives final colonized inoculum ready for application. 
SECTION: Inoculum
24. 
SECTION: Inoculum
23. Once dry, using proper PPE (mask, safety glasses, hearing protection, long shirt/pants and safety boots) grind barley using a Wiley mill with a 4mm sieve to reduce particulate size for application.
25. 
24.