TITLE: Phytophthora pluvialis culture cultivation, maintenance, and detached needle assay protocol
AUTHORS: Sophie Eccersall, Leann Seesom Vinson, Rebecca McDougal, Claudia-Nicole Meisrimler

[DESCRIPTION]
Phytophthora pluvialis is an oomycete that primarily infects Pinus radiata and Pseudotsuga menziesii causing the destructive foliar disease red needle cast (RNC). Recent observations show that P. pluvialis can also infect western hemlock inducing resinous cankers. High-throughput and reproducible infection assays are integral to find key information on tree health and oomycete pathogenicity. In this protocol, we describe the propagation and spore induction of P. pluvialis, followed by detached needle assays for verification and quantification of virulence of P. pluvialis in P. radiata needles. These needle assays can be employed for high-throughput screening of tree needles with diverse genetic backgrounds.
In downstream analysis, Quantitative PCR (qPCR) was utilised to assess relative gene expression, as exemplified by candidate RxLR effector protein PpR01.
Additional techniques like RNA sequencing, metabolomics, and proteomics can be combined with needle assays and can offer comprehensive insights into P. pluvialis infection mechanisms





[BEFORE_START]
All steps and procedure of the protocol should be completed in laminar flow hood wherever possible

[GUIDELINES]
*This protocol was developed using a single isolate of Phytophthora pluvialis isolate (NZFS 4015) although other isolates have been trialed. It is known that some variation can exist between isolates, especially if they have been passaged in the lab over a long period. It can be beneficial to passage through pine needles or apple/pear prior to prepping for zoospore production to re-invigorate isolates every three to four months. The use of freshly isolated strains from the field can also be helpful for zoospore production.

This protocol can also be used to induce zoospore release in Phytophthora cinnamomi.

[STEPS]
SECTION: A. Growth of P. pluvialis from existing culture plates
1. Grow P. pluvialis isolate on 20% cV8 agar (modified from Jeffers, 2006) at ~18 ˚C.

SECTION: A. Growth of P. pluvialis from existing culture plates
4. Seal plates with parafilm, and store upside down in an incubator at 17-21 ˚C in the dark. For zoospore production, allow plates to grow for 7-10 days, or until mycelium leading edge has filled the plate. Once mycelium has filled the plate, begin the replating procedure for maintenance.


 

SECTION: A. Growth of P. pluvialis from existing culture plates
3. Subculture existing culture onto fresh cV8 agar plates using the wide end of a pipette tip to create three plugs, and place in a triangular position onto fresh cV8 agar plates with tweezers (see figure 1). 
   

SECTION: A. Growth of P. pluvialis from existing culture plates
2. Sterilise tweezers with 70% ethanol and flame.
SECTION: B. Zoospore preparation
5. Sterilise tweezers and scalpel with ethanol and flame.
SECTION: B. Zoospore preparation
6. Cut ten 1cm×1cm cubes of mycelium grown on a cV8 agar plate (fully grown, 7-10 days) and place into petri dishes containing ~15 mL sterilised cV8 broth. Make sure all cubes are evenly dispersed and floating in broth (see figure 2A).

SECTION: B. Zoospore preparation
7. Seal plate in parafilm, and store in 17-21 ˚C in the dark for two days. Mycelium starts to grow beyond the cut edge of the agar, floating in the cV8 broth (see figure 2A). P. pluvialis isolate 4015 grows the best at 21 °C, but anything in the range of 17-21 °C works well).

   

SECTION: Zoospore preparation step 2, day three
8. Remove broth as thoroughly as possible from plates. 
SECTION: Zoospore preparation step 2, day three
9. Rinse multiple times with SDW to ensure all traces of broth have been removed. This step is critical. The next step is starving the Phytophthora of essential nutrients to encourage the growth of sporangia.
SECTION: Zoospore preparation step 2, day three
10. Soak mycelial mats in SDW for an hour. The mats should be pale pink/orange in colour.

SECTION: Zoospore preparation step 2, day three
11. Remove SDW ensuring all traces of calcium carbonate from the broth have been removed, and replaced with ~15 mL of sterile pondwater (see figure 3A).

   

SECTION: Zoospore preparation step 2, day three
12. Seal plates in parafilm, and store at 17-21 ˚C for 2-3 days in the dark (depending on the appearance of sporangia, see figure 3). Immature sporangia are spherical in shape and will not release zoospores (see figure 3B). Mature sporangia have a  characteristic semi papillate, ovoid shape (see figure 4A). 

   

SECTION: Zoospore production step 3, day six/seven
14. Place under bright light for at least five minutes.
SECTION: Zoospore production step 3, day six/seven
13. If mature sporangia are present (see figure 4A, B), place plates into a 4 ˚C fridge for at least one hour at minimum. Plates must be cold to the touch.
SECTION: Zoospore production step 3, day six/seven
16. If no zoospores have been released, place back under the light for intervals of 10 minutes, followed by viewing under the light microscope (10× lens) to check for release.




SECTION: Zoospore production step 3, day six/seven
15. Place under microscope at 10× magnification to observe zoospore release (see video 1 & 2).


 

 

SECTION: Zoospore production step 3, day six/seven
17. If zoospores have still not released, repeat chilling and light treatment. If no release occurs after 
30 min, leave plates for another half to full day in the fridge (plates can be left in the fridge overnight).
SECTION: Zoospore production step 3, day six/seven
18. If zoospore release is successful, prepare needle assays. P. pluvialis zoospores have a short life span of ~2 hours, use quickly.
SECTION: Zoospore production step 3, day six/seven
19. Once zoospore solution has been siphoned off, plates can be reinvigorated with cold sterile pondwater and light treatment to release a second and third wave of zoospores.
SECTION: Zoospore production step 3, day six/seven
20. Use a haemocytometer (Marienfeld Superior, Neubauer- improved cell counting chamber) and 10-20 µL of zoospore solution to count spores under a light microscope at 10× magnification. Count at least 6 samples per plate and average. Aim for a working concentration of 5 x 103 zoospores/mL. This concentration can be adjusted as needed.




SECTION: Zoospore production step 3, day six/seven
20.1. Troubleshooting. Release of zoospores is crucial to effective detached needle assays. If there is a lack of mature sporangia, place the plates with SPW and mycelial mats under a bright lamp for 
24 hours. Check for the presence of mature sporangia. Following this, if there are still no mature sporangia, place the same plates into the fridge overnight, and then back under the lamp. Occasionally, this will need to be done several times to push the sporangia to mature and allow for zoospore release. 




SECTION: C. Detached Needle Assays
21. Harvest fresh pine needles (of any age, although best results are seen on needles of at least one year old).
SECTION: C. Detached Needle Assays
22. Take 5 mL of zoospore solution and place into 50 mL falcon tubes. Be gentle when handling the zoospore solution - P. pluvialis zoospores encyst very easily when knocked.
SECTION: C. Detached Needle Assays
23. Place needles fascicle end down into zoospore solution.
SECTION: C. Detached Needle Assays
24. Seal, cover tubes in foil and leave overnight standing upright.
SECTION: C. Detached Needle Assays
25. 18-24 hours later, prepare petri dishes by layering 3-4 sterilised paper towels into a dish.
SECTION: C. Detached Needle Assays
26. Thoroughly dampen the paper towels with sterile H2O.

27. Place needles incubated with spores as flat as possible into the dishes, seal with parafilm (see figure 5).

  

 

SECTION: C. Detached Needle Assays
28. Place into an incubator (temperature set at 17˚C; 12 hours daylight, 12 hours darkness), and check at 3 days after infection (DAI), 7 DAI and 12 DAI intervals (see figure 6).

   

SECTION: D. Symptom monitoring and scoring
29. Measure and record the total number, length, and severity of lesions (see figure 6 and 7).

  

SECTION: D. Symptom monitoring and scoring
30. Note that symptoms may appear differently depending on the age of the needles. Needles from young plantlets show more yellowing and less discrete olive bands.
SECTION: E. Passaging Isolates
31. Take infected needles from assay plates or collected in the field from infected trees and rinse with 70% ethanol, follow by washing with sterile H2O, repeat 3-times.
SECTION: E. Passaging Isolates
32. Pat dry with sterile paper towel and place on antibiotic cV8 agar plates (cV8 agar recipe, with the addition of rifampicin (10 mg/mL), carbenicillin (100 mg/mL), nystatin (25mg/mL)).
SECTION: E. Passaging Isolates
33. Allow mycelium to grow on plates, this can take up to 7 days (see figure 8).

 
  

SECTION: E. Passaging Isolates
34. As soon as possible, transfer plugs of mycelium onto new non-antibiotic cV8 plates. 
SECTION: E. Passaging Isolates
34.1. Passaging isolates can be used for spore production. Approximately 21 days after infection, new sporangia are visible growing out from the lesions on the pine needle (see figure 9). These sporangia can also be used for zoospore release by placing them in cold SPW and placing them under bright light. 

   

SECTION: Confirmation of infection via qPCR
35. Extract RNA from mycelium, non-infected and infected needle samples at the preferred time point using users RNA extraction kit of choice.
SECTION: Confirmation of infection via qPCR
36. Measure extracted RNA concentration either via Quibit™ fluorometer (recommended) or equivalent.
SECTION: Confirmation of infection via qPCR
37. Using the measured RNA concentrations, dilute all samples to 50 ng/µL.
SECTION: Confirmation of infection via qPCR
38. Perform reverse transcription on the RNA samples using an appropriate kit of choice. You will need a cDNA concentration of 50 ng/µL for each sample. 
SECTION: Confirmation of infection via qPCR
39. Serially dilute the primers for the RxLR PpR01 and actin from 1:106.
For primer sequences, see materials section.
SECTION: Confirmation of infection via qPCR
40. Perform the qPCR according to manufacturer's instructions. If using the Thermofisher™ PowerTrack™ SYBR Green Master Mix, use the following settings:
 
     Step      Temperature      Time      Cycle        Enzyme activation      95℃      2 minutes      1        Denaturation      95℃      15 seconds      40        Extension      60℃      60 seconds   



 
  
 
SECTION: Confirmation of infection via qPCR
41. Analyse qPCR results with the appropriate software.