TITLE: Culturing of infective agents from infected wheat leaves
AUTHORS: Benjamin Schwessinger, Erin Hill, Peter Solomon

[DESCRIPTION]
This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics". 

You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen.  You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments.

In total, each group will obtain five pots each:


 


This specific protocol is a step by step guide to culture infective agents out of infected wheat leaves. This process will take three weeks to complete, therefore the protocol is applicable for week 2-5. 

The final goal is to isolate the disease causing agent, if at all possible, via culture media. Of course not all pathogens are culturable and the protocol is geared towards filamentous pathogens which are the most severe pathogens of wheat in Australia. So the specific approach to culturing and the identity of the disease causing agent defines the possibility of the organism being cultured. Something to keep in mind when interpreting the results.

Protocols progress overview:

Week 2 you will identify suitable infected leaf material, surface sterilise it, and culture cut leaf pieces on water agar to enable microbial growth.
Week 3 you will identify organisms that have grown out of the leaf onto the agar and subculture them onto high nutrient agar plates.
Week 4 you will take pictures of the subcutlured organisms and try to classify it them based on morphological features. 
Week 5 you will take pictures of the subcutlured organisms and try to classify it them based on morphological features. 


[BEFORE_START]
You must study the protocol carefully before you start. If anything is unclear post questions directly here on protocols.io.

[GUIDELINES]
You must have read, understood, and follow the health and safety instructions provided in the "Overview Mini-Research Project #1 BIOL3106/6106" provided on Wattle (ANU learning portal). 

You must have signed and returned one copy of the "Student Safety Declaration Form For Practical Class Work" before starting the any laboratory work. 

You must have read and understood the Hazard Sheets (Risk assessment) of all chemicals listed bellow in the "Safety Warnings" section. These Hazard Sheets are provided on Wattle as part of the  "Overview Mini-Research Project #1 BIOL3106/6106" document.

[STEPS]
SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
2. Carefully, study the plants in each pot and select two leaves with symptoms of infected plants (if possible) and control leaves of uninfected plants. Select two leaves for each treatment group.
SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
3. Perform the following steps [3.1 to 4.4] for each treatment group [#1-5] independently and consecutively. This will reduce the risk of cross contamination. Sterilise your equipment after each step. Start with the uninfected control samples treatment group #5 [TG#5].
SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
5. Incubate the plate at 16-20oC for 7 days 10080 min .

SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
3.1. Clearly, label the provided falcon tube with 1% hypochlorite solution with the treatment group number [eg TG#5] and your group identifier.
SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
3.2. Using the provided scissors, Cut the leaves into about 1 cm long pieces and place the pieces into the labelled falcon tube with 1% hypochlorite solution.

This step aims to perform a surface sterilisation of the leaf to avoid contamination of fungi and bacteria that are attached to the leaf surface. Fungi and bacteria within the leaf will not be killed with this treatment. 
SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
3.3. Incubate the leaf pieces for about 1 min  1 min with slight movement tipping the falcon tube upside down multiple times.

SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
3.4. Discard the 1% hypochlorite solution into the provided beaker while making sure the leaves stay within the falcon tube.
SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
3.5. Wash the leaves within the falcon tube with about 10 ml sterilised water for about 1 min 1 min each. The water is provided to you on the bench in a flask. Discard the water into the provided beaker while making sure the leaves stay within the falcon tube..

SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
3.6. Repeat step 3.5 two more times.  

SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
4. Now make sure that you work under the clean area close to the bunsen burner. 

 

The following steps will place the leaf pieces on the water agar plates (TWA) to try to culture the infective agent from the leaf material, if at all possible with this specific culture approach.
SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
4.1. Obtain one TWA plate and label it with the treatment group name, your research group name, and the date. 
SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
4.2. Using sterile forceps, place 4-5 leaf pieces equally distributed around the plate on the surface of the water agar. 
SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
4.3. Carefully close the plate and wrap it in clingfilm. 
SECTION: Week 3: Subculture of microbial outgrowth from leaves.
6. We would expect that there is filamentous or bacterial growth at the edges of the leaf if the infectious agent is culturable on agar and under the tested conditions. You will now aim to subculture one representative microbial outgrowth per treatment group on rich PDA media.
SECTION: Week 3: Subculture of microbial outgrowth from leaves.
7. Perform the following steps [7.1 to 7.10] for each treatment group [TG#1-5] independently and consecutively. This will reduce the risk of cross contamination. Sterilise your equipment after each treatment group. Start with the uninfected control samples TG#5.


 

SECTION: Week 3: Subculture of microbial outgrowth from leaves.
7.1. Unwrap the plate but keep the lid on.
SECTION: Week 3: Subculture of microbial outgrowth from leaves.
7.2. Close to the sterile bunsen burner area, remove any condensation that might have formed on the lid and place the lid back on top of the plate.
SECTION: Week 3: Subculture of microbial outgrowth from leaves.
7.3. Take a picture of the plate on a white background or a light box. Place a ruler and a label that clearly identifies the treatment group next to the plate before taking a picture. This will allow you to clearly identify the plate later on and add a scale bar to the picture digitally if required.
SECTION: Week 3: Subculture of microbial outgrowth from leaves.
7.4. For each treatment group, identify one representative out growth you intend to subculture.
SECTION: Week 3: Subculture of microbial outgrowth from leaves.
7.5. Obtain a PDA plate and label it with the treatment group name, your research group name, and the date.
SECTION: Week 3: Subculture of microbial outgrowth from leaves.
7.6. Close to the sterile bunsen burner area, cut out the representative outgrowth with a sterile scalpel. 
SECTION: Week 3: Subculture of microbial outgrowth from leaves.
7.7. Place the cut out piece in the center of the PDA plate using sterile forceps. 
SECTION: Week 3: Subculture of microbial outgrowth from leaves.
7.8. Close the plate and seal it with a mircotape. 
SECTION: Week 3: Subculture of microbial outgrowth from leaves.
8. Incubate the new PDA plate at 16-20oC for 7 days 10080 min 

SECTION: Week 4: Carefully assess and record representative microbial growth
9. We would expect extensive growth of the subcultured microbes on the rich PDA media. You will have to assess and record the microbial growth. This will include taking pictures of each plate.
SECTION: Week 4: Carefully assess and record representative microbial growth
10. Perform the following steps [10 to 10.5] for each treatment group [#1-5] independently and consecutively. Start with the uninfected control samples TG#5.
SECTION: Week 4: Carefully assess and record representative microbial growth
10.1. Unwrap the plate but keep the lid on.
SECTION: Week 4: Carefully assess and record representative microbial growth
10.2. Remove any condensation that might have formed on the lid and place the lid back on top of the plate. You can perform this step on your bench as you will discard the plate after this weeks prac.
SECTION: Week 4: Carefully assess and record representative microbial growth
10.3. Take a picture of the plate on a white background or a light box. Place a ruler and a label that clearly identifies the treatment group next to the plate before taking a picture. This will allow you to clearly identify the plate later on and add a scale bar to the picture digitally if required.
SECTION: Week 3: Subculture of microbial outgrowth from leaves.
7.10. Discard the old TWA plate in the appropriate waste bin.
SECTION: Week 4: Carefully assess and record representative microbial growth
10.4. Carefully assess the type of microbial growth on the plate including provided microscopes and describe the characteristics of the microbes in your lab notebook. You can compare them with the disease compendium, other provided literature, and online resources. 
SECTION: Week 4: Carefully assess and record representative microbial growth
10.5. Add your plates to the collection of plates for the whole class at one of the outer benches. This allows us to compare the subcultures obtained across the whole class. This way we can compare the replicates of each TG across the whole class becoming more confident. In addition, we will provide reference PDA plates for some of the pathogens.
SECTION: Completion and figure generation
12. This represents the end of the lab part of this protocol. We provide instructions on how to make figures as part of online resources on Wattle.
SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
1. You will receive five pots with wheat plants as detailed in the "Description" section. Please label each pot with your group name, the date, and the treatment group. 
SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
4.5. Sterilise your forceps as indicated by the demonstrator before moving to the next treatment group in    .

SECTION: Week 3: Subculture of microbial outgrowth from leaves.
7.11. Sterilise your forceps as indicated by the demonstrator before moving to the next treatment group i .

SECTION: Week 2: Selection of representative leaf samples, surface sterilisation, and initial culturing.
4.4. Take a picture of your plate for your records and include it in lab archives.
SECTION: Week 3: Subculture of microbial outgrowth from leaves.
7.9. Take a picture of your plate for your records and include it in lab archives.
SECTION: Week 5: Carefully assess and record representative microbial growth
11. We might get the cultures back for one last inspections depending on microbial growth during the prac. If so we will repeat the same as week 4 [10.1-10.5].