TITLE: Wheat disease symptoms observation, capture, description, and evaluation
AUTHORS: Erin H Hill, Benjamin Schwessinger

[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 capture and describe the disease symptoms the provided wheat plants display in week 2 and 3. You can then compare the observed disease symptoms with the provided compendium and other online sources to try to identify the causal infective agent.

The final goal is to achieve the following:
obtain high quality images of your diseased leaves for each treatment group that are representative. These pictures can be taken with your mobile phone for the complete attached leaves lined up on black cardboard paper or via the stereo microscope as demonstrated by the support staff.
provide a detailed description of the symptoms observed on the leaves. This includes the following: size, shape, form, color, microscopic structure, spore shape and color, and timing of disease symptom development post infection.
Compare your observed disease symptoms with the uninfected negative control, the provided compendium, and online sources.

This protocol is applicable for week 2 and 3. 

Protocols progress overview:

Week 2 observe, capture, and describe wheat leaf disease symptoms.
Week 3 observe, capture, describe, and evaluate wheat disease symptoms.
 
 
 
 
 
 
 
 
 
 
 
 

[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: Disease symptom capture
1. You will receive five pots with wheat plants as detailed in the "Description" section. Please label each pot with your research group name, the date, and the treatment group. 
SECTION: Disease symptom capture
2. Carefully, study the plants in each pot. Select four to five leaves for each treatment group that show representative symptoms [TG#1-4] or not in the case of the negative control [TG#5].
SECTION: Disease symptom capture
3. Perform the following steps [3.1 to 3.11 and 4] for each treatment group [#1-5].
SECTION: Disease symptom capture
3.2. Obtain one black cardboard paper. Label it with your research group name, the date, and the treatment group.
SECTION: Disease symptom capture
3.1. Layout your cardboard paper vertically [aka short side facing you] in front of you. 
SECTION: Disease symptom capture
3.3. Mark the bottom side closest to you of your black cardboard paper with a horizontal line approximately 1 cm from the edge. Make this line parallel to the edge. This will be called the baseline.
SECTION: Disease symptom capture
3.4. For your each treatment group, one after another, cut the selected leaves at the base.
SECTION: Disease symptom capture
3.6. Line up your cut leaves with the leaf apex [tip of the leaf] pointing away from the baseline and the leaf base aligned to the baseline. 
SECTION: Disease symptom capture
3.7. Mark the position of the tip of the leaf with a pen. This mark is called the tip-mark.
SECTION: Disease symptom capture
3.8. Attach one strip of double-sided tape at the bottom baseline. 
SECTION: Disease symptom capture
3.9. Attach one strip of double-sided tape parallel to the bottom baseline at a distance about 1 cm below the tip-mark. 
SECTION: Disease symptom capture
3.10. Attach your cut leaves to the cardboard with the leaf base affixed to the lower baseline double-sided tape and the tip affixed to the double-sided tape close to the tip-mark.
SECTION: Disease symptom capture
3.11. Place a ruler next to your leaves and take a clear picture with a camera. Having clear labels and a ruler will allow you to easily identify the specific sample later on and add a scale bar to the picture digitally if required. You will know how long 1 cm is based on the ruler. Ideally, you take the picture holding your camera completely parallel to your leaves mounted to the cardboard. 
SECTION: Disease symptom capture
3.5. Cut your selected leaves for the treatment group you are currently working on.
SECTION: Disease symptom capture
4. In addition, you can also take pictures of the disease symptoms with the stereo microscope. Please see a demonstrater for hands on instructions. The microscopes will be connected directly to a laptop. Alternatively you can connect via the Leica Airlab app (Google Play or App Store).

Make sure to label your pictures as follows so we can share them online after class and you can clearly identify your pictures. 

YYYYMMDD_ResearchGroupName_TreatmentGroupNumber_PictureNumber

For example:

20230206_BSPS_TG1_2.jpg

Pictures that do not follow this naming convention will not be shared with the class.
SECTION: Disease symptom capture
5. Repeat all steps for each treatment group in week 2 and week 3.