Methodology
How Clonar assesses disease resistance
A phenotyping result must be reproducible. Each trial reproduces the three conditions for disease, controls the sources of variation and records the procedures, from the isolate used to the greenhouse temperature.
Disease triangle
Conditions required for disease to occur in the trial
Resistance assessment requires the three conditions for disease to be present at the same time: healthy plant, aggressive pathogen and favorable environment. Without any one of them, the trial does not differentiate the clones.
- Healthy host: seedlings standardized in age, size and health status, so that the differences observed are due to the genotype.
- Aggressive pathogen: isolates from the collection with confirmed pathogenicity and known aggressiveness.
- Favorable environment: temperature, humidity and leaf wetness period adjusted to each pathogen.
The stages
Six stages and the control applied to each
The sequence of stages is the same for all diseases; the protocol for each stage varies with the pathogen.
Choice of isolates
Selection by origin, host and aggressiveness recorded in the collection. For clones intended for several regions, more than one isolate is used.
Control: isolate code recorded in the reportSeedling standardization
Healthy seedlings of the same age and size, acclimatized before inoculation.
Control: off-standard plants discardedRandomization and replicates
Clones and controls randomly assigned within blocks, so that differences in light and humidity in the greenhouse are not confounded with genetics.
Control: trial map archivedInoculum prepared and quantified
Method suited to each pathogen (in the wood, in the soil or on the foliage), with inoculum concentration checked on the day.
Control: inoculum batch and concentrationEnvironment favorable to infection
Temperature, humidity and leaf wetness period adjusted to the pathogen and recorded throughout the trial.
Control: continuous environmental recordMeasurement, analysis and report
Standardized scales and direct measurements, trained evaluators, statistical analysis and classification of each clone.
Control: controls validate the trialExperimental design
Randomization of plant positions within blocks
Inside a greenhouse, light, temperature and humidity vary from one bench to another. If a clone always occupied the most humid position, the effect of the environment would be confounded with the effect of the genotype.
- Blocks: each block contains every clone in the trial, once.
- Randomization: the position of each clone within the block is randomized.
- Controls: one clone of known resistance and one known susceptible clone in every block. If the susceptible control shows no symptoms, the trial is invalidated and repeated.
Illustration of the principle, with 6 clones and 4 blocks. The number of clones, blocks and plants per plot is defined in each proposal.
Rating scale · myrtle rust example
Rating scale for myrtle rust of eucalyptus
For foliar diseases, we use rating scales with an objective description of each class, reproducible among trained evaluators. Select a score to see the symptom and the classification.
Illustrative 0 to 4 scale for myrtle rust of eucalyptus (Austropuccinia psidii), adapted from Junghans et al. (2003), Theoretical and Applied Genetics.
From measurement to classification
Criteria for classifying the clones
1. Measurement
Depending on the disease: lesion length in the wood, percentage of affected leaf area, wilt incidence or scale score.
2. Statistical analysis
Means per clone, variability among replicates and comparison with the controls of the same trial.
3. Classification
Each clone is classified as resistant, tolerant or susceptible relative to the controls, with a recommendation for the program.
Reproducibility
What is recorded in every trial
Records that allow the trial to be repeated, in the same laboratory or in another, and the results to be compared.
- Code, origin and history of each isolate used
- Inoculum batch and concentration on the day of inoculation
- Trial map with the randomized position of each plant
- Temperature and humidity throughout incubation
- Assessment dates, evaluators and photos per clone
Next step
Protocol discussion before contracting
The technical team presents the trial design to the client's breeding team and adjusts the protocol to the objective of the program.
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