To begin, take canker inoculated poplar leaves kept in plastic bags. Carefully remove the plastic protective films from the leaves and gently remove the mycelium innoculants from the leaf surfaces. Observe and identify the leaf pathotypes of each poplar clone by examining the shape and color of necrotic spots.
Then place leaves in a ruler on a black background. Photograph the lower surface of the leaves with a camera, or scan them using a scanner to obtain images with a resolution of over 300 dots per inch. Open the images of the diseased leaves in the software ImageJ, 1.54G.
Set the scale in ImageJ according to the ruler visible in the leaf images. Then using the Wand tool in ImageJ, identify the lesions on the leaves and measure the area of each necrotic spot. After measuring all necrotic spots, record the area values and export these values as a spreadsheet for further analysis.
If the lesions around the mycelium covered peer sites are significantly larger than those around the PDA covered sites, classify them as disease sites. If the mycelium covered peer sites show changes like lesion color, hyphae-like structures pycnidia and conidia, define these as disease sites. Calculate the average area of effective PDA lesion spots for each poplar hybrid clone.
Divide the mycelium inoculated spots into two categories, onset and non-onset. Then calculate the tested poplars clone disease incidence rate. Calculate the average area of the diseased spots on leaves.
Based on the average areas of diseased spots across all tested poplar clones, set a five level disease grading standard. Calculate the disease index of each poplar clone using the five level disease grading standard. Next, using the Shapiro-Wilk test, verify the normal distribution of the numbers of poplar clones across different resistance levels.
Based on the disease index, classify all tested poplar clones into five or seven resistance groups. Very high resistance, high resistance, resistance, no resistance, and no susceptibility, susceptibility, high susceptibility, and very high susceptibility. Inoculation of stem canker pathogen induced necrotic lesions, and even pycnidia-like structures on poplar leaves.
Moreover, the leaf positions and light conditions of the leaves impact the disease severity of the leaves.
