In Vivo Leaf Inoculation: An Alternative Method to Assess the Disease Resistance of Hybrid Clones in Poplar Breeding of Stem Canker Disease

The scope of this research protocol is to examine the methodology of pathogen inoculation and disease-resistant assessment methods using the poplar hybrid breeding. We are approaching the in vivo leaf inoculation method as an alternative to te traditional methods with the aim of advancing the breeding of poplar stem canker disease. Using the leaf inoculation method, researchers can screen the disease resistance of large scale hybrid blooms in the second years of hybridization.

Realizing earlier on sliding selections in poplar hybrid breeding of stem canker disease. The most advantageous aspects of the leaf inoculation method are its high efficiency and high throughput. First, the leaf method can sharply shorten the period of disease resistance selection, from five to seven years after hybridization to two years after hybridization.

Second, the method can assess the disease resistance of large scale hybrid clones within several weeks, The leaf inoculation method will advance the hybrid breeding of poplar Botryosphaeria dothidea and Cytospora chrysosperma canker in China, and the poplar hybrid breeding of canker in North America. Moreover, the method can be used to find disease-resistant strains and improve the development of poplar molecular breeding. In addition to poplar hybrid breeding of stem canker disease, the leaf method can be used to screen the disease resistance of natural or mutant poplar populations and the susceptibility of poplar stem canker pathogens.

Moreover, the method can provide pathotypes for the mining of disease-resistant genes and the genetic analysis of disease resistance. Begin by adding potato extraction, dextrose, and agar in water to prepare potato dextrose agar or PDA culture medium for fungal strains. Heat the solution at 100 degrees Celsius for 10 minutes to completely dissolve the components.

Pour PDA medium into 25 milliliter tubes. Sterilize all tubes at 121.1 degrees Celsius for 30 minutes. Then, cool the sterilized tubes on a clean bench to 50 to 60 degrees Celsius.

Pour the medium into 9.0 centimeter diameter culture plates and cool them at room temperature. Next, cut the mycelium of the fungal pathogen cultured in PDA medium at 28 degrees Celsius for seven days into approximately 0.5 centimeter square cubes. Inoculate the fungal cubes at the center of the PDA plates, ensuring the mycelium sides face the medium.

Then, culture the inoculated fungal pathogens in a thermostatic incubator for seven to 10 days. After incubation, cut the PDA medium containing the fungal mycelium into square cubes with sides 1.0 to 1.2 centimeters long. Select the newly matured fully extended leaves from the top of the main branches of one-year-old poplar clones.

Ensure the leaves are free from pests, diseases, and mechanical damage. For one-year-old branches of perennial popular hybrid clones, select the top fifth to seventh leaves of the selected branches as the inoculated materials. Spray the selected poplar clones or branches with clean water.

After drying, wipe the selected leaves with 75%alcohol either one hour or one day before the inoculation manipulations. To begin, prepare fungal culture for canker pathogen and poplar material for leaf inoculation. For small leaves, inoculate two previously prepared square fungal mycelium cubes and PDA cubes onto the upper surface of the top fifth to seventh leaves of poplar clones.

Ensure that the mycelium faces the leaves, and the inoculation sites are located at the center of the half leaves. Inoculate four square fungal mycelium cubes and four PDA cubes onto the upper surface of the top fifth to seventh leaves of large poplar leaves. Then, wrap the inoculated leaves with transparent adhesive tape.

Gently press the tape to make it adhere to the leaves, preventing the movement and water loss of the mycelium cubes during the experiment. Next, using a five milliliter syringe needle, pierce the leaves and the mycelium cubes at five sites until the needle penetrates the cubes from the upper to the lower surface of the poplar leaves. Place one piercing site at the center and the other four one to two millimeters near the four vertices of the cubes.

Inspect the inoculated leaves within three days for any location shifts or water loss of the mycelium innoculants. Observe the onset of necrotic lesions around the piercing wound sites on the lower surface of the leaves. At the end of the experiment, pick off all inoculated leaves and place them in plastic sample bags.

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 pierce sites are significantly larger than those around the PDA-covered sites, classify them as disease sites. If the mycelium-covered ppierce sites show changes like lesion color, hyphy-like structures, pignidia, and canidia, 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 poplar's 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 pignidia-like structures on poplar leaves.

Moreover, the leaf positions and light conditions of the leaves impact the disease severity of the leaves.