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

Summary

We provide a step-by-step protocol for assessing poplar resistance to stem canker pathogens using an in vivo leaf inoculation method. This method is especially suitable for large-scale assessment of Cytospora chrysosperma and Botryosphaeria dothidea canker disease resistance in poplar breeding progeny in China.

Abstract

Stem canker diseases caused by the pathogen Cytospora chrysosperma (Pers.) Fr.) and Botryosphaeria dothidea (Moug. ex Fr.) Ces. & de Not. are the two major forest diseases in the poplar plantations in China, sometimes which can destroy all the poplar seedlings or severely damage mature poplar forests. Hybrid breeding is the most direct and efficient method of controlling and managing tree diseases. However, assessing disease resistance or selecting disease-resistance clones based on In vitro stem inoculation is inefficient, time-consuming, and expensive, limiting the development of hybrid breeding of poplar stem canker disease. In this study, we proposed an alternative method to assess disease resistance to stem canker pathogens through in vivo leaf inoculation. The test materials used in this method can be on 1-year-old poplar saplings or the annual branches of perennial poplars in the greenhouse or the field. The critical step of this alternative method is the selection of inoculating leaves: the 5-7^th newly matured leaves might be the most suitable. The second critical step of the leaf inoculation method is to make wounds on plant leaves through needle pierces, providing sufficient lesions to measure disease severity. For the adequate number of leaves produced in the early stage of poplar breeding, this in vivo leaf inoculation contributes to the rapid, accurate, and large-scale screening of the disease-resistance poplar clones to stem canker pathogens. Moreover, this leaf inoculation method will also serve as an efficient method for screening pathotypes of stem canker disease pathogen C. chrysosperma, B.dothidea, or other poplar stem canker pathogens.

Introduction

Poplar stem canker diseases, mainly caused by two necrotrophic pathogens, Cytospora chrysosperma (Pers.) Fr. and Botryosphaeria dothidea (Moug. ex Fr.) Ces. & de Not., severely threaten the development and survival of poplar plantations in the Northeast, North, and Northwest (Three-north) of China. Hybrid breeding is the most direct and efficient method to control and manage tree diseases; however, compared with the progress in breeding for high-yield, fast-growing, or other poplar hybrid clones, research on resistance breeding for poplar canker disease is scarce. Only limited studies of disease-resistance hybrid breeding have been reported^1,2,3, and no canker-resistance hybrid clone has been cultivated in poplar afforestation.

The crucial step of regular hybrid breeding is the clone selection based on the phenotype acquisition of hybrid progeny. However, acquiring the acquisition of pathological phenotypes (pathotypes) is time-consuming, laborious, tiring, expensive, and experts-depending process. For woody plants, it is more challenging due to the time-, labor-, and economy-consuming nature caused by their long lifecycle, slow growth, and massive body. For example, in poplar, the canker resistance screening of hybrid progeny was conducted 5-7 years after hybridization through conventional in vitro stem inoculation methods^1,2,3. Moreover, limited by this low-efficiency and high-consuming disease-resistant screening method, researchers re-selected the canker-resistant clones from a small subgroup (for example, the selected high-yield or fast-growing poplar clones), not from all hybrid progeny. Therefore, using the regular stem inoculation methods, it is not sure to identify the authentic resistant clones and can not reveal the disease-resistant diversity of the breeding progeny, thus limiting the exploration of disease-resistance-related genes or gene modules. Compared to the rapid development of poplar fast-growing/high-yield breeding, those breeding programs can obtain hybrids through phenotypic selection or even genomic selection in the first two years of breeding⁴, the poplar canker-resistance breeding developed slowly. The disease-resistant screening (or detection) of hybrid progeny, or the pathogen inoculation method, has become the crucial speed-limiting step in poplar canker disease-resistance breeding.

In this protocol, we introduce a novel inoculation method for poplar stem canker pathogens, in vivo leaf inoculation method. Using this method, we can quickly and efficiently test the canker disease resistance of dozens of poplar species (cultivars or clones) within 5-7 days. The validation assay illustrated that the in vivo leaf inoculation is consistent with the traditional in vitro stem inoculation on the resistance detection of stem canker disease, suggesting that the leaf inoculation method is suitable for large-scale resistance screening of poplar canker diseases, such as the whole genotype selection of resistance progeny in the breeding of stem canker disease. This method solves the pathological problem of selecting resistant offspring in the hybrid breeding of poplar canker diseases.

Protocol

1. Fungal culture for canker pathogen

1. Prepare the potato dextrose agar (PDA; potato extraction 6.0 g, dextrose 20.0 g, agar 20.0 g) culture medium for fungal strains; dissolve the above materials in water up to 1000 mL, completely dissolve the medium at 100 °C for 10 min.
2. Pour PDA medium into 25 mL tubes (each containing 15.0 mL); sterilize all tubes at 121.1 °C for 30 min. Pour the medium into culture plates (9.0 cm in diameter) and cool the plates at room temperature (RT).
3. Cut the mycelium of the fungal pathogen, which is cultured in PDA medium at 28 °C for 7 days, into ~0.5 cm square cubes; inoculate the fungal cubes at the center of PDA plates (the mycelium sides face the medium).
4. Culture the fungal pathogens in a thermostatic incubator (28 °C, in dark) for 7-10 days.
5. Cut the PDA medium with fungal mycelium into square cubes (side length 1.0-1.2 cm).

2. Preparation of poplar materials

1. For 1-year-old poplar clones (cultivated > 3 months), select the newly matured, fully extended leaves (always 5-7^th leaves, from the top of the sapling/branches) of the main branches as the inoculated materials, ensure they are free from pests, diseases, and mechanical damages.
2. For 1-year-old branches of perennial poplar hybrid clones (cultivated > 2 months), select the top 5-7^th leaves of the selected branches as the inoculated materials. Ensure the leaves are healthy and have the same light condition (shade or light) among all the tested poplar clones.

3. Pretreatment of inoculation leaves

1. Spray the poplar leaves with clean water, and after drying, wipe the selected leaves with 75% alcohol 1 h or 1 day before the inoculation manipulations.

4. In vivo leaf inoculation

1. For small leaves (leaf width < 8.0 cm), inoculate two square fungal mycelium cubes and PDA (or water agar, WA) cubes (1.0-1.2 cm in side length) onto the upper surface of the top 5-7 leaves of poplar clones; the mycelium faces the leaves. Ensure that the inoculation sites are located at the center of the half leaves, ~1-2 cm apart from the central veins, avoiding obscuring the secondary veins. Each clone inoculates 12 sites on 6 leaves (10 sites for mycelium and 2 sites for PDA inoculation).
2. For large leaves (leaf width ≥ 8.0 cm), inoculate four square fungal mycelium cubes and PDA (or WA) cubes onto the top 5-7 poplar leaves. Each clone inoculates 12 sites at 3 leaves (10 sites for mycelium and 2 sites for PDA inoculation.
3. Wrap the inoculated leaves with transparent adhesive tape (6.0 cm in width) and gently press them to make them adhesive to the leaves, preventing the moving and water loss of the mycelium (or PDA) cubes during the experiment.
4. Pierce the leaves and mycelium (or PDA) cubes at five sites until the needles penetrate the cubes from the upper to the lower surface of the poplar leaves. One site lies at the center, and the other four lie 1-2 mm near the four vertices of the cubes.
   NOTE: A 3 person team collaboration is recommended for fungal inoculation manipulation. The inoculation manipulation of a poplar hybrid population (>100 genotypes) can be conducted in 4 h through teamwork. The piercing manipulations were conducted after all the tested leaves were inoculated and wrapped to alleviate the impact of different piercing times on the disease severity.
5. Inspect the location shift and water loss of the mycelium inoculants, and observe the onset of necrotic lesions around the pierce wound sites from the lower surface of leaves until 3^rd day of inoculation.
   1. Within 3 days of inoculation, observe the location shift and water loss of the mycelium cubes. Define and mark the moved and dried mycelium cubes as ineffective inoculations and discard them from the final identification of pathotypes while defining the other cubes as effective inoculations.
      NOTE: This protocol defines the tested poplar clones with more than five effective inoculation cubes as invalid inoculated clones; therefore, each poplar clone provided at least 30 effective inoculation sites, which will develop into 30 independent necrotic spots.
6. Pick off all inoculated leaves at the end of the experiment (~5-7 days after inoculation), bring them back into the laboratory in plastic sample bags, and store them at 4 °C.

5. Leaf pathotype acquisition and assessment of poplar resistance to stem canker diseases in the laboratory

1. Carefully remove the transparent adhesive tapes and mycelium (or PDA) inoculants from the leaves.
2. Observe and identify the leaf pathotypes of each poplar clone, including the shape and color of necrotic spots, and may include the fungal hyphae-like structure, pycnidia, and conidia that formed on the surface of the leaves around the pierce sites.
3. Photograph the leaves (with an additional ruler) with a camera, or scan the leaves (with an additional ruler) using a scanner to obtain images with a resolution of over 300 dpi, and then save the images in JPEG, TIFF, or PNG format.

6. Identification and statistical analysis of disease occurrence

1. Open the images of diseased leaves in the software ImageJ 1.54g (http://imagj.org).
2. Set the scale according to the ruler in the leaf images.
3. Identify and measure the lesions with the wand (tracing) tool.
4. Record and export the area values as a spreadsheet after the areas of all necrotic spots are measured.
5. Set the criteria for disease determination:
   1. If the area of lesion around the mycelium cubes covered pierce sites is significantly larger than that covered by PDA cubes, then define these sites as disease sites.
   2. If the morphological characteristics of the mycelium cubes covered pierce sites significantly changed when compared with the PDA-covered sites, including colors of the lesion, producing hyphae-like structure, pycnidia, and conidia, define the site also as a disease site.
6. Calculate the average areas of each poplar hybrid clone's effective PDA lesion spots. According to the average areas, divide all the mycelium inoculated spots of one poplar clone into two categories: onset and non-onset. Then, calculate the tested poplar clone's disease incidence rate (Formula 1: Disease incidence rate (%) = Number of diseased prick sites/Total number of efficient prick sites × 100).
7. Calculate the average area of the diseased spots in leaves (n = 50). According to the values of average areas of diseased spots of leaves in all tested poplar clones, set a 5-level disease grading standard.
8. Calculate the disease index of each poplar clone (Formula 2) based on the above disease grading standard (Formula 2: Disease index = ∑(Number of prick sites by level × Values of severity at all levels)/(Value of the highest severity level × Total number of efficient prick sites) × 100).
9. Verify the normal distribution of the numbers of poplar clones across different resistance levels using the Shapiro-Wilk test using any appropriate data analysis software.
10. According to the disease index, identify all tested poplar clones as five (or seven) groups: very high resistance (VHR), high resistance (HR), resistance (R), no resistance and no susceptibility (NRNS), susceptibility (S), high susceptibility (HS), and very high susceptibility (VHS) group⁵.

Results

In this protocol, the schematic workflow was conducted in 48 poplar hybrid clones infected by the stem canker pathogen C. chrysosperma (Figure 1). The poplar hybrid clones are part of the hybridization progeny of P. deltoides, cultivated in the nursery at the Chinese Academy of Forestry (CAF), Beijing.

The stem canker pathogen C. chrysosperma isolate CZC is a typical fungal strain (with middle pathogenicity) used for the physiological re...

Discussion

This protocol provides a rapid and efficient inoculation method for poplar canker resistance pathogens, which is suitable for the research fields requiring large-scale disease resistance screening, such as hybrid breeding of poplar canker resistance and pathogenicity screening of stem canker pathogens.

The first key point of the method is to evaluate disease resistance by newly matured leaf inoculation instead of matured stems/branches. As a result, the selection of stem canker disease-resist...

Materials

Name	Company	Catalog Number	Comments
Aluminum foil	Biosharp	BS-QT-027B
C. chrysosperma isolate	China Forestry Culture Collection Center	CFCC86775	Separation and preservation by our laboratory
Cytospora chrysosperma	Plant Physiology Laboratory, Institute of Forestry New Technology, Chinese Academy of Forestry	NCBI accession number: MK994101 for rRNA-ITS and MN025273 for EF1α gene   CGMCC number:40575	Separation and preservation by our laboratory
Epson Perfection V370 Photo	Epson	V370	Scanner; Scan the leaves into image
PDA (Potato Dextrose Agar)	Solarbio	P8931	Provide nutrition for fungal growth
PE plastic film			To fix fungi on the leaves
Populus alba var. Pyramidalis	Plant Physiology Laboratory, Institute of Forestry New Technology, Chinese Academy of Forestry		Cultivated by our laboratory
SPSS	IBM		Data analysis software
Thermostatic incubator	Shanghai Kuntian Laboratory Instrument Co., Ltd	KTD-6000	Provide an environment for fungal growth
Tough TG-6 camera	OLYMPUS		To take photos of diseased leaves