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

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.......

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