Poplar Adventitious Roots Induced by Stem Canker Pathogens: An Experimental System for Studying Roots Biology and Light Response-Related Processes

Summary

Here, we present a protocol to induce the production of adventitious roots (ARs) through the phloem- or epidermis-girdle fungal pathogen inoculation pathway, which is suitable for the study of root biology and the light response-related physiological processes in poplar.

Abstract

Valsa sordida and Botryosphaeria dothidea are two crucial necrotrophic fungal pathogens that damage many plant hosts, particularly species in the genus Populus. These two fungal pathogens occur mainly in poplar branches, stems, and twigs, causing classic symptoms such as canker lesions, canopy dieback, and wilting. Pathogen inoculation is the most efficient pathway to study the mechanism of plant disease. Besides the canker lesions around the inoculation sites on the stems, a novel developmental phenomenon, copious adventitious roots (ARs) with bright red color, were observed in poplar species after stem canker pathogen inoculations. In this study, we described the method for inducing ARs using fungal pathogens in poplar trees. The crucial step of this method is the pathogen inoculation after (phloem or epidermis) girdling manipulation. The second crucial step is the application of the moisturizing material. Compared to the moisturizing manipulation with Parafilm, wrapping the inoculated sites with household polyethylene (PE) plastic wrap can produce colorful, numerous, and robust ARs in 20 days after girdling-inoculation. Finally, white ARs sprouted from the inoculated rings in the poplar stems after shading treatment (wrapping the stems with aluminum foil). This method introduces a novel experimental system for studying root development and morphogenesis, which is crucial for understanding the biology of root development, morphogenesis, and response under disease stress. Furthermore, when combined with shading treatment, this study can provide a convenient experimental system for investigating light response-related processes, for example, the biosynthesis of flavonoids, anthocyanins, or other related metabolites, and genes or transcription factors involved in these processes.

Introduction

Poplar stem canker diseases caused by necrotrophic fungal pathogens, Valsa sordida and Botryosphaeria dothidea, are the two crucial tree diseases in north China that severely damaged the development of ecological and economic plantations of poplar species. Poplar canker diseases always occur on the bark of the trunks and branches, while canker lesions are their typical symptoms. After the onset of diseases, the expanding canker lesions progressively damaged the phloem, cambium, and xylem of hosts. Further, they affected the transport of assimilated products and water through the vascular system. However, how the canker pathogens impede the phloem and....

Protocol

1. Induction of poplar ARs through girdling inoculation

1. Culture of fungal canker pathogen
   1. Dissolve 6 g of potato extract, 20 g of dextrose, and 20 g of agar in 1000 mL water to prepare the potato dextrose agar (PDA) medium. Sterilize the medium at 121.1 °C for 30 min and pour the medium into Petri dishes (9cm in diameter), each dish containing about 20 mL of PDA medium.
   2. Inoculate the activated fungal mycelium cube (~0.5 cm) in the center of the PDA Petri dish.
   .......

Discussion

Poplar species are apt to produce ARs or lateral Roots (LRs) from stem cuttings, which contributes to their reproduction and as the model for root biology studying in wood plants^7,8. Moreover, research indicated that inoculation of specific microorganisms, such as beneficial bacteria (Agrobacterium rhizogenes^9,10; Plant growth-promoting rhizobacteria [PGPR]¹¹), endophytes .......

Materials

Name	Company	Catalog Number	Comments
Agar	Solarbio	A8190	Provide nutrition for fungal growth
Aluminum foil	biosharp	BS-QT-027B	To provide shading for the infected area
Girdling knife	MoGong Hardware tool firms		Girdle the epidermis of poplar stems/branches
Grafting tape	CAPI	5cm	To fix fungi on the plants
PD (Potato extraction, Dextrose)	Solarbio	P7360	Provide nutrition for fungal growth
PE plastic film	MiaoJie	413703	To fix fungi on the plants
Petri dishes	Bkman biological Co.,Ltd	90mm	Prepare the PDA medium
Thermostatic incubator	Shanghai Kuntian Laboratory Instrument Co., Ltd	KTD-6000	Provide an environment for fungal growth