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 xylem transport remains unclear.

To reveal the transport mechanisms of carbohydrates and water in poplars infected by canker pathogens, we proposed the phloem or epidermis girdling inoculation methods^1,2, which combined classic garden girdle manipulation and pathogen inoculation method (mycelia block wounding inoculation). These methods can simulate the infestation process and the blockage of water and carbohydrates induced by canker pathogens.

Our research illustrated that fungal pathogens caused poplar canopy dieback by initially inducing carbon starvation, not hydraulic failure^1,3,4,5. Surprisingly, we have observed a special rhizogenesis on poplar stems that were associated with the inoculation of stem canker pathogens: copious red adventitious roots (ARs) grow from the low end of the upper stems (opposite to the upper edge of the phloem or epidermis girdling rings). Moreover, our experiments illustrated that the production of ARs is universal in poplar-canker pathogen interaction. They can be produced from kinds of poplar species or clones at different ages (1-, 2- or even 6-years old) and can be induced by different canker pathogens (V. sordida and B. dothidea) or their isolates. In addition, we have studied the color mechanisms of poplar ARs, and results showed it is associated with the biosynthesis of flavonoids and anthocyanins, as well as gene expression regulation of light-related genes (or gene modules) under lighting conditions⁶. Therefore, these poplar ARs induced by pathogens can be used as a stable and ideal experimental system for the study of plant-pathogen interaction, root biology, and the function and expression of light-related genes.

In this study, we will introduce and provide the protocol to establish a poplar ARs experimental system through girdling inoculation pathway; moreover, we point out the crucial factors that affect the formation of ARs and expound on the potential application of the girdling inoculation in the study of poplar root biology and other light response-related physiological processes.

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.
   3. Incubate the inoculated PDA plates in the dark at 28 °C for 7-10 days.
   4. Cut the incubated PDA medium with fungal mycelium into straps (1.2 cm in width; about 3-6 cm in length).
2. Preparation of poplar materials
   1. Select 1-2 years-old, vigorous-growing poplar saplings.
   2. Select mature stems/branches from poplar saplings (1-2 cm in diameter, free of diseases and pest infestation).
   3. Wash the inoculation regions (about 30 cm high above the ground or the base of the branches) of poplar stems/branches; sterilize the stems/branches with 75% alcohol solution.
3. Induction of ARs through phloem girdling-inoculation
   1. Carefully girdle the epidermis and phloem of the sterilized poplar stems/branches, remove the girdled phloem rings (1 cm in width, including partial cambium), and expose the white, inner cambium/xylem tissues.
      NOTE: Steps 1.3.2-1.3.4 detail alternative methods of inoculation.
   2. Completely cover the girdling region with mycelia straps (1.2 cm in width) as phloem girdling inoculation treatment (GP). The fungal hyphae face the exposed xylem.
   3. Scrape and damage the exposed inner cambium tissue with sterilized knives, and then inoculate the PDA straps (1.2 cm in width) on the girdling regions as girdling cambium-removal treatment (GR).
   4. Inoculate the girdling regions directly with the uncultured PDA medium straps (1.2 cm in width) as a girdling control (GC).
   5. Wrap the inoculated stems/branches with stretchable, colorless, and transparent grafting tape (or PE plastic film). Wrap 4 layers to keep moisture in.
   6. Tie the inoculated stems/branches with (metal, plastic, or woody) sticks (over 50 cm) to keep them from windbreak.
   7. Cultivate the girdled poplar materials with regular irrigation during the experiment.
   8. Observe from outside and record the formation of poplar ARs 14-30 days after inoculation.
4. Induction of ARs through epidermis girdling-inoculation
   1. Select and prepare inoculated materials as described in step 1.2.
   2. Carefully girdle the epidermis of poplar stems/branches.
   3. Remove the epidermis rings (1.0 cm in width) and expose the green phloem tissue.
   4. Slightly and vertically scrape the phloem tissue four times and expose the inner structure of the phloem.
   5. Inoculate the girdled epidermis region with the fungal mycelium (eGP) and PDA straps (eGC). Perform inoculation manipulations similar to steps 1.3.2-1.3.4.
   6. Wrap the inoculated stems with grafting tape (or PE plastic film) as described in step 1.3.5.
   7. Manageme poplars and observe ARs as described in steps 1.3.6-1.3.8.

2. Establishment of an experimental system for the research of light-related genes through girdling inoculation method

1. Induce poplar ARs using the phloem girdling inoculation method (steps 1.3.2-1.3.4).
2. Alternatively, induce poplar ARs using the epidermis girdling inoculation method as described in step 1.4.5.
3. Wrap the pathogen-inoculated regions of poplar stems/branches (15 cm in height) with aluminum foil (shading treatment, S) or without aluminum foil wrap (lighting treatment, L).
4. Tie the stems/branches to >50 cm long sticks to keep them from windbreak. Cultivate and manage the poplars regularly and keep them well irrigated during the experiment as described in steps 1.3.6-1.3.7.
5. Remove the aluminum foil from the stems at ~20 days after inoculation.
6. Observe and photograph the aluminum foil shaded (S) and unshaded poplar ARs (L) immediately.
7. Cultivate the shaded poplar ARs in sunlight or other artificial light sources/conditions.
8. Remove the grafting tapes (or PE plastic films) and harvest the poplar ARs at 1-5 days after light exposure.
9. Wrap all the AR samples with aluminum foil. For the poplar ARs that underwent shading treatment, harvest the samples in the dark.
10. Soak the AR samples in liquid nitrogen and store them at -80 °C for further investigation.
11. Harvest the light-exposed or unexposed poplar ARs (conducted in the dark) after wrapping them with aluminum foil, and store them at 4 °C for morphological and other in situ assays.

Results

The workflow of stem canker pathogens inducing adventitious roots through girdling inoculation is shown in Figure 1. The experiments conducted here showed that both stem canker pathogens, V. sordida, B. dothidea, and their isolates (from different hosts, regions, or pathogenicity) can induce the formation of ARs in poplar species. In this protocol, we used V. sordida isolate CZC, CFCC86775, and B. dothidea isolates SD50 and SD81 to produce ARs in P. al...

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