Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

Summary

This protocol presents a detailed summary of strategies to inoculate plant roots with soil-borne microbes. Exemplified for the fungi Verticillium longisporum and Verticillium dahliae, three different root infection systems are described. Potential applications and possible downstream analyses are highlighted, and advantages or disadvantages are discussed for each system.

Abstract

The rhizosphere harbors a highly complex microbial community in which plant roots are constantly challenged. Roots are in close contact with a wide variety of microorganisms, but studies on soil-borne interactions are still behind those performed on aboveground organs. Although some inoculation strategies for infecting model plants with model root pathogens are described in the literature, it remains difficult to get a comprehensive methodological overview. To address this problem, three different root inoculation systems are precisely described that can be applied to gain insights into the biology of root-microbe interactions. For illustration, Verticillium species (namely, V. longisporum and V. dahliae) were employed as root invading model pathogens. However, the methods can be easily adapted to other root colonizing microbes - both pathogenic and beneficial. By colonizing the plant xylem, vascular soil-borne fungi such as Verticillium spp. exhibit a unique lifestyle. After root invasion, they spread via the xylem vessels acropetally, reach the shoot, and elicit disease symptoms. Three representative plant species were chosen as model hosts: Arabidopsis thaliana, economically important oilseed rape (Brassica napus), and tomato (Solanum lycopersicum). Step-by-step protocols are given. Representative results of pathogenicity assays, transcriptional analyses of marker genes, and independent confirmations by reporter constructs are shown. Furthermore, the advantages and disadvantages of each inoculation system are thoroughly discussed. These proven protocols can assist in providing approaches for research questions on root-microbe interactions. Knowing how plants cope with microbes in the soil is crucial for developing new strategies to improve agriculture.

Introduction

Natural soils are inhabited by an astonishing number of microbes that can be neutral, harmful, or beneficial to plants¹. Many plant pathogens are soil-borne, surround the roots, and attack the subterranean organ. These microorganisms belong to a wide variety of clades: fungi, oomycetes, bacteria, nematodes, insects, and some viruses^1,2. Once environmental conditions favor infection, susceptible plants will become diseased and crop yields decline. The effects of climate change, such as global warming and weather extremes, will increase the proportion of soil-borne plant pathogens

Protocol

1. Media for fungal cultures and plant inoculation systems

1. Liquid Potato Dextrose Broth (PDB): Prepare 21 g/L PDB in ultrapure water in a heat-stable flask.
2. Liquid Czapek Dextrose Broth (CDB): Prepare 42 g/L CDB in ultrapure water in a heat-stable flask.
3. Medium for the Petri dish inoculation system: Prepare a heat-stable flask with 1.5 g/L Murashige and Skoog medium (MS) and 8 g/L agar in ultrapure water.
   NOTE: Avoid sugar in this medium as it will lead to excessive.......

Discussion

Due to the tremendous yield losses caused by soil-borne phytopathogens¹, an improvement of farming strategies or crop varieties is required. The limited insight into the pathogenesis of soil-borne diseases hinders the development of more resistant plants. Underlying pathomechanisms need to be explored, for which a robust methodological platform is required. Reported inoculation procedures have shown that multifactorial events in root-microbe interactions can be well dissected by combining differen.......

Materials

Name	Company	Catalog Number	Comments
Agar (Gelrite)	Carl Roth	Nr. 0039	all systems described require Gelrite
Arabidopsis thaliana wild-type	NASC stock	Col-0 (N1092)
Autoclave	Systec	VE-100
BlattFlaeche	Datinf GmbH	BlattFlaeche	software to determine leaf areas
Brassica napus wild-type	see Floerl et al., 2008	rapid-cycling rape	genome ACaacc
Cefotaxime sodium	Duchefa	C0111
Chicanery flask 500 mL	Duran Group / neoLab	E-1090	Erlenmeyer flask with four baffles
Collection tubes 50 mL	Sarstedt	62.547.254	114 x 28 mm
Czapek Dextrose Broth medium	Duchefa	C1714
Digital camera	Nikon	D3100 18-55 VR
Exsiccator (Desiccator )	Duran Group	200 DN, 5.8 L	Seal with lid to hold chlorine gas
Fluorescence Microscope	Leica	Leica TCS SP5 II
HCl	Carl Roth	P074.3
KNO3	Carl Roth	P021.1	≥ 99 %
KOH	Carl Roth	6751
Leukopor	BSN medical GmbH	2454-00 AP	non-woven tape 2.5 cm x 9.2 m
MES (2-(N-morpholino)ethanesulfonic acid)	Carl Roth	4256.2	Pufferan ≥ 99 %
MgSO4	Carl Roth	T888.1	Magnesiumsulfate-Heptahydrate
Murashige & Skoog medium (MS)	Duchefa	M0222	MS including vitamins
NaClO	Carl Roth	9062.1
Percival growth chambers	CLF Plant Climatics GmbH	AR-66L2
Petri-dishes	Sarstedt	82.1473.001	size ØxH: 92 × 16 mm
Plastic cups (500 mL, transparent)	Pro-pac, salad boxx	5070	size: 108 × 81 × 102 mm
Pleated cellulose filter	Hartenstein	FF12	particle retention level 8–12 μm
poly klima growth chamber	poly klima GmbH	PK 520 WLED
Potato Dextrose Broth medium	SIGMA Aldrich	P6685	for microbiology
Pots	Pöppelmann GmbH	TO 7 D or TO 9,5 D	Ø 7 cm resp. Ø 9.5 cm
PromMYB51::YFP	see Poncini et al., 2017	MYB51 reporter line	YFP (i.e. 3xmVenus with NLS)
Reaction tubes 2 mL	Sarstedt	72.695.400	PCR Performance tested
Rotary (orbital) shaker	Edmund Bühler	SM 30 C control
Sand (bird sand)	Pet Bistro, Müller Holding	786157
Soil	Einheitserde spezial	SP Pikier (SP ED 63 P)
Solanum lycopersicum wild-type	see Chavarro-Carrero et al., 2021	Type: Moneymaker
Thoma cell counting chamber	Marienfeld	642710	depth 0.020 mm; 0.0025 mm2
Ultrapure water (Milli-Q purified water)	MERK	IQ 7003/7005	water obtained after purification
Verticillium dahliae	see Reusche et al., 2014	isolate JR2
Verticillium longisporum	Zeise and von Tiedemann, 2002	strain Vl43