In vivo and In vitro Infection of Potato Roots with Plant Parasitic Nematodes for the Assessment of Induced Structural Changes

Summary

The protocol describes infection of Solanum tuberosum roots with plant parasitic nematodes under in vivo greenhouse conditions and potato in vitro transgenic roots for histochemical analysis of root structure through optical microscopy.

Abstract

Soil-dwelling plant parasitic nematodes (PPNs) are important potato pests that cause lesions and/or change plant roots structure, leading to reduced crop fitness and productivity. Research on the cellular and subcellular mechanisms of PPNs infection and development can resort to field plants or seedlings under greenhouse conditions. Field studies are more representative of natural environments but are subjected to the unpredictability of environmental conditions that can heavily influence research outcomes. Greenhouse studies allow higher control over environmental variables and higher safety against contaminants or pathogens. However, in some hosts, genetic diversity becomes an important factor of variability and influences the host-parasite complex response. We have developed in vitro co-cultures of transgenic roots with PPNs as a reliable alternative that occupies less space, requires less time to obtain, and is free from contamination or from host genetic variability. Co-cultures are obtained by introducing aseptic PPNs to host in vitro transgenic roots. They can be maintained indefinitely, which makes them excellent support for keeping collections of reference PPNs. In the present work, a protocol is detailed for the controlled infection of in vivo potato roots with the root lesion nematode and for establishing in vitro co-cultures of potato transgenic roots with the root-knot nematode. The in vitro co-cultures provided a laboratory proxy for the natural potato infection condition and produced nematode life stages irrespective of season or climate conditions. Additionally, the methodology used for structural analysis is detailed using histochemistry and optical microscopy. The acid fuchsin dye is used to follow nematode attack sites on roots, while differential staining with Periodic acid-Schiff (PAS) and toluidine blue O highlights nematode structures in potato internal root tissue.

Introduction

Root and tuber crops rank 4^th among the world's most important staple foods. Potato (Solanum tuberosum L.) is one of the most important cultivated tubers. It had its origin in the Andes mountains of South America, but after being introduced to Europe in the 16^th century quickly became the most common food source for the population with a lower income. Today, potatoes make up 1.7% of the world's caloric intake¹. Crop production is heavily affected by plant pests and pathogens, of which plant parasitic nematodes (PPNs) can cause average yield losses that rise up to 12%². Plant parasitic nemat....

Protocol

1. Infection of greenhouse-grown potato plants

NOTE: Greenhouse trials are performed with suspensions of PPNs in mixed life stages or second-stage juveniles (J2), depending on the specific life cycle of the PPN pest. For this protocol, suspensions of mixed life stages of the root lesion nematode (RLN) Pratylenchus penetrans were used. PPNs can either be reared in the lab or requested from certified reference laboratories.

1. Multiplication and maintenance of root lesion nematodes
   NOTE: Sterilized carrot disks are used for the multiplication and maintenance of RLNs¹⁴. Use comm....

Discussion

The study of the mechanisms of infection and disease development in plants attacked by soil-dwelling PPNs is difficult because these phytoparasites generally infect the inner tissues of the root system and induce unspecific symptoms in the shoots. Despite the controlled environmental conditions of the greenhouse, sprouting potato tubers and the growth of potato plants are still favored in the spring and summer months, reducing the experimental period available to one season per year. Also, a substantial number of pots ha.......

Materials

Name	Company	Catalog Number	Comments
2,4-Dinitrophenylhydrazine	Sigma-Aldrich	D199303
2-Hydroxyethyl methacrylate	Sigma-Aldrich	17348
Acetic acid	Sigma-Aldrich	695092
Acid Fuchsin	Sigma-Aldrich	F8129
Benzoyl peroxide	Sigma-Aldrich	B5907
borosilicate glass beaker	Sigma-Aldrich	Z231827
Carbenicillin disodium salt	Sigma-Aldrich	C3416
Cefotaxime sodium salt	Sigma-Aldrich	C7039
Dimethyl sulfoxide	Sigma-Aldrich	472301
Ethanol	Supelco	1.00983
Fertilizer	Compo Expert
Flower pot 5 L	VWR	470049-676
Glutaraldehyde	Sigma-Aldrich	354400
Glycerol	Sigma-Aldrich	G7893
Hydrochloric acid	Sigma-Aldrich	258148
Kanamycin monosulfate	Sigma-Aldrich	BP861
LB Broth with agar	Sigma-Aldrich	L3147
MCE syringe filter	Millipore	SLGSR33SS
PARAFILM M sealing film	BRAND	HS234526B-1EA
Pararosaniline hydrochloride	Sigma-Aldrich	P3750
Periodic acid	Sigma-Aldrich	P0430
Phyto agar	Duchefa Biochemie	P1003
Scalpel blade no. 24	Romed Holland	BLADE24
Schenk & Hildebrandt Basal salt medium	Duchefa Biochemie	S0225
Schenk & Hildebrandt vitamin mixture	Duchefa Biochemie	S0411
Schiff′s reagent	Sigma-Aldrich	1.09033
Sodium metabisulfite	Sigma-Aldrich	161519
Sodium phosphate dibasic	Sigma-Aldrich	S9763
Sodium phosphate monobasic	Sigma-Aldrich	S5011
Soil / Substrate	Compo Sana
Stainless Steel Tweezers	Sigma-Aldrich	22435-U
Sucrose	Duchefa Biochemie	S0809
Toluidine Blue O	Sigma-Aldrich	198161