Name: In vivo and In vitro Infection of Potato Roots with Plant Parasitic Nematodes for the Assessment of Induced Structural Changes (Video) | JoVE Journal
Uploaded: 2025-02-28T14:10:00.000+00:00
Duration: 10 min 35 s
Description: Learn in vivo and in vitro infection protocols for assessing plant parasitic nematode damage in Solanum tuberosum roots. These controlled methods enable precise histochemical analysis of infection sites and life stages.

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This research was partly funded by Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia (FCT), through grants NemACT, DOI: 10.54499/2022.00359.CEECIND/CP1737/CT0002 (JMSF),&#160;CEECIND/00040/2018,&#160;DOI:&#160;10.54499/CEECIND/00040/2018/CP1560/CT0001&#160;(CSLV)&#160;and&#160;SFRH/BD/134201/2017&#160;(PB);&#160;project&#160;PratyOmics,&#160;DOI:&#160;10.54499/PTDC/ASP-PLA/0197/2020; and structural funding UIDB/00329/2020 | cE3c (DOI:&#160;10.54499/UIDB/00329/2020)&#160;+&#160;LA/P/0121/2020&#160;|CHANGE&#160;(DOI:&#160;10.54499/LA/P/0121/2020), and GreenIT (DOI: 10.54499/UIDB/04551/2020 and DOI: 10.54499/UIDP/04551/2020)..

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<li>Giri, A., Narasu, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transgenic+hairy+roots%5BTitle%5D)+AND+%22Biotechnol+Adv%22%5BJournal%5D)">Transgenic hairy roots.</a> Biotechnol Adv. 18 (1), 1-22 (2000).</li>
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<li>Faria, J. M. S., Vicente, C. S. L., Rusinque, L., Camacho, M. J., Inácio, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant-Nematode+co-cultures+in+the+screening+of+sustainable+nematicides+against+soil-dweling+parasitic+nematodes+of+plants%5BTitle%5D)+AND+%22Revista+de+Ci%C3%AAncias+Agr%C3%A1rias%22%5BJournal%5D)">Plant-Nematode co-cultures in the screening of sustainable nematicides against soil-dweling parasitic nematodes of plants.</a> Revista de Ciências Agrárias. 45 (4), 436-439 (2022).</li>
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</ol>

We have nothing to disclose.

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...

Root and tuber crops rank 4th 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 16th 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 nematodes are responsible for some of the most damaging diseases to crops in modern agriculture. Soil-dwelling PPNs impose heavy losses to farmers because they affect plant roots and interfere with crop productivity by reducing production and/or injuring products, turning them unmarketable3. These dangerous phytoparasites use their stylet (a needle-like mouthpart) to puncture root cells and feed on cell content. Some PPNs feed from outside the roots, others enter the root and cause tissue damage (migratory), while others enter the roots and become sedentary, heavily changing root structure to facilitate feeding4. The main PPNs affecting potato are the potato cyst nematodes, Globodera spp., root-knot nematodes (RKN), Meloidogyne spp., root lesion nematodes, Pratylenchus spp., the false root-knot nematode Nacobbus aberrans, and the potato rot nematode Ditylenchus destructor. For these PPNs, different feeding habits induce different structural changes in host root tissues5,6. Research on the mechanisms of PPN infection and host response is often performed through field or greenhouse trials to maintain reference PPN culture collections or to perform large scale experiments7,8. Testing under natural conditions is strongly influenced by environmental variation and biotic or abiotic stress factors. Greenhouse bioassays are a closer alternative to a natural condition while allowing a relative control of environmental variation and limiting the influence of abiotic and biotic stress. However, host genetic diversity can still be a challenge for trials that require a finer control of biological variability. These limitations can be overcome by resorting to in vitro plant tissue cultures. These are versatile laboratory systems with many advantages for PPNs disease research. For soil-dwelling PPNs, in vitro cultures of transgenic roots are a useful tool for research in laboratory conditions9,10. 
Transgenic roots, or hairy roots (HR), are obtained after infection of plant material with Rhizobium rhizogenes (Riker et al. 1930) Young et al. 200111. This gram-negative bacterium induces the transfection of its Ri plasmid into the host genome and changes the regulation of plant hormone biosynthesis, promoting the formation of root tissue12. Transgenic roots can be maintained indefinitely under asepsis in a culture medium. The advantages of using HR for studying PPNs are a high growth rate in the absence of plant growth regulators that influence nematode infection and development, a high ratio of biomass production per unit time, and cellular integrity and longevity, which determine a higher genetic and biochemical stability6. By resorting to in vitro transgenic roots, PPNs genotypes can be maintained indefinitely under laboratory conditions, infection and PPNs development can be easily followed, host genetic variability can be reduced, manipulation of host molecular makeup can be directly linked to nematode response, and host and parasite structural changes can be more accurately followed6,13. For studies on PPN diseases of potato, in vitro transgenic root co-cultures allow carrying out experiments independently of season or potato tuber dormancy. 
In this protocol, the traditional methodology of PPNs maintenance and in vivo infection of potato plants are detailed. For the structural analysis of infected roots, an improved methodology based on the establishment of in vitro co-cultures of transgenic potato roots with PPNs is also detailed as an alternative that allows a higher control of environmental and host genetic variability. To follow PPNs infection and development in the root tissue, histochemistry is employed to aid in PPNs observation under optical microscopy. The overall aim of this protocol is to optimize the study of PPN-host interactions, ensuring more controlled and reproducible conditions for experimentation while facilitating detailed structural and developmental analyses of nematodes in the root tissue.

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

in vivo and in vitro infection of potato roots with plant parasitic nematodes for the assessment of induced structural changes

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

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.

Carrot disks can be used to multiply and maintain several types of migratory PPNs23. For the RLN, this technique is generally used to maintain reference collections of nematode species or isolates. Using carrot disks, an average 100x increase in nematode populations can be obtained in a period of 3 months (Figure 1). However, nematode numbers vary widely (between 30x and 200x), mainly owing to nematode genetic diversity and/or variation in nutritional contents of carr...

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.

Soil-dwelling plant parasitic nematodes (PPNs) are important potato pests that cause lesions and/or change plant roots structure, leading to reduced crop ...

in-vivo-vitro-infection-potato-roots-with-plant-parasitic-nematodes

Research

JoVE Journal

Biology

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

The goal of this presentation is to demonstrate in vivo and in vitro techniques for the rearing of entomopathogenic nematodes. In vivo methods consider the rearing of these nematodes with an insect host, whereas the in vitro methods utilize rich agar media.

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The present protocol describes a reliable and straightforward method for culturing, collecting, and screening Ditylenchus dipsaci.

Culturing and Screening the Plant Parasitic Nematode Ditylenchus dipsaci

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.

Inoculation Strategies to Infect Plant Roots with Soil-Borne Microorganisms

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

Studying Cell Death Initiation Using a Digital Microscope

The protocol describes the in vivo and in vitro pinewood nematode infection of Pinus pinaster and their volatilome analysis through Gas Chromatography (GC) and GC coupled to Mass Spectrometry (GC-MS).

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles

We present two methods for farmers to mass-propagate entomopathogenic nematodes as bio-insecticides. The first method uses polyacrylamide gel as the nematode culturing substrate, while the second uses polymer-coated cotton wool as the culturing substrate. Both methods rely on in vivo propagation of nematodes within insect cadavers.

Grower-Based In Vivo Propagation of Entomopathogenic Nematodes

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

Learn in vivo and in vitro infection protocols for assessing plant parasitic nematode damage in Solanum tuberosum roots. These controlled methods enable precise histochemical analysis of infection sites and life stages.

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

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