Microscopy Method to Compare Cyst Nematode Infection of Different Plant Species

Abstract

The cyst nematode, Globodera pallida, is an obligate, biotrophic pathogen of potato, causing up to 80% yield loss. In the present study, a non-destructive imaging technique was used to compare the development and behavior of G. pallida in its host Solanum tuberosum and in the non-host S. sisymbriifolium. We used microscopy-rhizosphere chambers coupled with the fluorescent stain PKH26, and compared this with destructively sampled acid fuchsin staining. No significant difference (P ≥ 0.90) in G. pallida numbers was found whether stained with PKH26 or acid fuchsin for either plant species indicating no toxic effect from the vital stain. PKH26 labelled J2s successfully located and penetrated roots of both S. tuberosum and S. sisymbriifolium. Two days after inoculation, PKH26 stained G. pallida was clearly observed migrating intercellularly through root tissues of both S. tuberosum and the non-host S. sisymbriifolium. Overall, more nematodes were observed in S. tuberosum than in S. sisymbriifolium roots. No live J2s were observed in S. sisymbriifolium roots stained with either acid fuchsin or PKH26 after 8 days. Understanding the time line of development of G. pallida in S. sisymbriifolium is important towards comprehensive understanding of plant defense responses.

High content analysis methods enable high throughput nematode discovery screening for viability and movement behavior in a multiplex sample in response to natural product treatment

“…The assay we developed utilized two different fluorescent dyes, the lipophilic membrane PKH26 dye 9 which was used to pre-stain large pools of worms collected from nematode cultures maintained on plant hosts grown in germination pouches 10 , and the viability dye SYTOX Green 11 which had been previously shown to stain non-viable nematodes 12 . By fluorescently pre-staining the nematodes with the permanent fluorescent marker PKH26, we could produce high contrast images that allowed identification and localization of individual worms in treatment wells.…”

Prospecting fungal parasites of the potato cyst nematode Globodera pallida using a rapid screening technique

“…Our methods were developed to provide rapid results to determine whether a fungus can influence the infectious stage of plant parasitism. Micro‐ROCs coupled with PKH26 fluorescent stain was earlier reported as an effective tool to analyze G. pallida colonization in potato roots . Direct, non‐destructive microscopic observations of PKH26 stained J2s from plants inoculated or not with C. globosum were made and compared to numbers counted when assessed with the standard acid fuchsin staining method and found to not statistically differ.…”

“…Potato plants were grown in microscopy rhizosphere chambers (micro‐ROCs) ; in brief, potato ( Solanum tuberosum cv. 'Desiree) were gown in tissue culture media for 1 week and transplanted in to micro‐ROCs (Advance Science Tools LLC, WA, USA) and kept in the greenhouse at 18 ± 2 °C with 16 h light and 8 h dark cycle for 2 weeks.…”

“…Cysts of G. pallida were obtained from infested fields of Idaho, USA and reared under greenhouse conditions . Second stage juveniles (J2s) of G. pallida were hatched, surface sterilized as described by Kooliyottil et al . A live stain PKH26 (Sigma Aldrich, USA) was used to stain the sterile G. pallida J2s .…”

Use of GFP-tagged Trichoderma harzianum as a tool to study the biological control of the potato cyst nematode Globodera pallida

“…Globodera pallida, obtained from an infested potato field in Shelley ID, was propagated on the susceptible potato cultivar 'Desiree' under greenhouse conditions of 18 C AE 2 C and 16:8-h light: dark period (Dandurand and Knudsen, 2016;Kooliyottil et al, 2016). After 16 weeks of growth, cysts were recovered from soil using the Fenwick method of flotation (Fenwick, 1940) and stored at 4 C prior experimental use.…”