Cytology of infection, development and expression of resistance to Plasmodiophora brassicae in canola

Abstract

The timing and expression of resistance to four isolates of Plasmodiophora brassicae, collected from research sites where pathotypes 2, 3, 5 and 6 (Williams' system) had been dominant when characterised in 2006, were assessed in four new commercial cultivars of canola (Brassica napus) with resistance to clubroot. Each of the resistant cultivars was highly resistant to all four of the isolates, and there was no difference in their response to infection. Root hair infection occurred at high levels, but pathogen development occurred more slowly than in a susceptible cultivar (control). Secondary infection and development in cortical cells was severely inhibited in each of the resistant cultivars; only a few bi-nucleated plasmodia were observed at 12 days after inoculation (DAI), and plasmodia were rarely observed at 18 and 24 DAI. In contrast, development in the susceptible cultivar had progressed to resting spores by 24 DAI. A dense ring of accumulated reactive oxygen species (ROS) was observed in the endodermis, pericycle and vascular cambium of noninoculated controls and inoculated plants of the resistant cultivars. However, the ROS ring disappeared rapidly in infected plants of the susceptible cultivar. Plasmodia invaded the stele of susceptible roots by preferentially colonising the xylem parenchyma cells. Expansion and enlargement of lignified xylem cells was observed by 35 DAI. The absence of any specific points of ROS accumulation or lignification of epidermal or cortical cells in the resistant cultivars indicates that a hypersensitive response is not the main mechanism of resistance in these lines. The uniform response of these resistant cultivars to the four isolates of P. brassicae indicates that the resistance in each cultivar may be conditioned by a gene(s) from a single source that confers broad resistance, because most sources of resistance to P. brassicae are pathotype specific.

Live cell imaging of Plasmodiophora brassicae—host plant interactions based on a two‐step axenic culture system

“…We have employed Nile red, which is cell permeable and binds to polar and nonpolar lipids in live cells, for labeling and visualization of intracellular lipid droplets of P. brassicae by confocal microscopy using excitation/emission wavelengths of 488 nm/585–615 nm. Our results are indeed consistent with past reports, demonstrating that neutral, nonpolar lipid droplets are abundant in secondary plasmodia and resting spores as well as in primary plasmodia of P. brassicae (Deora et al, ; Williams & McNabola, ; Williams & Yukawa, ). In contrast, lipid droplets were barely detected in cellular compartments of host Arabidopsis and canola cells of infected root tissues.…”

“…To facilitate the examination of structures and development of P. brassicae in B. napus callus tissues, we employed two fluorescence probes, Nile red and CFW, for detection of lipid droplets and chitin cell walls, respectively. Nile red, a lipophilic fluorescent marker, has been used previously to stain intracellular lipid droplets, abundant in secondary plasmodia and resting spores of P. brassicae (Deora et al, ; Williams & McNabola, ). CFW, selectively binding to β1–3 and β1–4 polysaccharides such as those found in cellulose and chitin, was used to label chitin in the cell walls of mature resting spores of P. brassicae (Moxham & Buczacki, ; Schwelm et al, ).…”

“…Clubroot research therefore requires dedicated tools for microscopic visualization of P. brassicae . Previous studies using TEM demonstrated that abundant intracellular lipid droplets were present in secondary plasmodia and resting spores of P. brassicae (Deora et al, ; Williams & McNabola, ; Williams & Yukawa, ). This key finding has led us to identify new histological probes for the observation of P. brassicae structures during the infection process.…”

“…Many studies on host plant– P. brassicae interactions have been performed under artificial inoculation conditions in growth chambers and greenhouses, that is, flooding potting mix with resting spore suspensions (Sharma, Gossen, & McDonald, ), growing a host plant in slurries of resting spores (Hwang et al, ), and using sand‐liquid culture (Agarwal, Kaul, Faggian, & Cahill, ; Deora, Gossen, & McDonald, ). However, observations of the infection process, especially during the early stages of infection, have proven difficult due to adherent soil and sand particles not easily removed by washing, subsequent root hair or root surface damage during root sample preparation and the presence of soil‐borne bacteria, fungi and oomycetes on and/or inside host tissues, increasing the complexity of this system for elucidating the P. brassicae life cycle.…”

Clubroot caused by Plasmodiophora brassicae Wor.: a review of emerging serious disease of oilseed rape in the Czech Republic

“…The zoospore penetration occurs in the root hairs of a resistant host (the primary phase of the pathogen life) (Hwang et al 2012). The secondary phase of infection in the root cortex also follows, but doi: 10.17221/87/2015-PPS formed plasmodia do not mature and resting spores are not created (Hwang et al 2011b;Deora et al 2013). Slight suppression of primary infection and significant suppression of secondary infection occur during the resistance response, the disruption of the cell walls is lesser and the secondary thickening of the stem is not stopped (Donald et al 2008).…”