Page 195 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

genetic diversity of individual resting spores from a single clubbed canola root of a collection from
Normandin, Quebec, pathotype 5X, was assessed. The ‘x’ designation indicates that the pathotype can
overcome first generation clubroot resistance in canola. An enzymatic method was developed to remove
cell walls from resting spores to produce protoplasts. The ~4,000 individual protoplasts were barcoded
and DNA of each cell was sequenced. Elbow plot analysis indicated the presence of at least two clonal
groups. Complementing this, silhouette clustering, which evaluates the proximity of points within clusters
to those in neighboring clusters, confirmed the presence of at least two clones. Hierarchical clustering
identified five distinct clones, consisting of 829 cells (Clade 1), 1120 cells (Clade 2), 1140 cells (Clade 3),
183 cells (Clade 4), and 445 cells (Clade 5). Principal component analysis supported the presence of five
clones. Heat maps were generated to visualize the genetic diversity across these clones and to compare
the single-cell data with bulk sequences from five field collections, including the original Normandin 5X.
Clade 3, which had the highest number of cells, showed a high similarity to the original field collection.
The identification of 2 to 5 clades among 4,000 resting spores demonstrates that the P. brassicae
population in a single club is genetically diverse. Large differences among the clones supported our
hypothesis from a previous study that entire genotypes are retained over time in the population of
P. brassicae. The genetic diversity this represents has important implications for breeding for resistance,
as no single-gene source of resistance is likely to be durable. Furthermore, the dissimilarity among
genotypes suggests they are not siblings from sexual reproduction, indicating that pathogen increase
within an infected host plant is predominantly or exclusively clonal.

[O180] METAGENOMICS-BASED MICROBIAL COMMUNITY PROFILING IN THE QUEST FOR
1
1,2
POTATO WART BIOLOGICAL CONTROL AGENTS. Ishraq Akbar , Yichao Shi , Bart. T. L. H. van de
Vossenberg , Theo A. J. van der Lee , Sean Li , Linda Jewell , Hai D.T. Nguyen , and Wen Chen .
1
1,2
5
3
3
4
1 Ottawa Research and Development Centre, Agriculture and Agri-Food Canada, 960 Carling ave.,
Ottawa, ON K1A 0C6 Canada; Department of Biology, University of Ottawa, Ottawa, 60 Marie Curie Prv.,
2
Ottawa, ON K1N 6N5, Canada; Wageningen University & Research, Droevendaalsesteeg 1, Plant
3
Science Group, 6708PB, Wageningen, The Netherlands; CFIA, Canadian Food Inspection Agency,
4
Charlottetown Laboratory, 93 Mount Edward Road, Charlottetown, Charlottetown, Prince Edward Island
C1A5T1Canada; and St. John’s Research and Development Centre, Agriculture and Agri-Food Canada,
5
308 Brookfield Road, St. John's, NL A1E 6J5 Canada
Correspondence to wen.chen@agr.gc.ca

Potato wart is a soil-borne disease characterized by cauliflower-like growths on potatoes, caused by the
obligate biotrophic chytrid fungus Synchytrium endobioticum. Recent outbreaks of this quarantine
pathogen in Prince Edward Island severely impacted local agricultural economy. The current containment
strategy involves strict phytosanitary measures combined with the use of potato varieties resistant to
specific pathotypes, of which there are 40 in total. However, these measures require taking land out of
production, and the resistant varieties are not universally effective. To aid in developing an alternative and
sustainable, long-term solution for managing this disease, our research investigates soil fungistasis,
leveraging native soil and endophytic microbial communities. We hypothesize that introducing S.
endobioticum inocula disrupts the ecological equilibrium of the phytomicrobiome, prompting potato plants'
defense systems to recruit and enrich specific beneficial microorganisms from the soil and rhizosphere to
combat the pathogen. To test this hypothesis, we employed Nanopore sequencing to profile and compare
the bacterial communities in healthy and diseased soil and/or potato tuber samples from three locations
endemic with potato wart disease. We used whole genome amplification (WGA) to increase DNA
quantities for sequencing—a technique regularly employed for S. endobioticum inoculum detection.
Microbiomes were recovered by metabarcoding the full length of the bacterial 16S rRNA gene region and
performing shotgun metagenomic sequencing. The preliminary results confirmed the effectiveness of our
Nanopore sequencing protocols by reliably identifying nearly all species in mock communities. High
sequencing depth provided comprehensive representation of community diversity. Nevertheless,
significant shifts were observed in the community compositional structure between original and WGA-
treated samples. Comparative network analysis further indicated that WGA led to reduced connectivity,
modularity, and feed-forward loop motifs, emphasizing the necessity for careful interpretation of microbial
interplay patterns in the search for S. endobioticum antagonists. Insights into the microbial dynamics in
soils used for potato production, particularly those affected by potato wart infestation, will enable us to
identify and isolate potential biocontrol agents for this pathogen and develop strategies to manage and
mitigate the impact of the disease.

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