Page 270 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

incubation, a sample with bacterial community is isolated for DNA extraction using the Takara NucleoSpin
96 Tissue kit. Following DNA extraction, we check the quality of randomly selected DNA extracts. The
DNA of each sample is then tested for the presence of different bacteria using high-throughput real-time
polymerase chain reaction (PCR) (Takara SmartChip system), capable of screening 216 samples for a
primer panel composed of 24 different targets. The samples are also being tested for community structure
using Illumina short read sequencing and PacBio single molecule, real-time (SMRT) technology (Kinnex
16S rRNA kit for full-length 16S sequencing). The results showed that most of samples were free from
harmful pathogens but contained many commensal bacteria that are common in the environment,
including those promoting plant growth. This method allows us to continue to monitor bacteria associated
with Canadian grain as part of grain quality and safety assurance.

[P134] DNA EXTRACTION METHODS AND COMPARATIVE GENOMICS FOR
PARASTAGONOSPORA SPP. Janice Bamforth and Sean Walkowiak . Microbiology and Grain
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1 1
Genomics, Grain Research Laboratory, Canadian Grain Commission, 196 Innovation Drive, Winnipeg,
MB, Canada, R3T 2N2
Correspondence to: Janice.bamforth@grainscanada.gc.ca

Septoria leaf and glume blotch are destructive crop diseases caused by fungal pathogens formerly
classified under the genus Septoria. Recent taxonomical revisions have led to the reclassification and
renaming of these fungi, some of which are now under the genus Parastagonospora of taxonomical
family Phaeosphaeriaceae. The Canadian Grain Commission (CGC) houses a substantial fungal culture
collection comprising over 6700 specimens, including isolates morphologically identified as species from
the former genus Septoria that were isolated from Canadian wheat, durum and barley. In this study, we
characterized 10 Parastagonospora isolates from the CGC fungal culture collection. DNA extraction
methods were explored to prepare fungal extracts suitable for sequencing using the Oxford Nanopore
platform. Freezing fungal mycelia at -140°C served as a viable substitute for liquid nitrogen for the initial
grinding of mycelia when preparing DNA extracts. DNA was extracted using a cetyltrimethylammonium
bromide (CTAB) and chloroform-isoamyl alcohol method as well as purification and size selection using
the Blue Pippin. Whole genome sequencing was performed using Oxford Nanopore Technologies long-
read platforms and genome assemblies were generated using Flye and polished with Illumina short-reads
with BWA-MEM and Pilon. To determine the relationship between these isolates and similar fungi that
have been previously characterized, we aligned the newly assembled genomes to 389 genomes from
taxonomical families containing Septoria-related genomes obtained from NCBI using NUCmer.
Comparative genomic analysis revealed that the CGC isolates clustered discretely with either
Parastagonospora nodorum (n = 5), or Parastagonospora avenae f. sp. Tritici (n = 5), which are causative
agents of glume blotch. Both species were isolated from wheat and barley and only the latter was isolated
from durum. None of the cultural isolates were found to be similar to NCBI genomes from other
Phaeosphaeriaceae, and taxonomical families Mycosphaerellaceae, Massarinaceae where former
Septoria spp. have been reclassified, including Zymoseptoria tritici, the causative agent of Septoria Tritici
Blotch. In summary, the CGC has a large fungal culture collection of historically important fungi. Here we
sequenced and assembled the genomes of Parastagonospora spp., and genomes will be made available
to enable further research on diseases which causes reduced yields in cereals in Canada and worldwide.

[P135] ASSESSING CHANGES IN AGGRESSIVENESS OF FUSARIUM AVENACEUM ISOLATES
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FOLLOWING PASSAGE THROUGH PEA AND WHEAT. Anas Eranthodi , Michelle Hubbard , David
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1 1
3
Overy , Linda Harris , Timothy Schwinghamer , Syama Chatterton , and Nora Foroud . Lethbridge
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3
Research and Development Centre, Agriculture and Agri-Food Canada, 5403-1st Avenue South,
Lethbridge, AB, Canada, T1J 4B1; Swift Current Research and Development Centre, Agriculture and
2
3
Agri-Food Canada, 1 Airport Road, Swift Current, SK, Canada, S9H 3X2; and Ottawa Research and
Development Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON, Canada, K1A
0C6
Correspondence to: syama.chatterton@agr.gc.ca; nora.foroud@agr.gc.ca

Fusarium avenaceum is a causative agent in pulse root rot and cereal head blight. In a recent study, we
found that F. avenaceum isolates from pea were more aggressive on pea and wheat than isolates
obtained from lentil or wheat. It is thus hypothesized here that cereal-pulse rotation can influence F.
avenaceum aggressiveness and that completion of the life cycle in one crop type might influence
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