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. 2023 Jan 5:13:1033437.
doi: 10.3389/fgene.2022.1033437. eCollection 2022.

Whole genome sequencing of Clarireedia aff. paspali reveals potential pathogenesis factors in Clarireedia species, causal agents of dollar spot in turfgrass

Bochra Amina Bahri  1   2 , Rajiv Krishna Parvathaneni  1   2 , Willis Turner Spratling  1 , Harshita Saxena  2 , Suraj Sapkota  1 , Paul L Raymer  2   3 , Alfredo D Martinez-Espinoza  1
Affiliations

Whole genome sequencing of Clarireedia aff. paspali reveals potential pathogenesis factors in Clarireedia species, causal agents of dollar spot in turfgrass

Bochra Amina Bahri et al. Front Genet. .

Abstract

Dollar spot is one of the most damaging diseases in turfgrass, reducing its quality and playability. Two species, Clarireedia monteithiana and C. jacksonii (formerly Sclerotinia homoeocarpa) have been reported so far in the United States To study the Clarireedia genome, two isolates H2 and H3, sampled from seashore paspalum in Hawaii in 2019 were sequenced via Illumina paired-end sequencing by synthesis technology and PacBio SMRT sequencing. Both isolates were identified as C. aff. paspali, a novel species in the United States Using short and long reads, C. aff. paspali H3 contained 193 contigs with 48.6 Mbp and presented the most completed assembly and annotation among Clarireedia species. Out of the 13,428 protein models from AUGUSTUS, 349 cytoplasmic effectors and 13 apoplastic effectors were identified by EffectorP. To further decipher Clarireedia pathogenicity, C. aff. paspali genomes (H2 and H3), as well as available C. jacksonii (LWC-10 and HRI11), C. monteithiana (DRR09 and RB-19) genomes were screened for fifty-four pathogenesis determinants, previously identified in S. sclerotiorum. Seventeen orthologs of pathogenicity genes have been identified in Clarireedia species involved in oxalic acid production (pac1, nox1), mitogen-activated protein kinase cascade (pka1, smk3, ste12), appressorium formation (caf1, pks13, ams2, rgb1, rhs1) and glycolytic pathway (gpd). Within these genes, 366 species-specific SNPs were recorded between Clarireedia species; twenty-eight were non-synonymous and non-conservative. The predicted protein structure of six of these genes showed superimposition of the models among Clarireedia spp. The genomic variations revealed here could potentially lead to differences in pathogenesis and other physiological functions among Clarireedia species.

Keywords: Clarireedia paspali; PacBio and Illumina technologies; effectors; gene prediction; genome annotation; pathogenicity; phylogeny; protein structure prediction.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Morphology, molecular identification, and symptoms of C. aff. paspali. (A) Front and (B) back of a 5-day old C. aff. paspali culture (H3 isolate) on PDA media. (C) Molecular characterization of H2 (no. 1) and H3 isolates (no. 2) of C. aff. paspali along with two reference isolates DS-SP of C. monteithiana (no. 3) and DS-CB of C. jacksonii (no. 4) on the ITS region (from left to right) showing no differences in band sizes between Clarireedia species. A Thermo Scientific GeneRuler 1 kb DNA Ladder was used. (D) Pathogenicity tests showing signs and symptoms of C. aff. paspali infection and (E) foliar tip dieback and aerial mycelia growth. (F) Non-inoculated control.
FIGURE 2
FIGURE 2
Genome estimation using k-mer analysis with 17-, 19- and 21- mers. The k-mers show a peak at a similar location which gives an estimate of 35 X genome coverage and a genome size of 48 MB for C. aff. paspali (H3 isolate).
FIGURE 3
FIGURE 3
Benchmarking Universal Single Copy Orthologs (BUSCO) analysis of gene models using the eukaryote obd10 (n = 255; species = 70) and the fungal obd10 (n = 758; species = 549) BUSCOs. Gene models generated in this paper from the Augustus pipeline were used for the C. aff. paspali (H3 isolate). Published gene models were used for C. jacksonii (LWC-10), C. monteithiana (DRR09), and R. sydowiana (CBS115975), and S. sclerotiorum (1980 UF-70).
FIGURE 4
FIGURE 4
Upset plot of orthologous gene clusters between C. aff. paspali and other related species. One to several genes can be present within each ortho gene cluster. S. sclerotiorum, C. jacksonii, C. monteithiana, and C. aff. paspali are represented by the genome of isolates 1980 UF-70, LWC-10, DRR09, and H3, respectively.
FIGURE 5
FIGURE 5
Genetic relationship of C. aff. paspali with related species. (A) DNA similarity matrix between the Clarireedia species. C. jacksonii (CJ), C. monteithiana (CM) and C. aff. paspali (CP) were represented by LWC-10 and HRI11, DRR09 and RB-19, H3 and H2 isolates, respectively. (B) Neighbor-joining tree of Clarireedia spp. and related species conducted from combined virulence and conserved gene sequences. Numbers associated with branches are bootstrap percentage. Two isolates for each Clarireedia species were used. S. sclerotiorum (1980 UF-70) and R. sydowiana (CBS115975) were used as representatives of the Sclerotiniaceae family and the Rutstroemiaceae family, respectively. Z. tritici (CBS115943) was used as an outgroup. Numbers associated with nodes are estimated divergence times. When known, the host origin of the isolate was indicated between brackets.
FIGURE 6
FIGURE 6
Three-dimensional protein models of six virulence genes [(A) gpd, (B) nox1, (C) nox2, (D) pka1, (E) pph1, and (F) smk3] from S. sclerotiorum aligned with the respective protein structures from C. jacksonii, C. monteithiana, and C. aff. paspali. The common structures between all species are in green. The species-specific structures of S. sclerotiorum that are distinct from all Clarireedia (C. jacksonii, C. monteithiana and C. aff. paspali) are highlighted in yellow. The single region in GPD (A) protein differentiating C. aff. paspli from C. jacksonii and C. monteithiana is color-coded in pink.
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