Genomic differences between the new Fusarium oxysporum f. sp. apii (Foa) race 4 on celery, the less virulent Foa races 2 and 3, and the avirulent on celery f. sp. coriandrii

- Based on significantly increased expression of Foa race 4 genes in planta vs.
- PCR primers for diagnosis of either Foa race 2 or 4 and the Foci were identified..
- Foa race 4 and Foa race 2, which are in different somatic compatibility groups, did not form CATs with each other..
- Conclusions: There was no evidence that Foa race 2 was involved in the recent evolution of Foa race 4.
- Foa race 2 and 4 are CAT-incompatible.
- However, horizontal chromosome transfer could account for the major difference in the accessory genomes of Foa race 4 and the Foci and for their differences in host range..
- Foa race 1 isolates are virulent on cv.
- Foa race 2 was first reported in 1976 in California [8, 9] and subsequently spread to other production areas in North America in the 1980’s.
- importantly, Foa race 3 was in a different somatic compatibility group than Foa race 2.
- Following the discovery of Foa race 2, resistance was identified in celeriac (A.
- Here, we further report that Foa race 4 has a broader host range than celery.
- it can also cause disease on coriander, although the Foci are more virulent on coriander than Foa race 4.
- We also identify i) previously undescribed effec- tors and pathogenicity factors that are up-regulated in Foa race 4 in planta.
- Foa race 1 was not sequenced because it is polymorphic and contemporary celery cultivars in the USA are not susceptible.
- Foa race 4 is highly virulent on all three cultivars.
- Longstanding is a secondary host of Foa race 4.
- coriander is also a secondary host of Foa races 2 and 3, but these races are less virulent than Foa race 4 (Fig.
- Foa race 4 was also analyzed with a Bionano optical map.
- Previously, using ten conserved genes in the FOSC, we placed Foa race 2 in FOSC Clade 3 and Foa races 1, 3 and 4 in FOSC Clade 2 [3].
- apii Foa race 4 FoaR4 Camarillo, Ventura Co.
- apii Foa race 3 FoaR3 Unknown b Before 1981 b NRRL 38295.
- apii Foa race 2 FoaR2 Santa Maria, Santa Barbara Co.
- of core, accessory and total genomes, the data in Table 3 indicate that Foa race 2 is the most dissimilar strain com- pared to the four members of the FOSC Clade 2 (P <.
- Identification of host-specific versus lineage-specific contigs in the accessory genome of Foa race 4 and Foci3 – 2.
- We used a gene-independent, Illumina-mapping method to further examine the differences between the genomes, particularly in the accessory contigs in Foa race 4 versus Foci3–2.
- Compared to the Foa race 4 reference (Fig.
- 3 and Additional file 8), Foa race 3 has coverage over 99.7%.
- of the genome, Foci3–2 and FociGL306 have 94.5 and 94.2% coverage, respectively, and Foa race 2 has only 78.9% coverage.
- In Foa race 4, the host-specific superscaffolds 17, 14, and 13, which represent 35% of the analyzed accessory genome, have less Illumina coverage in the Foci (from 49 to 57%) than from the Foa race 3 and 4 strains (from 87 to 95%) (Additional file 8).
- these contigs account for 60% of the length of the genome and contain 98.1% of the Sordariomycete BUSCO genes in Foa race 4.
- Of 6159 predicted genes in the accessory genome of Foa race 4, 64% had a homolog in the Foa race 3 accessory genome (Additional file 11).
- Of those, 90% were syntenic with Foa race 4.
- Fewer homologs of Foa race 4 accessory genes were identified in the accessory genomes of the Foci strains.
- For example, accessory genomes of Foci3–2 and FociGL306 had homologs with 50 and 46%, respect- ively, of those in Foa race 4.
- of those, 84 and 82% were syntenic with Foa race 4.
- Foa race 2 and Fol4287 had the fewest homologs (15 and 14%, respectively), and of those, the least synteny (41 and 35%, respectively) with Foa race 4.
- Compared to Foa race 4, Foa race 3 has more homologs than any of the other strains, but only for 59 to 73% of the gene models (Additional file 14).
- Foa race 2 and Fol4287 had the fewest (5 to 20%) and similar percentages (i.e.,.
- Foa race 2 is indicated with an arrow and the Foa races 3 and 4 and the Foci isolates are indicated with a star.
- QuantSeq mRNA libraries (Lexogen, Inc.) prepared from Foa race 4 during plant infection (in planta) and growth in liquid media (in vitro) with Foa races 2, 3, and 4.
- With Foa race 4, 76 genes were expressed signifi- cantly less in planta (adjusted P <.
- Amongst the 80 up-regulated Foa race 4 genes in planta,.
- 0.1% of the Foa race 4 counts in planta, and consequently were classified as lower priority for review.
- All have an identical DNA sequence in Foa races 3 and race 4, but 65% have a duplication within Foa race 4 compared with race 3 (Additional file 16).
- Forty- three percent of these predicted effectors had an identical sequence in both Foci isolates and Foa race 3 and 4..
- The 114 aa predicted protein of the other three putative effectors in Foa race 4 (PGN.15680/PGN.06376/.
- each has an identical predicted protein in Foa race 3.
- The single Foa race 2 predicted protein is 96 and 94% identical to a Table 3 ANchor DIstances between the complete, core and accessory genomes of the Foa and Foci strains a.
- Foa race 2 Foa race 4 Foa race 3 Foci3 – 2.
- Complete assembly Foa race 4 1.69E-02.
- Foa race 3 1.69E-02 2.63E-04.
- Core genome Foa race 4 1.42E-02.
- Foa race 3 1.42E-02 1.94E-05.
- Accessory genome Foa race 4 2.62E-02.
- Foa race 3 2.66E-02 8.74E-04.
- The two SIX1 orthologs in Foa race 3 and 4 are similar distances apart (46,973 and 50,005 bp, respectively), which suggests the ancestral SIX1 was duplicated and inverted in the same chromosome.
- Remarkably, a total of seven effectors (of 23 up- regulated and highly expressed in planta) were within 2.5 kbp downstream of a mimp in the Foa race 4 refer- ence assembly.
- 3 Coverage of Foa race 4 (FoaR4) and Foci 3 – 2 reference assemblies by Illumina reads from Foa and Foci strains.
- Each row corresponds to coverage from a single isolate, which is noted to the right of the graph (Foa race 2, FoaR2.
- Foa race 3, FoaR3).
- 4 Synteny of the accessory genomes of Foa race 4 and other strains.
- 0.05) increased expression in planta in celery crowns that were infected with Foa race 4 compared to Foa race 4 grown in vitro.
- The plots illustrate the most synteny between Foa races 4 and 3 in the accessory genome, less synteny between Foa race 4 and the F.
- coriandrii (Foci) strain 3 – 2, and the least synteny between Foa race 4 and either Foa race 2 or the f.
- There are the following number of genes in each of the selected accessory Foa race 4 superscaffolds (SS): SS2, 523.
- In these selected accessory contigs, Foa race 3 (A) has homologs of 59 to 73% of these Foa race 4 gene models, depending on the contig (Additional file 14).
- Both Foa race 2 (C) and Fol 4287 (D), which are in FOSC Clade 3, have the fewest homologs, with only a total of 11 to 12% of the homologs in the four accessory contigs in Foa race 4.
- Table 4 Thirty-five up-expressed potential effectors or pathogenicity/virulence factors in Foa race 4 in planta a.
- Second, the other isolate of Foa race 4 (FoaR4V-313–2.2) and one of the isolates from coriander (FoaR4V-7.5B) differ by a single common SNP in an ef1 intron (GenBank Acces- sions MT295485, MT295484).
- Third, the twenty-two pathogenic isolates of Foa race 2 have an identical haplotype..
- Each of the Foa race 1 isolates had a unique two-locus haplo- type and were never implicated as the primary pathogen in a contemporary California cultivar..
- Table 4 Thirty-five up-expressed potential effectors or pathogenicity/virulence factors in Foa race 4 in planta a (Continued).
- d Based on comparisons with progressiveMauve of Foa race 4 with the Fol 4287 reference.
- We identified a new and more specific primer pair (FOAR2-76 k) that can be used to identify the Foa race 2 haplotype.
- they also amplified with both the older and the new Foa race 2 primers.
- Because we have isolated Foa race 2 and race 4 from the same celery plant (data not shown), we assessed whether the two strains could anastomose via either hyphae or conidial anastomosis tubes (CATs).
- The results indicate that Foa races 3 and 4 and the Foci strains are in the same somatic compatibility group, and that Foa race 2 is in a different somatic compatibility group..
- Consequently, Foa race 4 and Foci readily form hetero-CATs, but Foa races 4 and 2 are CAT-incompatible..
- In the U.S., Foa race 2 has been an economic- ally important pathogen of celery since the mid-1970 ’ s;.
- from a Foa race 3-like strain, which may have arisen from one of the polyphyletic Foa race 1 strains.
- In contrast, Foa race 4 did not form CATs with Foa race 2.
- Nonetheless, we cannot rule out the possibility that Foa race 4 acquired a smaller DNA fragment from a member of the FOSC or another microorganism..
- Second, we demonstrated that Foa race 4 and Foci3–2, which are in the same somatic com- patibility group, form hetero-CATs as readily as either strain forms homo-CATs.
- In addition to causing disease in the greenhouse, Foa race 4 can cause disease of coriander in the field.
- That is, coriander is a secondary, symptomatic host of Foa race 4..
- Both SIX1 homologs are highly expressed in the crowns of Foa race 4-infected celery but are essentially not expressed when any of the three Foa races are grown in vitro.
- Interestingly, SIX1 in Foa race 4 and the Foci are on host- specific accessory contigs.
- the two homologs in Foa race 2 are located in a single accessory 3.3 Mb chromosome-sized contig (no.
- (Additional file 19), there is 100% identity of the Foa race 4 homolog with strains of the multiple ff.
- Consequently, horizontal chromo- some transfer of a pathogenicity chromosome is presum- ably responsible for the main difference between Foa race 4 and the Foci.
- and found an amplicon in Foa race 3.
- we then discovered that the Foa race 4 PGN.06282 was present in the raw reads (but not the final assembly) of Foa race 3..
- Foa race 2, Foa race 4 and the Foci were compared with all ge- nomes included in the whole genome phylogenetic tree..
- had the same two-locus haplotype as the Foa race 2, 19 non-pathogenic FOSC from celery, six non-pathogenic F..
- Additional file 8 Classification of contigs in the Foa race 4 accessory genome as either lineage- or host-specific.
- Additional file 10 Conserved synteny of BUSCOs in Foa race 4 and other Foa, Foci and a reference.
- Additional file 12 Synteny between FociGL306 and Foa race 4 in the conserved and accessory genomes.
- Additional file 14 Foa race 4 putative accessory chromosomes:.
- percentage of genes that have homologs in other strains Additional file 15 Homologs of gene models from Fol4287 chromosome 14 in Foa race 4 and FociGL306.
- Additional file 20 Diagnostic PCR primers for Foa race 2 haplogroup, Foa race 4, and Foci

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