Identification of pathogenicity-related genes in Fusarium oxysporum f. sp. cepae

Abstract

Pathogenic isolates of Fusarium oxysporum, distinguished as formae speciales (f. spp.) on the basis of their host specificity, cause crown rots, root rots and vascular wilts on many important crops worldwide. Fusarium oxysporum f. sp. cepae (FOC) is particularly problematic to onion growers worldwide and is increasing in prevalence in the UK. We characterized 31 F. oxysporum isolates collected from UK onions using pathogenicity tests, sequencing of housekeeping genes and identification of effectors. In onion seedling and bulb tests, 21 isolates were pathogenic and 10 were non-pathogenic. The molecular characterization of these isolates, and 21 additional isolates comprising other f. spp. and different Fusarium species, was carried out by sequencing three housekeeping genes. A concatenated tree separated the F. oxysporum isolates into six clades, but did not distinguish between pathogenic and non-pathogenic isolates. Ten putative effectors were identified within FOC, including seven Secreted In Xylem (SIX) genes first reported in F. oxysporum f. sp. lycopersici. Two highly homologous proteins with signal peptides and RxLR motifs (CRX1/CRX2) and a gene with no previously characterized domains (C5) were also identified. The presence/absence of nine of these genes was strongly related to pathogenicity against onion and all were shown to be expressed in planta. Different SIX gene complements were identified in other f. spp., but none were identified in three other Fusarium species from onion. Although the FOC SIX genes had a high level of homology with other f. spp., there were clear differences in sequences which were unique to FOC, whereas CRX1 and C5 genes appear to be largely FOC specific.

Keywords: Fusarium basal rot; Fusarium oxysporum f. sp. cepae; Secreted In Xylem (SIX); effector genes; onion; pathogenicity.

Figure 1

Pathogenicity of 32 Fusarium oxysporum isolates on onion seedlings (cv. Napoleon and Hazera Seeds standard susceptible line, HZS). Data shown are the percentage survival values relative to germination after 42 days in a glasshouse. Error bars represent the least significant difference (LSD) (5%) level for each onion cultivar. An ‘S’ indicates the value below which there is a significant difference from control plants.

Figure 2

Pathogenicity of 32 Fusarium oxysporum isolates on onion bulbs (cv. Napoleon). Data shown are the percentage bulb areas diseased on bisected bulbs after 9 weeks at 20 °C. Error bar represents the least significant difference (LSD) (5%) level. An ‘S’ indicates the value above which there is a significant difference from the uninoculated control bulbs.

Figure 3

Maximum likelihood tree of Fusarium isolates from onion and other hosts based on a concatenated alignment of translation elongation factor 1α (EF‐1α) (GenBank accession numbers KP964857–KP964909), RNA polymerase II second largest subunit (RPB2) (GenBank accession numbers KP964804–KP964856) and β‐tubulin (TUB2) (GenBank accession numbers KP964910–KP964962) genes. Numbers represent bootstrap values from 1000 replicates. Scale bar indicates 0.01 substitutions per site. The tree is rooted through L5 (F. avenaceum) and this branch has been collapsed because of its distance from F. oxysporum. BI refers to a sequence derived from the genomes on the Broad Institute Fusarium database (Broad Institute/MIT, 2007).

Figure 4

Maximum likelihood trees of Fusarium isolates from onion and other hosts based on (a) SIX 7, (b) SIX9, (c) SIX10, and (d) SIX12 gene sequences. Numbers represent bootstrap values from 1000 replicates. Scale bars indicate the number of substitutions per site. All FO lycopersici refers to the genome sequenced isolates listed in Table 1 as well as additional identical sequences obtained from a BLAST search. All FO cubense refers to the genome sequenced isolate II5 as well as identical BLAST hits. Sequences of other NRRL isolates were extracted from genome sequences (Broad Institute/MIT, 2007). All FO canariensis and FO lini isolates (with the exception of FOLIN, SIX12) are as described by Laurence et al. (2015).

Figure 5

Amino acid alignment of putative RxLR effectors from Fusarium oxysporum and F. proliferatum. The signal peptide (as predicted by SignalP) is shaded in light grey, whereas the RxLR domain is shown in bold (bold and italics for an incomplete RxLR domain). Amino acids that often occur after an RxLR domain (dEER) are underlined. Sequences from HDV247 and CL57 were obtained from the Broad Institute Fusarium database (Broad Institute/MIT, 2007).

Figure 6

Neighbour‐joining tree of Fusarium oxysporum CRX1 and CRX2 genes and their homologues. Numbers represent bootstrap values from 1000 replicates. The scale bar indicates 0.02 substitutions per site. Sequences from HDV247 and CL57 were obtained from the Broad Institute Fusarium database (Broad Institute/MIT, 2007). The sequence from Fa05001 was obtained from an assembled genome (GenBank accession number GCA_000769215).

Figure 7

Quantitative expression of a set of putative effector genes in onion roots following inoculation with Fusarium oxysporum f. sp. cepae (FOC) isolate FUS2. Expression was calculated relative to translation elongation factor 1α (EF‐1α) and β‐tubulin (TUB2). Error bars show the standard error of the mean (SEM) of three replicates; hpi, hours post‐inoculation. Asterisks indicate expression levels significantly different from 8 hpi based on analysis of variance (ANOVA) followed by Tukey's test (*P < 0.05, **P < 0.01, ***P < 0.001). + indicates that the expression of C5 was significantly higher at 72 hpi relative to 16 hpi (P < 0.05).