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Identification of Pathogenicity Groups and Pathogenic Molecular Characterization of Fusarium oxysporum f. sp. sesami in China

    Affiliations
    Authors and Affiliations
    • Yinghui Duan
    • Wenwen Qu
    • Shuxian Chang
    • Chun Li
    • Fangfang Xu
    • Ming Ju
    • Ruihong Zhao
    • Huili Wang
    • Haiyang Zhang †
    • Hongmei Miao †
    1. Henan Sesame Research Center, Henan Academy of Agricultural Sciences, Zhengzhou, Henan, 450002, P. R. China
    Published Online:6 Apr 2020https://doi.org/10.1094/PHYTO-09-19-0366-R

    Abstract

    Fusarium oxysporum f. sp. sesami is an extremely destructive pathogen, causing sesame Fusarium wilt disease worldwide. To clarify the pathogenicity and the genetic characters of F. oxysporum f. sp. sesami, we systematically investigated 69 F. oxysporum isolates collected from major sesame-growing areas in China. Among these isolates, 54 isolates were pathogenic and 15 were nonpathogenic according to pathogenicity testing on sesame seedlings. For the pathogenic isolates, three F. oxysporum f. sp. sesami pathogenicity groups were defined based on the three differential sesame hosts for the first time. A translation elongation factor 1α gene tree was constructed to determine the genetic diversity of the F. oxysporum isolates but could not separate F. oxysporum f. sp. sesami isolates from the nonpathogenic isolates and other F. oxysporum formae speciales. Ten secreted-in-xylem (SIX) genes (one family of effectors) were identified in F. oxysporum f. sp. sesami isolates by a search with the genome data, and were subsequently screened in the 69 F. oxysporum isolates. Compared with the SIX gene profiles in other F. oxysporum formae speciales, the presence and sequence variations of the SIX gene homologs directly correlated with the specific pathogenicity of F. oxysporum f. sp. sesami toward sesame. Furthermore, eight of these F. oxysporum f. sp. sesami SIX genes were significantly expressed in sesame plants as infection of the F. oxysporum f. sp. sesami isolate. These findings have important significance for understanding the pathogenic basis of F. oxysporum f. sp. sesami isolates, and will contribute to improve the diagnostics to effectively control Fusarium wilt disease in sesame.

    Sesame (Sesamum indicum L.) is one of the oldest oilseed crops and is widely cultivated in tropical and subtropical regions of Asia, Africa, and South America (Ashri 1998). Sesame production is seriously threatened by Fusarium wilt disease caused by Fusarium oxysporum f. sp. sesami, which was first found in the United States (Armstrong and Armstrong 1950) and was subsequently reported in China, Korean, Egypt, India, Sudan, and other main sesame production countries (Cho and Choi 1987; Li 1989; Verma et al. 2005). In China, F. oxysporum f. sp. sesami has been spread in most sesame-growing areas. The fungus commonly infects the roots of sesame plants, causing damping-off at the seedling stage, leaf chlorosis and abscission, stem necrosis, internal vascular browning, and eventually whole-plant wilting and death at the adult stage (Li et al. 2012; Su et al. 2012). Recently, a number of F. oxysporum f. sp. sesami isolates have been identified from wilted sesame plants and their morphological and pathogenic characteristics were further described (Li et al. 2012; Qiu et al. 2014; Su et al. 2012). However, only a single study has attempted to analyze the genetic diversity of F. oxysporum f. sp. sesami isolates by an amplified fragment length polymorphism (AFLP) method, which showed an ambiguous correlation between molecular characteristic and pathogenicity (Li et al. 2012). Thus far, a study on pathogenic differentiation, genetic diversity, and pathogenic basis of F. oxysporum f. sp. sesami isolates is not yet available.

    F. oxysporum is well known as a ubiquitous soilborne plant pathogen that infects a wide range of plant hosts worldwide and always causes significant economic losses (Gordon and Martyn 1997; Michielse and Rep 2009). As a species complex, F. oxysporum comprises a diversity of morphologically indistinguishable nonpathogenic and pathogenic isolates (Snyder and Smith 1981). Conventionally, pathogenic isolates are assigned to intraspecific groups designated as formae speciales based on their host specificity (Snyder and Hansen 1940). Furthermore, several formae speciales can be subdivided into races based on their pathogenic specificity to different genotypes, lines, or cultivars of the same host species (Armstrong and Armstrong 1981).

    To understand the pathogenic differentiation and genetic diversity in the F. oxysporum species complex (FOSC), numerous phylogenetic studies have been carried out using standard molecular approaches such as DNA fingerprinting by AFLP, restriction fragment length polymorphism, and random amplified polymorphic DNA; and locus genotyping based on sequences of internal transcribed spacer (ITS), intergenic spacer, and housekeeping genes, including translation elongation factor 1α (EF-1a), β-tubulin (Tub), RNA polymerase II, and so on (Baayen et al. 2000; Epstein et al. 2017; Lievens et al. 2008; Recorbet et al. 2003; Taylor et al. 2016). These studies revealed that the FOSC, including both phytopathogenic and clinical isolates, could be divided into four evolutionary clades (O’Donnell et al. 2004). Furthermore, the phylogenetic classification was attempted as the molecular method for identification of pathogenic F. oxysporum isolates, which was classically based on pathogenicity testing (Lievens et al. 2008). However, standard molecular approaches have not been successful in distinguishing between pathogenic and nonpathogenic isolates or between different formae speciales (Taylor et al. 2016).

    The genetic basis of pathogenicity in the FOSC has been found to involve specific virulence genes, which are required for virulence in host plants (Hogenhout et al. 2009). Recently, a number of putative effector genes were found to localize on the accessory lineage-specific chromosomes in F. oxysporum f. sp. lycopersici (Ma et al. 2010). Among them, 14 effector genes were identified and designated as secreted-in-xylem (SIX) genes. These genes encode small, cysteine-rich proteins, and are secreted into the xylem sap during infection of tomato plants (Houterman et al. 2007; Lievens et al. 2009; Rep and Kistler 2010; Rep et al. 2004; Schmidt et al. 2013). Moreover, five F. oxysporum f. sp. lycopersici SIX genes—SIX1 (Avr3), SIX3 (Avr2), SIX4 (Avr1), SIX5, and SIX6—were found to contribute directly to host-specific pathogenicity (Gawehns et al. 2014; Houterman et al. 2009; Ma et al. 2015; Niu et al. 2016; Rep et al. 2004), while SIX1, SIX3, and SIX4 were also recognized as avirulence genes by corresponding resistance genes I-3, I-2, and I in tomato (Houterman et al. 2009; Ma et al. 2015; Rep et al. 2004; Takken and Rep 2010). At present, a large number of homologs of F. oxysporum f. sp. lycopersici SIX genes have been subsequently detected in other F. oxysporum formae speciales, including betae, canariensis, cepae, conglutinans, cubense, dianthi, fragariae, lilii, lycopersici, medicaginis, melonis, niveum, passiflorae, phaseoli, physali, pisi, raphani, vasinfectum, and zingiberi (Chakrabarti et al. 2011; Covey et al. 2014; Czislowski et al. 2018; Fraser-Smith et al. 2014; Laurence et al. 2015; Lievens et al. 2009; Simbaqueba et al. 2018; Taylor et al. 2016; Thatcher et al. 2012; Williams et al. 2016). These homologs of SIX genes commonly form a particular combination within individual F. oxysporum formae speciales or races (Chakrabarti et al. 2011; Czislowski et al. 2018; Lievens et al. 2009). Several studies have also demonstrated that the presence and sequence variations of SIX genes could be used as ideal loci for pathogenicity-based molecular diagnostics to distinguish between F. oxysporum formae speciales or races (Chakrabarti et al. 2011; Fraser-Smith et al. 2014; Lievens et al. 2009).

    In this study, we systematically investigated the pathogenicity of 69 F. oxysporum isolates collected from major sesame-growing areas in China, and subdivided them into three pathogenicity groups based on the three differential sesame hosts with different genetic backgrounds of resistance to Fusarium wilt, which were selected from more than 500 sesame germplasm accessions worldwide using resistance evaluation techniques (Miao et al. 2019; Qiu et al. 2014). To further explore the relationship between the pathogenicity and genetic diversity of F. oxysporum isolates from sesame, molecular characterization of EF-1a and several SIX genes was determined. Particularly, the homologs of 10 SIX genes were first identified in F. oxysporum f. sp. sesami isolates. The presence and sequence variations of these SIX gene homologs were demonstrated to correlate with specific pathogenicity of F. oxysporum f. sp. sesami toward sesame. The results revealed the availability of SIX genes for reliable discrimination of F. oxysporum f. sp. sesami isolates from nonpathogenic isolates and other formae speciales.

    MATERIALS AND METHODS

    Collection of Fusarium isolates.

    A large number of wilted sesame plants were collected from major sesame-growing areas in China between 2007 and 2011. The Fusarium isolates were separated and purified using the method described by Su et al. (2012). A small section, 2 to 3 cm in length, was cut from the infected tissues and surface sterilized in 70% ethanol for 1 min followed by 1.25% NaClO for 10 min, then rinsed in sterile distilled water three times. The vascular tissue were removed from the sterilized section and placed on potato dextrose agar (PDA) medium with streptomycin at 100 μg/ml, and incubated for 3 to 4 days at 28°C. After the morphological characteristics of Fusarium-like colonies, hyphae, and microconidia were observed, the isolates were purified by single-spore isolation, and maintained on PDA plates. A representative subset of 69 F. oxysporum isolates and three F. solani isolates from sesame was selected for subsequent pathogenicity testing, gene amplification, and phylogenetic analysis (Table 1).

    TABLE 1. Fusarium isolates used for pathogenicity testing, phylogenetic analysis, and identification of secreted-in-xylem genes in this study

    Pathogenicity testing.

    The aforementioned 72 Fusarium isolates were assessed for their pathogenicity toward sesame seedlings as described by Qiu et al. (2014). Each isolate was cultured on PDA for 5 to 7 days at 28°C. One agar plug (5 mm) from the colony edge was placed in a culture flask containing potato dextrose broth (PDB) and incubated at 28°C and 120 rpm on a rotary shaker for 4 days. Microconidial suspensions were filtered through three layers of sterilized gauze and centrifuged at 5,000 rpm for 8 min. The microconidial pellet was washed three times in sterile distilled water to remove the PDB. Then, the spores were resuspended to a final concentration of 4 × 106 conidia/ml for inoculation.

    Three sesame cultivars (Yuzhi 11, Ji 9014, and HJ16) were chosen for the pathogenicity evaluation. Three independent replicates for each variety were set for each treatment. Ten seeds for each sesame cultivar were grown in 12-cm-diameter plastic pots with infected medium containing a mixture of conidial suspensions, sterilized vermiculite, and soil (1:3:3 [vol/vol/vol]). The final concentration of F. oxysporum f. sp. sesami isolates was set at 5 to 7 × 105 conidia/ml. Inoculated plants were positioned in growth chambers with day and night of 15 and 9 h at 28 and 23°C, respectively, and 70% relative humidity. The 10-fold-diluted liquid Murashige-Skoog medium was watered regularly for seedling growth. Sterile distilled water was used as control.

    Fusarium wilt disease symptoms of each treatment were recorded after 28 days inoculation. The symptoms of roots, stems, and leaves were scored using a categorical disease severity scale, where 0 = no visible symptoms, 1 = slight wilting of seedling, 2 = severe browning of principal root, wilting of plant, and slow growth, and 3 = death of the whole plant. The disease index (DI) data were further calculated using the formula described by Qiu et al. (2014). The significant difference in pathogenicity between Fusarium isolates was analyzed using analysis of variance in SAS software (SAS Institute Inc.). The pathogenicity levels of Fusarium isolates were also evaluated using the grade scale as follows: 0 ≤ DI ≤ 20 = nonpathogenicity or weak pathogenicity, 20 < DI ≤ 50 = moderate pathogenicity, and 50 < DI ≤ 100 = high pathogenicity.

    DNA extraction.

    The mycelia of each Fusarium isolate were collected on three layers of sterilized gauze, and the excess liquid was removed using filter papers. Immediately, the mycelia were frozen in liquid nitrogen and stored at −80°C for genomic DNA extraction. Genomic DNA was extracted using a Qiagen DNeasy Plant Minikit (Qiagen) according to the manufacturer’s instructions.

    PCR amplification of EF-1a gene.

    The EF-1a gene was used to distinguish taxa at the infraspecies level within the 69 F. oxysporum isolates from sesame. The EF-1a gene sequences were amplified by PCR with a primer pair designed using Primer Premier 5.0 (PREMIER Biosoft) (Table 2). PCRs were set up in a 20-μl mixture containing 1 × PCR Buffer, 0.1 mM dNTPs, 0.2 μM of each primer, 1.0 U of Taq polymerase (Vazyme), and approximately 50 ng of DNA. PCR amplifications were performed on an Eppendorf Mastercycler (Eppendorf) under the following conditions: 95°C for 2 min; 35 cycles of 95°C for 20 s, 55°C for 30 s, and 72°C for 1 min; with a final 8-min extension at 72°C. The products were visualized by electrophoresis on 1% agarose gel using a Gel Doc XR+ Imaging System (Bio-Rad) and purified using a SanPrep Column DNA Gel Extraction Kit (Sangon Biotech), then sequenced on an ABI 3730 XL DNA Analyzer (Applied Biosystem) by forward and reverse primers. The EF-1a gene sequences in the 69 F. oxysporum isolates were deposited into GenBank with the accessions MN417138 to MN417206. Additionally, EF-1a gene sequences in other species were obtained from the publicly available genomes of F. oxysporum (https://www.ncbi.nlm.nih.gov/genome/genomes/707) and the NCBI nucleotide database through a BLAST search on a reference sequence in F. oxysporum f. sp. sesami isolate FS08027 (Supplementary Table S1).

    TABLE 2. Primer pairs used for the amplification and quantitative detection of target sequences (genes) in this study

    Identification of SIX genes.

    The SIX genes were identified in F. oxysporum f. sp. sesami by a BLAST search with E-value ≤ 1E-10 from our preliminary genome assemblies of the isolates FS08027, FS09095, and FS10175 (unpublished data) based on the reference sequences of SIX1 to -14 from F. oxysporum f. sp. lycopersici isolates 4287, MN25, and BFOL-51 (Lievens et al. 2009; Schmidt et al. 2013). All F. oxysporum f. sp. sesami SIX genes were further screened in the 69 F. oxysporum isolates and 3 F. solani isolates using PCR amplification. The specific primers of F. oxysporum f. sp. sesami SIX genes were designed (Table 2), and PCRs were set up as described above. PCR amplification was performed with standard conditions as follows: 95°C for 2 min; 35 cycles of 95°C for 20 s, 55 to 61°C annealing (see Table 2 for temperatures) for 30 s, and 72°C for 1 min; with a final cycle of 8 min at 72°C. The amplicons were visualized, purified, and sequenced as described above. The GenBank accessions MN417207 to MN417222 were provided for the SIX1 to -14 gene sequences in the Fusarium isolates from sesame. The homologs of SIX genes were also searched in other species by blasting the reference sequences of F. oxysporum f. sp. lycopersici SIX1 to -14 against the aforementioned nucleotide datasets (Supplementary Table S1). The sequences of the SIX genes in both F. oxysporum f. sp. sesami and the other species were aligned using DNAMAN 6 software (Lynnon Corporation), and the sequence similarities of SIX genes were outputted by the observed divergence method based on the sequence alignments.

    Phylogenetic analysis.

    Phylogenetic analyses were performed on the EF-1a and SIX gene datasets. The sequences were aligned by the Muscle method, and the maximum-likelihood (ML) trees were constructed using MEGA 5.2 software (https://www.megasoftware.net/) with the computed most appropriate models: Kimura two-parameter for EF-1a, SIX6, SIX11, and SIX14; Kimura two-parameter plus invariant sites for SIX1 and SIX9; Kimura two-parameter plus γ distribution for SIX7, SIX8, and SIX13; Jukes-Cantor for SIX3; and Jukes-Cantor plus γ distribution for SIX10. For the EF-1a tree, the F. foetens isolate NRRL31841 was used as the outgroup taxon (Schroers et al. 2004). Branches were tested for the inferred tree by bootstrap analysis on 1,000 random trees.

    Infection treatment and expression profile assay of F. oxysporum f. sp. sesami SIX genes.

    The sesame cultivar Yuzhi 11, susceptible to isolate FS08027, was used in the infection treatment. Seed were grown in pots with sterilized vermiculite in growth chambers, as described above. Four-week-old seedlings with two pairs of true leaves were gently pulled out of the potting substrate, and the roots were carefully washed in water. The root mass was dipped into a microconidial suspension of 4 × 106 conidia/ml for 10 min; then, the seedlings were planted in pots with sterilized vermiculite. The roots were sampled at 12, 24, 48, 72, 120, and 168 h postinoculation (hpi). The microconidia and mycelia of the FS08027 strain were also collected as described above. Three independent replicates were carried out in the experiment. All samples were frozen in liquid nitrogen and stored at −80°C for total RNA extraction.

    Total RNA was extracted from samples using RNAiso Plus Reagent (TaKaRa), and genomic DNA was removed by DNase I treatment. First-strand cDNA was synthesized using a RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. Primer pairs of F. oxysporum f. sp. sesami SIX genes were designed using Primer Premier 5.0 for Real-time PCR detection. Real-time PCR was performed on a Mastercycler ep realplex (Eppendorf), with the PCR mixture in a total volume of 20 μl containing 10 μl of FastStart Essential DNA Green Master (Roche), 2.0 μl of fivefold-diluted first-strand cDNA, and 0.2 μM each primer. The cycling conditions were as follows: 95°C for 10 min and 40 cycles of 95°C for 15 s, 56 to 60°C for 20 s (Table 2), and 72°C for 15 s. The specific product of each gene was confirmed using the melt-curve analysis. The transcriptional level of F. oxysporum f. sp. sesami SIX genes was analyzed against the F. oxysporum f. sp. sesami Tub gene using the 2−ΔCT method (Pfaffl 2001). The significant difference between transcriptional levels in microconidia, mycelia, and infected roots was analyzed using Student’s t test in SPSS 16.0 (SPSS Inc.).

    RESULTS

    Pathogenicity testing and classification of pathogenicity group of F. oxysporum f. sp. sesami isolates.

    On the basis of the geographical locations, morphological characteristics, and ITS sequences, 69 F. oxysporum isolates from sesame were selected for pathogenicity testing. Meanwhile, three F. solani isolates originally separated from diseased sesame plants were also evaluated for their ability to cause wilt disease. In the sesame seedling test, significantly different pathogenicity was observed in the F. oxysporum isolates on three sesame cultivars (Yuzhi 11, Ji 9014, and HJ16) (Fig. 1). Of the 69 F. oxysporum isolates, 50 isolates caused significant seedling wilt on all three sesame cultivars; 1 isolate (FS09095) was highly pathogenic to Ji 9014 and HJ16 but presented nonpathogenicity or weak pathogenicity to Yuzhi 11; and 3 isolates (FS09060, FS09069, and FS10175) were specifically pathogenic to HJ16. Therefore, in total, 54 F. oxysporum isolates could be assigned to f. sp. sesami. In addition, the remaining 15 F. oxysporum isolates and 3 F. solani isolates did not cause significant disease symptoms on sesame.

    Fig. 1.

    Fig. 1. Pathogenicity of 69 Fusarium oxysporum isolates and 3 F. solani isolates toward the seedlings of three sesame cultivars: Yuzhi 11, Ji 9014, and HJ16. Data on the disease index (DI) were calculated from three independent biological replications at 28 days postinoculation. Error bars indicate the standard error of the mean. The least significant difference (α = 0.05) was analyzed using analysis of variance in SAS software. The dotted line separates the DI values into two parts, and the upper is significantly different from the uninoculated control (CK).

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    Based on the different pathogenicity toward three sesame cultivars, the F. oxysporum f. sp. sesami isolates were subdivided into three pathogenicity groups. F. oxysporum f. sp. sesami pathogenicity group 1 was defined for the isolates with pathogenicity specifically on HJ16, F. oxysporum f. sp. sesami pathogenicity group 2 was pathogenic to Ji 9014 and HJ16 but nonpathogenic to Yuzhi 11, and F. oxysporum f. sp. sesami pathogenicity group 3 was highly pathogenic to all three cultivars (Table 3). Surprisingly, 50 of the 54 F. oxysporum f. sp. sesami isolates were assigned to F. oxysporum f. sp. sesami pathogenicity group 3, which was distributed across almost all the collection locations in China between 2007 and 2011. In contrast, the three isolates of pathogenicity group 1 were collected from Hebei, Henan, and Anhui Provinces in 2009 and 2010, and the only isolate of pathogenicity group 2 was collected from Anhui Province in 2009.

    TABLE 3. Homologs of secreted-in-xylem (SIX)1 to -14 identified in Fusarium oxysporum and F. solani isolates from sesame

    Phylogenetic relationship of F. oxysporum f. sp. sesami isolates.

    To determine the phylogenetic relationship of F. oxysporum f. sp. sesami and nonpathogens from sesame, the ML tree was constructed based on EF-1a sequences, including those in F. oxysporum isolates from sesame and other plants and nonpathogenic isolate Fo47. The phylogenetic analyses showed that the FOSC isolates in this study were divided into four clades, and the 69 isolates from sesame were placed in clades I, III, and IV (Fig. 2). Clade I covered all 54 pathogenic isolates in F. oxysporum f. sp. sesami pathogenicity groups 1, 2, and 3 as well as the 2 nonpathogenic isolates, FS09046 and FS10184b1. Isolate FS09046 presented an EF-1a sequence identical to that of the F. oxysporum f. sp. sesami isolates, which was also identical to the isolates of ff. spp. vasinfectum, niveum, and fragariae. Additionally, 1 nonpathogenic isolate, FS11476a, shared an identical EF-1a sequence with the biocontrol isolate Fo47 and was placed in clade III (Fuchs et al. 1997), while the other 12 nonpathogenic isolates from sesame were clustered in clade IV with the isolates of f. sp. cubense race 4 isolates.

    Fig. 2.

    Fig. 2. Maximum-likelihood tree inferred from the translation elongation factor 1a (EF-1a) gene sequences in Fusarium isolates from sesame and other hosts. Scale bars indicate the number of substitutions per site. Numbers above the branch nodes represent bootstrap values from 1,000 replications. The sequence of EF-1a from Fusarium foetens isolate NRRL 31841 acts as an outgroup to root the tree. The isolates in the same pathogenicity group of F. oxysporum f. sp. sesami (Fos) with an identical EF-1a sequence are compressed into a branch, and their number is indicated in parentheses. The F. oxysporum f. sp. sesami isolates are indicated by solid black circles and nonpathogenic isolates from sesame are indicated by open circles.

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    Identification of SIX genes in F. oxysporum f. sp. sesami.

    The genome sequences of the three F. oxysporum f. sp. sesami isolates (FS10175, FS09095, and FS08027) were queried based on F. oxysporum f. sp. lycopersici SIX1 to -14 sequences to search for SIX genes in F. oxysporum f. sp. sesami. As a result, the homologies of 10 F. oxysporum f. sp. lycopersici SIX genes—SIX1, SIX3, SIX6, SIX7, SIX8, SIX9, SIX10, SIX11, SIX13, and SIX14—were identified in the F. oxysporum f. sp. sesami isolates. Furthermore, the presence and sequence of these SIX gene homologs were verified by PCR screening in the 69 F. oxysporum isolates and 3 F. solani isolates from sesame (Table 3). All 10 F. oxysporum f. sp. sesami SIX genes were present in the 3 isolates in F. oxysporum f. sp. sesami pathogenicity group 1 and 29 isolates in F. oxysporum f. sp. sesami pathogenicity group 3, and the majority of the SIX genes (except one or two of the SIX1, SIX3, SIX9, SIX10, and SIX14 genes) were present in the other 21 isolates in F. oxysporum f. sp. sesami pathogenicity group 3 and the 1 isolate in F. oxysporum f. sp. sesami pathogenicity group 2. Noticeably, five SIX genes (SIX6, SIX7, SIX8, SIX11, and SIX13) were identified in all 54 F. oxysporum f. sp. sesami isolates. In contrast, all SIX genes were absent in the 15 nonpathogenic F. oxysporum and 3 F. solani isolates, with the exception of two nonpathogenic isolates, FS10090a and FS10176b, which possessed the SIX8 gene.

    No polymorphisms in eight F. oxysporum f. sp. sesami SIX genes were individually detected either within an F. oxysporum f. sp. sesami isolate or between the different F. oxysporum f. sp. sesami isolates, with the exception of SIX8 and SIX13 (Table 3). The sequence variations of SIX8 and SIX13 were observed in a single F. oxysporum f. sp. sesami isolate, which led to the identification of five distinct SIX8 gene homologs (SIX8a1, SIX8a2, SIX8a3, SIX8a4, and SIX8b) and two SIX13 gene homologs (SIX13a and SIX13b) (Supplementary Figs. S1 and S2). Another SIX8 gene homolog (SIX8a5) was identified in the nonpathogenic isolates FS10090a and FS10176b. Similar to other F. oxysporum f. sp. sesami SIX genes, each homolog of F. oxysporum f. sp. sesami SIX8 and SIX13 had an identical sequence between the different F. oxysporum f. sp. sesami isolates.

    The homologs of SIX genes were also identified from another 26 F. oxysporum formae speciales isolates and Verticillium dahliae isolate JR2 (Supplementary Table S2) (Czislowski et al. 2018). The sequence alignments generated from the coding DNA sequences of these SIX genes showed that SIX genes in F. oxysporum f. sp. sesami shared high sequence similarity to those in other formae speciales, ranging from 63.4 to 100% nucleotide identity (Supplementary Table S3). Notably, F. oxysporum f. sp. sesami SIX8a4 and SIX14 shared identical sequences with corresponding SIX genes in F. oxysporum f. sp. niveum, and F. oxysporum f. sp. sesami SIX9 also had an identical sequence with the SIX9 genes in ff. spp. vasinfectum, niveum, and raphanin and Fo5176. However, comparing the presence and sequence variations of SIX genes between different F. oxysporum isolates, F. oxysporum f. sp. sesami exhibited a specific combination of SIX genes, which was distinguishable from the SIX gene profiles in other formae speciales screened in this study (Table 3; Supplementary Table S2). In addition, among the F. oxysporum f. sp. sesami isolates, F. oxysporum f. sp. sesami pathogenicity group 2 was distinguishable from F. oxysporum f. sp. sesami pathogenicity groups 1 and 3 by the absence of both SIX8a3 and SIX13b, whereas F. oxysporum f. sp. sesami pathogenicity groups 1 and 3 exhibited a similar profile of SIX genes.

    Evolutionary relationship of SIX genes in F. oxysporum f. sp. sesami.

    The phylogenetic trees were constructed for each of 10 SIX genes identified in F. oxysporum f. sp. sesami to further determine the evolutionary relationships of the SIX genes. The results showed that the SIX gene sequence divergence was clearly observed between the different F. oxysporum formae speciales, and F. oxysporum f. sp. sesami was also separated from the other formae speciales in the SIX1, SIX3, SIX6, SIX7, SIX10, SIX11, and SIX13 gene trees but not in the SIX8, SIX9, and SIX14 gene trees owing to the identical SIX gene sequences described above between F. oxysporum f. sp. sesami and several other formae speciales (Fig. 3). In addition, the five homologs of F. oxysporum f. sp. sesami SIX8 were clustered into four subclades in the SIX8 gene tree, while F. oxysporum f. sp. sesami SIX8a1 and SIX8a2 was placed in a subclade including SIX8a5 in the nonpathogenic isolates FS10090a and FS10176b and the homologs of SIX8 in f. sp. cubense race 4 isolates (Fig. 3E; Supplementary Fig. S1), and presented more distant evolutionary relationships with one another compared with the SIX8 homologs in other formae speciales. Unlike F. oxysporum f. sp. sesami SIX8, the two homologs of F. oxysporum f. sp. sesami SIX13 were sorted into one subclade, and F. oxysporum f. sp. sesami SIX13a seemed to be more similar to F. oxysporum f. sp. medicaginis SIX13b than to F. oxysporum f. sp. sesami SIX13b (Fig. 3I; Supplementary Fig. S2).

    Fig. 3.

    Fig. 3. Maximum-likelihood trees showing the sequences of secreted-in-xylem (SIX) gene sequences in Fusarium oxysporum isolates from sesame, other hosts, and a Verticillium dahliae isolate. A, SIX1; B, SIX3; C, SIX6; D, SIX7; E, SIX8; F, SIX9; G, SIX10; H, SIX11; I, SIX13; and J, SIX14. Isolates in other F. oxysporum and V. dahliae used in this study are listed in Supplementary Table S2, and the homologs of SIX genes were obtained for each isolate by a BLAST search from the public nucleotide datasets, including the nucleotide database in GenBank and the selected genome data described in Supplementary Table S1. Isolates in a pathogenicity group of F. oxysporum f. sp. sesami (Fos) with an identical sequence of SIX gene homologs are compressed into a branch. The F. oxysporum f. sp. sesami isolates are indicated by solid black circles and the nonpathogenic isolate from sesame is indicated by an open circle.

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    The topologies of the SIX gene trees were further compared with the EF-1a tree. F. oxysporum f. sp. sesami and other formae speciales were clustered together in the SIX1, SIX3, SIX10, SIX11, and SIX13 gene trees but were placed into distant clades in the EF-1a tree (Figs. 2 and 3). For example, F. oxysporum f. sp. sesami presented closer relationships to F. oxysporum f. sp. melonis in the SIX1 gene tree but they were separated into clades I and II in the EF-1a tree. The results indicate that the SIX gene phylogeny is discordant to the EF-1a phylogeny.

    Expression profiles of F. oxysporum f. sp. sesami SIX genes upon infection with F. oxysporum f. sp. sesami.

    To explore the expression profiles of the 10 F. oxysporum f. sp. sesami SIX genes identified in this study, the transcriptional levels of all of the F. oxysporum f. sp. sesami SIX gene homologs were evaluated in sesame roots infected by F. oxysporum f. sp. sesami pathogenicity group 3 isolate FS08027 using real-time reverse-transcription PCR (Fig. 4). The expression of each F. oxysporum f. sp. sesami SIX gene was not different between microconidia and mycelia cultured in vitro. Compared with that in microconidia, the expression of F. oxysporum f. sp. sesami SIX8a1, SIX8a2, SIX8b, SIX13a, and SIX14 was significantly upregulated at 24 to 168 hpi and F. oxysporum f. sp. sesami SIX7, SIX10, and SIX13b were upregulated at 48 to 168 hpi, whereas F. oxysporum f. sp. sesami SIX6, SIX9, and SIX11 were upregulated after 72 hpi. Furthermore, F. oxysporum f. sp. sesami SIX6, SIX8a1, SIX8a2, SIX8b, SIX9, SIX10, SIX11, SIX13a, and SIX14 showed more transcript accumulation as compared with F. oxysporum f. sp. sesami SIX7 and SIX13b. In contrast, the low expression level of F. oxysporum f. sp. sesami SIX1 did not significantly change at any time point, and F. oxysporum f. sp. sesami SIX3, SIX8a3, and SIX8a4 had no transcripts detected in planta during F. oxysporum f. sp. sesami infection.

    Fig. 4.

    Fig. 4. Expression profiles of the homologs of Fusarium oxysporum f. sp. sesami (Fos) secreted-in-xylem (SIX) genes in sesame roots inoculated with F. oxysporum f. sp. sesami isolate FS08027. F. oxysporum f. sp. sesami β-tubulin gene was used as a reference gene. Transcriptional levels of F. oxysporum f. sp. sesami SIX genes were calculated relative to β-tubulin. Error bars indicate the standard errors of three biological replications. Asterisks indicate significant differences (* and ** indicate P < 0.05 and 0.01, respectively) compared with microconidia using Student’s t test.

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    DISCUSSION

    Sesame Fusarium wilt is one of the major problems facing sesame producers in China owing to its economically destructive effects (Jyothi et al. 2011). Current effective control means are involved in the disease prevention prior to infection with pathogenic F. oxysporum isolates (Lievens et al. 2008). The ability to reliably identify and distinguish pathogenic F. oxysporum isolates from sesame is of key importance for implementation of disease management. In this study, we determined the structure of pathogenic differentiation of F. oxysporum f. sp. sesami and demonstrated the correlation between the presence of SIX genes and the pathogenicity of F. oxysporum f. sp. sesami on sesame.

    To systematically investigate the pathogenicity of F. oxysporum isolates from sesame, three sesame cultivars were selected as the differential hosts from more than 500 worldwide sesame germplasms, which were previously assessed for levels of resistance to Fusarium wilt under natural field and greenhouse conditions (data not shown). Based on the significantly different pathogenicity toward the differential cultivars, three pathogenicity groups were defined for F. oxysporum f. sp. sesami isolates for the first time. Of three F. oxysporum f. sp. sesami pathogenicity groups, the large number of isolates in pathogenicity group 3 with a high level of pathogenicity were the most geographically widespread across China, in contrast to the few isolates in F. oxysporum f. sp. sesami pathogenicity groups 1 and 2. Therefore, F. oxysporum f. sp. sesami pathogenicity group 3 was indisputably prevalent in past years in China.

    Our phylogenetic tree based on EF-1a sequences divided the FOSC isolates into four clades, which showed that there was considerable genetic diversity between the isolates from sesame. In contrast, the nonpathogenic isolates from sesame were demonstrated to be phylogenetically diverse. F. oxysporum f. sp. sesami isolates were topologically monophyletic, because all of the F. oxysporum f. sp. sesami isolates were placed in one clade with an identical EF-1a sequence. In addition, F. oxysporum f. sp. sesami isolates also shared the identical EF-1a sequence with two nonpathogenic isolates from sesame and another three formae speciales isolates. This suggested that the tree inferred from the EF-1a gene failed to distinguish between the pathogenic and nonpathogenic isolates from sesame and between isolates of different formae speciales. In previous reports, poor correlation has been found between sequence variation of housekeeping genes and host-specific pathogenicity in other F. oxysporum isolates (O’Donnell et al. 1998).

    Recent findings indicated that pathogenicity in F. oxysporum f. sp. lycopersici was conferred by multiple effector genes on a small accessory chromosome (Ma et al. 2010). Among these effector genes, several SIX genes have been further demonstrated to facilitate the pathogenicity of F. oxysporum f. sp. lycopersici (Gawehns et al. 2014; Houterman et al. 2009; Ma et al. 2015; Niu et al. 2016; Rep et al. 2004). The hypothesis of the current study was that homologs of SIX genes might exist in F. oxysporum f. sp. sesami with a conserved function, as proposed in other formae speciales (Simbaqueba et al. 2018; Taylor et al. 2016). For the first time, 10 SIX genes were identified in F. oxysporum f. sp. sesami isolates. F. oxysporum f. sp. sesami SIX gene sequences were identical and highly similar within and between F. oxysporum f. sp. sesami isolates, respectively, as well as highly conserved with those in other formae speciales. Previously, the high conservation of SIX gene sequences in formae speciales suggested that SIX genes on the accessory chromosome were gained by horizontal gene transfer between F. oxysporum strains (Czislowski et al. 2018; Fraser-Smith et al. 2014; Ma et al. 2010). In our gene trees, further evidence was that there was discordance between the evolutionary relationships of the SIX genes and EF-1a gene within the FOSC, which supported horizontal gene transfer as the evolutionary origin of F. oxysporum f. sp. sesami SIX genes.

    The SIX gene profiles in F. oxysporum f. sp. sesami isolates were significantly different from the combinations of SIX genes in other formae speciales and absence of SIX genes in the nonpathogenic isolates from sesame. This revealed that there was a clear correlation between the F. oxysporum f. sp. sesami SIX gene profile and pathogenicity toward sesame, suggesting that F. oxysporum f. sp. sesami SIX genes could be used as candidate loci for the molecular differentiation of F. oxysporum f. sp. sesami isolates from nonpathogenic isolates and the isolates of other formae speciales. Previously, gene SIX6 was demonstrated to distinguish F. oxysporum f. sp. vasinfectum isolates from related colocalized nonpathogenic F. oxysporum isolates and from nonnative countries (Chakrabarti et al. 2011). The polymorphisms of SIX3 and the presence of SIX4 were used to distinguish F. oxysporum f. sp. lycopersici races 1, 2, and 3 (Lievens et al. 2009), and the variation of the SIX gene profile could also reflect the differences in pathogenicity of F. oxysporum f. sp. cubense races (Czislowski et al. 2018; Fraser-Smith et al. 2014). In this study, F. oxysporum f. sp. sesami pathogenicity group 2 was distinguished from F. oxysporum f. sp. sesami pathogenicity groups 1 and 3 based on the absence of both SIX8a3 and SIX13b but F. oxysporum f. sp. sesami pathogenicity groups 1 and 3 seemed to be indistinguishable owing to their similar SIX gene profiles. Therefore, the suitability of SIX genes to distinguish F. oxysporum f. sp. sesami pathogenicity groups should be further assessed in a wider range of F. oxysporum f. sp. sesami isolates.

    The F. oxysporum f. sp. lycopersici race evolution has been reported in a stepwise manner, in which one race evolves from another and the virulence of a race accumulates sequentially in clonal lineages (Biju et al. 2017). Point mutation and gene deletion as well as transposon movement in SIX genes played major roles in the F. oxysporum f. sp. lycopersici race evolution by the loss of the avirulence function to evade resistance-gene-mediated resistance in a gene-for-gene relationship (Biju et al. 2017). For F. oxysporum f. sp. sesami isolates, pathogenicity groups 1, 2, and 3 were not only considered to originate from an ancestor because of their monophyletic evolution but also presented gradually higher virulence. Meanwhile, the gene-for-gene relationship was also observed between the differential cultivars and the F. oxysporum f. sp. sesami pathogenicity groups that could be indicated as the F. oxysporum f. sp. sesami races, because one dominant resistance gene locus was identified in Yuzhi 11 contributing to resistance to the isolates FS10175 and FS09095, respectively (data not shown). Therefore, the underlying hypothesis was that the F. oxysporum f. sp. sesami races had evolved in a similar manner to the F. oxysporum f. sp. lycopersici races. In this hypothesis, although the essential virulence genes in F. oxysporum f. sp. sesami functioned in host-specific pathogenicity, the sequence variations of avirulence genes were necessary for the emergence of diverse F. oxysporum f. sp. sesami races. Considering the direct correlation between the SIX genes and the specific pathogenicity of F. oxysporum f. sp. sesami, F. oxysporum f. sp. sesami SIX genes were inferred to be recognized as candidates for effector genes. As expected, the homologs of F. oxysporum f. sp. sesami SIX genes were significantly expressed in sesame plants during the F. oxysporum f. sp. sesami infection, with the exception of F. oxysporum f. sp. sesami SIX1, SIX3, SIX8a3, and SIX8a4. This supported the idea that most of the F. oxysporum f. sp. sesami SIX genes played important roles in the virulence genotype. However, the concrete roles of these F. oxysporum f. sp. sesami SIX genes were unknown. More studies such as gene knockout and complementation could be performed in order to reveal the role of SIX genes. In addition, owing to the indistinguishable SIX gene profiles between F. oxysporum f. sp. sesami pathogenicity groups 1 and 3, novel effectors should be expected to be found in F. oxysporum f. sp. sesami, because the putative effectors C5 and CRX1 were associated with pathogenicity in F. oxysporum f. sp. cepae (Taylor et al. 2016).

    Overall, the results of the current study subdivided F. oxysporum f. sp. sesami isolates into three pathogenicity groups in China. In F. oxysporum f. sp. sesami, the SIX genes were demonstrated to be associated with the specific pathogenicity toward sesame, and can be used as available candidate loci for molecular diagnosis on the basis of pathogenicity. Further studies are required to clearly define the F. oxysporum f. sp. sesami races and rigorously validate the hypothesis that SIX genes or novel genes in F. oxysporum f. sp. sesami function as effectors to facilitate pathogenicity on sesame and that their sequence variations have resulted in the emergence of diverse F. oxysporum f. sp. sesami races by the loss of the avirulence function or other evolutionary pathways.

    ACKNOWLEDGMENTS

    We thank Y. Su from Yunnan Academy of Agricultural Sciences for assisting us in the identification and classification of Fusarium isolates.

    The author(s) declare no conflict of interest.

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    The author(s) declare no conflict of interest.

    Funding: Key Laboratory of Specific Oilseed Crops Genomics of Henan Province, the earmarked fund for China Agriculture Research System (CARS-14), International Cooperation and Exchanges Project of Henan Province (182102410040), Science-Technology Foundation for Outstanding Youth Scientists of Henan Academy of Agricultural Sciences (2018YQ26), the Plan for Scientific Innovation Talent of Henan Province (184200510002), the Distinguished Professor Program of Institutions of Higher Learning in Henan Province (DPPIHL2017), and the Innovation Scientists and Technicians Troop Construction Projects of Henan Province (ISTTCPHP2016).