Comparative genomics of geographically distant Fusarium fujikuroi isolates revealed two distinct pathotypes correlating with secondary metabolite profiles

Abstract

Fusarium fujikuroi causes bakanae ("foolish seedling") disease of rice which is characterized by hyper-elongation of seedlings resulting from production of gibberellic acids (GAs) by the fungus. This plant pathogen is also known for production of harmful mycotoxins, such as fusarins, fusaric acid, apicidin F and beauvericin. Recently, we generated the first de novo genome sequence of F. fujikuroi strain IMI 58289 combined with extensive transcriptional, epigenetic, proteomic and chemical product analyses. GA production was shown to provide a selective advantage during infection of the preferred host plant rice. Here, we provide genome sequences of eight additional F. fujikuroi isolates from distant geographic regions. The isolates differ in the size of chromosomes, most likely due to variability of subtelomeric regions, the type of asexual spores (microconidia and/or macroconidia), and the number and expression of secondary metabolite gene clusters. Whilst most of the isolates caused the typical bakanae symptoms, one isolate, B14, caused stunting and early withering of infected seedlings. In contrast to the other isolates, B14 produced no GAs but high amounts of fumonisins during infection on rice. Furthermore, it differed from the other isolates by the presence of three additional polyketide synthase (PKS) genes (PKS40, PKS43, PKS51) and the absence of the F. fujikuroi-specific apicidin F (NRPS31) gene cluster. Analysis of additional field isolates confirmed the strong correlation between the pathotype (bakanae or stunting/withering), and the ability to produce either GAs or fumonisins. Deletion of the fumonisin and fusaric acid-specific PKS genes in B14 reduced the stunting/withering symptoms, whereas deletion of the PKS51 gene resulted in elevated symptom development. Phylogenetic analyses revealed two subclades of F. fujikuroi strains according to their pathotype and secondary metabolite profiles.

Fig 1. Phylogenetic relationships and chromosome characteristics of F. fujikuroi isolates and related species.

(A) World map modified after https://commons.wikimedia.org/wiki/File:BlankMap-World6.svg. Colored dots present the origin of the different F. fujikuroi isolates (see Table 1). (B) Maximum likelihood tree showing phylogenetic relationships of F. fujikuroi isolates and other species representing the Asian, African and American clades of the Fusarium fujikuroi complex (FFC), as well as F. oxysporum, and the distantly related species F. langsethiae and F. avenaceum. It was calculated based on the protein sequences of 5,181 single copy genes that are shared among all analyzed species. (C) and (D) Separation of chromosomes of the nine F. fujikuroi isolates and F. oxysporum V-64-1 as outgroup on a CHEF gel. Chromosomes of Schizzosaccharomyces pombe and Saccharomyces cerevisiae were used as size standards.

Fig 2. Phenotypic characteristics of the nine F. fujikuroi isolates and F. oxysporum V64-1 as outgroup.

Colony morphology of the strains grown on solidified complete medium from the top (A) and the bottom (B) of the plates. Variation in pigmentation of the strains grown in liquid synthetic medium containing 6 mM glutamine (C) (optimal for bikaverin), 60 mM glutamine (D) or 6 mM NaNO₃ (E) (optimal for fusarubins). (F) Microscopic images of microconidia and macroconidia.

Fig 3. Content and arrangement of genes in different gene clusters with variations in the single isolates.

The PKS51 (A) and PKS40 (B) gene clusters are present only in strain B14. (C) The PKS13, PKS17, PKS18, and PKS8 gene clusters at the end of chromosome 11 are missing in strain NCIM 1100. Arrows in blue represent genes belonging to a specific gene cluster. Green arrows represent genes that have closely related homologs in two or more isolates while light-gray arrows represent genes that do not have closely related homologs in other isolates. Ψ indicates a pseudogene.

Fig 4. Content and arrangement of genes in different gene clusters with variations in the single isolates.

(A) The PKS5 cluster seems to be functional only in strain B14. The PKS5-encoding gene is truncated or missing in the other isolates. (B) The PKS19 (fujikurin) gene cluster is present in some and totally missing in other isolates. (C) PKS16 at the end of chromosome 11 in F. fujikuroi IMI 58289 is truncated in stains C1995 and E282, and missing in strain NCIM 1100. Arrows in blue represent genes belonging to a specific gene cluster. Green arrows represent genes that have closely related homologs in two or more isolates while light-gray arrows represent genes that do not have closely related homologs in other isolates. Ψ indicates a pseudogene.

Fig 5. Gibberellic acid (GA) and fumonisin production and gene expression in the ten analyzed strains.

A) GA (CPS/KS) and fumonisin (FUM2) gene expression studies by Northern blot analysis after 3 days of growth in synthetic medium with 6 mM glutamine. GA (B) and fumonisin (C) production levels after 7 days of growth in synthetic medium with 6 mM glutamine.

Fig 6. Pathogenicity assay of the F. fujikuroi and F. oxysporum isolates.

(A) Symptoms in the rice stems that were inoculated with the fungal isolates and water only (control). The uppermost stem level of the rice seedling with control treatment is indicated by a thin dotted bar. (B) Symptoms in the rice roots after pathogen infection. (C) The plant heights and internode lengths of pathogen-infected rice seedling at 6 dpi. Error bars show standard deviations. The same letter above bars indicates no significant difference. n.d., not detected.

Fig 7. Shoot and root growth of rice seedlings 5, 7, and 9 days post inoculation (dpi) of F. fujikuroi B14 and gene deletion strains dervied from B14.

(A-C) Shoot growth. (D) Root growth. (A) Shoot and root growth 5 dpi with or without exogenous supply of fumonisin FB₁ (1 μM), fusaric acid (FSA; 10 μM) or GA₃ (10 μM). (B) Shoot growth 7 dpi. (C) Shoot growth 9 dpi. Mock: neither fungal inoculation nor toxin treatement; B14 (stunting pathotype) and B20 (bakanae pathotype) wild-type strains; Δfub1 (PKS6, fusaric acid) deletion strain; Δfum1 (PKS11, fumonisins) deletion strain; Δfub1/Δfum1 double deletion strain; ΔPKS51 (PKS51, unknown product).

Fig 8. Population analysis of field isolates in Korea.

(A) Rice seedlings 9 dpi of F. fujikuroi field isolates obtained from Korea. Control: no fungal inoculation, 1: B14, 2: B20, 3: JA19, 4: JA35, 5: JA23, 6: JD8, 7: OS122, 8: B41, 9: B3, 10: B66, 11: V76, 12: V74, 13: 16-R18, 14: B27, 15: B17, 16: R19, 17: 16-R24. Pathogenicity test with 15 additional isolates revealed a clear separation between the bakanae and stunting pathotype. (B) A phylogenetic tree constructed by the NJ method using the nucleotide sequences of combined TEF1 and RPB2 from the two pathotypes of field isolates, determined by pathogenicity test (A), and from the worldwide isolates used in this study. The phylogenetic tree supports the separation of the two pathotypes into two different phylogenetic subclades.