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.
Fusarium fujikuroi causes bakanae disease of rice. Infected seedlings appear to be taller and more slender when compared to healthy seedlings due to its ability to produce gibberellic acids (GAs). The disease is responsible for high yield losses, and its incidence varies with regions, rice cultivars grown and the aggressiveness of the fungal isolates. However, not all infected seedlings show bakanae symptoms: one of the isolates, B14, causes stunting and early withering of infected seedlings. The reason for the two pathotypes is not well understood. Researchers thought that the stunting phenotype was mostly caused by fungal-derived secondary metabolites such as fusaric acid, but there is no experimental evidence yet. B14 differs from the other strains by the presence of more PKS gene clusters, low expression of GA genes, lack of detectable levels of GAs and the production of high amounts of fumonisins in rice. Analysis of additional field isolates revealed a strong correlation between the pathotype (bakanae or stunting) and either GA or fumonisin production. Based on phylogenetic analyses, F. fujikuroi strains can be divided into two phylogenetically distinct subclades according to their pathotype and secondary metabolite profiles. This study provides new insights into the genomic variations and the population structure inside the species F. fujikuroi which will help to develop disease control strategies for this rice pathogen.
Citation: Niehaus E-M, Kim H-K, Münsterkötter M, Janevska S, Arndt B, Kalinina SA, et al. (2017) Comparative genomics of geographically distant Fusarium fujikuroi isolates revealed two distinct pathotypes correlating with secondary metabolite profiles. PLoS Pathog 13(10): e1006670. doi:10.1371/journal.ppat.1006670
Editor: Bart Thomma, Wageningen University, NETHERLANDS
Received: May 29, 2017; Accepted: September 26, 2017; Published: October 26, 2017
Copyright: © 2017 Niehaus et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The data on the new genomes, including annotation, was submitted to the European Nucleotide Archive, study accession PRJEB14872 available at: http://www.ebi.ac.uk/ena/data/view/PRJEB14872.
Funding: The study was supported by the Deutsche Forschungsgemainschaft (DFG), projects TU101/17-2 (BT); GU1205/2-2 (UG); HU730/9-3 (HUH). Furthermore, the work was supported by the Austrian Science Fund FWF, Special Research Project "Fusarium" F3705/DFGME1682/6-1 (UG) and by the grant from the Strategic Initiative for Microbiomes in Agriculture and Food, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea and by the Soonchunhyang University Research Fund (NO: 20130591) (SHY). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The heterothallic ascomycete Fusarium fujikuroi Nirenberg is a member of the Fusarium fujikuroi species complex (FFC), a monophyletic lineage which includes at least eleven mating populations (MPs A-K) that are sexually infertile with one another, and numerous distinct anamorphic species .
F. fujikuroi (MP-C) is the causal agent of the rice disease bakanae (“foolish seedlings”), one of the most notorious seed-borne diseases with increasing economic importance in the major rice-growing countries in the world, including all rice-growing Asian and African countries, California, and more recently, Italy [1–3]. The fungus was one of the first fungal pathogens to be described, and bakanae is one of the oldest known diseases of rice being reported more than 100 years ago by Japanese scientists [4,5]. The most prominent symptoms of the disease are chlorotic, elongated and thin seedlings that are often several inches taller than healthy plants, and empty panicles leading to yield losses ranging from ca. 30–95% [6–9]. However, not all infected seedlings show the bakanae symptoms: sometimes they may be stunted or appear symptomless . The incidence and severity of the bakanae or stunting disease symptoms varies with regions and isolate. The pathogen is dispersed predominantly with infected seeds, infected crop residues from the previous season in the soil, or by conidia on diseased stems which can be transmitted by rain and wind . Disease control has become increasingly difficult due to rapidly developing fungicide resistance in the fungal population .
The enormous elongation of infected plants is caused by the ability of the pathogen to produce gibberellic acids (GAs), a family of plant hormones . Fungal GAs are structurally identical to those synthesized by higher plants, but the respective biosynthetic pathways, genes and enzymes differ [12–14]. Previously we have shown that the ability of the fungus to produce GAs contributes to the efficient colonization in the rice roots . In contrast to typical bakanae symptoms, it is unknown how the stunting phenotype of infected rice seedlings is triggered by F. fujikuroi. In addition, stunting of infected rice plants can also be caused by other Fusarium species, such as F. proliferatum, which can also be isolated from rice, though less abundantly than F. fujikuroi .
Recently, the first high-quality draft genome sequence of F. fujikuroi IMI 58289 has been published and the genetic capacity for biosynthesis of a whole arsenal of natural compounds has been demonstrated . The genomes of additional F. fujikuroi isolates revealed some diversity regarding genome composition and virulence [17,18].
Here we present the genome sequences of eight additional F. fujikuroi strains, all but one isolated from infected rice from different geographic regions. The isolate FSU48 was obtained from maize. We provide a comparative analysis of the genome features, chromosome polymorphism, the ability to produce micro- and macroconidia, virulence, metabolome and transcriptome analyses under in vitro and in planta conditions. By the use of high-performance liquid chromatography coupled to a Fourier transform mass spectrometer (HPLC-FTMS) and genome-wide RNA-sequencing (RNA-seq), we demonstrate that in addition to species-specific common features there are differences between the isolates in all these aspects. Most importantly, we describe two pathotypes of F. fujikuroi on rice at the genomic, transcriptomic, and phylogenetic levels. Whereas most of the F. fujikuroi isolates caused typical bakanae symptoms with elongated chlorotic internodes, the isolate B14 caused stunting and withering of rice seedlings. We show that variations in the production of secondary metabolites (SMs), such as GAs and fumonisins, are crucial factors for the development of the bakanae or stunting pathotype, and that these two pathotypes are phylogenetically distinct groups among the field population of F. fujikuroi.
Results and discussion
Genome structure, gene families and the potential to produce secondary metabolites—common and different features
F. fujikuroi is broadly distributed world-wide in all rice-growing countries. To gain insight to the level of variation regarding genome structure, morphology, asexual spore formation, virulence, expression profiles and secondary metabolism under laboratory conditions as well as on rice, we chose nine isolates from different areas of the world for comparative analysis of all these parameters (Table 1, Fig 1A).
The high quality genome sequence of strain IMI 58289 that was assembled into twelve scaffolds corresponding to the twelve chromosomes  was used as master genome for structural annotation and for comparative analysis. The rice isolate V64-1 from Ruanda appeared to be a F. oxysporum strain when the genome was sequenced and analyzed. Therefore, this strain was used as outgroup in this study. A phylogenetic tree including all so far sequenced F. fujikuroi isolates and other FFC members, as well as more distantly related Fusarium species is shown in Fig 1B. The tree was generated based on the protein sequences of 5,181 single copy genes by the fast approximate likelihood ratio test to calculate branch support (aLRT) , which is a fast and accurate alternative to the time-consuming bootstrap analysis. Strain KSUX10626  seems to be outside the F. fujikuroi clade.
Although all newly sequenced strains except for V64-1 clearly belong to the species F. fujikuroi, visualization of chromosome content of the ten strains by pulse field gel electrophoresis (PFGE) combined with clamped homogeneous electric fields (CHEF) indicated that all strains contained multiple chromosomes of varying sizes (Fig 1C). However, the precise number of chromosomes in each strain could not be determined because several chromosomes had a similar size and could not be distinguished.
Table 2 summarizes physical genome features of the newly and previously (IMI 58289) sequenced strains. The genome size for the F. fujikuroi strains is in the range of 43.9 Mb (IMI 58289) to 46.1 Mb (E282 and FSU48). The outgroup genome F. oxysporum V64-1 is smaller (49.1 Mb) than the reference F. oxysporum strain 4287 (61.4 Mb). Most of these differences are likely due to a different read coverage and different completeness of assemblies for repetitive regions. Despite the varying number of protein-encoding genes (14,817 for IMI 58289 to 16,088 for E282) which partially may be due to a manual gene structure validation, most key genome features of the newly sequenced strains are similar to those of F. fujikuroi IMI 58289 .
The completeness of the new draft genomes was explored by comparing each predicted proteome to two different, highly conserved eukaryote protein sets by BLAST [20,21]. Orthologs for all conserved proteins were found in all proteomes with the exception of a missing ortholog to ‘T-complex protein 1 subunit theta’ and an ortholog to ‘translation factor eIF6’ in F. fujikuroi B20. In F. oxysporum V64-1, an ortholog to ‘MTO1 –mitochondrial translation optimization’ is missing suggesting that also these genomes are more than 99% complete. In addition, the protein sets were subjected to BUSCO analysis and subsequently compared to published Fusarium proteome sets . Of the 3,725 single-copy orthologs searched (library Sordariomyceta_odb9), 97.9%–99.2% were detected as complete and single-copy in the F. fujikuroi strains and F. oxysporum V64-1 which even exceeds the completeness of F. graminearum PH1 (94.7%) and F. oxysporum (94.2%) (S1 Table).
Species of the FFC are heterothallic . To determine the mating type of the nine F. fujikuroi isolates, we searched the genomes for the presence of genes either of the MAT1-1 or MAT1-2 mating types. Five of the strains contain the genes MAT1-1-1, MAT1-1-2 and MAT1-1-3, all belonging to the MAT1-1 mating type, while the other four contain the MAT1-2 mating type locus with the HMG-box type transcription factor (TF)-encoding gene MAT1-2-1 and an additional gene, MAT1-2-3, that has been recently identified as specific to the Hypocreales  (S2 Table).
Differences between the isolates were found regarding the size of gene families prevalent in the genome of B14, which is the only one of the ten examined strains causing stunting of rice seedlings (see below). The number of transporter-encoding genes (954), Zn(II)2Cys6 fungal type TFs (567), cytochrome P450- (173) and dehydrogenase-encoding genes (404) were higher when compared to the other F. fujikuroi strains. Also the number of polyketide synthase (PKS) genes (18 plus two truncated pseudogenes) was larger than in the genomes of the other strains (Table 3).
The strain NCIM 1100 encodes less PKS (eight plus one truncated pseudogene) and nine terpene cyclase (TC) genes which results in the inability to produce gibepyrones, fujikurin and certain terpenes besides other unknown products (S3 Table and below).
To gain a deeper insight into the variation at the chromosome ends between the ten analyzed strains, we used a PCR approach with primer pairs from the two ends of each chromosome based on the genome sequence of strain IMI 58289 . This analysis revealed differences between the isolates either on one side or on both sides of the chromosomes (S1 Fig). The most variations were found for subtelomeric regions of chromosomes 3, 5, 7, 10 and 11. Two of the key enzyme-encoding genes of SM gene clusters, NRPS31 (apicidin F) and PKS16 (unknown product), are located at the far end of chromosome 1 and 11, respectively, and the PCR analysis clearly showed differences in the presence of these SM genes (S1 Fig).
Variation in growth, colony morphology and asexual spore formation
All isolates showed differences in colony morphology and pigmentation on solidified and in liquid media, respectively, indicating metabolic variation between them (Fig 2A–2E). There were differences in the ability to produce the red pigments, bikaverin and fusarubins, despite the presence of the respective gene clusters in all F. fujikuroi isolates (Fig 2C–2E). Previously, we have shown that both PKS-derived pigments are only produced under low nitrogen conditions. However, while bikaverin biosynthesis is induced at acidic pH, the perithecial pigments fusarubins are produced only under alkaline pH conditions [24,25]. Strain C1995 showed no coloration under bikaverin-production conditions (Fig 2C), and C1995, B14, FSU48 and NCIM 1100 showed no pigmentation under fusarubin production conditions (Fig 2E). To verify that the biosynthetic genes are not expressed in these isolates, we performed Northern blot analyses using the bikaverin and fusarubin biosynthetic genes as probes (S2 Fig). FSR2 (encoding an O-methyltransferase in the fusarubin gene cluster) was expressed in IMI 58289, but not in the remaining strains suggesting that the fusarubin genes are only slightly expressed in m567, MRC2276, B20, E282 and F. oxysporum V64-1, or alternatively that the red pigmentation in these strains under fusarubin production conditions (Fig 2F) might be due to the expression of the bikaverin biosynthetic genes.
The species F. fujikuroi is described to produce slender macroconidia with three to five septa and oval or club-shaped microconidia, mostly without or with one septum . However, the strains used in this work differ in their ability to produce asexual spores, i.e. micro- and macroconidia (Fig 2F). While most of the strains produce both types of conidia, others produce predominantly (B14 and NCIM 1100) or exclusively (IMI 58289) microconidia or predominantly macroconidia (E282), respectively, on V8 agar under light conditions. Strain m567 hardly forms any spores. In A. nidulans, asexual reproduction has been extensively studied for several decades [26–28]. Sequential activation of three major regulators, BrlA, AbaA and WetA, is necessary for the fungus to undergo asexual development. In addition, there are a number of fluffy regulatory genes (FLBA–FLBE) which regulate BRLA expression. Recently, it has been shown that an AbaA-WetA pathway is conserved in the distantly related species Fusarium graminearum [29–31]. There are highly conserved homologs for FlbB, FlbC, FlbD, FlbE and the central regulators WetA and AbaA also in the genomes of the F. fujikuroi isolates. However, no close relative to BrlA, which is responsible for the vesicle formation during conidiogenesis in A. nidulans, has been found in any Fusarium genome.
The potential to produce a broad spectrum of secondary metabolites
In recent years, several new SMs have been identified in F. fujikuroi due to the deciphering of the fungal genome and the application of molecular techniques to activate silent gene clusters, e.g. those for apicidin F, fujikurins, beauvericin, trichosetin, and reversely N-prenylated tryptophan (r-N-DMAT) [32–36] as well as for the sesquiterpenes eremophilene and guaia-6,10(14)-diene [37,38]. In addition, well known SMs such as fusaric acid, fusarins, fusarubins and gibepyrones have been linked to the respective biosynthetic gene clusters [39–42].
Bioinformatic analysis of the nine F. fujikuroi strains revealed several differences in the presence of PKS, NRPS, dimethylallyltryptophan synthase (DMATS) and TC gene clusters between them and also compared to closely related FFC members, such as F. mangiferae and F. proliferatum  (S3 Table). Altogether, there are 17 NRPS-, 23 PKS-, three DMATS-, and twelve TC-encoding genes present in the nine F. fujikuroi strains, i.e. 55 unique core-enzyme-encoding genes that could give rise to 54 distinct SMs (the fusaric acid cluster encodes two core enzymes, PKS6 and NRPS34). Only twelve of the 23 PKS-, 13 of the 18 NRPS-, eight of the twelve TC- and two of the three DMATS-encoding genes are present in the genomes of all F. fujikuroi isolates. The Korean strain B14 has three additional PKS genes (PKS40, 43 and 51) not present in any other analyzed F. fujikuroi strain (S3 Table, Fig 3A and 3B). PKS51 is also not present in any other member of the FFC, while PKS40 is present in F. proliferatum ET1 and F. verticillioides, and PKS43 in F. mangiferae, respectively (S3 Table). In addition, B14 is the only F. fujikuroi isolate with a complete PKS5 cluster. This yet uncharacterized cluster is either absent or contains pseudogenes in the other isolates of this species, but seems to be functional in F. proliferatum and F. mangiferae (Fig 4A). The fujikurin gene cluster (PKS19) has been previously described as F. fujikuroi-specific , but was recently discovered in two newly sequenced strains of F. proliferatum . However, the fujikurin cluster is absent in three of the nine analyzed F. fujikuroi isolates (S3 Table, Fig 4B). The apicidin F gene cluster (NRPS31) which is located at the far end of chromosome 1 in strain IMI 58289  was shown to be unique for F. fujikuroi . This cluster is present in all isolates but one, B14, most likely due to chromosome rearrangements in the subtelomere regions (S1 and S3A Figs). While the entire fumonisin gene cluster (PKS11) is present in most members of the FFC except for F. mangiferae (S3 Table), nine genes of the cluster, the homologs of FFUJ_09248 –FFUJ_09256, are missing in F. fujikuroi B20 (S3B Fig) and C1995.
The Indian isolate NCIM 1100 has only 14 PKS-encoding genes (S3 Table). The absence of five PKS genes/clusters is due to large parts of chromosome 11 missing at both ends in this strain compared to IMI 58289 (Figs 3C and 4C, S1 Fig). The right border of chromosome 11 contains PKS8 (FFUJ_12090), PKS13 (FFUJ_12020), PKS17 (FFUJ_12066), and PKS18 (FFUJ_12074), while PKS16 (FFUJ_11199) and adjacent genes are absent at the left border of this chromosome (Figs 3C and 4C, S3 Table). Also the discontinuous distribution of some other SM gene clusters among the F. fujikuroi isolates is likely the result of gene loss and gene gain events, respectively, at the chromosome ends. However, gene clusters located in central parts of chromosomes, e.g. PKS19 (fujikurin) and PKS11 (fumonisin) clusters, are also only present in some and absent in other strains, while the genes at the right and left borders of the clusters show colinearity between all isolates. It is not clear whether these gene clusters are the result of horizontal gene transfer  or cluster duplication and loss (birth and death) as shown for the fumonisin gene cluster .
Thanks to the genome sequencing and subsequent identification of putative gene clusters , SM products have now been assigned to 22 of the 54 predicted PKS-, NRPS-, DMATS-, and TC-derived SM gene clusters in isolates of F. fujikuroi, and an additional two (ferrirhodin and depudecin) in members of the FFC. One reason for the inability to identify more products is probably due to the fact that many of the SM genes are not or only minimally expressed under laboratory conditions . Only 15 of these predicted clusters with known products are present in the genomes of all nine F. fujikuroi isolates indicating the genetic diversity among isolates of one species. The genes which are commonly present in all F. fujikuroi genomes are four NRPS genes required for synthesis of the siderophores ferricrocin and fusarinine and the mycotoxins beauvericin and fusaric acid (together with PKS6); six PKS genes required for synthesis of fusarubins, bikaverin, fusaric acid (together with NRPS8), fusarins, trichosetin, and fumonisins; six TCs required for synthesis of GAs, phytoene, eremophilene, (–)-α-acorenol, (–)-guaia-6,10(14)-diene, and (+)-koraiol, and one DMATS responsible for biosynthesis of r-N-DMAT (S3 Table).
To study the variability in the production of the most prominent SMs, we cultivated the strains under three standardized culture conditions (6 mM and 60 mM glutamine, 6 mM NaNO3). The optimal conditions for the production of the different metabolites were previously shown to vary considerably regarding nitrogen availability and pH in strain IMI 58289 . Therefore, we analysed these SMs in the three media by high-performance liquid chromatography coupled to a Fourier transform mass spectrometer (HPLC-FTMS) or by HPLC with a diode array detector (HPLC-DAD) in the case of GAs. We compared the production levels (Table 4) with transcriptome profiles of the SM biosynthetic genes generated by RNA-seq for two of these conditions (6 mM and 60 mM glutamine) (Table 5A and 5B). For most of the SMs, we observed similar regulatory mechanisms regarding nitrogen availability as previously described for strain IMI 58289. However, there were strain-specific differences.
The most prominent and species-specific SMs are the GAs causing the F. fujikuroi-specific bakanae disease of rice. Although some recently sequenced species of the FFC such as F. mangiferae and F. proliferatum contain one or even two GA gene clusters, these species produce either no or only very small amounts of GAs . In this work, we examined whether all analyzed F. fujikuroi strains produce GAs, and whether the GA levels can be correlated to the virulence on rice.
Previously, the regulation of GA biosynthesis has been extensively studied for strain IMI 58289. It has been shown that GA gene expression is strictly regulated by nitrogen availability in an AreA- and AreB-dependent manner [47–49]. To examine whether low nitrogen conditions are optimal for GA production also in the other isolates, we performed HPLC-DAD analysis of all strains and analysed the expression by Northern blot analysis in addition to RNA-seq data. Accordingly, we observed the highest GA yields and GA gene expression levels under low nitrogen conditions for eight of the nine F. fujikuroi strains. The only exception among the F. fujikuroi isolates is strain B14 which showed no production of GAs and no visible expression of the GA genes despite the presence of the complete GA gene cluster in the genome (Table 5A and 5B; Fig 5A and 5B). Besides the very low expression of GA genes, strain B14 differs from the other strains by high expression of fumonisin biosynthetic genes under low nitrogen conditions (Table 5A, Fig 5A and 5C). Recently, we have shown that the fumonisin gene cluster in F. fujikuroi IMI 58289 is almost silent. Consequently, only very low amounts of fumonisins are produced in comparison to F. verticillioides. In F. fujikuroi IMI58289, the genes are only expressed and fumonisins are only produced, when the cluster-specific TF gene FUM21 is constitutively and strongly expressed . Here, similar results were observed for eight of the nine F. fujikuroi isolates (except for B14) which all produce either no or only very small amounts of fumonisins and showed very low expression levels.
In addition, B14 is the only isolate producing fujikurins under alkaline and low nitrogen conditions (Table 4). Previously, the fujikurin gene cluster (PKS19) was shown to be silent under all conditions tested in strain IMI 58289, and the products have been identified only after simultaneous over-expression of both the cluster-specific TF gene and the PKS19 gene itself [15,34].
In most of the strains, fusarins, fusaric acid, and apicidin F (except for B14) were only produced under high nitrogen conditions (60 mM glutamine) in accordance with the higher expression levels of the corresponding biosynthetic genes. The genes of the beauvericin cluster were recently shown to be silent under all conditions tested and activated only after deletion of the histone deacetylase gene HDA1 and knock-down of the histone methyltransferase gene KMT6 in strain IMI 58289 [33,38]. Most of the other strains analyzed here showed no or very low expression levels and no beauvericin production. The only exception was strain B20 that highly expressed the biosynthetic genes under both nitrogen conditions (low and high amounts of glutamine) and produced high levels of beauvericin at high nitrogen (Table 4; Table 5A and 5B) suggesting that the chromatin status around the beauvericin cluster differs in B20.
Virulence assay on rice
To examine whether the isolates differ in their virulence on rice, and whether the different levels of GAs or the strain-specific production of other SMs (e.g. fumonisins in B14) correleate with the extent of symptom development, we performed assays with both germinating seeds and rice seedlings.
To determine the ability of all ten strains to impair the seed germination, rice seeds were soaked for 18 h in spore suspensions of the respective strains. The percentage of germinated seeds was counted after 14 days post inoculation (dpi). Strains B14 and B20 induced a high percentage of seedling death indicating a high potential to kill the host seedlings (S4 Table). As B14 does not produce much GAs, the aggressiveness of this strain cannot be caused by these phytohormones.
Next, we performed a rice seedling assay to compare the virulence of the different isolates to assess their ability to induce bakanae symptoms. The surface-sterilized seeds were first germinated, and the young healthy seedlings were then inoculated with spores. All strains except for B14 and F. oxysporum V64-1 (outgroup) induced the formation of slender, elongated and yellowish stems (Fig 6). Wheras V64-1-infected seedlings behaved like the water control, B14-infected seedlings were stunted and showed withering instead of the typical bakanae symptoms. The total seedling length and the length of the internodes were smaller than, or comparable to uninfected seedlings (water control). Previously, it has been reported that heavily infected seedlings can also be stunted and can show severe crown and root rot . The type of symptoms and severity of disease depends on the fungal isolate and is thought to be affected by the proportions of GA and fusaric acid produced by the fungus, which potentially cause elongation of the plants or stunting, respectively [52,53]. However, this assumption has never been proved experimentally.
In planta expression of SM gene clusters and product analysis
To examine which of the SM gene clusters are similarly expressed in rice in all isolates, and which of them are specifically expressed or not expressed in strain B14, and therefore could be relevant for disease symptoms (especially stunting), we performed RNA-seq for all isolates also from rice seedlings at 7 dpi (Table 5C). The expression patterns were compared with those under in vitro conditions at high (60 mM glutamine) and low (6 mM glutamine) nitrogen (Table 5A and 5B).
In general, the GA biosynthetic genes (shown for the key enzyme gene CPS/KS) are the most highly expressed genes in rice roots except for B14 in which they are hardly expressed (Table 5C).
To check whether the expression profiles of SM genes correspond to the in planta production levels, we also performed SM analysis of infected rice plants by HPLS-FTMS at 7 dpi. In accordance with gene expression, the in planta GA production levels also differed between the isolates: B14-infected rice plants contained no detectable GA amounts in rice compared to the bakanae strains (Table 6). The most obvious difference of strain B14 compared to the other isolates was the high expression of the fumonisin genes resulting in significant levels of fumonisins (FB1 and FB2) in planta, similar to those observed in vitro (Table 5C; Table 6). Furthermore, while all isolates showed similar expression patterns for fusaric acid biosynthetic genes (PKS6; NRPS34) and similar fusaric acid production levels (Table 5C, Table 6), B14 was the only strain with low expression of the fusarin C genes. As expected, no fusarins were detectable in rice roots inoculated with this strain (Table 6).
In addition, the yet uncharacterized PKS51 gene cluster, which is only present in B14, was exclusively expressed in planta, suggesting that the unknown product of this gene cluster might play a role during infection (Table 5C). Besides B20, B14 also gave high expression for beauvericin genes, but only low expression of gibepyrone (PKS13) and acorenol (STC6) biosynthetic genes.
In conclusion, the very low expression of GA genes and the lack of detectable GA levels in B14-infected rice seedlings after 7 dpi are most likely responsible for the absence of bakanae symptoms in this isolate. Instead, the high levels of fumonisins which are exclusively produced only in this isolate, may overrule the growth-stimulating effect of the GAs and cause stunting/withering.
Do the fumonisins, fusaric acid and/or the PKS51 product cause the stunting symptom development of F. fujikuroi B14?
To find out whether one of the gene clusters that are specifically expressed in B14 during infection on rice (Table 5C) might indeed cause the stunting effect of this isolate, the key enzyme-encoding genes for fumonisins (FUM1 = PKS11), and the yet uncharacterized B14-specific PKS51 gene were deleted in this strain. In addition, we also deleted the key gene for fusaric acid biosynthesis (FUB1 = PKS6) due to the speculation that fusaric acid production might cause the stunting pathotype .
The 5-day-old healthy rice seedlings were soaked in the conidial suspension of B14 or the mutant strains. At 5 dpi, the B14-infected seedlings were already stunted compared to the water control and showed withering symptoms which were even more obvious at 7 dpi and 9 dpi (Fig 7A). In contrast, the Δfum1 and Δfub1 strains of B14 appeared healthy at 7 dpi and caused delayed withering symptoms at 9 dpi only. The roots of all infected seedlings were heavily colonized with fungal mycelia at 9 dpi (Fig 7D). The double deletion mutants of B14 lacking both FUM1 and FUB1 behaved like the mock control and did not induce stunting at 5 dpi (Fig 7A) or occasionally upto 9 dpi. However, in some cases, they caused a similar delayed disease development as the single deletion strains at 9 dpi (Fig 7B and 7C). The add-back strains, which were generated by introducing the native copy of FUM1 and FUB1 into Δfum1 and Δfub1 mutants, respectively, caused typical stunting/withering symptoms similar to B14 (S4A Fig). The delay of stunting/withering symptom development by Δfum1 and Δfub1 strains indicates that the production of fumonisins and/or fusaric acid, in combination with the non-detectable levels of GAs, play an important role for the development of this specific pathotype in B14.
For comparison, the genes FUM1 and FUB1 were also deleted in strain B20 (bakanae strain). Unlike the B14-derived mutants, all of the gene deletion strains of B20 we examined caused typical bakanae symptoms (elongated, slender and chlorotic shoots), as did B20. No withering of the seedlings or mycelial colonization on roots were visible suggesting that the absence of fumonisin production by B20 and B20-1 derived mutants protect the rice seedlings from development of B14-like symptoms (S5 Fig).
Surprisingly, rice seedlings inoculated with the ΔPKS51 mutant strains showed an earlier and more severe symptom development compared to those with B14. The stunting/withering symptoms were clearly shown already at 5 dpi (Fig 7A). The more severe symptoms caused by the ΔPKS51 strains may suggest a possible role of the PKS51 product as an avirulence determinant. A similar role was described for the product of the Magnaporthe grisea PKS-NRPS gene ACE1 (Avirulence Conferring Enzyme1) which is also specifically expressed only on rice. Its yet unknown product is probably recognized by rice cultivars carrying a specific resistance gene [54,55]. However, further investigations will be needed to show whether the product of PKS51 acts in a similar way in F. fujikuroi strain B14.
To further study the impact of these SMs on disease symptom development, fumonisin FB1, fusaric acid, or GA3 were exogenously supplied to rice seedlings infected with wild-type or mutant strains (Fig 7A). Addition of FB1 and fusaric acid to rice seedlings infected with Δfum1 and Δfub1, respectively, restored the WT phenotype and resulted in stronger withering symptoms compared to the deletion strains without the toxins. However, the even more reduced virulence of the double deletion strain (Δfum1/Δfub1) was not clearly complemented by exogenous supplies of both fumonisin and fusaric acid (Fig 7A). Interestingly, exogenous supply of GA3 did not cause bakanae symptoms on rice seedlings inoculated with the wild-type B14 strain, while addition of GA3 to the Δfum1/Δfub1 double deletion strain caused stem elongation of rice seedlings at 5 dpi, similar to the bakanae symptom caused by B20 (Fig 7A). Therefore, conversion of B14 into a bakanae pathotype by addition of GAs was only possible after deleting the key genes for the production of fumonisins and fusaric acid.
Furthermore, exogenous supply of culture filtrate from strain B14 to seedlings infected with B14, B20 or B20 Δcps/ks caused stunting and withering symptoms while the culture filtrate of the B14 Δfum1/Δfub1 mutant caused milder symptoms (S4B and S4C Fig). Addition of culture fluid to B14-infected seedlings resulted in even more severe stunting than B14 alone (S4B Fig). Exogenous supply of B14 culture filtrate to seedlings inoculated with B20 or the GA-deficient B20 Δcps/ks mutant revealed severe stunting symptoms in both cases irrespective of the ability to produce GAs (S4C and S4D Fig). These data support our suggestion that fumonisins and fusaric acid play an important role for symptom development.
To further investigate the role of fumonisins for causing stunting and withering, we performed the rice seedling pathotest with two F. verticillioides strains from corn which were shown to produce high amounts (more than 3,000 μg/g) of fumonisins and no GAs in rice seedlings. Both F. verticilloides isolates (Os35 and Os40)  caused withering at 7 dpi although the plants were not stunted compared to those of the mock control (S6A Fig). In addition, mycelia of both F. verticillioides strains colonized the roots of infected rice seedling as much as those of B14 (S6B Fig). This strong root colonization was not observed for roots infected with B20. Previously, it has been already reported that B14 triggered severe inhibition of root growth, and that its own growth rate in rice roots was more than 4 times higher compared to that of B20.
Taking together the results of FUM1 and FUB1 deletion, exogenous addition of pure toxins or culture filtrate of B14 to rice seedlings inoculated with wild-type or mutant strains, and the pathotests with GA-deficient, highly fumonisin-producing F. verticillioides isolates provide strong indications that the biosynthesis of both fumonisins and fusaric acid and the lack of GA biosynthesis in B14 play cruicial roles for causing the stunting/withering phenotype on rice seedlings. However, additional factors are probably involved in inducing the stunting symptoms.
Comparisons of TF-encoding genes between B14 (stunting) and B20 (bakanae) strains of F. fujikuroi
Besides SMs, stunting/withering might be caused also by the different sets of TFs present in the genome of B14 and the bakanae strains, or by different expression levels of TF-encoding genes in rice. Therefore, we compared the expression levels of TF-encoding genes between B14 and the other eight F. fujikuroi isolates (S5A Table). There are 37 genes which are present in most of the nine genomes and which were specifically up-regulated during infection of rice (S5B Table). B14 had slightly higher expression levels only for three of them: FFB14_03090, FFB14_05980 and FFB14_01631. In addition, B14 has 28 strain-specific TFs which are not present in the genomes of the other strains (S5C Table). The most highly expressed gene in planta was FFB14_06367 encoding the pathway-specific TF of the putative PKS51 gene cluster. The high expression of PKS51 and the adjacent genes, including the TF-encoding gene, supports our assumption that this unique gene cluster is involved in determining the severity of disease symptom development.
Population analyses revealed the segregation of field isolates into the two different pathotypes
Because B14 was the only isolate causing the stunting pathotype among the analyzed ten strains, we attempted to determine how often this pathotype can be found in rice fields. Therefore, we inoculated rice seedlings with 15 field isolates, which were collected from rice grains and air above rice paddy fields in Korea between 2014 and 2016. Among the 15 isolates, we identified additional nine field isolates causing stunting and early withering symptoms similar to B14 while six isolates caused typical bakanae symptoms (Fig 8A).
To determine whether these isolates can be phylogenetically distinguished from each other, we generated a phylogenetic tree using the nucleotide sequences of the RPB2 and EF1A genes  from the new field isolates, the ten isolates used in this study, and some closely related Fusarium species of the FFC. The F. fujikuroi clade, which was clearly separated from those of other closely related species such as F. proliferatum and F. verticillioides, contained two strongly supported subclades (with 76% bootstrap support: BS). Interestingly, the subclade with B14 consists of all of the field isolates causing stunting symptoms, while the other subclade contained B20 and the other F. fujikuroi strains used in this study and all bakanae-type field isolates (Fig 8B). This result indicates that the two pathotypes of F. fujikuroi exist as phylogenetically distinct groups within the population.
To examine if there is a correlation between the pathotype and the presence of one of the SM clusters specific to B14 or B20 (PKS51 for B14, unknown product, and NRPS31 for B20, apicidin F) among the field isolates of each pathotype, we performed a PCR amplification using primer sets derived from PKS51 (unknown product) and NRPS31 (apicidin F), respectively. The PKS51-specific primer set amplified a fragment from all of the stunting-type isolates examined including B14, but not from the bakanae-type isolates. Similarly, the NRPS31-specific primer set amplified a fragment only from bakanae-type isolates examined (S7A Fig).
Based on the correlation between the phylogenetically distinct pathotypes on the one hand, and the presence of either the PKS51 or the apicidin F (NRPS31) gene clusters, we examined additional field isolates from Korea using these primer sets. Among a total of 151 isolates examined, the B14-specific PCR amplification pattern was found in 69% of rice isolates, 100% of corn isolates, and 85% of airspora isolates. A phylogenetic tree generated from all of these isolates revealed a clustering of the stunting-type and bakanae-type isolates similar to that for the field isolates examined by pathogenicity tests (S7B Fig). However, it is currently unclear why the stunting-type population is predominant among the field isolates in Korea.
To gain more information on the SM profile of these new stunting-type isolates, we examined the expression levels of the key genes of GA (CPS/KS) and fumonisin (FUM1) biosynthesis in the field isolates grown in liquid culture with 6 mM glutamine by use of quantitative real-time PCR (qPCR). The CPS/KS transcript levels from all the stunting-type isolates examined were similar to or even lower than that in B14, while those from all the bakanae-type isolates, including B20, were 8- to 24-fold higher than those in the stunting-type strains (S8A Fig). Furthermore, all stunting-type isolates showed clear expression of FUM1 in contrast to the bakanae isolates (shown for B20) (S8B Fig). These data indicate that both subgroups of isolates differ in a whole set of characteristic features (presence of either the PKS51 or NRPS31 genes, expression of either GA or fumonisin genes) which correlate with either the stunting or the bakanae pathotype. We also examined the production levels for both SMs in some of the new B14-like strains in vitro and in planta. As expected, the stunting-type isolates produced no or 10 to 15 times less GAs than the bakanae strain IMI 58289, while only the stunting-type strains produced fumonisins in submers cultures (S9A and S9B Fig). The analysis of GAs and fumonisins in rice roots and shoots revealed no GAs at all in seedlings infected with stunting-type isolates, while only the latter produced significant amounts of fumonisins in planta (S9C and S9D Fig). A similar correlation between low GA and high fumonisin levels on the one hand, and a pathotype called “dwarfism” on the other hand, was also described for the Italian F. fujikuroi isolate CSV1 .
In conclusion, we provide genome sequences of eight new F. fujikuroi isolates and one F. oxysporum isolate, all collected in different rice growing regions worldwide. We show that all these strains differ in genome and chromosome size, number of genes in major gene families such as TFs, transporters, SM biosynthetic genes and others. In addition, the isolates differ in colony morphology, pigmentation and the number and type of asexual spores (micro- and/or macroconidia). Major differences were identified in subtelomeric regions of the chromosomes where several SM gene clusters are located. Besides the differences in the presence of gene clusters, we observed variations in the ability to express SM genes and to produce the respective metabolites under in vitro and in planta (rice) conditions. Among the nine F. fujikuroi isolates analyzed, eight cause typical bakanae symptoms on rice seedlings due to their ability to produce GAs. Only one isolate, B14, does not cause elongation of infected plants, but instead causes stunting combined with early withering of rice seedlings. This isolate is the only one which does not produce GAs under in vitro and in planta conditions. Instead, B14 produces high amounts of fumonisins both under in vitro and in planta conditions. Furthermore, it is the only strain containing a putative gene cluster with PKS51 as key gene which is highly expressed in rice. To examine the determinant(s) of the stunting/withering pathotype, several key enzyme-encoding candidate genes were deleted in B14. The data demonstrate that the formation of fumonisins, and probably also fusaric acid, on the one hand, and the lack of GA production on the other hand, contribute to the stunting/withering pathotype, because the deletion of the respective key genes resulted in reduced virulence. Furthermore, the unknown PKS51 gene cluster seems to produce a SM which acts as an attenuator of disease, because the deletion of the PKS51 gene caused early withering of infected seedlings. Examination of more field isolates from Korean rice fields revealed a correlation between the pathotype and the ability to produce either fumonisins or GAs which is supported by the clear separation into two distinct phylogenetic clades.
Material and methods
Fungal and bacterial strains
The fungal strains used in this work and their origin are shown in Table 1. Strain IMI 58289 was derived from Commonwealth Mycological Institute, Kew, United Kingdom. Strains m567 and FSU48 were provided by the Fungal Stock Center at the University Jena, Germany, C1995 by J.F. Leslie, Kansas State University, E282 by S. Tonti, University Bologna, Italy, MRC2276 by W.C.A. Gelderblom, South Africa, and NCIM 1100 was provided by the National Collection of Industrial Microorganisms, India. Strains B14 and B20 were provided by S.-H. Yun, Korea. Strain V64-1 was kindly provided by T. Kyndt from the University Ghent, Belgium.
Escherichia coli strain Top10 F’ (Invitrogen, Groningen, The Netherlands) was used for plasmid propagation. The uracil-auxotrophic Saccharomyces cerevisiae FGSC 9721 (FY 834) was provided by the Fungal Genetics Stock Center (Kansas State University) and used for yeast recombination cloning.
Genome sequencing and assembly
F. fujikuroi B20: Illumina TrueSeq genome sequencing by TheragenEtex, Suwon, Korea. The assembly was performed using Celera Assembler version 7.0 , ‘overlap minimum length’ set to 150 bases. The assembly resulted in 318 scaffolds with a 21-fold coverage of the TrueSeq large reads.
For all other strains sequencing was carried out by shot gun sequencing of an 8 kb library with paired end 100 bp read length using Illumina HiSeq 2000 by Eurofins MWG Operon, Germany. The assemblies were performed by ALLPATHS-LG  and the scaffolds were error corrected by mapping all Illumina shotgun paired-end data and further scaffolded using SSPACE  (Table 2). The data on the new genomes, including annotation, was submitted to the European Nucleotide Archive, study accession PRJEB14872 available at: http://www.ebi.ac.uk/ena/data/view/PRJEB14872. Sample accession numbers are listed in Table 1. RNA-seq data are available at: https://www.ncbi.nlm.nih.gov/gds/?term=GSE89480.
Draft gene models for all genomes were generated by three de novo prediction programs: 1) Fgenesh  with different matrices (trained on Aspergillus nidulans, Neurospora crassa and a mixed matrix based on different species); 2) GeneMark-ES  and 3) Augustus  with Fusarium ESTs and RNA-seq based transcripts as training sets. Annotation was aided by exonerate  hits of protein sequences from F. fujikuroi IMI 58289 and F. oxysporum 4287 to uncover gene annotation gaps and to validate de novo predictions. Transcripts were assembled on the RNA-seq data sets using Trinity . The different gene structures and evidences (exonerate mapping, RNA-seq reads and transcripts) were visualized in GBrowse  allowing manual validation of coding sequences with a focus on SM cluster genes and other genes of interest. The best fitting model per locus was selected manually and gene structures were adjusted by splitting or fusion of gene models and redefining exon-intron boundaries if necessary. tRNAs were predicted using tRNAscan-SE . The predicted protein sets were searched for highly conserved single (low) copy genes to assess the completeness of the genomic sequences and gene predictions. Orthologous genes to all 246 single copy genes were searched for all proteomes by BLASTp comparisons (eVal: 10−3) against the single-copy families from all 21 species available from the FunyBASE . Additionally, the proteomes were searched for the 248 core-genes commonly present in higher eukaryotes (CEGs) by BLASTp comparisons (eVal: 10−3) . We also used BUSCO Version 3.0.1 in the Ubuntu virtual machine with the lineage specific profile library Sordariomyceta_odb9 (3.725 BUSCO groups), downloaded from http://busco.ezlab.org. The analysis was performed in gene set (protein) assessment mode running the python script run_BUSCO.py . All genomes were analyzed using the PEDANT system . To avoid misleading ortholog information based on similarity and bi-directional best hits, QuartetS  was applied to retrieve a reliable ortholog matrix which was used for all comparative representations.
Phylogenetic tree analyses
The phylogenetic tree of Fusarium species was calculated based on the protein sequences of 5,181 single copy genes that are shared among all analyzed species. Orthologs of the sequences were aligned separately using MAFFT . After that, we concatenated the alignments and removed columns with gaps using Gblocks . The evolutionary history was inferred using the Maximum Likelihood method PhyML  with default parameters and the amino acid substitution model LG. Branch support was tested using approximate likelihood ratio test (aLRT) based on the Shimodaira-Hasegawa-like (SH-like) procedure . The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.
We calculated single copy genes in clustering proteins of all genomes and selecting clusters with exactly one representative from each genome. Protein clusters were calculated using usearch  (e-value cutoff: 0.01) and mcl  (inflation value: 2).
For phylogenetic analysis of field isolates of F. fujikuroi in Korea, TEF1α and and RPB2 were amplified from fungal genomic DNAs using the primer sets fRPB2-7cF/fRPB2-11aR  and EF1/EF2 , respectively (S6 Table). All nucleotide sequences from PCR products were edited with Lasergene (ver. 6.0; DNASTAR, Madison, WI, USA) and aligned using ClustalW . Maximum parsimony (MP), neighbor-joining (NJ), and unweighted pair group method with arithmetic mean (UPGMA) analyses were performed using MEGA (ver. 4.02) with 1,000 bootstrap replications.
Contour-clamped homogeneous electric field (CHEF)-gel analysis
Protoplasts from Fusarium strains were prepared as described previously. The protoplast suspension was mixed with 1.2% InCert agarose (Lonza Group AG, Basel, Switzerland) and then loaded on a CHEF gel as described in . Chromosomes of Schizosaccharomyces pombe and S. cerevisiae were used as a molecular size marker (Bio-Rad, Munich, Germany).
In silico identification of secondary metabolite clusters
To identify SM clusters in each genome, the InterPro scan results of the PEDANT analysis were used as described . Essentially, predicted proteins with homology to a domain of a signature SM core enzyme (e.g. PKS, NRPS, TC or DMATS) were considered a marker for a gene cluster. A cluster was verified if any neighboring genes with homology to typical SM cluster enzymes, like P450 monooxygenases, oxidases, methyltransferases, MFS or ABC transporters or TFs, were identified. The extent of each putative gene cluster was then adjusted by comparison to previously published data and to homologous clusters in other Fusarium species.
For SM production experiments and RNA preparation under in vitro conditions, strains were first cultivated for 3 days in 300 mL Erlenmeyer flasks with 100 mL Darken medium  on a rotary shaker at 180 rpm at 28°C. 500 μL of this culture were then used to inoculate 100 mL of ICI (Imperial Chemical Industries, UK) media  containing either 6 mM glutamine, 60 mM glutamine, or 6 mM NaNO3 for 3 days (RNA extraction) or 7 days (SM analysis), respectively. For RNA extraction, mycelia were flash-frozen with liquid nitrogen prior to lyophilization. For in planta expression studies by RNA-seq, lyophilized roots of infected rice plants were used for RNA preparation.
Plant material for RNA sequencing
For the generation of infected rice samples for transcriptome analysis by RNA-seq, rice seeds of the cultivar GSOR 100, Nipponbare, and Dongjin were used. The seeds of the former two were provided by Genetic Stocks-Oryza (GSOR) Collection, USDA ARS Dale Bumpers National Rice Research Center, Hwy, Arkansas, USA.
RNA isolation, RNA sequencing and quantification
Total RNA was extracted from mycelia grown for 3 days in liquid ICI media (containing either 6 or 60 mM glutamine) and from infected rice seedlings after 7 dpi using TRIzol Reagent (Life Technology, Karlsruhe, Germany) and purified using an RNeasy Plant MinElute Cleanup Kit (Qiagen, Hilden, Germany). The quality of DNase-treated RNA (28S:18S > 1.0; RIN≥ 6.5; OD260/280 ≥1.8; OD260/230 ≥ 1.8) was determined using an Agilent Bioanalyzer. The high quality RNA was sent to BGI Tech Solutions Co., Limited (Hong Kong) for library construction and sequencing by Illumina HiSeq2000 technology.
RNA-seq reads were mapped on the reference genome using tophat2 (v2.0.8). The interval for allowed intron lengths was set to min 20 nt and max 1 kb [84–86]. We used cufflinks to determine the abundance of transcripts in FPKM (fragments per kilobase of exon per million fragments mapped) and calculated differentially expressed genes using cuffdiff [85,86]. The gene models were included as raw junctions. Genes with a minimum of fourfold increase or decrease in expression (|log2 of the FPKM values +1| ≥ 2) between the two experimental conditions were considered as regulated. The RNA-seq data has been deposited in NCBI's Gene Expression Omnibus  and are accessible through GEO Series accession number GSE89480.
Generation of vectors for targeted gene deletions and complementations
The DNA constructs for deletion of FUM1 (FFB14_08440, FFB20_01984), FUB1 (FFB14_01651FFB20_13404), and PKS51 (FFB14_06372) from the genomes of F. fujikuroi strains B14 or B20 were created using a split-marker recombination procedure as previously described . To delete FUM1, the 5' and 3' flanking regions of the FUM1 ORF were amplified with the primer pairs JFUM1f5/JFUM1rt5 and JFUM1ft3/JFUM1r3, respectively (in the first round of PCR), fused to the hygromycin B resistance gene (hygB) cassette, which was amplified from pBCATPH  with the primers HygB-for and HygB-rev (in the second round of PCR), and used as a template to generate split markers with the new nested primer sets, JFUM1fn/pUH-BC/H3 and JFUM1rn/pUH-BC/H2, respectively (in the third round of PCR) (S6 Table). Similarly, DNA constructs for targeted deletions of the other genes were created using the strategy described above. For the deletion of FUB1, the primer pairs JFUMB1f5/JFUM1Brt5 and JFUB1ft3/JFUB1r3 were used for amplification of 5' and 3' flanking regions of the FUB1 ORF, respectively, and JFUB1fn/pUH-BC/H3 and JFUB1rn/pUH-BC/H2 were used as the nested primer sets, respectively. For the deletion of PKS51, the primer pairs B14_6372For5/B14_6372rev5t and B14_6372for3t/B14_6372rev3 were used in the first round of PCR, and B14_6372forN/ pUH-BC/H3 and B14_6372revN/ pUH-BC/H2 for the third round of PCR. Additionally, for double deletion of FUM1 and FUB1, we generated a knock-out construct through which the 5′- and 3′-flanking regions of the FUM1 ORF were fused to a geneticin resistance gene cassette (gen) amplified from pII99 using the primer pair Gen-for and Gen-rev, as described above. The resulting construct was transformed into the deletion strain of FUB1.
For the complementation of each deletion mutant of FUM1 or FUB1 derived from B14, intact copies of each gene were amplified from the genome of B14 using the primers JFUMf5/JFUMr3 and JFUB1f5/JFUBr3, respectively, and directly added into protoplasts of each deletion strain along with pSK660 including the geneticin resistance (gen) gene for co-transformation as previously described . All primer sequences are listed in S6 Table.
Pathotests on rice
The effect of the single isolates on rice seedling germination was studied by infecting the seeds with conidia of each isolate. For spore formation, the fungal strains were cultured on PDA plates for 7 days at 28°C under light/dark (12 h/12 h) conditions. The plates were flooded with sterile water to obtain a conidial suspension (1 x 10−6). Seeds were soaked in the suspension for 18 h. Inoculated and non-inoculated (control) seeds were sown into 100 mL plastic pots. Eight days after sowing the number of germinated seeds was assessed. The number of dead, chlorotic and elongated seedlings was measured 15 days after sowing.
For pathogenicity assays, healthy rice seeds were surface-sterilized by submersion in 70% of ethanol followed by 1% of sodium hypochlorite. Sterilized seeds were germinated in Murashige and Skoog (MS) agar  at 26°C for 5 days. The Fusarium isolates were first grown on oatmeal agar for one week. The pathogenicity assays were performed as previously described . Agar plugs from the oatmeal plates were placed on top of 3 cm of sterilized vermiculite in a glass tube (18 cm x 1.6 cm). The agar plugs were then covered with 3 cm of vermiculite. Five-day-old seedlings were transferred to the surface of the vermiculite layer to avoid the direct contact between seedlings and fungal inocula. Before covering the tubes with a cap, 4 mL of Yoshida solution was gently added to each test tube to help retain high humidity  and placed at 26°C for 3, 5, 7 or 9 days. Their heights and internode lengths were measured and photographs of the seedlings and infected roots were taken. The symptom development caused by wild-type and mutant strains was examined in five independent pathogenicity tests. For pathotests with exogenous supply of culture filtrates, the wild-type B14 strain or its mutant lacking FUM1 and FUB1 (Δfum1/Δfub1) was inoculated into 50 mL of PDB (potato dextrose broth) and incubated for 5 days. The fungal liquid cultures were filtered through 2 × cheesecloth followed by filtration through 0.25 μm membranes. The culture fluid was dried to 5 ml by lyophilization (10-fold concentration). For inoculation assay, 500 μl of the concentrated culture filtrate was exogenously supplied to a single rice seedling.
F. fujikuroi B14 and B20 strains were transformed as previously described for F. graminearum . Vector integration events were confirmed by diagnostic PCR (S10 Fig) using specific primers as indicated (S6 Table).
PCR mixtures contained 25 ng of template DNA, 50 ng of each primer (S6 Table), 0.2 mM deoxynucleoside triphosphates, and 1 U of Biotherm Taq polymerase (Genecraft, Lüdinghausen, Germany). The cDNA synthesis was performed using Superscript II (Invitrogen, Groningen, The Netherlands) and 1.5 μg of total RNA as the template, according to the manufacturer's instructions. The qPCR was performed using iTaq Universal SYBR Green Supermix (BioRad) and Superscript II cDNA as template, in a Biorad thermocycler iTaq. In all cases, the qPCR efficiency was between 90–110% and the annealing temperature was 60°C. Gene expression was measured in three biological replicates from each time point, and the relative expression levels were calculated using the ΔΔCt method . The expression of a translation elongation factor α gene (EF1A), amplified by a primer pair (EF1-PS1 and EF1-PS2) (S6 Table), was used as an endogenous reference for data normalization.
Chemical analysis of secondary metabolites in vitro and in planta
For analyses of the SMs, the strains were grown in submerged cultures as described above. After 7 days, mycelia were removed from the culture by filtration through Mirachloth (Calbiochem, Merck KGaA, Darmstadt, Germany). The culture filtrates were filter-sterilized using 0.45 μm syringe filters (BGB, Schloßböckelheim, Germany).
Fusaric acid and beauvericin were obtained from Sigma-Aldrich (Deisenhofen, Germany), GAs from Serva (Heidelberg, Germany) and methylparaben (MePa) was obtained from Fluka (Steinheim, Neu-Ulm, Germany) in analytical grade. The remaining standard substances were obtained as described in previous work [24,25,34,35,42,93–95]. All solvents were obtained in gradient or analytical grade from Sigma-Aldrich, VWR (Darmstadt, Germany) or Merck Schuchardt (Hohenbrunn, Germany). Water was purified by a Milli-Q Gradient A 10 system (Millipore, Schwalbach, Germany).
Liquid culture samples were prepared as following: 10 μL of the culture filtrate and 10 μL of MePa (100 μg/mL) as internal standard were added to 80 μL of water. For in planta analysis, ten rice samples were combined and freeze-dried. The dried samples were treated with liquid nitrogen and pestled simultaneously, larger pieces were cut first with a scalpel. The samples were weighed and extracted with 1.5 mL of the following mixture: ethyl acetate:methanol (MeOH):dichloromethane, 3:2:1. Precellys ceramic beads 1.4/2.8 (Peqlab, Erlangen, Germany) were added to the samples, and the mixture was vortexed for 1 min. Afterwards, the samples were shaken for 1 h on a rotary shaker with 150 rpm. After a short centrifugation step (3 min, 2900 g), 500 μL of the supernatant were transferred to a new vial and the solvent was evaporated to dryness under constant nitrogen flow. The residue was dissolved in 100 μL MeOH and put to an ultrasonic bath for 10 min. After vortexing the samples, they were centrifuged again with 5000 g, and 50 μL of the supernatant were collected. Afterwards, the samples were dried again under nitrogen flow and 1.5 mL of MeOH/water, 3/1 (v/v), + 0.1% formic acid (FA) were added. The extraction procedure described above was repeated. 10 μL of each extract were mixed with 10 μL MePa and 80 μL water for analysis. Some extracts were diluted again 1:10; the corresponding values are labelled.
The liquid culture samples were analyzed as following: A Shimadzu LC-20AD HPLC (Shimadzu, Kyoto, Japan) with a SIL-20ACXR autosampler coupled to a Sciex QTRAP 5500 (Sciex, Darmstadt, Germany) mass spectrometer was used. Separation was carried out on a Synergi Hydro-RP column from Phenomenex (Aschaffenburg, Germany) with 50 × 2.0 mm and 2.5 μm particle size, protected by a KrudKatcher classic filter (Phenomenex), and on a Nucleodur C18 Gravity-SB column from Macherey-Nagel (Düren, Germany) with 50 × 2.0 mm and 3 μm particle size, protected by a KrudKatcher classic filter (Phenomenex), at 50°C. MeOH + 1% FA + 5 mM NH4Ac was used as eluent A, water + 1% FA + 5 mM NH4Ac was used as eluent B. A flow volume of 450 μL/min was applied, and the gradient started at 5% A. This condition was held for 1.5 min. The gradient rose up to 98% A in 10.5 min, and finally the column was rinsed for 3 min with 98%. After that, the column was equilibrated for 2.5 min with 5% A. The integrated valco valve was used, discarding the first 2 min of the run, and the samples were cooled to 7°C. 5 μL of each sample was injected.
Advanced scheduled multiple reaction monitoring (MRM) mode was used for acquisition. Both positive and negative ionization mode were applied. The curtain gas (CUR) was set to 35 psi, the collision gas was set to medium. The temperature of the heater gas (TEM) in the ion source was set to 450°C. Nebulizer gas (GS1) and auxiliary gas (GS2) were adjusted to 35 and 45 psi, respectively.
In the positive MRM mode, the ion spray voltage (IS) was set to 4500 V, and the target scan time of this experiment was adjusted to 0.3 s, resulting in a cycle time of 0.5 s. The positive ionization mode was applied for the relative quantification of O-methyl-fusarubin, fusaric acid, gibepyrone A, apicidin F, beauvericin, fumonisins (FB1+FB2), fusarins, trichosetin and fujikurin A. The cell exit potential (CXP) was set to 11 V, the entrance potential (EP) was set to 10 V. For the negative MRM mode, the IS was set to -4500 V and the target scan time was adjusted to 0.2 s, resulting in a cycle time of 0.5 s. The negative ionization mode was applied for the relative quantification of MePa, and the gibberellins GA3, GA4 and GA7. The CXP was set to 11 V, the EP was set to 10 V.
MRM transition for the quantification were as follows: FB1−722.4–223.1; FB2−706.4–336.3; GA3−345.2–143.0; GA4−331.3–243.1; GA7−331.3–243.1. The calibration curve for all the standards was prepared in a concentration range of 0.0001–1 μg/ml.
For bikaverin determination in liquid culture samples, HPLC-UV measurements were carried out on a Shimadzu LC-20AT pump system with a Shimadzu SIL autosampler and a photodiode array (PDA). A Gemini 5 u C6-Phenyl 110A, 250 × 4.60 mm, 5 μm column (Phenomenex) was used, with water + 1% FA as eluent A and acetonitril + 1% FA as eluent B. The column oven was set to 40°C. The gradient started with 10% B with 1.35 μL/min. After 3 min, the gradient rose up to 100% B during 17 min. The column was washed with 100% B for 6 min, and afterwards the column was equilibrated with 10% B again for 4 min. The wavelength for PDA analysis ranged from 220–600 nm. 100 μL of the sterile culture filtrate were injected. Peak areas were determined at 508 nm, and bikaverin obtained from Sigma Aldrich was used as standard substance.
In planta sample analysis was performed with a different HPLC-system but the same mass spectrum. An Agilent 1260 HPLC system (Santa Clara, USA) was used, and the expected retention time for scheduled MRM analysis needed to be adjusted (S7–S10 Tables). Furthermore, bikaverin was analyzed in positive ionization mode.
S2 Fig. Northern blot analysis for comparison of bikaverin (BIK2) and fusarubins (FSR2) gene expression.
The strains were grown for 3 days in synthetic medium with either 6 mM glutamine (bikaverin) or 6 mM NaNO3 (fusarubins) as nitrogen source.
S3 Fig. Content and arrangement of genes in gene clusters with variations in the single isolates.
(A) The apicidin F (NRPS31) gene cluster is present in most isolates but missing in strain B14. (B) The fumonisin (PKS11) gene cluster is present in most of the strains, but several genes of the cluster are missing in strain B20. 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.
S4 Fig. Rice seedlings inoculated with F. fujikuroi wild-type and mutant strains with or without addition of culture fluids to the pathosystem.
(A) Shoot growth of rice seedlings inoculated with B14 (wild type) and the B14 deletion and complemented strains 9 days after inoculation. (B, C, D) Shoot growth of rice seedlings inoculated with strain B14 (B), B20 (C) or the GA-deficient mutant strain of B20 (B20 Δcps/ks) (D) with or without the exogenous supply of the culture filtrate of B14 or its Δfum1/Δfub1 mutant. Mock: no fungal inoculation; Δfum1 and Δfub1 –deletion strains for the fumonisin and fusaric acid key genes, respectively; FUM1C and FUB1C –complemented strains carrying an intact copy of FUM1 or FUB1, respectively.
S5 Fig. Shoot and root growth of rice seedlings after inoculation with B20 mutants.
Rice seedlings (A-C) and roots (D) inoculated with the gene deletion strains derived from the F. fujikuroi B20 strain. Mock: no fungal inoculation; Δfum1, Δfub1, and Δcps/ks–deletion strains for the fumonisin, fusaric acid and giberellic acid key genes, respectively.
Shoot (A) and root (B) growth of rice seedlings 7 days after inoculation of high fumonisin-producing F. verticillioides strains. Mock: no fungal inoculation; B14: the F. fujikuroi B14 strain; FvOS35: the F. verticilliodies OS35 strain; FvOS40: the F. verticilliodies OS40 strain.
S7 Fig. Differentiation of field isolates for their B14-like or B20-like pathotype by PCR and phylogenetic analysis.
(A) Diagnostic PCR amplification using the primer pairs derived from PKS51 (unknown product, 382 bp) and NRPS31 (apicidin F, 434 bp), respectively. (B) Phylogenetic tree constructed by the NJ method using the nucleotide sequences of combined TEF1 and RPB2 from additional field isolates, determined by the diagnostic PCR of (A).
S8 Fig. Relative transcript levels for the key genes of GA (CPS/KS) and fumonisin (FUM1) biosynthesis.
The transcript levels of CPS/KS (A) and FUM1 (B) were determined by qPCR using total RNA from several F. fujikuroi field isolates grown in ICI liquid medium containing 6 mM glutamine for 7 d. Amplification levels of CPS/KS and FUM1 in the B14 strain were used as a reference (set to 1.0). The same letter above bars indicates no significant difference.
Biosynthesis of gibberellic acids (GA3, GA4, GA7) and fumonisins (FB1, FB2) of stunting-type F. fujikuroi isolates compared to bakanae strains under in vitro (A, B) and in planta (C, D) conditions. GA (A) and fumonisin (B) production levels after 7 days of growth in synthetic medium with 6 mM glutamine. The strains were grown in triplicates. GA (C) and fumonisin (D) production levels in rice roots and shoots 7 dpi. For in planta analyses, ten plants per isolate were used.
Confirmation of the gene deletions (A-F) or complementations (G and H) by PCR. Left panel in each figure: gene deletion or complementation schemes, right panel: PCR gel picture. The genomic positions of the primer pairs (S6 Table) used in the PCR amplification and expected size of PCR products (designated a or b) are indicated in the deletion schemes and the gels, respectively. In most cases, two independent strains with a gene deletion or a gene add-back (designated 1 and 2) were used in PCR along with their wild-type (WT) progenitor and those carrying a single gene deletion (SD). For gene complementation, we used a co-transformation strategy as previously described .
S1 Table. BUSCO single-copy analysis, performed in gene set (protein) assessment mode on the library Sordariomyceta_odb9.
S2 Table. Distribution of the mating type loci among the F. fujikuroi isolates.
S3 Table. Presence of secondary metabolite gene clusters in the analyzed Fusarium strains.
S4 Table. Rice seed germination assay.
(A) Expression data and list of all genes with a transcription factor specific InterPro ID in the corresponding protein sequence. (B) Expression data and list of 37 genes which are present in most of the 9 genomes and which are specifically up-regulated during infection of rice. (C) Expression data and list of 28 B14 strain-specific TFs which are not present in the genomes of the other strains.
S6 Table. PCR primers used in this work.
S7 Table. MRM transitions for liquid culture analysis in positive ionization mode, depicting also transition specific variables.
S8 Table. MRM transitions for liquid culture analysis in negative ionization mode, depicting also transition specific variables.
S9 Table. MRM transitions for in planta analysis in positive ionization mode, depicting also transition specific variables.
S10 Table. MRM transitions for in planta analysis in negative ionization mode, depicting also transition specific variables.
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